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ENGINEERING STRATEGIES FOR
REGENERATIVE MEDICINE
 ENGINEERING
STRATEGIES FOR
REGENERATIVE
MEDICINE
Edited by

Tiago G. Fernandes
Maria Margarida Diogo
Joaquim M. S. Cabral
Academic Press is an imprint of Elsevier
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Published by Elsevier Inc. All rights reserved.
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 Contributors

Pedro S. Babo 3B’s Research Group, I3Bs—Research Institute on Biomaterials,

Biodegradables and Biomimetics, University of Minho, Headquarters of the
European Institute of Excellence on Tissue Engineering and Regenerative Med-
icine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra,
Guimarães; ICVS/3B’s—PT Government Associate Laboratory, Braga/Guim-
arães, Portugal
Alexey Bersenev Cell Therapy Laboratory at Yale-New Haven Hospital, Yale
University, New Haven, CT, United States
Adara Bochanis Department of Pharmaceutical Sciences, University of Con-
necticut, Storrs, CT, United States
Joaquim M.S. Cabral Department of Bioengineering and iBB-Institute for Bio-
engineering and Biosciences; The Discoveries Centre for Regenerative and Pre-
cision Medicine, Lisbon Campus, Instituto Superior Técnico (IST), Universidade
de Lisboa, Lisbon, Portugal
João P. Cotovio Department of Bioengineering and iBB-Institute for Bioengi-
neering and Biosciences; The Discoveries Centre for Regenerative and Precision
Medicine, Lisbon Campus, Instituto Superior Técnico (IST), Universidade de
Lisboa, Lisbon, Portugal
Pedro Silva Couto Advanced Centre for Biochemical Engineering, Department
of Biochemical Engineering, University College London, London, United
Kingdom
Maria Margarida Diogo Department of Bioengineering and iBB-Institute for
Bioengineering and Biosciences; The Discoveries Centre for Regenerative and
Precision Medicine, Lisbon Campus, Instituto Superior Técnico (IST), Universi-
dade de Lisboa, Lisbon, Portugal
Jonathan S. Dordick Department of Chemical and Biological Engineering and
Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic
Institute, Troy, NY, United States
Tiago G. Fernandes Department of Bioengineering and iBB-Institute for Bioen-
gineering and Biosciences; The Discoveries Centre for Regenerative and Preci-
sion Medicine, Lisbon Campus, Instituto Superior Técnico (IST), Universidade
de Lisboa, Lisbon, Portugal
Manuela E. Gomes 3B’s Research Group, I3Bs—Research Institute on Bioma-
terials, Biodegradables and Biomimetics, University of Minho, Headquarters
of the European Institute of Excellence on Tissue Engineering and Regenera-
tive Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da
Gandra; ICVS/3B’s—PT Government Associate Laboratory, Braga/Guim-
arães; The Discoveries Centre for Regenerative and Precision Medicine, Head-
quarters at University of Minho, Guimarães, Portugal

vii
viii Contributors

Manuel Gomez-Florit 3B’s Research Group, I3Bs—Research Institute on Bioma-

terials, Biodegradables and Biomimetics, University of Minho, Headquarters of
the European Institute of Excellence on Tissue Engineering and Regenerative
Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra,
Guimarães; ICVS/3B’s—PT Government Associate Laboratory, Braga/Guim-
arães, Portugal
Ana I. Gonçalves 3B’s Research Group, I3Bs—Research Institute on Biomateri-
als, Biodegradables and Biomimetics, University of Minho, Headquarters of the
European Institute of Excellence on Tissue Engineering and Regenerative Med-
icine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra,
Guimarães; ICVS/3B’s—PT Government Associate Laboratory, Braga/Guim-
arães, Portugal
Gyuhyung Jin University of Wisconsin-Madison, Chemical and Biological Engi-
neering, Madison, WI, United States
Soowan Lee Department of Pharmaceutical Sciences, University of Connecticut,
Storrs, CT, United States
Sean P. Palecek University of Wisconsin-Madison, Chemical and Biological
Engineering, Madison, WI, United States
Qasim A. Rafiq Advanced Centre for Biochemical Engineering, Department of
Biochemical Engineering, University College London, London, United Kingdom
Theodore P. Rasmussen Department of Pharmaceutical Sciences; Institute for
Systems Genomics, Storrs/Farmington; University of Connecticut Stem Cell
Institute, University of Connecticut, Storrs, CT, United States
Rui L. Reis 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Bio-
degradables and Biomimetics, University of Minho, Headquarters of the Euro-
pean Institute of Excellence on Tissue Engineering and Regenerative Medicine,
AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra;
ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimarães; The Dis-
coveries Centre for Regenerative and Precision Medicine, Headquarters at Uni-
versity of Minho, Guimarães, Portugal
Márcia T. Rodrigues 3B’s Research Group, I3Bs—Research Institute on Bioma-
terials, Biodegradables and Biomimetics, University of Minho, Headquarters of
the European Institute of Excellence on Tissue Engineering and Regenerative
Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra;
ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimarães; The
­Discoveries Centre for Regenerative and Precision Medicine, Headquarters at
University of Minho, Guimarães, Portugal
André L. Rodrigues Department of Bioengineering and iBB-Institute for Bioen-
gineering and Biosciences; The Discoveries Centre for Regenerative and Preci-
sion Medicine, Lisbon Campus, Instituto Superior Técnico (IST), Universidade
de Lisboa, Lisbon, Portugal
Shahin Shams Department of Biomedical Engineering, University of California,
Davis, Davis, CA, United States
Eduardo A. Silva Department of Biomedical Engineering, University of Califor-
nia, Davis, Davis, CA, United States
 Engineering strategies for
regenerative medicine

In Greek mythology Prometheus was a Titan who was credited with the
forging of the first humans out of clay, and later on with providing man-
kind the gift of fire after tricking and stealing it from the gods. For that
action, once believed to allow the progress and advancement of civiliza-
tion, he was eternally punished by Zeus, the king of the Olympian Gods.
According to this myth, Prometheus was chained to a rock and each day
an eagle, the symbol of Zeus, was sent to feed on his liver, which would
then grow back overnight to be eaten again the following day.
This myth is often used to illustrate the remarkable capacity of certain
organs and tissues to regenerate themselves. In humans, this capacity if
frequently incomplete and, after damage or necrosis, comes fibrosis or the
formation of scar tissue [1]. However, some organisms like salamanders
and newts are able to regenerate entire limbs, tails, and a variety of other
body structures [2]. Regenerative medicine, an interdisciplinary field that
applies engineering and life science principles to promote regeneration,
can potentially restore diseased and injured tissues and whole organs in
humans [3]. This convergence between engineering and medicine thus
aims to maintain, recover, and improve the function of damaged tissues
and organs, as well as the production of new engineered tissues that may
be used to improve the quality of life in chronic patients. Therefore, re-
generative medicine integrates several domains, particularly the biology
and engineering of stem cells, tissue and organ engineering, genetic and
cellular therapies, biomaterial science and technology, micro- and nano-
technologies, and bioprocess engineering [4].
Regenerative medicine also offers unique technologies and treatments
for unmet clinical needs. Some of the potential applications include trans-
plantation of stem cells, progenitors, tissues, or organs [5], stimulation of
endogenous repairing mechanisms [6], the use of cells as gene expression
vehicles for the controlled delivery of cytokines and other biological mole-
cules [7], and cellular engineering/synthetic biology [8]. This represents a
market predicted to reach $67.5 billion by 2020, according to the report by
Allied Market Research [9]. For example, immunotherapy is also quickly
moving to the bedside, and advanced manufacturing has the potential to
recapitulate the native microenvironment and produce cellular surrogates
with high degrees of functionality [10].

ix
x Engineering strategies for regenerative medicine

In this context, this book starts by providing an in-depth overview of

pluripotent stem cell biology and the engineering tools that can harness
their full potential for regenerative medicine applications. Cotovio and
coworkers have explored in Chapter 1 some of these questions and sum-
marized key biological features of human pluripotent stem cells (hPSCs),
some of the foreseen applications in cellular therapy, disease modeling
and pharmaceutical testing, the bioengineering approaches used to ex-
plore stem cell systems at the cellular, multicellular and multiorgan lev-
els, and how this progress could potentially revolutionize clinical practice
and drug development. Following this, Couto, Bersenev, and Rafiq have
discussed process development and manufacturing approaches for mes-
enchymal stem/stromal cell therapies in Chapter 2. In fact, one of the cell
types under evaluation in numerous clinical trials are human mesenchy-
mal stem/stromal cells (hMSCs) that possess both immunomodulatory
properties and in vitro differentiation capability. These properties have
been explored for conditions such as graft vs host disease, acute myocar-
dial infarction and Crohn disease, among others. One serious limitation,
however, is the inability to generate clinically relevant number of cells to
meet the dose criteria, while retaining the product’s critical quality attri-
butes. Therefore, several challenges that manufacturers face during each
stage of the hMSC handling, from isolation to expansion, downstream
processing, and finally product formulation, are explored in this chapter.
In Chapter 3, Rodrigues and coworkers have discussed how biomate-
rials and tissue engineering approaches can be used to help the regener-
ation of musculoskeletal tissues. Since these tissues are highly prone to
injuries, conditions afflicting them have a great impact on the quality of
life of patients worldwide. Particularly, with the increase in life expec-
tancy and the maintenance of an active lifestyle by the aging population,
the cumulative musculoskeletal conditions potentiate disability through-
out life. Therefore, the economic impact of the United States alone was
estimated to be $796.3 billion in 2009–11 [11]. The authors have discussed
how the exploitation of materials and scaffolds to modulate cell responses
potentially contribute to the functional regeneration of bone, cartilage,
and tendon after injury. In the following chapter (Chapter 4), Shams and
Silva have further highlighted how materials can be used in regenerative
medicine. Particularly, they looked at d ­ elivery strategies using biomateri-
als for gene therapy applications focusing on vascular regeneration and
neurodegenerative diseases.
Stem cell-based microscale platforms, particularly the ones using
­patient-specific hPSCs, may prompt the development of phenotypic drug
discovery assays that would allow the identification of personalized drug
responses. In Chapter 5, Rodrigues and coworkers have outlined several
microscale high-throughput screening platforms that have been employed
in the regenerative medicine field. They have also provided examples of
 Engineering strategies for regenerative medicine xi

specific studies employing stem cells and derived progeny for screening
drug compounds and microenvironmental cues influencing cell behavior.
The impact of microscale technologies on early identification of poten-
tial new drug candidates may ultimately decrease both attrition rates and
capital investment in the drug development process, and further help the
progress of regenerative medicine applications.
In the following chapter (Chapter 6), Jin and Palecek have provided
an overview on how hPSC-derived cardiomyocytes have emerged as a
promising cell source to regenerate damaged heart tissue. Ischemic heart
disease is one of the leading causes of death worldwide and even though
some therapeutic options are available following heart attack, such as
pharmacological therapies, bypass surgery, valve surgery, stent implanta-
tion, and pacemaker implantation, most of these treatments are designed
to prevent recurring heart failure, attenuate symptoms, and assist cardiac
function. Since they do not provide restoration of physiological heart func-
tion, numerous efforts in the field of cardiac regenerative medicine have
been made to replace necrotic cardiomyocytes, thereby restoring the func-
tion to the damaged heart. Particularly, understanding the effects and un-
derlying mechanism of inductive factors on cardiomyocyte differentiation
from hPSCs may provide important clues to address some of the exist-
ing challenges and finally provide regenerative therapies for devastating
and lethal heart conditions. Lastly, one additional application explored in
Chapter 7 by Lee, Bochanis, and Rasmussen is the use of hPSCs as models
for early embryogenesis, and to precisely evaluate the risk of fetal expo-
sure to teratogens. The development of in vitro assays for the detection
of teratogens and developmental toxins is highlighted in this chapter, as
well as the current state of PSC-based platforms for the detection of com-
pounds that pose a risk to human developmental programs, and for the
ascertainment of teratogenic mechanisms at cellular and molecular levels.
These examples summarize some of the most compelling and innova-
tive approaches being explored in regenerative medicine. While the list
is not overly comprehensive, it for sure provides a collective impression
that medicinal practice can now focus on regenerating diseased tissues
and recover lost functionality. It was our aim to provide relevant and up-
dated content on these topics, and we hope that the reader will be more
informed about this exciting field.
Tiago G. Fernandes
Maria Margarida Diogo
Joaquim M.S. Cabral
Department of Bioengineering, Instituto Superior Técnico,
The University of Lisbon, Lisbon, Portugal
xii Engineering strategies for regenerative medicine

References
[1] Atala A, Irvine DJ, Moses M, Shaunak S. Wound healing versus regeneration: role of the
tissue environment in regenerative medicine. MRS Bull 2010;35(8):597–606. https://doi.
org/10.1557/mrs2010.528.
[2] Brockes JP, Kumar A. Plasticity and reprogramming of differentiated cells in amphibian
regeneration. Nat Rev Mol Cell Biol 2002;3(8):566–74. https://doi.org/10.1038/nrm881.
[3] Mao AS, Mooney DJ. Regenerative medicine: current therapies and future directions.
Proc Natl Acad Sci 2015;112(47):14452–9. https://doi.org/10.1073/pnas.1508520112.
[4] Tewary M, Shakiba N, Zandstra PW. Stem cell bioengineering: building from stem cell bi-
ology. Nat Rev Genet 2018;19(10):595–614. https://doi.org/10.1038/s41576-018-0040-z.
[5] Bajaj P, Schweller RM, Khademhosseini A, West JL, Bashir R. 3D biofabrication strat-
egies for tissue engineering and regenerative medicine. Annu Rev Biomed Eng
2014;16(1):247–76. https://doi.org/10.1146/annurev-bioeng-071813-105155.
[6] Kami D, Gojo S. Tuning cell fate. Organogenesis 2014;10(2):231–40. https://doi.
org/10.4161/org.28816.
[7] Nowakowski A, Drela K, Rozycka J, Janowski M, Lukomska B. Engineered mesen-
chymal stem cells as an anti-cancer trojan horse. Stem Cells Dev 2016;25(20):1513–31.
https://doi.org/10.1089/scd.2016.0120.
[8] Pawlowski M, Ortmann D, Bertero A, Tavares JM, Pedersen RA, Vallier L, Kotter MRN.
Inducible and deterministic forward programming of human pluripotent stem cells
into neurons, skeletal myocytes, and oligodendrocytes. Stem Cell Rep 2017;8(4):803–12.
https://doi.org/10.1016/j.stemcr.2017.02.016.
[9] Allickson J. Emerging translation of regenerative therapies. Clin Pharmacol Ther
2017;101(1):28–30. https://doi.org/10.1002/cpt.549.
[10] Lipsitz YY, Timmins NE, Zandstra PW. Quality cell therapy manufacturing by design.
Nat Biotechnol 2016;34(4):393–400. https://doi.org/10.1038/nbt.3525.
[11] Yelin E, Weinstein S, King T. The burden of musculoskeletal diseases in the United
States. Semin Arthritis Rheum 2016;46(3):259–60. https://doi.org/10.1016/
j.semarthrit.2016.07.013.
 C H A P T E R

1
Pluripotent stem cell biology
and engineering
João P. Cotovioa,b, Tiago G. Fernandesa,b,
Maria Margarida Diogoa,b, Joaquim M.S. Cabrala,b
a
Department of Bioengineering and iBB-Institute for Bioengineering
and Biosciences, Instituto Superior Técnico (IST), Universidade de
Lisboa, Lisbon, Portugal, bThe Discoveries Centre for Regenerative and
Precision Medicine, Lisbon Campus, Instituto Superior Técnico (IST),
Universidade de Lisboa, Lisbon, Portugal

1 Stem cells

In 1868 Ernst Haeckel [1], a notable biologist from Germany, came up

with the term Stammzelle to describe the unicellular ancestor from which
all multicellular organisms evolved, a concept very different from the
one existing today. Later, he used the same term to describe the fertilized
egg, or the zygote, capable of giving rise to all cell types of an organ-
ism [2]. This was the cradle for the English term “stem cell” (Fig. 1.1)
[3]. After previous studies on the continuity of the germ plasm and on
the origin of the hematopoietic system, Till and McCulloch proposed
in the 1960s what are still today the two gold standard features of stem
cells: (1) undifferentiated cells that are capable of self-renewal and (2) the
production of specialized progeny through differentiation [4]. To accom-
plish such attributes, it is now known that stem cells undergo asymmet-
ric cell division, by which the cell divides to generate one stem cell and
one differentiating cell. Therefore, it may be added that stem cells are of
major importance in the maintenance of homeostasis through a balance
between self-renewal and differentiation [5–7].
From conception to death, as cells develop derived from embryonic tis-
sue, they become progressively restricted in their developmental potency,
reaching the point when each cell can only differentiate into a s­ ingle ­specific

Copyright © 2020 Tiago G. Fernandes, Maria Margarida Diogo & Joaquim M. S. Cabral.
Published by Elsevier Inc. All rights reserved.
https://doi.org/10.1016/B978-0-12-816221-7.00001-X
2 1. Pluripotent stem cell biology and engineering

FIG. 1.1 Stem cell research timeline. Key events and technological breakthroughs in
stem cell research. EC, embryonal carcinoma; hESCs, human embryonic stem cells; hiPSCs,
­human-induced pluripotent stem cells; iPSCs, induced pluripotent stem cells; mESCs, mouse
embryonic stem cells.

cell type. In the beginning, the earliest cells in ontogeny are totipotent, giv-
ing rise, in mammals, to all embryonic and extraembryonic tissues, that is,
only a totipotent cell can originate an entire organism [8, 9]. Through em-
bryogenesis, when the pluripotent state is reached, a pluripotent stem cell
(PSC) can originate all the cells from all the tissues of the body, although
the contributions to the extraembryonic membranes or placenta are limited
[8]. On the other hand, a multipotent stem cell is restricted to the genera-
tion of the mature cell type of its tissue of origin and finally, a unipotent
stem cell displays limited developmental potential, giving rise to only a
single-cell type [8]. In an adult organism, stem cells can be found in most
tissues throughout the body, even within relatively dormant tissues. These
stem cells experience low or no division in normal homeostasis, remaining
quiescent for extended periods of time. However, these cells can respond
efficiently to stimuli upon initiation of homeostasis or injury [10].
Altogether, in both plant and animal kingdoms, the multicellularity of
highly regulated tissues is dependent of the generation of new cells for
growth and repair. Therefore, biological systems are driven by a balance
between cell death and cell proliferation, preserving form and function in
tissues. From this point of view, stem cells are the units of the following
attributes: development, regeneration, and evolution [9, 11].

1.1 Pluripotent stem cells

Pluripotency can be defined as a transient property of cells within the
early embryo, where PSCs have the capacity to form tissues of all three
germ layers of the developing embryo and, later, the organs of the adult
organism—ectoderm, mesoderm, and endoderm—and still the germ lin-
eage. As previously mentioned, PSCs typically provide little or no contri-
bution to the trophoblast layers of the placenta [8, 12].
The first PSCs to be isolated and investigated in culture were derived
from mouse teratocarcinomas—a tumor of germ cell origin that maintain a
 1 Stem cells 3

wide variety of diversely differentiated tissues—known as embryonal car-

cinoma (EC) cells [8, 13]. Nevertheless, PSCs can be isolated from several
sources through development [14], as murine [15, 16] and human blasto-
cyst [17] or even from the postimplantation epiblast [18, 19] or germ line
[20, 21]. Also, pluripotency can be recapitulated in vitro by reprogramming
somatic cells to become induced pluripotent stem cells (iPSCs) [22–24].
There are specific molecular mechanisms that characterize PSCs an-
chored by a selected set of core transcription factors essential to estab-
lish pluripotency. As part of the core pluripotency transcription factors
encoding genes are octamer-binding transcription factor 4 (OCT4),
SRY-box 2 (SOX2), and NANOG. In certain circumstances, the loss of
SOX2 or NANOG or their substitution can be tolerated [8, 12]. Despite
that, PSCs can be classified into different states of pluripotency based
on the molecular signatures, with the terms “naïve” and “primed” be-
ing introduced to describe early and late phases of ontogeny, respec-
tively [25].
Pluripotency can be suggested by such molecular signatures, but
only functional assays can reveal the developmental potential of a cell.
Functional assays to assess pluripotency include: differentiation into three
germ layers in vitro, teratoma formation in vivo, chimaera formation, ger-
mline transmission through blastocyst injection, tetraploid complementa-
tion, and single-cell chimaera formation [8]. For human pluripotent stem
cells (hPSCs), teratoma formation remains the gold standard of functional
assays.

1.1.1 Embryonic stem cells

Mammalian embryogenesis starts with a single totipotent cell, the zy-
gote. After the first cell division, the two-cell embryo is composed by two
equal blastomeres. In the earlier stages, including two- and four-cell em-
bryos, cells are still considered totipotent. Later, in what is called blasto-
cyst, it is possible to distinguish the extraembryonic trophectoderm (TE)
on the outside and the inner cell mass (ICM) [14, 26]. It is in the ICM that
pluripotent cells first arise. The ICM cells, cultured in conditions that al-
low indefinite self-renewal and maintenance of the pluripotent state, are
known as embryonic stem cells (ESCs) and were first derived from mouse
(mESCs) in 1981 by Martin Evans [15] and Gail Martin [16]. These cul-
tures proved to have all the properties previously established for EC cell
cultures, as well as a completely normal karyotype [27]. Only by the year
1998, Thomson derived the first ESC lines from human blastocysts [17],
the so-called human embryonic stem cells (hESCs).
Throughout normal development, the amount of ESCs is limited and
their existence is constrained in the time course of development, being
present for only a short period of time. In contrast, tissue culture allows
the generation and maintenance of millions of ESCs indefinitely, preserv-
ing their pluripotent state [27].
4 1. Pluripotent stem cell biology and engineering

1.1.2 Induced pluripotent stem cells

The molecular mechanisms “by which the genes of the genotype bring
about phenotypic effects”—the epigenetics concept—was captured by
Conrad Waddington [28] in the iconic image of the epigenetic landscape
that influences cellular fate during development, analogously to the move-
ment of a marble (Fig. 1.2). Since then, the possibility that cells can change
their identity has fascinated scientists [29]. This notion was first suggested
by Sir John Gurdon in 1958, establishing that in vivo plasticity of the differ-
entiated state can be induced artificially by directly manipulating cells and
their environment [30]. It was demonstrated that the marble can be rolled
back to the top of the hill, that is, committed or differentiated cells can be
reprogrammed back to a wider developmental potential (dedifferentiation).
As the possibility to reprogram cells, not by transplanting their nuclei,
but by introducing pluripotency factors into cells became a reality, cells
with a gene expression profile and developmental potential similar to ESCs
were generated in 2006. This accomplishment was reached using mouse
somatic cells together with a cocktail of four transcription factors [23]. The
resulting reprogrammed cells were termed iPSCs and were generated after
retrovirally introducing four transcription factors encoding genes, OCT4,
SOX2, Kruppel-like factor 4 (KLF4) and the MYC proto-oncogene, bHLH
transcription factor (MYC)—the “Yamanaka factors.” After successfully
generating mouse iPSCs, in 2007 iPSCs were generated from human fibro-
blasts, using the same four factors and alternatively NANOG and Lin-28
homolog A (LIN28) instead of KLF4 and MYC [22, 24]. It was the estab-
lishment of human induced pluripotent stem cells (hiPSCs). Since then,
cellular reprogramming became a robust method to convert differentiated
cells to a PSC state [31, 32].

FIG. 1.2 Cell fate plasticity. Contemporary version of Waddington landscape depicting
an analogy between a marble rolling downhill as development leads undifferentiated cells
to a mature state. Cellular reprogramming has shown that it is possible to make the marble
roll back to the top of the hill as mature cells can be reprogrammed back to a wider devel-
opmental potential.
 1 Stem cells 5

Afterwards, besides the initially used retroviral or lentiviral vectors,

nonintegrating methods have been developed and include reprogram-
ming using episomal DNAs, adenovirus, Sendai virus, PiggyBac trans-
posons, minicircles, recombinant proteins, synthetically modified mRNAs,
microRNAs (miRNAs), and more recently, small molecules [31]. These new
techniques, in addition to lower variability between cell lines, can lead to
safer reprogramming of iPSCs and to more suitable cells for clinical ap-
plications by avoiding insertional mutagenesis and transgene reactivation.

1.2 Applications of hPSCs

Since the isolation of ESCs from human embryos, the use of PSCs as
a potential tool for research and medicine has been growing (Fig. 1.3).
Besides that, after finding that somatic cells can revert all the way back
to an ESC state through transcription factor activation, manipulation of
signaling pathways aiming for cell differentiation has been studied con-
tributing to hiPSCs applications in biomedicine. Accordingly, several pro-
tocols have been described for in vitro direct differentiation of neurons,
hematopoietic cells, hepatocytes, smooth muscle cells, and cardiomyo-
cytes, among other cell types across the three germ layers [33].
An obvious application of hPSCs in medicine is in cell therapy.
Regenerative medicine strategies based on the use of stem cells to promote
regeneration or to replace damaged tissues after cellular transplantation
has been shown to successfully induce functional recoveries [31]. In fact,
several clinical trials were already established using hPSC-based therapies
[34]. In the particular case of hiPSCs, an important advantage of ­using

FIG. 1.3 Clinical applications of human induced pluripotent stem cells for precision med-
icine. After isolation of patient somatic cells, these cells can be cultured and reprogrammed
into patient-specific induced pluripotent stem cells (iPSCs). This is a promising cell source
for cell therapy as it is possible to silence mutations carried by patient-specific cells using
novel gene-editing tools (like CRISPR-Cas9) for the generation of corrected iPSCs that can be
used in regenerative medicine approaches. Also, the differentiation of patient-specific iPSCs
makes disease modeling and drug screening a possibility.
6 1. Pluripotent stem cell biology and engineering

these cells is the capability to generate autologous differentiated cells,

that is, patient-specific cells, theoretically suppressing the risk of immune
rejection. For instance, the first clinical study using hiPSC-derived prod-
ucts was performed in 2014 by Masayo Takahashi and Yasuo Kurimoto,
in which these two Japanese physicians successfully transplanted autolo-
gous retinal pigment epithelium sheets derived from hiPSCs into a woman
with macular degeneration [35]. Besides all this progress in ­hPSC-based
therapies, the acquisition of chromosomal aberrations, due to the repro-
graming process and subsequent culture, represents one of the disadvan-
tages of these cells [36]. Moreover, due to hPSC tumorigenicity, it is critical
to ensure that the transplanted product does not contain undifferentiated
cells with the potential to generate teratomas [31].
Another important biomedical application of hPSCs is in disease mod-
eling [37]. It is expected that in vitro hPSC-based disease models help to
identify the pathological mechanisms underlying human diseases. Both
hESCs and hiPSCs have been used for modeling human genetic diseases,
establishing isogenic cell lines with novel gene-editing tools (e.g., CRISPR-
Cas9), to induce disease-causing mutations or to silence mutations carried
by patient-specific cells [38, 39].
Modeling of human diseases is motivated by the necessity of develop-
ing novel therapeutic agents allowing the diseases to be treated, allevi-
ated, or cured. Therefore, drug screening and toxicological assays is also
considered as a potential application of hPSCs [37]. Animal models have
been used in drug screening but differences from the actual human set-
ting lead to an inaccurate forecasting of their effects. Moreover, animal
models are not suitable for high-throughput screening of small-molecule
­libraries [38, 40]. Until now, many drug screens have been conducted us-
ing hiPSC-based models and potential drug candidates have been iden-
tified. Also, it is not only important to assess efficacy but also toxicity,
predicting the likelihood of candidate drugs to cause serious side effects
[31]. A specific patient has a specific genetic background and this fact im-
plies different responses to medication for each individual. Accordingly,
hiPSC-based drug screening is the key for a personalized therapy, an
emerging approach known as precision medicine [40].
Just as new technologies are being developed, the greater will be the
potential applicability of hPSCs in the emerging fields of regenerative
medicine, disease modeling, and drug screening.

2 Stem cell systems

For a widespread use of stem cells in biomedical applications, it is es-

sential to understand the complexity and the dynamics of the stem cell
biological system as well as the information that flows between each layer
 2 Stem cell systems 7

FIG. 1.4 Layers of complexity within biological systems. Different layers of complexity of
the biological systems from the cell level to the whole organism and respective engineering
approaches. These approaches can help to understand each biological layer, and to a great
extent, they can help to create tissues that resemble the in vivo anatomy and physiology of
the human body for biomedical applications.

of it. In this section, a reductionist view of the different layers of complex-

ity of the biological systems is presented (Fig. 1.4), exploring this question
from the cell level to the whole organism.

2.1 The cell: The functional unit of the system

The cell by itself can be considered the functional unit of a biological
system. Furthermore, within each cell and as a first layer of regulation,
there are collections of molecular species that interact and constitute what
are called gene regulatory networks (GRNs), a term first coined in sea
urchin developmental studies [41]. The GRNs, based on the microenvi-
ronmental signals (input), are able to control gene expression levels and,
consequently, gene product abundance (output). Thus, the cellular GRN
is responsible for the development potential of the cell by regulating its
transcriptional activity [42, 43]. For example, the pluripotent state of a
PSC is determined by a set of pluripotency transcription factors, namely
OCT4, SOX2, and NANOG. These transcription factors are the core of the
pluripotency GRN and, therefore, they cooperate and regulate different
elements in the genome, including their own promoters [12, 44].
Besides that, additional factors that constitute regulatory inputs to
the pluripotency GRN can include proteins, both activators and repres-
sors, which will regulate gene expression at the transcription initiation
stage. Moreover, another form of transcriptional regulation is based on
the epigenetic information, namely chemical modifications or changes in
the 3D organization of the chromatin [45]. These regulatory mechanisms
are responsible for the unique epigenetic plasticity of PSCs shaping the
transcriptional events during lineage commitment. Finally, RNA-based
regulatory mechanisms also take part in the pluripotency GRN. Examples
 3 Stem cell engineering 19

the so-called bioink, to precisely positioning them in a layer-by-layer

­manner [127, 157]. Indeed, this technology has already been applied to
many stem cell derivatives [158] and a good example of that is the bio-
printing of hiPSC-derived hepatic tissue [148]. In this work, the assembly
of hiPSC-derived hepatocytes with other supportive cells, featured in the
human liver, was performed using a microscale hexagonal architecture.
This strategy led to improved morphological organization, upregulation
of liver-specific genes, and enhanced functionality.

3.2.2 Delivery of biochemical signals

Most cellular functions, from the embryo to adulthood, depend on the
continuous supply of nutrients and soluble factors to the cells. Analogously,
bioengineers have used different strategies to properly deliver such fac-
tors to stem cell-derived tissues and organoids [127]. One possible solu-
tion is the integration of endothelial cells (ECs) or their progenitors in the
engineered constructs or tissues [126]. As an illustrative example, recent
progresses have been made in liver organoids with the first report using
a progenitor population of adult mouse liver [149] and, in a subsequent
study, a human ­liver-like organoid structure called liver bud being gener-
ated by mixing three cell populations: hepatic endodermal cells derived
from hiPSCs, human ECs and human mesenchymal cells [137]. This ap-
proach could recapitulate the early steps of hepatogenesis ending in a vas-
cularized human liver bud organoid that showed improved functionality
in producing key liver enzymes. More recently, the same authors were
able to generate vascularized liver bud organoids by only using hiPSCs
as a cell source for the tree cell types [144]. It is important to highlight
that this kind of approach also improves the biophysical properties of the
tissue by facilitating oxygen delivery to the cells.
In fact, vascularization facilitates the delivery of nutrients and soluble
factors through the tissue, but desired soluble factors can be delivered as
well using special platforms designed for this intent. Traditionally, these
soluble signaling molecules are presented in a uniform fashion to the cells
cultured in vitro but, once again, spatiotemporal control of biochemical
signals is crucial. For example, delivery of such cues can be achieved by
means of light-triggered activation of caged molecules that are masked by
a photodegradable moiety in a hydrogel [150, 156]. Also, microbeads and
nanoparticles, in all possible forms, play an important role in the spatio-
temporal control of biochemical signals. These platforms can be loaded
with required signaling molecules and conjugated on the surface of the
cells [127]. The interesting part is that they can release its content in a bio-
responsive manner, that is, the content is only released when proper mo-
lecular cues are present.
Microfluidic devices can also be used to deliver soluble ligands mim-
icking signaling gradients present in vivo. This technology not only has
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