Essentials in Ophthalmology

Series Editor: Arun D. Singh

Jorge L. Alió
Jorge L. Alió del Barrio, and
Francisco Arnalich-Montiel Editors

Corneal
Regeneration
Therapy and Surgery
Essentials in Ophthalmology

Series Editor
Arun D. Singh

More information about this series at http://www.springer.com/series/5332

Jorge L. Alió
Jorge L. Alió del Barrio
Francisco Arnalich-Montiel
Editors

Corneal Regeneration
Therapy and Surgery
Editors
Prof. Jorge L. Alió MD, PhD, FEBO
Professor and Chairman of Ophthalmology
University Miguel Hernandez
Vissum-Instituto Oftalmologico de Alicante
Alicante
Spain

Jorge L. Alió del Barrio Francisco Arnalich-Montiel

University Miguel Hernandez Vissum Corporation
Vissum-Instituto Oftalmologico Madrid
de Alicante Spain
Alicante
Spain

ISSN 1612-3212     ISSN 2196-890X (electronic)

Essentials in Ophthalmology
ISBN 978-3-030-01303-5    ISBN 978-3-030-01304-2 (eBook)
https://doi.org/10.1007/978-3-030-01304-2

Library of Congress Control Number: 2018963751

© Springer Nature Switzerland AG 2019

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or
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The use of general descriptive names, registered names, trademarks, service marks, etc. in this
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The publisher, the authors, and the editors are safe to assume that the advice and information in
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This book is dedicated to our families and our patients, who
are those who enlighten our daily life with happiness and ideas
and who make our lives useful.
Acknowledgement

Prof. Jorge L. Alió would like to thank his supporting co-editors Jorge L. Alió
del Barrio, and Francisco Arnalich-Montiel for their contributions to this
work.

Alicante, Spain Prof. Jorge L. Alió

vii
Preface

 orneal Regeneration, the Present and the Future

C
of an Emerging Surgical Solution for Corneal Disease

Regeneration of human tissue is one of the most challenging and promising

topics in surgery and indeed in the whole of medicine. Eye care today, based
mainly on surgical options and medication, is finding the way to provide bet-
ter care for our patients affected by ocular disease using biological methods
of therapy, including tissue bioengineering which offers a new alternative that
may lead to a totally different perspective in the way in which we consider the
treatment of eye disease today.
Eye disease severely affects our quality of life. The possibility of success-
fully treating many ocular diseases which lead to visual impairment with
methods which are today the standards of care that do not involve surgical
options based on tissue substitution (transplantation), use of long-term ther-
apy or even mutilation caused by the elimination of diseased tissue and
replacement by new tissue (like in pseudophakia) is indeed extremely attrac-
tive. When accomplished, well-developed tissue regeneration therapy will be
considered as one of the major steps forward in human medicine.
This alternative is now being pursued by researchers and innovative clini-
cians and surgeons finding a way to obtain new methods that are more ana-
tomical and customized than those that we have today, even though these are
successful.
The cornea is a privileged tissue for studies on tissue regeneration. It is the
forefront of the eye, and due to its transparency, it provides the first step for
vision. Corneal blindness is one of the leading causes of blindness in the
world and can be cured only by tissue substitution, which is not always pos-
sible or even available. Corneal transplantation has been evolving over the
last 20 years into different modalities which include lamellar grafts of the
anterior and posterior surfaces that are, in fact, today, the most popular ways
to treat corneal disease. However, we are all aware of the limitations of cor-
neal grafting based on the availability of tissue and the biological hazard
always associated to these procedures.
The cornea, due to its accessibility, is where the most important advances
in corneal regeneration therapy and surgery are currently being developed. In
this book, the reader will find the most advanced and promising authors,
offering the alternative step that corneal regeneration may provide to clinical

ix
x Preface

practice today. From animal models to real applications based on data

obtained in clinical human studies, corneal regeneration is taking a great step
ahead towards an application that will change corneal surgery, creating a new
revolution. From ocular surface regeneration, corneal stroma regeneration
and corneal endothelial cell therapy, we may consider that there is an imme-
diate future in which not only shall we be able to regenerate these three parts
of the cornea (surface, stroma and endothelium), but, considered altogether,
we could regenerate the whole cornea based on autologous or cell bank tis-
sue. This is in part recreation and involves many challenges, from biological,
ethical, organizational and probably financial costs. Of all these, the funda-
mental use of autologous stem cells for these purposes harbours huge benefits
in terms of ethics and availability although we can already imagine the
emerging role that cell therapy banks can offer in the future.
We welcome the reader to this book which is the first of its type on this
topic and the first to offer in a systematic and improved way the developments
that we have obtained today in corneal regeneration. We hope that this will
open and stimulate the imagination of the reader and researchers in order to
go further and faster in this new and most promising way and stage of eye
surgery.
Just to finish this preface, I would like to thank all the participants, authors
and investigators of different origins who have participated directly or indi-
rectly in this book. Without their generous contribution, their ideas and their
hard work over many years in the background, something like this would not
have been possible. They have enlightened the future and this book aims to be
the platform to support all this light that will take our practice to a higher level
for the benefit of our corneal diseased patients.

Alicante, Spain Prof. Jorge L. Alió

Perspectives on Regenerative Medicine

It is now widely recognized that regenerative medicine is sure to open novel

treatment pathways deep into the twenty-first century, as it follows along the
line of organ and tissue transplantation, as well as stem cell-related transla-
tional research [1]. In regard to the former, it is set to replace organ replace-
ment therapies, such as heart, liver and kidney transplantations, via the use of
sophisticated cell therapies and/or tissues and organs created by tissue engi-
neering procedures [2, 3]. In regard to the latter, specific haematopoietic stem
cell transplantation, mesenchymal stem cell transplantation, cultured skin
sheets, corneal epithelial stem cell sheets, etc., can now be observed. In regard
to the cells used for regenerative medicine, there are two different strategies.
The first involves the direct use of stem cells in culture, while the second
involves the use of tissue-specific well-differentiated cells in culture. In
today’s regenerative medicine, two different concepts and strategies exist:
one is the direct transfer of cells and/or tissues that, expectedly, possess nor-
mal cell functions, while the other aims at releasing miracle substances,
including some cytokines, from the transferred cultured cells to the recipient
damaged/diseased organ, an example of which can now be seen in the heart-­
related field of regenerative medicine.
In the corneal field, upcoming medical advancements include superior
methods of donor corneal graft preparation prior to surgery. Previous cornea-­
related surgical procedures, such as keratophakia or epikeratophakia, as well
as more modern corneal endothelial grafting techniques, such as DSAEK and
DMEK, are prime examples [4]. From the aspect of pioneering corneal regen-
erative medicine techniques, autologous cultured corneal epithelial [5, 6] and
oral mucosal epithelial cell sheets [7, 8], as well as synthetic artificial corneal
grafts [9], have all been used in the clinical setting. Moreover, mesenchymal
stem cells have recently been applied for the reduction of corneal scarring
and restoration of corneal transparency, as have autologous adipose-derived
adult stem cells for the treatment of keratoconus [10, 11], a disorder that
results in a progressive thinning of the cornea. In addition, a novel cultured
corneal endothelial cell injection therapy for the treatment of bullous kera-
topathy [12] has recently been introduced. Some of these are truly ‘stem cell-­
specific’ therapies, while others are regenerative medicine-oriented treatments
that involve the use of cultured well-differentiated cells.
It is widely known that there are extremely important issues that must
firmly be addressed when considering the introduction of regenerative medi-
cine products to the clinical stage worldwide. This is clearly illustrated by the

xi
xii Perspectives on Regenerative Medicine

fact that although an impressive number of novel and significant academic

research findings have been published in the field of corneal regenerative
medicine, very few cell products have been officially approved by govern-
ment agencies. In many countries worldwide, the approved medical products
are categorized into three large groups, including pharmaceutical agents,
medical devices and regenerative medicine products. Of those, regenerative
medicine products fall into the newest category and are strictly regulated by
laws and/or guidelines set by each government. The primary point of govern-
ment laws and guidelines is to eliminate vague and/or false regenerative med-
icine products as well as to strictly control the products from the aspect of
safety and efficacy. While it is true that academic research scientists tend to
focus on the efficacy, regulatory authorities place the most importance on
safety, which is a vital perspective of regulatory science. Since most regen-
erative medicine products deal directly with in vitro human cells, the possi-
bility exists that recipients risk adverse events via dangerous unexpected
chemicals and/or viruses, thus illustrating why strict government regulation is
vitally important. With that aside, it remains undeniable that product efficacy
is a primary aspect [13, 14]. To reach that goal, the applied concept of ‘quality
by design’ [15] assures equal quality and flawless repeatability of the prod-
ucts, which is essential. To verify efficacy and safety in a clinical trial, one
must set the primary endpoint from not only the viewpoint of clinicians and
researchers but also via a general consensus of our society, which is the res-
toration of visual function. Moreover, the concept of a well-organized surro-
gate endpoint is important. In some ways, there are several important points
that slightly differ between the process of research and development and the
product development. Cutting-edge medical products originating from
embryonic stem cells, induced pluripotent stem cells and direct conversion
cells will assuredly emerge, as they may be key to unlocking the door to
future treatment pathways, thus once again illustrating the importance of
regulatory science, especially in regenerative medicine.

Shigeru Kinoshita
Department of Frontier Medical Science and Technology for
Ophthalmology
Kyoto Prefectural University of Medicine
Kyoto, Japan

References
1. Editorial. Advancing regenerative medicine. Nature Med. 2014;20(8):795.
2. Langer R, Vacanti JP. Tissue engineering. Science. 1993;260(5110):920-6.
3. Dimmeler S, Ding S, Rando TA, Trounson A. Translational strategies and challenges in
regenerative medicine. Nature Med. 2014;20(8):814-21.
4. Tan DT, Dart JK, Holland EJ, Kinoshita S.  Corneal transplantation. Lancet.
2012;379(9827):1749-61.
5. Pellegrini G1, Traverso CE, Franzi AT, Zingirian M, Cancedda R, De Luca M. Long-
term restoration of damaged corneal surfaces with autologous cultivated corneal epithe-
lium. Lancet. 1997;349(9057):990-3.
Perspectives on Regenerative Medicine xiii

6. Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G. Limbal stem-

cell therapy and long-term corneal regeneration. N Engl J Med. 2010;363(2):147-55.
7. Nishida K, Yamato M, Hayashida Y, et  al. Corneal reconstruction with tissue-engi-
neered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med.
2004;351(12):1187-96.
8. Sotozono C, Inatomi T, Nakamura T, et al. Visual improvement after cultivated oral
mucosal epithelial transplantation. Ophthalmology. 2013;120:193-200.
9. Fagerholm P, Lagali NS, Merrett K, et al. A biosynthetic alternative to human donor tis-
sue for inducing corneal regeneration: 24-month follow-­up of a phase 1 clinical study.
Sci Transl Med. 2010;2(46):46-61.
10. Alio Del Barrio JL, El Zarif M, de Miguel MP, et  al. Cellular therapy with human
autologous adipose-derived adult stem cells for advanced keratoconus. Cornea.
2017;36(8):952-60.
11. Alio Del Barrio JL, El Zarif M, Azzar A, et  al. Corneal stroma enhancement with
decellularized stromal laminas with or without stem cell recellularization for advanced
keratoconus. Am J Ophthalmol. 2018;186:47-58.
12. Kinoshita S, Koizumi N, Ueno M, et al. Injection of cultured cells with a ROCK inhibi-
tor for bullous keratopathy. N Engl J Med. 2018;378:995-1003.
13. Charo RA, Sipp D.  Rejuvenating regenerative medicine regulation. N Engl J Med.
2018;378(6):504-5.
14. Marks P, Gottlieb S. Balancing safety and innovation for cell-based regenerative medi-
cine. N Engl J Med. 2018;378(10):954-9.
15. Lipsitz YY, Timmins NE, Zandstra PW. Quality cell therapy manufacturing by design.
Nat Biotech. 2016;34:393-400.
Contents

Part I Corneal Regeneration: The Concept, the Facts, the Potential

1 Corneal Anatomy ����������������������������������������������������������������������������   3

Miguel Gonzalez-Andrades, Pablo Argüeso, and Ilene Gipson
2 Corneal Healing ������������������������������������������������������������������������������  13
Veronica Vargas, Francisco Arnalich-Montiel,
and Jorge L. Alió del Barrio
3 Corneal Tissue Engineering������������������������������������������������������������  23
Mohammad Mirazul Islam, Roholah Sharifi,
and Miguel Gonzalez-Andrades

Part II The Stem Cell

4 Stem Cells: Concept, Properties, and Characterization��������������  41

Natalia Escacena-Acosta, Javier Lopez-Beas,
Christian Claude Lachaud, Mehrdad Vakilian,
Juan Rigoberto Tejedo, Vivian Capilla-González,
Francisco Javier Bedoya, Franz Martin, Abdelkrim Hmadcha,
and Bernat Soria
5 Corneal Stem Cells: Identification and Methods of Ex Vivo
Expansion�����������������������������������������������������������������������������������������  57
Christian Claude Lachaud, Abdelkrim Hmadcha,
and Bernat Soria
6 Corneal Epithelial Stem Cells: Methods for Ex Vivo
Expansion�����������������������������������������������������������������������������������������  77
Gustavo S. Figueiredo, Hardeep Singh Mudhar,
Majlinda Lako, and Francisco C. Figueiredo
7 Corneal Stromal Stem Cell: Methods for Ex Vivo Expansion ����  99
Olena Al-Shymali, Jorge L. Alió del Barrio,
and James L. Funderburgh
8 Corneal Endothelial Cells: Methods for Ex Vivo Expansion ������ 109
Stephen Wahlig, Matthew Lovatt, Gary Swee-­Lim Peh,
and Jodhbir S. Mehta

xv
xvi Contents

9 Corneal Regeneration: Use of Extracorneal Stem Cells�������������� 123

Christian Claude Lachaud, Abdelkrim Hmadcha,
and Bernat Soria
10 One Cell, Two Phenotypes: Capturing Pluripotency for Corneal
Regeneration������������������������������������������������������������������������������������ 145
Trevor Sherwin, Carol Ann Greene, Colin R. Green,
and Kushant R. Kapadia
11 Corneal Stem Cell-Based Therapies���������������������������������������������� 155
Yuzuru Sasamoto, Yoshinori Oie, and Kohji Nishida

Part III Regenerative Surgery and Therapy of the Ocular Surface

Epithelium

12 Ocular Surface Epithelium: Applied Anatomy ���������������������������� 175

Harminder Singh Dua and Dalia G. Said
13 Classical Techniques for Limbal Transplantation������������������������ 191
Rafael I. Barraquer and Juan Alvarez de Toledo
14 Simple Limbal Epithelial Transplantation: An Update���������������� 213
Nandini Venkateswaran and Guillermo Amescua
15 Cell Therapy Using Ex Vivo Cultured Limbal Cells:
CLET and Equivalent���������������������������������������������������������������������� 221
Paolo Rama and Giulio Ferrari
16 Cell Therapy Using Cultivated Oral Mucosal Epithelial
Transplant (COMET)���������������������������������������������������������������������� 225
Roberto Fernández Buenaga and Sajjad Ahmad
17 Cell Therapy Using Extraocular Mesenchymal Stem Cells �������� 231
Teresa Nieto-Miguel, Sara Galindo, Marina López-­Paniagua,
Inmaculada Pérez, José M. Herreras, and Margarita Calonge
18 Cell-based Therapy Using Induced Plutipotent Stem Cell���������� 263
Ricardo Pedro Casaroli-Marano
19 Cultivated Limbal Stem Cell Transplantation: Indications
and Technique���������������������������������������������������������������������������������� 277
Joséphine Behaegel, Sorcha Ní Dhubhghaill,
and Marie-José Tassignon
20 Optimizing the Ocular Surface for Regenerative Surgery:
What Is Important and What Is Essential for the Outcome�������� 291
Kai B. Kang and Ali R. D’jalilian
21 Stem Cell Spheres for Corneal Regeneration�������������������������������� 299
Salim Ismail, Jennifer J. McGhee, Ye Li, Jeremy John
Mathan, Jinny Jung Yoon, Himanshu Wadhwa,
Stephanie U-Shane Huang, and Trevor Sherwin
22 Eye Platelet-Rich Plasma (E-PRP) for Corneal Regeneration���� 317
Alejandra E. Rodríguez and Jorge L. Alió
Contents xvii

Part IV Regenerative Surgery of the Corneal Stroma

23 Applied Anatomy of the Corneal Stroma�������������������������������������� 349
Harminder Singh Dua and Dalia G. Said
24 Confocal Microscopy of the Cornea in a Clinical Model
of Corneal Stromal Expansion Using Adipose Stem Cells
and Corneal Decellularized Laminas in Patients
with Keratoconus ���������������������������������������������������������������������������� 363
Mona El Zarif, Karim Abdul Jawad, and Jorge L. Alió
25 Limbal Stromal Stem Cells in Corneal Wound Healing:
Current Perspectives and Future Applications ���������������������������� 387
Noopur Mitragotri, Mukesh Damala, Vivek Singh,
and Sayan Basu
26 Cell Therapy of the Corneal Stroma Using Ex Vivo Cultured
Extraocular Cells ���������������������������������������������������������������������������� 403
Jorge L. Alió del Barrio

Part V Regenerative Surgery of the Corneal Endothelium

27 Corneal Endothelium: Applied Anatomy�������������������������������������� 419

Francisco Arnalich-Montiel
28 Corneal Endothelium: Isolation and Cultivation Methods���������� 425
David Mingo-Botín, Marie Joan Therese D. Balgos,
and Francisco Arnalich-Montiel
29 Corneal Endothelial Cell Transplantation: Animal Models�������� 437
Brad P. Barnett and Albert S. Jun
30 Cell Based Therapy for Corneal Endothelial Regeneration�������� 455
Noriko Koizumi and Naoki Okumura
31 Corneal Endothelium Regeneration: Future Prospects �������������� 463
Wei-Ting Ho, Hsin-Yu Liu, Fung-Rong Hu, and I-Jong Wang

Part VI Bioengineering Cornea Surgery

32 Umbilical Cord Stem Cells in the Treatment of Corneal

Diseases �������������������������������������������������������������������������������������������� 477
Mohammed Ziaei, Jie Zhang, Dipika V. Patel,
and Charles N. J. McGhee
33 Dysfunctional Corneal Endothelium:
Delivery of Cell Therapy ���������������������������������������������������������������� 485
Stephen Wahlig, Gary Swee-Lim Peh, Matthew Lovatt,
and Jodhbir S. Mehta
Index���������������������������������������������������������������������������������������������������������� 499
Contributors

Sajjad Ahmad, MB, BS, FRCOphth, PhD  Cornea and External Diseases

Department, Moorfields Eye Hospital, London, UK
Jorge L. Alió, MD, PhD, FEBO  Professor and Chairman of Ophthalmology,
University Miguel Hernandez, Vissum-Instituto Oftalmologico de Alicante,
Alicante, Spain
Jorge  L. Alió  del Barrio, MD, PhD, FEBOS-CR University Miguel
Hernandez, Vissum-Instituto Oftalmologico de Alicante, Alicante, Spain
Olena Al-Shymali, MD  Vissum Corporation, Alicante, Spain
Juan  Alvarez  de Toledo, MD, PhD, FEBO-CR  Centro de Oftalmología
Barraquer, Anterior Segment, Department, Barcelona, Spain
Institut Universitari Barraquer, Universitat Autónoma de Barcelona, Barcelona,
Spain
Guillermo  Amescua, MD Bascom Palmer Eye Institute, University of
Miami, Department of Ophthalmology, Miami, FL, USA
Pablo  Argüeso, PhD Massachusetts Eye and Ear and Schepens Eye
Research Institute, Department of Ophthalmology, Harvard Medical School,
Boston, MA, USA
Francisco  Arnalich-Montiel, MD, PhD, FEBOS-CR Vissum Corporation,
Madrid, Spain
Marie Joan Therese D. Balgos, MD  Research and Development Department,
Cornea, Cataract and Refractive Surgery Department, Vissum Alicante,
Alicante, Spain
Brad  P.  Barnett, MD, PhD Wilmer Eye Institute at Johns Hopkins,
Department of Ophthalmology, Baltimore, MD, USA
Rafael I. Barraquer, MD, PhD  Centro de Oftalmología Barraquer, Anterior
Segment Department, Barcelona, Spain
Sayan  Basu, MBBS, MS Center for Ocular Regeneration (CORE), LV
Prasad Eye Institute, Banajara Hills, Hyderabad, Telangana, India

xix
xx Contributors

Francisco Javier Bedoya, PhD, MD  Department of Cell Regeneration and

Advanced Therapies, Andalusian Center of Molecular Biology and
Regenerative Medicine-CABIMER, Junta de Andalucía-University of Pablo
Olavide-University of Seville-CSIC, Seville, Andalusia, Spain
Joséphine  Behaegel, MD Faculty of Medicine and Health Sciences,
Department of Ophthalmology, Visual Optics and Visual Rehabilitation,
University of Antwerp, Campus Drie Eiken, Antwerp, Belgium
Department of Ophthalmology, Antwerp University Hospital, Edegem, Belgium
Center for Cell Therapy and Regenerative Medicine, Antwerp University
Hospital, CCRG-Oogheelkunde, Edegem, Belgium
Department of Ophthalmology, Brussels University Hospital, Jette, Belgium
Roberto Fernández Buenaga, MD, PhD  Cornea, Cataract and Refractive
Surgery Department, Vissum Madrid, Madrid, Spain
Margarita  Calonge, MD, PhD CIBER-BBN (Biomedical Research
Networking Centre in Bioengineering, Biomaterials and Nanomedicine),
Carlos III National Institute of Health, Valladolid, Spain
IOBA (Institute of Applied Ophthalmobiology), University of Valladolid,
Valladolid, Spain
Vivian  Capilla-González, PhD Department of Cell Regeneration and
Advanced Therapies, Andalusian Center of Molecular Biology and
Regenerative Medicine-CABIMER, Junta de Andalucía-University of Pablo
Olavide-University of Seville-CSIC, Seville, Andalusia, Spain
Ricardo Pedro Casaroli-Marano, MD, MSc, PhD  Department of Surgery,
School of Medicine and Hospital Clinic de Barcelona, University of
Barcelona, Barcelona, Spain
Institute of Biomedical Research (IIB-Sant Pau) and Barcelona Tissue Bank,
Banc de Sang i Teixits, Barcelona, Spain
Mukesh  Damala, MSc Biochemistry Center to Ocular Regeneration
(CORE); and Brien Holden Eye Research Center, LV Prasad Eye Institute,
Hyderabad, Telangana, India
School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India
Harminder Singh Dua, MBBS, DO, MS, MD, PhD  Academic Section of
Ophthalmology, Division of Clinical Neuroscience, University of Nottingham,
Nottingham, UK
Department of Ophthalmology, Queens Medical Centre, University Hospitals
NHS Trust, Nottingham, UK
Mona El Zarif, Master in Optometry, OD  Optica General Sarl, Department
of Optometry and contactology, Saida, Lebanon
Vissum Instituto Oftalmologico de Alicante, Alicante, Spain
Natalia  Escacena-Acosta, PhD Department of Cell Regeneration and
Advanced Therapies, Andalusian Center of Molecular Biology and
Regenerative Medicine-CABIMER, Junta de Andalucía-University of Pablo
Olavide-University of Seville-CSIC, Seville, Andalusia, Spain
Contributors xxi

Giulio  Ferrari, MD, PhD San Raffaele Hospital, Department of

Ophthalmology—Cornea and Ocular Surface, Milan, Italy
Francisco  C.  Figueiredo Newcastle University, Institute of Genetic
Medicine, International Centre for Life, Newcastle upon Tyne, Tyne and
Wear, UK
Royal Victoria Infirmary, Newcastle upon Tyne, UK
Gustavo S. Figueiredo, MB, ChB, PhD  Newcastle University, Institute of
Genetic Medicine, International Centre for Life, Newcastle upon Tyne, Tyne
and Wear, UK
James  L.  Funderburgh, PhD University of Pittsburgh, Department of
Ophthalmology, Pittsburgh, PA, USA
Sara Galindo, PhD  CIBER-BBN (Biomedical Research Networking Centre
in Bioengineering, Biomaterials and Nanomedicine) Carlos III National
Institute of Health, Madrid, Spain
IOBA (Institute of Applied Ophthalmobiology), University of Valladolid,
Valladolid, Spain
Ilene Gipson, PhD  Massachusetts Eye and Ear and Schepens Eye Research
Institute, Department of Ophthalmology, Harvard Medical School, Boston,
MA, USA
Ali  R.  D’jalilian, MD University of Illinois at Chicago, Department of
Ophthalmology and Visual Sciences, Chicago, IL, USA
Miguel  Gonzalez-Andrades, MD, PhD Massachusetts Eye and Ear and
Schepens Eye Research Institute, Department of Ophthalmology, Harvard
Medical School, Boston, MA, USA
Carol  Ann  Greene, PhD Department of Ophthalmology, New Zealand
National Eye Centre, Faculty of Medical and Health Sciences, The University
of Auckland, Auckland, New Zealand
Colin  R.  Green, BSc, MSc, PhD, DSc Department of Ophthalmology,
New Zealand National Eye Centre, Faculty of Medical and Health Sciences,
The University of Auckland, Auckland, New Zealand
José  M.  Herreras, MD, PhD CIBER-BBN (Biomedical Research
Networking Centre in Bioengineering, Biomaterials and Nanomedicine)
Carlos III National Institute of Health, Valladolid, Spain
IOBA (Institute of Applied Ophthalmobiology), University of Valladolid,
Valladolid, Spain
Department of Ophthalmology, Clinic University Hospital, Valladolid, Spain
Abdelkrim Hmadcha, PhD  Department of Cell Regeneration and Advanced
Therapies, Andalusian Center of Molecular Biology and Regenerative
Medicien-CABIMER, Junta de Andalucía-University of Pablo Olavide-­
University of Seville-CSIC, Seville, Andalusia, Spain
xxii Contributors

Wei-Ting  Ho, MD  Department of Ophthalmology, Far Eastern Memorial

Hospital, New Taipei City, Taiwan
Fung-Rong  Hu, MD Department of Ophthalmology, National Taiwan
University Hospital, Taipei, Taiwan
College of Medicine, National Taiwan University, Taipei, Taiwan
Stephanie  U-Shane  Huang, Bachelor of Science (Hons)  Department of
Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and
Health Sciences, The University of Auckland, Auckland, New Zealand
Mohammad  Mirazul  Islam, MSc, PhD  Massachusetts Eye and Ear and
Schepens Eye Research Institute, Department of Ophthalmology, Harvard
Medical School, Boston, MA, USA
Salim Ismail, BSC, MSc (1st Class Hons)  Department of Ophthalmology,
New Zealand National Eye Centre, Faculty of Medical and Health Sciences,
The University of Auckland, Auckland, New Zealand
Karim Abdul Jawad, Bachelor of Science  Optica General Sarl, Department
of Optometry and Contactology, Saida, Lebanon
University of Nicosia, Department of Life and Health Sciences, Nicosia, Cyprus
Albert S. Jun, MD, PhD  Wilmer Eye Institute at Johns Hopkins, Department
of Ophthalmology, Baltimore, MD, USA
Division of Cornea, Cataract and External Eye Diseases, Baltimore, MD, USA
Kushant R. Kapadia, BSc, PGDipSci, MSc  Department of Ophthalmology,
New Zealand National Eye Centre, Faculty of Medical and Health Sciences,
The University of Auckland, Auckland, New Zealand
Christian  Claude  Lachaud, PhD Department of Cell Regeneration and
Advanced Therapies, Andalusian Center of Molecular Biology and
Regenerative Medicine-CABIMER, Junta de Andalucía-University of Pablo
Olavide-University of Seville-CSIC, Seville, Andalusia, Spain
Majlinda  Lako, PhD, MSc, BSc Newcastle University, Institute of Genetic
Medicine, International Centre for Life, Newcastle upon Tyne, Tyne and Wear, UK
Javier Lopez-Beas, PhD  Department of Cell Regeneration and Advanced
Therapies, Andalusian Center of Molecular Biology and Regenerative
Medicine-CABIMER, Junta de Andalucía-University of Pablo Olavide-­
University of Seville-CSIC, Seville, Andalusia, Spain
Kai  B.  Kang, MD University of Illinois at Chicago, Department of
Ophthalmology and Visual Sciences, Chicago, IL, USA
Noriko  Koizumi, MD, PhD Department of Biomedical Engineering,
Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe,
Kyoto, Japan
Ye  Li, MBBS (Student) Department of Ophthalmology, New Zealand
National Eye Centre, Faculty of Medical and Health Sciences, The University
of Auckland, Auckland, New Zealand
Contributors xxiii

Hsin-Yu  Liu, MD Department of Ophthalmology, National Taiwan

University Hospital, Taipei, Taiwan
Marina  López-Paniagua, PhD CIBER-BBN (Biomedical Research
Networking Centre in Bioengineering, Biomaterials and Nanomedicine)
Carlos III National Institute of Health, Valladolid, Spain
IOBA (Institute of Applied Ophthalmobiology), University of Valladolid,
Valladolid, Spain
Matthew  Lovatt, PhD Tissue Engineering and Stem Cell Group, The
Academia, Singapore Eye Research Institute, Singapore, Singapore
Franz Martin, PhD, MD  Department of Cell Regeneration and Advanced
Therapies, Andalusian Center of Molecular Biology and Regenerative
Medicine-CABIMER, Junta de Andalucía-University of Pablo Olavide-­
University of Seville-CSIC, Seville, Andalusia, Spain
Jeremy  John  Mathan, BMedSc (Hons), MBChB Department of
Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and
Health Sciences, The University of Auckland, Auckland, New Zealand
Charles N. J. McGhee, DSc, FRCOphth  Department of Ophthalmology,
New Zealand National Eye Centre, Faculty of Medical and Health Sciences,
The University of Auckland, Auckland, New Zealand
Jennifer  J.  McGhee, BSc Department of Ophthalmology, New Zealand
National Eye Centre, Faculty of Medical and Health Sciences, The University
of Auckland, Auckland, New Zealand
Jodhbir  S.  Mehta, BSc, MBBS, FRCSCEd Singapore National Eye
Centre, Department of Cornea and External Disease, Singapore, Singapore
David Mingo-Botín, MD, PhD  Cornea Unit, Department of Ophthalmology,
Hospital Universitario Ramón y Cajal, Madrid, Spain
Noopur  Mitragotri, MSc Biochemistry Center to Ocular Regeneration
(CORE); and Brien Holden Eye Research Center, LV Prasad Eye Institute,
Hyderabad, Telangana, India
Hardeep Singh Mudhar, BSc, PhD, MBBCHir, FRCPath  Royal Hallamshire
Hospital, Department of Histopathology, Sheffield, South Yorkshire, UK
Sorcha Ní Dhubhghaill, MD, PhD  Faculty of Medicine and Health Sciences,
Department of Ophthalmology, Visual Optics and Visual Rehabilitation,
University of Antwerp, Campus Drie Eiken, Antwerp, Belgium
Department of Ophthalmology, Antwerp University Hospital, Edegem, Belgium
Center for Cell Therapy and Regenerative Medicine, Antwerp University
Hospital, CCRG-Oogheelkunde, Edegem, Belgium
Teresa Nieto-Miguel, PhD  CIBER-BBN (Biomedical Research Networking
Centre in Bioengineering, Biomaterials and Nanomedicine), Carlos III
National Institute of Health, Valladolid, Spain
IOBA (Institute of Applied Ophthalmobiology), University of Valladolid,
Valladolid, Spain
xxiv Contributors

Kohji Nishida, MD, PhD  Department of Ophthalmology, Osaka University

Graduate School of Medicine, Suita, Osaka, Japan
Yoshinori Oie, MD, PhD  Department of Ophthalmology, Osaka University
Graduate School of Medicine, Suita, Osaka, Japan
Naoki  Okumura, MD, PhD Department of Biomedical Engineering,
Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe,
Kyoto, Japan
Dipika  V.  Patel, PhD, MRCOphth Department of Ophthalmology, New
Zealand National Eye Centre, Faculty of Medical and Health Sciences, The
University of Auckland, Auckland, New Zealand
Gary Swee-Lim Peh, PhD  Tissue Engineering and Stem Cell Group, The
Academia, Singapore Eye Research Institute, Singapore, Singapore
Inmaculada  Pérez, PhD  IOBA (Institute of Applied Ophthalmobiology),
University of Valladolid, Valladolid, Spain
Paolo Rama, MD  San Raffaele Hospital, Department of Ophthalmology—
Cornea and Ocular Surface, Milan, Italy
Alejandra  E.  Rodríguez, PhD  Laboratory of the Research, Development
and Innovation Department, Vissum Innovation, Alicante, Spain
Dalia  G.  Said, MB, BCh, MSc, MD, FRCS Academic Section of
Ophthalmology, Division of Clinical Neuroscience, University of Nottingham,
Nottingham, UK
Department of Ophthalmology, Queens Medical Centre, University Hospitals
NHS Trust, Nottingham, UK
Research Institute of Ophthalmology (RIO), Cairo, Egypt
Yuzuru  Sasamoto, MD, PhD Division of Genetics, Department of
Medicine, Brigham and Women’s Hospital, Boston, MA, USA
Roholah  Sharifi, PhD Massachusetts Eye and Ear and Schepens Eye
Research Institute, Department of Ophthalmology, Harvard Medical School,
Boston, MA, USA
Trevor  Sherwin, BSc (Hons), PhD Department of Ophthalmology, New
Zealand National Eye Centre, Faculty of Medical and Health Sciences, The
University of Auckland, Auckland, New Zealand
Vivek Singh, MSc, PhD  Center to Ocular Regeneration (CORE); and Brien
Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad, Telangana,
India
Bernat  Soria, MD, PhD, FRCP Department of Cell Regeneration and
Advanced Therapies, Andalusian Center of Molecular Biology and
Regenerative Medicine-CABIMER, Junta de Andalucía-University of Pablo
Olavide-University of Seville-CSIC, Seville, Andalusia, Spain
Centro de Investigación Biomédica en Red de Diabetes y Enfermedades
Metabólicas Asociadas (CIBERDEM), Madrid, Spain
Contributors xxv

Marie-José Tassignon, MD, PhD  Faculty of Medicine and Health Sciences,

Department of Ophthalmology, Visual Optics and Visual Rehabilitation,
University of Antwerp, Campus Drie Eiken, Antwerp, Belgium
Department of Ophthalmology, Antwerp University Hospital, Edegem, Belgium
Juan  Rigoberto  Tejedo, PhD Department of Cell Regeneration and
Advanced Therapies, Andalusian Center of Molecular Biology and
Regenerative Medicine-CABIMER, Junta de Andalucía-University of Pablo
Olavide-University of Seville-CSIC, Seville, Seville, Andalusia, Spain
Centro de Investigación Biomédica en Red de Diabetes y Enfermedades
Metabólicas Asociadas (CIBERDEM), Madrid, Spain University Pablo de
Olavide, Seville, Spain
Mehrdad  Vakilian, PhD Student Department of Cell Regeneration and
Advanced Therapies, Andalusian Center of Molecular Biology and
Regenerative Medicine-CABIMER, Junta de Andalucía-University of Pablo
Olavide-University of Seville-CSIC, Seville, Andalusia, Spain
Veronica Vargas, MD  Vissum Instituto Oftalmologico de Alicante, Alicante,
Spain
Nandini Venkateswaran, MD  Bascom Palmer Eye Institute, University of
Miami, Department of Ophthalmology, Miami, FL, USA
Himanshu  Wadhwa, MBChB, BMedSc (Hons) Department of
Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and
Health Sciences, The University of Auckland, Auckland, New Zealand
Stephen  Wahlig, BSc Tissue Engineering and Stem Cell Group, The
Academia, Singapore Eye Research Institute, Singapore, Singapore
I-Jong  Wang, MD, PhD  Department of Ophthalmology, National Taiwan
University Hospital, Taipei, Taiwan
College of Medicine, National Taiwan University, Taipei, Taiwan
Jinny  Jung  Yoon, PhD, MBChB Department of Ophthalmology, New
Zealand National Eye Centre, Faculty of Medical and Health Sciences, The
University of Auckland, Auckland, New Zealand
Jie Zhang, PhD  Department of Ophthalmology, New Zealand National Eye
Centre, Faculty of Medical and Health Sciences, The University of Auckland,
Auckland, New Zealand
Mohammed  Ziaei, FRCOphth  Department of Ophthalmology,
New Zealand National Eye Centre, Faculty of Medical and Health Sciences,
The University of Auckland, Auckland, New Zealand
 Part I
Corneal Regeneration: The Concept, the
Facts, the Potential
 Corneal Anatomy
Miguel Gonzalez-Andrades, Pablo Argüeso,
and Ilene Gipson
1.1 Introduction
The cornea is a uniquely translucent, avascular
tissue located on the anterior segment of the eye
(Fig. 1.1). It is surrounded and maintained by the
adjacent corneoscleral limbus and the connective
tissue of the conjunctiva with its adnexa [1]. It
plays a vital role in visual function by providing
(1) the major refractive component of the visual
system [2], (2) a translucent tissue that allows
light passage to the lens and retina, and (3) a bar-
rier that protects the eye from fluid loss and the
external environment. These crucial functions
result from the structure of the cornea, which is
composed of three anatomical layers: epithelium,
stroma, and endothelium (Fig. 1.2). The limbus is
the reservoir for the adult stem cell population
that replenishes the cornea and is the site of ter-
mination of the vasculature and entry of the
nerves that provide an extraordinarily rich inner-
vation environment (Fig. 1.2).
The embryology of the cornea is based on
inductive interactions that take place in the cra-
nial ectoderm [3]. The corneal epithelium origi-
nates from the cross talk of the neural ectoderm
with the optic vesicle [4]. The corneal stromal
cells or keratocytes and the corneal endothelium
M. Gonzalez-Andrades (*) · P. Argüeso · I. Gipson
Massachusetts Eye and Ear and Schepens Eye
Research Institute, Department of Ophthalmology,
Harvard Medical School, Boston, MA, USA
e-mail: Miguel_gonzalez@meei.harvard.edu
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_1
1
derive from neural crest cells that migrate
between the primitive corneal epithelium and the
lens [5–7].
The anatomical dimensions of the human cor-
nea are variable among individuals (Fig.  1.1).
The average corneal horizontal diameter (white-­
to-­white distance or limbus to limbus) is greater
than the vertical one (11.71 ± 0.42 mm and 10.63
± 0.63 mm, respectively) [8, 9]. The central cor-
neal thickness ranges from 0.50 to 0.60 mm [10–
12], which increases progressively toward the
periphery, where thickness can be 26% greater
than central point values [12].
1.2 The Corneal Epithelium
The corneal epithelium is a nonkeratinized strati-
fied squamous epithelium that constitutes the
outermost layer of the cornea. The epithelium is
comprised of 5–7 cell layers including flattened
apical squamous cells, subapical cells that have
winglike structure – hence they are called “wing
cells” – and a basal cell layer that is columnar in
shape (Fig.  1.3a). The corneal epithelium is
unique among other stratified squamous epithelia
in that it is of a very uniform thickness and has a
unique smooth wet surface that, along with the
tear film, provides its refractive power. Cells of
each layer have a specialized structure and func-
tion, and apical and basal cells show specific pro-
tein expression patterns.
3
4 M. Gonzalez-Andrades et al.
Fig. 1.1  The cornea (1) is located on the anterior seg-
ment of the eye. It is surrounded by different structures:
the sclera (2), the iris (3), the ciliary body (4), the lens (5),
and the zonule of Zinn (6). The anatomical dimensions of
the human cornea are variable. The average corneal hori-
Fig. 1.2  The cornea is composed of three anatomical lay-
ers: epithelium (1), stroma (3), and endothelium (5). Two
distinguishable areas flank the stroma: the Bowman layer
(2) and Descemet’s membrane (4). The limbus (green
circle) is the reservoir for the adult stem cell population
that replenishes the cornea. There are several cells
The most apical cell layer of the corneal epi-
thelium expresses membrane-associated mucins
MUC1 and MUC16 that form the surface glyco-
calyx [13, 14] (Fig. 1.3b). The apical cell mem-
brane of this cell layer exhibits folds or ridges
termed microplicae [15] (Fig.  1.3c). The
membrane-­associated mucins emanate from the
tips of these microplicae to form the glycocalyx
(Fig. 1.3d). The glycocalyx not only contributes
to formation of an optimal tear film, but it pro-
zontal diameter (orange arrow) is greater than the vertical
one (blue arrow). The central corneal thickness ranges
from 0.50 to 0.60 mm in the central area (dotted red line),
which increases progressively toward the periphery (dot-
ted green line)
involved in corneal epithelial regeneration: corneal stro-
mal stem cells (CSSCs), limbal epithelial stem cells
(LESC), suprabasal corneal epithelial cells (SCEC), tran-
sient amplifying cells (TAC), terminally differentiated
cells (TDC) (Conj., conjunctiva)
vides a barrier to the penetrance of large mole-
cules and pathogens [16, 17] with MUC16
functioning as the major component of the bar-
rier preventing, for example, rose bengal dye and
pathogen penetrance [16–18]. Apical cells exhibit
tight junctions or zonula occludens that consti-
tutes the paracellular barrier for fluid loss and
external molecules and microorganisms.
The basal cell layer is specialized and pro-
vides tight anchorage of the epithelium to the
1  Corneal Anatomy
a b
c d
Fig. 1.3 (a) The corneal epithelium is comprised of 5–7
cell layers. (b) The most apical cell layer of the corneal
epithelium expresses membrane-­associated mucins, such
as MUC16 (red staining), that form the surface glycocalyx
(cell nuclei in blue). (c) The apical cell membrane of this
underlying stroma. To provide these anchoring
functions, the basal cells secrete the basement
membrane, composed of type IV collagen, type
VII collagen, laminin, fibronectin, nidogen, and
perlecan, among other proteins [19–22]. Further,
they assemble specialized anchoring junctions,
hemidesmosomes, which link the cells’ cytoskel-
eton by way of alpha6 beta4 integrin to the type
VII collagen anchoring fibrils that penetrate into,
and intersperse between, collagen fibrils in the
anterior stroma [23].
Corneal epithelial cells express the cytokeratins
3 and 12, and all are connected to their neighbors
via intercellular desmosomes [24]. The cytokera-
tin intermediate filaments attach to the desmo-
somes’ placque [25] and, along with adherens
junctions and hemidesmosomes, confer to the cor-
5
cell layer exhibits folds or ridges termed microplicae
(MP). (d) The membrane-­associated mucins emanate from
the tips of these microplicae to form the glycocalyx.
(Modified from Argueso et al. [18] and Gipson et al. [2])
neal epithelium its cohesion and resistance to abra-
sion. The corneal epithelium also has gap junctions
for intercellular communication purposes, and
they are found throughout the epithelium but are
of different character apical to basal [26].
The cornea is the most densely innervated tis-
sue in the body [27, 28] (Fig. 1.4). Corneal inner-
vation is not only responsible for corneal
sensitivity but also provides trophic factors that
are essentials for wound healing and corneal
regeneration [29–31]. Intraepithelial nerve termi-
nals innervate all corneal epithelial layers [32].
Nerve terminals are branches from a continuous
subbasal nerve plexus, which originate from the
anastomosis of epithelial leashes in the central
and paracentral cornea. Epithelial leashes are
subbasal nerve fibers branched from stromal
6 M. Gonzalez-Andrades et al.
INT
SNP SNP
Epithelial
leashes
Corneal
stroma
Fig. 1.4  The cornea is the most densely innervated tissue
in the body. Intraepithelial nerve terminals (INT) inner-
vate all corneal epithelial layers. Nerve terminals are
branches from a continuous subbasal nerve plexus (SNP),
which originate from the anastomosis of epithelial leashes
nerves that penetrate into the cornea from the
corneoscleral limbus [32].
The corneal epithelium is renewed within
periods lasting between 7 and 14 days (for review
see [33]). The basal cells of the corneal epithe-
lium possess limited mitotic activity, presenting a
density of 6000  cells/mm2 [34, 35]. These cells
derive from limbal epithelial stem cells (LESC)
that reside in the corneoscleral limbus in an
undifferentiated state [36]. The capacity of LESC
for self-renewal is limitless and their mitotic
activity is low. These cells may represent less
than 10% of the total limbal basal cell population
[36, 37]. Different markers such as ABCG2,
ΔNp63α, N-cadherin, cytokeratin 19, vimentin,
KGF-R, and ABCB5 have been used to identify
putative LESC [36, 38–41]. The LESC divide
and give rise to transient amplifying cells capable
of a finite number of cell divisions [22]. Finally,
the cells migrate centripetally to differentiate into
suprabasal corneal epithelial cells, which give
rise to terminally differentiated cells of the cor-
neal epithelium surface [42]. The mechanism
underpinning this process of corneal epithelial
homeostasis is poorly defined. Recently a new
Long ciliary
nerves (Va)
Corneal Limbus
epithelium
Stromal
bundle
in the central and paracentral cornea. Epithelial leashes
are subbasal nerve fibers branched from stromal nerves
that penetrate into the cornea from the corneoscleral
limbus
hypothesis has been put forward by Connon et al.
which suggests that differences in substrate stiff-
ness beneath the epithelial cells (a centripetal
stiffness gradient) can drive both the inward
migration and differentiation of the limbal stem
cells via mechanotransductional forces [43].
Corneal epithelial cells sensing these changes
have been shown to undergo differentiation in
response to substrates of differing stiffness [44].
The limbus is a specialized region that acts as a
1.5 mm-wide transition zone between the cornea
and sclera [45]. It is densely vascularized, inner-
vated, and protected from UV light damage by
melanin pigmentation [39]. The limbus is com-
posed of an epithelium (which lies between the
corneal epithelium and the conjunctival epithe-
lium, basal cells of which house a population of
limbal epithelial stem cells – LESC – in an undif-
ferentiated state [36]) and a stroma that contains
vessels [46] and different types of cells such as
melanocytes [47] and mesenchymal stem cells
[48]. Two specialized structures in the basal epi-
thelium of the corneoscleral limbus make
epithelial-­stromal interactions possible through a
basement membrane along the limbus. The pali-
1  Corneal Anatomy 7

sades of Vogt are papilla-like structures located in
the subepithelial connective tissue that provide a
protective environment for LESC [49]. Among
these palisades are limbal epithelial crypts, finger-­
shaped projections where the LESC are located.
These structures protect the cells from shear stress
and supply them with nutrients from the neighbor-
ing blood vessels [22, 40]. At the palisades of Vogt,
LESC might interact with corneal stromal stem
cells, i.e., neural crest-derived mesenchymal stem
cells (MSC), that maintain the LESC niche and
can differentiate into functional keratocytes, which
in turn might regenerate the normal corneal stroma
[48, 50–52]. The limbal stroma is not transparent,
and its collagen fibrils are not as orderly arranged
as the adjacent corneal stroma, and it begins to
resemble the scleral stroma in both the arrange-
ment and the size of its collagen fibers [53]. Cell-
matrix interactions, partially mediated by local
variations in the composition of the extracellular
matrix and the heterogeneity of the basement
membrane, provide an optimum microenviron-
ment for limbal basal cells [22, 36, 39].
1.3 The Corneal Stroma
The corneal stroma is a specialized connective
tissue layer, between the corneal epithelium and
endothelium. It constitutes 90% of the human
corneal thickness, representing the structural axis
of the cornea. It is flanked by two distinguishable
areas: the Bowman layer on the anterior side and
Descemet’s membrane on the posterior side
(Fig.  1.2). The corneal stroma is considered an
immunologically privileged structure because of
the absence of blood and lymph vessels [54].
This feature maintains optimal transparency and
increases the chances of survival of the donor
cornea when it is transplanted into the host.
The corneal stroma is composed of extracellu-
lar matrix and its embedded cells (Fig. 1.5a). The

Fig. 1.5 (a) The corneal

a
stroma is populated by
keratocytes embedded
between the collagen
lamellae. (b)
Keratocytes, which are
quiescent in adults, have
a flat dendritic
morphology which gives
them a large area. (c)
Collagen fibers are
grouped together to form
lamellae that are
oriented mainly parallel
to the tissue surface at
all depths
b c
8 M. Gonzalez-Andrades et al.
extracellular matrix synthesized by these cells is
mainly formed by collagen, primarily type I col-
lagen, although type V and VI collagen are also
present. Type I collagen accounts for 68% of the
dry weight of the cornea. Other types of colla-
gen fibers present in much lower amounts in the
corneal stroma are types III, XII, and XIV [22,
55]. Collagen fibers are grouped together to form
lamellae that are oriented mainly parallel to the
tissue surface at all depths [56] (Fig.  1.5c). This
pattern of orientation is an important factor affect-
ing the transparency and strength of the cornea
[57–62]. In addition, the stroma’s unique unifor-
mity of collagen fibril diameter, controlled by the
precisely regulated amounts of type I and type
V collagen, contributes to transparency. A third
factor contributing to stromal transparency is the
amorphous ground substance or matrix surround-
ing the collagen fibrils. This material is primarily
dermatan and keratan sulfate proteoglycans, which
regulates interfibrillar distance between the colla-
gen fibrils thus contributing to the regular spacing
of the fibrils that is required for transparency [52,
63]. Water makes up 78% of the corneal volume.
The stroma is populated by keratocytes embed-
ded between the collagen lamellae, occupying
approximately 3% of the stromal volume [52]
(Fig.  1.5b). Keratocytes, which are quiescent in
adults, have a flat dendritic morphology which
gives them a large area; they make contact with each
other through gap junctions [64]. The keratocyte
density changes according to the stromal region,
decreasing in number from anterior to posterior
[65, 66]. The functional activities of keratocytes
include the synthesis of extracellular matrix and
stromal remodeling; these cells produce collagen
lamellae and proteoglycans including keratocan,
decorin, lumican, and mimecan [67]. Keratocytes
express mesenchymal and hematopoietic markers
and can be identified by expression of the follow-
ing proteins: keratocan, aldehyde dehydrogenase,
crystallins, CD133, CD34, and cytokeratin 3 [67,
68]. The expression of these markers is affected in
response to injury, which induces keratocytes to
adopt a fibroblast or myofibroblast scar-forming
phenotype that is reversible depending on the envi-
ronment [68, 69]. Keratocytes, as well as epithe-
lial cells, have developed a mechanism to increase
transparency and minimize fluctuations in the
refractive index, reducing light scatter by express-
ing a series of water-soluble proteins called crys-
tallins [70]. These proteins control optical changes
and reduce the dispersion of light that occurs in the
cytoplasm of both cell types [71].
Other cell types resident in the corneal stroma that
respond to injury and pathogens are bone marrow-
derived cells. These cells are involved in the anti-
gen presentation and immune response and include
dendritic cells and macrophages [72–76]. Dendritic
cells are also considered resident cells in the cor-
neal epithelium, where they received the name of
Langerhans cells (similar to the dendritic cells found
in the epidermis of the skin) or intraepithelial cor-
neal dendritic cells [76, 77]. Immune cells can influ-
ence the avascularity status of the cornea, which is
based on the balance of anti-angiogenic factors that
­counterbalance pro-angiogenic/lymphangiogenic
factors upregulated during wound healing [78, 79].
The two specialized acellular areas that border
the corneal stroma are Bowman’s layer and
Descemet’s membrane. Bowman’s layer is
8–14 μm in thickness and consists primarily of col-
lagen fibers type I, III, and V produced by epithelial
cells and intertwined with each other apparently
without any architectural pattern [46, 80].
Descemet’s membrane is a uniquely thick base-
ment membrane produced by the corneal endothe-
lium. It is composed of type IV, type VIII, and type
XII collagen, perlecan, nidogen, netrin, fibronectin,
and laminin, among other proteins [81, 82]. This
membrane is composed of an anterior banded layer
developed in the fetus and a posterior nonbanded
layer that is continuously synthetized by the endo-
thelial cells during adult life [83, 84].
1.4 The Corneal Endothelium
The corneal endothelium is a simple low cuboi-
dal epithelium [81, 85]. These cells show a hex-
agonal morphology on their apical surface that
abuts the aqueous humor (Fig. 1.6), whereas their
basal surface, in contact with Descemet’s mem-
brane, is irregular [86–88]. The human corneal
endothelium shows an increased cell density in
the paracentral and peripheral areas compared
1  Corneal Anatomy
Fig. 1.6  The corneal endothelium is a simple low cuboi-
dal epithelium, whose cells show a hexagonal morphol-
ogy on their apical surface that abuts the aqueous humor
(in vivo confocal image of the corneal endothelium of a
healthy person). (Courtesy of Dr. Caro-Magdaleno)
with the center [89]. Connections between the
different cells are based on adherens junctions,
tight junctions, and desmosomes [46, 90].
The functional activity of corneal endothelial
cells is to enable the exchange of ions and fluid
through active transport between the corneal
stroma and the anterior chamber, where the aque-
ous humor is located [91]. This functional activity
ensures corneal transparency and avoids stromal
edema [85, 92]. One of the main ionic pumps
expressed on the lateral membrane of the endo-
thelial cells is Na(+)/K(+)-ATPase, which is one
of the markers used to differentiate them from
other corneal cell types. Another marker com-
monly used for this purpose is ZO-1, even when
it is also present in the corneal epithelium [93].
Recently, some researchers have proposed new
markers that can be used to purify this cell popu-
lation, such as CD98, CD166, CD340, CLRN1,
MRGPRX3, HTR1D, GRIP1, and ZP4 [93, 94].
The mitotic activity of endothelial cells is min-
imal, with an annual loss of 0.6% approximately.
This cell loss is compensated by polymegathism
and polymorphism, by which the cells increase in
size and change shape, respectively [95–97]. The
regenerative capacity of the corneal endothelium
is very limited, and proliferation is reduced by
factors associated with age, central topographical
9
location, cell-cell contacts, and lack of growth
factor stimulation [98, 99]. Recent findings sug-
gest that endothelial progenitor cells exist in the
corneal periphery and can differentiate into func-
tional corneal endothelial cells [100–104].
Compliance with Ethical Requirements  Miguel
Gonzalez-Andrades, Pablo Argüeso, and Ilene
K.  Gipson declare that they have no conflict of
interest. No human or animal studies were car-
ried out by the authors for this article.
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 Corneal Healing
Veronica Vargas, Francisco Arnalich-Montiel,
and Jorge L. Alió del Barrio
2.1 Introduction
The cornea covers the anterior 1/6 of the total
surface of the globe and, due to its refractive
power and transparency, is the most important
optical element of the eye.
It has three major cell types, epithelial cells,
stromal keratocytes, and endothelial cells, and
consists of five layers, epithelium, Bowman’s
layer, stroma, Descemet membrane, and endothe-
lium [1], and a surgical layer called Dua’s layer.
We are going to describe briefly each one of them.
2.1.1 Corneal Epithelium
It is the first layer of the cornea and consists of a
nonkeratinized stratified squamous layer, and it
is richly innervated. It has 4–6 cell layers, a
thickness of 50um approximately, and three dif-
ferent kinds of cells, squamous cells, wing cells,
V. Vargas
Vissum Instituto Oftalmologico de Alicante,
Alicante, Spain
F. Arnalich-Montiel
Vissum Corporation, Madrid, Spain
J. L. Alió del Barrio (*)
University Miguel Hernandez, Vissum-Instituto
Oftalmologico de Alicante, Alicante, Spain
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_2
2
and basal epithelial cells. These cells have junc-
tional complexes between each other to prevent
the passage of foreign agents. They have an aver-
age lifespan of 7–10  days. Apoptosis of these
cells is induced by ultraviolet radiation, hypoxia,
and mechanical friction [2]. Because it is the
outer layer of the cornea, it can be easily injured.
2.1.2 Bowman’s Layer
It is an acellular layer composed by collagen
fibrils types I and III, with a mean thickness of
12 μm; it does not regenerate after injury [3].
2.1.3 Stroma
It constitutes 90% of the corneal thickness, and it
is composed by collagen types I, III, V, VI, XII,
and XIV; the uniform arrangement of the collagen
fibers is essential for the corneal transparency. Its
primary cells are the keratocytes which are usually
quiescent in the normal corneal; but in response to
stromal injury, they get activated and undergo
transdifferentiation into myofibroblasts [3].
2.1.4 Descemet Membrane
It is the basement membrane of the endothelium.
It has a thickness of 10 μm, and it is mainly com-
posed of collagen types IV and VIII [3].
13
14
2.1.5 Endothelium
It is a single layer of hexagonal cells. Young
adults have a cell density of 3500  cells/mm2
approximately, and they do not regenerate. Its
main function is to regulate corneal hydration
through ion transport systems in order to main-
tain the corneal transparency [1, 3].
The corneal healing process is complex and
crucial because its transparency may be jeopar-
dized during the process leading to scar forma-
tion and blindness.
2.2  ytokines and Growth
C
Factors Involved in Corneal
Healing
Corneal wound healing would not be possible
without the interaction of cytokines, growth fac-
tors, integrins, and proteases; so, first we are
going to describe the function of each one of
them:
2.2.1 Cytokines
Interleukins 1α (IL-1α) and 6 initiate the pro-
cess of epithelial wound healing [2]; they are
proinflammatory cytokines, and its levels have a
correlation with the severity of the corneal
injury [4].
IL-6 stimulates epithelial migration and may
influence the production of fibrotic material by
activated keratocytes [5].
IL-1α has several functions during corneal
healing, among them are [6]:
• Mediation of keratocyte apoptosis
• Epithelial cell proliferation by inducing the
expression of keratinocyte growth factor
(KGF) and hepatocyte growth factor (HGF)
on keratocytes
• Stimulation of epithelial wound healing in
conjunction with epidermal growth factor
(EGF), both accelerating epithelial wound
closure
V. Vargas et al.
2.2.2 Integrins
They are transmembrane receptors; their main
function is to connect cells to the extracellular
matrix, and they play an important role in the
healing process. The α2β1 integrin, expressed in
the epithelial cells that migrate following a cor-
neal wound, interacts with type I collagen and
upregulates the production and synthesis of
matrix metalloproteinases [6].
2.2.3 Matrix Metalloproteinases
(MPPs)
These zinc-dependent endopeptidases influence
cell migration by disengaging cell-cell and cell-­
matrix adhesion and extracellular matrix (ECM)
degradation. They also modulate the activity of
growth factors and cytokine receptors [7].
2.2.4 Urokinase-Type Plasminogen
Activator (uPA)
It is a serine protease synthesized by corneal epi-
thelial cells and corneal fibroblasts.
It mediates the release of inflammatory cyto-
kines from fibroblasts, activates MMP-9 [8], and
may stimulate cell migration [7].
2.2.5 Growth Factors
Depending on the situation, growth factors can
promote or inhibit the migration, proliferation,
and differentiation of cells [9]. They may act in
paracrine or autocrine mechanism [10].
2.2.6 E
 pidermal Growth Factor
(EGF)
It is a polypeptide produced by macrophages and
a variety of epithelial cells. It has a mitogen effect
on epithelial cells, and it is present in the tear film
[9]. After an epithelial wound, the EGF in tears
2  Corneal Healing
promotes cell proliferation [6]. It acts using both
paracrine and autocrine systems [10].
2.2.7 T
 ransforming Growth Factor-β
(TGF-β)
It is a polypeptide with three isoforms β1, β2,
and β3 and is produced by platelets, mononu-
clear, and inflammatory cells. Its receptors are
located at the stromal cells, limbus, and cen-
tral corneal epithelium. It acts through an
autocrine mechanism [10] and has several
functions [9]:
• Stimulates the production of proteoglycans,
fibronectin, and collagen.
• Limits inflammatory responses.
• Regulates (inhibits) epithelial cell
proliferation.
• Induces proliferation and migration of stromal
fibroblasts.
• Increases extracellular matrix synthesis.
• Antagonizes the mitogenic action of HGF,
EGF, and KGF in corneal epithelium [5].
• TGF-β1 and TGF-β2 trigger the transforma-
tion of fibroblasts to myofibroblasts.
• TGF-β3 mitigates scar formation after injury.
2.2.8 H
 epatocyte Growth Factor
(HGF)
It is a glycoprotein produced by mesenchymal
cells (fibroblasts and macrophages) [11]; its
function is to regulate the normal proliferation of
central and peripheral epithelial cells [6] and epi-
thelial differentiation. It targets epithelial cells in
a paracrine manner [7, 10]; it is secreted by the
lacrimal gland, and most of its receptors (c-Met
[12]) are in the central epithelial cells [9, 11],
although they can also be found in the endothe-
lium and keratocytes [12].
It induces epithelial cell migration and prolif-
eration and inhibits apoptosis [11].
A study demonstrated that under an inflam-
matory environment, HGF promotes corneal
15
epithelial cell proliferation and inhibits the
function and activation of inflammatory cells
in  vitro; the same study showed that topical
administration of HGF in an in  vivo model
of corneal injury suppresses ocular inflamma-
tion [12].
2.2.9 F
 ibroblast Growth Factor
(FGF)
It is produced by epithelial and corneal cells; has
a mitogenic effect in epithelial, stromal, and
endothelial cells; promotes cell migration; and
inhibits TGF-B1 expression in the stroma [9].
2.2.10 Keratinocyte Growth Factor 1
(KGF-1)
It is a member of the FGF family and functions in
a paracrine fashion [7, 10]. It is mainly expressed
in the corneal stroma, and its receptor is expressed
in limbal epithelial cells. It stimulates epithelial
cell proliferation [6].
2.2.11 Platelet-Derived Growth
Factor (PDGF)
Produced by epithelial cells, it is made up of a
combination of four polypeptide chains: A, B, C,
and D (PDFG-AA, PDFG-BB, PDFG-AB,
PDFG-CC, PDGF-DD). They regulate migration
and proliferation of keratocytes [7] through an
autocrine and paracrine mechanism [10].
2.2.12 Nerve Growth Factor (NGF)
It is a soluble protein that belongs to a family
of neurotrophic factors; it is normally released
in the aqueous humor and tear film [13]. During
corneal healing, it stimulates epithelial cell
migration [7], proliferation, innervation [9],
and keratocyte differentiation into myofibro-
blasts [7].
16
2.2.13 Insulin-Like Growth Factor
(IGF)
This growth factor and its receptors are expressed
in the corneal epithelial cells and in keratocytes.
It regulates cell migration and differentiation; it
acts synergistically with substance P enhancing
cell proliferation and wound closure [7].
2.3 Epithelial Wound Healing
Of all the corneal layers, the epithelium is the
quickest to heal. The healing process consists of
four phases: (1) latent, (2) migration, (3) prolif-
eration, and (4) attachment (see Fig. 2.1).
One phase can begin before the previous one
is completed, but they always follow the sequence
of the phases [5].
1. Latent Phase
It is the time between the injury and the com-
mencement of reepithelization [6].
Damaged cells undergo apoptosis and are
shed into the tear film, intercellular junctions
are dismantled, and the cell-substrate junc-
tions are replaced with weaker attachments
[5]. Cytoskeletal proteins like talin, vinculin,
actin, and integrin are synthesized. The latter
dissociates from the hemidesmosomes and
desmosomes to distribute evenly on the cellu-
lar surface serving as an adherent molecule to
Migration
• Celular
reorganization • Cells at wound
• Protein edge flattens
synthesis and migrate
Lag phase
Fig. 2.1  Epithelial healing phases
V. Vargas et al.
the extracellular matrix (ECM) [2, 6], which
is formed by fibronectin to allow cell migra-
tion [5].
2. Epithelial Cell Migration
The cells adjacent to the injury site flatten and
migrate toward the injured area to reestablish
the integrity of the epithelium [6]. The ECM
facilitates this migration, and the limbal epi-
thelial stem cells are activated by growth fac-
tors and cytokines.
The cell migration occurs at a constant rate
of 0.05–0.06  mm/h and slows down as the
defect gets smaller [5]; it is initiated by EGF
[7], and HGF and KGF facilitate it [9, 11].
The expression of the latter growth factors is
upregulated by PDGF [9].
There is no mitotic activity in this phase [5].
3. Proliferation and Differentiation
During this phase, the epithelial cell density is
restored.
Limbal epithelial stem cells proliferate dur-
ing wound healing and give rise to transient
amplifying cells that differentiate and migrate
to the center of the cornea [14]. There is mitotic
activity in the cells outside the wound area [5],
and it is delayed within the wound area [2].
Daughter cells begin to differentiate into
wing cells and then squamous cells, and the
intercellular junctions are reformed (the zonula
occludens are the first ones in being reestab-
Attachment
• Epithelial cell
density is • Epithelial layer is
restored adhered to the
underlying
substrate
Proliferation and
differentiation
2  Corneal Healing 17

lished) restoring the epithelial barrier function. Upon proper healing, once that the EBM
A new basement membrane is formed [5]. This recovers its barrier function, the levels of TGF-β
stage is stimulated by HGF [11]. and PDGF fall, myofibroblasts undergo apoptosis
(promoted by HGF which induces degradation of
4. Attachment the ECM, specially fibronectin that is an anchor
In this phase, the epithelial layer strongly adheres for myofibroblasts [11]), keratocytes reoccupy
to the underlying substrate through hemidesmo- the anterior stroma, ECM is reabsorbed, and cor-
somes [5]. The permanent attachment of the neal transparency is restored [16].
hemidesmosomes depends if the basement
membrane remained intact at the time of wound-
ing [15]. If the anterior stroma is compromised, 2.4.1 Haze Formation
this phase can last a long period of time [5].
A prolonged persistence of myofibroblast after
healing and an excessive number of them corre-
2.4 Stromal Wound Healing late to corneal opacity [11]. Mature myofibro-
blasts express vimentin, desmin, and α-smooth
The first stromal healing response after injury is muscle actin (which directly correlates to corneal
the apoptosis of anterior keratocytes. This process wound contraction), which are opaque due to the
is triggered by cytokines like IL-1, Fas ligand, and decreased intracellular crystallin production [16,
necrosis tumor factor α [7, 16]. The keratocytes 19]. Myofibroblasts elaborate a disorganized
that are adjacent to the wounded area activate and ECM that interferes with the adequate distribu-
differentiate into fibroblasts due to an increased tion of collagen fibers [19]. TGF-β favors the
expression of actin [11]. Once they arrive at the deposition of excessive ECM [7]. This new ECM
wounded area, they repopulate that region that has accumulates aberrant proteins and forms scars
been depleted of keratocytes through apoptosis that may persist for a long time due to the slow
and differentiate into myofibroblasts [11]. These turnover of these proteins. These scars have com-
cells deposit a provisional ECM and generate con- ponents that are absent in a healthy stroma like
tractile forces to close the wound [7, 17]. collagen types III, VIII, XIV, and XVIII and
The keratocyte transformation to fibroblast embryonic fibronectin [7] (see Fig. 2.2).
and myofibroblast is triggered by TGF-β1, TGF-­ This type of abnormal healing of the cornea
β2, and PDGF [7, 11, 17, 18] can be seen after surface ablation surgeries like

Fig. 2.2  In stromal

healing process, after
injury, keratocytes
transform to fibroblasts, Keratocyte Fibroblast Myofibroblast
which transform to
myofibroblasts. If there
is a pathological
remodeling process
(large amount of Remodeling
myofibroblasts and a
disorganized ECM),
haze develops; if not, the
myofibroblasts undergo
apoptosis, and the Pathological Physiological
cornea keeps its
transparency

Haze Apoptosis
18
photorefractive keratectomy (PRK). During the
first few weeks after PRK, a mild haze (early
haze) can be seen, which is secondary to corneal
fibroblasts that are opaque in the same manner
as myofibroblast, but the ECM they produce is
more organized than the one produced by the
myofibroblasts. This type of haze usually disap-
pears with time unlike the pathological haze
(late haze) that is secondary to myofibroblasts,
which appears 2–3 months [19] after the correc-
tion of (usually) high refractive errors and can
last years [11].
The epithelial basement membrane (EBM)
plays an important role in this scenario. A defec-
tive EBM allows the penetration of TGF-β and
PDGF from the epithelium into the stroma induc-
ing myofibroblast generation that, as mentioned
before, produces a disorganized ECM that blocks
the keratocytes that are in the posterior stroma
from moving into the subepithelial area to con-
tribute to the EBM regeneration [16].
Mitomycin C inhibits the development of
myofibroblasts, and its use has reduced the inci-
dence of late haze after PRK [19].
In LASIK, the creation of a flap preserves the
integrity of the EBM [19], so haze is not a risk
because the stromal remodeling is not as signifi-
cant as it is in PRK [7], except at the edge of the
flap where the EBM is damaged [19] and some
myofibroblasts may transiently appear [7].
2.5 Clinical Applications
of Growth Factors
Some growth factors have been clinically used to
enhance corneal healing with good results, espe-
cially in cases of neurotrophic keratitis:
2.5.1 Nerve Growth Factor
A study showed that topically applied NGF
closed persistent epithelial defects in 45 eyes
with neurotrophic keratitis. There are three
clinical stages in neurotrophic keratitis: in stage
1, there is a punctate corneal keratopathy with
tear film abnormalities; in stage 2, there is a
V. Vargas et al.
persistent epithelial defect without corneal
ulcers; and in stage 3, there is stromal edema,
corneal ulcers, and perforation. The patients
included in this study had stages 2 and 3; they
received one drop of NGF (50 μl) every 2 h for
2  days and one drop six times a day until the
epithelial defect healed. Reported side effects
like ocular pain, conjunctival hyperemia, and
photophobia were secondary to the improve-
ment of the corneal sensitivity. Just three
patients that had a previous trigeminal nerve
resection and secondary corneal anesthesia
required a retreatment [20].
It has also been reported that one drop of NGF
(10 μL) helps restoring the corneal transparency
and thickness at the site of the surgical wound
within 3 weeks following cataract (phacoemulsi-
fication) surgery [21].
Cases of autoimmune peripheral keratitis
have also been successfully treated with
NGF [22].
2.5.2 Insulin-Like Growth Factor
Topical treatment with insulin-like growth factor-
1 (IGF-1)-derived peptide (SSSR) combined
­
with substance P-derived peptide (FGLM-amide)
triggered epithelial cell migration [23]. This
combination has been used successfully to treat
epithelial defects secondary to neurotrophic kera-
titis in patients without limbal stem cell defi-
ciency [24] and in patients with neurotrophic
keratitis secondary to diabetes mellitus and neu-
rosurgical damage [23].
2.5.3 Epidermal Growth Factor
It has been used to treat epithelial defects of mul-
tiple etiologies; a study of nine eyes with epithe-
lial defects reported a complete reepithelization
in seven eyes after the use of a soft hydrogel con-
tact lens impregnated with EGF, while two eyes
did not respond to the therapy and had a signifi-
cant ocular inflammation; no ocular toxicity was
documented after the use of the EGF-impregnated
contact lens [25].
2  Corneal Healing
2.5.4 A
 utologous Eye Platelet-Rich
Plasma (E-PRP)
E-PRP is an autologous preparation of plasma
rich in platelets; the alpha granules in platelets
contain several growth factors like EGF,
PDGF-AB, IGF-1, and TGF-β. They modulate
wound healing once they are released from acti-
vated platelets [26].
Alió et al. [27] used E-PRP for the treatment of
dormant ulcers (neurotrophic keratopathy, her-
petic keratopathy, and immunological ulcers) at a
dose of six times a day in addition to routine med-
ication. Clinical improvement was found in 92%
of the eyes; a complete resolution of the ulcer was
achieved in 50% of the cases. In the same clinical
study, perforated eyes or high risk of perforation
due to deep chronic corneal ulcers was treated
with amniotic membrane transplantation com-
bined with a clot of autologous E-PRP; 71% of
the eyes had a complete resolution of the ulcer.
2.6 Physiological Mechanism
of Endothelial Regeneration
Endothelial cell density slowly declines over
time; however, corneal decompensation seldom
occurs. Besides normal aging or endothelial dis-
ease, there may be many exogenous stresses that
could potentially damage the corneal endothe-
lium over a person’s lifetime. These may reduce
even further the endothelial cell density below a
level critical to the maintenance of corneal
deturgescence, as the physiological pump-barrier
function fails and corneal edema establishes.
This critical density has been estimated to be
10–15% of the normal cell count, which falls
between 300 and 500 cell/mm2 [28].
The most common interventions that might
stress an individual’s corneal endothelium
[29] are:
1. Contact lens wear, causing corneal hypoxia
and leading to polymegathism and
pleomorphism
2. Intraocular surgeries such as cataract, glau-
coma, and refractive IOL surgery.
19
3. Penetrating trauma
4. Anterior segment infections or inflammations
including infectious or sterile endotheliitis
5. Pharmacologic toxicity leading to toxic
anterior segment syndrome due to irrigating
solutions or viscoelastic devices incorrectly
compounded or manufactured; ophthalmic
instrument contaminants; ocular medica-
tions with incorrect pH, osmolality, or pre-
servatives; contaminated water sources; and
intraocular lenses polishing, cleaning, and
sterilizing compounds
The endothelium has a rather restricted
response to injuries and is not thought to undergo
mitosis in vivo, so when cells are lost, the gap is
compensated by active sliding and enlargement
of adjacent cells [30]. This results in the restora-
tion of the endothelial barrier at the expense of
reducing cell density. The process of resurfacing
the injured area is completed in three stages that
can take several weeks [31]. First, there is an ini-
tial coverage of the wound by migration of adja-
cent endothelial cells, which forms a temporary
incomplete barrier with reduced pump sites and
immature tight junctions. Following this, the bar-
rier (i.e., tight junctions) and subsequently the
number and quality of pump sites normalize, the
endothelial cells adopt an irregular polygonal
shape, and the corneal thickness typically returns
to normal at the same time that the transparency
is restored. Finally, during the following months,
the endothelial cells reacquire their characteristic
hexagonal form.
There are a number of reasons that prevent
endothelial cells from dividing and are currently
still a matter of research and discussion. It is
thought that the densely packed cells exhibit
strong contact inhibition, mediated by p27kip1, a
cyclin-dependent kinase inhibitor that arrests
cells in the non-mitotic G1 phase [32, 33]. In
addition to this, it has been described that in the
aqueous humor, there are low concentrations of
growth factors that promote mitosis, while inhib-
itory growth factor such as TGF-ß is found in
abundance [32]. Finally, the high rate of cellular
metabolism with chronic ultraviolet light expo-
sure results in nuclear oxidative DNA damage
20
that can promote a state of stress-induced senes-
cence [34].
The current knowledge about the negligible
regenerative capacity of the corneal endothelium
has been challenged after the popularization of
endothelial keratoplasty (EK), specifically with
the Descemet membrane endothelial keratoplasty
(DMEK). Reports of spontaneous regeneration
of the corneal endothelium are increasingly
appearing in the literature, but it remains unclear
whether this clinical improvement is due to
enlargement and spreading of the cells or true
cell division.
Spontaneous endothelial barrier reformation
has been described in different surgical scenar-
ios. Corneal clearance after failed endothelial
keratoplasty due to graft detachment has been
reported in Fuchs endothelial corneal dystrophy
(FECD) patients [35] and also in normal subjects
after the accidental removal of the Descemet
membrane in the course of cataract surgery [35].
Bare areas of uncovered corneal stroma have
been repopulated with endothelial cells after
free-floating endothelial roll injection, procedure
known as Descemet membrane endothelial trans-
fer (DMET) in FECD but not in pseudophakic
bullous keratopathy [36]. Endothelial repopula-
tion has been also seen after DMEK graft decen-
tration that leaves an arc or annulus of bare
stroma or in recently described hemi-DMEK or
quarter-DMEK, where the graft is bisected or
divided into four pieces, respectively, prior to
implantation, which raises an interesting ques-
tion regarding the amount of endothelial graft
material needed for transplantation [35]. Even
more, the outcomes of planned descemetorhexis
without grafting in FECD were surprising, as
clear cornea was observed in more than 65% of
patients (n = 31/47), with improvement detected
as early as 1 month postoperatively [35]. The rea-
son why recipient endothelium does not show
capacity to repair itself without surgery is proba-
bly because in the cases of FECD and PBK, the
damaged endothelial cells function very poorly
but are not so dysfunctional as to detach from the
Descemet membrane. They therefore occupy
space and by contact inhibition prevent ingrowth
of healthy cells in their place.
V. Vargas et al.
Pharmacological treatment may also have a
role on endothelial barrier reformation. Since
Okumura and colleagues in 2009 identified a
specific ROCK inhibitor Y-27632 capable of
promoting adhesion, survival, and proliferation
of primate-derived corneal endothelial cells
in vitro [37], other studies have shown that this
drug is able to enhance corneal endothelial
wound healing in  vivo in animal models, both
when injected intracamerally with a cell suspen-
sion and when applied topically as eye drops
[38, 39]. Following on from the animal data, pro-
spective studies with higher number of patients
are needed to demonstrate its efficacy, although
some preliminary studies have shown that rho-
associated protein kinase (ROCK) inhibitors,
either as Y-27632 or ripasudil, exert a certain
effect and may have a role as supplementation to
salvage procedures when there is no corneal
edema resolution [40–42].
There is not a single mechanism that explains
the reformation of the endothelium in vivo, and
the relevance of each mechanism is still specula-
tion. Cellular enlargement and migration to repair
a defect is probably the most important one. In
the cases of corneal endothelial transplantation,
when a gap in the barrier is filled, it is often not
possible to tell whether migrating donor or recip-
ient cells filled the gap. The centripetal pattern of
corneal resolution in hemi-DMEK and quarter-­
DMEK implies that the cells on the donor graft
are responsible for clearing [43]. Paracrine effect
of growth factors secreted by the transplanted
donor graft may also contribute to endothelial
cell migration, and this mechanism has been con-
sidered key in DMET success [44]. Finally, true
proliferation may also happen in the reformation
of endothelium in  vivo, although there is not
enough evidence. Corneal endothelial mitosis
was described in patients in vivo as early as in the
1980s when mitotic spindles were detected using
specular microscopy and thymidine incorpora-
tion in transcorneal frozen eyes [44]. Recently a
study identified a rapidly proliferating subpopu-
lation of cells in the endothelium without any
sign of cell aging, with the ability to form spheres
and express stem cell and neural crest markers
[44, 45]. These results are suggestive, but
2  Corneal Healing
although it is tempting to think that there may be
an endothelial stem cell population, without
human studies using cellular labeling in vivo, it is
difficult to conclude with certainty if the corneal
endothelial cells are proliferating.
Take-Home Messages
• Corneal healing is a complex process in
which numerous growth factors and
cytokines are involved.
• Of all corneal layers, the epithelium is
the quickest to heal.
• During the stromal healing process,
haze may appear due to numerous myo-
fibroblasts and a disorganized ECM.
• After injury, endothelial cells adjacent
to the injury site slide and enlarge in
order to fulfill the defect.
• There might be endothelial cell prolif-
eration, but we need more evidence to
support it.
• Some growth factors have been success-
fully used to enhance corneal healing.
Compliance with Ethical Requirements  Veronica
Vargas, Francisco Arnalich-Montiel, and Jorge L.
Alio del Barrio declare that they have no conflict
of interest. No human studies were carried out by
the authors of this article. No animal studies were
carried out by the authors for this article.
Financial Disclosure
None of the authors have any financial disclosure
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42. Koizumi N, Okumura N, Ueno M, Nakagawa H,
Hamuro J, Kinoshita S.  Rho-associated kinase
inhibitor eye drop treatment as a possible medi-
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Melles GRJ.  Hemi-Descemet membrane endothelial
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44. Lam FC, Bruinsma M, Melles GRJ. Descemet mem-
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2016;186(10):2736–50.
 Corneal Tissue Engineering
Mohammad Mirazul Islam, Roholah Sharifi,
and Miguel Gonzalez-Andrades
3.1 Introduction
Mankind has been always fascinated with the
idea of restoring any damaged tissue or organ. In
the Ancient Egypt, handmade prostheses were
made of hardwood or cartonnage to restore the
function of lost toes [1]. Regarding corneal func-
tional restoration, the French ophthalmologist
Pellier de Quengsy was the first one proposing in
1789 a replacement of an opaque cornea using a
piece of glass surrounded by a silver ring [2].
However, the paradigm of corneal blindness
treatment does not change until 1905, when
Eduard Zirm performed the first corneal trans-
plant to a patient implanting a donor cornea [3].
Corneal transplant is still the most used and reli-
able treatment for some corneal diseases [4].
Corneal diseases are one of the most important
causes of blindness in developing countries,
accounting for 4–8  million people that suffer
bilateral corneal blindness [5, 6]. In 2015, only in
the USA, more than 48,792 corneal transplants
were carried out, which was 53% more than the
transplants performed in 2005 [7]. In the USA,
donor corneas are readily available for transplan-
M. M. Islam · R. Sharifi · M. Gonzalez-Andrades (*)
Massachusetts Eye and Ear and Schepens Eye
Research Institute, Department of Ophthalmology,
Harvard Medical School, Boston, MA, USA
e-mail: miguel_gonzalez@meei.harvard.edu
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_3
3
tation [8], although the whole world suffers a
severe scarcity of donor corneas. This results in
10 million untreated patients worldwide, with an
additional 1.5 million new patients every year to
the waiting list [9].
Although the cornea is considered as an
immune-privileged site of the body because of
its avascularity, physiopathological changes such
as corneal neovascularization or inflammation
disrupt this immune status, which subsequently
increases the risk of graft rejection after perform-
ing a corneal transplant. Even in non-­vascularized
and non-inflamed host eyes (low-risk cases), one
in three of transplanted corneas eventually leads
to rejection [10]. In high-risk cases, such as auto-
immune diseases, chemical burns and infections,
50–70% transplants undergo rejection even with
high doses of immunosuppressive drugs [11, 12].
Another major complication after corneal trans-
plant is donor-derived infections [13]. Herpes sim-
plex virus type-1 (HSV-1) DNA found from donor
corneas before and after corneal transplantation
confirmed the spread of HSV-1 through the trans-
plant from donor to recipient [14]. In this context,
the microbial testing, the administration and the
shipping of a donor cornea can cost $3000 in the
USA [15], becoming an unaffordable treatment
option for most of the people who live in develop-
ing nations. 90% of the visually impaired people
live in low-income countries, and most of them
(53% of the world population) do not have access
to transplantation facilities [16, 17].
23
24
Therefore, there is a great need of finding new
therapeutic strategies to address the three major
drawbacks of corneal transplantation: the scarcity
of donors, the risk of rejection and the transmission
of infectious diseases after implantation into the
host. In this milieu, corneal tissue engineering (TE)
emerges with the ambition of generating artificial
corneas or other types of tissue-engineered products
that lead to an optimal corneal regeneration, over-
coming those major disadvantages of allogeneic
corneal transplants. TE was defined in 1988, in a
workshop on TE organized by the University of
California, Los Angeles, as “the application of prin-
ciples and methods of engineering and life sciences
toward fundamental understanding of structure-
function relationships in normal and pathological
mammalian tissues and the development of biologi-
cal substitutes to restore, maintain, or improve tis-
sue functions” [18–20]. Engineering effectual
building blocks and assembling them to perform in
a unified architecture is the bedrock for the genera-
tion of fully functional biological substitutes. Such
constructs should also be able to communicate with
the other tissues and organs that surround it to coor-
dinate a unified function [21]. This demands pro-
found knowledge in material science, including
material interactions with cells and their microenvi-
ronment. Especially, this is crucial in ophthalmol-
ogy where besides physical and chemical properties
of the tissues, optical characteristics as well as
architectural design dictate the ultimate outcome.
3.2 Corneal Structure-Function
Relationships
Firstly, we need to understand the structure-­
function relationships in the cornea, in order to
develop an optimal corneal substitute. The cornea
is the outermost part of the eye and plays an impor-
tant role for vision by transmitting the light to the
retina while protecting the interior components of
the eye from external aggressions. The cornea is
composed of three primary cellular layers, the out-
ermost epithelium layer, a middle stroma contain-
ing keratocytes, and an innermost single layer of
endothelial cells called endothelium [22]. Two
acellular layers separate these cellular layers:
Bowman’s layer and Descemet’s membrane. The
M. M. Islam et al.
extracellular matrix (ECM) of the corneal stroma
is mainly composed by collagen and proteogly-
cans disposed in a highly specific arrangement.
This specific matrix arrangement grants the cornea
its characteristic transparency and physical struc-
ture, which allow an optimal vision and support
the intraocular pressure without deforming [23].
ECM is also intimately associated with corneal
innervation. Corneal nerves cross through the cor-
neal stroma toward the epithelium. Epithelial
innervation plays a vital role in functional activi-
ties of the cornea such as preserving the viability
and differentiation of the corneal epithelium, apart
from their role in tear production and blinking
[24]. Moreover, the optimal composition and
porosity of the corneal stroma allow the diffusion
of nutrients and other solutes from the posterior to
the anterior region of the cornea [25, 26]. Corneal
nutrition is complemented by the tears [27]. The
local immunity of the cornea is conditioned to its
avascularity. Antigen-presenting cells like den-
dritic cells are present in the cornea. These cells
are involved in T-cell-mediated immune responses
associated with corneal graft rejection. Natural
killer cells also participate in the allograft rejection
[28, 29]. Graft rejection starts when the host
immune system is activated against antigens in the
donor corneal tissue through different pathways
(for review see Refs. [30, 31]).
3.3 Development of Tissue-­
Engineered Corneal
Substitutes
Once we understand the structure-function rela-
tionships in the cornea, we can generate a tissue-­
engineered corneal substitute to restore, maintain,
or improve corneal functions, using different
building blocks: cells, scaffolds and bioactive
molecules.
3.3.1 Cells
As it was discussed earlier, healthy cellular layers
are necessary for the precise function of the
human cornea. In case of cell injury or loss, stem
cells need to regenerate and repopulate the dam-
3  Corneal Tissue Engineering 25

aged area. Stem cells are indispensable players Table 3.1  Human cells other than limbal stem cells eval-
uated for corneal epithelial regeneration
for the regeneration of any part of the body. In the
cornea, different types of stem cells reside in the In
vitro/in
limbus area with the capacity to regenerate the Source of cells vivo References
corneal epithelium [32] and the stroma [33, 34]. Conjunctival In vivo Ang et al. [55]; Sangwan
Limbal stem cells (LSCs) can differentiate into stem cell (human) et al. [56, 57];
corneal epithelial cells after isolating and cultur- Subramaniam et al. [58];
Tan et al. [59]
ing them from small biopsies of healthy limbal
Oral mucosal In vivo Burillon et al. [60];
areas [35–37]. Different substrates or carriers can epithelial cells (human) Inatomi et al. [61];
be used to culture and deliver LSCs in cases of Nakamura et al. [62];
limbal stem cell deficiency (LSCD) including Nishida et al. [63];
human amniotic membranes [38], fibrin sub- Takeda et al. [64];
Utheim [65]
strates [39] and collagen-based materials [40].
Nasal mucosal In vivo Chun et al. [44]; Kim
This therapeutic approach receives the name of epithelial cells (human) et al. [45]
cultured limbal epithelial transplantation (CLET). Dental pulp In vivo Gomes et al. [46];
This expansion of LSCs for transplantation stem cells (rabbit) Monteiro et al. [47]
requires certified good manufacturing practices Hair follicle In vivo Meyer-Blazejewska et al.
bulge-derived (mouse) [48]
facilities and procedures, which limit the expan- stem cells
sion of this therapeutic approach because of the Wharton’s jelly In vitro Garzon et al. [49]
high cost, especially in developing countries stem cells
[41]. Embryonic stem In vitro Ahmad et al. [66]; Zhang
In cases of suffering bilateral LSCD with no cells et al. [67]
Umbilical cord In vivo Reza et al. [50]; Reza
healthy limbal area to obtain an optimal biopsy, stem cells (rabbit) et al. [68]
only allogeneic limbal tissue can be used for per- Bone marrow-­ In vivo Ma et al. [20]; Rohaina
forming CLET.  To avoid the use of allogeneic derived MSC (Rat) et al. [52]
cultured cells and its inherent risk of immune Orbital In vivo Lin et al. [53]
rejection, other types of stem cells that do not fat-derived MSC (mouse)
Dermal In vitro Hayashi et al. [54]
reside in the limbal area are emerging as possible
fibroblast-­
future cell source for autologous CLET derived iPS cells
(Table  3.1). Conjunctival epithelial cells from Corneal limbal In vitro Hayashi et al. [54]
biopsies cultured and expanded in vitro on con- epithelial
tact lens were used to treat one patient, who cell-derived iPS
cells
improved visual acuity with no recurrence of cor-
neal vascularization [42]. The possibility of treat-
ing LSCD using in vitro cultivated oral mucosa epithelial-­like cells on fibrin-agarose-based stro-
autograft has been widely studied in at least 20 mal substitutes [49]. Transplantation of human
clinical trials in different countries. Accumulated umbilical cord stem cells in LSCD rabbit eyes
results from those clinical trials showed that 242 resulted in healthy corneal surface with positive
patients received this treatment with a success marker expression for corneal epithelial cells
rate of 72% [43]. Nasal mucosal epithelial cells [50]. Human bone marrow-derived mesenchymal
also showed promising results when transplanted, stem cells (MSC) were able to differentiate to
in two different clinical trials [44, 45]. Cultured corneal epithelial cells in vitro and in vivo, show-
human immature dental pulp stem cells recon- ing their capability to replace limbal epithelial
structed the eye surface in limbal stem cell-defi- stem cells [20, 51, 52]. Orbital fat-derived MSC
cient rabbits [46, 47]. Hair follicle bulge-derived also promoted corneal tissue regeneration
stem cells from transgenic mice also showed cor- through corneal epithelial differentiation [53].
nel epithelial cell differentiation in a LSCD Human adult dermal fibroblast-­derived induced
model [48]. Human Wharton’s jelly stem cells pluripotent stem (iPS) cells and human adult cor-
also showed potential to differentiate into corneal neal limbal epithelial cell-derived iPS cells were
26
also tested for differentiation into corneal epithe-
lial cells, which revealed that corneal epithelial
differentiation efficiency was higher in limbal-
derived iPS cells [54].
Keratocytes quiescently reside within colla-
gen lamellae in the stroma of a healthy cornea,
synthesizing ECM components, such as colla-
gen and proteoglycans [69, 70]. In the damaged
or injured cornea, keratocytes transform into
mitotically active fibroblasts [71, 72] and start
producing unorganized ECM which ultimately
turn into fibrotic tissue, which might lead to
vision loss [73]. Keratocytes can be isolated and
cultured under specific conditions using corneal
biopsies, which can be digested or directly cul-
tured applying an explant-based technique [74,
75]. Keratocytes can also be obtained from
MSC isolated from limbal biopsies [33, 34].
They can synthetize aligned collagen and kera-
tan sulfate proteoglycans, being able to recon-
stitute a fibrotic area in in vivo models, without
inducing inflammation, vascularization, or
rejection [76–78]. iPS cells can be also differen-
tiated to neural crest cells and then cultured on
corneal tissue to promote keratocyte differentia-
tion [79].
The corneal endothelium is a monolayer of
cells that lines at the posterior corneal surface,
which are responsible for pumping out excess
amount of water from the corneal stroma and
prevent it from swelling [80, 81]. Its failure usu-
ally requires a donor endothelial transplant
because of the very limited proliferative capac-
ity of these cells to self-regenerate the damaged
area. However, under specific conditions, endo-
thelial cells can proliferate and cultured in vitro.
Shigeru Kinoshita and co-worker described a
new promising approach based on the inhibition
of ROCK (Rho kinase), which enhances endo-
thelial cell proliferation, promotes cell adhe-
sion, suppresses apoptosis and promotes wound
healing. In 2013, they have started a clinical
trial to evaluate cultured human endothelial
cells in combination with a ROCK inhibitor as
treatment for corneal endothelial dysfunction.
Recently, they reported their initial results, sug-
gesting that this therapeutic option is safe and
effective [82].
M. M. Islam et al.
3.3.2 Scaffolds
Engineered 3D scaffolds not only can substitute a
damaged cornea, providing mechanical and
structural stability, but also provide the appropri-
ate microenvironment for the cells to regenerate
the tissue. Conceptually, scaffold is an engi-
neered template, which can mimic the ECM of
the native tissue and imitate the in  vivo setting,
supporting the cells to proliferate, migrate and
create their own microenvironment [83].
The ideal corneal scaffold should (1) be trans-
parent for restoration of vision; (2) be biocompat-
ible and support cellular adhesion, proliferation
and migration; (3) have similar biomechanical
properties to the human cornea to maintain its
shape, critical for an optimal vision, and har-
monically respond to the intraocular pressure
fluctuations; (4) preserve its smooth surface in
order to avoid scattering of light; (5) have bio-
degradation properties that match the time of
tissue remodeling and biointegration; (6) have a
refractive index similar to the cornea; (7) possess
appropriate porosity and diffusion for nutrients,
while serving as a microbial barrier; and (8) be
cost-effective in terms of manufacturing process
and implementation [84].
The scaffolds explored in ophthalmology for
corneal substitution can be categorized into three
classes: synthetic, natural-based and hybrid
materials. Polyethylene glycol [85], acrylate-­
based polymers [86], polyesters [87],
polydimethylsiloxane [88], polyvinyl alcohol-
based polymers [89] and polyamides [90] are the
main studied synthetic materials for corneal sub-
stitutes. Although these synthetic polymers have
tunable chemical and mechanical properties that
can be matched to the medical needs, their bio-
mimetic properties required for cell adhesion,
proliferation and effectual integration with the
host tissue need significant improvement before
their translation in the clinical settings. In addi-
tion to their non-biodegradable nature, their
inability to carry cells and biointegrate during
tissue healing and remodeling stands as their
main challenge [91]. The continued progress in
the engineering of novel biomaterials, along
with personalized modifications and the design
3  Corneal Tissue Engineering
of hybrid materials composed of synthetic and
natural polymers, might address such shortcom-
ings and can facilitate their widespread applica-
tions in the clinic.
On the other hand, natural-based biomateri-
als present intrinsic biocompatibility and bio-
degradability along with appreciable degree of
biomimetic properties and biological functions.
The most studied natural-based corneal scaffolds
are protein- or polysaccharide-based scaffolds.
Collagen is one of the most studied protein-
based scaffolds for artificial cornea. This stems
from collagen’s biocompatibility, low toxicity,
and well-studied structural, physical, chemi-
cal and immunological characteristics alongside
with maintaining arginine-glycine-aspartic acid
sequences in its structure that promotes cell adhe-
sion to the scaffold [92]. Although collagen-­based
scaffolds demonstrated such promising proper-
ties [93–95], their biomimetic characteristics still
require an enhancement to totally match those
found in specific native tissues [96]. Although
improving mechanical and biomimetic properties
of hydrogel is an ongoing challenge, engineering
new hybrid scaffolds and integrating the biologi-
cal cues might be a key to unlock their potential
as a true corneal substitute. Gelatin [97], fibrin
[98] and silk [99] are among the other protein-
based biopolymers that have also been explored
as possible candidates for corneal substitute.
While each class possesses different characteris-
tics, their mechanical properties are significantly
inferior compared to those of the native cornea
and unable to support integrity of injured cornea.
Polysaccharide-based materials (e.g., chitosan
[100], chondroitin sulfate [101], dextran [102],
hyaluronic acid [103], alginate [104], etc.) have
also been explored in corneal tissue engineer-
ing. Despite their superior mechanical and opti-
cal properties, they fall short in providing 3D
microenvironment for effective cell adhesion and
proliferation. Therefore, polysaccharide-­based
materials have not been yet able to offer an effec-
tive solution for corneal substitute.
Although both synthetic and natural-based
scaffolds offer an initiative window to develop an
effective artificial cornea, such scaffolds lack the
complexity of the 3D microenvironment of the
27
corneal native tissue in terms of not only physical
and chemical properties but also composition gra-
dients, alignment, directionality and microar-
rangement manifested in the human corneal
stroma. The emulation of such biomimetic char-
acteristics is very crucial in tissue engineering and
dramatically dictates biointegration of artificial
corneas with the host tissue and defines the ulti-
mate clinical outcome. Different strategies have
been described in the literature to address some of
those challenges such as the use of self-­assembly
or auto-generation of artificial matrixes in vitro.
Peptide amphiphiles (PA) are engineered syn-
thetic molecules constituted from a hydrophilic
peptide sequence and hydrophobic long chain,
which can self-assemble to generate nanofibers.
The non-covalent interaction of such nanofibers
via intermolecular forces can lead to the forma-
tion of 3D networks. The immense programma-
bility of PA to hold different functional groups
enables to generate 3D scaffolds, such as collagen
hydrogels, that, in principle, can mimic ordering
and complexity of native tissue [105–108].
Integrating such self-assembled structures within
various hydrogel offers a precious tool to intro-
duce highly complex multifunctional hydrogels
for ophthalmic surgery. Another approach to
introduce such nanoscale organization is auto-­
generation. This concept is based on engineering
an in vitro culture system that stimulates synthesis
of an in vivo-like stromal matrix that can lead to
the generation of highly organized collagen-based
corneal stroma [109, 110]. Moreover, it allows to
seed epithelial and endothelial cells on the synthe-
sized scaffold to create a functional, organotypic
cornea. In this regard, human mesenchymal stem
cells derived from the limbal stroma were cul-
tured in specific culture media, leading to rapid
expansion and differentiation into keratocytes and
ultimately generating an organized thick lamellar
stroma-like tissue containing aligned collagen
and keratan sulfate proteoglycans [76]. The con-
structs synthesized by these cultured cells, how-
ever, also present poor mechanical properties and
need significant improvement prior to their appli-
cation in ophthalmic surgery.
Incompetence of bioengineered scaffolds as yet
to fulfill the required properties of corneal substi-
28
tute is the main driving force to study other strate-
gies in parallel such as the use of modified
xenografts. Xenogeneic tissues and organs often
contain cellular antigens, which can be recognized
as foreign by the host tissue and consequently
leads to an inflammatory response or an immune-
mediated rejection [111]. Decellularization of
donor tissue to remove the inhabiting cells and its
cellular debris from the ECM of the tissue is a
practical strategy to obtain acellular scaffolds of
the ordinal tissue and bypass such adverse immune
response [112]. Besides intimate resemblance of
microarchitecture of xenogeneic corneas with
humans, their availability, lower cost and compa-
rable optical and mechanical properties are the
main momentum to envisage their application for
human corneal substitution. Moreover, it can sup-
port construction and host-guest tissue remodeling
and bypass the stimulation of inflammation while
avoiding scar tissue formation [113–116].
Different animals have been used as source of cor-
neal tissue for decellularization process. Due to
availability and the structural similarities between
the porcine and human cornea, the domestic pig is
the most commonly used animal to obtain decel-
lularized corneal xenografts [117]. Although vari-
ous chemical and physical techniques have been
explored to decellularized animal corneas, they
often alter the chemical, physical and biological
properties of the ECM via cleaving the collagen
fibers and disrupting the matrix ultrastructure or
partially eliminating key matrix constituents such
as glycosaminoglycans and growth factors and
adversely affect its natural properties [112, 118].
Such structural disruptions along with chemical,
mechanical and biological variations between por-
cine and human cornea are the main challenges,
preventing their successful translation into the
clinic [119].
3.3.3 Bioactive Molecules
and Other Environmental
Conditions
There are several soluble factors directly involved
in the process of proliferation and differentiation
of corneal cells. In this context, significant efforts
M. M. Islam et al.
have been carried out to understand the control
mechanisms of self-renewal and fate decision of
LSCs. There are growing evidences supporting
that LSCs are highly regulated by their stem cell
niche. LSC niche is a specific microenvironment
that comprises cellular and noncellular compo-
nents that regulate the stem cell pluripotency,
proliferation, differentiation, survival and local-
ization. LSC niche is located at the palisades of
Vogt of human corneoscleral limbus. Different
growth factors also play important role in the
differentiation of the stem cell to progeny. In
this regard, insulin-like growth factor I (IGF-I)
has been identified as the main factor respon-
sible for LSC differentiation into mature cor-
neal epithelial cells after injury. Furthermore,
some researchers have demonstrated that IGF-I
showed synergistic effect with the neuropeptide
substance P in proliferation and wound healing
of corneal epithelium [120]. Corneal epithe-
lium also produced fibroblast growth factor and
epidermal growth factor (EGF) to support LSC
proliferation devoid of affecting differentiation
[121]. EGF heparin-­binding EGF and amphireg-
ulin have been also shown to stimulate epithe-
lial wound repair by binding to a common EGF
receptor [122]. Hepatocyte growth factor is also
an important factor expressed by epithelial cells
and keratocytes after corneal epithelial injury,
which influences the proliferation, migration and
apoptosis of corneal epithelial cells [123–125].
Keratinocyte growth factor also plays impor-
tant role in epithelial wound healing through
MAP kinase and PI3K/p70 S6 signaling cascade
[126]. Moreover, transforming growth factor-β
(TGF-β) expressed by corneal epithelium and
stromal cells has mixed effect on corneal cells,
inhibiting ­epithelial cell proliferation [127] and
stimulating fibroblast proliferation [128]. TGF-β
also showed to influence myofibroblast differen-
tiation of cultured primary keratocytes and cor-
neal fibroblast cell line [129]. Platelet-derived
growth factors expressed by differentiated cor-
neal epithelium in  vitro regulate the prolifera-
tion and migration of corneal fibroblast [130].
Nerve growth factor (NGF) is a neurotrophic
factor expressed in the corneal epithelium that
promotes cell proliferation and wound healing.
3  Corneal Tissue Engineering 29

NGF improved epithelium restoration in patients 3.4 Clinical Experiences

with neurotrophic ulcers [131] and after cataract
surgery [132]. NGF also showed nerve regenera-
tion in a mechanical nerve injury mouse model
established by laser-assisted in situ keratomileu-
sis [133]. Opioid growth factor (OGF) is another
growth factor expressed by basal and suprabasal
layers of epithelium that binds OGF receptor to
inhibit DNA synthesis, cell migration and tissue
repair of the corneal epithelium [134]. Important
growth factors with their key physiological func-
tions are summarized in Table 3.2.
Not only growth factors but also culture con-
ditions are key in the proliferation and differen-
tiation of corneal cells. Concentration of carbon
dioxide in culture conditions critically alters cell
differentiation. It was shown that 7% CO2 in the
culture positively influences the differentiation
of embryonic stem cell to corneal epithelial pro-
genitor cells [67]. Hypoxic condition is also an
important factor that influences the differentia-
tion of limbal stem cells by downregulating
Polo-­like kinase 3 (Plk3) signaling activity at the
transcription level [151]. Co-culturing condi-
tions also positively influence cell growth, as
survival and proliferation of LSCs are promoted
when these are co-cultured with bone marrow
MSC [152].
and International
Regulations
Very few tissue-engineered products have been
translated into the clinic. Some of the organs
where tissue-engineered substitutes have been
successfully applied are the trachea [153], blood
vessels [154], the urinary bladder [155], and the
cornea [4]. Regarding the cornea, some notable
mentions are as follows: (1) autologous limbal
stem cells were collected from the healthy con-
tralateral eye and expanded on a fibrin substrate
and finally transplanted in 112 patients with
LSCD. Restoration of a transparent cornea with
a restored corneal epithelium was achieved in
76.6% of eyes and 21 patients achieved perma-
nent visual recovery of at least 0.6 [36]; (2) as a
phase 1 clinical trial, femtosecond laser cut ante-
rior corneal stroma was decellularized and trans-
planted in patients with keratoconus. Four out
of nine patients received a decellularized stroma
seeded with autologous adipose-derived adult
stem cells. Haze or scarring was not observed
by 3-month postoperative follow-up, and
patients got visual improvement after 6 months
of the graft [156]; (3) recombinant human col-
lagen (RHC)-based acellular artificial corneas

Table 3.2  List of growth factors that influence corneal regeneration

Growth factors Key function References
Epidermal growth Cell migration, proliferation and wound healing of Zieske et al. [122]; Nakamura et al. [135]
factor corneal epithelial cells
Hepatocyte Cell migration, proliferation and wound healing. It Wilson et al. [123]; Daniels et al. [124];
growth factor inhibits apoptosis of corneal epithelial cells Yanai et al. [136]
Keratinocyte Epithelial homeostasis and wound healing Chandrasekher et al. [137]
growth factor
Insulin-like Cell growth, energy metabolism, migration, Lee et al. [136]; Trosan et al. [138]; Yanai
growth factor differentiation, proliferation and survival of et al. [139]
corneal epithelial cells
Transforming Inhibition of corneal epithelial cell proliferation. It Pancholi et al. [127]; Haber et al. [128];
growth factor-β stimulates stromal fibroblast proliferation Andresen et al. [140]; Kay et al. [141]
Platelet-­derived Migration and proliferation of keratocytes Denk and Knorr [130]; Kamiyama et al.
growth factors [142]; Daniels and Khaw [143]
Thymosin-β4 Wound healing in corneal epithelial defects. It Sosne et al. [144, 145]; Dunn et al. [146]
decreases inflammation and inhibits apoptosis
Nerve growth Epithelial and stromal healing, anti-inflammatory Lambiase et al. [147]; Lambiase et al.
factor effect and recovery of corneal nerves [148]; Joo et al. [149]
Opioid growth Inhibitory effect on corneal epithelial cell Zagon IS et al. [150]
factor proliferation, migration, and tissue organization
30
have been transplanted in a clinical trial on ten
patients; nine of them had keratoconus and one
patient with permanent mid-stromal scar. The
implants promoted regeneration of corneal epi-
thelium, stroma, and nerves from host cells. The
transplanted cornea remained stable for 4 years
without any rejection and without sustained
immune suppression. Implanted patients had a
4-year average corrected visual acuity of 0.37
[4, 157]; (4) acellular interpenetrating polymer
networks of RHC and 2-­methacryloyloxyethyl
phosphorylcholine (MPC) have been trans-
planted in three patients with corneal ulcers and
recurrent corneal erosions. The implants pro-
vided relief from pain and discomfort, restored
corneal integrity, and improved vision in two out
of three patients [158]. Another clinical trial has
been completed with this materials on January
2017 (CT.gov identifier:NCT02277054). The
result showed that all patients improved from
pain and discomfort within 1–2  weeks after
transplantation. Corneal sensitivity regained,
and overall vision improved significantly in half
of the study patients, and even if the vision was
not enhanced, transplants made the cornea sta-
ble for further surgery to improve vision [159];
and (5) there is a randomized, controlled, open-
label clinical trial going on in different Spanish
hospitals (CT.gov identifier:NCT01765244)
to test a fibrin-agarose corneal substitute com-
bined with allogeneic corneal epithelial cells
and keratocytes [160].
In the translation of tissue-engineered prod-
ucts into the clinic, the different regulatory agen-
cies have created a regulatory framework that
controls and guarantees the correct and ethical
use of these therapies in humans, protecting not
only the patients but also the clinicians who are
applying the treatment. The European Medicines
Agency (EMA) includes tissue-engineered prod-
ucts under the definition of advanced therapy
medicinal products, which are defined as medi-
cines for human use that are based on genes,
cells, or tissue engineering [161]. According to
European Parliament regulations and the EMA
guidelines [51], “Tissue engineered product
means a product that: contains or consists of
engineered cells or tissues, and is presented as
having properties for, or is used in or adminis-
M. M. Islam et al.
tered to human beings with a view to regenerat-
ing, repairing or replacing a human tissue. A
tissue engineered product may contain cells or
tissues of human or animal origin, or both. The
cells or tissues may be viable or non-viable. It
may also contain additional substances, such as
cellular products, bio-molecules, biomaterials,
chemical substances, scaffolds or matrices.” This
definition together with other aspects contem-
plated in the European regulations is shown in
Fig. 3.1. Regarding the clinical trials previously
mentioned, only decellularized stromas seeded
with autologous adipose-derived adult stem cells,
autologous epithelial limbal stem cells cultured
on fibrin scaffolds, and fibrin-agarose corneal
substitutes with allogeneic cultured corneal cells
would be considered tissue-engineered products
according to EMA guidelines.
The Food and Drug Administration (FDA) is
responsible for the regulation of medical prod-
ucts, including tissue-engineered products, in
the USA.  FDA regulated medical products
under the separate categories of devices, bio-
logics, and drugs. According to FDA, human
tissue intended for transplantation, such as a
donor cornea, is regulated as a human cell, tis-
sue, and cellular and tissue-based product or
HCT/P [162]. Tissue-­engineered products usu-
ally consist in the combination of two or more
components that belong to different categories
in the FDA regulation, falling into the category
of combination products [163]. Tissue-
engineered products based on biomaterials in
conjugation with cells would fall in this cate-
gory. Recently, FDA announced new guidance
documents for comprehensive regenerative
medicine policy, defining the regulatory
requirements for devices used in the recovery,
isolation and delivery of regenerative medicine
advanced therapies (RMATs), including combi-
nation products and the description of those
regenerative medicine therapies that may be
eligible for RMAT designation, including cell
therapies, therapeutic tissue-engineered prod-
ucts, human cell and tissue products, and com-
bination products using any such therapies or
products, as well as gene therapies that lead to
a durable modification of cells or tissues
(including genetically modified cells) [164].
3  Corneal Tissue Engineering 31

- Regenerating,
Tissue engineered product Function** - Repairing or a human tissue
- Replacing

Composition
*Non-substantial manipulations: cutting;
- Human and/or
Origin grinding; shaping; centrifugation; soaking in
- Animal antibiotic or antimicrobial solutions; sterilization;
Engineered irradiation; cell separation; concentration or
purification; filtering; lyophilization; freezing;
cells and/or - Viable and cryopreservation; and vitrification.
tissues Viability - Non-viable

- Pharmacological,
Main mechanism of action - Immunological or
- Metabolic

Subjected to substantial manipulation*

to obtain its necessary properties to
achieve its function**
Requirements to be “engineered” And/or
Not intended to be used for the same
essential function or functions in the
recipient as in the donor
- Cellular products,
- Bio-molecules,
- Biomaterials,
Additional substances - Chemical substances,
- Scaffolds or
- Matrices

Fig. 3.1  Definition of tissue-engineered product according to the European regulations [51]

3.5 Conclusions References

Here we briefly highlight the corneal structure-­
function relationships and the principles to
develop a biological substitute of the human cor-
nea by tissue engineering, including some treat-
ment options for corneal diseases based on
specific tissue-engineering strategies. Moreover,
we explained the concepts and regulations neces-
sary to understand the future clinical impact of
tissue engineering in ophthalmology. The next
chapters of this book will elaborately explain the
use of cell and tissue-engineering therapies to be
surgically applied to different corneal diseases.
Compliance with Ethical Requirement
Statements  Mohammad Mirazul Islam,
Roholah Sharifi, and Miguel González-Andrades
declare that they have no conflict of interest. No
human or animal studies were carried out by the
authors for this article.
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 Part II
The Stem Cell
 Stem Cells: Concept, Properties,
and Characterization
4
Natalia Escacena-Acosta, Javier Lopez-Beas,
Christian Claude Lachaud, Mehrdad Vakilian,
Juan Rigoberto Tejedo, Vivian Capilla-González,
Francisco Javier Bedoya, Franz Martin,
Abdelkrim Hmadcha, and Bernat Soria

4.1 Concept ium of these two properties is the capacity to colo-

nize and reconstitute a tissue (as in hematopoietic
The biology of stem cells (SCs) is one of the most SC transplantation). Currently, proliferation and dif-
important areas in current biomedical research. ferentiation use opposite intracellular pathways, and
Currently, they have an important role in regenera- most times forcing one of them will block the other.
tive medicine and many potential applications. Due
to involvement of SCs in development and their
4.2 Classification
potential to give rise to different cell types, SCs are
essential in the field of developmental biology. SCs
The SC can be classified according to their dif-
are characterized by the capacity to proliferate and
ferentiation potential or based on their source [1]:
generate daughter cells that keep the same phenotype
(self-renewal, symmetric division) and the capacity
to differentiate into other cell types (differentiation, 4.2.1 Source
asymmetric division). While the first property allows
reaching enough cell mass to fulfill a physiological (i) Embryonic
function, the former allows obtaining postmitotic At the first days of development, an outer
differentiated cell types or somatic cells. A corolar- layer of cells committed to becoming part

N. Escacena-Acosta · J. Lopez-Beas · C. C. Lachaud

M. Vakilian · V. Capilla-González
Department of Cell Regeneration and Advanced
Therapies, Andalusian Center of Molecular Biology
and Regenerative Medicine-CABIMER, Junta de
Andalucía-University of Pablo Olavide-University of
Seville-CSIC, Seville, Andalusia, Spain
J. R. Tejedo · F. J. Bedoya · F. Martin · B. Soria (*)
Department of Cell Regeneration and Advanced
Therapies, Andalusian Center of Molecular Biology
and Regenerative Medicine-CABIMER, Junta de
Andalucía-University of Pablo Olavide-University of
Seville-CSIC, Seville, Andalusia, Spain
Centro de Investigación Biomédica en Red de
Diabetes y Enfermedades Metabólicas Asociadas
(CIBERDEM), Madrid, Spain
University Pablo de Olavide, Seville, Spain
e-mail: bernat.soria@cabimer.es
A. Hmadcha (*)
Department of Cell Regeneration and Advanced
Therapies, Andalusian Center of Molecular Biology
and Regenerative Medicine-CABIMER, Junta de
Andalucía-University of Pablo Olavide-University of
Seville-CSIC, Seville, Andalusia, Spain
e-mail: karim.hmadcha@cabimer.es

© Springer Nature Switzerland AG 2019 41

J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_4
42
of the placenta (trophectoderm identity)
separates from the inner cell mass (ICM),
the part of the blastocyst that is fated to
become the embryo, amnion, and yolk sac.
These pluripotent cells from inner cell mass
(ICM) of embryo, ICM-derived cells, are
ES cells.
(ii) Placental and Umbilical Cord Stem Cells
The human placenta, cord blood, and umbil-
ical cord are a rich source of hematopoietic
stem cells (HSCs) and mesenchymal stem
cells (MSCs), which possess enormous
regeneration potential. These SCs are multi-
potent, meaning that they can differentiate
into many different types of cells.
(iii)  Adult Stem Cells
Adult stem cells or tissue-specific stem
cells are unspecialized cells that have been
found in a particular small area of many tis-
sues. Adult stem cells are capable of long-
term renewal and differentiation into
specialized cell types. During the last years,
isolations of adult stem cells from different
sources have been reported. Bone marrow-
derived stem cells are still the most fre-
quently investigated cell type. Mesenchymal
stem cells (MSCs) are an example of tissue
or “adult” stem cells. They are “multipo-
tent,” meaning they can produce more than
one type of specialized cell of the body, but
not all types. MSCs make the different spe-
cialized cells found in the skeletal tissues.
MSCs are considered clonogenic and non-
hematopoietic and are characterized by
their easy isolation and for presenting the
ability to differentiate into multilineage cell
types, including osteoblasts, chondrocytes,
endothelial cells, and even neural-like cells.
MSCs constituted a heterogeneous cell
population that contains progenitor cells at
different maturation stages. MSCs were
derived and characterized using different
methods; to best match a more uniform
characterization of MSCs, the Mesenchymal
and Tissue Stem Cell Committee of the
International Society for Cellular Therapy
(ISCT) proposed minimal criteria to define
human MSC [2].
N. Escacena-Acosta et al.
4.2.2 Potentiality
(iv) Totipotential
Only the zygote or morula cells may be con-
sidered totipotential since they may give rise
to either the inner cell mass (that will dif-
ferentiate into any cell type of the fetus and
adult tissues) or the trophectoderm (which
contributes to the placental tissues) and gen-
erate a complete and viable organism.
(v) Pluripotential
They can self-renew and differentiate into
any of the three germ layers, ectoderm,
endoderm, and mesoderm, from which all
tissues and organs develop. Embryonic stem
cells (ESCs) and induced pluripotent stem
cells (iPSCs) are currently the only two
types known of pluripotent SCs.
(vi) Multipotential
They are self-renewing and developing into
closely related cell types but are more limited
than pluripotent cells. Mesenchymal stem cells
(MSCs) and hematopoietic stem cells (HSCs)
are a typical example of multipotent cells.
(vii) Unipotential
They are progenitors with the capacity to
divide and generate more cell mass of the
same cell type (e.g., the hepatocyte).
4.3 Properties
and Characterization
For decades, researchers have been studying the
biology of SCs and the molecular mechanisms
governing the pluripotency and differentiation of
the SCs, by a complex interconnected relation-
ship between an intrinsic transcriptional program
and extrinsic signaling pathways.
4.3.1 Self-Renewal
Self-renewal program of SCs is conferred by a
specific transcriptional network and cell cycle
regulation. Coordinated transcription factor net-
works act as the master regulatory mechanisms
of SC pluripotency and differentiation. Three
4  Stem Cells: Concept, Properties, and Characterization 43

key pluripotent transcription factors OCT4, NANOG-OCT4-SOX2 transcriptional core in

SOX2, and NANOG constitute the essential tran-
scriptional core maintaining the self-renewing
undifferentiated SCs [3]. The core pluripotency
circuitry is orchestrated to promote expression
of genes that maintain pluripotency while
repressing genes inducing differentiation. The
POU domain transcription factor OCT4 and
SRY-­ related HMG-box transcription factor
SOX2 are critical for the maintenance of pluripo-
tency. Although the homeodomain-containing
transcription factor NANOG is also essential for
the establishment of SCs from blastocysts, it is
however not absolutely required for the mainte-
nance of pluripotency under optimized culture
conditions [4, 5]. The core of pluripotency regu-
lators works in a combinatorial manner to con-
trol a pluripotent-specific program of gene
expression and repression to maintain pluripo-
tency. Recent studies have identified many addi-
tional transcription factors within the SC
pluripotency circuitry. Their interaction with the
ESCs was shown to be essential to induce a sta-
ble intrinsic pluripotency network to confer an
indefinite self-renewal.
Several signaling pathways that can support
pluripotent cell maintenance have been identi-
fied, including Wnt/β-catenin, FGF/ERK, TGF/
SMAD, and PKC signaling [6]. SCs require
extrinsic growth factors for the maintenance of
pluripotency in culture, and the signaling path-
ways controlled by these factors act to maintain
the intrinsic transcription factor network required
for pluripotency (Fig.  4.1). Human SCs require
basic fibroblast growth factor (bFGF) and insulin
or insulin-like growth factor (IGF) signaling to
support self-renewal. bFGF activates the
mitogen-­ activated protein kinase (MAPK) as
well as the Activin/Nodal signaling pathways.
IGF activates the Ras and PI3K/AKT while sup-
pressing MEK/ERK pathways, leading to high
GSK3β activity and low β-catenin-mediated
transcriptional activation to enhance propagation

bFGF→Activin/Nodal
WN
T
P P P P

P
P P P P
P Extracelular
lin

P P P P
P
su

P β-Catenin P P SSEAs
In

P
F/

SSEA-3/4
IG

P
MAPK P Cytoplasm
P P
P SMADs
P P
P P 2/3
GSK3
MEK/ERK

Nucleus Stem cells (SCs)

PI3K/AKT markers
Self-renewal
ESRRB
miR-302/367
P
SMADs
P
P

1/5/8 NANOG
P

P
P

IncRNA-ES1/ES2
P

P
BM

LINC-ROR
SOX2 OCT4
TRA-1-60
TRA-1-81
Pluripotency TF network

AP

Fig. 4.1  Overview of cell markers for identifying human phosphatase (AP), SSEAs (SSEA-3 and SSEA-4), TRA-­
stem cells. Schematic representation summarizing the 1-­60, and TRA-1-81) defining stem cell populations and
expression of specific pluripotency factors (OCT4, critical pathways involved in the maintenance of pluripo-
NANOG, and SOX2) and cell-surface markers (alkaline tency and self-renewal
44
and survival of human ESCs and prevent their
differentiation [7]. Therefore, modulation of key
extrinsic pathways such as TGF-β/Activin/
Nodal/SMAD2/3 and the BMP (bone morphoge-
netic protein) signaling pathway is important to
maintain SC identity [8].
4.3.2 Differentiation
The cross talk between different signaling path-
ways occurs constantly in SCs and results in a
cooperative regulation of self-renewal and/or dif-
ferentiation. The cell fate decision is accom-
plished through intricate mechanisms, involving
a network of cross talk between signaling path-
ways, externa/internal cues, gene expression reg-
ulation, and epigenetic modifications.
In recent years, a large number of growth fac-
tors have been identified, which depending on
their biological activity are capable of differenti-
ating SCs selectively toward mesodermal, endo-
dermal, or ectodermal lineages. As such, inducer
factors can play important roles during embryo-
genesis and can be used to direct or support the
differentiation of SCs. All of these approaches
increasingly underline the need to identify
inducer agents that can regulate the differentia-
tion of SCs and reliably direct the differentiation
of SCs toward specific cell types, which offer the
possibility of a renewable source of replacement
cells and tissues to treat a myriad of diseases [9].
Hence, during in  vitro differentiation experi-
ments, a common approach is to treat the cells by
proper differentiation factors that promote an exit
from the self-renewal program and drive cell dif-
ferentiation. Under appropriate conditions, SCs
generate a progeny consisting of derivatives of
the three embryonic germ layers: mesoderm,
endoderm, and ectoderm.
Some critical stages common to all protocols
of differentiation should be considered when
using the SC model for lineage-specific differ-
entiation. First, the earliest event in the devel-
opment is the formation of three germ layers:
ectoderm, mesoderm, and endoderm. The poten-
tial use of SCs to replace a functional loss of
­particular tissues may depend on efficient differ-
N. Escacena-Acosta et al.
entiation protocols to derive a specific lineage or
cell type progenitor that is required. Therefore,
protocols have been developed for directly dif-
ferentiating SCs toward a specific germ layer. At
this point, it is possible to promote the efficient
and reproducible development of the cell type
of interest. Protocols should recapitulate in vitro
the global developmental program taking place
during the early embryogenesis. Recombinant
growth factors and small-molecule compounds
are commonly used to mimic the signaling path-
ways which are activated during embryonic
development. Finally, differentiated cells that
have been developing must display functional
properties that are characteristic of mature
cell populations both in culture and when
transplanted.
4.3.3 Immunomodulation
and Immunogenicity
Prior to clinical application, translational research
will be necessary in order to ensure that differen-
tiated progenies generated are functional and
stable without the ability to dedifferentiate, at the
same time that is evaluated one of the major con-
cerns for the clinical application of SC-derived
grafts, such as the risks of immune rejection and
tumorigenicity. For a long time, SCs were con-
sidered to have immune-privileged properties
due to their low expression of major histocom-
patibility complex (MHC) class I, MHC class II,
and costimulatory molecules [1]. However, the
induction of SCs to become more differentiated
cells in vitro can result in their loss of immuno-
logical privilege.
Indeed, transplanted allogeneic and/or xeno-
geneic SCs and their derivatives are not immune-­
privileged. iPSCs would provide a source of
pluripotent cells for autologous cell therapy
without the concern of immune rejection.
­
However, iPSC-derived cells may express mole-
cules of embryonic origin associated with repro-
gramming and other abnormalities, so that even
autologous iPSCs may be targets of immune
rejection [9, 10]. Hence, immunogenicity has
emerged as a significant problem not only with
4  Stem Cells: Concept, Properties, and Characterization
allogeneic SCs but also potentially with autolo-
gous SC sources.
Previous studies have demonstrated that tradi-
tional immune suppression can prolong the sur-
vival of graft of SC derivatives but not prevent
immune rejection for a long-lasting transplanted
cell survival [10–12]. In addition, the immune
suppressants are highly toxic for patients and
have serious collateral damage such as increased
risk for cancer and infection. Therefore, it is
important to develop safe and effective strategies
to induce immune tolerance of SC-derived cells.
To address this bottleneck, recent studies are
focusing on addressing the immunomodulatory
capability of MSCs, due to its ability to prevent
immune rejection after implantation of both
autologous and allogeneic MSCs, through their
intrinsic low immunogenicity and immunomodu-
latory properties. Over the last few years, research
has been able to propose other cell types with
immunomodulatory properties such as mesothe-
lial cells capable of producing a wide number of
cytokines, growth factors, and extracellular
matrix components possessing anti-inflammatory
and immunosuppressive properties [13].
4.3.4 Characterization: SC
Signature
Once a set of defined culture conditions is estab-
lished, SCs can continue to proliferate and repli-
cate themselves indefinitely while maintaining
the capacity to generate any type of cell in the
body. The identification of established markers of
pluripotency in SCs ensures that downstream cell
proliferation and differentiation studies are con-
ducted on high-quality, undifferentiated starting
cell populations [14].
4.3.5 C
 ell Membrane Markers
and Intracellular Markers
Stage-specific embryonic antigens (SSEAs) are
cell-surface molecules used to describe the SC
differentiation state; SSEA-3 and SSEA-4 are
highly expressed in undifferentiated human SCs
45
and are downregulated upon differentiation [14–
16]. TRA-1-60 and TRA-1-81 antigens expressed
on the surface of human pluripotent SCs are
widely used as markers for identifying pluripo-
tency [14, 15, 17]. Besides this, “stemness” of
cells can also be identified by functional assays.
There are several additional marker systems
available to identify SCs; these include specific
enzyme activities, such as alkaline phosphatase
(AP) and telomerase. The ubiquitous alkaline
phosphatase (AP) is a hydrolase enzyme respon-
sible for dephosphorylating molecules such as
nucleotides and proteins. As such, the pluripotent
status of SCs can be characterized by a high level
of AP expression [14, 18]. In the same way, SCs
display high levels of telomerase activity and
hTERT expression, both of which are rapidly
downregulated during differentiation [14, 19,
20]. Hence, telomerase activity and telomere
maintenance are considered a general signature
of SCs.
OCT4 and NANOG were the first proteins
identified as essential for both early embryo
development and pluripotency maintenance in
SCs [15, 21]. Besides what we discussed so far,
OCT4-NANOG-SOX2 constitute the core pluri-
potency factors required to sustain SC self-­
renewal and pluripotency. Emerging studies have
identified additional molecular markers of pluri-
potency, including SALL4, DAX1, ESSRB, TBX3,
TCL1, RIF1, NAC1, and ZFP281 [22].
4.3.6 N
 oncoding RNAs (lncRNAs
and miRNAs)
Recent identification of specific combinations of
noncoding RNA (ncRNA) associated with pluri-
potency has highlighted the importance of long
and small RNA regulators for the maintenance
of pluripotency and controlling cell fate [23, 24].
In particular, microRNAs (miRNAs) and long
noncoding RNAs (lncRNAs) have emerged as
eminent players to drive major cellular processes
in SCs (Fig. 4.1). Over the past few years, miR-
NAs are emerging as novel regulators of small
endogenous RNA of about 21–25 nucleotides in
length that play essential roles in modulating SC
46
pluripotency maintenance, differentiation, and
reprogramming of somatic cells to an SC-like
state. The miR-302/367 cluster is essential for
the self-­renewal maintenance of human SCs
and particularly in the primed state of pluripo-
tency. These clusters belong to the embryonic
SC-specific cell cycle-regulating (ESCC) fam-
ily of miRNAs, related to cell cycle regulation
[25, 26]. SCs exhibit an unusually short G1 cell
cycle and high proportion of cells in the S-phase
to maintain their proliferation ability and pluri-
potency [27]. This short duration is associated
with a unique mechanism of cell cycle regula-
tion, which is highlighted by a lack of MAPK,
cyclin D, and pRB control and has been shown
miR-302-367 cluster (Fig. 4.1) holds the ability
of regulating cyclin D1 [28]. In the last decade,
expression studies have identified newly dis-
covered members of gene regulatory networks,
pluripotent long noncoding RNAs (lncRNAs),
which are endogenous cellular RNAs of >200
nucleotides in length and involved in the main-
tenance and induction of SC pluripotency. It has
become apparent that lncRNAs can transcrip-
tionally or posttranscriptionally regulate gene
expression by diverse molecular mechanisms.
A number of lncRNAs, such as lncRNA-ES1,
lncRNA-ES2, and Linc-ROR, are involved in
the maintenance of pluripotency of SCs or iPSCs
[29]. The miRNA-lncRNA cross talk contrib-
utes to modulate gene expression patterns on
all levels (transcriptional, posttranscriptional,
and posttranslational) to regulate the induction,
maintenance, and directed differentiation of SCs
and iPSCs.
4.4 Stem Cell Culture
Serum-free media (SFM) presents an alterna-
tive to serum-containing media and offers sev-
eral advantages, which include better control
of media composition, reduced cost, and avoid
the risk of contamination by possible infectious
agents found in serum. SFM is considered as a
chemically defined medium, characterized by
the absence of animal serum; however it could
contain undefined animal-derived products, such
N. Escacena-Acosta et al.
as serum albumin, hydrolysates, growth fac-
tors, hormones, carrier proteins, and attachment
factors.
Chase et al. described a serum-free MSC cul-
ture medium that supports robust expansion and
phenotype preservation of human MSCs
(hMSCs) as compared with cells expanded in tra-
ditional serum-containing medium [30]. Later
on, these authors developed a xeno-free medium
based on a set of growth factors, including
platelet-­derived growth factor-BB (PDGF-BB),
basic fibroblast growth factor (bFGF), and trans-
forming growth factor beta (TGF-β1) that have
been shown to support the expansion of hMSCs.
Several experiments were done using hMSCs
isolated from bone marrow and adipose tissue.
This medium revealed a promoting effect on cell
proliferation, maintaining the major functional
and phenotype properties of the tested cells.
Ishikawa et  al. performed a proliferation com-
parative study with three types of medium
(DMEM, MSCGM, STK2) used to culture
hMSCs and found a remarkably higher prolifera-
tion in hMSCs cultured in STK2, which is a novel
serum-free medium developed for hMSC expan-
sion [31].
On the other hand, and for clinical application
of hMSCs, it is critical to develop an effective
cryopreservation medium, to optimize protocols
to preserve hMSCs for clinical use, and to ensure
a cell backup for repeated transplantations. An
ideal cryoprotectant should be nontoxic and non-­
immunogenic for both the cells and the recipient,
preserving cell viability and characteristics,
being chemically inert and highly water soluble
in low temperature, having a low molecular
weight, and being affordable.
Al-Saqi et  al. reported the cryopreserva-
tion of hMSCs obtained from the bone marrow
and adipose tissue in a SFM, showing that the
cells maintained a high viability and expressed
specific surface markers and have the ability to
differentiate to mesodermal lineages [32]. Skog
et al. developed a chondrogenic xeno-free differ-
entiation medium for human bone marrow MSCs
[33]. These authors compared the success of this
medium to commercially available chondro-
genic differentiation media and confirmed that
4  Stem Cells: Concept, Properties, and Characterization 47

the xeno-free differentiation medium was more pended in medium to be re-plated for more
efficient than the commercially available chon- passages or in PBS to be analyzed.
drogenic medium. Finally, Iwamoto et  al. dem- • Cryopreservation: An adequate cryopreserva-
onstrated that liver fibrosis was reduced by bone tion medium is crucial to maintain MSCs – the
marrow-derived cells cultured in SFM, improv- harvest cells are cryopreserved in CryoTubes
ing liver function in cirrhotic mice [34]. with the SFM and 10% DMSO and transferred
Based on these observations, we describe to −80 °C with a slow-freezing cryopreserva-
below the crucial steps for improving the culture tion process. After 24  h the cells are trans-
of adipose tissue-derived MSCs using a serum-­ ferred to liquid nitrogen.
free medium (SFM): • Quality Analysis of Mesenchymal Stem Cells
Cultured in a SFM: Human MSCs (hMSCs)
• Isolation: Adipose tissue-derived MSCs are cultured in SFM have been isolated and
obtained from subcutaneous adipose tissue, expanded; they need to pass a quality control
which is acquired from selective liposuc- to assure that these cells maintain MSC char-
tion or abdominoplasty surgeries. The tis- acteristics and can be useful for therapeutic
sues come from patients with medium age, applications. To determine the quality of the
with good health, and with no diabetes or cultured SFM human MSCs, their viability,
other complications. The tissue is minced proliferation, morphology, phenotype, and
and washed with sterile Hanks’ Balanced multilineage differentiation potential are ana-
Salt Solution (HBSS) until the eluent is clear lyzed, comparing these parameters with
and then digested with collagenase type II hMSCs cultured in SCM.
for 45  min at 37  °C with intermittent shak- • Cell Viability and Cell Proliferation: The via-
ing. After centrifugation, three layers are bility and cell proliferation are essential and
obtained; the upper yellow layer corresponds can be determined by a counting cell prolifer-
to the fat, the middle red layer to the blood ation assay, expecting an equal or higher den-
cells, and the pellet cells to the adipocytes, sity and proliferation for hMSCs cultured in
which are resuspended in medium. (For SFM than for those cultured in SCM.
example, for human bone marrow MSC iso- • Cell Morphology: MSC cultured in SFM
lation, 10 to 20  ml of bone marrow is aspi- should be successfully expanded during sev-
rated from the iliac crest of normal donors eral passages and maintain their morphological
in a medium age. Heparinized bone marrow characteristics, demonstrating a homogenous
is mixed with PBS, centrifuged, and layered morphology with no gross changes in respect
on with Percoll solution. After centrifugation with the MSC cultured in control medium.
the interface is collected, washed with PBS, • Immunophenotype Analysis: It is crucial that
and resuspended in medium). hMSCs expanded in SFM retain their pheno-
• Expansion: The cells obtained are cultured typic characteristics. For that, the surface
according to the manufacturer instructions for expression markers are characterized by flow
SFM use. Often, the culture dish needs to be cytometry, using monoclonal FITC or PE con-
treated with substrates such as fibronectin or jugated antibodies: CD3, CD14, CD34, CD45,
vitronectin. The cells are plated and main- CD80, CD14, HLA, CD73, CD90, and
tained at 37  °C in a humidified environment CD105. Thus, cells should be positive in more
containing 5% CO2, changing the medium than 95% for CD73, CD90, CD105, and
every 3  days until they reached 80% conflu- HLA-I and negative in less than 5% for CD3,
ence. These cells are defined as “passage 0.” CD34, CD14, CD45, CD80, and HLA-II.
• Harvesting: The harvest protocol replaces • Potential of Differentiation: The multilin-
trypsin-EDTA with TrypLE for the serum-free eage differentiation potential is investigated
culture, to ensure the process is animal post-­harvest by inducing hMSCs to differen-
component-­ free. Then the cells are resus- tiate toward the adipogenic, chondrogenic,
48
and osteogenic lineages. After 14–21  days
in lineage-­ specific differentiation media,
hMSCs cultured in SFM should maintain
their capability for osteogenic differentiation,
being positive for alkaline phosphatase; have
the potential for adipogenic differentiation,
forming lipid vacuoles positive for Oil Red
staining; and demonstrate chondrogenic dif-
ferentiation ability, being positive for Alcian
Blue staining.
4.5 Advanced Therapies
with Stem Cells
The advances in cellular and molecular biotech-
nology have introduced a new era of innovative
medicines with complex structures, and the active
pharmaceutical element does not only refer to
inert chemical synthesis substances. In recent
years, it has increased the interest in products that
use a biological approach to restore, maintain, or
improve tissue function, due to their potential to
treat diverse chronic diseases or conditions with
unmet medical needs. In this regard, products
and/or therapies derived from biological or bio-
technological approaches should not be confused
with those derived from advanced therapies.
Advanced therapy medicinal products
(ATMPs) are those that incorporate living cel-
lular component, tissues, or genes, and their
characteristics clearly differentiate them from
conventional drugs obtained by chemical synthe-
sis. These differences range from early stages of
development to its clinical use. Therefore, many
of the concepts we have about conventional drugs
cannot be extrapolated to the advanced therapy
medicines and require that they be considered, at
all levels, as a very different class of drugs.
Currently, most of the medicinal production of
advanced therapies is conducted by academics,
academic spin-offs, not-for-profit organizations,
and small- and medium-sized enterprises (SMEs).
Even so, ATMPs must follow the same develop-
ment and requirements as for pharmacological
compounds, which consists of several phases
including preclinical and clinical studies. Toxicity
and the biological activity are evaluated, as well
N. Escacena-Acosta et al.
as the safety and efficacy in the clinical phases.
Cell therapy drugs are the only ones in which the
active component is living cells, and therefore,
the cells are the product and not what they pro-
duce. It is difficult to define their pharmacologi-
cal characteristics since they produce a variable
and often unknown quantity of bioactive mole-
cules. Therefore, the definition of the composi-
tion of a cellular product, its mechanisms of
action, pharmacokinetics, toxicity, and efficacy
represent challenges which have never been
anticipated before in conventional pharmacology
[35, 36]. For this reason, the transfer of basic
research to clinic is subject to requirements on
which good manufacturing practices (GMP),
clean room facilities, qualified personnel, quality
controls, and aseptic process are involved. In this
way, the holder of an authorization to market
medicinal products for advanced therapy must
guarantee a system that ensures that each product
and its components can be traced throughout the
process, including procurement, manufacturing,
packaging, storage, transportation, and delivery.
European Medicines Agency (EMA) tracks
research into the use of stem cells in medicines
thoroughly and is responsible for assessing mar-
keting authorization applications for medicines
containing stem cells.
The development of ATMPs is an active
and fast-growing field and also requires new
approaches in terms of regulatory aspects.
Considered as medicinal products ATMPs should
be subjected to the same regulatory principles
applied on any other drugs. It is important for
all linked professionals to familiarize themselves
with the rules, standards, directives, regulations,
and guidelines related with these pharmaceuti-
cal innovative products. The regulation builds
on directives (Directive 2001/20/EC; Directive
2001/83/EC; Directive 2012/26/EU) concerning
medicinal products for human use [37], qual-
ity and safety standards for human tissues and
cells [37, 38], clinical trials, as well as regula-
tions concerning centralized marketing authori-
zation procedures [39, 40]. On 30 October 2007,
the European Council formally adopted the spe-
cific regulation on ATMPs (Regulation (EC) No
1394/2007) [41], which came into force in the
4  Stem Cells: Concept, Properties, and Characterization
GLPC
Experimental observations
Pre-clinical research
GCP/GMP
Clinical research Phase I-II
Clinical research Phase III
GMP/GVP
Authorization
Marketing
Fig. 4.2  Compliance with GMP is mandatory for all
medicinal products that have been granted a marketing
authorization. Likewise, the manufacture of investiga-
tional medicinal products must be in accordance with
European Union on 30 December 2008. It rep-
resents a regulatory framework to achieve har-
monized market availability within the European
Union (Fig. 4.2).
4.6 Definitions
Due to the innovative concept of these products, a
new Committee for Advanced Therapies (CAT)
was established at the EMA. The CAT is respon-
sible for all regulatory procedures concerning
ATMP in the EU, among which the referred prod-
uct falls within definition of ATMP, applicable to:
• Somatic cell therapy medicinal product
(SCTMP)
• Gene therapy medicinal product (GTMP)
• Tissue-engineered product (TEP)
• Combined advanced medicinal product
The definitions of a gene and a somatic cell
therapy medicinal product according to Directive
2001/83/EC, Annex I, Part IV, as amended
(implementing Directive 2009/120/EC) [42] are
presented below. A legal definition of TEP was
for the first time introduced with the Regulation
(EC) No 1394/2007.
Table 4.1 summarizes the principal character-
istics that biological medicinal products have to
fulfill.
49
Regulation 1394/2007/EC
Active substance
Directive 2004/23/EC
Directive 2006/17/EC
Directive 2001/83/EC
Investigational
medicinal product
Directive 2009/120/EC
(IMP)
Directive 2003/94/EC
Regulation (EU) No 536/2014
Registered product
GMP. GLPC, good laboratory practices compliance; GCP,
good clinical practices; GMP, good manufacturing prac-
tices; GVP, good pharmacovigilance practices
Based on these definitions, it is clear that a
classification of a medicinal product as a GTMP
depends on the addition of a recombinant nucleic
acid sequence and that its mode of action is
related with this gene sequence. Viral and nonvi-
ral vectors (which includes plasmid DNA), as
well as genetically modified viruses and cells, are
all examples of this type of product.
SCTMP and TEP are two types of cell-based
medicinal products, which may contain similar
components. To ensure your product falls into
some of the above definitions, the following cri-
teria must be taken into account:
(i) Degree of “engineering” to which cells are
subjected in order to achieve their intended
mode of action in humans.
It is important to know if your manu-
facturing process includes some type of
substantial manipulation of the cells and
tissues, which can alter their biological
characteristics, physiological functions,
or structural properties. For example, cell
culturing leading to expansion is consid-
ered a substantial manipulation. Annex I of
the ATMP Regulation listed manipulations
which shall not be considered as substantial
manipulations.
(ii) Intended to be used (same essential function
or nonhomologous use in the recipient and
the donor).
50 N. Escacena-Acosta et al.

Table 4.1  Principal characteristic of different biological medicinal products

Biological
medicinal
products Characteristics Observations
Gene therapy (a) It contains an active substance which contains or consists Gene therapy medicinal
medicinal product of a recombinant nucleic acid used in or administered to products should not include
(GTMP) human beings with a view to regulating, repairing, vaccines against infectious
replacing, adding, or deleting a genetic sequence diseases
(b) Its therapeutic, prophylactic, or diagnostic effect relates
directly to the recombinant nucleic acid sequence it contains
or to the product of genetic expression of this sequence
Somatic cell (a) Contains or consists of cells or tissues that have been
therapy medicinal subject to substantial manipulation so that biological
product (SCTMP) characteristics, physiological functions, or structural
properties relevant for the intended clinical use have been
altered or of cells or tissues that are not intended to be
used for the same essential function(s) in the recipient and
the donor
(b) I s presented as having properties for or is used in or
administered to human beings with a view to treating,
preventing, or diagnosing a disease through the
pharmacological, immunological, or metabolic action of
its cells or tissues
Tissue-­engineered Tissue-engineered product means a product that Article 2, 1(b)
products (TEP)  Contains or consists of engineered cells or tissues
 Is presented as having properties for or is used in or
administered to human beings with a view to regenerating,
repairing, or replacing a human tissue
A tissue-engineered product may contain cells or tissues of
human or animal origin or both. The cells or tissues may be
viable or nonviable. It may also contain additional substances,
such as cellular products, biomolecules, biomaterials,
chemical substances, scaffolds, or matrices
Products containing or consisting exclusively of nonviable
human or animal cells and/or tissues, which do not contain
any viable cells or tissues and which do not act principally by
pharmacological, immunological, or metabolic action, shall
be excluded from this definition

Combined It must incorporate, as an integral part of the product, one or Article 2 1(d)
advanced therapy more medical devices within the meaning of Article 1(2)(a) of
medicinal product Directive 93/42/EEC or one or more active implantable
medical devices within the meaning of Article 1(2)(c) of
Directive 90/385/EEC, and its cellular or tissue part must
contain viable cells or tissues, or its cellular or tissue part
containing nonviable cells or tissues must be liable to act
upon the human body with action that can be considered as
primary to that of the devices referred to

Same essential function is related with the
preservation of the original function in the
same anatomical or histological environment
in the donor and the recipient. Replacement
of a tissue as its whole or functional unit
(such as cornea) is regarded as a use for the
same essential function. Conversely, cells or
tissues applied in a new physiological micro-
environment will be considered a nonhomol-
ogous use.
4  Stem Cells: Concept, Properties, and Characterization
Since in many cases the classification of these
products is not an easy task, the CAT can be con-
sulted and will issue a classification recommen-
dation within 60 days after receiving the request,
according to Article 17 of the ATMP Regulation.
4.7  oloclar®, the First Stem Cell
H
Product Approved
in the European Union
According to the previous definitions, stem cells
are categorized as ATMPs when these cells
undergo a substantial manipulation or are used
for a different essential function. They can be
SCTMPs if they treat, prevent, or diagnose a dis-
ease (pharmacological, immunological, meta-
bolic action) or TEPS to regenerate, repair, or
replace a human tissue.
Although stem cell treatment is not a new con-
cept, Holoclar® (ex vivo expanded autologous
human corneal epithelial cells containing stem
cells) is the first ATMP to be approved and for-
mally registered in the Western world. It is a cell-­
based tissue-engineered therapy that replaces
epithelial cells in a damaged cornea. Adult
patients with moderate to severe limbal stem cell
deficiency (LSCD) due to corneal surface injury,
such as after chemical burns, may benefit from
this innovative medicine. In 1997, the research
team of Holoclar reported the successful ocular
surface regeneration in two patients with LSCD
after transplantation of an in  vitro engineered
neo-epithelium from autologous limbal stem
cells [43]. In 2010, 112 patients with corneal
damage were included in a study obtaining satis-
factory effects in 77% of the cases [44]. In 2013,
the team published further results from 152
patients treated with Holoclar for burn-related
eye damage. In February 2015, the commercial
use of Holoclar was authorized within the
European Union. Therefore, the release of this
product to the market has taken 25 years.
The pathway to regulatory approval requires a
considerable amount of expertise, time, and
investment. The EMA’s specialist experts in the
CAT rigorously evaluate quality, safety, and effi-
cacy of product. Few ATMPs have been approved
51
in Europe and the USA due to the novelty, com-
plexity, and extreme diversity of these innovative
products. An optimal and current knowledge of
regulatory issues is necessary to advance in the
intricate path of the development of a cellular
medicine.
4.8 Stem Cells and Cancer
The capacities of SCs to self-renew and to dif-
ferentiate into multiple lineages make them a
powerful tool in regenerative medicine.
Unfortunately, these properties also make stem
cells capable of tumorigenesis, representing an
obstacle to the safe use of stem cell-based
therapies.
Normal SCs share many characteristics with
cancer stem cells (CSCs), a population of cells
within the tumor capable to replicate the original
tumor after transplantation into a mouse model
[45]. CSCs were first identified in human acute
myeloid leukemia (AML) after their transplanta-
tion into immunodeficient mice [46]. Lapidot and
colleagues demonstrated that the peripheral
blood of patients with AML contains approxi-
mately 1:250,000 CSCs, which are capable of
engraftment in the bone marrow of mice after tail
vein injection and initiating a human AML.  A
subsequent study showed that CSCs presented a
more undifferentiated phenotype than mature
blood cells, being more closely related to hema-
topoietic SCs [47]. Thus, it was postulated that
the population of cancer cells with stem cell-like
features gives rise to the majority of the tumor
cells with more differentiated phenotypes. In the
last decade, the cancer stem cell theory has
gained increasing attention from researchers, and
evidences have been provided for the existence of
CSCs in most, if not all, tumors [48–53].
The theory that cancer arises from normal
SCs uncovered the tumorigenic property of SCs.
For instance, human ESCs spontaneously dif-
ferentiate into an array of various tissue types
of the embryo when transplanted into immuno-
suppressed mice, forming complex teratomas
[54, 55]. First evidences were found in 1967,
when normal germinal SCs from the gonadal
52
ridge were transplanted into the testicles of adult
male mice and developed teratomas [56, 57].
Pluripotency of iPSCs is also associated with
tumorigenesis [58]. Recently, a study found that
iPSCs survive and differentiate into three neural
lineages when transplanted into injured spinal
cord mice. Despite initial recovery of motor func-
tion, grafted mice developed tumors and exhib-
ited gradual deterioration of hind limb movement
during long-term observation [59]. Studies have
provided evidences that adult SCs may also trans-
form into oncogenic cells. For example, neural
stem cells within the subventricular zone, a main
SC compartment in the adult brain, are known
to proliferate in response to the platelet-­derived
growth factor (PDGF), generating glioma-­ like
masses [60]. In accordance with this study, Chen
and colleagues identified a subset of cells within
the subventricular zone (i.e., neural SCs) as a
source of new cancer cells responsible for tumor
growth. The chemical depletion of this subset of
cells with ganciclovir and temozolomide impedes
tumor development [61].
Understanding the mechanisms responsible
of SC tumorigenesis has become a primary
goal in the field of regenerative medicine. In
this context, the interaction of genetic and epi-
genetic mechanisms seems to play important
roles in the tumorigenic potential of SCs [62,
63]. Genetic modifications are defined as
changes in the genomic DNA sequence, includ-
ing point mutation, single nucleotide polymor-
phism, or copy number variability. Point
mutations are the main DNA changes that can
accumulate in somatic cells. In most cases,
processes of base mispairing during DNA rep-
lication cause these point mutations. On the
other hand, epigenetic changes regulate gene
expression without modifying the underlying
DNA sequence. The epigenetic changes include
DNA methylation, histone modifications, chro-
matin remodeling, and changes in noncoding
RNAs. A study of a subset of cancer methyl-
ated genes revealed that these genes are fre-
quently methylated in human ESCs, indicating
a role for DNA methylation in the control of
gene expression in human stem cells and sug-
gesting a role in tumorigenesis [64].
N. Escacena-Acosta et al.
4.9 Conclusions
The use of living cells as a medicinal product for
clinical application has become increasingly more
realistic. SCs possess the capacity of self-­renewal
and differentiate into other cell types, being a valu-
able tool in regenerative medicine. However, the
potential tumorigenic property of SCs has ques-
tioned their safety in cell therapy. For this reason,
clinical trials to validate the safety of using SCs for
health problems need to be carried out before
treating patients, not only to assure that SCs do not
induce a graft-versus-host disease but also that
cells do not form tumors. In this context, one chal-
lenge is to develop strategies to control the mecha-
nisms inducing SC oncogenicity, thus increasing
the safety when using these cells for regenerative
medicine. The efforts already achieved in this field
will definitely improve the clinical use of SCs;
probably we are reaching a new era of a safe and
more efficient stem cell-based therapy.
Acknowledgments Authors are supported by the non-
profit Fundación Progreso y Salud, Consejería de Salud,
Junta de Andalucía; FEDER cofunded grants from Instituto
de Salud Carlos III and the Ministry of Economy, Industry
and Competitiveness (Red TerCel: RD12/0019/0028 and
RD16/00259; CIBERDEM: CB07/08/0006; PI14/01015,
PI16/00259, PI17/02104, and CD16/00118); and Junta de
Andalucía (PAI-BIO311, CTS-576, CTS 11-727, PI-0109-
2014, PI0007/2016, and PI0272/2017). CIBERDEM is an
initiative of the Instituto de Salud Carlos III.
Competing Interests  The authors declare no
conflict of interest.
Informed Consent  No human studies were car-
ried out by the authors for this article.
Animal Studies  No animal studies were carried
out by the authors for this article.
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 Corneal Stem Cells: Identification
and Methods of Ex Vivo Expansion
Christian Claude Lachaud, Abdelkrim Hmadcha,
and Bernat Soria
5.1 Introduction
The cornea is the clear convex membrane at the
front of the eye. The maintenance of its integrity
and transparency is critical for an optimal light
transmission and refraction into the internal eye.
The integrity and functionality of the corneal epi-
thelial, stromal, and endothelial layers are main-
tained in a very large extent by their cellular
components [1, 2]. Massive injuries or diseases
affecting any of those corneal layers lead ineluc-
tably to a loss of corneal function, tissue scarring,
and corneal opacification [3–6]. This is particu-
larly true for the stromal and endothelial layers
due to their almost inexistent regenerative capac-
ities [1, 7]. To date, most corneal disorders with
C. C. Lachaud · A. Hmadcha
Department of Cell Regeneration and Advanced
Therapies, Andalusian Center of Molecular Biology
and Regenerative Medicine-CABIMER, Junta de
Andalucía-University of Pablo Olavide-University of
Seville-CSIC, Seville, Andalusia, Spain
e-mail: christian.lachaud@cabimer.es
B. Soria (*)
Department of Cell Regeneration and Advanced
Therapies, Andalusian Center of Molecular Biology
and Regenerative Medicine-CABIMER, Junta de
Andalucía-University of Pablo Olavide-University of
Seville-CSIC, Seville, Andalusia, Spain
Centro de Investigación Biomédica en Red de
Diabetes y Enfermedades Metabólicas Asociadas
(CIBERDEM), Madrid, Spain
e-mail: bernat.soria@cabimer.es
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_5
5
severe visual impairment can only be corrected
by transplantation of a donated corneal tissue.
Due to the shortage of donors, there is a growing
interest in developing alternative strategies such
as a tissue engineering of artificial corneal equiv-
alents created from stem cells and scaffolds. In
this way, among the different types of stem cells
proposed so far, are found corneal layer-specific
stem/progenitor cells.
The limbal epithelial stem cells (LESC) were
the first type of stem/progenitor cells discovered
in the cornea. Since far, their use has revolution-
ized the field of ocular surface regenerative
medicine [7–9]. The best example is the suc-
cessful regeneration of unilateral epithelial
defects with the transplantation of a patch of
denuded amniotic membrane (AM) re-epitheli-
alized with limbal tissue biopsies collected from
the contralateral healthy eye [10]. A further
refinement of this application using an AM car-
rier with cultivated limbal epithelial stem cells
(LESC) has been possible, thanks to progresses
in the procedures permitting their isolation and
ex  vivo expansion [9, 11]. More recently were
identified a population of ABCG2+/PAX6+ pro-
genitors with mesenchymal stem cell (MSC)
characteristics, clonal growth capacity, and ker-
atocyte differentiation potential in the periph-
eral corneal stroma [12]. Converging evidence
suggest that these corneal stromal stem cells
(CSSC) might be natural ­keratocyte progenitors
and thus are considered useful for corneal
57
58
stroma regeneration. In a similar way, it is also
the identification of immature corneal endothe-
lial cells (CEnC) in the transitional zone
between the peripheral corneal endothelium and
the trabecular meshwork [13, 14]. Consistent
with their expression of stem and progenitor
markers, these putative corneal endothelial pro-
genitor cells (CEnPC) also display superior pro-
liferation capacities than CEnC collected from
the central cornea [14–17]. Different laborato-
ries are actually focusing efforts on their ex vivo
expansion and use in experimental animal mod-
els of corneal endothelial injury [14, 18–24].
Below we review the different findings on the
molecular and biological properties of corneal
layer-specific stem/progenitor cells. We detail for
limbal epithelial stem cells, corneal stromal stem
cells, and corneal endothelial progenitor cells
which are the isolation procedures used and also
review the different approaches of cultures
reported so far, the culture systems used for their
expansion and their experimental or/and clinical
applications.
Figure 5.1 shows a schematic representation
of a cross section of the peripheral human
cornea
5.2  imbal Epithelial Stem Cell
L basal epithelium, the limbal crypts found between
(LESC)
The first evidence of the re-epithelialization
capacities of the peripheral corneal epithelium
arose from studies performed in rabbits, where
after the debridement of the entire corneal epithe-
lium, epithelial cells of suggested “conjunctival
origin” were shown to propagate onto the entire
corneal surface and regenerate the transparent
corneal epithelium [25, 26]. The regenerative
capacity of the corneal epithelium was further
found to be attributed to epithelial cells located in
the limbus, the transitional border area between
the cornea and the conjunctiva [27].
The existence of a population of epithelial
stem cells in the human limbus was first sug-
gested in the mid early 1980s on the basis of a
differential expression of paired cytokeratins 3
and 12 (CK3 and CK12), which expression
C. C. Lachaud et al.
increase as corneal epithelial cells (CEC) become
more mature in the central cornea and upper epi-
thelium layers [28]. Of special interest, Schermer
et al. by analyzing the CK3 expression pattern in
the rabbit corneal epithelium and stratified cor-
neal epithelial cells in culture led to the conclu-
sion that CK3-negative cells identify a population
of immature epithelial cells which is mostly con-
centrated into the basal layer of the peripheral
limbal zone and that are responsible for the pro-
duction of more mature CK3-positive epithelial
cells [29]. The identification of the limbal basal
epithelium as the niche of corneal epithelial stem
cells niche was rapidly confirmed by “pulse-­
chase” experiments with tritiated thymidine (3H-­
T) or bromodeoxyuridine (BrdU) onto ex  vivo
cultured whole cornea [30, 31]. By using this
approach, the authors could detect a population
of label-retaining cells in the limbal basal epithe-
lium. These low-cycling small rounded cells with
high nuclear cytoplasmic ratio were found to pro-
liferate in response to corneal surface wounding,
thus suggesting they correspond to a population
of epithelial stem cells for corneal epithelium
renewal [30, 31]. Additionally, these experiments
also evidenced the slow centripetal migration of
LESC toward the central cornea [30]. LESC were
shown to be located into invaginations of the
the palisades of Vogt. This limbal niche was
shown to provide a unique protective environ-
ment for LESC with an underlying stroma highly
vascularized and innervated [32].
Stem cell properties of LESC, including their
markers’s signature [33], their asymmetric divi-
sion kinetic under normal homeostasis or experi-
mental injury models [29–31, 34], extracellular
matrix composition of their basal membrane
niche [35–37], and proliferative responsiveness
to growth factors [38], have also been object of
intense research.
Of particular interest are studies which have
provided significant advances on the identifica-
tion of intracellular and surface markers that
are preferentially or specifically expressed in
LESC.  Pellegrini et  al. demonstrated that the
transcription factor p63 is restricted to basal
cells of the limbal epithelium, but not to transient
5  Corneal Stem Cells: Identification and Methods of Ex Vivo Expansion 59

EA
RN
CONJUNCTIVA CO

ANTERIOR
CHAMBER

SCLERA
S
IRI

LENS

Conjunctiva Limbus Peripheral cornea Central cornea

Epithelium

Bowman’s layer

Stroma

Descemet’s membrane
Endothelium

Anterior chamber

Transitional zone
Trabecular
meshwork

Terminally differentiated cell (squamous) Limbal stromal MSC Blood vessel

Post mitotic cell (wing cell) Keratocyte Nerve

Transit amplifying cell (TA) Corneal endothelial cell (CEnC) Collagen fibril

Limbal epithelial Stem cell (LESC) Corneal endothelial progenitor (CEnPC)

Fig. 5.1  Schematic representation of a cross section of
the peripheral human cornea. The limbus at the periphery
of the cornea is the niche for lineage layer’s specific cor-
neal stem cells (CSC) and progenitors. Limbal epithelial
stem cells (LESC) reside in the basal layer of the limbal
crypt, an invagination of the limbal epithelium.
Underneath the limbal epithelial niche, in the limbal
stroma, are residing corneal stromal stem cells (CSSC).
Immature corneal endothelial progenitor cells (CEnPC)
with expression of neural crest markers were identified in
the transitional zone, a small area between the peripheral
corneal endothelium and the trabecular meshwork

amplifying (TA) cells in suprabasal corneal epi-
thelium layers, thus suggesting to be a specific
marker for epithelial stem cells [39]. In a simi-
lar fashion, Chen et al. further reported that the
ATP-­ binding cassette subfamily G member 2
(ABCG2), a member of the ATP-binding cas-
sette (ABC) transporters, was expressed in basal
epithelial cells of the limbal epithelium, but not
into basal cells of the central corneal epithelium
[40]. In the same year, Watanabe et al. indicated
that the human limbal epithelium contains side
population (SP) cells expressing the cell mem-
brane marker ABCG2 [41], being SP+ cells a
subset of tissue cells which can efficiently efflux
60
the dye Hoechst 33342 via the multidrug trans-
porter ABCG2, a characteristic which is almost
restricted to somatic and cancer stem cells [42].
Soon after, de Paiva et al. reported that ABCG2
is a marker identifying clonogenic limbal epithe-
lial cells [43]. Of special interest, Ksander et al.
recently reported that the ATP-binding cassette,
subfamily B, member 5 (ABCB5) is a gene criti-
cal for corneal epithelium development, homeo-
stasis, and regeneration and which expression is
restricted to LESC [44].
Taking advantages on these findings, Kim
and colleagues recently demonstrated the pos-
sibility of purifying LESC from highly prolif-
erative limbal epithelium-derived cell cultures
by fluorescent activated cell sorting (FACS) of
ABCG2+/ABCB5+ double-positive cells. These
purified ABCG2+/ABCB5+ LESC could effi-
ciently differentiate into CK3+ CEC once cul-
tured into CnT30 medium [45]. Of interest, they
reported culture media conditions and the sub-
strate (mixture of matrigel and fibronectin)
favoring their expansion, thus providing signifi-
cant advances for studying in vitro their biologi-
cal mechanisms or factors maintaining their
stemness or promoting their epithelial differen-
tiation. Additionally, these novel insights lay the
basis toward the definition of an optimized
ex vivo expansion protocol of LESC for corneal
diseases associated with LSCD.
5.3 Methods of Isolation
and Expansion
5.3.1 Limbal Epithelium Tissue
Preparation
By majority, laboratories isolate limbal tissue
from remaining corneoscleral rims proceeding
from donor cornea transplantation. Corneoscleral
rims are obtained after excision or trephination of
the corneal button used for keratoplasty. Limbal
rim tissue is obtained after removal of excess
sclera, conjunctiva, iris remnants, and endothe-
lium [46, 47]. Limbal rim tissues are usually
enzymatically digested with dispase II for several
hours at 4  °C to allow the detachment of the
C. C. Lachaud et al.
l­imbal epithelium from the stromal layer by dis-
solving basement membrane proteins, mainly
laminin-5 and also in minor extent collagen IV
[45, 46, 48–50]. Alternatively, the use of trypsin
was also compared to dispase II, showing how-
ever higher effectiveness with the use of dispase
II in terms of epithelial cell recovery, viability,
and colony-forming efficiency [51]. In a similar
fashion, a comparative study proved a better
preservation, integrity, and recovery of the limbal
epithelium sheets with dispase II with respect to
collagenase [49]. Nonetheless, it was however
found that collagenase treatment leads to the
recovery of greater numbers of epithelial progen-
itor cells together with closely associated mesen-
chymal cells, providing cultures with higher
numbers of holoclones (stem cells derived) and
meroclones (transient amplifying cells derived)
on 3T3 feeder fibroblasts [49, 52].
From this isolation stage, different procedures
for isolating or obtaining LESC from limbal epi-
thelial tissue have been reported, being mostly
culture explant systems or adherent culture of
limbal epithelial cells suspension within a media
conditioned by feeder layer cells. Alternatively,
LESC were also co-cultured separately with
inactivated 3T3 feeder cells in a transwell system
(see Fig.  5.2 for summary techniques and
approaches for LESC isolation and expansion).
5.3.2 C
 ulture Media and the Use
of Feeder Cells
Media and culture conditions currently used for
expanding limbal epithelial cells have been pro-
gressively optimized with the objective of
improving their long-term proliferation and
favoring clonal LESC outgrowth. Of particular
importance was the introduction of lethally irra-
diated 3T3 feeder layer cells as a critical culture
component supporting CEC and LESC expan-
sion [53, 54].
Most laboratories have used as basal expan-
sion media a mixture of DMEM and F12 (either
1:1 or 2:1) supplemented with 10% of either
fetal calf serum (FCS) [55, 56], human serum
(HS) [46], or knockout serum replacement
5  Corneal Stem Cells: Identification and Methods of Ex Vivo Expansion 61

Corneal button for

Explant culture
keratoplasty
onto 3T3 feeder fibroblasts

Air lifting Submerged

corneoscleral scrapping
donor’s eye rim endothelium Surgical isolation
Limbal tissue
Cell
suspension
Limbal
epithelium
Enzymatic Mechanical
digestion separation
(trypsin, colagenase)
Partial enzymatic
Expansion ? digestion
LESC
(dispase II)
Explant culture
Sorting ABCG2+/ABCB5+ LESC Limbal stroma

LESC

Fig. 5.2  Procedures for obtaining human limbal epithe-
lial stem cells (LESC). Corneoscleral disc removed from
donor’s eye is further subjected to the excision (trephina-
tion) of the central cornea button (used for transplanta-
tion). The resulting corneoscleral rim is further cleaned
off residual scleral, conjunctival, iris, and endothelial tis-
sues. The corneoscleral rim is then cut into small pieces
that can be directly subjected to explant culture (airlifting
or submerged) or further enzymatically processed with
dispase II and/or collagenase to digest basement mem-
brane ECM molecules and help separate limbal epithe-
lium and stroma layers. Full enzymatic digestion of limbal
epithelium tissue allows obtaining limbal epithelial cells
suspension, which can be further cultured at low density
onto 3T3-inactivated mouse fibroblasts to stimulate their
proliferation and the outgrowth of LESC clones. ABCG2+/
ABCB5+ highly proliferating LESC can be ultimately
purified by FACS [45]

(KSR) [56]. Media are usually supplemented
with insulin, transferrin, and selenium. Limbal
epithelial cell proliferation is additionally stimu-
lated with 1–10  ng/ml epidermal growth factor
(EGF), and hydrocortisone is incorporated as an
important agent inhibiting epithelial-to-mesen-
chymal transition (EMT) [57]. Additionally, cul-
ture media have incorporated several other
important compounds stimulating LESC prolif-
eration: basic fibroblast growth factor (bFGF or
FGF-2), adenine, cholera toxin, leukemia inhib-
iting factor (LIF), and triiodothyronine (T3) [54,
55, 57]. Of particular importance is the concen-
tration of calcium in the media, which was
shown to be preferentially low to medium (0.03–
0.4 mM), as limbal epithelial cells cultured with
high calcium concentrations undergo rapid dif-
ferentiation and stratification [58, 59]. Recently,
Miyashita et  al. reported that a combined
s­ upplementation of culture media with 10 ng/ml
of keratinocyte growth factor (KGF) and 10 μM
of the ROCK inhibitor Y-27632 allowed a long-
term expansion of LESC [60].
CEC are also prone to differentiate and
undergo a stratification process upon culture in a
media containing FBS [61]. This issue has stimu-
lated a special interest in developing serum-free
media formulations able to sustain CEC prolifer-
ation in a more stable epithelial progenitor phe-
notype. In this way, Castro-Muñozledo et  al.
formulated a complete culture media in which
FBS was substituted with a low content of serum
albumin and supplemented with TFG-α and EGF
and that could sustain the growth of CEC onto
3T3 feeder layer cells. Parallely, different labora-
tories also successfully cultured human limbal
epithelial cells in a keratinocyte serum-free
medium (KSFM) supplemented with bovine
62
pituitary extract (BPE) and EGF [55, 62]. Seeber
et  al. reported that human CEC cultured under
serum-free media supplemented with ascorbic
acid, calcium, hydrocortisone, and retinoic acid
retains barrier properties and native epithelium-­
like morphology [63].
Of particular importance is the development
of efficient culture systems avoiding potential
animal-derived infectious agents. In this direc-
tion, recent advances have been made with the
introduction of commercial human fibroblast cell
lines able to efficiently substitute 3T3 feeder
layer cells [51, 64] and also of synthetic culture
media compounds [51, 65] able to sustain human
CEC proliferation. Yokoo et al. reported the pos-
sibility of growing corneal limbal epithelial cells
in a serum-, feeder cells-, and BPE-free culture
medium containing B27 supplements and EGF,
making of this culture system a valuable approach
for future clinical use [66] .
5.3.3 Explant Culture
The explant culture was the first type of culture
introduced in the laboratory. It is the simplest
way of culturing cells and offers the advantage of
preserving much of the morphostructural and
biochemical properties of tissues and of the stem
cell niche [67, 68]. In this way, this approach was
proved very valuable for in vitro culture of limbal
epithelium explants and LESC expansion [45, 46,
54, 55, 69–77]. Indeed, cultured limbal explants
onto denuded AM are currently applied for re-­
epithelializing the ocular surface in patients with
total LSCD [8, 9]. Different laboratories have
compared the efficiency of the explant culture
approach with single-cell suspension culture sys-
tems in term of recovery of LESC, growth poten-
tial, morphology, and stemness markers [54, 55,
77]. Although the explant system was reported to
require higher amount of time to reach conflu-
ence, both approaches however produced quite
similar numbers of slow-cycling BrdU label-­
retaining cells and percentages of small cells
expressing progenitor epithelial cells markers
[54]. By contrast, Zhang et  al. reported better
outcomes using the single-cell suspension
C. C. Lachaud et al.
approach in term of LESC recovery [55]. In
agreement with Kim et  al.’s results, Li et  al.
recently reported almost similar outcomes with
both approaches in terms of LESC purity and
clonal growth capacity, thus reinforcing the use-
fulness of the explant culture for LESC expan-
sion [77].
5.3.4 Single-Cell Suspension
Culture
The number of laboratories using the cell suspen-
sion culture of limbal epithelial cells has
increased notably in recent years. This tendency
may be largely explained by progresses made in
their enzymatic isolation procedure and the
development of optimized culture media promot-
ing and sustaining their growth potential. By
overall majority, single-cell suspension were
obtained by trypsinization (0.25% trypsin/EDTA)
of purified limbal epithelium tissues at 37 °C for
incubation times ranging from 10 min to 2 h [46,
48, 78]. Of note, the report by Kim et  al. that
LESC can be purified from limbal epithelial cells
culture by FACS of ABCG2+/ABCB5+ cells [45]
opens thus the possibility of using this approach
to obtain FACS-purified ABCG2+ and/or ABCB5+
single-cell suspensions of freshly isolated LESC
and perform their ex  vivo expansion in an
improved way.
5.3.5 Sphere-Forming Assay
Alternatively, the suspension culture approach
has also been used to evaluate the sphere-forming
capacity of CEC [79, 80]. In this way, Mimura
et al. cultured CEC suspensions from the limbus
and central cornea under serum-free and feeder
layer-free culture conditions in a medium con-
taining methylcellulose gel matrix to impede
CEC reaggregation and sphere formation at the
clonal level. Authors reported a higher rate of pri-
mary and secondary sphere-forming capacities
from epithelial cells of the limbal region
compared to those of the central cornea.
­
Consistent with their stemness, cells in primary
5  Corneal Stem Cells: Identification and Methods of Ex Vivo Expansion 63

spheres expressed mitogenesis (BrdU incorpora-
tion) and stem/progenitor (p63, p75(NTR), and
nestin) markers [79]. By using a similar approach,
Chang et al. also reported similar sphere-forming
capacities and efficiencies of CEC isolated from
both the limbal and central corneal region [80].
with basal cells retaining LESC phenotype (p63,
CK15, integrin α6) and suprabasal cells express-
ing the differentiated CECs marker CK3 [84].
5.3.7 Amniotic Membrane

The use of cryopreserved amniotic membrane

5.3.6 C
 ulture onto ECM-Coated (AM) as temporary patch for ocular surface
Surface, Carrier, and Scaffolds emergencies such as thermal and chemical burns
has proved to be extremely useful to prevent the
The incorporation of components of the limbal occurrence of limbal stem cell deficiency (LSCD)
epithelial niche in culture systems has been by preserving remaining healthy limbal stem
address to evaluate their mitogenic potential onto cells and accelerating corneal re-­epithelialization,
cultured LESC.  Of particular importance are thus allowing to regain a healthy ocular surface
extracellular matrix (ECM) molecules compo- ocular [10, 85, 86]. In patients with poor prog-
nents of the limbal epithelium basal membrane nostic indicators because of a massive loss of the
(BM) and their interactions with their cognate native LESC pool, the sole use of AM is however
LESC-surface receptors. The underlying BM of insufficient and requires the co-transplantation of
the corneal epithelium as well as those from other autologous LESC from limbal biopsies collected
epithelial tissues is basically composed by a mix- from the contralateral healthy cornea or from
ture of collagens, laminins, and heparan sulfate donor’s limbal biopsies for treating patients with
proteoglycans [81]. Laminins are important BM total bilateral LSCD [87, 88]. Further improve-
components and are composed of three distinct ment of this approach using tissue-engineered
types of chains, alpha, beta, and gamma. Different corneal epithelial equivalents created by in vitro
genetic variants of laminin chains produce a large re-epithelialization of the denuded AM with cul-
variety of heterotrimeric isoforms with tissue-­ tured limbal epithelial cell suspension or limbal
specific distribution pattern [82]. Ebihara et  al. biopsy explants was shown to provide successful
evaluated the growth capacity of human CECs in outcomes in animal transplantation models [66,
response to laminin-5, a major component of the 71, 73] and in humans [89–91]. Progresses made
corneal BM. Coating of plastic dishes with exog- toward the purification of LESC and of the pres-
enous laminin-5 was shown to increase CEC ervation of their stemness in culture are sug-
adhesion but however failed to improve their pro- gested advantageous for tissue engineering of
liferation [83]. Of particular interest, Polisetti more functional AM-based corneal epithelial
et al. performed qPCR and confocal microscopy equivalents.
image analysis of laminin chains expression on
microdissected LEPC clusters and in human lim-
bal epithelium sections, respectively [84]. They 5.3.8 Silk Fibroin
could identify the expression pattern of different
laminin isoforms with the laminin-α5 specific to Laminar fibrous scaffolds or porous films made of
epithelial progenitor cells. From this analysis, the silk fibroin (SF) are suggested very promising bio-
authors tested the activities of laminin-511 and logic substrates for tissue engineering of corneal
laminin-521 onto cultured LESC and show how epithelial equivalents and a possible alternative to
both of them could significantly increase their the use of AM in ocular surface surgery [92]. SF
adhesion and proliferation. Incorporation of lam- used in experimental tissue engineering is usually
inin-­511 to fibrin-based hydrogels could effi- obtained from the silkworm (Bombyx mori). This
ciently improve LESC adhesion and led to the protein is suggested to be extremely useful for
establishment of stratified epithelium-like tissue ­ corneal regeneration, because of its high
64
b­iocompatibility, biodegradability, transparency,
and mechanical strength, among other properties
(for specific review see [92, 93]). The production
of nano-/micro-SF fibers by electrospinning and
the possibility to control their deposition pattern in
diverse fibrous arrangements and thickness allow
the creation of customizable laminar fibrous mesh-
works, with adequate topography and porosity
favoring LESC adhesion, migration, and expan-
sion [94]. In most cases, the SF-based scaffolds
tested experimentally for the adhesion and expan-
sion of LESC have been thin porous films or mem-
branes obtained by pouring SF solutions into a
mold [95–100]. Of particular importance, thin SF
films are transparent, and the possibility of incor-
porating poly(ethylene glycol) (PEG) in SF solu-
tions, followed by its removal from the films,
allows creating pores for metabolites diffusion
[77, 99, 101]. Consistent with its high biocompat-
ibility, rabbit and human limbal epithelial cells
grown onto porous SF films were shown to be able
to form a stratified epithelial sheet [99, 101]. This
construct was shown to regenerate the corneal epi-
thelial surface in a rabbit model of LSCD [77].
5.4  orneal Stromal Stem Cell
C Limbal stromal tissue was obtained after scrap-
(CSSC)
The identification by Du et al. in 2005 of a popu-
lation of cells in the peripheral corneal stroma
with MSC characteristics and keratocyte differ-
entiation potential has opened up the promise of
using these corneal stromal stem cells (CSSC) in
tissue engineering of a corneal stroma equivalent
[12]. These CSSC were shown to express the
stem and progenitor cell markers ABCG2 and
PAX6, being this later a transcription factor
required for embryonic ocular development [12,
102]. Culture of CSSC with basic fibroblast
growth factor (bFGF or FGF-2) was found to pro-
mote their differentiation toward cells up express-
ing the keratocyte markers: keratocan, aldehyde
dehydrogenase 3A1, and keratan sulfate [12,
102]. Supporting these findings, Yamagami et al.
reported how stromal cells of the peripheral
human corneal stroma had a fourfold increased
sphere-forming capacity than those from the
C. C. Lachaud et al.
c­entral cornea [103]. Other studies also con-
firmed the presence of cells with CSSC charac-
teristics in the peripheral corneal stroma and
provided additional evidences of their stemness
characteristics, multilineage differentiation
toward adipocytes and osteocytes, and of their
keratocyte differentiation potential [104–107].
Hasmani et al. analyzed with flow cytometry the
co-expression of CD34 and CD105  in cultured
limbal and peripheral stromal cells. They show
how the CD34+/CD105+ cells subset displays the
highest stemness characteristics and transdiffer-
entiation ability into CK3+ and CK19+ epithelial
cells phenotypes, suggesting the presence of
bipotent mesenchymal and epithelial stem cells
in the limbal and peripheral stromal layer [108].
5.4.1 M
 ethods of Obtaining Corneal
Stromal Stem Cells
Different methods for obtaining human CSSC
have been reported so far [12, 104, 105, 107–
109]. By majority, laboratories have isolated
hCSSC from corneoscleral rims, which include
limbal and peripheral cornea stromal tissue.
ping off the epithelial and endothelial cells. Small
pieces of limbal stroma were then enzymatically
digested with collagenase [65, 104, 109, 110]. In
their work, Wu et  al. isolated hCSSC from
collagenase-­ digested human limbal tissue.
Primary cultures of limbal stromal cells were
established at clonal low density. With this
approach, cultures produced outgrowth colonies
of small polygonal cells consisting in CSSC,
which were further subcultured in stem cell
growth medium to foster their expansion [107].
Hashmani et  al. indicated that hCSSC can be
directly purified from heterogeneous corneal
stromal cells by FACS of the CD34+/CD105+
cells subset [108]. In their work, Du et al. demon-
strated that hCSSC can be sorted based on their
ability to efflux the dye Hoechst 33342, thus rep-
resenting a demarcated population termed “side
population or SP.” Cultured SP+ cells were shown
to display clonogenic proliferation capacity and
to express the stem/progenitor markers ABCG2
5  Corneal Stem Cells: Identification and Methods of Ex Vivo Expansion
Limbal epithelium
stroma
scraping
(collagenase)
endothelium
Limbal tissue
Plastic adherent
culture
Partial enzymatic digestion
(dispase II)
Fig. 5.3  Schematic summary of methods for isolating
and expanding corneal stromal stem cells (CSSC). Limbal
tissue is first excised from whole cornea or corneoscleral
rim. Limbal stromal tissue is then obtained after scrapping
off the epithelium and endothelium layers. Alternatively,
limbal stroma can also be obtained through partial enzy-
matic digestion of limbal tissue which dispase II to digest
basement membrane proteins, thus facilitating the poste-
and PAX6. When cultured in media supple-
mented with bFGF, SP+ cells could efficiently
upregulate their expression of several keratocyte
markers including keratocan, aldehyde dehydro-
genase 3A1, and keratan sulfate [12, 105].
Figure 5.3 is a schematic summary of methods
for isolating and expanding corneal stromal stem
cells.
5.4.2 Methods of Expanding
Corneal Stromal Stem Cells
5.4.2.1 Plastic Adherent Culture
CSSC are in nature similar to mesenchymal stem
cells (MSCs), which possess high adherence
capacities [12]. As such, the culture of corneal
stromal cells of murine, rabbit, bovine, and human
origin has been mostly performed into classic
plastic-treated tissue culture dishes to allow CSSC
expansion [12, 65, 102, 104–117]. The capacity of
hCSSC to undergo clonal growth under low-den-
sity culture has been indicated to be helpful to
select rapidly proliferating outgrowth hCSSC col-
onies (either by colony picking or in limited dilu-
tion cloning culture) from primary cultures and
65
Explant culture
Enzymatic
Limbal stromal
digestion cells
Culture onto
scaffolds
Suspension
culture
rior mechanical detachment of the epithelium and endo-
thelium layers. The resulting limbal stromal tissue can be
then directly placed in explant culture or fully disaggre-
gated with collagenase to release stromal cells suspen-
sions containing CSSC. Cell suspension of limbal stromal
cells have been cultured using three different methods: (i)
plastic adherent culture, (ii) suspension culture for spheres
formation, or (iii) culture onto scaffolds (carrier)
thus allow initiating subcultures with highly
enriched hCSSC [12, 102, 107, 118]. In most
cases, hCSSC expansion has been performed in
culture growth media supplemented with fetal
bovine serum (FBS) and growth factors [12, 105].
Du et al. stimulated hCSSC clonal growth in a low
FBS-containing media (2%) containing epidermal
growth factor (EGF), platelet-­derived growth fac-
tor BB (PDGF), and leukemia inhibitory factor
(LIF) among other media additives [12].
Funderburgh et  al. used similar growth medium
incorporating basic fibroblast growth factor
(bFGF) [102]. By contrast, other studies used high
FBS-containing media (10–20%) without growth
factors supplementation [104, 108]. Interestingly,
Sidney et  al. compared the ability of different
media formulations to sustain hCSSC growth and
concluded that a stem cell media formulation con-
sisting in a DMEM-F12 basal media supplemented
with 20% serum replacement, basic fibroblast
growth factor, and LIF is the most appropriate for
the clinical production of hCSSC [110].
5.4.2.2 Explant Culture
The possibility of obtaining CSSC from explant
culture of stromal tissue pieces is also attractive
66
as this approach does not require proteolysis (not
stressful for the cells) and thus preserve their
niche environment. Factors released from the
explanted tissue are also suggested important for
stimulating the posterior outgrowth of spreading
cells [68]. An adequate tissue culture explant sys-
tem allowing the fixation of tissue pieces on the
substrate is a critical issue, necessary for cell
adhesion and spreading. This is particularly true
for tissues with a high ECM content and harbor-
ing a low amount of cells, which the case for the
corneal stroma. In many instances, this initial
step of culture is challenging and may require
surface coating of plastic surface, using small
pieces of tissue in a minimum amount of media
to cover the pieces and increase their stability
[68]. In that way, Ghoubay-Benallaoua et  al.
reported the successful establishment of corneal
stromal fibroblastic cells through explant culture
of human limbal stromal tissue [65].
5.4.2.3 Suspension Culture
The suspension culture or sphere-forming assay
of corneal stromal cells has been addressed by
several laboratories in order to quantify their con-
tent in CSSC.  A Japanese group focused great
attention on this approach, showing how human
corneal stromal cells could efficiently formed
spheres containing cells expressing mesenchy-
mal and neural markers [19, 119]. Of special
interest, and consistent with the report by Du
et al., the authors also reported how cells isolated
from the peripheral cornea stroma have signifi-
cantly higher sphere-forming capacities than
those from the central cornea [19, 103, 119–121].
The media formulation used in their studies con-
sisted in a DMEM/F12 supplemented with the
growth supplements B27, 20  ng/ml EGF, and
40 ng/ml bFGF and the addition of methylcellu-
lose to create a gel matrix preventing cell reag-
gregation, thereby ensuring clonogenic
sphere-forming capacity. Basu et al. reported the
isolation of limbal biopsy-derived stromal cells
(LBSC) from human cadaveric corneoscleral
rims. These LBSC cultured in low-attachment
plates were able to generate spheres expressing
stem cells (ABCG2, Nestin, NGFR, Oct4, PAX6,
and Sox2) genes [109]. Ghoubay-Benallaoua
C. C. Lachaud et al.
et al. also use the sphere-forming assay to evalu-
ate the expansion capacity of hCSSC. They ini-
tially generated floating hCSSC spheres under
feeder-free conditions using the xeno-free E8
culture media and show their retention of prolif-
erative capacities upon serial subculture. They
suggest this method to be convenient for the pro-
duction of hCSSC for therapeutic purposes [65].
5.5 Corneal Endothelial
Progenitor Cell (CEnPC)
A major function of corneal endothelial cells
(CEnC) is to regulate the hydration state of the
cornea to maintain an optimal corneal stromal
matrix organization and transparency [1]. This
function is principally assumed by a sodium- and
potassium-dependent adenosine triphosphatase
(Na+/K+ATPase) pump which actively remove an
excess water content inside the stroma into the
anterior chamber. A normal pump function of the
corneal endothelium is primarily linked to its cel-
lularity or density, which in healthy subjects
decrease from 6000 CEnC/mm2 soon after birth
to 2300 CEnC/mm2 in 85  years old persons [1,
122]. CEnC are arrested in the G1  cycle, with
limited evidence of in vivo proliferation capaci-
ties in healthy or injured corneas. Instead, the
corneal endothelium compensates its gradual loss
of cellularity along life by hypertrophy of adja-
cent cells to restore a continuous functional
endothelium monolayer [1, 123–126]. This bio-
logic phenomenon is however critically accentu-
ated in patients suffering from corneal endothelial
dystrophies, such as the Fuchs endothelial cor-
neal dystrophy (FECD) where the abnormally
high rate of CEnC death leads to a rapid decline
of corneal endothelium cellularity to a point
(below 500 cells/mm2) at which it does not sup-
port a normal function, thereby causing corneal
edema and opacity [127–131].
Different studies have provided cumulating
evidences supporting the existence of immature
CEnC with progenitor/stem cell characteris-
tics within the Schwalbe’s ring region, a small
transitional area located between the periph-
eral corneal endothelium and the trabecular
5  Corneal Stem Cells: Identification and Methods of Ex Vivo Expansion
meshwork [13, 14, 23]. These stem/progeni-
tor cells have been suggested to originate cells
of both the trabecular meshwork (TM) and
corneal endothelium. Two studies conducted
onto healthy human corneas shortly cultured
in vitro reported the presence of cells express-
ing nestin and displaying alkaline phosphatase
(AP) activity and bromodeoxyuridine (BrdU)
incorporation in the trabecular meshwork (TM)
and transitional zone, but not in the central
corneal endothelium [13, 14]. Interestingly,
and by comparison, in corneas experimentally
wounded cells of the trabecular meshwork
started to express the stem cell marker Oct-3/
Oct-4. In addition, CEnC of the peripheral cor-
neal endothelium also started to express stem
cells differentiation markers (Pax-6, Sox-2),
two findings supporting the presence of stem/
progenitor cells at the posterior limbus [14].
In line of this concept, different studies also
reported how CEnC isolated from the periph-
eral area possess higher sphere-forming capaci-
ties than CEnC of the central cornea [132]. Of
particular interest, a comparative microanatomy
study of flat-mounted corneas from young and
old donors provided evidences of the occur-
rence of a continuous slow centripetal migra-
tion of CEnC.  Limbal peripheral endothelial
cells were found to be less differentiated than
central CEnC and to display higher expression
of the stem cell markers nestin and telomerase
[15]. Recently, Katikireddy et  al. reported the
presence in the healthy human corneal endothe-
lium of a subpopulation of cells with distinc-
tive higher proliferating capacities in vitro and
expressing stem cells (SOX2, OCT4, LGR5,
p63) and neural crest (PSIP1, p75(NTR), nes-
tin, PAX3, SOX9, AP2B1 (AP-2beta)) genes.
This subset of neural crest-derived progenitors
could differentiate into Na+/K+ ATPase CEnC
cells [16].
Thus, converging evidence points to the pres-
ence of a reservoir of corneal endothelial progen-
itor cells (CEnPC) at the boundary of the corneal
endothelium. These findings have propitiated
hope on the possibility of using these peripheral
CEnPC for tissue engineering of corneal endo-
thelial equivalents.
67
5.5.1 Methods of Obtaining
and Culturing CEnC
5.5.1.1 Adherent Culture of Single Cells
The first approach is used to isolate and culture
hCEnC consisted in digesting the entire cornea
with trypsin or collagenase. With this method, a
heterogeneous population of epithelial, stromal,
and endothelial cells was brought in cultures,
which inevitably developed outgrowth stromal
fibroblasts [133]. The development of novel tech-
niques allowing a selective collection of the cor-
neal endothelium by endothelial scrapping or
Descemet’s membrane stripping allowed obtain-
ing purified hCEnC single-cell suspensions and
cultures with a very low content of contaminating
fibroblasts outgrowth [125, 133–135]. To date,
the different in vitro culture protocols reported to
promote hCEnC expansion have however reached
success with diverse degrees among laboratories.
These divergent outcomes appear to be largely
linked to their capacity of inhibiting hCEnC to
undergo an endothelial-to-mesenchymal transi-
tion (EndMT), a mechanism triggering their phe-
notypic conversion into fibroblastic cells (for
specific review see [136]). Among reported
EndMT-inducing factors for hCEnC are their
repeated trypsin-EDTA enzymatic disruption
along sequential subculture steps. Another criti-
cal factor is fetal bovine serum (FBS) or culture
media containing pro-fibrotic growth factors,
such as the transforming growth factor-beta
(TGF-β) and also in lower extent to basic fibro-
blast growth factor (bFGF) [136, 137]. The
EndMT activity of FBS may be largely explained
by its high concentrations in the growth factors
bFGF, EGF, PDGF, and IGF-1 [138]. In support
of this premise is a previous report indicating
how hCEnC cultured in media with bFGF
increase significantly their EndMT [139]. Of par-
ticular interest, Zhu et al. reported that an experi-
mental cell-cell junction rupture of monolayered
hCEnC cultures with trypsin-EDTA treatment
followed by their culture with either FGF,
­TGF-­β1, or EGF are two factors synergistically
inducing hCEnC EndMT [17, 140].
A major issue in the process of hCEnC cul-
ture is also their arrest of division once they
68
have reached confluence, a phenomenon termed
“contact inhibition.” In this way, and by using
interfering siRNA, Zhu et al. demonstrated that
the knockdown of p120 catenin (p120) into
confluent hCEnC cultures was able to unlock
their contact inhibition by inducing trafficking
of p120 to their nucleus and activation of
RhoA-ROCK signaling. With this method,
monolayered hCEnC cultures increased signifi-
cantly their cellularity [140]. Later on, Zhu
et  al. demonstrated that a combined siRNA
knockdown of p120 and Kaiso in cultured
hCEnC monolayers could induce even more
efficiently their loss of contact inhibition, gen-
erating significantly higher density cultures
than by knockdowning only p120 [17, 141].
Using this method, the authors report tissue
engineering of eight corneal endothelial equiv-
alents from hCEnC isolated from one corneo-
scleral rim [17].
5.5.1.2 Explant Culture
Techniques such endothelial cells scrapping or
Descemet’s membrane stripping have permitted
obtaining purified corneal endothelium, enabling
thus the possibility of using its explant culture for
expanding nearly pure populations of CEnC
[142–145]. With this method, pieces of corneal
endothelium that could successfully adhere onto
the substrate initiate their spreading within few
days and start to produce early outgrowth cells
migrating centrifugally to generate a surrounding
dense cell monolayer [142]. This approach offers
the advantages of avoiding the use of digestive
enzymes which substantially degrade cell surface
molecules (adhesion proteins, receptors), thereby
damaging cells and making them potentially
more susceptible to undergo EndMT. The explant
culture is therefore recognized by many research-
ers as a more natural approach of cell culture.
The case report by Walshe et al. showing that a
serial explant culture was able to provide conflu-
ent hCEnC monolayer over seven subsequent
subculture steps, for a total period of 6  months
[146], suggests that future improvements of this
procedure of culture will allow to expand hCEnC
into clinically relevant numbers in reduced peri-
ods of time.
C. C. Lachaud et al.
5.5.1.3 Suspension Culture: Sphere-­
Forming Assay
The modality of culturing cells under suspension
into anti-/low-adherent culture plates or semi-
solid media offers several advantages over adher-
ent plastic surface cultures as rigid matrices were
reported to affect stem cell behaviors [147]. In
that sense, and taking in account the neural crest
origin of the corneal endothelium, several labora-
tories logically came to testing the growth capac-
ity and stemness characteristics of hCEnC under
the sphere-forming assay [18, 22, 148]. Of par-
ticular interest, it was shown that hCEnC
expanded under this condition retained higher
telomerase activity and telomere length and
lower senescence that their plastic adherent coun-
terparts, thus indicating that this approach may
enrich their content in hCEnC progenitors and
also maintain or/and improve their progenitor
characteristics [148].
5.5.1.4 Culture onto Biological
and Biosynthetic Substrates
The limited proliferation capacities of hCEnC
usually occurring after few rounds of subculture
have stimulated the search for novel culture sub-
strates able to sustain their proliferation in a more
efficient way. In line with this idea, different lab-
oratories cultured hCEnC onto tissue cultures
plates coated with biologic polymers, natural
components of the animal tissues extracellular
matrix (ECM). The different results obtained
could be interpreted as somehow disappointing
in this way, as although collagens I and IV and
fibronectin could significantly increase hCEnC
adhesion; they however failed to produce any sig-
nificant proliferation activity [20, 149, 150].
Only laminin V coating could moderately
improve hCEnC proliferation [151]. Conversely,
ECM-derived fibrous macroproteins might be
useful to improve the adhesion of hCEnC onto
biologic or synthetic scaffolds. In this way, Kim
et  al. reported an improved adhesion of rabbit
CEnC onto SF films coated with collagen type I
and their retention of normal polygonal morphol-
ogy [152]. Additionally, different laboratories
evaluated the capacities of hCEnC to adhere onto
natural biological scaffolds, potential surrogates
5  Corneal Stem Cells: Identification and Methods of Ex Vivo Expansion 69

of the Descemet membrane, and to form on their Table 5.1  Markers expressed in corneal stem/progenitor
cells
surface a corneal endothelium-like monolayer.
These include non-ocular decellularized tissues SC
type Markers Exp° Ref
such as the human amniotic membrane [153] and
LESC CK3/CK12 − [28]
corneal tissues such as whole corneas devoid of CK14 + [48]
their corneal endothelium (for specific review see CK15 + [48,
[154]). Additionally extracorneal ocular tissues 159]
such as the decellularized anterior lens capsule P63 + [39]
were also used [142, 155]. ABCG2 + [40, 43,
48]
ABCB5 + [44, 45]
5.5.1.5 The Use of Cell-Conditioned CXCL12, ISL1, COLA2, + [48]
Media for CEnC Expansion NCAM1, ACAN, FOXA2,
The application of cell-conditioned media for CX32, MSX1
stimulating the growth of hCEnC has also CSSC ABCG2 + [12,
105]
attracted interest. Among the different cell types
Pax6 + [12,
from which were obtained conditioned media 102]
showing significant mitogenic activity onto CD34, CD105 + [108]
hCEnC are found embryonic stem cells [21], CEnPC Alkaline phosphatase + [13, 14]
human amniotic epithelial cells [156], corneal Telomerase + [14, 15]
stromal cells [157], bone marrow-derived endo- Nestin + [14–17]
thelial progenitor cells [157], and bone marrow- SOX2, OCT4, LGR5, p63, + [16]
p75(NTR), PAX3, SOX9,
derived mesenchymal stem cells [157, 158]. AP2B1
Interestingly, the different cell type-conditioned cMyc, KLF4, Nanog, Oct4, + [17]
media tested onto hCEnC were also reported to Rex1, Sox2, SSEA4, AP2α,
preserve their functional phenotype. AP2β, FOXD3, HNK1,
MSX1, P75NTR, Sox9
Table 5.1 shows markers expressed in corneal
Abbreviations: CEnPC corneal endothelial progenitor
stem/progenitor cells
cell, CK cytokeratin, CSSC corneal stromal stem cell,
CEnPC corneal endothelial progenitor cell, Exp expres-
sion, LESC limbal epithelial stem cell
5.6 Conclusions
a challenge which is under intense research.
This chapter has focused on recovering past and Other issue of relevant interest is also the devel-
current findings on the identification of corneal opment of efficient xeno-free media for amplify-
stem/progenitor cells in the corneal epithelium, ing corneal stem/progenitor cells in a safe way.
stroma, and endothelium layers. We recovered Other important focus of efforts is around the
the different procedures which have been used characterization and optimization of scaffolds
for their isolation and ex vivo expansion, much of mimicking the native properties of corneal base-
them being still at the bench, and few others ment membranes, Bowman’s layer, and the
based on the use LESC being successfully trans- Descemet membrane. Solving these issues is of
lated to human corneal surface applications. major importance to progress toward the manu-
Despite clear progresses and successes in this facture of a full-thickness corneal graft.
direction, many challenges remain on the way
until experimental applications using corneal Acknowledgments Authors are supported by the non-
stromal stem cells (CSSC) and corneal endothe- profit Fundación Progreso y Salud, Consejería de Salud,
and Junta de Andalucía; FEDER co-funded grants from
lial progenitor cells (CEnC) can be traduced in Instituto de Salud Carlos III and the Ministry of Economy,
effective clinical therapies. Controlling the Industry and Competitiveness (Red TerCel:
in vitro expansion of corneal endothelial progeni- RD12/0019/0028 and RD16/00259; CIBERDEM:
tor cells and the long-term expansion of LESC is CB07/08/0006; PI14/01015, PI16/00259, PI17/02104 and
70 C. C. Lachaud et al.

CD16/00118); Junta de Andalucía (PAI-BIO311, CTS-­ 1 4. McGowan SL, Edelhauser HF, Pfister RR, Whikehart
576, CTS 11-727, PI-0109-2014, PI0007/2016 and DR. Stem cell markers in the human posterior limbus
PI0272/2017). CIBERDEM is an initiative of the Instituto and corneal endothelium of unwounded and wounded
de Salud Carlos III. corneas. Mol Vis. 2007;13:1984–2000.
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Competing Interests  The authors declare no corneal endothelial periphery: new evidence for con-
conflict of interest. tinuous centripetal migration of endothelial cells in
humans. Stem Cells. 2012;30(11):2523–34.
Informed Consent  No human studies were car- 16. Katikireddy KR, Schmedt T, Price MO, Price FW,
Jurkunas UV.  Existence of neural crest-derived
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lial dystrophy corneal endothelium. Am J Pathol.
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 Corneal Epithelial Stem Cells:
Methods for Ex Vivo Expansion
Gustavo S. Figueiredo, Hardeep Singh Mudhar,
Majlinda Lako, and Francisco C. Figueiredo
6.1 Introduction
The corneal epithelium is the only layer in the
cornea that is able to regenerate in vivo, and cor-
neal integrity and function are dependent on this.
The corneal epithelium is a stratified squamous
epithelium, where a few layers of flattened epi-
thelial cells lie on top of a basement membrane.
It is thought that this ability of the epithelium to
regenerate relies on a small population of stem
cells, located in the basal region of the limbus.
Limbal epithelial stem cells (LSCs) are found
within the limbus, at the corneoscleral junction.
These cells (LSCs) share many features with
other adult stem cells including their small size
and high nuclear to cytoplasm ratio [1]. They also
do not express markers of differentiation such as
cytokeratins 3 (CK3) and 12 (CK12). Limbal
epithelial stem cells are generally slow cycling
G. S. Figueiredo · M. Lako
Newcastle University, Institute of Genetic Medicine,
International Centre for Life,
Newcastle upon Tyne, Tyne and Wear, UK
H. S. Mudhar
Royal Hallamshire Hospital, Department of
Histopathology, Sheffield, South Yorkshire, UK
F. C. Figueiredo (*)
Newcastle University, Institute of Genetic Medicine,
International Centre for Life,
Newcastle upon Tyne, Tyne and Wear, UK
Royal Victoria Infirmary, Newcastle upon Tyne, UK
e-mail: francisco.figueiredo@newcastle.ac.uk
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_6
6
during homeostasis but can become highly pro-
liferative in periods of injury.
Fuchs et  al. (2000) were among the first to
describe the idea of stem cells being found in a
“niche” or a specific location within an organ in
which the microenvironment supports stem cell
viability and provides protection to them [2]. The
limbal niche is a term which describes a protec-
tive environment containing a reservoir of LSCs,
ready to migrate from the peripheral limbus cen-
tripetally towards the centre of the cornea. This is
a continuous process but is exaggerated during
periods of stress to the cornea, such as during a
wound healing response, guided by signals gen-
erated by the limbal niche.
A previous study from Dua et al. in 2005 and
subsequently confirmed by a further study from
Tseng’s group in 2007 both demonstrated that the
LSC niche is located within the palisades of Vogt
at the corneoscleral limbus [3, 4]. Due to their
ridge-like structure, vascularity and pigmenta-
tion, the palisades of Vogt have long been thought
to play an important role in the renewal and
regeneration of the corneal epithelium [5].
The stem cell niche provides an environment
that protects the stem cells from stimuli that may
adversely promote differentiation or apoptosis.
Tseng’s group also showed as an example that
melanocytes present in the limbal basal layer
help produce and transport melanin pigments
into epithelial cells to minimise damage by ultra-
violet irradiation [4]. Thus, in a normal eye, LSCs
77
78
are reasonably protected from external insult.
However, with damage to the palisades of Vogt
following chemical or thermal burn injuries, the
stem cells fail or become dysfunctional, and a
sequelae of symptoms and signs ensues, often
accompanied by inflammation and clinically
defined as limbal stem cell deficiency (LSCD).
The notion that the corneal epithelium is able
to regenerate was first hypothesised by Thoft and
Friend in 1983, when they explained their “X, Y,
Z hypothesis of corneal epithelial maintenance”
[6]. They proposed that the proliferation of basal
cells (X), added together with the centripetal
migration of cells (Y), was equal to the epithelial
cell loss from the surface (Z). Subsequently,
Sharma and Coles indicated in 1989 that corneal
epithelial cell mass could be renewed by cells
from the limbal epithelium alone [7].
At the limbus, mitotic cell division occurs in
those cells in contact with the basement mem-
brane. These putative LSCs can divide symmetri-
cally to regenerate and asymmetrically to produce
daughter cells, termed transient amplifying cells
(TAC), which are greater in number and retain a
high proliferative ability in health, to replace
superficial epithelial cells that have been natu-
rally desquamated. Aside from the vertical and
horizontal movement of cells that characterises
the corneal epithelium, centripetal movement of
cells from the limbus to the centre of the cornea
has also been previously demonstrated as
described above [6]. Early clinical observations
involving migration of limbal pigment post-
trauma [8], epithelial microcysts at the site of
corneal sutures [9] and limbal cells marked with
India ink demonstrated a centripetal movement
of epithelial cells from the limbus towards the
centre of the cornea [10] and evidence of epithe-
lial regeneration following limbal transplantation
[11]. This led to the long-established hypothesis
that the limbus contains an important driving
force towards epithelial regeneration and repair
[5, 12]. Evidence of this centripetal movement is
also seen clinically in the healing of central cor-
neal abrasions, where a leading edge of three or
more sheets of epithelium arising from an intact
peripheral epithelium migrates centrally until
they meet to close the epithelial defect [13, 14].
G. S. Figueiredo et al.
As cell division occurs, the epithelial cells
(TAC) migrate centripetally and in a superficial
direction (i.e. horizontally and vertically),
becoming more differentiated in the process, to
eventually form terminally differentiated cells
(TDC). Transient amplifying cells have the
capacity to divide exponentially when the epithe-
lium is wounded. Terminally differentiated cells
form the majority of the corneal epithelium.
Lavker et al. showed in 1991 that the most super-
ficial layers of the corneal epithelium contain
postmitotic cells and are therefore unable to
regenerate through mitotic cell division [15].
These cells (TDCs) are constantly sloughed off
the ocular surface through blinking and normal
wear and tear, and as they are incapable of divid-
ing, this stimulates the cycle of cell division,
migration and differentiation [1]. Corneal epithe-
lial cells have been shown to have a rapid turn-
over of between 1 week and 10 days [16].
6.2 Limbal Stem Cell Deficiency
Limbal stem cell deficiency (LSCD) is a painful,
blinding, chronic condition characterised by a
failure of the limbus in its function as a stem cell
niche and also as a physical barrier to prevent
vascularised conjunctival tissue from migrating
into the cornea. Consequently, failure of limbal
stem cells can occur due to either a loss of limbal
stem cells or their dysfunction [17]. It is a disease
therefore that results in conjunctivalisation of the
corneal surface with migration of a thick, vascu-
larised, conjunctival tissue (pannus) leading to
scarring and vascularisation of the cornea and
subsequent permanent visual impairment,
together with recurrent or persistent epithelial
erosions and ulcerations that result in stromal
opacity and chronic pain and discomfort.
6.2.1 Aetiology
LSCD can occur as part of a hereditary group of
conditions, such as aniridia, among others, or as
an acquired condition, including chemical and
thermal burns (the most frequent causes of
6  Corneal Epithelial Stem Cells: Methods for Ex Vivo Expansion
LSCD), inflammatory diseases of the anterior
segment (e.g. ocular cicatricial pemphigoid,
Stevens-Johnson syndrome and chronic limbitis)
or extensive microbial infections. It can also
result from potentially damaging ocular treat-
ments such as extensive cryotherapy; radiation;
surgery at the limbus; subconjunctival or topical
anti-proliferative agents, e.g. mitomycin C
(MMC) for ocular tumours and adjunct therapy
in glaucoma surgery; and long-term use of pre-
served topical medications (e.g. for glaucoma).
LSCD has also been shown to result from pro-
longed contact lens use, although this is rare
[17]. In the absence of an obvious aetiology, it is
a condition that may be described as idiopathic,
or of an unknown cause, although this is
extremely rare. In most cases of acquired LSCD,
it is likely that both LSCs and their niche are
affected [18].
6.2.2 Epidemiology
The incidence and prevalence of LSCD are not
well understood. As has already been described,
there are a variety of conditions known to cause
LSCD, and these conditions are largely
uncommon.
Among the hereditary conditions, the reported
prevalence of aniridia ranges from 1 in 40,000 to
1 in 100,000 people [19].
Globally, chemical burns account for 7–18%
and thermal burns for 16% of eye injuries leading
to an emergency visit [20]. However, it is
unknown what proportion of these patients will
develop LSCD.  The UK national incidence of
severe chemical corneal injuries is unclear [21,
22]. A recent national British Ophthalmological
Surveillance Unit (BOSU) study estimated the
incidence of severe chemical injuries, with the
potential to lead to total LSCD, as 12 per 60 mil-
lion or 0.02 per 100,000 per year [23].
The incidence of Stevens-Johnson Syndrome
(SJS) is 2.6 to 7.1 cases per 1 million person-­
years in the USA and 1.1 cases per 1 million
person-years in Germany [24]. The incidence of
ocular mucous membrane pemphigoid is esti-
mated at 1.16 cases per million population in
79
France and 0.87 cases per million population in
Germany [25].
The leading causes of LSCD in the UK are
chemical/thermal injury, SJS, aniridia and ocular
mucous membrane pemphigoid. The combined
incidence of these conditions is 1:25000  in the
UK, and Shortt et al. stipulated that assuming that
10% of these patients develop LSCD, this results
in 240 new cases of LSCD per year in the UK
alone [26].
In view of its low incidence and prevalence
but significant debilitation from the condition,
LSCD is designated an orphan disease by the
European Medicines Agency. This status pro-
vides incentives for trial sponsors to develop
medicines that would otherwise fail a test of eco-
nomic viability. In 2015, the European Medicines
Agency recommended for Holoclar® (Chiesi
Farmaceutici S.p.A., Parma, Italy) to be given
approval for the marketing authorisation for the
production of an autologous limbal stem cell
product for patients with moderate to severe
LSCD due to ocular surface burns (see
Sect. 6.4.5).
6.2.3 Semiology
Limbal stem cell deficiency may affect patients
of all ages, depending on the original aetiology.
Patients typically present with discomfort or pain
and decreased vision. They may also have suf-
fered from recurrent breakdowns in their corneal
epithelium resulting in recurrent corneal erosions
or even corneal ulceration and consequent sec-
ondary infection, resulting in corneal thinning or
even perforation. These features, over time, lead
to permanent conjunctivalisation of the epithe-
lium and may also lead to corneal neovasculari-
sation at the deeper stromal layer, often
accompanied by scar formation. The irregular
conjunctival epithelial surface over the cornea
also leads to an unstable tear film, reduced visual
acuity and photosensitivity; it may produce fila-
ments and further corneal erosions. Epithelial
keratinisation may also occur in the presence of
an adverse ocular surface environment, including
tear deficiency.
80
Limbal stem cell deficiency can be subclassi-
fied into partial or total LSCD (TLSCD), depend-
ing on whether the whole limbus or only a
segment of the limbus is involved, or unilateral or
bilateral, depending on whether both eyes are
affected [27].
Limbal stem cell deficiency presents with typ-
ically classical clinical features, which are depen-
dent on the severity of the condition (i.e. partial
or total LSCD). With an abnormal (dysfunc-
tional) limbus, conjunctivalisation of the cornea
occurs and is the hallmark of the condition, as
described above. Dua et al. showed in 2003 that
the most significant clinical manifestation of con-
junctivalisation of the cornea is a persistent epi-
thelial defect [28]. This can lead to other
complications such as corneal ulceration and
even perforation. In LSCD, late fluorescein stain-
ing of the corneal epithelium can be observed,
corresponding to the area of conjunctivalisation
as a result of partial or total LSCD (Fig. 6.1).
This characteristic finding is the result of the
fluorescein dye seeping through to the basement
membrane of the cornea due to the loss of cell-­
cell tight junctions in the conjunctivalised cor-
neal epithelium. Late staining of these cells
lacking in these tight junctions represents either a
total replacement of the corneal epithelium by
conjunctival-type epithelium or a mosaic pattern
of corneal and conjunctival epithelial cells. A
clear demarcation line between corneal and con-
junctival phenotypic epithelial cells can some-
times be clearly observed in cases of partial
LSCD as conjunctival cells stain with fluorescein
much more easily (Fig.  6.1). This stippled, late
fluorescein staining on the cornea can occasion-
ally be seen to form “whorls”, giving a typical
Fig. 6.1 Colour
Partial
photograph showing
conjunctivalisation
clinical signs of partial
of corneal surface
limbal stem cell
deficiency not involving
the visual axis
Vascularised,
opaque section of
cornea
G. S. Figueiredo et al.
appearance on slit lamp biomicroscopy.
Sometimes a clear area of corneal thinning may
be observed, along with other areas of corneal
scarring, which may be superficial (anterior stro-
mal) or deep (mid-deep stromal) or both. The
palisades of Vogt at the limbus are easily observed
through slit lamp biomicroscopy, in normal eyes,
as “fronds” of tissue at the limbus; however, they
become much more disorganised and less obvi-
ous or flattened in cases of LSCD (Fig. 6.2).
In total limbal stem cell deficiency (TLSCD),
there is complete obliteration of the limbus 360°
around the cornea, resulting in conjunctivalisa-
tion of the entire corneal surface (Fig.  6.3). In
such cases, the characteristic areas and demarca-
tion line of late epithelial staining seen in cases of
partial LSCD are not observed. Chronic inflam-
mation and redness of the anterior segment of the
eye often result.
As described above, limbal stem cell defi-
ciency can be classified into partial or total
LSCD, unilateral or bilateral, and this classifica-
tion defines further management of the patient.
Total LSCD is defined as the complete absence of
normal corneal epithelial cells clinically by slit
lamp biomicroscopy but also on histopathologi-
cal examination (corneal impression cytology,
CIC); in contrast, partial LSCD contains some
normal corneal epithelial cells as previously
described.
6.2.4 Management
The management of partial LSCD is essen-
tially medical, including the use of inten-
sive preservative-­
free ocular surface topical
Well-defined area of
delayed fluorescein
staining with a clear
demarcation line
Area of clear cornea
in partial LSCD
6  Corneal Epithelial Stem Cells: Methods for Ex Vivo Expansion 81

a b

Fig. 6.2  High magnification colour photographs showing presence of palisades of Vogt (arrows) in a healthy eye (a)
and absence of palisades of Vogt (arrow) in a limbal stem cell deficiency eye caused by chemical burn (b)

a drops (AlloSE) [27, 29, 30]. These patients often

b consequent reduced visual acuity, partial LSCD
Fig. 6.3  Colour photograph showing clinical signs of
total limbal stem cell deficiency, resulting in conjuncti-
valisation of the entire corneal surface involving the visual
axis (a) and total late epithelial staining without demarca-
tion line (b)
l­ubrication and the judicial use of topical steroid
therapy (often as pulse therapy), and, in some
cases, these can be combined with autologous
serum eye drops (ASE) or allogeneic serum eye
do not require surgical treatment if the visual axis
is not affected. In severe cases, as the limbal area
is more affected, the visual axis is often involved,
which leads to a very low vision. In addition,
these patients suffer from pain and photopho-
bia, due to recurrent epithelial defects, chronic
ocular surface inflammation, corneal conjucti-
valisation, central corneal opacity and superficial
vascularisation, which make the patient function-
ally blind [18]. If the visual axis is affected with
can be treated by surgically excising the abnor-
mal epithelium (i.e. epitheliectomy) to allow the
denuded cornea to be re-epithelialised from the
remaining intact limbal epithelium [13, 14, 31].
This may be combined with an amniotic mem-
brane transplantation to help reduce the risk of
recurrence of conjunctivalisation [32]. In addi-
tion, topical preservative-­free lubricants, steroid
eye drops and ASE/AlloSE are often used [26].
Total LSCD has traditionally been more com-
plex to manage, with a variety of conservative
measures aimed at controlling symptoms and
maintaining the integrity of the ocular surface,
which as a result may also produce some form of
visual improvement. These measures included
copious use of topical preservative-free lubricants
and ASE/AlloSE [26, 33–35]. More challenging
cases, with a persistent epithelial defect that is
82
unresponsive to medical treatment, a few addi-
tional treatment options such as tarsorrhaphy [26,
36–41], botulinum toxin-induced ptosis [42–45],
bandage contact lenses [26, 46–50] or human
amniotic membrane transplantation [51–55] could
be used to temporarily protect the diseased ocular
surface. In most cases of LSCD, patients are often
managed in more specialised tertiary centres with
treatment including a number of these options in
combination. Lamellar (partial thickness) or pen-
etrating keratoplasty (PKP, full thickness) corneal
transplantation involves partial or total replace-
ment of the central cornea, preserving the limbus.
Therefore, although it replaces an opaque cornea
with a clear cornea, it does not restore LSC func-
tion, and, in time, the corneal transplant also
becomes opaque and fails due to persistent LSCD
and consequent recurrent conjunctivalisation. This
is therefore not a suitable treatment option for this
condition unless the LSCs are replaced prior to
corneal transplantation [56].
Where there is a lack of healthy, functional
stem cells such as in total LSCD, the ideal treat-
ment would be to replace them, so that they can
then proliferate, repopulate their niche and
provide the natural LSC resource capable of
­ protocol is still in use, although there have been
maintaining normal corneal epithelial homeosta-
sis. Replacement of limbal stem cells was first
proposed by Kenyon and Tseng in 1989, by per-
forming whole limbal tissue grafts [11, 57].
Whole tissue grafts were taken either from a
patient’s contralateral healthy eye (autograft) in
unilateral cases of LSCD or from a living related
or cadaveric donor (allograft) in cases of bilateral
LSCD.  In allogeneic tissue transplantations,
post-operative systemic immunosuppression was
necessary, which had its own drawbacks particu-
larly in terms of the risk of opportunistic infec-
tions and iatrogenic malignancy [11, 58–66].
Additionally, they are associated with poor long-­
term survival outcomes, as they often fail due to
allograft rejection [19, 67]. The large amount of
tissue required to be transplanted in these opera-
tions poses enormous risk to the donor eye in
autografts and living-related allografts in terms
of developing LSCD [68].
In 1997, Pellegrini et al. described the first two
cases of autologous transplantation of ex  vivo
G. S. Figueiredo et al.
expanded limbal stem cells in patients with uni-
lateral TLSCD, where a much smaller biopsy
was taken from the healthy fellow eye and
expanded ex  vivo in culture before being trans-
planted into the affected eye with negligible or no
risk to the donor eye [69]. They described the
cases of two patients who had suffered severe
alkali burns in one eye, with the fellow eye
remaining healthy. A 1  mm2 biopsy was taken
from the limbus of the uninjured eye, fragmented
and treated with trypsin (0.05% trypsin and
0.91% edetic acid). The cells were then plated on
mitotically inactivated 3T3-J2 mouse fibroblast
cells and cultured in 5% carbon dioxide in a
medium that had been used previously to culture
skin epithelial cells. After 16 or 19 days, the cells
were released from the plastic dish using a neu-
tral protease, Dispase II, and placed either on a
petrolatum gauze or a soft contact lens. The
injured eye was then prepared for transplantation
by surgically removing the diseased conjuncti-
valised epithelial surface by superficial keratec-
tomy (i.e. dissection with a blunt knife or
scissors), with a 360° conjunctival peritomy,
extending at least 2 mm beyond the limbus. This
some important modifications since, as described
by the Newcastle Limbal Stem Cell Group [70].
Current procedures involve the use of an animal-­
free culture method that adheres to Good
Manufacturing Practice (GMP) principles, using
human autologous serum (HAS) in place of fetal
bovine serum (FBS); transferrin and glutamine
have been removed from the culture medium; and
the 1  mm2 limbal biopsy is immediately plated
onto human amniotic membrane (epithelium side
up), instead of being fragmented, trypsinised and
cultured on 3T3-J2 mouse fibroblast cells.
Following transplantation of the human amniotic
membrane (HAM) with the ex  vivo expanded
stem cells, a further HAM is sutured in place on
top of the transplanted stem cells and a bandage
contact lens placed on top of the second HAM for
protection [70].
Two-year follow-up and analysis in the
Pellegrini case series showed these autologous
limbal stem cell transplants (ALSCT) to have
been successful, and this report remains the
6  Corneal Epithelial Stem Cells: Methods for Ex Vivo Expansion
benchmark against which all other limbal stem
cell transplants are based on. In the following
years, a number of studies have refined or adapted
this technique and reported on their outcomes.
This was extensively reviewed by Baylis et al. in
2010, who reported an overall success rate of
77% for autografts and 73% for allografts [56].
They also showed that failures appeared to hap-
pen in the first 1–2  years post-transplant, based
on studies by Sangwan et  al. (2006), Pauklin
et  al. (2010) and Rama et  al. (2010) [71–73].
Significantly, Rama et al. reported that those LSC
cultures that had more than 3% of cells staining
positive for p63 bright resulted in 78% success
clinically, whereas those with less than 3% cells
staining p63 positive were only 11% successful
clinically [73].
Prior to successful ALSCT, especially in
patients who have suffered a thermal injury to
the eye, it is important to ensure the eyelids are
well positioned and apposed, without any lag-
ophthalmos or any exposure of the corneal sur-
face, and that Schirmer’s test of tears production
is within normal limits to maximise success of
ALSCT. It is greatly important that preoperative
­inflammation is managed with topical steroids,
whether pulsed with high doses or on a low-
maintenance dose, and that the ocular surface is
well lubricated using preservative-free topical
therapy, particularly (although not essential)
with ASE in advance of the ALSCT. It is essen-
tial that a careful assessment of the “unaffected”
other eye in unilateral cases is made to ensure
that it is in fact “healthy”. As a minimum, CIC
and, if available, IVCM should be performed in
both eyes to confirm the TLSCD diagnosis in the
affected eye and to exclude any evidence of lim-
bal stem cell damage in the unaffected contralat-
eral healthy eye so as to not iatrogenically induce
limbal stem cell failure post-biopsy, although
this has not yet been reported since the introduc-
tion of small limbal biopsies by Pellegrini et al.
in 1997.
Following successful ALSCT, it is important
to maintain a bandage contact lens for a suit-
able period of time, usually 8–12  weeks post-­
operatively, to protect the ocular surface, the
donor LSCs and the HAM covering the LSCs
83
and to allow the eye to settle. During this period,
the eye is treated with intensive eye drops in
the form of preservative-free ocular lubricants,
preservative-­free topical steroid drops and
preservative-­free topical antibiotic drops, often in
combination with ASE. This treatment is gradu-
ally tapered over the initial few weeks to months,
aiming to stop all topical steroid treatment by
12 months [70].
Whilst ex vivo expanded stem cell or whole
tissue transplantation alone may improve vision
in these patients, a combination with corneal
transplantation is often necessary to achieve
optimal visual recovery. Corneal transplanta-
tion without prior regeneration of the corneal
epithelium by stem cell transplantation inevi-
tably fails in patients with severe or TLSCD
[74]. Following successful ALSCT, the deeply
scarred cornea can be subsequently replaced by
corneal transplantation (penetrating or lamel-
lar keratoplasty) often as part of a two-stage
approach [75, 76]. Basu et  al. demonstrated a
two-stage approach as having favourable cor-
neal transplantation survival outcomes com-
pared with a single-stage approach [76].
6.3  orneal Epithelial Cell
C
Culture
Corneal epithelial culture can be carried out either
in a single cell suspension culture with an inacti-
vated 3T3-J2 mouse fibroblast feeder layer or as
an ex  vivo expansion of limbal explant cells on
HAM. The single cell suspension method allows for
cells to be counted so a specific number of cells can
be used, and experiments are therefore better con-
trolled, improving reproducibility and comparison
in scientific experiments. It is therefore a method
widely used in laboratory science. However, the
single cell suspension method involves the use of
animal feeder cells which potentially carry the risk
of disease transmission. The ex vivo expansion
method of limbal explants on HAM that we devel-
oped is a xeno-­free culture system that eliminates
all animal feeders and components, which therefore
satisfies MHRA regulations for animal-free prod-
ucts to be used in human clinical trials as described
84
in a review by the World Health Organization and
the Advisory Committee on the Safety of Blood,
Tissues and Organs (SaBTO) of the donation of
starting material for cell-based advanced therapies
[77–80]. The ex vivo expansion of limbal explant
culture system is therefore more widely used in
human clinical trials [71, 81].
6.3.1 M
 ethod of 3T3 Fibroblast
Coculture (Cell Suspension)
6.3.1.1 Initiation of 3T3 Cultures
from Frozen Stock
A Cryovial of frozen mouse strain J2-3T3 fibro-
blast stock is thawed in a water bath at 37 °C. Once
defrosted, the 3T3 fibroblasts are suspended in
5 ml of 3T3 medium and centrifuged at 1000 RPM
for 3 min. The cell pellet is then resuspended in
10 ml 3T3 medium (Dulbecco’s Modified Eagle
Medium [DMEM] with 10% FBS) in a 75  cm2
(T75) cell culture flask.
6.3.1.2 Maintenance and Subculture
of 3T3 Feeder Layer
The 3T3-J2 fibroblast cultures are examined
every day and the medium exchanged on alter-
nate days. Once 60–75% confluent, the cells are
passaged by firstly aspirating the 3T3 medium
from the flask and gently rinsing the cells twice
with Dulbecco’s Phosphate-Buffered Saline
(DPBS). The 3T3 cells are then released from the
base of the cell culture flask by incubation with
trypsin 0.05% for 10 min at 37 °C. After ensuring
the cells are free in suspension, the cells are
removed into a centrifuge tube, and the trypsin is
inactivated by adding 3T3 medium. The resulting
suspension is then centrifuged at 1000 RPM for
3 min at room temperature. The resulting pellet is
then resuspended, and cell count and viability are
assessed. One hundred thousand viable cells are
then added to 10 ml 3T3 medium and the suspen-
sion transferred to a new 75 cm2 cell culture flask.
6.3.1.3 Preparation of Frozen Stocks
To maintain a supply of 3T3 mouse fibroblasts,
frozen stocks are prepared from the early pas-
sages. This is done by releasing subconfluent
G. S. Figueiredo et al.
cells by trypsinisation as described above, cen-
trifuging the cell suspension to a pellet and
resuspending the cell pellet in 1  ml of 90%
FBS, 10% dimethyl sulphoxide (DMSO,
Sigma-Aldrich). The 3T3 fibroblasts are then
frozen at −80 °C.
6.3.1.4 Inactivation by Irradiation
of 3T3 Cells and Preparation
of Feeder Layers
For successful coculture of LSCs, the 3T3
mouse fibroblast layer must first be mitotically
inactivated to prevent their proliferation/over-
growth and subsequent suffocation of LSCs in
culture. To do this, the cells are irradiated at
60  Gy. The medium is then removed and the
cells rinsed twice with DPBS before their
release by trypsinisation as described above.
The suspended cells are then transferred to a
centrifuge tube and the trypsin inactivated by
the addition of 3T3 medium. The resulting sus-
pension is then centrifuged at 1000  RPM for
3  min at room temperature. The resulting cell
pellet is resuspended in 3T3 medium, and cell
count and viability are assessed. The cells are
then plated in a 9.6 cm2 tissue culture well at a
final density of 2.4  ×  104 cells per cm2 with
0.5  ml 3T3 medium and incubated for 24  h at
37 °C to allow for their adhesion to the culture
well before the addition of LSCs.
6.3.2 L
 imbal Explant Culture
on a Human Amniotic
Membrane Substrate
6.3.2.1 Preparation of Limbal Epithelial
Culture Medium
Limbal epithelial culture medium is prepared
2  days prior to the limbal biopsy. Dulbecco’s
Modified Eagle Medium (DMEM, low glucose
with GlutaMAX and pyruvate) is used as the
main basic ingredient in a 3:1 mixture with 25%
Ham’s F12 and GlutaMAX nutrient medium (i.e.
125 ml Ham’s F12 was added to 375 ml DMEM).
From this mixture, 1  ml is removed, and seven
supplements (all Sigma-Aldrich, UK) are added
in sequence as per Table 6.1.
6  Corneal Epithelial Stem Cells: Methods for Ex Vivo Expansion 85

Table 6.1  Table showing supplementation of basic lim- can be stored at 4  °C and leftover autologous
bal epithelial culture medium
serum stored at −20 °C in case further complete
Supplement Concentration Volume LSC medium is required during the culture
Hydrocortisone 0.4 μg/ml 500 μl process.
Insulin 5 μg/ml 250 μl
Triiodothyronine 1.4 ng/ml 35 μl
Adenine hydrochloride 24 μg/ml 300 μl 6.3.2.2 Preparation of HAM Construct
Cholera toxin 8.4 ng/ml 84 μl Large HAMs (3 × 3 cm) suitable for clinical use
Epidermal growth factor 10 ng/ml 50 μl are obtained in the UK from NHS Blood and
Transplant (NHSBT) Tissue Services based at
Speke, Liverpool, UK, and stored at −80 °C. The
The mixture is then filtered in a 0.2 μm pore HAM construct consists of a sheet of 3 × 3 cm
size 500 ml fast PAS Nalgene® sterile filter unit HAM that is trimmed to size and wrapped around
(Sigma-Aldrich, UK) to make up the basic LSC a sterile (autoclaved) glass coverslip, epithelial
medium. side up with the edges trapped between a second
For the preparation of complete LSC medium, coverslip underneath (Fig. 6.4).
human autologous serum is then prepared using A wash solution of 49.5  ml DPBS with
the patient’s own blood that has been obtained 0.5  ml penicillin-streptomycin is made, and
24 h previously and allowed to clot overnight at 2 ml of the wash solution is then added to four
4 °C. To do this, the blood is initially collected (at wells of a six-­well plate, and 2 ml of complete
least 60 mls) in sterile 50 ml centrifuge tubes and LSC medium is added to the remaining two
centrifuged at 1600 × g at room temperature for wells of the six-well plate (Fig. 6.5a). Two ster-
10 min. The supernatant (serum) is then carefully ile (autoclaved) glass coverslips are carefully
removed into two new sterile 50  ml centrifuge taken and washed in sequence in the prepared
tubes and centrifuged again at 1600 × g at room wells of the six-well plate so that each glass
temperature for 10 min. The supernatant (serum) coverslip is rinsed twice in the wash solution
is again carefully removed into a new sterile and once in complete LSC medium. The glass
50 ml centrifuge tube. Eleven microlitres of the coverslips are then placed on a flat surface
prepared autologous serum is then added to 99 ml inside the laminar flow hood, ready to receive
of the previously prepared basic LSC medium the HAM.  The HAM comes frozen wrapped
(i.e. 10% autologous serum), and the mixture is round a nitrocellulose paper and is prepared by
filtered once again in a 0.2 μm pore size 500 ml thawing at room temperature immediately prior
fast PAS Nalgene® sterile filter unit (Sigma-­ to use before washing it sequentially in a similar
Aldrich, UK) to make up the complete LSC manner (twice in the wash solution and once in
medium. The complete LSC medium is then ali- LSC medium) in the six-well plate. The HAM is
quoted into 15 ml centrifuge tubes. There are two then carefully unwrapped from its nitrocellulose
10  ml LSC medium aliquots to which 100  μl backing (Fig. 6.5b) and stretched out epithelial
penicillin-streptomycin (or 50  μl gentamicin in side up, over a pre-washed glass coverslip on a
penicillin-allergic patients) is added, and these flat surface carefully using sterile suture tying
are used for plating the HAM construct and sub- ophthalmic micro forceps (Duckworth & Kent,
sequent plating the limbal biopsy. A further 13
aliquots of 6 ml LSC medium each are prepared Human amniotic
without antibiotics as per MHRA requirements to membrane (HAM)
be used for the medium exchanges. Three micro-
biological samples are sent per LSC medium Sterile glass
preparation: a sample from the basic LSC coverslips
medium, autologous serum and the complete
Fig. 6.4  Diagram showing human amniotic membrane
LSC medium. Complete LSC medium aliquots (HAM) construct with HAM trapped between two sterile
are stored at 4 °C. Leftover LSC basic medium glass coverslips
86
a b
c d
Fig. 6.5  Colour photographs showing preparation of
human amniotic membrane (HAM) construct. (a) Washing
HAM in DPBS and LEC medium, (b) unravelling HAM
UK) so that its edges just overhang the edges of
the coverslip. HAM orientation (i.e. epithelial
side up) is checked several times with a ster-
ile lint-free surgical spear (EyeTec, UK). The
edges of the HAM are then trimmed with an
overhang of 2–3 mm from the edges of the glass
coverslip (Fig. 6.5c). The glass coverslip is then
lifted off the flat surface by carefully sliding
the blade of a scalpel with a number 22 surgi-
cal scalpel blade under the glass coverslip and
G. S. Figueiredo et al.
from its nitrocellulose paper backing, (c) stretching out
and trimming edges of HAM, (d) folding edges of HAM
around glass coverslip
gently l­ifting it, maintaining the same orienta-
tion, so that the HAM edges neatly wrap around
the edges of the coverslip (Fig. 6.5d). The HAM
is then stretched out over the edges of the cov-
erslip so that it lies taught over the coverslip.
Care is always taken to prevent the HAM from
drying out by moistening it with complete LSC
medium as required throughout the procedure.
Two or three drops of complete LSC medium
are then added to the surface of the second glass
6  Corneal Epithelial Stem Cells: Methods for Ex Vivo Expansion
coverslip, and the HAM/first coverslip construct
is slowly lowered from one edge to the opposite
edge of the second coverslip so that there are no
trapped air bubbles between the two coverslips
and the edges of the HAM are neatly tucked
between the coverslips. Capillary suction from
the drops of complete LSC medium adheres the
two glass coverslips together. If any of the HAM
edges are loose, the whole procedure is repeated
as the coverslips are separated and the HAM/first
coverslip is gently lowered onto the second cov-
erslip again until all edges are neatly tucked in.
The HAM/coverslip construct is then placed in a
well of a new sterile six-well plate and incubated
in 2 ml complete LSC medium in an incubator at
37  °C and 5% CO2. A 24  h check of the HAM
construct is made the following day, prior to the
limbal biopsy scheduled for the day after, and if
any HAM edges are loose, it is replated.
6.3.2.3 Limbal Explant Biopsy
The limbal biopsy is a small, 1 by 2 mm partial
thickness autologous biopsy of the limbus from
the patient’s healthy contralateral eye in cases of
unilateral LSCD, superiorly at the 12 o’clock
position or inferiorly at the 6 o’clock position,
where it is known there are more LSCs [82].
The limbal explant biopsy is performed in a
dedicated ophthalmic theatre under local or gen-
eral anaesthetic (i.e. patient preference) as day
case procedure. It involves exposing the limbus
superiorly, followed by performing a linear
superficial keratotomy (150  μm deep) using a
diamond knife (Duckworth & Kent, UK) paral-
lel to the limbus (either side, close to 12 o’clock
superiorly) in an area previously determined (by
the surgeon performing the operation) on slit
lamp assessment as being rich in well-character-
ised palisades of Vogt. Then, a careful lamellar
dissection posteriorly is performed using a
2.25 mm, 60° circular edge head blade (Beaver-
Visitec International Ltd., UK) going beyond
the posterior edge of the limbus. This is fol-
lowed by two relieving incisions at the two lat-
eral edges of the biopsy and finally a posterior
dissection of the limbal biopsy that is accompa-
nied by a peritomy of the conjunctiva. The pos-
terior and superior corner edge of the biopsy is
marked with a surgical marker pen away from
87
the palisades of Vogt, and the biopsy is then
placed in a well of a sterile six-well plate, coated
in ocular viscoelastic device (Healon GV®,
Abbott Medical Optics Inc., Sweden) to prevent
it from desiccation during immediate transpor-
tation to the GMP facility [70].
6.3.2.4 Limbal Explant Culture
Once the limbal explant biopsy was received in
the GMP facility, it is immediately transferred to
the clean room, where it would be plated onto
the HAM construct. The HAM construct is
inspected a final time, and medium is removed
from the culture well. The six-well plate contain-
ing the HAM construct is tilted approximately
15° to allow all of the medium to be drained
from the surface of the HAM, whilst the limbal
explant biopsy is washed in complete LSC
medium. To do this, the biopsy is carefully
picked up with sterile non-toothed ophthalmic
micro-tying forceps (Duckworth & Kent, UK),
preventing any potential damage to the biopsy
and maintaining its orientation throughout (epi-
thelial side up). The biopsy is washed thor-
oughly, with complete LSC medium, to facilitate
adhesion to the HAM. It is then dried by gently
“running” it on a sterile, flat surface, until com-
pletely dry. The six-­well plate containing the
HAM construct is then laid flat, and the explant
biopsy carefully placed in the centre of the HAM
and allowed to rest for 2  min before 1.3  ml of
complete LSC medium is gently added. The con-
struct is then carefully inspected to make sure
that limbal biopsy is completely adherent to the
HAM before it is incubated. The culture medium
is exchanged on the third culture day and every
second/third day thereafter until the culture
reaches >90% confluence. The explant culture is
incubated at 37 °C in 5% CO2.
6.3.3 T
 he Use of Human Amniotic
Membrane as a Substrate
for the Culture of Limbal
Stem Cells
Human amniotic membrane (HAM) has long
been used in ophthalmic procedures to treat ocu-
lar surface diseases and burns [55], due to its
88
ability to promote re-epithelialisation via growth
factors (such as EGF, KGF and HGF) and its
basement membrane [83], and to inhibit fibrosis
through suppression of TGFβ signalling [84].
Fresh HAM consists of an epithelial layer (devi-
talised by the freezing process), a stroma and a
thick basement membrane. The HAM stroma
exhibits anti-inflammatory effects by suppress-
ing interleukin 1α and interleukin 1β in cultured
limbal epithelial cells [85] and preventing poly-
morphonuclear cells infiltrating into the corneal
stroma [86]. In addition, the basement mem-
brane and extracellular matrix components of
HAM, when used as a substrate, have shown
similar properties to conjunctival and corneal
epithelium [87, 88].
Human LSCs have commonly been expanded
ex vivo on human amniotic membrane for both
clinical and research purposes [70, 89, 90].
HAM shows low or no immunogenicity [91–94].
It is not a defined substrate and has some poten-
tial disadvantages, most importantly its biologi-
cal variability and the potential to carry or
transmit infections, but despite this, HAM
remains the gold standard and most widely used
substrate for the expansion of LSCs in vitro and
in clinical trials [56, 95, 96]. Consequently, there
has been a significant drive to better characterise
HAM in order to develop a better substrate for
the in  vitro expansion of LSCs [96–98]. One
such approach has been to decellularise HAM,
as this process can remove all cellular, poten-
tially immunogenic components from a tissue,
whilst preserving key extracellular matrix
(ECM) components and the basement mem-
brane, ensuring cell attachment and expansion.
The decellularisation process may create a more
consistent, defined substrate that does not elicit
an adverse immunological response in vivo [99–
101]. Several studies have suggested that LSCs
cultured on decellularised HAM expand more
quickly but are also more differentiated com-
pared to fresh frozen HAM [102–104]. The
improved cell proliferation has been attributed to
better cell adhesion to the substrate compared to
fresh frozen HAM, due to exposure of the base-
ment membrane, unobstructed by a devitalised
epithelial cell layer.
G. S. Figueiredo et al.
Decellularisation of human tissues has been
extensively investigated and applied to a wide
array of tissues for different clinical applications.
A wide range of agents have been used to decel-
lularise tissues, including sodium dodecyl sul-
phate (SDS) [99], dispase, thermolysin, trypsin,
ethylenediamine tetra-acetic acid (EDTA) and
ethanol, among others [95, 105]. In 2013,
Saghizadeh et  al. described a new, simpler and
faster decellularisation method that involved
using a cotton bud soaked in sodium hydroxide
0.5  M solution to rub the epithelial side of the
HAM to debride its epithelial cells in a period of
5–10 s [95]. It was demonstrated that the HAM
retained the native ECM structure following
decellularisation with sodium hydroxide; how-
ever, LSCs were not cultured on the substrate.
Decellularisation may better define HAM;
however, the risk of disease transmission remains
in a non-sterile substrate. With this in mind, Hogg
et al. demonstrated in 2015 that γ-irradiation is an
effective method of terminal sterilisation in the
production of decellularised skin dermis for
direct allogeneic transplantation [106].
A characteristic of HAM is its biological vari-
ability, both in thickness and, consequently, elas-
ticity [107, 108]. In 2012, Jones et  al. [109]
applied a theory first demonstrated in 2006 by
two separate groups that showed that human
mesenchymal stem cells are sensitive to substrate
rigidity and matrix elasticity which when altered
can give rise to specific stem cell lineages [110,
111]. Jones et  al. demonstrated that LSCs cul-
tured on uncompressed (i.e. with reduced stiff-
ness) collagen gels were less differentiated than
those cultured on compressed collagen gels
[109]. The same group then showed the differ-
ences in corneal stiffness, where in a healthy,
clear cornea, the central cornea is stiffer than the
peripheral cornea, which drives the migration
and differentiation of LSCs centripetally towards
the centre [112]. Molladavoodi et  al. supported
this finding when demonstrating that human cor-
neal epithelial cells had lower rates of migration
in compliant tissues [113]. It has therefore been
hypothesised that the mechanical properties
(stiffness) of the corneal anterior surface repre-
sent a major factor regulating corneal epithelium
6  Corneal Epithelial Stem Cells: Methods for Ex Vivo Expansion
homeostasis [112]. In particular, it has been pro-
posed that the mechanical environment of the
limbus (i.e. being more compliant) may be funda-
mental for the maintenance of LSC stemness,
whereas the stiffer matrix of the central cornea is
instrumental in driving centripetal cell migration
through durotaxis and inducing epithelial cell
differentiation [112]. Lepert et al. used Brillouin
microscopy as a reliable method of measuring
corneal stiffness and demonstrated similar find-
ings [114]. Chen et  al. demonstrated that
γ-irradiation can cross-link collagen-chitosan
scaffolds [115]. Mi et al. showed that UVA cross-­
linked plastically compressed collagen (PCC)
gels had a greater breaking force than uncross-­
linked PCC [116].
In our own experiment [117], comparing the
ex vivo culture method described here using four
different substrates, namely, fresh HAM, NaOH
decellularised HAM, SDS decellularised HAM
and γ-irradiated SDS decellularised HAM, we
were able to show that there was a significantly
more efficient rate of ex vivo LSC expansion on
decellularised HAM compared with fresh HAM,
with cultures grown on SDS decellularised HAM
reaching confluence fastest overall. Cultures on
SDS decellularised HAM preserved a greater
expression of putative stem cell (ΔNp63 and
ABCG2) and cell proliferation markers (Ki67)
and lower expression of markers of cell differen-
tiation (CK12 and CK13) than the limbal
explants cultured on fresh HAM or NaOH decel-
lularised HAM.  Gamma-irradiation enables us
to start with a sterile substrate and did not appear
to affect either the rate of ex vivo expansion from
limbal explants, the preservation of stem cell
properties in the cultured cells or the stiffness of
the substrate. Using Brillouin shift measure-
ments, we have shown that decellularised HAM
without γ-irradiation was found to have slightly
reduced stiffness compared to fresh HAM and
that cells cultured on decellularised HAM had
greater expression of stem cell markers. HAM
stiffness is not influenced by our decellularisa-
tion or sterilisation methods and, in turn, that
LSC differentiation does not appear to be influ-
enced primarily by substrate stiffness; however,
HAM stiffness did appear to affect cell ­migration
89
rate. Nevertheless, it must also be recognised
that the degree of stiffening achieved with
γ-irradiation may not be sufficient to influence
the behaviour of LSCs in culture. SDS decellula-
rised HAM may be more efficacious as a sub-
strate for the ex  vivo expansion of limbal
epithelial cells for use in clinical trials, and in
particular, the use of a γ-irradiated decellularised
HAM allows the user to start the manufacturing
process with a sterile substrate, making it poten-
tially safer [106, 117, 118].
6.3.4 Assessment of Culture Growth
6.3.4.1 Histology and Immunostaining
Histological analysis includes an assessment of
both the macroscopic and microscopic appear-
ances of the HAM and the ex vivo expanded epi-
thelial cells with haematoxylin and eosin staining,
including whether a monolayer of epithelium is
present, with a quantitative description of the
thickness of the epithelium and the presence or
absence of goblet cells. It also includes whether
there were any abnormal features relating to the
size of the cells and their nucleus or cytoplasm.
The cells are stained by immunocytochemistry for
putative limbal stem cell markers ΔNp63 (Dako
Ready-to-Use pre-diluted [Agilent Technologies,
Glostrup, Denmark]; antigen retrieval using Dako
high pH  8 target retrieval solution) and ABCG2
(clone BXP-21 [Chemicon, EMD Millipore,
Billerica MA, USA], used at a dilution of 1:50
with pH  6 retrieval using Bond ER1 [Leica
Biosystems, Wetzlar, Germany]), a marker of cor-
neal epithelium CK12 ([Abgent, San Diego CA,
USA], diluted 1:100  in 0.2% calcium chloride
buffer; trypsin retrieval at pH 7.8), two markers of
conjunctival epithelium CK13 ([Abcam,
Cambridge, UK], dilution 1:50, trypsin antigen
retrieval at pH 7.8) and MUC5AC (clone CLH2
[Novocastra, Newcastle-upon-Tyne, UK] dilution
of 1:100, with pH  6 retrieval using Bond ER1
[Leica Biosystems, Wetzlar, Germany]) and a
marker of stem cell proliferation Ki67 (Dako
Ready-to-Use pre-diluted [Agilent Technologies,
Glostrup, Denmark]; antigen retrieval using Dako
low pH  6 Dako target retrieval solution).
90
Visualisation of staining is with avidin-biotin
complex (ABC) (Vector Labs, Peterborough, UK)
with diaminobenzidine brown reaction product.
Taking the longest section of tissue, 100 nuclei
are then counted in the centre of the section, and
at either end (i.e. three fields in total), a number of
immuno-positive cells are noted and a percentage
of positive cells calculated for each section. An
average of the percentage positive cells is then
calculated and the proportion of cells expressing
each marker determined.
6.3.4.2 Measuring Explant Outgrowths
Limbal explant outgrowth expansion is marked
on the underside of the culture well every
2–3 days, at every medium exchange (Fig. 6.6).
At the termination of the culture, the sequential
outgrowth markings are transferred to an acetate
sheet, which is then placed over a graph paper to
calculate the area covered by the limbal explant
outgrowth by counting the number of small
squares and multiplying by 4 to give the total sur-
face area in mm2 covered by expanded cells on
the HAM. This is then plotted as a graph of the
total surface area of the outgrowth (in mm2)
against time in culture [119].
6.3.4.3 Cell Counting and Cell Viability
In cultures that are not to be used for transplanta-
tion, as soon as a confluent monolayer of ex vivo
Fig. 6.6  Colour photograph showing limbal explant in
culture on human amniotic membrane construct.
Outgrowth expansion was marked on the underside of the
culture well (arrow)
G. S. Figueiredo et al.
expanded epithelial cells covering at least 90% of
the HAM surface area was observed microscopi-
cally, the epithelial cells can be dissociated from
the HAM for counting and further investigation
with histological or polymerase chain reaction
(PCR) techniques. For this, the limbal explant is
mechanically removed from the HAM using
­sterile non-toothed ophthalmic micro-tying for-
ceps (Duckworth & Kent, UK), and the ex vivo
expanded epithelial cells and the underlying
HAM are washed twice with 2 mls DPBS, trans-
ferred to a 15 ml centrifuge tube and incubated
for 10 mins at 37 °C with 4 mls of trypsin 0.05%
solution (Gibco, USA). The trypsin is then inacti-
vated with 8 mls of LSC medium and the tryp-
sinised culture centrifuged for 3  min at 1300
RPM at room temperature. The inactivated tryp-
sin is then aspirated and the cells resuspended in
1 ml of DPBS. Twenty microlitres of the cell sus-
pension is transferred to a 1.5  ml cryo tube
together with 180 μl 0.04 % Trypan blue solution
for cell counting.
The number of viable cells in 10 μl is counted
in a dual-chamber haemocytometer (Scientific
Laboratory Supplies) under a light microscope.
Cell viability is assessed by the Trypan blue
exclusion test, where a 10 μl sample of the cell
suspension is mixed with 25  μl Trypan blue
0.04% (Sigma-Aldrich) and 15 μl DPBS (Gibco).
Cells that absorb Trypan blue solution do not
have intact cell membranes and are therefore
considered non-viable; viable cells have intact
cell membranes and therefore exclude the dye.
Blue, non-viable cells are excluded from the final
cell count.
6.3.4.4 Colony-Forming Efficiency
Assays
In cultures that are not to be used for transplanta-
tion, a colony-forming efficiency (CFE) assay is
a method of determining the ability of limbal epi-
thelial cells to form colonies, where a predeter-
mined number of cells are suspended in culture
over a fixed period of time and the final total
number of colonies derived from those initial
cells is counted.
Cells from confluent limbal explant cultures
are separated from the HAM by trypsinisation, as
6  Corneal Epithelial Stem Cells: Methods for Ex Vivo Expansion
described above. Five hundred cells are used in
CFE assays.
First, mitotically inactivated 3T3-J2 mouse
fibroblasts are suspended in 3T3 medium and
plated in a 9.6 cm2 tissue culture well at a final
density of 2.4 × 104 cells per cm2 and placed in
a tissue culture incubator at 37  °C overnight to
allow the establishment of a 3T3-J2 feeder layer.
The following day, 500 viable limbal epithelial
cells from each explant culture are plated onto
the prepared 3T3-J2 feeder layer along with 2 ml
of LSC medium. The culture is then placed in
an incubator at 37 °C and the medium changed
every 2–3  days. The CFE is measured on the
12th day of culture by removal of epithelial
medium followed by two brief irrigations with
DPBS.  The culture is then fixed in 3.7% form-
aldehyde (VWR International, UK) in DPBS for
10 min at room temperature. The formaldehyde
solution is then removed and the culture washed
with PBS. Colonies are then stained by incuba-
tion with 1% Rhodamine B (Sigma-Aldrich, UK)
in methanol for 10  min at room temperature.
Colonies are then counted under a dissecting
microscope (SMZ645, Nikon, Japan), and CFE
is calculated by the formula as follows: (number
of colonies/number of cells plated) × 100.
6.4 The Manufacturing
of a Corneal Epithelial Cell
Product for Autologous
Transplantation
6.4.1 Good Manufacturing Practice
In the past two decades, medicine has moved
more and more towards the development of
advanced therapy medicinal products (ATMPs),
with new, experimental products being devel-
oped, based on genes (gene therapy), cells (cell
therapy) and tissues (tissue engineering).
With this drive towards the development of
ATMPs came challenges as to how these prod-
ucts should be regulated to ensure public safety.
In view of these challenges, in 2007, Regulation
(EC) No. 1394/2007 of the European Parliament
and of the Council on Advanced Therapy
91
Medicinal Products was adopted [120]. The
regulation imposed strict rules on the develop-
ers of ATMPs to ensure the production of ATMPs
adheres to Good Manufacturing Practice (GMP)
principles. The World Health Organisation
(WHO) version of GMP guidelines is widely
used in the pharmaceutical industry and phar-
maceutical regulators. This set of practice guide-
lines encompasses the manufacturing, testing and
quality assurance of a product, to ensure that it is
safe for human consumption.
Good Manufacturing Practice is thus defined,
as “a system for ensuring that products are con-
sistently produced and controlled according to
quality standards. It is designed to minimize the
risks involved in any pharmaceutical production
that cannot be eliminated through testing the
final product”. GMP covers all aspects of pro-
duction from the starting materials, premises and
equipment to the training and personal hygiene
of operators. Detailed, written procedures are
essential for each process that could affect the
quality of the finished product. All materials,
production environments and relevant documen-
tation must be quality controlled, and there must
be systems to provide documented proof that
correct procedures are consistently followed at
each step in the manufacturing process every
time a product is made.
6.4.2 Quality Control
There are three phases of quality control for the
autologous limbal stem cell product.
1. Phase I
(a) Preparation of media: a 2 ml sample from
the basic limbal epithelial culture medium,
a 1 ml sample from the autologous serum
used in medium preparation and a 1  ml
sample from the complete limbal epithe-
lial culture medium are sent in blood cul-
ture bottles (BacT/ALERT®PF Plus,
bioMerieux, France) to a local microbiol-
ogy department.
(b) Preparation of HAM: the prepared HAM
is checked 24 h after plating to ensure it
92
remains attached at all edges and also to
ensure the medium remains clear, without
any sign of contamination.
2 . Phase II
(a) Cell culture: at day 0, the day of the lim-
bal biopsy, the culture medium is removed
from the HAM in culture, and 0.5  ml is
sent for mycology screening, whilst the
remaining medium is sent in a blood cul-
ture bottle as before to a local Department
of Microbiology. Finally, a further 1  ml
medium sample is sent in a blood culture
bottle 72 h prior to the product release.
(b) A macroscopic and microscopic examina-
tion of the LSC culture system is per-
formed 24 h prior to the expected release
of the product to ensure the product meets
the release criteria and that the medium is
clear and free of contamination.
(c) A further 1  ml of culture medium is
taken immediately prior to the release
of the product for retrospective quality
analysis by an independent microbiology
­laboratory for European Pharmacopoeia
standard sterility testing (Ph Eur 2.6.1.).
3. Phase III
(a) At the time of transplantation in the oper-
ating theatre, the posterior edge excess
product is sent to an external and indepen-
dent histopathology laboratory for histo-
logical assessment (haematoxylin and
eosin staining and transmission electron
microscopy), in addition to testing with
immunocytochemistry for the expression
of putative LSC markers p63 and ABCG2,
cell proliferation marker Ki67 and low
expression of CK12 (corneal) or CK13
(conjunctival) differentiation markers.
(b) A growth curve demonstrating the culture
growth is recorded for every culture
process.
6.4.3 Release of the Manufactured
Product
There are a set number of criteria the product has
to satisfy before it is deemed ready for release for
G. S. Figueiredo et al.
transplantation into the patient’s diseased ocular
surface with TLSCD:
1. ALSC must be shown to be at least 90% con-
fluent by visual assessment.
2. ALSC must be morphologically sound (an
evenly distributed layer of epithelial pheno-
type, i.e. small and regular with primitive
cytoplasm and large nuclei) macroscopically.
3. The donor must have negative results for all
infectious diseases screening from a blood
sample taken on the day of limbal biopsy.
4. Day 0 medium must be negative for aerobes
and anaerobes after 5  days BacT/ALERT
culture.
5. Day 0 medium mycology screen must be
negative.
6. A medium sample taken within 72 h of release
must be negative after 48  h BacT/ALERT
culture.
6.4.4 A
 utologous Limbal Stem Cell
Transplantation
Whilst the explant culture remains in the incubator
in the GMP facility after having been released for
transplantation, the procedure for ALSC transplan-
tation begins in the ophthalmic operating theatre.
This is often performed under general anaesthesia.
The initial part of the procedure involves perform-
ing a complete superficial keratectomy to excise
the superficial conjunctivalised scar tissue (pannus)
covering the patient’s own cornea and going well
beyond the limbus 360°. This excised corneal pan-
nus is analysed histologically for the presence of
vascularisation and inflammation, goblet cells,
keratinisation and metaplasia and is immunos-
tained for CK12 and CK13 to confirm that the epi-
thelial cells present are conjunctival in nature.
Once the pannus has been excised, the explant
culture is removed from the incubator in the GMP
facilities, carefully packed and transported by
hand to the ophthalmic operating theatre. There,
the product construct, consisting of HAM covered
in the epithelial monolayer, is unpacked and
handed over in sterile condition to the operating
surgeon and carefully removed from the culture
6  Corneal Epithelial Stem Cells: Methods for Ex Vivo Expansion
well and separated from the glass coverslips. It is
then placed onto the eye (epithelial side up), care-
fully lining up the explant biopsy at the 12 o’clock
position at the limbus of the patient’s diseased eye.
It is then sutured in place using four separate
Ethilon® nylon 10-0 suture (Ethicon, Scotland,
UK), and the excess product posteriorly is care-
fully dissected and sent for histological analysis as
a secondary quality control measure to confirm the
transplantation of non-­conjunctival corneal epithe-
lium containing LSCs. A second HAM (stroma
facing down) is placed over the culture HAM/
LSCs as a bandage, in effect sandwiching the
expanded epithelial monolayer between two HAM
sheets [70]. The second HAM is then sutured in
place using a combination of separate 10-0 nylon
sutures (Ethicon, UK) and a continuous 11-0 nylon
suture (Ethicon, UK), and a large bandage contact
lens (22 × 9.0 mm; Bausch & Lomb, UK) is placed
on the eye to protect the ALSC transplant and
HAM for at least 6–8  weeks. This method was
originally described by Kolli et al. [70].
All patients receive post-operative frequent
preservative-free topical steroid eye drops (pred-
nisolone acetate 1%), preservative-free antibiotic
eye drops (chloramphenicol 0.5% preservative-­
free) and preservative-free ASE (or AlloSE)
drops. All drops are gradually reduced and subse-
quently stopped within 12  months, with the
exception of ASE (or AlloSE) that is kept indefi-
nitely at least four times per day.
6.4.5 European Marketing
Authorisation
for the Production of Corneal
Epithelial Stem Cells
In 2015, the European Medicines Agency recom-
mended for Holoclar® (Chiesi Farmaceutici S.p.A.,
Parma, Italy) to be given approval for the marketing
authorisation for the production of an autologous
limbal stem cell product for patients with moderate
to severe LSCD due to ocular surface burns, defined
as eyes with corneal neovascularisation in at least
two quadrants, with central corneal involvement
and severely impaired visual acuity. This is the first
approved stem cell therapy product in Europe. In
93
the UK, this product was subsequently given
approval by the National Institute for Health and
Care Excellence (NICE) in August 2017 [121].
Compliance with Ethical Requirements Gustavo
S.  Figueiredo, Hardeep S.  Mudhar, Majlinda Lako and
Francisco C. Figueiredo declare that they have no conflict
of interest. All procedures followed were in accordance
with the ethical standards of the responsible committee on
human experimentation (institutional and national) and
with the Helsinki Declaration of 1975, as revised in 2000.
Informed consent was obtained from all patients for being
included in the study. No animal studies were performed
by the authors for this chapter.
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 Corneal Stromal Stem Cell:
Methods for Ex Vivo Expansion
Olena Al-Shymali, Jorge L. Alió del Barrio,
and James L. Funderburgh
7.1 Introduction: The Cornea,
Corneal Stroma, and Stem
Cells
The cornea is an important barrier between the eye
and the outer world; it represents a tough and well-
organized connective tissue. The transparent cor-
nea is a significant refractive element providing
two-thirds of the eye’s total refractive power. The
cornea consists of three cellular layers and three
basement membranes. The stratified squamous epi-
thelium is the outermost layer of the cornea limited
posteriorly by the Bowman’s membrane. The
innermost layer is represented by a single layer of
metabolically active endothelial cells in direct con-
tact with the anterior chamber aqueous and is sepa-
rated by the Descemet’s membrane from the
corneal stroma. Although each layer of the cornea
plays an indispensable role in its transparency,
organization, and function, the content of our chap-
ter will be mostly about the corneal stroma.
O. Al-Shymali
Vissum Corporation, Alicante, Spain
J. L. Alió del Barrio
University Miguel Hernandez, Vissum-Instituto
Oftalmologico de Alicante, Alicante, Spain
J. L. Funderburgh (*)
University of Pittsburgh, Department of
Ophthalmology, Pittsburgh, PA, USA
e-mail: jlfunder@pitt.edu
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_7
7
The corneal stroma is the centermost tissue
that makes up approximately 90% of the corneal
volume [1]. The corneal stroma is composed of
keratocytes interspersed in a unique extracellular
matrix (ECM) made of multiple lamellae of long
parallel collagen fibrils which spread throughout
the full diameter of the cornea. The uniform size,
organization, and tight packing of the stromal
collagen ensures corneal transparency. The latter
relies as well on the collagen fibril spacing regu-
lated by tissue-specific keratan sulfate and chon-
droitin sulfate proteoglycans in the ECM [2–6].
Three percent of the corneal stromal volume is
occupied by the keratocytes, neural crest-derived
mesenchymal cells. The number of dividing kera-
tocytes decreases after birth and eventually these
cells withdraw from the cell cycle and become
quiescent in adult mammals [7, 8]. However, fol-
lowing wounding, keratocytes adjacent to the site
go through apoptosis, and keratocytes peripheral
to the wound become mobile, mitotically active
fibroblasts [9–11] that repair the damage by
secreting scar tissue [12]. The fibrotic ECM
found in stromal scars cause incident light scat-
tering, thus resulting in visual impairment.
Millions of people worldwide suffer blindness or
deterioration of their visual function due to cor-
neal scarring [13–15].
Nowadays, keratoplasty in its different options
is the most commonly applied treatment for cor-
neal damage and scarring, especially in the devel-
oping world [16]. However, its limitations such
99
100 O. Al-Shymali et al.

as immune response reactions, donor tissue avail- adult stem cell marker ABCG2 [21, 22]. The lat-
ability, and central size-restricted transplants ter is an ATP-binding cassette transporter G fam-
open the way for the development of new proce- ily member and represents a specific marker for
dures such as the use of stem cells  which different kinds of stem cells such as neural,
have gained a lot of attention in the recent years. hematopoietic, cardiac, muscle, mesenchymal,
islet, and keratinocyte stem cells [23]. This trans-
porter protein is able to efflux the fluorescent dye
7.2 Characteristics of Corneal Hoechst 33342; thus adult stem cells expressing
Stromal Stem Cells (CSSC) ABCG2 are isolated as a “side population” (SP)
by fluorescence-activated cell sorting (FACS)
7.2.1 Progenitor Potential [24]. FACS works by separating a population of
of Stromal Cells cells into subpopulations depending on certain
fluorescence characteristics and patterns. In order
Adult keratocytes of the corneal stroma typically to have a sufficient amount of human stromal
adopt a fibroblastic morphology and produce a cells for FACS analysis, the primary cultures
scar-like ECM during their expansion in  vitro were expanded in a stem cell growth medium
[17]. However, it was shown recently that early-­ (SCGM), in which stem cells can proliferate
passage stromal cells mimic a nonfibrotic repair without losing their differential potential [23].
phenotype and have the potential to re-express the Eventually FACS allows the separation of an
characteristics of a differentiated keratocyte [18]. ABCG2-expressing cell population, corneal stro-
Funderburgh et al. [19] showed in an adult bovine mal stem cells (CSSC), from a heterogenous
model that corneal stromal cells have different population of cells.
capacities of transferring a keratocyte phenotype
after extensive proliferation and only 3% of them
were detected to grow clonally. This population of 7.2.3 Embryonic Origin of CSSC
cells shared some genes typical to mesenchymal
stem cells but not to keratocytes. Nevertheless, Although corneal epithelium is a derivative of
upon culturing in a low-mitogen media, these embryonic ectoderm, the endothelium and cor-
clonal cells developed dendritic morphology and neal stroma are derived from the neural crest.
expressed molecular markers of differentiated The expression of genes typical for descendants
keratocytes such as keratocan, aldehyde dehydro- of the neural ectoderm such as PAX6, Six2,
genase 3A1 (ALDH3 A1), and keratan sulfate. In Six3, and Notch1 by CSSC suggests a neural
addition to the elevated expression of mRNA for crest lineage for these cells. However, some
several gene characteristics of stem cells, the pro- authors proposed a bone marrow origin for all
genitor cells expressed elevated mRNA for genes, adult MSCs as bone marrow-derived cells have
expressed during ocular development including been detected in the corneal stroma [25]. Other
PAX6, Six2, Six3, and Notch1 [20]. The stable authors suggest a perivascular origin (pericytes)
progenitor phenotype of these stem-like cells has for MSC and other adult progenitor cells [26].
been proven by their sustention of keratocyte dif- This hypothesis is difficult to test in human
ferentiation potential after their expansion through CSSC because of the stem cell phenotypic plas-
more than 50 cumulative population doublings ticity. However, in a mouse model, Yoshida et al.
during consecutive subculture. [27] proved a neural crest origin for the multipo-
tent progenitor cell population in the stromal
limbus. These cells, analogous to human CSSC,
7.2.2 Primary Isolation of CSSC are CD45(−) supposing that they do not have a
in Humans bone marrow origin; however, they express
embryonic neural crest markers Twist, Slug,
The isolation of adult stem cells from the corneal Snail, and Sox9 [27]. As the CSSC and mouse
stroma was done using the well-documented stromal progenitor cells have similar character,
7  Corneal Stromal Stem Cell: Methods for Ex Vivo Expansion 101

we can insinuate that these stromal stem cells are Notch1, in addition to genes present in early cor-
not of perivascular or bone marrow origin, but neal development, PAX6 and Six2. CSSC
they are derivatives of ocular neural crest. also expressed genes associated with pluripotent
cells, SSEA4, SOX2, REX1, NANOG, KLF4,
and OCT4, and genes associated with neural
7.2.4 Mesenchymal Stem development such as NESTIN, NGFR, and
Cell Phenotype of CSSC CDH2 [20]. Yet, upon differentiation of CSSC,
they expressed high levels of keratocan,
The term mesenchymal stem cells (MSCs) was ALDH3A1, PTDGS, PDK4, and CXADR, all
introduced by Caplan in 1991 as a collective term genes highly expressed in keratocytes [35].
for nonhematopoietic stem cell populations [28].
Afterward, the Mesenchymal and Tissue Stem
Cell Committee of the International Society for 7.2.7 CSSC Differentiation
Cellular Therapy (ISCT) suggested minimal cri-
teria to define human MSC [29]. First, when 7.2.7.1 Differentiation into Non-ocular
maintained in standard culture conditions, MSC Cells
must be plastic-adherent. The second criterion is Human CSSC as with most MSC populations
the trilineage differentiation potential in  vitro. demonstrate a multipotent differentiation capac-
Lastly, MSC expressing stem cell markers ity. Upon culturing these cells in chondrogenic
CD105, CD90, and CD73, should lack the media, mRNAs for collagen II, aggrecan, and
expression of CD14, CD19, CD34, CD45, cartilage oligomeric protein (COMP), all
CD11b, CD79alpha, and HLA-DR surface mol- cartilage-­specific ECM molecules were detected.
ecules. MSC characteristics of cells isolated from Under these chondrogenic conditions, a signifi-
the human limbal stroma were reported by some cant amount of ECM was deposited and stained
authors [30–32]. Branch et al. [33] identified cor- strongly with toluidine blue, a characteristic
neal stromal cells according to the ISCT criteria resulting from proteoglycan accumulation typi-
as a source of MSCs. During their clonal expan- cal of cartilage. In a like manner, when incubated
sion, most of the CSSC expressed MSC markers in a medium reported to induce neurogenesis,
such as CD73, CD90, and CD166 and did not CSSC showed mRNA upregulation of glial fibril-
express the hematopoietic progenitor cell antigen lary acidic protein (GFAP) and neurofilament
CD34 [20]. protein [23]. The ability of CSSC to differentiate
to osteoblast, chondroblast, and adipocyte lin-
eages was demonstrated by conventional staining
7.2.5 CSSC Properties [33]. When CSSC were stimulated for adipogen-
esis, the transcriptions of the adipocyte markers
Like any MSC, CSSC exhibit clonal growth, self-­ PPARG, PLIN, and FASN were all significantly
renewal properties, and a potential for differenti- upregulated. Osteogenic stimulation of CSSC
ation into multiple distinct tissue types [23]. upregulated significantly the following markers
BMP-4 and BMP-6 and insignificantly
OPG. Similarly, upon chondrogenic stimulation,
7.2.6 CSSC Gene Expression CSSC demonstrated a significant upregulation of
COMP and ACAN.
Du et al. [34] used gene array technology to dis-
cover genes useful in distinguishing keratocytes 7.2.7.2 Differentiation into Keratocytes
and CSSC.  They identified a panel of genes
highly expressed in CSSC that are weakly In Vitro
expressed in keratocytes and vice versa. The Keratocytes when activated in acute wound heal-
CSSC included genes specific to MSC such as ing transfer to fibroblasts and secrete contractile
ABCG2, CXCR4, BMi1, CD166, cKIT, and ECM components such as α-smooth muscle actin
102
[36]. However, CSSC maintain the ability
to acquire a keratocyte phenotype. After incuba-
tion in a keratocyte differentiation medium
(KDM) that is a serum-free medium containing
fibroblast growth factor 2 (FGF2), CSSC upregu-
lated expression of mRNA for keratocan, a pro-
teoglycan core protein that in adults is uniquely
expressed in the corneal stroma. The same applies
for keratan sulfate, a glycosaminoglycan unique
in abundance in the corneal stroma, and
ALDH3A1, a protein present in elevated amounts
in keratocytes in vivo [23].
CSSC can be cultured in  different media
resulting in differentiation into keratocytes with
some variations in the produced ECM character-
istics depending on the cultures used (see Sect.
7.3.2).
In Vivo
One study demonstrated the ability of CSSC to
adopt keratocyte phenotype in vivo in a murine
model [37]. After the direct injection of human
CSSC into a mouse corneal stroma, human
corneal-­specific ECM accumulated, including
the proteoglycans keratocan and lumican, and
replaced mouse matrix components. The
injected human stem cells remained viable for
months, apparently having permanently become
quiescent keratocytes. Consequently, these
results suggest that keratocyte represents the
default lineage for the CSSC. The consequence
of this is that CSSC keep their stem/progenitor
character in vivo due to their limbal microenvi-
ronment; however when they enter the stroma,
the CSSC automatically differentiate into
keratocytes.
In a recent study, scaffold-free tissue engi-
neering methods were used, in which human
CSSC were cultured on substrates with aligned
microgrooves, which directed parallel cell align-
ment and matrix organization, similar to a native
corneal stromal lamella [38]. These cells pro-
duced cellular and collagenous tissue sheets that
were afterward transplanted into mouse corneal
stromal pockets. Subsequently, these engineered
corneal stromal tissues became transparent, and
the human CSSC persisted expressing human
corneal stromal matrix molecules [38].
O. Al-Shymali et al.
7.2.8 C
 orneal Limbal Stem Cell
Niche
7.2.8.1 Localization of CSSC
Based on the immunostaining for ABCG2 and
PAX6 proteins, CSSC were observed in the lim-
bus, a transitional zone between the cornea and
sclera [23]. These stained cells were found subja-
cent to the epithelial basement membrane in the
anterior stroma, in regions where the basement
membrane has ripples and folds (Fig. 7.1). These
anatomical features, named the palisades of Vogt,
form a niche for limbal epithelial stem cells
(LESC), a population of slow-cycling stem cells
[39]. The limbal stroma beside the palisades has
particular characteristics such as a blood supply
and melanocytes (Fig. 7.2). LESCs maintain the
self-renewing corneal epithelium. LESC defi-
ciency drives in-growth of conjunctival cells
across the cornea leading to its opacity and visual
loss. However, corneal transparency could be
restored in some individuals by the transplanta-
tion of corneal LESC either from collateral eyes
or from other individuals [40–42].
Fig. 7.1  Localization of the corneal stromal stem cells in
the limbus. PAX6-positive (red) and ABCG2-positive
(green) cells were observed in the human anterior stroma.
Arrows show stromal cell staining with both markers near
the folded region of the epithelial basement membrane
known as palisades of Vogt. The white bar represents
50 μm (Image from Du et al. [23], with permission)
7  Corneal Stromal Stem Cell: Methods for Ex Vivo Expansion
Fig. 7.2  Anatomical and cellular features of the stromal
stem cell niche. The section shows the anterior region of
the limbus. Epithelium is thickened over regions of undu-
lations in the epithelial basement membrane known as the
palisades of Vogt. Limbal epithelial stem cells (LESCs)
are localized in limbal basal epithelium. Unlike the central
cornea, limbal stroma is vascularized, containing melano-
cytes (black) and mesenchymal keratocytes (blue).
Corneal stromal stem cells (green) are located subjacent
to the basement membrane near LESCs (Image from
Pinnamaneni and Funderburgh [35] with permission)
7.2.8.2 LSSC-CSSC Interactions
Several studies have confirmed the presence of
limbal mesenchymal cells with stem cell proper-
ties that are closely associated in vivo with limbal
epithelial cells [32, 35, 43–48]. These  two cell
types were co-isolated in collagenase digests of
tissue from the limbal region [32, 44]. Both cell
types express stem cell genes but exhibit different
protein phenotypes, both in vivo and ex vivo [32,
44]. It is hypothesized that MSCs in this zone
(termed “niche cells”) help preserve the stem
phenotype of the LESC [30]. Different studies
demonstrated that LESC and limbal MSC express
N-cadherin, suggesting this cell-cell junction
protein provides interaction between these cell
populations [43, 45, 49, 50]. This interaction was
documented by Dziasko et  al. in a three-­
dimensional reconstruction of the LESC niche
using serial block-face scanning electron micros-
copy (SBFSE) [48]. These images showed long
processes of stromal cells extending through
focal basement membrane interruptions and
forming contact with basal epithelial cells.
The identity of the LESC-associated anterior
stromal cells as CSSC was confirmed by Basu
et al., as they isolated the epithelial-­mesenchymal
cell aggregates from mock biopsies with collage-
nase treatment [51]. Afterward, they compared
the mesenchymal cells from these isolates to
103
CSSC in terms of their expression of stem cell
genes, clonal growth, formation of spheres, and
differentiation ability to functional keratocytes.
Consequently, this study showed that in vitro, the
anterior limbal mesenchymal “niche cells” could
not be distinguished from the previously charac-
terized CSSC [51].
7.2.8.3 Function of CSSC
According to several studies, in the limbal niche
in vivo, CSSC ensure the ideal support system for
maintaining the active population of LESC [32,
43, 44, 52–57]. Upon the co-isolation of CSSC
and LESC by collagenase digestion, LESC showed
improved expansion and clonogenicity [43] as
well as the formation of more holoclones in the
presence of CSSC [44]. The disruption of the
reunion of these co-isolated cells led to reduced
sphere and holoclone formation by LESC [58].
Furthermore, LESC showed better expansion
when cultured with limbal CSSC than with mesen-
chymal cells from the central stroma [44]. These
studies and the anatomical closeness of these two
cell populations lend support to the idea that the
principal role of CSSC in vivo is the homeostatic
maintenance of the LESC in the limbal niche.
7.2.9 Immune Reactions of CSSC
The immune-modulatory properties of MSC and
their therapeutic effectiveness in immune-­
incompatible individuals were demonstrated in
some studies and clinical trials [59–61]. In addi-
tion to exhibiting characteristic of adult MSC,
CSSC can modify the cellular immune response.
Mouse corneal stroma in vivo was injected with
human CSSC on one hand and with human cor-
neal fibroblasts on the other. One month after
injection, the corneas injected with CSSC were
transparent, while those injected with fibroblasts
had a slight haze [37]. CSSC in the mouse cornea
did not experience a xenogeneic T-cell-mediated
immune response rejection; however, the injected
fibroblasts produced a notable increase in CD45+
cells after 1 week. Similarly, CD3+ T cells were
detected in fibroblast-injected corneas, but not in
CSSC-injected corneas, 2  weeks after injection
104 O. Al-Shymali et al.

[37]. Cytometric analysis of immune cells in the serum (FBS) or human serum (HS). CSSC in HS
cornea showed a fast influx of neutrophils into had significantly larger colonies compared to
the stroma of the mouse after both injections. Yet, clones in FBS [51]. CSSC cultured as a pellet in
it was transient in the cornea injected with CSSC a serum-free medium containing FGF2 differen-
and the tissue cleared from the cells within tiate to keratocytes exhibiting a characteristic
1  week. Still, in mouse cornea injected with gene expression pattern and accumulation of
human fibroblasts, the population of inflamma- ECM with tracts of aligned collagen fibrils, like
tory cells increased significantly [37]. Moreover, those seen in the stroma in vivo [34]. When cul-
limbal MSC cultured from both human and rab- tured on a substrate of aligned poly(ester-­
bit corneas showed the ability to suppress T-cell urethane-­ urea) (PEUU) nanofibers, CSSC
proliferation in  vitro [55]. These studies and produced layer of highly parallel collagen fibrils
results support an immunomodulatory function with uniform diameter and regular interfibrillar
for CSSC and the potential use of allogeneic spacing similar to that of native stroma [65].
CSSC in different cell-based or tissue-engineered CSSC incubation in PEUU in the presence of
therapeutic applications. both fibroblast growth factor 2 (FGF2) and trans-
forming growth factor-beta 3 (TGF-β3) resulted
in a more abundant multilayered lamellae with
7.3 CSSC Isolation and Culturing orthogonally oriented collagen fibrils, in a pattern
mimicking the human corneal stromal tissue [66,
7.3.1 CSSC Isolation from Limbal 67]. Moreover, CSSC showed to secrete highly
Biopsy organized stroma-like ECM by responding to a
specific pattern of topographical cues when cul-
Nowadays, limbal stem cells can be obtained tured in a silk or polycarbonate substrata with
from limbal biopsies of living human patients shallow parallel grooves [68–71].
[40, 62–64]. After obtaining the tissue samples, In a late study, authors compared the properties
they can be manipulated in two ways in order to of cultured CSSC in seven different media formu-
obtain the MSC (CSSC) [51]. The first consists of lations: two FBS-containing media, DMEM with
an initial removal of epithelial cells using the 10% FBS and M199 with 20% FBS; stem cell
neutral protease Dispase, and then stromal cell medium (SCM) with basic fibroblast growth fac-
dispersion is achieved by collagenase digestion. tor (b-FGF) and leukemia inhibitory factor (LIF);
The second approach consists of cell isolation a medium developed for the growth of endothelial
using only collagenase in which cells of both epi- cells (EGM); a serum-free medium (K-SFM); and
thelial and mesenchymal morphologies are pro- two media designed for the culture of hematopoi-
duced in the primary cultures. The epithelial cells etic cells, one semisolid (MethoCult) and one liq-
are lost after expansion, and cultures of homoge- uid (StemPro-34) [72]. The SCM medium was
nous mesenchymal cells are obtained. In fact, considered the most potential for cell therapy
limbal stem cells proliferated much faster while applications. Yet, as it was not completely free of
using collagenase-only isolation than the dispase/ animal-derived products (xeno-free), it was still
collagenase isolation [51]. not perfect to produce CSSC for clinical use [72].
However, in a more recent study, Matthyssen
et al. used DMEM as a basal medium for human-
7.3.2 CSSC Culturing isolated CSSC cultivation and recruited it with
four supplements: FBS, XerumFree (XF), human
After their isolation, CSSC can be expanded AB (HAB) serum, and human platelet lysate
without loss of differentiation potential in a low-­ (HPL) [73]. The supplements were added at three
glucose culture medium containing ascorbate, different concentrations, thus giving rise to 13 dif-
EGF, PDGF, insulin, and dexamethasone in addi- ferent culture conditions. The rate of expansion of
tion to a low level of serum, either fetal bovine CSSC was demonstrated to be significantly higher
7  Corneal Stromal Stem Cell: Methods for Ex Vivo Expansion
with 10% HPL medium compared to the others,
as it generated the best growth kinetics for CSSC
proliferation, in the same time preserving their
viability, cellular phenotype, and differentiation
potential. The xeno-free HPL supplement sur-
passed HAB, XerumFree, and FBS for human
CSSC expansion [73].
7.4 Confocal Microscopy
of Limbal Niche
Recently, Mathews et  al. characterized the
microarchitecture of the limbal niche in healthy
individuals using in vivo confocal microscopy
(IVCM) [74]. The limbal superficial epithelial
cells appeared as polygonal cells with a bright
nuclei and cytoplasm and a perinuclear dark
halo. Wing cells in the suprabasal layer, in con-
trast, displayed highly reflective well-demar-
cated cell borders and dark intracellular
regions.  Basal  cells in the palisades of Vogt
appeared hyperreflective with indistinguishable
a b
Fig. 7.3  Comparison of (a) in vivo confocal microscopic
image of superior limbus in a healthy subject with (b)
confocal microscopic image of immunostained [CD90-­
FITC (green), CD105-Alexa 633 (red), propidium iodide
(blue)] tangential cryosection of limbus from a deceased
donor. Unique hyperreflective (HR) structures were
observed in the anterior limbal stroma subjacent to the
basal epithelial cells by IVCM. The location and morpho-
105
borders. Subjacent to the basal cells,  dendritic
cells with bright cell bodies and processes were
noted. At a mean depth of 50.2 ± 8.7 μm in the
anterior limbal stroma clusters of hyperreflective
structures were revealed subjacent to the basal
epithelium [74]. Up to a mean depth of 98 ± 12.8,
these clusters continued in an individual manner
to form eventually a continuous nonhomoge-
neous structure with different brightness intensi-
ties (Fig.  7.3a). Deeper, they appeared to
surround blood vessels. Continuous hyperreflec-
tive linear structures were found in the interpali-
sade regions, extending from the clusters in the
anterior stroma. No such hyperreflective struc-
tures were observed upon the examination of the
central stroma using IVCM [74].
On examination of tangential sections of
cadaver limbus stained with hematoxylin-eosin,
highly compact clusters and a large number of
individual cells were seen in the anterior stroma,
extending into the interpalisade region [74].
Upon immunostaining for MSC-specific ­markers,
CD90- and CD105-positive cells were observed
logical features of these HR structures were similar to the
clusters of CD90- and CD105-positive mesenchymal stem
cells observed in donor limbal sections. LE limbal epithe-
lium, St stroma. IVCM image is 400 × 400 μm (Courtesy
of Saumi Mathews, Jaya Devi Chidambaram, Shruti
Lanjewar, Jeena Mascarenhas, Namperumalsamy
Venkatesh Prajna, Veerappan Muthukkaruppan, Gowri
Priya Chidambaranathan)
106
subjacent to the limbal basal epithelium and
between the interpalisade ridges during the
microscopic analysis of the tangential sections
[74] (Fig. 7.3b). Depending on the similarity in
the location, organization, and distribution of
both MSC clusters and the hyperreflective clus-
ters, it may be concluded that CSSC form these
hyperreflective clusters in the anterior limbal
stroma (Fig.  7.3). In patients with limbal stem
cell deficiency, during IVCM analysis of their
limbal stroma beyond the basal epithelium, the
hyperreflective niche structures were substituted
by bright fibrotic structures [75].
7.5 Conclusions
Corneal stromal stem cells are a recently discov-
ered source for autologous stem cells. They are
mesenchymal cells of neural crest origin. They are
located in the limbal niche where they insure the
homeostatic maintenance of the limbal epithelial
stem cells. CSSC are clonal growing cells with
self-renewal properties and a potential for differ-
entiation into multiple distinct tissue types. In
vitro, CSSC differentiate into functional kerato-
cytes that produce extracellular matrix compo-
nents similar to that of the human cornea. Since
CSSC display immunosuppressive properties,
their rejection risk should be minimal. Therefore,
they have a promising future in different cell-­based
or tissue-engineered therapeutic applications.
Conflict of Interest Olena Al-Shymali: no conflict of
interest
Jorge L. Alio del Barrio: no conflict of interest
James L. Funderburgh: no conflict of interest
Informed Consent  No human studies were carried out
by the authors for this article.
Animal Studies  No animal studies were carried out by
the authors for this article.
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 Corneal Endothelial Cells: Methods
for Ex Vivo Expansion
Stephen Wahlig, Matthew Lovatt,
Gary Swee-­Lim Peh, and Jodhbir S. Mehta
8.1 Introduction
The human cornea consists of three main cell
types: outer multilayered corneal epithelial cells,
a stromal layer containing keratocytes embedded
within a highly ordered array of collagen fibrils,
and a one-cell-layered corneal endothelium.
Descemet’s membrane, synthesized by the endo-
thelium, is situated between the stroma and endo-
thelial layer. The monolayered corneal
endothelium is composed of a mosaic of hexago-
nal endothelial cells, which serves a critical func-
tion in modulating fluid and nutrient transport
into the corneal stroma [1, 2]. The endothelium
also serves as a “pump,” generating an ionic gra-
dient to promote movement of excess fluid from
the stroma to the anterior chamber, thereby regu-
lating stromal hydration. Endothelial cell density
decreases with age, at an approximate rate of
0.6% per year for adults [3]. Despite this attri-
tion, corneal endothelium function is spared until
a critical cell density is reached, between 500 and
1000 cells/mm2 [4–6]. When excess endothelial
cells are lost, from hereditary dystrophies or iat-
S. Wahlig · M. Lovatt · G. S.-L. Peh
Tissue Engineering and Stem Cell Group, The
Academia, Singapore Eye Research Institute,
Singapore, Singapore
J. S. Mehta (*)
Singapore National Eye Centre, Department of
Cornea and External Disease, Singapore, Singapore
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_8
8
rogenic trauma, for example, the fragile dynam-
ics of corneal hydration may be disrupted. This
weakened functional capacity can result in stro-
mal edema and decreased transparency that will
eventually progress to corneal blindness if left
untreated [7, 8]. Corneal endothelial cells
(CEnCs) have limited ability to regenerate in vivo
in response to cell loss and instead utilize cell
enlargement and migration to maintain func-
tional integrity [9]. For this reason many endo-
thelial pathologies must be treated with
transplantation, making the cornea the most
transplanted tissue in the human body [10].
Despite corneal transplants having a high initial
success rate greater than 90% at 1  year, this
decreases to 74% and 64% at 5- and 10-year
intervals, respectively [11]. In addition, the
global supply of donor corneal graft material is
insufficient to meet the growing demand. From
2011 to 2015, the number of total US corneal
grafts increased 17%, while donation number
only increased 14.6% [12]. The most recent inter-
national data suggests there are 70 individuals
requiring surgery for every 1 transplant recipient,
with 12.7 million people currently awaiting
transplantation [13]. The need for alternative
therapies is readily apparent as donor shortages
become more pronounced in a rapidly aging
global population.
Cellular and regenerative therapies are an
attractive substitute for cadaveric transplants.
Although human corneal endothelial cells
109
110 S. Wahlig et al.

(HCEnCs) were initially thought to lack
­proliferative capacity, a report in 1979 first dem-
onstrated in  vitro endothelial cell proliferation
using an explant culture methodology [14]. Since
that discovery there have been steady improve-
ments in our ability to culture HCEnCs in vitro
while maintaining an appropriate endothelial
phenotype [7–18]. Preliminary analysis estimates
that a pair of donor corneas could yield up to 80
tissue-­engineered endothelial constructs, gener-
ating significant economic advantages over cur-
rent cadaveric transplants [19, 20].
Hence, a viable treatment alternative to reverse
the effects of corneal endothelial dysfunction
will first require a robust approach for the isola-
tion and subsequent expansion of primary
HCEnCs. This chapter will review the biology of
HCEnCs and the potential for in vitro expansion
of these cells. We will then discuss the develop-
ment of in  vitro culture protocols, including
HCEnC isolation techniques, culture conditions,
and characterization of endothelial cells after
expansion.
8.2  uman Corneal Endothelial
H existing pathology, drug use, cause of death, and
Cells
8.2.1 N
 ative Human Corneal
Endothelial Cells
EMT in HCEnC culture, it is essential to monitor
cellular phenotype during expansion. Beyond
standard microscopic observations of morphol-
ogy, endothelial phenotype is classically deter-
mined initially through the expression of
function-associated markers such as the Na+/K+
ATPase and ZO-1 tight junction protein [28],
although more specific cell markers such as cell
surface peroxiredoxin 6 (sPrdx-6) [29] have been
recently described and will be discussed in more
detail later in this chapter.
In addition to the deleterious effects of EMT,
there are significant known variations in the cul-
ture yield based on donor characteristics. One
particular donor factor is age: young donor cells
generally possess a greater proliferative capacity
than their older counterparts [25, 30]. Young
donor cells are also less prone to undergo EMT
during culture [15, 31]. One recent study was
able to successfully culture endothelial cells from
61% of donors younger than 30 but only 18% of
donors older than 60 [15]. This observation has
been partly explained by increased replicative
senescence in cells from older donors [32]. Other
causes of interdonor variability include pre-­
graft storage conditions [33–35]. Due to this vari-
ability, even a highly robust culture system will
yield significantly different quantities of HCEnCs
based on available donor corneas.

HCEnCs are arrested in G1 phase and do not

undergo active mitosis within the eye, due mainly 8.2.2 Progenitor Cells
to inhibition from cell-cell contacts [21–23].
However, current culture protocols, using a vari- 8.2.2.1 Existence of Endothelial
ety of media and growth factors, are able to induce Progenitor Cells
in vitro proliferation of isolated HCEnCs. The pri- HCEnCs are normally nonproliferative in  vivo,
mary challenge with in vitro endothelial culture is instead compensating for cell loss with migration
a tendency for HCEnCs to develop a fibroblastic and enlargement. While corneal limbal epithelial
morphology, a process termed endothelial-­ progenitor cells have been well-described and
mesenchymal transition (EMT) [24, 25]. These used for regenerative therapies [36, 37], such a
fibroblastic-looking cells lack the regular hexago- stem or progenitor cell population has not been
nal architecture and pump functionality of healthy clearly identified for the corneal endothelium. Yet
endothelium [4]. Multiple biochemical pathways some data suggests the existence of peripherally
have been noted to induce EMT across cell types, located HCEnCs with the ability to proliferate
including Wnt, Notch, and TGFβ; activation of in  vivo. In 1982 a unique cell subpopulation,
Wnt signaling, in particular, is correlated with termed “Schwalbe’s line cells,” was discovered in
EMT in HCEnCs [26, 27]. Given the relevance of the anterior trabecular meshwork (TM) bordering
8  Corneal Endothelial Cells: Methods for Ex Vivo Expansion
the corneal periphery [38]. Later work demon-
strated that laser stimulation of the TM caused a
fourfold increase in cellular division, with 60%
of the dividing cells located in the area of
Schwalbe’s cells [39]. Following this discovery
of anatomically distinct cells in the peripheral
cornea, a series of in  vitro studies offered evi-
dence of possible HCEnC progenitor cells. Stem
cell markers telomerase, alkaline phosphatase,
and nestin have been observed in the endothelial
periphery, while BrdU fluorescence assays dem-
onstrated baseline cell division activity near the
anterior TM [40–42]. From these observations, it
has been hypothesized that progenitor cells in the
corneal periphery could potentially proliferate in
response to endothelial damage, producing prog-
enies that migrate centrally toward the wounded
areas. This is supported by anatomical analyses
describing unique cell clusters in the extreme
periphery with adjacent cells positioned in cen-
tripetal rows [40]. In addition to this peripheral
cell subpopulation, neural crest-like cells have
drawn significant interest recently. Corneal endo-
thelial cells are developmentally derived from
neural crest [43]. One potential progenitor
HCEnC subpopulation defined by expression of
neural crest stem cell markers HNK-1 and p75
neurotropin receptor (p75NTR) has been discov-
ered in the transitional zone between peripheral
endothelium and trabecular meshwork [44, 45].
Morphological analysis of ex vivo HCEnCs has
identified another p75NTR-positive HCEnC sub-
population, which expresses stem markers LGR5
and PAX2 [46, 47]. However, since each of these
studies utilizes a different method for selection of
neural crest-like cells, there may be several dis-
tinct p75NTR+ HCEnC subpopulations within
the corneal endothelium. Additionally, while
these cells express stem cell markers and are
capable of proliferation in  vitro, their function
in  vivo is still unknown. To date, each of these
potential progenitor cells have yet to be isolated,
defined definitively, or clearly characterized.
8.2.2.2 Pluripotent/Multipotent Stem
Cells
In addition to precursors derived from corneal
tissue, other cell sources including human embry-
111
onic stem cells (ESCs) [45, 48, 49], mesenchy-
mal stem cells (MSCs) [50], and induced
pluripotent stem cells (iPSCs) [51] appear to pos-
sess the capacity for differentiation into HCEnC-­
like cells. These stem cells have been isolated
and are already being utilized in a variety of
human clinical trials in the fields of neurology,
rheumatology, and cardiology, among others
[52], unlike the undefined corneal endothelial
progenitor cells. They are also significantly eas-
ier to produce in large quantities compared to pri-
mary endothelial cells, avoiding the difficulties
of ex  vivo HCEnC culture. The challenge with
stem cells lies in defining optimal conditions to
direct differentiation of stem cells toward a cor-
neal endothelial phenotype. Use of lens epithelial
cell-conditioned medium (LECCM) induces
human umbilical cord blood MSCs to form an
HCEnC-like phenotype, migrating toward dam-
aged endothelial cells and forming a monolayer
[50]. However, this MSC-derived monolayer
does not resemble the regular hexagonal form of
native HCEnCs, highlighting the difficulty in
inducing a specific phenotype from multipotent
cells. One solution is to generate neural crest
cells, the embryological progenitor of HCEnCs
[43], as an intermediate cell type during the dif-
ferentiation process. Transformation of human
ESCs into neural crest-like cells can be induced
with SMAD inhibition, yielding cells positive for
the neural crest marker PAX6 [53]. Using this
SMAD inhibition technique, other groups have
developed protocols for further differentiating
these intermediary neural crest-like cells into
HCEnC-like cells that express endothelial mark-
ers ZO-1, Na+/K+ ATPase, and collagen 8A1 [48,
54]. Human ESCs have also been utilized in vivo;
a two-phase method using corneal stromal cell
co-culture and LECCM differentiated human
ESCs into HCEnC-like cells, capable of forming
a functional endothelial monolayer in a rabbit
model [49]. However, the restricted availability
and ethical controversies surrounding human
ESCs may limit future applications of these pro-
tocols [55].
Induced pluripotent stem cells (iPSCs) lack
these ethical concerns and may reduce the likeli-
hood of posttransplantation immune rejection
112
compared to autologous donor cells. Recently,
human iPSCs have been shown to differentiate
into neural crest cells, which then assumed an
endothelial phenotype upon ROCK and TGFβ
inhibition [51]. Although promising, this human
iPSC approach still requires validation in an ani-
mal model. Use of iPSCs may also be hindered
by the risk of tumorigenesis [56, 57] as well as
potential contamination with undesired cell types
through off-target differentiation [58].
Optimization of iPSC programming and differen-
tiation to address these issues is necessary before
clinical applications can be achieved.
8.2.3 HCEnC Isolation Protocols
The first step in establishing an HCEnC culture is
isolation of the corneal endothelial cells from
donor tissue. One approach is the explant tech-
nique. The endothelium and attached Descemet’s
membrane (DM) are cut into smaller pieces and
placed endothelial side-up in culture dishes,
wherein cells gradually migrate from the explant
to the culture surface [14, 59]. Another method
utilizes a scalpel or other surgical instruments to
directly scrape cells off the DM [60, 61]. HCEnCs
can also be isolated by a peel-and-digest method.
The DM and adhered endothelium are removed
from the cornea (Fig.  8.1) and then digested
either nonenzymatically in EDTA or enzymati-
cally in collagenase, dispase, or trypsin to isolate
the HCEnCs (Fig.  8.2) [7]. It should be noted
here that the DM/endothelium peeling process
must be approached with care as improper tech-
nique can result in detachment of stromal fibers,
which may in turn release keratocytes into cul-
ture during enzymatic digestion. Since kerato-
cytes have a higher proliferative index than
HCEnCs, they will overwhelm the culture with
fibroblastic cells. A vacuum suction holder can
be used to assist during the DM peeling, improv-
ing successful culture yield [62, 63]. Use of
L-valine-free media to selectively inhibit kerato-
cyte growth has also been described [64].
However, given the difficulty in eliminating
L-valine from the complex media needed for
HCEnC culture, this method is not generally pre-
S. Wahlig et al.
ferred. Since selective media is rarely used, isola-
tion of the DM/endothelial layer must be
performed by an experienced technician with
skillful, gentle manipulation to avoid stromal
contamination.
8.3 Endothelial Cell Culture
8.3.1 Growth Media
Selecting the appropriate culture conditions for
expansion of isolated HCEnCs is a difficult task
as evidenced by the numerous studies attempting
to find an ideal culture medium [15, 16, 24].
Inducing HCEnC expansion is a fine balance
between maximizing proliferation and maintain-
ing a functional polygonal monolayer with
appropriate cell-cell connections and pump
activity. The different stages of in vitro HCEnC
growth are illustrated in Fig.  8.3. Comparisons
of various culture media derived from different
basal media identified OptiMEM-I and Ham’s
F12/M199-­ based formulations as superior for
facilitation of HCEnC proliferation [24]. A criti-
cal component of these culture media is addi-
tives: the OptiMEM-I based media is
supplemented by nerve growth factor (NGF),
epidermal growth factor (EGF), chondroitin sul-
fate, and bovine pituitary extract (BPE), while
the Ham’s F12/M199 media is supplemented by
basic fibroblast growth factor (bFGF), insulin,
transferrin, and selenite [25, 65, 66]; both media
are supplemented with ascorbic acid and serum.
Analysis of these individual growth factors does
not always demonstrate a significant benefit;
NGF and EGF appear to have minimal effect on
endothelial proliferation [25, 65, 67]. BPE mod-
erately stimulates HCEnC growth, but this affect
is blunted in older donor corneas [25, 67].
Additionally, pituitary extract is composed of
unknown quantities of individual factors, which
is undesirable when attempting to create a well-
defined, robust culture platform. Although bFGF
does augment endothelial cell proliferation [68,
69], it also functions as a differentiation factor
that contributes to EMT through activation of
Wnt signaling [4, 27].
8  Corneal Endothelial Cells: Methods for Ex Vivo Expansion 113

a b

c d

e f

Fig. 8.1 Isolation of the Descemet’s membrane and
endothelium. (a) Cadaveric corneoscleral rim is placed in
a suction holder for the peeling process. (b) Trypan blue is
used to identify the margins of the endothelium; the blue
ring at the periphery is transition zone/extreme peripheral
endothelium that is not removed. (c) Dead endothelial
cells can be identified by blue staining (white arrow-
heads). (d) DM is carefully peeled off the stroma and
spontaneously scrolls. (e) Trypan blue stains exposed
stroma, illustrating the circle of removed DM. (f) The
removed DM/endothelium is collected in Liberase for
digestion

Among the nongrowth factor additives, chon- [59, 70]. Ascorbic acid, specifically L-ascorbic
droitin sulfate is a proteoglycan with known pro-­ acid 2-phosphate (Asc-2P), both inhibits EMT
proliferative effects on vascular endothelial cells and promotes HCEnC proliferation [69, 71]. The
that also enhances HCEnC growth and adhesion benefits of Asc-2P are thought to be due to
114 S. Wahlig et al.

a b

c d

Fig. 8.2  Digestion of the Descemet’s membrane/endo-
thelium. (a) Microscopic views of the scrolled DM shows
the monolayer of hexagonal endothelial cells. (b) After
45  min of Liberase digestion, the endothelial cells are
beginning to separate from the DM (marked by white
arrows). (c) By the fourth hour of digestion, nearly all
cells have separated from the DM, as indicated by the near
transparent DM. Many of the floating CEnCs can be seen
clustering together to form larger clumps (arrows). (d)
HCEnC clumps are partially broken up with TrypLE and
are subsequently seeded onto an adherent tissue culture
plate. Scale bar = 100 μm

p­ rotection from oxidative DNA damage [69] in
addition to upregulation of hepatocyte growth
factor production [71]. Insulin, transferrin, and
selenite each have a modest proliferative benefit
and allow for lower concentrations of animal
serum to be used; insulin in particular acts by
synergistically improving the growth-promoting
effect of bFGF [66]. However, while these media
additives can enhance HCEnC proliferation, this
benefit is often offset by a loss of endothelial
functionality through EMT [72].
In addition to growth factors, small molecule
media additives can be used for targeted modula-
tion of biochemical pathways thought to control
EMT. The most significant development has been
the use of the Rho-associated kinase (ROCK)
8  Corneal Endothelial Cells: Methods for Ex Vivo Expansion 115

a b

c d

Fig. 8.3  Morphology of cultured primary human endo-
thelial cells. (a) Elongated HCEnC phenotype during
early proliferation, 3 days after passaging (Donor age 3,
Passage 3). (b) Subconfluent HCEnC culture, partially
assuming a flattened polygonal morphology (Donor age 3,
P4). (c) Confluent HCEnC culture, demonstrating regular
polygonal monolayer (Donor age 3, P3). (d) Evidence of
replicative senescence, including intracellular vacuoliza-
tion and irregular morphology (Donor age 13, P3). Scale
bar = 100 μm

inhibitor Y-27632 to maintain HCEnC morphology An alternative method of balancing endothelial

during expansion. ROCK inhibition has been dem-
onstrated to enhance cell adhesion to prevent EMT
while simultaneously increasing proliferation and
suppressing apoptosis; this is thought to be due to
intracellular cytoskeleton modulation [17, 73].
Another major biochemical target for HCEnC
expansion is TGFβ, which is known to inhibit cor-
neal endothelial proliferation [74, 75] and promote
EMT, likely through Smad2/Smad3 signaling [76,
77]. The specific TGFβ receptor kinase inhibitor
SB431542 suppresses in vitro fibroblastic transfor-
mation of HCEnCs while maintaining expression
of ZO-1 and Na+/K+ ATPase [76].
proliferation and EMT is a dual-media expansion
protocol. This method initially uses a proliferative,
growth factor-supplemented media to expand the
HCEnCs. Once 80–90% confluence is reached, a
growth factor-free maintenance media is used to
prevent EMT prior to passaging [18]. Even after
the third passage, the HCEnCs continue to form a
hexagonal/polygonal monolayer and expressed
high levels of endothelial markers Na+/K+ ATPase
pump, ZO-1, GPC4, and CD200 [18, 78]. This
dual-media solution can be further optimized with
addition of a ROCK inhibitor, increasing HCEnC
yield by two- to threefold [79].
116
Translating these propagated HCEnCs into
clinical applications will require development of
a culture system adherent to good manufacturing
practice (GMP) guidelines. Research-grade
reagents like collagenase and trypsin must be
exchanged for GMP-compliant alternatives such
as Liberase™ and recombinant TrypLE™ Select.
In addition to substituting individual components
with GMP-grade equivalents, a continued con-
cern is the use of reagents that are animal-derived
and/or of undefined composition, specifically
bovine serum. Use of animal products increases
the risk of microbial contamination as well as
immunogenic complications, a potential obstacle
for applications in human transplantation [80].
Xeno-free growth conditions have been
described: one study comparing human serum
with bovine serum reported no differences in pro-
liferation of cultured HCEnCs [81], although
morphological variability has been noted [19].
Protein concentrates released from activated
human platelets can also serve as a bovine serum
substitute, producing morphologically normal
HCEnCs albeit with a reduced proliferative
capacity [82]. However, these xeno-free alterna-
tives have yet to surpass bovine serum in the
majority of published HCEnC culture protocols.
Further modifications to eliminate serum entirely
would also be desirable, as serum contains com-
plex and highly variable mixtures of assorted
proteins that can differ tremendously from batch
to batch [83]. Serum can also introduce microbi-
ological contaminants into cell culture [84].
Despite these concerns, serum has proven so crit-
ical to endothelial cell culture that it remains a
necessary component of HCEnC culture systems,
although use of GMP-grade serum products like
EquaFetal can minimize the risks described
above [19].
8.3.2 Culture Surfaces
In addition to culture media, there is a growing
interest around the role of culture substrates and
their role in modulating cell growth. Ideally, the
culture substrate should mimic Descemet’s mem-
brane in order to replicate the in  vivo environ-
S. Wahlig et al.
ment of corneal endothelial cells. A variety of
natural and synthetic substrates have been trialed,
including collagen IV, laminin, Matrigel, and
FNC Coating Mix, a proprietary reagent contain-
ing fibronectin, collagen, and albumin [25, 79,
85, 86]. All of these substances appear compati-
ble with HCEnC culture, although direct com-
parison between collagen IV, Matrigel, laminin,
and fibronectin noted that collagen IV is most
conducive to HCEnC adherence and expansion
[25]. A similar study demonstrated superior hex-
agonal morphology as well as ZO-1 and actin
expression with a collagen IV substrate [87].
More recently, the specific laminins in DM have
been identified as laminin-511 and laminin-521
[88]. These laminin isotypes induced superior
HCEnC adhesion, proliferation, and maintenance
of endothelial phenotype compared to collagen I
and fibronectin [88]. As collagen IV and laminin-
­511 and laminin-521 are both major components
of the human DM, these observations support the
hypothesis that materials similar to native DM
produce desirable culture results [89]. In addition
to the surface proteins used, adjusting the elastic
modulus of culture substrates to mimic the
mechanical properties of Descemet’s membrane
also improves endothelial morphology [87].
Many of these substrates are animal-derived,
and as mentioned above, this increases the risk
for infectious and immunogenic complications in
clinical applications [80]. Recombinant proteins
like laminin-511 and laminin-521 avoid this con-
cern, but are prohibitively expense [19]. One
potential xeno-free culture substrate is a pericel-
lular matrix from human decidua-derived mesen-
chymal cells (PCM-DM). This PCM-DM,
composed of collagen IV and fibronectin among
other proteins, has been shown to produce
HCEnC monolayers at a density even higher than
collagen IV or fibronectin alone [90]. Although
its derivation from human fetal membranes poses
a challenge for upscaling and reproducibility, the
potency and xeno-free nature of PCM-DM is
attractive for endothelial culture.
A further refinement of the culture substrate
has been the use of nano-topographical patterns
in the substrate surface, intended to mimic the
biophysical interaction between endothelial cells
8  Corneal Endothelial Cells: Methods for Ex Vivo Expansion
and the DM. Standard tissue culture polystyrene
(TCPS) patterned with 1 μm or 250 nm structures
has been shown to demonstrate increased cell
density, regular morphology, and upregulated
ZO-1 and Na+/K+ ATPase expression [91].
Specifically, the 1  μm pillar pattern produced a
threefold increase in proliferation, an impressive
improvement given the low-risk nature of pat-
terning culture substrates [91].
8.3.3 Functional Cell Markers
Typically, cultured HCEnCs have been identified
through detection of ZO-1 tight junction protein
and Na+/K+ ATPase expression in addition to direct
morphological visualization [28]. However, these
markers are primarily expressed while in a conflu-
ent monolayer and regress when the monolayer is
disrupted throughout the culture process [92].
Detection of bona fide endothelial cells is useful
both for quantifying the quality of propagated
HCEnCs and potentially for purifying cultures of
contaminating fibroblasts as discussed earlier. For
this reason, recent studies have attempted to gener-
ate a panel of defining HCEnC markers [78, 93,
94]. A number of endothelial surface markers have
been reported, including CD200 and GPC4 [78],
CD 166 [29, 94, 95], CD98 and CD340 [94], neu-
ral cell adhesion molecule (NCAM) [96], and
sPrdx-6 [29, 96]. Gene expression analyses have
also uncovered a set of genes overexpressed in
HCEnCs, such as SLC4A11, COL8A2, and
CYYR1 [93] and CLRN1, MRGPRX3, HTR1D,
GRIP1, and ZP4 [97]. Certain marker sets provide
additional phenotypic information; for example,
CD9, CD49e, CD44, and CD73 are reportedly
associated with fibroblastic HCEnCs [94], while a
signature of CD166+, CD105−, and CD44− is
thought to mark an endothelial subpopulation that
is particularly resistant to EMT [98]. These “nega-
tive” HCEnC markers are consistent with prior
characterizations, as CD73 is associated with lung
fibrosis, while CD44 is used to differentiate mature
fibroblasts from iPSCs [99, 100]. This also holds
true for the positive HCEnC markers; CD166, or
activated leukocyte cell adhesion molecule
117
(ALCAM), is highly expressed among vascular
endothelial cells [101], while GPC4 augments the
Wnt5a pathway that contributes to endothelial cell
migration [102, 103].
A more recent comprehensive transcriptomic
analysis highlights the importance of markers,
reporting significant differences in marker levels
between cultured HCEnCs and primary endothe-
lial cells [104]. For example, CD200 and GPC4
were mainly expressed in primary HCEnCs, while
SLC4A11 was present at significantly higher lev-
els in ex vivo cultured HCEnCs [104]. The differ-
ence in gene expression between cultured and
primary HCEnCs highlights the potential for cul-
ture protocols to influence endothelial phenotype
markers. Numerous variables including culture
media, donor age, and passage number may con-
tribute to differential marker expression among
functional endothelial cells. An exhaustive explo-
ration of the factors contributing to differential
marker expression is not realistic, so determina-
tion of a broader panel of endothelial markers
would be helpful for identifying functional
HCEnCs across culture protocols.
Along with discovery of endothelial markers,
there has also been progress in developing spe-
cific antibodies to these markers to isolate and
enrich HCEnCs. Monoclonal antibodies
TAG-1A3 and TAG-2A12 have been found to
identify HCEnCs, targeting CD166/ALCAM and
sPrdx-6, respectively [29]. Notably, TAG-2A12
bound only to HCEnCs (Fig. 8.4) and not to other
cells screened, including lung fibroblasts, human
ESCs, and neural crest cells. Unlike TAG-2A12,
commercially available Prdx-6 antibodies dem-
onstrate cross-reactivity with stromal fibroblasts,
highlighting the importance of the targeted epit-
ope and specific antibody used when attempting
to isolate HCEnCs using cell surface markers
[29]. While these results are encouraging, there is
still not a definitive array of HCEnC markers. As
mentioned above, the specific functional HCEnC
markers will likely vary depending on the culture
protocol. Standardized markers should be devel-
oped for each particular cell therapy protocol to
facilitate HCEnC enrichment and ensure quality
control.
118 S. Wahlig et al.

a b

Fig. 8.4  Immunofluorescence histology of marker proteins in endothelial cells. Primary cultured HCEnCs stain with
TAG-2A12 antibody (a) and express proteins ZO-1 (b) and Na+/K+ ATPase (c). Scale bar = 50 μm

8.4 Conclusion enticing prospect of endothelial-like tissue

The cornea is one of the most transplanted tissues
in the world, and demand continues to outpace
the supply of donor tissue. A cellular therapy,
composed of human corneal endothelial cells
expanded in vitro, is an intriguing potential solu-
tion to this problem. While human endothelial
cells are usually non-mitotic, newly developing
culture techniques can induce proliferation while
maintaining the native phenotype necessary for
function in vivo. Although there is not yet a stan-
dardized protocol for isolation and expansion of
HCEnCs, advances in cell extraction, culture
media, and specially designed culture substrates
have significantly improved culture yields. The
derived from stem cells, avoiding many of the
difficulties with primary HCEnC culture, is also
an object of future research. With continuous
improvement in these areas of in  vitro culture,
this is an especially exciting time for corneal
endothelial cell therapies.
Conflict of Interest  No conflicting relationship exists for
any author.
Financial Disclosure  No financial disclosures.
Compliance Statements  J. S. Mehta, M. Lovatt,
G. Peh, and S. Wahlig declare that they have no
conflict of interest. All procedures performed by
8  Corneal Endothelial Cells: Methods for Ex Vivo Expansion
the authors were followed and were in accor-
dance with the ethical standards of the responsi-
ble committee on human experimentation
(institutional and national) and with the Helsinki
Declaration of 1975, as revised in 2000. Informed
consent was obtained from all patients for being
included in the study. All institutional and
national guidelines for the care and use of labora-
tory animals were followed.
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 Corneal Regeneration: Use
of Extracorneal Stem Cells
Christian Claude Lachaud, Abdelkrim Hmadcha,
and Bernat Soria
9.1 Introduction
A major issue for regenerative therapies of dam-
aged or diseased tissues is identifying suitable
SCs able to recreate a functional tissue. Ideally,
this task should be performed with the use of SCs
of autologous and endogenous origin, thereby
avoiding immune rejection risk and ensuring
optimal regenerative outcomes [1]. This approach
is however currently unviable for most corneal
disorders, such as corneal dystrophies, where
endogenous stem and/or progenitor cells and
their differentiated cellular progenies are pre-
cisely affected or lacking or because effective
technologies based on their use are still not avail-
C. C. Lachaud · A. Hmadcha
Department of Cell Regeneration and Advanced
Therapies, Andalusian Center of Molecular Biology
and Regenerative Medicine-CABIMER, Junta de
Andalucía-University of Pablo Olavide-University of
Seville-CSIC, Seville, Andalusia, Spain
e-mail: christian.lachaud@cabimer.es
B. Soria (*)
Department of Cell Regeneration and Advanced
Therapies, Andalusian Center of Molecular Biology
and Regenerative Medicine-CABIMER, Junta de
Andalucía-University of Pablo Olavide-University of
Seville-CSIC, Seville, Andalusia, Spain
Centro de Investigación Biomédica en Red de
Diabetes y Enfermedades Metabólicas Asociadas
(CIBERDEM), Madrid, Spain
e-mail: bernat.soria@cabimer.es
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_9
9
able [2, 3]. For instance, the transplantation of a
donor’s corneal tissue is at present the only thera-
peutic option to regain vision for most corneal
disorders causing vision loss and blindness.
Unfortunately, the availability of donated ocular
tissues is however globally much lower than the
demand, a frustrating situation which has forced
the research of alternative approaches, such as
tissue engineering of artificial corneal tissues
with scaffolds and SCs.
SCs are unspecialized cells with the unique
ability to undergo asymmetric cell division. By
means of this mechanism, SCs can maintain their
self-renewal potential while generating differen-
tiating progenies [4, 5]. Embryonic SCs (ESCs)
are pluripotent SCs giving rise to the three germ
layers of the developing embryo. Upon an ade-
quate stepwise differentiation protocol, embryo-­
derived ESC lines can virtually give rise to any
differentiated cell type of the adult body, includ-
ing corneal cells [6]. Somatic SCs residing in
fetal and adult tissues have by contrast a more
restrictive differentiation potential in vivo, which
is committed to produce cellular components of
the tissues where they are housed, making them
cells with progenitor characteristics [7].
Cumulative evidence has however indicated how
adult SCs have a broader spectrum of differenti-
ating capacities than initially thought [8]. As
such, and under new in  vitro chemical environ-
ments, several types of adult SCs such as
­mesenchymal SCs (MSCs) are able to acquire
123
124
differentiated characteristics of neurons or hepa-
tocytes, among others, two cell types which are
derived from the embryonic ectoderm and endo-
derm germ layers, respectively [8, 9]. Such abil-
ity to switch their phenotype across distinct cell
lineages is known as transdifferentiation poten-
tial, a property which has attracted growing inter-
est for using adult SCs in the development of
novel therapies for corneal regeneration [10, 11].
To date, human embryonic and human-­
induced pluripotent SCs, as well as MSCs, are
the types of SCs which have been more broadly
applied for experimental corneal regeneration. In
slight contrast, fetal and adult stem/progenitor
cells have been in general more restrictively
applied to the regeneration of a single corneal
cellular layer (epithelium, stroma or endothe-
lium), principally on the basis of their phenotypic
similarities with the type of corneal cells to
regenerate. As such, epithelial SCs from different
extracorneal sources such as the skin or hair fol-
licle have been specifically applied for corneal
epithelium regeneration [12–14], whereas endo-
thelial progenitor cells of haematopoietic origin
have been tested for repairing the corneal endo-
thelium [15, 16].
We herein recover the different types of extra-
corneal SCs which have been experimentally
and/or clinically applied for regenerating the cor-
neal epithelium, stroma and endothelium layers
and detail their applications.
9.2 Corneal Epithelium
Regeneration
Different types of extracorneal SCs have been
proposed for corneal epithelium regeneration,
either because of their ability to differentiate into
cells with corneal epithelium cells characteristics
or indirectly due to their secretion of factors
inducing in situ corneal epithelium cells regen-
eration. They can be basically classified into four
categories: (i) pluripotent SCs (embryonic or
induced by artificial genetic reprogrammation),
(ii) epithelial stem/progenitor cells (oral mucus,
hair follicle, epidermis and amnion), (iii) MSCs
(umbilical cord, placenta, amnion, bone marrow,
C. C. Lachaud et al.
adipose tissue and gingiva) and (iiii) multipotent
SCs of neural crest origin (dental pulp and
follicle).
Table 9.1 shows extracorneal stem cells used
for corneal epithelial regeneration.
9.2.1 Pluripotent Stem Cells
9.2.1.1 Embryonic Stem Cells
In 2001, Yu et al. addressed for the first time the
ability of ESC to differentiate into corneal epithe-
lial cells (CEC) by using a Transwell™ coculture
system incorporating rabbit primary
CEC.  Authors indicated how soluble factors
secreted by the cocultured rabbit CEC could
induce mouse ESC to differentiate into epithelial-­
like cells with developed microvilli and tight
junctions and express the CEC markers cytokera-
tins 3 y 12 (CK3/12) [17]. Thereafter, Wang et al.
reported that D3 mouse ESC-GFP cells pre-­
induced with retinoic acid (RA) could form a cor-
neal epithelium-like tissue expressing the CEC
markers CK3 and p63 after their seeding onto
deepithelialized corneoscleral slices [18]. By rec-
reating a corneal niche microenvironment, human
ESC (hESC) cultured onto collagen IV-coated
tissue culture dishes containing conditioned
media by limbal fibroblasts could acquire epithe-
lioid morphologies and express CK12 and p63
[19]. Interestingly, a study reported how murine
ESC transduced with pax6, a transcription factor
critical for eye development, could efficiently
generate monolayered corneal epithelial-like
cells expressing CK12, E-cadherin and CD44
which could adhere and remain alive onto the
surface of injured mouse corneas [20]. The same
laboratory further reported almost similar results
with non-human primate ESC cells cultured onto
collagen IV-coated surfaces. In this case, the
authors reported their moderated capacity to gen-
erate a corneal epithelium-like tissue (positive for
pax6, CK3/12 and p63) onto injured mouse cor-
neas [21].
The importance of the corneal epithelium
niche environment to direct the differentiation of
ESC towards corneal epithelial-like cells was
further reinforced by other studies [22–24].
­
9  Corneal Regeneration: Use of Extracorneal Stem Cells 125

Table 9.1  Extracorneal stem cells used for corneal epithelial regeneration
SC type Main procedures Principal results References
ESC Transwell coculture with rabbit CEC Epithelial-like, exp° CK3/12 [17]
Preinduction with RA, seeding on Epithelium-like, exp° CK3 and p63 [18]
corneoscleral slices
Culture on collagen IV coating, CM by Epithelioid, exp° CK3 and p63 [19]
limbal fibroblasts
Transduction with pax6 Epithelial-like, exp° CK12, E-cadherin, CD44 [20]
Application on mouse injured corneal Integration on mouse injured corneal surface
surface
Culture on collagen IV coating Limited capacity to form an epithelium [21]
Graft on mouse injured corneal surface in vivo
Exp° CK3/12, pax6, p63
Spontaneously differentiated hESC seeded Expansion, formation multilayers [22]
onto Epithelial-like, exp° CK3, pax6
Bowman’s membrane of deepithelialized
human cornea
Use limbal epithelial SC (LESC) CM Generation LESC-like cells. Multilayers onto [23]
Seeding on APCM, implantation into rabbit APCM, restore transparency in vivo
LSCD model
Culture in KSFM with 7% CO2. Airlifting CEC-like, formation multilayered sheet [24]
culture
Diff° into CEC-like cells and CEnC-like Multilayered epithelium-like onto APCM [24]
cells Restore corneal transparency in rabbit
Generation corneal equivalent onto APCM
iPSC iPSC from human corneal limbal epithelium Diff° into CEC-like CK12, pax6 [25]
cells
Use of stromal inducing activity of PA6
fibroblasts
Coculture with corneal limbal stromal cells Diff° into CEC-like CK12 [26]
Media containing bFGF, EGF and NGF
Culture on collagen IV coating Diff° into CEC-like CK3/12, p63 [27]
Media with inhibitor TGF-β (SB-5051249
and Wnt (IWP-2) + bFGF
Partial reprogrammation into LESC-like Diff° into limbal epithelial cell-like CK3/12 [28]
cells
OMEC Culture onto denuded AM. Graft onto Restore epithelium and corneal transparency [29]
injured surface rabbit corneas
COMET in humans with LSCD Restore epithelium and corneal transparency [30]
COMET in human with Stevens-Johnson Restore epithelium and corneal transparency [31]
syndrome
Culture OMEC into SFM onto collagen IV Generation OMEC sheets [32]
coating
HFSC Culture in limbal fibroblasts CM, collagen Diff° into CEC-like CK12, exp° α6-integrin [13]
IV coating and CK15
Epithelial cell sheets onto fibrin gels
Transplantation in mouse injured surface Successful re-epithelialization [33]
corneas
SESC Culture onto denuded AM. Graft in goat Restore epithelium and corneal transparency [12]
injured cornea (LSCD)
HAEC Culture onto collagen corneal shields Adhesion onto third part of corneas [34]
Transplantation onto deepithelialized rabbit
corneas
Culture with CM by immortalized hCEC Diff° into CEC-like CK3/12 [35]
Airlifting culture onto deepithelialized Multilayered epithelium-like, exp° CK3/12 [36]
rabbit corneas
(continued)
126 C. C. Lachaud et al.

Table 9.1 (continued)
SC typeMain procedures Principal results References
CLEC Organotypic culture umbilical cord lining Formation stratified epithelium in vitro [37]
Cultivation onto denuded AM Formation stratified sheet of CK3/12 cells [38]
BM-MSC Cultivation onto denuded AM Graft adhesion and survival. Lower [39]
Transplantation injured corneal surface (rat) inflammation and neovascularization. No diff°
into CK3+ cells
Coculture system with rat CSSC. Graft in Acquire CK12 exp°. Graft reduces corneal [40]
rat LSCD model opacity and neovascularization
IV and IP injection in rat with injured Limited corneal homing of MSC. MSC [41]
corneal surface reduces corneal opacity and inflammation via
TSG-6
IV injection in mice with injured surface Corneal homing. Induces corneal epithelial [42, 43]
cornea regeneration
Culture onto denuded AM with Corneal healing, reduces corneal opacity [43]
KGF-2 + AS
Grafting onto injured corneal surface
Diff° and culture onto denuded AM with Increased CEC markers ABCG2 and p63 [44]
limbal epithelial CM. Grafting into LSCD Graft improve corneal regeneration
model
Growth on nanofiber scaffolds. Grafting Improves corneal healing. Reduces [45]
onto injured corneal surface (rabbit) inflammation, thickness, neovascularization
Seeding and culture onto nanofiber Graft restores antioxidant enzymes [46]
scaffolds
Grafting onto injured corneal surface
(rabbit)
Subconjunctival injection in a model of Diabetic corneal epithelial wound healing via [47]
corneal epithelial wound healing in type 1 TSG-6
diabetic mice
ADSC Adherent subculture Exp° p63, ABCG2, upregulated CK12 [48]
collagen IV coating, use of CM by hCEC Epithelioid, exp° CK12 [49]
Seeding onto PLA-based nanofibers Adhesion graft onto injured rabbit corneas [45]
scaffold
Topical application onto rabbit injured Adhesion onto cornea [50]
surface cornea Reduces neovascularization and corneal
opacity
HWJSC Culture and seeding onto fibrin-agarose Generation multilayered sheet onto scaffolds [51]
scaffolds Exp° CK3712, CX43, PKG, ZO-1
IDPSC Transplantation onto rabbit injured surface Reconstruction epithelium, exp° LESC and [52]
corneas CEC markers
hIDPSC sheets. Graft onto rabbit injured Reconstruction epithelium, exp° LESC and [53]
surface corneas CEC markers
Abbreviations: Diff differentiation, Exp expression, ADSC adipose-derived SCs, AM amniotic membrane, ALDH1A1
aldehyde dehydrogenase 1 family, member A1, ALDH3A1 aldehyde-3-dehydrogenase 1 family, AS autologous serum,
bFGF basic fibroblast growth factor, BM-MSC bone marrow mesenchymal SCs, CEC corneal epithelial cells, CLEC
cord lining epithelial cells, CM conditioned medium, COMET cultivated oral mucosa epithelial transplantation, DPSC
dental pulp SCs, ESC embryonic SCs, HAEC human amniotic epithelial cells, HFSC hair follicle SCs, HSHS platelet-­
poor horse serum, HWJSC human Wharton’s jelly SCs, IGF-1 insulin-like growth factor 1, iPSC immature dental SCs,
LESC Limbal epithelial SCs, LSCD limbal stem cell deficiency, KS keratan sulphate, OMEC oral mucosa epithelial
cells, PEA poly-(ethyl acrylate), PLGA polylactic-co-glycolic acid, RA retinoic acid, SESC skin epidermal SCs, SFM
serum-free media, TGF-β1 transforming growth factor beta 1, UC-MSC umbilical cord mesenchymal SCs

Indeed, spontaneously differentiated hESC 3–4 stratified layers of pax6+/CK3+ CEC-like

seeded onto the Bowman’s membrane of deepi- cells [22]. Additionally, the use of a culture media
thelialized human cornea buttons were shown to conditioned by limbal epithelial SCs (LESC) was
successfully expand and establish a differentiated shown to be enough by itself to direct hESC
9  Corneal Regeneration: Use of Extracorneal Stem Cells 127

towards LESC-like cells [23]. Their subsequent thus suggesting that their specific epigenomic
seeding onto an acellular porcine cornea matrix status might be responsible of their higher pro-
(APCM) could generate a stratified epithelium pensity to differentiate along the CEC lineage
with basal cells retaining LESC markers. [25].
Transplantation of this neo-corneal tissue graft By replication of the limbal epithelial stem
into a rabbit model of LSCD could restore the cell niche, Yu et  al. reported the generation of
ocular surface damage and prevent corneal neo- CK12+ CEC-like cells from mouse iPSC cocul-
vascularization [23]. Zhang et al. recently gener- tured with limbal stromal cells in a media supple-
ated hESC-derived corneal epithelial progenitor mented with a mix of bFGF, EGF and NGF [26].
cells (CEPC) through culture into a mixture of Mikhailova et al. could efficiently direct human
DMEM/F12 and keratinocyte serum-free medium iPSC into p63+ and CK3/12+ CEC-like cells,
(KSFM) (1:1) under distinct carbon dioxide through sequential culture onto collagen
(CO2) concentrations. The concentration of 7% IV-coated surface, followed by their suspension
of CO2 yielded highest numbers of ABCG2+ and culture in a media containing two small mole-
p63+ CEPC. Purified hESC-derived CEPC could cules (TGF-β inhibitor SB-505124 and Wnt
efficiently generate multilayered epithelioid cells inhibitor IWP-2) and bFGF and a final matura-
by airlifting culture [24]. In the same year, and tion step into the corneal epithelial media CnT-30
using the same protocols as previously published [27]. Of special interest, Cieslar-Pobuda et  al.
[23, 54], Zhang et al. tested the functionality of generated CK3/12+ limbal epithelial cells from
their hESC-derived CEPC to engineer a full-­ induced LESC-like cells by partially reprogram-
thickness corneal equivalent by coculturing them mation of HDFs with three limbal-specific tran-
together with hESC-derived corneal endothelial-­ scription factors: TCF4, CEBPD and ΔNp63α.
like cells onto an APCM. Of note, CEPC gener- Such reprogrammation shortcut avoiding a com-
ated onto the APCM a multilayered corneal plete return to the pluripotent step is suggested to
epithelium equivalent with cells expressing the circumvent the potential teratoma formation
corneal epithelium markers ABCG2+ and CK3+ associated with the use of iPSC-derived corneal
cells. Of particular interest, the recellularized epithelial-like cells [28].
APCM corneal substitute increased progressively
their transparency over the 8 weeks of follow-up
after transplantation in rabbit eyes, thus indicat- 9.2.2 Epithelial Stem Cells
ing the functionality of the hESC-derived neo-­
epithelium and endothelium [55]. 9.2.2.1 Oral Mucus
The oral mucus is a non-keratinized stratified
9.2.1.2 Induced Pluripotent Stem Cells squamous epithelium displaying morphostruc-
Hayashi et  al. reported the first generation of tural and phenotypical similarities with the cor-
iPSC from both human adult dermal fibroblast neal epithelium. As such, Nakamura et  al.
(HDF) and human adult corneal limbal epithelial published in 2003 a pioneer study reporting the
cells (HLEC) by using the retrovirus-mediated cultivation of oral mucosal tissue biopsies onto a
transfection of Yamanaka’s four factors [56], denuded amniotic membrane (AM) as carrier
namely, Oct3/4, Sox2, c-Myc and Klf4 [25]. [29]. The re-epithelialized AM could success-
Colonies of pax6+/CK12+ CEC-like cells could fully graft onto injured corneas of rabbits pro-
be obtained by subjecting both HDF- and HLEC-­ duced by lamellar keratectomy and generate a
derived iPSC to a stromal cell-derived inducing corneal neo-epithelium substitute re-establishing
activity differentiation method based on the sec- corneal transparency [29]. Soon after, in 2004,
retome activity of mouse stromal cells (PA6 Nishida et al. generated autologous cultured oral
cells). Significantly better corneal epithelial dif- mucosa epithelial cell (OMEC) sheets from
ferentiation was however achieved with the patients with bilateral LSCD, by culturing OMEC
HLEC-derived iPSC, which still retained a small into temperature-responsive cell-culture inserts
proportion of differentially methylated regions, placed into larger culture plates containing 3T3
128
feeder cells. Transplantation of OMEC sheets
onto their corneas’ ocular surface has shown to
successfully restore their corneal epithelium and
cornea transparency [30]. This landmark clinical
study was further followed by the report of the
successful restoration of corneal functionality in
patients with Stevens-Johnson syndrome and
chemical eye injury after a sequential transplan-
tation of cultivated oral mucosal epithelial trans-
plantation (COMET) and penetrating keratoplasty
(PKP) [31]. Of special interest, a recent study
reported the generation of tissue-engineered oral
mucus epithelial cell sheets in a xenogeneic-free
culture system for potential application into ocu-
lar surface surgery. The best protocol consisted in
generating epithelial cell sheets onto collagen
IV-coated culture inserts in a chemically defined
media previously conditioned by inactivated
human foreskin fibroblasts and containing 10 ng/
ml recombinant EGF [32].
9.2.2.2 Hair Follicle
In 1990, Costarelis et al. identified a population
of slow-cycling SCs in the bulge area of the hair
follicle [57]. These follicular bulge epithelial SCs
were further shown to contribute to the epidermis
regeneration during wound healing [58–60].
Later on, Blazejewska et al. reported for the first
time that hair follicle SCs (HFSC) could transdif-
ferentiate in vitro into corneal epithelial-like cells
upon exposure to limbal environment [13]. Of
particular interest, the authors reported formation
of holoclones (stem cell-derived) only from cells
of the bulge area. HFSC obtained either by enzy-
matic digestion or explant culture of the bulge
area were expanded onto 3T3 feeder cells.
Subcultivated HFSC were found to form regular
epithelioid cell layers onto collagen IV- and
laminin-­5-coated surfaces, but not onto laminin 1
or fibronectin. Additionally, HFSC could be effi-
ciently differentiated into CK12+ CEC-like cells
when cultured in a media conditioned by limbal
fibroblasts, but not by peripheral corneal or 3T3
fibroblasts. HFSC-derived CEC-like cell sheets
generated onto fibrin gels precoated with lam-
inin-­5 displayed morphostructural hallmarks of
the native corneal epithelium, with basal cells
expressing the epithelial SC markers α6-integrin
C. C. Lachaud et al.
and CK15 and superficial squamous epithelioid
cells expressing dermal (CK10) and corneal
(CK12) differentiated markers [13]. A more
definitive evidence of the re-epithelialization
potential of HFSC for the corneal epithelium was
further reported in a study where a fibrin gel car-
rier seeded with mouse HFSC was transplanted
onto the injured ocular surface of mouse corneas
from which were removed their limbal and cor-
neal epithelium [33]. Altogether, these studies
therefore identify HFSC as strong candidate cells
for being applied to human LSCD.
9.2.2.3 Epidermis
The skin epidermis is a complex multilayered
stratified epithelium with a strong regenerative
potential provided from a pool of skin epidermal
SCs (SESC) residing in the basal layer. Howard
Green’s laboratory established the first successful
long-term in vitro expansion of human epidermal
keratinocytes obtained from organotypic culture
of epidermis biopsies onto inactivated 3T3 embry-
onic fibroblasts [61]. This landmark study laid the
basis for the successful grafting of in vitro gener-
ated skin substitutes onto massive full-­thickness
burn wounds in humans [62]. Of interest, the skin
and corneal epithelium share common lineage
origin, the basal layer of the embryonic skin, and
their respective SCs, namely, epidermal and lim-
bal epithelial SCs, are both two types of keratino-
cyte SCs [10]. In line of their phenotypic
similarities, Yang et  al. demonstrated that SESC
isolated from goat skin and cultured onto denuded
human AM could restore a transparent corneal
epithelium after transplantation onto the damaged
corneal surface of goats with total LSCD [12, 63].
9.2.2.4 Amniotic Membrane
The amniotic membrane encloses the amniotic
cavity housing the foetus. Its inner side in contact
with the amniotic fluid is a continuous layer of
flattened cells of ectodermal origin, the amniotic
epithelium. Different studies have suggested that
human amniotic epithelial cells (HAEC) repre-
sent putative extracorneal SCs for corneal surface
regeneration. In this way, He et  al. reported for
the first time in 1999 the feasibility of achieving
corneal epithelium repair with HAEC. This was
9  Corneal Regeneration: Use of Extracorneal Stem Cells
done by seeding and culturing HAEC onto the
concave surface of collagen corneal shields. The
cellularized shields were finally transplanted
onto the deepithelialized surface of rabbit cor-
neas. HAEC were found to adhere and be retained
onto the third part of the corneas transplanted
[34]. Of interest, Yao et al. further reported that
HAEC cultured into a conditioned media secreted
by spontaneously immortalized hCEC [64] could
differentiate into CK3/12+ CEC-like cells [35].
Fatimah et  al. also reported how subcultured
HAEC strongly express CK18 and β1 integrin
and how their expression of CK19 and CK3
increased upon subculture in a normal media
containing 10 ng/ml EGF, while in turn they lost
their expression of the stem cell marker p63.
Zhou et al. recently reported how the seeding and
airlifting culture of HAEC onto the Bowman’s
membrane of deepithelialized rabbit corneal
stroma could generate 4–5 stratified layers of
CK3/12+ cells mimicking the native corneal epi-
thelium. Transplantation of the HAEC-corneal
stroma onto deepithelialized rabbit cornea could
restore corneal transparency and a normal cor-
neal epithelium within 4 weeks [36].
9.2.2.5 Umbilical Cord Lining
Epithelium
The umbilical cord lining is a multilayered epi-
thelium [65]. An initial report described how
umbilical cord lining epithelial cells (CLEC)
were able to form a stratified epithelium upon
organotypic culture [37]. Reza et  al. cultivated
human CLEC onto a denuded AM carrier. They
report their initial expression of SC markers
(HES1, ABCG2, BMI1, CK15) and their ability
to adhere, grow and form a stratified sheet of
CK3/12+ CEC-like cells. Transplantation of the
neo-epithelial tissue onto rabbit eyes with limbal
stem cell deficiency restored a normal clear cor-
neal surface, showing the promise of this cell
type for ocular surface repair [38].
9.2.3 Mesenchymal Stem Cells
Mesenchymal SCs (MSCs) are multipotent
mesodermal progenitors residing in vascularized
129
connective tissues, principally at the perivascular
niche [66], and as such, they can be harvested
from virtually all vascularized tissues and organs
[67, 68]. Reports in the late 1980s described the
ability of mesenchymal cells to acquire simple
epithelial markers (CK18 and CK19) upon
in vitro culture into serum-containing media [69].
Of interest, the process of mesenchymal-to-­
epithelial transition (MET) was shown to be nec-
essary for the reprogrammation of adult
fibroblasts into iPSC [70]. Whether adult MSC
possesses such transdifferentiative ability and
can convert into corneal epithelial cells has
attracted great interest for corneal surface regen-
erative therapies. To date, MSCs of distinct origin
have been tested, including the bone marrow, adi-
pose tissue and fetal tissues.
9.2.3.1 Bone Marrow MSC
Bone marrow-derived MSCs were the first type
of MSC investigated for corneal regeneration.
Ma et  al. in 2006 reported that a denuded AM
seeded with human BM-MSC could induce the
re-establishment of a corneal epithelium-like tis-
sue onto injured rat corneas. BM-MSC survived
onto the allograft and could inhibit neovascular-
ization and inflammation, although they failed to
transdifferentiate into CK3+ corneal epithelial-­
like cells [39]. Further studies also reported the
ability of BM-MSC to acquire some of the CEC
markers (CK3/CK12, beta1-integrin, C/
EBPdelta, ABCG2 and p63) upon directed differ-
entiation culture and their ability to induce the
regeneration of the corneal epithelium in animal
models of ocular surface epithelial injury [40–47,
71–73]. A detailed analysis of the studies pub-
lished so far clearly demonstrated how BM-MSC
can accelerate the healing and regeneration of the
injured corneal epithelium. However, the diver-
gent outcomes published so far question whether
such regenerative mechanism is directly medi-
ated through their transdifferentiation into CEC
or is instead rather indirectly mediated by their
secretion of anti-inflammatory and regenerative
factors sustaining endogenous corneal epithe-
lium regeneration [11].
In support of their indirect contribution are
several reports indicating how BM-MSC stimu-
130 C. C. Lachaud et al.

late corneal epithelium regeneration through how topically transplanted human ADSC could
secretion of transforming growth factor beta 1 adhere and migrate onto injured corneas and
(TGF-β1) [73] and tumour necrosis factor-­ reduce inflammation and fluorescein positive epi-
inducible gene 6 protein (TSG-6), being this later thelial defects. ADSC could partially revert the
able to induce pro-inflammatory macrophages course of ocular surface neovascularization and
(M1) to adopt an anti-inflammatory phenotype opacity in the partial LSCD model [50].
(M2) [41, 47]. Additionally, BM-MSC transplan-
tation onto injured rabbit corneas after alkali 9.2.3.3 Fetal Tissue MSC: Placenta,
burns has also shown to reduce the expression of Amniotic Membrane
pro-­inflammatory and angiogenic markers and Umbilical Cord
(iNOS, MMP9, alpha-SMA, TGF-β1 and VEGF) The accessibility of discarded fetal tissues such
in subepithelial corneal stromal cells [46]. as fetal placenta, umbilical cord or amniotic
membrane, from which can be harvested clini-
9.2.3.2 Adipose Tissue MSC cally relevant numbers of MSC, has attracted
Several studies addressed the potential of human great interest for regenerative therapies of cor-
subcutaneous adipose tissue-derived MSC (usu- neal disorders. In this way, Garzón et al. isolated
ally termed h-ADSC) to acquire CEC character- human Wharton’s jelly SCs, which are MSC in
istics in  vitro [48, 49] and/or to induce corneal nature [75]. They report how their seeding and
epithelium regeneration in animal models of ocu- culture at high density onto a fibrin-agarose scaf-
lar surface injury [45, 46, 50, 74]. Martinez-­ fold could led to the formation of a sheet of 4–5
Conesa et  al. reported how early cultured layers of cells expressing a set of markers
h-ADSC express the SC markers p63 and ABCG2 [CK3/12, connexin 43 (CX43), plakoglobin
and upregulate their expression of CK12 upon (PKG) and zonula occludens 1 (ZO1)] consistent
time culture, suggesting they possess CEC differ- with CEC [51]. Of special interest, Nam et  al.
entiation capacity [48]. In this way, h-ADSC cul- isolated MSC from human placenta and umbili-
tured onto a collagen IV-coated surface and into a cal cord and expanded them initially in a mini-
conditioned media (CM) by human CEC were mum essential medium-alpha +10% fetal bovine
found to undergo a phenotypical switch into epi- serum. Serum-free conditioned media by P-MSC
thelioid cells expressing CK12. Such transdiffer- or UC-MSC were then produced and found to
entiation however didn’t occur when using limbal exert clonal growth expansion of human limbal
fibroblast CM [49]. In their work, Holan et  al. epithelial cells through secretion of the matrix
established cultures of rabbit limbal epithelial molecule protein transforming growth factor-­
SCs (LESC) and ADSC, which were then seeded beta-­induced protein (TGF-βIp) [76].
for separately onto poly(L-lactic) acid (PLA)-
based nanofibers laminar scaffolds. Of interest,
they report how both LSC and ADSC scaffolds 9.2.4 Immature Dental Pulp Stem
could adhere onto the injured surface of rabbit Cells
corneas and promote corneal epithelium regen-
eration and restore corneal transparency with Vascularized tissues of the oral cavity have been
similar efficiency [45]. The same laboratory fur- identified as a rich source of SCs with MSC char-
ther described how the ADSC scaffolds trans- acteristics. Oral sources for isolating MSC
planted onto injured rabbit corneas led to a include the gingiva, periodontal ligament, devel-
restoration of antioxidant enzymes in the regen- oping tooth (papilla and follicle) and exfoliated
erated epithelium, as well as to a downregulation deciduous teeth (pulp) [77, 78]. Few studies have
of pro-inflammatory and angiogenic markers addressed the potential of oral MSC for corneal
(iNOS, MMP9, alpha-SMA, TGF-β1 and VEGF) epithelium regeneration. Monteiro et al. isolated
[46]. Galindo et al. developed models of partial human immature dental pulp SCs (hIDPSC, also
and total LSCD in rabbits. The authors report termed SHED) from exfoliated teeth [52]. The
9  Corneal Regeneration: Use of Extracorneal Stem Cells
hIDPSC were shown to express MSC and ESC
markers. Of interest, undifferentiated hIDPSC
also expressed the limbal SC markers p63 and
ABCG2. The healing potential of hIDPSC was
evaluated in a rabbit model of total limbal SC
deficiency (TLSCD) induced by sequential
chemical burn and keratectomy. Transplanted
hIDPSC onto the deepithelialized corneas could
reconstruct the corneal epithelium by direct cel-
lular incorporation, and hIDPSC expressed lim-
bal SCs and corneal epithelial cells markers [52].
In 2010, the same laboratory generated undiffer-
entiated hIDPSC compact sheets onto a thermo-­
responsive polymer, poly-N-isopropyl acrylamide
(PNI-PAAm) culture surface. Transplantation of
the hIDPSC sheet onto the same rabbit model of
TLSCD as previously described [52] led to the
reconstruction of the epithelium, with cells cor-
responding to hIDPSC and with higher expres-
sion of the CEC marker CK3 in suprabasal cells,
whereas basal cells showed highest expression of
the LESC markers ABCG2 and p63 [53]. In fur-
ther support of this concept, Tsai et  al. also
reported the ability of hIDPSC to acquire expres-
sion of the CEC marker CK3/12 after 7 days of
coculture in a transwell system with immortal-
ized human corneal epithelial (HCE-T) [79].
9.3 Corneal Stromal
Regeneration
The corneal stroma harbours specialized fibro-
blasts, the keratocytes, which are critical to main-
tain a specific arrangement of extracellular matrix
(ECM) molecules, necessary to confer transpar-
ency. Keratocytes are neural crest-derived mes-
enchymal cells [80]. Although they are quiescent
in the healthy cornea, corneal trauma can how-
ever partially induce their reactivation into myo-
fibroblasts and their secretion of de novo ECM
fibrotic components leading to fibrosis/scarring
and loss of transparency. Additionally, corneal
stromal diseases also originate when keratocytes
become dysfunctional and/or are progressively
lost through apoptosis, such as in keratoconus,
one of the most common form of corneal dystro-
phy. Keratoplasty is currently the only treatment
131
available for severe keratoconus forms where
corneal opacity has developed. SC-based thera-
pies are suggested promising, as they may offer
the possibility of producing healthy keratocytes
to be reincorporated in the diseased corneal
stroma or for biomedical engineering applica-
tions such as the manufacture of corneal stromal
equivalents. In this way, we below recover the
use of different types of extraocular SCs with
keratocyte differentiation potential proposed in
the last decade.
Table 9.2 shows extracorneal stem cells used
for corneal stromal regeneration.
9.3.1 E
 mbryonic Stem Cells
and Induced Pluripotent Stem
Cells
The derivation of phenotypically and function-
ally differentiated keratocytes from human
embryonic SCs (hESC) has been reported quite
recently [81–83]. Chan et  al. in 2013 generated
and purified hESC-derived neural crest progeni-
tors (NCP) expressing the cell surface nerve
growth factor receptor (NGFR), by coculturing
them onto the PA6 embryonic fibroblasts, a cell
line with reported neuronal-inducing activity
[81]. Culture of NGFR+ NCP as compact 3D pel-
lets in serum-free media containing ascorbate has
shown to upregulate their expression of the kera-
tocytes genes AQP1, B3GNT7, PTDGS and
ALDH3A1 and keratocyte phenotype as demon-
strated by their 10,000-fold upregulation of kera-
tocan (KERA), a proteoglycan principally found
in the corneal stroma [100]. Additionally, culture
medium from pellets was also found to contain
high molecular weight keratocan modified with
keratan sulphate, a unique molecular component
of corneal stroma [81]. Later on, the same labora-
tory modified slightly their previous protocol, in
a two-step procedure omitting the monolayered
culture of purified hESC-derived NGFR+, which
were instead directly put into 3D pellets culture
in a defined serum-free keratocyte differentiation
medium (KDM) consisting of advanced DMEM,
with 10 ng/ml bFGF and 0.1 mM ascorbic acid-­
2-­phosphate to induce a keratocyte phenotype as
132 C. C. Lachaud et al.

Table 9.2  Extracorneal stem cells used for corneal stromal regeneration
SC typeSummary procedures Principal results References
ESC Cocultured with PA6 fibroblasts. 3D pellet culture Exp° AQP1, B3GNT7, PTDGS, [81]
in SFM + ascorbate ALDH3A1, keratocan, KS
3D pellet culture. SFM + RA + ascorbic Exp° keratocan, KS [82]
acid-2-phosphate
iPSC 3D pellet culture in DMEM + bFGF + ascorbic Exp° keratocan, ABCB5, vimentin and [83]
acid-2-phosphate HNK1
DPSC 3D pellet culture in DMEM + ascorbate-2-­ Exp° type I collagen, keratocan, KS [84]
phosphate, bFGF and TGF-β1. Intrastromal Restore transparency
injection in mouse corneas
UC-MSC Standard adherent culture. Intrastromal injection Exp° ALDH3A1, lumican and [85]
in lumican−/− null mice keratocan
Standard adherent culture. Intrastromal injection Reduced stromal GAGs and corneal [86]
in mucopolysaccharidosis VII mice haze
BM-MSC Intrastromal injection in keratocan null mice Diff° into keratocan+ keratocytes [87]
Culture with keratocyte CM Diff° into keratocytes-like, keratocan+, [88]
lumican+ and ALDH1A1+
Generation GFP+-BM-MSC. Intrastromal Increased stromal keratocyte markers [89]
injection in injured rat corneal stroma (keratocan, ALDH and CD34). Diff°
into GFP+/keratocan+ cells
Culture with mouse corneas extract + IGF-1 Diff° into keratocan+, lumican+ [71]
keratocytes-like
Topical corneal application of CM by BM-MSC Enhanced stromal keratocyte survival [90]
in injured surface cornea
IV injection in injured mouse cornea Reduced corneal swelling, [91]
inflammation and opacity. Reduced
TNF-α, IL-1β and IL-6
ADSC Intrastromal injection in rabbit cornea Rearranged into multilayered [92]
keratocytes. Exp° ALDH3A1+ and
keratocan+
Culture into fibrin gels or 3D pellet culture into Diff° in vitro into keratocan+, [93]
SFM + ascorbate ALDH3A1+
Culture onto PLGA scaffold. Intrastromal graft Good intrastromal implantation. [94]
ADSC-PLGA construct Restore corneal transparency
Cocultured with human keratocytes Diff° in vitro into keratocan+, [95]
ALDH3A1+
Seeding on PEA-based porous membranes. Good intrastromal implantation inside [96]
Creation of corneal stromal equivalent the stroma of rabbit corneas
Seeding onto acellular human corneal stroma Good intrastromal graft integration. [97]
sheet. Graft into rabbit cornea Diff° of h-ADSC into human
keratocan+
Phase-1 study intrastromal implantation of Good graft implantation at 6 months [98]
h-ADSC seeded human cornea matrix for Moderate amelioration visual
advanced keratoconus parameters
Culture in DMEM + bFGF, RA, Diff° in vitro into keratocan+, [99]
ascorbate-2-phosphate ALDH3A1+
Abbreviations: Diff differentiation, Exp expression, ADSC adipose-derived SCs, ALDH1A1 aldehyde dehydroge-
nase 1 family, member A1, ALDH3A1 aldehyde-3-dehydrogenase 1 family, bFGF basic fibroblast growth factor,
BM-MSC bone marrow mesenchymal SCs, CM conditioned medium, DPSC dental pulp SCs, ESC embryonic
SCs, GAGs glycosaminoglycans, HSHS platelet-poor horse serum, IGF-1 insulin-like growth factor 1, iPSC
immature dental SCs, KS keratan sulphate, PEA poly-(ethyl acrylate), PLGA polylactic-co-glycolic acid, RA reti-
noic acid, SFM serum-free media, TGF-β1 transforming growth factor beta 1, UC-MSC umbilical cord mesen-
chymal SCs
9  Corneal Regeneration: Use of Extracorneal Stem Cells
evidenced by their increased secretion of kerato-
can and keratan sulphate proteoglycans after
3  weeks of differentiating culture [82]. Naylor
et  al. reported the derivation of AP2a+ and
p27NTR+ neural crest cells (NCCs) from human-­
induced pluripotent SCs (hiPSC). 3D pellet cul-
ture of the hiPSC-derived NCCs into a keratocyte
differentiation (KD) media could efficiently
direct their differentiation into keratocytes-like
cells expressing keratocan, ABCB5, vimentin
and HNK1. Additionally, hiPSC-derived NCCs
seeded onto donor’s corneal tissue could migrate
inside the corneal stroma and developed a
keratocytes-­like morphology and expressed kera-
tocyte markers [83].
9.3.2 M
 esenchymal Stem Cells
(MSCs)
A special interest has been focussed onto adult
MSC as extracorneal SCs for obtaining kerato-
cytes, which are indeed cells with MSC-like fea-
tures [101]. The sources of MSC used to generate
keratocyte-like cells include the bone marrow,
adipose tissue, umbilical cord and dental pulp.
9.3.2.1 Bone Marrow MSC
The first evidence of the keratocyte differentia-
tion capacity of MSC of bone marrow origin was
reported by Liu et al. [87]. They showed how a
corneal intrastromal transplantation of ex  vivo
Dil-labelled mouse BM-MSC in keratocan null
(Kera−/−) mice led to their correct stromal inte-
gration and differentiation into Dil+/keratocan+
keratocytes [87]. Park et al. reported how human
BM-MSC cultured in a keratocyte-conditioned
media (KCM) could efficiently differentiate into
keratocyte-like cells, as evidenced by their loss of
α-SMA expression and up-expression of kerato-
can, lumican and aldehyde dehydrogenase 1 fam-
ily, member A1 (ALDH1A1) [88]. Demirayak
et al. created a penetrating injury in rat corneas.
Rat GFP+ BM-MSC were further injected into
the anterior chamber, and their homing to the
injury site was monitored. At 8  weeks post-­
transplantation, rats displayed increased kerato-
cyte markers (keratocan, ALDH and CD34), and
133
stromal BM-MSC-derived GFP+/keratocan+ cells
were found homed at the site of injury [89]. In
other study, purified mouse BM-MSC were cul-
tured with mouse corneas extract (MCE) obtained
through repeated freezing-thawing cycles and
with or without 20 ng/ml insulin-like growth fac-
tor 1 (IGF-1). Combination of MCE and IGF-1
has shown to be the best condition for differenti-
ating BM-MSC into K12+, keratocan+ and lumi-
can+ keratocyte-like cells [71]. To study the
anti-inflammatory role of MSC for corneal
stroma injury, Jiang et  al. performed a mouse
model of corneal injury by epithelial scraping
after ethanol injury. They showed that topical
corneal surface application of a conditioned
media by BM-MSC could significantly enhance
stromal keratocyte survival [90]. By using the
same mouse corneal injury model, Yun et  al.
demonstrated that an intravenous transplantation
of 1.106 human BM-MSC was able to reduce cor-
neal swelling and inflammation through reduced
leukocytic infiltration. BM-MSC treatment
resulted in decreased corneal opacity and reduc-
tion of the inflammatory markers TNF-α, IL-1β
and IL-6 [91].
9.3.2.2 Adipose Tissue MSC
MSC isolated from processed lipoaspirate (PLA)
cells of the human subcutaneous adipose tissue
(termed ADSC or ASC) are a type of extracorneal
MSC that has received specific attention for
establishing regenerating therapies of corneal
stromal disorders. Arnalich-Montiel et  al.
reported for the first time that human PLA cells
could satisfactorily incorporate for up to 10
weeks in the corneal stroma of immune-­
competent rabbits [92]. In a second set of experi-
ments, they further showed that PLA cells
injected in rabbit corneas with partial stromal
ablation could home and rearrange into the typi-
cal multilayered keratocytes organization and
express ALDH and keratocan [92]. In their study,
Ma et  al. report their creation of a thin disc of
polylactic-co-glycolic acid (PLGA) as a scaffold
that was further cellularized with autologous rab-
bit ASC.  The rASC-PGLA stromal equivalent
constructs were further grafted in a rabbit corneal
stromal pocket. Implanted rASC-PGLA con-
134
structs became progressively transparent and
with histological features similar to the native
corneal stroma [94]. Alió del Barrio et al. reported
the creation of corneal stromal equivalents by
in  vitro cellularization of poly-(ethyl acrylate)
(PEA)-based macroporous membranes with
human ADSC.  The authors reported how PEA
scaffolds containing 10% hydroxyethyl acrylate
(PEA-HEA10) display optimal outcomes in
terms of h-ADSC colonization rate and lack of
extrusion at 3 months post implantation inside
the stroma of rabbit corneas. [96]. In the same
year, the same laboratory successfully recolo-
nized in vitro decellularized sheets of human cor-
neal stroma with h-ADSC, which were further
intrastromally implanted in rabbit corneas.
Stromal equivalents demonstrated an excellent
biointegration, transparency and survival, and
differentiation of h-ADSC into human kerato-
can+ cells was patent at 3 months post implanta-
tion. The combined use of h-ADSC and donor´s
corneal stroma may allow tissue engineering of
bioequivalents for corneal stromal disorders [97].
Alió del Barrio et  al. recently published results
from a human Phase-1 study addressing the effi-
cacy of the implantation of a human corneal stro-
mal lamina cellularized or not with autologous
h-ADSC for the treatment of advanced keratoco-
nus [98]. Intrastromal implantation of decellular-
ized and recellularized laminas were similarly
well accepted after 6 months, and both approaches
provided a similar moderated recuperation of
visual parameters and keratometric values, thus
suggesting h-ADSC didn´t exerted significant
improvements over that of the stromal implant
alone [98].
In addition to these preclinical studies, other
studies also addressed the in  vitro ability of
human subcutaneous adipose tissue MSC [93,
95, 99] to differentiate into keratocytes. Human
ADSC-derived keratocyte-like cells (keratocan+/
ALDH3A1+) could be obtained through different
methods. In one study, they were obtained by cul-
turing h-ADSC into fibrin gels or as pellet cul-
tures into serum-free or reduced-serum media
containing ascorbate [93]. Alternatively Zhang
et  al. generated keratocyte-like cells from
h-ADSC cocultured with human primary kerato-
C. C. Lachaud et al.
cytes in a transwell system and using a kerato-
cytes media consisting of DMEM media
containing 10 ng/ml FGF2 0.1 mM ascorbate-­2-­
phosphate 1% heparin-stripped platelet-poor
horse serum (HSHS) [95]. Cultured h-ADSC into
DMEM containing 10  ng/ml FGF2 0.1  mM
ascorbate-2-phosphate and retinoic acid (RA)
were shown to upregulate keratocyte markers. A
concentration of 1 μM RA produced the highest
upregulation of keratocan and ALDH3A1 [99]
9.3.2.3 Umbilical Cord MSC
Liu et al. performed an intrastromal implantation
of Dio- and/or Dil-labelled human umbilical cord
MSC (h-UMSC) into lumican−/− null mice.
Transplanted h-UMSC in lumican−/− mice prolif-
erated transiently inside the stroma and expressed
lumican. Corneal transcript expression for
ALDH3A1, lumican and keratocan was also
increased in response to h-UMSC transplantation
[85]. Later on, an intrastromal transplantation of
h-UMSC was also performed into mucopolysac-
charidosis VII mice which develop corneal cloud-
ing due to glycosaminoglycans accumulation. Of
note, h-UMSC implantation reduced corneal
haze, restored keratocyte morphology and
decreased stromal content of chondroitin and
heparan sulphate [86].
9.3.3 O
 ral Stem Cells: Dental Pulp
Stem Cells
In their work, Syed-Picard et al. isolated human
adult dental pulp cells (hDPCs) from extracted
third molars [84]. Collected pulp tissue was sub-
jected to collagenase I treatment to liberate
DPCs, which were then expanded on plastic
adherent plates into a DMEM media containing
20% FBS. Subcultured hDPCs between passages
2 and 4 were subjected to pellet culture for
3  weeks into a DMEM basal media containing
ascorbate-2-phosphate, FGF2 and TGF-β1 [84].
After 2 weeks of culture in keratocyte differentia-
tion medium, hDPCs displayed increased type I
collagen and keratocan expression and secreted
keratan sulphate-containing proteoglycans.
Intrastromal injection of Dio-labelled hDPC in
9  Corneal Regeneration: Use of Extracorneal Stem Cells
healthy mouse corneas revealed good implanta-
tion outcomes over time, without alterations of
corneal transparency or thickness. Of special
interest, 2 weeks after implantation, Dio+ hDPCs
were found to have produced specific human col-
lagen type I and keratocan [84].
9.4 Corneal Endothelium
Regeneration
The search for extracorneal SCs able to differ-
entiate into functional CEnC or of adult cells
displaying similar functions (surrogate) has
focussed significant attention in the last decade.
In that way, different laboratories have experi-
mentally evaluated the suitably of obtaining
CEnC by in vitro ocular lineage restriction of
their precursor SCs, such are ESC or postnatal
neural crest progenitors. The use of iPSC to
generate CEnC has also been object of interest.
On the other hand, other laboratories also eval-
uated the suitability of adult stem or progenitor
cells from distinct lineages, such as MSC, vas-
cular endothelial progenitor cell (VEPC) and
skin-derived precursors (SKPs). Finally, other
possibilities would be to use differentiated
cells from tissues which are morphostructur-
ally and functionality closely similar to the
corneal endothelium such are some simple epi-
theliums such as the vascular endothelium or
mesothelium.
We below detail in more extent the use of the
different types of extracorneal SCs that have been
proposed for corneal endothelium regeneration.
Table 9.3 shows extracorneal stem cells used
for corneal endothelium regeneration.
9.4.1 Embryonic and Induced
Pluripotent Stem Cells
Zhang et al. addressed for the first time in 2014
the corneal endothelial differentiation capacity of
human ESC using a two-step induction proce-
dure. A transwell-based coculture system incor-
porating human corneal stromal cells was first
used to direct hESC towards periocular mesen-
135
chymal progenitors (POMPs). POMPs cultured
into a lens epithelial cell-conditioned medium
could generate N-cadherin+ CEnC-like cells
expressing the transcription factors FoxC1 and
Pitx2. Seeding of hESC-derived CEnC-like cells
onto a porcine corneal stromal lamella was fur-
ther performed to create a corneal endothelium
equivalent. Implantation of the construct into
eyes of rabbit with corneal endothelium dysfunc-
tion has shown to gradually increase corneal
transparency [54]. Soon after, McCabe et al. also
reported the successful directed differentiation of
hESC into CEnC-like cells by promoting their
initial differentiation into neural crest progenitors
(NCPs) in a culture media containing the Smad
inhibitors Noggin and SB431542. NCPs were
further differentiated towards CEnC-like cells in
a corneal endothelium culture media containing
B27 supplement, 10  ng/ml platelet-derived
growth factor BB (PDGF-BB) and 10  ng/ml
dickkopf WNT signalling pathway inhibitor 2
(Dkk-2). hESC-derived CEnC-like cells have
shown to express the corneal endothelium mark-
ers COL8A1, COL8A2 and Na+/K+ATPase α1
(ATPA1) [102]. In their work, Chen et al. show
that treatment with all-trans retinoic acid (RA) of
embryoid body (EB) generated from mouse-­
induced pluripotent SCs (iPSC) could induce
their differentiation into neural crest cells [104].
Replating of the differentiated EB onto gelatin-­
coated plates into a conditioned medium pro-
duced by primary lens epithelial cells has shown
to produce outgrowth monolayered cells express-
ing the CEnC markers ATPA1, ZO-1 and AQP1
[104]. Following a similar two-step differentia-
tion protocol, Song et al. generated hESC-derived
EB, which were further induced to NCPs by
using a culture media containing Noggin and
SB431542. Differentiating EBs were further cul-
tured on adherence onto chondroitin sulphate and
laminin-­coated plates in a medium supplemented
with PDGF-BB, Dkk-2 and TGF-β or alterna-
tively in a CM produced by bovine primary
CEnC, this later producing the best differentia-
tion into CEnC-like cells expressing COL8A1,
ATPA1, ZO-1, AQP1 and S100A4 [103]. Zhao
and Afshari reported the successful generation of
CEnC-like cells through in  vitro ocular lineage
136 C. C. Lachaud et al.

Table 9.3  Extracorneal stem cells used for corneal endothelium regeneration
SC type Summary procedures Principal Results References
ESC Use of lens epithelial cells CM Polygonal, CDH2+, Vimentin+. Functional [54]
in vivo
SF media + B27, PDGF-BB, Dkk-2 Polygonal-shaped, COL8A1+, COL8A2+, [102]
ATPA1+
Chondroitin sulphate and laminin coating Polygonal, COL8A1+, ATPA1+, ZO-1+, AQP1+ [103]
Use of CM by bovine primary CEnC and S100A4+
Three-step diff° protocol Functional full-thickness cornea substitute [55]
(TECS)
iPSC EB formation. Treatment with RA Polygonal ATPA1+, ZO-1+, AQP1+ [104]
Gelatin coating, lens epithelial cells CM
Three-step diff° protocol. Suppression Polygonal ZO-1+, ATPA1+, CDH2+ [105]
TGF-β and ROCK signalling
NCSC Explant culture neural tube. Laminin and Polygonal, ATPA1+, ZO-1+ [106]
chondroitin-6 sulphate coating Generation of CE-like monolayer onto bovine
Use of rat primary CEnC CM APCM
SKP Spheres into SFM containing B27, EGF Polygonal, Atp1a1+, Cdh2+ and Pitx2+. Seeded [107]
and bFGF. Media with RA, BIO and on
Y-27632 Type I atelocollagen, functional in vivo
Chondroitin sulphate and laminin coating Polygonal, express genes (atp1a1, ZO-1, [108]
Coculture with hCEnC line B4G12 cdh2, ca2, col4a2, col8a2) and proteins
(ZO-1, ATP1A1)
BM-EPC Transwell coculture with human CEnC Polygonal, AQP1+, NSE+, functional in cat [15]
corneas
UCB-­ Collagen I coating into EGM-2 media Integrate into rabbit injured CE using [16]
EPC magnetic field
UCB-­ Use of human lens epithelial cells CM Not polygonal, intercellular expression ZO-1, [109]
MSC CDH2
BM-MSC BM-MSC-CM improves CEnC growth Improves polygonal shape of human CEnC [110]
AT-MC Culture in MRPM. Seeding onto human Polygonal. ZO-1, CDH2, ATP1A1, COL4A2, [111]
lens capsules (HLCs) COL8A2, SLC4A4. Adhere onto HLCs
APCM acellular porcine cornea matrix, AT-MC adipose tissue mesothelial cells, BM-EPC bone marrow endothelial
progenitor cells, bFGF basic fibroblast growth factor, BM-MSC bone marrow mesenchymal SCs, CDH2 N-cadherin,
CEnC corneal endothelial cells, CM conditioned medium, EGF epidermal growth factor, ESC embryonic SCs, IPSC
immature dental SCs, MRPM mesothelial retaining phenotype media, NCSCs neural crest SCs, PDGF-BB platelet
growth factor BB, RA retinoic acid, SFM serum-free media, SKP skin-derived precursor, UCB-EPC umbilical cord
blood endothelial progenitor cells, UCB-MSC umbilical cord blood mesenchymal SCs

restriction of human iPSC, using a three-step
induction protocol [105]. The iPSC were first
directed towards eye field SCs (EFSC) in a
serum-free media containing bFGF, N2 and B27
growth supplements and small molecule inhibi-
tors (SB431542, LDN193189 and IWP-2). The
iPSC-derived EFSC were then directed towards
HNK-1+/p75NTR+ ocular neural crest SCs
(NCSCs) onto Matrigel-coated surface in a
serum-free media containing N2, B27, L-ascorbic
acid 2-phosphate and CHIR99021 (GSK-3
Inhibitor). NCSCs were finally directed towards
ZO-1+, ATPA1+ and N-cadherin+ CEnC-like cells
by suppressing TGF-β and ROCK signalling
[105]. Zhang et  al.’s laboratory recently pub-
lished a relevant study demonstrating successful
tissue engineering of a full-thickness cornea sub-
stitute (TECS) through sequential cellularization
of an acellular porcine cornea matrix (APCM)
with limbal epithelial-like cells and CEnC-like
cells generated from hESC, using for these later a
similar approach as previously published in 2014
[54]. Of special interest, TECS implanted within
rabbit eyes increased their transparency after few
weeks and developed significant lower neovascu-
larization than implanted APCM scaffolds with-
out cellularization, that instead became
increasingly cloudy [55].
9  Corneal Regeneration: Use of Extracorneal Stem Cells
9.4.2 Neural Crest Stem Cells
Ju et  al. isolated P75 and HNK-1 dual positive
neural crest SCs (NCSCs) from an explant cul-
ture of the neural tube of rat embryos.
Differentiation of NSCs into ATPA1+ and ZO-1+
polygonal CEnC-like cells was achieved by cul-
turing NSCs onto plastic culture plate coated
with laminin and chondroitin-6 sulphate in an
inductive media containing a CM by rat primary
CEnC.  Seeding and culture of the NSC-derived
CEnC-like cells onto an APCM could generate a
corneal endothelium-like monolayer [106].
9.4.3 Skin-Derived Precursors
Skin-derived precursors (SKPs) reside in the
adult dermal layer and were shown to represent a
pool of primitive cells sharing hallmarks with
embryonic neural crest SCs, the population from
which are derived CEnC [112]. In light of this
concept, Inagaki et al. reported a work addressing
the ability of SKPs isolated from neonatal mouse
facial skin to differentiate along the corneal
endothelial cells lineage [107]. For this issue, the
dermal skin layer was separated from the epider-
mis after dispase II enzymatic digestion and was
then fully disaggregated using collagenase. SKPs
were propagated as floating spheres in low attach-
ment plate and in a serum-free media containing
B27 supplements, EGF and bFGF. SKP spheres
expressed the neural crest markers p75NTR and
N-cadherin. Adherent culture of SKPs onto 0.1%
gelatin-coated plate in a corneal endothelium-­
inducing medium (CEIM) containing 5% FBS,
insulin, all-trans retinoic acid, the GSK 3-beta
inhibitor BIO, TGF-β2 and the ROCK inhibitor
Y-27632 was able to direct their differentiation
into polygonal CEnC-like cells expressing the
corneal endothelium genes Atp1a1 (Na+/
K+ATPase α1), Cdh2 (N-cadherin) and Pitx2.
Culture of the SKP-derived CEnC onto type I
atelocollagen sheets was performed to generate a
corneal endothelium (CE) equivalent.
Transplantation of the CE equivalent into a rabbit
model of bullous keratopathy has shown to sub-
stantially reduce corneal thickness and maintain
137
transparency by comparison to the carrier with-
out cells [107]. In 2017, Shen et al. published an
excellent preclinical study based on the use of
human adult SKPs isolated from eyelid operation
[108]. SKPs were isolated from the dermal layer
after enzymatic digestion into Liberase DH solu-
tion. SKPs were induced to proliferate in a
serum-free DMEM/F12-based media supple-
mented with 2% B27, bFGF and EGF and sub-
cultured for 2–4 passages. Their differentiation
was performed for 8 days into a transwell cocul-
ture system with SKPs cultured onto a chondroi-
tin sulphate- and laminin-coated surface and
immortalized B4G12 hCEnC cells in the upper
insert. By this method, hSKPs progressively
underwent a fibroblastic-to-polygonal morpho-
logic shift, which was associated with increased
CEnC genes (Atp1a1, ZO-1, N-cadherin, CA2,
Col4a2 and Col8a2) and proteins (ZO-1,
ATP1A1). Functional evaluation of hSKP-­
derived CEnC-like cells was demonstrated after
implantation as a cell suspension in the anterior
chamber of rabbit and monkey eyes, from which
were scrapped their corneal endothelium to
expose a decellularized Descemet’s membrane.
Eyes treated with CEnC-like cells significantly
maintain good corneal thickness and clarity, by
comparison to non-treated operated eyes that
developed severe stromal oedema and corneal
opacity [108].
9.4.4 Vascular Endothelial
Progenitors: Bone Marrow
and Cord Blood
Shao et al. reported the isolation of human fetal
bone marrow-derived endothelial progenitor cells
(BEPC) [15]. Using a coculture transwell system,
corneal endothelial differentiation of BEPCs was
induced in a non-contact fashion with human pri-
mary CEnC and their conditioned medium. Upon
stimulation, the induced BEPC-derived CEnC-­
like cells adopted polygonal morphologies and
expressed AQP1, neuron-specific enolase (NSE)
and tight junctions. BEPC-derived CEnC-like
cells seeded and grown onto an APCM could
form a corneal endothelium equivalent with the
138 C. C. Lachaud et al.

capacity to restore the clarity in cat’s corneas CEnC.  For this purpose, hBM-MSC were first
after stripping of their Descemet’s membrane and expanded in a DMEM containing 10% FBS, and
corneal endothelium [15]. In a next study, Shao confluent hBM-MSC were then used to condition
et  al. isolated EPCs from the mononuclear cell a basal growth medium designed for culturing
(MNC) fraction of human umbilical cord blood. CEnC cultures (OptiMEM-I containing 8% FBS,
Selective outgrowth of umbilical cord EPCs was EGF, ascorbic acid and chondroitin sulphate).
performed through culture of adherent MNCs Application of this conditioned medium to
onto collagen I-coated plates into the EGM-2 hCEnC culture has shown to modify their mor-
endothelial culture media. EPCs positive for von phology to a more and regular polygonal mor-
Willebrand factor (vWF), Ac-LDL, CD133 and phology similar to that displayed in  vivo by
CD34 were obtained with this method. CEnC. The hBM-MSC-­conditioned medium also
Trypsinized EPCs were further labelled with stimulates CEnC in vitro proliferation to a higher
CD34 immunomagnetic nanoparticles and degree than the conditioned medium obtained
infused into the anterior chamber of rabbit eyes from 3T3 mouse fibroblasts [110].
after surgical removal of the Descemet’s mem-
brane and corneal endothelium. Their apposition
and adhesion onto the denuded anterior corneal 9.4.6 Adipose Tissue Mesothelial
surface were achieved by placing during several Cells
hours a magnet on top of the eyes. EPC trans-
plantation could progressively reduce corneal Mesothelial cells are squamous epithelial-like
oedema and opacity [16]. cells of mesodermal origin [113]. They are found
lining the surface of visceral organs house into
coelomic cavities and also the walls of these cavi-
9.4.5 Mesenchymal Stem Cells ties. Although mesothelial cells are not demon-
strated SCs, cumulating evidence is however
9.4.5.1 Umbilical Cord Blood MSC demonstrating their in  vitro capacity to acquire
The first experimental evaluation of the capacity hallmarks of several differentiated mesodermal
of mesenchymal SCs (MSCs) to differentiate into cell lineage such as vascular smooth muscle cells,
CEnC-like cells was published by Joyce et al. in adipocytes and osteocytes, and thus the existence
2012. MSCs were isolated from human umbilical of mesothelial progenitors has been proposed
cord blood (UCB-MSC) and established under [114–116]. Because the mesothelium and corneal
adherent culture. The authors reported significant endothelium are two types of simple epithelium,
morphological modification of UCB-MSC cul- it is therefore possible that mesothelial cells are
tured in a culture media supplemented with 20% endowed with some similar functions of
FBS previously conditioned by human lens epi- CEnC.  Our laboratory reported that mesothelial
thelial cells. Although the differentiated UCB-­ cells represent a putative cellular surrogate to
MSC expressed ZO-1 and N-cadherin at regenerate the corneal endothelium [111]. In this
intercellular contact, they however failed to adopt way, we reported that freshly isolated murine adi-
the typical polygonal morphology of pose tissue mesothelial cells (ATMCs) express
CEnC.  UCB-MSC could successfully adhere different genes expressed in CEnC in similar
in vitro onto areas of damaged corneal endothe- (N-cadherin) or inclusively higher levels (col4a2,
lium of human corneas but however didn’t adopt slc4a4, car-2 and atp1a1). Only col8a2, a highly
polygonal morphologies similar to CEnC [109]. specific CEnC marker, was expressed in lower
extent in the ATMCs. Subcultured ATMCs were
9.4.5.2 Bone Marrow MSC shown to fully adhere and form a continuous
Nakamura et al. evaluated whether the secretome monolayer onto decellularized human lens cap-
of human bone marrow-MSC (hBM-MSC) is sules [111]. Further studies are currently under
able to enhance the proliferation of human development to assess whether human ATMCs
9  Corneal Regeneration: Use of Extracorneal Stem Cells
have similar phenotype with CEnC and could
serve for tissue engineering of a functional cor-
neal endothelium equivalent.
9.5 Concluding Remarks
The approach of using extracorneal SCs for
regenerating the cornea is an incipient field which
is however experimenting a very fast develop-
ment, principally for tissue engineering applica-
tions of corneal tissue equivalents. A global
examination of the different types of SCs of
extracorneal origin proposed so far for corneal
regeneration leads to the conclusion that using
SCs belonging to a similar or closely related lin-
eage to that of the corneal cell type to replace
appears to be the most promising cell replace-
ment strategy to use in the near term. Given that
much of the progresses accomplished until now
in this matter are globally related to the regenera-
tion of the corneal epithelium, increased efforts
are thus required to accelerate strategies for
regenerating the corneal stroma and endothelium.
Tissue engineering of a corneal stromal equiva-
lent appears actually the most challenging goal to
achieve in corneal regeneration, principally
because it will require to identify or develop ade-
quate scaffolds mimicking more closely the
extremely complex biochemical and morpho-
structural properties of the native stromal extra-
cellular matrix and also to better define which is
the best type of SCs able to replace the lost or
diseased keratocytes. In that way, autologous
adult MSCs seem a very promising cellular phe-
notype able to provide neo-keratocytes. As stated
above, regenerating the corneal endothelium with
SCs of extracorneal origin is also a great priority.
The simple epithelium-like structure of the cor-
neal endothelium makes of its tissue engineering
with laminar scaffolds and SCs a goal less com-
plex to achieve by comparison to the corneal
stroma. Using extracorneal SCs or progenitors
from monolayered cells tissues, such as vascular
endothelium progenitors or squamous epithelial
cells from the amniotic membrane or adult sim-
ple epitheliums, is a promising approach to recre-
ate a tissue surrogate of the native corneal
139
endothelium. Whether these cells are plastic
enough to adapt to the anterior chamber microen-
vironment and acquire functions of native cor-
neal endothelial cells is a critical issue to better
determine. Altogether, the use of extracorneal
SCs of autologous origin for tissue engineering
of corneal tissue equivalents is expected to pro-
vide great achievements in the next decades and
might converge towards the manufacture of a
full-thickness corneal autograft.
Acknowledgement  Authors are supported by the nonprofit
Fundación Progreso y Salud, Consejería de Salud, Junta de
Andalucía; FEDER cofounded grants from Instituto de
Salud Carlos III and the Ministry of Economy, Industry and
Competitiveness (Red TerCel: RD12/0019/0028 and
RD16/00259; CIBERDEM: CB07/08/0006; PI14/01015,
PI16/00259, PI17/02104 and CD16/00118); and Junta de
Andalucía (PAI-BIO311, CTS-576, CTS 11-727, PI-0109-
2014, PI0007/2016 and PI0272/2017). CIBERDEM is an
initiative of the Instituto de Salud Carlos III.
Competing Interests  The authors declare no
conflict of interest.
Informed Consent  No human studies were car-
ried out by the authors for this article.
Animal Studies  No animal studies were carried
out by the authors for this article.
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 One Cell, Two Phenotypes:
Capturing Pluripotency
for Corneal Regeneration
Trevor Sherwin, Carol Ann Greene, Colin R. Green,
and Kushant R. Kapadia
10.1 Introduction
A major goal for corneal restoration and regen-
eration over the last decade has been the produc-
tion of bioengineered corneas in  vitro for
transplant, with many research laboratories
investigating a variety of scaffold materials aimed
at preserving the physiological and optical prop-
erties of the cornea [1]. However, advances in
cell reprogramming [2, 3] and in gene therapy [4,
5] have made the possibility of in  vivo corneal
engineering a distinct possibility. Here we
describe the methods by which we have been
able to induce in vivo keratocytes to produce pro-
teins normally associated with other cell pheno-
types, which may prove to be simple and effective
examples of in vivo corneal engineering.
10.2 Homoeostasis in the Stroma
The corneal stroma and associated keratocytes
arise from the neural crest during embryonic
development. The stroma of the early cornea is
largely composed of type II collagen which is
T. Sherwin (*) · C. A. Greene · C. R. Green
K. R. Kapadia
Department of Ophthalmology, New Zealand
National Eye Centre, Faculty of Medical and Health
Sciences, The University of Auckland,
Auckland, New Zealand
e-mail: t.sherwin@auckland.ac.nz
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_10
10
slowly replaced by the keratocytes in later devel-
opment with type I collagen [6, 7], the most
abundant collagen of the adult human cornea [8].
The spacing of collagen fibrils and thickness of
collagen lamellae and their arrangement within
the stroma are critical to the passage of light and
thus the transparency of the cornea. Thus, post-­
partum, the extracellular matrix of the corneal
stroma is thought to turn over very slowly in
order not to disturb the delicate balance of consti-
tution and spacing of the lamellae and fibrils. The
keratocyte thus has long been thought of as a
largely quiescent cell residing in the stroma ready
to jump into action if needed, such as following
trauma to the cornea [9].
10.3 Keratocyte Activation
(Fig. 10.1)
Quiescent keratocytes close to a traumatic
wound soon become activated and acquire the
cellular properties necessary to deal with the
wound [10]. As the cells directly traumatized by
the wound and those in close association will die
soon after the damage, there is a need to derive
new cells and for them to be able to migrate into
the wound site. Thus, upon activation following
trauma, the previously quiescent keratocyte
acquires the fibroblastic phenotype able to
undergo rapid cell division and the mechanistic
molecules that promote migration. Upon arrival
145
146
a b
Fig. 10.1  Cultured human keratocytes display typical
stellate morphology of compact cell body with long very
fine dendritic extensions that interconnect between kera-
tocytes to form a syncytium (a). Upon wounding stimuli,
keratocytes differentiate into fibroblasts with the more
spindle-shaped cell bodies revealed by tubulin staining. A
large myofibroblast lies amongst the fibroblasts and
at the wound edge, these fibroblastic cells
remodel their actin cytoskeletons and become
the giant myofibroblast with prominent stress
fibres that are capable of straddling and contract-
ing the wound edges. The fibroblasts also down-
regulate the recognized proteins of the
differentiated keratocyte and start expressing the
enzymatic proteinases required to remodel the
damaged collagen fibres and lamellae [11, 12].
They also lay down type III collagen which can
be responsible for scarring and, as a conse-
quence, visual defects [13, 14].
T. Sherwin et al.
accentuates the difference in cell size between these two
differentiated keratocyte lineages (b). The myofibroblast
can be molecularly distinguished from the surrounding
keratocytes by using smooth muscle actin staining to
reveal the stress fibres (red) that underlie the contractile
nature of the myofibroblast. Cell nuclei are stained with
DAPI (blue). Scale bar = 50 μ
The fate of the myofibroblast is unclear fol-
lowing wound closure. Evidence suggests that
they disappear; however, whether this is through
targeted apoptosis or by dedifferentiation still
remains to be elucidated [12].
10.4 Keratocyte Differentiation
Given the keratocyte activation following trauma,
it is strange to think of the keratocyte as a quies-
cent cell when it is capable of such responses [9].
10  One Cell, Two Phenotypes: Capturing Pluripotency for Corneal Regeneration 147

Furthermore, it follows that we should actually
think of the keratocyte as a cell of greater potency
and differentiation potential than previously
thought [15]. The archaic view of the terminally
differentiated cell being the dead end of a one-­way
street, with the cell neither being able to transdif-
ferentiate into another cell type or to dedifferenti-
ate into an earlier stage, has been shown to be
misguided [16]. For instance, the now well-
accepted epithelial to mesenchymal transition pro-
cesses [17] that occur in corneal epithelial
development, fibrosis and pathogenesis show that
transdifferentiation occurs in the cornea [18]. The
potential of cells to dedifferentiate into much ear-
lier and much more potent stages has also been
demonstrated elegantly by the production of
induced pluripotent stem cells by the induction of
transcription factors by genetic manipulation [19]
but also subsequently by using small molecule
chemical stimuli [20]. Thus, it is not beyond the
realms of belief to imagine that keratocyte cells
may well be inducible into other phenotypes
should the right stimulus present itself.
10.5 Untapped Potential
of Keratocytes (Figs. 10.2
and 10.3)
Serendipitously, we were able to show that kera-
tocytes possessed greater differentiation potential
than previously thought [21]. In a series of exper-
iments where we isolated keratocytes and induced
them to form spheres (one parameter indicative

a b

c d

Fig. 10.2  Neuronal reprogramming of corneal kerato-
cytes and fibroblasts. Keratocytes within human corneal
slices cultured in neuronal reprogramming medium
expressed the early neuronal marker, nestin (a), within
3  days in culture, and by day 8, the cells expressed the
mature neuronal marker, map-2 (b). Similar experiments
with corneal fibroblasts revealed that they too are amena-
ble to neuronal reprogramming, expressing nestin (c) and
map-2 (d) in cell culture. These results suggest that the
corneal fibroblast may not be a terminally differentiated
cell type as once thought
148 T. Sherwin et al.

Fig. 10.3 Chondrogenic
a b
reprogramming of
keratocytes. Keratocytes
cultured for 3 weeks in
chondrogenic
differentiation medium
containing TGFβ3 and
dexamethasone formed
spheres (b) which
labelled for collagen
type II. Keratocytes in
the control medium
failed to form spheres
(a). Human corneal
slices (d) cultured for
2 weeks in the same
chondrogenic
reprogramming medium
(with control medium-­ c d
treated cornea shown in
(c)) and rat corneas
treated in vivo (f) (with
control medium-treated
cornea shown in (e)) for
at least 2 weeks also
labelled positively for
collagen type II

e f

of stem or progenitor potential) by growing them Initially we planned to use this observation to
in neuronal-specific media, we were able to show return to cultured slices of corneal tissue and
the production of cells with neuronal-specific induce the neuronal phenotype with the aim of
protein markers [21] (Fig. 10.2). showing definitively that the cells capable of
10  One Cell, Two Phenotypes: Capturing Pluripotency for Corneal Regeneration
p­ roducing neuronal markers would be located in
the stroma underlying the limbus.
However, we were surprised to find that upon
culture of corneal slices in our neuronal-specific
media, all of the keratocytes were capable of
expressing the neuronal proteins. Furthermore, if
we continued culturing the corneal slices under
our specific conditions, the reprogrammed kerato-
cytes expressed early, intermediate and mature
neuronal markers in the same timeframe and
sequence as occurs during the differentiation of
immature neuronal cells to mature neuronal cells.
Concommitant with this change in protein expres-
sion, we also saw a change in morphology from
keratocyte to neural phenotype. We were able to
reproduce this effect in both rodent and human tis-
sues, including in vivo treatments in rodents with a
simple eye drop delivery of the reprogramming
factors, but also show that the molecules that
induced the neuronal switch were species specific
[21]. These findings enabled us to propose that the
keratocyte cell retains a memory of its neural crest
origins that can be tapped into for therapeutic pur-
poses. We further tested this hypothesis to deter-
mine if other cells that descend from the neural
crest also retain some memory of their lineage,
and we were able to show that neural crest-derived
cartilaginous cells were also capable of switching
on neural protein expression [22].
10.6 F
 urther Untapped Potential
of Keratocytes
This serendipitous finding of a neural crest mem-
ory in keratocytes led us to hypothesize that we
could turn on other molecules that are expressed
by neural crest cells during the developing cor-
nea. We aligned this hypothesis with the observa-
tion that type II collagen is expressed in the
developing cornea but is later replaced by type I
collagen [6–8]. Knowing also that certain carti-
laginous tissues are derived from the neural crest
and that type II collagen is a cartilage-specific
collagen, we applied molecules used to induce
cartilage cell differentiation to our keratocytes to
assess whether the keratocytes would be capable
of once again turning on type II collagen.
149
We identified that the TGFβ family were pow-
erful promoters of chondrogenic differentiation
and also stimulators of collagen production [23].
Noting that TGFβ1 and TGFβ2 are associated
with matrix production aligned with scarring, we
tested TGFβ3 as it acts without instigating fibro-
sis or scar tissue formation [24, 25]. We tested
TGFβ3 coupled with the synthetic steroid dexa-
methasone, a combination which has been shown
to be effective in promoting chondrogenesis [26],
and found that this combination of factors pro-
duced strongly positive and consistently repro-
ducible results for matrix production.
10.7 Potential Therapeutic
for the Treatment
of Keratoconus
After successfully inducing the production of the
developmental type II collagen in isolated kerato-
cytes (Fig. 10.2), we investigated the potential (if
translatable to keratocytes within the stroma) for
retarding not only the loss of stroma in kerato-
conic corneas but also the possibility of restoring
some lost matrix and increasing the thickness of
the corneal stroma in this ectatic disease.
Furthermore, we hypothesized that if we could
stimulate the production of type II collagen
within a keratoconic cornea, then this would
present an opportunity to also reshape the cornea
at the same time as strengthening it to restore
both integrity and optical functionality. This has
the potential to become the first regenerative
treatment for keratoconus. To this aim we started
to ask a series of experimental questions [27].
10.7.1 Can We Induce the Production
of the Developmental Type II
Collagen in Human Tissue?
Following initial promising studies on isolated
keratocytes, we used our combination of TGFβ3
and dexamethasone treatment to see if we could
induce collagen II production in keratocytes in
situ by incubating slices of human corneal tissue
in an organotypic system. Production of collagen
150
II was not evident in either the control or the
treated corneas after 1  week of incubation.
However, slices incubated in the induction
medium for 3 weeks showed the de novo appear-
ance of collagen II, while there was no corre-
sponding labelling of collagen II apparent in the
control samples. It was important to note that the
deposition of type II collagen that we observed
was uniform and evenly distributed along the
existing collagen lamellae. No clumping of pro-
tein deposition was seen, and no disruption of the
lamellar spacing was observed (Fig. 10.3).
Furthermore, no labelling for collagen III
(associated with scar formation) or αSMA (asso-
ciated with fibrosis) was observed in either the
control or treated corneas, supporting the use of
TGFβ3 as a non-scar-producing factor.
10.7.2 Can We Induce Collagen II
Production in Keratoconic
Tissue?
Having observed the induction of collagen II in
situ in human corneal slices under organotypic
conditions, we investigated the use of this tech-
nology to potentially restore matrix production in
a b
Fig. 10.4  Collagen type II induction in keratoconic cor-
neal tissue. Slices of keratoconic cornea were cultured for
3 weeks in the control medium (a) and chondrogenic
medium (b) and labelled for collagen type II. There was
T. Sherwin et al.
keratoconic tissue. Such production of a corneal
developmental collagen may return the cornea to
a less dystrophic stage and restore the integrity of
the collagen lamellae. Such a treatment may rep-
resent the first restorative therapeutic treatment
for keratoconus. However, the possibility
remained that the keratocytes within keratoconic
tissue might already be significantly affected by
the disease process and could no longer be
induced to produce the developmental collagen II.
We examined this by using human kerato-
conic buttons obtained from corneal transplant
surgery. Corneal buttons were divided into half
and placed into either collagen II induction
medium or into control medium. For compari-
son, normal tissue was also induced. Again,
after 3 weeks, collagen II production had been
switched on in the treated corneas despite the
presence of keratoconus. The number of kerato-
cytes was much lower than in comparison to
normal tissue, so it was not surprising that the
labelling for collagen II was lower than that
observed in normal corneas under induction
conditions. Nonetheless, significant deposition
occurred with the collagen II in keratoconic cor-
neas evenly distributed along lamellae and with-
out clumping (Fig. 10.4).
clear and strong labelling for collagen type II (green)
within the stromal matrix of corneal tissue cultured in the
chondrogenic medium
10  One Cell, Two Phenotypes: Capturing Pluripotency for Corneal Regeneration 151

10.7.3 Does Collagen II Production Elasticity

0.001
Alter the Biomechanical Untreated cornea
Properties of Dystrophic 0.0009
Treated cornea
Corneas? 0.0008
0.0007

ER (Gpa)
We were fortunate to obtain a pair of keratoglo- 0.0006
bus corneas that were initially donated for trans- 0.0005
plant but were unable to be used for this purpose 0.0004
due to the presence of keratoglobus. Treatment of
0.0003
these corneas for extended periods in our induc-
0.0002
tion media was followed by the measurement of
0.0001
biomechanical properties in our collaborator’s
laboratory. Subsequent tests of both the elasticity 0
Rat cornea (after Keratoglobus
modulus and the hardness of the corneas follow- in vivo cornea (after ex
ing type II collagen induction showed a signifi- treatment) vivo treatment)
cant increase in both parameters. This showed
that the deposition of type II collagen was Hardness
0.0005
­associated with a recovery of corneal elasticity, 0.00045
Untreated cornea
that is, the ability to restore shape after deforma- Treated cornea
0.0004
tion and, in corneal stiffness, the ability to resist
0.00035
deformation (Fig. 10.5).
H (Gpa)

0.0003
0.00025

10.7.4 Can We Induce the Production 0.0002

of the Developmental Type II 0.00015

Collagen in an In Vivo Model?
Having shown that the use of TGFΒ3 and dexa-
methasone was capable of inducing the production
of collagen II in both normal human and kerato-
conic corneas, we realized that to advance the
technology we needed to transfer the treatment
into animal models that could be tested in vivo.
Experimentation using the collagen II induc-
tion media on rat tissue in vitro showed exactly
the same results as those observed for human tis-
sue in vitro.
Thus, after obtaining ethical approval for
in vivo experimentation, we were able to treat rat
corneas once daily with our TGFβ3 and dexa-
methasone in an eye drop formulation (a tenfold
concentration of the factors was used to compen-
sate for washout by the tear film and the reduced
time of contact).
Production of collagen II in the in vivo rat cor-
neas followed the same pattern as seen in human
corneas ex vivo when examined by immunohis-
tochemistry, with collagen II being deposited
throughout the depth and the breadth of the
0.0001
0.00005
0
Rat cornea (after Keratoglobus
in vivo cornea (after ex
treatment) vivo treatment)
Fig. 10.5  Results from ex  vivo corneal biomechanical
testing via nanoindentation. Elastic modulus (Er) and
hardness (H) measures of treated and control rat corneas
treated in  vivo for 3  weeks reveal a marked increase in
both parameters in the treated corneas. Similarly, in a pair
of ex  vivo treated and untreated keratoglobus corneas,
there is a clear increase in elastic modulus (Er) and hard-
ness (H) in the treated cornea
c­ ornea but in close association with the existing
lamellae and without observed clumping.
Slit lamp examination of the cornea revealed
normal stromal appearance without any hint of
stromal opacity, and fundoscopy revealed easy
imaging of the optic nerve and retina to illustrate
the clarity of the tissue in front of the fundus,
including the cornea.
The rat corneas were also investigated for any
changes in biomechanical properties. Once again,
152
a 3-week treatment with the collagen II induction
factors produced a significantly higher elasticity
modulus and corneal stiffness when compared to
control corneas. Interestingly, in vivo rat corneas
treated for only 1 week with the induction factors
did not show increased biomechanical properties.
This aligns nicely with our immunohistochemis-
try data which shows that collagen II deposition
is not apparent until week 2.
10.7.5 Can We Switch Off Collagen II
Production?
We also processed tissue from treated and con-
trol corneas from the in vivo rat experiments for
PCR analysis. We looked at the levels of colla-
gen II mRNA after induction in treated and con-
trol corneas for a 1-week and a 3-week treatment
period. Following treatment, we withdrew the
factors from the culture medium and cultured for
a further 4 weeks. For comparison (as an internal
control), we also examined the mRNA levels of
collagen I.
Interestingly, after 1  week of treatment, the
mRNA for collagen II was maximally induced
which coincides with the protein deposition mea-
surable by week 2 after treatment. Upon withdrawal
of treatment for 4 weeks, the levels of collagen II
mRNA were returning to baseline indicating that
the induction of collagen II production is controlla-
ble. Interestingly in our treated samples, collagen I
production is also upregulated at week one and may
be a helpful byproduct of the treatment.
10.7.6 Is the Technology Transferable
to a Large Animal Model?
Next, we asked if the technology was transferable
to a large animal model as we wanted to know if
we could induce collagen II production in an
in vivo cornea of similar size and anatomy to that
of a human. We also wanted to examine the
­longevity of the treatment effect. We commenced
sheep models where we treated one eye for 3
weeks with our TGFβ3 and dexamethasone for-
mulation and used the other eye as the control.
The sheep tissue was then collected directly after
T. Sherwin et al.
a 3-week treatment period, while two further
cohorts of sheep were treated for 3 weeks or
6  weeks and then grazed out for a further
6  months prior to collection. Similarly to the
human and rat studies, the sheep corneas showed
deposition of collagen II in only the treated group
with the collagen deposition being uniformly
even along the pre-existing lamellae.
To assess changes in the biomechanical prop-
erties of treated sheep corneas, sheep eyes were
collected from controls, from 3-week-treated ani-
mals and from 3- or 6-week-treated animals left
for by 6 months with no treatment prior to collec-
tion. The intraocular pressure was raised to
20  mmHg in all eyes to normalize the pressure
reading; then the use of a Tono-Pen was engaged
to determine changes in corneal resistance. The
Tono-Pen normally records intraocular pressure,
but the readings are confounded in part by corneal
biomechanics. In our study, the intraocular pres-
sure was set as a constant, and changes in read-
ings therefore solely reflect changes in corneal
biomechanics. In all 11 treated eyes tested, the
resistance was greater than non-treated eyes by an
average of 23%. Interestingly the increased resis-
tance of treated sheep corneas was still present
after a 6-month lay-off period, indicating that the
treatment has a lasting effect of at least 6 months.
10.7.7 Is It Toxic?
Full systemic toxicity testing has been analysed
for the sheep, and no evidence of toxicity has
been found in any of the body fluids or tissue.
Analysis of the corneal and retinal tissue post-
treatment also showed no sign of inflammation of
any tissues of the eye following treatment [28].
10.7.8 Can We Reshape the Cornea?
We have turned our attention to whether our col-
lagen II protocol could be used to reshape the
cornea. We envisage the treatment being applied
in combination with a reshaping contact lens or
stromal inserts which could hold the cornea in a
new conformation while the collagen II deposi-
tion occurs. With the increased corneal stiffness
10  One Cell, Two Phenotypes: Capturing Pluripotency for Corneal Regeneration
and increased elasticity of the corneas following
our treatment, we hypothesize that we may be
able to hold the newly reshaped cornea for an
extended period after treatment. This would,
therefore, not only see this therapeutic as being
the only restorative treatment for keratoconus in
terms of regenerating the corneal stroma but may
also provide a mechanism to restore some visual
acuity or function.
To this aim we have performed some prelimi-
nary experiments in the sheep model where we
have inserted compression sutures in the cornea
to induce a classical bowtie astigmatism, then
treated with our TGFβ3 and dexamethasone for-
mulation. The hypothesis being that the induced
astigmatism would remain for an extended period
after the removal of the compression suture. Pilot
experiments were highly encouraging in that
even after only 1 week of compression suture and
treatment a proportion of the induced astigma-
tism (reshaping) was retained compared to the
control eye which showed no retention of the
induced shape change following a compression
suture without treatment.
10.8 Conclusion: Restorative
Treatment for Keratoconus?
We have identified a combination drug therapy
that is capable of turning on the corneal develop-
ment collagen II expression in living corneas
from three species rat, sheep and human.
Surprisingly, both components are used at a very
low concentration, 2000-fold less than for current
clinical ocular applications of dexamethasone
and around 0.5% of normal circulating blood lev-
els for TGFβ3. The collagen II is deposited in a
manner in which the transparency of the cornea is
maintained, while the cornea becomes stiffer but
also more elastic. The potential of this drug com-
bination therapy is to provide a unique restorative
drug therapy for keratoconus. However, as the
drug therapy works in normal as well as
­dystrophic corneas, it could be used to stiffen
induced reshaping of normal corneas and thus is
a potential therapeutic for myopia, a concept cur-
rently being explored.
153
Compliance with Ethical Requirements Carol Greene,
Colin Green and Trevor Sherwin are coinventors listed on
patent PCT/NZ2016/050033, application date: 05 Mar
2015. Kushant Kapadia has no conflict of interest to declare.
All procedures followed were in accordance with the ethi-
cal standards of the responsible committee on human
experimentation (institutional and national) and with the
Helsinki Declaration of 1975, as revised in 2000. Informed
consent was obtained from all patients for being included in
the study. All institutional and national guidelines for the
care and use of laboratory animals were followed.
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 Corneal Stem Cell-Based Therapies
Yuzuru Sasamoto, Yoshinori Oie,
and Kohji Nishida
11.1 C
 urrent Stem Cell-Based
Therapy
Currently, stem cell-based therapy is the way to
restore a cornea with stem cell deficiency. Stem
cells are well investigated in the corneal epithe-
lium (we’re going to refer to stem cells in corneal
stroma and endothelium later). Corneal epithelial
stem cells reside in the basal layer of limbus and
are thus called limbal stem cells (LSCs) [1]. They
give rise to transient amplifying cells (TACs) and
provide terminally differentiated epithelial cells
by proliferation and migration toward the apical
central cornea (X, Y, Z hypothesis) [2, 3]. They
play an important role in the maintenance and
wound healing of the corneal epithelium. Severe
loss of LSCs leads the defect of corneal epithelial
regeneration, and this situation is called limbal
stem cell deficiency (LSCD). Other than artificial
cell-free devices such as Boston type I kerato-
prostheses, transplantation of LSCs or their sub-
stitute containing tissue is the only way to
overcome the deficiency of stem cells. We mainly
Y. Sasamoto
Division of Genetics, Department of Medicine,
Brigham and Women’s Hospital, Boston, MA, USA
Y. Oie · K. Nishida (*)
Department of Ophthalmology, Osaka University
Graduate School of Medicine, Suita, Osaka, Japan
e-mail: knishida@ophthal.med.osaka-u.ac.jp
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_11
11
focus on the current and future stem cell-based
therapies for corneal epithelium and also refer to
future stem cell-based therapy for corneal stroma
and endothelium.
11.2 C
 urrent Stem Cell-Based
Therapy: Corneal Epithelium
There are various causes of LSCD, from con-
genital diseases (e.g., aniridia and multiple
endocrine deficiency) to acquired diseases (e.g.,
Stevens-­Johnson syndrome [SJS], ocular cicatri-
cial pemphigoid [OCP], chemical or thermal
burns, and contact lens overwear). LSCD
patients suffer from severe visual impairment
with conjunctival epithelial ingrowth, neovascu-
larization of corneal stroma, and corneal opacifi-
cation [4, 5]. Partial LSC abnormality is
presumed to be one of the causes of pterygium, a
fibrovascular tissue which grows over the limbus
onto the cornea [6]. Patients with pterygium suf-
fer from impaired vision due to high astigma-
tism, altered tear film, binocular diplopia,
irritation, etc. Partial LSCD is also brought by a
surgical excision of limbus in patients with ocu-
lar surface neoplasia.
The effort to treat LSCD with LSCs starts
after the establishment of the concept of LSCs in
1986 [1]. After the development of conjunctival
limbal autograft (CLAU), keratolimbal allograft
(KLAL), living-related conjunctival limbal
155
156
allograft (Lr-CLAL), cultivated limbal epithe-
lial transplantation (CLET), and simple limbal
epithelial transplantation (SLET) were developed
one after the other, and cultivated oral mucosal
epithelial transplantation (COMET) was devel-
oped as a transplantation using alternative source
of LSCs.
11.2.1 Conjunctival Limbal Autograft
(CLAU)
Conjunctival autograft was performed for the
treatment of LSCD in the early 1980s [7]. After
the establishment of the concept of LSCs in 1986
by Schermer et al. [1], Kenyon et al. introduced
CLAU, which contains limbal stem cells in the
graft [8]. This was the first stem cell-based ther-
apy for LSCD.
11.2.1.1 Indications
Indication of CLAU includes corneal vascu-
larization, scarring, and long-term ocular sur-
face failures including epithelial defects,
keratinization, symblepharon, inflammation
and ocular pain, and reduced visual acuity in
patients with LSCD [8]. As CLAU uses the
contralateral uninjured limbal tissue as a graft,
its indication is limited to the unilateral
LSCD.  CLAU is also adopted as one of the
surgical treatments of pterygium because
destruction of LSCs resulting from chronic
exposure to ultraviolet radiation has been
thought to play a major role (besides CLAU,
bare sclera technique, free conjunctival auto-
graft, and amniotic membrane (AM) are the
other surgical options) [9–12]. Visually sig-
nificant induced astigmatism, threat of
involvement of the visual axis, severe symp-
toms of irritation and cosmesis are the surgical
indications of pterigium. CLAU is also used to
promote epithelialization and prevent fibro-
vascular proliferation and conjunctivalization
of the cornea after excision of neoplasia when
the ocular surface neoplasia extensively
involves the limbal area [13, 14].
Y. Sasamoto et al.
11.2.1.2 Procedure
LSCD (Fig. 11.1)
Abnormal corneal epithelium and fibrovascular
pannus are stripped by blunt dissection using for-
ceps, spatula, and/or scarifier and polish the
exposed stromal surface to improve clarity. Two
grafts from limbus and adjacent conjunctiva are
excised from the contralateral eye. The grafts are
transferred to their corresponding sites in the
recipient eye and sutured at the both corneal and
conjunctival edge [8]. Simultaneous or subse-
quent penetrating keratoplasty (PK) or deep
lamellar keratoplasty should be considered when
the stromal scarring hampers improvement of
visual acuity.
Combination of CLAU with AM transplanta-
tion (AMT) may result in a higher success rate
because AM provide a basement membrane for
epithelial migration and healing and reduce
inflammation, scarring, and vascularization [15].
AM, with the basement membrane side up, is
grafted onto the defect of the donor site and onto
the recipient corneal and limbal sclera before
transplantation of grafts.
Traditionally, CLAU has involved the removal
of two 60° large free grafts from the healthy fel-
low eye [16]. There is a report suggesting only
Fig. 11.1  Limbal stem cell deficiency. Limbal stem cell
deficiency caused by Stevens-Johnson syndrome. Corneal
surface is totally covered by vascularized conjunctival
epithelium
11  Corneal Stem Cell-Based Therapies
Fig. 11.2  Pterygium. Thickened vascularized pterygium
invaded into pupillary zone of patient’s cornea at 9 o’clock
position
one 60° graft together with AMT and fibrin glue
successfully reconstructed a corneal surface [17],
but it’s still controversial [18, 19].
Pterygium (Fig. 11.2)
The head of pterygium is scraped from the cor-
neal surface followed by excision of the body of
pterygium from the surrounding conjunctiva and
removal of episcleral scarring. A graft excised
from the limbus and conjunctiva of the contralat-
eral eye is sutured onto the recipient bed [20, 21].
Fibrin glue can be used instead of suture [22–24].
Applying mitomycin C (MMC) and/or AMT on
the limbus and bare sclera is optional [11,
25–27].
Ocular Surface Neoplasia
After the excision of neoplastic region in the cor-
nea and conjunctiva combined with the intraop-
erative use of 0.02% MMC, CLAU obtained from
the contralateral eye is transplanted on the exci-
sion site and sutured onto the sclera. Graft should
be enough large to cover the bare area [14].
11.2.1.3 Outcome
First series of CLAU was reported by Kanyon
et al. in 1989 [8]. They performed CLAU in 21
patients with unilateral LSCD.  Most patients
experienced prompt reduction and permanent
157
resolution of chronic external ocular inflamma-
tion. After 6-month follow-up or more, visual
acuity was improved in 17 cases (81%), and sta-
ble epithelial adhesion without recurrent erosion
or persistent epithelial defect was recorded in 20
cases (95.2%). There was no immune rejection as
expected. They did not encounter infection, lim-
bal graft failure, and sloughing of the grafts.
Meallet MA et al. reported that CLAU com-
bined with AMT improved visual acuity, corneal
clarity, and successful ocular surface reconstruc-
tion in all five patients (mean follow-up period:
22  months) [15]. In the other study of CLAU
combined with AMT, the overall cumulative graft
survival was obtained in eight of ten eyes (80%)
(mean follow-up period: 33  months) [28].
However, comparative study showed no differ-
ence in visual acuity and ocular surface recon-
struction between CLAU with AMT and without
AMT [29].
Recurrence rate of pterygium treated with
CLAU was reported to be 2.14% (5 recurrences
in 234 procedures) after 2-year follow-up.
Recurrences were observed in 4 of 58 (6.9%)
cases with recurrent pterygium and 1 of 178
(0.57%) cases with primary pterygium in this
study [30]. In the study of 10-year follow-up, 2 of
29 (6.9%) cases had recurrent pterygium after
CLAU [31].
Systematic review shows that CLAU signifi-
cantly reduces the risk of pterygium recurrence
compared with pterygium excision with bare
sclera technique (odds ratios [OR]  =  0.08,
p  <  0.01), bulbar conjunctival autograft
(OR = 0.10, p < 0.01), and intraoperative MMC
(OR = 0.22, p < 0.01) [32]. There was no signifi-
cant difference between CLAU and AMT
(OR = 0.66, p = 0.39). Another systematic review
shows that CLAU significantly reduced the risk
of pterygium recurrence compared with con-
junctival autograft (relative risk [RR]  =  0.09,
p = 0.02) [33].
As for combination therapy, development of
recurrence is less in CLAU with AMT group than
MMC with AMT group (0% vs 20%) for the treat-
ment of recurrent pterygium [34]. There is no signifi-
158
cant difference in recurrence rates between CLAU
with MMC and conjunctival autograft with MMC
in primary and recurrent pterygium [11]. Also,
there is no significant difference in recurrence
rates between CLAU with MMC and AMT with
MMC in recurrent pterygium [35].
It is still controversial whether the application
of CLAU in addition to the excision of ocular
neoplasia is necessary to prevent recurrence [14].
11.2.2 Keratolimbal Allograft (KLAL)
As CLAU requires relatively large biopsy of lim-
bus, this procedure frequently brings LSC deple-
tion in the healthy donor eye. In addition, CLAU
cannot apply to the patients with bilateral
LSCD. KLAL was developed to overcome these
situations [36]. KLAL uses cadaveric donor lim-
bal tissue, so that there is no concern about the
LSC depletion in the contralateral eye and this
procedure is applicable to bilateral LSCD
patients. However, systemic immunosuppression
is required to avoid graft rejection.
11.2.2.1 Indications
KLAL is applicable to both unilateral and bilat-
eral LSCD.
11.2.2.2 Procedure (Fig. 11.3)
Corneoscleral rims obtained from two cadaveric
donor eyes are sectioned into two equal halves,
and they are thinned by removing the posterior
two thirds of corneoscleral tissue. On the recipi-
ent eye, a 360° limbal peritomy is performed.
After the removal of abnormal fibrovascular pan-
nus and epithelium, three of the four dissected
crescents are transplanted on the recipient eye
with the donor corneal edge overlying the recipi-
ent limbus and are secured with sutures [37].
Fibrin glue can be used instead of suture [38].
Simultaneous AMT is optional [39]. Other than
topical immunosuppression using corticosteroid
and cyclosporine A, oral immunosuppressants,
including prednisone, cyclosporine A, and aza-
thioprine, should be considered [37].
Simultaneous or subsequent keratoplasty can be
considered when the stromal scarring hampers
improvement of visual acuity.
Y. Sasamoto et al.
11.2.2.3 Outcome
Holland et al. reported that regeneration of normal
ocular surface obtained in 23 of 31 patients
(74.2%) and visual acuity improvement was
obtained in 27 of 31 patients (87.1%) and there
were no adverse reactions in patients with bilateral
LSCD caused by aniridia [37]. Of note, 20 eyes
(64.5%) underwent subsequent PK. A systematic
review shows that 20 of 29 eyes (69%) had best-
corrected visual acuity (BCVA) ≥20/200 at last
follow-up (median follow-up period: 42 months)
and 32 of 36 eyes (80%) underwent simultaneous
or subsequent PK [40]. Solomon A. et al. reported
long-term outcome of KLAL and AMT with or
without PK, but simultaneous KLAL and PK may
be associated with a less favorable outcome [39].
In eyes with unilateral LSCD, Shimazaki et  al.
reported that KLAL resulted in a worse clinical
outcome than CLAU [41].
11.2.3 Living-Related Conjunctival
Limbal Allograft (Lr-CLAL)
Lr-CLAL was developed to reduce the risk of
rejection by KLAL [42, 43]. The limbal and
conjunctival tissues are harvested from a
patient’s living relative. Systemic immunosup-
pression is still required at least in HLA-
incompatible cases.
11.2.3.1 Indications
Indications of Lr-CLAL are similar to that of
KLAL (both unilateral and bilateral LSCD), but
Lr-CLAL is applicable to the patients who has a
willing living relative. A good HLA match is
preferable (Ophthalmology. 2001 Daya SM,
Ophthalmology 1999, Srinivas KRaoDO).
For the eyes with severe LSCD, combination
of KLAL and Lr-CLAL can be considered
(Cincinnati Procedure) [47].
11.2.3.2 Procedure
Two grafts from limbus and adjacent conjunc-
tiva are excised from the donor eye [44].
Transplantation onto the recipient cornea is
same as CLAU.  Simultaneous AMT, PK, and
the use of fibrin glue instead of suture are
optional [24, 45].
11  Corneal Stem Cell-Based Therapies 159

a e

b f

c g

d h

Fig. 11.3  KLAL procedure. (a) Preoperative status; (b) surface. (d–f) Limbal graft was created using cadaver
peritomy was performed to separate abnormal fibrovascu- sclerocorneal tissue using trephine, golf blade, and scis-
lar tissue inside the cornea from conjunctiva on the sclera. sors. (g, h) Limbal tissue from donor was sutured to host
(c) Fibrovascular pannus was removed from the corneal limbus
160
11.2.3.3 Outcome
Daya et al. reported the outcome of ten eyes of
Lr-CLAL (mean follow-up period: 26.2 months)
[44]. Systemic immunosuppression was adminis-
tered in all the patients. Restoration of corneal
epithelium and reduction of vascularization were
obtained in eight eyes (80%). Visual improve-
ment was achieved in seven eyes (70%). Allograft
rejection occurred in two eyes (25%), which had
two class I HLA mismatches and underlying
diagnosis of SJS. Gomes et al. reported the out-
come of Lr-CLAL with AMT in ten LSCD eyes
caused by SJS (mean follow-up period:
1.67 months) [45]. Nine patients (90%) were not
HLA-compatible with their respective living-­
related donors and required systemic immuno-
suppression. Ocular surface reconstruction was
obtained only in two eyes (20%), infection
occurred in four eyes (40%), and visual acuity
improvement was achieved in four eyes (40%).
When compared to KLAL, Lr-CLAL had a
higher gain in vision (p = 0.029), decrease in con-
junctivalization (p  =  0.009), and increase in
Schirmer’s values (p = 0.009) in eyes with ocular
burns [46]. The other group showed that CLAU
achieved better long-term corneal surface results
compared to KLAL and Lr-CLAL [33]. Although
they showed no significant difference between
KLAL and Lr-CLAL, they indicate that the out-
come of Lr-CLAL may be improved by HLA
matching and systemic immunosuppression.
Biber et  al. reported clinical outcome of 24
eyes which performed combination of KLAL and
Lr-CLAL (mean follow-up period: 43.4 months)
[47]. Keratoplasty was undergone in 79.2% eyes
after ocular surface reconstruction. Stable or
improved ocular surface was achieved in 87.5%,
and improved visual acuity was obtained in 75%.
11.2.4 Cultivated Limbal Epithelial
Transplantation (CLET)
CLAU potentially damages the donor cornea due
to a large excision [48]. To overcome this disad-
vantage, Pellegrini et  al. developed autologous
cultivated limbal epithelial transplantation
(CLET) for the treatment of unilateral LSCD
Y. Sasamoto et al.
[49]. They utilized cells harvested from the small
biopsy specimen of contralateral limbus.
Allogenic CLET was developed for the eyes with
bilateral LSCD [50]. This technique is advanta-
geous in regard to the risk of donor cornea dam-
age when compared to KLAL and Lr-CLAL.
11.2.4.1 Indications
Autologous CLET is applicable to the patients
with unilateral LSCD, and allogeneic CLET is
applicable to the patients with bilateral LSCD.
11.2.4.2 Procedure
One of the standard procedures was reported by
Rama et  al. [51]. A 1–2  mm2 limbal biopsy is
taken from contralateral eye, and primary limbal
keratinocytes are cultivated on fibrin and feeder
layer of lethally irradiated 3T3-J2 cells for
14–16 days. After a 360° limbal peritomy and the
removal of fibrovascular corneal pannus in the
recipient eye, the fibrin-cultured epithelial sheet
is transplanted on the prepared corneal wound
bed. The conjunctiva is then sutured over the
peripheral fibrin sheet. Xeno-free explant culture
system on human AM was developed to reduce
the risk of transmission of zoonotic infection
from mouse 3T3 cells and fetal bovine serum in
the medium [52].
Use of systemic immunosuppression is con-
troversial after allogenic CLET. When the central
cornea scarring exists, simultaneous or subse-
quent keratoplasty is optional [53].
11.2.4.3 Outcome
Long-term CLET study of patients with unilateral
LSCD caused by ocular surface burns was reported
by Rama et  al. in 2010 (mean follow-up period:
2.91  ±  1.99  years) [51]. They cultivated limbal
keratocyte on fibrin and 3T3-J2 feeder cells. Of the
107 eyes treated, 73 eyes (68.2%) were classified
as success, and 60 eyes (56.1%) had improved
visual acuity. Sangwan et  al. reported long-term
outcome of CLET using a xeno-free explant cul-
ture system and human AM as a substrate for the
treatment of LSCD caused by ocular surface burns
(mean follow-up period: 3 ± 1.6 years) [52]. Of the
200 eyes treated, completely epithelialized, avas-
cular, and clinically stable corneal surface was
11  Corneal Stem Cell-Based Therapies
obtained in 142 (71%) eyes, and visual acuity was
improved in 121 eyes (60.5%).
Basu et al. reported the outcome of the combi-
nation of CLET and PK in 47 patients (mean
follow-up was 4.2 ± 1.9 years) [54]. Visual acuity
of 20/40 or better was attained by 71.4% of the
eyes. However, as simultaneous PK lead higher
recurrence rate of LSCD (58.3%) than subse-
quent PK (14.3%), they suggest that simultane-
ous PK should be avoided. Allogeneic CLET,
followed by PK when needed, can restore the
ocular surface and improve vision in patients
with bilateral LSCD [55]. Qi et al. administered
systemic steroid and topical steroid and cyclo-
sporine A after allogenic CLET and rejection
were seen in 10 eyes of 42 eyes (23.8%) during
the follow-up of 17.8 ± 3.8 months [56].
A couple of systematic reviews show the
effectiveness of CLET.  Holland reported long-­
term outcome of 720 eyes treated with either
autologous (641 eyes) or allogenic CLET (79
eyes) [57]. Overall success rate was 72%, and
improved visual acuity was obtained in 63%.
Haagdorens et  al. reported clinical outcomes of
1029 cases of autograft and 135 cases of allografts
[58]. Overall success rate was 70%, and improved
visual acuity was obtained in 55%. Zhao et  al.
reported clinical outcomes of 572 cases of CLET
with human AM as the substrate [59]. The suc-
cess rate was 67%, and improved visual acuity
was achieved in 62%.
11.2.5 Simple Limbal Epithelial
Transplantation (SLET)
CLET was developed to overcome the disadvan-
tage of CLAU, which may damage the donor cor-
nea due to a large excision. However, CLET
requires high-cost clinical-grade laboratory sup-
port for the graft cultivation and two steps of sur-
gical procedures (limbal excision and
transplantation). To overcome these situations,
Sangwan et  al. developed SLET by combining
the benefits of CLAU and CLET [60]. This tech-
nique utilizes small portion of autologous donor
limbus for transplantation and does not require
clinical-grade laboratory.
161
11.2.5.1 Indications
Patients with unilateral LSCD.
11.2.5.2 Procedure [60, 61]
A 1-clock hour limbal tissue is excised from the
contralateral donor eye. After the removal of
vascular pannus covering the cornea, human
AM graft is placed over the bared ocular sur-
face and secured with fibrin glue. The donor tis-
sue is then cut into small pieces, placed on the
human AM, and fixed with fibrin glue.
Keratoplasty can be combined with SLET to
improve vision.
11.2.5.3 Outcome
Basu et  al. reported the outcome of SLET in
125 patients with unilateral LSCD caused by
chemical or thermal burns (median follow-up
period: 1.5 years) [61]. Successful outcome (a
completely epithelialized, clinically stable,
and avascular corneal surface) was observed in
95 eyes (76%), and two-line improvement in
visual acuity was seen in 94 eyes (75.2%).
Randomized study shows that a stable epitheli-
alized corneal surface was obtained in all
patients and there was no significant difference
in the vision improvement between CLAU and
SLET [62].
11.2.6 Cultivated Oral Mucosal
Epithelial Transplantation
(COMET)
KLAL, Lr-CLAL, and allogenic CLET are per-
formed for the treatment of bilateral LSCD, but
those techniques have disadvantages arisen
from their allogenic manner, that is, rejection,
adverse events associated with immunosuppres-
sion, and disease transmission. Autologous
COMET was developed to eliminate these con-
cerns [63]. Cultivated oral mucosal epithelial
cells contain epithelial stem cells which work as
an alternative source of LSCs on the ocular
surface.
11.2.6.1 Indications
Patients with bilateral LSCD.
162 Y. Sasamoto et al.

11.2.6.2 Procedure [63, 64] (Fig. 11.4)
3-by-3-mm specimen of oral mucosal tissue is
excised from the interior buccal mucosal epithe-
lium. Dissociated oral mucosal epithelial cells are
seeded onto a human AM or fibrin-coated culture
plate or a temperature-responsive dish, under
which 3T3 cells are cultured as feeder layers and
incubated for 14  days to obtain epithelial cell
sheet. Three to five layers of cultivated oral muco-
sal epithelial sheets are placed directly onto the
corneal surface after excision of conjunctival and
subconjunctival scar tissue. Although xenogenic
feeder cells have a potential risk to transmit virus
infection, autologous feeder layer can be a resolu-
tion [65]. Transportation technique for cultivated
cell sheet has been developed for multicenter clini-
cal trial [66]. To improve corneal stromal transpar-
ency, secondary keratoplasty can be considered.

a b

c d

e f

Fig. 11.4  COMET procedure. (a) Preoperative status; host ocular surface. (e) Continuous suture using 10-0
(b) fibrovascular pannus was removed from cornea. (c) nylon was added to limbus. (f) Ocular surface was covered
Cultivated oral mucosal epithelial cell sheet was harvested by cell sheet and host conjunctiva
with temperature reduction. (d) Cell sheet was placed on
11  Corneal Stem Cell-Based Therapies
11.2.6.3 Outcome
Satake et al. reported a long-term outcome of 40
bilateral LSCD eyes treated by COMET (mean
follow-up period: 25.5 months) [64]. Stable ocular
surface was achieved in 57.5% eyes, and improved
visual acuity was obtained in 59% eyes. A second-
ary keratoplasty was performed to improve visual
acuity in seven eyes. Another long-term clinical
report shows stable ocular surface was achieved in
15 of 20 eyes (75%) and improved visual acuity
was obtained in 14 eyes (70%) (mean follow-up
period: 31.9 months) [67].
11.2.7 Complications (Fig. 11.5)
Postoperative complications include recurrence
or persistence of epithelial defects, continued
conjunctivalization (recurrence of pterygium),
Fig. 11.5  Complications. (a) Fungal keratitis caused by Candida parapsilosis following limbal transplantation. (b)
Rejection following allogenic limbal transplantation. Rejection line was observed (arrow head)
163
subconjunctival hemorrhage, corneal thinning (or
melting, perforation), inflammation, and infec-
tious keratitis. In the donor eyes of CLAU and
Lr-CLAL, biopsy-related epithelial abnormali-
ties, filamentary keratitis, and subconjunctival
hemorrhage have been reported [43, 48, 68].
However, LSCs repopulate the donor area within
1 year, and removal of 2 pieces of 2 clock hours
each of donor limbus does not bring any adverse
effect in the donor cornea [48, 69].
In KLAL, CLAL, and allogenic CLET, there
are risks of transplant rejection and systemic
immunosuppression-related adverse events such
as infection, anemia, hyperglycemia, and renal
and liver function abnormalities [70–72]. In those
allogenic techniques, there are some reports of
disease transmissions, such as tumor (melanoma
and breast carcinoma) and cytomegalovirus dis-
ease [73–75].
164
Development of superficial corneal neovascu-
larization under the epithelial sheet is the major
concern in COMET [63, 76].
3T3 cells and fetal bovine serum for cell cul-
ture can increase the risk of transmission of zoo-
notic infection in CLET and COMET [77].
11.3 F
 uture Stem Cell-Based
Therapy: Corneal Epithelium
As each current method has its pros and cons, no
gold standard exists so far. Especially for the
treatment of bilateral LSCD, the necessity of sys-
temic immunosuppression is a big concern.
COMET can solve this barrier, but the recon-
structed ocular surface does not allow a full,
long-term improvement of visual acuity and is
prone to problems associated with
neovascularization.
Therefore, the researchers are seeking the
other sources of stem cells to overcome the
situation.
The cell sources being investigated can be cat-
egorized into four groups: (i) corneal epithelial
cells induced from pluripotent stem cells, (ii) cor-
neal epithelial cells induced from the other dif-
ferentiated cell source (direct reprogramming),
(iii) corneal epithelial alternatives from the other
surface ectoderm-derived cells, and (iv) purified
LSCs.
11.3.1 Corneal Epithelial Cells
Induced from Pluripotent
Stem Cells
Researchers are trying to generate corneal epithe-
lial cells or LSCs from three different sources of
pluripotent stem cell, that is, embryonic stem
cells (ESCs), induced pluripotent stem cells
(iPSCs), and mesenchymal stem cells (MSCs).
Corneal epithelial-like cells were successfully 11.3.2 Corneal Epithelial Cells
induced from ESCs in limbal fibroblast-­
conditioned medium [78]. However, there are
some obstacles such as immunogenicity, uncon-
trolled growth which results in increased tumori-
genicity, and ethical issues arisen from its Direct reprogramming, the way to convert fully
embryonic origin [79]. differentiated mature cells into the other cell
Y. Sasamoto et al.
iPSCs were developed by introducing
Yamanaka four factors into the differentiated cells
such as fibroblasts and blood cells and were
shown that they have the similar characteristics as
ESCs [80]. This technique can overcome the ethi-
cal problem. Hayashi et  al. reported a way to
induce corneal stem and progenitor cells from
iPSCs by mimicking whole-eye development [81,
82]. They successfully generated a corneal epithe-
lial cell sheet, and its transplantation recovered
the LSCD model eye in rabbit (Fig.  11.6).
However, generation of iPSCs and induction of
corneal epithelial cells take long time and have
high cost. Creation of HLA-typed iPSC banks can
be one of the solutions [83]. It is calculated that 50
iPSC lines of the highest-ranked homozygous
HLA types can provide a zero HLA mismatch for
79% of potential UK recipients. One strategy is to
induce corneal epithelial stem and progenitor
cells from top-ranked HLA-typed iPSCs and store
them in advance, and then we can thaw and cul-
ture them when needed. There still remain some
issues to be solved about tumorigenicity, immu-
nogenicity, and mutagenicity of iPSCs [84, 85].
MSCs are multipotent adult stem cells which
reside in multiple tissues, including the umbilical
cord, bone marrow, and adipose tissue. MSCs not
only can overcome the ethical issues of ESCs but
also can be obtained much easier than ESCs and
iPSCs. MSCs also have less immunogenicity
even when being used as allogenic transplanta-
tion and may have less mutagenicity [86, 87].
MSCs were shown to differentiate into corneal
epithelial-like cells in LSC niche environment
in  vitro [88–90] and in  vivo [91]. Dental pulp
stem cells, which share the characteristics of
MSCs and ESCs, can generate a cell sheet and
reconstruct the corneal epithelium in a rabbit
model of LSCD [92]. There still remains a con-
cern of tumorigenicity [93].
Induced from the Other
Differentiated Cell Source
(Direct Reprogramming)
11  Corneal Stem Cell-Based Therapies
b Before
iCEC
Control (sham)
Fig. 11.6 Ocular surface reconstruction using iPSC-­
derived corneal epithelial cell sheet in rabbit model. (a) A
corneal epithelial cell sheet derived from human iPSCs.
(b) Barrier function assay using fluorescein staining for
iPSC-derived corneal epithelial cell sheet transplanted
165
7d 14d
rabbit LSCD model on days 0, 7, and 14 post-surgery.
iPSC-derived corneal epithelial cell sheet successfully
recovered a healthy corneal barrier function. iCEC iPSC-­
derived corneal epithelial cells. (Reproduced from
Hayashi et al. [82])
166 Y. Sasamoto et al.

types while bypassing a pluripotent state, can 11.4 F

 uture Stem Cell-Based
shorten the induction time of corneal epithelial Therapy: Corneal Stroma
cells from the other types of cells and reduce the
risk of tumorigenicity [94]. Ouyang et al. induced Corneal stroma consists of collagens, proteogly-
LSC-like cells by transduction of PAX6 in skin cans, glycoproteins, and a small amount of stro-
epithelial stem cells in  vitro [95]. Corneal mal cells known as keratocytes. PK and deep
epithelial-­like cells can also be induced from anterior lamellar keratoplasty have been per-
fibroblasts and oral mucosal epithelial cells by formed for the treatment of corneal stromal dis-
overexpression of gene sets including PAX6 [96, eases [106].
97]. Although gene delivery has a potential of Corneal stromal stem cells are a population of
insertional mutagenesis, transgene-free methods neural crest-derived MSC which resides in the
such as Sendai virus, direct mRNA, or protein limbal stroma, subjacent to the epithelial base-
delivery can solve this issue [98]. Interestingly, ment membrane [107]. Corneal stromal stem
hair follicle epithelial stem cells and limbal fibro- cells injected into the stroma can restore the
blasts have a potential to differentiate into cor- transparency and thickness of lumican-defective
neal epithelial-like cells without any gene murine corneas [108].
modification [99, 100]. Keratocyte-like cells have been induced from
ESCs [109, 110], iPSCs [111], MSCs [112], and
human dental pulp stem cells [113] in  vitro.
11.3.3 Corneal Epithelial Alternatives MSCs are shown to have an ability to treat cor-
from the Other Surface neal stromal diseases in animal models. MSCs
Ectoderm-Derived Cells injected into the mouse corneal stroma can sur-
vive for more than 3  months, and these cells
Other than oral mucosa, there are some other sur- assumed a keratocyte phenotype [114]. Also,
face ectoderm-derived cells that can be alterna- MSCs injected into the anterior chamber can
tives of corneal epithelium. Conjunctival migrate to the stroma and differentiate into func-
epithelial cell cultivated ex  vivo (EVCAU) is tional keratocytes [115].
already performed as a clinical trial for the treat-
ment of bilateral LSCD [101]. Clinical improve-
ment was observed in 83.3%, and visual acuity 11.5 F
 uture Stem Cell-Based
was improved in 75% cases. Nasal mucosal epi- Therapy: Corneal
thelial cells also have a potential to reconstruct Endothelium
functional ocular surface epithelial cells [102].
Loss or dysfunction of corneal endothelium leads
to bullous keratopathy (BK). Major causes of BK
11.3.4 Purified LSCs are cataract surgery, peripheral iridotomy, and
Fuchs endothelial corneal dystrophy (FECD)
Ksander et al. reported that cell membrane pro- [116]. As there were no efficient ways to prolifer-
tein ABCB5 is a marker of LSCs [103]. They ate corneal endothelial cells, BK has been treated
showed that LSCs can be purified by anti-ABCB5 by corneal transplantation such as PK, Descemet
antibody. Transplantation of purified ABCB5-­ stripping automated endothelial keratoplasty
positive LSCs can restore the corneal epithelium (DSAEK), and Descemet membrane endothelial
in a LSCD model mouse. As ABCB5-positive keratoplasty (DMEK) [117–119]. Recently,
dermal stem cells are reported to induce allograft ROCK inhibitor was shown to enhance prolifera-
tolerance to HLA-mismatched transplants, tion, promote cell adhesion, and suppress apopto-
ABCB5-positive LSCs may be a good candidate sis of corneal endothelial cells [120, 121]. Pilot
for allogenic transplantation in patients with data from a clinical trial which injected cultured
bilateral LSCD [104, 105]. corneal endothelium in combination with a
11  Corneal Stem Cell-Based Therapies
ROCK inhibitor into the anterior chamber has
shown its safety and effectiveness [121].
Autologous or allogenic cultured corneal endo-
thelial cell transplantation can be applied to the
treatment of BK in the future.
The existence of corneal endothelial stem
cells had been discussed for a long time [122,
123]. Recently, multipotent neural crest-derived
progenitor (NCDP) cells were isolated from cor-
neal endothelium, and corneal endothelial cells
were induced from NCDPs [124]. As normal
NCDPs were detected not only in normal but also
in Fuchs endothelial corneal dystrophy (FECD)
endothelial tissue, this cell population has prom-
ise for potential autologous cell therapies.
Human corneal endothelium-like cells have
been induced from ESCs [125–127], iPSCs
[128], MSCs [129], adipose-derived stem cells
[130], and corneal stroma stem cells [131].
11.6 Summary and Conclusions
Clinical studies have shown certain degrees of
success in several stem cell-based therapies espe-
cially in the treatment of LSCD.  However, we
still need to establish more evidence to decide
which procedure is preferable for a specific con-
dition. In addition to the therapies currently per-
formed, an increasing number of novel techniques
using various stem cells are being investigated.
ESCs, iPSCs, and MSCs are increasing their
presence for the induction of not only corneal
epithelial cells but also keratocytes and corneal
endothelial cells. These techniques can be one of
the promising future therapies, but there are many
obstacles to be solved, e.g., ethical issues, tumor-
igenicity, immunogenicity, and mutagenicity.
Besides possible future therapies shown
above, building an entire functional cornea,
which has organized epithelium, stroma, and
endothelium, may be the next breakthrough. Very
recently, a corneal organoid which contains these
three components has been induced from human
iPSCs [132], and an artificial cornea which con-
sists of limbal epithelial cell-like cells and cor-
neal endothelial cell-like cells derived from
human ESCs and acellular porcine cornea matrix
167
has been developed [133]. Drastic development
of technologies is expediting the research of cor-
neal stem cell therapy.
Compliance with Ethical Requirements Yuzuru
Sasamoto and Yoshinori Oie declare no conflict of inter-
est. Prof. Kohji Nishida received a Grant for Translational
Research Network Program supported by the Ministry of
Education, Culture, Sports, Science and Technology of
Japan. All procedures followed were in accordance with
the ethical standards of the responsible committee on
human experimentation (institutional and national) and
with the Helsinki Declaration of 1975, as revised in 2000.
Informed consent was obtained from all patients for being
included in the study. All institutional and national guide-
lines for the care and use of laboratory animals were
followed.
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 Part III
Regenerative Surgery and Therapy of the
Ocular Surface Epithelium
 Ocular Surface Epithelium:
Applied Anatomy
Harminder Singh Dua and Dalia G. Said
Key Points
• Anatomically the ocular surface con-
sists of the conjunctiva, limbal and cor-
neal epithelium covered by the tear film.
Functionally, the blink reflex and the
lacrimal drainage apparatus are also
important components.
• The tear film affords an optical polish to
the corneal surface and also plays an
important role in ocular surface defence.
• The corneal epithelium is made of five
layers of cells. The basal cells have
H. S. Dua (*)
Academic Section of Ophthalmology, Division of
Clinical Neuroscience, University of Nottingham,
Nottingham, UK
Department of Ophthalmology, Queens Medical
Centre, University Hospitals NHS Trust,
Nottingham, UK
e-mail: harminder.dua@nottingham.ac.uk
D. G. Said
Academic Section of Ophthalmology, Division of
Clinical Neuroscience, University of Nottingham,
Nottingham, UK
Department of Ophthalmology, Queens Medical
Centre, University Hospitals NHS Trust,
Nottingham, UK
Research Institute of Ophthalmology (RIO),
Cairo, Egypt
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_12
12
mitotic activity and are transient ampli-
fying cells. The adhesion complexes of
the basal epithelium are stronger  and
manifold at the periphery than centre;
hence the central epithelium abrades
easily.
• Turnover of corneal epithelium is main-
tained by stem cells that reside in pali-
sades of Vogt and limbal epithelial
crypts.
• In physiological homeostasis, the basal
epithelial cells can maintain the epithe-
lial cell mass, but the limbal stem cells
play a key role in response to injury and
disease.
• Damage or destruction of the limbus
leads to limbal stem cell deficiency that
can alter the nature of the corneal epi-
thelium and affect vision.
• The conjunctiva is an integral part of the
mucosal immune system. Conjunctival
epithelium is of variable thickness and
is endowed with goblet cells that secrete
the major component of the  mucins of
the tear film.
• The cornea is the most sensitive struc-
ture of the human body and is supplied
by a dense network of sensory and tro-
phic nerves from the fifth cranial nerve.
It also has an autonomic nerve supply,
which is less well defined in humans.
175
176
The term ‘ocular surface’ (OS) has anatomical
and functional connotations. Anatomically it
includes the mucosal lining of the lid margins
and conjunctiva and the limbal and corneal epi-
thelium. Functionally the OS includes the eye
lids and associated blink reflex, the tear film
together with the various glands that secrete tear
components and the lacrimal drainage system.
The transparent cornea is the key component of
the OS, permitting the regular passage and focusing
of light on its way to the retina. The OS functions as
a unit to maintain the health and optical qualities of
the cornea. The OS also interfaces with the envi-
ronment and hence is endowed with an array of
innate and adaptive immune mechanisms, coupled
with an intricate sensory neuronal input providing
both trophic and protective defence responses,
emphasizing the importance of vision, in evolution-
ary terms, for the survival of the species.
The cornea extends from limbus (corneo-­
scleral junction) to limbus with a horizontal diam-
eter of 11.5–12 mm and approximately 1 mm less
vertically. It appears to be oval as the sclera
encroaches on the superior and inferior limbus;
however, if viewed from its inner aspect, the cor-
nea appears circular with an average diameter of
11.7  mm. It is prolate in shape with a steeper
radius of curvature centrally than peripherally.
Similarly, its thickness increases from about
0.53 mm centrally to approximately 0.71 mm at
the periphery [1, 2]. Despite the highly organized
and regular structure of the corneal epithelium, it
is not a perfect optical surface. The optical polish
is provided by the tear film, which is crucial for
both vision and corneal health.
The cornea is made of five layers, namely,
from anterior to posterior, the epithelium,
Bowman’s layer, stroma, Descemet’s membrane
(DM) and endothelium. There is increasing evi-
dence to support the presence of a distinct layer,
the pre-Descemet’s layer (Dua’s layer), just ante-
rior to the DM [3].
12.1 Corneal Epithelium
Ocular surface epithelium can be considered in
three distinct regions: the corneal epithelium, the
limbal epithelium and the conjunctival epithelium.
H. S. Dua and D. G. Said
The corneal epithelium is a non-keratinized
uniform stratified squamous epithelium derived
from the surface ectoderm at 5–6 weeks of gesta-
tion. It extends from limbus to limbus and is con-
tinuous with the limbal epithelium. It is formed
of 4–6 layers and ranges in thickness from 40 to
50 microns. Like all ‘covering epithelia’, its
functions include selective diffusion of gases
(oxygen from the atmosphere), nutrients and
waste products of cell metabolism and at the
same time preventing excessive loss of fluid;
secretion of a variety of substances as part of nor-
mal physiology and in response to insult, such as
mucins and antimicrobial peptides; physical pro-
tection against abrasive forces; and a barrier
function between the cornea and the outside
world. All layers of the epithelium are extremely
well organized in the interest of maintaining
transparency of the cornea. The absence of blood
vessels and relative sparsity of intracellular
organelles also contribute to corneal epithelial
transparency. The corneal epithelium is made of
three types of cells that demonstrate polarity
between the external and internal environment
[4] (Fig. 12.1).
Superficial Flat Cells  The superficial 2–3 lay-
ers of epithelium are made of polygonal flat cells
(50 × 5 microns) with flat nuclei (Fig. 12.1a, b).
The anterior surface of the anterior most layer of
cells shows well-developed ‘apical microvilli
“finger like projections” and micro plicae “ridge
like projections”’, which increase the surface
area of the cell membrane and are covered by a
charged glycocalyx layer secreted by the epithe-
lial plasma membrane. This strengthens the adhe-
sion of the mucinous layer of the tear film to the
epithelial cell membrane. Deficiency in this layer
leads to tear film instability due to poor mucin
adhesion that can be detected by a rapid tear film
breakup time [5]. Wing cells (suprabasal cells):
Beneath the superficial layers, there are 2–3 lay-
ers of wing cells which have a central oval or
polyhedral cell body from which wing-like lat-
eral processes extend on either side (20 microns
in diameter), convex anterior surface and a con-
cave posterior surface. Both superficial cells and
wing cells are postmitotic. Basal cells: The deep-
est layer of the corneal epithelium, ‘the basal
12  Ocular Surface Epithelium: Applied Anatomy
a b
d e
Fig. 12.1 (a) Transmission electron micrograph (TEM) of
the stratified corneal epithelium showing columnar cells in
the basal layer; wing cells in the middle layers and flat
squamous cells in the superficial layer. (b) Scanning elec-
tron micrograph (SEM) of the anterior surface of the super-
ficial squamous cells showing the polyhedral outline. The
cell outlines are clearly visible. The dark ‘holes’ seen at the
junction of cells are fixation artefacts. (c) TEM of corneal
epithelial cells illustrating hemidesmosomal attachments
(arrows) with the underlying substratum (amniotic mem-
brane). (d) TEM of basal epithelial cells that have been
layer’, comprises of a single layer of columnar
cells (20 microns tall) that sit on a basement
membrane, secreted by the basal cells. These
cells are mitotically active (transient amplifying
cells) (see below) [4, 6].
Cell Attachments  Like all epithelia, the corneal
epithelial cells exhibit specialized cell-cell and
cell-basement membrane attachments that inte-
grate individual cells into a cohesive sheet of epi-
thelium. The cell membranes of adjacent
superficial cells close to the apex are tightly
attached or fused together along the circumfer-
ence to form tight junctions or zonula occludens.
This forms an effective seal that restricts passage
of small molecules, water and electrolytes. The
main protein of this junction is occludin. Occludin
is closely associated with another cell protein
called ZO-1, which in turn connects with the
intracellular cytoskeletal proteins. Occludin and
ZO-1 are targeted by invading pathogens that
must breach this barrier to gain access to the epi-
thelium. Fluorescein dye normally does not pen-
etrate this barrier, and hence the normal
177
c
f
separated from the underlying stroma showing the detached
hemidesmosomes. (e) SEM of the multilayered corneal epi-
thelium and the basement membrane from which the cells
have been stripped off. (f) TEM of two basal epithelial cells
from a case of recurrent corneal erosion syndrome. The
boundary between the two cells (arrowhead) is clearly vis-
ible. The hemidesmosomes are abnormal (arrow) and rest
on collagenous debris (star) between the basal cells and
Bowman’s membrane. (Adapted from author’s own publi-
cation Browning et al. [16], Fig. 12.1f is from author’s own
publication Dua et al. [15])
epithelium does not stain. However, if this barrier
is breached or poor, fluorescein dye can outline
cells in intact epithelial sheets as is seen when
there is active migration of epithelium during
wound healing or when conjunctivalization of the
cornea occurs [7, 8].
Below the tight junctions and in all underlying
cell layers, the space between adjacent cells is
filled with a protein called E-cadherin, a glyco-
protein, which ‘cements’ the cells together to
form anchoring junctions or zonula adherens.
Zonula adherens in turn link with intracellular
cytoskeletal actin filaments. Desmosomes or
macula adherens are another well-defined type of
cell-cell adhesion structure made of electron-­
dense localized (spots) plaques on adjacent cell
walls that are linked by transmembrane cadherin
proteins called desmogleins and desmocollins.
These, like in zonula adherens, link with intracel-
lular cytoskeletal filaments. These attachments
give some flexibility to the cells, allowing indi-
vidual cells to change shape, within limits, whilst
178
retaining solidarity with the epithelial sheet.
Communicating channels, called gap junctions,
also exist between cells. These are not adherens-­
type junctions. Specialized molecules called con-
nexins form a microscopic channel called a
connexon. Connexons from adjacent cells line up
to form a continuous channel between the two
cells, called the gap junction. These channels
allow molecules less than a thousand kilo dal-
tons, but not proteins, to pass through [4, 9].
Specialized adhesion complexes exist between
the basal cells and underlying basement mem-
brane and Bowman’s layer. About 25 percent of
the cytoplasmic surface of the basal cell mem-
brane of the central basal epithelial cells is
occupied by electron-dense plaques called
­ construct above it, facilitating cell migration
hemidesmosomes, into which cytoskeletal fila-
ments (keratin) insert (Fig. 12.1c, d). Posterior to
and corresponding to the hemidesmosome is a
much narrower electron-dense line which gives
origin to fine fibrils, the anchoring fibrils, made
predominantly of collagen type VII.  Anchoring
fibrils traverse the lamina lucida and lamina
densa of the basement membrane and insert into
anchoring plaques located in the anterior one to
two microns of the Bowman’s layer. The major
protein of the anchoring plaques is laminin. Here,
the type VII collagen of the anchoring fibrils and
plaques is intricately interwoven with the type I
collagen of the Bowman’s layer. The key trans-
membrane protein of the adhesion complexes is
integrin. In addition to these specific attachments,
the cell membranes of all suprabasal cells and of
the apical surface of basal cells show undula-
tions, which interdigitate with adjacent cells
making them ‘fit’ with each other like the pieces
of a jigsaw puzzle. In epidermolysis bullosa there
is a genetic defect of type VII collagen causing
the epithelium to lose attachment to the substrate,
all over the body [10].
The intracytoplasmic structural element of all
epithelial cells, known as the cytoskeleton, is
made of intermediate filament proteins, the cyto-
keratins. Cytokeratin (CK) 3 (basic)/CK12
(acidic) pair are specific to corneal epithelium
[11, 12]. Cytoskeletal filaments attach to the dif-
ferent adhesion structures described above. Other
cytoskeletal filaments are actin filaments and
H. S. Dua and D. G. Said
microtubules. The latter are especially seen in
mitotic cells where they provide directional guid-
ance to the separating chromosomes.
Basement Membrane  The basement mem-
brane (BM), approximately 0.11–0.33 microns
thick, is secreted by the basal epithelial cells
and is made of several proteins mainly type IV
collagen, laminin and nidogens (glycopro-
teins),  fibrin and fibronectin [13, 14].
Structurally, by transmission electron micros-
copy, it demonstrates a pale anterior part, the
lamina lucida, and a dense posterior part, the
lamina densa. It serves to provide a substrate for
cell adhesion, like a foundation for the epithelial
and importantly providing a basis for cell polar-
ity and maintenance of cell stratification [6]. In
diabetes, where the BM is thickened and dupli-
cated, the anchoring filaments are not long
enough to penetrate anterior Bowman’s layer
causing the epithelium to be loosely attached
and readily come off, making diabetics suscep-
tible to corneal abrasions. A similar mechanism
has been reported in recurrent corneal erosion
syndrome, where there is an accumulation of
subepithelial collagenous debris that weakens
the attachment of the anchoring filaments [15]
(Fig.  12.1e, f). When the BM is damaged, as
after laser surgery, it takes a week or longer for
new BM to be formed, but fibronectin provides
a temporary matrix for the cells to migrate on.
Attachment of epithelium after laser keratec-
tomy is strong making the removal of epithe-
lium for retreatment more difficult [6, 16].
The corneal epithelium is self-renewing.
Superficial corneal epithelial cells are continu-
ously undergoing apoptosis,  shedding and are
replaced by cell migration from the deeper layers
of the epithelium with a complete turnover of the
epithelium occurring in 5–7  days [17, 18].
However, following corneal transplantation, epi-
thelial rejection can occur for up to a year, sug-
gesting that donor cells are not ‘turned around’
for at least a year in humans. It is likely that the
basal epithelium survives for much longer [19].
Like all self-renewing systems, the drive for
12  Ocular Surface Epithelium: Applied Anatomy
regeneration and replacement of cells is provided
by stem cells located at the limbus.
12.2 Limbus and Limbal
Epithelium
The palisades of Vogt with their inter-palisade rete
ridges, the limbal epithelial crypts, the specialized
connective tissue architecture and the different
epithelial immunophenotypes, supported by a
highly organized vascular and nervous supply,
provide the anatomical basis and the physiological
microenvironment for the limbus to sustain the
limbal epithelial stem cells. Stem cells (SC) are
progenitor cells that are r­esponsible for cellular
replacement and tissue regeneration. They are the
ultimate source cells from which arise almost all
other cells that constitute a given organ served by
the SC.  Stem cells are slow cycling and live as
long as the organ or organism they serve. They
constitute a very small proportion of the total cell
mass of the organ. Their major attributes are
potency and plasticity. Potency is the ability of a
single SC to differentiate along different lineages
of cell types, for example, the fertilized ovum
(totipotent SC), the mother of all SC, can produce
a whole baby and part of the placenta. With subse-
quent cell divisions, some of the potency is lost
though the cells retain qualities of SC. There thus
exists a hierarchy of SC  – totipotent, pluripotent
and multipotent. Plasticity is the ability of SC
serving one organ to start serving a different organ
when transplanted into that organ. This attribute,
a b
Fig. 12.2 (a) The limbal palisades of Vogt are clearly
seen as radially oriented slender lines in a pigmented indi-
vidual. They blend with the conjunctival epithelium
peripherally and transition into dot-like circular palisade
structures centrally. (b) Impression cytology of the limbal
179
also termed transdifferentiation, is the basis of the
clinical application of SC derived from one source
to regenerate and rejuvenate another failing organ.
Corneal epithelial stem cells are unipotent or com-
mitted progenitors that in their physiological role
only differentiate to corneal epithelial phenotype,
though in experimental conditions can be made to
transdifferentiate into hair follicles. Stem cells
usually reside in a defined SC niche, which repre-
sents the anatomical and physiological microenvi-
ronment of the SC. The inter-palisade rete ridges
of the palisades of Vogt and the limbal epithelial
crypts constitute the limbal SC niche [20–24].
Palisades of Vogt  The palisades of Vogt are radi-
ally oriented fibrovascular ridges that are most
prominent along the upper and lower limbus and
readily visible in pigmented individuals due to the
increased melanin content of the epithelial cells
(Fig. 12.2a). The ‘valley’ between adjacent pali-
sades is packed with epithelial cells that constitute
the inter-palisade (epithelial) rete ridge.
Impression cytology studies show that the limbal
epithelial cells are smaller, with a higher cell den-
sity and a greater nucleus to cytoplasm ratio com-
pared to central corneal epithelial cells [25, 26]
(Fig. 12.2b, c). The connective tissue of the pali-
sade contains arteries and veins, radially oriented
as narrow hairpin loops, nerves and lymphatics.
The arterial supply is from the anterior ciliary
arteries derived from arteries of the rectus mus-
cles. The epithelium covering the palisade is two
to three cells thick. The epithelial cells in inter-
palisade rete ridges are 10–15 cells thick [27, 28].
c
and peripheral corneal epithelium. The limbal epithelial
cells are small, densely packed and show a greater
nucleus-to-cytoplasm ratio compared to the peripheral
and (c) central epithelial cells. (From author’s own publi-
cation Dua et al. [52])
180 H. S. Dua and D. G. Said

Basal cells of the rete ridges are (CK) 5/14+, palisade rete ridge and extend into the surround-
CK19+ and CK3/12+ [4, 12]. These cells also ing substantia propria peripherally, centrally
show staining for vimentin, epidermal growth fac- towards the cornea or circumferentially in a
tor receptors and alpha-enolase and are connexin clockwise or anticlockwise direction [26, 38]
43 (gap junction protein) negative [29–32]. An (Fig. 12.3). LEC are classed as minor and major,
ATP binding cassette transporter protein, ABCG2, with the major ones ranging from 40 to over 200
is considered to be a marker of a subpopulation of microns. They are widest at their origin and taper
cells that contain SC. ABCG2-­positive cells have to a narrow-rounded end at their termination. The
been located in the basal cells of the limbus [33, number of LEC range from 3 to 13 with an aver-
34]. These cells also show a specific isoform of age of 9 per eye. LEC are packed with epithelial
P63 (ΔNp63α), a transcriptional factor believed phenotype of basal, suprabasal and central cells.
to correlate with SC [35]. Other putative stem cell Basal cells are small with prominent nucleoli and
markers like nestin, cytokeratin 15 and SOX-9 dense marginated chromatin, which is different
transcription factor have also been identified in from the more superficial cells (Fig.  12.4a, b).
relation to limbal stem cells [36, 37]. The cell membrane of the basal cells in contact
with the basement membrane have complex
finger-­like processes that match corresponding
Limbal Epithelial Crypts  Limbal epithelial invaginations in the basement membrane and
crypts (LEC) are distinct solid cords of epithelial express multiple hemidesmosomes, allowing for
cells that arise from the posterior end of the inter-­ a firm attachment between these putative SC and

Fig. 12.3  Serial sections of a limbal epithelial crypt (LEC) from start to termination. The LEC is seen to arise from the
posterior end of the inter-palisade rete peg and extend into the underlying stroma, tapering to a point at its termination
12  Ocular Surface Epithelium: Applied Anatomy
a b
Fig. 12.4 (a) Transmission electron micrograph (TEM)
of the basal cells of the limbal epithelial crypt (LEC)
showing prominent nucleoli and marginated chromatin.
(b) Central cells of the LEC. (c) Showing attachments of
the basal cells of the LEC. Cytoplasmic extensions of the
a b
Fig. 12.5  In vivo confocal microscopic (IVCM) features
of (a) the limbal palisades showing basal cells of the rete
pegs (arrow heads) and stroma of the palisades (arrows). (b)
The reflectivity of these structures reverses when the IVCM
the substrate (Fig. 12.4c). The peripheral corneal
basal cell attachments too are stronger than the
central epithelial attachments. Hence following
injury, a peripheral rim of corneal epithelium
(and the limbal epithelium) remains attached
even when a large area of the central cornea is
denuded. Cells from this rim migrate centripe-
tally as convex sheets that close the defect and
restore epithelial integrity. Lines of contact
between these sheets stain with fluorescein and
appear as the pseudo-dendrites seen during heal-
ing of corneal epithelial defects [7]. In vivo con-
focal microscopy (IVCM) is a good clinical tool
to examine the eye for assessment of both normal
anatomy and evaluation of limbal stem cell defi-
ciency [39, 40]. The stroma of the palisades
appears as a hyper-reflective band and the basal
limbal epithelial cells in the rete pegs appear
dark, outlined with a bright border (Fig. 12.5a).
In the plane deeper to the basal cells. The stroma
181
c
cell membrane are seen extending into corresponding
invaginations of the basement membrane with multiple
hemidesmosome attachments (arrows). (Adapted from the
author’s own publication Shanmuganathan et al. [38])
c
plane is moved to the stroma underlying the cells. (c) A
cluster of hyper-reflective cells is seen in deep stroma, rep-
resenting the limbal epithelial crypt cells. (Adapted from
the authors’ own publication Miri et al. [39])
of the rete pegs appears hyper-reflective (bright),
and the stroma of the palisades, in the corre-
sponding plane, appears hypo-reflective (dark)
(Fig.  12.5b). Deeper in the stroma, at places,
clusters of bright cells are seen, probably repre-
senting limbal epithelial crypts (Fig.  12.5c). In
vivo demonstration of crypts with optical coher-
ence tomography is possible and helps in assess-
ment of limbal stem cell deficiency [41, 42].
Immunophenotypically, the LEC cells show
SC characteristics. Cytokeratin 3, which is a dif-
ferentiation marker, is present in central corneal
epithelium but absent from limbal and LEC
cells. CK19, which is a predominant cytokeratin
of conjunctival cells, is seen in the LEC. Vimentin,
an intermediate filament that is largely expressed
in stromal cells and less differentiated cells, is
present in clusters of limbal basal cells and in
basal and suprabasal LEC cells. P63 and ABCG2,
182
putative SC markers, are expressed maximally in
the LEC (Fig.  12.6a). Desmoglein 3 is a cell
membrane protein that has a role in the forma-
tion of desmosomes and in cell-cell adhesion.
Decreased expression of desmoglein 3, as seen
in the LEC, is associated with increased colony
formation efficacy. Tenascin C, an extracellular
matrix protein, is expressed prominently by the
BM of LEC.  It modulates cell adhesion and is
b c
Fig. 12.6 (a) A limbal epithelial crypt (LEC) stained for
the transporter protein ABCG2 showing strong staining of
all cells in the LEC and along the basal cells of the rete
peg. The staining becomes less intense towards the con-
junctival side of the LEC. (Adapted from author’s own
publication Dua et  al. [26]). (b–d) Immunoperoxidase
staining of LEC for connexin 43 (Cx43). The LEC and
H. S. Dua and D. G. Said
suggested to have a role in corneal growth,
development and maintaining corneal stem cell
“stemness” by anchoring SC [43, 44]. Ki67
(MIB-1), a marker of cellular proliferation, and
CD34, a haematopoietic stem cell marker, are
not expressed in the LEC cells nor in the limbus
adjacent to LEC bearing rete pegs. SC are known
to lack gap junctions (connexin (Cx) 43). This is
believed to confer a survival advantage to the SC
d
overlying epithelium are both positive for Cx43. (b) All
cells of the central corneal epithelium are positive for
Cx43. The rete peg basal cells some distance from the
LEC are negative for Cx43, whilst the adjacent peripheral
corneal epithelial cells are positive (d). (Adapted from the
author’s own publication Shanmuganathan et al. [38])
12  Ocular Surface Epithelium: Applied Anatomy
a b
Fig. 12.7  In vivo confocal microscopic (IVCM) features
of limbal stem cell deficiency. (a) The superficial layer
shows hyper-reflective cells with ill-defined margins repre-
senting conjunctivalization of the cornea (*). The adjacent
normal corneal epithelium (+) shows dark cells with well-
defined margins. Arrowheads mark the junction of the two
epithelial phenotypes. (b) The subsuperficial cells of the
in that it isolates them from their progeny and
factors related to infection and stress in an adja-
cent cell cannot pass into the healthy cell.
However, LEC cells express abundant Cx43.
This could be related to the range of non-gap
junction functions attributed to Cx43, which
includes a role in suppression of tumour growth
and in differentiation and migration. Thus, the
finding of Cx43+ cells in the LEC would be con-
sistent with it being a putative niche for stem
cells, and the Cx43+ cells could be niche cells
that support the division of stem cells [45–47]
(Fig. 12.6b).
The corneal epithelial cell mass is main-
tained by the SC of the limbus/LEC, the prog-
eny of which step out of the niche as transient
cells (TC) and transient amplifying cells (TAC,
basal cells), which migrate centripetally on the
corneal BM.  Postmitotic cells migrate anteri-
orly as the wing cells and the superficial cells.
This is embodied in the X, Y, Z hypothesis of
corneal epithelial maintenance, where X = pro-
liferation of basal epithelial cells, Y = contribu-
tion to the cell mass by centripetal movement of
peripheral cells and Z = the epithelial cell loss
from the surface [48]. Healthy central islands
of corneal epithelium survive despite total loss
of limbal epithelium indicating that the central
epithelial cell mass can be sustained by the
basal cells (TAC). It is postulated that SC play
183
c
transition zone of the conjunctivalized area (*) demonstrate
prominent cell nuclei with no cell borders. The adjoining
area (+) of normal corneal epithelial cells shows bright,
well-defined borders. Arrowheads mark the junction. (c)
Cystic spaces (arrowhead) and goblet cells, singly or in
rosettes (arrow), are seen in conjunctivalized area. (Adapted
from the author’s own publication Dua et al. [19])
a crucial role in healing and regeneration of
epithelium after injury but have a minimal role
in physiological homeostasis of the corneal epi-
thelium, which can be served by the basal cells
[19, 49].
Clinically, the limbal stems cells can be dam-
aged by a variety of causes ranging from con-
genital defects to chronic inflammation and
chemical burns. The corneal epithelium is
replaced by conjunctival epithelium (in mild
cases) or by an extensive fibrovascular pannus
(in severe cases) [50, 51]. Conjunctival epithe-
lium on the cornea on IVCM demonstrates
hyper-reflective metaplastic cells with ill-defined
margins and goblet cells distributed singly or in
rosettes (Fig. 12.7a–c). The fact that these eyes
can be successfully treated by limbal transplan-
tation or ex vivo cultured limbal SC is a strong
indication of the importance of the limbus and
direct clinical confirmation of the limbal loca-
tion of SC [52–54].
12.3 Conjunctiva
The conjunctiva is the highly specialized muco-
sal lining of the ocular surface that extends from
the lid margin, posterior to the grey line. It lines
the posterior surface of the lids as the palpebral
or tarsal conjunctiva folds over to cover the eye
184
a b
Fig. 12.8 (a) Haematoxylin- and eosin-stained section of
the normal conjunctiva showing a multilayered epithelium
with goblet cells sitting on a basement membrane with
underlying substantia packed with resident immune cells,
vessels and connective tissue cells. (b) Immunoperoxidase
ball as the bulbar conjunctiva. The folds between
the palpebral and bulbar conjunctiva are termed
the superior, inferior, medial and lateral fornices.
Palpebral conjunctiva is firmly attached to the
underlying tarsal plates but the forniceal and bul-
bar conjunctiva is loosely attached to the under-
lying substantia propria, except close to the
limbus, where for about 2–3 mm the bulbar con-
junctiva is firmly attached. Hence in grasping the
globe with forceps, it should be applied immedi-
ately next to the limbus where grasping the con-
junctiva provides a grasp on the globe as well.
The substantia propria, the loose connective tis-
sue underlying the conjunctival epithelium, is
highly vascularized and contains diffuse and
localized collections of lymphoid tissue made of
lymphocytes, plasma cells, mast cells and neutro-
phils (Fig. 12.8a). In inflamed states, the vessels
of the substantia propria express high endothelial
venules and lymphocyte homing receptor pro-
tein, vascular adhesion protein-1 for attachment
and egress of circulating white cells [55, 56]
(Fig. 12.8b). Unlike the corneal epithelial base-
ment membrane, the conjunctival basement
membrane shows fenestrations through which
immune cells can migrate from the substantia
propria to the surface during inflammation [57]
(Fig.  12.9a, b). Tufts of vascular proliferation
covered by thickened epithelium constitute a
papillary reaction and collections of lymphoid
cells constitute a follicular reaction, both of
H. S. Dua and D. G. Said
staining of blood vessels in a section of inflamed conjunc-
tiva. The endothelial cells stain strongly for vascular adhe-
sion protein-1. (Adapted from the author’s own publication
Haynes et al. [55])
which are non-specific responses of the conjunc-
tiva to chronic inflammation.
Conjunctival Epithelium  The conjunctival epi-
thelium is non-keratinized and squamous in
nature, two to three cells thick in all parts and
closely integrated with goblet cells, which are
spread throughout the conjunctiva but especially
concentrated in the nasal palpebral and fornicial
regions. At the lid margin, it is thicker and can be
10–12 cells thick. Conjunctival cells have abun-
dant cytoplasmic organelles, unlike corneal
­epithelial cells. Along the basolateral surface of
the basal cells of the conjunctiva are intraepithe-
lial CD8+ lymphocytes that are a feature of most
mucosal epithelia [58] (Fig. 12.10). Goblet cells
are columnar, glandular cells found among most
mucosal epithelial cells including the conjunctiva.
They are part of the mucosal barrier in innate
defence and their secretion serves as an important
part of the tear film to afford lubrication to the
ocular surface that interfaces with the environ-
ment. They have a distinct morphology with
mucin vesicles and granules filling the apical part
of the cell, and the nucleus and other cell organ-
elles packed at the bottom. When the mucin is dis-
charged, the cell assumes the shape of a ‘goblet’.
Secretion of the mucin occurs by holocrine (the
cell membrane bursts to release the secretion
effectively implying destruction of the cell) or
apocrine (the apex of the cell pinches off to release
12  Ocular Surface Epithelium: Applied Anatomy
a b
Fig. 12.9 (a) Scanning electron micrograph (SEM) of
basement membrane of the conjunctival epithelium show-
ing multiple fenestrations. (b) SEM of the denuded con-
Fig. 12.10  Section of normal human conjunctiva stained
with the alkaline phosphatase anti-alkaline phosphatase
technique for the human mucosal lymphocyte antigen-1
(HML-1). HML-1 positive cells are seen associated with
the basal epithelial cells as red dots. These cells are seen in
mucosal surfaces elsewhere in the body as well. (Adapted
from the author’s own publication Dua et al. [58])
the secretory granules or mucin-filled vesicles and
some cytoplasm, but the cell survives). The con-
junctiva serves two major functions of protection
and lubrication. For OS defence the epithelial
cells are capable of directly phagocytosing bacte-
ria and secrete substances such as antimicrobial
peptides that are a­ntibacterial and antiviral.
Lubrication is provided by mucin, which, as
185
junctiva a few hours after maintaining in tissue culture
medium showing mononuclear cells that have migrated
through the fenestrations
­ entioned above, is an integral part of the tear
m
film. Extensive injury to the conjunctiva like
chemical burns or chronic inflammation results in
loss of goblet cells as well with consequent mucin
deficiency and rapid tear film breakup time. The
ducts of the lacrimal and Meibomian glands open
into the conjunctiva. Extensive scarring of the
conjunctiva can lead to obstruction of the gland
orifices, depriving the OS of lacrimal secretions
leading to dry eyes [4, 9].
Tear Film and Conjunctiva  The tear film is a
dynamic complex structure served by numerous
cells and glands and continuously replenished
and evenly distributed across the OS by the
blinking action of the eyelids. The hydrophobic
surface of the corneal epithelium is rendered
hydrophilic by the binding of mucin to the epi-
thelial cells. This allows the aqueous component
to form a relatively thick layer on the mucin bed,
upon which the Meibomian oil (meibum) spreads
as a thin layer, delaying both evaporation and the
breakup of the tear film. The tears contain a
number of proteins, growth factors, vitamins,
electrolytes and a few cells that collectively con-
tribute to the nourishment, defence and health of
the ocular surface [59]. These include immuno-
globulins, lysozyme, lactoferrin and a number of
antimicrobial peptides. Adaptive cell-mediated
and humoral responses by the resident and circu-
186
lating lymphocytes contribute to the protection
provided by the conjunctival mucosal immune
system [60, 61].
The cornea receives its nutrients from the
aqueous humour, the tear film and the limbal ves-
sels. Oxygen from the atmosphere dissolved in
the tear film reaches the cornea; hence contact
lenses can potentially interfere with this and
cause adverse consequences. Equally noxious
agents such as carbon monoxide from smoke can
also dissolve in tears and reach the cornea.
Deficiency of tears causes dry eyes with trouble-
some symptoms and blurring of vision that
affects the quality of life of the patient. Surgical
interventions to restore sight loss such as corneal
and limbal transplantation generally do not sur-
vive for long in a dry environment [53].
12.4 Corneal Innervation
The cornea is the most sensitive structure in the
human body, being a hundred times more sensi-
tive than the conjunctiva [62] and over 400 times
more than the skin [63].
Corneal innervation is predominantly sensory
from the ophthalmic division of the trigeminal
nerve via the long posterior ciliary nerves. These
nerves arborize to form the peri-limbal plexus,
which has both sensory and autonomic nerves and
is predominantly vasomotor in function [64].
From the peri-limbal plexus, demyelinated radial
nerves enter the mid-thickness of the corneal
stroma, about 11  in each quadrant. These travel
centrally and superficially to form a sub-­Bowman’s
plexus. Smaller twigs penetrate Bowman’s layer
and end in the subbasal plane of the epithelium, in
distinct terminal bulbs [65]. From the bulb-like
structures finer neurites emerge as naked endings
(without the Schwann cells) to form the subbasal
plexus. The neurites are generally oriented such
that they converge to an area between the upper
two thirds and the lower one third where they form
H. S. Dua and D. G. Said
a distinct whorl pattern [65, 66] (Fig. 12.11). From
the subbasal plexus, terminal nerve endings enter
the epithelium and terminate both inter and intra-
cellularly [67].
Autonomic innervation consists essentially of
sympathetic nerves from the superior cervical
ganglion (SCG). Fibres destined for the eye form
the sympathetic root of the ciliary ganglion.
Some fibres directly merge with the long ciliary
nerves and others pass through the ciliary gan-
glion, without synapse, to emerge in the short
ciliary nerves. Though there are few sympathetic
fibres in the cornea in humans [68, 69], they are
an important component of the peri-limbal nerve
plexus.
OS innervation serves sensory, trophic and
vasomotor functions. The limbal vascular arcade
responds to corneal nerve stimuli by vasodilation
(circumcorneal congestion) which facilitates dia-
pedesis of white blood cells (triple response)
which invade the cornea to combat noxious and
infective agents. In situations where the innerva-
tion is compromised, infective organisms can
flourish without the host response in a unique
pattern of infection called infectious crystalline
keratopathy [70]. Loss of sensory innervation of
the cornea leads to neurotrophic keratopathy
wherein the OS suffers from tear film instability,
epithelial irregularity, persistent epithelial defects
and stromal melting. Damage to corneal innerva-
tion peripheral to and involving the trigeminal
ganglion results in loss of sensory and trophic
function with risk of epithelial loss and stromal
lysis. Damage central to the ganglion results in
sensory loss, whilst trophic functions are pre-
served [71, 72]. The prognosis in such cases is
likely to be good.
Declaration of Interest None of the authors have any
conflict of interest related to the subject matter and con-
tent of the chapter. HS Dua is the consultant for Dompe,
Santen, Thea and Shire. He has shares in NuVision
BioTherapeutics and GlaxoSmithKline. No human or ani-
mal studies were carried out by the authors for this
chapter.
12  Ocular Surface Epithelium: Applied Anatomy 187

a b

c d

e f

Fig. 12.11  Photomicrographs of whole mount of normal
human cornea stained by the acetylcholinesterase tech-
nique. (a) Subbasal nerve plexus with characteristic
branching (arrows) and union/reunion (arrowheads). (b)
A stromal nerve (arrowhead) penetrates Bowman’s mem-
brane (arrow) and ends in a terminal bulb from which a
leash of neurites of the subbasal plexus emerge. (c) A
thick stromal nerve (arrow) emerges anterior to the
Bowman’s membrane and ends in a blub (arrowhead). (d)
A stromal nerve bifurcates immediately after penetrating
Bowman’s membrane and each branch ends in a terminal
blub (arrowheads) which give rise to slender neurites. (e)
A single sub-Bowman’s nerve (arrow) gives rise to multi-
ple branches (arrowheads), each of which ends in a termi-
nal bulb on penetrating Bowman’s membrane. (f) A higher
magnification of (e) showing the terminal bulbs (arrow-
heads) and the leash of neurites emerging from the bulbs.
(Adapted from the author’s own publication Al-Aqaba
et al. [65])
188 H. S. Dua and D. G. Said

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 Classical Techniques for Limbal
Transplantation
13
Rafael I. Barraquer and Juan Alvarez de Toledo

13.1 Historical Background In 1977, Thoft proposed “conjunctival trans-

plantation” for unilateral chemical burns [6].
Limbal transplantation for the management of This technique did not result in a functional cor-
severe ocular surface disease was proposed as neal surface, as it probably did not include cor-
early as 1964 by José I. Barraquer, during a discus- neal epithelial stem cells (CESC) in the donor
sion at the first World Cornea Congress [1]. He tissue. He further described a “kerato-­
described the use of “epithelial conjunctivo-­ epithelioplasty” procedure in 1984 [7], later
corneal limbus taken from the other eye” for the modified by Turgeon et  al. in 1990 [8]. This
treatment of superficial burns of a single affected involved the application of several thin disks or
eye [2]. In 1966, Strampelli and Restivo Manfredi lenticels from cadaveric peripheral cornea. These
published a cases of highly vascularized and grafts probably included only a small amount of
opaque corneas improved after a transplant of a donor CESC.  However, they represent the first
complete ring from the limbus of the fellow eye attempt at using allografts for ocular surface
[3]. The technique was described in more detail, reconstruction, which would allow treating bilat-
together with a second case, at the 2nd International eral diseases.
Corneo-Plastic Conference in London in 1967 [4]. Relevant research about epithelial regenera-
Joaquin Barraquer also performed some cases in tion and homeostasis can be traced back to the
Barcelona (Fig. 13.1) and later published his tech- 1940s. Studies showed increased frequency of
nique (Figs. 13.2 and 13.3) [5]. mitoses in the basal layer of peripheral cornea
using mitotic figure counts and radiated thymi-
dine [9–11]. However, the crucial role of the
R. I. Barraquer (*) “limbal stem cells” – or, more accurately, CESC –
Centro de Oftalmología Barraquer, Anterior Segment was understood only during the 1970s and 1980s,
Department, Barcelona, Spain following the works of Davenger and Evenson
Universitat Internacional de Catalunya, [12], Schermer [13], Kinoshita [14], and Potten
Barcelona, Spain and Loeffler [15].
Institut Universitari Barraquer, Universitat Autónoma In 1989, Kenyon and Tseng published a
de Barcelona, Barcelona, Spain series of limbal transplantations acknowledg-
J. Álvarez de Toledo ing the CESC theory [16]. They employed two
Centro de Oftalmología Barraquer, Anterior Segment arcuate segments of conjunctival and periph-
Department, Barcelona, Spain
eral corneal tissue from the fellow eye to treat
Institut Universitari Barraquer, Universitat Autónoma unilateral limbal stem cell deficiency (LSCD).
de Barcelona, Barcelona, Spain

© Springer Nature Switzerland AG 2019 191

J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_13
192 R. I. Barraquer and J. Álvarez de Toledo

a b

c d

Fig. 13.1 (a) Right eye (RE) of a female patient who had
a chemical burn in her left eye. This RE was amblyopic due
to childhood unilateral aphakia. The image shows the status
of the ocular surface 2 months after removal of a 360° ring
of limbal conjunctiva. (b) Left eye (LE) of the patient
affected by a chemical burn before autologous limbal trans-
plantation. (c) Immediate postoperative result of the CLAU
15 days after surgery. (d) One year after a penetrating kera-
toplasty performed to rehabilitate the visual function. (Case
courtesy of Prof. Joaquin Barraquer, performed in 1981)

Their technique continues being the standard
treatment for most unilateral severe ocular sur-
face disease, especially where ex  vivo expan-
sion techniques are not available. In 1994, Tsai
and Tseng first described a proper limbal
allograft technique using cadaveric donor tis-
sue [17]. One year later, Kwitko et al. reported
the use of conjunctiva from a living-­related
donor – siblings or other living relatives – for
treating bilateral LSCD [18]. Their technique
was modified by Kenyon and Rapoza to include
limbus and conjunctiva from the living ­relative,
being the first description of a living-­related
conjunctival-limbal autograft (lr-CLAL) pro-
cedure [19].
As an increasing number of techniques were
being published with different names, in 1996
Holland and Schwartz proposed a classification
based on the anatomical location of the donor
­tissue and whether the source is or not the same
13  Classical Techniques for Limbal Transplantation 193

a b

c d

Fig. 13.2 (a) Schematic design of a ring-shaped CLAU
(Emilio Iglesias MD, PhD [5], 1981). A ring of limbal tis-
sue including 5 mm. of limbal conjunctiva and peripheral
corneal epithelium is obtained from the donor and
anchored in the recipient’s eye. (b) A complete peritomy
and (c) a peripheral keratectomy marked with a non-­
perforating superficial 9 mm. trephination are performed
in the recipient’s eye. (d) Dissection of the peripheral lim-
bal area is performed with a crescent blade

a b

c d

Fig. 13.3 (a) Complete removal of the fibrovascular tissue is then anchored with sutures in the conjunctival and cor-
that covers the limbal area and cornea. (b) Recipient’s eye neal circumferences. (d) Limbal area starts repopulating the
is prepared with a bare ocular surface. (c) The ring autograft epithelial layer from the stem cells present in this area
194 R. I. Barraquer and J. Álvarez de Toledo

individual [20]. This should allow a better under-
standing of each technique and its outcomes and
improvement in the communication between sur-
geons. A committee of the Cornea Society recently
reviewed and modified this classification and pro-
posed that all corneal surgeons use the same
nomenclature [21].
In this chapter, we will review the classical
surgical techniques for ocular surface rehabilita-
tion, excluding ex  vivo expansion techniques
which will be described elsewhere.
13.2 Classification of Surgical
Procedures
The techniques for ocular surface reconstruction
were named as they emerged along several
decades, using an array of terms including autol-
ogous and allograft conjunctival transplantation,
kerato-epithelioplasty, homotransplantation of
limbal cells, limbal transplantation, and autolo-
gous and allograft limbal transplantation. As this
was prone to cause confusion, the Cornea Society
established in 2008 a steering committee to pro-
pose a more rational nomenclature. The ensuing
classification – and set of abbreviations – is based
on the anatomical origin of the transplanted tis-
sue (conjunctival, limbal, other mucosal), the
relation between donor and recipient (autograft,
cadaveric, living relative or non-relative
allografts), and whether the tissue was cultivated
ex vivo (Table 13.1). This classification and com-
mon nomenclature contribute to clarify the com-
munication, allowing a more accurate comparison
of the outcomes of each procedure. This classifi-
cation can be reviewed to eventually incorporate
novel techniques.

Table 13.1  Classification of surgical and tissue-engineered procedures for ocular surface rehabilitation
Procedure Abbreviation Donor Transplanted tissue
Conjunctival transplantation
 Conjunctival autograft CAU Fellow eye Conjunctiva
 Cadaveric conjunctival allograft c-CAL Cadaveric Conjunctiva
 Living-related conjunctival allograft lr-CAL Living relative Conjunctiva
 Living-nonrelated conjunctival allograft lnr-CAL Living non-relative Conjunctiva
Limbal transplantation
 Conjunctival-limbal autograft CLAU Fellow eye Limbus/conjunctiva
 Cadaveric conjunctival-limbal allograft c-CLAL Cadaveric Limbus/conjunctiva
 Living-related conjunctival-limbal allograft lr-CLAL Living relative Limbus/conjunctiva
 Living-nonrelated conjunctival-limbal allograft lnr-ClAL Living non-relative Limbus/conjunctiva
 Keratolimbal autograft KLAU Fellow eye Limbus/cornea
 Keratolimbal allograft KLAL Cadaveric Limbus/cornea
Other mucosal transplantation
 Oral mucosa autograft OMAU Recipient Oral mucosa
 Nasal mucosa autograft NMAU Recipient Nasal mucosa
 Intestinal mucosa autograft IMAU Recipient Intestinal mucosa
 Peritoneal mucosa autograft PMAU Recipient Peritoneum
Ex vivo cultivated conjunctival transplantation
 Ex vivo cultivated conjunctival autograft EVCAU Recipients eye(s) Conjunctiva
 Ex vivo cultivated cadaveric conjunctival allograft EVc-CAL Cadaveric Conjunctiva
 Ex vivo cultivated living-related conjunctival allograft EVlr-CAL Living relative Conjunctiva
 Ex vivo cultivated living-nonrelated conjunctival EVlnr-CAL Living non-relative Conjunctiva
allograft
Ex vivo limbal transplantation
 Ex vivo cultivated limbal autograft EVLAU Recipients eye(s) Limbus/cornea
 Ex vivo cultivated cadaveric limbal allograft EVc-LAL Cadaveric Limbus/cornea
 Ex vivo cultivated living-related limbal allograft EVlr-LAL Living relative Limbus/cornea
 Ex vivo cultivated living-nonrelated limbal allograft EVlnr-LAL Living non-relative Limbus/cornea
Other ex vivo cultivated mucosal transplantation
 Ex vivo cultivated oral mucosa autograft EVOMAU Recipient Oral mucosa
13  Classical Techniques for Limbal Transplantation
The first feature to be considered is the ana-
tomical type of transplanted tissue for ocular sur-
face reconstruction. These include most
frequently conjunctiva and/or limbus. However,
other tissues such as oral [22], nasal [23], perito-
neal [24], or intestinal (rectal) mucosa have been
used in some cases. Oral mucosa is probably the
extraocular mucosal tissue most frequently used
for ocular surface reconstruction, especially for
recurrent pterygia, symblepharon, and recon-
struction of conjunctival fornices, as well as in
biological keratoprosthesis procedures.
Conjunctival tissue is recognized as an impor-
tant contributor to the ocular surface homeosta-
sis. A healthy conjunctiva is absolutely necessary
in cases of LSCD, due to its crucial role in the
production of mucins  – from the goblet cells  –
and of cytokines. Conjunctival tissue can be har-
vested from the same eye, from the patient’s
fellow eye, or from a donor. It can be obtained
from either the bulbar or fornix conjunctiva. The
latter has been reported as a greater source of
conjunctival stem cells [25]. However, bulbar
conjunctiva is more commonly used due to an
easier harvesting.
Limbal tissue has been shown to be the locus
of CESC. These appear to be present around the
limbus in a nonuniform distribution, as a number
of discrete niches related to Vogt’s palisades.
When the tissue is obtained from limbal conjunc-
tiva alone (conjunctival-limbal graft), it will
include few if any CESC.  On the other hand, a
keratolimbal tissue segment including the periph-
eral and superficial cornea will contain most of
the CESC present in the harvested limbal sector.
The first type is mainly used for the reconstruc-
tion of a conjunctival defect adjacent to the cor-
nea, as in pterygium or limbal tumor surgery, or
in cases with localized LSCD in which there is a
wide, healthy, and functional limbus in the
remaining sectors of the limbus. In conjunctival-­
limbal grafts, the CESC from the donor site are
preserved. The keratolimbal tissue is the pre-
ferred tissue in cases with severe or complete
LSCD, as it not only repairs the limbal area but
contributes to the repopulation of the corneal sur-
face with new epithelial corneal cells. As CESC
are removed from the harvested site in keratolim-
bal grafts, this should be taken into account
195
regarding the potential consequences for the
donor eye – the patient’s fellow eye or from a liv-
ing relative.
The relation between donor and recipient is a
key parameter for the success and long-term
prognosis in ocular surface transplantation, as
can be represented by the degree of histocompat-
ibility. In this respect, the best source of tissue is
the autologous origin, as no immune reaction will
be present. This is, nevertheless, not an option in
cases with bilateral disease. The second best
source is a compatible living donor: a parent,
brother, or sister with at least half of the major
histocompatibility antigens identical [26]. In this
scenario, HLA types I and II should be deter-
mined for all the relatives and potential family
donors. In cases of non-related living or cadav-
eric donors, some degree of tissue matching
should be attempted to improve the results. Ex
vivo expansion techniques might offer the possi-
bility of modifying the immunogenicity of the
cultivated cells [27] to decrease the immune
rejection to the grafted cells [28].
13.3 Conjunctival Transplants
Conjunctival tissue has been widely used as
repairing tissue for many ocular surface diseases
such as pterygium [29], symblepharon [30], post-­
glaucoma surgery complications [31] like bleb
perforations and valve or tube extrusions, tumor
excision [32], and other conjunctival defects. In
most cases conjunctiva is used as an autograft
(CLAU), from the same eye or from the patient’s
fellow eye. In cases of bilateral disease, conjunc-
tiva from a living-related donor or a cadaveric
donor can be employed. Nevertheless, conjuncti-
val allograft represents nowadays only a small
percentage of conjunctival transplants.
At the donor site, conjunctiva must be care-
fully dissected and separated from Tenon’s cap-
sule. Inclusion of this layer will induce retraction
and fibrosis of the graft in the postoperative
period. It is advised to mark the perimeter of the
graft with a marker pen before harvesting
(Fig. 13.4). This will allow a better size match-
ing to the recipient bed (Fig. 13.5). The graft can
be secured in place with biological (fibrin) glue
196
Fig. 13.4  Marking of the conjunctival autograft with a
pen to delimitate the area of the conjunctiva to be
dissected
Fig. 13.5  Conjunctival autograft is placed over the cor-
nea with stromal face up, and fibrin glue bioadhesive is
applied onto the bare sclera and on the autograft. Care
must be taken in positioning the limbal area of the donor
graft in the limbal area of the recipient when it is flipped
and adjusted
(Fig.  13.6), several 10-0 nylon interrupted
stitches, or a continuous running suture. The
donor site can be closed with the same tech-
niques (Fig. 13.7). However, a purely conjuncti-
val (non-­Tenon) donor site of moderate size in
an otherwise healthy upper conjunctival sector –
which will be protected by the upper eyelid  –
may be left to spontaneous reepithelialization.
The conjunctival autograft technique is mostly
employed in pterygium surgery but can be used
for repairing a perforated filtering bleb or the
extrusion of a glaucoma drainage device
(Fig. 13.8).
R. I. Barraquer and J. Álvarez de Toledo
Fig. 13.6  Conjunctival graft is placed with epithelium
facing up and adjusted in position with its limbal area 1 to
2 mm. away from the recipient’s corneal limits to avoid
postoperative dellen formation when the grafts naturally
swell until revascularization occurs. Peripheral borders
are also ironed, extended, and contacting with the recipi-
ent’s conjunctiva
Fig. 13.7  Donor area is covered again with the conjunc-
tival tissue attached with fibrin glue to avoid scarring and
symblepharon formation that may rarely occur in some
races
Conjunctival allograft is used only in cases in
which an autograft tissue cannot be obtained, and
amniotic membrane is not suitable or indicated.
A combined procedure using CLAU and lr-­
CLAL has been recently described in two cases
of unilateral severe chemical/thermal burn. The
rationale for this approach is the combined use of
conjunctiva from the patient’s other eye to
account for the conjunctival and goblet cell
­deficiency and an HLA-compatible conjunctival
allograft from a living-related donor with a good
ABO and HLA matching to cover the larger
defect. This combination reduces the antigen
load, and no immune episodes were documented
during the follow-up period  – although both
13  Classical Techniques for Limbal Transplantation 197

a b

c d

Fig. 13.8 (a) Extrusion of a Baerveldt implant through fellow eye and a thin layer of a collagen implant
the conjunctiva in a patient affected of secondary glau- (Tutopatch®). (c) Dehiscence of the conjunctival wound
coma due to anterior chamber epithelial ingrowth. (b) and recurrence of the valve extrusion. (d) Definitive repair
Reconstruction of the extrusion with conjunctiva from the with a new Tutopatch® implant and oral mucosal graft

patients were under immunosuppression with trolled using a long-term immunosuppression

oral tacrolimus and mycophenolate mofetil.
Using a conjunctival or limbal allograft
involves a number of risks including immune
rejection, systemic toxicity due to the long-term
immunosuppressive drugs, the possibility of
inducing a LSCD in the donor eye [33], trans-
mission of a donor infection [34], even the
recently described transmission of a conjunctival
neoplasia [35]. Immune rejection can be con-
protocol under a strict monitoring and supervi-
sion by a specialist [36]. Multiple drug protocols
include combinations of oral steroids, double
immunosuppression with tacrolimus and myco-
phenolate mofetil, valganciclovir, and TMP/
SMX for prophylaxis of cytomegalovirus and
Pneumocystis carinii. These are usually contin-
ued for 3 years after the surgery if ocular condi-
tions are stable.
198
LSCD has never been described in a healthy
donor eye when at least half of the limbal circum-
ference had been respected. However, the potential
donor eye must be carefully inspected at the slit
lamp to rule out any subclinical LSCD. The pres-
ence of any kind of malignancy or infectious trans-
missible disease disqualifies a potential allograft
donor, because the required immunosuppression
would enhance risk of transmission and could favor
the growth of dormant metastatic cells. However,
in another reported case of transmission of a donor-
derived breast carcinoma [37], the activation of the
malignant cells in the recipient coincided with the
reduction of the oral immunosuppression.
13.4 Limbal Autograft Techniques
Conjunctival-limbal and keratolimbal autografts
(respectively, CLAU and KLAU) are the ocular
surface rehabilitation procedures with the best
prognosis [38]. The use of autologous tissue
avoids the risk of immune rejection, as well as
the traumatic maneuvers involved in harvesting a
postmortem tissue and preserving the tissue in
culture media  – as is the case in a majority of
allograft techniques, except for the relatively rare
living-related sources of tissue.
The donor tissue in CLAU comprises con-
junctiva with some peripheral limbal epithelial
cells (Fig. 13.9a, b), while in KLAU, it includes
the limbal conjunctiva and peripheral superficial
corneal  – stroma and epithelium  – including
Vogt’s palisades (Fig. 13.9c, d).
In CLAU, donor conjunctiva is dissected care-
fully separating Tenon’s capsule to its limbal
insertion, and a superficial sheet of limbal
(peripheral corneal) epithelium is cut with scis-
sors, without including Bowman’s membrane.
This technique is mainly used for pterygium sur-
gery (Fig.  13.10). However, there is little hard
evidence on whether the limbal component of
these grafts represents any significant benefit
compared to a standard conjunctival graft.
In KLAU, a portion of the peripheral superfi-
cial corneal stroma and epithelium is included in
the donor tissue. After dissecting the conjunctiva
centripetally, a 150–200 μm groove is performed
with a blade at the limbal sclera; lamellar dissec-
R. I. Barraquer and J. Álvarez de Toledo
tion of the peripheral superficial corneal stroma
is then performed, dissecting approximately 1.0–
1.5 mm of the peripheral cornea. This donor tis-
sue will include most of the CESC niches present
at the local Vogt’s palisades. A KLAU can be
obtained from the same eye (Fig. 13.11), when a
translocation of the limbus is used for treating a
localized LSCD, or from the fellow eye in cases
with severe unilateral LSCD (Fig. 13.12).
CLAU or KLAU can be combined with a
lamellar (Fig.  13.13) or penetrating keratoplasty
(Fig.  13.14), to improve ocular surface stability
and avoid neovascularization of the graft in cases
of LSCD associated with corneal opacities. After
dissecting all the superficial corneal neovascular-
ization and limbal conjunctiva, eliminating all the
perilimbal fibrotic tissue, a lamellar or penetrating
keratoplasty is performed following the standard
technique. Once the suture is completed, a 90°
(3-h) autograft is obtained from the fellow eye and
anchored with monofilament sutures (10-0 nylon
or 11-0 polyester), usually at the upper limbus.
A combined conjunctival autograft and kera-
tolimbal allograft (CLAU + KLAL), sometimes
referred as “the modified Cincinnati procedure”
[39], uses two fragments of recipient’s ­conjunctiva
obtained from the fellow eye and two sectors of a
cadaveric donor keratolimbal ring. The conjunc-
tival grafts are placed superiorly and inferiorly,
while the keratolimbal allograft sectors are
placed nasally and temporally.
Another way of transplanting CESC from a
donor fellow eye or from an allograft has been
proposed as “single epithelial limbal transplanta-
tion” (SLET) [40]. In this technique, a human
amniotic membrane (hAM) is grafted on the bare
ocular surface with fibrin glue. Small fragments
of limbal tissue from the fellow eye or a fresh
cadaveric corneoscleral rim are placed on the
cornea covered with the hAM graft in a circular
fashion avoiding the visual axis. A layer of fibrin
glue is applied to fixate the small pieces of grafted
tissue, and finally a bandage soft contact lens is
fitted over the cornea. These multiple fragments
of CESC-containing tissue will originate a new
epithelial multilayer, stimulated by the known
beneficial effects of hAM has on cell growth.
In cases of autografts, the patient’s main con-
cern relates to the possible consequences of
13  Classical Techniques for Limbal Transplantation 199

a b

c d

Fig. 13.9 (a) Delimitation of the area of a CLAU. Only included. (c, d) KLAU dissection includes peripheral
an epithelial part of the limbal area is removed. (b) superficial corneal stroma, epithelium, and superficial
Dissection of the conjunctiva is done splitting the Tenon’s limbal sclera to ensure the inclusion of all the niches of
capsule, which will induce retraction of the graft if the limbal epithelial stem cells

removing a limbal area from the healthy eye. In
the CLAU procedure, the preferred area for har-
vesting the donor tissue is the superior or supero-­
temporal conjunctival quadrant  – of either the
fellow or the same eye  – due to the protection
offered by the upper lid. Scarring and fibrosis of
the donor area, even symblepharon in the upper
fornix, can occur when the donor area is not
properly repaired, especially in certain ethnic
groups predisposed to scarring, and in cases
when postoperative steroid treatment poorly
complied by the patient.
Rare and relatively minor complications have
been reported after KLAU [41], including infec-
tions of the donor site, filamentary keratitis, neg-
ative fluorescein staining, and subconjunctival
hemorrhage. Astigmatism can be induced if the
corneal stroma is removed too deep or too cen-
trally. It has been documented that the CESC
repopulate completely the donor area within
1  year after the procedure [42]. In only one
instance, a case of Mooren’s ulcer has been
reported after a CLAU procedure for pterygium
surgery [43].
200 R. I. Barraquer and J. Álvarez de Toledo

a b

c d

Fig. 13.10 (a) Recurrent pterygium after four surgeries the lateroversion. (c) Result after careful removal of the
with multiple corneal adherences. (b) Symblepharon of symblepharon, CLAU, and superficial keratectomy. (d)
the infero-nasal fornix with involvement of the inferior Slit lamp appearance of the cornea with moderate thin-
lacrimal punctum, retraction of the eyelid, and diplopia in ning of the inferior stroma after the keratectomy

a b

c d

Fig. 13.11 (a) Keratolimbal autograft (KLAU) in a
patient with a unilateral chemical burn in his RE. One day
after surgery with two autografts positioned in the supe-
rior and inferior limbus. (b) Centripetal reepithelialization
from the limbal autografts (fluorescein staining). (c)
Complete epithelial layer with no fluorescein staining
6  days after the surgery. (d) Stable ocular surface and
improvement in corneal transparency 9  years after
KLAU. Patient’s BCVA reached 0.9 with RGP CL
13  Classical Techniques for Limbal Transplantation 201

a b

c d

Fig. 13.12 (a) Limbal stem cell deficiency (LSCD) in
the only eye of a patient in which more than 20 intravitreal
anti-VEGF have been injected through the upper nasal
sclera. Toxic effect of the antiseptic or anesthetic drugs
applied during the injections had been involved in the
etiopathogenesis of this case of LSCD. (b) KLAU from
the inferior quadrant of the same eye was used because of
the absence of the other eye (lost in his infancy) and
grafted in the supero-nasal area. (c) A complete healthy
epithelium covers the cornea 2 months after the KLAU.
(d) Corneal superficial stroma is still with a tenuous opac-
ity but BCVA and subjective symptomatology improved

a b

c d

Fig. 13.13 (a) Corneal opacity and neovascularization limbal conjunctiva with a CLAU from the fellow eye, a
after previous failed pterygium surgery. (b) Lamellar ker- lamellar corneal graft was sutured in the corneal bed. (d)
atectomy was performed manually until reaching a trans- Final result 6 months after the procedure with a stable
parent corneal stromal plane. (c) After reconstructing the ocular surface and a transparent corneal graft
202
a b
c d
Fig. 13.14 (a) Total superficial corneal neovasculariza-
tion after a previous conjunctival flap performed to treat a
bacterial keratitis with risk of perforation. (b) Combined
penetrating keratoplasty with KLAU in the superior lim-
KLAU are generally very successful provided
some guidelines are strictly followed. The placement
of a healthy keratolimbal tissue from the fellow eye
replenishes the pool of CESC of the recipient eye,
promotes increase in transparency of the corneal
stroma improving vision, and enhances ocular com-
fort. However, KLAU will not work if the ocular sur-
face of the recipient’s eye is severely inflamed, and
there is a tear film deficiency or eyelid malposition.
All these factors should be medically or surgically
corrected to ensure the survival of the CESC and a
correct regrowth of a corneal epithelial layer. Corneal
stroma should also be uninflamed; intrastromal neo-
vascularization should be treated or decreased before
the KLAU is performed. Ocular surface reconstruc-
tion procedures must always be rationally staged in
order to achieve an optimal final result.
A large series of 125 eyes treated with autolo-
gous SLET found an overall success of 76% after
1.5 years of follow-up, with progressive conjuncti-
valization in 18.4% of treated eyes [44]. Main fac-
tors of failure were acid injury, severe symblepharon,
R. I. Barraquer and J. Álvarez de Toledo
bus. (c) KLAU fixed in position with good revasculariza-
tion. (d) Transparent corneal graft and stable ocular
surface 1 year after the surgical procedure
SLET combined with keratoplasty, and postopera-
tive loss of the transplants, which highlights the
importance of performing the procedure in quiet
eyes without inflammation and with previously
repaired eyelid or conjunctival malposition.
13.5 Limbal Allograft Techniques
In cases with total bilateral LSCD, allogenic
CESC transplantation is the only way to repopu-
late the affected ocular surface with these pro-
genitor cells of the corneal epithelium. The first
descriptions of successful limbal allografts were
reported in the mid-1990s. Turgeon et al. reported
on 13 patients in which kerato-epithelioplasty was
performed to stabilize the ocular surface affected
with persistent epithelial defects [8]. Tsai and
Tseng reported a series of 16 eyes with several
causes of LSCD (chemical burns, Stevens-Johnson
syndrome, congenital sclerocornea, Terrien’s
degeneration, and chronic conjunctivitis) in which
13  Classical Techniques for Limbal Transplantation 203

a limbal ring-shaped allograft was grafted as a cularization and scarring of the ocular surface
source of CESC [17]. (Fig. 13.16).
Limbal (keratolimbal) allograft or KLAL can
be performed in different modalities: (1) sectorial
KLAL, which corresponds to the first description
of kerato-epithelioplasty by Thoft; (2) ring-­shaped
KLAL; (3) a combined ring-shaped KLAL with
lamellar or penetrating keratoplasty; and (4) mul-
tiple fragments of limbal allogenic tissue covered
by hAM and fibrin glue (SLET allograft).
Sectorial KLAL (Fig. 13.15) consists in graft-
ing two arcuate segments of limbal tissue from a
donor (living related or cadaveric) after perform-
ing a 360° peritomy and removal of the pannus
that usually covers the cornea in these cases.
When the recipient Bowman’s membrane is
absent, a hAM can be transplanted under the lim-
bal allografts, covering the corneal stroma to pro-
mote the epithelial repopulation, decrease the Fig. 13.15 Schematic representation of a sectorial
stromal inflammation, and inhibit the neovascu- KLAL. After removal of the pannus and fibrovascular tis-
sue in the recipient’s cornea and limbal area, two
larization. Sectorial KLAL is indicated in cases
3-o’clock-hour-wide limbal allografts are placed in the
with total bilateral LSCD without extensive vas- superior and inferior limbus

a b

c d

Fig. 13.16 (a) Bilateral LSCD in a patient due to chronic New epithelial centripetal growth from the two limbal
contact lens abuse. Persistent epithelial defects and super- allografts. (d) Complete reepithelialization and recovery
ficial stromal scarring. (b) Sectorial KLAL with two lim- of the corneal transparency observed 1 year after the
bal grafts placed in the vertical meridian of the limbus. (c) reconstructive surgery
204 R. I. Barraquer and J. Álvarez de Toledo

Ring-shaped KLAL is the most widely used

technique to treat bilateral LSCD (Fig. 13.17). A
ring of keratolimbal tissue is obtained from a
fresh cadaver eye (Fig. 13.18), with the inner and
outer diameters appropriately marked with dif-
ferent trephines. Ring width and thickness should
be enough to include the corneal-scleral limbal
area including the CESC niches. As in the secto-
rial technique, a complete dissection of the super-
ficial corneal and limbal pannus in the recipient’s
eye is mandatory, hAM can be applied to pro-
mote reepithelialization, and a stable tear film
and good eyelid function is required to achieve a
long-term success (Fig. 13.19).
Ring-shaped KLAL may also be combined Fig. 13.17  Schematic representation of a ring-shaped
KLAL. After removal of the pannus and fibrovascular tis-
with simultaneous lamellar or penetrating kerato-
sue in the recipient’s cornea and limbal area, a ring-shaped
plasty (Fig.  13.20). This multiple procedure is limbal allograft including limbal conjunctiva, superficial
indicated when the central cornea is in poor con- sclera, and cornea is placed in the limbal area and fixed
dition or rapid visual recovery is demanded by the with sutures

a b

c d

Fig. 13.18 (a) Harvesting of a ring-shaped KLAL from a
fresh cadaveric eye. Conjunctiva is cut 3–5 mm. from the
limbus and reflected over the corneal surface. (b) The
inner diameter of the ring is marked with an 8–9 mm. tre-
phine which penetrates 150–200 μ in the corneal stroma.
(c) The outer scleral diameter is also marked and tre-
phined with a 13–14 mm. trephine. (d) A careful lamellar
dissection is performed to obtain a ring of limbal tissue
containing all the pool of the donor’s limbal stem cells
13  Classical Techniques for Limbal Transplantation
a b
c d
Fig. 13.19 (a) Bilateral chemical burn with superficial
pannus and central corneal leukoma. (b) Ring-shaped
KLAL 4 days after surgery. Only the outer border of the
tissue was sutured with 8-0 Vicryl sutures, and no suture
was used to fix the inner border. (c) Complete epithelial
Fig. 13.20 Schematic representation of a ring-shaped
KLAL combined with central lamellar or penetrating ker-
atoplasty. After removal of the pannus and fibrovascular
tissue in the recipient’s cornea and limbal area, a central
lamellar or penetrating keratoplasty is performed and
fixed with 8-0 nylon sutures. Then, a ring-shaped limbal
allograft (A) including limbal conjunctiva, superficial
sclera, and cornea is placed in the limbal area and fixed
with 8-0 Vicryl sutures in the scleral side. The central graft
(B) is sutured with eight more sutures, and the previously
placed eight sutures are removed and changed for another
eight sutures that include the corneal graft, the recipient’s
peripheral cornea, and the rink KLAL
205
layer with epithelial healing lines that are visible with
fluorescein in the central cornea. (d) Final result 1 year
after surgery with stable epithelium and clear cornea.
Patient is under oral cyclosporine A
patient (Fig. 13.21). After removing all the limbal
fibrotic tissue and superficial neovascularization,
a penetrating or deep lamellar corneal graft is
anchored with eight 10-0 nylon single sutures; the
ring-shaped allograft is then placed and sutured
with several 10-0 nylon or 8-0 Vicryl sutures to
the sclera in the outer borders of the graft. After
that, eight more nylon sutures are placed includ-
ing the central donor graft, the recipient’s periph-
eral cornea, and the inner border of the limbal
allograft. The previous eight sutures that were
used to secure the central corneal graft at the
beginning are then removed, and eight new nylon
sutures that attach all the three tissues are placed
again. Finally, conjunctiva of the donor and the
recipient is secured with 8-0 or 9-0 Vicryl.
A single large-diameter lamellar or penetrat-
ing keratoplasty, trephined eccentrically in the
donor (Fig.  13.22), will include a sector of the
donor’s limbal area with its stem cells. This pro-
cedure, named limbo-keratoplasty by its authors
[45] in 1994, is technically less demanding than
206 R. I. Barraquer and J. Álvarez de Toledo

Fig. 13.21  Combined ring-shaped KLAL with penetrat- Typical postoperative intra-tissular hemorrhage that
ing keratoplasty in a case of Stevens-Johnson syndrome. occurs before re-connection of the blood microcirculation
Limits of the donor’s conjunctiva are highlighted (arrows). has been completed

a b

c d

Fig. 13.22 (a) Limbo-keratoplasty of 9 mm of diameter
performed in the single eye of a congenital aniridia-­
affected patient. (b) Around 40% of the donor limbus is
included in the graft when trephined eccentrically. (c)
Epithelial irregularities in the inferior cornea seen under
retro-illumination. (d) Normal corneal epithelium pattern
with fluorescein in the superior and central cornea; epithe-
lial defects and conjunctival cell staining pattern in the
inferior cornea, demonstrating that LSCD is present in the
inferior limbus. Graft failed 3 years after surgery due to
recurrence of LSCD

the previously described but has more chance of
failure due to the smaller proportion (up to 40%)
of the limbal zone included in the graft. With the
recent advances in immunosuppression, better
long-term results have been reported with this
procedure in non-severe special indications like
gelatinous corneal dystrophy [46].
Success with allogeneic SLET  – with donor
tissue  – has also been reported. The minimal
amount of tissue needed to assure a correct
13  Classical Techniques for Limbal Transplantation
growth of epithelial cells has been established by
in vitro studies in about 0.3 mm2 of a live limbal
fragment including a CESC niche [47].
Depending on the donor source (cadaver or live
tissue), the growth potential is different, being
necessary a larger amount of cadaveric tissue
(0.5 mm2) to obtain a similar proliferative rate as
with the live tissue. This limbal allograft tech-
nique represents an in  vivo or in situ stem cell
and epithelium expansion which bypasses the
need for the sophisticated and expensive cultur-
ing technology of ex vivo cell expansion, particu-
larly in countries where these are not available.
Oral immunosuppression is a key factor in the
long-term success rate of limbal allograft tech-
niques. The limbal tissue antigen load is much
larger than that of a standard penetrating
­keratoplasty, due to the presence of different cell
types including Langerhans cells. Limbal
allografts are at high risk of immune rejection
also due to the vascularity of the limbal area,
which negates the immune privilege of the central
cornea. Most ophthalmologists are not familiar
with the required immunosuppression protocols
and their general side effects. Despite the fact that
these are not serious in the majority of cases [48],
specific knowledge and experience are required
for a proper management. As these protocols have
to be frequently adjusted to the postoperative sta-
tus of each patient, the cooperation of specialist in
immunosuppression  – usually an internist  –
becomes a requirement in their monitoring.
A recently reported protocol involves two oral
immunosuppressants (mycophenolate mofetil
and tacrolimus) combined with 1  mg/kg oral
prednisone, all of which should be started 1 week
before surgery. Tacrolimus levels are adjusted to
8–10 ng/ml the first month postoperative and to
5–8 ng/ml at 6 months postoperative. Oral pred-
nisone is slowly tapered and discontinued after
3 months. One year after the procedure, mono-
therapy can be considered, and 3 years after sur-
gery, oral medication can be stopped if the
ocular surface is stable [36] (but see below).
Other protocols consist in combinations of aza-
thioprine and cyclosporine A with prednisone.
Absolute contraindications for oral immuno-
suppression include patients with a history of pre-
vious malignancy 5 years before, nonadherence to
207
a strict clinical or laboratory follow-up or medica-
tions, and significant health issues like diabetes,
uncontrolled hypertension, renal insufficiency,
severe heart diseases, or other organ failures. Age
over 70 is also a contraindication, and patients
among 60–70 years will be selected for immuno-
suppression depending on their general status.
Discontinuation of the oral immunosuppression
remains a controversial issue. Acute limbal
allograft rejection has been described recently [49]
in a series of six patients more than 3 years after a
KLAL procedure, suggesting that donor cells are
still present, thus at risk of acute rejection. Long-
term or indefinite i­mmunosuppression should be
considered despite a good midterm result.
Advanced topical treatments to promote and
protect epithelial cell growth constitute a signifi-
cant aspect in the postoperative period.
Autologous serum or growth factor-enriched
plasma drops, amniotic membrane extracts, topi-
cal immunosupressives [50], and epithelializa-
tion promoters like carboxylmethyl glucose
poly-sulfate should be rationally used to enhance
the epithelial healing and maintain epithelial
layer homeostasis.
Regarding the long-term outcomes, a recent
report found KLAL to achieve a true ocular sur-
face stability in 72.7% of cases with a mean fol-
low-­ up of 9.1  years, when the appropriate
selection criteria and proper immunosuppression
are applied, and the procedure is repeated as
needed [51]. Including this last option obviously
conditions the meaning of that long-term “suc-
cess,” as the duration of each individual trans-
plant is actually shorter. In a deep review of the
evidence-based published results of limbal trans-
plantations [52], there were statistically signifi-
cantly better results with the use of autologous
tissue, but no statistically significant differences
between the types of limbal allograft technique
(KLAL and lr-CLAL, p = 0.328). When lr-CLAL
is performed under a close HLA matching (0-1
HLA mismatches), the graft survival is increased
[26]. Other authors also reported better results
when using living-related donor tissue [53].
Patients with Stevens-Johnson syndrome and
patients with concurrent hAM transplantation
had poorer prognosis and long-term surface
improvements [52].
208
Donor age seems not to be a critical factor for
a successful clinical result. Limbal stem cell cul-
tures obtained from older donors show a mini-
mum required of >3% of p63+ cells considered
as the minimum value to predict a favorable out-
come [54]. Routine hypothermic storage in liquid
media at 4  °C is generally used to preserve the
donor limbal tissue, but novel methods of preser-
vation like hypothermic airlifted conditions [55]
have demonstrated better maintenance of epithe-
lial structure, cell phenotype, and higher viability
of the stem cell pool.
13.6 Other Mucosal
Transplantation
Oral mucosa is the human mucosal tissue more
frequently used to repair ocular surface defects.
Its ease of harvesting, the large amount of tissue
a b
c d
Fig. 13.23 (a) Castroviejo’s electro-keratome with the
motor in the handle, the head in which the oscillating
blade is mounted, and the gauge to obtain the desired
thickness of the graft. (b) After injecting saline solution
under the submucosal area of the inferior lip to create
intra-tissular tension, the electro-keratome is firmly
R. I. Barraquer and J. Álvarez de Toledo
that can be obtained, and its histological and ana-
tomical characteristics have made its use as a rou-
tine procedure in diseases like pterygium for
decades. Aside from its ophthalmological indica-
tions, oral mucosa is the new gold standard tissue
used in urethral reconstructive surgery [56].
Recently, interest in this tissue has re-emerged as
a source of cultivated epithelial cells to be utilized
in ex vivo cell expansion techniques [57, 58].
Oral mucosa has been used in many procedures
to repair symblepharon and primary or recurrent
pterygium, as a component of biological kerato-
prostheses, fornix reconstruction, and other recon-
structive techniques of the ocular surface and
oculoplastic procedures. The larger experience with
the oral mucosal tissue relates to pterygium surgery.
With an appropriate dissection of a thin graft with
an electro-keratome (such as Castroviejo’s), excel-
lent cosmetic and anatomical long-term results can
be obtained (Fig.  13.23). In this technique, a thin
advanced through the surface of the mucosa. (c) An excel-
lent and thin graft is then removed with scissors from the
lip. (d) Graft is placed in the inferior conjunctiva of a
patient affected of ocular pemphigoid to reconstruct the
inferior fornix
13  Classical Techniques for Limbal Transplantation
layer (0.2–0.5 mm thick) of oral mucosa of up to 50
by 20 mm is obtained from a clamped and serum-
hydrated inner lower lip and placed on the conjunc-
tival or fornix site to be reconstructed. This method
of mechanized harvesting the graft favors the
absence of submucosal tissue and fat lobules, avoid-
ing tissue retraction. The cosmetic appearance is
improved compared to a full-thickness graft
obtained manually with scissors.
13.7 Conclusions
Classic ocular surface reconstruction techniques
are still the surgical procedures of choice in many
ocular surface conditions involving conjunctival
defects or LSCD. An adequate knowledge of the
anatomy and pathophysiology of the condition,
together with familiarity of all surgical options and
armamentarium, is a requisite for the selection of
the best technique and for success. Autograft tech-
niques are preferable whenever possible, and an
adequate management of the tissues in both the
donor and recipient areas is the clues for excellent
results avoiding complications. When allografts are
required, it is paramount to establish a close col-
laboration with the specialist in systemic immuno-
suppression, as the application of adequate
treatment protocols appears to substantially
improve the long-term results. Clear information of
the advantages and possible complications of the
options should be given to the patients and to
potential living-­related donors, also highlighting
the benefits of being the source of tissue for their
affected relatives. Some techniques that may have
been considered old-fashioned at a certain point,
like oral mucosal transplantation, can be very use-
ful to solve complex situations. As long as the tech-
nological, availability, and cost issues of ex  vivo
cell expansion and regeneration limit their practical
application, the classical techniques remain a very
valid option offering excellent results when indi-
cated and performed in an appropriate manner.
Compliance with Ethical Requirements Rafael
I. Barraquer and Juan Alvarez de Toledo declare that they
have no conflict of interest. No human or animal studies
were carried out by the authors for this chapter.
209
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 Simple Limbal Epithelial
Transplantation: An Update
14
Nandini Venkateswaran and Guillermo Amescua

14.1 Causes and Manifestations
of Limbal Stem Cell
Deficiency
The corneal epithelium plays an integral role in pro-
tecting the cornea, maintaining corneal optical prop-
erties as well as corneal transparency. The corneal
epithelium undergoes constant shedding and regen-
eration, and the limbal stem cells that are critical for
the renewal of healthy corneal epithelium are located
at the corneal limbus. Within the limbal region, cor-
neal stem cells are located specifically in niches
within radial fibrovascular ridges called the pali-
sades of Vogt. Limbal stem cell deficiency (LSCD)
is a condition defined by the delay or failure of nor-
mal corneal epithelialization. The absence of normal
corneal epithelium can lead to conjunctivalization,
superficial corneal neovascularization, and poor
healing of the corneal surface, ultimately causing
corneal scarring and secondary blindness [1–3].
LSCD can occur due to a myriad of primary and
secondary causes. The process is driven largely by
destruction of the microenvironment of the stem
cell niche [2]. Primary causes include conditions
such as aniridia, multiple endocrine deficiency, epi-
dermal dysplasias, congenital erythrokeratoder-
mia, and dyskeratosis congenita. Secondary causes,
which are typically more common, include chemi-
cal and/or thermal burns, long-term contact lens
use, inflammatory eye diseases such as Stevens-
Johnson syndrome, ocular cicatricial pemphigoid,
atopic conjunctivitis, microbial keratitis, neuro-
trophic keratitis, history of multiple surgical inter-
ventions, radiation or systemic chemotherapy
exposure, or the use of topical antimetabolites such
as 5-fluorouracil or mitomycin C [1].
The diagnosis of LSCD is largely clinical.
Ophthalmologists arrive at the diagnosis based
on a patient’s clinical history and ophthalmic
examination. Patients often present with symp-
toms of pain, decreased vision, and photophobia.
On examination, there is loss of normal limbal
anatomy, a “whorled-like” corneal epithelium
with fluorescein staining, as well as corneal con-
junctivalization, persistent epithelial defects, and
corneal scarring [1, 2] (Fig. 14.1). Corneal impres-

N. Venkateswaran · G. Amescua (*)

Bascom Palmer Eye Institute, University of Miami, Fig. 14.1  Clinical photograph of a patient who devel-
Department of Ophthalmology, Miami, FL, USA oped 160° limbal stem cell deficiency after sustaining a
e-mail: Gamescua@med.miami.edu chemical burn

© Springer Nature Switzerland AG 2019 213

J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_14
214 N. Venkateswaran and G. Amescua

sion cytology can reveal goblet cells and confo- 14.3 S

 imple Limbal Epithelial
cal microscopy can show loss of the palisades of Transplantation
Vogt [4].
Simple limbal epithelial transplantation (SLET)
is a novel, surgical approach heralded by
14.2 Treatments for Limbal Stem Sangwan in 2010 that permits the in vivo expan-
Cell Deficiency sion of harvested limbal stem cells [7]. In SLET
surgery, a 2 × 2 mm donor limbal graft from the
The treatment of LSCD is primarily geared unaffected eye is carefully harvested, typically
toward restoring the normal, phenotypic corneal from the superior quadrant. Once the limbal graft
epithelium and rehabilitating the ocular surface. is harvested, it is left in balance salt solution. In
Prior to any surgical intervention, the ocular sur- the recipient eye, a 360° conjunctival peritomy is
face must be optimized and augmented to made and the fibrovascular pannus secondary to
improve surgical outcomes. Active, comorbid LSCD is mechanically removed. A fresh amni-
autoimmune diseases must be managed with the otic membrane transplant is secured onto the cor-
proper medications such as immunomodulatory nea with fibrin glue. The donor limbal graft is cut
agents with the goal of having a quiescent ocular into eight to ten small pieces, and these pieces are
surface. Exposure or neurotrophic keratopathy evenly distributed and placed epithelial side up
should be treated with botox-induced ptosis and/ on the amniotic membrane transplant and secured
or tarsorraphies. Mechanical irritation secondary with fibrin glue. A bandage contact lens is then
to lid or lash abnormalities should be co-­ placed over both the basal amniotic membrane
managed with oculoplastics, and surface protec- and overlying limbal transplants. Patients are
tion can be achieved with scleral lenses or subsequently followed to monitor surface healing
mucous membrane grafts. Aqueous tear defi- and assess for LSCD recurrence. Patients are
ciency should be addressed with punctal occlu- treated with topical antibiotics and a course of
sion, lubricating drops, or autologous serum tapered topical steroid drops. The bandage con-
tears [5]. tact lens is typically removed within 1 week of
For surgical approaches, main consider- the postoperative period [7]. Recently, Amescua
ations include the extent of limbal involvement et al. in 2014 posed a modification to the above
(sectoral vs total) and laterality of disease (uni- SLET surgery procedure, implementing the use
lateral or bilateral) [2]. It is important to note of a double-layer cryopreserved amniotic mem-
that corneal transplants almost universally fail brane, which is readily available in the United
in cases of LSCD [6]. Mechanical debridement States. The limbal transplants with this modified
as well as amniotic membrane grafting can ini- technique are sandwiched within two layers of
tially be pursued for partial LCSD.  However, amniotic membrane, as opposed to only being
full recovery is often limited by the degree of laid on the layer of amniotic membrane that is
remaining corneal stem cell reserves, and for secured to the corneal surface. This double layer
more extensive cases, limbal stem cell trans- of amniotic membrane aims to protect and repli-
plantation must be pursued [1]. Various tech- cate a fetal environment for the limbal stem cells
niques have been described and differ based [6]. Epithelialization after SLET can occur as
upon the anatomical source of transplanted tis- early as 2  days postoperatively. Mittal et  al.
sue (conjunctival, keratolimbal, or mucosal), showed that epithelialization stems from the lim-
the use of autologous or allogeneic tissue bal transplants over the amniotic membrane
(cadaveric or living-related) source, as well as transplant and progresses over 2 weeks. The lim-
cell culture techniques, such as cultivated lim- bal transplants disappear within 1–2 months [8],
bal epithelial transplantation (CLET) [2, 3]. in some cases it can take upto 4–6 months.
14  Simple Limbal Epithelial Transplantation: An Update
14.4 Advantages of Simple
Limbal Epithelial
Transplantation
SLET offers several advantages over other LSCD
surgical approaches [7]. Conjunctival limbal
autograft (CLAU) requires the removal of three
to six clock hours of limbus and adjacent con-
junctiva from a donor eye that is then transplanted
to the recipient eye [9]. While CLAU is a one-­
time surgery similar to SLET and has a reported
success rate of 77–100%, it requires harvesting a
much larger area of donor limbus and conjunctiva
as compared with SLET to achieve success [10,
11]. The procedure itself is also technically
demanding and poses the risk of destabilizing the
ocular surface of the donor eye [12].
Alternative limbal transplantation procedures
can be classified according to the source and
anatomic location of the donor tissue. These can
include cadaveric conjunctival limbal autografts
(c-CLAL), conjunctival limbal autografts from
living-related donors (lr-CLAL), and finally
cadaveric keratolimbal allografts (KLAL) [13].
These procedures are typically reserved for cases
of bilateral LSCD where autologous transplanta-
tion is unable to be performed. These procedures
in particular require patients to use long-term
immunosuppression, typically three agents
including a corticosteroid, an antimetabolite,
and a T-cell inhibitor, in order to decrease the
chances of immunological rejection. The use of
immunosupression comes with increased sys-
temic side effects, and these patients need to be
monitored by an expert in the field of immuno-
suppression [14].
In comparison to the above techniques, culti-
vated limbal epithelial transplantation (CLET)
allows for the ex vivo expansion of less than one
clock hour of harvested donor limbus cells [15].
Success rates range from 50 to 100%; however,
the feasibility of this technique is limited by the
time and cost needed to cultivate cells as well as
the need for facilities to allow for cell harvesting
and expansion. In the United States, there is no
Food and Drug Administration-approved facility
that will allow for the ex vivo cultivation of lim-
bal stem cells at this time [16, 17].
215
14.5 Indications for Simple
Limbal Epithelial
Transplantation
The indication for a limbal transplantation proce-
dure largely relies on the status of the limbal stem
cells and the conjunctiva. Staging paradigms
have classified ocular surface disease based upon
the percentage of involvement of the limbus,
involvement of the conjunctiva, and level of
active inflammation of the conjunctiva [14]. Stem
cell transplantation is typically difficult in
patients with a dry, inflamed ocular surface with
conjunctival scarring [13].
SLET, although a relatively new surgical pro-
cedure, has shown increasing promise in success-
fully treating LSCD.  The procedure was
originally described for unilateral 360° LSCD,
but it has also shown benefit in patients with par-
tial LCSD [7]. It has been most successful in
patients with unilateral LSCD secondary to
chemical burns or previous surgery (i.e., ocular
surface tumor removal) with a well-lubricated
ocular surface (Figs.  14.2, 14.3, and 14.4). A
large retrospective, multicenter, interventional
series by Vazirani et al. found SLET was success-
ful in 83.8% of cases of unilateral LSCD with a
wet ocular surface, with a survival probability of
greater than 80% at a median of 1 year of follow-
­up [18]. Outcomes are limited in patients with a
poorly optimized ocular surface in the setting of
active chronic inflammatory conditions such as
Stevens-Johnson syndrome or ocular cicatricial
pemphigoid [18].
Sangwan first implemented SLET surgery in a
cohort of six patients with unilateral or total
LSCD secondary to ocular surface burns. The
unaffected fellow eye served as the donor eye in
all cases. Postoperatively, all six eyes had stable,
avascular, epithelialized corneal surfaces by 6
weeks, which were maintained at a mean of 9
months. Visual acuity improved to 20/60 from
worse than 20/200 on presentation in 66.6% of
cases [7]. In Amescua et al.’s series of four patients
with unilateral LCSD treated with SLET using a
double-layer cryopreserved amniotic membrane,
100% of cases had graft success. In addition to
achieving a stable epithelium, 100% of patients
216 N. Venkateswaran and G. Amescua

a b

Fig. 14.2 (a) Clinical photograph of a patient with lim- epithelium with corneal clearing and no evidence of
bal stem cell deficiency after a chemical burn. (b) Two recurrence of limbal stem cell deficiency
years postoperatively, the patient has achieved a stable

a b c

Fig. 14.3 (a) Clinical photograph of a patient with 360° operatively, the patient has achieved corneal clarity with
limbal stem cell deficiency after a chemical burn. (b) mild residual haze and no further corneal
Clinical photograph of a patient postoperatively after sim- neovascularization
ple limbal epithelial transplantation. (c) Two years post-

a b c

Fig. 14.4 (a) Clinical photograph of a young patient who plant on the corneal surface. (c) Five months postopera-
developed limbal stem cell deficiency following at alkali tively, the patient has noted marked improvement in visual
burn. (b) Postoperatively, pieces of the donor limbal graft acuity with a stable ocular surface
are evenly distributed on an amniotic membrane trans-
14  Simple Limbal Epithelial Transplantation: An Update
had improvement in visual acuity to 20/50 or bet-
ter from initial acuities of 20/200 or worse and
had complete resolution of ocular symptoms.
Donor eyes notably had no damage [6].
Basu et al., in a prospective case series of 125
patients including adults and children with
LSCD, found that 76% of cases maintained epi-
thelialized, avascular corneas at a median follow-
­up of 1.5 years after undergoing SLET surgery,
and 67% of cases achieved a final vision of
greater than 20/60, which was statistically sig-
nificant [12]. SLET can also be used as an effec-
tive treatment modality for unilateral ocular
surface burns, with a success rate of 70% at a
median follow-up of 1.1 years as noted by Gupta
et al. [19]. In children particularly, SLET has also
shown promise in treating severe, unilateral
chemical burns [20].
Several case reports have also described the
success of SLET in various other clinical set-
tings. Cases of concomitant SLET at the time of
extensive ocular surface squamous neoplasia
excision have shown complete restoration of
limbal stem cells and maintenance of a stable
ocular surface without evidence of LSCD [21,
22]. The use of SLET to treat LCSD refractory to
CLET in pediatric patients suggests the benefits
of using SLET in this population as a first- or
second-line treatment approach, as compared
with CLET which has shown a suboptimal suc-
cess rate in children as compared with adults
[23, 24]. Studies have also demonstrated that sta-
bilization of the ocular surface with SLET can
a b
Fig. 14.5 (a) Clinical photograph of a patient who sus-
tained limbal stem cell deficiency after Pseudomonas
scleritis. (b, c) The patient underwent a penetrating kera-
217
facilitate anatomical and visual success of subse-
quent deep anterior lamellar keratoplasties in
pediatric patients with severe chemical injuries
[25]. Arya et al. have also reported the success of
SLET in restoring a stable, avascular epithelium
in patients with acid injuries as well as severe
dry eye disease [26].
14.6 Complications and Failures
of Simple Limbal Epithelial
Transplantation
SLET is an overall well-tolerated procedure.
Complications to the donor eye are infrequent
and can include subconjunctival hemorrhages,
pyogenic granuloma formation, or LCSD. In the
recipient eyes, SLET surgery can cause compli-
cations including corneal perforation at the time
of fibrovascular pannus debridement, postoper-
ative keratitis, detached amniotic membrane,
loss of limbal transplants, or symblepharon for-
mation [12].
SLET, although largely successful, can also be
associated with failures. Basu et  al. found pro-
gressive conjunctivalization in 18.4% of cases,
and factors associated with failure included his-
tory of acid injury, thermal burns, severe sym-
blepharon, SLET combined with keratoplasty,
and postoperative loss of transplants (Fig. 14.5)
[12]. Gupta et  al. as well as Vazirani et  al. also
found that the presence of symblepharon and
combined SLET with penetrating keratoplasty
c
toplasty and simple limbal epithelial transplantation with
subsequent corneal clearing and restoration of visual
function
218
a b
Fig. 14.6  Clinical photographs of a patient who devel-
oped limbal stem cell deficiency and conjunctival scarring
after a corneal burn from a fireworks injury. Despite lysis
of symblepharon, simple limbal epithelial transplantation,
and ocular surface reconstruction with a mucous mem-
was associated with SLET failure [18, 19]. These
risk factors for failure should be taken into con-
sideration when selecting the patient candidates
for SLET procedure surgery (Fig. 14.6).
14.7 Conclusions
SLET surgery has proven to be a safe, reproduc-
ible, and effective surgical intervention that allows
for the long-term regeneration of the ocular sur-
face in cases of unilateral LCSD in patients with a
wet ocular surface and adequate control of the
ocular surface inflammation. The outcomes of
SLET have also shown that by just restoring the
corneal epithelial phenotype, patients can achieve
significant improvements in visual acuity [6]. The
procedure is easy to replicate, does not require
laboratory support for cell cultivation, poses mini-
mal risk to the donor eye, and has proven to be
successful in both pediatric and adult cases of
LSCD. SLET offers several advantages to CLAU
and CLET, especially in the pediatric population;
however, there have been no randomized control
trials comparing these different treatment modali-
ties. Ultimately, assessment of larger cohort of
patients treated with SLET with long-term fol-
N. Venkateswaran and G. Amescua
brane graft, there was only marginal improvement in the
ocular surface. (a) Limbal stem cell deficiency and cor-
neal scarring after chemical/thermal burn. (b) Recurrance
of Limbal stem cell deficiency after ocular surface recon-
struction with mucous membrane graft and SLET
low-up can help clinicians better understand the
utility of this procedure in the treatment of LCSD.
Acknowledgments We would like to thank Dr. Anat
Galor MD MSPH for assistance in obtaining clinical pho-
tographs used in this publication.
Compliance with Ethical Requirements
Conflict of Interest Nandini Venkateswaran and
Guillermo Amescua declare that they have no conflict of
interest.
Informed Consent  No human studies were carried out
by the authors for this article.
No animal studies were carried out by the authors for
this article.
References
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Dhubhghaill S, Koppen C, Tassignon MJ, et al. Limbal
stem cell deficiency: current treatment options and
emerging therapies. Stem Cells Int. 2016;2016:9798374.
2. Atallah MR, Palioura S, Perez VL, Amescua
G.  Limbal stem cell transplantation: current per-
spectives. Clinical Ophthalmol (Auckland, NZ).
2016;10:593–602.
3. Yin J, Jurkunas U.  Limbal stem cell transplan-
tation and complications. Semin Ophthalmol.
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38.2017.1353834. Epub 2017 Nov 27.
14  Simple Limbal Epithelial Transplantation: An Update
4. Nubile M, Lanzini M, Miri A, Pocobelli A, Calienno
R, Curcio C, et  al. In vivo confocal microscopy
in diagnosis of limbal stem cell deficiency. Am J
Ophthalmol. 2013;155(2):220–32.
5. Espana EM, Di Pascuale M, Grueterich M,
Solomon A, Tseng SC.  Keratolimbal allograft in
corneal reconstruction. Eye (London, England).
2004;18(4):406–17.
6. Amescua G, Atallah M, Nikpoor N, Galor A, Perez
VL. Modified simple limbal epithelial transplantation
using cryopreserved amniotic membrane for unilat-
eral limbal stem cell deficiency. Am J Ophthalmol.
2014;158(3):469–75.e2.
7. Sangwan VS, Basu S, MacNeil S, Balasubramanian
D.  Simple limbal epithelial transplantation (SLET):
a novel surgical technique for the treatment of uni-
lateral limbal stem cell deficiency. Br J Ophthalmol.
2012;96(7):931–4.
8. Mittal V, Jain R, Mittal R.  Ocular surface epitheli-
alization pattern after simple limbal epithelial trans-
plantation: an in  vivo observational study. Cornea.
2015;34(10):1227–32.
9. Kenyon KR, Tseng SC.  Limbal autograft transplan-
tation for ocular surface disorders. Ophthalmology.
1989;96(5):709–22; discussion 22–3.
10. Tan DT, Ficker LA, Buckley RJ. Limbal transplanta-
tion. Ophthalmology. 1996;103(1):29–36.
11.
Arora R, Dokania P, Manudhane A, Goyal
JL.  Preliminary results from the comparison of
simple limbal epithelial transplantation with con-
junctival limbal autologous transplantation in severe
unilateral chronic ocular burns. Indian J Ophthalmol.
2017;65(1):35–40.
12. Basu S, Sureka SP, Shanbhag SS, Kethiri AR, Singh
V, Sangwan VS. Simple limbal epithelial transplanta-
tion: long-term clinical outcomes in 125 cases of uni-
lateral chronic ocular surface burns. Ophthalmology.
2016;123(5):1000–10.
13. Holland EJ, Schwartz GS.  The evolution of epithe-
lial transplantation for severe ocular surface dis-
ease and a proposed classification system. Cornea.
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14. Biber JM, Skeens HM, Neff KD, Holland EJ. The cin-
cinnati procedure: technique and outcomes of com-
bined living-related conjunctival limbal allografts and
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ure. Cornea. 2011;30(7):765–71.
15. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged
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16. Shortt AJ, Tuft SJ, Daniels JT. Ex vivo cultured limbal
epithelial transplantation. A clinical perspective. Ocul
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Ramachandran C, Basu S, Sangwan VS,
Balasubramanian D.  Concise review: the coming of
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Stem Cells Transl Med. 2014;3(10):1160–8.
18. Vazirani J, Ali MH, Sharma N, Gupta N, Mittal V,
Atallah M, et  al. Autologous simple limbal epithe-
lial transplantation for unilateral limbal stem cell
deficiency: multicentre results. Br J Ophthalmol.
2016;100(10):1416–20.
19. Gupta N, Joshi J, Farooqui JH, Mathur U.  Results
of simple limbal epithelial transplantation in uni-
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20. Mittal V, Jain R, Mittal R, Vashist U, Narang
P.  Successful management of severe unilateral
chemical burns in children using simple limbal epi-
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Guarnieri A, Moreno-Montanes J.  Concomitant
simple limbal epithelial transplantation after surgical
excision of ocular surface squamous neoplasia. Am J
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23. Bhalekar S, Basu S, Lal I, Sangwan VS.  Successful
autologous simple limbal epithelial transplanta-
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2016;10(6):Nd06–7.
 Cell Therapy Using Ex Vivo
Cultured Limbal Cells: CLET
and Equivalent
Paolo Rama and Giulio Ferrari
15.1 Introduction: Epidemiology
of LSCD
Limbal stem cell deficiency (LSCD) is defined as
a loss or deficiency of the corneal epithelial cells,
which are located at the limbus [1–4]. Many ocular
diseases can induce LSCD. These include congen-
ital anomalies, such as aniridia; traumatic diseases,
such as chemical/thermal injuries; inflammatory
disease, such as Stevens-­Johnson or Lyell syn-
drome; toxic injuries, such as factitious keratitis or
contact lens overuse; and infections [5, 6]. It
should be noted that, although these disorders
eventually induce stem cell damage, the entire
ocular surface is also affected (typically the eye-
lids, conjunctiva, corneal nerves, stroma, and lac-
rimal system). Such a complex pathophysiology
of ocular surface disease in LSCD should be kept
in mind when treating LSCD.
Because the corneal epithelium is constantly
renewed by shedding older cells, LSCD causes
corneal epithelial turnover breakdown and
delayed wound healing at first. Then, secondary
ingrowth of the conjunctiva onto the cornea
occurs. Conjunctival migration, or “conjunctival-
ization”, protects the cornea from infections and
stromal melting, although at the cost of severe
P. Rama (*) · G. Ferrari
San Raffaele Hospital, Department of
Ophthalmology—Cornea and Ocular Surface,
Milan, Italy
e-mail: rama.paolo@hsr.it
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_15
15
visual impairment, because the conjunctival epi-
thelium is opaque and vascularized.
The estimated prevalence of LSCD is 1–9 sub-
jects in 100,000. As such, it is considered a rare
disease, and it is listed on the Orphanet database
(ORPHA: 171673) in Europe. There is evidence,
however, for a higher prevalence when considering
different geographical areas. For instance, in India
it has been estimated that prevalence of severe ocu-
lar surface disorders associated with LSCD is
1.25% [7], perhaps as a consequence of a higher
incidence of industry/domestic ocular injury.
15.1.1 Treatments for LSCD
15.1.1.1 Non-surgical Treatments
Medical therapy cannot heal LSCD, but it is an
adjunctive treatment of the ocular surface that might
improve clinical findings in the early stage. Therapy
includes frequent topical application of lubricants,
bandage contact lenses, and topical steroids in case
of chronic or persistent inflammation.
15.1.1.2 Surgical Treatments
Amniotic membrane grafting can work only in
partial LSCD by promoting recovery from the
undamaged area of the limbus.
Corneal transplantation has also been
attempted, although LSCD is now unanimously
considered a contraindication to corneal trans-
plantation, either penetrating or lamellar, because
221
222
donor graft re-epithelialization will not take place,
which results in defective epithelization, graft
conjunctivalization, and eventually graft failure.
Unilateral limbal stem cell deficiency has
been successfully treated for years by directly
grafting a portion of the healthy limbal tissue
taken from the contralateral eye, although poten-
tial risks to the unaffected eye are associated
with this procedure, especially if the donor eye is
also affected by partial LSCD, as it is frequently
the case. In addition, no quality control can be
provided on these implants other than clinical
inspection. In summary, LSCD used to be an
area of significant and unmet medical need.
Simple limbal epithelial transplantation
(SLET) is a recently described surgical technique
[8] where autologous limbal stem cells are col-
lected from the unaffected eye and implanted on
the LSCD eye in one surgical session. Specifically,
the donor tissue is cut into small pieces and trans-
planted using amniotic membrane scaffold and
fibrin glue. SLET has shown good results at the
1.5-year follow-up; it is a low-cost procedure and
remains an option where tissue shipping and lab-
oratory processing may not be feasible.
15.1.1.3 CLET
To overcome risks for the donor eye, and ensure
that the correct amount of stem cells is adminis-
tered, autologous cultivated limbal epithelial
transplantation (CLET) has been developed.
CLET was originated from the work of
Rheinwald and Green, who managed to obtain a
layer of stratified epithelium by cultivating a sin-
gle keratinocyte on a layer of murine fibroblasts
(3T3 cells) [9]. Later, Pellegrini et al. showed that
autologous grafts of cultivated cells obtained
from a 1 mm2 limbal biopsy were able to restore
the corneal surface in two patients with complete
loss of the corneal limbus epithelium [10]. The
culture procedure was then standardized [11],
and to date more than 270 grafts have been trans-
planted in various centres throughout Italy, with
long-term stability reported in more than 150
patients and with a success rate in 70–80% of
cases [12, 13]. The procedure was then further
refined to fulfil regulatory standards and was
finally approved by the European Medicines
Agency (EMA) in February 2015 for the treat-
P. Rama and G. Ferrari
ment of corneal burns (Holoclar; Holostem
Terapie Avanzate, Modena, Italy).
The protocol for CLET includes biopsy on
the fellow eye and grafting of the ex  vivo
expanded limbal stem cells on a fibrin layer. The
biopsy is usually taken from the superior quad-
rant of the limbus, but it can be done in any
quadrant when we deal with partial LSCDs. The
procedure is done under topical or local anaes-
thesia, and no bandaging is required. The biopsy
is then shipped to the laboratory, where it is pro-
cessed and stem cells are extracted and culti-
vated. The stem cell product undergoes a number
of quality checks (for sterility, expression of
stemness markers, etc.). Expression of p63 is
used to distinguish LSCs from transiently ampli-
fying cells. Deductively, potency is addressed by
quantification of p63-bright cells. Release speci-
fications were set at 2.5–10.0% p63-bright cells.
In fact, a previous study showed that cultures in
which p63 cells accounted for more than 3%
resulted in successful clinical outcome in nearly
80% of cases [12]. The stem cells are then placed
on a fibrin sheet and shipped back to the hospi-
tal. The surgical procedure is done under locore-
gional or general anaesthesia. First, the pannus is
removed with careful cauterization to prevent
bleeding under the fibrin sheet. Then, stem cells
are transferred on the cornea on their fibrin glue
layer and the lids temporarily closed with a
Steri-Strip. Postsurgical therapy includes corti-
costeroid treatment, progressively tapered, to
keep the inflammation under control; persistent
or recurrent inflammation has been the first
cause of failure in previous studies [12, 13].
CLET has several advantages compared with
in vivo grafting of limbal tissue.
First, less invasive surgery is required on the
contralateral eye, which is frequently partially
involved by the same disease-inducing
LSCD. This greatly reduces potential risks aris-
ing from the surgical procedure, including infec-
tions and induction of LSCD in the fellow eye.
Second, it allows regrafting if failure of the
primary implant should occur.
Third, stringent quality controls on CLET
warrant that the minimal amount of stem cells
required to achieve clinical success is trans-
planted each time.
15  Cell Therapy Using Ex Vivo Cultured Limbal Cells: CLET and Equivalent
Finally, banking of autologous stem cells
could pave the way to genetic manipulation of
stem cells so to correct genetic anomalies (i.e.
aniridia, Meesmann dystrophy) or reduce chances
of graft rejection (major histocompatibility com-
plex modulation).
In summary, monolateral or partial bilateral
LSCD can be successfully treated with CLET.
Notably, CLET presents a number of advan-
tages over allogenic stem cell transplantation (i.e.
when cell recipient and donor are not the same
individual). Specifically, CLET does not require
systemic, lifelong immunosuppression.
Moreover, published data suggest long-term sta-
bility of transplanted epithelium in CLET [12],
while questions remain over long-term efficacy
of allogenic stem cell transplantation, since a
number of studies [14–16] have failed to detect
persistent expression of donor DNA in corneal
implants already 3 months after allogenic trans-
plantation. It should be noted that CLET is not an
option in case of bilateral LSCD, which can
instead be addressed with allogenic
transplantation.
15.1.1.4 Restoration of the Ocular
Surface Ecosystem
We would like to highlight the importance of
restoring the appropriate ocular surface milieu
in LSCD patients, as this is instrumental to the
success of any cell therapy. Indeed, stem cell
deficiency is concomitant with extensive sub-
version of the entire ocular surface, including
the conjunctiva, lids, and lachrymal systems.
Hence, conjunctival scarring causing ankylo-
blepharon/symblepharon should be surgically
corrected, as well as lid position anomalies. It
is also fundamental to maintain a good lubrica-
tion of the ocular surface, as severe dry eye is
the norm in these patients and can severely
impact any cellular implant. Finally, ocular sur-
face inflammation should be controlled at all
times. Recalcitrant inflammation is a frequent
occurrence in LSCD patients, typically in those
affected with chemical burns, Stevens-Johnson,
and ocular cicatricial pemphigoid. Bursts of
inflammation should be promptly controlled
with appropriate topical immunomodulating
agents.
223
15.2 C
 onclusion: CLET and Future
Perspectives
The recent approval of CLET from the European
Medicines Agency has paved the way to stem cell
therapy for ocular surface disorders. Now that a
regulatory pathway has been established, further
developments can be foreseen. First, transplanta-
tion of induced pluripotent stem cells (iPSCs)
could allow treatment of bilateral stem cell defi-
ciency patients, where no stem cells are available
to biopsy. Second, gene editing of stem cells
(autologous or allogenic) can be envisaged. This
approach would be ideal to definitively heal cer-
tain corneal epithelial dystrophies (e.g.
Meesmann epithelial dystrophy). In addition,
modulation of the expression of major histocom-
patibility complex (MHC) molecules could allow
to control/modulate graft rejection.
In summary, CLET discloses a number of
opportunities in areas where there is a current
significant and unmet medical need.
Technology advancements are making cell
reprogramming and gene editing easier, faster,
and less toxic. Nevertheless, long-term risks for
toxicity or malignant transformation and the high
costs of GMP production deserve close attention
and will eventually determine future develop-
ments of CLET.
Figure 15.1 shows severe limbal stem cell
deficiency secondary to chemical burn, and
Fig. 15.2 shows stable epithelium with clear cor-
nea 1 year after CLET.
Fig. 15.1  Severe limbal stem cell deficiency secondary
to chemical burn
224
Fig. 15.2  Stable epithelium with clear cornea 1  year
after CLET
15.3 Compliance with Ethical
Requirements
Paolo Rama and Giulio Ferrari declare that they
have no conflict of interest. No human or animal
studies were carried out by the authors for this
chapter.
References
1. Notara M, Alatza A, Gilfillan J, et al. In sickness and
in health: corneal epithelial stem cell biology, pathol-
ogy and therapy. Exp Eye Res. 2010;90(2):188–95.
2. Ordonez P, Chow S, Wakefield D, et  al. Human
limbal epithelial progenitor cells express
alphavbeta5-­ integrin and the interferon-induc-
ible chemokine CXCL10/IP-10. Stem Cell Res.
2013;11(2):888–901.
3. Cotsarelis G, Cheng SZ, Dong G, et al. Existence of
slow-cycling limbal epithelial basal cells that can be
preferentially stimulated to proliferate: implications
on epithelial stem cells. Cell. 1989;57(2):201–9.
4. Thoft RA, Wiley LA, Sundarraj N. The multipotential
cells of the limbus. Eye (Lond). 1989;3(Pt 2):109–13.
P. Rama and G. Ferrari
The limbus is the transition area placed between the
conjunctiva and the cornea.
5. Dua HS, Saini JS, Azuara-Blanco A. Limbal stem cell
deficiency: concept, aetiology, clinical presentation,
diagnosis and management. Indian J Ophthalmol.
2000;48(2):83–92. Review
6. Daniels JT, Notara M, Shortt A, et  al. Limbal epi-
thelial stem cell therapy. Expert Opin Biol Ther.
2007;7(1):1–3. Review.
7. Vemuganti GK, Sangwan VS. Interview: affordability
at the cutting edge: stem cell therapy for ocular sur-
face reconstruction. Regen Med. 2010;5:337–4.
8. Basu S, Sureka SP, Shanbhag SS, et al. Simple lim-
bal epithelial transplantation: long-term clinical
outcomes in 125 cases of unilateral chronic ocular
surface burns. Ophthalmology. 2016;123(5):1000–10.
9. Rheinwald JG, Green H. Serial cultivation of strains
of human epidermal keratinocytes: the formation
of keratinizing colonies from single cells. Cell.
1975;6(3):331–43.
10. Pellegrini G, Traverso CE, Franzi AT, et  al. Long-­
term restoration of damaged corneal surfaces with
autologous cultivated corneal epithelium. Lancet.
1997;349(9057):990–3.
11. Pellegrini G, Golisano O, Paterna P, et  al. Location
and clonal analysis of stem cells and their differenti-
ated progeny in the human ocular surface. J Cell Biol.
1999;145(4):769–82.
12. Rama P, Matuska S, Paganoni G, et al. Limbal stem-­
cell therapy and long-term corneal regeneration. N
Engl J Med. 2010;363(2):147–55.
13. Pellegrini G, Rama P, Matuska S, et  al. Biological
parameters determining the clinical outcome of
autologous cultures of limbal stem cells. Regen Med.
2013;8(5):553–67.
14. Chen P, Zhou Q, Wang J, et  al. Characterization of
the corneal surface in limbal stem cell deficiency and
after transplantation of cultured allogeneic limbal
epithelial cells. Graefes Arch Clin Exp Ophthalmol.
2016;254:1765–77.
15. Henderson TR, Coster DJ, Williams KA, et  al. The
long term outcome of limbal allografts: the search for
surviving cells. Br J Ophthalmol. 2001;85:604–9.
16. Daya SM, Watson A, Sharpe JR, et al. Outcomes and
DNA analysis of ex vivo expanded stem cell allograft
for ocular surface reconstruction. Ophthalmology.
2005;112:470–7.
 Cell Therapy Using Cultivated Oral
Mucosal Epithelial Transplant
(COMET)
Roberto Fernández Buenaga and Sajjad Ahmad
16.1 Main Principles
in the Management of LSCF
There are five main principles in the management
of LSCF:
1. Understanding and controlling inflammation:
It is important that ocular surface inflamma-
tion is controlled prior to any surgical man-
agement, using either topical or systemic
immune suppression. After an acute chemical
burn, inflammation can take as long as
12–18  months to control. Patients with
mucous membrane pemphigoid will require
systemic immune suppression.
2. Correcting lid malposition and fornix adhe-
sions: These are important factors that con-
tribute to ocular surface trauma and
inflammation that exacerbate the disease pro-
cess and prevent successful ocular surface
reconstruction. These therefore need to be
corrected in the first instance, prior to any sur-
gical procedure for the LSCF.
R. F. Buenaga (*)
Cornea, Cataract and Refractive Surgery Department,
Vissum Madrid, Madrid, Spain
S. Ahmad
Cornea and External Diseases Department,
Moorfields Eye Hospital, London, UK
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_16
16
3. Managing dry eye disease: LSCF is not
uncommonly associated with tear film abnor-
malities. This can be as a result of obstruction
of lacrimal duct outflow such as in mucous
membrane pemphigoid, loss of goblet cells
such as in Stevens-Johnson syndrome, or mei-
bomian gland dysfunction resulting from any
cicatrizing conjunctivitis. These abnormalities
need to be improved as much as possible prior
to consideration of any ocular surface recon-
structive procedure. Surgical measures such
as punctal occlusion and permanent partial
tarsorrhaphies may also be needed to achieve
this.
4. Partial versus total LSCF: One of the main
goals of ocular surface reconstruction is to
improve vision. In both partial and total
involvement of the limbus and therefore cor-
neal surface, central corneal involvement is an
important reason for surgical reconstruction.
There are many patients with partial LSCF
who do not have central corneal involvement
and some patients with total LSCF with cen-
tral corneal sparing who do not need ocular
surface reconstruction procedures. It must
also be noted that in partial LSCF, healthy
portions of the limbus can be utilised for sur-
gical reconstructive methods (either auto-­
CLET or auto-SLET).
5. Unilateral versus bilateral LSCF: Knowledge
of aetiology in LSCF is important in deter-
mining the surgical options, mainly whether
225
226
autograft or allograft procedures should be
employed. In unilateral disease, most com-
monly in injurious or iatrogenic causes of
LSCF, an autograft procedure would be
employed. In bilateral disease, such as genetic
and inflammatory diseases, COMET or
allograft procedures would be utilised.
Following the principles above in managing
each patient individually enables successful
determination of which surgical ocular surface
reconstructive procedure to employ.
16.2 Early Oral Mucosal Studies
The use of oral mucosa for ocular surface recon-
struction was first attempted in 1963 by Ballen
and co-workers [1]. These oral mucosal grafts
included both epithelium and subepithelial tis-
sues and developed early fibrosis and intense cor-
neal vascularisation. Later on, in 1986, Gipson
and co-workers [2] used oral epithelium (freed of
connective tissue to avoid vascularisation), but
they found that these epithelial cells could not
survive in central avascular areas of the cornea.
16.3 The Development of COMET
Taking into consideration the issues of these pre-
vious studies, Nakamura and co-workers [3]
developed a method to culture rabbit oral muco-
sal epithelial cells on amniotic membrane as a
carrier (Fig. 16.1). Small oral biopsies (approxi-
mately 2–3  mm) were obtained from the oral
cavity. The biopsy specimens were then incu-
bated with enzymatic reagents to separate epi-
thelial cells from the underlying connective
tissue. The resultant single-cell suspensions of
oral mucosal epithelial cells were cocultured for
2–3  weeks on a denuded amniotic membrane
carrier, with inactivated 3T3 fibroblasts. Toward
the end of the culture period, an air-lifting tech-
nique was used to facilitate epithelial differentia-
tion and stratification. The resulting cultured
grafts were transplanted in rabbit studies. The
eight rabbit corneas studied were all clear and
R. F. Buenaga and S. Ahmad
a
b
Fig. 16.1  Schematic illustration of culture methods for
oral mucosal epithelial cells. Figure (a) shows an explant
culture on amniotic membrane. The arrows indicate out-
growth of expanding epithelial cells around the explant.
Figure (b) shows a cell suspension culture with single epi-
thelial cells on the amniotic membrane proliferating to
give rise to multiple colonies or clumps of expanding cells
epithelialised 10  days after surgery. The oral
mucosal epithelial cells showed 5–6 layers of
stratification and appeared very similar to in vivo
normal corneal epithelium. The authors detected
the presence of nonkeratinised, mucosal-specific
keratin 4, keratin 13, and importantly corneal
epithelial specific keratin 3, whereas keratinisa-
tion-related keratin 1 or keratin 10 was not
detected. Epithelial cells were very similar in
appearance to those of normal corneal epithe-
lium, had numerous desmosomes, and were
attached to a basement membrane with hemides-
mosomes. These phenotypic characteristics were
also confirmed in other studies later on [4, 5].
Other investigators have also reported a
method of oral mucosa culture without the use of
animal feeder cells (like fibroblasts) and/or
animal-­ derived products in the culture media,
thus avoiding the risk of animal pathogen trans-
mission and reducing the risk of rejection [6].
16.4 COMET: Surgical Procedure
A 360° conjunctival peritomy 3  mm from the
limbus is performed. All perilimbal scarred or
inflamed subconjunctival tissue down to bare
sclera is removed. The corneal pannus can be
completely removed by blunt dissection or
superficial keratectomy using surgical scissors
or a blade. The subconjunctival spaces can be
treated with MMC 0.04% for 5 min followed by
vigorous wash with saline solution. The culti-
vated oral mucosal epithelial sheet is placed over
16  Cell Therapy Using Cultivated Oral Mucosal Epithelial Transplant (COMET)
the corneal surface and secured in place with
10-0 nylon sutures at the limbus. The integrity of
the cultivated epithelium is confirmed by fluores-
cein staining at the end of the surgery, and the
ocular surface is protected with a silicone hydro-
gel bandage contact lens.
16.5 Clinical Results of COMET
Re-establishment of a stable and transparent cor-
neal epithelium, regression of corneal conjuncti-
valisation/vascularisation, and resolution of
persistent epithelial defects (PEDs) have been
considered as criteria for clinical success in
LSCF.  In a short-term follow-up study, ten eyes
with PED due to LSCF from several ocular sur-
face chronic conditions were transplanted with
COMET. At the fourth postoperative week, seven
eyes (70%) had achieved complete epithelialisa-
tion and three eyes showed a small epithelial
defect. At the 24th postoperative week, PED had
improved in all patients except one patient who
did not undergo the 24th week visit [7]. In another
study with longer mean follow-up of 25.5 months,
40 eyes underwent COMET surgery [8]. Corneal
surface stability revealed an early decline in trans-
planted COMET over the first 6 months, remain-
ing stable thereafter (1 year, 64.8%; 2 years, 59%;
and 3 years, 53.1%). Persistent epithelial failure
despite COMET showed a very strong correlation
with preoperative PED (most of them eyes with
SJS, chemical and thermal injuries). Median sur-
vival in these eyes was only 27 days. Fibrovascular
invasion of the corneal surface extending over the
pupillary was observed in eight eyes after surgery.
This was especially remarkable in eyes with
OCP.  Reconstruction of a functional fornix was
combined with the corneal reconstruction at the
time of surgery in 15 eyes. This was achieved in
11 out of the 15 cases at the final examination
visit. In terms of vision, visual acuity was
improved in many patients; however, the survival
curve showed a gradual decline afterwards.
Complications like melting, perforation, or
infectious keratitis occurred in ten eyes, and all of
these eyes had PED despite the COMET treat-
ment. Finally, two eyes showed no response to
227
medical or surgical therapy (including repeated
COMET or tectonic grafts) requiring eviscera-
tion finally.
Nakamura and co-workers [9] reported better
results in 19 eyes after a longer mean follow-up
of 55 months. All of the cases demonstrated total
re-epithelialisation of the corneal surface within
2–7  days after surgery. BCVA was improved
more than two lines in 15 eyes (79%), and VA at
the postoperative 36th month was improved in
eight eyes (42%). All eyes manifested various
degrees of postoperative corneal neovascularisa-
tion, but it gradually abated, and its activity was
stable at 6 months after surgery. Regarding com-
plications, seven eyes (37%) manifested PEDs
once during the follow-up periods. Ocular hyper-
tension was detected in three eyes (16%). Finally,
one eye developed a postoperative corneal infec-
tion caused by a methicillin-resistant S. aureus.
Other authors [10] reported similar outcomes in
20 eyes with a mean follow-up of 31.9 months.
Clinical success (defined as stable ocular surface)
was 70.5% at 4  years and BCVA improved in
70% of the eyes. Corneal perforation occurred in
one patient with SJS complicated by severe lid
abnormality that induced a persistent epithelial
defect. This patient did not comply with the fol-
low-­up protocol and the cornea perforated requir-
ing a tectonic graft which was successful and the
situation settled.
16.6 Sequential Procedures:
COMET + Keratoplasty
Severe stem cell deficiency is sometimes accom-
panied by significant corneal opacity and/or
endothelial dysfunction. When the COMET pro-
cedure has been successful to restore the ocular
surface, a corneal graft, usually penetrating kera-
toplasty (PKP), may be considered as a two-step
approach in order to achieve a better corneal clar-
ity. In these patients, a keratoplasty without a pre-
vious epithelial transplantation (COMET in this
case) would result in persistent epithelial defect
leading to many possible complications such as
graft melting, perforation, and/or conjunctival
invasion compromising the visual axis.
228
There are a few publications reporting
COMET+ keratoplasty outcomes. Inatomi et al.
[11] reported two cases treated with COMET fol-
lowed approximately 6  months later by a PKP
triple procedure. The mean follow-up was
22.5 months. No complications such as persistent
epithelial defects, rejections, or recurrence of
cicatrisation were observed. In the first case
(chemical burn), the BCVA improved from hand
motion preoperatively to 20/100 after both proce-
dures and remained stable during the follow-up.
In the second case (SJS), the preoperative visual
acuity was hand motion and improved to 20/125
after both procedures and remained stable after-
wards. Satake et al. [8], in their series of 40 eyes
undergoing COMET surgery, performed a sec-
ondary keratoplasty in 7 eyes (4 eyes had PKP, 2
eyes underwent anterior lamellar keratoplasty,
and 1 eye underwent deep anterior lamellar kera-
toplasty). The mean period between COMET and
keratoplasty and the mean follow-up after kerato-
plasty were 12.6 months (range, 6–19.6 months)
and 22.2  months (range, 9.4–42.5  months),
respectively. Corneal epithelium was maintained
in six eyes; however, two of these eyes showed
gradual conjunctival invasion and neovasculari-
sation of the corneal surface at 18 months. Clarity
of the cornea with epithelial stability was
observed in 4 eyes (57.1%) after keratoplasty.
Visual acuity in four eyes with no recurrence of
conjunctival invasion was still better than 20/125
at the end of the follow-up. Complications
included recurrent herpes keratitis in one eye and
an endothelial rejection in another one.
Baradaran-Rafii et  al. [12] recently reported
their results in 14 patients with previous success-
ful COMET surgery who underwent a PKP
7.6  ±  1.3  months later. Mean follow-up period
after PKP was 28.2 ± 8.0 months. Complete cor-
R. F. Buenaga and S. Ahmad
neal epithelialisation occurred 5–7  days after
PKP in all eyes. Thirteen eyes had stable ocular
surface without epithelial defect at final examina-
tion. One eye (case 6) developed persistent cor-
neal epithelial defect followed by bacterial
keratitis 3 months after surgery. It was success-
fully treated by topical antibiotics and finally
healed with graft vascularisation. Overall graft
survival rate was 92.6%, and rejection-free graft
survival rate at 12, 24, and 40 months was 76.9,
69.2, and 69.2%, respectively. Mild stable periph-
eral superficial vascularisation (grade 1) began
1–2 months after surgery in most of the cases and
peaked at 3–6 months. During follow-up period,
four eyes had episodes of endothelial graft rejec-
tion, which was controlled with treatment.
16.7 Conclusions
COMET is a valid alternative in the management
of bilateral LSCF with visual axis involvement.
For a sub-group of patients with LSCF, such as in
graft versus host disease, where immune suppres-
sion is not possible, autologous treatment modali-
ties such as COMET are necessary. It is therefore
an important management option for LSCF. The
major benefits of COMET are its autologous
nature and its mucosal origin. In bilateral eye dis-
eases, such as mucous membrane pemphigoid,
Stevens-Johnson syndrome, and graft-versus-­host
disease are most likely indications for COMET. In
these diseases, where fluctuating inflammation is
a key component of the disease process, alloge-
neic limbal transplantation is a less favourable
treatment than COMET which is autologous. The
main indications for COMET are therefore bilat-
eral LSCF with visual axis involvement where
immune suppression is not possible (Fig. 16.2).
16  Cell Therapy Using Cultivated Oral Mucosal Epithelial Transplant (COMET) 229

Corrected lid malposition and significant

fornix adhesions

Optimise the tear film

Visual axis NO Reconstruction not

involved? necessary

Yes

Total or NO Consider partial

more than epitheliectomy with AM
1/3 LSCF? overlay

Yes

Bilateral NO
Perform CLAU or auto-SLET
disease?

Failure

Yes
Use commercial available
auto-CLET

Can systemic
immune- NO
COMET
suppression
be used?

Yes

Kerato-limbal allograft or allo- Failure

Allo-CLET
SLET from cadaveric tissue

Fig. 16.2  Proposed decision-making flow chart

230
Compliance with Ethical Requirements  Sajjad Ahmad
and Roberto Fernandez Buenaga declare that they have no
conflict of interest. No human or animal studies were car-
ried out by the authors for this chapter.
References
1. Ballen PH.  Mucous membrane grafts in chemical
(eye) burns. Am J Ophthalmol. 1963;55:302–12.
2. Gipson IK, Geggel HS, Spurr-Michaud SJ. Transplant
of oral mucosal epithelium to rabbit ocular surface
wounds in vivo. Arch Ophthalmol. 1986;104:1529–33.
3. Nakamura T, Endo K, Cooper L, et al. The success-
ful culture and autologous transplantation of rabbit
oral mucosal epithelial cells on amniotic membrane.
Invest Ophthalmol Vis Sci. 2003;44:106–16.
4. Nakamura T, Inatomi T, Cooper LJ, et al. Phenotypic
investigation of human eyes with transplanted
autologous cultivated oral mucosal epithelial sheets
for severe ocular surface diseases. Ophthalmology.
2007;114(6):1080–8.
5. Satake Y, Dogru M, Yamane GY, et al. Barrier function
and cytologic features of the ocular surface epithelium
after autologous cultivated oral mucosal epithelial
transplantation. Arch Ophthalmol. 2008;126(1):23–8.
6. Kolli S, Ahmad S, Mudhar HS, et  al. Successful
application of ex  vivo expanded human autologous
R. F. Buenaga and S. Ahmad
oral mucosal epithelium for the treatment of total
bilateral limbal stem cell deficiency. Stem Cells.
2014;32(8):2135–46.
7. Sotozono C, Inatomi T, Nakamura T, et al. Cultivated
oral mucosal epithelial transplantation for persistent
epithelial defect in severe ocular surface diseases
with acute inflammatory activity. Acta Ophthalmol.
2014;92(6):e447–53.
8. Satake Y, Higa K, Tsubota K, et  al. Long-term out-
come of cultivated oral mucosal epithelial sheet
transplantation in treatment of total limbal stem cell
deficiency. Ophthalmology. 2011;118:1524–30.
9. Nakamura T, Takeda K, Inatomi T, et  al. Long-term
results of autologous cultivated oral mucosal epithelial
transplantation in the scar phase of severe ocular sur-
face disorders. Br J Ophthalmol. 2011;95(7):942–6.
10. Prabhasawat P, Ekpo P, Uiprasertkul M, et al. Long-­
term result of autologous cultivated oral mucosal
epithelial transplantation for severe ocular surface
disease. Cell Tissue Bank. 2016;17(3):491–503.
11. Inatomi T, Nakamura T, Kojyo M, et al. Ocular sur-
face reconstruction with combination of cultivated
autologous oral mucosal epithelial transplantation
and penetrating keratoplasty. Am J Ophthalmol.
2006;142(5):757–64.
12. Baradaran-Rafii A, Delfazayebaher S, Aghdami N,
et  al. Midterm outcomes of penetrating keratoplasty
after cultivated oral mucosal epithelial transplantation
in chemical burn. Ocul Surf. 2017;15(4):789–94.
 Cell Therapy Using Extraocular
Mesenchymal Stem Cells
Teresa Nieto-Miguel, Sara Galindo,
Marina López-­Paniagua, Inmaculada Pérez,
José M. Herreras, and Margarita Calonge
Abbreviations
ABCG2 ATP-binding cassette subfam-
ily G member 2
ALDH3A1 Aldehyde dehydrogenase 3
family member A1
AM Amniotic membrane
APCs Antigen-presenting cells
AT-MSCs Adipose tissue-derived mesen-
chymal stem cells
BM-MSCs Bone marrow-derived mesen-
chymal stem cells
C/EBPδ Cytosine-cytosine-adenosine-­
adenosine-thymidine/
enhancer-binding protein-δ
CAT Catalase
CCL Chemokine (C-C motif) ligand
These authors contributed equally: Teresa Nieto-Miguel,
Sara Galindo and Marina López-Paniagua
T. Nieto-Miguel · S. Galindo · M. López-Paniagua ·
M. Calonge (*)
CIBER-BBN (Biomedical Research Networking
Centre in Bioengineering, Biomaterials and
Nanomedicine), Carlos III National Institute of
Health, Valladolid, Spain
IOBA (Institute of Applied Ophthalmobiology),
University of Valladolid, Valladolid, Spain
e-mail: calonge@ioba.med.uva.es
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_17
17
CCL2/MCP-1 Chemokine (C-C motif) ligand
2/monocyte chemoattractant
protein-1
CD Cluster of differentiation
CINC-1/CXCL1 Cytokine-induced neutrophil
chemoattractant 1
CK Cytokeratin
CLET  Cultivated limbal epithelial
transplantation
Cox-2 Cyclooxygenase-2
Cx43 Connexin 43
CXCR4 C-X-C chemokine receptor type 4
DP-MSCs Dental pulp-derived MSCs
EGF Epidermal growth factor
GM-CSF  G r a n u l o cy t e - m a c r o p h a g e
colony-­stimulating factor
GMP Good manufacturing practices
I. Pérez
IOBA (Institute of Applied Ophthalmobiology),
University of Valladolid, Valladolid, Spain
J. M. Herreras
CIBER-BBN (Biomedical Research Networking
Centre in Bioengineering, Biomaterials and
Nanomedicine), Carlos III National Institute of
Health, Madrid, Spain
IOBA (Institute of Applied Ophthalmobiology),
University of Valladolid, Valladolid, Spain
Department of Ophthalmology, Clinic University
Hospital, Valladolid, Spain
231
232
GPX Glutathione peroxidase
GvHD Graft versus host disease
HLA-DR Human leukocyte antigen-DR
ICAM-1  Intercellular adhesion mole-
cule 1
IDO Indoleamine-2,3-dioxygenase
IFN-γ Interferon gamma
Ig Immunoglobulin
IGF-I Insulin-like growth factor-I
IL Interleukin
iNOS Inducible nitric oxide synthase
iPSC Induced pluripotent stem cells
iPSC-MSCs Induced pluripotent stem cell-­
derived mesenchymal stem
cells
IVCM In vivo confocal microscopy
KGF-2 Keratinocyte growth factor-2
LESCs Limbal epithelial stem cells
LSCD Limbal stem cell deficiency
M1 Macrophages type 1
M2 Macrophages type 2
MCP-1 Monocyte chemotactic protein 1
MDA Malondialdehyde
MHC Major histocompatibility
complex
MIP-1α Macrophage inflammatory
protein-1 alpha
MMP Matrix metallopeptidase
MPO Myeloperoxidase
MSCs Mesenchymal stem cells
MSCT Mesenchymal stem cell
transplantation
NaOH Sodium hydroxide
NF-kB Nuclear factor-kappa beta
NK Natural killer cells
NO Nitric oxide
NT Nitrotyrosine
PanCK Pan-cytokeratin
Pax6 Paired box 6
PCNA Proliferating cell nuclear
antigen
PD-1 Programmed death-1
PDGF Platelet-derived growth factor
PD-L1 Programmed death ligand-1
PEDF Pigment epithelium-derived
factor
PGE2 Prostaglandin E2
RT-PCR Reverse transcription-poly-
merase chain reaction
T. Nieto-Miguel et al.
SDF-1α/CXCL12 Stromal cell-derived factorα1/
C-X-C motif chemokine 12
SGPT Serum glutamic-pyruvic
trans­aminase
SOD Superoxide dismutase
SSEA4 Stage-specific embryonic
antigen-4
TER Transepithelial electrical
resistance
TGF-β Transforming growth factor
beta
TLR Toll-like receptors
TNF-α Tumor necrosis factor alpha
Treg Regulatory T cells
TSG-6 Tumor necrosis factor-a-stim-
ulated gene/protein-6
TSP-1 Thrombospondin-1
UC-MSCs Umbilical cord-derived mes-
enchymal stem cells
VCAM-1 Vascular cell adhesion protein 1
VEGF Vascular endothelial growth
factor
WJ-MSCs Mesenchymal stem cells
derived from the Wharton’s
jelly of the umbilical cord
XOX Xanthine oxidase
ZO-1 Zonula occludens-1
17.1 Introduction
Maintenance of corneal epithelium integrity is
essential for preserving corneal transparency and
visual function. For that reason, a population of
stem cells residing in the basal epithelial layer of
the corneoscleral limbus, the so-called limbal
epithelial stem cells (LESCs), is continuously
renewing the corneal epithelium layers [1–4].
Destruction or dysfunction of these limbal epi-
thelial stem cells or their niche induces a syn-
drome known as limbal stem cell deficiency
(LSCD), which can be caused by a wide variety
of ocular surface injuries and disorders such as
chemical or thermal burns, multiple previous eye
surgeries, cicatrizing-autoimmune pathologies,
severe dry eye syndrome, infections, congenital
disorders, etc. LSCD is characterized by a defi-
cient regeneration of the corneal epithelium that
17  Cell Therapy Using Extraocular Mesenchymal Stem Cells
eventually leads to persistent epithelial defects,
ingrowth of conjunctival tissue onto the cornea
surface, neovascularization, and persistent ocular
surface inflammation. All these events usually
result in loss of vision and a chronic pain syn-
drome [5, 6].
Transplantation of cultivated limbal epithelial
cells (CLET) harvested from the contralateral
healthy eye is the current treatment of choice for
unilateral cases of LSCD, in which it is possible
to harvest autologous limbal tissue from the
healthy fellow eye and cultured it to expand the
stem cell population before transplantation [7]. In
bilateral cases, which in fact are more frequent, it
is necessary to resort to allogeneic limbal tissue
from deceased donors due to the scarcity of
healthy tissue in patient’s eyes, transforming an
autologous transplant into an allogeneic one.
Allogeneic transplantations require 1 year of sys-
temic immunosuppression in order to avoid
immune rejection, which consequently increases
the risk of the patient’s morbidity and the medical
expenditures, although it avoids the costs related
with the extraction of a biopsy [7, 8]. The use of
alternative sources of extraocular cells could help
to overcome the dependence on and the limita-
tions of limbal epithelial cells, which are difficult
to obtain and culture and that, in case of being
allogeneic, can induce immune rejection [9–11].
Mesenchymal stem cells (MSCs) constitute a
subset of stromal cells that can be isolated from a
variety of tissues including bone marrow, adipose
tissue, dental pulp, umbilical cord, and limbal
stroma of the human eye, among others [9, 12].
They are partially defined by their adherence to
plastic supports when cultured in standard condi-
tions; their multipotent differentiation potential
to bone, cartilage, and adipose tissue in vitro; and
the expression of a specific profile of surface
antigens, including positivity for cluster of dif-
ferentiation (CD) 73, CD90, and CD105 and
negativity for CD34, CD45, CD11b or CD14,
CD19 or CD79α, and HLA-DR markers [13].
The use of human MSCs in cell-based thera-
pies has tremendously increased over the last
decade in the field of regenerative medicine.
There is a growing body of literature supporting
that the therapeutic effects of MSCs not only rely
on their differentiation ability to repair damaged
233
tissue but also on their immunomodulatory and
anti-inflammatory properties, their capability to
migrate to injured and inflamed tissues, and their
capacity to modulate local environment, activate
endogenous progenitor cells, and secrete trophic
factors. In addition, compared with other types of
stem cells, MSCs have several advantages, such
as availability and easy harvesting from a variety
of tissue types, few ethical concerns, elevated
proliferative capacity in vitro, and low immuno-
genicity [12, 14, 15].
For all these reasons, MSCs have emerged as
very attractive candidates for cell-based therapies
in numerous clinical applications including auto-
immune and inflammatory diseases (e.g., graft
versus host disease (GvHD), Crohn’s disease, and
rheumatoid arthritis), skeletal disorders, heart dis-
eases, diabetes, neurological disorders, etc. [12,
15]. In ophthalmology, the potential therapeutic
applications are also many, and, according to the
ClinicalTrial.gov research database, there are cur-
rently several ongoing clinical trials to test the
efficacy of MSCs in the treatment of a variety of
ocular disorders such as retinitis pigmentosa, age-
related macular degeneration, glaucoma,
non-arteritic ischemic optic neuropathy, and dif-
ferent ocular surface disorders [16]. This book
chapter is specifically focused on the preclinical
and clinical advancements on the use of extraocu-
lar MSCs for corneal epithelium regeneration.
17.2 E
 xtraocular MSCs for Corneal
Epithelium Regeneration
17.2.1 Therapeutic Potential
of Extraocular MSCs
in Corneal Epithelium
Regeneration
There are abundant in vitro and in vivo studies on
the use of extraocular MSCs for corneal epithe-
lium regeneration. All published studies have
revealed promising results in animal models and
have shown significant corneal regeneration,
improved corneal transparency, and a rapid heal-
ing process associated with the restoration of
vision [10, 17–19]. However, the studies per-
formed to unravel the mechanisms underlying the
234 T. Nieto-Miguel et al.

Fig. 17.1  Properties of Differentiation toward

mesenchymal stem cells other cell types Support of tissue-specific
(MSCs). MSCs have the progenitor cells
capacity to differentiate
into other cell types; to Selective migration
support tissue-specific to the site of injury
progenitor cells; to
provide a regenerative
microenvironment; to be
non-immunogenic; to
have
immunomodulatory,
Regenerative
anti-fibrotic, and
microenvironment
anti-apoptotic
properties; and to
migrate to the site of
MSCs
injury

Anti-fibrotic and
anti-apoptotic effect lmmunomodulation

Non-immunogenic cells

beneficial effects of MSCs on the damaged ocu- Transdifferentiation Capacity of Bone

lar surface have shown that multiple mechanisms
might contribute simultaneously to their thera-
peutic action. Although it remains uncertain if
MSCs can transdifferentiate into corneal epithe-
lial cells [20], these cells have shown a capacity
of secreting trophic and growth factors capable of
stimulating resident stem cells, reducing tissue
injury, an ability to exert anti-inflammatory and
immunomodulatory effects, and a capability to
migrate into injured tissues [11, 17, 19–21]
(Fig. 17.1).
17.2.1.1 Transdifferentiation
Capacity into Corneal
Epithelial Cells
Corneal epithelium derives from the surface ecto-
derm during embryonic development [22].
Although it seems pretty clear that MSCs can dif-
ferentiate not only into mesodermal but also into
non-mesodermal cell lineages including neuroec-
todermal and epithelial cells [23–25], the poten-
tial transdifferentiation capacity of MSCs into
corneal epithelial cells is unclear and remains
under investigation [20].
Marrow-Derived MSCs (BM-MSCs) into
Corneal Epithelial Cells
There are several studies published on the trans-
differentiation capacity of human BM-MSCs into
corneal epithelial cells. It has been shown that
human BM-MSCs cultured on amniotic mem-
brane (AM) and transplanted onto the surface of
an alkali-burned rabbit cornea migrated to the
cornea and expressed the corneal epithelial cell
marker cytokeratin (CK) 12 when located in the
corneal epithelium [26]. Subsequently, the poten-
tial of human BM-MSCs to differentiate into cor-
neal epithelial cells was confirmed when they
were cultured first on pig Bowman’s membrane
and later under air-lifting conditions for creating
a multilayer of cells. After 4 weeks of differentia-
tion induction, Hou et  al. saw that part of the
BM-MSCs expressed the corneal and limbal epi-
thelial markers CK12 and CK19, respectively,
but not the limbal stem cell marker ATP-binding
cassette subfamily G member 2 (ABCG2) [27].
Afterward, Rohaina et al. also showed that while
CK3 and p63 were not detected in human
BM-MSCs, upon being cultured in limbal media
17  Cell Therapy Using Extraocular Mesenchymal Stem Cells
for 10  days, they significantly increased their
expression for the corneal and limbal epithelial
markers CK3 and p63, respectively [28]. They
also demonstrated that transplantation of induced
BM-MSCs stratified on AM onto a nude LSCD
rat model remarkably improved corneal regener-
ation in terms of corneal transparency and vascu-
larization. Although they did not track
transplanted cells, they observed CK3 and p63
protein expression in the newly regenerated cor-
nea [28]. A recent study has also revealed that
human BM-MSCs positive for the early embry-
onic stem cell marker stage-specific embryonic
antigen-4 (SSEA4) had higher potential to differ-
entiate into corneal epithelial cells. Even though
differentiated cells did not reach the typical tran-
sepithelial electrical resistance (TER) of corneal
epithelium, they presented an epithelial-like mor-
phology and were positive for the corneal and
limbal epithelial markers CK3, CK12, CK8,
CK14, CK15, β1-integrin, and E-cadherin [29].
In addition, another in  vivo study has demon-
strated too that human BM-MSCs administered
to mice by subconjunctival injection migrated to
the corneal tissues and displayed morphological
characteristics of epithelial, stromal, and endo-
thelial cells [30].
Regarding rabbit BM-MSCs, Gu et al. showed
that when they were co-cultured with limbal epi-
thelial stem cells, or their conditioned medium,
they changed their fibroblastic morphology to a
more flattened epithelial-like shape and tran-
siently increased their expression of the corneal
epithelial-specific marker CK3 [31]. They also
saw that when they were suspended in fibrin gels
and further transplanted onto alkali-burned rabbit
corneas, rabbit BM-MSCs induced a successful
ocular surface reconstruction, and some of those
that located in the corneal epithelium expressed
the corneal epithelial-specific marker CK3,
implying BM-MSC differentiation into corneal
epithelial cells [31]. Interestingly, another study
evidenced that, under standard culture condi-
tions, rabbit BM-MSCs were negative for the
limbal and corneal epithelial markers ABCG2,
p63, and connexin 43 (Cx43), but positive for
CK3/12 and α-enolase, suggesting, for the first
time, that CK3 and CK12 markers were not
235
highly specific for differentiated corneal epithe-
lium [32].
Concerning BM-MSCs isolated from rats,
Jiang et al. demonstrated that co-cultivation of rat
BM-MSCs with corneal stromal cells induced the
expression of the corneal epithelial-specific
marker CK12 and the acquisition of morphology
and cell structures (tight junctions) typical of epi-
thelial cells. Transplantation of the induced rat
BM-MSCs cultured on AM for 7 days ­demonstrated
that those cells significantly improved the recon-
struction of the cornea surface in an alkali-­burned
LSCD rat model [33]. Zhang et al. also supported
that rat BM-MSCs had the ability to transdifferen-
tiate into corneal epithelial cells when they cul-
tured them on a xenogeneic acellular corneal
matrix in  vitro for 7  days and showed that cells
acquired an epithelium-like shape and expressed
the corneal epithelial marker CK3 [34].
In regard to mouse BM-MSCs, a recent
in vitro study demonstrated that when they were
cultured with corneal extract in the presence of
insulin-like growth factor-I (IGF-I), mouse
BM-MSCs differentiated to cells with features of
corneal epithelial cells and keratocytes and main-
tained MSC properties. They observed that the
expression of the corneal-specific markers CK12,
keratocan, and lumican was upregulated after
culture with corneal extract and that adding IGF-I
to the culture medium significantly increased the
expression of those genes [35].
In contrast to the evidence supporting the dif-
ferentiation potential of BM-MSCs into corneal
epithelial cells, a study conducted by Ma et  al.
revealed that human BM-MSCs transplanted
over AM onto a chemically burned rat cornea sur-
vived and successfully reconstructed the dam-
aged rat corneal surface but that the therapeutic
effect did not come from epithelial differentiation
of BM-MSCs but as result of inhibition of neo-
vascularization and inflammation [36]. Likewise,
another study showed that rabbit BM-MSCs sys-
temically administered in a rabbit corneal alkali-­
burned model, engrafted to injured cornea,
promoted wound healing by stimulating and syn-
ergizing with native cells and by differentiating
not into corneal or limbal epithelia cells but into
myofibroblast [37].
236
Although the outcome of many in vitro experi-
ments supports the idea that BM-MSCs are able
to transdifferentiate into corneal epithelial cells
under certain conditions, in  vivo data published
up to date has shown contradictory and inconclu-
sive results pointing out that further investiga-
tions are required to confirm whether BM-MSCs
can or cannot acquire a corneal epithelial cell
phenotype.
Transdifferentiation Capacity
of Subcutaneous Adipose Tissue-Derived
MSCs (AT-MSCs) into Corneal Epithelial
Cells
Up to now, results from the few studies specifi-
cally performed on AT-MSCs differentiation into
corneal epithelial cells do not bring enough evi-
dence to support the hypothesis of their real
transdifferentiation into functional corneal epi-
thelial cells since all of them are only based on
in vitro experiments. While a first study showed
that orbital fat-derived stem cells had more poten-
tial to differentiate into corneal epithelial cells
than MSCs derived from subcutaneous AT due to
their same developmental origin during embry-
onic development [38], a subsequent study found
that human AT-MSCs expressed the progenitor
markers p63 and ABCG2 and the cytokeratins for
differentiated epithelial cells CK12 and CK76.
Furthermore, the study showed that CK12 expres-
sion spontaneously and progressively increased
by cell adhesion over time in culture, suggesting
that AT-MSCs had the potential to acquire cor-
neal epithelial-like characteristics under the
appropriate conditions [39]. Those results were
further confirmed by Nieto-Miguel et  al. when
they demonstrated that human AT-MSC grown
under basal culture conditions expressed the cor-
neal epithelial markers CK3 and CK12 and that
AT-MSCs acquired a more epithelial-like mor-
phology and an upregulated expression of CK12
when they were cultured in corneal epithelial
cell-conditioned medium for 15 days [40]. Later,
the transdifferentiation capacity of AT-MSCs was
also advocated when another in  vitro study
showed that paired box 6 (Pax6) stable transfec-
tion stimulated differentiation of murine
AT-MSCs into corneal epithelial-like cells by
T. Nieto-Miguel et al.
induction of the expression of the corneal
epithelium-­specific marker CK12 [41].
Transdifferentiation Capacity of MSCs
Derived from the Wharton’s Jelly
of the Umbilical Cord (WJ-MSCs) into
Corneal Epithelial Cells
Base on the accessibility, abundance, lack of ethi-
cal issues, and capability of transdifferentiating
into epithelial cells [42], Garzón et  al. chose
human Wharton’s jelly MSCs to generate a three-­
dimensional heterotypic human cornea with
fibrin-agarose scaffolds. They showed that
WJ-MSCs were able to differentiate into corneal
epithelial-like cells with results similar to the
native corneas in the expression of corneal epi-
thelial key markers such as CK3/12, plakoglobin,
zonula occludens 1 (ZO-1) and Cx43 [43]. On
the other hand, Sidney et al. studied the expres-
sion of specific epithelial cell markers at different
passages and activity stages of corneal stromal
cell and compared it to the one in WJ-MSCs.
Results demonstrated that in vitro culture of stro-
mal cells not only caused phenotypical changes
but also induced expression of specific cytokera-
tins. Despite not being from epithelial origin,
WJ-MSCs were positively stained for CK3/12,
CK19, panCK, and E-caherin, while corneal stro-
mal cells only were stained for CK3/12 and
CK19. Reverse transcription-polymerase chain
reaction (RT-PCR) analyses revealed signifi-
cantly decreased gene expression of CK3, CK12,
and CK19  in WJ-MSCs compared to corneal
stromal cells and showed detectable levels of
both CK5 and E-cadherin genes, suggesting that
there might be some specificity to the cytokera-
tins expressed in stromal cells isolated from dif-
ferent tissues [44].
Transdifferentiation Capacity of Dental
Pulp-Derived MSCs (DP-MSCs) into
Corneal Epithelial Cells
There are only few studies in which it has been
explored the potential capacity of DP-MSCs to
differentiate into corneal epithelial cells [45–48].
In the first published study, Monteiro et al. dem-
onstrated that human immature dental pulp stem
cells expressed in vitro limbal epithelial stem cell
17  Cell Therapy Using Extraocular Mesenchymal Stem Cells
markers such as ABGC2, Cx43, integrin-β1, p63,
and vimentin, while they did not express or
weakly expressed the corneal epithelial-specific
markers CK3 and CK12, respectively [45]. The
subsequent transplantation of DP-MSC sheets
onto the ocular surface of a mild and severe
LSCD rabbit model also confirmed that DP-MSCs
successfully induced corneal epithelium recon-
struction and had the capacity to transdifferenti-
ate into corneal epithelial cells in vivo [45, 46].
Later, it was demonstrated that stem cells from
human exfoliated deciduous teeth also had the
potential to be induced to differentiate into cor-
neal epithelial-like cells in  vitro. Tsai et  al.
showed that both direct and indirect co-culturing
with corneal epithelial cells upregulated the
expression of the corneal epithelial marker CK3
and the corneal epithelial progenitor marker
CK19 [48]. Furthermore, when Kushnerev et al.
used soft contact lenses to deliver MSCs derived
from pulp tissue explants to an ex vivo model of
human epithelial-debrided corneas in organ cul-
ture, they saw that, once the cells had transferred,
they began to express the corneal epithelial-­
specific markers CK3 and CK12, supporting that
DP-MSCs differentiated into corneal epithelial-­
like cells once they attached to Bowman’s mem-
brane [47].
Although the results derived from several
in vitro experiments support the idea that MSCs
are able to differentiate into a corneal epithelial-­
like cell phenotype under certain conditions, it
remains uncertain whether the expression of cer-
tain epithelial markers like cytokeratins repre-
sents a real functional differentiation or just the
result of MSC culturing [20, 44]. Moreover, the
fact that results from several in  vivo studies do
not support the data shown by in  vitro experi-
ments suggests the need of performing additional
in vivo studies to address whether MSCs can or
cannot transdifferentiate into functional corneal
epithelial cells.
17.2.1.2 Trophic Activity
The Mesenchymal and Tissue Stem Cell
Committee of the International Society for
Cellular Therapy proposed the minimal criteria
to define human MSCs in 2006 [13]. Nevertheless,
237
even before an official definition about MSCs
was established, it was known that mesenchymal
progenitor cells obtained from bone marrow
could secrete different cytokines which had spe-
cific functions inside tissues under physiological
conditions and whose expression was modified
under inflammation situations [49]. This fact,
together with results obtained from different
studies in which BM-MSCs enhanced corneal
wound healing in spite of the absence of MSC
migration to damaged tissue [50, 51], contributed
to establish the concept currently known as “tro-
phic effect of MSCs.”
At the moment, it is known that MSCs secrete
a large panel of soluble bioactive molecules
which can cause direct intracellular signaling
(autocrine effect), direct extracellular effects, or
indirect effects by causing another cell in the
vicinity to secrete the functionally active agent
[52, 53]. The main functions of these bioactive
molecules are (1) to inhibit apoptosis and limit
the field of damage or injury (anti-apoptosis
mechanism), (2) anti-inflammatory action, (3) to
regulate angiogenesis (pro- and anti-angiogenic),
(4) to stimulate and support the growth and dif-
ferentiation of local stem cell, and (5) oxidative
stress protection [52, 54, 55]. The trophic effects
of MSCs have been also detected in corneal epi-
thelium regeneration, showing that different fac-
tors secreted by MSCs can be implicated in a set
of processes, such as corneal re-epithelialization,
and anti-inflammatory and anti-angiogenic
actions that finally end in total or partial corneal
tissue regeneration.
Corneal Wound Healing,
Re-epithelialization, and Anti-apoptosis
Mechanism
Corneal wound healing is a complex multistage
process. For corneal wounds to heal optimally, a
cascade of multicellular interactions and tissue
remodeling must proceed in an orderly fashion.
This process can be enhanced by several tissue
repair growth factors secreted by cells and those
recruited to the wound immediately after tissue
injury [34, 56]. Currently, it is known that
BM-MSCs secrete factors as vascular endothelial
growth factor (VEGF), epidermal growth factor
238
(EGF), pigment epithelium-derived factor
(PEDF), and transforming growth factor beta
(TGF-β) 1 [34], with the EGF and TGF-β1 play-
ing an important role in the proliferation and
migration of corneal epithelial cells [57–59] and,
therefore, in the stimulation of corneal epithe-
lium regeneration. When the effect of rat MSCs
versus rat LESCs was compared for in vitro heal-
ing corneal epithelium wounds, it was observed
that the secretion of EGF and TGF-β1 was higher
by MSCs than by LESCs. This difference would
indicate that MSCs could be better than LESCs
for corneal wound healing [34]. However, differ-
ent studies showed that rabbit BM-MSCs,
AT-MSCs, and LESCs secreted, spontaneously
or after stimulation with lipopolysaccharides,
levels of TGF-β without significant differences
among them [60, 61]. In addition, when these
three types of cells were cultured on nanofiber
scaffold and transplanted onto a de-epithelialized
rabbit corneal surface, the transfer of stem cell-­
seeded nanofiber scaffolds onto the damaged
ocular surface significantly improved corneal
healing, in part because the number of apoptotic
caspase 3+ cells decreased in the groups treated
with stem cell-seeded nanofibers with respect to
untreated injured corneas or with respect to cor-
neas treated with cell-free nanofiber scaffolds
[60]. This fact support the important implication
of numerous trophic and growth factors secreted
by stem cells, such as MSCs or LESCs, on wound
healing. In addition, the best re-epithelialization
was observed in corneas treated with nanofiber
scaffolds cultured with BM-MSCs or LESCs,
reporting that MSCs from different sources can
have different trophic effects on wounded corneal
epithelium [60]. On the other hand, it is known
that MSCs promote the survival and proliferation
of LESCs in the tissue, and that these effects may
be mediated in a paracrine manner, due to the fact
that cell proliferation rate and expression of
EGFs were significantly higher in LESCs co-­
cultured with MSCs than in LESCs cultured
alone [62]. In addition, the expression of matrix
metallopeptidase (MMP)-9 by damaged human
corneal epithelial cells is strongly inhibited by
human MSCs [63]. Taking into account that
MMP-9 interferes with normal re-­epithelialization
T. Nieto-Miguel et al.
of the wounded cornea [64], this fact supports the
hypothesis that MSCs transplanted after corneal
and limbal epithelium debridement could pro-
mote the proliferation of LESCs and decrease the
induced apoptosis in corneal epithelial cells.
Recently, it has been also shown that tumor
necrosis factor-a-stimulated gene/protein-6
(TSG-6), secreted by murine BM-MSCs, pro-
moted corneal epithelial wound healing in dam-
aged corneal epithelium of diabetic mice through
activation of LESCs [65].
Anti-inflammatory Action
In 2006, it was reported the first evidence that
human BM-MSCs could be used for the treat-
ment of damaged corneal epithelium through an
anti-inflammatory mechanism, since pro-­
inflammatory molecules which were detected in
injured corneas treated with AM alone, such as
CD45, interleukin (IL) 2, and MMP-2, were not
detected in the damaged corneal surfaces treated
with AM plus MSCs [36]. Interestingly, the ther-
apeutic effect of the transplantation could be
associated with the anti-inflammatory trophic
effect of MSCs, although this hypothesis must
be contrasted. Some years after, it was observed
that topical application of MSC-derived condi-
tioned media contributed to restoration anti-­
inflammatory property of the rat corneal
epithelium in damaged animals [66]. These
authors suggested that the exogenous high con-
centration of IL-6 secreted by MSCs to extracel-
lular medium, and subsequently administrated to
damaged corneas, could suppress dendritic cell
activation in injured ocular surface [66–68]. In
accordance with this, several research groups
have supported that soluble factors secreted by
the MSCs played an important role as anti-­
inflammatory therapy in wounded corneas, since
decreased corneal inflammation can be observed
in spite of the absence of MSC migration to
damaged tissue [51, 69], establishing that the
anti-­inflammatory mechanism of MSCs could be
mediated by paracrine pathways. Several works
have supported that trophic factors secreted by
MSCs could suppress the infiltration of inflam-
matory cells and CD68+ macrophages by sup-
pressing the expression of the macrophage
17  Cell Therapy Using Extraocular Mesenchymal Stem Cells 239

inflammatory protein-1 alpha (MIP-1α) [51, 70, as pro-angiogenic molecules in the cornea, while
71] and that factors secreted by MSCs are capa- TSP-1 is a powerful anti-angiogenic factor that
ble of inhibiting production of pro-inflammatory acts inhibiting VEGF-induced angiogenesis [76–
tumor necrosis factor alpha (TNF-α) by macro- 78], maintaining the characteristic avascular cor-
phages in locally burned corneas [51, 72, 73]. In neal situation. Another molecule secreted by
addition, it is known that MSCs, activated by MSCs that has been proposed as anti-angiogenic
inflammatory signals, secreted the anti-­factor after corneal damaged is the TSG-6, since
inflammatory protein TSG-6 [69, 74]. it can decrease the neovascularization in corneas
Specifically, it is known that chemical injury to with their epithelium debrided [75]. On the other
corneal epithelial cells activated human MSCs to hand, it is known that MSCs and LESCs secrete,
secrete anti-inflammatory protein TNF-α that spontaneously or after stimulation with lipo-
stimulated TSG-6 expression, molecule which polysaccharides, levels of VEGF without signifi-
interacts through the CD44 receptor on resident cant differences among them, suggesting that
macrophages, attenuating the inflammatory cas- there is not additional risk of inducing corneal
cade that is initiated by them [74] and reducing neovascularization by MSCs [34, 60]. Therefore,
inflammatory damage to the cornea [69]. A when cultured rabbit BM-MSCs, AT-MSCs, or
meticulous study showed that intraocular injec- LESCs on nanofiber scaffold were transplanted
tion of TSG-6 into the anterior chamber reduced on a de-­epithelized rabbit corneal surface, the
markedly the expression of different pro-­ neovascularization decreased with all three types
inflammatory cytokines and chemokines (IL-6, of stem cells. Surprisingly, however, the greatest
IL-1β, cytokine-induced neutrophil chemoat- decrease was found in injured corneas treated
tractant 1 (CINC-1/CXCL1), and monocyte che- with nanofiber scaffolds seeded with BM-MSCs
moattractant protein-­ 1 (CCL2/MCP-1)), and or LESCs [60].
levels of MMP-9, as well as the infiltration of
neutrophils and proteases into the cornea after Stimulation and Support of the Growth
corneal and limbal epithelial debridement, being and Differentiation of Local Stem Cells
the effects of TSG-6 dose-dependent [75]. These Several studies have reported that MSCs can
results strongly support the hypothesis that induce re-epithelialization on wounded corneas.
TSG-6 produced by human BM-MSCs in This fact can be explained by the transdifferentia-
response to injury signals can protect the corneal tion of MSCs transplanted or by other mecha-
surface from the excessive inflammatory nism. A group of authors observed that when
response [75]. de-epithelialized rat corneas were treated with
rabbit BM-MSCs, AT-MSCs or LESCs cultured
Anti-angiogenic Function on nanofiber scaffold, the wound healing and the
In 2008, it was proposed that MSCs could have epithelial expression of CK3 and CK12 signifi-
anti-angiogenic actions that could be mediated cantly enhanced on the cornea [60]. They thought
by paracrine pathways, in accordance with a that MSC transdifferentiation is not the main
work in which it was shown that thrombospon- mechanism of the re-epithelialization effect, sug-
din 1 (TSP-1), a powerful anti-angiogenic factor, gesting that important effects represented by the
was upregulated at high levels in response to cor- production of numerous trophic and growth fac-
neal epithelium injury treated with topical MSC-­ tors that can support the growth of residual cor-
derived conditioned media [66]. Currently, it is neal epithelial cells and LESCs [60].
known that human BM-MSCs secreted different
molecules in basal conditions, such as VEGF, Oxidative Stress Protection
MMP-2, and TSP-1 [75]. In addition, MSCs Currently, it is known that reactive oxygen species
secrete high amounts of VEGF when these cells are increased in corneal epithelium after alkali cor-
are stimulated under inflammatory environment neal injury [79, 80]. However, rabbit BM-MSCs
[66, 68, 72, 75]. VEGF and MMP-2 play a role cultured on nanofiber scaffold can reduce alkali-
240
induced oxidative and nitrosative stress in de-epi-
thelized corneas. It is believed that MSCs cultured
on nanofiber scaffolds protect the formation of
toxic peroxynitrite, lower apoptotic cell death, and
decrease matrix metalloproteinase and pro-inflam-
matory cytokine production, resulting in reduced
corneal inflammation as well as neovascularization
and significantly accelerated corneal healing [80].
In addition, it is known that the expression of anti-
oxidant enzymes superoxide dismutase (SOD) and
xanthine oxidase (XOX) is restored in the regener-
ated corneal e­ pithelium similar to healthy corneas
after BM-MSCs or LESCs nanofiber treatment
[61]. In accordance with these results, some authors
postulated that MSCs can exert direct antioxidant
activities through the secretion of antioxidant mol-
ecules, due MSCs secrete the extracellular antioxi-
dant molecule SOD3 [81].
In summary, paracrine mechanisms of MSCs
may exert a significant impact in promoting cor-
neal wound repair, which involves the joint par-
ticipation of different soluble factors that
modulate inflammation and angiogenesis as well
as improve tissue regeneration (Table  17.1).
However, the involved biophysiological factors
and the underlying mechanism in corneal wound
healing remain unclear [82].
Recently, important reviews reporting the tro-
phic effects of MSCs, obtained from different
sources, had been published [53, 83]. Concretely,
in corneal regeneration, the potential less thera-
peutic effects of AT-MSCs in comparison with
BM-MSCs or LSCs could be due to the lower dif-
ferentiation potential of AT-MSCs and to the dif-
ferent spectrum of growth and immunoregulatory
factors produced by these cells [53, 83], although
this fact is currently in controversy. On the other
hand, several authors have reviewed that com-
monly referred to as the MSC secretome, describ-
ing exhaustibly soluble factors, and reporting
new mechanisms related with factors released in
extracellular vesicles, for example, exosomes and
microvesicles [83–85]. Exosomes are one of sev-
eral groups of secreted vesicles, which have a
size of 40–100  nm, and can be distinguished
from microvesicles and apoptotic bodies by size
and morphology [86]. They can fuse with cellular
membranes and it has been suggested that exo-
T. Nieto-Miguel et al.
Table 17.1  Trophic activity of mesenchymal stem cells
(MSCs)
Trophic
factors
secreted by
MSCs Effect on corneal regeneration
EGF Induction of proliferation and
migration of corneal epithelial cells
IL-6 Anti-inflammatory effect, suppressing
dendritic cells
SOD3 Antioxidant effect
TGF-β Induction of proliferation and
migration of corneal epithelial cells
TNF-α Stimulation of TSG-6 expression
TSG-6 Activation of LESCs. Inhibition of
local activated macrophages, decreased
expression of pro-inflammatory
cytokines, chemokines, and MMP-9.
Decreased neutrophil infiltration on the
cornea and decreased corneal
neovascularization
TSP-1 Anti-angiogenic effect
Trophic Effect of inhibition action on corneal
factors regeneration
inhibited by
MSCs
IL-2 Anti-inflammatory effect
MMP-2 Anti-inflammatory effect
MMP-9 Increase of proliferation of LESCs and
decreased apoptosis of corneal
epithelial cells
VEGF Anti-angiogenic effect
Abbreviations: EGF epidermal growth factor, IL interleu-
kin, LESCs limbal epithelial stem cells, MMP matrix
metallopeptidase, MSCs mesenchymal stem cells, SOD
superoxide dismutase, TGF-β transforming growth factor
beta, TNF-α tumor necrosis factor alpha, TSG-6 tumor
necrosis factor-a-stimulated gene/protein-6, TSP-1 throm-
bospondin 1, VEGF vascular endothelial growth factor
somes are secreted by a variety of cell types;
between them we can find the MSCs, transport-
ing genetic material and proteins [87, 88]. They
are thought to mimic the roles played by MSCs
from which they originate [88, 89]. Currently, the
effect of MSC exosomes on tissue wound healing
had been reported [90]. Although currently there
are no studies in which this action was checked in
corneal epithelium, it could be other possible via
for trophic action of MSCs [83].
17.2.1.3 Immunomodulatory Ability
Over time, several studies have confirmed that
MSCs have important immunomodulatory prop-
17  Cell Therapy Using Extraocular Mesenchymal Stem Cells
erties [82]. This question was proposed some
years ago thought studies in which MSCs directly
inhibited proliferation of T cells [91, 92]. The
immunomodulatory action of MSCs is often
attributed at the action of different components
that this type of cells can secrete. To effectively
influence immunoregulation, MSCs require to be
activated by an inflammatory microenvironment
and stimulation by pro-inflammatory cytokines,
such as interferon gamma (IFN-γ) and TNF-α
[82]. This immunomodulatory action can be
characterized by a final immunosuppression
action or by a final immunostimulatory effect
[82, 93]. In addition, MSCs can suppress mecha-
nisms regulated by innate and adaptive immune
cells [82, 93]. Regarding innate immune reaction
regulation, MSCs secrete molecules such as
granulocyte-macrophage colony-stimulating fac-
tor (GM-CSF), IL-6, and prostaglandin E2
(PGE2). GM-CSF elicits the anti-inflammatory
state of macrophages type 2 (M2) which have
decreased the capacity of antigen presentation
function and do not secrete proinflamatory mol-
ecules [73, 94]. IL-6 secreted by MSCs inhibit
neutrophil action, preventing respiratory bursts
[95, 96], while PGE2 secreted by these cells
inhibit mast cell degranulation [97]. In addition,
PGE2 together with TGF-β or indoleamine-­2,3-­
dioxygenase (IDO) can decrease natural killer
cell proliferation and the pro-inflammatory cyto-
kine production carried out by these cells, while
PGE2 with IL-6 inhibit dendritic cell maturation,
cytokine expression, and migration to lymph
nodes [98, 99]. In adaptive immune response, the
programmed death-1 (PD-1) and programmed
death ligand-1 (PD-L1) factors, both secreted by
MSCs, can inhibit B cell proliferation and reduce
production of immunoglobulin (Ig) G, IgM, and
IgA [100–103]. In addition, MSCs inhibit T cell
proliferation and regulate cytokine expression,
being both mechanisms regulated through mole-
cules secreted by MSCs, such as nitric oxide
(NO), IL-10, PD-1, PD-L1, TGF-β, etc. [72, 91,
104, 105]. For example, MSCs can decrease the
expression level of IFN-γ (proinflamatory mole-
cule) from Th1 cells and increase the expression
levels of IL-4 and IL-10 from Th2 cells, both
with anti-inflammatory effects [72, 106, 107].
241
Moreover, MSCs promote expansion and differ-
entiation of regulatory T cells (Treg), which
inhibit T cell response [82]. On the other hand,
MSCs have pattern-recognition receptors which
can regulate specific immunostimulatory proper-
ties of these cells, such as toll-like receptors
(TLR). For example, in response to TLR3 signal-
ing, MSCs maintain an anti-inflammatory pheno-
type, while in response to signaling through
TLR4, MSCs adopt a pro-inflammatory pheno-
type, upregulating production of IL-8, IL-1, and
CCL-5 [93, 108–110].
Finally, MSCs show reduced expression of
major histocompatibility complex (MHC) class I
and do not express MHC II neither the classic co-­
stimulatory molecules [72]. In addition, MSCs
reduce the expression of MHC class II and co-­
stimulatory molecules such as CD80, CD86, and
CD40 on the surface of other cells [82], facts that
wake it possible for MSCs to likely have a non-­
immunogenic phenotype, showing immunoprivi-
leged features. Therefore, MSCs can potentially
be used allogenically or xenogenically in a vari-
ety of tissue disease states [54].
Immunosuppression
and Immunostimulation Induced by MSCs
on Corneal Epithelium
All these immune modulatory characteristics of
MSCs play a determinant role in corneal epithe-
lium regeneration. It is known that when human
corneal epithelial cells are cultured under a pro-­
inflammatory environment (IFN-γ and TNF-α
stimulation), they increased their expression of
intercellular adhesion molecule 1(ICAM-1),
MHC-I, and HLA-DR (MHC-II) [111]. However,
MSCs effectively decreased IFN-γ/TNF-α-­
induced ICAM-1, MHC-I, and MHC-II expres-
sion on this type of cells [111]. Since these
adhesion molecules play an important role in the
infiltration of activated leucocytes in the injured
corneal epithelium [112, 113], this fact could be
one of the mechanisms through MSCs decrease
the infiltration of leukocytes in damaged corneas.
Currently, it is suggested that MSCs reduce the
expression of adhesion molecules in corneal epi-
thelial cells through nuclear factor-kappa beta
(NF-kB) signaling pathways [111], due to the
242
specific inhibition of NF-kB nuclear transloca-
tion which decreases the expression of molecules
such as ICAM-1 and vascular cell adhesion pro-
tein 1 (VCAM-1) on epithelial cells [66, 114–
116]. On the other hand, contrary to the
expectations, MSCs significantly attenuated
cytokine-induced IDO expression on human cor-
neal epithelial cells [111], molecule which is
strongly linked to prolong corneal allograft sur-
vival [117]. Since some years, the
­immunomodulatory effect of MSCs on corneal
epithelium also was studied by in  vivo experi-
ments. Currently, it is known mechanical injury
on mice corneal-­limbus epithelium induced an
inflammatory reaction characterized by produc-
tion of IFN-γ and inducible nitric oxide synthase
(iNOS) in the ocular surface [118]. In addition,
IL-6 is also expressed when damaged ocular sur-
faced is treated with allogeneic limbal transplan-
tation. However, both inflammatory situations
are decreased when nanofiber scaffolds with
BM-MSCs are transplanted in a mice model of
damaged corneal/limbal epithelium, suggesting
that inflammation inhibition was partially regu-
lated by MSC immunomodulatory effects, since
MSCs inhibited IFN-γ secretion and T cell prolif-
eration in  vitro [118]. On the other hand, it has
been reported the immunoregulatory effect of
rabbit BM-MSCs and AT-MSCs cultured on
nanofiber scaffold in a rabbit model of corneal
de-epithelialization, which showed a strong tis-
sue infiltration with executor cells of adaptive (T
lymphocytes) and innate (iNOS-expressing cells)
immunity [60]. The MSC transplantation signifi-
cantly decreased all of the harmful manifesta-
tions of the injury; although less pronounced
therapeutic effects of AT-MSCs were observed in
comparison with BM-MSCs. The effects of MSC
transplantation were related with the expression
of genes for the immunoregulatory molecules
IDO-2, cyclooxygenase-2 (Cox-2), and iNOS by
these cells, since these molecules secreted by this
type of cells like to inhibit proliferation of acti-
vated T cells, inhibit natural killer (NK) cells, and
suppress mast cells in the presence of low con-
centration of IFNα/IL-1, respectively [93, 97].
On the other hand, the effect of BM-MSC intra-
venous injection in a mouse model of autoim-
T. Nieto-Miguel et al.
mune ocular inflammation (autoimmune uveitis)
has been tested, observing an increase of Treg
cells and M2  in peripheral blood and lung,
respectively [119]. However, since high levels of
M2 lasted longer than Treg, it was reported that
M2 were effectively protected against experi-
mental autoimmune uveitis independently of
Treg, indicating a direct role of M2 in the MSC-­
induced tolerance [119].
This fact was confirmed by other authors, who
co-cultured MSCs with macrophage isolates
from mouse peritoneum. When macrophages
where cultured alone, they differentiated into
classical macrophages type 1 (M1) (pro-­
inflammatory); however, when these polarized
M1 were exposed to human umbilical cord-­
derived MSCs (UC-MSCs), these macrophages
began to express markers typical of M2 (anti-­
inflammatory) [120], although the effect of
UC-MSCs on M1 was inhibited when UC-MSCs
were modified, removing from their surface
extracellular matrix, dermatan sulfate, and hyal-
uronan [120]. In addition, currently, it is known
that the presence of extracellular matrix and hyal-
uronan in the surface of UC-MSCs is vital for
these MSCs to suppress inflammation and escape
host rejection when they were transplanted in a
model of epithelial corneal alkali burn [120].
Another action that the hyaluronan of MSCs can
regulate is the induction of regulatory T cells
[120, 121]. In conclusion, currently it is sug-
gested that UC-MSC glycocalyx play an impor-
tant role in host immune modulation.
Effect of Immunomodulatory Properties
of MSCs in Corneal Transplantation
Currently, MSCs have been widely used as
immunotherapy in solid organ transplantation.
Over time, several authors have reported that
MSCs show potential therapeutic effect for cor-
neal allograft transplantation, decreasing the
immune rejection and promoting corneal
allograft survival [82]. The first study developed
in this field was carried out in 2009, supporting
that the survival of corneal xenografts was not
significantly prolonged by rat commercial MSC
application [122]. However, some years later, a
research group found that intravenous injection
17  Cell Therapy Using Extraocular Mesenchymal Stem Cells
of rat BM-MSCs prolonged corneal allograft
survival when cells were administered immedi-
ately after transplantation [123], while preopera-
tive infusion was ineffective, in accordance with
the results previously reported [122, 123]. In this
work, when mononuclear cells were isolated
from the spleen and lymph nodes of both MSC-­
treated and vehicle-treated rats, previously trans-
planted with allogenic cornea, the proliferation
of T cells from MSC-treated rats was signifi-
cantly reduced compared with T cells from
vehicle-­ treated postoperative rats, suggesting
that MSCs may prevent rejection at least in part
by reducing effector T cell proliferation in vivo.
It was also observed that media obtained from T
lymphocytes isolated from rats treated postop-
eratively with MSCs showed a decrease quantity
of Th1 pro-inflammatory cytokines and elevated
IL-4 secretion. In addition, Treg were upregu-
lated in mononuclear cultures isolated from rats
treated with MSCs. Consequently, it was sug-
gested that MSCs can decrease corneal allograft
rejection, postulating that this effect could be
mediated by immunomodulatory reactions, as
inhibition of pathogenic T cell responses, an
anti-­inflammatory shift in the Th1/Th2 balance,
and activation of Treg cells [123]. In addition,
currently, it is known that intravenous-injected
mice BM-MSCs to allografted mice recipients,
3 h after surgery, increase allograft corneal sur-
vival by inhibiting antigen-presenting cell (APC)
maturation and induction of alloreactive T cells
[119, 124]. On the other hand, the ability of rat
BM-MSCs from three distinct sources to pro-
long rat corneal allograft survival has been
tested. MSCs obtained from same strain of recip-
ient (syngeneic transplant, syn-MSCs), from
same strain of donor (Allo-MSCs) or from a
third-type strain (third-­ MSCs) were injected
intravenously in rat recipient. A high percentage
of untreated and syn-MSC-treated allografts
were rejected (80% and 100%, respectively).
Conversely, corneal allograft survival was sig-
nificantly prolonged in allo-MSC (90%)- and
third-MSC (80%)-treated allograft recipients
[125]. This fact could be explained by an inferior
number of corneal natural killer T cells coupled
with a higher proportion of splenic regulatory T
243
cells showed in animals treated with allo- or
third strain-MSCs compared to animal corneas
treated with syn-MSCs. It was also shown a
decrease of CD4 T and B cell activated in cor-
neas from animals receiving either of the MSC
types compared with untreated animals [125].
All these results induce to conclude that the
effect of MSCs on inhibiting the immune rejec-
tion response can depend of MSC source.
However, in spite of the data previously
reported, the in vivo effect of MSCs on inhibiting
the immune rejection response is controversial
[82], and it is known that other parameters, as the
moment of MSC application with respect to tis-
sue transplant or the total cell doses applied, can
play an important role about the effect of MSCs
on corneal transplantation [82, 93]. When the
effect of local and systemic injection of human
AT-MSCs into two rabbit models of corneal
allograft rejection (normal- and high-risk model)
was studied, in contrast to the expectation, it was
observed that local injection of AT-MSCs, at the
time of surgery, induced an increase of inflamma-
tion, corneal edema, and higher levels of infiltra-
tion of lymphocytes in both models, inducing a
decrease in graft survival [126]. This results
could not be explained by to the use of human
MSCs could create a xenogeneic environment,
because the results were similar when trans-
planted MSCs were isolated from human or rab-
bit. It has been speculated that MSCs could be
recognized by the adaptive immune system [127].
In the current scenario in which an inflammatory
environment is already established, the secretion
of IL-6 and IL-8 by AT-MSCs can exacerbate the
chemotaxis of leukocytes to the injected zone,
thus increasing inflammation. In this case, the
action of pro-inflammatory cytokines IL-6 and
IL-8 seems to be higher than the immunosup-
pressive effects of IDO and NO also secreted by
AT-MSCs [126].
In conclusion, although the immunomodula-
tory effect of MSCs has been shown in repeated
occasions and some action mechanisms are
known at the moment (Table 17.2), more research
is necessary in order to clarify all immunomodu-
latory actions that MSCs can perform in dam-
aged corneal epithelia.
244
Table 17.2  Immunomodulatory ability of mesenchymal
stem cells (MSCs)
Immunomodulatory
factors secreted by General effects on corneal
MSCs regeneration
Cox-2 Inhibition of natural killer
GM-CSF Induction of anti-­inflammatory
state of macrophages
IL-6 Inhibition of neutrophil action or
exacerbation of the chemotaxis
of leucocytes to damaged cornea
IL-10 Inhibition of T cell proliferation
iNOS Inhibition of mast cells
NO Inhibition of T cell proliferation
PD-1 Decrease of production of IgG,
IgM, and IgA
Inhibition of B and T cell
proliferation
PD-L1 Decrease of production of IgG,
IgM, and IgA
Inhibition of B and T cell
proliferation
PGE2 Inhibition of mast cells
degranulation. PGE2 + TGFβ or
IDO: decrease of natural killer
cell proliferation. PGE2 + IL-6:
inhibition of dendritic cells
TGF-β Inhibition of T cell proliferation
Immunomodulatory Effect of inhibition action on
factors inhibited or general immune response
downregulated by detected on corneal regeneration
MSCs
IDO Increase of T cell proliferation
IFN-γ Decrease of ICAM1 and MHCI
and II expression on corneal
epithelial cells. Consequently,
decrease of leucocyte infiltration
on corneal inflamed epithelium
TNF-α Decrease of ICAM1 and MHCI
and II expression on corneal
epithelial cells. Consequently,
decrease of leucocyte infiltration
on corneal inflamed epithelium
Abbreviations: Cox-2 cyclooxygenase-2, GM-CSF
granulocyte-­macrophage colony-stimulating factor, IDO
indoleamine-2,3-dioxygenase, IFN-γ interferon gamma,
Ig immunoglobulin, IL interleukin, iNOS inducible nitric
oxide synthase, MSCs mesenchymal stem cells, NO nitric
oxide, PGE2 prostaglandin E2, PD-1 programmed death-
­1, PD-L1 programmed death ligand-1, TGF-β transform-
ing growth factor beta, TNF-α tumor necrosis factor alpha
17.2.1.4 Tissue Homing Capacity
Currently, it is known that MSCs have capacity to
migrate specifically to inflamed tissues, in accor-
dance with general the premise that injury and
T. Nieto-Miguel et al.
inflammation induce stem cell mobilization,
migration, and colonization [82, 128]. Although
the tissue homing capacity of MSCs has been
robustly demonstrated by several authors along
the time [55, 82, 83, 129–133], at the moment,
the mechanisms involved in the regulation of
MSC homing capacity still remain unclear, while
it is thought that this property is mainly regulated
by growth factors and/or chemokines expressed
by damaged tissues and by extracellular matrix
receptors expressed on the surface of MSCs [55,
82, 134]. For example, the interactions of stromal
cell-derived factor 1/C-X-C motif chemokine 12
(SDF-1α/CXCL12) and C-X-C chemokine
receptor type 4 (CXCR4) directly mediate the
trafficking of transplanted MSCs, while the pres-
ence of inflammatory cytokines as platelet-­
derived growth factor (PDGF), TGF-β1, TNF-α,
and SDF-1α/CXCL12 increases MSC motility
[83, 135–137]. One of the several tissues where
MSCs can migrate is to the inflamed corneal epi-
thelium; due to the fact that damaged corneal epi-
thelium expresses specific chemoattractants that
can mobilize MSCs to this region.
BM-MSC migration into peripheral blood,
bone marrow, and cornea was monitored after
intravenous administration in a rabbit model of
alkali burn, observing that successful engraft-
ment of MSCs occurred in all treated animals,
although different cell migration patterns were
observed. When MSCs were administrated to no
immunosuppressed animals, MSCs rapidly
migrated to bone marrow and corneo-limbal
wound (epithelium, stroma, and corneal endothe-
lium), although, subsequently, the presence of
MSCs decreased over time. However, when
MSCs were transplanted in immunosuppressed
animals, MSCs slowly migrated to bone marrow.
In this case, MSCs remained long time in periph-
eral blood, and presence of MSCs in cornea,
mainly in stomal area, slowly increased over
time, observing high infiltration of MSCs after
1 month. Consequently, currently it is known that
bone marrow state plays an important role in
BM-MSC migration capacity when these cells are
systematically injected. Other experiments showed
that intravenously injected mice BM-MSCs, in a
mouse model of corneal epithelial thermal
17  Cell Therapy Using Extraocular Mesenchymal Stem Cells
c­auterization, could migrate specifically to the
injured corneas, persisting MSC infiltration at
least for 50  days, while MSCs did not migrate
into healthy contralateral corneas [138]. In addi-
tion, it was shown that when exogenous MSCs
were not applied in damaged animals, increased
levels of endogenous MSCs were found in the
peripheral blood 48  h after the stimulus of cor-
neal injury, a fact that correlated with the pres-
ence in blood circulation and corneal damaged
tissues of high levels of SDF-1/CXCL12 factor
and substance P molecules which reported to
possess stem cell chemoattractant properties
[139–141]. It was speculated that the injured cor-
neal tissue was the source of these MSC che-
moattractants [138]. Recently, a new study has
supported the important role of SDF-1/
CXCL12  in corneal epithelium healing when
MSCs are implicated in this repair function. In an
in  vitro model of wound healing, in which a
scratch was made through rat limbal MSC mono-
layer, the presence of SDF-1/CXCL12 stimulated
MSC migration from wound edge to damaged
culture area in order to repair cell monolayer
[89]. In another study in which systemically
MSC administration was used as therapy for the
treatment of corneal damage, specifically in cor-
neal transplantation, it was shown that BM-MSCs,
intravenously administrated, preferentially home
to the inflamed ocular surface and draining lymph
nodes, while these cells did not migrate to the
healthy corneas [124].
Some authors have suggested that after sys-
temic MSC administration, most of the trans-
planted cells migrate to the main organs, such as
the lung, liver, kidney, and spleen, and, therefore,
MSCs have lower capacity to migrate to areas
with tissue damage [82, 142], as the damaged
corneal epithelium. Consecutively, some
researchers began to perform topical MSC trans-
plantations. Some years ago, rat BM-MSC migra-
tion to damaged corneal epithelium after topical
application was evaluated, finding MSCs in cor-
neal epithelium 3 weeks after the injury. This fact
support that MSCs were successfully engrafted
underneath the epithelial cell layers up to 3 weeks
after topical application [66]. The migration
capacity of topically administrated MSCs to
245
injured corneal epithelium was also shown by
another research group, who administrated
human AT-MSCs in suspension (topical drops) in
a rat model of corneal burn [143]. On the other
hand, when human MSCs obtained from amni-
otic membrane were administrated subconjuncti-
vally on damaged corneal epithelia of rabbits, it
was observed that MSCs migrated to limbal-­
corneal tissues at day 28 after administration
[144]. In addition, human BM-MSCs in damaged
corneal tissue of mice after its subconjunctival
administration could migrate to corneal epithe-
lium, in which MSCs appeared at different epi-
thelial layers. In addition, these cells showed
different morphologies from cubic to flattened,
depending on their position within the tissue
[30]. Also, when MSCs were cultured on amni-
otic membrane, with or without keratinocyte
growth factor-2 (KGF-2) and/or with human
serum, and this cell-substratum complex was
transplanted on rat with damaged corneal epithe-
lium, MSCs generally could migrate to injured
cornea [145]. In a recently developed study, in
which human AT-MSCs cultured on amniotic
membrane were transplanted on rabbits in which
a partial or a total limbal stem cell deficiency had
been previously induced, AT-MSCs could migrate
from amniotic membrane to ocular surface, since
MSCs were found in the inflamed areas of the
superior and inferior limbal stroma and corneal
8  weeks after transplantation [18]. This cell
migration could be regulated by the signaling
pathway mediated by SDF-1/CXCL12 and its
receptor CXCR4 expressed by MSCs [146].
However, MSC migration to inflamed tissues has
not been supported by all studies performed in
this area [50, 51].
At the moment, cell mechanisms involved in
MSC migration to damaged tissues remain
unclear, as well as the different physiological and
pathological situations that regulate MSC migra-
tion [82]; although in general, results reported by
several authors suggested that MSCs have the
capacity to migrate to injured corneal epithelium,
enhancing epithelial wound healing and reducing
corneal opacification and neovascularization,
independently of the administration route. In
conclusion, scientific community accepts that
246
homing capacity of MSCs, together with their
tropic and immunomodulatory actions, plays an
important role in corneal epithelium regeneration.
17.2.2 Extraocular MSCs
in Experimental Models
of Corneal Epithelial Damage
17.2.2.1 Experimental Models
of Corneal Epithelial
Damage
In order to study different potential therapies to
treat diseases, it is necessary to deepen in the
knowledge and the understanding of diseases.
Experimental models represent one of the best
tools that help to accomplish this goal.
There are a lot of different kinds of experi-
mental models of corneal epithelial damage with
diverse characteristics, such as the size of the
affected area in the cornea and/or the limbus, the
severity of the model, and the methodology used
to develop it, among others. Experimental mod-
els with central corneal damage but without lim-
bal injury are considered corneal burn or corneal
injury models. Corneal burn models are induced
by a chemical agent, whereas corneal injury is
induced by mechanical damage. Experimental
models that include limbal damage are desig-
nated as LSCD models. The most used agent dur-
ing the induction of both corneal burn and LSCD
models is sodium hydroxide (NaOH). However,
ethanol, n-heptanol, and mechanical scraping
have been also used to induce corneal epithelial
damage.
There are several studies that describe the
use of MSCs to treat corneal epithelial injuries
using different experimental models and meth-
odology. One of the principal factors that could
condition the results of these works is the
administration route: systemically (intravenous
or intraperitoneal), using different carriers
(amniotic membrane, fibrin, biopolymers, etc.),
topically, or even by subconjunctival injection.
In addition, some researchers have studied the
therapeutic effect of endogenous MSCs in the
ocular surface after induced corneal epithelial
damage.
T. Nieto-Miguel et al.
17.2.2.2 Mobilization of Endogenous
MSCs
Different researchers have demonstrated that
endogenous MSCs can carry out a therapeutic
effect in the cornea when a corneal epithelial
damage has been induced. In a partial LSCD
model, endogenous MSCs could reduce the epi-
thelial corneal defects and opacity in rabbits with
normal bone marrow function [147], while this
did not happen in bone marrow-suppressed rab-
bits. Furthermore, MSCs and hematopoietic stem
cells were able to migrate from bone marrow to
the injured cornea in rabbits with competent bone
marrow, but it did not occur in the bone marrow-­
suppressed animals [147]. Additionally, a signifi-
cant increment in the number of circulating
endogenous MSCs was observed in animals with
a partial LSCD [138]. This increment in circulat-
ing MSCs was accompanied by an increase of
SDF-1/CXCL12 and substance P levels both in
peripheral blood and in corneal tissue. These fac-
tors then contribute to the mobilization of the
endogenous cells [138]. Therefore, these data
demonstrate that endogenous MSCs can migrate
from the bone marrow to the cornea in order to
trigger their therapeutic effect in corneal epithe-
lial damage.
17.2.2.3 Intravenous Administration
of MSCs
There is evidence that MSCs administered by
intravenous injection can help during the wound
healing process and reduce the clinical signs of
the corneal epithelial damage in different experi-
mental models. BM-MSCs intravenously admin-
istered reduced significantly the epithelial defects
in corneal injury and partial LSCD models [19,
138]. Furthermore, the neovascularization devel-
oped in a partial LSCD model was also reduced
by the intravenous administration of BM-MSCs
[37]. In addition, corneal opacity has been widely
studied in experimental models of corneal epithe-
lial damage, showing that intravenous MSCs
were able to diminish it in both corneal injury
and LSCD models [19, 37, 69, 148]. Some of
these studies showed that MSCs effectively
reduce corneal opacity in corneal epithelial dam-
age, but their effect was variable depending on
17  Cell Therapy Using Extraocular Mesenchymal Stem Cells
the cell donor; MSCs from some donors were
more efficient at reducing corneal opacity than
cells from other donors [148]. Additionally, intra-
venous induced pluripotent stem cells (iPSC)-
derived MSCs (iPSC-MSCs) could also reduce
corneal opacity in a LSCD model [149].
Therefore, all these data together indicate that the
intravenous administration of MSCs in experi-
mental models of corneal epithelial damage can
contribute to the reduction of clinical signs.
However, MSCs administration in those studies
was performed in the acute phase of the corneal
epithelial damage. This fact could contribute to
restrain the progression of the damage in an early
phase. In contrast, there are a lot of patients suf-
fering LSCD or corneal epithelial damage in a
chronic phase; hence it would be necessary to
study the effect of the MSCs intravenously
administered in corneal epithelial damage or
LSCD models well established in a chronic
phase.
To trigger the therapeutic effect of MSCs in
the corneal epithelium when they are intrave-
nously administered, it would be necessary that
the cells migrate from the peripheral blood to the
injured cornea or that they carry out their actions
from the distance. Some authors observed the
presence of intravenously administered MSCs in
the injured cornea [37, 138, 150]. Furthermore,
they also described that the systemic adminis-
tered cells were found in the peripheral blood
and migrated to the bone marrow [37]. Although
some authors advocate the migration of the
MSCs intravenously administered to the injured
cornea, others have located fewer than 10 iPSC-
MSCs in the cornea [149] or have not found data
that supports the migration of MSCs [69]. In this
case, the authors postulated that the therapeutic
effect of the MSCs was produced from the dis-
tance [69].
The anti-inflammatory properties of MSCs
have been widely described in many tissues, such
as the cornea. This anti-inflammatory effect
could be beneficial for the regeneration of the
corneal epithelium after damage. In this regard,
some authors have reported the anti-­inflammatory
role of the MSCs after their intravenous adminis-
tration in LSCD models. Less infiltration of
247
inflammatory cells in the corneal stroma and less
disorganized cornea were observed when
BM-MSCs were intravenously administered in
LSCD models [150]. Furthermore, the level of
myeloperoxidase (MPO), a molecule released by
activated neutrophils, was lower in the injured
corneas treated with intravenous BM-MSCs than
in the untreated corneas [69, 148], indicating less
infiltration of neutrophils in the cornea of the
treated animals [149]. Nevertheless, a variable
effect on the BM-MSC capacity to reduce the
MPO levels has been observed depending on the
donor of the cells [148]. Moreover, a direct rela-
tion between the level of TSG-6 expression in the
BM-MSCs and the capacity of these cells to
reduce MPO has been described [148]. In addi-
tion, the expression of the pro-inflammatory
cytokines TNF-α, IL-1β, and IL-6, CCL2, CCL3,
and CCL4 was reduced in the corneas with LSCD
and treated with MSCs [69, 149]. Also the expres-
sion of tenascin C, a proangiogenic and profi-
brotic molecule, was reduced in the corneas
administered with MSCs [69]. Additionally, the
anti-inflammatory factors TGF-β and IL-1Ra
grew up in the corneal tissues after the intrave-
nous administration of BM-MSCs in LSCD mod-
els [138]. Thus, MSCs seem to have the capacity
to release these anti-inflammatory factors in the
injury site. Furthermore, in order to demonstrate
if the therapeutic effect of MSCs is mediated by
TSG-6, some authors knocked down the expres-
sion of TSG-6  in MSCs, and these cells were
intravenously administered without showing
therapeutic effect in the cornea [69, 149]. These
data could indicate that the TSG-6 is responsible
for the beneficial effect of the MSCs in the cor-
nea. With all these data, we can conclude that the
administration of intravenous MSCs promotes an
anti-inflammatory effect in the corneal epithe-
lium when it has been damaged. Furthermore,
this effect seems to be induced to a large degree
by the factor TSG-6, secreted by MSCs.
Regarding corneal epithelium phenotype, there
are data to support that MSCs can help to the pro-
genitor residence cells to recover their phenotype
and function through the release of trophic and
paracrine factors. The expression of the stem cell
markers ABCG2, p63, Hes1, and C/EBPδ, as well
248
as the epithelial cell markers CK12 and CK19,
increased in the corneas of partial LSCD models
treated with intravenous BM-MSCs in compari-
son with the non-­administered animals [37, 138].
In addition, the cell proliferative activity marker
proliferating cell nuclear antigen (PCNA) showed
its expression not only in the basal limbus but
also in the peripheral corneal epithelium in the
MSC-treated group [37]. Therefore, MSCs pro-
mote a positive effect in the recovery of the phe-
notype of the corneal and limbal cells after a
corneal epithelial damage.
17.2.2.4 Intraperitoneal
Administration of MSCs
The knowledge about the effect of MSCs intra-
peritoneally administered in experimental mod-
els of corneal epithelial damage is limited. As in
the case of the intravenous administration, a
decrease in corneal opacity and MPO levels was
observed after intraperitoneal administration of
MSCs in a rat LSCD model [69]. The expression
of pro-inflammatory cytokines and chemokines
IL-1β, CXCL1, CCL2, CCL3, and CCL4 as well
as tenascin C was also decreased in the group
with intraperitoneal administration of MSCs. As
in the intravenous administration, the MSCs
administered intraperitoneally were not detected
in the cornea [69]. Therefore, the therapeutic
effect of the MSCs was produced from the dis-
tance. However, IL-17 levels in corneal tissues
were lower in a mice model of LSCD treated with
intraperitoneal BM-MSCs in comparison with
untreated animals [151]. Thus, the authors con-
cluded that BM-MSCs have an anti-inflammatory
effect on the IL-17 secreting cells by blocking
both non-Th17 cells and Th17 cells [151]. These
data demonstrate that the MSCs intraperitoneally
administered have an anti-inflammatory effect in
corneal epithelial damage. However, there is no
data to support the migration from the peritoneal
area to the damage cornea, so their effect could
be produced from the distance. It is important to
note that, as in the case of the intravenous admin-
istration, all the studies with MSCs intraperitone-
ally administered were performed in an acute
phase of the epithelial damage, and this fact
could condition the results. Therefore, further
T. Nieto-Miguel et al.
studies applying the cells in a chronic phase of
the corneal epithelial damage or in chronic LSCD
models are necessary to deepen on the knowl-
edge of the beneficial effects of the MSCs sys-
temically administered.
17.2.2.5 Ocular Topical
Administration of MSCs
Topical administration is the easiest way to apply
a drug on the ocular surface; however, it has some
drawbacks, such as low retention time, high
washing rate, and low permeability of the corneal
epithelium, among others. The administration of
topical AT-MSCs in corneal burn models reduced
the epithelial defects similarly to the effect of the
treatment with topical serum [143]. The lack of
differences in the therapeutic effect between the
serum group and the AT-MSC-treated group
could represent a drawback. However, the mild-
ness of the corneal burn model, along with the
small number of animals and the short follow-up
time of the study, could have contributed to the
lack of differences between the treatment groups.
In addition, the corneas of a LSCD model topi-
cally treated with BM-MSCs did not show posi-
tive healing results [80]. Thus, the rapid washing
away of the topically administered BM-MSCs to
the ocular surface could have avoided their thera-
peutic action. In contrast, neovascularization and
corneal opacity decreased in the corneas treated
with BM-MSCs or with BM-MSCs conditioned
medium in a rat LSCD model, mainly in the
BM-MSC-treated group [66]. Additionally,
whereas the anti-angiogenic factor TSP-1
increased, the pro-angiogenic factor MMP-2
decreased only in the BM-MSC-treated corneas
[66]. All these data indicate a poor beneficial
effect of the MSCs topically administered on the
clinical signs of a corneal epithelial damage.
AT-MSCs topically administered were found
in the corneal epithelium and stroma of a corneal
burn model developed in rat [143]. Therefore,
cells were able to migrate from the corneal sur-
face to deeper layers of the corneal epithelium
and stroma. However, this migration experiment
was only performed in both eyes of one rat; more
data should be obtained from more animals to
draw any valid conclusions.
17  Cell Therapy Using Extraocular Mesenchymal Stem Cells 249

The inflammation of the corneal tissue was plantation of MSCs seeded on AM to the ocular
lower in corneal burn and LSCD models treated surface [18, 28, 33, 36, 145]. Additionally, the
with AT-MSCs or BM-MSCs than in other exper- expression of the pro-angiogenic factor MMP-2
imental groups [66, 143]. In addition, the expres- was reduced in the corneas treated with
sion of the pro-inflammatory factors IL-2 and BM-MSCs [36]. Furthermore, in order to check
IFN-γ decreased in the corneas treated with visual function of the rats, a head tracking method
BM-MSCs. However, the expression of the anti-­ showed that the animals transplanted with
inflammatory factors IL-10 and TGF-β increased BM-MSCs had better visual capacity than the
in the BM-MSC-treated corneas. Although, the untreated groups [36]. Nevertheless, transplanta-
level of IL-6 was mild, it was higher in the tion of BM-MSCs on AM did not show signifi-
BM-MSC-treated group than in the vehicle-­ cant improvement of the clinical signs in a
treated group [66]. This data point out that IL-6 corneal burn model, whereas BM-MSCs subcon-
could mediate the immunomodulatory action of junctivally injected showed therapeutic action
MSCs. In general, BM-MSCs applied topically [50]. However, it is important to note that the cell
to the damaged corneas for 2  h showed better dose administered by subconjunctival injection
results than conditioned medium administration. was higher than the cell dose seeded on the AM,
It could indicate that cell-to-cell contact has addi-thus it could have influenced the results. In gen-
tive effects to the action of soluble factors or thateral, the transplantation of MSCs seeded on AM
the continuous secretion of factors induces better to the ocular surface of experimental models with
outcomes than the administration of these factors corneal epithelial damage not only improves the
by the conditioned medium. These data along wound healing process but also reduces the neo-
with the poor therapeutic effect of the topical vascularization and corneal opacity.
MSCs observed in the clinical signs could indi- Some authors have demonstrated that MSCs
cate that the low retention time, the high washing migrate from AM to corneal tissues in experi-
rate, and the low permeability of the corneal epi- mental models of corneal epithelial damage [18,
thelium are responsible for these results. 36, 145]. Therefore, MSCs transplanted to the
Furthermore, there are not enough data about the ocular surface using AM as carrier are able to
migration capacity of the MSCs topically admin- migrate to the corneal tissues and exert its benefi-
istered to the ocular surface; thus, further investi-cial effect there.
gations in this field are necessary. The anti-inflammatory properties of the MSCs
have been also confirmed when they are trans-
17.2.2.6 MSCs Seeded on Amniotic planted on AM to the ocular surface of experi-
Membrane mental models with corneal epithelial damage.
The AM is the innermost layer of the placenta, Less inflammatory cells and less disorganization
and it is increasingly used for ocular surface of the cornea were observed in LSCD models
reconstruction in a variety of ocular pathologies, after the MSC transplantation using AM as car-
even as carrier for the ex vivo expansion of stem rier [18, 28, 33]. In addition, the expression of the
cells in order to treat corneal epithelial damage or leukocyte marker CD45 and the pro-­inflammatory
LSCD.  AM have several beneficial properties, cytokine IL-2 was significantly lower in eyes
such as low immunogenicity, stimulation of the transplanted with BM-MSCs [36]. All these
epithelialization, and anti-fibrotic, anti-­results indicate that the MSCs seeded on AM and
inflammatory, anti-angiogenic, and antimicrobial transplanted to the ocular surface can reduce the
effects [152]. In this field, the AM has widely infiltration of inflammatory cells in the experi-
used as carrier to transplant MSCs to the ocular mental models of corneal epithelial damage.
surface of experimental models with corneal epi- However, the mechanism that triggers the anti-­
thelial damage. The improvement of epithelial inflammatory action of the MSCs transplanted on
defects, neovascularization, and corneal opacity AM to the ocular surface has not been deeply
was observed in LSCD models after the trans- studied.
250
Regarding corneal markers, the recovery of the
corneal epithelial markers CK3 and E-cadherin in
damaged corneas has been demonstrated after the
transplantation of AT-MSCs or BM-MSCs seeded
on AM [18, 28]. Nevertheless, other authors did
not find the expression of the corneal epithelial
markers CK3, CK12, and panCK in the epithe-
lium of the BM-MSCs treated eyes [33, 36].
Therefore, they postulated that the therapeutic
effect of BM-MSCs may not come from differen-
tiation of these cells into corneal epithelial cells.
Moreover, the expression of the limbal stem cell
markers CK15 and p63 was partially restored in
the limbal epithelium of the AT-MSC-treated eyes
[18, 28]. Additionally, the rats transplanted with
BM-MSCs showed positive expression of
CK19 in the limbal epithelium, while the untreated
rats did not express this marker [145]. However,
the location of the CK19 marker is controversial
because its expression has been described not
only in the limbal epithelium but also in the con-
junctival epithelium [153]. These works alto-
gether show poor evidence of MSCs having effect
in the recovery of the corneal and limbal pheno-
types. Therefore, further works are necessary to
elucidate this issue.
In the case of MSC transplantation using AM
as carrier, the cell dose is lower than in the admin-
istration by other routes. Furthermore, the dose of
the transplanted MSCs is not totally controlled in
several of the studies performed using AM as car-
rier, because some authors seed a number of cells
on the AM, but then they keep them in culture for
some days. Additionally, MSCs seeded on AM are
administered in a less acute phase of the disease
than with other administration ways; this fact
could help to know the effect of the MSCs in a
chronic phase of the corneal epithelial damage.
17.2.2.7 Amniotic Membrane Pocket
AM has also been used in order to create a pocket
in which BM-MSCs were administered in a
LSCD model developed in rabbits [32]. However,
there were no differences in clinical signs among
the different experimental groups [32]. The cor-
neal epithelial markers CK3/12 were more
strongly expressed in the corneas of the
BM-MSC-treated group than in those of the
T. Nieto-Miguel et al.
untreated group. Moreover, the expression level
of the epithelial marker Cx43 and the limbal stem
cell markers ABCG2 and β1-integrin was higher
in the cornea of the BM-MSC-treated group than
in the untreated group [32].
17.2.2.8 M  SCs Seeded on Fibrin
Matrix
Fibrin gel is prepared from fibrinogen and throm-
bin, obtaining a matrix able to house cells and
suitable for transplantation. The transplantation of
fibrin matrix could reconstruct the corneal epithe-
lium in a LSCD model not only when it contained
BM-MSCs but also without cells [31].
Furthermore, BM-MSCs were located in the cor-
neal epithelium 4 weeks after transplantation. In
addition, the expression of the corneal epithelial
marker CK3 was observed in both experimental
groups; however the expression was more regular
and continuous in the cornea treated with
BM-MSCs. In addition, some of the BM-MSCs
transplanted to the ocular surface expressed CK3
[31]. However, other authors have described that
MSCs cultured in normal conditions express CK3
[32, 40]; thus the expression of CK3 in the trans-
planted BM-MSCs might not be indicating a real
differentiation of MSCs into corneal epithelium.
17.2.2.9 Contact Lens Carriers
Other carriers used to transplant MSCs to the ocu-
lar surface are contact lenses. The effect of human
AT-MSCs seeded on scleral contact lenses applied
over the cornea of a LSCD model has been stud-
ied [154]. Epithelial defects, neovascularization,
and corneal opacity were reduced in the animals
treated with AT-MSCs in contrast to the group
with contact lenses without cells. The authors also
observed less disorganization and fewer inflam-
matory cells in the stroma of the AT-MSC-treated
corneas [154]. Thus, contact lenses could be an
adequate substrate to seed stem cells and to trans-
plant them to the ocular surface in order to treat
corneal epithelial damage.
17.2.2.10 Poly-L-Lactic Acid Carriers
Biopolymers are one of the alternative carriers
that have been investigated in order to find repro-
ducible substrates that allow cell adhesion and
17  Cell Therapy Using Extraocular Mesenchymal Stem Cells
proliferation, as well as the re-epithelialization of
the ocular surface when they are transplanted to
the ocular surface of experimental models with
corneal epithelial damage. The most studied bio-
polymer in this kind of experimental model is
based on poly-L-lactic acid. Epithelial defects,
neovascularization, corneal opacity, and corneal
thickness showed a reduction in corneal burn and
LSCD models transplanted with poly-L-lactic
acid carriers containing MSCs, both BM-MSCs
and AT-MSCs [60, 61, 80, 155]. However,
AT-MSCs showed less pronounced therapeutic
effect in LSCD models than BM-MSCs [60, 61].
In addition, the expression of the pro-angiogenic
factor VEGF was also reduced in the BM-MSC-­
treated corneas [60, 61, 80, 155]. All these data
demonstrate that MSCs seeded on poly-L-lactic
acid nanofibers and transplanted to the ocular
surface can mitigate the clinical signs in experi-
mental models of corneal epithelial damage.
Furthermore, BM-MSCs show higher therapeutic
effect than AT-MSCs when they are transplanted
using poly-L-lactic acid carriers.
There are no data about the migration capacity
of the MSCs from the poly-L-lactic acid carriers
to the ocular surface. It would be very interesting
to perform further experiments to elucidate the
migration of the cells transplanted in this kind of
substrates.
Regarding corneal inflammation, MSCs
transplanted to the ocular surface on poly-L-lac-
tic acid carriers were able to decrease the pres-
ence of CD3+ cells and macrophages in the
corneal tissues of LSCD models [60, 80].
Furthermore, the expression of the pro-inflam-
matory cytokines TGF-β, IL-6, IL-8, IL-1β,
IL-2, and IFN-γ also decreased in the corneas
with epithelial damage and treated with
BM-MSCs seeded on poly-L-­lactic acid carriers
[61, 80, 155]. On the other hand, the increment
of the oxidative stress in corneal epithelial dam-
age has been demonstrated [80]. In this regard,
the BM-MSCs seeded on nanofibers reduced the
expression of the oxidative stress markers iNOS,
caspase-3, nitrotyrosine (NT), and malondialde-
hyde (MDA) and on the other hand promoted the
increment of the protective enzyme aldehyde
dehydrogenase 3 family member A1 (ALDH3A1)
251
[60, 61, 80, 155]. Furthermore, the expression of
XOX and the levels of the antioxidants SOD,
glutathione peroxidase (GPX), and catalase
(CAT) were similar between the BM-MSC-
treated corneas and the healthy control corneas,
while SOD, CAT, and GPX showed lower levels
in AT-MSC-treated corneas than healthy corneas
[61]. Moreover, in the injured corneas treated
with BM-MSCs the expression of MMP-9 was
reduced or suppressed [61, 80, 155]. Therefore,
all these results demonstrate the anti-inflamma-
tory properties of MSCs, as well as their benefi-
cial role against the oxidative stress produced in
a corneal epithelial damage. Nevertheless, the
treatment was applied always in an acute phase
of the damage. In this regard, it has been
described that the sooner the treatment is applied
the better the healing is, mainly in an inflamma-
tory and oxidative environment. However, in the
clinical setting, there are a lot of patients in a
chronic phase of the disease, and then it is neces-
sary to know the behavior of these cells in those
patients. Therefore, further experiments in a
chronic phase of corneal damage are necessary
to overcome this issue. In addition, as in the AM
experiments, the number of stem cells that can
be transplanted using this kind of substrates is
very low in contrast to the cell dose that can be
administered by injection. It is important to note
that the cell dose transplanted in these carriers to
the ocular surface was not totally controlled
because the cells were kept in culture for some
days before transplantation.
Regarding corneal phenotype, the expression
of the corneal epithelial markers CK3 and CK12
increased in the MSC-treated groups, mainly in
the BM-MSC-treated group [60].
17.2.2.11 Polyamide 6/12 Carriers
Polyamide nanofiber scaffolds containing LESCs
+ BM-MSCs have been transplanted to the ocular
surface of a LSCD model [118]. A decrease of
the iNOS, IL-2, and IFN-γ levels was observed in
the treated corneas in comparison with the
untreated eyes [118]. This anti-inflammatory
effect could be originated by both cell types, or
just by one of them, but the design of the study
does not allow to know.
252
17.2.2.12 Subconjunctival Injection
of MSCs
Subconjunctival injection is a technique widely
used in ophthalmology. Although this way of
administration allows to apply a local treatment
with a high cell dose, the volume administered
have to be low. A reduction in epithelial defects,
neovascularization and corneal opacity has been
observed after subconjunctival administration of
MSCs in corneal burn experimental models [50,
51, 156, 157]. Furthermore, BM-MSCs adminis-
tered by subconjunctival injection immediately
after injury produced a decrease of the epithelial
defects in the diabetic mice, showing similar reep-
ithelialization rate of the nondiabetic mice [65]. It
has also been demonstrated that the expression of
the pro-angiogenic factors VEGF and MMP-9 is
lower in the BM-MSC-treated corneas, whereas
the anti-angiogenic TSP-1 factor increase its
expression in the corneas of the BM-MSC-treated
groups [50, 51, 156]. Therefore, MSCs, both
BM-MSCs and AT-MSCs, subconjunctivally
administered have a positive effect in the reduc-
tion of the clinical signs in experimental models
with corneal epithelial damage.
In regard to the migration of the MSCs sub-
conjunctivally administered, although some
authors have located the BM-MSCs in the wound
edge of the cornea [65], others located the
BM-MSCs in the injection site but not in the cor-
nea [50, 51]. These data seem to indicate that the
MSCs subconjunctivally administered tent to
keep in the injection site and do not migrate to the
central cornea. Nonetheless, the therapeutic
effect of the cells has been demonstrated; thus the
MSCs trigger their beneficial role from the injec-
tion site without migrating to the damaged area.
The MSCs subconjunctivally administered
also showed an anti-inflammatory role in the ocu-
lar surface. Less infiltration of inflammatory cells,
macrophages (CD68+ cells), and leukocytes
(CD45+ cells) was observed in the MSC-­treated
eyes of experimental models with corneal epithe-
lial damage [50, 51, 65]. In addition, the expres-
sion of MPO was lower in the BM-MSC-­treated
corneas than in the PBS-treated corneas [65].
Furthermore, the expression of the chemotactic
factor MIP-1α, the monocyte chemotactic pro-
T. Nieto-Miguel et al.
tein-1 (MCP-1), the pro-inflammatory cytokines
TNF-α and IL-1β, and the protein MMP-2 was
lower in the corneas of the BM-MSC-treated
groups in contrast to the untreated groups [51, 65,
156]. Additionally, the anti-inflammatory factor
TGF-β showed an increment in the BM-MSC-­
treated corneas [156]. Moreover, the expression
of TSG-6 was elevated in the corneas treated with
BM-MSCs, whereas no changes were observed in
other factors that could be also related with the
immunomodulatory effect of MSCs [65]. In addi-
tion, BM-MSCs knocked down for TSG-6 did not
show therapeutic effect in the diabetic corneas
with epithelial damage [65]. Therefore, TSG-6
seems to be one of the mediators of the therapeu-
tic effect of the MSCs when they are subconjunc-
tivally injected into a damaged cornea.
An increase in the expression of the epithelial
markers Cx43 and β-catenin in the AT-MSC-­
treated corneas was described, whereas differ-
ences were not seen in the epithelial marker
E-cadherin and in the limbal stem cell marker p63
[157]. Therefore, the role of the MSCs subcon-
junctivally administered in the recovery of the
corneal and limbal phenotypes is not yet clear.
17.2.2.13 Combined Administration
of MSCs
AT-MSCs have also been administered to the cor-
nea of a partial LSCD model using combined
routes: topically, subconjunctivally, and in a stro-
mal pocket [158]. The AT-MSC-treated group
showed attenuated epithelial defects, neovascu-
larization, and cornea opacity in contrast with the
untreated corneas. The expression of the prolif-
eration marker Ki67 increased in the AT-MSC-­
treated corneas, whereas a reduction of the
secreted VEGF factor was observed in these cor-
neas. Twenty-eight days after damage and treat-
ment, the AT-MSCs administered were found in
the cornea. In regard to inflammation, the authors
noted inflammatory cells in the untreated cor-
neas, while the animals treated with AT-MSCs
presented a normal corneal stroma without
inflammation. Furthermore, the indicator of dam-
age serum glutamic-pyruvic transaminase
(SGPT) was reduced in the corneas treated with
AT-MSCs. Therefore, the combined administra-
17  Cell Therapy Using Extraocular Mesenchymal Stem Cells
tion of MSCs, using three local applications, is
effective in the treatment of corneal epithelial
damage in experimental models.
In conclusion, both BM-MSCs and
AT-MSCs have shown therapeutic effects at
reducing clinical signs in corneal epithelial
damage or LSCD experimental models using
different administration routes. Nevertheless,
all of these ways of administration have limi-
tations; the intravenous or intraperitoneal
administrations inject the MSCs very far from
the cornea, the transplantation of MSCs on
carriers do not allow high cell dose, in the top-
ical administration the cells are washed away
very quickly, and the volume administered by
subconjunctival injection has to be low.
Despite these drawbacks, all these administra-
tion routes of MSCs are effective in the treat-
ment of experimental corneal epithelial
damage. Additionally, migration of MSCs
from the application site to the cornea has
been confirmed using intravenous and intra-
peritoneal injection or transplantation of the
cells on a carrier. However, subconjunctival
injection does not allow the migration of the
cells to the central cornea, and further experi-
ments are needed to know the migration of
topically administered MSCs. Furthermore,
the anti-inflammatory properties of these
cells, as well as their beneficial role against
the oxidative stress triggered by the epithelial
corneal damage, have been demonstrated. In
addition, one of the principal factors impli-
cated in the anti-inflammatory effect of MSCs
is TSG-­6. Although some authors advocate the
transdifferentiation of extraocular MSCs to
corneal epithelial cells, only one of them have
demonstrated that MSCs expressed CK3 in the
corneal epithelium when they were trans-
planted to the ocular surface of an experimen-
tal corneal epithelial damage [31]. But since
some authors have shown that MSCs express
CK3  in basal conditions [18, 32], the CK3
expression might not indicate a real transdif-
ferentiation. Therefore, transdifferentiation of
MSCs into corneal epithelial cells has not be
confirmed in  vivo, but all the results seem to
indicate that MSCs contribute to the recovery
253
of the corneal epithelium by secreting factors
that act at a paracrine level promoting prolif-
eration and differentiation of the resident stem
cells in the tissues.
17.2.3 Extraocular MSCs for Corneal
Epithelial Damage in Humans
MSCs have been used successfully to repair and
regenerate tissues [159]. In the 1980s, it was con-
cluded that MSCs could differentiate into osteo-
blasts, chondrocytes, adipocytes, and myoblasts,
and, consequently, they have been used to repair
infracted myocardium [160], in hepatic cirrhosis
[161], in gastrointestinal cancer [162], in peri-
odontal therapy [163], or for bone deformities
and fractures [164]. It was also demonstrated that
these cells could contribute to the regeneration of
ectodermal tissues, such as bronchial and alveo-
lar epithelial cells [165], glomerular and tubular
cells, or gastrointestinal cells [166]. This process
of differentiation will depend on induced and
control factors that cohabit in the environment
where they are located [167].
In the field of ophthalmology and vision-­
related therapies, some approaches have been
made using MSCs, mainly at the ocular surface.
But even though there are publications using
these cells in animal models [10, 17–19, 60, 82],
there is practically no studies in the clinical area.
MSCs have been used to treat the deleterious
consequences of chronic GvHD, one of the most
dreaded complications of otherwise successful
allogeneic hematopoietic stem cell transplanta-
tion to treat hematological malignancies [168,
169]. Some years ago, a study published the
beneficial effect of intravenous injection of
MSCs to treat the severe ocular surface involve-
ment that often occurs in this disease. This study
described improvement in clinical dry eye
scores in 54.55% of the 22 patients enrolled,
also accompanied by in increased levels of regu-
latory CD8+CD28− lymphocytes but not
CD4+CD25+, which led authors to conclude that
the immunomodulatory effect by which MSC
transfusion improved the refractory dry eye dis-
ease could be due to regulating the Th1/Th2 bal-
254
ance by triggering the generation of CD8+CD28−T
lymphocytes [169].
There is also a single case report in which a
patient suffering from keratoconus and a persis-
tent corneal epithelial defect unresponsive to
conventional therapy was treated by topical
administration of adipose tissue-derived MSCs,
achieving corneal epithelial healing after
1 month [170].
MSCs have been experimentally used to treat
not only the acute phase of ocular chemical
burns [171] but also the chronic devastating con-
sequences of severe ocular chemical injuries. To
this later aim, there exists now enough experi-
mental evidence showing that MSCs are benefi-
cial in dealing with the chronic sequelae of
chemical injuries, helping to provide corneal
epithelium with the lost regenerative capacity
due to the destruction of the limbal stem cells
and usually their niche entirely [10, 17–19]. But,
although MSCs are shown useful in animal mod-
els of LSCD, they have not been yet fully
approved to clinical use, as there is only one
clinical trial on the use of MSCs for LSCD in
humans. This exploratory proof-of-concept clin-
ical trial, performed in our institution [172],
explored the safety and efficacy of bone marrow-
derived MSCs for bilateral LSCD of any etiol-
ogy in which the more widely accepted
autologous cultivated limbal epithelial trans-
plantation (CLET) was not feasible due to the
complete absence of healthy limbus where to
remove a biopsy from [7–9]. In this randomized
controlled clinical trial, we compared patients
subjected to CLET, as we had enough previous
experience in this procedure [8, 173], with
patients subjected to MSC transplantation
(MSCT), enrolling though a small but significant
number of patients due to its exploratory nature.
Using an extensive evaluation endpoint analysis,
there was enough evidence showing that MSCT
was as safe and as effective as CLET, with a
73–86% success rate after 12  months [172]
(Fig. 17.2). These results, although must be rep-
licated in a larger clinical trial, provide hope to
those patients that are not eligible to have autolo-
gous limbal transplantation procedures due to
extensive bilateral disease.
T. Nieto-Miguel et al.
17.3 Future Perspectives
and Conclusions
Extraocular MSCs represent an available, non-­
immunogenic source of stem cells that has proved
to possess a potential therapeutic value in corneal
epithelium regeneration based on results obtained
in both preclinical and clinical studies, although
only a few clinical studies have been performed
yet, as this approach is quite novel. However,
MSCs isolated from different tissues by different
techniques may possess different properties and
levels of activity, which may directly influence
their efficacy. In addition, most of the MSC isola-
tion, propagation, and characterization protocols
vary among laboratories, and clinical translation
of MSC-based therapies implies that their manu-
facturing process should comply with good man-
ufacturing practices (GMP) in order to preserve
the quality and safety standards of the final cell
product. Therefore, the development of standard-
ized protocols for the preparation, characteriza-
tion, and evaluation of the biological potential
activity (potency assays) of MSCs is essential to
better understand MSCs function and clinical
utility and to improve comparison of results
between research and clinical centers. To achieve
this goal, cooperation between basic researchers,
physicians, industry, and regulatory authorities is
fundamental.
Although several scientific groups, including
ours, have reported promising results on the use
of MSCs for corneal epithelial regeneration, the
precise mechanism of how these cells exert their
therapeutic effects on the ocular surface remains
unclear and deserves further investigation.
Additional research in LSCD animal models is
needed to address whether MSCs can or cannot
transdifferentiate into corneal epithelial cells, to
determine how long MSCs can survive in the cor-
nea secreting factors that reduce tissue injury,
and to answer questions such as which is the
most appropriate cell dose and best route of
administration for applying MSCs onto the ocu-
lar surface.
Our proof-of concept clinical trial have shown
that MSCs transplantation onto the ocular sur-
face of patients suffering from LSCD can safely
17  Cell Therapy Using Extraocular Mesenchymal Stem Cells
Fig. 17.2  This 48-year-old man had a bilateral chemical
injury 4 years before, for which multiple amniotic mem-
brane transplantations were performed, as per standard of
care. Both eyes show the typical end-stage picture of a
severe chemical burn: residual corneal vascularized opaci-
fication that is due to a full-thickness stromal scarring.
Not seen in these pictures are bilateral cataracts, many
areas of synechiae between the iris and posterior cornea
and glaucoma. Thus, three surgical procedures, i.e., stem
cell transplantation, further penetrating keratoplasty, and
another non-corneal surgery (cataract removal), would be
required to recover vision. He underwent MSC transplan-
tation in his left eye. The upper row displays left eye
255
4  weeks before cell transplantation. The middle row
shows transplant in place with the cells facing down and
the amniotic membrane (arrows) facing the scleral contact
lens 2 days after surgery. Lower row displays successful
transplant after 12 months. In vivo confocal microscopy
showed a conjunctival-like phenotype in the central cor-
nea before transplantation (upper row). Twelve months
after surgery, a corneal-like phenotype was present in his
left central cornea (lower row). The continued improve-
ment of the central corneal epithelial phenotype 12 month
after transplantation indicates that this patient would have
a better chance for a successful penetrating corneal trans-
plant to improve his vision
256
and effectively restore the corneal epithelium.
Nevertheless, to keep moving forward it is
needed further research and to decipher the
mechanism by which transplanted MSCs
improve corneal epithelial cell phenotype. To
accomplish this, it is first essential to agree on
LSCD diagnostic criteria and clinical evaluation
endpoints so that the data collected from differ-
ent clinical centers applying cell-based therapies
for LSCD treatment can be effectively
compared.
Acknowledgment  Financial Support: Carlos III National
Institute of Health, Spain (CIBER-BBN, CB06/01/003
MINECO/FEDER; Spanish Network on Cell Therapy,
TerCel RD12/0019/0036); Ministry of Economy and
Competitiveness and European Regional Development
Fund, Spain (SAF2015-63594-R MINECO/FEDER, EU);
Regional Center for Regenerative Medicine and Cell
Therapy, Castilla y León, Spain.
Compliance with Ethical Requirements  T Nieto-
Miguel, S Galindo, M López-Paniagua, I Pérez, JM
Herreras and M Calonge declare that they have no
conflict of interest.
All procedures followed were in accordance
with the ethical standards of the responsible com-
mittee on human experimentation (institutional
and national) and with the Helsinki Declaration
of 1975, as revised in 2000. Informed consent
was obtained from all patients for being included
in the study.
All institutional and national guidelines for
the care and use of laboratory animals were
followed.
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 Cell-based Therapy Using Induced
Plutipotent Stem Cell
Ricardo Pedro Casaroli-Marano
18.1 Introduction
The structural integrity and the maintenance of
the physiology of the cornea are key elements for
its transparency. Corneal epithelium has an
important role in corneal transparency, contribut-
ing not only to the absence of blood vessels but
also to a process of permanent repair, which is
essential to preserve its normal status [1–3].
Thus, homeostasis of the corneal epithelium
maintains a state of structural integrity of the
ocular surface that is essential for vision.
Continuous renewal of the corneal epithelium
has been shown to be possible thanks to a cellular
subpopulation located in the basal layers of the
sclerocorneal limbic region. These cells – known
as limbal stem cells (LSCs)  – have progenitor
characteristics and can maintain a constant epi-
thelial renewal dynamic. Their key properties are
that they are oligopotent cells that present a high
proliferative potential with a slow cell cycle,
while showing great capacity for self-renewal by
asymmetric division [2, 3]. Due to these particu-
larities, they are attributed a crucial function in
R. P. Casaroli-Marano (*)
Department of Surgery, School of Medicine and
Hospital Clínic de Barcelona, University of
Barcelona, Barcelona, Spain
Institute of Biomedical Research (IIB-Sant Pau) and
Barcelona Tissue Bank, Banc de Sang i Teixits,
Barcelona, Spain
e-mail: rcasaroli@ub.edu
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_18
18
maintaining ocular surface integrity and renewal
of the corneal epithelium. In addition, the sclero-
corneal limbus not only serves as a reservoir for
LSCs but also functions as a structural barrier
against uncontrolled and abnormal growth of
conjunctival epithelial cells replacing the corneal
epithelium and inducing neovascularization
[1–3].
The microenvironment of the sclerocorneal
limbus provides a specialized niche for ocular
surface homeostasis, making the limbic ocular
region of special interest in the cell renewal pro-
cess. Thus, aggressions that affect the integrity of
the sclerocorneal limbic region, leading to the
disappearance, reduction, or even functional
deterioration of LSCs, may produce a pathologi-
cally maintained condition of limbal stem cell
deficiency (LSCD) with significant changes to
the ocular surface. These changes include the
appearance of persistent corneal defects, epithe-
lial keratinization, and conjunctivalization phe-
nomena, with corneal neovascularization under a
persistent inflammatory process that reduces cor-
neal transparency and promotes vision loss [1–4].
In these situations, replacing the diseased cornea
by keratoplasty would result in graft failure
because the physiological mechanism of corneal
regeneration by epithelial renewal is damaged by
the functional absence of the LSCs [4].
Currently, cell-based therapy is the most
appropriate approach for the replacement and
repair of the ocular surface in cases of LSCD
263
264
[5, 6]. The cultured limbal epithelial transplanta-
tion (CLET) or the cultured oral mucosal epithe-
lial transplantation (COMET) approaches
involve obtaining healthy autologous tissue by
minimally invasive biopsy after ex  vivo cell
expansion. Both approaches present very encour-
aging clinical results for the recovery of the ocu-
lar surface and may contribute to the restoration
of vision in appropriately selected patients. In
unilateral disease, autologous treatment with
CLET is preferred, although this source of pro-
genitor cells is not always available. If both eyes
present with severe disease, allogeneic LSCs or
the COMET technique can be used [5–7].
The development of cell therapies with adult
tissue progenitors, such as adult mesenchymal
stem cells (MSCs) or induced pluripotent stem
cells (IPSCs), represents a significant advance in
the treatment of certain eye diseases [2, 8].
Whether associated with the emerging approaches
of tissue bioengineering or not, these offer a
physiological treatment option that is more ratio-
nal and less invasive for the regeneration of the
ocular surface. In this chapter, future perspectives
regarding the use of IPSCs as a cell source in
these new approaches in regenerative medicine
for the ocular surface are outlined and discussed.
18.2 L
 imbal Stem Cells (LSCs):
Epithelial Renewal
and Molecular Mechanisms
One of the essential elements for maintaining an
adequate renewal and regeneration process in the
corneal epithelium, apart from maintaining the
structural integrity of the sclerocorneal limbus, is
the balance of the microenvironment, or niche,
where the LSCs are located. The palisades of
Vogt – a series of radially oriented fibrovascular
ridges in the region of the human sclerocorneal
limbus – and, more recently, the limbal epithelial
crypts have been described as the natural niche
for LSCs [9, 10]. Histologically, palisades of
Vogt correspond to projections of the corneal
stroma into the basal epithelial layer. Interspersed
between them, limbal epithelial crypts are regions
where the epithelium projects deep into the
R. P. Casaroli-Marano
stroma; its lowermost aspect, which is farthest
from the surface, is probably where the largest
population of LSCs is located [9, 11].
The niche provides a variety of specific factors
that contribute to the survival and dynamics of
LSCs. On the other hand, they provide certain
components of the extracellular matrix and con-
tribute to the growth factors and cytokines that
determine a special proliferative state and a pro-
cess of organized cell growth. The niche is also
responsible for maintaining a state of stemness in
LSCs, with controlled inhibition of cell differen-
tiation observed together with asymmetric divi-
sion [1, 12]. This asymmetric division allows one
of the daughter cells to retain its stemness and
replenish the stem cell pool, while the other
daughter cell enters differentiation.
It has been demonstrated that LSCs can dif-
ferentiate into postmitotic cell populations
through an oriented process of cell differentia-
tion. This obeys a continuous centripetal move-
ment from the deepest peripheral epithelial layers
(basal layers of the sclerocorneal limbus) to the
superficial and more central layers (superficial
corneal epithelium). This ordered mechanism of
cellular proliferation and differentiation ensures
constant renewal of the corneal epithelium and
maintenance of its integrity [10, 13–15].
However, a cellular subpopulation may be located
in the central cornea that has the capacity to gen-
erate undifferentiated progenitor cells with
regenerative epithelial capacity [10, 15, 16]. This,
in turn, indicates the possibility that accessory
mechanisms exist for the renewal and mainte-
nance of the corneal epithelium.
Research has also demonstrated that the clo-
nogenic capacity of epithelial cells in the
sclerocorneal limbic region is organized under
a system of progenitor cells stratified in com-
partments [10, 12, 15, 17]. In the initial
­compartment, the undifferentiated cell subpop-
ulation presents characteristics of progenitor
cells with high self-renewing capacity; how-
ever, this property decreases as cells migrate
through the following compartments. In the
final compartment, a cellular subpopulation
exists with terminal differentiation characteris-
tics associated with low or no self-­ renewal
18  Cell-based Therapy Using Induced Pluripotent Stem Cell
capacity. These cells are incorporated as differ-
entiated corneal epithelial cells on the s­ urface
of the central cornea. In this sense, and in con-
trast to epithelial cells of the central cornea, it
is considered that epithelial cells of the sclero-
corneal limbic region can form holoclones with
greater clonogenic potential [12]. Likewise,
epithelial cells isolated from the basal layers in
the limbal region exhibit a high in vitro prolif-
erative potential during expansion or in
response to corneal injury and show an undif-
ferentiated phenotype lacking expression of
corneal epithelial differentiation markers [17].
Conjunctiva (A) Limbus (B) Peripheral cornea (C)
LSC
LSCs TACs
Conjuntival
epithelial
and Proliferation zone
goblet cells
C1 C2
CK5, CK14 ABCG2, ABCG5 ABCG2, p63
Muc5AC p63, p75, Bmi-1 CK19, a9
C/EPBδ, Notch-1 Vimentin
CK15, CK19, a9
Fig. 18.1  Differentiation of limbal stem cells (LSCs)
into corneal epithelial cells during the repair of corneal
epithelium, according to Thoft hypothesis (XYZ hypoth-
esis) [13]. LSCs differentiate in an ordered manner that is
carried out progressively in compartments and regions of
cellular “maturation.” The sclerocorneal limbic region (B)
corresponds to the LSC “reservoir” and works as a spe-
cialized microenvironment (niche), providing a barrier
against the growth of the conjunctival epithelial cells (A)
and their blood vessels in peripheral (C) and central (D)
corneal tissue. LSCs are characterized as a cell subpopula-
tion with stemness, high proliferative capacity, and self-­
renewal proprieties (compartment C1). Transient
amplifying cells (TACs) appear to be another cell subpop-
ulation presenting some degree of differentiation and lim-
ited capacity for self-renewal (intermediate compartment
265
Taking the above into consideration, it is
accepted that LSCs give rise to a subpopulation
of transient amplifying cells (TACs) and that
these cells migrate and differentiate under an
ordered centripetal movement from the limbic
region toward the central corneal surface, where
they generate differentiated corneal epithelial
cells that eventually disappear. This coordinated
mechanism is responsible for the renewal of the
corneal epithelium and its continued regenera-
tion (Fig. 18.1).
At present, the identification of specific mark-
ers and the detailed study of cellular and molecu-
Central cornea (D)
Corneal epithelial cells
PMCs TDCs
Differentiation zone
C3
Nestin, Involucrin CK3
Cx43, a2, a6 CK12
E-cadherin
C2). The proliferation zone (compartments C1 and C2) is
also characterized by a specialized environment (niche)
responsible for maintaining LSC oligopotency. Postmitotic
cells (PMCs) and terminally differentiated cells (TDCs)
are in the differentiation zone (compartment C3) and are
characterized by their terminal differentiation state that
lacks self-renewal capability. Repair of the corneal epithe-
lium is established by events in which LSCs proliferate,
migrate, and differentiate from the suprabasal layers of
the sclerocorneal limbus (epithelial crypts), resulting in
TACs (XY movement, B to C), and with centripetal
migration forming TDCs as cell differentiation proceeds
toward to the superficial layers of the central cornea
(movement YZ; to D). The main putative cell markers are
shown in the right part of the diagram
266 R. P. Casaroli-Marano

lar relationships in the niche are of great interest stroma mesenchymal cells, melanocytes, vascu-
to improving our understanding of the therapeutic lar cells, and nerve endings, as well as extracel-
potential of LSCs. The transcription factor p63 lular elements, such as extracellular matrix
(ΔNp63α), specifically its α-isoform, has been (ECM) proteins and intracellular signaling mol-
described as one of the most characteristic mark- ecules [10, 14, 15]. The precise and coordinated
ers for the identification of LSCs in humans [4, function of all cellular and extracellular compo-
18]. Based on the presence of p63 cells, it has also nents in the niche are essential for its correct
been possible to establish clinically relevant qual- physiology when regulating and modulating the
ity criteria for the success of CLET techniques [4, proliferation, migration, and differentiation of
18, 19]. It has been concluded that the clinical the LSCs. Thus, homeostasis of the sclerocorneal
success of transplantation is dependent on the limbic region should involve a complex interrela-
existing p63+ cell population in the cultures tionship between niche elements (i.e., the ECM
obtained by ex vivo expansion. Successful clini- and cellular components) and the LSCs.
cal results have been observed more frequently in Corneal stroma mesenchymal cells appear to
cases in which transplanted cell cultures con- exert a regulatory role on LSCs, contributing to
tained between 5% and 10% p63+ cells. Cases in the maintenance of their stemness [10, 15]. The
which the percentage of p63+ cells was slower niche is also rich in vascular and nerve endings
than 3% were associated with clinical failure [19]. that are essential for maintaining the survival and
Recently, our group has been able to verify that, trophic function of LSCs [15, 23]. Also, certain
in limbic biopsies obtained from donors older than major ECM proteins, such as laminin, fibronectin,
65 years, cell cultures can be generated with a high and collagens [24], are not only essential for the
percentage of p63+ cells, making them viable for ultrastructural conformation of the niche but also
allogeneic transplant [20]. However, because the for participating in the events related to the regu-
LSC population in the cornea is scarce, character- lation and control of LSC migration and differen-
ization and cellular behavior studies imply an tiation. Thus, the structural and molecular
added difficulty, both for in vitro methodologies as characteristics of the limbal niche make it a
well as for experimental models. Thus, there is still unique environment for LSC behavior and stem-
no consensus on the definition of specific markers ness. Likewise, the intracellular signaling mediat-
for this cell population. Despite this, different puta- ing molecules and the molecular pathways
tive markers have been described based on pro- involved in cell behavior are crucial for maintain-
teomic and immunolocalization studies. These ing proliferation and differentiation of LSCs [24].
have used a reduced panel of possible markers, The signals that trigger the activation, prolif-
with ΔNp63α, ATP-binding cassette subfamily G eration, and migration of LSCs into TAC com-
member 2 (ABCG2), member 5 (ABCG5), Ccaat- partments are not fully understood. It has been
(cytosine-­c ytosine-­a denosine-adenosine- suggested that certain growth factors and cyto-
thymidine)-enhancer-binding protein delta-isoform kines, as well as chemotactic molecules that
(C/EBPδ), polycomb complex protein B lymphoma include ECM degradation products, all contribute
Mo-MLV (Bmi-1), cytokeratin 15 (CK15), and to the mechanisms of LSC corneal epithelial
Notch homolog 1, translocation-associated renewal and homeostasis.
(Drosophila) (Notch-1) being the main candidates.
The exclusion of cellular expression of cytokeratin
13 (CK3) and cytokeratin 12 (CK12), considered 18.3 Induced Pluripotent Stem
markers of corneal epithelial differentiation, is also Cells (IPSCs): Mechanisms
associated [21, 22] (Fig. 18.1). for Reprogramming
The LSC niche (i.e., the palisades of Vogt and
the limbal epithelial crypts) consists of various Together with clustered regularly interspaced
elements. These include cellular elements from short palindromic repeats (CRISPR)-associated
different characteristics, such as antigen-­ systems (i.e., Cas) for genome editing  – also
presenting and immune system cells, corneal called “genome surgery” [25, 26] – the technol-
18  Cell-based Therapy Using Induced Pluripotent Stem Cell
ogy for inducing pluripotent stem cells has been
one of the most significant advances in the con-
text of advanced therapies in regenerative medi-
cine over recent decades. Through progressive
development, it has been shown that transfection
and overexpression of a specific set of transcrip-
tion factors  – Oct4/Klf4/Sox2/cMyc or Oct4/
Sox2/Lin28/Nanog  – in differentiated somatic
cells from adult tissue (e.g., skin keratinocytes or
fibroblasts) can reprogram cell fate from an
immature cell state to recover its capacity for plu-
ripotency: IPSCs, with high clonal and prolifera-
tive potential [27–29]. These cells can be
differentiated into several other cell types of dif-
ferent embryonic origin, such as endo-, meso-, or
ectodermal lineages. This property has opened
possibilities to research different cellular events,
including the mechanisms of differentiation, pro-
liferation, senescence, and toxicity, as well as
other processes related to cell behavior. Unlike
embryonic stem cells, for which there are estab-
lished ethical problems related to their collec-
tion and use, IPSCs can readily be collected
from differentiated cells in adult tissue to create
an autologous source of progenitor cell popula-
tions. In turn, these can be used in both regen-
erative therapy and research into the mechanisms
of rare pathologies. Thus, cell modeling  –
through patient-specific IPSCs – can be used for
certain rare disorders to help better understand
their pathophysiology and develop treatments
[30–32].
IPSCs represent a promising resource for
future clinical application in advanced cell-based
therapy. Currently, the methodologies and
approaches related to IPSCs are being expanded
to increase the efficiency of their production and
to reduce their immunogenic and tumorigenic
potential. Cell therapy with patient-specific
autologous IPSCs has been shown not only to
mitigate potential immune reactions but also to
provide a large and continuous supply of cells for
transplantation [32, 33]. For clinical application
of IPSCs in humans, choosing the correct donor
cell type and the most appropriate methodology
for reprogramming is critical. The donor somatic
cell must be easily obtained and reprogrammed
and ideally be from the patient requiring treat-
ment. Thus, peripheral blood mononuclear cells
267
(PBMCs), together with fibroblasts and dermal
keratinocytes, as previously mentioned, represent
the preferred cellular candidates for inducing
pluripotency [29, 33–35].
The methodology for generating IPSC lines is
based on the approaches for cellular reprogram-
ming (Fig.  18.2). Since the early work of
Yamanaka [27], which used a highly efficient
viral reprogramming method (the Moloney
murine leukemia retrovirus) that incorporated a
retrovirus into the cell genome, variations of this
method have been developed. However, this tech-
nology presents restrictions for clinical applica-
tion in humans, partly due to the unknown
problems associated with using IPSCs, such as
the long-term repercussion of incorporating viral
elements in host DNA.  Cellular transfection
methods with high efficiency (>0.1%) are note-
worthy, such as integrating retrovirus and lentivi-
rus in the cellular genome (integrative
reprogramming) or using the adenovirus and
Sendai virus (non-integrative reprogramming)
[29, 36, 37]. Other approaches for non-­integrative
cellular reprogramming have been developed that
do not use viruses as cellular vectors, but these
have variable transfection efficiency. Methods
based on DNA plasmids (non-episomal) and epi-
somes are non-integrative approaches that show
medium transfection efficiency (0.01–0.1%) [29,
36]. In recent years, synthetic RNA or micro-­
RNA (miRNA) has been developed with supe-
rior efficiency to the average non-integrative
methodologies [29, 37]. Moreover, non-integra-
tive reprogramming methods appear to be pre-
ferred for clinical application [38] because they
limit the possibilities of internal mutagenesis,
cell cycle dysregulation, and tumor formation
[29, 38].
Direct cellular reprogramming in different
states, whether pluripotent or somatic, has recently
been developed. These are among the most promis-
ing approaches in regenerative medicine, with
great potential for secure and therapeutic clinical
application. Direct reprogramming is characterized
by a process in which mature and completely dif-
ferentiated somatic cells or immature cells that
retain some potential for differentiation are induced
to other cell types without needing to pass through
a state of pluripotency [36, 39]. For this purpose,
268
OKSM
Delivery
OSLN
methods
Virus
Retrovirus
Lentivirus
Adenovirus
Sendai virus
DNA
Adult somatic cell
Plasmids
Episomes
RNA
Synthetic RNA
miRNA
Protein
Peptides STF
Fig. 18.2  Strategies for cell reprogramming. The intro-
duction (transfection) and overexpression of certain tran-
scription factors in partial or terminally differentiated
adult (somatic) cells can induce pluripotency (i.e., IPSCs).
The first transcription factors shown to be capable of
inducing pluripotency were Oct4/Klf4/Sox2/cMyc
(OKSM) and Oct4/Sox2/Lin28/Nanog (OSLN). The
methods used for cellular transfection are based on the use
of viral and non-viral vectors, giving rise to integrative
(retrovirus and lentivirus) and non-integrative reprogram-
cells can be reprogrammed by transient overex-
pression of transcriptional factors during a rela-
tively short interval. Cells in this state are therefore
called partial IPSCs and can be modulated to
respond to different signaling environments (e.g.,
growth factors, cytokines, or inducing agents). In
appropriate environments, these cells have the
capacity to direct cell fate in reprogramming [39]
(Fig.  18.2). For corneal repair, direct reprogram-
ming would be of great advantage, not only elimi-
nating the need for the potentially risky pluripotency
stages but also reducing time and resource require-
ments when producing a specific IPSC line.
Another advantage of direct reprogramming is
that very few cells would be needed for cell ther-
apy of the ocular surface, meaning that difficulty
obtaining and producing an IPSC line would not
R. P. Casaroli-Marano
IPSC
Differentiated cell
Direct reprogramming
ming approaches (adenovirus, Sendai virus, DNA, epi-
somes, RNA, and peptides). Recently, direct
reprogramming strategies have been described that permit
the transfection of selective and specific transcription fac-
tors (STF) that allow somatic cells to acquire differentia-
tion capacity (partial induced pluripotency) to other cell
types, without necessarily going through a specific state
of pluripotency, thereby avoiding the risk of mutagenesis
and predisposition to teratogenesis
be limiting factors. However, there is still only
limited experience with generating LSCs by
transdifferentiation of readily accessible adult
somatic cells. The first evidence of the potential
for transdifferentiation was achieved by convert-
ing progenitor cells from the bulb of the hair fol-
licle into corneal epithelial cells. These cells
acquired several markers of LSCs, including
CK15, p63, and the low-affinity nerve growth
factor receptor (p75; neurotrophin receptor),
when the niche of the sclerocorneal limbic region
was mimicked; for this, the presence of corneal
stromal fibroblasts and laminin-5 were crucial
[40]. The limbal stem-like cells that were
obtained using this method were successfully
used to repair the damaged cornea of an experi-
mental model in which autologous stem cells
18  Cell-based Therapy Using Induced Pluripotent Stem Cell
were used [41]. In other research murine adult
stem cells from a hair follicle bulb were transdif-
ferentiated into corneal epithelial-like pheno-
types by forcing the overexpression of Pax6 and
were maintained in supplemented culture
medium with soluble factors obtained from the
sclerocorneal limbic region [42]. Also, corneal
epithelial cells could be generated from progeni-
tor skin cells and cultured in the presence of
growth factors [43]. Corneal epithelial-like phe-
notypes can also be obtained using MSCs from
adipose tissue when cultivated with an LSC cul-
ture medium supplemented with inducing agents
for cellular differentiation [44]. These are encour-
aging results that may open the door to novel
sources of autologous LSC supply.
Interestingly, the results obtained from a
recently developed direct transdifferentiation
approach have been described for LSCs and cor-
neal epithelium and was compared with a deriva-
tion method from IPSCs [45]. Human fibroblasts
were directly transdifferentiated into LSCs and
corneal epithelial cells by overexpressing three
limbal-specific transcription factors in lentivirus
transfection and maintaining them in a corneal-­
specific culture medium. They used ΔNp63α, C/
EBPδ, and transcriptional factor 4 (TCF4 or
immunoglobulin transcription factor 2; ITF-2) as
limbal-specific transcription factors. The resulting
differentiated cells exhibited morphological and
marker changes consistent with corneal epithelium,
demonstrating the potential for direct reprogram-
ming from human dermal fibroblasts, bypassing
the intermediate state of pluripotency. Although
cells derived from the IPSCs also showed specific
characteristics and markers, the expression of plu-
ripotency markers (e.g., Oct4 and Klf4) could not
be eliminated. This remains an advantage of direct
programming over using undifferentiated states of
pluripotency (IPSC state).
18.4 Translational Application
on the Ocular Surface
In the context of clinical application for the ocu-
lar surface, therapeutic approaches based on
IPSCs are promising. Their unlimited prolifera-
269
tive capacity and their specificity (autologous
cells) can provide sufficient quantities of cells,
while theoretically minimizing the risks of
immune reactions and graft versus host disease
associated with allogeneic transplant. This could
also mitigate or obviate the undesirable adverse
effects resulting from immunosuppression of the
receptor. For example, in neurodegenerative and
heart diseases, which have limited treatment
options because neuronal and cardiac tissues are
nonrenewable, IPSC-based strategies could be
encouraging [29, 33].
Clinically, the first trials of cell-based therapy
with autologous and allogenic IPSCs in humans
have recently been carried out for the treatment
of age-related macular degeneration [46, 47]. In
these clinical trials, retinal pigment epithelium
(RPE) cells derived from autologous IPSCs gen-
erated from dermal fibroblasts were first gener-
ated from the patient to avoid a potential
immunological rejection. Once implanted there
were no side effects, but the clinical trial was sus-
pended before treating the second patient,
because mutations were detected in the IPSCs.
This finding, coupled with the fact that the gen-
eration of patient-specific IPSCs is long and
expensive, has led to the use of allogeneic IPSCs;
these have not only been shown to be safe but
also to be HLA compatible with the haplotype of
the recipient. For HLA-A, HLA-B, and HLA-­
DRB1 antigens, it has recently been demon-
strated that human lymphocytes recognize RPE
cells derived from IPSCs that directly express
HLA class I/II antigens, though this has not been
observed with homozygous RPE donor cells with
identical haplotypes [48, 49]. This approach is
highly innovative, translational, and cost-­
effective because it can generate a bank of IPSCs
from cells with homozygous haplotypes, thereby
covering a greater number of candidate recipients
for cell-based therapies.
As discussed, LSCs make it possible to repair
and constantly regenerate the corneal epithelium.
These cells have the capacity for continuous self-­
renewal and to produce TACs that differentiate
terminally after a brief period of proliferation [1,
2, 10, 14, 15, 50]. However, there are limitations
to its therapeutic use in LSCD.  When a patient
270
has unilateral LSCD, even obtaining a biopsy
through a minimally invasive procedure can
cause damage to healthy tissue in either the con-
tralateral or affected eye. In cases with bilateral
LSCD, however, obtaining allogeneic cell popu-
lations can increase the risk of immune rejection
in the recipient. Thus, IPSCs represent a real
alternative, providing a permanent cellular
reserve that could be obtained directly from the
patient, thereby avoiding problems of immune
rejection and cell availability.
Initial studies have shown that it is possible to
generate corneal epithelial cell lineages from
IPSCs by blocking the TGF-β/Nodal and Wnt/β-­-
catenin signaling pathways to induce the early
developmental events of the ocular ectoderm [51].
To this end, Mikhailova and colleagues [52] stud-
ied a differentiation protocol without either a
feeder-layer system for cell growth or serum sup-
plements in cell culture medium. The two relevant
cellular signaling pathways were blocked with
small molecule inhibitors (SB431542 and GSK3β,
respectively), and intracellular signaling related to
the fibroblast growth factor (FGF) pathways was
stimulated. After a long period of induction to dif-
ferentiation, they generated relatively pure popu-
lations of corneal epithelial progenitor cells
capable of expressing the specific markers
ABCG2, CK15, p63, and PAX6. The terminal dif-
ferentiation toward mature corneal epithelial cells
(CK3 and CK12) was also achieved.
It has been shown that a reproducible protocol
can restate the canonic pathway of corneal epithe-
lial embryogenesis and reproduce it by inducing
pluripotency in the keratinocytes of the hair folli-
cles and dermal fibroblasts. This can be achieved
by infection with a lentiviral vector containing a
polycistronic cassette of Oct4/Sox2/c-­Myc/Klf4,
with a conditioned culture medium derived from
corneal fibroblasts to induce differentiation, and
supplemented with bone morphogenic protein 4
(BMP4) in the presence of collagen IV substrate
[53]. The organotypic induction assays, with rela-
tively pure cell populations that exhibit corneal
epithelial phenotypes, suggest an ability to strat-
ify into epithelium that is positive for the specific
corneal markers p63, CK14, and CK12.
Additionally, this model allowed the identifica-
R. P. Casaroli-Marano
tion of miRNA profiles as molecular switches for
ocular epithelial differentiation. Interestingly, an
increase in miR-450b-5p was shown to inhibit
PAX6 expression, and consequently the in  vitro
development of corneal epithelium, while a
detectable decrease in miR-­184 during early ocu-
lar development was shown to inhibit PAX6 and
CK3 expressions [53]. These findings allow us to
conclude that miRNA – a group of small noncod-
ing RNA molecules  – also participate in deter-
mining the fate of IPSCs. Overexpression or
depletion of individual miRNAs was associated
with human disease, suggesting that dysregula-
tion of the miRNA system and alterations in ele-
ments of their biogenesis could also be involved
in LSC dysfunction [54, 55].
Recently, it has become possible to produce a
pure and maintained population of LSCs derived
from IPSCs, with phenotypes and molecular
characteristics that are comparable to those of
somatic LSCs. Moreover, these preserve terminal
differentiation capacity under cell culture conflu-
ence conditions [56]. Four lines of human IPSCs
were used – AnaW04, DFC, B5CRE, and 29.3 –
the first obtained from hair follicle keratinocytes
and the last three derived from human dermal
fibroblasts. To initiate limbal compromise, the
IPSC lines were cultured in an environment simi-
lar to the human sclerocorneal limbal niche by
means of a feeder-layer system with irradiated
fibroblasts isolated from the peripheral cornea in
culture plates coated with collagen IV. The cul-
tures were maintained in modified SHEM culture
medium that is usually used for the ex  vivo
expansion of human LSCs [57]. The study dem-
onstrated that PAX6, CK14, and E-cadherin were
expressed after differentiation [56]. Although the
research aim had been to develop differentiated
cell lines for in vitro cytotoxicity tests for use in
preclinical validations, they clearly showed the
potential for IPSCs to differentiate into pheno-
types similar to LSCs, allowing their clinical use
to be considered.
Recent efforts have made significant advances
in the development of self-organized three-­
dimensional organoid approaches from IPSCs,
and these will surely have translational implica-
tions in the future [58]. These studies may affect
18  Cell-based Therapy Using Induced Pluripotent Stem Cell 271

toxicology and drug testing, human ocular dis- Finally, the possibility of differentiating IPSCs
ease modeling, and replacement treatments based in other corneal cell lines, such as stromal kerato-
on cell therapies [59–61]. cytes or corneal endothelium, has also been veri-
Hayashi and colleagues [62] described an fied. Due to its mesenchymal origin, the
in  vitro model of ocular morphogenesis using differentiation protocols must pass through an
human IPSCs maintained in differentiation media, intermediate cellular subpopulation, neural crest
which spontaneously and progressively formed cells (NCCs). These NCCs are embryonic migra-
an ocular primordium comprising four morpho- tory cells with the ability to differentiate into a
logically identifiable concentric zones  – self- wide variety of cell types in different tissues,
formed ectodermal autonomous multi-zone including the cornea, the peripheral nervous sys-
(SEAM). In this interesting research, the concen- tem, and the cutaneous melanocytes [65]. Using
tric SEAMs mimicked the development of a com- cell pellets compacted by centrifugation, NCCs
plete primitive eye because the cell locations in derived from IPSCs and cultured in a 3D matrix
different zones corresponded to lineages of the were shown to form satellite cells with pheno-
ocular surface ectoderm: the lens, the neurosen- types similar to keratocytes of the corneal stroma,
sory retina, and the RPE. Cellular characterization which expresses keratocan. However, a more
studies showed that the innermost SEAMs were detailed analysis by real-time qPCR suggested
positive for class III β-tubulin (TUBB3), a marker that they were different to in vivo stromal kerato-
for neuronal lineage, and for PAX6. Surprisingly, cytes [66]. Interestingly, when NCCs were intro-
the outermost SEAMs were positive for the epi- duced in the appropriate niche in  vitro
thelial/superficial ectodermal markers p63 and environment (human corneal tissue), they actively
E-cadherin. Cells isolated from the ectodermal migrated into the stromal collagen fibrils,
zone corresponding to the ocular surface could be acquired a fibroblastic-like phenotype, and pre-
expanded ex vivo to create corneal epithelium that sented an identical transcriptional behavior to
specifically expressed CK12 [63]. These epithe- in vivo corneal keratocytes [66]. Fukuta and col-
lial sheets, once implanted in an experimental leagues [67] also described a simple and robust
LSCD model, were able to recover the function of derivation method based on using defined cell
a damaged ocular surface. culture medium supplemented with insulin. They
An efficient approach for generating complex induced small molecules – blocking the Wnt/β-­-
three-dimensional corneal organoids from human catenin (GSK3β) and TGF-β/Avidin/Nodal
IPSCs has been described recently [64]. In this (SB431542) signaling pathways  – allowing
research, the F2-3F1 human IPSC line formed NCCs to be generated as an intermediate of
typical embryoid body colonies both on mouse IPSCs, retaining the ability to produce differenti-
embryonic fibroblast feeder layers and on ated cells in terminal stages. Induced NCCs were
matrigel-­ coated substrates. As the three-­successfully differentiated into corneal endothe-
dimensional structures matured progressively lial cells, among other cell types, using a condi-
in vitro, oval-shaped eye field primordial clusters tioned cell culture medium from human corneal
developed in self-organized miniature structures endothelial cell cultures and a selective inhibitor
that consisted of retinal cells, a corneal primor- of Rho-kinase (ROCK, Y-27632).
dium, and an outer covering resembling a primi-
tive eyelid. The mature organoids were developed
in complex 3D tissues, like mini-corneas, and 18.5 New Perspectives
simulated the events of early development, and Challenges for Clinical
in  vitro. These 3D structures, in more terminal Application
stages, expressed specific markers, such as PAX6,
p63, and CK12 [64]. These approaches could In recent years, the great potential of advanced
represent an alternative source of tissue for sev- cell-based therapies for the repair and regenera-
eral ocular cell types. tion of different ocular structures in a variety of
272
ophthalmic diseases has started to be realized,
mainly for corneal and degenerative retinal
pathology [68, 69]. The advances focus on new
technologies for embryonic, adult, and induced
pluripotent cells that are differentiated to various
cell phenotypes. Improved understanding of the
molecular mechanisms of pluripotency and its
clinical implications are producing ever richer
data on the way in which the signals of the limbal
niche promote differentiation of stem cells.
Particularly in the cornea, stem cell-based ther-
apy offers the most physiological, most rational,
and least invasive approach for regeneration. For
LSCD, this is already considered an advanced
therapeutic option.
There remain some important problems with
IPSCs that need to be resolved before they can be
used freely in cell therapy. Notably, strict guide-
lines on their safety and tolerability are required to
minimize the potential for harmful effects in recip-
ients. The main concerns for the clinical use of
IPSCs surround their nature and the methodology
used to obtain pluripotent cell lines. These must be
thoroughly evaluated before it can be judged
whether their clinical use will be safe. Assessment
should ensure the following: (a) they should not
produce tumors or cancers, (b) they should not
proliferate uncontrollably, (c) they should not gen-
erate immunogenic responses in recipient tissue
when using vectors (viral or not), (d) they should
not produce an aggressive response in the recipient
tissue, and (e) they must be located exclusively at
the specific application site of the target regener-
ated tissue [70]. Consistent with these, the most
recent methodologies for inducting pluripotency
by non-­integrative reprogramming – transient and
controlled transfection vectors, using DNA or
interference RNA, episomes – probably represent
the best options for creating a safe final differenti-
ated product. The methods of direct reprogram-
ming, which avoid pure pluripotent states, may
also offer significant advantages for the control of
cellular behavior in host tissue.
An interesting advantage that favors wide-
spread clinical application of IPSCs is that autol-
ogous ocular tissue may not always be available
where there is severe bilateral ocular disease.
Whereas cell populations obtained and applied
R. P. Casaroli-Marano
from allogeneic tissues have the potential to
cause immune reactions in recipient tissue, recent
advances in adult tissue autologous stem cells
(IPSCs and induced MSCs) herald the possibility
of compatible therapy based on deriving different
types of ocular tissues [68, 71–73]. The required
progenitor cells can be obtained easily from the
skin, adipose tissue, or peripheral blood by mini-
mally invasive methods. Although IPSCs have
not yet been used clinically to reconstruct the
ocular surface, several recent studies have dem-
onstrated the successful generation of LSC and
corneal epithelial cell populations from IPSCs.
However, further refinement of the corneal cell
derivation from IPSCs will be required before
this potentially unlimited source of corneal epi-
thelial stem cells meets the requirements for clin-
ical application.
Generally, the methods used to induce cellular
pluripotency and the ex vivo expansion of differ-
entiated cell lines requires the use of xenobiotics
or allogeneic products. These may include cells
of nonhuman origin for the co-culture approaches
in feeder-layer techniques, the supplementation
of cell culture media with serum of animal origin,
or the use of other molecules for the induction or
inhibition of in  vitro molecular phenomena.
Unfortunately, these may not meet the minimum
quality specifications required for clinical appli-
cation. These products potentially risk disease
transmission, tumorigenesis, or precipitating an
immune response in the recipient [4, 18, 33, 74,
75]. They also show an idiosyncratic biological
variability that can negatively affect the quality
of cell cultures and the final results of transplan-
tation. Therefore, there is currently a need to
investigate the options for using chemically
defined xenofree culture media and recombinant
molecules of human origin that meet clinical
grade specifications and protocols [57]. Thus, the
production of clinical grade cell lines, in the
absence of xenobiotics, and guided by Good
Manufacturing Procedures (GMP) regulations
and standard operating procedures, is important
and mandatory before we can produce IPSCs and
differentiated cells for clinical purposes. Another
important concept is the balance between cost
and safety [32]. To this end, a significant step for-
18  Cell-based Therapy Using Induced Pluripotent Stem Cell
ward in establishing the definitive clinical appli-
cability of this cell type may be to develop IPSC
biobanks in which cells originate from homozy-
gous haplotypes that show high frequency in the
population [76].
As confirmed, the in vivo or in vitro microen-
vironment (niche) is a crucial element for main-
taining the homeostasis and stemness of
progenitor cells. Improving our knowledge of the
properties of this niche for LSCs is a key issue as
we move forward, since this could contribute to
understand the mechanisms related to corneal
repair and the events associated with cell differ-
entiation. Indeed, the balance between cells and
their environment is due, in large part, to the
presence of sustained production and secretion of
paracrine factors, which are specific to each
microenvironment. Intercellular communication
is partially mediated by signals that come from
the ECM and secreted microvesicles (SMV) – the
exosomes are SMVs of endosomal origin that are
released as multivesicular bodies [77]  – which
are enriched by cytoskeletal molecules, signal
transduction proteins, and cytoplasmic enzymes
[77, 78]. Surprisingly, they can also transfer
functional RNA and miRNA to recipient cells
[78]. Exosomes and miRNA also have pleiotro-
pic biological functions that are still poorly
understood, but that may include roles in immune
activation, angiogenesis modulation, and cell
protection and repair [55, 79]. In addition, the
regenerative and cell-inducing plasticity propri-
eties have been proven based on exosomes
obtained from MSCs [80] and ESCs [72]. Thus, a
key future area of study in the field of corneal
regeneration will surround the corneal microen-
vironment, and particularly the role of exosomes
secreted by corneal cells to maintain the homeo-
stasis and regeneration of the ocular surface.
18.6 Conclusions
Advanced cell-based therapy, whether associ-
ated with tissue bioengineering approaches or
not, has experienced some surprising advances
over the last decade. An alternative emerging
source of cells for ocular surface treatment has
273
arisen in progenitor cells from adult tissue.
These provide high proliferative potential and
lower immunogenicity, although with certain
limitations for complete terminal differentiation.
Ex vivo cell expansion techniques based on the
patient’s own tissue represent a rational, physio-
logical, and minimally invasive approach for the
treatment of some prevalent ocular surface dis-
eases, including LSCD.  Indeed, autologous tis-
sue provides key advantages over allogenic
approaches, most notably in relation to immune
tolerance in the recipient tissue. These tissue
engineering techniques continue to evolve, with
methodological improvements continuing to
increase their efficacy and clinical applicability.
There have also been significant increases in our
understanding of the mechanisms of regenera-
tion and repair of the ocular surface. In turn, bet-
ter understanding of LSC behavior and the
limbal niche has shed light on improvements in
translational observations for clinical results. A
central concept is that the paracrine factors pro-
duced and secreted in the sclerocorneal limbic
region constitute specialized elements that are
required for cell survival and LSC self-renewal,
making them essential to the regenerative pro-
cess of corneal epithelial cells.
The advent of IPSCs holds great promise for
future clinical application. The eye, and the ocu-
lar surface (cornea) in particular, appears to have
the optimal characteristics for the application of
advanced therapy based on IPSC techniques.
Being a well-compartmentalized structure of
small dimensions, the eye requires only a small
number of cells for treatment and enjoys an
“immune privilege” state. Another advantage is
that processes are easy to monitor by image
­capture and recording after implantation on ocu-
lar surfaces. Likewise, the ocular surface of the
contralateral eye can serve as a control for the
evolution of outcomes in cases of bilateral dis-
ease. These are incontestable facts because, at
the time of writing, the only ongoing human
clinical trials using IPSCs were for the treatment
of age-related macular degeneration. Specifically,
this was through the implantation of RPE cells
derived from IPSCs that originated from the
patient’s own dermal fibroblasts [46].
274
Despite the promise of these new approaches,
there remain some unresolved points regarding
the safety and tolerability of differentiated cell
lines from IPSCs. Among these, concerns of the
cell cycle and the potential for tumorigenesis are
the most worrisome. Approaches for inducing
pluripotency by non-integrative or even direct
reprogramming have been integrated into the
methodological arsenal to allow for a more con-
trolled clinical application. There is also a need
for cell culture techniques that produce cell
expansion and maintenance with defined culture
media and supplements that are of clinical grade
requirements and free of xenoproducts. This
would not only improve the quality of the final
therapeutic product but would also increase its
safety in the recipient. These problems are cur-
rently the focus of ongoing research. Once
resolved, we could see a new era based on the
controlled clinical use of IPSCs for certain ocu-
lar diseases, particularly those of the ocular
surface.
Acknowledgments The author acknowledges support 12. Pellegrini G, Golisano O, Paterna P, Lambiase
from Fundació Marató TV3 (120630-31) and Fondo de
Investigaciones Sanitarias del Instituto de Salud Carlos
III (FIS14-PI00196 and FIS18-PI00355), from the
European Regional Development Fund (FEDER) of
European Union.
Compliance with ethical requirements: Ricardo Pedro
Casaroli-Marano declares that he has no conflict of inter-
est. All procedures followed were in accordance with the 14. Dua HS, Miri A, Alomar T, Yeung AM, Said DG. The
ethical standards of the responsible committee on human
experimentation (institutional and national) and with the
Helsinki Declaration of 1975, as revised in 2000. Informed
consent was obtained from all patients for being included 15. Yazdanpanah G, Jabbehdari S, Djalilian AR. Limbal
in the study. No animal studies were carried out by the
authors for this article.
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 Cultivated Limbal Stem Cell
Transplantation: Indications
19
and Technique

Joséphine Behaegel, Sorcha Ní Dhubhghaill,
and Marie-José Tassignon

Table 19.1  Aetiologies of limbal stem cell deficiency

19.1 Introduction Genetic Acquired
Aniridia [3] Chemical and thermal burns [4]
Limbal stem cell deficiency (LSCD) is a condi- Turner syndrome [5] Stevens-Johnson syndrome [6]
tion that results from a loss of limbal stem cells Dyskeratosis Ocular cicatricial pemphigoid
or a dysfunctional limbal microenvironment. congenita [7] [8]
The term refers to a highly heterogeneous group Xeroderma Graft versus host disease [10]
pigmentosum [9]
of pathologies ranging from acquired diseases
LADD syndrome [11] Infectious keratitis [12]
such as chemical burns to severe autoimmune KID syndrome [13] Iatrogenic causes:
conditions, iatrogenic trauma and genetic abnor- chemotherapy, radiation,
malities such as aniridia [1, 2] (Table 19.1). In surgery, cryotherapy [14–16]
all of these conditions, the reserve of corneal Dominantly inherited Ocular surface tumours [18]
keratitis [17]
limbal stem cells buried in the limbal niches is
Congenital epidermal Chronic contact lens wear [20]
dysplasia [19]
Bullous keratopathy [21]

J. Behaegel
Faculty of Medicine and Health Sciences,
Department of Ophthalmology, Visual Optics and
Visual Rehabilitation, University of Antwerp,
Campus Drie Eiken, Antwerp, Belgium
Department of Ophthalmology, Antwerp University
Hospital, Edegem, Belgium
Center for Cell Therapy and Regenerative Medicine,
Antwerp University Hospital, CCRG-Oogheelkunde,
Edegem, Belgium
Department of Ophthalmology, Brussels University
Hospital, Jette, Belgium
e-mail: josephine.behaegel@uzbrussel.be
S. N. Dhubhghaill
Faculty of Medicine and Health Sciences,
Department of Ophthalmology, Visual Optics and
Visual Rehabilitation, University of Antwerp,
Campus Drie Eiken, Antwerp, Belgium
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_19
Department of Ophthalmology, Antwerp University
Hospital, Edegem, Belgium
Center for Cell Therapy and Regenerative Medicine,
Antwerp University Hospital, CCRG-Oogheelkunde,
Edegem, Belgium
e-mail: Sorcha.NiDhubhghaill@uza.be
M.-J. Tassignon (*)
Faculty of Medicine and Health Sciences,
Department of Ophthalmology, Visual Optics and
Visual Rehabilitation, University of Antwerp,
Campus Drie Eiken, Antwerp, Belgium
Department of Ophthalmology, Antwerp University
Hospital, Edegem, Belgium
e-mail: Marie-jose.tassignon@uza.be
277
278
depleted. In the absence of these cells, lost epi-
thelial cells cannot be replenished resulting in
corneal epithelial defects. This breakdown of
the epithelial barrier results in a status of chronic
inflammation with eventual migration of con-
junctival cells to cover the defects and subse-
quent superficial vascularization of the cornea
[4, 22].
As a result, patients suffer from decreased
visual acuity, pain and photophobia. Traditional
corneal grafting techniques tend to fail in these
patients for two reasons:
1. The epithelial growth over a new transplant
is typically slow, and the new graft stroma
may be exposed by a persistent epithelial
defect. Even when the epithelium does close,
this is frequently due to conjunctival epithe-
lial cell overgrowth with compromised
transparency.
2. The increased corneal vascularization pres-
ent in these eyes will significantly increase
the risk of graft rejection over the long term
[23].
For both of these reasons, standard corneal
transplantation is not advised in these high-risk
patients.
The primary goal, therefore, in LSCD treat-
ment is to restore the limbal microenvironment
and to regain a corneal epithelial phenotype that
can self-sustain. This can be achieved by the
transplantation of healthy limbal epithelial cells.
The main difference between surgical approaches
is the quantity, source and postharvest manipula-
tion of the cells. Direct transplantation of “large-­
tissue” grafts includes keratolimbal allograft,
conjunctival-limbal autografts and living-related
conjunctival-limbal allografts, all of which
require large sections of limbal donor tissue [24–
26]. While autologous grafts perform better than
allogenic, there is a safety limit to how much tis-
sue the healthy fellow eye can donate. Large-­
tissue autologous grafts therefore can typically
only be performed once. Cultivated graft tech-
niques were developed to reduce this risk to
donor eyes by reducing the amount of donor tis-
sue required [27].
J. Behaegel et al.
Cultivated in  vivo expansion has several
advantages when compared with large-tissue
transplantations of donor tissue:
1. There is a reduction of the risks to donor eye
since only a small amount of donor tissue is
needed.
2. If necessary, a regraft can be performed [28].
3. There is less risk of donor rejection in case of
allogeneic transplantation since the antigen-­
presenting macrophages do not survive the
cultivation process [29].
4. The dose and the duration of systemic immu-
nosuppression are significantly reduced in
case of allogeneic transplantation since the
donor cells show limited long-term survival
[30–32].
As a result, many ocular centres have pivoted
to a cultivated approach.
19.2 Preoperative Assessment
19.2.1 Indications for Surgery
and Preoperative Evaluation
Cultivated limbal stem cell transplantation is
indicated for patients suffering from either total
or subtotal LSCD.  Prior to transplantation, the
eye should be carefully evaluated and prepared to
optimize the environment and to ensure the best
chance of a successful grafting as not all LSCD
patients are likely to benefit.
19.2.1.1 Patient
Management of LSCD begins with a careful eval-
uation of the extent of deficiency and associated
abnormalities [33]. Because LSCD is a term that
applies to the end result of a variety of patholo-
gies, it rarely presents as a single, isolated epithe-
lial problem. Patients typically have a complex
history of disease progression, treatments and
surgeries. Depending on aetiology and severity,
there can be involvement of the surrounding ocu-
lar tissues including the corneal nerves, corneal
stroma, endothelium, conjunctiva, eyelids and
lacrimal system.
19  Cultivated Limbal Stem Cell Transplantation: Indications and Technique
Tears
Epithelium
Stem cells
Ocular surface
Fig. 19.1 Successful limbal stem cell transplantation is
similar to growing a plant from seed (Analogy adapted
from Tseng and Tsubota [34])
Limbal stem cells may be considered as seeds,
while the ocular surface is the soil in which they
are embedded [34] (Fig.  19.1). The seeds will
only grow in an optimal environment with the
right nutrients. Therefore, prior to stem cell trans-
plantation, it is important to prepare the best
recipient bed for the cultivated cells in order to
restore the ocular surface defences.
The preoperative examination therefore begins
with the eyelids.
Eyelids
The eyelids are crucial for the preservation of
ocular surface integrity, as well as for maintain-
ing a healthy preocular tear film [35]. Many of
the pathologies that result in LSCD such as
chemical injury or mucous membrane pemphi-
goid will also cause severe conjunctival and eye-
lid disease. Even the highest-quality cultivated
cells will not be able to resist repeated mechani-
cal abrasions experienced with trichiasis, entro-
pion or exposure and will not engraft. Contact
lenses used to protect the new grafts can also be
difficult to fit around symblepharon, so prior to
CLET all lid abnormalities should be corrected to
the fullest extent possible.
279
Tear Film
Similarly, a healthy tear film is essential for sur-
vival of the stem cells. A tear film supports the
ocular surface by providing oxygen and nutrients
and protects against desiccating environments
[36]. Tear film can be optimized by the use of
nonpreservative teardrops or punctal occlusion
and by treating eyelid inflammation such as
blepharitis. While a mild dry eye is not an exclu-
sion criteria, a complete absence of any tear pro-
duction and/or the presence of corneal epithelial
keratinization identifies an extremely hostile
environment, and as a result these eyes perform
very poorly and are typically excluded from lim-
bal stem cell surgery.
Additionally, any inflammation of the ocular
surface should be evaluated and treated if neces-
sary. Inflammation disturbs the normal milieu of
the remnants of the limbal niche and may lead to
dysfunction or aberrant differentiation of the
stem cells [37].
Corneal Epithelium
At this point we examine the cornea itself. While
classification schemes for LSCD clinical trials
are currently debated, it is useful to note the
degree of clock hours that have been conjuncti-
valized or vascularized. The presence and mea-
surements of any epithelial defects should also be
noted. This can easily be documented by means
of ocular surface photography. It is important to
determine whether the visual axis is involved or
threatened, as no surgical treatment may be
required in case the central axis is free from
abnormal tissue. In case of partial LSCD, a
repeated debridement of the conjunctival epithe-
lium can be performed. This simple surgery peels
back the migrating conjunctival cells to buy time
for the healthy corneal epithelial cells to cover
the defect in a technique known as sequential
sector conjunctival epitheliectomy (SSCE) [38,
39]. This has the advantage of not requiring
donor tissues but is only an option for those cases
of milder disease where there are some remain-
ing stem cells to sustain an epithelial layer. In
total LSCD with central corneal involvement, a
limbal stem cell graft remains the treatment of
choice.
280
Corneal Stroma
In the simplest cases of LSCD where the pathol-
ogy is limited to the epithelium, the thick fibro-
vascular pannus can be peeled away to reveal a
clear stromal bed. These are the ideal patients,
where successful CLET surgery can result not
only in an improvement in the epithelial layer but
also an increase in visual acuity. In reality, how-
ever, this represents the minority of LSCD
patients. Injuries that cause LSCD are often
accompanied by deeper damage in the stromal
layer resulting in scarring. It is important for
these patients to understand that if there is signifi-
cant corneal stromal scarring, CLET surgery will
not be a sight-restoring procedure. The aim of
CLET in these cases is to improve the epithelium
as a first step and to facilitate a sight-restoring
corneal transplant as a second step.
Persistent epithelial defects and stromal lysis
can also result in regions of corneal thinning
which can be of particular concern during CLET
surgery. If the donor stromal bed is too thin,
removing the fibrovascular pannus and placing a
CLET can result in a cornea with severely dam-
aged biomechanics leading to further thinning
and perforation. A residual stromal bed of at least
350 μm is advised to avoid this serious adverse
outcome. When significant stromal thinning is
noted preoperatively, the surgical plan may have
to be adapted to include stromal replacement by
PK or lamellar surgery (deep anterior lamellar
keratoplasty, DALK) combined with the CLET
surgery, though this is not without its own risks.
Corneal Endothelium
A functional corneal endothelium is also known
to be essential for epithelial cell adhesion.
Without endothelial active pump function, even
healthy corneal epithelium can form bullae and
slough off [40]. Transplanted epithelium does not
fare any better. Clinical evaluation of the endo-
thelium can be difficult in these patients, as the
corneal pannus and stromal scarring can often
prevent visualization of the anterior segment and
a covering of conjunctival epithelium will not
form the same characteristic bullae as corneal
epithelium to aid diagnosis. Severe endothelial
dysfunction can be suspected by a highly thick-
J. Behaegel et al.
ened corneal stroma on preoperative imaging. If
this is the case, it may also be an argument for
performing a PK at the time of CLET.
Corneal Nerves
Corneal sensitivity should be tested and docu-
mented (in clinical studies with a Cochet-Bonnet
esthesiometer). In health, corneal epithelial cells
are highly innervated by the terminal nerve fibres
of the sub-basal plexus [41]. This innervation is
not only essential for the provision of nerve
growth factor (NGF), which plays a crucial role
in corneal healing processes, but is also a key
input to the lacrimal functional unit (LFU) gov-
erning tear film production [42, 43]. Loss of cor-
neal sensitivity can therefore cause a “double-hit”
to the newly seeded stem cells by depriving them
of NGF and damaging the tear film.
Glaucoma
As mentioned previously, LSCD typically occurs
in the larger context of severe anterior segment
disease. Raised intraocular pressure (IOP) and
glaucoma as a result are frequent comorbidities,
particularly in cases of aniridia. The postopera-
tive management of the transplant requires a pro-
longed period of topical corticosteroid use, and
even in normotensive patients, the possibility of a
cortisone IOP response should be considered.
Any increase in IOP should be treated prior to
considering CLET.  Topical glaucoma medica-
tions can cause superficial punctate keratopathy
in normal corneas [44] and are even more toxic to
cultured epithelial transplants. We recommend
preservative-free drops, laser or surgery for glau-
coma control prior to CLET.
Indications and contraindications may vary
among various centres. Bilateral LSCD is often
described as a contraindication since this requires
allogeneic stem cells. In our centre we include
both uni- and bilateral diseases and include all
aetiologies of LSCD.  Our contraindications are
listed in Table 19.2.
19.2.1.2 Donor
Both autologous and allogeneic donor eyes may be
included for limbal tissue donation. Autologous tis-
sue is always preferred as it confers no
19  Cultivated Limbal Stem Cell Transplantation: Indications and Technique
Table 19.2  Contraindications for CLET
Contraindications:
relative Contraindications: absolute
Clear visual axis Corneal keratinization
Inflammation of ocular Phthisis bulbi
surface (ocular surface/
eyelids)
Conjunctival pathology Absence of tear film
(symblepharon,
scarring)
Malposition of the Inability to follow post-op
eyelids treatment schedule/any
conditions hampering
treatment compliance
Glaucoma Uncontrollable raised IOP
Hypersensitivity to post-op
treatment
i­mmunoreactivity and does not require systemic
immunosuppression postoperatively. In case of
bilateral disease or other conditions prohibiting
autologous tissue donation, living-related or cadav-
eric donors can be considered. While the benefit of
human leucocyte antigen (HLA) testing for CLET
and limbal tissue donation has not been validated,
we nevertheless perform it in our centre as we
believe that it could confer an advantage. We
require a match of at least 50% on loci HLA-A,
HLA-B and HLA-DR for tissue donation in order
to reduce graft rejection. Only a small piece of
healthy limbal tissue is required for donation;
1 × 2 mm is sufficient for most culture protocols.
We exclude donation from eyes suffering from par-
tial limbal stem cell deficiency, to avoid additional
risks to an already compromised eye. Opinions
vary, as other groups do harvest stem cells from
compromised eyes with the goal of providing
autologous grafts even in bilateral disease [45].
When considering a limbal region for biopsy,
we estimate the overall health of the region by
visualizing the limbal palisades of Vogt (POV)
using slit lamp biomicroscopy and non-invasive
imaging modalities. In addition, all tissue donors,
whether autologous or allogeneic, are screened
for human immunodeficiency virus 1 and 2
(HIV-1 and HIV-2), hepatitis B (HBV) and C
(HCV) and syphilis prior to surgery. While autol-
ogous tissue will not transmit disease to the
recipient, this is performed to determine status of
the tissue that is to be handled and must be known
281
for traceability purposes. Unlike autologous tis-
sue, allogenic cells do confer additional infec-
tious risks to the recipient patient. As a result all
allogenic tissue donors require additional screen-
ing for human T-cell leukaemia-lymphoma virus
1 and testing with nucleic acid tests (NAT) for
HBV, HCV and HIV. Allogeneic donors undergo
screening questionnaires for Creutzfeldt-Jakob
disease (CJD) and rabies among others.
A maximum of two biopsies are permitted per
donor eye. This is due to the surgical damage to
the harvest site. Removing more than two biop-
sies may compromise the donor limbus and con-
fer a risk of developing localized iatrogenic
LSCD. Therefore, a previous history of two biop-
sies is an exclusion criterion for donation in our
protocol.
One final but important consideration in the
case of living-related allogeneic tissue donation
is the ethics of donation. This is often considered
in organ transplantation but frequently forgotten
in stem cell surgery. It can be difficult to verify
whether the donation is truly a voluntary act.
Family pressures, emotions and external influ-
ences can play a role in manipulating the donor’s
right to choose. They may be afraid to say that
they actually do not want to donate. We advise
discussing the donation with the possible donor
in a quiet space, without the recipient or other
family members. Should the possible donor not
wish to donate, we respect their wishes and com-
municate to the patient that the possible donor
was “not appropriate” for donation. Informing
the family that the possible donor simply did not
want to donate could have serious ramifications
in the family. The confidentiality of the possible
donor must be respected, and it is therefore not
ethical to let the details of their decision be
known.
Our indications and contraindications for tis-
sue donation are listed in Table 19.3.
19.2.2 Supplementary Examinations
In addition to slit lamp biomicroscopic evalua-
tion, we perform a number of additional preop-
erative tests during patient and donor screening.
282 J. Behaegel et al.

Table 19.3 (Contra-)indications for limbal tissue 19.2.2.3 Optical Coherence

donation
Tomography (OCT)
Indications Contraindications OCT is a non-invasive, non-contact imaging
Healthy cornea and Signs of limbal stem cell technique that we use for cross-sectional images
limbus deficiency
Age ≥ 18y Age < 18y
of the anterior segment including the cornea and
≥50% HLA matching <50% HLA matching on limbus. This imaging modality is an added value
on locus HLA-A, locus HLA-A, HLA-B and for preoperative assessment of the patient and the
HLA-B and HLA-DRB1a donor eye, intraoperatively during pannus dissec-
HLA-DRB1a
tion and postoperatively during the follow-up
Voluntary donationb History of two limbal biopsy
donations/large limbal graft period. OCT has been shown to be a more reli-
Positive serology testing a able tool for visualization of the palisades of
Questionnaire/history Vogt, when compared with slit lamp biomicros-
suggestive for potential copy [48], but when compared with in vivo con-
transmittable diseasesb focal microscopy (IVCM), the resolution of OCT
External pressure for tissue
donation b images is significantly lower. OCT examination
is, however, less time-consuming, non-contact
a
In case of an allogeneic (living-related and cadaveric)
donor and less operator dependent and has a wider field
b
In case of an allogeneic living-related donor of view making it easier to implement in practice.
Using Fourier domain-OCT (FD-OCT), we have
developed a technique for quantification of lim-
19.2.2.1 Schirmer Test bal POV, which can aid in targeting the biopsy
Schirmer filter paper testing is used to estimate site for tissue donation [48] (Fig. 19.2).
the quantity of the tear film [46]. This quantita- During preoperative evaluation, OCT images
tive method provides little information about the are also used to examine the stromal involvement
quality of the tear film, which may be more and thickness and to estimate the residual corneal
important than quantity, but it is a useful preop- stroma after pannus removal. If the thinnest
erative measure. If the wetting strips measure less region measures less than 350  μm, CLET may
than <10  mm/5  min, punctal plugs should be need to be combined with either a PK or DALK
inserted prior to stem cell surgery to enhance the in order to prevent postoperative corneal thinning
tear film stability. If the wetting strips show no and perforation. Knowing this preoperatively
moisture whatsoever, this may be a contraindica- also allows the opportunity to discuss the adapted
tion to surgery. surgery with the patient.
Intraoperatively, OCT can be used to assist the
19.2.2.2 Impression Cytology surgeon in the removal of the fibrovascular pan-
Immunohistochemistry carried out on corneal nus. This aids the surgeon in a more precise
impression cytology allows to distinguish removal of the diseased tissue, which augments
between corneal and conjunctival epithelial cells chances of sparing any viable corneal stroma and
and therefore aids in diagnosing LSCD.  While may reduce risks of extreme thinning and perfo-
the procedure is relative simple, the test is ration [49]. During the postoperative follow-up
regarded as painful by the patients and risks dis- period, OCT is useful for evaluation of the
rupting the epithelium. Moreover, the false-­ ­corneal epithelium, graft tracking and assessment
negative rate of impression cytology is high of the corneal thickness.
partly because of coexisting goblet cell defi-
ciency in many other surface disorders [47]. We 19.2.2.4 In Vivo Confocal Microscopy
no longer routinely perform impression cytology (IVCM)
during patient screening and postoperative evalu- IVCM is a contact imaging technique that pro-
ation, preferring to use in vitro confocal micros- vides high-resolution images of the ocular sur-
copy, as described below. face at the cellular level. This technique has
19  Cultivated Limbal Stem Cell Transplantation: Indications and Technique
a b
Fig. 19.2  Evaluation of a donor limbus: (a) Slit lamp
image of an inferior limbal rim. The black arrow indicates
the palisades of Vogt. (b) OCT image of the same subject
Fig. 19.3  IVCM findings of a stem cell-rich region of the
inferior limbus showing limbal palisade ridges (arrows)
and focal stromal projections (arrowheads)
gained wide popularity among corneal specialists
to evaluate various structures of the cornea, con-
junctiva and limbus [50–53]. It can be used in
preoperative patient and donor screening and in
the assessment of limbal stem cell deficiency.
Because of its high resolution, IVCM enables
differentiation between the stem cell niches.
Imaging of these niches helps to ensure the health
of the donor limbus and indicates the regions
containing highest density of stem cells. In prac-
tice, the donor is imaged at inferior and superior
limbus, as stem cells are predominantly located
in these regions. Documentation of the most stem
cell-rich regions enables a targeted biopsy har-
vesting, which could lead to improved clinical
outcomes (Fig. 19.3).
283
through the inferior limbal rim, identifying the limbal
niches (grey arrows). (c) OCT selection of the region of
interest
ICVM signs of LSCD include the presence
of goblet cells on the cornea, changes in the epi-
thelial phenotype, loss or abnormal palisades of
Vogt, neovascularization of the cornea and loss
of sub-basal nerves (Fig.  19.4). These signs
assist ophthalmologists in making the clinical
diagnosis of LSCD, and as such, ICVM can pro-
vide an alternative diagnostic technique to
impression cytology [54–56]. Postoperatively,
IVCM assists in evaluation and can show graft
integration and possible disease progression
prior to it being clinically visible at the slit
lamp.
All supplementary tests during patient and
donor assessment are listed in Table 19.4.
19.3 Procedure
Different protocols for cultivation and transplan-
tation of limbal stem cells have been described
worldwide. Variability is seen in patient selection,
culture techniques, donor materials, surgical
approaches and the postoperative treatment and
follow-up. Good clinical results have been
reported using different approaches, and to date
there is no consensus on the optimal protocol. The
complexity and expense of cell-based approaches
as well as the orphan nature of the disease make
large multicentre comparative trials effectively
impossible. In this chapter we will describe our
284 J. Behaegel et al.

a b

c d

Fig. 19.4  IVCM findings of LSCD: goblet cells on the cornea (a), corneal neovascularization (b), abnormal palisades
of Vogt (c) and metaplastic epithelial phenotype (d)

standardized approach, but the reader should con-
sider the alternative approaches [29, 57].
19.3.1 Culture Method
19.3.1.1 Scaffold
We use human amniotic membrane (HAM) as a
scaffold for cellular expansion of our limbal
stem cells. HAM is known to have anti-inflam-
matory and antimicrobial properties, and its low
immunogenicity makes it an ideal carrier for
ocular surface reconstruction. The membranes
are obtained from the Amniotic Membrane
Bank University Hospital Antwerp and are pre-
pared in a standardized way [58]. Briefly, 24 h
before use, the HAM is thawed and washed
three times with 0.9% sterile NaCl for 30 min.
They are then treated with thermolysin enzyme
(Sigma-Aldrich) in order to remove the devital-
ized amniotic epithelium [58, 59].
Before placing the cells in culture, the HAM is
stretched into two interlockable amnion rings
(Lumox cultureware, Greiner Bio-One, BeLux).
19  Cultivated Limbal Stem Cell Transplantation: Indications and Technique 285

Table 19.4  Supplementary tests tres of medium is placed on the fixed HAM and
Patient Donor 2,5  ml in the culture dish. These conditions are
Biomicroscopic Biomicroscopic evaluation maintained for the duration of the entire culture
evaluation period of 14  days, with the medium changed
Tear film (Schirmer Medical questionnaire tissue
test) donation
every 2–3 days. The medium is routinely checked
IOP IOP for aerobic bacteria, anaerobic bacteria, fungi,
OCT OCT (limbus) yeast, mycoplasma and endotoxin [58].
IVCM IVCM (limbus)
Flash visual evoked
potentiala
19.3.2 Surgical Procedure
Serology Serology
Nephrology screeningb
19.3.2.1 Biopsy
a
In case visual acuity is less than hand movements or sus-
pected optic neuropathy The biopsy harvesting is performed under topical
b
In case immunosuppression is required anaesthesia. Superficial limbal tissue of 1 mm by
2 mm is taken from either the superior or the infe-
rior limbus in a stem cell-rich region, as indicated
These rings are made of plastic and provide a by preoperative imaging. The edges of the biopsy
taut, wrinkle-free construct. are marked off with a 45° diamond blade, and a
The spongy layer of the HAM is removed with crescent knife is used to tunnel a depth of approx-
the help of two 25  mm cell scrapers (Sarsted, imately 100 microns. We do not use any forceps
Essen, Belgium). This provides a thinner, more during this procedure in order to avoid crush
transparent scaffold, with a more standardized injury to the limbal stem cells or niches within
proteomic profile [59, 60]. After removal of the the biopsy (Fig. 19.5). Immediately after dissec-
spongy layer, the inner ring (diameter, 32 mm) is tion, the harvested tissue is placed in culture
placed on top of the HAM, and the edges of the medium and transported to the laboratory where
membrane are pulled over the outer surface of the it is rinsed with culture medium supplemented
ring. The HAM attached to the inner ring is with antibiotics [58].
flipped over so that this is correctly oriented.
Then, the outer ring (diameter, 35 mm) is locked 19.3.2.2 Graft Transplantation
over it, sandwiching the membrane in place. The After the 14-day culture period, the limbal stem
final position of the HAM is with its basement cell graft is transplanted under general anaes-
membrane facing upwards [58]. thesia onto the stem cell-deficient eye. Surgery
begins with a 360 ̊ conjunctival periotomy at
19.3.1.2 Technique the limbus using conjunctival scissors
The 1  ×  2 mm biopsies are cultured using a (Fig.  19.6a). The fibrovascular pannus is then
xenogeneic-­ free culture protocol. The fixed
HAM are placed in a 55 mm culture dish, con-
taining 3.5 mL CnT-20 medium (progenitor cell-­
targeted, corneal epithelial medium; CELLnTEC,
Bern, Switzerland), supplemented with 1%
human AB serum (Sigma-Aldrich) and incubated
at 37 °C with 5% CO2 for 24 h. Before the limbal
biopsy is placed in culture, it is rinsed six times in
CnT-Prime medium containing antibiotics and
placed at the centre of the prepared HAM.
The cells are then cultured in the CnT-Prime
medium supplemented with 1% human AB serum
at the air liquid interface. One hundred microli- Fig. 19.5  Biopsy harvest of a donor limbus
286
dissected (Fig. 19.6b), under guidance of ante-
rior OCT imaging. After pannus dissection, the
composite graft is attached to the cornea by
applying fibrin glue (Tissucol®) to the surface
of the cornea (Fig. 19.6c). The graft is cut out
of the rings just beyond the limbs using a tre-
phine and m ­ icrosurgical scissors (Fig.  19.6d).
A secondary HAM is then placed over the graft
with its epithelial side facing downwards. This
membrane serves as a biological patch offering
additional mechanical protection to the graft
preventing primary dehiscence. The patch is
secured with four 10–0 nylon sutures to the
conjunctiva and is removed 5–7  days after
transplantation. To end, a bandage contact lens
is applied to the eye [58].
a b
c d
Fig. 19.6  Different0 steps during CLET: conjunctival
periotomy (a), dissection of the pannus tissue (b), applica-
tion of the graft using fibrin glue (c), releasing the graft
J. Behaegel et al.
19.4 Postoperative Management
19.4.1 Early Postoperative Treatment
Preservative-free drops are the gold standard in
the management of limbal stem cell patients. The
cellular toxicity associated with preservatives
includes a risk for both the stem cells and the sur-
face microenvironment [61]. Therefore eye drops
containing preservatives should be avoided wher-
ever possible. We prescribe all patients with 0.3%
ofloxacin drops 4×/day, 0.1% dexamethasone
drops 8×/day and 20% autologous serum drops
16×/day for 1  month postoperatively and then
gradually taper the drops down. In cases the
patient has any transmittable disease prohibiting
using a trephine (not shown) and scissors (d), releasing
the graft using a 13 mm trephine in diameter and end the
trephination with Vanas scissors
19  Cultivated Limbal Stem Cell Transplantation: Indications and Technique
serum drops (e.g. HIV), these are replaced by
preservative-free sodium hyaluronate eye drops.
Allogeneic limbal transplant patients need
additional immunosuppressive treatment in order
to prevent graft rejection. We use cyclosporine A
3 mg/kg/day which is commenced 1 week prior
to transplantation and continued for 2  months
thereafter. Blood trough levels should be main-
tained at approximately 100–150  ng/ml (unless
renal or liver function impairment occurs) and
then tapered to 25–50 mg/kg/day and maintained
at this dosage for a period of at least 1  year. If
required, perioperative IV methylprednisolone
can be administered (1–2 mg/kg/day, depending
on the degree of vascularization of the cornea and
limbus) and continued daily for 3 days. Depending
on host inflammatory response, the corticosteroid
treatment can be prolonged orally if required, but
it is generally recommended that the preoperative
inflammation is well controlled prior to surgery.
19.4.2 Additional (Surgical)
Management
The primary goal in limbal epithelial stem cell
transplantation is to restore the ocular surface.
While visual acuity may already improve follow-
ing CLET if the underlying stroma is clear, further
surgery is often required to restore corneal clarity
and improve visual outcomes (Fig. 19.7). In gen-
a b
Fig. 19.7  Total limbal stem cell-deficient eye after an alkali burn before (a) and after a two-stage approach: 25 months
post-CLET; 9 months post-PK (b)
287
eral it is accepted that a two-stage approach is pre-
ferred above a combined procedure since the latter
is associated with poorer clinical outcomes [62].
In our opinion certain ocular conditions necessi-
tate a combined approach. As mentioned previ-
ously, in patients with a residual stroma of less
than 350 μm as assessed by OCT imaging, a CLET
will be combined with either a full-thickness cor-
neal transplant or a DALK in order to reduce the
risk on postoperative complications. In addition,
evidence of a non-functional endothelium may be
an indication to perform PK during stem cell sur-
gery. It is important to note that every patient is
different, and treatment should be adapted based
on their individual needs and expectations.
In cases of graft failure, a second stem cell
transplantation can be offered to the patient. We
limit treatment to a maximum of two transplanta-
tions per patient. For safety reasons, we do not
harvest more than two biopsies from the same
donor eye. In the case of allotransplants, there is
also the possibility of host sensitization to the
alloantigens, and as a result we also limit these
transplants to a maximum of two per patient.
19.5 Conclusion
LSCD is a complex disorder requiring an individu-
alized approach and a step-by-step r­econstruction.
Transplantation of ex  vivo cultivated limbal stem
288
cells can offer corneal rehabilitation in both unilat-
eral and bilateral (sub)total stem cell-deficient eyes.
A comprehensive preoperative evaluation and care-
ful preparation of the ocular surface are crucial to
provide an optimal environment for the transplanted
cells. A well-­prepared ocular surface together with
a rigorous postoperative management and follow-
up will ensure the best chance on successful graft-
ing. Despite the safety of the CLET technique, the
treatment of LSCD remains challenging because of
the heterogeneity of the population and associated
comorbidities. In case of graft failure, there is still
the possibility to perform a second transplantation.
Acknowledgement  We would like to acknowl-
edge the important input of Nadia Zakaria who
initiated the culturing method as used in the clini-
cal trial and realised the GMP accreditation of the
ophthalmology branch of the CCRG unit. She is
employee at Novartis Institute of Biomedical
Research since 2017.
Compliance with Ethical Requirements  12. Bobba S, Di Girolamo N, Mills R, Daniell M, Chan
Joséphine Behaegel, Sorcha Ní Dhubhghaill and
Marie-José Tassignon declare that they have no
conflict of interest.
Informed Consent  All procedures followed
were in accordance with the ethical standards of
the responsible committee on human experimen-
tation (University Hospital of Antwerp) and with
the Helsinki Declaration of 1975, as revised in
2000. Informed consent was obtained from all
patients for being included in the study.
No animal studies were carried out by the
authors for this article.
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 Optimizing the Ocular Surface
for Regenerative Surgery:
What Is Important and What Is
Essential for the Outcome
Kai B. Kang and Ali R. D’jalilian
Summary
• Approaches to the optimization of ocu-
lar surface health for regenerative sur-
gery include improving the quality of
the tear film, controlling of local and
systemic inflammation, and repairing
eyelid abnormalities.
• Various tear supplements, hemoderiva-
tives, treatment for Meibomian gland
dysfunction, usage of contact lenses,
amniotic membrane, and punctal occlu-
sion as well as reduction in the toxicity
of glaucoma drops are essential in
improving tear film health.
• It is crucial to control local and system
inflammation prior to regenerative sur-
gery. Approaches to the control of local
inflammation may include the use of topi-
cal steroids, cyclosporine, tacrolimus, and
lifitegrast. Approaches to the control of
systemic inflammation include various
steroids and steroid-sparing agents.
Kai B. Kang and Ali R. D’jalilian declare that they have
no conflict of interest. No human or animal studies were
carried out by the authors for this article.
K. B. Kang · A. R. D’jalilian (*)
University of Illinois at Chicago, Department of
Ophthalmology and Visual Sciences,
Chicago, IL, USA
e-mail: adjalili@uic.edu
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_20
20
• Eyelid abnormalities should also be
repaired to fix exposure and misdirected
lashes prior to regenerative surgery.
20.1 Introduction
The preoperative planning for regenerative sur-
gery requires the optimization of patients’ ocular
surface health. A dry and hostile surface environ-
ment will lead to failure of regenerative surgery.
In this chapter, we outline several important
approaches the surgeon should take to maximize
the ocular surface health of patients. These
approaches include optimization of the tear film,
control of local and systemic inflammation, and
repair of eyelid abnormalities.
Figure 20.1 shows key components in the
optimization of ocular surface health.
20.2 Improving Tear Film Health
20.2.1 Tear Supplementation
To improve the success of regenerative surgery, it
is important to return the tear film and the ocular
surface to the normal homeostatic state by opti-
mizing the quality of the tear film. Several steps
may be taken to improve tear quality depending
on the root cause. One important step is to
291
292 K. B. Kang and A. R. D’jalilian

Optimization of ocular surface

Improving tear film health Controlling inflammation Repairing eyelid abnormalities

1. Local inflammation
1. Tear supplements 1. Exposure
a. Steroids
2. MGD treatment 2. Misdirected lashes
b. Cyclosporine
3. Autologous serum
c. Lifitegrast
4.Soft and scleral lenses
2. Systemic inflammation
5. Punctal occlusion
a. Steroids
6. Minimizing drop toxicity
b. Steroid-sparing agents

Amniotic membrane

Fig. 20.1  Key components in the optimization of ocular surface health

s­upplement tear with lubricants, which include
artificial tears, ointments, and gels. A dry ocular
surface may cause further osmotic stress and con-
sequent elevation in electrolyte concentration
leading to activation of cellular stress signaling
pathways and cellular damage [1]. Ocular lubri-
cants may provide both electrolytes and hypoto-
nicity to the tear film in order to overcome these
problems. For regenerative surgery, preservative-­
free lubricants are the best as preservatives
(detergent or oxidative) may further irritate and
damage epithelial cells and decrease cellular
survival.
20.2.2 MGD and Blepharitis
Treatment
Several antibiotics with anti-inflammatory
property are also available in the management of
Meibomian gland disease [9]. These antibiotics
include tetracyclines, e.g., doxycycline and
minocycline, and macrolides, e.g., azithromycin.
Several studies comparing the effectiveness of
topical or oral azithromycin to oral doxycycline
in the management of Meibomian gland dysfunc-
tion found that despite different mechanisms of
action, either antibiotics relieved the signs and
symptoms and restored normal lipid properties of
Meibomian gland secretions [10, 11]. However,
azithromycin may stimulate Meibomian gland
epithelial cell differentiation and may be prefer-
able in certain instances as a short 5-day course
showed overall better clinical response compared
to a 1-month treatment with doxycycline [12].

In addition, many therapeutic options are avail-

able and essential in improving Meibomian gland
health. For example, omega-3 fatty acid supple-
mentation (e.g., from fish oil and flaxseed oil) has
been shown to alter the composition of Meibomian
gland lipids and decrease pro-­inflammatory medi-
ators, e.g., IL-1 and TNF-­alpha [2, 3]. Clinically,
omega-3 fatty acid supplementation showed
improved tear production, TBUT, Schirmer score,
and ocular surface disease index [2, 4–8].
20.2.3 Hemoderivatives
Various hemoderivatives produced from the patient’s
own blood are valuable options to help in optimizing
the ocular surface health for patient undergoing
regenerative surgery. These include autologous
serum, platelet-rich plasma, and plasma rich in
growth factors (PRGF). Hemoderivatives are made
preservative-free and provide natural ­substitutes for
20  Optimizing the Ocular Surface for Regenerative Surgery: What Is Important and What Is Essential…
many proteins, vitamins, and lipids active in the
human tear. Autologous serum drops have been
shown to suppress apoptosis in the conjunctival and
corneal epithelium [13]. Concentrations between
20% and 100% of serum tears have been used.
Recently, a study of long-term outcomes of 50%
serum tears demonstrated improvement in fluores-
cein staining, Schirmer scores, and ocular surface
index scores [14]. Autologous serum tears are espe-
cially important in patients with severe aqueous tear
deficiency and deficiency in reflex tearing. This
includes patients with scarring of the lacrimal glands
as seen in Stevens-Johnson syndrome, mucous
membrane pemphigoid, or following alkali or ther-
mal injuries. Recent studies have also shown that
platelet-rich plasma and plasma rich in growth fac-
tors can be superior than autologous serum espe-
cially when used in patients with persistent epithelial
defects [15].
20.2.4 Soft and Scleral Contact
Lenses
Therapeutic soft contact lenses are often used
to protect and hydrate a dry corneal surface.
Soft contact lenses often provide immediate
relief of symptoms in patients with severe dry
eyes and help to protect the ocular surface
against mechanical trauma. For example, in
patients suffering from lid margin keratiniza-
tion, friction from blinking may cause damage
to the ocular surface. One way to prevent cor-
neal damage is to use a bandage contact lens
chronically. To achieve a better contact lens fit,
the clinician may consider a larger diameter
Kontur lens. Additionally, scleral lenses are
typically fluid-filled and vault over the cornea.
They rest over the limbus and provide the ocu-
lar surface with constant lubrication. These
lenses may protect the ocular surface by mini-
mizing mechanical and frictional forces on the
epithelium and maintaining a constant tear film
over the epithelium. Studies have shown that
scleral lenses improved patient comfort and
ocular surface health in those with severe ocu-
lar surface diseases [16–20]. Different options
of scleral lenses are currently available. For
293
example, the Boston scleral prosthetic device
(Prosthetic Replacement of the Ocular Surface
Ecosystem, PROSE, Boston, MA, USA) allows
for a highly customizable device that precisely
fits the curvature of the patient’s eye and may
be beneficial especially in the regenerative sur-
gery patient population with highly irregular
ocular surface [20].
20.2.5 Punctal Occlusion
Another approach to the management of a dry
ocular surface due to tear insufficiency is punctal
occlusion. Either intracanalicular placement of
punctal plugs or placement at the punctal level is
available. We recommend a trial with temporary
punctal plugs (e.g., collagen plugs) first, and if
proven effective for the specific patient, the clini-
cian may consider the placement of permanent
plugs (e.g., silicone or acrylic plugs).
20.2.6 Minimizing Drop Toxicity
It is also important to minimize the toxic effects
of topical agents in preparation for patients
undergoing regenerative surgery. Many patients
using glaucoma medications experience toxic
conjunctivitis after chronic use. For example,
topical beta-adrenergic blockers such as timolol
have been associated with punctate epithelial
keratopathy and epithelial erosions [21]; dorzol-
amide has been associated with mucopurulent
sterile conjunctivitis [22]; and brimonidine can
commonly cause allergic blepharoconjunctivitis
and follicular conjunctivitis [23]. For glaucoma
patients who are on multiple drops, the clinician
may consider surgery or oral agents to minimize
drop toxicity.
20.2.7 Amniotic Membrane
Amniotic membranes were first used in skin
transplants to treat ulcers and burns [24]. The
basement membrane of amniotic membrane has
collagen and laminin structures that are similar to
294
the conjunctiva [24]. It has a basement membrane
that facilitates epithelial cell migration, adhesion,
proliferation, and differentiation [25–27]. The
basement membrane of amniotic membrane also
has anti-inflammatory, antiangiogenic, and anti-­
fibrotic properties. These properties make amni-
otic membrane a great substrate to promote
ocular surface healing.
There are two major types of amniotic mem-
branes: cryopreserved and dehydrated.
PROKERA biological bandage (Bio-Tissue,
Doral, Florida) is held in place with a PMMA
ring. It has anti-inflammatory effect to quiet
acute phase ocular surface inflammation. In
patients who cannot tolerate PROKERA’s
PMMA ring, or in those with glaucoma filtering
blebs or tubes that make fitting difficult, dehy-
drated amniotic grafts, e.g., AmbioDisk (IOP
Ophthalmics, Costa Mesa, California), may
work [28]. This dehydrated form is placed on the
ocular surface and then covered with a bandage
contact lens. Its use is indicated in patient with
severely dry ocular surface. In addition, sutured
surgical amniotic membrane grafts are often
used in conjunction with regenerative surgery,
e.g., KLAL in inflamed eyes, as it has been
shown to suppress inflammation and facilitate
epithelization.
20.3 Controlling Inflammation
Another major step in optimizing patients’ ocular
surface is to control local and systemic inflamma-
tion. Regenerative surgery will not be successful
in an overall inflamed eye. Many agents are avail-
able to help control inflammation.
20.3.1 Local Inflammation
Steroids
Topical glucocorticoids are still the most effec-
tive and fast-acting therapy to suppress inflam-
mation on the ocular surface. Glucocorticoids
work to suppress arachidonic acid release from
phospholipids. At the tissue level, glucocorti-
coids may prevent or suppress local hyperther-
K. B. Kang and A. R. D’jalilian
mia, vascular congestion, edema, and pain
associated with acute inflammation. They also
suppress fibroblast proliferation, collagen depo-
sition, and scarring associated with late inflam-
matory response. For short-term or pulse therapy,
one may use commercial available steroids
(dexamethasone, prednisolone acetate, lotepred-
nol, difluprednate, fluorometholone, etc.).
Preservative-free forms, e.g., methylpredniso-
lone 1%, also act as good alternatives to commer-
cially available steroid preparations and are less
toxic to the epithelium [29, 30].
Cyclosporine/Tacrolimus
Calcineurin inhibitors act to reduce pro-­
inflammatory cytokines and activated lympho-
cytes [31]. Prepared in a glycerin, castor oil, and
polysorbate 80 emulsion, the vehicle for the
topical preparation cyclosporine 0.05% itself
provides good lubrication to the ocular surface.
Twice-daily dosing with this agent has negligi-
ble systemic absorption and side effects. A recent
systemic review on efficacy of cyclosporine
0.05% showed lower ocular surface disease
index scores, longer tear film breakup time,
improved Schirmer I scores, reduced corneal
fluorescein staining, and higher goblet cell den-
sities [31]. Although it is known that this agent
may only benefit a subgroup of patient, it contin-
ues to act as an important nonsteroidal agent in
the management of ocular surface disease.
Tacrolimus is another calcineurin inhibitor, and
the topical preparation of tacrolimus 0.03% and
other concentrations has shown reduced side
effect profile and good efficacy in control of
inflammation, especially in patients with atopic
diseases [32].
Lifitegrast
Topical lifitegrast is one of newest addition to the
current management regimen to reduce ocular
surface inflammation. It works to block lympho-
cyte function-associated antigen-intracellular
adhesion molecule-1 interaction, thus decreasing
T-cell recruitment [33]. We hope that in the
future, we will gain more experience and conduct
studies that look at the efficacy of this agent for
various disease processes.
20  Optimizing the Ocular Surface for Regenerative Surgery: What Is Important and What Is Essential…
20.3.2 Systemic Inflammation
Steroids
Uncontrolled ocular surface inflammation is a
threat to any regenerative surgery. Systemic
immunosuppressive therapy is often necessary to
further suppress inflammation prior to regenera-
tive surgery. Oral steroid is still the most rapid
and effective systemic immunosuppressant avail-
able. Due to its adverse effects when used in the
long term, including osteoporosis, hypertension,
hyperglycemia, weight changes, and mood
changes, only short courses of steroids are rec-
ommended prior to surgery to further decrease
ocular surface inflammation.
Steroid-Sparing Immunosuppression
In the preparation for regenerative surgery, ade-
quate control of systemic inflammation in
patients with autoimmune diseases such as
mucous membrane pemphigoid and Stevens-­
Johnson syndrome is essential. Long-term
immunosuppression can be achieved in these
patients with steroid-sparing agents. It is recom-
mended that these patients be managed in col-
laboration with a local organ transplant team
who has a broad experience with available
­immunosuppressive agents. Examples of avail-
able immunosuppressive agents include calci-
neurin inhibitors which mainly act on T cells by
inhibiting transcription of lymphokines, includ-
ing IL-2 [34]. Cyclosporine A (CsA) and tacroli-
mus (Prograf) are the most commonly used
agents. Adverse effects of calcineurin inhibitors
include hypertension, nephrotoxicity, neurotox-
icity, hyperglycemia, and hepatotoxicity [34,
35]. One advantage of tacrolimus over CsA is
that hirsutism and gingival hyperplasia are not
seen [34]. Another class of immunosuppressive
agents is antimetabolite that blocks the prolifera-
tion of dividing cells, including azathioprine
(Imuran) and mycophenolate mofetil (CellCept).
Complete blood counts and liver function tests
should be monitored on a regular basis for
patient on these medications as myelosuppres-
sive effects can be observed in these patients [36,
37]. Mycophenolate mofetil is often used as part
of a triple therapy with CsA and steroids. A third
295
class of immunosuppressive agent is sirolimus
(rapamycin), a macrolide immunosuppressant
that prolongs the cell cycle by inhibiting mTOR,
which acts on phosphorylation of many cell
cycle-dependent kinases. It is important to moni-
tor serum lipids and blood counts periodically in
patients who use sirolimus, as hyperlipidemia,
leukopenia, and thrombocytopenia may develop
[38]. In addition, various biological drugs that
target specific components of the immune sys-
tem may help patients who fail traditional immu-
nosuppressive agents. For example, TNF
inhibitors such as infliximab (Remicade) or
monoclonal CD-20 antibody rituximab (Rituxan)
have been shown to be effective in controlling
ocular inflammation.
20.4 Repair Lid Abnormalities
Abnormalities in the eyelids can lead to disrup-
tion of ocular surface health as normal eyelid
blinking, apposition, and closure are essential for
ocular surface protection. The ophthalmologist
often encounters abnormalities in eyelid struc-
tures and functions especially in patients under-
going regenerative surgeries as these patients
often suffer from cicatricial conditions or have a
history of chemical or thermal injuries. Prior to
any regenerative surgery, it is essential to repair
eyelid abnormalities in order to provide a pro-
tected and wet ocular surface.
20.4.1 Exposure
Several eyelid abnormalities may cause chronic
exposure of the ocular surface. For example,
patients may suffer from lagophthalmos due to
paralysis from facial nerve palsy, ectropion, and
malpositioned and keratinized lid margins.
Lagophthalmos can cause chronic desiccation of
the cornea; thus it is essential to repair lagoph-
thalmos. The management of lagophthalmos
depends on the underlying cause and requires the
participation of the oculoplastic surgeon. For
example, there are various causes for lagophthal-
mos due to ectropion. Involutional ectropion,
296
caused by horizontal lid laxity, requires a lateral
tarsal strip procedure [39]. Paralytic ectropion
secondary to facial nerve palsy may require a lat-
eral tarsorrhaphy. Lagophthalmos from facial
nerve palsy may require the placement of gold or
platinum weight [40]. Cicatricial ectropion may
require scar revision and anterior lamella length-
ening with skin grafts [41].
20.4.2 Misdirected Lashes
Misdirected lashes due to entropion, trichiasis,
and distichiasis may cause mechanical injury to
the conjunctival and corneal epithelium, lead-
ing to a chronic inflammatory state. It is essen-
tial to reduce misdirected lashes prior to
regenerative surgery. Long-term management
plan including cryotherapy, argon laser abla-
tion, full-thickness wedge resections, and repair
of eyelid entropion would be beneficial prior to
regenerative surgery.
20.5 Conclusion
One of the first steps in regenerative surgeries is
to ensure the presence of a healthy ocular surface
to allow for the long-term success of surgeries. It
is essential to avoid a dry and hostile surface
environment. In this chapter, we outline various
approaches the surgeon should take to optimize
the ocular surface health. Many medications are
available in our management armamentarium;
however, the surgeons should individualize the
treatment course based on each patient’s disease
mechanism.
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36; discussion 436–437. https://doi.org/10.1097/01.
SLA.0000033429.89424.F8.
36. Liang L, Sheha H, Tseng SCG. Long-term outcomes
of keratolimbal allograft for total limbal stem cell
deficiency using combined immunosuppressive
agents and correction of ocular surface deficits. Arch
Ophthalmol. 2009;127(11):1428–34. https://doi.
org/10.1001/archophthalmol.2009.263.
37. European Mycophenolate Mofetil Cooperative Study
Group. Mycophenolate mofetil in renal transplanta-
tion: 3-year results from the placebo-controlled trial.
Transplantation. 1999;68(3):391–6.
38. Kruh J, Foster CS.  Corticosteroid-sparing agents:
conventional systemic immunosuppressants.
Dev Ophthalmol. 2012;51:29–46. https://doi.
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39. Jordan DR, Anderson RL.  The lateral tarsal strip
revisited. The enhanced tarsal strip. Arch Ophthalmol.
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40. Reanimation of the paretic eyelid using gold weight
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Accessed 31 Dec 2017.
41. Brown BZ, Beard C. Split-level full-thickness eyelid
graft. Am J Ophthalmol. 1979;87(3):388–92.
 Stem Cell Spheres for Corneal
Regeneration
Salim Ismail, Jennifer J. McGhee, Ye Li,
Jeremy John Mathan, Jinny Jung Yoon,
Himanshu Wadhwa, Stephanie U-Shane Huang,
and Trevor Sherwin
21.1 S
 tem Cell Spheres
for Corneal Repair
Limbal cells isolated from human corneoscleral
tissue and cultured in vitro as stem cell-enriched
spheres have incredible potential as transplant-
able elements for corneal repair and regeneration.
Our laboratory has championed the use of mixed
populations of cells isolated from the limbus as
transplantable elements capable of reforming and
restoring the limbal niche. However, as these
spheres inherently contain mixed cell types of
different lineages in addition to the stem cells,
their characterization and regenerative abilities
need to be carefully assessed. We have extensive
experience of both in vitro and in situ evaluation
of these spheres in our laboratory. In this chapter,
we discuss the differing cell types that make up
our spheres and the roles they each play in the
onset and treatment of corneal dystrophies within
human tissue. We also outline our extensive char-
S. Ismail · J. J. McGhee Y. Li · J. J. Mathan
J. J. Yoon · H. Wadhwa S. U.-S. Huang
T. Sherwin (*)
Department of Ophthalmology, New Zealand
National Eye Centre, Faculty of Medical and Health
Sciences, The University of Auckland,
Auckland, New Zealand
e-mail: s.ismail@auckland.ac.nz;
c.mcghee@auckland.ac.nz; j.mcghee@auckland.ac.nz;
yli912@aucklanduni.ac.nz;
stephanie.huang@hudson.org.au;
t.sherwin@auckland.ac.nz
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_21
21
acterization of the behaviour and abilities of cul-
tured stem cell-enriched spheres and their true
potential as a stem cell therapy.
21.2 L
 imbal Stem Cells/
Keratocyte Progenitor Cells/
Stromal Stem Cells
The human eye is one of a number of organs
within the body that harbour a repository of adult
stem cells. Although there is increasing evidence
that these adult stem cells may possess the ability
to differentiate into other cell types, their main
purpose is considered to be the long-term mainte-
nance of the tissues that make up the eye.
Within the cornea, the limbal stem cell (LSC)
has long been accepted as the driver of corneal sur-
face repair and maintenance. Their identification
however has proved difficult due to the lack of a
single definitive limbal stem cell marker or even a
definitive panel of markers [1]. The well-­
characterized attributes of limbal stem cells are that
they first and foremost reside at the limbus [2], the
region at the periphery of the transparent cornea
and the opaque sclera. They are quiescent and slow
cycling but, when required, have the ability to
respond quickly and positively to ensure mainte-
nance of vision. Within the human limbal region,
LSCs have been reported to exist in the palisades of
Vogt [3, 4] and limbal epithelial crypts [5]. During
normal homoeostatic progression, LSCs give rise
299
300
to transient amplifying cells which migrate centrip-
etally along the basal epithelium and differentiate
to replace lost epithelial cells [6]. Therefore the pri-
mary purpose of LSCs is considered to be long-
term maintenance of the corneal surface.
Corneal stromal stem cells have proven even
more difficult to identify and isolate than corneal
epithelial stem cells. Despite the stroma making
up 90% of the thickness of the cornea, the major-
ity of its structure is composed of collagen
fibrils, glycoproteins and proteoglycans with a
consistent distribution of differentiated but qui-
escent keratocytes [7]. In 2005, a report demon-
strated the presence of a side population (SP) of
cells from within the corneal peripheral stroma
that appeared to possess stem cell properties
similar to that of limbal epithelial stem cells and
could be induced to express differentiation mark-
ers consistent with a keratocyte-like phenotype
[8] indicating the presence of a specific popula-
tion of adult stem cells for stromal repair and/or
maintenance. Corneal stromal stem cells are
considered to be mostly located in the anterior
stroma subadjacent to the basal side of the pali-
sades of Vogt [9, 10]. Current evidence appears
to suggest that this population of stromal stem
cells concentrated at the peripheral cornea play a
role in maintaining the keratocyte population
within the stroma. In addition, there is limited
evidence that they may also contribute to epithe-
lial regeneration by mesenchymal-­ epithelial
transdifferentiation [11].
Keratocyte progenitor cells (KPCs) have a
limited ability to self-renew and differentiate
[12]. This potency differentiates them from true
stem cells although they do possess the ability to
grow clonally in vitro and phenotypically become
keratocytes; however they differ from keratocytes
in that they do not rapidly lose their phenotype
with in vitro expansion. KPCs are found through-
out the stroma [8, 13, 14] but appear to be par-
ticularly concentrated at the peripheral cornea
[13] and therefore are often not easily distin-
guishable from true stromal stem cells. The pri-
mary role of KPCs is considered to be keratocyte
replenishment in corneal homoeostasis.
In addition to stem and progenitor cells that
play a direct role in the repair and maintenance of
S. Ismail et al.
the cornea, the limbus contains a niche [15]
which perpetuates the regeneration and repair
ability of these stem and progenitor cells [16, 17]
and houses a host of other cell types including
melanocytes, immune cells (Langerhans cells,
macrophages), vascular endothelial cells and lim-
bal stromal fibroblasts [18], all or some of which
may play a role in preserving the niche through-
out adulthood.
21.3 Limbal Stem Cell Deficiency
Limbal stem cell deficiency (LSCD) arises when
the normal population of limbal stem cells are
either partially or completely destroyed and/or
become dysfunctional; the phenotypic outcome
of which is a loss of corneal transparency and
ultimately blindness.
This destruction of LSCs has been reported to
occur in a number of ways including mechanical
injury, chemical or thermal burns as well as infec-
tions and autoimmune diseases that cause scar-
ring [19]. In addition, inflammatory diseases
such as Stevens-Johnson syndrome, contact lens
wear, UV irradiation and chemotherapy have also
been implicated in some cases of LSCD [20, 21].
The resultant characteristic absence or dys-
function of LSCs severely impacts the regenera-
tive ability of the corneal surface leading to a
range of persistent epithelial defects [22].
Commonly, conjunctival invasion is observed
which results in a loss of transparency coupled
with vascularization and inflammation [19, 22–
24]. Patients presenting with this condition suffer
from chronic pain, redness, irritation, scarring
and decreased vision due to opacity and irregular
astigmatism [25]. In severe cases, LSCD can
progress to stromal scarring including the loss of
resident keratocytes, ulceration and, on rare occa-
sions, perforation [25].
Given the vast spectrum of progression and
severity of LSCD symptoms that patients present
with, diagnosis is not straightforward [26]. The
most obvious and reliable clinical feature used
as a diagnostic indicator is conjunctivalization
identified by slit-lamp examination [27, 28].
Impression cytology is commonly used to col-
21  Stem Cell Spheres for Corneal Regeneration
lect cells from the corneal surface which are then
examined for the presence of goblet cells to pro-
vide evidence of conjunctivalization [29]. This
technique, however, is of limited sensitivity and
can result in false negatives [25]. Imaging tech-
niques such as in vivo laser scanning confocal
microscopy (IVCM) have been used to examine
cellular morphologic changes at the limbus [30,
31], while optical coherence tomography (OCT)
has been used to measure epithelial thinning
which is common in LSCD [32, 33]. IVCM can
provide high-resolution images which allow pre-
cise examination of limbal structures and can
identify the loss of these structures or morpho-
logical abnormalities at the limbus [34, 35].
OCT is faster and requires less patient co-opera-
tion, and although it can provide images of lim-
bal structure, they are not as high resolution as
IVCM [36]. However, neither of these tech-
niques is currently used in standard diagnostic
practice [25].
21.4 Treatments: Limbal
Transplants, Limbal Stem
Cell Expansion and Stromal
Stem Cells?
The basic premise of any treatment for LSCD is
the replacement of the lost stem cells and regen-
eration of the limbal structure damaged through
injury or disease.
In less severe cases or partial LSCD, more
conservative treatment approaches can initially
be taken. Non-surgical treatments include the use
of autologous serum drops, therapeutic contact
lenses and eye lubrication which are designed to
limit any further defects, promote healing of
existing defects and facilitate proliferation and
migration of existing stem cells to re-form a
healthy limbus [37–40]. Corneal scraping is
sometimes used to remove the conjunctival over-
growth and allow for the remaining healthy cor-
neal epithelial stem cells to repopulate the corneal
surface [41]. This treatment however is depen-
dent on sufficient remaining stems cells to
actively repopulate the epithelium before the
recurrence of conjunctivalization [41]. An adjunct
301
to corneal scraping to restrict further conjuncti-
valization and assist epithelial recovery is amni-
otic membrane transplantation. Immediately post
scraping, biologically sourced donor amniotic
membrane is attached to the affected epithelial
region [42]. The nutrient-rich amniotic mem-
brane provides an ideal microenvironment for the
local stem cells to migrate and proliferate in the
hope of epithelial and/or limbal restoration.
Should these initial treatments prove ineffec-
tive or if LSCD is at a severe stage of disease
progression, such that the resident stem cells are
overtly dysfunctional or too few in number,
replacement of the LSCs from an external source
is required. In severe cases of LSCD where only
one eye is affected, autologous limbal transplants
from the unaffected eye to the damaged eye were
historically the preferred treatment [43]. This has
the advantage of utilizing the patient’s own
healthy limbal tissue for treatment alleviating any
immunosuppression and infection-related issues
that would be of major concern in cases where a
limbal allograft is necessary [44–46]. The major
disadvantage of limbal transplant in either case is
that the large amount of limbal tissue required to
be excised from the healthy donor eye creates a
risk of that eye developing LSCD itself. To cir-
cumvent this major downside, smaller sections of
limbal tissue can be used to expand the stem cell
population ex  vivo on supporting matrices such
as amniotic membrane prior to grafting onto the
affected corneal epithelium [44, 47–49].
The corneal epithelium being the anterior-most
part of the cornea is primarily impaired in LSCD;
however in most severe cases such as with signifi-
cant chemical burns, the stroma can be defective,
and the resident keratocytes can also be lost. As
most of the treatments above target epithelial
recovery first and foremost, the impact that the
loss of keratocytes and their stem and progenitor
precursors may have on limbal recovery has not
been fully elucidated. In LSCD cases where stro-
mal scarring is evident, it is common for either a
full-thickness or partial-thickness corneal trans-
plant procedure to be performed immediately
prior to grafting of the ex  vivo expanded LSCs.
This has the combined desired result of replacing
the defective stroma and potentially facilitating
302 S. Ismail et al.

limbal recovery but is hindered by the age-old characterized and defined cell population for use
problem of donor tissue availability both for in limbal transplantation is desirable. With this
transplant and ex vivo expansion. end goal in mind, we have focused on isolating a
mixed population of cells from the cornea which
in the right circumstances have the potential for
21.5 Regenerative Capabilities: limbal reformation.
Regeneration of the Limbus? The location of the desirable cell types along
the corneal surface is an important consideration.
Although the dysfunction or loss of stem cells is With the limbus as the well-accepted site for lim-
considered the primary cause of LSCD, recent bal stem cells, we assessed the regenerative capa-
findings suggest that significant disturbances to bilities of cells present at the central and
the limbal niche could be responsible for the peripheral cornea. In 2008, we developed a
observed stem cell dysfunction [6] and as such, ‘donut’ excimer laser ablation model with which
LSCD should be considered a limbal niche defi- we showed that corneal epithelial repair occurs
ciency. It is therefore imperative to consider inwards from the peripheral cornea as well as
whether the above treatments for LSCD results in outwards from the central cornea suggesting the
the regeneration of the limbus complete with a presence of regenerative cells at the peripheral
limbal niche. and central cornea [51]. Even when the limbus
Ex vivo expansion on amniotic membrane was completely ablated, epithelial regrowth was
would likely result in cultivation of a mixture of observed, providing evidence that corneal repair
epithelial cells, transient amplifying cells and can occur independent of a functional limbus.
stem cells which collectively have the makings of Despite these findings, we have deemed it likely
the limbal niche, but current practices are unable that limbus-independent corneal regeneration is
to characterize, enumerate or manipulate their essentially a wound healing response for short-­
relative proportions amongst the expanded cells term repair (12 h post-ablation in our study) and
on the amniotic surface prior to grafting onto the that a healthy limbus is essential for the long-­
limbal stem cell deficient eye. In effect, this term maintenance of the cornea.
inability to standardize cell expansion results in With this in mind, we have looked at isolation
unknown numbers of LSCs being grafted, and of cells from the limbus and peripheral cornea
although it has a reported success rate of 70% in and assessed their potential to re-form limbal tis-
improving the patients’ vision [50], this unknown sue. Corneoscleral rims composed of peripheral
entity would certainly contribute to the variabil- cornea, limbus and scleral tissue are often dis-
ity in the success of this procedure. The long-­ carded after the central cornea is removed by the
term success of these treatments is yet to be surgeon for transplantation procedures. This
proven as there is little evidence of where the postsurgical ‘waste’ tissue is an ideal source of
transplanted cells eventually localize and whether limbal and stromal stem and progenitor cells that
the procedure eventually restores the limbus to its could be harvested for use in cell-based therapy.
pre-injury state is unclear given that the criteria The limbal/peripheral corneal region from this
used to determine ‘success’ are often subjective. tissue could be excised from the scleral tissue and
the cells extracted from extracellular tissue com-
ponents. The resultant cell extracts would likely
21.6 Isolation of Spheres represent the individual cellular components of
with Mixed Potential the in  vivo niche. The sphere-forming assay is
one such method that has been used to isolate
Given the challenges associated with the treat- cells with proliferative potential from tissue
ment of LSCD and the ambiguities of the regen- extracts in a non-adherent culture system. Since
erative capabilities of the cells used in current its inception in the early 1990s as ‘neurosphere
treatment practices, the development of a well-­ assay’ [52] in brain tissue and from the early
21  Stem Cell Spheres for Corneal Regeneration
2000s in ocular tissue, the sphere-forming assay
has become a widely used method for isolation
and enrichment for adult stem and progenitor
cells [53]. Cells cultured in sphere-forming assay
have some advantages to those cultured in tradi-
tional adherent monolayers. The most notable of
which is that they retain similar characteristics to
the tissue from which they have been isolated and
can accurately simulate the native behaviours of
this tissue [54].
Our laboratory routinely isolates limbal cells
for sphere-forming assay from corneoscleral rims
by mechanical removal of endothelial cells and
anterior epithelial cells prior to prolonged tissue
treatment with dispase II, collagenase and hyal-
uronidase to ensure the complete dissociation of
limbal cells from extracellular tissue compo-
nents. The single-cell extracts are subsequently
cultured in vitro in a mitogen-driven serum-free
culture system [55]. Typically, single cells come
together to form cell aggregates with well-defined
spherical borders which morphologically differ-
entiate true spheres from loose cell aggregates
(Fig. 21.1). In our experience, sphere formation
can occur within days to several weeks post
in vitro culture. The process of sphere formation
is not merely a passive process of cell aggrega-
tion but rather an active and dynamic one with
multiple facets coming together to form the stem
cell-enriched sphere. Although passive aggrega-
tion of cells that are in close vicinity to form
loose clusters may be the initial step in sphere
Fig. 21.1  Sphere-forming assay: cell isolates from human corneoscleral tissue cultured in sphere-forming conditions
form cell aggregates with well-defined spherical borders
303
formation, we have shown that active recruitment
may also occur whereby single cells that may not
have been part of a sphere or had previously
migrated out from a sphere are pulled into the
body of the sphere by extended pseudopodia
[55]. Similarly, some proliferating cells at or out-
side of the periphery of a sphere show rapid
migration back towards the sphere. Additionally,
cells within a sphere are also proliferative with
EdU-based proliferation assays that label DNA in
dividing cells showing up to 70% of spheres
composed completely of EdU-positive cells at
7  days in culture [55]. Despite sphere-forming
culture conditions promoting non-adherent cell
culture, we often observe cells attached to the
glass surface that form a cell network which
comes together over time to form semi-attached
spheres that subsequently become non-adherent
mature spheres. These observations combined
indicate that the spheres are not necessarily clo-
nogenic as previously thought [56] and are
formed by a combination of cell aggregation,
active recruitment and proliferation.
The culture conditions used in sphere-forming
assay are designed to promote the survival of
stem and progenitor cells and select out the more
differentiated cell types in extended culture.
Given the source of the cell extracts being limbus
and peripheral cornea and the knowledge that
sphere formation is non-clonal, spheres are likely
to contain cells of epithelial and stromal origin.
Long-term free-floating culture of spheres for up
304 S. Ismail et al.

to 4  months without passaging confirms their Collectively, these results suggest that spheres
ability for self-maintenance and ensures stem cell contain mixed cell populations both in terms of
enrichment. Although we have observed consid- cell lineage and stemness and/or differentiation
erable donor variation in both the ability of cell state. This heterogeneity of cell types observed in
extracts to form spheres and the longevity of sphere composition may reflect that observed in
spheres once formed, we have been able to main-
tain healthy spheres in culture for greater than
9 months in some cases. Characterization of the
cells within spheres by assessment of their ability
to respond to stimulus such as a collagen sub-
strate shows an ability to rapidly migrate and pro-
liferate with the greatest proliferation observed at
the periphery of the sphere and amongst migra-
tory cells, while the cells at the core of the sphere
remain relatively quiescent (Fig.  21.2) [55].
Immunocytochemical detection of cell differen-
tiation markers amongst these migrating cells
shows expression of the mesenchymal cell
marker vimentin and the epithelial cell marker
keratin K3/K76 confirming a likely mix of epi-
thelial and stromal origin cells (Fig.  21.3). By
contrast free-floating spheres and non-migratory
cells within collagen-stimulated spheres showed Fig. 21.2  Spheres respond to collagen stimulus with pro-
expression of the limbal basal epithelial marker liferation and migration: fluorescence ubiquitination cell
cycle indicator (Fucci) labelling of spheres placed on a
deltaNp63, the limbal niche marker notch1, the
collagen surface shows greatest proliferation (green/yel-
neural progenitor cell marker nestin and the low) at the periphery of the sphere and amongst migratory
repair cell (myofibroblast) marker α-smooth cells (red), while the cells at the core of the sphere remain
muscle actin (α-SMA) (Fig.  21.4) [55–57]. relatively quiescent

a b

Fig. 21.3  Mixed origin of sphere-derived cells: migra-
tory cells derived from spheres show expression of the
mesenchymal cell marker vimentin (a) and the epithelial
cell marker keratin K3/K76 (b) (green) confirming a
likely mix of epithelial and stromal origin cells. Panel a:
Cell nuclei labelled with DAPI (blue), proliferative cells
labelled with EdU (pink/red). Panel b: Cell nuclei labelled
with propidium iodide (red/orange). (Panel a reproduced
in part from reference: [57])
21  Stem Cell Spheres for Corneal Regeneration 305

a b

c d

Fig. 21.4 Sphere cell characteristics: free-floating
spheres and non-migratory cells within collagen-­
stimulated spheres show expression of the limbal basal
epithelial marker deltaNp63 (a, green), the limbal niche
marker notch1 (b, green), the neural progenitor cell
marker nestin (c, red) and the repair cell (myofibroblast)
marker α-smooth muscle actin (α-SMA) (d, green). Cell
nuclei labelled with DAPI (blue). (Panel b reproduced in
part from reference: [57])

the in  vivo limbal niche and facilitate the long-­
term maintenance of stem and progenitor cells.
When exposed to a collagen substrate, spheres
show a number of distinct and intriguing behav-
iours which need to be carefully assessed in order
to determine their suitability for use in stem cell
therapy. Upon placement onto a collagen sub-
strate, spheres cease to be free floating and attach
to the surface, and cells rapidly migrate outwards,
and this is often coupled with cell division.
Outgrowth of cells would appear to be random
and in all directions around the circumference of
the sphere in 75% of spheres (Fig.  21.5a).
However, in 25% of spheres, the response to col-
lagen stimulus would appear to be more deliber-
ate and targeted with an apparent directional
response (Fig.  21.5b). In all cases, a distinct
polarity seems to develop with cellular outgrowth
occurring in opposite directions from two ‘poles’,
while the areas adjacent to the polar outgrowth
306
a b
c d
Fig. 21.5 Sphere behaviour on collagen substrate:
spheres respond to collagen stimulus in a variety of ways
with approximately 75% reacting with random outgrowth
from all directions, (a) while approximately 25% show a
remain as cell exclusion zones (Fig.  21.5c). In
some cases, a few migratory cells venture out
into the apparent cell exclusion zones but do not
continue migrating outwards, instead turning
back towards the direction of polar outgrowth or
reversing towards the sphere. Over time
(2–10 days), this polar pattern of cell migration is
partially or completely lost with cell migration
into the exclusion zone ensuing from both the
sphere and the directional migratory cells
(Fig.  21.5d). Distance from the central sphere
also appears to be important for cell signaling as
cells furthest from the sphere seem to lose
directionality.
In rare cases (less than 10% of spheres anal-
ysed), a unique phenomenon of en masse migra-
tion occurs (Fig. 21.6). With what would appear
to be directed cell migration with polar outgrowth
S. Ismail et al.
directional response with cell migration along distinct
‘north and south poles’. (b) This directed cell migration
creates cell exclusion zones (c, arrow) which over time
may be partially or completely repopulated (d)
initially occurring, the entire sphere migrates as a
whole with cells from each pole displaying dis-
tinct locomotive behaviour. The cells at the front
of the motile sphere undergo cycles of
­lamellipodia extension and traction in a forward
direction, while the trailing cells at the opposite
pole show a highly coordinated pattern of retrac-
tion which serves to project the sphere forwards
[55]. The direction of en masse sphere migration
is not linear, with the migratory path able to be
changed from one initial direction to another pos-
sibly in response to microenvironmental signals.
The significance of this en masse migration is
unclear and must be carefully considered. The
advantage of such behaviour may allow spheres
to sense its environment and enable it to ‘relo-
cate’ to the most favourable location to re-estab-
lish the limbus. However, on the flip side, this
21  Stem Cell Spheres for Corneal Regeneration
Fig. 21.6  Collective sphere migration: en masse migra-
tion is observed in less than 10% of spheres where the
entire sphere migrates as a whole with cells from each
pole displaying distinct locomotive behaviour. Here the
sphere initially migrates slightly downwards and to the
right from 5 to 10  h post placement on a collagen sub-
strate. At 30–37  h, the sphere changes direction and
migratory pattern may also result in spheres mov-
ing away from their intended transplanted site
and moving to unintended areas of the cornea
where undesirable cell migration and prolifera-
tion may occur.
When multiple spheres are seeded in close
proximity to each other, the migratory cells from
each sphere interact to form both areas of out-
growth and cell exclusion zones which, when
organized in a circular pattern, form a ring-like
structure with a large central exclusion zone.
These observations, albeit largely qualitative, are
not isolated instances and have been observed
amongst spheres prepared from tissue collected
from multiple donors over a number of years.
Collectively, these observations demonstrate our
limbal and peripheral corneal spheres to be het-
erogeneous structures that are capable of dynamic
behaviours in response to positive stimuli. It is
our opinion that these behaviours are not innate
predetermined responses but rather determined
by the microenvironment that they are placed in
and as such, if correctly manipulated, could be
used to effectively regenerate the limbus.
307
begins moving upwards, and by 42–45  h post collagen
placement, the sphere begins to migrate out of view. This
indicates that sphere migration en masse can take place in
multiple directions. Red dot indicates original central
position of sphere; white reference line indicates original
horizontal position of centre of sphere
The dual role of corneal stem cells to replace
lost cells and to respond to injury warranted
investigation of this second function amongst our
sphere cultures in order to gauge their wound
healing ability. To this end, we conducted a series
of direct and remote wounding experiments to
evaluate the sphere response to physical injuries
and assess their capacity for wound healing
(Fig.  21.7). Mature spheres in culture for 3–4
months post tissue extraction that were most
likely to be stem cell enriched were placed onto a
collagen substrate and further stimulated 48 hours
post attachment by a direct compression injury to
the centre of the sphere using 10  μm glass
­capillaries with the aid of a micromanipulator.
Fluorescence-based staining for live/dead cells
confirmed an increase in cell death upon com-
pression injury when compared to uninjured
spheres. This increase in cell death elicited a dif-
ferentiation response from the cells within some
of the spheres whereby an observable increase in
the expression of the myofibroblast repair cell
marker α-SMA was detected 24 hours post injury
[58]. This increase in α-SMA-positive cells was
308
Fig. 21.7  Sphere response to injury: direct compression
injury of a sphere results in outward cell migration,
increased proliferation (EdU incorporation, red/pink) and
myofibroblast differentiation (α-smooth muscle actin
expression, green). Radiating concentric circles drawn at
100  μm intervals were used to quantify outward cell
migration, while a directional response was evaluated by
enumerating cell migration in quadrants 1–4. Cell nuclei
labelled with DAPI (blue). Scale bar = 100 μm
no longer observed in spheres at days 4, 7 and 14
post injury. Migrating cells from injured spheres
showed morphological features characteristic of
myofibroblasts indicating the stromal origin cells
within the spheres were upregulating α-SMA in
what could be considered a wound healing
response [58]. An important part of any wound
healing response is a rapid increase in cell divi-
sion to replace the dead cells at the injury site
with new healthy cells. In spheres with direct
compression injury, a significant increase in pro-
liferative cells as detected by EdU-based prolif-
eration assay was evident at 4  days post injury
when compared to uninjured spheres. This prolif-
erative response was also present at 7 days post
injury but had declined somewhat from the day 4
peak, and by day 14 the proliferative response
had returned to that of uninjured spheres. Cells
that had migrated outwards from the injured
sphere showed no difference in proliferation,
indicating that the increase in cell division was a
direct response to the cell death signals elicited
by the injured cells. Direct compression injury
S. Ismail et al.
also appeared to slow the outward migration of
cells from the sphere at 24  h post injury which
then resumed at 7 days post injury, presumably as
the effect of the injury had diminished. These
results clearly established that quiescent spheres
of 3–4 months in culture are capable of reacting
to injury similar to how one would expect cells at
the limbus to react. However, injuries to the eye
may not always be to the peripheral cornea or
limbal region, and therefore the ability of spheres
to respond to remote injury is also an important
consideration. To test whether our spheres pos-
sessed this ability, we conducted remote scrape
wounding of migrating cells at 50 and 100  μm
distances from each sphere circumference in the
direction that had the greatest level of outward
cell migration. Having previously established
that spheres are capable of directed cell migra-
tion on a collagen surface, we measured whether
there was an increase in directed outward cell
migration from the sphere in the direction of the
scratch injury. By measuring cell numbers in
100  μm concentric circles from the sphere, we
showed that scratch injury at the closest distance
to the sphere (50 μm away) resulted in a greater
number of outwardly migrating cells than from
those spheres that did not receive scratch injury
to migrating cells and those that received scratch
injury to cells at a greater distance (100  μm
away). This may be due to a lack of a strong
enough signal to provoke a wound healing
response from those cells injured at 100 μm from
the sphere. When looking at proliferative
response, scratch injury resulted in a similar or
greater number of dividing cells amongst the
migratory cells, while the presence of α-SMA-­
positive cells both within the spheres and amongst
migrating cells in remotely injured spheres
showed that a stromal cell response had been
triggered. No such response was seen in spheres
that had not received scratch injury [58].
Collectively, these results show that spheres have
the ability to respond to injury (both direct and
remote) by initiating migration, proliferation and
differentiation of cells into a repair cell pheno-
type similar to cells at the limbus. However, the
spheres did not show a specific directional
response to remote scratch injury in  vitro, sug-
21  Stem Cell Spheres for Corneal Regeneration
gesting other factors (physical and physiological)
may play a role in ensuring migratory and repair
cells reach the site of injury from the limbus.
Having established the properties and behav-
ioural characteristics of spheres in vitro and con-
firming their ability to respond favourably to
collagen substrate and to elicit a wound healing
response, it was pertinent to assess their ability to
repopulate human corneal tissue. Therefore we
investigated whether spheres could be transplanted
into decellularized (repeated freeze-­thaw) corneo-
scleral tissue at the position of the limbus and
whether the implanted spheres would respond in
the same way that was observed in vitro on a colla-
gen-coated surface. To facilitate this, we developed
a microsurgical technique to create wedge-shaped
incisions at the scleral border of the limbus [59] to
create a shallow trough into which spheres could be
implanted. The limbal tissue was confirmed to be
completely decellularized by a lack of live cell
staining prior to sphere implantation and essen-
tially served as a model of LSCD.  Implanted
spheres from mature sphere cultures could easily
be implanted under a dissecting microscope and
showed a propensity to attach to the tissue and
remain within the site of surgical incision for the
duration of the 241-­hour experiments (Fig. 21.8a).
In an ex  vivo setting where decellularized tissue
implanted with spheres was incubated in serum-
containing culture medium, spheres were observed
to exhibit a number of similar behaviours to those
observed on a flat expansive collagen-coated sur-
face. Firstly, spheres were able to respond to the
collagen fibrils surrounding them by actively send-
ing migratory cells outwards. Secondly, the active
a b
Fig. 21.8  Implantation of spheres into donor corneo-
scleral rims: surgical implantation of spheres into the lim-
bal region of decellularized corneoscleral rims (a) results
in multidirectional live (calcein AM, green) cell migration
309
organization of cell migration patterns emanating
from the spheres was observed with directed cell
migration, and thirdly, spheres placed in proximity
to each other showed migration of cells towards
each other (Fig. 21.8b) [57]. In addition, the migra-
tion pattern of cells showed an obvious preference
towards the corneal direction of the tissue as
opposed to the scleral direction. Proliferation was
evident as by day 7 post implantation, the entire
corneal surface of the tissue segment had become
repopulated as evidenced by live cell staining, and
the number of cells present could not have repopu-
lated the surface by migration alone. The limitation
to cell migration appeared to only be the extent of
the corneal tissue with cells observed all the way to
the furthest edge of the corneal surface (Fig. 21.8c).
This phenomenon was not observed on the scleral
side of the tissue where only limited migration had
occurred and most of it had remained in proximity
to the limbal region. These observations again
strengthen our earlier thoughts that the spheres do
not necessarily have predetermined responses but
rather respond appropriately according to the envi-
ronment into which they are placed. Therefore
when placed into the limbal region, they would
appear to behave in a similar way to how one would
expect limbal cells to behave by migrating towards
the corneal surface. Closer examination of the
migratory cells in situ revealed differing cellular
organization patterns at the corneal, limbal and
scleral surfaces with those at the limbal and scleral
region showing thin spindle-like elongated mor-
phology, while those at the corneal surface were
broader in appearance and at the leading migratory
edge showed branching cellular processes. Cells
c
at 3 days post implantation (b) with preferential and com-
plete repopulation of the available corneal surface within
7 days of implantation (c). (Reproduced in part from ref-
erence: [57])
310
appeared more organized at the limbal and corneal
surfaces, while those at the sclera did not show
such a uniform pattern of alignment. Cross-­
sectional analysis of the sphere-implanted tissue
showed mostly repopulation over the anterior cor-
neal surface with limited evidence of cell migration
deeper down into the tissue. At 14 days post sphere
implantation, the stem cell marker deltaNp63 could
be detected along with differentiation markers lam-
inin and vimentin. Whole-­ mount staining of
sphere-implanted tissue also demonstrated the
presence of the stem cell marker ABCG2 in some
cells and the limbal basal epithelial marker
notch1 in others, while the keratocyte marker kera-
tocan was not detected. Taken together, these
results would suggest that stem and progenitor cell
repositories as well as limbal components and that
of transient amplifying cells and differentiated cells
were being retained in implanted tissue. All com-
ponents would be present in native cellularized tis-
sue. The lack of keratocan detection may reflect the
lack of keratocyte differentiation from stromal cells
at 14 days post implantation and likely due to the
limited cellular infiltration of underlying tissue and
the serum-containing culture conditions, but the
presence of the mesenchymal cell marker vimentin
provides evidence that stromal origin cells are
being retained in sphere-implanted tissue. Gene
expression data on sphere-implanted tissue con-
firmed a reduction in keratocan and also laminin-1
14  days post implantation when compared to the
original sphere cells, while the proliferation marker
PCNA showed a significant increase at day 4 fol-
lowed by a reduction from the day 4 peak at day 7
and a return to basal levels at day 14 post sphere
implantation. This day 4 proliferation peak is iden-
tical to the peak in proliferative cells observed in
the earlier direct compression sphere wounding
experiments. It would seem that day 4 post place-
ment on a collagen stimulus is a critical time point
for cell proliferation. Gene expression data also
confirmed no change in vimentin expression and
stem cell marker (ABCB5, ABCG2 and p63)
expression which may suggest stability in the
maintenance of these cell types over time. The evi-
dence provided by these sphere implantation
experiments that agree with our in  vitro wound
healing work and suggests that the response that
S. Ismail et al.
spheres elicit upon implantation is that of a wound
healing response with migration and proliferation
towards the corneal surface. However, the entire
corneal bed was repopulated within 7 days, and the
repopulated surface could be maintained for at
least another 7 days which would suggest that cor-
neal homoeostatic processes may be beginning to
take place. Although the short time frame for this
does not allow us to definitively show this. Longer-
term sphere implantation experiments that allow us
to determine if keratocyte differentiation of stromal
cells and retention of stem cell populations that
facilitate corneal surface maintenance would be
valuable. However, these results show remarkable
promise for the use of our spheres in corneal
regeneration.
Sphere implantation experiments were con-
ducted using spheres prepared from normal donor
tissue and implanted into decellularized normal
donor tissue. However this does not address the
fact that in order to be an effective cell-based ther-
apy, spheres would be transplanted into patients
with ocular conditions amounting to abnormal tis-
sue structures. Therefore to test whether spheres
have the equivalent ability to repopulate diseased
tissue, our wedge-shaped incision/sphere implan-
tation technique was applied to diseased corneal
tissue. Corneal buttons from keratoconus patients
and a decompensated cornea from a patient receiv-
ing corneal transplant were used to implant spheres
into the central cornea. Again, the tissues were
decellularized by repeated freeze-thaw cycles and
confirmed to be free of host tissue cells by live cell
staining. This left an acellular diseased tissue
matrix which was much weaker and friable than its
corresponding normal donor equivalent. This yet
to be published data showed that implanted spheres
were able to adhere to the diseased tissue and
remain in position similar to that of normal tissue.
Over the course of 7 days, spheres were also able
to send out migratory cells over the t­issue surface.
The extent of migration was variable amongst dif-
ferent spheres, and in some cases, initial rapid
migration observed at day 3 post implantation had
reduced by day 7. However this observation was
not unique to the diseased tissue, and a similar
response was observed in concurrently implanted
normal corneal buttons suggesting the response
21  Stem Cell Spheres for Corneal Regeneration
was a sphere-specific or culture condition-specific
response. Due to the larger surface area of a cor-
neal button compared to a corneoscleral tissue seg-
ment, complete surface repopulation was not
observed by implantation of 3–4 spheres
(Fig. 21.9). The pattern of repopulation, however,
was similar in both normal and diseased tissue
both in terms of cell morphological patterns and
cell orientation patterns. Spheres implanted in
close vicinity to each other showed circumferen-
tial migration of cells including towards each other
where they appeared to form cellular bridges
between spheres when the cells from each sphere
interacted. The extent of cell migration over 7 days
gave clear evidence of cell proliferation as the
sheer numbers of cells present could not have pop-
ulated the migratory area by outgrowth alone.
Moreover the spheres maintained themselves in
terms of their morphological structure in most
cases. EdU-based proliferation assays confirmed
the presence of proliferative cells at day 10 post
implantation. Furthermore, placement of spheres
onto en face 10 μm sections of decellularized dis-
eased stromal tissue invoked a similar response of
attachment, cell migration and proliferation. In
these experiments, cell migration by day 14 was to
a b
Fig. 21.9  Implantation of spheres into diseased central
corneal tissue: sphere implantation into a decompensated
central corneal button (recovered from corneal transplant
surgery) assessed by calcein AM live cell staining (green)
311
the extent of the entire stromal section when mul-
tiple spheres were placed on the section. In all
cases, the sphere cell migration showed a propen-
sity to migrate across the stromal section preferen-
tially to the culture dish surface. These unpublished
results clearly demonstrated that the abnormal
structure of the diseased tissue was not a deterrent
to sphere attachment, migration and division
although there was no observable increase in any
of these measures in response to diseased tissue
compared to normal tissue either. Of note was the
fact that keratoconic stromal sections have a much
looser arrangement of collagen fibrils and this
altered structure did have a noticeable effect on the
cell migration pattern at days 7 and 14 post implan-
tation with cell orientation taking on a less orga-
nized and a more random orientation pattern which
was not observed in normal stromal sections.
Qualitative observation of cell morphology of
migratory cells showed limited evidence of a
keratocytic phenotype in a few cells from spheres
implanted into diseased tissue. Gene expression
data showed no apparent differences in stem cell
markers, epithelial cell markers, keratocyte mark-
ers or extracellular matrix markers between nor-
mal and diseased sections indicating the sphere
shows a pattern of cell migration outwards from implanted
spheres which predominantly increased from day 3 (a) to
day 10 (b) post implantation
312
response to be essentially the same in diseased
tissue as observed in normal tissue. Vimentin
expression showed an increase over time in both
normal and diseased sections and may reflect a
stromal cell migratory response over the largely
stromal sections. Whether these stromal cells
would differentiate into keratocytes was not
clear, and there was no evidence of this at 14 days
post implantation. Longer-term experiments
would help to determine if the migrated cells,
once established in their post-migration position
within the stroma, would become resident kerato-
cytes. Culture conditions promoting keratocyte
differentiation would also likely be required to
confirm this. Immunohistochemical staining for
α-SMA at days 1 and 4 post implantation showed
a few cells positive for this repair cell marker
indicating a small wound healing response. These
unpublished results confirm the ability of spheres
to respond to diseased tissue in a similar way to
their response to normal tissue and that the cells
show the correct markers and morphological
(epithelial or stromal) tendencies for the tissue
structures they were placed in.
Further investigations in this area need to
address whether the implanted spheres from nor-
mal cells are capable of altering the structure of
the diseased tissue. Our initial experiments here
Fig. 21.10  Response of a
spheres from
keratoconic cells:
spheres formed from
keratoconic cell isolates
respond more rapidly to
collagen stimulus than
spheres from normal
cells (a, time
point = 25 h post
implantation). The
sphere from keratoconic Normal
cells is unable to b
maintain itself, and
migrating cell numbers
are not retained over an
extended period of time,
while the sphere from
normal cells remains
intact (b, time
point = 266 h)
S. Ismail et al.
showed some evidence of collagen expression
from implanted cells indicating they may be
capable of laying down new matrix, but the
14-day time period is likely too short to deter-
mine whether this would be the beginnings of
structural repair to the diseased tissue. A second
consideration would be the cell-cell interaction
between normal donor and diseased recipient
cells. As all of our in  vitro and ex  vivo experi-
ments were conducted on decellularized matrix,
the effect of resident cells is largely unknown.
Best-case scenario would be that the transplanted
normal cells would be able to use signaling
mechanisms to reverse, alter or stop the abnormal
cellular response of host cells. Any remedial
effect of transplanted sphere cells may be depen-
dent on the severity of disease progression, and it
may be that there is a critical level of progression
or ‘tipping point’ where any therapeutic effects
exerted by transplanted cells would not be able to
reverse the disease progression. All of these ques-
tions remain to be answered.
We have recently looked at sphere-forming
ability in cells extracted from keratoconic tissue
and assessed their ability to respond to similar
stimuli to that of normal donor spheres
(Fig.  21.10). However, as keratoconic tissue is
usually obtained as central corneal buttons that
Keratoconic
21  Stem Cell Spheres for Corneal Regeneration 313

have been removed from patients prior to trans- remarkable amount of potential for cell-based
plant, there is a lack of peripheral corneal and therapy as they are enriched with stem and pro-
limbal cells present. In our previous experience genitor cells, they are responsive to stimuli such
looking at sphere-forming ability in normal cen- as collagen and tissue matrices and they possess
tral corneal buttons, we found that sphere forma- the ability to elicit a wound healing response
tion did indeed occur from central corneal cells and appear to show an appropriate level of
but at a diminished rate of 1:2 compared to migration, differentiation and division to repop-
sphere-forming ability in peripheral corneal/lim- ulate decellularized tissue surfaces. Additionally,
bal extracts [56, 60]. This sphere-forming abilitythey do not appear to adversely change their
is further diminished in keratoconic cell extractsbehavioural characteristics when applied to dis-
where we seldom observed sphere-forming cells eased tissue matrix.
from keratoconic tissue. On the rare occasions There are a number of ways in which to
where sphere formation was successful, the apply stem cell-based therapies which in the
spheres did not survive as long as those formed eye currently rely largely on ex vivo expansion
from normal tissue extracts in culture. When on amniotic membrane. In any case, none of the
placed onto collagen stimulus and compared current cell therapy applications address the
with spheres from normal donor tissue, kerato- cellular make-up of the expanded cell popula-
conic spheres appeared to respond much faster tions. Very few details exist of the numbers and
to the substrate with rapid cell migration and types of cells that are being grafted and where
division. This response however was not pro- the grafted cells end up both along the surface
longed, and over time the cells of the normal of the cornea horizontally and within the depth
spheres migrated out further and proliferated of the cornea vertically. Similarly there is little
more than the keratoconic spheres which could long-term evidence of whether these cells
not maintain their migratory and proliferative repopulate the limbus and maintain a fully
response. This may suggest that cells from kera- functional limbus over a prolonged period of
toconic tissue are in a perpetually heightened time. Stem cell-enriched peripheral corneal
state of wound healing response which cannot spheres formed by sphere-forming assay repre-
maintain correct corneal homoeostasis long sent a way to apply a well-defined, known cell
term. Again whether co-culture of keratoconic population for treating a range of corneal dys-
cells with normal cells would revert the diseased trophies. As cells are extracted as single cells
cells to a normal phenotype remains to be from tissue, they have the ability to be sepa-
ascertained. rated and sorted by laboratory methods such as
FACS prior to culture in sphere-forming assay,
and individual sphere cultures can be thor-
21.7 Conclusion: Potential oughly tested in vitro for their ability to respond
of Spheres for Treating LSCD appropriately prior to transplantation. Sphere
and Corneal Dystrophies cell populations could potentially be manipu-
lated for use in the treatment of different condi-
Our work with peripheral corneal- and limbal-­ tions to enhance the therapeutic effect, for
derived cells cultured in sphere-forming assay example, to maximize limbal components for
has shown that spheres cultured in this way are LSCD treatment or to enrich for epithelial stem
stem cell-enriched, discrete populations of cells cells to treat recurrent epithelial erosions.
that are not merely aggregates but dynamic enti- Although further assessment of the potential of
ties that represent a heterogeneous mix of lim- sphere-based cell therapy is required, current
bal niche components and possess unique evidence would suggest that it would be a use-
properties that allow them to react to mechani- ful adjunct to current treatments and represent
cal, environmental and chemical stimuli. To an improved method for long-term sustained
date, we have shown that these spheres hold a corneal regeneration.
314
Compliance with Ethical Requirements  10. Dhamodaran K, Subramani M, Ponnalagu M, Shetty
1. Conflict of Interest
Salim Ismail, Charles N. J. McGhee, Jennifer
J. McGhee, Ye Li, Jeremy John Mathan, Jinny
Jung Yoon, Himanshu Wadhwa, Stephanie
U-Shane Huang and Trevor Sherwin declare
that they have no conflict of interest.
2. Informed Consent/Human Studies
All procedures followed were in accordance
with the ethical standards of the responsible
committee on human experimentation (insti-
tutional and national) and with the Helsinki
Declaration of 1975, as revised in 2000.
Informed consent was obtained from all
patients for being included in the study.
3. Animal Studies
No animal studies were carried out by the
authors for this article.
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 Eye Platelet-Rich Plasma (E-PRP)
for Corneal Regeneration
Alejandra E. Rodríguez and Jorge L. Alió
22.1 Introduction
There are different situations, in which the ocu-
lar surface can be altered, from a mild form such
as dry eye or environmental exposure to a very
severe one that can even compromise vision,
reaching blindness as in the case of ocular cica-
tricial pemphigoid or Stevens-Johnson syn-
drome [1].
Some of the conditions that produce ocular
surface disorders are ocular dryness, dry kerato-
conjunctivitis, recurrent corneal erosions, persis-
tent epithelial defects, neurotrophic keratopathies,
ocular surface syndrome after LASIK, torpid cor-
neal ulcers, graft-versus-host disease and ocular
cicatricial pemphigoid [1], among others.
After the alteration or injury of the conjunc-
tival or corneal tissue, a biological process of
regeneration occurs to restore that damaged
ocular surface. This is a complex process involv-
ing the epithelial stem cells of the cornea and
the conjunctiva since they are responsible for
A. E. Rodríguez (*)
Laboratory of the Research, Development and
Innovation Department, Vissum Innovation,
Alicante, Spain
e-mail: alejandra@vissum.com
J. L. Alió
Professor and Chairman of Ophthalmology,
University Miguel Hernandez, Vissum-Instituto
Oftalmologico de Alicante, Alicante, Spain
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_22
22
homeostasis and the regeneration of the ocular
surface [2], as well as growth factors, cytokines
and elements of the extracellular matrix [3].
These natural physiological processes of res-
toration of damaged tissue are permanent activi-
ties that occur in our body without being aware of
it, but there are situations in which by the severity
of the damage or by the inability of the body
itself to resolve the situation, administration of
treatments is necessary to solve the problem.
Treatments range from the administration of
topical artificial tears to the practice of complex
ocular surface restoration surgeries such as cor-
neal transplants in their various modalities [1, 4–
7]. Within the wide range of therapeutic
possibilities available, conventional treatments
are sometimes not effective in solving the patho-
logical condition.
In this aspect, the administration of blood
products as a therapeutic option has acquired
great prominence, due to its biological composi-
tion and its ability to induce the regeneration of
affected tissues [8–10].
22.2 Platelets
Platelets were discovered in 1882 and have tradi-
tionally been presented as cytoplasmic remains
of megakaryocytes [11]. At present, they are con-
sidered to be anucleated cells that come from the
megakaryocytes of the bone marrow that, after a
317
318
complex mechanism of nuclear and cytoplasmic
maturation, become free platelets [12]. In a
healthy adult, the physiological range of platelets
is 150–400 × 103 cells/ml, with an average pro-
duction of around 1 × 1011 platelets daily [13].
The main function of platelets or thrombo-
cytes is to ensure haemostasis and prevent
blood loss when blood vessels rupture. In this
process, they adhere to the exposed sub-endo-
thelial collagen, form a platelet plug and then
activate [14].
Platelets also participate in the activation of
the coagulation cascade and secondary haemo-
stasis [15]. The accidental rupture of platelets
produces the formation of a certain amount of
thrombin, and if the generated thrombin is large
enough, the mechanisms that convert fibrinogen
into fibrin are triggered, forming the thrombus
composed of fibrin and erythrocytes. Thrombosis
causes myocardial infarction if the affected organ
is the heart [16] and apoplexy if it occurs in the
brain vessels [17].
Although central role is in the prevention of
bleeding, platelets contribute to various processes
that extend beyond haemostasis and thrombosis,
including the promotion of inflammatory and
immune responses, maintenance of vascular
integrity and contribution to healing of wounds.
Platelets can recruit leukocytes and progenitor
cells to sites of vascular injury and thrombosis;
they store, produce and release pro-inflamma-
tory products, anti-inflammatory and angiogenic
factors and microparticles in the circulation, in
addition to stimulating the generation of throm-
bin [18].
In electron microscopy, the different organ-
elles that contain the platelets are observed: mito-
chondria, peroxisomes, ribosomes, glycogen and
granules; the latter are divided into three types:
alpha, delta or dense granules and lambda [19].
The alpha granules of platelets contain more
than 30 bioactive proteins, and despite the
absence of the nucleus and DNA, platelets have
a system to perform protein synthesis and have
copies of mRNA for almost a third of known
proteins in the human genome; they process the
mRNA and efficiently translate different pro-
teins [20].
A. E. Rodriguez and J. L. Alió
These proteins perform tissue repair functions
and influence the vascular reactivity of blood
cells, angiogenesis and inflammation [21]. The
proteins secreted by the alpha granules also inter-
vene in the cellular defence against exogenous
agents through the production of signalling pro-
teins that attract macrophages.
Platelets are essential for the innate immune
response and to fight infections caused by viruses,
bacteria or other microorganisms. They help to
maintain and modulate inflammation and are an
important source of pro-inflammatory molecules
since they secrete P-selectin, tissue factor,
CD40L and metalloproteinases.
They also participate in skin diseases, aller-
gies, rheumatoid arthritis or liver disease, while,
paradoxically, platelet-rich autologous plasma
and platelet concentrates are used as an aid to
promote tissue repair and cell growth [22].
Under physiological conditions, platelets are
circulating in a non-active form, and on their sur-
face, they express a relatively small number of
molecules, which, in their activated state, will
facilitate their interaction with other platelets and
cells in their environment [13].
Platelets change shape with activation due to
complex modifications in the skeleton and cyto-
skeleton of their membrane. With activation, they
experience the release of granules, dense bodies
and the content of lysosomes.
This activation, also known as degranulation,
can be caused by a physical stimulus, chemical
or a combination of both and causes the alpha
granules to fuse with the platelet membrane,
where some of the secretory proteins (e.g. cer-
tain factors of growth) pass to the active state
and thus are released to the outside where they
bind to the receptors of the target cells (endothe-
lial cells, epithelial cells, osteoblasts, among
others) [23].
Growth factors released by activated platelets
initiate and modulate healing because they
induce chemotaxis, cell proliferation and differ-
entiation, neovascularization and deposition of
extracellular matrix [24]. Specifically, alpha
granules constitute the largest reservoir of
growth and mitogen factors that are released dur-
ing platelet activation. These factors are involved
22  Platelet-Rich Plasma (E-PRP) for Corneal Regeneration
in processes of chemotaxis, cell proliferation,
differentiation and angiogenesis, thus promoting
healing [22, 25].
22.3 Growth Factors
Growth factors (GF) are a group of substances of
peptide nature whose function is intercellular
communication at the molecular level. These are
capable of modifying cellular biological
responses as they intervene by regulating the pro-
cesses of migration, proliferation, cell differenti-
ation and metabolism and even apoptosis.
The main function of growth factors is that of
external control of the cell cycle. These promote
increased cell size by increasing the protein syn-
thesis of the cells on which they act [26].
The most important GFs coming from plate-
lets are transforming growth factor beta (TGF-β),
basic fibroblastic growth factor (FGFb), platelet-­
derived growth factor (PDGF), vascular endothe-
lial growth factor (VEGF), connective tissue
growth factor (CTGF), type 1 insulin-like growth
factor (IGF-1) and epidermal growth factor
(EGF) [27].
Growth factors act locally. Cell stimulation
can be done through an autocrine system, a
mechanism by which cells produce and respond
to the biological mediator, or by a paracrine sys-
tem in which the cell that generates the factor is
in the vicinity of the cells on which it acts.
Generally, GFs are synthesized in the form of
precursors, which is why a proteolysis process is
required for their release in the active form. The
mechanism of action that they carry out begins
when they join specific membrane receptors,
there being for each FC a receptor or set of spe-
cific receptors. The process is mediated by a sys-
tem of second messengers that activate a cascade
of signals. Thanks to this mechanism, the action
of the factors does not stop at the site of the
injury, although they have disappeared from the
environment, since they have activated the way of
second messengers.
Apart from the alpha granules of the platelets
that constitute a large reservoir of growth fac-
tors, there are other cell types that produce these
319
molecules, such as fibroblasts, osteoblasts, endo-
thelial cells, leukocytes, monocytes and macro-
phages [26].
We will describe below the most relevant GFs
involved in the ocular regeneration.
22.3.1 Platelet-Derived Growth
Factor (PDGF)
Platelet-derived growth factor (PDGF) is a known
modulator of fibroblast cell mitosis and chemo-
taxis [28]. It indirectly promotes angiogenesis
through macrophages through a mechanism of
chemotaxis [29].
In studies carried out, the expression of
PDGF-AA, PDGF-BB and PDGF-AB and their
corresponding receptors in the human cornea and
their participation in proliferation and chemo-
taxis in assays with cultures of human corneal
fibroblasts have been demonstrated, showing that
the PDGF-BB factor has a significantly higher
chemotactic effect compared to PDGF-AA or
PDGF-AB.  Due to the chemotactic effect that
PDGF presents in the healing of corneal wounds,
the hypothesis is hypothesized that said factor
regulates the chemoattractant of neutrophils,
IL-8 [30, 31].
Studies conducted with PDGFs and their
receptors (PDGFRs) in animal development and
disease have revealed their role as CF in organo-
genesis, cancer, vascular inflammation and tissue
fibrosis [32].
PDGF-BB is used to improve wound healing
in clinical practice for about 25  years.
Recombinant human PDGF-BB, called becapler-
min, has been introduced into the clinic as a topi-
cal treatment for chronic lower extremity
neuropathic diabetic ulcers [33].
It has also been used against pressure ulcers
and to accelerate healing in various surgical pro-
cedures. PDGF-BB-induced tissue repair mecha-
nisms appear to involve fibroblast proliferation,
collagen production and neovessel formation
[34] and may involve direct effects on mesenchy-
mal cells expressing PDGFR-α [35].
Its clinical efficacy has been demonstrated in
several phase III studies [36], and the combined
320
results suggest that the topical application of
PDGF-BB is safe and well tolerated. A study with
more than 900 patients confirmed that topical
treatment with PDGF improved the healing of
chronic foot ulcers of full-thickness diabetics [37].
The local administration of PDGF-BB has also
been tested in patients with severe periodontal
disease and has been found to increase biomark-
ers for bone metabolism and rotation [38] and
provide improved periodontal regeneration [39].
22.3.2 Transforming Growth Factor
Beta (TGF-Β)
The transforming growth factor beta (TGF-β) is
multifunctional and one of the most important
factors involved in modulating the behaviour of
ocular tissues. In addition, this ligand participates
in cell migration and proliferation, cell death,
protein synthesis during development and repair
of tissues among other physiological or patho-
logical processes [40].
TGF-β is an intriguing cytokine exhibiting
dual activities in malignant disease. On the one
hand, it is an important mediator of cancer inva-
sion, metastasis and angiogenesis, while on the
other hand, it exhibits antitumour functions [41].
In most cases, TGF-β improves the production
of the extracellular matrix and suppresses cell
proliferation, for example, that of epithelial cells
in the presence of other factors. On the other
hand, this GF is also able to induce the produc-
tion of other factors such as connective tissue GF
(CTGF), platelet-derived GF (PDGF), fibroblast-­
derived GF (FGF) and vascular endothelial GF
(VEGF), as well as stimulating its own produc-
tion. All of them play an important role in restor-
ing normal tissue after suffering an injury [27].
There are three isoforms of TGF-β, called
TGF-β1, TGF-β2 and TGF-β3, whose presence is
found in the tissues of mammals; however, its
expression is not uniform. As in other tissues,
overactivation of TGF-β underlies the pathogen-
esis of ocular diseases that can lead to impaired
vision and the alteration of ocular tissue homeo-
stasis. Therefore, the possibility of developing
therapeutic strategies for such diseases oriented
A. E. Rodriguez and J. L. Alió
towards the signalling pathway of TGF-β [40]
can be considered.
The TGF-β signalling pathway plays a key
role in the function of ocular fibroblasts, their dif-
ferentiation and their proliferation. The suppres-
sion of the TGF-β pathway has shown a decrease
in extracellular matrix deposition in ocular fibro-
blasts in vitro [42].
On the other hand, it has been demonstrated
that cyclosporine A inhibits TGF-β2 induced by
the myofibroblasts from human pterygium fibro-
blasts obtained from primary culture [43], so
these results could indicate the therapeutic poten-
tial of cyclosporine A against progression of the
pterygium.
22.3.3 Epidermal Growth Factor
(EGF)
The epidermal growth factor (EGF) plays a very
important role in the migration and proliferation
of the corneal epithelium to improve the pro-
cesses of tissue regeneration, as well as the stim-
ulation of the granulation tissue.
Studies show that the mitogen effect of EGF
on the proliferation of corneal epithelial cells
requires the suppression of expression of the
Pax-6 gene specific to the eye. Fibroblasts, pro-­
osteoblasts and pre-chondrocytes express a high
number of receptors for EGF [44].
On the other hand, it is known that EGF
increases the concentration of intracellular cal-
cium [Ca++]i and activates the protein kinase ERK
½ to stimulate the mucin secretion of conjuncti-
val goblet cells [45]. It is also known that scarring
of the corneal epithelium is stimulated by EGF
and is partially mediated through the 12/15-LOX-­
LXA4 pathway [46].
To achieve an ideal wound-healing process
that promotes healthy growth of tissue with less
scarring, a gel-based topical drug delivery system
composed of three different polymers chitosan,
dextran sulphate and polyvinylpyrrolidone K30
(CDP) was prepared. Applied topically, EGF evi-
denced clearly beneficial effects by accelerating
the wound healing process and producing less
scarring [47].
22  Platelet-Rich Plasma (E-PRP) for Corneal Regeneration
22.3.4 Vascular Endothelial Growth
Factor (VEGF)
Vascular endothelial growth factor (VEGF) has
been known for a long time as a potent stimulator
of blood vessel proliferation. It is an endothelial
cell mitogen, angiogenic factor and a potent
mediator of vascular permeability, in addition to
being involved in the growth of nerves [48].
In vitro experiments have shown that VEGF
and its receptors are expressed by neurons and
stimulate the proliferation of cortical neurons,
provide protection to central and peripheral neu-
rons against hypoxia-induced death and promote
axonal growth in peripheral neurons [49].
On the other hand, overexpression of VEGF is
a factor that contributes to the development of
disease. For example, solid tumours require an
increase in blood supply if they will continue to
grow beyond a certain size and tumours that
express VEGF are able to continue growing; this
process is called angiogenesis [50].
Overexpression of VEGF can also lead to vas-
cular disease in the retina and other parts of the
body. A decrease in the level of VEGF in the pul-
monary arteries has also been associated with the
condition of pulmonary emphysema [50].
Since cancer growth is stimulated by VEGF,
researchers have made numerous attempts to
decrease its expression to prevent angiogenesis
and tumour growth. Two drugs that have been
successful in slowing the progression of diseases
that depend on VEGF are bevacizumab and
ranibizumab [51].
22.4 Adhesion Molecule:
Fibronectin
Fibronectin is an adhesion protein, which is free
in plasma, and is a major constituent of the com-
position of the extracellular matrix. Plasma
fibronectin levels are approximately 300 ± 100
μg/mL [52].
This protein has been widely studied for its
ability to interact with various cells and macro-
molecules, influencing its behaviour and the bio-
chemical reactions of various systems, as well as
321
a wide variety of physiological processes such as
phagocytosis, proliferation, adhesion and cell
migration [53, 54].
Fibronectin is able to bind fibrin, collagen,
glycosaminoglycans, heparin, certain proteogly-
cans and cellular plasma membrane proteins such
as integrins. Therefore, it establishes connections
between molecules of the extracellular matrix
and between molecules of the cells with the
extracellular matrix. Fibronectin molecules can
appear forming insoluble protein fibres in the
connective tissues or soluble in the plasma of
body fluids, such as blood [55].
Among its main functions is to intervene in
the remodelling of tissues during embryogenesis
and participate in the healing process of a lesion
after the formation of a fibrin clot, generated by
the activation of the haemostatic system [56].
22.5 Properties of Blood
Derivatives with and Without
Platelets
The blood products have shown their ability to
improve healing and stimulate the regeneration
of different tissues, an effect that is due to the
growth factors and bioactive proteins that are
synthesized and present in the blood [21].
Platelet-rich plasma (PRP) is a blood deriva-
tive obtained by centrifugation of whole blood
characterized by having a high concentration of
platelets [29].
The most important growth factors found in
platelet-rich plasma, from which cells they derive
and which are their main functions, are described
in [57].
In addition to platelets, the plasma contains
some cell adhesion molecules such as fibrin, fibro-
nectin and vitronectin which provide structural
support for cell migration, proliferation and three-
dimensional growth in the tissues in which they
act [23]. On the other hand, the cytokines PF4 and
CD40L present in the plasma promote tissue repair
and influence the vascular reactivity of blood cells,
angiogenesis and inflammation [21].
Different in vitro assays have been performed
to study and try to understand the effect that dif-
322
ferent types of blood preparations based on plate-
lets produce in cells and tissues.
Hartwig et  al. studied the epitheliotrophic
capacity of platelet and serum concentrates in
human cells of the corneal epithelium. After
stimulation they observed a significant increase
in cell proliferation of the platelet concentrate.
The effects of migration and cell differentiation
were positive with both preparations but slightly
higher with serum [58].
Following this line of work, in 2005 they car-
ried out another study also on human epithelial
cells in an in vitro cell culture model. This time
they compared the epitheliotrophic capacity of
the cells when faced with fresh frozen plasma
(without platelets) and with serum. The results of
cell proliferation, migration and differentiation
were significantly better in serum than in fresh
frozen plasma (without platelets). In this study,
none of the preparations studied contained plate-
lets, but it was inferred that the greater epithelio-
trophic capacity of the serum could be due to the
higher concentration of proliferation mediators
such as the EGF and PDGF growth factors and
the higher vitamin A content [59].
In a subsequent trial, they compared the cor-
neal epitheliotrophic capacity using three differ-
ent blood preparations: serum, fresh frozen
plasma and a platelet concentrate that was acti-
vated with thrombin. In this case, the cell prolif-
eration results were better with the platelet
concentrate than with the rest, while the serum
showed better results of migration and cell dif-
ferentiation [60].
The plasma rich in growth factors (PRGF) is
another blood derivative, which as its name indi-
cates has a high concentration of growth factors;
and due to its preparation, it does not contain
platelets [61].
Studies conducted with cultures of human
fibroblasts obtained from the skin, synovium and
tendon revealed a significant proliferative
response in all these cells when stimulated with
different preparations of PRGF.  The maximum
proliferation occurred with concentrations of
PRGF from twice to four times the initial. They
also observed that PRGF increased the synthesis
of hyaluronic acid but that it did not alter the pro-
A. E. Rodriguez and J. L. Alió
duction of type I collagen and that the synthesis
of VEGF occurred only in the tendon cells. They
concluded that the biological effects of PRGF
may depend on the concentration of platelets
applied and the type of cells used [62, 63].
More recent trials have studied the biological
effects of PRGF and autologous serum in cul-
tures of corneal stromal human keratocytes (HK)
and conjunctival fibroblasts (HConF). The results
showed higher significant levels of all the growth
factors analysed in the PRGF when compared
with in the autologous serum. It was also observed
that PRGF produced a significant increase in the
biological results of both HK and HFCF cells and
reduced fibroblast differentiation to myofibro-
blasts induced by TGF-β1 in contrast to autolo-
gous serum. This latter finding suggests that
PRGF can improve the treatment of superficial
wound healing by minimizing scar formation
compared to AS while promoting cell prolifera-
tion and migration [64, 65].
Freire et al. did a study comparing the effects
of autologous serum, PRP and PRGF on cultures
of human corneal cells. All the compounds had
high levels of growth factors, but the EGF values​​
were higher with PRGF than with the rest. The
cell proliferation rates were higher with autolo-
gous serum and with PRGF [66].
Subsequently, the same authors conducted an
experimental trial in rabbits, in which they
induced a corneal erosion to be treated with the
same three previous blood derivatives. All the
compounds improved healing, but the corneas
treated with autologous serum and PRGF healed
more quickly [67].
Experimental studies were also carried out but
with clinical sense performing photorefractive
keratectomy (PRK) to C57BL/6 mice to assess
the healing process after the application of
plasma rich in growth factors. The control ani-
mals showed a higher corneal haze index, and the
epithelium that has been generated was hyper-
plastic, while those treated with PRGF presented
a greater regeneration of the corneal epithelium
and lower haze [68].
More recent studies have demonstrated the
ability of s-PRGF to promote the proliferation
and migration of rabbit epithelial and corneal
22  Platelet-Rich Plasma (E-PRP) for Corneal Regeneration 323

cells and to accelerate corneal re-epithelialization

rich fibrin preparation (PRF) with different blood
in in vivo assays [69]. derivatives. In this study, it determines the con-
There are other studies in which blood deriva- centrations of three pro-inflammatory cytokines
tives rich in platelets have consistently shown to (IL-1β, IL-6 and TNF-α), the anti-inflammatory
enhance the proliferation, migration and differen- cytokine IL-4 and the angiogenesis promoter
tiation of stem cells, with varied applications in VEGF. Their results revealed that the PRF could
tissue engineering and regenerative medicine be an immune regulation node with inflammatory
[70–73]. retro-control capacity. This concept could explain
In addition to the aforementioned blood prod- the reduction of postoperative infections when
ucts, based on plasma with or without platelets, the PRF was used as a surgical additive [78].
we find autologous serum (AS) that is also used Other trials have demonstrated the effects of
in the field of ophthalmology. Autologous serum, PRP on the release of cytokines from monocytes
although it does not contain platelets, presents and the generation of lipoxin A4 (LXA4) which
variable amounts of growth factors, which is the is an endogenous anti-inflammatory. In this case,
reason why it is included in this section [74]. PRP promoted significant changes in the release
In vitro toxicity studies using human corneal of cytokines and pro-inflammatory chemokines
epithelial cells have shown reduced toxicity when mediated by monocytes and increased lipoxin
compared to hypromellose without a preserva- A4. This suggests that PRP can suppress cyto-
tive, used to prepare artificial tears [75]. kine release, limit inflammation and thus pro-
Several experimental studies have also shown mote tissue regeneration [79].
their regenerative properties. Autologous serum Mazzocca et  al. evaluated in  vitro the anti-­
enhances the regenerative effect of mesenchy- inflammatory effects of PRP, ketorolac and meth-
mal stem cells in damaged rat corneas [76]. In ylprednisolone in human umbilical vein
addition, it promotes the migration and differen- endothelial cells. Although PRP and ketorolac
tiation of corneal epithelial cells [58, 59]. AS did not produce such a significant reduction in
also presents high amounts of EGF and induces markers of cellular inflammation as methylpred-
the proliferation of epithelial cells of the human nisolone, they reduced cellular inflammation
cornea [66], promoting corneal reepithelization compared to control. Therefore, they concluded
in rabbits’ eyes with previously debrided epithe- that PRP may have clinical application as an
lium [67]. injectable anti-inflammatory drug [80].
In addition, it has been observed that AS On the other hand, Schar et al. studied the lev-
increases the conjunctival expression of mucin els of various growth factors and the inflamma-
MUC5AC, increasing as a consequence the gob- tory cytokine IL-1β in platelet-rich plasma
let cell’s density in the conjunctiva, mainly in preparations with leukocytes (L-PRP). In this
patients with severe dry eye [77]. trial, the levels of IL-1β and VEGF were signifi-
cantly lower in the L-PRP than in the autologous
serum, demonstrating its lower pro-inflammatory
22.6 Inflammation and Blood and angiogenic power [81].
Derivatives Following this same line of work, Anitua et al.
performed the same assay, comparing a platelet-­
There are many research groups that have studied rich plasma scaffold with leukocytes (L-PRP) with
the markers of inflammation in different blood a PRGF scaffold without leukocytes. The concen-
preparations with platelets with variable results, tration of all the growth factors released was com-
mainly due to the differences in PRP preparation parable between both methods, with no differences
methods [73]. between them. But after 3 days of incubation in the
In fact, there are several who are in favour of L-PRP scaffolds, the VEGF levels decreased dras-
the presence of leukocytes in their formulations, tically. On the other hand, the inclusion of leuko-
such as Dohan et al. who compared their platelet-­ cytes in the L-PRP scaffolds produced a significant
324
increase in the pro-­inflammatory cytokines, IL-1β
and IL-16, with respect to the PRGF scaffolds that
presented very low values. Scaffolds of PRGF
remained stable during the 8  days of incubation
[82]. The subsequent addition of leukocytes to the
PRGF scaffolds stimulated a more pro-inflamma-
tory environment, secreting more quantities of
pro-­inflammatory cytokines such as IL-1β, IL-6,
IL-8, IL-16 and TNF-α, altering the properties of
fibrin [83].
The inactivation of plasma by heat has also
been studied at 56 °C in order to reduce the activ-
ity of complement and immunoglobulin E and in
this way be able to apply it in patients with auto-
immune diseases. The cell proliferation and
migration assays gave good results with the
plasma heated to 56 °C, compared to the unheated
plasma. On the other hand, most of the proteins
and morphogens involved in healing were ade-
quately preserved [84].
To evaluate the anti-inflammatory power of
the plasma rich in growth factors, the blood of
patients with graft-versus-host disease was con-
fronted with ocular surface fibroblasts in a pro-­
inflammatory environment (IL-1β and
TNF-alpha). The results concluded that even in
this environment, the plasma rich in growth fac-
tors had regenerative power and that it also had
an anti-inflammatory effect [85].
In a further step towards controlling the anti-­
inflammatory effects of blood derivatives, some
authors have studied the effects of allogeneic
blood products.
Stenwall et al. have investigated the composi-
tion of cytokines and the anti-inflammatory effects
of allogeneic serum preparations to assess their
best use as eye drops. For this purpose, the serum
of 15 donors was inactivated, and then the regula-
tory capacity of inducing T cells was studied [86].
Although the vast majority of blood deriva-
tives used in ophthalmology is of autologous ori-
gin, there are some authors who have treated
patients with serum of allogeneic nature.
Chiang et  al. applied allogeneic serum from
relatives to patients with persistent epithelial defect
in whom it was not feasible or appropriate to use
autologous serum, and none of the patients pre-
sented an adverse effect during the treatment [87].
A. E. Rodriguez and J. L. Alió
On the other hand, other researchers have
applied allogeneic serum in patients with severe
dry eye who suffered from graft-versus-host dis-
ease, in whom it was not possible to apply autolo-
gous serum. The patients improved their
symptoms and signs of dryness and had no
adverse effects [88, 89].
22.7 Antimicrobial Activity
of the Platelet-Rich Plasma
Searching more properties and biological effects
of platelet-rich plasma, several authors have stud-
ied its antimicrobial capacity.
In addition to the large amount of proteins
and growth factors, they have discovered that
platelet-­rich plasma releases immunomodulatory
agents with antimicrobial activity such as plate-
let microbicidal proteins (PMPs), thrombocydins,
RANTES, connective tissue-activating peptide
and platelet factor 4 (PF-4), among others [90, 91].
Other authors have directly studied the inhibi-
tory effect of platelet concentrates against differ-
ent cultures of microorganisms. Drago et  al.
demonstrated the inhibitory capacity of PRP
against strains obtained from the oral cavity of
Enterococcus faecalis, Candida albicans,
Streptococcus agalactiae and Streptococcus ora-
lis, but not Pseudomonas aeruginosa [92]. In
general, most authors agree on the inhibitory
effects of PRP against Staphylococcus aureus
and Staphylococcus epidermidis whether they are
methicillin-sensitive or methicillin-resistant [92–
94] and Streptococcus agalactiae [92, 94]; but
they differ in the antimicrobial activity of PRP
against Escherichia coli [94, 95] and Enterococcus
faecalis [92, 95]. All agree on the inability of
PRP to inhibit the growth of Pseudomonas aeru-
ginosa [92, 95].
22.8 O
 btaining Blood Derivatives
Used in Ophthalmology
The denomination and way of obtaining products
prepared with blood derivatives in the literature is
very diverse and leads to constant misunderstand-
22  Platelet-Rich Plasma (E-PRP) for Corneal Regeneration
ings in readers, scientists and reviewers [96]; so
we will try to clarify this aspect as much as
possible.
Initially, the term “PRP” was described by
Marx [23, 97] and, in general, refers to a two-step
centrifugation process, whereby a platelet-rich
plasma is obtained which is subsequently acti-
vated with calcium chloride or thrombin, imme-
diately before use. This PRP has a gel or clot
consistency or even more solid fibrin mesh and
had always been used in surgical procedures as
an adjunct to healing, in addition to direct appli-
cations on skin ulcers in diabetic patients.
Given that there is a huge variety of systems to
obtain autologous blood derivatives, with vari-
able amounts of platelets and growth factors, we
will focus on those used in the field of ophthal-
mology, which are those related to the topic of
this chapter.
22.8.1 E-PRP (Eye Platelet-Rich
Plasma)
The acronym E-PRP comes from its denomina-
tion in English as “Eye-PRP” (eye platelet-rich
plasma) and refers to the platelet-rich plasma that
is used in ophthalmology.
The term E-PRP was introduced by Alio et al.
[8] to differentiate it from the term “PRP” that
had been used in its previous publications related
to this topic [98–100] and that caused confusion
in the literature. The process of obtaining E-PRP
is different from that used by Marx et al. [23].
The E-PRP is very versatile since it is possible
to use it as eye drops (without activating), as a clot
(activated) or as an autologous membrane of fibrin
(activated). The autologous E-PRP in the form of
eye drops is used as ocular topical treatment for
various pathologies of the ocular surface [98–100]
and in the form of a clot or autologous fibrin mem-
brane in surgical procedures for the reconstruction
of the ocular surface [98, 101, 102].
E-PRP for ophthalmological use is carried out
through a one-step centrifugation process, and
the final rate of platelet concentration will depend
on whether it will be used as eye drops, as a clot
or as an autologous fibrin membrane.
325
Briefly, the process to prepare the E-PRP is:
–– Blood collection in ten sterile tubes, each of
10  ml with 1  ml of 3.2% sodium citrate
(Monovette, Stardest) to prevent coagulation.
–– Centrifugation in conventional laboratory cen-
trifuge: 10 min at 1600 rpm [9]. These condi-
tions may vary depending on the centrifuge
and the size of the tubes used.
–– Separation of platelet-rich plasma in a laminar
flow hood, in strict conditions of sterility and
with disposable material.
–– 4 ml of E-PRP are placed in each of ten sterile
glasses or plastic dropper bottles (in case it is
prepared as eye drops).
–– If activation is needed: 50  μl 10% calcium
chloride per 1  ml of E-PRP (clot or fibrin
membrane).
–– To have a more exhaustive control of the
obtaining process, a haemocytometer or blood
cell counter can be used to know the haemato-
logical parameters of the whole blood before
the centrifugation (haematocrit, RBC count,
leukocytes, platelets) and after the preparation
of the E-PRP (count of red blood cells, leuko-
cytes and platelets).
Under these conditions, the resulting E-PRP
has a platelet enrichment index of 1.6–2.5 times
above the baseline values ​​(whole blood). The
E-PRP eye drops is a 100% autologous com-
pound since it does not contain any additives.
In Fig. 22.1 the different phases of the E-PRP
preparation can be observed from the centrifuged
blood.
Patients are instructed on how to wash their
hands before using the product, and how to
­perform a correct application avoiding contact
with the dropper with any part of the eye. These
precautions are carried out in order to avoid fur-
ther contamination.
A bottle of E-PRP (4 ml) in use can be kept at
4  °C for a week, after which it should be dis-
carded. The rest of the E-PRP bottles should be
kept frozen at −20  °C for a maximum of
3 months.
This system of preparing platelet-rich
plasma is called “open” since it is necessary to
326 A. E. Rodriguez and J. L. Alió

Conservation:
1 week at 4° C
3 months at –20° C

4 ml

PRP E-PRP eyedrop

Cl2Ca

30
min
37° C
Centrifugated PRP
E-PRP clot
blood
Autologous
thrombin
Cl2Ca

60 min
37° C
PPP
Platelet poor Fibrin membrane
plasma ø: 18–22mm – Thickness: 1mm
Immediate use

Fig. 22.1  Diagram with the steps followed in the preparation of the different types of E-PRP: eye drops, clot and fibrin
membrane

open the tubes with blood for handling, which
is why they must be performed under sterile
conditions.
22.8.2 Closed Systems for Obtaining
PRP
There are other methods to obtain platelet-rich
plasma with a “closed” system; in these cases the
tubes are manipulated to obtain the PRP, but
without the opening of them. They use syringes
and needles.
These systems are commercial kits, consisting
of specially designed blood collection tubes, with
their own consumables. In general, you must pur-
chase the specific centrifuge for that commercial
kit, as it comes with the established ­centrifugation
parameters and cannot be modified by the user.
22.8.2.1 RegenKit® BCT
It is a closed system for the production of plasma
rich in platelets. The RegenKit® BCT belongs to
the company RegenLab USA. It is approved by
the FDA (Food and Drug Administration). The
name of the product is A-PRP, acronym of autol-
22  Platelet-Rich Plasma (E-PRP) for Corneal Regeneration
ogous platelet-rich plasma. Each Regen BCT
tube with 8  ml of blood generates 4–5  ml of
autologous plasma enriched with platelets, at a
concentration of 1.6 of the basal levels [96].
22.8.2.2 ANGEL®
ANGEL is a closed system to obtain platelet-
rich plasma from the supplier Arthrex. This
device is based on a centrifugal equipment plus
software that uses a system of tubes and pro-
grammed centrifuges, to obtain the PRP.  It
requires user experience and allows selecting the
degree of platelet concentration in a wide range
(3× to 18×). The device that acts as a plasma
fractionator is the centrifuge itself with a com-
plex set of fungibles. The content is not exposed
to the environment [103].
22.8.2.3 PRGF Endoret®
The preparation of this plasma rich in growth fac-
tors is carried out with the closed system PRGF
Endoret®, a commercial kit from BTI
(Biotechnology Institute, S.L., Miñano, Álava,
Spain). It is necessary to acquire the kit and the
specific centrifuge (BTI System IV, Vitoria,
Spain). The process is based on a double centrifu-
gation and activation technique.
Briefly the steps required are the following:
–– First centrifugation: to obtain the platelet-rich
plasma, using a specific centrifuge (580 g for
8 min).
–– Plasma collection and activation (products
provided in the kit).
–– Incubation of the plasma in glass tubes at
37 °C for 1 h.
–– Second centrifugation: 1000 g for 10 min.
–– Separation of plasma rich in growth factors
and storage in mini vials.
–– It can be stored up to 3  months frozen at
−20 °C [104].
22.8.3 Autologous Serum
Autologous serum, unlike plasma, is obtained
from blood without anticoagulant. Since all cel-
lular components are trapped in the clot, the
327
autologous serum lacks platelets and any other
cells. It is considered an “open” method since for
its preparation, it is necessary to open the tubes to
manipulate the blood.
Although the methods used to obtain autolo-
gous serum do not usually follow standardized
protocols, except very few [105], and the results
obtained from the literature are extremely dispa-
rate, the basic steps for obtaining autologous
serum would be:
–– Spontaneous coagulation of the blood
extracted without anticoagulant
–– Incubation for 1 h at room temperature
–– Centrifugation
–– Collection of supernatant fraction
–– Dilution
–– Conservation
For its preparation, blood is used without
anticoagulant. The blood is allowed to coagulate
spontaneously in the tubes for 1 h at room tem-
perature and subsequently centrifuged to release
the serum from the clot with red blood cells and
leukocytes. Some authors prefer to wait up to
2 h [105].
With respect to centrifugation, there are dis-
crepancies in the literature regarding the amount
of revolutions per minute (rpm) used and the time
of centrifugation used for their separation [67,
105–107]. A standard option would be 3000 rpm
for 15 min, which does not produce haemolysis
and separates an adequate amount of serum
(approximately half of the total blood volume)
while maintaining the unaltered properties [66].
Most authors perform the preparation of autol-
ogous serum with fresh serum, continuing with
the procedure immediately after its separation
[59, 105, 108–110]. But here there is no consen-
sus, and some authors choose to freeze the serum
at −80 °C to prepare it at another time [107].
In relation to the subsequent dilution that is
made to the AS, as expected, there is also no
agreement between the authors. Some of them
opt for dilution at 20% [105, 107, 111], and oth-
ers use it at 50% [75, 110, 112–114]. Even some
of them also use it 100% when they do not obtain
the expected results [75]. Most authors use 0.9%
328
physiological saline for dilution, while others use
balanced saline solution (BSS) [115] or sodium
hyaluronate [112].
On the other hand, some carry out a filtering
after its preparation to eliminate any possible
contamination. To do this, they use a syringe with
0.22 μm pore filters that ensure the elimination of
bacteria [105, 112, 116].
With respect to conservation, most agree that
keeping it for up to 3 months frozen at −20 °C
does not alter the properties of autologous serum
[105, 109, 110]. Some authors even assert that
storage at −20 °C for 9 months does not alter the
concentration of growth factors [74].
22.9 Clinical Applications
of Blood Derivatives
The ocular surface comprises the corneal and
conjunctival tissue including all its cellular lay-
ers. These structures interact with others, such as
the eyelids with their lacrimal and Meibomian
glands to maintain an adequate tear film and con-
tribute to eye homeostasis. There are many spe-
cific situations and pathologies that affect this
complex balance producing a decompensation
and altering the surface of the eye accordingly. In
this sense, the most frequent pathology that
affects the ocular surface is the dry eye syndrome
from its mild to very severe states in which the
vision of patients is compromised [117].
Common treatments include an immense vari-
ety of artificial tears, lubricants with lipid con-
tent, liposomal spray, inserts, anti-inflammatories,
immunosuppressant such as cyclosporine A or
extracellular matrix regenerators, in addition to
other procedures such as occlusion of lacrimal
punctures or the most recent treatments with
intense pulsed light (IPL) [118, 119].
Therapeutic strategies in ocular surface altera-
tions are increasingly focused on the use of
multiple-­action, less artificial treatments without
potential allergens such as preservatives or other
products that could induce toxicity in a very vul-
nerable ocular surface. These therapies must be
effective in reducing symptomatology and pre-
venting late complications, with little or no aller-
A. E. Rodriguez and J. L. Alió
gic response, without side effects and without
altering eye homeostasis.
Blood derivatives for the treatment of ocular
surface diseases have been used for a long time
with very promising results. One of the first
records in the field of ophthalmology belongs to
Rosenthal et al. [120], who in 1975 used a mix-
ture of autologous platelets, fibrinogen and
thrombin as an ocular surface adhesive for the
fixation of lamellar keratoplasties in rabbits.
One of the most popular blood preparations
and used to date in the field of ophthalmology is
autologous serum. Although there are older
records [121], it was when Fox et al. [116] and
Tsubota et  al. [5] published their work for the
treatment of dry eye with Sjögren’s syndrome
that the use of autologous serum became more
popular. Since that time, a great variety of studies
have been carried out to demonstrate its clinical
efficacy in the treatment of dry eye, epithelial
defects, dry eye post LASIK and other alterations
of the ocular surface [58, 75, 105, 107, 108,
122–125].
But it has been recently that several studies
have been published comparing the effects of
autologous serum with other blood derivatives
such as PRP or PRGF [66, 67, 126].
The first author to use platelet-rich plasma
(PRP) in the field of ophthalmology was Alio
et al. in 2007 to treat neurotrophic corneal ulcers
[98]. He then continued using E-PRP eye drops
for the treatment of dry eye [100], in the postocu-
lar surface LASIK syndrome [99] and other ocu-
lar surface alterations [8, 9].
In their last study with moderate to severe
cases of dry eye disease, Alio et al. evaluated the
use of autologous platelet-rich plasma (E-PRP)
eye drops as monotherapy. Three hundred sixty-­
eight patients with moderate to severe dry eye
disease (DED) were included. Subjects were
classified as evaporative DED (EDED) or aque-
ous deficient DED (ADDED). Two hundred
ninety-seven (80.7%) patients were women, and
71 (19.3%) were men. Two hundred thirty-two
(63%) patients had EDED, while 136 (37%) had
ADDED.  After 6  weeks of monotherapy treat-
ment with autologous PRP, dry eye symptoms
improved in 322 (87.5%) cases. A decrease of
22  Platelet-Rich Plasma (E-PRP) for Corneal Regeneration 329

corneal fluorescein staining (CFS) was observed a

in 280 (76.1%) patients. One hundred six (28.8%)
patients improved at least 1 line of BCVA.  The
scores in the Ocular Surface Disease Index
(OSDI) and the Oxford scale of corneal fluores-
cein staining decreased statistically after the
treatment (p  <  0.05). Regarding the number of
rounds of E-PRP treatment, of the 368 patients
treated, 237 (64.4%) received only one round of
PRP (6  weeks consecutively) and 131 (35.6%)
between two and five rounds [127]. b
Recently, Alio et al. published another work
to evaluate the efficacy of autologous platelet-
rich plasma (E-PRP) eye drops for the treat-
ment of chronic ocular surface syndrome (OSS)
following laser in situ keratomileusis (LASIK)
[128]. It was a prospective interventional clini-
cal study (Registration NCT03322917) what
included 156 eyes of 80 patients. 89.6% of the
patients who had positive CFS before treatment
showed a decrease in at least one quadrant
Fig. 22.2 (a) Eye of a patient suffering OSS post LASIK
to total disappearance on CFS.  Conjunctival before the E-PRP treatment. (b) Same patient than A after
hyperaemia improved in 93.3% of the patients receiving E-PRP during 6 weeks
with previous signs of ocular surface inflam-
mation. There was a significant improve-
ment in logMAR CDVA from 0.14  ±  0.19 to Fig. 22.3 shows the corneal ulcers of two
0.06  ±  0.12 (p  =  0.000), and 74 (71.4%) eyes patients post keratoplasty surgery, before and
improved at least 1 line in CDVA.  They con- after the treatment with E-PRP.
clude that monotherapy with autologous E-PRP We can see the eye of a patient before and
is a well-tolerated, safe and effective treatment after the treatment with E-PRP during 6 weeks.
for the management of post-­LASIK ocular sur- In addition to topical applications such as eye
face syndrome. drops, E-PRP can be prepared in the form of clot
In Fig.  22.2 we can see the eye of a patient or fibrin membrane and can be used as an adju-
suffering OSS post-LASIK before and after the vant in surgical processes of ocular surface
treatment with E-PRP during 6 weeks. reconstruction. In some cases of corneal perfora-
Autologous E-PRP has been also used for the tion, they have used the autologous fibrin mem-
treatment of dormant corneal ulcers secondary to brane with E-PRP clots at the site of injury to seal
corneal surgery and unresponsive to conventional the perforation and recover that cornea [101]. For
treatment [129]. A total of 44 eyes of 28 patients this purpose, both the fibrin membrane and the
were included in the study and treated with autol- E-PRP clot were prepared with the patient’s own
ogous platelet-rich plasma during 6 weeks, after blood just before the operation. Nylon 10-0
presenting corneal ulcer post keratoplasty, refrac- stitches were used to fixate the fibrin membrane
tive surgery, cross-linking or chronic postsurgical to the conjunctiva, and then the E-PRP clot was
corneal oedema. In all, 40 (90.9%) experienced placed over the corneal perforation, underneath
an improvement in their symptoms, 28 patients the fibrin membrane. A temporal partial tarsor-
(65.1%) improved their visual acuity and 26 rhaphy was performed at the end of the proce-
(59.09%) had a decrease in the size of the ulcer or dure. The fibrin membrane was present over the
even a total closure. corneal surface for the first 3–5  days and then
330 A. E. Rodriguez and J. L. Alió

a b

c d

Fig. 22.3 (a) and (c) show the corneal dormant ulcers of two patients’ post-keratoplasty surgery. In (b) and (d), the
corneas appear regenerated after 6 weeks of treatment with E-PRP

gradually disappeared. No evidence of infection
or inflammation was detected, and in all cases,
the corneal perforation was sealed.
In others cases, they have used E-PRP clots
together with a collagenous patch derived from
bovine pericardium (Tutopatch®) to repair perfo-
rated corneas [102]. Six patients with perforated
corneal ulcers were treated with this procedure,
and they were followed up for 3 months with no
evidence of relapses or perforations in five of
them.
Before the implementation of the use of
Tutopatch® or autologous fibrin membrane, Alio
et al. also used this surgical reconstruction tech-
nique together with amniotic membrane [98].
Other collaborators have also used this tech-
nique of surgical reconstruction of the ocular
surface with clots and autologous fibrin mem-
brane based on E-PRP [130, 131], as well as in
the form of eye drops to see the regeneration
after LASIK [132].
Kim et al. used PRP to treat epithelial defects
that did not resolve after corneal infections and
compared it with autologous serum. The healing
rates of the corneal epithelium were significantly
higher with PRP than with autologous serum
[133]. Lee et al. used the same PRP preparation
of Kim et  al. (2012) for recurrent corneal ero-
sions, being effective in decreasing the recur-
rence rate [134].
In addition, PRP has been injected into the
lacrimal glands of patients with severe lacrimal
dysfunction and severe dry eye, improving tear
production and decreasing associated keratitis
[135]. Other authors have successfully used PRP
as adjuvant during macular hole surgery in
patients with high myopia [136]. Platelet deriva-
tives have also been used in subconjunctival infil-
trations for the treatment of corneal causticity
[137].
The other blood derivative, PRGF, has been
used for multiple purposes by Anitúa et al. since
22  Platelet-Rich Plasma (E-PRP) for Corneal Regeneration
1999 [61] and has been employed since then in
several areas of medicine [138–140]. But it was
not until 2010 that the PRGF began to be used in
the field of ophthalmology. López-Plandolit et al.
used PRGF diluted 50% to treat persistent cor-
neal epithelial defects in 18 patients. The results
showed a recovery of the epithelial defects in
85% of the cases [141]. Subsequently, the same
authors evaluated the results of the PRGF diluted
to 20% in cases of dry eye. They observed a
reduction in symptoms and an improvement in
squamous metaplasia, although this was not sta-
tistically significant [141].
On the other hand, Merayo et  al. used PRGF
Endoret preparations for the treatment of patients
with dry evaporative eye [142] and in patients with
ocular surface disorders refractory to conventional
treatments with satisfactory results [143].
More recently, Sánchez-Ávila et  al. used the
PRGF to treat neurotrophic keratitis in stages 2
and 3. The majority of patients resolved the cor-
neal defect, and one patient presented an adverse
effect [144].
22.10 Characterization of E-PRP
Although E-PRP has been used widely in the
clinic with excellent results, there was not enough
information regarding the determination of the
concentrations of the most important growth fac-
tors and its adequate conservation for its later use
as a treatment for the different affections of the
ocular surface.
To achieve these objectives, the concentrations
of platelets, leukocytes and red blood cells in the
whole blood and E-PRP were determined, and the
main growth factors of E-PRP and also the fibro-
nectin adhesion protein were quantified. ELISA
immunoassays were performed for each molecule
(eBioscience, Bender MedSystems GmbH,
Vienna, Austria). In parallel, in order to determine
the biological stability of the autologous E-PRP
and demonstrate that it retained its biological
potential after storage at −20  °C, the concentra-
tions of the growth factors of fresh E-PRP and fro-
zen E-PRP to −20 °C for 3 months, with or without
subsequent centrifugation, were compared.
331
The data shown below are provided by our
own work developed in the Laboratory of
the Research, Development and Inno­
vation Department of Vissum Instituto
Oftalmológico de Alicante and Vissum
Innovation (Spain), and they are in process
of publication in a peer-reviewed journal.
22.10.1  Results of E-PRP
Characterization
Blood from different healthy volunteers was used
to prepare E-PRP.  The concentration of the
growth factors PDGF-BB, TGF-β1, EGF,
VEGF-A and fibronectin was determined in each
of the four treatments including fresh E-PRP,
fresh spin, frozen E-PRP at −20 °C for 3 months
and frozen at −20  °C for 3  months and spin
before the ELISA tests.
Platelets in the E-PRP concentrated almost
twice, according to the concentration index
obtained from 1.89. The red blood cells in the
E-PRP obtained were almost insignificant com-
pared to the whole blood. The reduction observed
was greater than 99%, and the leukocyte values
in E-PRP were very low compared to the whole
blood. The reduction was of 82% with respect to
baseline values. The increase of platelets and
reduction of red and white blood cells in the
E-PRP is statistically significant regarding to the
baseline (p ≤ 0.001).
In Table 22.1, we can see the values of the cell
count before and after the preparation of the
E-PRP.
Growth factor concentrations in E-PRP were
analysed on the day of the collection (fresh sam-
ples) and after have been stored frozen at −20 °C
for 3  months with or without posterior
centrifugation.
On the other hand, it was observed that all the
concentrations of the GFs increased considerably
in the fresh or frozen E-PRP with respect to the
plasmatic levels (Table 22.2). The exception was
the fibronectin whom values increased slightly in
the fresh or frozen E-PRP, with respect to the
basal plasma levels.
332 A. E. Rodriguez and J. L. Alió

Table 22.1  Blood cells count before and after the E-PRP preparation
Platelets × 103/μl Red cells × 106/μl White cells × 103/μl
Whole blood E-PRP Whole blood E-PRP Whole blood E-PRP
Mean 265.17 495.00 4.11 0.04 5.43 0.97
SD 69.75 95.14 0.32 0.01 0.70 0.30
SD standard deviation

Table 22.2  Comparison of levels of growth factors in plasma and in fresh and frozen E-PRP
Molecules Plasmaa Fresh E-PRPb Frozen E-PRPb
PDGF-BB <2000 pg/ml 5065.83 pg/ml 11961.67 pg/ml
TGF-β 1725 pg/ml 84165.71 pg/ml 74710.36 pg/ml
EGF 110–350 pg/ml 338.24 pg/ml 672.25 pg/ml
VEGF 47.3 pg/ml 274.25 pg/ml 494.49 pg/ml
Fibronectin 400 μg/ml 549.02 μg/ml 544.10 μg/ml
Values obtained from the eBioscience Kit protocol
a

Values obtained from our experiments

b

Fig. 22.4 Concentration EGF

* P value £ 0.05
of epidermal growth 900.00
factor (EGF) in E-PRP *
after the application of 800.00 **
four procedures
700.00
***
600.00

500.00 **
mg/ml

*
***
400.00

300.00

200.00

100.00

0.00
Fresh Fresh-spin Frozen Frozen-spin

The concentrations of EGF and PDGF-BB
obtained in the E-PRP were significantly higher
(p  <  0.05) after freezing it during 3  months at
−20 °C (Figs. 22.4 and 22.5).
The concentration levels reached by the
growth factor TGF-β1 were significantly higher
(p < 0.05) in the fresh and frozen at −20 °C, over
the fresh and spin and frozen at −20 °C and spin
(Fig. 22.6).
However, no significant differences were
observed among the treatments for the growth
factor VEGF-A and fibronectin (Figs.  22.7
and 22.8).
There was a positive and significant correla-
tion between the growth factor PDGF-BB and
platelets (p  <  0.05). In addition, there was also
strong and significant positive correlation
between TGF-β1 and VEGF-A (p < 0.01), which
showed a relationship between these two GF.
22.10.2  Discussion of E-PRP
Characterization
There are different opinions about which platelet
concentration index is appropriate to use platelet-­
rich plasma as a therapeutic tool in the field of
tissue regeneration.
On the one hand, there are those who prefer
high or very high concentration indices. In this
case we refer to those who use platelet concen-
trates more than double the baseline values [145]
22  Platelet-Rich Plasma (E-PRP) for Corneal Regeneration 333

PDGF-BB
* P value £ 0.05
18000.00
**
16000.00 *

14000.00

12000.00
mg/ml

10000.00

8000.00

6000.00 ***

4000.00

2000.00

0.00
Fresh Fresh-spin Frozen Frozen-spin

Fig. 22.5  Concentration of platelet-derived growth factor BB (PDGF-BB) in E-PRP after the application of four
procedures

Fig. 22.6 Concentration TGF-b1

* P value £ 0.05
of transforming growth 100000.00
factor beta (TGF-β) in *
E-PRP after the 90000.00
**
application of four ***
80000.00
procedures
70000.00

60000.00
mg/ml

50000.00 **
40000.00

30000.00

20000.00
*
10000.00 ***

0.00
Fresh Fresh-spin Frozen Frozen-spin

and affirm that more platelets provide a higher
concentration of GF. In these cases, complex sys-
tems for obtaining PRP with sophisticated equip-
ment or even several centrifuges are usually
necessary [135, 146].
On the other hand, there are researchers who
concentrate platelets moderately, usually after
only one centrifugation, and who use the enriched
plasma as obtained after centrifugation [8, 9,
136] or those who dilute it for clinical application
[133, 134].
And finally there are the authors who prefer
that their preparations do not contain platelets,
either because they use blood without antico-
agulant and obtain autologous serum (AS),
leaving the platelets trapped in the blood clot
together with leukocytes and fibrin [112, 147,
148], or those that prepare a plasma rich in
platelets but then activate it with calcium chlo-
ride or directly with thrombin, to obtain plasma
rich in growth factors (PRGF) [104, 141, 142].
Either in the case of autologous serum or in that
334 A. E. Rodriguez and J. L. Alió

Fig. 22.7 Concentration VEGE-A

of vascular endothelial 1000.00
growth factor A
900.00
(VEGF-A) in E-PRP
after the application of 800.00
four procedures
700.00

600.00

mg/ml 500.00

400.00

300.00

200.00

100.00

0.00
Fresh Fresh-spin Frozen Frozen-spin

Fig. 22.8 Concentration Fibronectin

of the plasma protein 600.00
fibronectin in E-PRP
after the application of 580.00
four procedures
560.00

540.00
mg/ml

520.00

500.00

480.00

460.00
Fresh Fresh-spin Frozen Frozen-spin

of PRGF, both preparations present variable
concentrations of GF [74, 104].
In our study, the results obtained after the cen-
trifugation of the blood show that the level of plate-
lets in the E-PRP is almost twice as high as the basal
values in​​ the whole blood, since the concentration
index reached was 1.89. Therefore, the centrifuga-
tion speed used for the preparation of the E-PRP
and the established time are adequate to ensure the
optimal supply of GF for clinical uses [146, 149].
Regarding the red blood cell count, the results
have shown that, after centrifugation, the values​​
in the E-PRP are almost insignificant compared
to the total blood counts since the percentage
found is only 0.9%, obtaining a reduction index
greater than 99%.
No papers were found in the consulted bibli-
ography that expressed the concentration of red
blood cells in the blood derivatives used for oph-
thalmology. Anitua et al. [82] analysed the com-
position of PRGF scaffolds and determined that
the concentration of red cells in the PRGF
Endoret was between 0.02 and 0.04 (×106/μl).
Other authors found concentrations of red blood
cells between 0.01 and 0.03 (×106/μl) in PRP
used for regenerative medicine [150]. All these
values are
​​ comparable to those of our study,
which turned out to be 0.04 ± 0.01 (×106/μl).
Regarding leukocytes, the values ​​found in the
E-PRP were very low compared to the whole
blood, showing a reduction of 82% with respect
to the baseline. The average concentration of
22  Platelet-Rich Plasma (E-PRP) for Corneal Regeneration
white blood cells in the E-PRP (0.97  ×  103/μl)
was comparable to that found by some authors in
PRP [146, 151] or slightly higher than that
described by others for PRGF [64, 82], although
none of the latter indicates the basal concentra-
tion in the whole blood from which they start.
These small values ​​of leukocytes found in the
E-PRP do not classify it as a blood derivative
“with” leukocytes. This denomination is suitable
for those prepared with high concentrations of
white blood cells, close to or equal to those in the
whole blood. Such is the case of L-PRF and
L-PRP [81–83], or the PRP prepared in two steps
traditionally used in other areas of medicine other
than ophthalmology [29, 152, 153]. Those PRPs
used in ophthalmology after a centrifugation, but
incorporating the buffy coat [133, 134, 146],
would also fall into this category.
As far as possible, leukocytes should be avoided
in PRP preparations due to their potential pro-
inflammatory effect since their presence increases
the concentration of the pro-­inflammatory cyto-
kines IL-1β, TNF-α and the metalloproteinases
MMP-9 and MMP-13 [154, 155].
On the other hand, it must be considered that
non-leukocyte blood derivatives, such as SA, in
addition to GF, have pro-inflammatory cytokine
values ​​such as IL-1β, significantly higher than
those present in platelet-rich plasma with leuko-
cytes (L-PRP) [81]. This is because at the time of
blood coagulation, the leukocytes are trapped in
the clot releasing their content including the
interleukins.
The growth factors chosen in this study were
the most representative in the field of ocular
regeneration.
The epidermal growth factor (EGF) partici-
pates actively in the migration and proliferation
of the corneal epithelium in tissue regeneration
processes [44]. The concentrations of EGF in the
frozen E-PRP samples were significantly higher
than the corresponding fresh ones, even doubling
their values. These values ​​of the frozen samples
have higher values than
​​ those obtained with the
PRGF of Anitua et  al. [64, 104] and with the
autologous serum of López-García et al. [74].
The concentrations of EGF in the frozen and
frozen-spin E-PRP were comparable, with no
335
variations observed between them. This indicates
that the subsequent centrifugation that removes
the platelets from the supernatant does not mod-
ify the EGF concentration, probably because all
of this EGF has been released after freezing.
Platelet-derived growth factor (PDGF) is a
modulator of the chemotaxis and cellular mitosis
of fibroblasts [28]. It is presented in three differ-
ent isoforms, PDGF-AA, PDGF-AB and
PDGF-BB, and for our study, we have chosen
PDGF-BB, since it has the highest chemotactic
power of the three [30, 31].
PDGF-BB followed the same pattern as the
previous EGF, observing much higher values ​​in
frozen E-PRP compared to fresh. This difference
was significant for the fresh spin, but not for the
fresh when compared with the respective frozen
E-PRP, although the concentrations found were
more than double in the latter. Possibly this lack
of statistical significance is due to the variability
of the data.
The concentrations obtained for PDGF-BB
were comparable to those found by other authors
[156, 157] and much higher than those of other
researchers [158]. On the other hand, there are
interesting studies that compare the GF of sam-
ples of PRP, PRGF and AS but determine the
PDGF-AB, so the results cannot be compared
[64, 74, 104]. Other authors do not specify what
fraction of PDGF they have analysed, so the find-
ings cannot be compared either [66].
When the different GFs were correlated with
the blood cells in the fresh E-PRP, it was observed
that there was a significant and positive correla-
tion between the platelets and the PDGF-BB
(r  =  0.841 and p  =  0.036). This is because this
factor is released during the degranulation of
platelets when they are in active form [159]. But
no correlation was found between the platelet
count in the E-PRP and the frozen PDGF-BB
(p = 0.203).
Several authors have also found a positive cor-
relation between the platelet count and PDGF-BB
[160–162], while others have found no correla-
tion between them in platelet-rich plasma prepa-
rations [156, 157, 163].
The transforming growth factor β1 (TGF-β1)
is a multifunctional GF and one of the most
336
important that intervene in the modulation of
the behaviour of ocular tissues [40]. It favours
the chemotaxis of fibroblasts, promotes the dif-
ferentiation of myofibroblasts and induces the
production of extracellular matrix by stimulat-
ing the production of collagen, fibronectin and
proteoglycans and decreases their degradation
by inhibiting metalloproteases and other proteo-
lytic enzymes. Depending on the context, it may
have pro-inflammatory or anti-inflammatory
effects [164].
The concentrations of TGF-β1 in the fresh and
frozen E-PRP were high, not observing statistical
differences between them. But subsequent cen-
trifugation drastically decreased the concentra-
tion of this factor.
Given the results obtained, it can be inferred
that TGF-β1 does not need to be activated to per-
form its function since there are no differences
between the fresh values ​​and after freezing at
−20 °C for 3 months. And since the decrease in
concentration is significant after the centrifuga-
tion of both preparations, fresh and frozen, it
could be assumed that the TGF-β1 molecules are
somehow adhered to the platelet membrane since
when they are eliminated the values descend
significantly.
When the results obtained were compared
with those of the bibliography, it was observed
that there was great variability between the dif-
ferent tests, either by the detection technique, the
blood processing method or the individual varia-
tions [165, 166]. Our TGF-β1 values in ​​ the
E-PRP were higher than those obtained by other
authors in AS [86] and in PRGF [104].
Some authors have found a positive correla-
tion between TGF-β1 and the initial concentra-
tion of platelets [161], but this fact has not
occurred in our study (r = 0.676 and p = 0.141),
although in view of the results found, it seems
that there is some relationship.
On the other hand, it was observed that there is
a significant and positive correlation between the
leukocytes and the concentration of TGF-β1  in
the frozen E-PRP (r = 0.932 and p = 0.007). But
this correlation, strangely enough, was not evi-
dent when the white blood cell counts were con-
trasted with the TGF-β1 concentrations in the
A. E. Rodriguez and J. L. Alió
fresh E-PRP (r = 0.408 and p = 0.421). Probably
this difference is due to methodological issues in
the realization of the ELISA technique.
This positive correlation found between leu-
kocytes and TGF-β1 is most likely because this
GF originates in macrophages, monocytes, neu-
trophils and some types of lymphocytes, as well
as in platelets [57].
A strong and positive correlation was also
found between TGF-β1 and VEGF-A (r = 0.943
and p  =  0.005). This fact could be explained
because the presence of VEGF, dose dependent,
induces cell migration and cell proliferation,
accompanied by upregulation of TGF-β1 mRNA,
improving the secretion of this factor and its bio-
activity [167]. On the other hand, it has also been
observed that cytokines derived from leukocytes,
together with the TGF-β1 released by platelets
and plasma-derived factors, are capable of
­inducing the expression of VEGF-A genes [168].
In this way, VEGF-A and TGF-β1 cooperate
because they have a strong relationship, to act in
processes of cellular chemotaxis and neovascu-
larization promoting the regeneration of tissues.
Vascular endothelial growth factor (VEGF-A)
is a potent angiogenic and endothelial cell mito-
gen that is present in platelets and in endothelial
cells stimulating the proliferation of blood ves-
sels [57].
With respect to the concentrations obtained of
VEGF-A, there were no significant differences
between any of the treatments, although a slightly
higher average concentration was found in the
frozen samples. This could be because the expres-
sion of the growth factor VEGF-A is modulated
by other factors such as EGF, TGF-α and β,
PDGF, the GF derived from keratinocytes or the
insulin-like GF type 1, as well as by cytokines. It
is also affected by stimuli such as hypoxia/isch-
emia mainly through the inducible hypoxia factor
(HIF-1) [169].
The concentrations found in the fresh E-PRP
were the same as those found by other authors in
fresh PRGF, while the values o​​ f the frozen E-PRP
at −20  °C for 3  months turned out to be twice
those of the PRGF stored under the same condi-
tions [104]. There is a study that compares the
concentrations of GF in samples of AS, PRGF
22  Platelet-Rich Plasma (E-PRP) for Corneal Regeneration
and PRP but does not specify the fraction of
VEGF analysed, and although the concentrations
are the same units as our study (pg/ml), the val-
ues ​​are of another order of magnitude, so they
cannot be matched.
In contrast, in another study comparing the
levels of VEGF in AS with PRGF, it was found
that the highest levels of VEGF corresponded to
PRGF [64]. The authors comment that although
higher VEGF values ​​were found, they did not
detect neovascularization in any of the patients
treated with PRGF in previous studies [141, 170].
Unlike the previous ones, we would be inter-
ested that the values of VEGF in the E-PRP are
not very high given that a high concentration of
them contributes to the development of disease,
since the overexpression of VEGF can lead to
vascular alterations of the retina [50] and has
even been associated with the development and
maintenance of tumours [51].
Fibronectin is an adhesion protein, which is
free in plasma, and is one of the main constitu-
ents of the extracellular matrix. This protein can
interact with various molecules and cells, estab-
lishing connections between them in the extracel-
lular matrix. This molecule is one of the first to
arrive when a corneal wound occurs filling the
bed of the lesion, thus producing cell migration
during the repair process of the corneal epithe-
lium [53–55].
In our trial, no significant differences were
found in the concentration of fibronectin, since
the values ​​of this protein remained constant in the
four types of procedures performed. The expla-
nation for this fact is because fibronectin is a
plasma protein and its presence does not depend
on platelet activation.
The fibronectin concentrations found in our
study are slightly higher than the blood plasma
reference values ​​ since it is approximately
300 ± 100 μg/ml [52]. These variations between
healthy individuals, probably physiological, may
be due to variations in age and sex [52].As it hap-
pened when trying to compare our results with
the literature to compare the levels of the GF
found, it is also difficult to do so for fibronectin
since the values found,
​​ although in the same con-
centration (μg/ml), are in an order of magnitude
337
inferior and the authors assure that their values​​
are normal as in our case [66, 104]. A reasonable
explanation for this situation could be the vari-
ability in the analytical detection techniques
used.
Although some authors found a decrease in
the levels of this protein after storage at −20 °C
for 3 months [104], this was not the case in our
case since the concentrations obtained were simi-
lar in fresh and frozen samples. Other authors
found a fragmentation of the fibronectin mole-
cule that produced a decline in its activity, but
this happened after 4–12 months of cryopreserva-
tion [171].
Except for PDGF-BB, no correlation was
found between the growth factors EGF, TGF-β1
and VEGF-A with the platelet count (p > 0.05).
These results coincide with those reported by
other authors who affirm that this fact could be
explained by the high individual variability that
exists in the cellular production and storage of
GF in platelets. The content of GF in individuals
could be influenced by cells other than platelets
such as leukocytes; this means that each individ-
ual would need different concentrations of plate-
lets to achieve a comparable biological effect.
Therefore, the platelet count in the whole blood
or PRP would not be a predictive factor of HR
values in​​ the PRP [157, 163]. Another possible
fact is that there are other mechanisms of interac-
tion, not dependent on platelets, among the
immense number of molecules present in the
blood plasma that modulate the release of growth
factors.
In this study, different procedures applied to
E-PRP have been evaluated in order to find out
how they affected freezing, storage time and cen-
trifugation in the concentration of the different
bioactive molecules of the plasma. According to
the results obtained, we could say that freezing at
−20  °C for 3  months provided higher levels of
the growth factors EGF and PDGF-BB and main-
tained the concentrations of TGF-β1, VEGF-A
and fibronectin.
This behaviour is because the process of freez-
ing and subsequent thawing of the E-PRP causes
a powerful activation of the platelets it contains
[158]. The PRP must be activated so that the
338
α-granules release their content, and this activa-
tion can occur by the addition of collagen, cal-
cium and/or thrombin, by contact with the glass
or by freezing cycles [150, 172]. With this last
procedure, it is possible to arrive until the total
liberation of its content thus providing great
amount of GF.
On the contrary, the centrifugation applied to
fresh and frozen E-PRP samples, contrary to the
previous idea that it could activate the platelet
content, did not give the expected results. All the
concentrations of the molecules studied in the
“centrifuged/spin” groups, except for TGF-β1,
were like their homologs without centrifuging,
and no statistically significant differences were
found.
In the particular case of TGF-β1, the concen-
trations of the centrifuged samples decreased sig-
nificantly with respect to their counterparts
without centrifuging, demonstrating that the
platelets had to be present to obtain values close
​​
to the initial ones.
The centrifugation parameters used (6000 rpm
for 5 min) did not produce the activation of the
platelets present in the E-PRP, but their irrevers-
ible aggregation as they were agglomerated in the
bottom of the conical tubes, making their dissolu-
tion impossible.
22.11 Conclusions
The interest in the therapeutic application of
platelet-rich plasma has increased in different
fields of medicine. It is known that this treatment
has properties that enhance and accelerate the
process of tissue regeneration through the stimu-
lation of cell growth and function. Due to its
properties, it has generated great advances in
many areas of regenerative medicine. This bio-
logical and autologous product does not generate
rejection and minimizes the possible adverse
effects.
Numerous studies have demonstrated the effi-
cacy of E-PRP as a treatment for several ocular
surface diseases due to their concentration of
platelets, which remain viable and retain the abil-
A. E. Rodriguez and J. L. Alió
ity to provide growth factors involved in the bio-
logical responses of cells.
E-PRP is very versatile since it can be applied
as an eye drop in various pathologies of the ocu-
lar surface as in the case of corneal ulcers, dry
eye and ocular surface dysfunction after LASIK
and as a clot or autologous fibrin membrane for
surgical reconstruction in cases of severe corneal
ulcers or corneal perforations.
But contrary to what might be expected, high
platelet concentrations (1  ×  106/μl), which pro-
duce very high levels of GF, are not necessary to
successfully treat corneal lesions [158]. With
much lower concentrations, the desired effects
are achieved. In our study, the average concentra-
tion of platelets was 495 × 103/μl, which is why
this concentration is adequate to obtain sufficient
levels of growth factors to treat the various ocular
surface affectations that require regenerative
therapy.
The finding of high concentrations of growth
factors EGF, PDGF-BB and fibronectin in the
E-PRP is the key to obtain the desired regenera-
tive effects on the ocular surface.
This occurs because high levels of fibronectin
together with high PDGF-BB values ​​stimulate
the migration of corneal epithelial cells in cor-
neal healing processes [173, 174]. On the other
hand, EGF increases proliferation and cell migra-
tion, promoting the reordering of actin filaments
[175], and is also able to stabilize the tear film
and prevent apoptosis [176]. All these molecules,
together with fibroblast GF (FGF), facilitate cor-
neal re-epithelialization while being modulated
by several cytokines [164].
Clinical studies in which platelet-rich plasma
was used as a treatment showed very good results
in patients with different pathologies of the ocu-
lar surface. All these findings are now reinforced
by knowing the concentration of the different
components of E-PRP in terms of cell elements,
growth factors and fibronectin.
Therefore, it is possible to conclude that the
E-PRP presents the necessary amounts of growth
factors and adhesion proteins, proving to be an
adequate tool as a regenerative therapy of the cor-
nea and ocular surface.
22  Platelet-Rich Plasma (E-PRP) for Corneal Regeneration
Compliance with Ethical Requirements  Conflict of
Interest
Alejandra E. Rodríguez and Jorge L. Alio declare that
they have no conflict of interest.
Informed Consent
All procedures followed were in accordance with the
ethical standards of the responsible committee on human
experimentation (institutional and national) and with the
Helsinki Declaration of 1975, as revised in 2000. Informed
consent was obtained from all patients for being included
in the study.
No animal studies were carried out by the authors for
this chapter.
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 Part IV
Regenerative Surgery of the Corneal
Stroma
 Applied Anatomy of the Corneal
Stroma
Harminder Singh Dua and Dalia G. Said
Key Points
• The corneal stroma is made of
Bowman’s layer anteriorly, the pre-­
Descemet’s layer (Dua’s layer, also
termed the Dua-Fine layer) posteriorly
and the stromal lamellae and kerato-
cytes, which form the bulk of the cor-
nea, in between.
• The function of Bowman’s layer is not
well known as it is absent in some spe-
cies and does not seem to affect the cor-
nea when ablated in laser refractive
surgery. Transplantation of Bowman’s
H. S. Dua (*)
Academic Section of Ophthalmology, Division of
Clinical Neuroscience, University of Nottingham,
Nottingham, UK
Department of Ophthalmology, Queens Medical
Centre, University Hospitals NHS Trust,
Nottingham, UK
e-mail: harminder.dua@nottingham.ac.uk
D. G. Said
Academic Section of Ophthalmology, Division of
Clinical Neuroscience, University of Nottingham,
Nottingham, UK
Department of Ophthalmology, Queens Medical
Centre, University Hospitals NHS Trust,
Nottingham, UK
Research Institute of Ophthalmology (RIO),
Cairo, Egypt
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_23
23
layer can strengthen the cornea and
arrest or retard progressing of ectasia.
• Stromal lamellae are made predomi-
nantly of type I collagen, which are very
compact and intertwined anteriorly,
comparatively less compact centrally
and posteriorly and relatively compact
again in the pre-Descemet’s layer.
• Organization of stromal collagen
lamellae is complex, being orthogonal
centrally and tangentially at the
periphery to accommodate the biome-
chanical stresses that the cornea is
subjected to.
• The pre-Descemet’s layer also contains
predominantly type I collagen but has
more elastin relative to other parts of the
cornea. It is distinctly connected with
the trabecular meshwork.
• The intricate arrangement and charac-
teristics of the cells and matrix struc-
tures collectively contribute to corneal
transparency.
• Elucidation of the corneal stromal anat-
omy, especially of the posterior stroma,
has enabled development and under-
standing of lamellar corneal surgery and
corneal pathology.
349
350
The corneal stroma is one of the most organized
connective tissue elements of the body. Lined by
cells on either side, it constitutes about 90% of
the corneal substance. Its structure is designed to
act as the wall of the eyeball to protect the tissues
inside and maintain intraocular pressure, to be
transparent to allow passage of light, to act as an
optical lens to provide two thirds of the focusing
power of the eye and to be resilient to withstand
external and internal forces that deform it yet
spring back to its basic shape. In the interest of
transparency, it is adapted to meet its energy
requirements without a vascular supply.
23.1 Biometrics and Structure
The corneal shape is that of an aspheric convex
lens. Both anterior and posterior surfaces of the
cornea contribute to its overall refractive power.
Externally, the clear cornea is horizontally oval
measuring 11–12 mm in the horizontal axis and
9–11 mm vertically, in adults. The oval shape is
due to the scleralization of the superior and infe-
rior approximately 0.5–0.75  mm. When viewed
from the posterior surface, the corneal outline
appears circular. The central cornea is between
500 and 550 microns, increasing gradually to
around 700 microns at the periphery. The radius
of curvature differs in the central optical zone
(3 mm diameter) where it is 7.5–8 mm, from the
peripheral zone where it is flatter giving the
aspheric optical effect. The total refractive power
of the cornea is 40–44 dioptres. The refractive
index of the cornea is 1.376 compared to air,
which is 1.00, and tears and aqueous which are
both approximately 1.336. The cornea transmits
about 90% of the incident light [1, 2].
23.2 Corneal Stroma
In broad terms, the stroma is organized in three
components based on anatomical, behavioural and
functional characteristics determined by the cells
within these components and in part adjacent to
them. The anteriormost component is Bowman’s
membrane or, perhaps technically more correctly,
H. S. Dua and D. G. Said
Bowman’s layer, upon which rests the basement
membrane of the corneal epithelium. The middle
component is the corneal stroma, which consti-
tutes 90% (500 microns) of the corneal thickness,
and posteriorly is the pre-­Descemet’s layer (Dua’s
layer), which is lined by Descemet’s membrane
(DM) and endothelium on its posterior surface.
Bowman’s Layer  It is also termed Bowman’s
membrane, Bowman’s zone or anterior limiting
lamina (Fig.  23.1a). It is an 8–10-micron-­ thick
acellular layer of primarily type I and type III col-
lagen fibres which are narrower than those found in
the stroma. These are randomly woven into a dense
and compact layer of very uniform thickness for
most part. It extends from limbus to limbus and
narrows towards its termination, which marks the
anterior limit of the cornea and the commencement
of the limbus. Like in the stroma, the space between
collagen fibres is filled with proteoglycans and gly-
cosaminoglycans (see below). The anterior surface
of Bowman’s layer is very smooth, while at its pos-
terior surface, the fibres extend into anterior stroma
with which they are intricately related. On in vivo
confocal microscopy, it appears as a wavy structure
with some prominent crisscrossing lines of colla-
gen in the interface of Bowman’s layer and anterior
stroma, called K-structures. These are believed to
be the basis of the mosaic pattern seen in the ante-
rior cornea [3, 4] (Fig. 23.1b). Although the turn-
over of Bowman’s layer is attributed to the
underlying stromal keratocytes, it does not regener-
ate when damaged by injury or photorefractive
keratectomy. Its function is not clear, but it is sug-
gested that it affords rigidity to the anterior cornea
and helps maintain the refractive shape and pro-
vides a firm, smooth and regular surface for the
corneal epithelium. However, some species of
mammals do not have a Bowman’s layer [5], and
these functions are still apparently well served.
Transplantation of Bowman’s layer in patients with
keratoconus is being performed to afford additional
strength and rigidity to the cornea in patients with
keratoconus [6].
Stroma  The stroma is made essentially of extra-
cellular matrix, which accommodates cells and
nerves.
23  Applied Anatomy of the Corneal Stroma
b c
Fig. 23.1 (a) Resin-embedded semi-thin section of the
anterior corneal stroma and epithelium. The stratified
squamous epithelium sits on Bowman’s layer, which
appears as homogenous structure of uniform thickness.
The underlying anterior stroma shows collagen fibres of
the stromal lamellae and keratocytes (toluidine blue
Extracellular Matrix  The extracellular matrix of
collagen and glycosaminoglycans accounts for
about 70% of the dry weight of the cornea. Type
I collagen is the most abundant with lesser
amounts of type III, type V and type VI [7, 8].
Collagen fibres are bundled together in about
200–250 lamellae of 2 microns thickness that run
from limbus to limbus, spanning the entire cornea
[9]. Fibres in the lamellae run parallel to each
other, and the lamellae run parallel to the surface
of the cornea, but individual lamellae subtend
large angles to adjacent lamellae [8]. The arrange-
351
stain). (b) In vivo confocal microscopy enface image of
Bowman’s layer with sub-basal nerves and K-lines. (c)
Transmission electron microscopy of the corneal stroma
showing the transversely (top) and horizontally cut lamel-
lae (bottom)
ment of collagen is predominantly orthogonal,
i.e. at right angles to each other (Fig. 23.1c), in
the mid and posterior cornea and the largely iso-
tropic, i.e. similar in all directions, in the anterior
stroma [10–12]. Furthermore, there is a preferen-
tial orientation of collagen along the superior-­
inferior and nasal-temporal directions which
seems to be an adaptation to absorb biomechani-
cal stress exerted by the rectus and orbicularis
muscles [13]. The arrangement of lamellae in the
anterior third is more oblique with some branch-
ing [14] and is undulating and interwoven [8, 15].
352
A complex branching pattern of collagen fibres is
seen in the anterior stroma where fibres extend
from Bowman’s layer, intertwine with the stro-
mal tissue and reinsert in Bowman’s layer. This
pattern diminishes systematically towards the
posterior stroma [16]. These features confer a
greater compactness to the anterior stroma.
There is also a difference in compactness of
the peripheral and central stroma in the middle
and posterior parts of the stroma. The force
required to separate the stromal tissue is less in
the centre than at the periphery of the cornea
[17]. Air injected in the stroma, as in the proce-
dure of deep anterior lamellar keratoplasty
(DALK), follows a distinct circular pattern of
movement in the peripheral cornea (Fig. 23.2a),
which corresponds to the 1.5–2 mm of compact
stroma at the periphery, which also corresponds
to the flatter peripheral part of the prolate cornea.
Histology of this area after air insufflation sug-
gests an interweave of collagen that is different
from that seen in the central cornea [18]
(Fig. 23.2b). There is a ‘circular annulus’ of col-
lagen fibres at the periphery of the cornea. The
fibres are oriented tangentially to the limbus
probably creating an anatomical landmark as
they transition from orthogonal to the tangential
orientation in the annulus adding to the compact-
ness and tighter attachment of fibres at the
periphery [11, 19–21]. Though collagen is by far
the most dominant extracellular matrix compo-
a b
Fig. 23.2  Corneal stromal architecture. (a) Air injected
in the stroma travels circumferentially along the limbus,
in the peripheral 1–1.5 mm of the cornea. This consistent
movement of air suggests a special construction of this
part of the stroma. (b) Histology of the cornea showing
H. S. Dua and D. G. Said
nent of the stroma, there is also a well-defined
network of elastin fibres [22, 23]. An annulus of
an elastic fibre system is also present at the
cornea-­scleral limbus [24, 25]. These structural
features of the peripheral part of the cornea
explain the circular movement of air at the
periphery and the limit of 8.5–9 mm diameter of
the type 1 big bubble in DALK [18] and the
greater force required to separate the peripheral
stroma [17].
Glycosaminoglycans  Glycosaminoglycans also
called mucopolysaccharides are made of repeat-
ing units of disaccharide that form long
unbranched structures. They are highly polar
(having negative and positive charges that do not
neutralize each other) and attract water. The
space between collagen fibres is filled with
ground substance made of the glycosaminogly-
cans keratan sulphate, dermatan sulphate, chon-
droitin and hyaluronan. The latter is most
abundant in the developing cornea and declines
thereafter. In the adult cornea, keratan sulphate is
the most abundant and along with dermatan sul-
phate and chondroitin can bind to proteins in the
stroma to form proteoglycans. The posterior cor-
nea contains more keratan sulphate, which is
more hydrophilic than dermatan sulphate, which
is more in the anterior stroma [26, 27]. This con-
tributes to the differential swelling of the cornea,
with greater swelling of the posterior stroma
compared to the anterior stroma [10].
the expanded peripheral cornea with wide interlamellar
spaces (arrows). This arrangement offers less resistance to
the passage of air compared to the central cornea.
(Reproduced from the author’s own publication Dua et al.
2018 [18])
23  Applied Anatomy of the Corneal Stroma
Keratocytes  Keratocytes constitute most of the
cells that reside in the stroma. Macrophages,
dendritic cells and transiting bone marrow-
derived cells are also present in very small num-
bers. The total cell mass is less than 4% of the
stromal mass [28, 29]. Keratocytes are akin to
the fibroblasts of other connective tissues, hence
also termed stromal fibroblasts. Keratocytes are
located between lamellae and have a central flat-
tish spindle-­shaped cell body from which mul-
tiple cytoplasmic processes extend in all
directions, making contact with similar pro-
cesses of neighbouring keratocytes (Figs. 23.1a
and 23.3). Cells are capable of communication
with each other by exchange of information via
gap junctions at the tips of keratocyte processes.
This enables them to act as one cell mass and
respond collectively to physiologic and patho-
logic stimuli. Keratocytes also express integrins
through which they interact with the surround-
ing extracellular matrix, and CD34, a trans-
membrane phosphoglycoprotein in abundance,
which also serves as an adhesion molecule,
binding to its ligand, L-selectin, in the matrix
[30, 31]. The keratocyte-matrix interaction reg-
ulates and modulates keratocyte behaviour such
as mitotic rate and activity. Normally they are
quiescent and with a very low proliferative rate.
Keratocytes have a well-developed cytoskeleton
of predominantly actin filaments, which give the
cells contractile ability.
a b
Fig. 23.3  Keratocytes. (a) The anterior stroma shows a
large number of keratocytes stained for CD34 (an adhe-
sion molecule) marker, which is seen on all keratocytes.
Bowman’s layer is acellular. The corneal epithelium is
artefactually detached from Bowman’s layer (immuno-
peroxidase stain). (b) Transmission electron micrograph
353
By in vivo confocal microscopy (IVCM), it has
been estimated that the corneal stroma contains
about 20,500 cells per cubic millimetre (Fig. 23.3c).
The density of cells in anterior stroma is 765 ± 262
cells/mm2, in mid-stroma around 347 ± 64.4 cells/
mm2 and in posterior stroma about 315 ± 57.2 cells/
mm2. The greatest density is in the anterior 10% of
the stroma. Keratocyte density is age related and
declines by about 0.45% per year being about twice
the amount in the anterior stroma. With IVCM,
keratocytes in the anterior stroma demonstrate
smaller nuclei than those in the posterior stroma
[32, 33]. The limbal stroma acts as a stem cell niche
giving residence to progenitor keratocytes or kera-
tocyte stem cells. The peripheral and limbal corneal
stromal cells express all features of mesenchymal
stem cells that have the potential to transdifferenti-
ate to an epithelial phenotype [34, 35].
Keratocytes function to produce and maintain
the extracellular matrix of collagen and glycos-
aminoglycans. Collagen secretion starts with
intracellular synthesis of pro-α-collagen. Three
hydroxylated and glycosylated molecules are
assembled into procollagen, which is secreted out
by the cells. In the extracellular space, procolla-
gen is converted to collagen molecules, several of
which form collagen fibrils, which in turn com-
bine to form tough collagen fibres with a charac-
teristic cross striation pattern, with a periodicity
of 67  nm. In the corneal stroma, the collagen
fibres are very uniform in diameter ranging from
22.5 to 35 nm [8, 36].
c
of a keratocyte process within the stromal lamellae. An
intracytoplasmic deposit (vacuole) of lipid material is
seen (arrow). (c) In vivo confocal microscopic image of
keratocytes in the corneal stroma. The nuclei bodies
appear white with some showing extensions of keratocyte
processes
354
Keratocytes contain water-soluble structural
proteins called crystallins of which aldehyde
dehydrogenase is the most prominent. Crystallins
help in maintaining the transparency of the kera-
tocyte and cornea [37, 38]. They also secrete
matrix metalloproteases, which degrade the
extracellular matrix in the physiologic role of (re)
modelling the stroma and in disease states, caus-
ing ulceration by excessive degradation or lysis
of the collagen (stromal melting). The function of
MMPs is balanced by the presence of tissue
inhibitors of MMPs (TIMPS). In response to
a b
d e
Fig. 23.4  Keratocyte variations in cornea transplanted
with amniotic membrane on the surface. (a) Transverse
section of the corneal stroma with the epithelized, trans-
planted amniotic membrane on the surface (haematoxylin
and eosin stain). Keratocytes are seen in the stroma of the
cornea and in the amniotic stroma where they have
migrated from the corneal stroma through breaches in
Bowman’s layer. (b) The corneal stroma shows kerato-
cytes (brown) stained with CD34 antibody. The kerato-
cytes in the amniotic stroma do not show any stain with
CD34 indicating that they have lost the resting keratocyte
H. S. Dua and D. G. Said
wounding or disease, keratocytes near the pathol-
ogy break their intercellular connections and then
behave independently and can transform to myo-
fibroblasts and activated fibroblasts [39]
(Fig.  23.4a–c). A common response of kerato-
cytes to injury including laser refractive surgery
and cross-linking is apoptosis, which is believed
to be a ‘defence’ reaction to limit spread of the
infection or injurious agent [40, 41]. In culture,
keratocytes can express cytokeratin 3, suggesting
possible mesenchymal-epithelial transition [42]
(Fig. 23.4d–f).
c
f
immunophenotype. (c) The section is stained for alpha
smooth muscle actin. All corneal stromal keratocytes are
negative, but the cells in the amniotic stroma are positive
indicating that they are keratocyte-derived myofibro-
blasts. (d) Cultured corneal keratocytes stained with anti-
­CD34 antibody (red). (e) Cultured corneal keratocytes
stained with anti-cytokeratin 3 antibody (green). (f)
Superimposition of the images showing that the same cell
expresses both CD34 and cytokeratin 3 – mesenchymal-­
epithelial transition. (a–c Adapted in part from authors’
own publication Said et al. [39])
23  Applied Anatomy of the Corneal Stroma
23.3 Stromal Nerves
The cornea is the most sensitive structure in the
human body, being 40 times more sensitive than
dental pulp and over 400 times more than the skin
[43]. Corneal innervation is predominantly sen-
sory, from the ophthalmic division of the trigemi-
nal (V cranial) nerve. The nasociliary branch of
the ophthalmic division of the trigeminal nerve is
the main nerve of the ocular surface. It enters the
orbit through the superior orbital fissure. Its
branches include the anterior and posterior eth-
moidal nerves, two or three long ciliary nerves
and a communicating branch to the ciliary gan-
glion. It terminates as the infra-trochlear and
nasal nerves. Six short ciliary nerves arise from
the ciliary ganglion and together with the long
ciliary nerves enter the suprachoroidal space by
penetrating the sclera around the optic nerve.
They pass anteriorly, supply the iris and ciliary
body and terminate in the pericorneal (limbal)
plexus. The limbal plexus thus has both sensory
and autonomic nerves and is predominantly vaso-
motor in function [44].
Eleven nerve bundles per quadrant, carrying a
mixture of sensory and autonomic fibres, pass
through the limbus and enter the cornea in the
middle third of the stroma (Fig. 23.5). Just before
or soon after entering the corneal stroma, the
nerve bundles lose their perineurium and con-
tinue radially and anteriorly between the collagen
lamellae towards the central area, giving rise to
branches that spread through the anterior and
mid-stroma. The posterior stroma lacks nerves
though some innervation of the corneal endothe-
lium has been described [45]. The axons of the
central stromal nerves are unmyelinated and run
in the anterior stroma parallel to collagen bun-
dles. Most of the axons in these bundles are about
0.5 μm in diameter but can be as thick as 2.5 μm
[46, 47]. There is a loose sub-Bowman’s plexus
of nerves from where fibres penetrate Bowman’s
zone, predominantly in the mid-periphery of the
cornea, and emerge in the sub-basal plane of the
epithelium where they end in single or multiple
bulblike structures which probably represent the
termination and folding of the nerve sheath [48].
A small population of axons terminate in the
355
stroma. A close anatomical relationship with
stromal keratocytes and macrophages has also
been described [46, 49]. Stromal nerves show
considerable anomalies in corneal pathology, a
fact that is increasingly being recognized with the
advent of IVCM [50] (Fig. 23.6).
Autonomic innervation is from postganglionic
fibres from the SCG that ascend in the plexus
around the internal carotid artery. Ocular fibres
leave in the cavernous sinus and enter the orbit
through the superior orbital fissure as the sympa-
thetic root of the ciliary ganglion. These fibres
either merge directly with the long ciliary nerves
or pass through the ganglion, without synapse, to
emerge in the short ciliary nerves. Human cor-
neas have sparse sympathetic innervation [51].
23.4 P
 re-Descemet’s Layer (Dua’s
Layer, PDL)
The development and practice of lamellar corneal
surgery especially DALK by the big-bubble tech-
nique brought to light a layer at the posteriormost
part of the stroma termed pre-Descemet’s layer
or Dua’s layer. The American Association of
Ocular Oncologists and Pathologists has recently
termed this the Dua-Fine layer (Fig. 23.7).
The PDL is less than 20 microns
(10.15 ± 3.6 m) thick and is made up of 5–8 com-
pact lamellae of predominantly type I collagen
like most of the stroma. The lamellae are orga-
nized in longitudinal, transverse and oblique
directions. It also has types IV and VI (long-­
spacing) collagen, which are more than in the
stroma (Fig. 23.7a–e). The width of the lamellae
is significantly narrower than in the correspond-
ing region of the stroma immediately anterior to
the PDL, where there are 3–5 layers for the same
width of tissue [52, 53]. The PDL is by and large
acellular though occasional keratocytes or kera-
tocyte processes can be seen in it [54]. The col-
lagen fibres in the PDL are also significantly
thinner than in the stromal lamellae anterior to it
[52]. The PDL also has a high elastin content,
more than any other part of the cornea and as
much as the trabecular meshwork [24, 25, 55].
This explains why, during DALK, the wall of the
356 H. S. Dua and D. G. Said

a b

c d

Fig. 23.5  Corneal stromal nerves. (a) Thick myelinated
corneal nerves are seen entering the peripheral cornea
from the limbus. These become invisible as they lose their
myelin towards the centre of the cornea. Inset: slit view
showing the nerves in cross section lying in the middle
third of the stroma (arrows). (b) In vivo confocal micros-
copy of the thick peripheral (limbal) entrance of a stromal
nerve. (c) The same nerve narrows towards the mid-­
periphery and (d) tapers to become thin and fine in the
centre of the corneal stroma on its way to the sub-­
Bowman’s plexus

big bubble expands and, on deflation, returns to
its original dimensions. The compactness of the
layer coupled with the elastin content also prob-
ably accounts for the fact that the layer is imper-
vious to air. On forceful injection, air in the
corneal stroma traverses through the stroma
(probably around keratocyte spaces) until it
reaches the PDL. Here it cannot pass through the
layer and hence cleaves it off the posterior stroma
to form the type 1 bubble in DALK. Injection of
air after excimer laser ablation of the PDL (in
experimental eyes) fails to produce a big bubble
indicating that the PDL is unique [56]. The PDL
can be visualized in  vivo between DM and the
last row of keratocytes by ultrahigh-resolution
optical coherence tomography [57]. There is a
distinct plane of cleavage between the PDL and
the posterior stroma, with the separation occur-
ring along the last row of keratocytes. This plane
is exploited in big-bubble DALK where the type
23  Applied Anatomy of the Corneal Stroma 357

a d

b e

c f

Fig. 23.6  Altered corneal nerves in bullous keratopathy.
In vivo confocal microscopy (IVCM) (a, b, c) and corre-
sponding whole-mount acetylcholinesterase-stained (d, e,
f) corneal stromal nerves. (a) Thickened nerve with
excrescences (arrows) and involuted excrescence (arrow-
head). (d) Corresponding image on whole-mount stain
showing that the excrescences (arrows) could represent
the site of origin of branches, which may be undergoing
involution (arrowhead). (b) A bifurcation of a thickened
stromal nerve is seen. Note the absence/decreased visibil-
ity of keratocytes. (e) Corresponding image on whole-­
mount stain showing aberrant hyper-regeneration of fine
nerves from the bifurcated trunk that are coiled and tortu-
ous. These were not visible on IVCM. (c) Loops and coils
of relatively thicker nerves in the stroma. (f) Corresponding
images on whole-mount stain showing a similar aberrant
pattern (Reproduced from the author’s own publication,
Al-Aqaba et al. [50])
358
a b
d e
Fig. 23.7  The pre-Descemet’s layer (Dua’s layer PDL
also termed the Dua-Fine layer by the American
Association of Ocular Oncologists and Pathologists). (a)
Light photomicrograph of the PDL that was separated
from the underlying stroma by the injection of air.
Descemet’s membrane (DM) has been stripped off the
PDL. (b) Transmission electron micrograph (TEM) of the
PDL showing that it is made of multiple thin and com-
pactly arranged lamellae. The DM has been peeled off. (c)
Higher-power TEM of the PDL and DM. The dark verti-
cal band in the middle of the image is the banded layer of
the DM. The non-banded layer and the endothelial cell are
1 bubble represents the separation of the PDL
together with DM and endothelium from the
­posterior stroma. The plane can also be surgically
accessed when air injection fails to create the big
bubble. Such separation is also manifest in cor-
neal pathology related to infection, offering a
plane for spread of fungi [58], and in chronic cor-
neal oedema where spontaneous dehiscence of
the PDL together with the DM occurs [59]. There
is evidence to suggest that acute hydrops in kera-
toconus is related to a rupture of the DM and
PDL [60] and that the PDL is part of descemeto-
celes [52]. There is also a clear plane of cleavage
between PDL and DM which becomes apparent
in DM detachments, DM peeling during
Descemet’s membrane endothelial keratoplasty
(DMEK) and in the type 2 bubble of DALK [52,
53]. The PDL is strong and resilient with a burst-
ing pressure (of type 1 bubble) of 500–700 mm of
mercury. This allows surgeons to perform phaco-
emulsification with lens implant under the PDL
H. S. Dua and D. G. Said
c
f
on the right and the PDL on the left. The PDL shows the
lamellar structure with some areas of darker long-spacing
collagen towards the DM interface. (d) Scanning electron
micrograph (SEM) of the PDL, separated from the deep
stromal bed anterior to it. The edge of the PDL is clearly
visible. (e) SEM of the cornea from which the EL with
DM has been separated. The two layers are scrolled
together with the endothelial cells outside. (f) SEM of
periphery of PDL showing a cluster of fenestrations
(arrow). These fenestrations are distributed all along the
periphery of PDL and allow air injected in the stroma to
pass through PDL to create a type 2 big bubble
during DALK with a type 1 bubble, also termed
the DALK triple procedure [61]. Another surgical
procedure termed pre-Descemet’s endothelial
keratoplasty (PDEK) involves use of the PDL
together with DM and endothelial cells as the
transplant tissue to replace host DM and endothe-
lium, as an alternative to DMEK with good
results [62].
At its periphery, all along the circumference,
the collagen fibres of the PDL start to fan out like
a three-dimensional crisscrossing sieve and con-
tinue as the core of the trabecular meshwork
beams, which too have the same collagen and
elastin content as the PDL. Trabecular cells rest-
ing on basement membrane are seen in the
peripheral 350 microns of the stroma of the PDL
[63]. Clusters of fenestrations are presented in
the PDL, within 500 microns central to the termi-
nation of DM. Fifteen to twenty fenestrations or
clusters of fenestrations are present along the cir-
cumference, on either side of the termination of
23  Applied Anatomy of the Corneal Stroma
the DM. These are present within 500 microns of
the termination of DM centrally and between ter-
mination of DM and the trabecular meshwork
peripherally. The fenestrations measure between
5 and 60 microns with a mean of 20.3 microns
[63] (Fig. 23.7f). These fenestrations explain the
passage of air into the anterior chamber during
DALK and the occurrence of a type 2 bubble
(and mixed bubble) wherein air passes through
these fenestrations to access the plane between
PDL and DM.
There is an embryologic basis for the
PDL. Separation of the lens vesicle from the sur-
face ectoderm initiates the formation of the cor-
nea, with the surface ectoderm forming the
two-layered epithelium [64]. The epithelium
secretes collagen and glycosaminoglycans that
fill the space between the epithelium and the lens
vesicle as the acellular primary stroma [65–68].
The first wave of migration of neural crest cells
from the rim of the developing optic cup passes
between the posterior layer of the primary stroma
and the lens vesicle to form the embryonic endo-
thelium. The third wave of neural crest cells
migrate into the middle of the primary stroma
and form the stromal keratocytes (second wave
forms the iris and pupillary membrane). The ker-
atocytes lay down the definitive or secondary
stroma. The primary stroma anterior to the
expanding secondary stroma is compressed and
forms the basis of Bowman’s layer. It is likely
that the compressed primary stroma posterior to
the secondary stroma forms the basis of the
PDL. The PDL is also likely to be influenced by
the endothelium because of its proximity to this
layer. The endothelium is known to contribute
collagens [69–71] and hyaluronan [72] to the
developing posterior stroma (PDL).
23.5 Corneal Transparency
Maintaining transparency to allow entry of light
to the eye is the most important function of the
cornea. Several factors both anatomical and
physiological contribute to this. The epithelium,
stroma and endothelium all have important roles,
and compromise in structure and/or function of
359
any of these singly or in combination can com-
promise transparency and affect vision. Intrinsic
transparency of the cornea is related primarily to
the architecture of the stromal collagen. The very
uniform diameter of the collagen fibres, the
equally uniform and consistent spacing of the
fibres, the relative state of dehydration (78%
water content), the unmyelinated nerves that pop-
ulate the stroma, the ‘transparent’ keratocytes
and the absence of blood vessels all make signifi-
cant contributions to transparency.
The mean diameter of the corneal fibres and
the interfibre distance is less than half of the
wavelength of visible light, which is 400–700 nm.
Thus any scattering of a light ray passing through
one fibre is cancelled by the scatter of the adja-
cent ray allowing light to pass through the cornea
[73]. Any alteration in the finely organized archi-
tecture of the collagen fibres, as occurs with
oedema and scarring, leads to random scattering
and back scatter (diffuse and irregular reflection),
limiting the amount and quality (focused) of light
entering the eye.
Despite the water-holding property of the gly-
cosaminoglycans, the cornea is maintained in a
state of relative dehydration, in the interest of
transparency. This is largely brought about by the
endothelial pump function of the endothelium.
Surface evaporation of water and the barrier
afforded by the epithelium also contribute to loss
of stromal water. Hence corneal thickness is
physiologically slightly more on waking. In
patients with early endothelial failure, e.g. Fuchs
endothelial dystrophy, the complaint of blurry
vision on waking, which can take a few hours to
clear, is related to, among other factors, the loss
of evaporation during sleep. Epithelial loss (abra-
sion) is associated with stromal oedema due to
breach of the epithelial barrier function. It is
interesting that localized endothelial dysfunction
or loss is associated with localized oedema of the
overlying stroma only. This suggests that the
endothelial pump function strictly serves the
stroma directly anterior to it with little if any lat-
eral effect.
The magnitude of the contribution of the endo-
thelium to maintaining transparency can be seen
in the fact that an excised cornea swells up to
360
1200 microns thickness with an increase in hydra-
tion from 78% to 98%, related to the swelling
pressure of the stroma, i.e. the ability of the polar
glycosaminoglycans to absorb water and separate
the collagen fibres. This pressure is countered by
the endothelial pump with a little help from the
intraocular pressure, in vivo. Increased hydration
of the cornea causes the cornea to become hazy,
while fibrosis/scarring causes opacity. Clinically
there is an interesting anomaly in that corneas suf-
fering from complete hydration for several months
due to endothelial failure show remarkable clear-
ance after endothelial keratoplasty. Equally, there
are some such corneas that retain a degree of
‘haze’ and poor quality of vision post-operatively
(authors’ experience), suggesting that prolonged
oedema can lead to structural changes in collagen
organization and/or abnormal collagen produced
by the stressed keratocytes [1].
Declaration of Interest None of the authors have any
conflict of interest related to the subject matter and con-
tent of the chapter. HS Dua is Consultant for Dompe,
Santen, Thea and Shire. He has shares in NuVision
BioTherapeutics and GlaxoSmithKline. No human or ani-
mal studies were carried out by the authors for this
chapter.
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66. Bron A, Tripathi R, Tripathi B.  Wolff’s anatomy of
the eye and orbit. 8th ed. London: Chapman & Hall;
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67. Dodson JW, Hay ED. Secretion of collagenous stroma
by isolated epithelium grown in vitro. Exp Cell Res.
1971;65:215–20.
68.
Cai CX, Fitch JM, Svoboda KK, Birk DE,
Linsenmayer TF. Cellular invasion and collagen type
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69. Hayashi M, Ninomiya Y, Hayashi K, Linsenmayer TF,
Olsen BR, Trelstad RL. Secretion of collagen types I
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1988;103:27–36.
70. Linsenmayer TF, Gibney E, Gordon MK, Marchant
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71. Quantock AJ, Young RD. Development of the corneal
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73. Maurice DM. The cornea and sclera. In: Davison H,
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biochemistry. 3rd ed. Orlando: Academic press; 1984.
 Confocal Microscopy of the Cornea
in a Clinical Model of Corneal
Stromal Expansion Using Adipose
Stem Cells and Corneal
Decellularized Laminas in Patients
with Keratoconus
Mona El Zarif, Karim Abdul Jawad,
and Jorge L. Alió
24.1 Introduction
Keratoconus is characterized by a progressive
decrease in corneal strength [1], related to a pro-
gressive loss of keratocytes of the human eye, the
number of keratocytes decrease from anterior to
posterior stroma [2], and the progressive thinning
of the corneal stroma [2, 3]. This definition is
valid for most patients with keratoconus, although
some variations in the phenotypic expression of
the disease might be present [4]. Most of kerato-
conus cases have thin corneas and a weak
mechanical resistance related to the progressive
M. El Zarif (*)
Optica General Sarl, Department of Optometry and
contactology, Saida, Lebanon
Vissum Instituto Oftalmologico de Alicante,
Alicante, Spain
K. A. Jawad
Optica General Sarl, Department of Optometry and
Contactology, Saida, Lebanon
University of Nicosia, Department of Life and Health
Sciences, Nicosia, Cyprus
J. L. Alió
Professor and Chairman of Ophthalmology,
University Miguel Hernandez, Vissum-Instituto
Oftalmologico de Alicante, Alicante, Spain
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_24
24
loss of keratocyte density [5]. Apoptosis of kera-
tocytes [2, 6] or release of enzymes is thought to
be the cause of keratocyte loss and consequently
loss of corneal stroma over time [2, 6]. The pro-
portion of keratocytes in the corneal stroma is
decreased with the progression of the disease [2].
In the end stages of keratoconus, the clinical
aspects of the thin and debilitated cornea are
associated with a sharp decrease in the amount of
keratocytes. There is severe corneal deformation
[2], and alteration in the location of the corneal
apex [7] consequently causes severe visual loss.
The treatment of keratoconus should be
directed towards the recovery of the normal char-
acteristics of the corneal stroma. This can be
achieved by substituting the diseased cornea with
a healthy cornea, by inserting into the cornea dif-
ferent materials to increase resistance such as
intracorneal rings, or biological tissues, or by
stimulating cells present in the corneal stroma to
produce collagen. Such recovery of the corneal
stroma could be induced by either stimulating the
cells that produce collagen or by increasing the
thickness with the use of corneal stroma or any
other tissue that could increase the corneal thick-
ness and optical performances while improving
the corneal thickness with the production of new
collagen, substituting the one lost due to the pro-
gression of the disease.
363
364
Based on the authors’ experience in corneal
regeneration [8–10], we proposed the use of cor-
neal decellularized lamina with or without ADAS
cells for the treatment of severe thin corneas suf-
fering from keratoconus [8]. The extremely thin
cornea that exists in severe cases could at the
same time further increase in thickness with the
use of a corneal lamina [9], cut by a femtosecond
laser from corneas of eye bank donors, that could
be implemented in its biological value with the
associated use of the above-described stem cells
with the purpose of increasing the corneal thick-
ness even further [8]. Along this chapter, we are
going to describe the evolution of confocal
microscopy following a clinical experiment, in
which autologous stem cells have been injected
in the corneal stroma [8]. Corneal human donors
unsuitable for corneal graft with or without the
impregnation of autologous stem cells, for the
purpose of treating of patients with advanced
stages of keratoconus [9, 10].
We are going to report herein the results of
this experiment with the use of confocal micros-
copy as an investigational tool to ascertain the
fate of the stem cells used along the experiment
and the impact that this cellular therapy might
have in the future treatment of keratoconus [8].
24.2 Patients and Methods
24.2.1 Patients
Fourteen consecutive keratoconus patients were
enrolled in the study and randomly distributed
into three study groups:
Group 1: Autologous ADASC implantation (5
patients)
Group 2: Decellularized human corneal stroma
transplantation (5 patients)
Group 3: Autologous ADASC + decellularized
human corneal stroma transplantation (4
patients)
Thirteen patients completed the study, only one
abandoned it due to independent personal
reasons.
M. El Zarif et al.
24.2.2 Methods
24.2.2.1 D  escription of the Confocal
Microscope Used: (HRT3
RCM Device
from Heidelberg)
With confocal microscopy using optical section-
ing, the microstructure of the cornea can be
observed under better conditions than with previ-
ously available devices. For instance, the utiliza-
tion of coherent light has allowed the improvement
of contrast and the quality of the images of the
stroma that is observed under the confocal micro-
scope [2].
Considered as a noninvasive technique, confo-
cal microscopy allows in vivo observation of the
microstructure of the cornea and a more accurate
quantification of keratocyte density (Fig.  24.1
and Table 24.1).
24.2.2.2 P  reparation of the Confocal
Microscope
While the device is turned off, the Rostock
Cornea Module (RCM) objective must be set at
+12 D (Fig. 24.2). Then, a drop of contact gel is
applied on the front surface of the microscope’s
lens of the RCM. The gel must not contain air
bubbles (Fig. 24.3). The TomoCap is then placed
on top of the RCM objective (Fig.  24.4). The
camera is placed on the side of the examined
eye [11].
After getting the consent of the patient, mak-
ing sure there is no allergy towards the gel and
anesthetic applied, and informing how the exami-
nation will be performed, the patient is prepared
by applying a drop of topical anesthetic in the eye
to be examined. Then, a gel tear substitute is
applied in both eyes. The gel is also applied on
the outer surface of the TomoCap in order to
improve the contact surface between the cap and
the eye of the patient. Heidelberg Engineering
recommends using the same high viscosity gel
for both applications on the eye of the patient and
the TomoCap in order to prevent any confusion
between the gels, since two different gels can be
used in each application. We recommend pre-
serving the gel at 4 °C for a period of time before
24  Confocal Microscopy of the Cornea in a Clinical Model of Corneal Stromal Expansion Using Adipose… 365

Fig. 24.1  The HRT3 1

RCM device from
Heidelberg [11] 7
2 8
3

4
9

10
11
5

6 12

Table 24.1  Illustration of the confocal microscope with

detailed description of its different parts [11]
1 Forehead rest 7 CCD camera
2 Headrest column with red 8 RCM objective
marks
3 External fixation light 9 Chin rest
4 Camera head 10 Adjustment screw
for camera position
5 Adjustment screw for 11 Adjustment screw
vertical and horizontal for chin rest
camera position
6 CCD camera cable 12 RCM objective
cable

its use and maintaining the environment of the

examination room at a relatively low
temperature.
Before proceeding with the examination, the
patient must remain stable, comfortable, and
aligned with the lens of the confocal microscope.
Afterwards, the patient is instructed not to move
the eyes, to keep on looking at the light inside the
lens, not to blink, and not to look outside the lens
in order not to hinder the observations made with
the microscope [11].
Afterwards, the CCD camera is adjusted per-
pendicularly to the optical axis of the scanning
laser camera. The RCM objective and the focal
plane are adjusted to the ocular surface of the
Fig. 24.2  RCM objective set at +12 D [11]
TomoCap, and then the focal plane position is set
to “0” by clicking on “reset” the focal section
(Fig. 24.5). Resetting the focal position to “0” at
the superficial epithelial cells of the examined
eye is crucial to measure the depth of the corneal
cell layer relative to the corneal surface.
Following this, the scanning laser camera is
slowly moved towards the patient’s eye
366
Fig. 24.3  Front surface of the contact lens of the micro-
scope where the gel is applied [11]
Fig. 24.4  TomoCap attached to the objective [11]
(Fig. 24.6). It is advised to apply gel on the exte-
rior face of the TomoCap to improve the contact
between the cap and the patient’s cornea. At this
point, the patient is asked to widely open the eye,
and the scanning camera is slowly moved towards
the eye until the TomoCap touches the patient’s
cornea. The RCM is turned clockwise or counter-
clockwise, and the focal plane is adjusted to the
desired cell layer once a contact between the
patient’s cornea and the TomoCap has been
made. The headrest must not be moved transver-
sally. Finally, an image is obtained by pressing
the foot switch [11].
M. El Zarif et al.
4
Fig. 24.5  Image seen after adjustment of the focal plane
to the ocular surface of the TomoCap [11]
1
Fig. 24.6 Reflection of the laser from the confocal
microscope in the center of the pupil indicating the right
alignment between the microscope and the eye of the
patient [11]
24.2.2.3 D  ifficulties in Handling
the Confocal Microscope
The use of the confocal microscope for the study
of the cornea is always somehow cumbersome
for both the patient and the examiner.
24  Confocal Microscopy of the Cornea in a Clinical Model of Corneal Stromal Expansion Using Adipose… 367

The patient must be able to collaborate with Unlike in normal epithelial tissues, it can be
the examiner in maintaining a stable posture, a ­difficult to distinguish the borders between the
good fixation, and a wide palpebral opening of epithelial cells, and the basal epithelium shows
the eye in order to allow contact between the distortions and irregularities (Figs.  24.9, 24.10,
TomoCap and the eye [11]. and 24.11) [13].
The feeling of contact of the TomoCap with
the eye of the patient may cause tearing and pho-
tophobia in some patients, which increases the
difficulty of the examination.

24.2.2.4 Confocal Sampling

of the Corneal Stromal
in Advanced Keratoconus
In confocal microscopic investigations of kera-
toconic patients, superficial epithelial cells that
are elongated in oblique direction, are consis-
tently found alongside highly reflective
(Fig. 24.7), pathological alterations are found in
principle at the fourth stage of the disease in the
central area of the cornea, as reported by various
authors [12].
In our experience, we also found (Figs.  24.7
and 24.8) that in the epithelial layer, superficial
cells are elongated and distorted in an oblique Fig. 24.8  Superficial epithelium cells at −1 μm from a
34-year-old keratoconic female patient show different
direction instead of them being presented as a sizes of superficial epithelial cells and an elongated one in
mosaic form of regular polygonal surface [13]. oblique direction

Fig. 24.7  Superficial epithelium cells at −3 μm from a Fig. 24.9  Corneal epithelium at 32  μm from a kerato-
34-year-old keratoconic female patient. It shows elon- conic female patient
gated cells in oblique direction, highly reflective with evi-
dent area of missing cells
368 M. El Zarif et al.

Fig. 24.10  Corneal epithelium at 50  μm from a kerato- Fig. 24.12  Bowman’s membrane of a 34-year-old female
conic female patient showing the nerve plexus, irregular with advanced keratoconus shows a visible dark striae at
and distorted epithelial cells and small area of missing cells 61 μm

Fig. 24.11  Basal epithelium cells at 61  μm from a Fig. 24.13  Basal epithelium, Bowman’s membrane, and
32-year-old keratoconic female patient. It shows highly subepithelial plexus nerve of a 31-year-old male patient;
reflective nodules related to scar tissue at the level of the fibers are disposed in oblique direction and display
Bowman’s membrane, with areas of missing cells fine irregularity

Extending from the basal membrane to the (Fig. 24.12) has also been described in other stud-
immediate stroma beneath it, ruptures in ies that found that there is a reflection of bright
Bowman’s membrane are represented by a highly background illumination underneath Bowman’s
reflective scar tissue [13]. Presence of striae membrane due to a disarrangement of collagen
24  Confocal Microscopy of the Cornea in a Clinical Model of Corneal Stromal Expansion Using Adipose… 369

Fig. 24.14 Preoperative corneal anterior stroma at Fig. 24.15  Deep corneal stroma at 396  μm from a
158 μm of a 35-year-old keratoconic female patient show- 50-year-old keratoconic female patient showing oblique,
ing oblique dark striae with a moderate number of reflec- vertical, and horizontal dark striae with a moderate num-
tive keratocytes ber of hyperreflective keratocytes

fibers [12]. Sometimes, the thin nerve fibers pres-

ent in the area of corneal protrusion, disposed in
an oblique horizontal pattern that is coherent with
the direction of the stretched epithelial cells
(Fig. 24.13) [13]. In addition, an ­elevation in the
anterior stroma and Vogt’s striae in the posterior
stroma’s reflectivity are consistent with pathologi-
cal processes of the corneal stroma [14]. One of
the main abnormalities of the stroma is the pres-
ence of microstriae that are evident in the whole
stroma of the cornea, which also appear as multi-
ple dark and thin lines in comparison with the
brighter stromal reflectivity (Fig.  24.14).
These microstriae could possibly result from the
degenerative process that leads to changes in the
extracellular collagen lamellae of the cornea [13].
Fig. 24.16  Anterior corneal stroma at 63  μm from a
These microstriae can appear as horizontal, verti-
33-year-old keratoconic female patient, with a reduced
cal, oblique, or reticular lines (Fig.  24.15), and number of keratocytes and thin nuclei content
they appear to extend from the most anterior
stroma to Descemet’s membrane [13].
Morphology of keratocytes changes from the shape ( Fig.  24.17), and more consistent and
anterior to the posterior stroma. They have thin bigger nuclei size in the mid of stroma
­
and consistent nuclei in the anterior stroma (Fig. 24.18). The morphology of the keratocytes
(Fig. 24.16), some stromal cells have a dendritic in the posterior stroma is similar to the one in the
370 M. El Zarif et al.

Fig. 24.19  Deep corneal stroma at 439  μm from a

Fig. 24.17  Preoperative mid-stroma at 217  μm from a
33-year-old keratoconic female patient, with a significant
31-year-old keratoconic male patient. It shows  kerato-
decrease in the number of keratocytes, and less reflective
cytes in dendritic form
and dense nuclei

Fig. 24.18  Mid-stroma at 220  μm from a 33-year-old

keratoconic female patient, with a reduced number of Fig. 24.20  Descemet’s membrane from a 31-year-old
keratocytes with more dense nuclei keratoconic male patient, showing an acellular layer with
a highly reflective zone

middle stroma, but they are smaller and less cell layer might show changes such as an increase
bright (Fig. 24.19). in cell area, a decrease in cell density, polymega-
Finally, the descemet membrane appears com- thism, and pleomorphism depending on the pro-
pletely acellular (Fig. 24.20) and the endothelial gression of the disease (Fig. 24.21) [13].
24  Confocal Microscopy of the Cornea in a Clinical Model of Corneal Stromal Expansion Using Adipose…
24.2.2.5 Methods of Cell Counting
Automated Cell Counting of Keratocytes
The automatic count that is calculated by confo-
cal corneal microscopy devices usually is not
precise, which leads to an increase in errors when
calculating the number of cells. Therefore, for the
purpose of this investigation, we preferred to per-
form a manual cell count of the keratocytes of the
corneal stroma [11].
Fig. 24.21  Endothelium at 531  μm from a 24-year-old
keratoconic male patient with mild pleomorphism
Fig. 24.22  Cell count performed with mid brightness and contrast; the keratocytes are marked in blue
371
Method for Manual Cell Counting
of Keratocytes
First, an image of the corneal stroma of a patient
is selected. The image has to have a good quality
of contrast and illumination, and must ­contain
keratocytes. An area known as the region of inter-
est (ROI) is determined. This region has plenty of
cells. It is advised to choose the same approxima-
tion of ROI (e.g., 0.1000 mm2) for all cell counts
in all layers of the corneal stroma [2, 11].
The cell count begins with mid brightness and
contrast. At this point, the cells that are more illu-
minated and more refringent [5, 6] are selected
with the marker of the device, which usually is
either white or light gray and have clear borders
[2]. The dark gray cells should not be taken into
consideration, because these cells do not belong
to the chosen plane. This high brightness is usu-
ally attributed to the nuclear content in these
cells, and they have irregular oval-shaped bodies
(Fig.  24.22) [5], while the difference in bright-
ness is due to the metabolic activation and the
direction of incidence of the laser light [5].
After the first count is done, the contrast is
increased to the maximum, and the brightness is
decreased to the minimum. Keratocytes that dis-
appear from the chosen plane are eliminated, and
only the ones that remain are used for the final
372 M. El Zarif et al.

Fig. 24.23  Elimination of keratocytes that do not belong to the plane under observation using low brightness and high
contrast settings

Fig. 24.24  Refining of the cell count using three-quarter high brightness and a quarter low contrast at which the image
appears in grayscale

count of cells even if they are faintly identified in
the image (Fig. 24.23).
Finally, the contrast is decreased to a quarter,
and the brightness is increased to three quarters
(Fig.  24.24). In this newly adjusted image, the
plane becomes grayscale, and the keratocytes
become highly illuminated, which allows a final
refinement of the cell count.
When the images are very dark, it is recom-
mended to increase the contrast to the maxi-
mum and the brightness to three quarters
(Fig.  24.25). This facilitates the count of the
24  Confocal Microscopy of the Cornea in a Clinical Model of Corneal Stromal Expansion Using Adipose… 373

Fig. 24.25  Maximum contrast and three-quarter brightness is used in the case of very dark images

Fig. 24.26 Count of ADAS cells and keratocytes,
1  month following surgery of a 33-year-old keratoconic
patient. The cells are marked in blue
keratocytes, and the cell count can be resumed
as mentioned above.
Cell Counting of ADAS Cells
The counting method for transplanted ADAS
cells is done similarly to the way normal kerato-
cytes of the corneal stroma are counted. The
only difference between the two methods is that
Fig. 24.27  Count of cells for the same patient 1  year
after surgery
in the first 6 months after the surgery, the ADAS
cells have a round shape and are more luminous
and refringent than normal keratocytes
(Fig. 24.26) [8]. After these 6 months, the ADAS
cells assimilate a shape similar to normal kera-
tocytes [8], thus  reducing any morphological
differences between them (Fig.  24.27).
Therefore, the cell count becomes the same for
both kinds of cells.
374
Cell Count on a Decellularized Lamina
The images provided by the confocal micro-
scope shows that the lamina appears totally acel-
lular in the first month (Fig. 24.28) but the newly
colonized transplanted lamina by the keratocytes
of the patient’s corneal stroma show that these
cells differ in morphology from the native kerato-
cytes of the cornea. This is caused by the reduced
transparency of the lamina right after it is
Fig. 24.28  Anterior aspect of a decellularized lamina of
a female, which appears to be without cells 1 month
post-operation
Fig. 24.29 Cell count of a 24-year-old keratoconic
patient 3 months post-operation on the posterior aspect of
the lamina
M. El Zarif et al.
­transplanted (Fig.  24.  29). Thus, the images of
these cells lack the depth seen in the normal cor-
neal keratocytes of the patient. Three months
after the surgery, the morphology of patients’
cells colonized on the lamina started to show
some  new keratocyte appearance (Fig.  24.29).
The shape of these cells at 6 months became
more similar to the keratocytes (Fig. 24.30) [9],
before fully developing into cells that have the
same morphology of keratocytes 1 year post-
operation (Fig.  24.31) [10]. Therefore, it is
Fig. 24.30  Cell count in the anterior aspect of the lamina
in a 29-year-old keratoconic patient, 6  months
post-operation
Fig. 24.31  Cell count on the anterior aspect of the lam-
ina of a 31-year-old patient, 1 year after the surgery
24  Confocal Microscopy of the Cornea in a Clinical Model of Corneal Stromal Expansion Using Adipose…
important to identify the change in morphology
of these cells throughout the months, which helps
in achieving a more accurate cell count.
Cell Count on a Recellularized Lamina
The cell count on a recellularized lamina is rela-
tively similar to the one made on a decellularized
one. In this case, a few ADAS cells can be seen
within the lamina at 1 month after the surgery in
two of the four patients. In addition, the recellular-
ized lamina consistently contained a higher num-
ber of keratocytes than in the decellularized one,
when observed 3–12 months after the surgery [10].
24.2.2.6 Corneal Density Calculation
To obtain cell density, the examiner must use the
cellular counting software in the confocal micro-
scope [11] and delimit a fixed area depending on
the images measured by the specialist, with a
periphery that contains abundant keratocytes.
Since it is difficult to achieve great corneal con-
tact between the TomoCap and the patient, the
specialist is required to select an area that con-
tains abundant cells.
The following data appear on the software
screen:
• Number of cells: Which are the number of
cells counted by the observer
• ROI (mm2): The area chosen by the same
observer
Density +/− std (cell in mm2): Density calcu-
lated by mm2 by the same device with an error of
more or less the number of cells in the area [6, 11].
For example:
Density ( cells in mm 2 ) = 200 ± 12
24.2.2.7 Difficulties in Cell Counting
During our experience in counting keratocytes,
we encountered difficulties until we established
specific criteria and methodology for the purpose
of cell counting. We recommend the following:
• The cells that appear in the captured images as
if they belonged to the same plane of 50
micron of depth cannot be counted. These
cells appear with a faded gray color. At this
375
point, it is advisable to lower the illumination
to 0% and increase the contrast to 100% as
indicated in (Fig. 24.23).
• If the images have low resolution, the contrast
must be modified towards 25%, and the illumi-
nation is raised to approximately 75%. By
doing so, this image is seen in the clear back-
ground, and the keratocytes appear with distin-
guished border and are clearly illuminated.
Therefore, they can be marked more easily. We
call this regulation the grayscale (Fig. 24.24).
• If the confocal images are very dark, it is
advisable to set the illumination to 75% and
the contrast to 100% (Fig. 24.25).
• If a line of separation between both structures
of attached cells is not clear, it is advisable to
interchange between the contrast and illumi-
nation until a discrimination of the orientation
of the nuclear content of both cells can be
made. If the content appears parallel, it is
more likely that they belong to the same
nucleus. However, if the nuclear contents of
the two cells are in different directions, then
they belong to two different cells.
• If the cells at the edge of the selected plane are
not seen in their complete shape, every two
halves of cells are counted as one single cell.
When observing the transplanted laminas, it
was noticed that their morphology is either decel-
lularized or recellularized; has an extracellular
matrix characterized by the presence of abundant
streaks, which are larger in size and are more pro-
found; and has changed during the next months
after the transplant. This changed morphology
remained constant afterwards.
24.3 Results
24.3.1 Confocal Microscopy
of the Corneal Stroma
Following the Stromal
Enhancement Intervention
24.3.1.1 Patients with  Transplanted
ADAS
One to three months following the implantation
surgery, the transplanted ADAS cells were round in
shape and more refringent [8], in comparison to the
376
Fig. 24.32  Confocal microscopy of corneal stroma from
a 33-year-old keratoconic female patient at 265  μm
1 month after transplantation of ADAS cells
Fig. 24.33  Confocal microscopy at 273  μm from a
33-year-old keratoconic female patient 3  months after
transplantation of ADAS cells. The blue dots show the
transplanted cells in the plane of the lamellar pocket. They
have a round morphology and are more refringent than the
original keratocytes of the patient’s stroma
fusiform morphology of the keratocytes [5], pres-
ent in the recipient cornea (Figs. 24.32 and 24.33).
However, the shape of ADAS cells changed from
round to fusiform 6 months after the operation [8],
and they assimilated a morphology similar to that
M. El Zarif et al.
Fig. 24.34  Morphological change in the shape of ADAS
cells, from round to fusiform in the same 273  μm, of a
33-year-old female patient 6 months after the operation
Fig. 24.35  Keratocytes in the surgical plane at 250 μm,
1 year after the transplant
of the original keratocytes of the patient’s cornea
(Fig. 24.34). The round morphology of these cells
is a strong indicator of their ability to survive, espe-
cially during the early postoperative period [8]. We
also found that the number of keratocytes increased
6 months after the operation [9]. One year after the
surgery, it is noticeable that the number of kerato-
cytes increased even more (Fig.  24.35) [10].
A gradual increase (P value = 0.07) in the density
24  Confocal Microscopy of the Cornea in a Clinical Model of Corneal Stromal Expansion Using Adipose… 377

Table 24.2  Evolution of the density of keratocytes of the patients over 1 year
Preop 6 months 12 months
Group
Group 1 Group 2 Group 3 Group 1 Group 2 Group 3 1 Group 2 Group 3
Anterior stroma 236.45 222.6 187.6 254.15 215.3 286.6 288.5 441.2a 467.4
(cells/mm2) (220, (163.3, (176.6, (210, (190, (210, (210, (392.5, (333.3,
280) 286.6) 198) 306.6) 273.3) 356.6) 340) 474) 594)
Posterior stroma 202.5 181.8 192.8 255.8 239.16 237.4 322.5 459.7a 416.5
(cells/mm2) (190, (136.6, (174, (190, (213.3, (163.3, (290, (376.6, (388,
225) 216) 203.3) 320) 265) 293.3) 340) 540) 447.5)
Keratocyte cellular density (mean value and range) for G 1 (autologous ADASC implantation; n:4), G 2 (decellularized
human corneal stroma implantation; n: 5) and G 3 (autologous ADASC recellularized human corneal stroma implanta-
tion; n: 4) before, 6 and 12 months after surgery [10]
a
Shows statistically significant (P ≤ 0.05) differences between the preoperative and 12 months postoperative values for
each parameter and for each study group separately (for mid-stroma, this comparison is between 6 and 12  months
post-operation)

Fig. 24.36  Posterior face of a decellularized lamina of a

31-year-old male, 1  month after the operation, seen at
289 μm. The lamina appears to be almost acellular
of cells was evident in the anterior, middle, and
posterior stroma of the patients 1 year after the
operation, in comparison to the density level preop-
erative (Table 24.2).
24.3.1.2 P  atients with Decellularized
Lamina
One month after the surgery, the transplanted lam-
ina generally remained acellular in most of the
patients (Fig. 24.36) [9]. However, two out of five
patients showed in the anterior aspect of the lam-
ina the beginning of keratocyte colonization,
which is a change normally seen 3 months after
Fig. 24.37  Anterior face of the decellularized lamina of
the same patient, 3  months after the operation, seen at
207 μm, showing early colonization of the keratocytes to
the lamina
the surgery (Fig.  24.37). On the other hand, the
posterior aspect of the lamina appeared to be
clearly colonized by some keratocytes at the same
time of examination in three of the five patients
(Fig. 24.38). Six months after the transplant, the
anterior aspect of the lamina was clearly colo-
nized by keratocytes in some patients (Fig. 24.39),
while the posterior aspect was more abundant in
these cells (Fig. 24.40). Moreover, 1 year after the
operation, both the anterior and posterior aspects
became fully colonized by keratocytes (Figs. 24.41
and 24.42) [10]. The density of cells on the lamina
378
Fig. 24.38  Posterior face of the lamina at 271  μm,
3 months after the operation clearly colonized by the sur-
rounding keratocytes
Fig. 24.39  Colonized anterior face of the lamina at
176 μm at 6 months
was similar to what is expected in a normal cor-
nea. We were able to demonstrate that there was a
significant increase in keratocytes within the lam-
ina and keratocyte density in both the anterior and
posterior stroma was statistically significant (P
value  =  0.008) 1 year after the operations were
performed (Table 24.2).
The collagen matrix morphology of the decel-
lularized lamina differs from the collagen matrix
M. El Zarif et al.
Fig. 24.40  Posterior face of the lamina that is highly
colonized by keratocytes from the cornea 6 months after
the operation
Fig. 24.41  Anterior face of the lamina showing high
quantity of keratocytes 12 months after the surgery
of the patient’s stroma. It shows clear and abun-
dant dark striae, some of which have consider-
able thickness and have vertical, horizontal, and
oblique directions (Figs. 24.43 and 24.44).
We observed at the periphery and border of
the lamina highly reflective structures that are
caused by the mechanical trauma during the
surgery (Figs. 24.45 and 24.46). The transition
zone between the lamina and the corneal stroma
24  Confocal Microscopy of the Cornea in a Clinical Model of Corneal Stromal Expansion Using Adipose…
Fig. 24.42  Posterior face of the lamina colonized by
high numbers of keratocytes 12  months after the
operation
Fig. 24.43  Anterior face of the decellularized lamina of
a 31-year-old male patient, 3 months postoperatively, seen
at 154 μm, showing striae that are darker and larger than
the ones present in a normal stroma
can be  easily differentiated (Fig.  24.47); also,
the ­lamina’s border has, in addition, a reflective
structure (Fig.  24.47). We observe as well a
clear keratocyte migration from the corneal
stroma to the decellularized lamina (Fig. 24.48).
Also, the lamina’s light reflectivity is higher
than the matrix’ corneal stroma (Figs.  24.44
and 24.48).
379
Fig. 24.44  Anterior face of the decellularized lamina of
a 35-year-old keratoconic female patient showing dark
and abundant striae. The matrix is clearer than the normal
stroma and more reflective
Fig. 24.45  Anterior face of the decellularized lamina of
a 34-year-old female, 1  month post-operation, shows
highly reflective peripheral structures on the lamina due to
mechanical trauma
Twelve months after the operation, both anat-
omy of the corneal anterior and posterior stroma
and the keratocyte density did not differ in patients
to the normal corneal stroma at the confocal
microscopy level (Figs. 24.49 and 24.50) [10].
380
Fig. 24.46  Decellularized lamina present at 272 μm, of a
31-year-old male, 3 months after surgery, showing reflec-
tive peripheral structures, due to the mechanical surgical
trauma, needle-shaped bodies, and some keratocytes with
morphological change
Fig. 24.47  Transition zone at 190 μm between the border
of the anterior face of the decellularized lamina and the
Fig. 24.49  Anterior stroma at 103 μm of a 24-year-old
corneal stroma, 3  months post-operation, for a 35-year-­
patient, 12 months after the surgery
old female showing highly reflective structures, with a
few dendritical keratocytes
24.3.1.3 P atients with Recellularized
Lamina
One month after the surgery, both the anterior
and posterior faces of the patients’ laminas
showed in two of the four patients the presence of
M. El Zarif et al.
Fig. 24.48  Transition zone at 306 μm between the poste-
rior face of the decellularized lamina and the recipient
stroma with a number of migrating keratocytes with den-
dritical shapes and the corneal stroma with a number of
keratocytes, 12  months post-operation of a 35-year-old
female patient
a few number of ADAS on their surface, which
are smaller than normal keratocytes in the ante-
rior face of the lamina (Figs.  24.51 and 24.52),
but they are more similar to them in the posterior
face, unlike the decellularized laminas, which
were completely acellular. At 3  months, the
24  Confocal Microscopy of the Cornea in a Clinical Model of Corneal Stromal Expansion Using Adipose…
Fig. 24.50  Posterior stroma at 410  μm of the same
24-year-old patient, 12 months after the surgery
Fig. 24.51  Anterior face of a recellularized lamina at
174  μm of a 50-year-old patient, 1  month after the
operation
n­ umber of cells increased slightly, and all the
patients showed a few number of cells similar to
keratocytes, some of them presenting with den-
dritical shapes. Six and twelve months after the
­operation, the number of cells that colonized the
laminas was higher in the recellularized laminas
than in the decellularized ones (Figs.  24.53,
24.54, 24.55, and 24.56) [9, 10].
The histology of the anterior and posterior
corneal stroma showed a very high number of
381
Fig. 24.52  Aspect of the posterior face of the lamina at
275 μm, 1 month post-operation
Fig. 24.53  Anterior face of the recellularized lamin at
195 μm, 6 months after the surgery
keratocytes [10], similar to that in a normal cor-
nea (Figs. 24.57 and 24.58). Results derived from
the anterior stroma (P  =  0.008) and posterior
stroma (P  =  0.008) showed a statistically
­significant increase in the keratocytes’ density of
the patients from the G2  +  G3 (Table  24.2). In
addition, we could demonstrate that there was a
significant increase in keratocytes within the
recellularized lamina more higher in the group 3
than the group 2.
382
Fig. 24.54  Posterior face of the lamina 6 months post-
operation with a high number of keratocytes
Fig. 24.55  Anterior face of the recellularized lamina at
215 μm 12 months after the surgery
24.4 Discussion
These sets of experiments represent the first
attempt at using stem cell therapy for human
­corneal stroma in patients affected by advanced
keratoconus.
We have previously reported that after the
injection of autologous stem cells (ADASC) in the
stroma of the cornea, there was an increase in the
M. El Zarif et al.
Fig. 24.56  Posterior face of the lamina at 299  μm,
12 months post-operation. It shows a very high number of
keratocytes
Fig. 24.57  Anterior corneal stroma with abundant kera-
tocytes in a 33-year-old female patient, 12  months after
the operation, seen at 106 μm
density of corneal keratocytes. This favored the
production of new collagen, which resulted in an
improved corneal thickness [9]. In  addition, this
study showed that the implantation of a decellular-
ized lamina, either colonized or not by ADASC,
also produced changes in the corneal topography,
thickness, and corneal r­ egularity that may be con-
sidered as therapeutic with a potential application
to the treatment of keratoconus [9, 10].
24  Confocal Microscopy of the Cornea in a Clinical Model of Corneal Stromal Expansion Using Adipose…
Fig. 24.58  Posterior corneal stroma at 507 μm with high
number of keratocytes for the same 33-year-old patient
In this innovative study, we have reported
the  outcomes of the use of corneal confocal
microscopy for the observation of ADASC in the
selected surgical plane in vivo, which allowed a
qualitative and quantitative assessment of them
along the experience. Moreover, it allowed moni-
toring of the progressive morphological changes
that occurred in the decellularized and recellular-
ized laminas throughout the observation and
assisted in determining the change in the cell
densities in the grafted tissue as well as in all the
corneal stroma.
Confocal corneal microscopy has shown to be
an essential tool in the assessment and “in vivo”
follow-up of the corneas implanted with mesen-
chymal cells for corneal regeneration purposes.
Corneal bioconfocal microscopy demonstrates
an increase in the cellularity of the anterior and
posterior stroma following the implantation of
ADAS cells into the corneal pockets created
using a femtosecond laser.
Relevant changes in the morphology were
observed in the implanted cells from the moment
of their implantation into the corneal pocket,
showing changes from clusters around the
­individual cells to their development into adult
keratocytes. These findings occurred in the ante-
rior, mid, and posterior stroma of the implanted
corneas. Moreover, changes inside the acellular
383
lamina were also observed. The decellularized
lamina was colonized by the patient’s own kera-
tocytes from the first month. After 12 months, the
lamina was completely recellularized.
Confocal microscopy of the cornea during the
study has been able to show us from the first post-
operative month the following findings: accumu-
lations of the mesenchymal cells implanted on
the lamina, cell morphological changes have
been clearly seen first on the posterior face fol-
lowed by the anterior face, and the number of
cells has increased statistically and significantly
between immediately after the surgery and a
12-month period.
Confocal microscopy is considered a noninva-
sive technique [6]. But difficulties can arise when
patients are being examined, especially when the
contact between the TomoCap and the cornea of
the patient must not cause any harm for the
patient. The fragility of the operated cornea is
also a concern when examining a patient with the
confocal microscope. Before performing the test
with the confocal microscope, it is advisable to
introduce a silicone hydrogel disposable contact
lens, which improves the contact between the eye
and the TomoCap. This lens prevents the gel from
leaking. In the absence of this lens, the gel dries
out with time or dissipates outside the eye. In
such case, the examination is interrupted, and
additional gel must be reapplied in the eye of the
patient. Also, the addition of this lens reduces
corneal sensitivity, which allows the patient to
keep the eye open for a longer time, which ulti-
mately facilitates the examination process. In
addition, sterilization must be maintained
throughout the process in order to prevent any
contamination in the patient’s eye, to reduce error
that may hinder the results obtained from the
observation, and to preserve the quality of the
images obtained by the confocal microscope
(Figs. 24.59 and 24.60).
One of the limitations of this study is the
method used for counting cells as it was
­performed with a manual method. Although it
was performed by two independent observers in
all cases following the same guidelines, this
method may introduce a certain amount of sub-
jectivity and interpretation bias. However, this
method yields at present moment more accurate
384
Fig. 24.59 Anterior face of decellularized lamina at
155 μm in a 33-year-old patient, 12 months after the sur-
gery; image taken with silicone hydrogel contact lenses
Fig. 24.60  Posterior face of decellularized lamina at
280 μm in a 33-year-old patient, seen 12 months after the
surgery; image taken with silicone hydrogel contact lenses
results than an automated cell counting micro-
scope which are affected by a large variability
and misinterpretations. We performed a refined
automatic cell count, and we observed a bias
towards high and abnormal number of cells that
were counted for the normal structure of the cor-
nea, and a considerable number of cells that
M. El Zarif et al.
Fig. 24.61  Confocal microscopy automatic cell counting
of corneal stroma, from a 33-year-old female with kerato-
conus at 151 μm seen 12 months after transplantation of
ADAS cells
Fig. 24.62  Anterior face of the recellularized lamina at
195 μm of a 35-year-old female, 6 months after the sur-
gery, demonstrates automatic cell counts with high and
abnormal number of cells
should have been counted within the corneal
stroma were not accounted for by the device
(Fig.  24.61) on both sides of the transplanted
lamina (Fig.  24.62). An artifacted result on the
lamina’s border in the transition zone between
the lamina and stroma was seen with this device
as well (Fig. 24.63).
24  Confocal Microscopy of the Cornea in a Clinical Model of Corneal Stromal Expansion Using Adipose… 385

d­ ecellularized laminas should be made, along-

side the growth of keratocytes in the corneal
stroma in both types of transplants, and relate it
to the improvement of the visual acuity of the
operated patients.
Furthermore, comparisons among different
morphologies of keratocytes should be estab-
lished, comparing non-dendritic cells, with oth-
ers that have a high dendritic shape and investigate
the biological implications that such morphologi-
cal changes may have.
A particular interest has been for the observa-
tion of hyper-reflective area, acellular or with very
few cells within the corneal stroma studied by the
confocal microscope. Further investigation should
verify if this fact is due to secondary reactions
caused by mechanical manipulation after surgery,
or to the formation of new collagen related to the
Fig. 24.63  Transition zone at 154 μm between the border ADAS cells implanted in the surgical plan.
of the anterior face of the decellularized lamina and the Comparisons of studies between different age,
corneal stroma, 6  months post-operation, for a 31-year-­
old male, showing highly reflective structures, the blue sex, and ethnic groups can yield many explana-
dots are generated by automatic count cells tions about prevalence and frequencies in the
population. We believe that it is necessary to
establish a more objective method of cell count-
24.5 Future Perspectives ing based on image processing programs, where
one can edit the contrast and illumination and use
In our study, confocal microscopy was very different specialized filters to optimize the visu-
­useful for keratocyte cell counting and determin- alization of keratocyte cells and to better calcu-
ing the evolution of the implanted cells in terms late their size.
of morphology and densities along the follow-up In addition, the use of digital cameras captur-
time of the experience. ing at high frames per second and high resolution
Although only 14 patients participated in this in the future can reduce the examination time and
study, sufficient statistical significance levels of provide a more reliable measurement of the
confidence were obtained that allow us to ­confirm thickness evolution of the cornea and the newly
differences among the clinical groups, which implanted corneal layers, with fewer errors
demonstrates the importance of the study estab- caused by the movement of the patient’s eye.
lished by the confocal microscope. However, it is Also, the development of a new TomoCap that
necessary to increase in future experiences the has a biconcave or planoconcave silicone hydro-
clinical number of participants in order to gel lens fixated to its top, with the lens made of a
strengthen the statistical significances found in specific thickness containing a viscous gel, may
the present study. shorten and provide more comfort in the confocal
Further research should be carried out to exam of the implanted patients. Also, it would be
answer questions like if all morphological very interesting to have a new  RCM objective
changes begin in the posterior stroma and then in that can be applied on the eye, while the patient is
the anterior, just as it was observed in our study, lying down on his back. This would restrict the
which seems to be a specific finding of this patient’s body and head movements. In this case,
investigation. a better fixation of the laser that is projected on
Comparisons between the recellularized the cornea can be made, which can provide better
laminas containing Bowman’s membrane and images of the different layers of the cornea and
­
386
less error while measuring the pachymetry of the
cornea. This innovative biomicroscope objective
would allow more comfortable and probably pre-
cise study of the confocal cornea microscopy
evolution of the experience, as it may allow to
study the collagen fibers the implanted laminas
as they are added to the collagen matrix structure
of the patient’s cornea.
Acknowledgments The authors would like to thank
Jorge L.  Alió del Barrio, MD, PhD; Ziad Abdul Jawad,
Od; Peggy Saba, Od; María P. de Miguel, PhD; Nehman
Makdissy, PhD; Francisco Arnalich, MD, PhD; and
Ibrahim Achkar, MD, for their essential contribution to
the clinical and cell biology part of this study.
References
1. Piñero D, Alio J, Barraquer R, Michael R, Jiménez
R.  Corneal biomechanics, refraction, and cor-
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2. Ku J, Niederer R, Patel D, Sherwin T, McGhee
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3. Alió J. Keratoconus: recent advances in diagnosis and
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A, Makdissy N, Harb W, et al. Cellular therapy with
human autologous adipose-derived adult stem cells for
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Khalil C, Harb W, et al. Corneal stroma enhancement
with decellularized stromal laminas with or without
stem cell recellularization for advanced keratoconus.
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Makdissy N, Khalil C, et  al. Regenerative medicine
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 Limbal Stromal Stem Cells
in Corneal Wound Healing:
Current Perspectives
and Future Applications
Noopur Mitragotri, Mukesh Damala, Vivek Singh,
and Sayan Basu
25.1 Introduction: Cornea
and Vision
The eye is a complex organ comprised of many
cell types. Each region has a specific optical
function to aid the transmission of light onto the
retina. The cornea is one of the most important
organs which help to achieve this purpose. The
human eye can be considered optically equiva-
lent to a photographic camera, where the cornea
and human crystalline lens focus the incident
rays of light on the retina just like the camera lens
focus the light on film. The iris works like the
shutter and the pupil as an aperture; the retina
acts like a film. In spite of having many similari-
ties, a major difference between the two systems
N. Mitragotri · V. Singh (*)
Center to Ocular Regeneration (CORE); and Brien
Holden Eye Research Center, LV Prasad Eye
Institute, Hyderabad, Telangana, India
e-mail: viveksingh@lvpei.org
M. Damala
Center to Ocular Regeneration (CORE); and Brien
Holden Eye Research Center, LV Prasad Eye
Institute, Hyderabad, Telangana, India
School of Life Sciences, University of Hyderabad,
Hyderabad, Telangana, India
S. Basu (*)
Center for Ocular Regeneration (CORE), LV Prasad
Eye Institute, Banajara Hills, Hyderabad,
Telangana, India
e-mail: sayanbasu@lvpei.org
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_25
25
is the mechanism involved in focusing an image
of an object. However, change in both the s­ ystems
is observed on the basis of changes in the power
of the lens by varying its curvature in the human
eye, i.e. the size of the lens can be adjusted,
whereas, in the cameras, there are a number of
lenses that change their relative position [1].
Figure  25.1 shows the high-­ resolution normal
eye image.
The sclera, limbus, ciliary body and vitreous
of the eye are also important for wound healing,
accommodation, movement, nourishment, main-
taining the shape of the eye, etc. Many extrinsic
and intrinsic factors affect the transparency of the
cornea, like the stromal scars or wound due to
infections, physical damage, chemical burns, etc.
Earlier, there was no other clinical therapy to
treat these stromal scars or wound, other than
photo keratectomy. However, many groups have
recently reported the use of stem cell- or cell-­
based therapy for treating these wounds in animal
models. This chapter will cover various modali-
ties along with our own method of using the lim-
bal stem cells in these cases.
25.1.1 Cornea
The cornea is present on the external surface of
the eye, and its optical clarity is important for
vision as it helps to transmit light onto the retina
as mentioned earlier. It is a transparent layer, and
387
388 N. Mitragotri et al.

Fig. 25.1  Anatomy of

a
the human eye: (a)
Normal human eye:
Image showing the
external appearance of a
normal human eye. (b)
Cornea: Normal
transparent cornea
giving the clear view of
intraocular iris and
limbal region. (c)
Limbus: High-resolution
image of normal limbus
located between the
conjunctiva and sclera,
the source of epithelial
and stromal stem cells b c

the transparency is maintained by the regular so it helps to maintain the structural frame-
arrangement of collagen. The corneal transpar- work. It also helps to maintain the avascularity,
ency is maintained by the three main and two transparency and immunity of the cornea.
auxiliary layers. Figure  25.2 shows different Stroma occupies more than 90% of the corneal
­layers of the cornea. Out of these five layers, thickness. It consists of an extracellular matrix,
­epithelium, stroma and endothelium are the most keratocytes (corneal fibroblasts) and nerve
important layers. Structure of cells in each layer fibres. This is how stroma is responsible for
plays an important role in corneal functioning. corneal strength and transparency [6]. When
there is a defect in the corneal stroma, it leads
(a) Corneal epithelium: It is the outermost layer to oedema, rupturing of the Descemet’s mem-
of the cornea which is approximately 50 μm brane and collagen arrangements which lead to
in thickness and has four to six layers of non-­ a loss of corneal transparency as shown in
keratinized, stratified squamous epithelial Fig. 25.3. Any problem in the stroma leads to
cells [2]. Epithelium with tear film maintains the obstruction of clear vision. Scarring of the
the optically smooth surface of the cornea corneal stroma because of surgery, trauma and
and acts as a barrier to external biological infections affects the vision of millions world-
and chemical insults [3, 4]. Epithelial cells wide [7]. Physical or chemical injuries also
can be identified by the keratin markers like result in forming scars which eventually leads
CK3 and CK12 as shown in Fig. 25.2. to vision loss or blindness.
(b) Bowman’s layer: The transparent film of tissue (d) Descemet’s membrane: It is the basement
layer behind the corneal epithelium is Bowman’s membrane of the corneal endothelium. It is
layer. It is 8–12 μm thick. It is an acellular, trans- 3–10 μm thick and made of collagen fibrils.
parent and non-regenerating layer which is It acts as a protective barrier against infec-
composed of strong layered collagen fibres. It tions and injuries and helps in maintaining
protects the subepithelial nerve plexus [5]. the corneal dehydration [5].
(c) Stroma: The major portion of the cornea is (e) Endothelium: The endothelium is the thin,
occupied by stroma. It is mainly composed of innermost layer of the cornea composed of
collagen and water. It is 450–500 μm thick and flat polygonal cells. They keep the cornea
25  Limbal Stromal Stem Cells in Corneal Wound Healing: Current Perspectives and Future Applications 389

a b

c d

Fig. 25.2  Cornea through histopathological view: (a)
Histopathological section of a normal cornea showing the
epithelium, stroma and endothelium layers. (b) Section of
a normal cornea showing the distribution of keratocytes
stained with the CK3 marker (red) and their nuclei coun-
terstained with DAPI (blue). (c) Cross-section of the cor-
nea with epithelial hyperplasia and stromal fibrosis. (d)
Abnormal cornea with hypertrophy, stromal oedema and
ruptured Descemet’s membrane. Scale: 200 μM

Fig. 25.3 
a
Vascularization of the
cornea leading to vision
loss. (a) Image of an
inflamed eye with
corneal scarring and
vascularization. (b)
Cornea with partial
limbal stem cell
deficiency with 6 clock
hour normal limbus and
6 clock hour
vascularization. (c)
Inflamed limbus with
neovascularization and
dysfunctional limbus
with early limbal stem b c
cell deficiency
390
dehydrated and clear. Generally, fluid gets
leaked slowly from inside the eye into the
stroma. The function of endothelium is to
pump excess water out of stroma to avoid
swelling and opacity [8]. The endothelial cell
density decreases according to age from
approximately 3000–4000 to 2500 cells/
mm2. Unlike epithelium, endothelial cells do
not demonstrate proliferative ability.
Endothelial cells do not undergo division.
Thus, if they get damaged by diseases or
trauma, they cannot be regenerated in vivo.
The corneal surface is more vulnerable to
infections and injuries as it is continuously
exposed to the environment. However, stroma
and endothelium also get affected. This leads to
optical damage or visual dysfunction or a combi-
nation of both.
25.2 The Necessity
of Regenerative Medicine
in Corneal Blindness
As the cornea plays the primary role in vision, any
damage to it hampers the vision. Though cataract
is responsible for nearly 20 million of the 45 mil-
lion blind people in the world, the next major
cause is trachoma, corneal scarring and vascular-
ization, blinding 4.9 million individuals [4].
Ocular trauma and corneal ulceration are underre-
ported mostly but are observed to be the signifi-
cant causes of corneal blindness responsible for
1.5–2.0 million new cases of monocular blindness
every year. In India, approximately 6.8 million
people are estimated to have vision less than
6/60 in at least one eye due to corneal diseases; of
these, about a million have bilateral involvement.
The number is expected to increase by 2020 to
10.6 million [9]. According to the National
Programme for Control of Blindness (NPCB) esti-
mates, currently, 120,000 people are blind because
of corneal disorders who are estimated to increase
by 25,000–30,000 every year [10]. In developing
countries, stromal dysfunction, infections, scar-
ring, injuries, etc. are the common causes of blind-
ness. Diseases affecting the cornea are a major
N. Mitragotri et al.
cause of blindness. The epidemiology of corneal
blindness is complicated and encompasses a wide
variety of infectious and inflammatory eye dis-
eases that cause corneal scarring, which ultimately
leads to blindness. To address the huge burden of
blindness in the developing world, there is a need
to pay some extra attention in developing new and
promising therapies like stem cell-based therapy.
The increasing interest in cell-based therapies is
because of increasing approvals and promising
and better outcomes leading to less clinical follow-
­up. Photo keratectomy has its own limitations and
complications mainly with the suturing, clinical
visits, long-term steroid usage, glaucoma, etc.
Diseases affecting the cornea are a major cause of
blindness. The epidemiology of corneal blindness
is complicated and encompasses a wide variety of
infectious and inflammatory eye diseases that
cause corneal scarring, which ultimately leads to
functional blindness which can be understood
from Fig.  25.4. Therefore, treatment of corneal
diseases is the necessity of the world. Corneal
transplantation is the standard of care as per cur-
rent clinical practice which is used to regain the
vision. But the supply of donor tissue is poor com-
pared to the demand. However, it is necessary to
look for the new alternative treatments/therapies
other than the commonly practised methods.
25.3 Limbus and the Stem Cells
The eye is the most sensitive organ of the body
and it is continuously functional. The layers of
corneal epithelium and stroma are continuously
under stress because of pollution, environmental
factors and microbes. Because of continuous
wear and tear, epithelial cells of the cornea are
consistently regenerated by the stem cells present
in the limbus.
Between the optically clear cornea and opaque
sclera, a transitional region of the stem cells
exists. This transitional zone is called limbus
(Fig.  25.1), which continuously regenerates the
corneal epithelial cells [11, 12] and stromal stem
cells [13–15]. It contains radially arranged ridge-­
like structures called the palisades of Vogt
(Fig.  25.1c). The corneal epithelial stem cells
25  Limbal Stromal Stem Cells in Corneal Wound Healing: Current Perspectives and Future Applications
a b
d e
Fig. 25.4  Representative images of the different clinical
causes for loss of corneal transparency. (a) Scarring: cor-
neal fibrosis. (b) Corneal oedema. (c) Corneal infection.
generate from the progenitor cells residing in
structures called limbal crypts, which extend
from the interpalisade ridges [16]. The palisades
protect from ultraviolet light-induced damage
[17] and a rich supply of blood vessels [18] to the
corneal stem cells. The palisades of Vogt provide
the niche for limbal epithelial stem cells (LESCs).
These stem cells give rise to transient-amplifying
cells (TACs) that have the potential to divide only
a limited number of times. They migrate centrip-
etally and are found in the limbal and peripheral
corneal basal epithelium. Initially, the TACs dif-
ferentiate into postmitotic cells of suprabasal cor-
neal epithelium and later to terminal differentiated
cells. This is how epithelium is regenerated and
preventing the overgrowth of conjunctival epithe-
lium onto the corneal epithelium [19]. The main
function of limbal epithelial stem cells (LESCs)
is to divide, differentiate and migrate centripe-
tally towards the cornea which essentially helps
to maintain the transparency of the cornea.
Recently, the superficial population of limbal
stromal stem cells has been recognized. The
391
c
(d) RAFT oedema with vascularization and scarring. (e)
Graft infection
thickest layer of the cornea is stroma; it contains
a population of adult stem cells having mesen-
chymal stem cell (MSC) characteristics. These
cells are quite in a proximity to the human limbal
epithelial stem cells. These stem cells not only
support epithelial cells but also suppress inflam-
mation by remodelling the affected stromal tissue
and help the cornea restore the transparency [20].
Limbal stem cells can be expanded to large quan-
tities and thus can be used for bioengineering the
epithelial-stromal tissue equivalents. This cell-­
based therapy can be used for treating ocular sur-
face defects [21].
25.4 Corneal Wound Healing
The human body has a natural mechanism to fight
against the external insults and injuries and also,
to repair the damaged parts of tissues and organs.
Epithelium, stroma and nerves maintain the
homeostasis of the cornea. A cascade mediated by
paracrine and autocrine interactions of cytokines,
392
growth factors and chemokines produced by the
epithelial cells, stromal cells, immune cells and
lacrimal gland along with the corneal nerves
involving interactions between the epithelial cells,
stromal keratocytes corneal nerves, lacrimal
glands, tear film and cells of the immune system
is responsible for corneal wound healing [22].
25.4.1 Epithelial Injury
Cytokines are released following epithelial
injury. It includes interleukin-1 (IL-1), TNF-α,
epidermal growth factor (EGF) and platelet-­
derived growth factor (PDGF), while the stromal
keratocyte response includes the release of IL-1-­
mediated Fas ligands to induce apoptosis [23,
24]. IL-1 is the modulator which regulates the
cascade of all the factors involved in healing [25].
It regulates hepatocyte growth factor and kerati-
nocyte growth factors which are mediators of
stromal and epithelial interactions produced by
keratocytes and myofibroblasts to regulate prolif-
eration and motility. IL-1 also controls the
expression of collagenase, metalloproteases and
other enzymes which leads to collagen remodel-
ling and healing [25, 26].
25.4.2 Myofibroblast Migration
and Differentiation
Myofibroblasts are the cells which play a crucial
role in wound healing process. They have con-
tractile pseudopodia and alpha-smooth muscle
actin (α-SMA) which are present in the anterior
stroma below areas of the epithelial layer. They
can be seen after disruption by stains against
these components after 1–2 weeks following
injury. They respond to the transforming growth
factor beta (TGF-β) and so are considered to be
the derivatives of keratocytes. They show
reduced transparency because of altered corneal
crystalline production and participate mainly in
collagen and extracellular matrix remodelling
through the production of collagen, glycosami-
noglycans, collagenases, gelatinases and matrix
metalloproteinases (MMPs) [27]. Myofibroblasts
N. Mitragotri et al.
are also important in the corneal haze formation
and regression due to stromal remodelling. Our
previous work in mice model has shown that
TGF-β and PDGF are the main factors which
convert corneal keratocyte into myofibroblast
[23, 24]. Our study also has shown that the bone
marrow cells may migrate to corneal stroma
after wound healing for the formation of
myofibroblasts.
25.4.3 Stromal Remodelling
In stromal wound healing, fibroblast growth fac-
tor-­2 (FGF-2) induces the expression of PAX-6
and ABCG2 progenitor markers, which leads to
the differentiation of corneal keratocytes in vitro.
These cells fulfil the criteria for MSC by express-
ing markers of MSCs. Remodelling is an impor-
tant mechanism contributing to the morphogenesis
of repair. Corneal epithelial cells express the
PDGF receptors [28]. PDGF is found in the epi-
thelial basement membrane, at very high levels,
and modulates corneal fibroblast proliferation,
chemotaxis and possibly their differentiation
[29]. TNF-α might also have a function in initiat-
ing the early wound healing responses.
Factors that are involved in corneal wound
healing are shown in Table 25.1.
Wound healing by limbal stem cells is similar
to that of the general wound healing mechanism,
and these cells suppress the inflammatory
responses. Epithelial and stromal damages trig-
ger a healing process mediated by progenitor
cells which are located in the limbus [30–33].
Limbal stem cells exert therapeutic effects
which induce the repairing by secreting soluble
factors. These factors suppress the inflamma-
tion and angiogenesis which is why expression
level of interleukin-2 and metalloproteinase is
observed to be decreased. In injured corneas,
the upregulation of IL-2, interferon γ (IF γ) and
vascular endothelial growth factor (VEGF) has
been reported. For healing of the wound, limbal
stem cells secrete certain growth factors to facili-
tate the survival of injured cells and fasten the tis-
sue regeneration procedure. Higher levels of
growth factors, including VEGF, EGF and TGF-β,
25  Limbal Stromal Stem Cells in Corneal Wound Healing: Current Perspectives and Future Applications 393

Table 25.1  List of the factors involved in corneal wound healing, describing their source of secretion and their
functions
Sl
# Factor Secreted by Function
1 Basic fibroblast growth factor Endothelial cells Mitosis/chemotaxis of epithelial cells
(bFGF)
2 Connective tissue growth TGF-β induced fibroblast cells Mediator for TGF-β, fibroblast,
factor collagen synthesis
3 Epidermal growth factor Epithelial cells, lacrimal gland Mitosis of epithelial cells, ECM
(EGF) production
4 Platelet-derived growth factor Corneal epithelial cells Myofibroblast proliferation and
(PDGF) migration [23, 24]
5 Transforming growth factor Corneal epithelial cells Stimulation of epithelial cells
alpha (TGF-α)
6 Transforming growth factor Corneal epithelial cells, lacrimal gland, Regulation of fibroblast proliferation
beta (TGF-β) endothelial cells, inflammatory cells [23, 24]
7 Keratinocyte growth factor Keratocytes, lacrimal gland Epithelial proliferation, mitosis,
(KGF) migration
8 Interleukin-1 (IL-1) Corneal epithelial cells, keratocytes, Stimulation of Fas and Fas L
macrophages, endothelial cells, production, fibroblast and MMP
fibroblasts, eosinophils production
9 Fas Keratocytes Binds with Fas L, involved with
autocrine suicide pathway
10 Tumour necrosis factor alpha Macrophages Protease synthesis, inducing
(TNF-α) apoptosis
11 Interleukin-4 (IL-4), T-lymphocytes Anti-inflammatory, inhibits
interleukin-10 (IL-10) production of TNF α, IL-1, IL-6 [57]

can be observed after the application of limbal
stem cells [34].
25.5 V
 arious MSc Sources
for Stem Cell-Based
Therapies for Cornea Wound
Healing
MSCs are non-haematopoietic, multipotent stem
cells having multi-lineage differentiation poten-
tial. They home to the injured cornea. Many
groups have been looking for the alternative ther-
apies using stem cells from adipose tissue, bone
marrow (BM), limbus, etc.
1. Bone marrow: It is the most commonly used
and explored source of MSCs. They can be
differentiated into mesoderm, neuroectoderm
and endodermal cells in  vitro. Two major
stem cell populations can be obtained from
bone marrow, viz. HSC, haematopoietic stem
cells, and MSCs. MSCs can be differentiated
into a variety of progenitor cells of connec-
tive tissues which can be demonstrated by
observing the expression of CD90, CD44,
CD73, CD105, CD45 and CD34 markers. It
has also been reported that BM-derived stem
cells/keratocytes are important for wound
healing after corneal haze. Several studies
have also demonstrated that MSCs suppress
the inflammatory cells [23, 24, 35]. It is
observed that severity of injury lessens dur-
ing the acute phase of injury because of
MSCs [36].
2 . Adipose tissue: MSCs are easily isolated from
the stromal vascular fraction and are more
accessible with higher yield. Adipose-derived
stem cells (ADSCs) have potential to differen-
tiate into fat, cartilage, bone and muscle under
specific culture conditions [37]. ADSCs can
be isolated by the lipoaspiration method. They
have potential to synthesize and secrete
keratocyte-­ specific proteins. ADSCs have
been shown to be helping in wound healing by
replacing and regenerating the new cells [38].
394
3. Dental pulp: Dental pulp stem cells (DPSCs)
are similar to the BM-MSCs and are derived
from the pulp of extracted teeth (generally the
third molar exfoliated deciduous). They
express some of the markers for BM-MSCs
and have similar differential potential [39].
DPSCs have potential to differentiate into
bone, fat, dental, cartilage, nerve and muscle
cells and are very friendly with most of the
biomaterials. DPSCs have been reported to
regenerate epithelial cells in animal models
with LSCD [40].
4. Umbilical cord: Umbilical cord MSCs
(U-MSCs) are very naive, proliferative and
immunosuppressive than the adult or BM-­
MSCs [41]. U-MSCs show the homing prop-
erty because of which they differentiate into
the corneal endothelial cells when practised
on the corneal injury.
5. Embryonic stem cells (ESCs): Preliminary
results have been reported by the company
Ocata Therapeutics (formerly ACT) on clini-
cal safety trials for the use of retinal pig-
mented epithelial (RPE) cells derived from
hESCs for dry macular degeneration and
Stargardt’s macular dystrophy [42, 43].
6. Induced pluripotent stem cells (iPSCs): The
iPSC inference of the limbal genealogy may,
in the long run, give a restorative wellspring
of limbal cells. Late advances in the recog-
nizable proof of key administrative qualities
in limbal improvement, separation and devel-
opment are probably going to fasten this
restorative open door [44]. There is also a
report of one Japanese patient who received a
transplant of a sheet of iPSC-derived RPE
[45]. This is the first of its kind in a human
study that may be the leader in many other
applications of iPSC derivatives. While it
might be expected that iPSCs will be used as
autologous therapies, there is a strong move-
ment for their use as allogeneic transplants or
as partially compatible HLA haplotyped
derivatives [46].
7. Neural stem cells: Many studies have shown
that human neural brain stem cells can be used
for the treatment of retinal degeneration
caused by dry macular degradation and for the
maintenance of photoreceptor cells.
N. Mitragotri et al.
25.5.1 Limitations of These Sources
of MSCs
1. Though natural body cells are used in the cell-­
based therapies, there are few limitations to it.
2. The role in corneal regeneration is very limited.
3. Long-term preservation of these cells.
4. Possible induction of an immunogenic
response.
5. Risky, painful and invasive procedures for the
isolation of BM-MSCs which always have a
possibility of infections.
6. BM-MSCs show reduced plasticity, growth with
the increase in donor age and passage number
and the number of cells acquired being low.
7. The exact role of U-MSCs is still unknown.
8. Graft survival rate is not up to the mark and
induction of inflammation and neovascular-
ization [47].
9. Neural stem cell therapies are still under clini-
cal trial phase I/II.  A lot of information and
confirmation for treating patients is yet to be
acquired.
Therefore, limbal stem cells are the preferred
source of stem cells for patients with corneal dis-
orders, burns and chemical injuries. These trans-
plants have the highest success ratio because of
renewing and restoring the capacity of the cor-
neal stroma and epithelium. This keeps the cor-
nea transparent. Even a tiny part of the limbus
can be expanded in  vitro. In our recently pub-
lished data of 125 patients, using the simple lim-
bal epithelial transplant surgery has shown a
success rate of more than 75% with 4  years of
follow-up. Similarly, cultivated limbal epithelial
transplantation by Pellegrini group and by our
group showed good success ratio in patients with
limbal stem cell deficiency.
25.6 Results of the Preclinical
Trials
In our previous preclinical studies, in the mouse
model of corneal opacity, stromal stem cells effec-
tively regenerated extracellular matrix and collagen
[48]. Mesenchymal stem cells have shown to be
restoring corneal transparency in lumican k­ nockout
25  Limbal Stromal Stem Cells in Corneal Wound Healing: Current Perspectives and Future Applications
mice. Mesenchymal stem cells obtained from lim-
bal biopsy also show immune-­suppressing prop-
erty and thus help to prevent inflammation, scarring
and immune rejection of transplanted tissue. It is
published that cells obtained from expanding lim-
bal biopsy cultures can be used as the cell-based
therapy. This has been proven by applying the lim-
bal stroma stem cells embedded in fibrin gel to the
surface of healing murine debridement wound
[49]. Our team has proved that stromal cells derived
from limbal biopsies differentiate into functional
keratocyte and regenerate the damaged stromal tis-
sue. Isolation of cells using collagenase resulted in
faster cell growth and proliferation. This led to the
upregulation of ABCG2, Nestin, OCT4, PAX6,
NGFR and SOX2 gene expression which is associ-
ated with adult and pluripotent stem cells.
Downregulated expression of genes ABCG2 and
Nestin was observed in the limbal stem cells cul-
tured on collagen substratum in serum-free kerato-
cyte differentiation medium (KDM) [50–52].
When the ability of cells cultured in KDM
was examined on mouse corneal debridement
model, limbal stem cells induced fibrotic matrix
deposition and thick ECM structures. Fifty thou-
sand limbal stem cells were applied to the wound
which was labelled with 3–3′-dioctadecyloxa-
carbocyanine. These labelled limbal stem cells
were found to be distributed throughout the
wounded area. Inflammation or rejection was not
observed during this time, and after 4  weeks,
limbal stem cells were observed to contain
human keratocan and type I collagen compo-
nents of the normal transparent stromal extracel-
lular matrix (ECM) [53].
As shown in Fig.  25.6, fibrotic markers hyal-
uronan, fibronectin, tenascin C, biglycan and
decorin were abundant showing scar formation in
the controls (without limbal stem cells).
Proteoglycan, decorin and the stromal matrix were
observed in the wounded corneas treated with lim-
bal stem cells. Additionally, mRNA for type III
collagen and SPARC (secreted protein acidic and
rich in cysteine) was upregulated after 2  days in
debrided corneas. Visible scarring was observed in
all the eyes which did not receive the limbal stem
cells through the low magnification photos.
Corneal scars scatter the light leading to
reduced visual acuity, but the eyes treated with
395
limbal stem cells did not show increased scatter-
ing compared to the normal preoperative corneas.
Neovascularization is another threat to the vision.
Corneal scarring is healed when treated with lim-
bal stem cells and the healed corneas do not
express CD31 marker (which is the hallmark
for  vascular endothelial cells/ blood vessels).
Basu et al. have shown that the cells isolated from
biopsy procedure can be used for autologous
transplants and these cells suppress the accumu-
lation of fibrotic scar tissue [53]. The study
proves that the MSCs obtained from the limbal
source are functionally similar to corneal stromal
cells with the evidence of sphere-forming capac-
ity, gene expression pattern and organization of
ECM.  This is how limbal stem cells induce
repairing of ablated tissue with transparent ECM
containing well-organized collagen.
This preclinical study was carried out to deter-
mine the mesenchymal stem cells present in the
human cornea have the ability to differentiate into
keratocyte which helps to prevent corneal scar-
ring. This was confirmed by various experiments
like clonogenic assay, sphere formation, gene
expression, immunostaining and optical coher-
ence tomography (OCT), described below in
Figs. 25.5 and 25.6. Human LMSCs were shown
to suppress the immune response, viz. when
human stromal cells are injected in the mouse
model, T-cell-mediated response was not induced.
This immune-suppressing quality of hLMSCs
makes them favourable and the first choice for
transplantation. They provide cell-­based therapy
for corneal scarring. A Lum−/− mouse, which had
a thin cornea, haze and abnormal collagen distri-
bution, was treated with limbal stem cells. Cell
rejection was not observed and the stromal cells
got distributed on the mouse stroma [48].
25.7 Human Limbal-Derived
Stromal/Mesenchymal Stem
Cells (hLMSCs): Our
Approach for Treating
Corneal Scar and Other
Corneal Pathologies
Therapeutically accepted and serologically
tested cadaveric donor corneas were obtained
396
Fig. 25.5  Repairing mechanism of the limbal stem cells
in a mouse model. (a) Whole-mount immunostaining of
the murine cornea that received hLMSCs, showing the
persistence of hLMSCs, DiO-labelled (green), 1 week
after the transplantation. (b) Histological sections of the
murine cornea showing positive expression of human
from the Ramayamma International Eye Bank,
L V Prasad Eye Institute, approved for process-
ing, storing and distributing human ocular tis-
sue. Corneas were washed with antibiotic
fortified buffered saline before extracting lim-
N. Mitragotri et al.
keratocan and type I collagen. The nuclei in all sections
were counterstained with DAPI (blue). Ctrl, control;
scale: 50 μM, 500 μM. (*Note – This figure is reproduced
with permission (copyright licence number
4307021062699) from Basu et  al. [53], Science
Translational Medicine journal)
bus from them. In aseptic conditions, a 360°
limbal ring was excised using optical magnifica-
tion and surgical instruments. Then it is washed
with buffered saline and minced into smaller
fragments. The minced tissue fragments were
25  Limbal Stromal Stem Cells in Corneal Wound Healing: Current Perspectives and Future Applications
Biglycan No cells
Decorin
Fibronectin
Tenascin C
binding protein
Hyaluronan
Fig. 25.6 Image showing the blockage of fibrotic
matrix deposition by hLMSCs in murine corneas.
Collage of the immunostaining analysis, showing the
negative expression of the fibrotic markers, decorin,
tenascin C and hyaluronan, in the murine corneas
treated with the limbal-derived stromal stem cells. The
collected into incomplete media (plain DMEM/
F12 media, Lonza, BE04-­687F/U1) and were
subjected to collagenase digestion by adding
20  μL of reconstituted collagenase IV
(17104019, Thermofisher) at a ­concentration of
10  IU/μL, to the tissue suspension. Incubation
of the limbal fragments with the collagenase
was carried out for 16  h at 37  ° C in 5% CO2
chamber.
After 16 h of incubation, enzymatic digestion
of collagenase was stopped by adding 2 mL of
397
LBSC
100 µm
100 µm
100 µm
nuclei in all sections were counterstained with DAPI
(blue). Scale: 100  μM. (*Note  – This figure is repro-
duced with permission (copyright licence number
4307021062699) from Basu et  al. [53], Science
Translational Medicine journal)
complete media fortified with 2% serum. The
digested tissues were then spun down at
1000  rpm for 3  min at room temperature, in
saline. Subsequently, the addition of complete
media was done to dissolve the pellet, and the
isolated hLMSC suspension was then placed in
culture flasks. Media were replaced after every
2 days. Primary cultures yield mostly epithelial
cells which were sub-cultured for three genera-
tions to obtain a pure population of the
hLMSCs.
398
The Application/Delivery of the hLMSCs  The
technique of delivering the hLMSCs established
by Funderburgh et al. involves (1) debridement
of the corneal epithelium using dry sponge, (2)
half-­
a-­million cells mixed in 0.05  mL of the
thrombin component of the fibrin glue and
allowing the two components to gel, and (3) a
bandage contact lens placed on the eye. Patients
in this study group were prescribed prophylactic
topical antibiotics without any corticosteroids.
Patients in the control group were given the stan-
dard medical therapy, including topical cortico-
steroids and debridement with fibrin glue but
without any cells.
The Funderburgh technique of using autolo-
gous and allogenic limbal stromal stem cells has
shown to enhance vision, promote epithelializa-
tion in the cornea, improve the corneal clarity and
reduce corneal scarring, thus obviating the need
for corneal transplantation in the eyes with cor-
neal burns, ulcers and scars (Fig. 25.7).
a b
d e
g h
Fig. 25.7  Events of tissue processing and application of
the hLMSCs during transplantation. (a) Debris and
unwanted layers of corneal tissues removed. (b) A 360°
limbal rim dissected out. (c) Collagenase-digested limbal
explants were cultured in T25 flasks. (d) Image of epithelial
N. Mitragotri et al.
25.8 Challenges in a Clinical Trial
1. The use of hLMSCs is still under trial.
Obtaining an adequate number of donor cor-
neas is still a big challenge.
2. The number of cells obtained per square mil-
limetre reduces with age of the donor.
3. Transportation is a major issue as the cells
have to be preserved or transported at a very
low temperature that requires definite logistics
and equipment.
4. As the cells will be used for patients, sterility
has to be maintained which requires cGMP
(Current Good Manufacturing Practices) labs
which are very expensive.
The demonstration of safety and efficacy of
the drug product intended for use in humans is
essential before it is approved by the Central
Drugs Standard Control Organization (CDSCO),
for import or manufacturing. The information
required for approval of an application to import
c
f
cells population after 14 days of culture. (e) Image of the
pure stromal cell population which can be obtained after
few passages. (f) Trypsinized cell pellet sent for transplan-
tation. (g) Mixing of the hLMSCs with fibrin glue. (h) Cells
with fibrin glue being applied onto the debrided cornea
25  Limbal Stromal Stem Cells in Corneal Wound Healing: Current Perspectives and Future Applications
or manufacture a new drug for marketing regula-
tions under Appendix I, IA and VI of Schedule Y
is described well. In India, for the approval of
new drug, all the necessary requirements along
with the NDA to FDA should be provided [54],
which were already obtained by our institute, for
this particular clinical trial. Before starting the
clinical trials (http://www.clinicaltrials.gov, Ref#
NCT02948023) using the hLMSCs, various doc-
uments have been produced and submitted to the
DCGI.  All procedures followed were in accor-
dance with the ethical standards of the responsi-
ble committee on human experimentation
(institutional and national) and with the Helsinki
Declaration of 1975, as revised in 2000. Informed
consent was obtained from all patients for being
included in the study.
1. Ethical committee approval/IRB approval
(Ref # LEC 01-14-015)
2. Data of the preclinical studies
3. ICSCR (Institutional Committee for Stem
Cell Research) approval
4. Form 44 of the CDSCO that includes the
study protocol, CV of the principal investiga-
tor, informed consent form, case report form,
master formula record, etc.
Requirements of cGMP Facility  In order to
conduct human clinical trials, there is a require-
ment of few sophisticated facilities and the neces-
sity to have standard SOPs. These are the main
regulatory criteria for ensuring the pharmaceuti-
cal quality of a product which will be used for the
patients. Good manufacturing production facili-
ties are always a prerequisite for cell- and tissue-­
based therapies. The facilities are designed as per
the purpose and are accredited by the national
regulatory body. The scientists and co-workers
work as per the standard protocols of quality
assurance and ethics committees to ensure the
safety of cellular products [55].
Cell-based medicinal products (CBMPs) are
handled with lots of precautions. Any method
associated with CBMPs requires a strict control
in cGMP facility. A cGMP facility is meant for
399
the production of pharmaceutical or cell products
[56]. It incorporates the assembling space, the
capacity stockroom for crude and completed
items and other research facility territories.
Manufacturing space, the storage warehouse for
raw and finished product and support laboratory
areas are included in the cGMP facilities. The
cGMP facilities are organized and designed
according to quality system for collection, pro-
cessing and storage of cell therapy products. For
cell-based products, terminal sterilization of the
product or removal/inactivation of microbial con-
taminants cannot be done. Thus, tested and certi-
fied starting materials and validated processes
have been followed, and sterility testing for the
product has been done [55].
The facilities must be planned and designed as
per Good Manufacturing Practice for
Pharmaceutical Manufacturers including quality
control and quality affirmation programmes,
which set up a quality system way to deal with
control gathering, preparing, stockpiling and the
arrival of cell treatment items and furthermore
address the accompanying components like:
1 . Facility (outline, access and maintenance)
2. Equipment (buy, utilization and support)
3. Raw material specifications (buy, stockpiling
and utilization)
4. Quality assurance (quality control, approval,
capability and documentation)
The quality assurance is based on these listed
elements: quality programme, organization and
personnel, standard operating procedures, envi-
ronmental control, equipment monitoring, sup-
plies and reagents, process controls, process
changes, process validation, labelling design and
control, storage requirements, records, tracking,
non-conformances and complaints management,
risk assessment, reporting and reviewing. In
areas where quality assurance interacts with hos-
pital departments and infrastructures, such as
housekeeping, engineering, information technol-
ogy and supply departments, the aim is to estab-
lish relevant service-level agreements in order
to  ensure an adequate level of regulatory
compliance.
400
The quality system approach is chance based.
For the facilities, it is of principal significance to
forestall potential defilement of both microbio-
logical and endotoxin contaminations because of
the imperfections in natural conditions, handlers,
culture holders, crude materials or cross tainting
with different items arranged in a similar genera-
tion plant. Care has to be taken with techniques
for compartment disinfection and control of
crude materials and other reagents. Utilization of
high-efficiency particulate absorbing (HEPA)
channel to anticipate airborne cross tainting, iso-
late treatment of materials from various patients
and so on has to be done. Consequently, the most
critical rooms of these facilities incorporate the
alleged clean rooms. These clean rooms are
grouped into four classes relying upon air and
dust particles, in view of the number of particles
of two sizes (≥0.5 μm, ≥5 μm). Different param-
eters, for example, temperature, moisture and
pressure, have been considered and checked on
account of their potential effect on microorgan-
ism expansion. The flow of materials and the per-
sonnel is kept seperate and unidirectional to limit
cross-contamination. All the activities are docu-
mented in real time. The specialized staff trained
in essential cleanliness measures required for
control in clean rooms. The specialized staff has
sufficient capability for both the lead and recon-
naissance of all exercises. Generation and dis-
semination of CBMPs or ATPs are controlled by
the applicable local and national authorities.
25.9 Summary
Corneal clarity is essential for clear vision. This
is often affected by various factors like mechan-
ical injuries, burns and scars, which turn out to
be the major causatives of visual impairment.
The underlying and natural mechanism of cor-
neal repair involves the layering of fibrotic tis-
sue that makes the cornea cloudy and opaque.
The standard and current care of therapy for any
such corneal stromal defects are penetrating
keratoplasty or lamellar keratoplasty, replacing
the whole cornea or the affected stromal region,
respectively. These clinical treatments are fully
N. Mitragotri et al.
optimized and have a very good success rate.
Yet, these corneal transplants need lifelong fol-
low-up along with standard medical care. With
the emergence of cell-based therapies, mainly
the stem cells, they have made a paradigm shift
from the conventional therapies to regenerative
medicine. Our previous work in mice has shown
that stromal stem cells obtained by culturing in
low serum media can prevent corneal scarring.
We have proven that these mesenchymal stem
cells of limbal origin, when given in 50,000
cells along with fibrin sealant, can heal the
mouse corneal scars in 4  weeks. We now have
taken this technology to the clinic, with the fully
equipped GMP facility at our institute. Our pilot
study has shown safety and efficacy in treating
various corneal pathologies mainly the corneal
scars.
Acknowledgements  We acknowledge Mr. Abhinav
Reddy Kethiri, Brien Holden Eye Research Center, and
Mr. Sridhar Rao Boyinpally, Department of Pathology, LV
Prasad Eye Institute, for helping in generating the images
for this chapter. We thank Champalimaud Foundation and
Tej Kohli Cornea Institute and LV Prasad Eye Institute for
providing the financial assistance and manpower for this
chapter.
Conflict of Interest  Noopur Mitragotri, Mukesh Damala,
Vivek Singh and Sayan Basu  declare that they have
no conflict of interest.
Informed consent was obtained along with the consent
for publication.
Note  No animal studies were carried out by the authors
for this article.
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 Cell Therapy of the Corneal Stroma
Using Ex Vivo Cultured Extraocular
Cells
Jorge L. Alió del Barrio
26.1 Introduction
The stroma constitutes more than 90% of the cor-
neal thickness. Many features of the cornea,
including its strength, morphology and transpar-
ency, are attributable to the anatomy and biome-
chanical properties of the corneal stroma [1].
Many diseases as corneal dystrophies, scars or
ectatic disorders induce a distortion of its anat-
omy or physiology leading to a transparency loss
and subsequent loss of vision. Despite the great
efforts in the last decade to try to replicate the
corneal stroma in the laboratory in order to look
for an alternative to classical corneal transplanta-
tion, this has not been accomplished yet due to
the extreme difficulty to mimic the highly com-
plex ultrastructure of the corneal stroma, obtain-
ing substitutes that either don’t achieve enough
transparency or strength properties [2].
In this scenario, in the last few years has
gained a lot of interest the cellular therapy of the
corneal stroma by the use of mesenchymal stem
cells from either ocular or extraocular sources,
capable to differentiate into adult keratocytes
in vitro and in vivo in animal models [1]. It has
J. L. Alió del Barrio (*)
University Miguel Hernandez, Vissum-Instituto
Oftalmologico de Alicante, Alicante, Spain
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_26
26
been demonstrated by several authors, including
reports from our research group [3–5], the capa-
bility of these stem cells to not only survive and
differentiate into adult human keratocytes in
xenogeneic scenarios without inducing any
inflammatory reaction but also to produce new
collagen within the host stroma [3, 6], to modu-
late preexisting scars by corneal stroma remodel-
ling [7, 8] and to improve corneal transparency in
animal models for corneal dystrophies by colla-
gen reorganization as well as in animal models
for metabolopathies by the catabolism of the
accumulated proteins [9–12]. Mesenchymal stem
cells have also shown immunomodulatory prop-
erties in syngeneic, allogeneic and even xenoge-
neic scenarios [12, 13]. Actually, the first clinical
data regarding the safety and preliminary efficacy
of the cellular therapy of the corneal stroma from
phase 1 human clinical trials is now available [14,
15], leading to a new and promising field for
future research that may end up providing a real
alternative treatment option for corneal diseases
in the near future.
Considering existing scientific evidence, it
seems that all types of mesenchymal stem cells
(MSCs) have similar behaviour in  vivo
(Table 26.1) and thus are able to achieve a kerato-
cyte differentiation and modulate the corneal
stroma with immunomodulatory properties [16].
It has also been recently reported that MSC
secretes paracrine factors such as vascular endo-
thelial growth factor (VEGF), platelet-derived
403
404
Table 26.1  Stem cells used for the regeneration of the corneal stroma
CSSC
Keratocyte differentiation in vitro demonstrated Yes
Keratocyte differentiation in vivo demonstrated Yes
Possible autologous use Yes/no
Evidence of keratocyte differentiation and their possible autologous applications
growth factor (PDGF), hepatocyte growth factor
(HGF), transforming growth factor beta 1
(TGFβ1), etc. that promote cell migration and
keratocyte survival by apoptosis inhibition and
upregulate the expression of extracellular matrix
(ECM) component genes in keratocytes, subse-
quently enhancing corneal re-epithelialization
and stromal wound healing [17]. MSC can be
obtained from many human tissues, including
adipose tissue, bone marrow, umbilical cord,
dental pulp, gingiva, hair follicle, cornea and pla-
centa [18, 19].
Corneal stroma stem cells (CSSCs) are a
promising source for cellular therapy as far as
their isolation technique and culture methods are
being optimized and refined [20], and presum-
ably they should have a more efficient keratocyte
differentiation as they are already corneal cells.
On the other hand, CSSC isolation technique is
more technically demanding considering the
small amount of tissue that is usually processed
and still requires a contralateral healthy eye (for
autologous use), which is not always available
(bilateral disease) or accepted by patients. An
allogeneic use would still require living or cadav-
eric donor corneal tissue. These drawbacks may
limit their use in clinical practice (mainly for
autologous use). On the other hand, extraocular
stem cells don’t present these limitations. Human
adult adipose tissue has shown to be an ideal
source of autologous stem cells, as it satisfies all
the requirements, easy accessibility to the tissue,
high cell retrieval efficiency and the ability of its
stem cells (h-ADASCs), to differentiate into mul-
tiple cell types (keratocytes, osteoblasts, chon-
droblasts, myoblasts, hepatocytes, neurons, etc.)
[3]. This cellular differentiation occurs due to the
effects of very specific stimulating factors or
environments for each cell type, avoiding the mix
of multiple kinds of cells in different niches.
Bone marrow MSCs (BM-MSCs) have the same
J. L. Alió del Barrio
BM-MSC ADASC UMSC ESC iPS
Yes Yes Yes Yes Yes
Yes Yes Yes No No
Yes Yes Yes/no No Yes
profile as ADASCs, but their extraction by bone
marrow puncture is a more complicated and pain-
ful procedure that requires general anaesthesia.
Umbilical MSCs (UMSCs) present an attractive
alternative, but their autologous use would be
expensive and is currently impossible as umbili-
cal cord is not generally stored after birth.
Embryonic stem cells have great potential but
also present important ethical issues. On the
other hand, the use of iPS technology [21] could
solve such problems, and their capability to gen-
erate adult keratocytes has already been proven
in vitro [22].
In this chapter we will review the different
types of extraocular stem cells (mesenchymal or
not) that have been suggested for the regenera-
tion of the corneal stroma, as well as the current
existing evidence either in  vitro or in  vivo.
Finally, we will review the different surgical
approaches that have been suggested (in vivo) for
the application of this stem cell therapy for the
corneal stroma regeneration.
26.2 E
 xtraocular Stem Cells Used
for Corneal Stroma
Regeneration
26.2.1 Bone Marrow Mesenchymal
Stem Cells (BM-MSCs)
Park et  al. reported that human BM-MSCs dif-
ferentiate in vitro into keratocyte-like cells when
they are grown in specific keratocyte differentia-
tion conditions [23]. They demonstrated a strong
expression of keratocyte markers as lumican and
ALDH, together with the loss of expression of
stem cell markers such as α-smooth muscle actin.
However, they couldn’t demonstrate an evident
expression of keratocan on these differentiated
cells [23]. Trosan et  al. showed that mice
26  Cell Therapy of the Corneal Stroma Using Ex Vivo Cultured Extraocular Cells 405

BM-MSC cultured in the presence of corneal
extracts and insulin-like growth factor-I (IGF-I)
efficiently differentiate into corneal-like cells
with expression of corneal-specific markers, such
as cytokeratin 12, keratocan and lumican [24].
The survival and differentiation of human
BM-MSCs into keratocytes have also been dem-
onstrated in vivo when these cells are transplanted
inside the corneal stroma of an animal model.
Keratocan expression was observed without any
sign of immune or inflammatory response [25].
inflammatory response. These findings were later
reproduced and confirmed by other authors in
several research papers [16].
The relatively easier extraction and isolation
techniques of these cells with respect to bone
marrow sources make these cells so far the best
extraocular candidates for clinical use for corneal
stroma regeneration.
26.2.3 Umbilical Cord Mesenchymal
Stem Cells (UCMSCs)

26.2.2 Adipose-Derived Adult Human mesenchymal stem cells isolated from

Mesenchymal Stem Cells neonatal umbilical cords have shown similar
(ADASCs) behaviour to other types of mesenchymal stem
cells when transplanted inside the corneal stroma
Human ADASCs (h-ADASCs) cultured in  vitro in  vivo, expressing keratocyte-specific markers
(Fig. 26.1) under keratocyte differentiation con- such as keratocan without inducing immune or
ditions express collagens and other corneal-­ rejection responses [27]. Liu et al. reported that
specific matrix components. This expression is the injection of these cells inside the corneal
quantitatively similar to that achieved by the dif- stroma of lumican-null mice improved corneal
ferentiated h-CSSCs [26]. transparency and increased the stromal thickness
The differentiation of h-ADASCs in func- with a reorganized collagen lamellae, also
tional human keratocytes has also been demon- improving the host keratocyte function through
strated in  vivo, for the first time, in a previous enhanced expression of keratocan and ALDH in
study of our group using a rabbit animal model these mice [10]. These data is encouraging,
[3]. These cells, once implanted intrastromally, although up to date, the autologous use of
express not only collagens type I and VI (the UCMSCs is not possible as far as the umbilical
main components of corneal extracellular matrix) cord from new births is not generally stored.
but also keratocyte-specific markers such as kera-
tocan or ALDH, without inducing an immune or
26.2.4 Embryonic Stem Cells (ESCs)

Current experience with these human pluripotent

Fig. 26.1  Microscopic appearance (phase-contrast pho-
tograph) of human ADASCs (×10 magnification)
stem cells for the corneal stroma regeneration is
much more limited. Chan et al. reported that dif-
ferentiation of these cells into a keratocyte lin-
eage can be induced in  vitro, demonstrating an
upregulation of keratocyte markers including
keratocan [28].
To the best of our knowledge, no studies
in vivo with these cells have been performed in
the field of regenerative medicine for the corneal
stroma. The use of these cells also raises many
ethical issues and, together with the lack of
in vivo data so far, discourages for their use in the
clinical setting at this time.
406
26.2.5 Induced Pluripotent Stem
Cells (iPSCs)
As already discussed, embryonic stem cells have
fallen into a certain abandonment partly because
of their ethical problems but especially because
of the appearance of the iPSC [21], which do not
present these problems as they derive from adult
cells. In 2012, the Japanese Shinya Yamanaka
and the British John B.  Gurdon received the
Medicine Nobel Prize for the discovery that
mature, specialized cells can be reprogrammed to
go back to an immature or stem cell state, with a
differentiation capacity to almost any cell line of
the organism. The iPSCs promise to be the future
of tissue and cellular engineering.
Regarding their application into the corneal
stroma regeneration, human iPSC has shown the
capability to differentiate into neural crest cells
(the embryonic precursor to keratocytes) and, by
culturing them on cadaveric corneal tissue, to
promote their keratocyte differentiation by
acquiring a keratocyte-like morphology and
expressing markers similar to corneal keratocytes
[22]. It has been also shown that iPSC-derived
MSCs exert immunomodulatory properties in the
cornea similar to those observed with BM-MSCs
[29]. To the best of our knowledge, no studies
have been published reporting the capability of
the iPSC to differentiate into adult keratocytes
in vivo in the animal model.
26.3 Corneal Stroma
Regeneration Techniques
All these types of stem cells have been used in
various ways in several research projects in order
to find the optimal procedure to regenerate the
human corneal stroma. These approaches could
be classified into seven basic techniques:
26.3.1 Ocular Surface Implantation
of Stem Cells
Surface implantation of MSC would be the opti-
mal approach for ocular surface reconstruction
J. L. Alió del Barrio
and corneal epithelium/limbal stem cell niche
regeneration, topic not treated here as it has been
already discussed in a previous chapter of this
book. However, surface implantation of MSC
would still have a place for the prevention or
modulation of anterior stromal scars after an ocu-
lar surface injury as a chemical burn. As already
discussed before, MSC secretes paracrine factors
that enhance corneal re-epithelialization and stro-
mal wound healing [17]. Thus, MSC benefits
onto the ocular surface may be justified more by
these paracrine effects rather than by a differen-
tiation of the MSC into epithelial cells, being the
evidence for the latest controversy. On this
regard, Di et  al. assayed subconjunctival injec-
tions of BM-MSC in diabetic mice, reporting an
increased corneal epithelial cell proliferation and
an attenuated inflammatory response mediated
by tumour necrosis factor-α-stimulated gene 6
(TSG6) [30].
Holan et  al. suggested the MSC application
onto the ocular surface by the use of nanofibre
scaffolds. They reported that BM-MSC growth
onto these scaffolds can enhance re-­
epithelialization and supress neovascularization
and local inflammatory reaction when applied
onto an alkali-injured eye in a rabbit model, and
these results were comparable to the ones
obtained with limbal epithelial stem cells and
both of them better than with ADASC [31]. The
same group suggested that these results can be
improved when these nanofibre scaffolds seeded
with rabbit BM-MSC are covered with cyclospo-
rine A (CsA)-loaded nanofibre scaffolds, observ-
ing an even greater scar suppression and healing
results with the combination of both nanofibres
(MSC and CsA) [32].
Topical application of a suspension of autolo-
gous ADASC has been reported in an isolated
clinical case report where authors describe the
healing of a neurotrophic ulcer nonresponsive to
conventional treatment [33]. The lack of further
scientific evidence for this delivery method since
2012 raises questions about its real efficacy.
Finally, Basu et  al. suggested the delivery of
MSC by the use of fibrin glue [20]. They resus-
pended CSSC in a solution of human fibrinogen,
and this was added onto a wounded ocular s­ urface
26  Cell Therapy of the Corneal Stroma Using Ex Vivo Cultured Extraocular Cells
with thrombin onto the wound bed. Subsequently
the fibrinogen gelifies. By this application, they
demonstrated the prevention of corneal scarring
in the mice model, together with the generation of
new stroma with a collagen organization indistin-
guishable from that of native tissue. Currently,
this group is enrolled in a clinical trial to validate
these findings, using autologous and heterologous
CSSC from limbal biopsies for cases of chemical
burns, neurotrophic ulcers and stablished scars,
preliminary reporting and improvement in visual
parameters, corneal epithelization, corneal neo-
vascularization and corneal clarity [34].
26.3.2 Intrastromal Implantation
of Stem Cells Alone
Direct injection of stem cells inside the corneal
stroma of an animal model has been assayed
in  vivo in some studies, demonstrating the dif-
ferentiation of the stem cells into adult kerato-
cytes without signs of immune rejection. In our
study, we also demonstrated by immunohisto-
chemistry the production of human extracellular
matrix when h-ADASCs were transplanted inside
the rabbit cornea [3]. As expected, collagens type
I and type VI were founded to be expressed in the
rabbits’ corneal stroma as well as in the trans-
planted h-ADASCs, and collagens III and IV, not
expressed normally in the corneal stroma, were
detected neither in the host corneal stroma nor in
the transplanted h-ADASCs. Du et al. [9] reported
a restoration of the corneal transparency and
thickness in lumican-null mice (thin corneas,
haze and disruption of normal stromal organiza-
tion) 3 months after an intrastromal transplant of
human CSSCs. They also confirmed that human
keratan sulphate was deposited in the mouse
stroma and the host collagen lamellae were reor-
ganized, concluding that delivery of h-CSSCs to
scarred human stroma may alleviate corneal scars
without requiring surgery [9]. Very similar find-
ings were reported by Liu et  al., using human
UMSCs in the same animal model [10]. Coulson-­
Thomas et  al. found that, in a mice model for
mucopolysaccharidosis, transplanted human
UMSCs participate both in extracellular glycos-
aminoglycan (GAG) turnover and enable host
407
keratocytes to catabolize accumulated GAG
products [11].
Recently, our group has published the first
clinical trial where the preliminary safety and
efficacy of the cellular therapy of the human cor-
neal stroma are reported [14, 35]. In this pilot
clinical trial, we implanted autologous ADASC
(obtained by elective liposuction) within a mid-­
stroma femtosecond laser-assisted lamellar
pocket in patients with advanced keratoconus
(and already candidates for corneal transplanta-
tion). No signs of inflammation or rejection were
observed, confirming all previous evidence
reported in the animal model, and we could also
demonstrate, by anterior segment optical coher-
ence tomography (AS-OCT), the production of
new collagen in the area of the MSC implantation
(Fig.  26.2), with a mean central pachymetric
improvement of 15 μm. This neo-collagen band
did not induce any clinical haze (Fig. 26.2), and
all patients moderately improved their visual
function (mean improvement of two lines in all
visual parameters), while keratometric values
remained stable (Fig.  26.2) [14, 35]. Further
studies with larger samples are required in order
to confirm this preliminary data, but encouraging
results were observed, opening a new and excit-
ing line of therapy for research. As we have dis-
cussed, the production of new extracellular
matrix by the implanted mesenchymal stem cells
occurs, but is not quantitatively enough to be able
to restore the thickness of a severely diseased
human cornea (as in extreme keratoconic cor-
neas). However, the direct injection of stem cells
may provide a promising treatment modality for
corneal dystrophies, for corneal stroma progres-
sive opacification in the context of systemic met-
abolic disorders and for the modulation of corneal
scarring.
26.3.3 Intrastromal Implantation
of Stem Cells Together
with a Biodegradable Scaffold
To enhance the growth and development of the
stem cells injected into the corneal stroma, trans-
plantation together with biodegradable synthetic
extracellular matrixes has been performed.
408 J. L. Alió del Barrio

Axial / Sagittal Curvature (Front)

90º
0º 60
8 9mm 12 º

+1.3
+1.3 +1.5

0º

30
15
4 +1.2 +1.2 +0.9

º
+0.5 +0.2
+0.8 -0.9 -0.9 +0.7

180º
0 -1.4 0.0

0º
-0.3
+0.2 -0.4 -1.5 +0.2
+0.1 -0.9
4 +0.1 -0.7 -0.6

0º
21

33
-0.3 -0.6

0º
-0.5

8 24 0º
0º 30

a b 270º
c
8 4 0 4 8

Fig. 26.2  Autologous h-ADASC corneal stroma implan-
tation for advanced keratoconus. (a) Slit lamp picture 1
year after the procedure and (b) topographic changes
(Pentacam) between pre-op and 12 months after surgery.
Observe the stability of the keratometric parameters; (c)
corneal confocal biomicroscopy pictures at the surgical
plane in the first postoperative month. Stem cell survival
is confirmed by the presence of cells showing a more
rounded morphology (white arrows); (d) AS-OCT picture
1 year postoperatively. Note the patched hyper-reflective
areas (red arrows) at the level of the stromal pocket com-
patible with areas of new collagen production

Espandar et al. injected h-ADASCs with a semi-
solid hyaluronic acid hydrogel into rabbit corneal
stroma, reporting better survival and keratocyte
differentiation of the h-ADASCs when compared
to their injection alone [6]. Ma et al. used rabbit
ADSCs with a poly(lactic-co-glycolic) (PLGA)
biodegradable scaffold in a rabbit model of stro-
mal injury, observing newly formed tissue with
successful collagen remodelling and less stromal
scarring [36]. Initial data shows that these scaf-
folds could enhance stem cell effects over cor-
neal stroma, although more research is required.
26.3.4 Intrastromal Implantation
of Stem Cells with a Non-­
biodegradable Scaffold
At the present time, no clinically viable human
corneal equivalents have been produced by tissue
engineering methods. The major obstacle to the
production of a successfully engineered cornea is
the difficulty with reproducing (or at least simu-
lating) the stromal architecture. The majority of
stromal analogs for tissue-engineered corneas
have been created by seeding human corneal
stromal cells into collagen-based scaffoldings,
which are apparently designed to be remodelled
[2]. In addition, new and improved biomaterials
compatible with human corneas have been devel-
oped leading to advanced scaffolds that can be
used to engineer an artificial cornea (keratopros-
thesis), such as poly(hydroxyethyl methacrylate)
hydrogels, collagen chondroitin sulphate hydro-
gels and polyurethanes [37]. The combination of
these scaffolds with cells can generate promising
corneal equivalents, and some studies have
already been published that use mainly corneal
cell lines, including stromal keratocytes, pro-
viding positive results regarding adhesion and
26  Cell Therapy of the Corneal Stroma Using Ex Vivo Cultured Extraocular Cells
cellular survival in  vitro [38]. However, limited
research has been done in  vivo and using stem
cells in combination with this kind of synthetic
scaffolds. Mimura et  al. used corneal fibroblast
precursors together with porous gelatin hydro-
gels in  vivo in a rabbit model, detecting an
increased expression of type I collagen, but the
authors later stated that these scaffolds were
weak and unstable [39]. In a previous study by
our group, we analysed the survival and biointe-
gration of scaffolds composed by macroporous
membranes of poly(ethyl acrylate) (PEA) colo-
nized by h-ADASCs, inside the rabbit corneal
stroma in  vivo [4]. Despite demonstrating stem
cell survival in vivo after 12 weeks, h-ADASCs
did not improve the extrusion rate of the biomate-
rial when compared with PEA membranes
implanted without stem cells. Our opinion is that
stem cells do not differentiate properly into kera-
tocytes in the presence of these synthetic bioma-
terials. They lose the potential benefits
demonstrated with biodegradable scaffolds or by
isolated injection and fail to encroach on the bio-
material despite a macroporous structure
(Fig. 26.3).
a b
Fig. 26.3 h-ADASC colonized poly(ethyl acrylate)
(PEA) membrane grafted within rabbit’s corneal stroma
in vivo; (a) SEM image of a poly(ethyl acrylate) (PEA)
macroporous membrane (scale bar 200 mm); (b) fluores-
cence of Vybrant CM-DiI labelled h-ADASC (white
arrows, magnification 400×); (c) anterior segment picture
409
26.3.5 Intrastromal Implantation
of Stem Cells
with a Decellularized Corneal
Stroma Scaffold
The complex structure of the corneal stroma has
not been yet replicated, and there are well-known
drawbacks to the use of synthetic scaffold-based
designs: strong inflammatory responses induced
upon their biodegradation, and nearly all polymer
materials cause a nonspecific inflammatory
response.
Recently, several corneal decellularization
techniques have been described, which provide an
acellular corneal extracellular matrix (ECM) [40].
These scaffolds have gained attention in the last
few years as they provide a more natural environ-
ment for the growth and differentiation of cells
when compared with synthetic scaffolds. In addi-
tion, components of the ECM are generally con-
served among species and are tolerated well even
by xenogeneic recipients. Moreover, keratocytes
are essential for remodelling the corneal stroma
and for the normal epithelial physiology [41].
This highlights the importance of transplanting a
c
d e
of PEA graft within the corneal stroma 3 months after sur-
gery; (d) histological sections stained with Masson’s tri-
chrome. Observe the absence of a real biointegration of
the implant that detaches from the surrounding tissue
along the histological processing; (e) PEA graft with
extrusion 3 months after surgery
410
cellular substitute together with the structural sup-
port (acellular ECM) to undertake these critical
functions in corneal homeostasis. To the best of
our knowledge, all attempts to repopulate decel-
lularized corneal scaffolds have used corneal cells
[42–44], but as already discussed, these cells have
significant drawbacks that limit their autologous
use in clinical practice (damage of the donor tis-
sue, lack of cells and more difficult cell subcul-
tures), thus the efforts to find an extraocular
source of autologous cells. In a previous study by
our group, we showed the perfect biointegration
of human decellularized corneal stromal sheets
(100 μm thickness) with and without h-ADASC
colonization inside the rabbit cornea in  vivo
b c
Fig. 26.4  Corneal stroma enhancement with decellular-
ized human corneal stroma with h-ADASC recellulariza-
tion in the rabbit animal model. (a) Haematoxylin-eosin
staining of a rabbit cornea with an implanted graft of
decellularized human corneal stroma with h-ADASC col-
onization (magnification 200×); (b) human cells (arrows),
J. L. Alió del Barrio
(Fig.  26.4a), without observing any rejection
response despite the graft being xenogeneic [5].
We also demonstrated the differentiation of
h-ADASCs into functional keratocytes inside
these implants in vivo, which then achieved their
proper biofunctionalization (Fig. 26.4b, c). Other
authors have later assayed the integration of
decellularized pig articular cartilage ECM colo-
nized with mice BM-MSC into the rabbit’s cor-
neal stroma in  vivo, reporting similar findings,
although the transparency of this decellularized
scaffolds was not clearly reported [45].
In the author’s opinion, the implantation of
MSC together with decellularized corneal ECM
would be the best technique to effectively restore
labelled with CM-DiI, around and inside the implant con-
firming the presence of living human cells inside the rab-
bit corneal stroma; and (c) same section showing human
keratocan and thus their differentiation into human kera-
tocytes (arrows) (magnification 400×). Abbreviations:
Epi, epithelium; Str, stroma
26  Cell Therapy of the Corneal Stroma Using Ex Vivo Cultured Extraocular Cells 411

the thickness of a diseased and severely weak-
ened human cornea, as the implantation of MSC
alone only achieves a limited new ECM forma-
tion and thickness restoration [3, 14]. Moreover,
through this technique, and by using autologous
MSC from a given patient, it is theoretically pos-
sible to transform allogenic grafts into functional
autologous grafts, thus avoiding any risk of rejec-
tion. Following this research line, we have
recently published the first clinical trial using
these decellularized human corneal stroma scaf-
folds (120  μm thickness and 9.0  mm diameter
laminas), with or without autologous ADASC
recellularization, in patients with advanced kera-
toconus [15, 35]. We could demonstrate a moder-
ate but significant improvement in all visual
parameters (about two lines of improvement),
together with a refractive sphere reduction, sig-
nificant anterior keratometric flattening
(Fig.  26.5f) and improvement of corneal aber-
rometry, especially the spherical aberration [15,
35]. A complete corneal transparency without
significant haze was obtained 3 months after the
procedure (Fig.  26.5a, b), and all pachymetric
parameters improved over 100  μm as expected,
anatomically restoring these advanced kerato-
conic corneas (Fig. 26.5e). Although no clinical
differences could be demonstrated between
decellularized and recellularized implants, the
host keratocyte recellularization of the implants
and increase in the keratocyte density within the
anterior and posterior stroma were more intense
in those patients where autologous ADASCs
were also implanted (Fig. 26.5c, d) [15, 35].
26.3.6 Anterior Chamber Injection
of Stem Cells
Demirayak et  al. reported that BM-MSC and
ADASC, suspended in phosphate-buffered solu-
tion (PBS) and injected into the anterior cham-
ber after a penetrating corneal injury in a mice
model, are able to colonize the corneal stroma
and increase the expression of keratocyte-spe-
cific markers as keratocan, with an increase in
the keratocyte density by confocal microscopy
[8]. However, it is very questionable the possible
side effects of this MSC injection into the ante-
rior chamber for the lens epithelium and trabecu-

a b c d

e
Axial / Sagittal Curvature (Front)

f 0º
90º
60
8 9mm 12 º

-0.4
-0.8 +2.2
0º

30
15

4 +2.7 -2.5 +2.7

º

-0.7 +1.2
+3.2 -3.7 -1.1 +1.7
180º

0 +1.5 -3.9 -0.1

0º

+1.6 -1.5 -4.0 +0.2

-1.2 -3.1
4 -0.2 -5.0 -0.4
0º
21

-2.5 -2.2
33

-2.8
0º

8 24 0º
0º 30
270º
8 4 0 4 8

Fig. 26.5  Corneal stroma enhancement with decellular-
ized human corneal stroma with or without autologous
h-ADASC recellularization. (a, b) Slit lamp pictures
1  week (a) and 3  months (b) after surgery. Observe the
complete corneal transparency restoration; (c) corneal
confocal biomicroscopy picture. The implanted lamina
showed a complete acellular pattern 3  months after sur-
gery; (d) intense recellularization signs of the implanted
lamina 12 months after surgery; (e) AS-OCT image 1 year
after corneal stroma enhancement; and (f) topographic
changes between pre-op and 12  months after surgery.
Observe the significant flattening of the keratometry
412 J. L. Alió del Barrio

lar meshwork, where it may induce scarring and stroma, despite being a xenogeneic transplant,
a subsequent glaucoma. Considering this, the no signs of rejection or inflammation have never
potential clinical use of this approach, in the been reported [3–12]. This is congruent with the
author’s opinion, might be limited. strong evidence regarding the immunomodula-
tory and immunosuppressive properties of MSC
that aid them in evading and surviving from host
26.3.7 Intravenous Injection of  immune rejection by inhibiting adhesion and
Stem Cells invasion, and inducing cell death of inflamma-
tory cells, partially thanks to a rich extracellular
The systemic use, by intravenous injection, of glycocalyx which contains tumour necrosis
MSC has also been tested. Intravenous mice factor-α-­ stimulated gene 6 (TSG6) [13, 48].
BM-MSC injected after an allograft corneal TSG6 has been demonstrated to play a critical
transplant has been shown to be able to colonize role in the immunosuppressive properties of
the transplanted cornea and conjunctiva (inflamed MSC [12, 29, 30]. Considering all this, the use
ocular tissues), but not the contralateral ungrated of heterologous MSC would simplify tremen-
cornea, simultaneously decreasing immunity and dously the clinical application of MSC, as clini-
significantly improving allograft survival rate cal application centres would not need any
[46]. Yun et al. recently reported similar findings specific equipment as potential MSC banks
with the intravenous injection of iPSC-derived could store and supply the stem cells for their
MSC and BM-MSC after a surface chemical use in patients. Actually, there are already avail-
injury, where they could observe that the corneal able low-cost systems capable of enhancing the
opacity, inflammatory infiltration and inflamma- preservation of MSC at hypothermic tempera-
tory markers in the cornea were markedly tures while maintaining their normal function,
decreased in those treated mice, without signifi- allowing to extend the time window for distribu-
cant differences between both MSC types [29]. tion between the sites of manufacture and the
In contrast, our group could not find any benefit clinic and reducing the wastage associated with
in corneal allograft survival and rejection rates the limited shelf life of cells stored in their liq-
after systemic injection of rabbit ADASC prior to uid state [49]. In addition, Funderburgh et  al.
surgery, during surgery and at various time points recently reported that MSC from different
after surgery in rabbits with vascularized corneas donors may have different immunosuppressive
(model more similar to human corneal trans- properties and, in consequence, different abili-
plants than those reported in mice). Actually, a ties to regenerate and relieve stromal scars [50].
shorter graft survival compared with the non-­ Considering this important finding, best donors
treated corneal grafts was observed [47]. could be selected in MSC banks in order to
expand and supply only those MSC with the
highest immunosuppressive and regenerative
26.4 Autologous Versus capacity, being then not necessary to use autolo-
Heterologous MSC gous cells. We should also consider that adult
keratocytes obtained from autologous MSC
A critical question for future clinical trials to may still carry the same genetic defect that leads
further assess the feasibility of the cellular ther- to the corneal disease in case of corneal dystro-
apy of the corneal stroma is whether the use of phies. In this scenario, the use of heterologous
autologous MSC is really necessary or, on the instead of autologous MSC would be interest-
other hand, heterologous MSC could achieve ing. Actually, a recent study observed gene
the same benefit without still adding any risk of expression differences between the iPSC-
inflammation or rejection. If we consider all derived keratocytes generated from fibroblasts
published evidence in the animal model where of both keratoconic and normal human cor-
human MSC was implanted into the corneal neal stromata, influencing cellular growth and
26  Cell Therapy of the Corneal Stroma Using Ex Vivo Cultured Extraocular Cells
proliferation, confirming that, at least in kerato-
conus cases, adult cells obtained from MSC
may still not be functionally normal [51].
26.5 MSC Exosomes
Exosomes are nanosized extracellular vesicles
which originate from the fusion of intracellular
multivesicular bodies with cell membrane and
are released into the extracellular spaces [48].
They have been implicated in the ability of MSC
to repair damaged tissue. Funderburgh et  al.
have recently shown that exosomes isolated
from the culture media of human CSSC had
similar immunosuppressive properties and also
significantly reduced stromal scarring in
wounded corneas in  vivo in the animal model
[52]. This finding suggests that for some dis-
eases, as prevention or reduction of cornel scars,
MSC exosomes may allow a non-cell-based
therapy [50]. Zhang et  al. suggested the exo-
somes released by transplanted UCMSC in the
diseased cornea are able to enter into host cor-
neal keratocytes and endothelial cells and
enhance their functions [48]. Actually, in their
in  vitro experiment in mucopolysaccharidosis
VII mice, they discovered that UCMSC secreted
exosomes assisted in the recycling process of
accumulated GAGs in the lysosomes in diseased
cells [11]. These findings open as well an excit-
ing new field for research as the use of exosomes
per se could overcome some of the limitations
and risks associated with the intrastromal cellu-
lar injection, as exosomes can potentially be
applied topically [27].
In conclusion, cellular therapy of the corneal
stroma is a novel treatment modality for stromal
diseases that still requires further studies in form
of clinical trials with larger samples in order to
definitely stablish its safety and efficacy for the
different stromal diseases; the initial data
obtained from the first pilot clinical trials is very
encouraging, showing very exciting data with
great potential. In the author’s opinion, the cre-
ation of cellular banks storing and expanding het-
erologous cells or their exosomes and their
shipping and delivery to the different clinical
413
centres for their use are the future for this promis-
ing treatment modality, although before we get
there, there is still a lot of research work to do.
Compliance with Ethical Requirements Conflict of
interest: The authors, their families, their employers and
their business associates have no financial or proprietary
interest in any product or company associated with any
device, instrument or drug mentioned in this article. The
authors have not received any payment as consultants,
reviewers or evaluators of any of the devices, instruments
or drugs mentioned in this article. All procedures followed
were in accordance with the ethical standards of the
responsible committee on human experimentation (insti-
tutional and national) and with the Helsinki Declaration of
1975, as revised in 2000. Informed consent was obtained
from all patients for being included in the study. No ani-
mal studies were carried out by the author for this
chapter.
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 Part V
Regenerative Surgery of the Corneal
Endothelium
 Corneal Endothelium: Applied
Anatomy
Francisco Arnalich-Montiel
27.1 Introduction
Current knowledge of the development, struc-
ture, and function of corneal endothelium allows
understanding of the effects of external trauma or
ocular and systemic disease on this apparently
simple monolayer of cells. We will review the
anatomy, physiology, and immunohistochemistry
of human corneal endothelium in this chapter.
27.2 Anatomy of the Corneal
Endothelium
27.2.1 Embryology
The major structures of the eye have different
embryological origins. In particular, the cornea is
derived from two distinct embryologic tissues:
the corneal epithelium from epidermal ectoderm
and the rest from neural crest cells [1].
Developmentally, the human corneal endothe-
lium cannot be considered true endothelium, as
its origin is quite distinct from that of vascular
endothelium. The latter derives from the heman-
gioblast, a mesoderm-derived progenitor cell that
F. Arnalich-Montiel (*)
Vissum Corporation, Madrid, Spain
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_27
27
gives rise to hematopoietic stem cells, and to
angioblasts, the precursors of endothelial cells. It
would be, therefore, more correctly referred to as
the posterior cell layer of the cornea or the poste-
rior epithelium [2].
The cornea is formed as a result of the last
series of major inductive events during eye devel-
opment at ∼5–6 weeks of human gestation, when
the surface ectoderm interacts with the lens vesicle
[3]. When the surface ectoderm and the lens vesi-
cle are completely separated, the space between
them is filled with perinuclear neural crest cells
(NCCs). These NCCs originate during gastrula-
tion at the neural plate border and migrate from
folds of the neural ectoderm as the neuroepithe-
lium closes to form the neural tube [4]. In humans,
the NCC migrates in three waves into the eye,
while in mice and chick, there appears to be only
two waves [5]. In humans, the first wave of NCCs
migrates into the space between the anterior sur-
face of the lens and the surface ectoderm destined
to form the corneal endothelium [5]. A second
wave of cells migrates between the corneal epithe-
lium and endothelium to become the keratinocytes
of the corneal stroma. The corneal epithelium syn-
thesizes components of the extracellular matrix for
the formation of primary stroma when the lens
vesicle detaches from the surface ectoderm, while
a third wave of NCCs migrates to the angle
between the posterior cornea (endothelium) and
the anterior edge of the optic cup, eventually con-
tributing to the ciliary body and iris stroma [5].
419
420 F. Arnalich-Montiel

The first wave of NCCs forms a loosely
arranged sheet of overlapping cells, and by the
8th week of human gestation, it thins to a mono-
layer of orderly arranged cuboidal cells [1].
Around the 78-mm stage, these cells flatten and
become tightly connected to one another by tight
junctions. By the 120- and 165-mm stages, the
endothelial monolayer is of uniform thickness,
spans the entire posterior corneal surface, and
fuses peripherally with the trabecular beams. The
endothelial cells stay arrested in the G1-phase of
mitosis due to different factors that include lack
of response to positive mitogenic stimulation
in  vivo, suppression of the cell cycle entry by
TGF-beta2 in aqueous humor, and contact inhibi-
tion induced by the formation of mature cell–cell
junctions [6]. At birth, the endothelial monolayer
is approximately 10 μm thick [7].
Immediately anterior to the flattened layer is a
discontinuous homogeneous acellular layer,
which in time becomes Descemet’s membrane
[3]. Three major processes can be distinguished
during the formation of Descemet’s membrane
[8]. First, there is a growth in thickness by lamel-
lar deposition during the prenatal period by the
endothelial cells starting at 8  weeks. Secondly
there is a prenatal differentiation into a striated
basement membrane through the gradual addi-
tion of short and thin cross-linking bridges sepa-
rated by 110-nm intervals that are disposed in a
plane perpendicular to the lamellae. This struc-
ture has a distinctive banded appearance when
viewed by electron microscopy and is approxi-
mately 3 μm thick at the time of birth. Finally in
the postnatal period, the membrane continues to
grow in thickness by deposition of a nonstriated,
nonlamellar material posterior to the striated pre-
natal layer, which has an amorphous texture
when viewed by electron microscopy, reaching
up to 10 μm thick.
27.2.2 Morphology
Human corneal endothelium consists on a mono-
layer of flat cells on an amorphous collagenous
membrane (Descemet’s membrane). These cells
adopt a honeycomb-like mosaic when viewed
from the aqueous side, showing a hexagonal pat-
tern in healthy corneas (see Fig. 27.1). At birth,

Auto
Number 61 SD 230 um2
CV 59 %
CD 2558 /mm2
Max 1420 um2
AVG 391 um2 Min 117 um2
CCT 577 um

Area (Polymegathism) Apex (Pleomorphism)

000-100 um2 0% 3 0%
100-200 um2 20 % 4 6%
200-300 um2 20 % 5 29 %
300-400 um2 20 % 6A 24 %
400-500 um2 21 % 7 24 %
500-600 um2 5% 8 12 %
600-700 um2 8% 9 6%
700-800 um2 2% 10- 0%
800-900 um2 2%
900- um2 3%

Fig. 27.1  Normal human corneal endothelium as seen with specular microscopy. CD cell count density, Avg average
cell area in microns, CCT central corneal thickness, CV coefficient of variation
27  Corneal Endothelium: Applied Anatomy
the endothelial monolayer is approximately
10  μm thick and tends to flatten over time and
stabilize at 4 μm [1]. The cellular morphological
features evolve with aging, as some cells grow
and others disappear; the mosaic shows poly-
megathism (different cell size) and polymor-
phism (different geometric cell morphology other
than a hexagon).
They are highly polarized cells, with an apical
surface devoided of surface villi, unlike the epi-
thelium. The interaction between tight junctions
and a submembranous network of actomyosin is
responsible for the hexagonality of the apical sur-
face. The lateral membranes, which support
enzymatic pumps such as the Na+/K+ -adenosi-
netriphosphatase (ATPase) pump, presented
extensive lateral interdigitations between adja-
cent cells [9]. The basal side of the endothelial
membrane contains numerous hemidesmosomes
that promote adhesion to Descemet’s membrane.
During life, endothelial cell density and hex-
agonality decline (Fig. 27.2). At birth endothelial
cell count is over 4000 cells/mm2, with a rapid
decline in the first 2 years of an 8% likely related
to growth in corneal diameter and hence surface
area [10]. From the second to eighth decades of
life, the cell density declines to around 2600
cells/mm2 [11]. At the same period of time, the
percentage of hexagonal cells declines from
approximately 75% to approximately 60% [12].
In this period, the central endothelial cell density
decreases at an average rate of 0.6% per year in
3000-4000 cell/mm2
18µ
Fig. 27.2  Changes in
corneal endothelial cell
density and size At birth
throughout life
421
normal corneas. A minimal numerical density of
400–500 cells/mm2 is required to sustain the
pumping activity of the endothelium [10].
27.3 Physiology of Corneal
Endothelium
The corneal endothelium has three major roles; it
functions as a barrier and as a water pump, and
also it is implicated in the production of
Descemet’s membrane.
The morphology and arrangement of the cor-
neal endothelium are optimized to fulfill its main
physiological function that of acting as a regula-
tor of the entrance and exit of fluids to or from the
cornea. In order to optimize the barrier function,
the hexagonal shape of the cells is the most effi-
cient to cover the surface of the posterior surface
of the cornea without leaving gaps between them
[7]. A hexagonal shape also minimizes the area
that the individual cell must occupy, allowing the
maximum number of cells per mm2 and thus
maximizing pump site density.
27.3.1 Barrier Function
The endothelial monolayer functions as a per-
meability barrier that restricts the movement of
water and solutes into the hydrophilic stroma
due to the presence of intercellular gap junc-
2500 cell/mm2 <2000 cell/mm2
> 40 µm
From 2° to 8th
decade Old age
422
tions (GP) and tight junctions (TJ) at the apical
membrane of endothelial cells. However, unlike
the corneal epithelium, which forms a tight bar-
rier, these junctions do not provide a complete
seal around the cell, so there is a constant leak
of aqueous humor into the stroma at a slow rate.
This constant leak also provides the major
source of nutrients for cells of the nonvascular
cornea [1].
27.3.2 Pump Function
The endothelial monolayer has a pump activity to
generate a state of relative stromal deturgescence
(78% water content). This state of stromal dehy-
dration allows the necessary arrangement of the
collagen fibers and glycosaminoglycans for tis-
sue transparency [13].
Therefore, endothelial activity consists on a
pump-leak mechanism in which there is dynamic
equilibrium between two opposite forces: (1) a
tendency of the stroma to swell due to the imbibi-
tion pressure (60 mm Hg) produced by the pro-
teoglycan matrix that surrounds each collagen
fiber of the stroma [1] and (2) a net outflow of
fluid from the corneal stroma into the aqueous
humor driven by ionic gradients located in the
basolateral side of the membrane. Although the
net outflow of fluid from the corneal stroma into
the aqueous humor is passive and it does not
require energy consumption, it does depend on
aerobic metabolism to maintain the favorable
osmotic gradient that allows this by means of
intracellular and membrane ion transport
systems.
The two main ion transport systems responsi-
ble for the osmotic gradient which allows move-
ment of fluid from a relatively hypoosmotic
corneal stroma toward a relatively hypertonic
aqueous humor are:
1. Membrane-bound Na+/K+ -ATPase sites
[14]. An osmotic gradient of sodium is present
between the aqueous humor and the stroma
and results in the influx of sodium ions from
the aqueous humor and in an efflux of potas-
sium ions to the aqueous humor.
F. Arnalich-Montiel
2. Intracellular carbonic anhydrase pathway
[14]. Carbon dioxide also diffuses into the
cytoplasm of the endothelial cells, and in
combination with water, the carbonic anhy-
drase produces bicarbonate ions that diffuse
or are transported into the aqueous humor
coupled with an efflux of water.
27.3.3 Secretory Function
Lastly, endothelial cells also have a secretory func-
tion and are responsible for Descemet’s membrane
formation and postnatal development. They
secrete collagen type VIII which is an important
component of Descemet’s membrane [15].
27.4 Immunohistochemistry
of Corneal Endothelium
Although the endothelial layer is only 5 μm thick,
there is a great three-dimensional complexity due
to the high polarization of the cell [16]. The CEC
when seen from the apical surface is usually seen
as flat cells with a polygonal pattern resembling
terra-cotta tiles. On the basal side, however, there
are numerous interdigitations between these cells
resembling more like an amoebic pattern.
The classic molecular markers are used to
characterize CEC such as Zonula occludens-1
(ZO-1), aquaporin 1, Na+/K+ pump, or neuron-­
specific enolase [17]. More recently CD 200 (a
member of the immunoglobulin superfamily of
proteins), glypican-4, CD 166 (an activated leu-
kocyte cell adhesion protein), and the antioxidant
enzyme peroxiredoxin-6 have been also used to
characterize corneal endothelium [18, 19].
However most of them have been found to be
nonspecific as they can be expressed in other cor-
neal cells, as well as other ocular and non-ocular
tissues. In a recent study, N-cadherin was the
only one of all the targets that was found exclu-
sively in CECs and not in other corneal cells [16].
Nevertheless the subcellular distribution features
of some of these proteins can be used as specific
markers to distinguish these cells from the other
corneal cell types (see Table 27.1).
27  Corneal Endothelium: Applied Anatomy 423

Table 27.1  Endothelial specific 3D mapping proteins

Fluorescence pattern
Target protein Function Endothelium Epithelium Stroma
Zonula occludens (ZO-1) Tight junction Zigzag line at the apical Continuous Cytoplasm
complex surface interrupted at the Y regular straight
junction line
ATPase, Na+/K+ transporting Active pump Flower shaped at the lateral Cell to cell Cytoplasm
beta 1 polypeptide membrane junction
Actin Microfilaments Submembranous network at Cytoplasm Cytoplasm
the apical surface exclusively
Myosin IIa Microfilaments Submembranous network at Cytoplasm Cytoplasm
the apical surface exclusively
Integrin α3β1 Cell adhesion Polarization at the basal Cell to cell Absent
surface junction
N-cadherin Cell adhesion Flower shaped at the lateral Absent Absent
membrane
Activated leukocyte adhesion Cell adhesion Flower shaped at the lateral Cell to cell Faint in the
molecule (ALCAM or CD membrane junction cytoplasm
166)
Neural cell adhesion Cell adhesion Flower shaped at the lateral Absent Cytoplasm
molecule 1 (NCAM or CD56) membrane
Peroxiredoxin-6 (Prdx-6/ Antioxidant Flower shaped at the lateral Absent Absent
antibody Tag2A12) enzyme membrane

Three differentiated staining patterns can be

identified:

1. The hexagonal apical surface pattern of the

cell can be well characterized by ZO-1 (see
Fig. 27.3), which is distributed along a jigsaw
pattern interrupted at the Y junction between
three cells, and by a submembraous network
of actin and myosin IIa, exclusively located at
the apical surface.
2. The lateral membrane begins immediately
beneath the apical surface with fingerlike pro-
cesses progressively expanding toward
Fig. 27.3  ZO-1 staining for rabbit corneal endothelial
Descemet’s membrane. This side of the cell cells (red) highlighting the hexagonal pattern with a zig-
can be revealed with similar staining patterns zag distribution. Nuclei were counterstained by Hoechst
by targeting N+/k + ATPase, CD166, Prdx-6, 33342 (in blue)
NCAM, and N-cadherin.
3. The basal surface shows a polarization of or by fibroblastic conversion, also known as
integrin α3ß1. endothelial-to-mesenchymal transition (EnMT)
that becomes evident in early passages. In a
Finding a specific marker for endothelial recent study, the markers proposed [20] were
cells is so important and is becoming highly CD56, or neural cell adhesion molecule
important in clinical research. It allows the (NCAM), a surface glycoprotein; CD248, or
identification of high-quality human corneal endosialin which belongs to the family of C-type
endothelial cells in culture, to distinguish this lectin transmembrane receptors; coxsackie-­
subpopulation from other subpopulations that adenovirus receptor (CAR) is part of the junc-
might be contaminated by stromal keratocytes tional adhesion molecules (JAMs); and CD109,
424
a cell surface antigen involved in the TGF-β
pathway. Another study identified five genes,
CLRN1, MRGPRX3, HTR1D, GRIP1, and
ZP4, as novel markers of corneal endothelial
cells, and the specificities of these genes were
successfully confirmed by independent experi-
ments at both the RNA and protein levels [21].
Compliance with Ethical Requirements Francisco
Arnalich-Montiel declares no conflict of interest. No
human or animal studies were carried out by the author for
this article.
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Kerkis I, editor.
 Corneal Endothelium: Isolation
and Cultivation Methods
David Mingo-Botín, Marie Joan Therese D. Balgos,
and Francisco Arnalich-Montiel
28.1 Introduction
The corneal endothelium is the innermost layer
of the human cornea, composed of a single layer
of hexagonal cells that are organized in a regular
mosaic on their basement membrane  – the
Descemet membrane. Corneal endothelial cells
maintain corneal transparency by controlling
deturgescence in the corneal stroma [1]. They act
as a partial or leaky barrier to nutrients and other
molecules in the aqueous humor in a mechanism
explained by the pump-leak hypothesis. The cor-
neal endothelial cells are able to accomplish this
with the use of tight junctions, simple diffusion,
facilitated diffusion, and active transport mecha-
nisms. In the absence of this mechanism, the
strict organization of the collagen fibrils in the
stroma, critical for corneal transparency, would
be compromised [1, 2].
D. Mingo-Botín
Cornea Unit, Department of Ophthalmology,
Hospital Universitario Ramón y Cajal, Madrid, Spain
M. J. T. D. Balgos
Research and Development Department,
Cornea, Cataract and Refractive Surgery Department,
Vissum Alicante, Alicante, Spain
F. Arnalich-Montiel
Vissum Corporation, Madrid, Spain
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_28
28
Human corneal endothelial cells (HCECs) are
derived from the cranial neural crest. They have a
limited capacity to proliferate in vivo, with majority
of cells arrested in the G1 phase of the cell cycle and
cannot go further in the cell cycle phase in spite of
endothelial cell injury or loss [3]. This has been
attributed to the following factors as reviewed by
Cui et al. – the presence of negative regulator mole-
cules (such p21, p27, p16, p15, p19, and p53), trans-
forming growth factor β (TGF-β) in aqueous humor,
contact inhibition of densely packed cells, or stress-
induced premature senescence [4]. Residual endo-
thelial cells thus enlarge and migrate to cover the
defects left by lost cells [1, 5]. To achieve corneal
deturgescence and transparency, a minimal func-
tional capacity is required, which depends on both
endothelial cell density and quality. There is an
inverse relationship between age and corneal endo-
thelial cell density [1, 6]. The high endothelial cell
densities found in infants, up to 5000 cells/mm2,
decrease during early childhood until gradual physi-
ologic decline of approximately 0.6% per year is
reached in adulthood. This can occur without
impairing corneal transparency. However, certain
endothelial dystrophies and traumatic endothelial
injury can dramatically accelerate this process, mak-
ing endothelial cell density fall below the threshold
of 300–500 cells/mm2 [2]. At this stage, irreversible
corneal edema occurs, leading to vision loss.
Corneal disease is a leading cause of visual
impairment worldwide. Corneal opacities make
up 4% of the leading causes of blindness, as per
425
426
the 2010 Global Estimates of the World Health
Organization [7, 8]. While nearly 80% of causes
of corneal blindness are avoidable or preventable,
when these measures have been unavailable or
unsuccessful, the only recourse is for replace-
ment of the diseased corneal tissue [9–11].
Indications for corneal transplantation vary
among countries, but the most common are
Fuchs’ dystrophy, keratoconus, bullous keratopa-
thy, and infections. Improvement in surgical
technique and available technology and the
establishment of eye banks all over the world
have contributed greatly to the establishment of
corneal transplantation as the standard treatment
for visual impairment due to corneal pathologies
[10]. A longer recovery time, the risk of develop-
ing high or irregular astigmatism, glaucoma, and
immune-mediated graft rejection are the main
concerns that have been associated with penetrat-
ing keratoplasty (PK). These are limited with the
use of endothelial and anterior lamellar kerato-
plasty, although both techniques require a higher
learning curve than PK. Endothelial keratoplasty
is also associated with a higher degree of postop-
erative endothelial cell loss than PK [4].
Furthermore, long-term graft survival after PK is
limited – with 5- and 10-year corneal graft sur-
vival estimates at 74% and 64%, respectively  –
and repeat grafting is likely necessary [12].
Corneal transplantation rates are hampered by
the number of available suitable cadaver corneas
for transplantation. A generally low cornea dona-
tion rate, coupled with strict criteria for screening
and exclusion of donor corneas (which include
serology tests, the presence of notable systemic
medical conditions, and the presence of structural
defects in the corneal tissue), has led to an enor-
mous gap between corneal supply and demand.
Global demand for cornea is at 12.7 million, yet
the range of corneal transplants that have been
performed is only at 100,000–185,000 [9]. This
imbalance will probably be aggravated by
increasing global population growth and a con-
comitant aging of the general population. To cope
with the shortage of donor cornea, steps have
been proposed to maximize one donor cornea for
use with multiple patients. Using anterior or pos-
terior lamellar grafts, a good-quality donor cor-
D. Mingo-Botín et al.
nea could be divided into three further parts – the
peripheral corneoscleral rim with epithelium and
stroma, an anterior lamellar stromal disc, and a
posterior lamellar disc with Descemet and endo-
thelium [13].
Another strategy being evaluated is to maxi-
mize the remaining corneal endothelial cells by a
topical treatment that would decrease endothelial
cell loss and promote the function of the remain-
ing cells. Koizumi et al. and Okumura et al. have
reported that the topical use of Rho-kinase
(ROCK) inhibitor Y-27632 in rabbit and primate
corneal injury models slowed the progression of
endothelial cell degeneration and led to restora-
tion of normal cell counts [14–17]. It has also
been partially successful in four human patients
with corneal endothelial dysfunction [17]. The
exact mechanism by which Rho-kinase inhibition
increases endothelial cell count is still unknown,
and other studies have presented conflicting data
[18]. Hence, more studies are needed on the
safety and efficacy of these molecules as possible
topical treatment for corneal endothelial disease.
28.2 H
 uman Corneal Endothelial
Cell Engineering
There is now considerable interest in the develop-
ment of suitable alternatives for donor endothe-
lial graft material through tissue engineering and
regenerative medicine. Intensive collaborative
and multidisciplinary research has been done on
the topic to realize the expansion of functional
HCECs in  vitro and to transplant them onto a
recipient cornea to recover normal or near-­normal
corneal endothelial function.
Although HCECs have limited proliferative
capacity in  vivo, it has been demonstrated that
after the release of cell-cell junctions, and in the
presence of appropriate growth factors, they are
able to proliferate ex vivo [3]. Thus, tissue engi-
neering gives us the opportunity to replace dys-
functional corneal endothelium without relying
on the limited available cadaveric donor cornea
supply.
Prior to transplantation, tissue-engineered
grafts would need to fulfill similar requirements
28  Corneal Endothelium: Isolation and Cultivation Methods
to those that endothelial grafts from an eye bank
must meet [19]. These grafts should be functional
and highly cellular to compensate for the cell loss
associated with surgery. They should be deliv-
ered on a biocompatible carrier that does not alter
the optical and biomechanical properties of the
cornea and allow a convenient surgical technique.
In addition to its availability and high cellularity,
other potential advantages of corneal endothe-
lium culture would be the production of autolo-
gous tissue (although it would be limited to
secondary endothelial diseases), eliminating
immunological rejection, and its ability to with-
stand cryopreservation [20].
Since the successful expansion of HCECs
reported by Mannagh and Irving in 1965 [21],
research in this field has focused especially on
optimizing the methods of culture as well as the
biomaterials used as carriers for transplantation.
As in other engineered tissues, there are a num-
ber of key variables that determine their prolifer-
ative and functional capacity. These include the
viability of the donor cells, the procedure for iso-
lation, the substrate, and the composition of the
culture medium.
28.2.1 Source of HCECs
There are three main sources of HCECs: primary
HCECs, established cell lines, and stem cells, which
may originate from presumed endothelial progeni-
tors or from adult stem cells from other locations.
(a) Primary HCECs are isolated from donor cor-
neoscleral rims remaining after corneal
transplantation or human cadaver corneas
that are ineligible for surgery. The viability
of these cells may be influenced by cell den-
sity, death to preservation time, preservation
medium and period, age, overall health of the
donor, and specific cause of death [22, 23].
In particular, the effects of donor age and
topographical source on the proliferative
capacity of primary HCECs have been studied
extensively. Using ex vivo cornea [24] and tis-
sue culture methods [23, 25–27], it has been
shown that, although older donor cells can
427
also divide, young donors have a larger cell
population that retain the capacity to prolifer-
ate in response to mitogenic stimuli. Likewise,
peripheral HCECs exhibit a higher replication
competence than cells from the central cornea,
regardless of donor age [28, 29].
(b) Stem cells are the ideal potential source for
tissue engineering, and they may be derived
from presumed endothelial progenitors or
from adult stem cells from other locations.
Progenitor cells in the corneal endothelium
were first located in a monkey model. Several
studies indicate the presence of corneal endo-
thelial progenitors, known as Schwalbe’s line
cells, at the transition zone between the ante-
rior extension of the trabecular meshwork
and endothelial periphery [30–33].
An examination of the microanatomy of
the endothelium showed that cells at the
extreme periphery were organized in small
clusters around the Hassall-Henle bodies,
arranged in centripetal rows. These cells
exhibited lesser differentiation than endothe-
lial cells in the cornea, with preferential
expression of stem cell markers (nestin,
telomerase, and occasionally breast cancer
resistance protein). This suggested a continu-
ous slow centripetal migration of endothelial
cells from these niches [31]. Similar results
were reported by another study. Manifest
telomerase reaction and BrdU-linked alkaline
phosphatase activity staining, which demon-
strates cellular division, were detected in
peripheral corneal endothelium and enhanced
after mechanical wounding of the cornea.
These point to a wound-induced cell response
which suggests that cells in the corneal endo-
thelium may be renewed by stem-­like cells
originating from the peripheral endothelium
and migrating toward the center, in order to
minimize endothelial cell loss [32]. This was
clinically observed in an in vivo model in an
observational case series of three human
patients with chemical eye injuries, as well as
in an in  vivo endothelial chemical injury
model in rabbit eyes. Corneal endothelial cell
density was found to increase at 6  months
post-injury in human eyes, compared to
428
immediately after and 1 month after. This was
accompanied by an improvement in corneal
edema and reduced corneal thickness. In the
rabbit eyes, corneal edema was noted to
decrease over time (although corneal endo-
thelium of rabbits has been known to regener-
ate) albeit without improvement in central
corneal opacity. The peripheral cornea was
clear. Vital staining with alizarin S red and
trypan blue showed cell damage centrally and
peripherally immediately after injury. Within
7 days of the injury, corneal endothelial cells
were already present in the periphery. Corneal
endothelial cells were only detected in the
center 14 days after injury [34].
Sphere-forming cells derived from HCECs
may also represent a potential source of pro-
genitor cells [35, 36] – they have been dem-
onstrated to be highly proliferative with a
propensity to later differentiate into HCEC-­
like cells. It appears that this sphere-forming
assay results in longer telomeres and higher
telomerase activity, and they are better formed
from endothelial peripheral cells [37].
(c) Immortalized cell lines are easily available
and have the advantage of allowing the large
quantities of homogeneous cells required for
many in vitro studies. The first line of HCECs
was developed in 2000 [38] by transfection
with simian virus 40 T antigen. Two clonal
cell lines were later separated from this cell
population [39]: B4G12 [40], which is con-
sidered a good model of differentiated HCEC,
and H9C1, with developing HCEC character-
istics. Other immortalized HCEC line used
transduction of human papilloma virus 16 E6/
E7 oncogenes [41]. To avoid the potential
carcinogenesis induced by viral oncogenes,
other attempts have been made by transduc-
tion with human telomerase reverse transcrip-
tase [42], Cdk4R24C/Cyclin D1 [43], and
immortalizing untransfected HCECs [44].
28.2.2 Isolation
Stromal keratocytes have much greater prolifera-
tive capacity in vitro than endothelial cells, and
D. Mingo-Botín et al.
therefore it is essential to prevent them from con-
taminating the HCEC culture. This was achieved
with the use of explant or scraping techniques
[45], although even and fibroblast growth-inhib-
iting media [46] or magnetic cell separation [47]
have been proposed. However the current refine-
ment of Descemet membrane (DM) extraction
techniques, similar to those used in DM endothe-
lial keratoplasty (DMEK) [48], allows for a much
cleaner extraction. Briefly, the donor corneo-
scleral button is positioned on a support, usually
with suction vacuum; peripheral and very shal-
low marking is performed by means of a needle
or a trephine, and, always under balanced salt
solution, the edges of the DM are raised to obtain
a plane of cleavage from which to pull gently
until the dissection is complete. After peeling the
DM with the endothelium, the cells are detached
and dissociated by enzymatic or nonenzymatic
methods. Currently this is the most widespread
strategy, and it is known as “peel and digest”
[22].
Enzymatic digestion methods mainly use
trypsin, collagenase, or dispase [19, 49]. Its main
drawback is that enzymatic digestion requires a
prolonged incubation time to separate the cells
from the matrix, which is accompanied by fur-
ther cell degradation and increases the possibil-
ity of contamination by stromal cells.
Nonenzymatic methods use EDTA to release
intercellular junctions, which also may promote
cell proliferation [50], but can induce some cell
degradation too. It is also possible to combine
methods, such as that described by Li et  al.,
where collagenase A is first used to detach cells
from DM and aggregates of HCECs are obtained
that could be preserved in serum-free medium
and maintain HCEC characteristics for at least 3
weeks. Trypsin and EDTA are then added to sep-
arate the cells, resulting in higher proliferative
response [51, 52]. To optimize the isolation of
viable cells, the use of a stabilization medium
(serum-supplemented with no added growth fac-
tors) for up to 6 days prior to isolation has also
been proposed. This could enable the selection
of viable cells and remove the negative impact of
the apoptotic neighboring cells [53], increasing
the yield of subsequent culture [54].
28  Corneal Endothelium: Isolation and Cultivation Methods
28.2.3 Substrate or Extracellular
Matrices
At the initiation of primary cell culture, adher-
ence to the substrate is important to initiate cell
growth, whereas detachment of a confluent cell
layer in later phases is necessary for transplanta-
tion purposes [55]. While it has been suggested
that matrix coating might not be mandatory for
culturing HCECs [56], it is known that proteins
in the basement membranes are implicated in the
processes of cell adhesion, proliferation, migra-
tion, morphogenesis, and differentiation [57, 58].
For this purpose, the ideal substrates would be
those similar to DM in their physiological and
mechanical characteristics. They should provide
a favorable microenvironment for endothelial
activity, be transparent and nontoxic, and be easy
to handle during transplantation [55]. Multiple
biological, synthetic, and biosynthetic substrates
have been used and described.
Extracellular matrix (ECM) proteins found in
DM include fibronectin, laminin, collagen types
I, IV, VI, and VIII, and proteoglycans [57, 59],
which can be employed as culture substrates. The
coatings based on these natural polymers can be
composed of single proteins such as collagen,
fibronectin, laminin, or gelatin) or combinations
of different proteins (such as ECM from cultured
bovine corneal endothelial cells, and FNC
Coating Mix®, containing a mixture of fibronec-
tin, collagen, and albumin, (Athena Environmental
Sciences Inc.).
Choi et al. evaluated adhesion, proliferation,
and phenotypic maintenance between uncoated
and culture plates coated with different ECM –
including collagen type I, collagen type IV,
fibronectin, laminin, and FNC Coating Mix [57].
All the coated cultured plates had increased inte-
grin expression as adhesion markers (especially
integrin ɑ2) compared to the uncoated dishes,
and centrifugation assays showed improved
adhesion strength in all except for the laminin-
coated plate. HCECs formed a compact mono-
layer after a week, without significant differences
between coated and uncoated plates; but cells
grown on collagen IV-, fibronectin-, and FNC-
coated plates showed more typical compact cel-
429
lular morphology. Na+/K+ATPase expression was
also significantly higher in collagen type I-,
fibronectin-, and laminin-coated culture plates.
Yamaguchi et al. investigated also the expression
of laminin-­5 and its receptor-α3β1 integrin in
HCECs cultures and concluded that it promoted
adhesion, migration, and moderate proliferation
[60].
Extracellular matrices from cultured bovine
endothelial cells have been extensively used for
HCEC cultures [61, 62]; however the use of ani-
mal components gives rise to the possibility of
xenogenic contamination and immunogenicity.
Numata et al. used pericellular matrix of decidua-­
derived mesenchymal cells (PCM-DM) as sub-
strate in order to avoid animal-derived ECM
sources. They demonstrated that this xeno-free
coating PCM-DM substrate enhanced cell adhe-
sion via integrin, promoted cell proliferation, and
suppressed apoptosis [63].
In the same way that collagen type I sheets have
been used both as ECM and as carrier [61], other
tissues have been tested with the same goal.
Decellularized human corneal stroma (seeded with
feline [64] or human [59, 65] corneal endothelial
cells) and bovine [66] corneal stroma (seeded with
HCECs) were shown to be possible scaffolds for
cultivation and transplantation of HCECs.
Other human-derived sources of tissue sub-
strate and carrier are amniotic membrane (AM)
and anterior lens capsule. The anti-inflammatory
and non-immunogenic properties of AM could be
favorable properties for this purpose [67, 68]. Its
main problems are that it is translucent (although
using only basement membrane could improve
this issue [68]), it is heterologous tissue, and
there might be high inter-donor variability which
could influence the outcome. AM can be used
cryopreserved or lyophilized [69]. The potential
use of de-epithelized human anterior lens capsule
coming from cataract surgeries has been explored
by Yoeruek et  al. [70], who found optimal cell
density and phenotype after using this scaffold
for culture. This has been later confirmed by
other investigators [71, 72], although its biologi-
cal origins still confer inter- and intra-donor vari-
ability with the theoretical risk of disease
transmission.
430 D. Mingo-Botín et al.

Synthetic and biosynthetic substrates have which can also vary depending on the donor
been developed to function as carriers, but samples. In order to reduce serum dependence,
their discussion is not included in the scope of additional supplementation with serum com-
this chapter. Within however, it is worth men- ponents is required. These serum components
tioning thermoresponsive plates grafted with include insulin, selenium, transferrin, lipids,
poly(N-isopropylacrylamide) (PIPAAm), essential amino acids, or ascorbic acid [77].
which can be hydrophobic or hydrophilic Mitogenic growth factors used include epider-
depending on the temperature and allow the mal growth factor (EGF), vascular endothelial
detachment of endothelial cell layer for trans- growth factor (VEGF), insulin-­ like growth
plantation [73, 74]. factor (IGF), and fibroblast growth factor
Table 28.1 gives examples of natural culture (FGF)  – which could also play a role as dif-
substrates or extracellular matrices. ferentiation factor [49] or promote fibroblastic
transformation [78]. Recently, leukemia inhib-
itory factor (LIF) has been shown to stimulate
28.2.4 Culture Media HCEC growth and preserve cell phenotype in
the presence of bFGF in a media without serum
Many attempts to optimize culture media have [79].
been made, using different basal media sup- Some of the more commonly cited formula-
plemented with several growth factors, serums, tions are summarized in Table 28.2.
and nutrients  – all aimed to stimulate HCEC Although a variety of protocols have proven to
expansion and help maintain cell phenotype. support continuous cell proliferation, the cells
Published protocols vary greatly, and each one often undergo morphological transformation
involves serum as an essential component. toward a fibroblast-like morphology after few
This causes major uncertainty because the use passes. This is known as endothelial-to-­
of serum introduces a complex mixture of mesenchymal transition (EMT) [78, 86]. It is
ingredients, at poorly defined concentrations believed that this phenomenon is promoted by
the use of trypsin-EDTA to break the cell-cell
junctions and bFGF in the culture medium via
Table 28.1  Examples of natural culture substrates or activation of the canonical Wnt signaling, espe-
extracellular matrices cially when TGF-­β1 signaling is also activated
Substrate Reference [86–90]. This highlights the importance of char-
Biological substrates acterization of the obtained endothelium with
 Decellularized Proulx et al. [64] each model  – and such an assessment should
corneal stroma Honda et al. [65]
Choi et al. [59]
evaluate morphology, protein expression (typi-
Bayyoud et al. [66] cally Na/K-ATPase and zonula occludens pro-
Yoeruek et al. [75] teins), and function in  vitro or in  vivo [19].
 Amniotic membrane Ishino et al. [67] Recently, a panel of novel markers correlated
Wencan et al. [68] with function in culture has been described [91].
 Lens capsule Yoeruek et al. [70]
Kopsachilis et al. [71]
Using RNA sequencing, it is also possible to
Bogerd et al. [72] compare transcriptomic profile between ex vivo
Natural polymers HCEC, primary HCEC cultures, and immortal-
 Collagen types I and Choi et al. [59], Choi et al. ized HCEC lines. With this technique, Frausto
IV [57], Numata et al. [63] et  al. found that, at a molecular level, primary
 Laminin Yamaguchi et al. [60]
HCECs resembled ex vivo HCECs more closely
 Bovine endothelial Miyata et al. [26]
ECM Mimura et al. [61] than immortalized lines [92]. Other specific
Hitani et al. [62]. HCEC markers, specially CD166, have been
 FNC Coating Mix ® Joyce et al. [76] identified through the use of monoclonal antibod-
Choi et al. [57] ies [93, 94].
28  Corneal Endothelium: Isolation and Cultivation Methods 431

Table 28.2  Basal media, supplements, and sera for HCEC culture
Basal medium Supplements Serum Reference
F99 (Ham’s F12 and M199, 1:1 ratio) 20 μg/ml ascorbic acid 2–5% Engelmann et al. [80]
20 μg/ml bovine insulin
2.5 μg/ml transferrin
0.6 ng/ml sodium selenite
10 ng/ml bFGF
MEM 5 μg/ml insulin 10% Blake et al. [81]
5 μg/ml transferrin
5 ng/ml sodium selenite
50 μg/ml gentamicin
100 U/ml penicillin
0.1 mg/ml streptomycin
0.25 μg/ml amphotericin B
150 μg/ml ECGS
DMEM 30 mg/l L-glutamine 15% Miyata et al. [26]
2.5 mg/l Fungizone
2.5 mg/l doxycycline
2 ng/ml bFGF
DMEM 2 ng/ml bFGF 10% Ishino et al. [67]
50 U/ml penicillin
50 mg/ml streptomycin
Opti-MEM-I 5 ng/ml EGF 8% Zhu et al. [23]
20 ng/ml NGF
100 μg/ml pituitary extract
20 μg/ml ascorbic acid
200 mg/l calcium chloride
0.08% chondroitin sulfate
50 μg/ml gentamicin
1/100 antibiotic/antimycotic
SHEM (Ham’s F12 and DMEM; 1:1 ratio) 0.5% DMSO 5% Li et al. [51]
2 ng/ml mouse EGF
5 μg/ml insulin
5 μg/ml transferrin
5 ng/ml selenium
0.5 μg/ml hydrocortisone
1 nM cholera toxin
50 μg/ml gentamicin
1.25 μg/ml amphotericin B
EGM-2® VEGF 10% Choi et al. [59]
(Lonza Group Ltd, Basel, Switzerland) EGF
IGF
bFGF
Ascorbic acid
Hydrocortisone
Gentamicin
Amphotericin B
Endothelial SFM® 10 ng/ml FGF-2 – Jäckel et al. [82]
(Life Technologies/Thermo Fisher Scientific, USA) Antibiotics
DMEM:F12 (1:1 ratio) 20% HAF – Feizi et al. [83]
MESCM 10% knockout serum, – Zhu et al. [84]
(DMEM and F12; 1:1 ratio) 5 μg/ml insulin
5 μg/ml transferrin
5 ng/ml sodium selenite
4 ng/ml bFGF
10 ng/ml hLIF
50 μg/ml gentamicin
1.25 μg/ml amphotericin
F99 (Ham’s F12 and M199, 1:1 ratio) 0.1 mg/ml phPL – Thieme et al. [85]
DMEM Dulbecco’s modified Eagle’s medium, DMSO dimethyl sulfoxide, ECGS endothelial cell growth supplement,
EGM endothelial growth medium, HAF human amniotic fluid, MESCM modified embryonic stem cell medium, SFM
serum-free medium, SHEM supplemented hormonal epithelial medium, phPL processed human platelet lysate
432
28.2.4.1 A  pproaches to Avoid
Endothelial-to-Mesenchymal
Transition
Peh et al. evaluated four different culture media:
Dulbecco’s modified Eagle’s medium (DMEM)
[67], Opti-MEM-I [23], DMEM/F12 [51], and
Ham’s F12/M199 [80] in FNC-coated plates using
HCECs from paired donor corneas in order to
avoid donor variability (Peh 2011) [52]. They
found that isolated hCECs could be established in
all four media for a short period of time, but only
Opti-MEM-I and Ham’s F12/M199 [23, 80] were
able to support the continual propagation of
hCECs and expression of Na+ K+/ATPase and
ZO-1. In all of the media, cells lost their character-
istic morphology beyond the third passage. In
order to overcome EMT transformation – due to
the growth factors included in the culture medium
and loss of contact inhibition by trypsin-EDTA
treatment – some authors suggest the use of a dual
media approach [78]. This dual dual media
approach involves the combined use of a prolifera-
tive medium [80] with greater expansion proper-
ties, with a stabilization medium [23] with better
capacity to preserve the cellular morphology of
primary HCECs in vitro. HCECs propagated using
this dual media approach were homogeneous,
retained their characteristic cellular morphology,
and expressed higher levels of corneal endothe-
lium-associated markers than in those cultured in
proliferative medium alone, which were heteroge-
neous and fibroblastic in appearance.
The addition of ROCK inhibitor Y27632,
extensively studied by the laboratory of Kinoshita,
seems to preserve proliferation and maintain
HCEC phenotype in vivo [17, 95]. The use of this
molecule is believed to inhibit EMT that occurs
after intercellular junctions are disrupted as in
wounding processes [88], maintaining the physi-
ologic suppression of RhoA activation with con-
tact inhibition. A different technique was
developed by Zhu et al. using interference RNA
technology to knockdown p120 catenin and
Kaiso transcription suppressor. This unlocks
mitotic contact inhibition but avoids disruption of
intercellular junctions or activating of canonical
Wnt signaling [84]. The resultant expanded
HCECs have a phenotype suggestive of a neural
crest progenitor reprogramming.
D. Mingo-Botín et al.
28.2.4.2 Animal-Free Approaches
Another important line of research is dedicated to
eliminating animal by-products from the HCEC
culture process. Given the therapeutic target of
these cultures and regulatory issues, it is neces-
sary to achieve HCEC propagation under xeno-­
free conditions in order to eliminate the risk of
animal disease transmission to humans. In this
regard, several laboratories have developed
serum-free and xeno-free media.
Jackel et  al. compared MEM with fetal calf
serum and endothelial serum-free medium (SFM)
and found that cells cultured in MEM were more
susceptible to apoptosis in the absence of exoge-
nous noxious stimuli, while HCEC cultured in
SFM seemed to be protected from cell death even
when the apoptosis-inducer staurosporine was
added [82]. Another serum-free medium, previ-
ously used in limbal stem cell cultures, is modi-
fied embryonic stem cell medium (MESCM). It
contains LIF and bFGF and allowed dramatic
expansion of the size of HCEC monolayers [84].
A different approach is to replace FBS with
human-derived sera. A promising candidate as
the source of nutrients and growth factors in basal
growth media is human platelet lysate [96].
Thieme et  al. compared FBS and processed
human platelet lysate (phPL) in immortalized
and primary HCEC cultures and found superior
viability in cultures with phPL [85]. Another
study compared the use of human amniotic fluid
obtained from diagnostic amniocentesis and
FBS, and the first exhibited greater stimulatory
effect on HCEC growth [83]. Recently, the group
of Peh et  al. have described a regulatory-­
compliant protocol based on their dual media
approach, collagen IV substrate, and EquaFETAL
serum (derived from specially controlled ani-
mals) and applied it to a rabbit model of bullous
keratopathy, demonstrating that it is possible to
translate tissue engineering into the clinic [97].
28.3 Conclusion
Great progress has been made in the isolation and
cultivation of HCEC, especially thanks to the
efforts of several laboratories to improve sub-
strates and culture media. However, a standard
28  Corneal Endothelium: Isolation and Cultivation Methods
culture method that satisfies all the requisites for
human use has not yet been achieved. The
advances in the knowledge of the HCEC cell
cycle along with better identification of endothe-
lial progenitors, as well as the use of xeno-free
culture systems, may allow in the not-so-distant
future the application of tissue engineering in
patients with corneal endothelial disease, thus
meeting the great world demand for this tissue.
Compliance with Ethical Requirements  David Mingo-
Botín, Marie Joan Therese Dr. Balgos, and Francisco
Arnalich-Montiel declare that they have no conflict of
interest. No human or animal studies were carried out by
the authors for this article.
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 Corneal Endothelial Cell
Transplantation: Animal Models
Brad P. Barnett and Albert S. Jun
29.1 Introduction
Over the past decade, endothelial transplantation
has replaced full-thickness corneal transplanta-
tion as the definitive treatment for endotheliopa-
thies. In addition to partial-thickness
transplantation, emerging alternative therapies
include the use of cultured cells either to form
grafts or as injectable cellular therapy, as opposed
to relying on donor tissues as the sole means of
cellular replacement. In this chapter we will
review the history of animal models used in
endothelial transplantation studies as well as gen-
eral insights from full-thickness penetrating kera-
toplasty in various animal models. Care will be
taken to discuss how research groups have
matched experimental aims to the animal model
best suited to achieve those aims. To assist future
researchers in determining the best animal model
for their purposes, the relative pros and cons of
each animal model will also be reviewed. A sum-
mary of all animal models used to date for endo-
thelial transplantation can be found in Table 29.1.
B. P. Barnett
Wilmer Eye Institute at Johns Hopkins, Department
of Ophthalmology, Baltimore, MD, USA
A. S. Jun (*)
Wilmer Eye Institute at Johns Hopkins, Department
of Ophthalmology, Baltimore, MD, USA
Division of Cornea, Cataract and External Eye
Diseases, Baltimore, MD, USA
e-mail: aljun@jhmi.edu
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_29
29
Historically, animal models have played a
vital role in the study of penetrating keratoplasty
and more recently in the study of endothelial
transplantation. Notably, Samuel Bigger was the
first to report on corneal transplant in an animal
model in 1837 in which he performed a penetrat-
ing keratoplasty on a domesticated gazelle [25,
26]. During this same period, Eduard Zirm and
others experimented on penetrating keratoplasty
in chickens and rabbits [27, 28]. Since that time,
animal models have been recognized as essential
to the understanding of the process of endothelial
graft rejection, in addition to aiding in the cre-
ation of possible therapeutics.
The majority of initial research efforts in this
area were tailored to help improve transplantation
success in human penetrating keratoplasty by bet-
ter understanding immunological graft rejection.
The rabbit eye is a popular choice in the study of
corneal grafting due its size, making it easily trans-
latable to human grafting. Moreover, the rabbit
displays various signs of rejection also seen in
humans such as retrocorneal membranes and epi-
thelial decompensation [27]. Despite its popular-
ity, various shortcomings of the rabbit model then
led investigators to rodent animal models for
endothelial transplantation [29, 30]. The rodent
model now serves as the staple in immunological
studies, whereas large animal models are primarily
dedicated to studying graft rejection in [31, 32].
In addition to full-thickness grafts, the appli-
cation of carriers for creation of human corneal
437
438 B. P. Barnett and A. S. Jun

Table 29.1  Summary of endothelial transplant animal models

Host Endothelial graft Procedure Author/reference
Mouse Human spheres with magnetic nanoparticles AC injection Mimura [1]
Mouse Mouse single cells AC injection Yamada [2]
Mouse Autologous mouse on Descemet’s membrane DSAEK Joo [3]
Rat Human spheres with magnetic nanoparticles AC injection Mimura [1]
Rat Human on rat devitalized cornea PKP Tchah [4]
Rabbit Human spheres with magnetic nanoparticles AC injection Mimura [1, 5, 6]
Rabbit Human on collagen carrier DSAEK Mimura [7]
Rabbit Rabbit on Descemet’s membrane DSAEK Lange [8]
Rabbit Human on collagen carrier DSAEK Lai [9]
Rabbit Human on poly(N-isopropylacrylamide) DSAEK Lai [10]
Rabbit Human on human stroma DSAEK Honda [11]
Rabbit Rabbit on silk fibroin carrier DMEK Vazquez [12]
Rabbit Human on amniotic membrane PKP Ishino [13]
Rabbit Bovine on homografts PKP Gospodarowicz [14, 15]
Rabbit Rabbit on homografts PKP Jumblatt [16]
Cat Autologous cat on human devitalized cornea PKP Proulx [17, 18]
Cat Bovine on homografts PKP Bahn [19]
Monkey Monkey on collagen carrier DSAEK Koizumi [20, 21]
Monkey Human neonatal on denuded human cornea PKP Insler [22–24]
AC anterior chamber, PKP penetrating keratoplasty, DSAEK Descemet stripping automated endothelial keratoplasty,
DMEK Descemet’s membrane endothelial keratoplasty

endothelial cell sheets has been reported by many
laboratories during previous decades (Fig. 29.1).
In early animal transplantation studies of cul-
tured corneal endothelial cells, endothelial cells
from humans were implanted on Descemet’s
membranes from rabbits [14–16], cats [15], mice
[3], and cattle [8, 14, 16] or on gelatin mem-
branes [7] to generate full-thickness corneal
grafts utilizing denuded homo-, allo-, or xeno-
grafts that were then transplanted with penetrat-
ing keratoplasty. Infant and human neonatal
corneal endothelial cells have been employed
with success to create such grafts [22–24]. More
recent efforts have focused upon partial-­thickness
endothelial transplantation in animal models as
well as cellular therapeutics designed to be
injected into the anterior chamber [1, 5–7, 11].
29.2 In Vivo and Postmortem
Assessment
Methods of assessing the viability of a graft and
the presence of rejection are essential to any ani-
mal model. Efforts to ascertain the above can be
achieved by both in vivo and postmortem analy-
sis. Starting with in  vivo analysis, the slit-lamp
exam is useful for serially monitoring the trans-
parency of a graft, in addition to looking for
inflammation and possible immune rejection.
Not unlike clinical ophthalmology, using
pachymetry to monitor corneal thickness is also a
useful way to monitor for corneal edema as a
marker of endothelial cell function. Anterior-­
segment optical coherence tomography also pro-
vides a means of measuring corneal thickness
and provides precise cross-sectional images.
Specular microscopy can also be employed to
provide endothelial cell count and morphology.
Postmortem techniques allow for histological
examination which provides a whole host of
information not readily available with in  vivo
assessment modalities. For example, histological
analysis enables direct verification of the pres-
ence of inflammatory cells, fibrosis, and
­neovascularization [32, 34, 35]. Moreover, it
allows the researcher to assess for repopulation
of the graft with the host’s stromal keratocytes
and to assess for the presence of nerve regrowth
into the graft [32, 34, 35].
In this chapter we will discuss both transplan-
tation of graft tissue and the burgeoning field of
29  Corneal Endothelial Cell Transplantation: Animal Models 439

a b

c d

e f

Fig. 29.1  Morphology of human corneal endothelial
cells (HCECs) cultured in monolayer (a) and HCECs
seeded onto a carrier to generate a bioengineered endothe-
lial graft (b–f). (a) 62-year-old donor HCECs demonstrate
the typical hexagonal morphology. (b–f) Cultured HCECs
were seeded onto various carriers to generate grafts for
penetrating keratoplasty (b, c) or Descemet stripping with
automated endothelial keratoplasty (d–f). Scale
bars = 100 μm. (Used with permission [33])
440 B. P. Barnett and A. S. Jun

cell therapy in which cells or clusters of cells are also be employed [44]. Unlike indirect methods
transplanted into the anterior chamber to repopu- of cell labeling, direct methods do not provide
late the endothelium. Broadly, within any field of any information about cell viability, as the fluo-
experimental transplantation, a means of tracking rescent agent will still produce signal in a dead
the fate of transplanted cells is necessary. cell. In addition, in the setting of dividing cells,
Historically, the primary means of determining direct methods suffer from the dilution of con-
the fate of transplanted cells was through histo- trast as the total amount of contrast agent remains
logical assessment. Histologically Y chromo- constant, while the number of cells increases.
somes could be detected when grafting male cells Moreover, in certain instances contrast agent is
in a female host [14, 15, 36], and in the case of unequally distributed between the parent and
xenogeneic transplantation, native proteins only daughter cell, with the parent cell retaining the
present in the graft species could be detected in majority of contrast [41].
the detection of a human protein when a human Indirect cell labeling methods require genetic
graft is transplanted in swine [37]. Transgenically alteration of the cell to introduce a reporter gene
expressed reporter genes have also been employed [45]. The reporter gene may code for a fluorescent
for histological analysis such as beta-­or bioluminescent protein, an enzyme, or a recep-
galactosidase and alkaline phosphatase [38, 39]. tor. In the event of the latter, it is the interaction
The use of histological analysis to track cell between the substrate and the encoded reporter
fate has a number of shortcomings. First and protein that provides an optical signal that enables
foremost it prevents real-time serial monitoring in  vivo detection. As reporter genes will be
of graft function and correlation of graft viability expressed by parent and daughter cell alike, no
with other measures of corneal health. With an dilution effect exists. Moreover, as the genes will
acknowledgment of these limitations, various only be expressed by live cells, a means of moni-
groups have developed in vivo cell imaging tech- toring in  vivo viability is also achieved. Various
niques. Due to the optical clarity of the cornea, reporters utilized for assessing the health of cor-
fluorescent, bioluminescent, and other cell track- neal grafts include the use of bioluminescent
ing techniques that are detected optically are imaging of firefly luciferase and fluorescent imag-
ideal for monitoring corneal endothelial trans- ing of green fluorescent protein [3, 13, 46].
plants. In order to create a cell that is fluorescent
or bioluminescent, direct or indirect cell methods
can be employed. 29.3 Clinical Criteria of Corneal
Compared to indirect methods, direct cell Graft Rejection
labeling methods are more straightforward and
do not involve genetic modification of the cell Rejection criteria differ and may not be translat-
[40]. In direct cell labeling, an agent is introduced able from tissue to tissue. For example, skin graft
into or attached to cells prior to transplantation. rejection is an obvious event with marked tissue
Such labeling can be achieved with transfection sloughing and discoloration. Conversely, corneal
agents [40, 41], electroporation [42], and anti- rejection is commonly more subtle and character-
body labeling [43]. Following transplantation, ized largely by opacification of tissue without
the labeling agent can be imaged as a surrogate sloughing, although sloughing has been wit-
marker of the grafted cells. For optical imaging, nessed in small xenografts [32] . Short-term cor-
quantum dots and other fluorescent semiconduc- neal opacification has been observed in both mice
tor nanocrystals can provide a robust fluorescent and rats. Applying clinical criteria for graft rejec-
signal when either loaded into cells or attached to tion, this temporary opacification can ultimately
the surface with antibodies. Fluorochromes can be significant enough to qualify as complete
also be employed in a similar manner [40]. The rejection [47]. However, reversal of this presenta-
use of contrast agents visible on optical coher- tion has been observed after anywhere from
ence tomography, such as gold nano-rods, could many days to weeks later [32, 47].
29  Corneal Endothelial Cell Transplantation: Animal Models
Some researchers feel that a mild degree of
corneal opacification does not necessarily imply
a terminal event. In a seminal study, grafts that
were undergoing rejection as measured by
opaqueness did not necessarily lead to terminal
graft failure. For these experiments, grafts were
obtained from C57Bl6 mice that transgenically
expressed green fluorescent protein. These grafts
were then transplanted into a Balb/c mouse host.
In grafts that developed permanent opacification,
all donor endothelium were lost as no green fluo-
rescent protein was seen with confocal micros-
copy [48]. Interestingly, some grafts displayed
transient opacification between 10 and 21  days
but then ultimately recovered clarity. These grafts
retained donor endothelium despite a transient
opacification episode [48]. In this study and a
series of follow-up studies, it was found that a
minimum of 48 percent endothelial cell coverage
was present at all times in the mouse corneas that
eventually cleared [48, 49]. Taken together, in the
murine model, although corneal opacity is cer-
tainly a strong predictor of graft failure, it is
important to assess both the grade of corneal
opacity and wait a sufficient time after transplan-
tation (>8 weeks in the mouse) before diagnosing
irreversible graft rejection [48, 49].
In addition to frank opacification of corneal
transplants, ophthalmologists regard the presence
of epithelial rejection lines and the even more
ominous endothelial rejection, or Khodadoust,
lines as indicative of rejection [50]. Comparable
rejection episodes are seen in animal experiments.
In these studies, the development of rejection
lines was predictive of total graft rejection, espe-
cially when not adequately treated with immuno-
suppression [51, 52]. To emphasize this deleterious
outcome, in the presence of Khodadoust lines, all
sheep eventually developed completely scleral-
ized and opaque white cornea [32].
Although presence of corneal rejection lines
or corneal opacification is useful for assessing
graft rejection, for endothelial transplantation the
most reliable diagnostic tests involve direct eval-
uation of the endothelial monolayer. To this end,
specular microscopy and confocal ophthalmos-
copy can be utilized in the large animal models.
In mice and rats, microscopy of corneal flat
441
mounts has been the primary means of evalua-
tion. In the future, in vivo specular and confocal
microscopy will likely be widely available for
murine models. Indeed, researchers have already
begun using such techniques in smaller order ani-
mals such as a recent study utilizing specular
microscopy in the Chinese tree shrew [53], an
animal comparable in size to the rat. Moreover,
new techniques designed to adequately image
corneal endothelial cells in rats such as micro-­
optical coherence tomography are currently
being employed shortly after sacrifice in other-
wise intact rat eyes [54]. In the not too distant
future, it is likely similar techniques will be
employed for serial in vivo assessment of murine
endothelial cells.
29.4 I nsights in Corneal Graft
Rejection from Animal
Models
The brain, testes, and the eye are unique in terms
of transplant sites as they are immune-privileged.
Due to this fact, the anterior chamber has proven a
hospitable location for host-incompatible grafts
that would otherwise be rejected in other graft loca-
tions [55, 56]. This well-accepted immune privi-
lege has been coined anterior c­ hamber-­associated
immune deviation (ACAID). ACAID has been
exploited not only in the case of endothelial cell
transplantation, but has also been explored for allo-
geneic pancreatic islet cell transplantation in
baboons [57] with early clinical studies being per-
formed in humans by Perez’s group in an ongoing
study.
In addition to allotransplantation, the eye has
also proven as a potential site for xenogeneic
grafts. For example, when human endothelial
cells on a collagen carrier were transplanted into
rabbits, slit-lamp microscopy revealed no overt
signs of rejection such as fibrin deposition or
keratic precipitates for the observed period of 1
month (Fig. 29.2) [33]. It remains unclear to what
degree using collagen carriers as opposed to
human stroma helped prevent rejection in this
instance. Endothelial transplantation in this set-
ting is unique as the xenogeneic transplanted
442 B. P. Barnett and A. S. Jun

a
(µm)
1400

1200
Corneal thickness

1000

800 *
†
600
† † † †
400

200

0
preop 0.5 1 3 7 14 21 28
Time (days)
b c

Fig. 29.2  Change in central corneal thickness (a) and cor-
neal appearance through slit lamp (b, c) after transplantation
of HCEC seeded on collagen carrier DSAEK grafts. (a)
There was a significant difference in rabbit corneal thickness
between groups (*p  <  0.05 and †p  <  0.01) that received
DSAEK grafts of HCECs on collagen carrier (filled circles)
or carrier alone (open circles) after stripping of Descemet’s
membrane. Twenty-eight days after transplantation, slit-
lamp photography of the DSAEK group revealed a thin cor-
nea without stromal edema (b), while rabbits that received
transplantation of collagen carrier alone had severe corneal
edema (c). (Used with permission [33])

cells do not directly contact host cells, but instead
face the anterior chamber.
Some evidence exists in mice that partial-­
thickness grafts are better tolerated than full-­
thickness grafts in terms of rejection. Hayashi
et  al. demonstrated in the mouse that chimeric
corneal endothelial cell allografts produced from
cultured allogeneic corneal endothelial cells
showed little in the way of rejection [58].
Specifically, no mixed lymphocyte reaction or
delayed hypersensitivity to assess rejection was
detected. In contrast, full-thickness corneal
allografts exhibited a high rate of rejection.
Conversely, when allogeneic epithelium and
29  Corneal Endothelial Cell Transplantation: Animal Models
stroma from BALB/c mice were reconstituted
with immortalized C3H/HeN mouse corneal
endothelial cells and transplanted into C3H/HeN
recipients, a similar rate of rejection occurred as
compared to full-thickness allografts. Taken
together, Hayashi’s group suggested these results
demonstrate that endothelial cells alone neither
help promote tolerance nor induce allograft rejec-
tion. Instead, they argued it is the non-endothelial
component of the full-thickness allografts that
induce rejection [58].
Numerous groups have proposed mechanisms
of corneal endothelial allograft acceptance. This
tolerance has been attributed to immunologic
ignorance of draining lymph node T-inducer
priming cells and active immunosuppression
with T-cell regulatory induction and T-cell anergy
[59–63]. Mice exhibiting long-term acceptance
of full-thickness corneal transplantation reveal
how active immunosuppression is induced [2, 47,
59]. However, active immunosuppression failed
to be induced in corneal endothelial cell
allografts, as demonstrated by adoptive transfer
of splenocytes from mice that had accepted chi-
meric corneal endothelial cell allografts and
regraft challenge in corneal endothelial cell
allograft-accepted mice [2, 47, 59].
ACAID induction was demonstrated by
Niederkorn’s group and Amano’s group after the
injection of corneal endothelial cells into the
anterior chamber [58, 64]. ACAID induction was
not demonstrated however after corneal endothe-
lial cell allograft transplantation [58]. These
results indicate that corneal endothelial cell graft
survival without rejection was not associated
with immunosuppression or T-cell anergy.
Immunologic ignorance appears to be the pri-
mary reason for rejection-free acceptance of chi-
meric corneal endothelial cell allografts [58].
29.5 Choice of Animal Model
In general ethics dictates the use of the animal of
lowest order that will achieve the research aims.
Primary applications of animal models are to
assess basic processes of and potential therapeu-
tics for graft rejection and to develop novel trans-
443
plant approaches including cellular therapeutics
and bioengineered corneal grafts. In the latter
case, selection of the animal model that most
closely approximates human anatomy and physi-
ology is desired to easily facilitate the transfer of
developments from animal model to humans
when creating corneal endothelium. The primary
goal of using animal models is to assess the pro-
cess and potential therapeutics for graft rejection,
in addition to exploring and evaluating cellular
therapeutics as well as bioengineered corneal
grafts. Particular attention is necessary to evalu-
ate the multiple factors impacting the rejection of
corneal grafts in humans. Despite not being HLA
matched, most human corneal grafts demonstrate
a substantial acceptance rate1 year out from
grafting [65]. Principles to optimize corneal
transplantation in cat, rabbit, and the outbreed
sheep model have proven translatable to optimiz-
ing transplant in humans [32, 65, 66].
The potential for expanding focused immuno-
logical studies related to rejection of corneal grafts
has been attainable with the use of the rat model
[29] and particularly the use of the mouse model
[32]. Due to their extensive use in immunological
research, much is known about these processes in
rodents. Moreover, numerous strains are available
including both inbred and congenic strains. In
recent years, numerous gene knockout strains
including conditionally deleted strains as well as
mice that transgenically express cell reporters
have been developed. Using these various models,
researchers can tailor the rodent strain chosen to
the specific research question at hand.
However, clinically both the mouse model and
the rat model exhibit important anatomic vari-
ances when compared to human grafts. Due to
the small size of rodents, advanced surgical skill
is necessary to avoid the mouse or rat being sub-
jected to procedural trauma that thereby induces
the innate immune response. Regardless, when
looking at the models, particularly the mouse
model, both have provided a wealth of under-
standing of the pathogenesis of immune-related
rejection and specifically the value of indirect
allorecognition [60, 67].
For ethical and cost considerations, primate
transplantation is generally reserved for treat-
444
ments that have been vetted in animal models of
lower order. Larger animals provide not only ease
of surgical intervention but also the ability to
employ clinical techniques familiar to the oph-
thalmologist, such as slit-lamp biomicroscopy
and confocal microscopy, in order to assess for
graft rejection and investigate changes in
endothelium.
29.6 Types of Animal Models
29.6.1 Rodent
Primarily due to the size of the eyes, small
rodent animals such as the mouse and rat are
not the preferred models for preclinical studies,
even though they have been utilized to study
corneal endothelium tissue engineering [3, 4,
68]. The rabbit model has been effective for
functional in vivo assessments due to the acces-
sibility, cost, small size, and ease of handling.
However, controlled preclinical studies on
endothelial tissue engineering are impeded by
the capacity of the rabbit corneal endothelium
to proliferate extensively in vivo. For this rea-
son, rodent models are increasingly being
employed.
Corneal transplantation in rodents has proven
invaluable in the understanding of xenorejection
and allorejection. In a study by Amano’s team,
human corneal endothelial cells were seeded
onto a carrier to make a full-thickness graft, and
penetrating keratoplasty was performed in immu-
nodeficient nude rats [68]. In the setting, the
xenogeneic graft was well tolerated with few
signs of rejection. As opposed to nude rats, in the
work by She et  al. of murine corneal grafts, a
robust immune response was seen. In this model,
a 2  mm donor graft is transferred to a 1.5  mm
host bed and secured with 8–12 sutures [30].
Several groups have adopted this model with an
aim of understanding the immune mechanisms at
play during graft rejection [30, 47, 69–75]. The
variety of genetically manipulated strains, as well
as the plethora of scientific reagents available
specifically for mice, have proven quite helpful in
this aim.
B. P. Barnett and A. S. Jun
The mouse model, like other species, demon-
strates rejection through corneal opacification.
On the other hand, the rat model presents addi-
tional signs like corneal edema, corneal vascular-
ization, and inflammatory cell invasion.
Comparing a grading system incorporating these
other signs of rejection against the use of the
degree of opacification alone has indicated that
the degree of opacification is correlated with the
extent of rejection [30, 47, 69–75]. Corneal
opacification alone is inadequate to identify non-
reversible rejection. Temporary corneal opacifi-
cation secondary to the extent of procedural
trauma often occurs in the both the rat and mouse
model within days of graft placement. Instead the
degree of opacification and the length of time
opacification present are necessary in order to
determine nonreversible rejection.
A vast amount of information on the process
of immune graft rejection has been obtained by
studying the mouse model. Nevertheless, the dis-
similarity of the mouse immune system to that of
the human, as well as the inherent size discrep-
ancy between the species, has made the use of
larger animal models essential.
29.6.2 Rabbit
The rabbit model was the earliest model to be
used in a standardized study of corneal grafts and
secondary lymphoid tissue [76]. Beginning in the
1930s, corneal grafts were effectively placed in
rabbits [66, 77] with subsequent reports presented
in literature [78, 79]. In addition to penetrating
keratoplasty, rabbits were also employed for pos-
terior lamellar keratoplasty [80], limbal epithelial
grafts [81], and limbal stem cell grafts [82, 83].
Toward the end of the 1970s, the first tissue-­
engineered cornea was constructed by
Gospodarowicz and Greenburg [14, 15]. They
seeded cultured bovine corneal endothelial cells
onto a cell carrier and transplanted into rabbits
after endothelium removal [14, 15]. In the fol-
lowing years, Amano’s team pushed the field fur-
ther toward clinical application when they
utilized cultured human corneal endothelial cells
on a carrier to make full-thickness grafts [84, 85].
29  Corneal Endothelial Cell Transplantation: Animal Models
In 2004, using collagen carriers for human cor-
neal endothelial cells, Mimura et  al. [33] per-
formed DSAEK in the rabbit (Fig. 29.3a–c, e–g).
They followed this study by seeding cultured
human corneal endothelial cell suspensions onto
human stroma and again performed DSAEK in
the rabbit [11].
Additional groups have also explored cultured
corneal endothelial cells to create grafts that were
then transplanted with DSAEK in a rabbit model.
Using human endothelial cells seeded onto a
poly(N-isopropylacrylamide) and gelatin-based
carrier, Lai et  al. demonstrated uniform corneal
transparency within 2  weeks of DSAEK in the
rabbit [10]. Similarly, Vasquez et  al. employed
silk fibroin films to culture both rabbit and human
corneal endothelial cells. Utilizing a DMEK-like
procedure, artificial silk fibroin endothelial grafts
were transplanted in rabbits [12]. At the 6-week
mark following transplantation of the grafts in
the rabbit model, both normal thickness and
transparency of the cornea returned. Optical
coherence tomography of the cornea with the silk
fibroin endothelial graft compared to the unaf-
fected contralateral eye demonstrated compara-
ble thickness of the cornea. The silk fibroin graft
was found to be well incorporated into the host
tissue and displayed proper intercellular junction
and cellular pump function [12]. Moreover, his-
tological analysis revealed the silk fibroin graft
was attached and well integrated with host tissue
with little inflammatory reaction. Additionally,
rabbit corneal endothelial cells displayed appro-
priate monolayer morphology and further
expressed ZO-1 and Na+-K+ ATPase as deter-
mined by immunohistochemistry [12].
Another study undertaken in the rabbit
involved making a 7 mm full-thickness trephina-
tion in the rabbit host [13]. To the excised button,
Ishino et  al. then performed a DSAEK-like
lamellar procedure with a graft of human corneal
endothelial cells on an amniotic membrane car-
rier. The corneal button was then sewn back into
the host. With this technique, graft transparency
was maintained for 1 week.
Devitalization is a novel approach as an option
other than use of fresh native tissue. Devitalization
functions to remove living cells from the carrier
445
to prevent the endothelial, stromal, or epithelial
cells of the carrier from contaminating the graft.
This process of native cell destruction is antici-
pated to decrease carrier immunogenicity [86,
87]. Preservation of corneal transparency was
recognized by Amano et al. when liquid nitrogen
gas was employed to decellularize the corneal
stroma [88]. Engelman et  al. discovered that
denuding Descemet’s membrane without com-
promising cell adhesion could be accomplished
in a single freeze-thaw cycle [89]. Utilizing
ammonium hydroxide chemical debridement
yielded comparable results [17, 18] where well-­
organized native collagen fibers, essential for
corneal transparency, were preserved. These
examples of devitalized stromal carriers provided
desirable tissue-engineered endothelium adher-
ence and were favorable for the recipient after
full-thickness transplantation in the rabbit.
In lieu of full-thickness or even partial-­
thickness transplantation, some groups have
championed the use of cellular therapeutics con-
sisting of single cells or clusters of cells injected
into the anterior chamber (Fig. 29.3d, h). Prior to
anterior chamber injection, various techniques
have been utilized to remove the diseased
endothelium from the host. Cryoablation has
­
been explored, but this procedure has demon-
strated irreversible damage to keratocytes [90].
Alternative techniques include Descemet’s mem-
brane stripping, enabling cells injected into the
anterior chamber to directly contact the host
stroma [68]. With this technique, proper head
positioning for a sufficient time is required in
order to allow the transplanted cells to settle and
adhere on the posterior corneal surface and to
minimize adherence to the trabecular meshwork,
iris, or lens.
To help ensure proper localization of trans-
planted cells to the posterior cornea, Mimura
et al. [91] and Patel et al. [92] have recommended
loading iron particles or superparamagnetic
microspheres into cells prior to transplantation.
These techniques have been previously explored
for different applications, namely, labeling cells
with an iron-based MRI contrast agent [41, 42].
Following injection into the anterior chamber,
these iron-loaded cells are attracted to the poste-
446 B. P. Barnett and A. S. Jun

a b

c d

e f

g h

Fig. 29.3  Slit-lamp (a–d) and histologic (e–h) photo-
graphs of corneas posttransplantation of bioengineered
HCEC grafts or injected into the anterior chamber. (a, e)
Slit-lamp photography and histology of rabbit corneas
after full-­thickness transplantation of PK grafts generated
from cultured HCECs on rabbit corneal stroma denuded
of endothelium. (b, f) Slit-lamp photography and histol-
ogy of a rat cornea after penetrating keratoplasty with a
graft comprised of HCECs seeded onto rat corneal stroma
denuded of endothelium. (c, g) Slit-lamp photography and
histology of rabbit cornea post DSAEK with a graft com-
prised of cultured HCECs seeded onto human amniotic
membrane. (d, h) Slit-­lamp photography and histology of
rabbit cornea after in  vivo corneal endothelial stripping
followed by cultured HCECs injected into the anterior
chamber. Scale bars = 50 μm. (Used with permission [33])
29  Corneal Endothelial Cell Transplantation: Animal Models 447

rior corneal surface by an external magnet. Using the graft. A similar exclusion of immune ele-
this approach, complete resurfacing of the poste- ments is also achieved in cell microencapsulation
rior cornea, both in  vitro [92] and in  vivo, has for allotransplantation and xenotransplantation
been demonstrated in the rabbit model [92]. [37]. Due to its biodegradable nature, with time
However, safety factors of long-term iron require the collagen disc was broken down thereby pro-
further study. viding direct contact of the transplanted endothe-
As opposed to single cell suspensions, in a lial cells to the anterior chamber in the proper
serious of work from Amano et  al., endothelial apical-basal orientation [10].
cells were coaxed to form spheroid bodies in cul- Various factors have been postulated to con-
ture [5, 6, 93]. These spheroid bodies were then tribute to graft rejection in the rabbit. Not unlike
injected into the anterior chamber [93]. Prone human transplants, the presence of vasculariza-
position postsurgery was necessary for these tion at the host-graft junction at the time of trans-
spheroid bodies to attach to the posterior corneal plant is associated with increased rates of
surface. Subsequent restoration of endothelial rejection [95]. The rate of graft rejection has also
function as noted by resolution of corneal edema been shown to increase when a skin graft from
was observed [93]. the donor was transplanted to the host following
The rabbit model has also been explored for prior corneal transplantation from the same donor
transplantation of human corneal endothelial [52].
cells on electron beam-grafted poly(N-­ Rabbits are known to have rapid and extensive
isopropylacrylamide)-based cell culture carriers. clotting of the aqueous when the anterior cham-
Following stripping of endothelium and trans- ber is entered [52]. Although possibly under-­
plant of these grafts, corneal thickness signifi- identified, a similar occurrence has not been
cantly decreased, as compared to control groups reported in other species. Blocking this anterior
[10]. In this study, the researchers encountered chamber fibrin response with intracameral hepa-
frequent tearing of the graft during transfer due to rin during surgery and topical heparin postsur-
its fragile nature [10]. In order to overcome this gery has proven effective in decreasing the rate of
fragility, a circular poly(vinylidene fluoride) rabbit allograft rejection [76].
membrane was utilized to stabilize the cell sheet In addition to identifying the now eponymous
during transfer. The use of this poly(vinylidene endothelial rejection lines, Khodadoust uncov-
fluoride) membrane protected the graft during ered a variety of factors that influence graft rejec-
transfer by enabling adherence to the posterior tion in the rabbit. He demonstrated that the size
corneal stroma in the proper orientation [10]. of the graft and the location of the graft contrib-
Use of a gelatin disc to facilitate transfer of an uted to rejection. For example, with 7 mm grafts
endothelial graft into a rabbit after Descemet in the rabbit, widespread rejection has been
stripping has been described [10]. Importantly observed [76]. However, smaller grafts of
collagen was not used as a carrier containing 4–5 mm did not appear to induce rejection with
attached cells. Instead these collagen discs faced grafts maintaining continued transparency [52].
the anterior chamber and sandwiched the endo- In this same study, he also found that grafts
thelial graft to the recipient stroma. The porous placed closer to the limbus demonstrated higher
nature of the gelatin disc enabled transfer of rejection rates, especially when vascularization
nutrients from the aqueous humor to the graft and occurred [52].
allowed cellular waste products from the endo- In the rabbit model, Khodadoust also demon-
thelial cell sheet to escape into the anterior cham- strated the use of heparin to prevent fibrin forma-
ber [9, 94]. tion and early suture removal to prevent
Although not directly investigated, it is also neovascularization impacted rejection rates.
possible that this semi-porous collagen disc also Although lamellar grafts had a better prognosis
prevented interaction of immune cells circulating than penetrating grafts, rejection was commonly
in the anterior chamber to directly interact with seen in both when sutures were left in place long
448
enough to induce vascularization [34]. With care-
ful surgical technique to minimize trauma, appro-
priate suture removal, and the use of topical
heparin to prevent clotting during the procedure,
Khodadoust achieved successful grafts in 95% of
7 mm grafts and 100% of 5 mm grafts [66].
Although not fully understood, it has been
proposed that neovascularization of the graft
leads to rejection as the efferent arm of the
immune response can be carried directly into the
graft [50]. An alternate or potentially simultane-
ous mechanism of graft rejection involves the
increased afferent cell traffic to the draining
lymph node when graft neovascularization occurs
thereby further driving vasculogenesis and lym-
phangiogenesis [96]. Regardless of the mecha-
nism, early suture removal has proven to help
prevent neovascularization. In regard to suture
choice, various groups have used interrupted and
continuous sutures of varying sizes (8-0 to 10-0)
[97]. Certain groups have suggested that continu-
ous sutures pose less of a nidus for neovascular-
ization [97]. Most studies have employed
polyglactin or another dissolvable suture.
Nevertheless, as many researchers have seen vas-
cularization crossing the host-graft junction in as
little as 9–10  days [97], the use of nylon or
another non-dissolvable suture with early suture
removal may be advantageous.
In the rabbit, typically rejection is first marked
by neovascularization of the graft [50]. In a series
or early experiments, double exchange of corneal
grafts between pairs of rabbits with selective
removal of one or more layers of cornea was
undertaken [51]. This enabled the regrafting of
donor-host chimeric grafts to the original donor.
In these experiments, it was the epithelium that
was rejected first [51]. Accordingly, the follow-
ing vascularization epithelial rejection lines are
commonly seen during rejection. Subsequent to
the development of an epithelial rejection line, an
endothelial rejection line develops in the rabbit.
This Khodadoust line consists of a combination
of mononuclear cells and dying endothelial cells
[51]. A terminal event in rabbit allograft rejection
is the development of a retrocorneal membrane.
This membrane is formed when the failing endo-
thelium is engulfed by inflammatory cells and
spindle-shaped fibroblastoid cells [98, 99].
B. P. Barnett and A. S. Jun
29.6.3 Pig
The pig model has been of subject of interest in
ophthalmic research due to its anatomic and
physiologic parallels to the human eye.
Additionally, the affordable cost and easy acces-
sibility of pig tissue further support interest in
this model. The cornea of an adult pig has a hori-
zontal diameter that typically measures from 14.5
to 16.5 mm [100, 101] with a central pachymetry
of approximately 665 um [102, 103]. The endo-
thelial cells of pigs exhibit minor proliferative
ability [104] with endothelial cell counts averag-
ing approximately 4000 cell/mm2 [105]. Various
stem cell [106] and other corneal endothelial cell
culture studies [46] have utilized the pig cornea,
and the majority of the development of refractive
corneal procedures have been developed in the
pig model [107–109]. Despite the wide interest in
and popularity of the pig model, this model is not
commonly utilized for full-thickness or partial-­
thickness corneal transplantation studies.
29.6.4 Cat
Although less common than previously described
animal models, corneal transplantation has been
explored in the cat. Various groups have utilized
cat corneal endothelium to create tissue-­
engineered grafts [17, 18, 110]. Cats have also
been used as the recipient of both xenogeneic and
allogeneic endothelial transplants [14, 15, 17–19,
100, 110, 111]. The average adult cat has a cor-
neal diameter that ranges from 15.5 to 18  mm
[100] with central pachymetries ranging from
545 to 650  μm [112–116]. Not unlike humans,
cat endothelial cells generally do not replicate
[112, 117, 118]. On average cats have endothelial
cell counts approximating 2300–2900 cells/mm2
[112–116]. In contrast to the pig model, posterior
lamellar transplant [119] and penetrating kerato-
plasty [19, 31, 120–123] have been successfully
achieved.
Brunette et  al. [124] favored the cat model
over the pig model for full-thickness corneal
transplantation, as the desired surgical technique
was easier to accomplish. Additionally, Brunette’s
groups have performed a series of studies to
29  Corneal Endothelial Cell Transplantation: Animal Models 449

determine the minimal endothelial biopsy size heparin to prevent anterior chamber clotting [32].
necessary to construct a full corneal endothelium. Allograft rejection occurred on average at 20 days
In the cat model, a 5 mm biopsy (representative of survival time with related significant vascular-
of about 8% of the cat’s endothelial eye surface) ization [32]. The presence of fibrin was the first
was necessary to reconstruct a full corneal endo- reported sign of clinical rejection followed by
thelium [125]. Brunette’s group also revealed keratic precipitates [67].
that the functional endothelium of grafted cor-
neas was minimally impacted by injected corneal
endothelial cells in the cat model. With or with- 29.6.6 Monkey
out central endothelial scraping, feline corneal
endothelial cells supplied with Y-27632 and For obvious reasons, the ideal research model for
marked with 3,30-dioctadecyl-5,50(4-­ human application is the primate. There have
sulfophenyl)oxacarbocyanine were injected in been reports of engineered corneal endothelium
cats. Results revealed that the healthiest endothe- in monkeys [20, 21, 126] as well as endothelial
lium was achieved with injection of Y-27632 transplants of both human and pig xenografts
without corneal endothelial cell scraping [124]. [22–24] and monkey allografts and homografts
Use of the cat model has been instrumental in [20, 21]. The corneal diameter in the full-grown
assessing endothelial cell counts to evaluate graft rhesus monkey approximates 10.6  mm with a
survival and graft rejection. Evaluation of the pachymetry of 470 μm at the center of the cornea
allograft 1 month post procedure revealed an [127]. Although described endothelial cell counts
average of 30% cell loss over the entire donor approximating 4000 cells/mm2 [128, 129] exceed
area. Conversely, homograft endothelial cell loss those found in humans, replication in animal
was 15% [31]. Other cat model experiments con- models has not been demonstrated [24, 117, 130].
centrated on removal of the endothelium from the The availability and cost of primates in both
donor before grafting, and no endothelium regen- North America and Europe are limiting factors in
eration was observed in the host [31]. Further sup- the use of this model.
porting these outcomes, corneal homograft cell In Wuzhishan pigs, the survival of pig-rhesus
counts decreased by about one-half over a course corneal xenografts was explored after donor bone
of 6 weeks to 9 months after the graft was placed marrow transplantation. The pigs were utilized as
[98] with a notable polymegathism increase. A donors, while the rhesus monkeys received corneal
rationale for this progressive cell loss following xenotransplantation. The study group was com-
homografting has not been identified [50]. prised of 12 rhesus monkeys separated into two dif-
ferent groups. The treatment group was given
cyclophosphamide intravenously with subsequent
29.6.5 Sheep autologous bone marrow transplantation. The con-
trol group received cyclophosphamide intrave-
Although sheep models of endothelial cell trans- nously alone. Slit-lamp microscopy was used to
plantation are lacking, sheep are the latest large evaluate the xenografts. The recipient’s immuno-
animal model to be utilized for research focusing logical status was analyzed after transplantation by
on corneal graft rejection [32]. In a Morino out- assessing for a mixed lymphocyte reaction, the
bred strain, 12  mm grafts were transferred presence of serum immunoglobulin and comple-
between animals. Fixation of the graft was ment, as well as chimerism formation [131].
obtained with 9-0 polyglactin sutures. Of note, The graft survival time for the bone marrow
the use of smaller 10-0 polyglactin sutures fre- transplant group was 36.0 ± 4.7 days and 17.1 ±
quently broke in the sheep model and thus was 3.2 days for the control group. The bone mar-
abandoned. In the sheep model, a weaker inflam- row transplant arm revealed decreased serum
matory response was observed compared to other immunoglobulin and complement levels as well
animal models thereby negating the need for as decreased mixed lymphocyte reaction.
450
Additionally, the corneal xenografts in the bone
marrow transplant arm demonstrated negligible
inflammatory cell infiltration and no eosinophil
infiltration. These results indicate that autolo-
gous bone marrow transplantation may sup-
press immune reaction thereby prolonging
corneal xenograft survival and chimerism for-
mation [131].
Another study in primates undertaken by
Koizumi et al. involved cultured corneal endothe-
lial cells seeded onto a type I collagen carrier.
They transplanted these grafts through DSAEK
in monkeys [20, 21]. Although the transplanted
sheets detached from the host cornea within 1
week post grafting, the corneas recovered trans-
parency within 6 months posttransplantation.
Although not directly addressed by Koizumi’s
group, these results have three potential explana-
tions. The first possibility is endothelial cells
separated from the detached graft and repopu-
lated the cornea. Another more likely possibility
is the native host peripheral endothelial cells
repopulated the corneal endothelium and the
graft itself had no impact on endothelial recovery.
A final possibility, ripe for exploration, is that the
transplanted graft provided cytokines or other
factors that coaxed the native endothelial cells
into division, thereby repopulating the cornea.
29.7 Conclusion
Endothelial transplantation and more broadly
corneal transplantation have been explored in a
variety of animal models. In general, the various
animal models can be divided into small inbred
animal models and large outbred models. Smaller
animal models have the benefit of being rela-
tively inexpensive and easier to maintain. In addi-
tion, numerous inbred strains are available with
various mutations and novel reporters transgeni-
cally expressed. Larger animal models enable the
use of surgical techniques employed clinically
with humans and moreover enable the use of
grafts that are closer in size to human grafts. The
choice of animal model is largely guided by the
experimental aims, and ethics dictates the use of
the animal of lowest order that will achieve these
B. P. Barnett and A. S. Jun
aims. To date, the various animal models have
proven complementary in providing researchers
and clinicians with a means of developing new
surgical techniques as well as assessing the func-
tion of various corneal grafts and novel cellular
therapeutics. Moreover, the various animal mod-
els have proven invaluable in providing insight
into the immunological processes that take place
during corneal graft rejection. The future remains
bright for endothelial transplantation. We cer-
tainly are indebted to the clinical and scientific
pioneers and various animals for their contribu-
tions to the field of corneal endothelial cell
transplantation.
Compliance Statements Brad P.  Barnett and Albert
S. Jun declare that they have no conflict of interest.
No human studies were carried out by the authors for
this article.
No animal studies were carried out by the authors for
this article.
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 Cell Based Therapy for Corneal
Endothelial Regeneration
30
Noriko Koizumi and Naoki Okumura

30.1 Introduction resolved. One alternative to corneal endothelial

keratoplasty is regenerative medicine, which may
The corneal endothelium plays an essential role represent the next-generation therapy for treating
in the maintenance of corneal transparency corneal endothelial dysfunction. In that regard,
through its barrier and pump functions. However, current research efforts are aimed at developing
the proliferative ability of human corneal endo- cell-based therapies for corneal endothelial dys-
thelial cells is severely limited in vivo, and severe function. One promising approach involves cell-­
cell loss due to dystrophy, trauma, or surgical sheet transplantation and cell-injection therapy
intervention often causes irreversible corneal using cultivated corneal endothelial cells
endothelial dysfunction [1]. Traditionally, a full-­ (Fig. 30.1).
thickness corneal transplantation has been the
only therapeutic choice for the restoration of
clear vision. The recent evolution of surgical pro- 30.2 Corneal Endothelial Cell
cedures for corneal endothelial keratoplasty, such Biology
as such as Descemet’s stripping endothelial kera-
toplasty (DSEK) or Descemet’s membrane endo- In vivo human corneal endothelial cells are
thelial keratoplasty (DMEK), has enabled less thought to be arrested at the G1 phase of the cell
invasive treatment of corneal endothelial dys- cycle [2, 3], a phase that is negatively regulated
function with better visual recovery. Despite the by the CIP/KIP family (p21 and p27), the INK4
good visual recovery obtained with corneal endo- family (p15, p16, and p19), and the p53 family of
thelial keratoplasty, the severe shortage of donor proteins (p53, Tap63) [4–6]. Several other fac-
grafts is a serious worldwide problem for per- tors, such as transforming growth factor beta
forming this operation. Additional problems (TGF-β) in the aqueous humor, the contact inhi-
associated with corneal transplantation, such as bition of cells [7], and stress-induced premature
allograft rejection, primary graft failure, and senescence [8, 9] have also been reported to
gradual decrease of cell density, have yet to be inhibit the in vivo proliferation of corneal endo-
thelial cells. Although the in  vivo existence of
stem/progenitor cells has yet to be proven, these
N. Koizumi (*) · N. Okumura endothelial cells are known to retain their prolif-
Department of Biomedical Engineering, Faculty of erative ability in vitro [10, 11]. One suggestion is
Life and Medical Sciences, Doshisha University,
Kyotanabe, Kyoto, Japan that peripheral corneal endothelial cells in cell-­
e-mail: nkoizumi@mail.doshisha.ac.jp culture conditions have a higher proliferative

© Springer Nature Switzerland AG 2019 455

J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_30
456 N. Koizumi and N. Okumura

Fig. 30.1  The concept Sheet transplantation

of cell based therapy as
a future therapeutic
modality for corneal
endothelial disease Expansion culture of human
corneal endothelial cells

Donor cornea
Cell-injection therapy

Cell-suspension of
cultivated corneal
endothelial cells

ability than do central corneal endothelial cells;
another is that a high density of stem/progenitor
cells of corneal endothelium exists in the periph-
eral region [12]. These cell clusters located in the
extreme peripheral region are considered poten-
tial stem cell niches [13]. Other reported findings
also appear to indicate that human corneal-­
endothelial stem/progenitor cells are regulated by
the leucine-rich repeat-containing G protein-­
coupled receptor 5 (LGR5) via the Hedgehog and
Wnt signaling pathways [14].
30.3 C
 orneal Endothelial Cell
Culture for Clinical
Application
18]. The establishment of optimal conditions for
the cultivation of human corneal endothelial cells
is critical for the clinical application of a cell
based therapy for corneal regeneration. Recently,
the usefulness of a TGF-β receptor inhibitor [17]
and p38 MAP kinase inhibitor [19] in the cell
culture medium was reported in terms of prevent-
ing the EMT and cellular senescence. Other tech-
niques, including the use of Rho-associated
protein kinase (ROCK) inhibitor [20–22] or the
use of a culture substrate with a specific extracel-
lular matrix similar to the Descemet’s membrane,
were also reported to promote the cell prolifera-
tion and to support human corneal endothelial
cell cultures that show high cell density [23].

The in  vivo and in  vitro proliferative ability of
human corneal endothelial cells is, in general,
severely controlled, as is observed in monkey
corneal endothelial cells [15, 16]. Thus, a com-
plete understanding of the biological characteris-
tics of cultivated human corneal endothelial cells
is essential for developing translational research
for cell based therapy. However, most researchers
have encountered difficulties in consistently
expanding a clinically applicable quantity and
quality of cultivated human corneal endothelial
cells with healthy biological characteristics,
because human corneal endothelial cells readily
enter into an epithelial mesenchymal transition
(EMT) to develop a fibroblastic phenotype [17,
30.4 Cultivated Corneal
Endothelial Cell Sheet
Transplantation
A cultivated human corneal endothelial cell
sheet, with [24–29] or without [30] carrier mate-
rials, has now been created, and it demonstrates
in vivo functionality in both rabbit and monkey
models. We previously reported the temporary
attachment of a cultivated monkey corneal endo-
thelial cell sheet (on type 1 collagen) onto the
Descemet’s membrane. This sheet slipped off
into the anterior chamber and, surprisingly,
resulted in the recovery of corneal transparency
[25, 26]. This quite unexpected finding led us to
30  Cell Based Therapy for Corneal Endothelial Regeneration
speculate that cultivated primate corneal endo-
thelial cells migrate and proliferate to some
extent in vivo, and a subsequent series of experi-
ments provided us with a potential new concept
for the treatment of corneal endothelial dysfunc-
tion. We postulated that, rather than transplanting
a cultivated corneal endothelial sheet, we could
transplant individual endothelial cells that show a
renewed ability to proliferate in  vivo.
Interestingly, a similar event was reported in clin-
ical practice with human corneal endothelial cells
following a DMEK procedure [31] or the sponta-
neous clearing of the cornea after Descemet’s
rhexis without endothelial grafting [32].
30.5 Cell-Injection Therapy
When compared to transplantation of cultivated
corneal endothelial sheets, the direct injection of
Fig. 30.2  Surgical procedure of cell-injection therapy. (1)
Patients’ corneal endothelial cells were gently scraped off
with a silicone needle, (2) Co-injection of cultured corneal
endothelial cells and a ROCK inhibitor into the anterior
457
cultivated corneal endothelial cells into the ante-
rior chamber might provide several advantages,
including a better optical quality of the postop-
erative cornea, less direct manipulation of the
cultivated cells, a minimally invasive surgery,
and the potential for a mass application from one
donor cornea. Attempts to develop an effective
method to deliver cultivated corneal endothelial
cells to the posterior cornea have included the use
of magnetic attachment of iron-powder [33] or
superparamagnetic microspheres incorporated
into cultivated corneal endothelial cells [34] in a
rabbit transplantation model and in an organ cul-
ture model of the human eye.
The recent discovery of the effects of ROCK
inhibitor on human corneal endothelial cells has
also shed some light on the development of a new
concept for treatments of corneal endothelial dys-
function, such as eye drop treatments and the cell-
injection therapy [35–37] (Fig. 30.2). Preclinical
chamber, (3) Patient in the face-down position, to allow the
corneal endothelial cells to sink down to the anterior cham-
ber side of the cornea, (4) Regeneration of the corneal endo-
thelium by the injected cultured corneal endothelial cells
458
experiments involving rabbits and monkeys
showed that a cell-injection therapy, combined
with the use of a ROCK inhibitor and followed by
3 h in a face-down position, promoted cultivated
corneal endothelial cell adhesion onto the poste-
rior cornea, ultimately resulting in the recovery of
corneal transparency in both animals [38]. In fact,
the monkey eyes receiving allogeneic cultivated
monkey corneal endothelial cell injection with
ROCK inhibitor exhibited complete corneal trans-
parency post injection, and the transparency and
normal corneal thickness were maintained
throughout a one-year observation period. The
safety and efficacy of cell-injection therapy was
also confirmed by the transplantation of cultivated
human corneal endothelial cells into a monkey
corneal endothelial dysfunction model in preclini-
cal research [39].
Based on the data obtained from the estab-
lished culture protocol for human corneal endo-
thelial cells and from animal experiments, a
first-in-man clinical trial of cell-injection therapy
was initiated in December 2013 under official
approval from the Regenerative Medicine
Committee of the Japanese government [40, 41].
The initial results of this cell-injection therapy
appear to be promising for the treatment of
patients with corneal endothelial dysfunction
Fig. 30.3  Cell-injection therapy for bullous keratopathy
patient. Slit lamp microscopy images of the first patient, a
Japanese female with corneal endothelial decompensation
induced by argon laser iridotomy (left). Preoperative
visual acuity was 0.04 due to the edema in the corneal
epithelium and stroma. After mechanical removal of an
8  mm diameter section of the corneal endothelium, cul-
N. Koizumi and N. Okumura
caused by Fuchs endothelial corneal dystrophy,
pseudophakic bullous keratopathy, and the argon-
laser iridotomy-­ induced bullous keratopathy,
which is a quite common cause of corneal endo-
thelial dysfunction in Japan (Fig. 30.3). Notably,
the replacement of corneal endothelial cells of
failed grafts with cultivated corneal endothelial
cells was also effective in recovering the clarity
of the full-­thickness graft (Fig. 30.4). Similar to
the observation in animal experiments, a beauti-
ful monolayer of attached cells was observed on
the Descemet’s membrane when viewing the
postoperative patient’s eyes with specular micros-
copy (Fig. 30.5). Clinically, reasonable recovery
of corneal endothelial functions and vision were
observed in all cases, without any postoperative
adverse reactions such as elevated intraocular
pressure, uveitis, or allograft rejection.
30.6 Toward the Product
Development of Cell-­
Injection Therapy
Cell based therapy for corneal endothelial dys-
functions is a “long-cherished dream” of cornea
surgeons, as well as researchers. Recently devel-
oped cell-injection therapies will deliver many
tured human corneal endothelial cells, together with a
ROCK inhibitor, were injected into the anterior chamber.
The postoperative visual acuity recovered to 1.0, together
with the associated recovery of corneal transparency
(right). (Reproduced from Okumura et  al. J
Ophthalmology, 2017. CC BY 4.0 https://creativecom-
mons.org/licenses/by/4.0/)
30  Cell Based Therapy for Corneal Endothelial Regeneration 459

90° 90°
° 60 OD ° 60 OD
9mm 120 ° 9mm 120 °
0°

0°
15

15
30

30
°

°
180°

180°

0°
0°
0°

0°
33

33
21

21
0°

0°

24 0° 24 0°
0° 30 0° 30
T 270° N T 270° N

Fig. 30.4  Endothelial replacement with cell-injection months after cell-injection surgery, the graft edema
therapy in a failed graft eye. Pre-treatment image of a improved and corneal clarity was recovered (right).
patient with a failed graft due to allograft rejection. Severe (Reproduced from Koizumi, Ganka. Ophthalmology,
corneal edema with opacity was observed (left). Eighteen 2017)

novel findings and knowledge regarding cell based
therapy. Clinical evidence of the safety and effi-
cacy of cell-injection therapy should be accumu-
lated and evaluated very carefully, and technical
improvements in surgery and cell culture might be
necessary for the development of cell-­ injection
therapy products. Product development by phar-
maceutical or biotechnology companies for mass
application of this therapy will require technical
breakthroughs for frozen preservation of culti-
vated human corneal endothelial cells for creating
a master cell bank and for the transport/distribu-
tion of cell products from company cell processing
centers to hospitals around the world. Recent
research is also now aimed at establishing less
invasive evaluation methods for cultivated human
corneal endothelial cells and the technical refine-
ment of cell production under ­ GMP-­ compliant
systems [39, 42–47]. If cell products are devel-
oped by companies and made available in many
countries, cell based therapy might represent a
paradigm shiftin corneal endothelial treatments in
the near future.
Conflict of Interest  Noriko Koizumi and Naoki Okumura
declare that they have no conflict of interest.” (If any
460 N. Koizumi and N. Okumura

Number 212
CD /mm2 2526
AVG um2 396
SD um2 182
CV % 46
Max um2 1251
Min um2 110

CCT 548 um

Area (Polymethism) Apex (Pleomorphism)

[um2] % 50 100 % 50 100
0-100 0 3 0
100-200 13 4 12
200-300 21 5 30
300-400 22 6A 26
400-500 22 7 19
500-600 9 8 10
600-700 8 9 2
700-800 3 10- 0
800-900 1
900- 1 Customize

Fig. 30.5  Specular microscopy of the post cell-injection 2500 cells/mm2 were observed with non-contact specular
eyes. Eighteen months after cell-injection therapy, homo- microscopy (same patient as in Fig.  30.4). (Reproduced
geneous corneal endothelial cells with a density more than from Koizumi, Ganka. Ophthalmology, 2017)

authors do have a conflict of interest it must be spelled out
specifically).
Informed Consent All procedures followed were in
accordance with the ethical standards of the responsible
committee on human experimentation (institutional and
national) and with the Helsinki Declaration of 1975, as
revised in 2000. Informed consent was obtained from all
patients for being included in the study.
Animal Studies  No animal studies were carried out by
the authors for this article.
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 Corneal Endothelium
Regeneration: Future Prospects
Wei-Ting Ho, Hsin-Yu Liu, Fung-Rong Hu,
and I-Jong Wang
The human corneal endothelium lines the
­innermost layer of the human cornea and consists
of a monolayer of hexagonal cells. This single
layer of cells is the key to maintain corneal trans-
parency and health by regulating the hydration of
cornea. Based on the pump-leak mechanism [1],
the endothelial cells are permeable to nutrients
and other metabolites from the aqueous humor
while actively pump out ions to move water
osmotically into the anterior chamber.
At birth, endothelial density (ECD) is approx-
imately 6000 cells/mm2 [2], and declines thereaf-
ter as the eyes grow [3, 4]. In young adults, the
ECD is about 3500 cells/mm2.The ECD gradu-
ally stabilizes in adulthood and drops at a rate of
0.6% annually [3, 4]. The human corneal endo-
thelial cells (HCECs) are considered to have lim-
ited ability in regeneration under normal
condition, despite induced mitosis are observed
W.-T. Ho
Department of Ophthalmology, Far Eastern Memorial
Hospital, New Taipei City, Taiwan
H.-Y. Liu
Department of Ophthalmology, National Taiwan
University Hospital, Taipei, Taiwan
F.-R. Hu · I.-J. Wang (*)
Department of Ophthalmology, National Taiwan
University Hospital, Taipei, Taiwan
College of Medicine, National Taiwan University,
Taipei, Taiwan
e-mail: ijong@ntu.edu.tw
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_31
31
in cultured corneas [5, 6]. Contact inhibition
­preventing transition to the S-phase and low ratio
of positive versus negative growth factors con-
centration in the anterior chamber both prevent
the endothelial cells from dividing [5, 7–9].
31.1 H
 uman Corneal Endothelial
Cell Dysfunction and Current
Managements
Human corneal endothelial cell dysfunction will
result in increased imbibition of water and thus
corneal edema [10]. Among the relatively few pri-
mary corneal endothelial diseases, Fuchs’ endo-
thelial corneal dystrophy (FECD) is the most
common [11]. In FECD, the basement membrane
of the endothelium, Descemet’s membrane
becomes diffusely thickened and develops guttae
which are visible with slit-lamp and specular
microscopy. FECD can be inherited and tends to
be bilateral [12]. The endothelial cells dysfunc-
tion often becomes evident in middle age or later.
In secondary corneal endothelial diseases,
there is a recognizable cause that contributes to
the malfunction of corneal endothelium. These
includes contact lens-induced endotheliopathy,
iatrogenic endotheliopathy and endotheliopathy
secondary to ocular inflammation [13, 14].
Contact lens use increases the polymegathism
and pleomorphism of endothelial cells. These
changes are considered to be hypoxia-related
463
464
[15]. Endothelial decompensation has been
reported after ocular procedures or surgeries. The
impact on endothelium after laser iridotomy and
cataract extraction has been reported and exten-
sively studied [16–18]. Mechanism proposed for
endothelial damage during laser iridotomy
include direct focal injury, thermal damage,
mechanical shock waves, inflammation and iris
pigment dispersion. During cataract surgery,
endothelial injury may occur due to many fac-
tors, such as corneal distortion, collision of lens
fragments with corneal endothelium [19], local-
ized temperature rise [20] and increased oxida-
tive stress by free radicals [21]. After initial cell
loss, the ECD will continue to decrease for at
least 10 years after surgery [22].
For mild endothelial dysfunction, observation
with lubrication and hyperosmotic topical medi-
cations may be sufficient. Once moderate or
severe endothelial decompensation occurs, surgi-
cal replacement with donor cells is required to
restore corneal transparency. Both penetrating
keratoplasty (PK) and endothelial keratoplasty
(EK) are treatment choices for endothelial dys-
function [23, 24]. EK may be superior in terms of
less invasive manipulation of recipients and
quicker recovery. Thus, the surgical treatment for
endothelial diseases dramatically evolved toward
EK in recent years. EK procedures have evolved
recently and now contain different techniques,
including Descemet’s stripping endothelial kera-
toplasty (DSEK) and Descemet’s membrane
endothelial keratoplasty (DMEK). Despite DSEK
and DMEK implement the concept of selective
tissue transplantation, risk of greater endothelial
loss due to donor tissue manipulation and techni-
cal difficulty still pose challenges in performing
EK.  To date, there is no strong evidence from
randomized controlled trials of difference in
long-term endothelial cell loss and final visual
outcome between EK and PK [25].
31.2 Strategy for Corneal
Endothelial Cell
Regeneration
and Reconstruction
Regarding keratoplasty, the difficulties such as
limited donor availability and post-operative com-
W.-T. Ho et al.
plications drive the need for investigating alterna-
tives to surgery. Strategies targeting corneal
endothelial dysfunction can be divided into three
parts: preventing corneal endothelial loss, in vivo
stimulation of human corneal endothelial cell pro-
liferation and/or function, cell replacement with
cultivated human corneal endothelial cells.
31.2.1 Preventing Corneal
Endothelial Cells Loss
31.2.1.1 Sulforaphane
Jurkunas et al. described the involvement of oxi-
dative stress in the pathogenesis of FECD and led
to the identification of sulforaphane. By upregu-
lating major antioxidant response element-­
dependent antioxidants, sulforaphane can
ameliorate oxidative stress-induced apoptosis in
ex  vivo FECD sample and in Fuchs’ corneal
endothelial cell lines. Therefore, it may prevent
further cell loss in FECD [26, 27].
31.2.1.2 R  TA 408 Ophthalmic
Suspension
RTA 408 Ophthalmic Suspension (Reata
Pharmaceuticals; Irving, Texas) is currently in a
Phase II trial for prevention of endothelial cell
loss after cataract surgery through reducing oxi-
dative stress-induced damage [28].
31.2.2 In Vivo Stimulation of Corneal
Endothelial Regeneration
31.2.2.1 Pharmacological Treatment
Growth Factors
Growth factors have been detected in aqueous
humor and Descemet’s membrane, and even cor-
neal endothelial cells themselves. Fibroblast
growth factor (FGF) [29–31], insulin-like growth
factor [31], insulin-like growth factor binding
protein and hepatocyte growth factor (HGF) [31]
have all been reported as potential positive
growth factors while TGF -β appeared to negative
growth factor [32–35]. Among the many poten-
tial growth factors, FGF-2 has been proven to
stimulate endothelial cell proliferation. However,
the application of FGF-2 as therapeutics is
31  Corneal Endothelium Regeneration: Future Prospects
l­imited by endothelial mesenchymal transforma-
tion mediated by FGF-2 [36, 37]. In 2015, Trefoil
Therapeutics have announced supporting data for
the utility of engineered FGF-1 in corneal endo-
thelial regeneration with less undesired side
effects and is now moving toward an investiga-
tional new drug program [38, 39].
ROCK Inhibitors
Rho is a GTPase which is activated by guanine
nucleotide exchange factors (GEFs). RhoA then
activates ROCK, a serine/threonine kinase that
phosphorylates various substrates. ROCK signal-
ing involves a wide spectrum of essential cellular
events, such as cell adhesion, motility, prolifera-
tion, differentiation, and apoptosis [40].
Therefore, it has become a potential therapeutic
target for many diseases.
Okumura N et al. demonstrated that the use of
Y-27632, a selective ROCK inhibitor, can pro-
mote the proliferation of corneal endothelial cells
through stimulation of cell cycle regulators [41].
Subsequent researchers also confirmed the use of
ROCK inhibitor can improve the survival of cor-
neal endothelial cell culture and promote corneal
endothelial wound healing in both rabbit and pri-
mate animal models [42, 43].
In 2013, results of the first clinical trial to
determine the effect of ROCK inhibitor eye drops
in human was published. Total 8 patients with
corneal endothelial dysfunction scheduled for
EK (4 FECD cases and 4 bullous keratopathy
cases) were enrolled [44]. Patients underwent
transcorneal freezing, creating a central endothe-
lial wound of 2-mm diameter, followed by
10 mM ROCK inhibitor eye drops 6 times daily
for 7 days. A clear trend of reduction in central
corneal thickness was observed in the group with
central corneal edema group, but not in the dif-
fuse edema group. Despite the small case number
and the absence of control group in this pilot
research, the positive results prompted several
researchers to test the efficacy of ROCK inhibitor
eye drop as a treatment for early-stage FECD fol-
lowing Descemet’s membrane removal [45–47].
Further studies to determine whether corneal
endothelial cells can repopulate on the Descemet’s
membrane with guttae and validate the effective-
ness of ROCK inhibitors eye drops in larger
cohorts as treatment for FECD are necessary.
465
Another potential indication for ROCK inhibi-
tor eye drop is acute corneal endothelial damage,
such as following cataract surgery. Okumura
et  al. (2015) reported 3 cases of half or more
endothelial loss during cataract surgery whose
corneal edema reduced after ROCK inhibitor eye
drop for 6  months [48]. Moloney et  al. (2017)
also described the use of a ROCK inhibitor,
Ripasudil, as a salvage agent in FECD cases who
failed to regain corneal clarity after descemeto-
rhexis without grafting [49].
31.2.2.2 Surgical Treatment
Simple Descemetorhexis With or Without
Grafting
Unexpectedly, spontaneous recovery of corneal
endothelial function after complications arising
from EK or noncorneal surgeries have led to the
development of new surgical approaches for cor-
neal endothelial reconstruction which challenge
the conventional paradigms on endothelial wound
healing. Recovery of corneal clearance and
pachymetry values were found in EK cases with
endothelial graft detachment from limited area to
subtotal detachments [50–53]. Shah et al. (2012)
reported the endothelial migration and repopula-
tion after Descemet’s stripping without endothe-
lial graft [45]. Giving evidence in the above
matter, newer approaches have been tried based
on the hypothesis that endothelial cells have
capacity to migrate and repopulate. In 2012,
Descemet’s membrane endothelial transfer
(DMET) was reported, which was similar to
DMEK but without the unrolling part. Dirisamer
et al. enrolled 7 FECD patients and 5 pseudopha-
kic bullous keratopathy (PBK) patients in a pro-
spective DMET study [54]. At 6  months
post-operatively, all eyes with FECD showed re-­
endothelialization and clinical improvement
while none of the eyes with PBK had clinical
improvement. This observation may suggest a
depletion of recipient endothelial cells such as
PBK requires anatomical positioning of endothe-
lial transplant while dysfunctional recipient
endothelial cells such as FECD may need only to
remove the Descemet’s membrane and its guttae
without transplant. Hemi- and quarter-DMEK
have also been proposed [55, 56]. The reported
outcome showed gradual clearance of cornea
466
from center to periphery in first 12 months post-­
operatively with a low but steady ECD years after
surgery.
Some studies have tried to explore the possi-
bility of the regenerative capacity of endothelial
cells further by descemetorhexis without donor
material. The rationale behind this surgery was to
remove the dysfunction endothelial cells in the
center and to allow the healthy endothelial cell in
the periphery to be free from contact inhibition,
thus to migrate and repopulate. FECD with cen-
tral guttae was the specific treatment target with
various outcomes reported [47, 57–59], mainly in
case reports or case series. All patients undergone
descemetorhexis were FECD and one case was a
dual combination of FECD and posterior poly-
morphous corneal dystrophy proven by
Descemet’s membrane specimens [45]. In those
reporting spontaneous corneal clearance after
descemetorhexis without grafting, most were
observed between post-operative month 1 to
month 6 [49, 58]. Moloney G. et al. (2017) also
reported using topical Ripasudil as salvage treat-
ment in those who failed to clear. Among the 3
cases using topical Ripasudil, 2 eyes resulted
complete corneal clearance after 2 weeks of sal-
vage therapy while 1 eye still failed to clear and
received EK at the end [57]. In those with unsat-
isfactory outcomes, residual stromal edema, slow
visual recovery and even posterior stromal scar-
ring with irregular topography were observed
[59–61]. Despite that simple descemetorhexis
proves to stimulate corneal endothelial wound
healing in vivo, further clinical study is required
to determine the adequate patient population, the
safe extent of tissue removal and the potential
role of topical Ripasudil of the surgery.
31.2.3 Ex Vivo Expansion of Corneal
Endothelial Cell
and Transplantation
31.2.3.1 Corneal Endothelial
Regeneration with Tissue-­
Engineered Cell Sheet
Regenerated HCECs can be transplanted into the
eye by using several strategies. One strategy aims
W.-T. Ho et al.
at ex vivo expansion of HCECs on the carriers or
scaffolds to form tissue-engineered cell sheet,
which can be used for further transplantation.
Since its development, different materials have
been used as carriers or scaffolds for the cells
[62]. The first category is naturally grown mem-
branes, which can be dated back to late 1970s
when Gospodarowicz and Greenburg cultured
bovine CECs onto bovine and rabbit denuded
corneal stroma [63]. Subsequently, other natu-
rally grown membranes were also used as the
HCEC carrier and has been tested in the animal
model to restore the corneal transparency, includ-
ing denuded Descemet’s membrane and amniotic
membrane [64, 65].
The second category is biological polymers.
Compared with naturally grown membranes, bio-
logical polymers are composed of definite ele-
ments, have more standardized properties, and
can avoid contamination by infectious agents or
undesirable biological substances. Biological
polymers used as HCECs carriers include gelatin
and cross-linked type I collagen [66, 67]. In addi-
tion, silk fibroin has also been used as the sub-
stratum of HCECs [68]. Because silk fibroin has
the advantage of optical transparency, nonimmu-
nogenicity, controllable degradation rate, and
robust mechanical properties, it has been devel-
oped as an artificial endothelial graft for DMEK
surgery [69].
In addition to naturally grown membranes and
biological polymers, synthetic materials comprise
the third category of carriers for HCECs.
Examples include polyvinylidene fluoride and
biodegradable polymers, such as poly-l-lactic and
poly-dl-lactic-co-glycolic acid [70, 71]. Moreover,
synthetic materials can be blended with biological
polymers to generate carriers. For instance, chito-
san and polycaprolactone (PCL), both of which
are biodegradable biomaterials approved by Food
and Drug Administration (FDA), can be mixed
into 75:25 (chitosan:PCL) blends that have better
transparency than 100% PCL, and better cell
growth after seeding bovine CECs than 100% chi-
tosan [72]. Another group of synthetic materials is
thermos-responsive polymers. Made of homopol-
ymer of poly(N-­isopropyl acrylamide) or copoly-
mer of N-isopropyl acrylamide with
31  Corneal Endothelium Regeneration: Future Prospects
2-carboxyisopropylacrylamide, the carrier can
undergo phase transition from hydrophobic to
hydrophilic state when the temperature is below
the critical temperature, which can allow the cul-
tured HCECs detached from the carrier and form
the cell sheet ready for transplantation. The fabri-
cated cell sheet has been tested for the ability to
reconstruct corneal endothelium in vivo [73, 74].
31.2.4 Cell-Based Therapy
Although various materials can be used as carri-
ers or scaffolds to generate HCECs cell sheet,
which can be further used for corneal endothe-
lium reconstruction, this strategy has some chal-
lenges. For example, the sheet material may
reduce corneal transparency after transplantation,
change the cell behavior, or may not be biocom-
patible in the eye and cause excessive intraocular
inflammation. Moreover, to transplant a thin cell
sheet onto the posterior corneal surface, sophisti-
cated surgical skill is indispensable. Direct trans-
plantation of cultivated corneal endothelial cells
through “cell injection into the anterior chamber”
ideally bypass the difficulties in managing the
fragile graft in EK.  To this attempt, Okumura
et al. demonstrated that direct injection of CECs
combined with a ROCK inhibitor into the ante-
rior chamber can regenerate a corneal endothelial
monolayer and restore corneal clarity without
causing elevated intraocular pressure or exces-
sive inflammation in rabbit and primate models
[75, 76]. Another study also reported the efficacy
of cell injection in restoring corneal clarity in a
rabbit model [77].
After proving the efficacy and the safety of
cell injection therapy, there are still challenges to
be overcome. As aforementioned, HCECs are
considered to have limited proliferative potential.
To promote the efficiency of ex vivo expansion of
HCECs, various techniques have been developed,
including isolation of HCECs, extracellular
matrices coating, and culture media. After being
peeled off from the research grade cornea or
residual corneal-scleral rim after keratoplasty, the
Descement’s membranes are subjected to enzy-
matic digestion to dislodge the HCECs, such as
467
trypsin, collagenase A, and dispase. Compared
with others, collagenase A treatment can preserve
cell junctions and form cell aggregates with
higher viability [78]. After enzymatic digestion,
the cell aggregates are seeded onto the culture
plate. Evidences suggest that culture plates
coated with extracellular matrices, such as type
IV collagen, fibronectin, FNC mix, laminin-511
and -521 improve the proliferation of HCECs
[75, 79, 80]. Furthermore, supplemented media
containing EGF, insulin, transferrin, basic fibro-
blast growth factor, nerve growth factor, and pitu-
itary extracts have been used during the culture
[79]. In addition, other strategies have also been
developed to further overcome the limited prolif-
erative capacity. For example, Zhu et al. demon-
strated that the knockdown of p120 catenin can
promote HCEC proliferation by disrupting the
contact inhibition [81]. ROCK inhibitors have
also been used to enhance HCEC proliferation
both in vitro and in vivo [41, 82]. To further sus-
tain cell growth and prevent cell senescence,
mesenchymal stem cell-conditioned medium was
used in HCEC culture [83]. The development of
these culture techniques has markedly improved
the efficiency of the ex vivo expansion of HCECs.
Although HCEC proliferation can be enhanced
through incubation with growth-stimulating
agents, a major concern is that the cells may
undergo phenotypic changes and exhibit various
side populations during culture, particularly in the
presence of these agents [84]. The so-called endo-
thelial–mesenchymal transition (EnMT), which
resembles epithelial–mesenchymal ­ transition
(EMT), is characterized by cell junction destabili-
zation, loss of apical–basal polarity, cytoskeletal
rearrangement, expression of alpha-­smooth mus-
cle actin, and secretion of type I collagen [85].
These characteristics may compromise the func-
tion of HCECs, hampering the use of ex  vivo
expanded cells in tissue engineering. To further
optimize the efficacy of cell injection therapy,
there are several goals to be accomplished:
31.2.4.1 I dentification of the EnMT or
Side Population Markers
The hallmark of EMT includes loss of epithelial
markers, such as E-cadherin, and gain of mesen-
468 W.-T. Ho et al.

chymal markers including N-cadherin and ical data is required to completely assess the
vimentin [86]. Due to its neural crest origin, long-term safety and effectiveness of cell-­
CECs constantly express N-cadherin and vimen- injection therapy in the future.
tin [87, 88], rendering these typical EMT markers
unsuitable for defining the EnMT process.
Studies have identified that the morphological 31.2.5 Stem Cell Approach
and functional change of CECs during EnMT are to Regenerate Corneal
accompanied with the activation of some EMT-­ Endothelium
triggering pathways, including FGF-2/PI3K,
Wnt/β-catenin, TGF-β1/Smad2, and Notch sig- Limited donor resource has always been an issue
naling pathway during the EnMT process [81, 84, in corneal transplantation. Although ex  vivo
89–91]. To more comprehensively identify side expansion of HCECs combined with transplanta-
populations, Hamuro et  al. used a panel of cell tion by tissue-engineered sheet or direct cell
surface markers to discriminate different HCECs, injection can ameliorate this problem, the cul-
and identified that effector cells, ie. functional tured HCECs rarely maintain normal phenotype
HCECs, express CD166  +  CD105-­ CD44-­ beyond higher passage and will undergo EnMT
CD26-CD24- among heterogeneous HCECs eventually [98]. To address this unmet need, stem
[92]. The same group further analyzed microRNA cells-based therapies have been proposed because
or exosome proteins to identify functional of its capabilities of unlimited proliferation, self-­
HCECs [93–95]. renewal and differentiation into functional cells.
The sources of stem cells for corneal endothelial
31.2.4.2 Increase of the Percentage regeneration can be classified according to the
of Functional HCECs cell potency.
Currently, several strategies have been developed
to increase the functional HCECs percentage. 31.2.5.1 Pluripotent Stem Cells
Peh et al. used dual media approach to culture the Embryonic stem cells (ESCs) harbor the pluripo-
cells, one is Ham’s F12/M199-based medium tency that can differentiate into three germ layers.
supplemented with 5% FBS and 10 ng/ml bFGF To induce the differentiation, ESCs are first
(M4 medium), and the other is human endothelial-­ induced to differentiate into neural crest cells and
SFM-­based medium supplemented with 5% FBS then into CECs, mimicking the embryonic devel-
(M5 medium). Compared with M4 medium opment process [99, 100]. The differentiated
alone, cells grown in M4 followed by mainte- cells are further validated by cell morphology,
nance in M5 exhibited better phenotype and CEC markers, and transcriptomics analysis,
higher expression of CEC specific markers [96]. which show high similarity between ESC-­derived
Alternatively, small chemicals targeting different CECs and primary HCECs [99, 100].
EnMT signaling pathway have been shown to The second source of pluripotent stem cells
suppress EnMT and preserve the phenotype and comes from induced-pluripotent stem cells
function of CECs, including TGFβ inhibitor, (iPSCs). Developed by Yamanaka et  al., iPSCs
Notch inhibitor, and MMP inhibitor [89, 91, 97]. have the advantage over ESCs that it can be cus-
These approaches, combined with selection tomized, ie. derived from the patient’s own cells,
and enrichment of functional HCECs by surface obviating the risk of immune rejection, and it
markers, microRNA, exosome proteins, may avoids the ethical issue that using cells from the
extensively improve the treatment effect of cell embryo [101]. Using iPSCs as the tool, CECs or
injection therapy. In 2013, the Kinoshita group corneal organoids can be generated by sequential
reported the first cohort of patients treated with round of differentiation program [102, 103].
cell injection therapy (UMIN000012534) and a Another difference from ESCs is that iPSCs har-
positive outcome was observed. Despite current bor epigenetic memories, as iPSCs generated
evidence has suggest a great potential, more clin- from a somatic cell type exhibit tendency for
31  Corneal Endothelium Regeneration: Future Prospects
spontaneous re-differentiation into the same cell further differentiation, these progenitor cells can
[104]. This property may be utilized to generate be served as a promising source for regenerating
iPSCs derived from CECs that can be further re-­ corneal endothelium.
differentiated into functional cells.
31.2.5.2 Multipotent Stem Cells
Multipotent stem cells, with more restricted dif-
ferentiation capacity compared with pluripotent
cells, are implicated to regenerate corneal endo-
thelium. Bone marrow-derived endothelial pro-
genitor cells (BEPCs), co-cultured with CECs
using transwell system, can develop polygonal
cell shape and markers of CECs [105]. Likewise,
human umbilical cord blood mesenchymal stem
cells (hUCB MSCs) can be induced to differenti-
ate into CEC [106]. Direct injection of hUCB
MSCs into anterior chamber has also been shown
to repair corneal endothelial defects in rabbit
model [107].
31.2.5.3 CEC Progenitor Cell
HCECs are largely believed to have limited pro-
liferative capacity in vivo. However, there are sev-
eral evidences indicating that corneal endothelium
stem cells may locate adjacent to the endothelial
periphery in the posterior limbus. First, HECEs
derived from the peripheral part of the cornea
showed more vibrant mitotic activity compared
with cells from central part [108]. Besides, several
stem cell markers were detected in the trabecular
meshwork and the transition zone of the human
posterior limbus, including Oct-3/4, Wnt-1,
telomerase, alkaline phosphotase, nestin, Pax-6,
and Sox-2 [109, 110]. Moreover, cultured HCECs
can form spherical colonies in sphere forming
assay, suggesting the existence of precursor cells
[111]. These cells may contribute to replacement
of HCECs, but its slow proliferation in physiolog-
ical environment may be insufficient to compen-
sate for the cell loss that leads to corneal edema.
Nevertheless, isolating these precursors and
expanding in vitro are attracting choices for cor-
neal endothelial regeneration. Katikireddy et  al.
have isolated a subpopulation of cells from human
corneal endothelium exhibiting rapid prolifera-
tion and neural crest markers, and can be cultured
in  vitro up to 80 passages [112]. Coupled with
469
31.3 Conclusion
The field of CEC regeneration evolves rapidly.
Newer surgical techniques greatly improve the
safety and treatment outcome. Emerging medica-
tions can prevent CEC loss or may enhance cell
proliferation in  vivo. Advances in cell culture
technology also help ex vivo expansion of CECs
that once considered poorly proliferated.
Different modalities, either tissue-engineered
cell sheet or direct cell injection, facilitate the
implantation of CECs onto the posterior cornea
and regeneration of CEC monolayer. Finally,
stem cell-based treatment may fundamentally
solve the issue of organ shortage or even contrib-
ute to customized regenerative medicine. These
developments pave the way for better treatment
and improvement in live quality for those afflicted
by corneal endothelial diseases.
Conflict of Interest Wei-Ting Ho, Hsin-Yu Liu, Fung-
Rong Hu and I-Jong Wang declare that they have no con-
flict of interest.
Informed Consent  No human studies were carried out
by the authors for this article.
Animal Studies  All institutional and national guidelines
for the care and use of laboratory animals were followed.
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 Part VI
Bioengineering Cornea Surgery
 Umbilical Cord Stem Cells
in the Treatment of Corneal
Diseases
Mohammed Ziaei, Jie Zhang, Dipika V. Patel,
and Charles N. J. McGhee
32.1 Introduction
The inbuilt capacity of self-renewal and differen-
tiation of stem cells has rapidly established stem
cell-based therapies as a major new direction in
the investigation of novel therapies for human
disease. The field of ophthalmology, and in par-
ticular corneal surgery, has witnessed great prog-
ress in the development of stem cell therapy for
tissue engineering applications in recent years.
This is of key importance as specialists deal with
the ever-increasing burden of corneal disease and
a chronic global shortage of suitable donor mate-
rial in their efforts to treat >1.5 million new cases
of monocular corneal blindness annually [1].
32.2 Stem Cells
Stem cells are cells with three basic properties:
they are capable of self-renewal, they have the
capacity to undergo differentiation to become spe-
cialized progeny cells, and they have the potential
to renew the tissue that they populate [2].
M. Ziaei · J. Zhang (*) · D. V. Patel · C. N. J. McGhee
Department of Ophthalmology, New Zealand
National Eye Centre, Faculty of Medical and Health
Sciences, The University of Auckland,
Auckland, New Zealand
e-mail: jie.zhang@auckland.ac.nz
© Springer Nature Switzerland AG 2019
J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_32
32
Stem cells can be classified according to their
origin or potency. On the basis of trans-­
differentiation potency stem cells can be either
totipotent, pluripotent, multipotent, oligopotent
and unipotent.
Totipotent stem cells can differentiate into
embryonic and extra-embryonic cell types and
thus can construct a complete, viable organism.
Pluripotent stem cells are descendants of totipo-
tent cells and have the potential to differentiate
into any of the three germ layers. Multipotent
stem cells can differentiate into a limited number
of cells i.e. those of a closely related family of
cells.
Oligopotent stem cells can differentiate into
only a few cell lineages whereas unipotent cells
possess self-renewal properties but can produce
only one cell type.
Human stem cells can be isolated from the fol-
lowing four sources:
1. Embryonic tissue: these stem cells are pluripo-
tent, capable of differentiating into embryonic
tissue such as ectoderm, mesoderm and endo-
derm and are harvested from the blastocyst of
5-day-old pre-implantation embryos [3].
2. Foetal tissue: these stem cells are multipotent
cells capable of differentiating into a limited
number of cell types, as dictated by the degree
of prior differentiation and can be isolated
from two distinct sources; the foetus proper
and supportive extra-embryonic tissues, such
477
478
as amniotic fluid, umbilical cord, placenta and
amnion.
3. Adult tissue: these stem cells are multipo-
tent cells capable of differentiating into a
more limited number of cell types than foe-
tal stem cells and have been isolated from
the corneal limbus, stroma, and transition
zone (periphery of Schwalbe’s line to the
anterior portion of the trabecular mesh-
work) [4].
4. Induced pluripotent stem cells (iPSCs): adult
somatic cells can be genetically repro-
grammed to an embryonic stem cell-like state
by forcing expression of key transcription fac-
tors such as OCT4, SOX2, KLF4, and c-Myc;
important for maintaining the defining proper-
ties of embryonic stem cells. The resulting
cells can give rise to all tissue types [5].
Fig. 32.1  Cross-section of the human umbilical cord. A
artery, V vein, WJ Wharton’s jelly, UCL umbilical cord
lining, SA, IV, and PV subamnion, intervascular, and peri-
vascular zones of Wharton’s jelly, VW blood vessel wall.
Hematoxylin and eosin staining, scale bar 200 μm. Stem
cells found in the umbilical cord: Cord lining: Mucin-­
expressing cord lining epithelial cell (CLEC-muc).
M. Ziaei et al.
32.3 H
 uman Umbilical Cord Stem
Cells
The umbilical cord was the first foetal tissue to be
explored for the presence of stem cells. The cord
contains two arteries and a vein, wrapped in a
sheath of proteoglycan rich, mucoid, porous con-
nective tissue (Wharton’s jelly), and covered by a
simple epithelial layer believed to be derived
from amniotic membrane epithelium (Fig. 32.1).
A reservoir of blood is present in the umbilical
cord vein post-partum.
Umbilical cord-derived stem cells represent a
class of stem cells that have a number of advan-
tages over other traditional sources of stem cells.
The advantages and disadvantages of umbilical
cord-derived stem cells are summarized in
Table 32.1 [7].
Umbilical vein: Umbilical cord blood derived mesenchy-
mal stem cells (UCBMSCs) & Human umbilical cord
blood endothelial progenitor cells (UCB EPCs). Wharton’s
jelly: Umbilical Cord Mesenchymal Stem Cells
(UCMSCs). (Reproduced from [6] with permission.
CC-BY 4.0 https://creativecommons.org/licenses/by/4.0/)
32  Umbilical Cord Stem Cells in the Treatment of Corneal Diseases 479

Table 32.1  A summary of the advantages and disadvantages of therapy utilizing stem cells derived from the umbilical
cord [7]
Advantages
Easy, non-invasive and ethically acceptable collection of cells.
Widely available stem cell banks in the public and private setting.
Cryopreservation of cells possible.
Multiple cell lineages available in the umbilical cord.
Low risk of teratoma formation.
UCMSCs exhibit a gene expression profile more similar to that of embryonic stem cells and possess faster
self-renewal compared to other mesenchymal stem cells.
A substantial number of UCMSCs can be derived after ex vivo expansion.
UCMSCs can be considered for autologous/allogeneic use.
UCMSCs retain their multipotency for longer periods than other mesenchymal stem cells.
UCMSCs are considered to have low immunogenicity with good immunosuppressive properties in vitro and in vivo
and therefore pose a lower risk for rejection and graft-vs-host disease.
UCMSCs can act as a feeder layer for other pluripotent stem cells, and exert a non-tumorigenic effect on other cell
types.
Disadvantages
The differentiation of UCMSCs can be partial for some cell lineages.
Transplantation can lead to transfer of rare infectious or genetic diseases.
Banking of umbilical cord derived stem cells is expensive, lacks stringent regulation and long-term health of stored
cells remains unknown.
Current methods for isolation and culture of stem cells from the umbilical cord are cumbersome and result in a low
yield of primary cells.

Currently, there are numerous methods for 32.4 Corneal Regeneration

isolation and culture of stem cells from the
umbilical cord. These approaches are mainly
based on enzymatic treatment or explantation
techniques in order to isolate different stem cell
populations from the cord, with subsequent cul-
ture in specific media to encourage differentia-
tion of cell types. Stem cells can be derived from:
• Umbilical cord blood: a source of primarily
hematopoietic lineage cells with a small mul-
tipotent cell population (0.00003% of all
extracted nucleated cells).
• Umbilical cord lining membrane: a source for
umbilical cord blood derived mesenchymal
stem cells (UCBMSCs) which have the poten-
tial to differentiate into multiple cell types, in
addition to generating ectodermal and endo-
dermal lineages by crossing the germline bar-
rier [8].
• Wharton’s jelly: a rich source of umbilical
cord mesenchymal stem cells (UCMSCs),
approximately 400,000 cells per umbilical
cord compared to 1 mesenchymal stem cell in
10,000 nucleated bone marrow cells [9].
32.4.1 Epithelial Regeneration
Corneal epithelium is an ectoderm-derived, non-­
keratinized, stratified layer with a unique cyto-
keratin expression pattern and a reported
thickness of between 48–53 μm [10]. In health,
the corneal epithelium is capable of rapid regen-
eration and is maintained by the integrity and
functionality of a specialized stem cell popula-
tion, known as limbal stem cells (LSCs). The
LSCs are located in the basal region of the lim-
bus, a narrow transition zone between the periph-
eral cornea and conjunctiva [11].
Regeneration of the corneal epithelial sur-
face has traditionally been thought to involve
division, migration, and maturation of LSCs.
When this process breaks down due to destruc-
tion of the delicate micro-environmental niche
of the LSC, limbal stem cell deficiency (LSCD)
results. Advanced LSCD has a reported inci-
dence of 3.81  ×  10−6 cases per million,
per  annum and is associated with devastating
visual loss [12, 13].
480
A variety of corneal limbal stem cell grafting
procedures have been utilized to treat LSCD. They
involve either direct transplantation of limbal tis-
sue or transplantation of in vitro expanded cells
on a variety of biological or synthetic carrier
materials. These techniques include conjunctival
limbal autograft (CLAU), living-related conjunc-
tival allograft (Lr-CAL), keratolimbal allograft
(KLAL), autologous ex  vivo cultivated limbal
epithelial transplantation (CLET), simple limbal
epithelial transplantation (SLET), cultivated oral
mucosal epithelial transplantation (COMET) and
transplantation of peripheral corneal cells.
Currently these techniques are challenging to
perform, expensive, and have variable long term
success rates.
Cells derived from umbilical cord stem cells
are capable of forming a stratified epithelial layer
when seeded on artificial matrices such as colla-
gen gels populated with fibroblasts as feeder
cells. Researchers have therefore investigated the
feasibility of umbilical cord-derived stem cells
for ocular surface reconstruction.
Researchers have transdifferentiated UCMSCs
into corneal epithelial cells on a 3D human artifi-
cial anterior cornea model and confirmed the
capacity of UCMSCs to form a stratified epithe-
lial layer with favourable ultrastructural charac-
teristics such as expression of epithelial markers
(cytokeratin 3/12, plakoglobin), tight junction
protein zonula occludens 1 (ZO-1), and gap junc-
tion protein Connexin 43.
A novel cell type of the human umbilical
cord lining membrane that expresses mucin1,
hence termed mucin-expressing cord lining epi-
thelial cell (CLEC-muc) on human amniotic
membrane has also been used successfully in an
in vivo rabbit model of LSCD. Complete regen-
eration of the ocular surface was observed in 4
out of the 6 animals treated with CLEC-mucs
with all controls demonstrating signs of total
limbal stem cell failure at 10-weeks postopera-
tively. In the treatment group, one eye devel-
oped mild superficial inflammation whilst
another developed severe n­eovascularization.
Molecular analysis of transplanted eyes revealed
a similar gene expression profile to intact nor-
mal cornea controls [14].
M. Ziaei et al.
The success of the above in  vivo studies has
led to trials for the treatment of persistent corneal
epithelial defects in humans. Unpublished reports
suggest that allogeneic CLEC-muc sheets cul-
tured on contact lens-shaped plastic can lead to
corneal epithelialization in 95% of patients with
chronic corneal ulcers but further details remain
elusive at this stage and are keenly awaited.
The use of umbilical stem cell derived epithe-
lial cells would circumvent the problem of the
limited amount of limbal tissue available for
transplantation, especially in cases of bilateral
LSCD, but further study is still required.
One significant barrier that needs to be over-
come to allow the clinical application of umbili-
cal stem cell reconstruction of the ocular surface
is the lack of cell surface marker exploitation
techniques. These can be used for enriching spe-
cific cell populations when compared to other
stem cell populations (e.g. hematopoeitic stem
cells). There is also the lack of a standardized
cultivation process that consists of only clinical-­
grade, xeno-free, products in good manufactur-
ing practice (GMP) grade conditions [15].
Further work is also required to determine the
best composition of the substrate matrix used to
differentiate mesenchymal stem cells into epithe-
lial cells. Current techniques which attempt to
recreate the microenvironmental niche of the host
tissue by seeding the stem cells on a fibrin–aga-
rose stromal substitute containing human kerato-
cytes, fail to allow full in vivo differentiation of
an epithelial layer which suggests that a number
of different cell signals are required for complete
cell differentiation and tissue morphogenesis.
32.4.2 Stromal Regeneration
The corneal stroma is a precisely organized avas-
cular tissue which comprises approximately 90%
of corneal thickness and provides the mechanical
integrity and transparency of the cornea. It con-
tains 200–250 distinct lamellae, each layer
arranged at approximately right angles relative to
fibers in adjacent lamellae.
Keratocytes are the major cell type in the
stroma and are embryologically derived from
32  Umbilical Cord Stem Cells in the Treatment of Corneal Diseases
mesenchymal cells lying between the corneal
epithelium and endothelium. Keratocytes are
capable of synthesizing collagen molecules, gly-
cosaminoglycans, and matrix metalloproteases
(MMPs), and are crucial in maintaining local
homeostasis [16]. Keratocytes, unlike corneal
epithelial cells, are difficult to propagate ex vivo
as they have a low proliferative capacity and tend
to irreversibly transform into fibroblasts.
Recent work has suggested that UCMSC
transplantation may be a feasible alternative to
keratoplasty in treating congenital disorders of
the cornea secondary to keratocyte dysfunction
such as mucopolysaccharidoses. Intrastromal
injection of UCMSC has been shown to be effec-
tive in treating Mucopolysaccharidosis VII (Sly
syndrome) and Lumican deficiency in genetically
modified mouse models [17]. Stem cell treatment
led to a reduction in corneal haze as assessed by
in  vivo confocal microscopy with an overall
decrease in the corneal content of glycosamino-
glycans. Transplanted stem cells survived the
host immune response and ultimately differenti-
ated into resident stromal cells without the need
for immunosuppressive therapy. Of particular
note early treatment of affected mice led to sig-
nificantly improved corneal integrity, suggesting
that prophylactic treatment may prevent the cor-
neal haze seen in congenital corneal dystrophies.
UCMSCs have also been shown to be useful
in treating acquired disorders of the cornea such
as corneal opacification secondary to iatrogenic
trauma. In a mouse model created by a central
keratectomy wound, UCMSC treatment resulted
in partial recovery of corneal transparency and
thickness [18].
The use of umbilical stem cell derived stromal
substitutes is an intriguing therapeutic possibility,
especially given the longevity of mesenchymal
stem cell derivatives in corneal tissue. However,
further work is required to refine techniques to
create a reproducible and homogenous cell popu-
lation that can be used for clinical purposes.
Currently it remains uncertain how long UCMSC
derived cells survive in the cornea and whether
they have potential to cause long term adverse
effects e.g. through modulation of the local
immune response that can occur through the inhi-
481
bition of inflammatory cell adhesion/invasion
[19]. Another potential difficulty with intrastro-
mal injection of stem cells or transplantation of
stroma-like tissue sheets in hazy or scarred cor-
neas is the possibility of an inflammatory or scar-
ring response in an already compromised cornea.
Ultimately a great deal of additional research
is required to fully elucidate the therapeutic
potential for curative therapy in animal models
before consideration of treatment of corneal stro-
mal disease in humans.
32.4.3 Endothelial Regeneration
Human corneal endothelial cells (HCECs) con-
sist of a 4 μm thick monolayer of hexagonal cells
lining the posterior aspect of the cornea. To per-
form their essential pump function, a minimal
HCEC functional capacity is required, which
depends on both HCEC density and quality.
Endothelial cells do not undergo mitosis in vivo
as they are arrested in the G1-phase of the cell
cycle [20]. As a result, damaged endothelial cells
are rapidly replaced by enlargement of the sur-
rounding cells and centripetal migration into the
injured region. As the endothelial cell density
falls below a threshold level of 400–800 cells/
mm2, the cornea fails to maintain its transparency
through loss of deturgescence and subsequent
corneal edema [21].
In recent years, investigators have evaluated
the feasibility of transforming UCMSCs into
HCECs. Corneal endothelial cells are embryo-
logically differentiated from mesenchymal cells
that migrate between the surface epithelium and
lens placode [22]. Researchers have used lens
epithelial cell-conditioned medium (LECCM) to
mimic the effect of the lens during corneal endo-
thelial differentiation.
Ex vivo models have shown that UCBMSCs
could be transformed into HCECs through
LECCM treatment and exhibit a close phenotypi-
cal resemblance to HCECs (monolayer of
closely-packed, elongated cells), possess specific
cell markers such as N-cadherin and ZO-1 and
“home in” and attach to damaged HCEC with
great efficiency [23].
482
An in vivo rabbit model has also demonstrated
the ability of expanded human umbilical cord
blood endothelial progenitor cells (UCBEPCS)
to restore function and transparency to decom-
pensated corneas. After a central 9 mm surgically
created Descemetorhexis was performed,
UCBEPCS bound with immuno-magnetic CD34
nanoparticles were injected into the anterior
chamber and a magnet was placed on the rabbits
eyelid for 12 hours to attract the injected cells to
the host stroma [24]. The injected cells were
noted to migrate towards the magnet and the
treated corneas expressed aquaporin-1 (AQP1),
indicating intact pump function. At 4  months
postoperatively, the experimental group had little
to no corneal edema, and corneal thickness mea-
surements comparable to normal eyes, suggest-
ing restoration of endothelial pump function.
The aforementioned studies are fascinating as
they open up the possibility of a cell-based treat-
ment option for endothelial dysfunction, which is
the most common reason for corneal transplanta-
tion in industrialized countries [25]. However,
there are many obstacles that need to be over-
come before such therapy can become a clinical
reality.
Currently our knowledge regarding the molec-
ular pathways that govern development and dif-
ferentiation of corneal endothelial cells is limited.
In particular, identification is required of specific
micro-environmental conditions that enable
inductivity of HCEC from umbilical stem cells to
replace damaged or diseased cells. Although
advances have been made with the discovery of
the role of TGF-β2 in HCEC development, unsur-
prisingly the process appears to be complex with
differentiation requiring the interaction of a num-
ber of signals from other cell types from the ante-
rior segment [26].
It is also worth noting that whilst the ability
of injected HCEC to deturgesce the cornea has
been borne out in the short term, the longevity
of effect of such treatment remains undeter-
mined. Additionally, studies to date have not
rigorously examined the characteristics and
functions of transplanted endothelial cells.
Ideally, transmission electron microscopy
(TEM) studies are required to allow fine charac-
terization of the reconstructed endothelial ultra-
M. Ziaei et al.
structure and to confirm the presence of a
confluent and adherent layer of cells and pro-
gressive closure of the tight junctions between
adjacent cells [27]. The use of corneal endothe-
lial cell biomarkers such as ZO-1 and N-cadherin
to identify corneal endothelial cells in  vitro is
problematic as they are also expressed in other
lineages, and a step toward a better characteriza-
tion of endothelial cells might come from the
identification of novel cell markers such as
Wnt5a, S100A4, S100A6 and IER3 [28].
32.5 Conclusion
Umbilical stem cells have a number of qualities
which make them attractive in the field of regen-
erative medicine. They are easy to access, com-
prise large numbers of newborn stem cells with
excellent proliferative capacity and cell extrac-
tion does not lead to any morbidity or mortality
to either mother or infant (nor do they carry the
contentious ethical issues of embryo derived
cells). This makes them an economically viable
cell source for translational clinical applications.
The potential for developing cornea-like bioma-
terials has been greatly advanced with the use
and availability of stem cells, with preclinical
studies suggesting umbilical stem cells to be a
practical and realistic alternative to traditional
stem cell sources in the treatment of corneal dis-
ease. The field is still in its infancy, and further
studies are required to determine the long-term
outcome of these transplants.
Specifically, additional research is required
regarding the phenotypical and transformational
quality control of expanded cells before applica-
tion to the eye and the microbiological safety of
such an intervention. There are also issues regard-
ing the assessment of the functional capacity of
differentiated cells in vitro.
Whilst there are many avenues that are yet to
be explored, the evidence suggests that human
umbilical stem cells are excellent candidates for
regenerative medicine and may well play an inte-
gral role in stem cell translational therapy.
Preliminary data support the potential use of
human umbilical stem cell derived tissue in the
treatment of human limbal stem cell deficiency,
32  Umbilical Cord Stem Cells in the Treatment of Corneal Diseases
keratocyte dysfunction and corneal endothelial
disease. Developments in this field offer hope of
future cellular therapy for the millions whose
lives are affected by visually-impairing corneal
disease, currently untreatable or only amenable
to corneal transplantation.
Compliance with Ethical Requirements Mohammed
Ziaei, Jie Zhang, Dipika V. Patel, and Charles N.  J.
McGhee declare that they have no conflicts of interest. No
human or animal studies were carried out by the authors
for this article.
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 Dysfunctional Corneal
Endothelium: Delivery of Cell
33
Therapy

Stephen Wahlig, Gary Swee-Lim Peh,
Matthew Lovatt, and Jodhbir S. Mehta

33.1 Introduction an estimated 12.7 million individuals globally are

awaiting corneal transplantation, less than 2%
Corneal endothelial transplantation has seen a receive their needed treatment [8].
significant series of advances over the past sev- In the face of this shortage, cell based therapies
eral decades. Instead of full thickness penetrating provide an alternative source of tissue for treat-
keratoplasty (PK), partial thickness techniques ment of endothelial dysfunction. While originally
like Descemet’s stripping automated endothelial thought to be non-proliferative, human corneal
keratoplasty (DSAEK) and Descemet’s mem- endothelial cells (HCEnCs) have been success-
brane endothelial keratoplasty (DMEK) are now fully propagated in vitro [9, 10]. These develop-
preferred for treating endothelial pathology. ments are expanded upon in greater detail within
These newer methods have improved the recov- [CITE CELL CULTURE CHAPTER XXX]. In
ery speed and visual outcomes for patients with addition to propagation of these primary corneal
endothelial dysfunction [1]. However, graft rejec- endothelial cells (CEnCs), a robust method of
tion is still a concern: graft viability is only 83.3% delivering HCEnCs to the dysfunctional endothe-
at 3  years [2] and 76% at 5  years [3] for non-­ lium is necessary for clinical applications. While
Fuch’s dystrophy indications. While DMEK is some have attempted to mimic a donor corneal
thought to have reduced graft rejection compared graft using scaffolds seeded with HCEnCs, novel
to PK, some centers have reported rejection rates approaches like cellular injection also show prom-
up to 5.1% after 1  year [4], with early steroid ise as regenerative therapies (Fig. 33.1) [11, 12].
tapering thought to contribute to these elevated This chapter will initially review the pre-clinical
rates [5]. In addition, demand for graft tissue is animal models used to evaluate endothelial thera-
rapidly outstripping our limited supply. The num- pies, then proceed to discuss the principles and
ber of transplants in the United States has efficacy of several vehicles for delivery of propa-
increased over 60% since 2005 [6, 7]. Although gated endothelial cells to a dysfunctional cornea.

S. Wahlig · G. S.-L. Peh · M. Lovatt 33.2 Animal Models

Tissue Engineering and Stem Cell Group, The of Endothelial Dysfunction
Academia, Singapore Eye Research Institute,
Singapore, Singapore
Prior to initiation of human clinical trials,
J. S. Mehta (*)
Singapore National Eye Centre, Department of promising therapies must demonstrate safety
Cornea and External Disease, Singapore, Singapore and efficacy in  vivo. To create animal models

© Springer Nature Switzerland AG 2019 485

J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2_33
486 S. Wahlig et al.

a
Endothelial dysfunction

Healthy cornea Edematous cornea

b
HCEnC isolation HCEnC expansion
Donor tissue

c Removal of
DM and endothelium
Detached monolayer
1
Critical Endothelial
temperature keratoplasty

Removal of
DM and endothelium
2

HCEnC Endothelial
seeding keratoplasty

3
Legend Removal of endothelium
Cornea
Descemet’s membrane
HCEnC Cell
Endothelial cell (CEnC)
dissociation injection
Dysfunctional CEnC
Cell scaffold

Fig. 33.1  Overview schematic of endothelial cell ther-
apy. (a) The dynamics of corneal hydration are altered by
endothelial dysfunction or cell loss, resulting in swelling
and edema. (b) To generate cells for therapeutic applica-
tions, HCEnCs are harvested from donor corneas via DM
stripping and digestion and subsequently expanded
in vitro. (c) Expanded HCEnCs can potentially be deliv-
ered via (1) direct implantation of the cultured monolayer,
(2) seeding cells onto an engineered scaffold to be
implanted via a DSAEK-like technique, or (3) injected
directly into the anterior chamber

for this in  vivo evaluation, dysfunction of the
native endothelium must first be induced. This
can be accomplished by mechanical scraping of
the endothelium [13–15], cryoinjury [16],
phacoemulsification [17], Nd:YAG laser abla-
tion [18], or induction of bullous keratopathy
with injection of benzalkonium chloride (BAK)
[19]. While these procedures carry some risk of
inducing side effects like anterior chamber
inflammation or glaucoma, they are all effective
at disrupting normal endothelial pump function
and producing an edematous cornea [18]. The
most commonly used animal model for corneal
endothelial cell therapy is the rabbit [20–25].
Murine [16] and rat [26] models have also been
described, but the small size of these animals’
eyes causes difficulty with surgical manipula-
tion and therapeutic delivery. Rabbit eyes are
similar in size to humans, which makes them
ideal models for ocular therapy [27]. However,
the rabbit cornea possesses CEnCs)that retain
extensive proliferative capacity in  vivo, such
33  Dysfunctional Corneal Endothelium: Delivery of Cell Therapy
that even damage to 50% of the central cornea
can be repaired within 10  days [28]. As such,
several in vivo trials are now using primate [12,
24, 29] and feline [30] models, which like
human eyes lack a proliferative endothelium
[28, 31]. While rabbits are more cost effective
to purchase and maintain than primates or
felines, trials in rabbit models must include
careful analysis of negative controls to ensure
any observed endothelial regeneration is not the
product of native CEnC proliferation.
33.3 Direct Monolayer
Implantation
HCEnCs can be propagated in vitro as an adher-
ent culture, producing a polygonal monolayer
morphologically similar to the in  vivo corneal
endothelium. One delivery strategy involves har-
vesting this cultured cell monolayer and associ-
ated extracellular matrix (ECM) for direct
transplantation without need for a scaffold or fur-
ther modification. The first implementation of
this approach utilized culture surfaces coated
with the thermoresponsive polymer poly(N-­
isopropylacrylamide) (PNIPAAm), that detach
the HCEnC monolayer when cooled below a
critical temperature [32]. This method was subse-
quently used to transplant functional HCEnC
monolayers into a rabbit model, reducing corneal
edema [36]). Use of thermoresponsive polymer
culture surfaces requires a careful balance
between HCEnC attachment for monolayer for-
mation and effective stimulated detachment.
Improvements to this approach have come
through development of superior polymers,
including poly(vinyl methyl ether) (PVME) and
N-isopropylacrylamide-co-glycidylmethacrylate
(NGMA) [34, 35]. Both PVME and NGMA have
tuneable physical properties (e.g. film thickness,
stiffness) that in turn modulate HCEnC attach-
ment and ease of detachment [34, 35].
One of the advantages of this approach is the
ultra-thin nature of the monolayer, since the abil-
ity of corneal endothelium to maintain deturges-
cence (stromal dehydration) is reduced with
increasing membrane thickness [36]. This
487
method does not require additional carrier mate-
rial to be transplanted, thus reducing the risk of
immunogenic complications. However, the
ultra-­thin nature of the monolayer causes
extreme difficulty with manipulation and han-
dling of the implant, potentially damaging the
delicate endothelial cells and limiting the practi-
cality of its use. As compensation for the mono-
layer fragility, gelatin carriers have been used to
provide stability during surgical manipulation
[37]. This carrier is implanted with the mono-
layer and provides mechanical support from
within the anterior chamber until it eventually
degrades. The pores in the carrier facilitate nutri-
ent transport while the gelatin is in place [37–
39]. However, the need for a gelatin carrier
circumvents the benefits of direct monolayer
transplantation compared to seeding HCEnCs
directly onto an engineered carrier scaffold.
However, we feel that an endothelial monolayer
is too fragile and its transplantation too techni-
cally complex for widespread clinical use.
Instead, HCEnCs seeded on a tuneable scaffold
is the most feasible approach.
33.4 Biological Cell Carriers
Cultured CEnCs can be seeded onto a biocom-
patible scaffold, generating a tissue engineered
construct prior to implantation into the anterior
chamber. A variety of scaffolds tested in animal
models are detailed in Table 33.1. Implantation
of the seeded scaffold is nearly identical to the
existing Descemet Stripping Automated
Endothelial Keratoplasty (DSAEK), greatly sim-
plifying clinical integration of these constructs.
The HCEnC density on the scaffold can also be
precisely controlled to optimize graft function
while minimizing the quantity of required cells.
Of note, such a scaffold needs to satisfy certain
criteria such as biocompatibility, optically trans-
parency, easy handling for surgical manipula-
tion, and demonstration of similar mechanical
properties to the native cornea. Biological scaf-
folds can mimic the native endothelial environ-
ment, which should promote superior graft
integration.
488 S. Wahlig et al.

Table 33.1  Animal studies of carrier implantation

Animal Surgical Follow-up
Carrier material CEnC source model technique duration Reference
Biological
100-μm-thick Human Stroma/ Human Rabbit DSEK 4 weeks Peh et al. [55]
DM Lenticules
Human Denuded Amninotic Human cell line Feline PK 3 months Fan et al. [41]
Membrane (C3B)
Human Stromal Lenticules Human Rabbit DSEK 4 weeks Honda et al. [11]
Human Denuded Amniotic Human Rabbit PK 1 week Ishino et al. [42]
Membrane
Bovine DM Rabbit Rabbit PK 4 months Lange et al. [90]
Synthetic
10-μm-thick Silk Fibroin Rabbit Rabbit DSEK 6 weeks Vazquez et al. [91]
Membrane
6–8-μm-thick Aloe Vera-Silk Rabbit Rabbit DSEK 4 weeks Kim do et al. [20]
Fibroin Membrane
200-μm-thick Gelatin None Rabbit DSEK 8 weeks Niu et al. [23]
Membrane
20-μm-thick Spherically Curved Monkey Monkey DSEK 4 weeks Kimoto et al. [29]
Gelatin Membrane
50-μm-thick PEG-based None Sheep N/Aa 4 weeks Ozcelik et al. [69]
Hydrogel
700-μm-thick Gelatin Disc None Rabbit N/Aa 3 days Lai et al. [39]
Collagen I Membrane Monkey Monkey DSEK 2 years Koizumi et al. [14]
700–800-μm-thick Gelatin Disc Human Rabbit DSEK 3 months Hsiue et al. [32]
40–50-μm-thick Collagen I Human Rabbit DSEK 1 month Mimura et al. [92]
Membrane
100-μm-thick Hydrogel Lens Rabbit Rabbit PK 2 months Mohay et al. [57]
100-μm-thick Hydrogel Lens Feline Feline PK 2 months Mohay et al. [57]
1–5-μm-thick Gelatin Rabbit Rabbit PK 7 months McCulley et al. [77]
Membrane
DSEK technique implies use of a partial-thickness graft, while PK technique implies use of a full-thickness graft. CEnC
corneal endothelial cell, DSEK Descemet’s Stripping Endothelial Keratoplasty, DM Descemet’s membrane, PEG
poly(ethylene glycol), PK Penetrating Keratoplasty
a
Scaffolds were implanted into the anterior chamber without alteration of host endothelium

One of the earliest biological scaffolds utilized
was Descemet’s membrane. As the natural sub-
strate for endothelial cells, DM is an intuitive cell
carrier choice. Rabbit CEnCs were seeded onto
bovine DM, which was sutured onto a rabbit cor-
neal button and implanted using a PK approach
into a rabbit endothelial deficiency model with
resolution of corneal edema [40]. Since animal
DM and xeno-transplantation introduces potential
immunogenic complications, and human DM is
already in limited supply, this is not a realistic car-
rier for clinical practice. Another option, amniotic
membrane, is already used for a variety of sys-
temic therapies [40]. Human amniotic membrane
has functioned as a scaffold for human endothe-
lial cells, with the constructs sutured to corneal
buttons and successful transplanted via PK into
both rabbit and feline model [41, 42]. However,
the quality of amniotic membrane may not be
consistent, and it is not fully transparent. As such,
it is not likely to be a carrier of choice for a stan-
dardized tissue engineering protocol.
Another intuitive option is the use of pre-­
existing corneal tissue. Corneal tissue is both
transparent and highly compatible with endothe-
lial cells, although donor cells should be ­eliminated
to reduce immunogenic complications [25, 43].
Decellularized bovine and porcine stroma has
served as scaffolds for CEnCs with formation of
regular polygonal monolayers that demonstrate
pump functionality in vitro [44–48]. Reassuringly
for xenotransplantation, porcine constructs did not
33  Dysfunctional Corneal Endothelium: Delivery of Cell Therapy 489

induce an immune response in a rabbit model [25]. generally easier to mass produce and can be mod-
One challenge with these animal corneas is unde- ified to acquire desired properties for implanta-
sirable alteration of tissue properties after decel- tion. The first synthetic scaffolds were simple
lularization. A variety of protocols, including biomaterials like gelatin and soft hydrogel con-
freeze/thaw cycles, high pressure exposure, and tact lenses [56, 57]. While capable of adhering to
chemical detergents, have been used for removal endothelial cells, these early attempts were com-
of host cells [45, 49, 50]. Some of these techniques plicated by graft rejection in animal models [57].
incompletely remove host cellular material, while To reduce the incidence of these immunologic
others alter the stromal architecture. The com- complications, synthetic carriers can be formed
monly used ionic detergent SDS increases corneal from bioactive substances. Hyaluronic acid, an
swelling and diminishes transparency, although ECM protein with desirable biocompatibility, has
this can be reversed through immersion in 100% been processed to form a transparent hydrogel
glycerol [51]. In addition to the need for a stan- discs for HCEnC transplantation [58], although
dardized decellularization protocol, transplanta- some reports indicate that endothelial cells do not
tion of animal tissue may raise regulatory concern. adhere well to this carrier [59]. Collagen is a par-
Despite the use of implanted decellularized por- ticularly attractive substrate, as it is a major com-
cine tissue for approved cardiac valvular indica- ponent of Descemet’s membrane [60]. Monkey
tions [52, 53], this will be an obstacle to CECs cultured on collagen I sheets have been
implementation in humans. successfully transplanted into a monkey endothe-
Use of human corneal tissue circumvents lial deficiency model, demonstrating preserved
many of these concerns around xenotransplanta- corneal clarity at 2  years [13, 14]. For further
tion. A construct generated from non-­improvement in graft transparency, collagen III
decellularized human stromal discs and HCEnCs could be substituted for collagen I [61]. These
has been implanted into a rabbit model with min- scaffolds appear highly biocompatible in humans,
imal post-operative inflammation [11]. This tech- as demonstrated by clinical trial results of a col-
nique only harvested 2–3 stromal discs per donor lagen III anterior corneal implant which reported
cornea, indicating that tissue shortages will still no rejection episodes over 4 years [62]. Collagen
be an obstacle with the use of human stroma [11]. sheets can be easily and reproducibly manufac-
More recently, He et  al. published a method of tured using techniques like plastic compression,
producing thin decellularized human stromal acquiring the mechanical properties necessary
lamellae with a femtosecond laser [54]. Using the for surgical handling [63]. However, while highly
immortalized cell line HCEC-B4G12, these stro- biocompatible and easy to produce, pure collagen
mal lamellae were able to support an endothelial scaffolds have considerably reduced mechanical
cell density > 6000 cells/mm2 with minimal cell strength compared to native cornea [64].
loss upon surgical handling. These thin lamellae Another method to promote graft integration
are also compatible with primary human cells, as is the use of bioactive surface coatings. Silk
transplantation of HCEnC-seeded grafts have fibroin, a transparent biomaterial with high ten-
successfully reversed corneal edema in a rabbit sile strength, demonstrates significantly improved
model (Fig. 33.2), although the use of native DM endothelial attachment and morphology after
in these constructs restricts the number of obtain- coating with collagen or FNC [65, 66]. This idea
able grafts per donor cornea [55]. of combining properties from multiple biomateri-
als has yielded an enormous variety of blended
polymer substrates. These blends combine bioac-
33.5 Synthetic Cell Carriers tive materials (e.g. collagen, chitosan) with
mechanically strong substances (e.g. phosphoryl-
A variety of synthetic carriers have been pro- choline, polycaprolactone) [20, 67–71]. By
posed as endothelial scaffolds. Although syn- adjusting the relative levels of each material in a
thetic materials do not possess the innate blend, scaffold properties can be tuned for opti-
biocompatibility of biological scaffolds, they are mal function [20, 72].
490 S. Wahlig et al.

a b

c d

e f

Fig. 33.2  Descemet’s stripping endothelial keratoplasty into a Tan EndoGlide (b) Scleral tunnel is created (d) Host
(DSEK) in a primate model using a biologic graft (denuded DM and endothelium is peeled and removed (e) Graft is
Descemet’s membrane) seeded with HCEnCs. (a) inserted into the anterior chamber (f) Unfolded graft is in
Transparent graft prior to implantation (b) Graft is loaded place on the posterior cornea, supported by an air bubble

In addition to the materials used to produce
the scaffold, reproduction of the corneal microar-
chitecture has helped synthetic scaffolds mimic
the transparency and biomechanical properties of
native cornea. Instead of the aligned collagen
fibrils that form corneal stroma, an electrospin-
ning technique creating a poly (glycerol
sebacate)-poly (ε-caprolactone) (PGS-PCL)
nanofibrous scaffold with similar physical and
chemical properties to native cornea [73, 74].
33  Dysfunctional Corneal Endothelium: Delivery of Cell Therapy
This PGS-PCL nanofibrous scaffold also sup-
ports HCEnC monolayer formation and does not
alter human granulocyte activation, encouraging
evidence of its biocompatibility [75].
An array of production modifications can also
enhance the viability of synthetic scaffolds. Use
of curved molds produces a 3D scaffold shape
similar to the cornea, reducing complications of
graft folding and non-adherence that can arise
with flat scaffolds [29]. Scaffolds can be func-
tionalized by integrating heparin, which binds
growth factors that are slowly released to pro-
mote HCEnC growth [23]. Another useful modi-
fication is the use of topographical patterning.
This approach mimics the biophysical interaction
between endothelial cells and Descemet’s
Membrane. A gelatin methacrylate (GelMA+)
based hydrogel was patterned with 1  μm and
250 nm cylindrical wells and pillars, which dem-
onstrated improved endothelial homogeneity and
ZO-1 expression (Fig. 33.3) [76]. In addition, the
GelMA+ polymer was manipulated through
a model, intending to produce a monolayer on the
b monolayer formation, necessitating a higher
Fig. 33.3  Scanning electron microscopy (SEM) images
of GelMa scaffold with 1  μm topographical patterning,
depicted in oblique (a) and overhead (b) views
491
chemical and physical cross-linking to signifi-
cantly improve its mechanical properties [76].
The development of synthetic scaffolds has given
rise to tremendously customizable materials,
which show promising compatibility with
HCEnCs and the mechanical requirements of
transplantation. Of note, while many of these
polymers have demonstrated encouraging repro-
duction of corneal properties, head to head com-
parisons between synthetic scaffolds is still
needed to clarify the optimal carrier material.
33.6 Cellular Injection
Direct HCEnC injection of single cells into the
anterior chamber is one of the original approaches
tested for endothelial cell therapy; a collection of
animal trials are listed in Table 33.2. The simplic-
ity of HCEnC injection is its strongest feature:
corneal regeneration therapy could be imple-
mented globally, even in areas without access to
highly skilled corneal surgeons (Fig.  33.4). A
1980 publication by McCulley et  al. describes
injection of cultured rabbit CECs into a rabbit
posterior cornea for replacement of the removed
endothelium [77]. However, the endothelium did
not regenerate, and ectopic CEC deposits were
visible on the iris and lens in addition to the cor-
nea. This initial failure highlights a major chal-
lenge with the direct injection approach:
localizing cells to the posterior cornea, rather
than other anterior chamber structures. Ectopic
cell localization diminishes efficient endothelial
number of injected cells. It is plausible that unat-
tached cells would follow aqueous flow into the
trabecular meshwork (TM). Ectopic cellular
deposits in the TM could produce an obstruction
and increase intraocular pressure (IOP), poten-
tially causing secondary glaucoma [21, 78].
One simple approach to cell localization is
having the recipient assume a face down position,
so that gravity brings the injected cells down onto
the DM [79]. Studies in a rabbit model of bullous
keratopathy have been successful in using cellu-
lar injection, followed by 6  h in the face down
position, to generate a new endothelial layer and
492 S. Wahlig et al.

Table 33.2  Animal studies of cell injection

CEnC Animal Cell ROCK Localization Follow-up
source model number inhibitor technique duration Reference
Monkey Monkey 5 × 105 Yes Face-down 1 year Okumura et al.
Human Monkey 5 × 105 Yes Face-down 3 months [12]
Rabbit Rabbit 5 × 105 Yes Face-down 2 weeks Okumura et al.
Human Rabbit 1 × 106 Yes Face-down 48 h [87]
Feline Feline 2 × 105 Yes Face-down 1 month Bostan et al. [30]
1 × 106
Rabbit Rabbit 2 × 105 Yes Face-down 2 weeks Okumura et al.
Monkey Monkey 2 × 105 Yes Face-down 3 months [24]
Human Rabbit 150 No Face-down 1 month Mimura et al.
spheres [80]
Human Rabbit 1 × 107 No Face-down 1 month Mimura et al.
150 [79]
spheres
Rabbit Rabbit 5 × 105 No Magnet 1 year Mimura et al.
[22]
Rabbit Rabbit 5 × 105 No Magnet 2 months Mimura et al.
[21]
CEnC corneal endothelial cell

reduce corneal edema [80]. However, even slight
movements can interfere with HCEnC localiza-
tion, so this is not a perfect method of preventing
ectopic endothelial deposition. Another potential
technique for localization integrates magnetic
particles into HCEnCs, and application of an
external magnetic field attracts the cells towards
the DM.  Initial studies successfully used ferro-
magnetic iron particles loaded in HCEnCs in a
rabbit model, but this approach carries some con-
cern of ocular toxicity [21, 22]. Ferromagnetic
material can retain magnetic properties even after
the external field is removed, potentially causing
self-aggregation and poor endothelial formation
[81, 82]. In response, superparamagnetic micro-
spheres and nanoparticles have been developed
which do not demonstrate this ‘memory’ of the
external magnetic field [78, 83]. These nanopar-
ticles also have minimal impact on HCEnC func-
tion or viability after uptake [84]. However, more
work is needed to determine the optimal device
for magnetic field generation, as both commer-
cially available permanent magnets [21, 22] and
custom electromagnets [83] have been used in
existing animal trials. The size and shape of the
magnet must also be defined; although rabbit
experiments have been successful using a small
(10 mm diameter × 5 mm length cylinder) mag-
net [21], whether this is appropriate for humans
is unknown. Additionally, the duration of mag-
netic field application needs clarification, as the
2–7  days used in an ex  vivo human eye model
[78] could cause difficulties with patient compli-
ance if the magnet is bulky or uncomfortable. A
variety of alternative techniques have been pro-
posed for future consideration, including suspen-
sion of HCEnCs in a viscous material prior to
injection to limit uncontrolled dispersal of free-­
floating cells. Yet to date the only two viable
methods are the gravity and magnetic nanoparti-
cle techniques, both of which have demonstrated
success with in  vitro and in  vivo experiments.
However, the gravity approach is significantly
simpler and more practical, with no need to inte-
grate magnetic particles into delicate HCEnCs or
use an external magnet. The long term fate and
safety implications of these nanoparticles after
injection is also not well understood. Still, local-
ization of HCEnCs must be further be optimized
to minimize cell seeding numbers and potential
side effects of the injection approach.
In addition to localizing cells to the posterior
cornea, the HCEnCs must attach to the corneal
surface to form a functional monolayer. Inhibition
of Rho-associated kinase (ROCK) has been
hypothesized to modulate HCEnC cytoskeleton
33  Dysfunctional Corneal Endothelium: Delivery of Cell Therapy 493

a b

c d

Fig. 33.4  Cell injection using a rabbit endothelial dys- ough removal of host endothelium (d) HCEnCs are
function model. (a) Clump of endothelial cells (stained injected into the anterior chamber. The rabbits are then
blue) kept in a tube prior to injection (b) Rabbit endothe- kept in a prone position for 3–4 h to facilitate cell adher-
lium is scraped off (c) Trypan blue is used to ensure thor- ence to host DM

function, and is noted to improve endothelial cell
adhesion to substrates, as well as proliferation,
migration, and wound healing [85]. Co-injection
of monkey CEnCs with the prototypic ROCK
inhibitor Y-27632  in a primate model reduced
corneal thickness significantly more than injec-
tion of CEnCs alone [12, 24]. However, conflict-
ing data has emerged from trials in a feline model,
which suggested that Y-27632 alone may be more
beneficial than co-administration of CEnCs and
ROCK inhibitor [30]. In this model, increasing
the number of injected CEnCs led to formation of
abnormal multilayered endothelium that
expressed non-endothelial markers collagen I
and alpha smooth muscle actin (α-SMA), sug-
gesting that injection of excess cells may reduce
treatment efficacy [30]. Further studies with
HCEnC and ROCK inhibitor injection are neces-
sary to clarify the relative contribution of each
therapy to endothelial regeneration. In 2013
494
Okumura et al. were approved to begin a human
clinical trial of HCEnC injection therapy with
ROCK inhibition.
As clinical trials of HCEnC injection begin,
there are still a number of safety and efficacy
questions that must be answered. Even with
ROCK inhibition, only 40–50% of injected cells
were adherent to the cornea [12]. Not only does
this hinder endothelial regeneration by reducing
the effective cell seeding density, free floating
cells can potentially pass into venous circulation
via aqueous flow and cause systemic side effects
(eg immune rejection, pulmonary embolism).
Use of ROCK inhibitor-based therapy in the US
will also require an FDA-approved pharmaceuti-
cal; netarsudil is currently awaiting FDA approval
while ripasudil has been approved in Japan since
2014 [86]. Additionally, though multiple in vivo
studies have shown no increase in IOP [21, 22,
24] despite ectopic localization of non-adherent
HCEnCs [30], long term monitoring for intraocu-
lar hypertension will also be necessary to confirm
safety in humans. Also, effectiveness of the
injected HCEnCs relies on an intact host DM for
endothelial cell adherence [24, 87]. Fuch’s endo-
thelial dystrophy, the most common indication
for endothelial transplantation in the United
States [8], produces a dysfunctional DM marked
by excressences known as guttata [88]. Although
removal of the pathological DM is necessary for
visual recovery in Fuchs dystrophy, it may hinder
the efficacy of an injection based cell therapy
[89]. Given these limitations, there may be a role
for both scaffold and injection based delivery
methods for regenerative endothelial therapies.
33.7 Conclusion
Current supply of graft tissue for corneal trans-
plantation is insufficient to address endothelial
dysfunction globally. Major advances in HCEnC
expansion in vitro have opened the door for cel-
lular therapies to meet this need [CITE CELL
CULTURE CHAPTER 8]. In order to apply this
technology clinically, a robust method for delivery
of therapies to the posterior corneal surface must
be developed. Application of endothelial mono-
layers is possible, although technically difficult
S. Wahlig et al.
given the monolayer fragility. In response, a vari-
ety of biological and synthetic substrates have
been created to maximize ease of handling and
post-operative integration within the host cornea.
One of the most promising delivery approaches is
direct injection of expanded HCEnCs into the
anterior chamber. This injection technique can be
easily utilized by ophthalmologists globally, even
those without specialized training in corneal sur-
gery. While we are still awaiting results from the
first human trial for endothelial cell therapy, this
emerging technology holds significant promise
for future clinical applications.
Compliance Statements J.  S. Mehta, M.  Lovatt,
G. Peh, and S. Wahlig declare that they have no conflict
of interest. All procedures performed by the authors
followed and were in accordance with the ethical stan-
dards of the responsible committee on human experi-
mentation (institutional and national) and with the
Helsinki Declaration of 1975, as revised in 2000.
Informed consent was obtained from all patients for
being included in the study. All institutional and
national guidelines for the care and use of laboratory
animals were followed.
Conflict of Interest: No conflicting relationship exists
for any author.
Financial disclosure: No financial disclosures.
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 Index

A B
Adipose derived adult mesenchymal stem cell Barrier function, corneal endothelium, 421
(ADASC), 373, 405 Basic fibroblast growth factor (bFGF), 112
Adipose tissue-derived MSCs (AT-MSCs), 236 Biodegradable polymers, 466
Adipose tissue mesothelial cell (ATMC), 138 Biological cell carriers, 487
Adult stem cells, 42, 478 Blindness, 390
Advanced therapy medicinal products (ATMPs), 48, 91 Bone marrow-derived endothelial progenitor cells
Allogeneic limbal transplant, 287 (BEPC), 137, 489
Alpha smooth muscle actin (α-SMA), 304, 307, Bone marrow-derived MSCs (BM-MSCs), 234
308, 493 Bone marrow mesenchymal stem cell
Amniotic membrane (AM), 63, 128, 429 (BM-MSC), 404
Amniotic membranes, 293 Bone morphogenic protein 4 (BMP4), 270
Amniotic membrane transplantation, 301 Bovine pituitary extract (BPE), 112
ANGEL®, 327 Bowman’s layer, 13, 388
Animal model, 152 Bullous keratopathy (BK), 166
cat, 448
choice of, 443
monkey, 449 C
pig, 448 Calcineurin inhibitors, 294
rabbit, 444 Cancer stem cells (CSCs), 51–52
rodent, 444 Canonical Wnt signaling, 430
sheep, 449 Carbon dioxide, 422
Anterior chamber associated immune deviation Cataract extraction, 464
(ACAID), 441, 443 CD44, 117
Anterior chamber injection, 411 CD56, 423
Anterior lens capsule, 429 CD109, 423
Anti-angiogenic function, 239 CD 166, 422
Anti-inflammatory action, 238 CD200, 117, 422
Aquaporin 1 (AQP1), 482 CD248, 423
Aqueous deficient DED (ADDED), 328 Cell-based medicinal products (CBMP), 399
Artificial cell-free device, 155 Cell-based therapy, 467–468
Autologous eye-platelet rich plasma (E-PRP), 19 Cell carriers
Autologous limbal stem cell product, quality biological, 487
control for, 91 synthetic, 489
Autologous limbal stem cell transplants (ALSCT), 82 Cell-injection therapy, 457
Autologous serum eye drops (ASE), 81 for bullous keratopathy patient, 458
Autologous transplantation in failed graft eye, 459
European marketing authorisation, 93 ROCK inhibitor, 457
good manufacturing practice, 91 safety and efficacy of, 459
limbal stem cell, 92–93 specular microscopy, 460
manufactured product, 92 surgical procedure, 457
quality control, 91–92 Cell migration, 306

© Springer Nature Switzerland AG 2019 499

J. L. Alió et al. (eds.), Corneal Regeneration, Essentials in Ophthalmology,
https://doi.org/10.1007/978-3-030-01304-2
500 Index

Cell therapy Connexon, 178

with adult tissue progenitors, 264 Cornea, 419
clinical application, 271 anatomical dimensions, 3
using cultivated oral mucosal epithelial transplant biometrics and structure, 350
(COMET), 225–228 Bowman’s layer, 388
using ex vivo cultured limbal cells, 221–224 Descemet’s membrane, 388
using induced pluripotent stem cell, 263–273 diseases, 23
Cell types, 109 embryology, 3
Cellular injection endothelium, 388
animal studies of, 492 epithelium, 388
endothelial dysfunction, 491 integrity and transparency, 57
CEnPC, see Corneal endothelial progenitor cell (CEnPC) stroma, 388
Chitosan, 466 transparency, 388
CLAU, see Conjunctival limbal autograft (CLAU) Corneal endothelial cell (CEnC), 58, 109, 481, 485, 486
CLET, see Cultivated limbal epithelial transplantation biology, 455–456
(CLET) cell-injection therapy, 457–458
CLRN1, 424 for bullous keratopathy patient, 458–459
Collagen, 27, 489 in failed graft eye, 459
Collagen II production, 152 product development, 458
dystrophic cornea biomechanical property, 151 specular microscopy, 460
in human tissue, 149 surgical procedure, 457
in vivo model, 151 culture for clinical application, 456
in keratoconic tissue, 150 sheet transplantation, 456–457
Collagen type-1, 351 Corneal endothelial cell loss, prevention
COMET, see Cultivated oral mucosal epithelial RTA 408 ophthalmic suspension, 464
transplantation (COMET) sulforaphane, 464
Committee for Advanced Therapies (CAT), 49 Corneal endothelial progenitor cell (CEnPC), 58
Confocal microscopy function, 66
cell counting methods obtaining methods
ADAS cell, 373 adherent culture of single cells, 67
automated cell counting, 371 application, 69
decellularized lamina, 374 culture onto biological and biosynthetic substrate,
difficulty, 375 68
manual cell counting, 371–373 explant culture, 68
recellularized lamina, 375 suspension culture, 68
corneal density calculation, 375 study, 66
corneal stroma Corneal endothelium, 14, 26, 425
automatic cell counting, 384 changes in cell density and size, 421
patients with decellularized lamina, 377–381 3D mapping proteins, 423
patients with recellularized lamina, 380–383 embryology, 419–420
patients with transplanted ADAS, 375–376 functional activity, 8
sample, 367–371 human, 419, 420
handling difficulty, 366 immunohistochemistry of, 422–424
preparation, 364–366 mitotic activity, 9
Confocal ophthalmoscopy, 441 morphology, 420–421
Conjunctival epithelium physiology
goblet cells, 184 barrier function, 421
innervation, 186 pump function, 422
mucin, 184 secretory function, 422
tear film, 185–186 ZO-1 staining for rabbit cells, 423
Conjunctival invasion, 300 Corneal epithelial cells (CEC), 13, 77, 422, 479
Conjunctival limbal autograft (CLAU), 156 apical cell layer, 4
indication, 156 basal cell layer, 4
LSCD, 156 hypothesis, 6
ocular surface neoplasia, 157 innervation, 5
outcome, 157 limbus, 6
pterygium, 157 mass, 183
Conjunctival transplantation, 191, 195–198 regenerate, 78
Conjunctivalisation, 301 stem cell based therapy, 155
Connexins, 178 Corneal epithelial stem cells, 179
Index 501

Corneal graft rejection bone marrow, 393

clinical criteria, 440 dental pulp, 394
from animal models, 441 embryonic, 394
Corneal healing process induced pluripotent stem cell, 394
attachment, 17 neural stem cell, 394
cytokines, 14 umbilical cord, 394
endothelial regeneration, physiological mechanism stromal remodelling, 392
of, 19–21 Corneoscleral rims, 158, 302, 303, 427
epithelial cell migration, 16 Coxsackieadenovirus receptor (CAR), 423
growth factor Cryoablation, 445
autologous eye-platelet rich plasma, 19 Cultivated limbal epithelial transplantation (CLET),
epidermal, 14, 18 160–161, 222, 223
fibroblast, 15 biopsy harvesting, 285
hepatocyte, 15 culture method
insulin-like growth factor, 16, 18 scaffold, 284
keratinocyte growth factor 1, 15 technique, 285
nerve growth factor, 15, 18 graft transplantation, 285
platelet-derived growth factor, 15 post-operative management
transforming growth factor-β, 15 additional, 287
latent phases, 16 early, 286
proliferation and differentiation, 16 pre-operative assessment
Corneal layers, 176 indications, 278–281
Corneal neovascularization, 23 supplementary examinations, 281–283
Corneal opacification, 440, 441, 444 Cultivated oral mucosal epithelial transplantation
Corneal scraping, 301 (COMET), 161–163
Corneal stroma. See also Corneal stromal stem cells clinical results, 227
(CSSC), 7–8 development, 226
Corneal stromal stem cell (CSSC), 57, 404 keratoplasty, 227–228
with basic fibroblast growth factor, 64 surgery, 226–227
culturing, 104–105 Cultured limbal epithelial transplantation (CLET), 25
differentiation Current stem cell based therapy, 155
into keratocytes, 101–102 conjunctival limbal autograft, 156
into non-ocular cells, 101 indication, 156
embryonic origin, 100 LSCD, 156
expanding methods ocular surface neoplasia, 157
explant culture, 65 outcome, 157
plastic adherent culture, 65 pterygium, 157
suspension culture, 66 cultivated limbal epithelial transplantation, 160–161
function, 103 cultivated oral mucosal epithelial transplantation,
gene expression, 101 161–163
immune reactions of, 103 keratolimbal allograft, 158
interactions, 103 living-related conjunctival limbal allograft, 158–160
isolation from limbal biopsy, 104 postoperative complication, 163–164
limbal niche, confocal microscopy of, 105–106 simple limbal epithelial transplantation, 161
localization of, 102 Cyclophosphamide, 449
mesenchymal origin, 101 Cysclosporine A (CsA), 295
obtaining methods, 64 Cytokeratin(s), 178, 181
primary isolation, in humans, 100 Cytokeratin intermediate filaments, 5
progenitor potential of, 100 Cytokine, 392
properties, 101 Cytoskeleton, 178
Corneal wound healing, 237
clinical trial, 398–400
epithelial injury, 392 D
hLMSC, 395–398 Decellularization, 88, 489
limbus and the stem cell, 390–391 Decellularized lamina
MSC limitations, 394 anterior face, 377–379
myofibroblast migration and differentiation, 392 collagen matrix morphology, 378
preclinical trial, 394–395 posterior face, 377–379
stem cell-based therapy, 393 reflective peripheral structures, 380
adipose tissue, 393 transition zone, 380, 385
502 Index

Degranulation, 318 Endothelial transplantation

Demoglein, 177 animal models, 438
Dendritic cells, 8 cat, 448
Dental pulp-derived MSCs (DP-MSCs), 236 choice of, 443–444
Dental pulp stem cell (DPSC), 394 monkey, 449
Descemet membrane (DM), 8, 13, 112, 388, 420, 428, pig, 448
463, 488 rabbit, 444–448
Descemet membrane endothelial transfer rodent, 444
(DMET), 20, 465 sheep, 449
Descemet’s membrane endothelial keratoplasty central corneal thickness, changes in, 442
(DMEK), 464 corneal graft rejection
Descemet stripping automated endothelial keratoplasty clinical criteria, 440–441
(DSAEK), 445, 487 from animal models, 441–443
Descemet’s stripping endothelial keratoplasty (DSEK), in vivo and post-mortem assessment, 438–440
464, 490 Enzymatic digestion, 428
Descemetorhexis, 465 Epidermal growth factor (EGF), 14, 18, 28, 320, 335
Desmocollins, 177 Epidermal stem cell, 128
Desmoglein 3, 182 Epithelial basement membrane (EBM), 18
Desmosomes, 177 Epithelial cell culture
Devitalization, 445 assessment
Direct cell labelling methods, 440 cell counting and viability, 90
Direct cellular reprogramming, 267 colony forming efficiency assays, 90–91
Direct compression injury, 308 histology and immunostaining, 89–90
Direct monolayer implantation, 487 measure explant outgrowth expansion, 90
Donut excimer laser ablation model, 302 human amniotic membrane, 87–89
Dry eye disease, 225 limbal explant culture, 87
DSAEK, see Descemet stripping automated endothelial biopsy, 87
keratoplasty (DSAEK) culture medium preparation, 84
Dua-Fine layer, 355 HAM construct preparation, 85
Dua’s layer, 13 single cell suspension method, 83
Dulbecco's phosphate-buffered saline (DPBS), 84 3T3 fibroblast co-culture
from frozen stock initiation, 84
frozen layers preparation, 84
E frozen stocks preparation, 84
E-cadherin, 467 maintenance and subculture, 84
Ectatic disorder, 403 Epithelial keratinisation, 79
Embryonic stem cell (ESC), 41, 123, 394, 404, 405, 468 Epithelial mesenchymal transition (EMT), 456, 467
corneal endothelium regeneration, 135 Epithelial regeneration, 479–480
corneal epithelium regeneration, 124–127 Epithelial stem cell, corneal epithelium regeneration
corneal stroma regeneration, 131–133 amniotic membrane, 128
Embryonic tissue, 477 epidermis, 128
Endosialin, 423 hair follicle, 128
Endothelial cell, see Human corneal endothelial cell oral mucus, 127–128
(HCEnC) umbilical cord lining, 129
Endothelial cell density (ECD), 425, 463 Epithelial wound healing, 16–17
Endothelial cell therapy, 486 Ethylenediamine tetra-acetic acid (EDTA), 428
Endothelial decompensation, 464 European Medicines Agency (EMA), 30, 93
Endothelial dysfunction Ex vivo expansion, 83
animal models, 485–487 corneal endothelial regeneration, 466–467
carrier implantation Exosomes, 413
animal studies of, 491–494 Extracellular matrix (ECM), 24, 351, 429
biological cell carriers, 487–489 Extracorneal stem cell
synthetic cell carriers, 489–491 corneal endothelium regeneration, 135
cellular injection, 491, 493 embryonic and induced pluripotent stem cells,
animal studies of, 492 135–136
direct monolayer implantation, 487 mesenchymal stem cells, 138
management, 463–464 mesothelial cell, 138–139
Endothelial keratoplasty (EK), 426, 455, 464 neural crest stem cells, 137
Endothelial-mesenchymal transition skin-derived precursor, 137
(EMT), 110, 430, 467 vascular endothelial progenitor, 137–138
Endothelial regeneration, 481–482 corneal epithelium regeneration, 124
Index 503

epithelial stem cell, 127–129 G

immature dental pulp stem cell, 130–131 Gap junctions (GP), 178, 421–422
mesenchymal stem cell, 129–130 Gelatin, 489
pluripotent stem cell, 124–127 Gelatin disc, 447
corneal stroma regeneration, 131 Gene therapy medicinal product (GTMP), 49
embryonic stem cells and induced pluripotent Glucocorticoids, 294
stem cells, 131–133 Glycocalyx, 4
mesenchymal stem cells, 133–134 Glypican 4, 422
oral stem cell, 134–135 Gold nano-rods, 440
Extraocular mesenchymal stem cell Good manufacturing practice, 91
for corneal epithelial damage, 253–254 GPC4, 117
experimental model GRIP1, 424
amniotic membrane, 249–250 Growth factors (GF), 14, 464
with central corneal damage, 246 epidermal growth factor, 320
combined administration, 252–253 function, 319
contact lens carrier, 250 platelet-derived growth factor, 319–320
fibrin matrix, 250 synthesize, 319
intraperitoneal administration, 248 transforming growth factor beta, 320
intravenous administration, 246–248 vascular endothelial growth factor, 321
mobilization of endogenous, 246 Guanine nucleotide exchange factors (GEFs), 465
ocular topical administration, 248–249
polyamide 6/12 carrier, 251
poly-L-lactic acid carrier, 250 H
subconjunctival injection, 252 Hair follicle stem cell (HFSC), 128
therapeutic potential HCEnC, see Human corneal endothelial cell (HCEnC)
immunomodulatory ability, 240–244 Hemangioblast, 419
in vitro and in vivo studies, 233 Hematopoietic markers, 8
tissue homing capacity, 244–246 Hemidesmosomes, 178
transdifferentiation capacity, 234–237 Hemoderivatives, 292
trophic activity, 237–240 Heparin, 447
Extraocular stem cell Hepatocyte growth factor (HGF), 15, 28, 464
adipose derived adult mesenchymal stem cell, 405 Herpes simplex virus type-1 (HSV-1), 23
bone marrow mesenchymal stem cell, 404 Holoclar®, 51
embryonic stem cell, 405 Homeostasis, in stroma, 145
induced pluripotent stem cell, 406 HTR1D, 424
umbilical cord mesenchymal stem cell, 405 Human adult adipose tissue, 404
Eyelid abnormalities, 295 Human adult corneal limbal epithelial cell (HLEC), 127
Eye-platelet rich plasma (E-PRP), 325–326, 332 Human adult dental pulp cells (hDPCs), 134
Human adult dermal fibroblast (HDF), 127
Human amniotic membrane (HAM), 85–89, 198, 284
F Human bone marrow-MSC (hBM-MSC), 138
Ferromagnetic material, 492 Human corneal endothelial cell (HCEnC), 485, 487, 489,
Fetal calf serum, 432 492
Fibrin gel, 250 cellular and regenerative therapies, 110
Fibroblast growth factor (FGF), 15, 464 culture media, 430–432
Fibronectin, 321 culture conditions
Fluorescence ubiquitination cell cycle indicator (Fucci), functional cell marker, 117–118
304 growth media, 112–116
Fluorochromes, 440 surface, 116–117
Foetal tissue, 477 endothelial progenitor cell, 110–112
Food and Drug Administration (FDA), 30 extracellular matrix, 429, 430
Fuchs endothelial corneal dystrophy (FECD), 20, 167, immortalized cell lines, 428
463, 465, 466 injection, 491, 494
Future stem cell based therapy, 164 isolation, 112, 428
corneal endothelium, 166–167 morphology of, 439
corneal epithelial cell native, 110
differentiated cell source, 164–166 primary, 427
from pluripotent stem cell, 164 sources, 427–428
corneal stroma, 166 stem cells, 427–428
purified LSC, 166 substrates, 429–430
surface ectoderm-derived cell, 166 tissue engineering, 426–432
504 Index

Human eye, 299 automated cell counting, 371

Human limbal-derived stromal/mesenchymal stem cell collagen II production, 150
(hLMSC), 395–398 differentiation, 146
Human mesenchymal stem cells (hMSCs), 46 manual cell counting, 371
Human umbilical cord morphology of, 369
cross-section of, 478 treatment, 149–153
stem cells, 478–479 untapped potential of, 147, 149
Human umbilical cord blood mesenchymal stem cells Keratocyte differentiation medium (KDM), 102
(hUCB MSCs), 469 Keratocyte progenitor cells (KPC), 300
Hyaluronic acid, 489 Kerato-epithelioplasty, 191
Hydrogel, 489 Keratolimbal allograft (KLAL), 158
Keratoplasty, 99, 227–228
KLAL, see Keratolimbal allograft (KLAL)
I
Immature dental pulp stem cells, 130
Immune cells, 8 L
Immunohistochemistry, 422 Lagophthamos, 295
Immunosuppression, 241 Lens epithelial cell-conditioned medium
Impression cytology, 282 (LECCM), 111, 481
In vivo confocal microscopy (IVCM), 105, 282 LESC, see Limbal epithelial stem cell (LESC)
In vivo laser scanning confocal microscopy (IVCM), 301 Leucine-rich repeat-containing G protein-coupled
In vivo stimulation receptor 5 (LGR5), 456
pharmacological treatment Lid abnormality repair, 295–296
growth factors, 464 Lifitegrast, 294
ROCK inhibitors, 465 Limbal cells, 299
surgical treatment, 465–466 Limbal epithelial crypts (LEC), 180–183
Indirect cell labeling methods, 440 Limbal epithelial stem cell (LESC), 6, 57, 77
Induced pluripotent stem cell (iPSC), 111, 394, 406, identification, 58
468, 478 isolation methods
corneal endothelium regeneration, 135 amniotic membrane, 63
corneal epithelium regeneration, 127 culture media, 60–62
corneal stroma regeneration, 131–133 culture onto ECM coated surface, 63
mechanisms for reprogramming, 266–269 explant culture, 62
Inflammation feeder cells, 60–62
local, 294 silk fibroin, 63
systemic, 295 single-cell suspension culture, 62
Insulin-like growth factor (IGF), 16, 18, 28 sphere-forming assay, 62
Integrins, 14 tissue preparation, 60
Interleukin 1α (IL−1α), 14 properties, 58
International Society for Cellular Therapy, 237 Limbal explant biopsy, 87
Intracellular carbonic anhydrase pathway, 422 Limbal explant culture, 87
Intrastromal implantation, 407–409 Limbal niche, 77
Intravenous injection, 412 Limbal stem cell (LSC), 155, 191, 263–266,
299–300, 479
Limbal stem cell deficiency (LSCD), 25, 78, 232, 277,
J 300, 479
Junctional adhesion molecules (JAMs), 423 aetiology, 78, 277
cause and manifestation, 213–214
characteristics, 80
K conjunctivalisation, 301
Keratin, 304 destruction, 300
Keratinocytes growth factor, 28 epidemiology, 79, 221
Keratinocyte growth factor 1 (KGF-1), 15 grafting procedures, 480
Keratoconus impression cytology, 300
characteristic, 363 in vivo laser scanning confocal microscopy, 301
methods (see Confocal microscopy) management, 80–83
patients, 364 regeneration of limbus, 302
treatment, 363 semiology, 79–80
Keratocyte(s), 8, 26, 131, 353, 480 slit lamp examination, 300
activation, 145 treatment, 214, 221–223, 301–302
Index 505

Limbal stem cell failure (LSCF), management, 225–226 Negative HCEnC markers, 117
Limbal transplantation, 195 Nerve growth factor (NGF), 15, 18, 28
allograft, 202–208 Neural cell adhesión molecule (NCAM), 423
autograft, 198–202 Neural crest cell (NCC), 271, 419, 420
classification, 194–195 Neural crest-derived progenitor (NCDP), 167
conjunctival, 195–198 Neural crest progenitors (NPCs), 135
history, 191–194 N-isopropylacrylamide-co-glycidylmethacrylate
Limbus, 6 (NGMA), 487
Living-related conjunctival limbal allograft (Lr-CLAL), Non-enzymatic digestion, 428
158–160
Local inflammation
cyclosporine/tacrolimus, 294 O
lifitegrast, 294 Occludin, 177
steroids, 294 Ocular surface (OS), 176
Lr-CLAL, see Living-related conjunctival limbal ecosystem restoration, 223
allograft (Lr-CLAL) epithelium
LSCD, see Limbal stem cell deficiency (LSCD) basement membrane, 178–179
cell attachment, 177–178
conjunctiva, 183–186
M function, 176
Macula adherens, 177 limbus and limbal, 179–183
Massive injury, 57 superficial flat cell, 176
Matrix metalloproteinases (MPPs), 14 implantation, 406–407
Medical therapy, 221 optical polish, 176
Membrane-bound Na+, K+ -ATPase sites, 422 reconstruction of, 226
Mesenchymal stem cell (MSC), 25, 42, 101, 123, 233 translational application on, 269–271
autologous vs. heterologous, 412 transparent cornea, 176
corneal endothelium regeneration, 138 Oligopotent stem cells, 477
corneal epithelium regeneration Opioid growth factor (OGF), 29
adipose tissue, 130 Optical coherence tomography (OCT), 282, 301, 438, 445
bone marrow, 129 OptiMEM-I based media, 112
fetal tissue, 130 Oral mucus, 127
mesenchymal-to-epithelial transition, 129 Oral mucosa transplantation, 208
corneal stroma regeneration Oral mucosa uses, 226
adipose tissue, 133 Oral stem cell, 134
bone marrow, 133 Osmotic gradient, 422
umbilical cord, 134 Oxidative stress protection, 239
exosome, (see also Extraocular mesenchymal stem
cell), 413
immune modulatory characteristics, 241 P
immunomodulatory properties, 242–244 Palisades of Vogt, 6–7, 179, 180
types, 403 Palpebral conjunctiva, 184
Misdirected lashes, 296 Penetrating keratoplasty (PK), 426, 437, 464
Mitomycin C inhibits, 18 Peptide amphiphiles (PA), 27
Modified embryonic stem cell medium (MESCM), 432 Pericellular matrix from human decidua-derived
MRGPRX3, 424 mesenchymal cells (PCM-DM), 116
Mucin-expressing cord lining epithelial cell (CLEC-­ Periocular mesenchymal progenitors (POMPs), 135
muc), 480 Photorefractive keratectomy (PRK), 18
Mucopolysaccharides, 352 Placental stem cell, 42
Mucosa transplantation, 208–209 Platelet(s)
Multipotent, 42 alpha granule, 318
Multipotent stem cell, 42, 111, 469, 477 blood derivative
Mycophenolate mofetil, 295 inflammation, 323–324
Myofibroblast, 392 properties, 321–323
discovery, 317
electron microscopy, 318
N function, 318
National Programme for Control of Blindness (NPCB), 390 physiological condition, 318
Native human corneal endothelial cells, 110 Platelet-derived growth factor (PDGF), 15, 28,
N-cadherin, 468, 481, 482 319–320, 335
506 Index

Platelet-rich plasma (PRP), 321 Sclerocorneal limbus, 263

antimicrobial activity, 324 Secretory function, corneal endothelium, 422
autologous serum, 327–328 Serum free media (SFM), 46, 432
blood derivative, 324 cell morphology, 47
clinical applications, 328–331 cryopreservation, 47
closed system, 326 expansion, 47
ANGEL®, 327 harvest protocol, 47
PRGF Endoret®, 327 immunophenotype analysis, 47
RegenKit® BCT, 326 isolation, 47
E-PRP, 325–326 multilineage differentiation potential, 47
Pluripotency of iPSCs, 52 quality analysis, 47
Pluripotent stem cell, 42, 111, 477 viability and cell proliferation, 47
corneal epithelium regeneration Severe stem cell deficiency, 227
embryonic stem cell, 124–127 Silk fibroin (SF), 63, 489
induced, 127 Silk fibroin graft, 445
p75 neurotropin receptor (p75NTR), 111 Simple limbal epithelial transplantation (SLET), 161,
Poly (glycerol sebacate)-poly (ε-caprolactone) 214, 222
(PGS-­PCL), 490 advantages, 215
Poly(N-isopropyl acrylamide), 430, 466, 487 complication, 217–218
Polyamide nanofiber scaffold, 251 indication, 215–217
Polycaprolactone (PCL), 466 Skin-derived precursors (SKPs), 137
Poly-L-lactic acid carrier, 250 SLET, see Simple limbal epithelial transplantation (SLET)
Polymegathism, 421 Slit lamp examination, 300
Polymorphism, 421 Slit lamp microscopy, 438, 441
Polysaccharide-based materials, 27 of rabbit corneas, 446
Poly (vinylidene fluoride) membrane, 447, 466 of rat corneas, 446
Poly(vinyl methyl ether) (PVME), 487 Soft contact lenses, 293
Post-mortem techniques, 438 Somatic cell therapy medicinal product (SCTMP), 49
Pre-Descemets layer (PDL), 355–359 Somatic stem cell, 123
PRGF Endoret®, 327 Specular microscopy, 438, 441
Primordium, 271 Sphere(s)
Proliferating cell nuclear antigen (PCNA), 310 cell characteristics, 305
Pterygium, 157 direct compression injury of, 307, 308
Pump function, 422 en masse migration, 306, 307
Pump-leak mechanism, 422 from keratoconic cell, 312
implantation
into diseased central corneal tissue, 311
Q into donor corneoscleral rims, 309
Quality assurance, 399 migratory cells, 304
Quality system approach, 400 on collagen substrate, 304–306
Quantum dots, 440 Sphere formation, 303
Sphere forming assay, 68, 302, 303
Stage-specific embryonic antigens (SSEAs), 45
R Stem cells, 179
Recombinant human collagen (RHC), 29 anterior chamber injection of, 411
Regenerative therapy, 123 advanced therapies with, 48–51
RegenKit® BCT, 326 and cancer, 51–52
Rho-associated kinase (ROCK), 114, 426, 456–458, 465, CEC progenitor cell, 469
467, 492, 494 cell membrane markers, 45
Ripasudil, 465, 466 characterization, 45
Rostock Cornea Module (RCM) object, 364 classification
RTA 408 ophthalmic suspension, 464 potentiality, 42
source, 41
concepts, 41
S culture, 46–48
Scaffold, 26–28 intracellular markers, 45
bioengineered, 27 intrastromal implantation of, 407–409
in ophthalmology, 26 intravenous injection of, 412
peptide amphiphiles, 27 marker, 310
synthetic and natural based, 27 multipotent, 469, 477
Schirmer filter paper test, 282 non-coding RNAs, 45
Schwalbe’s line cells, 110, 427 ocular surface implantation of, 406–407
Index 507

oligopotent, 477 Tenascin C, 182

pluripotent, 468, 477 Terminally differentiated cells (TDC), 78
properties, 477 Tight junctions (TJ), 422
differentiation, 44 Tissue engineered product (TEP), 49
immunomodulation and immunogenicity, 44 Tissue engineering (TE)
self-renewal program, 42 clinical experiences, 29–30
spheres corneal substitute
isolation of sphere with mixed potential, 302–313 bioactive molecules, 29
keratocyte progenitor cells, 300 cells, 25
limbal stem cell, 299–300 scaffolds, 26–28
limbal stem cell deficiency, 300–301 definition, 24
regeneration of limbus, 302 international regulations, 29–30
stromal stem cells, 300 Tonopen, 152
treatment, 301–302 Topical glucocorticoids, 294
sources Total limbal stem cell deficiency (TLSCD), 80
adult tissue, 478 Totipotent stem cells, 42, 477
embryonic tissue, 477 Trabecular meshwork (TM), 491
foetal tissue, 477 Traditional corneal grafting techniques, 278
induced pluripotent stem cells, 478 Transcription factor p63 (ΔNp63α), 266
totipotent, 477 Transforming growth factor beta (TGF-β), 15, 28, 320
transplantation, 215 Transient amplifying cell (TAC), 78, 265, 391
types, 123 Trypsin, 428
Steroids
local inflammation, 294
systemic inflammation, 295 U
Steroid-sparing immunosuppression, 295 Umbilical cord, 478
Stevens-Johnsons syndrome (SJS), 79 blood, 138, 479
Stroma, 13 lining membrane, 479
Bowman’s layer, 350 stem cells in, 478
corneal transparency, 359–360 Umbilical cord blood endothelial progenitor cells
extracellular matrix, 351–352 (UCBEPCS), 482
glycosaminoglycan, 352 Umbilical cord lining epithelium, 129
keratocyte, 353–355 Umbilical cord mesenchymal stem cell
nerves, 355 (UCMSC), 405, 480
pre-descemets layer, 355–359 corneal opacification, 481
Stromal keratocytes, 428 in Lumican deficiency, 481
Stromal regeneration, 480–481 in mucopolysaccharidosis VII, 481
Stromal stem cells, 300 Umbilical cord stem cell, 42
Stromal wound healing, 17–18 Unipotential stem cell, 42
Structure-function relationships, 24 Urokinase-Type Plasminogen Activator (uPA), 14
Subconjunctival injection, 252
Sulforaphane, 464
Superficial corneal epithelial cells, 178 V
Superparamagnetic microspheres, 445 Vascular endothelial growth factor (VEGF), 321, 336
Synthetic cell carriers, 489 Vimentin, 304, 310, 312, 468
Systemic immunosuppressive therapy, 295 Vogt’s striae, 369
Systemic inflammation, 295
Systemic toxicity test, 152
W
Wharton’s jelly, 479
T Wharton’s jelly of the umbilical cord (WJ-MSCs), 236
Tacroliums, 294 Wing cells, 3
TAG-2A12, 117
Tear film health
amniotic membrane, 293 X
hemoderivatives, 292 Xenogeneic grafts, 441, 444
MGD and blepharitis treatment, 292
minimizing drop toxicity, 293
punctal occlusion, 293 Z
soft and scleral contact lenses, 293 ZO-1, 481, 482
tear supplementation, 291 ZP4, 424