STUDIES ON FEEDER LAYERS AND TRANSCRIPTION BASED MARKERS EXPRESSION TO ESTABLISH BUFFALO EMBRYONIC STEM CELLS

THESIS SUBMITTED TO THE NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL (DEEMED UNIVERSITY) IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF

DOCTOR OF PHILOSOPHY IN DAIRYING (ANIMAL BIOTECHNOLOGY)
BY

Dharmendra Kumar
M. V. Sc. (Animal Biotechnology)

ANIMAL BIOTECHNOLOGY CENTRE NATIONAL DAIRY RESEARCH INSTITUTE (DEEMED UNIVERSITY) KARNAL-132 001 (HARYANA), INDIA

2008
Regn. No. 1120502

This Thesis is Devoted to My Dear Maa and Bauji for their Constant Encouragement and Love

ACKNOWLEDGEMENT In the last few years I have had the pleasure to work in an excellent scientific environment with dynamic people. Looking back over this period, I have the feeling that it was a productive and intensive stage of my life. However, to come to this end would not have been possible without the help of many people both from the scientific community and outside of the academic environment. It is a pleasant aspect that I have now the opportunity to express my gratitude for all of them. I want to thank all those who have been generous to me and helped me by any means during past years.

It is a great privilege to express my gratitude to my Major Advisor Dr. R.S. Manik, Principal Scientist, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, India, for his guidance encouragement, moral support and faith in my ability. I consider myself fortunate to work under his supervision. His constant moral boosting and marvelous scientific thoughts brought a perfect shape to this manuscript. My special thanks go to Dr. M.S. Chauhan, Senior Scientist and Dr. P. Palta, Principal Scientist, ABTC, for their constant encouragement and continuous help in terms of suggestion, guidance and moral support during this study. Their logical way of discussion and personal guidance have provided a good basis for understanding various problems and their solutions. I am also thankful to Dr. S.K. Singla, Principal Scientist, ABTC, who gave inspiration and support throughout this work.

I am extremely thankful to all my Advisory Committee members, Dr. M.S. Chauhan, Senior Scientist, ABTC, Dr. A.K. Mohanty, Senior Scientist, ABTC, Dr. T.K. Mohanty, Senior Scientist, DCB Division, Dr. S, Pandita, Principal Scientist, DCP Division and Dr. V.S. Raina, Principal Scientist, DCB Division for their valuable suggestions and constructive criticism during the period of thesis work.

I thank Dr. Sushil Kumar, former Director and Dr. A.K. Srivastava, Director, NDRI, Karnal, Dr. S.L. Goswami, Joint Director (Research) and Officer In-Charge, Animal Biotechnology Centre for providing the necessary facilities for conducting this study. I thank Dr. D. Malakar, Dr. T.K. Datta, Dr. S. De, Dr. A.K. Mohanty and Dr. J.K. Kaushik for providing their valuable suggestions. I wish to express my hearty thanks to my Ph.D. friends- Riaz sir, Manoj, Pradeep (Chullan), Paras, and lab mates Aman, Ruchi, Mushrifa, Ambika, Ayan, Panda, Vijayanand, Ankit, Naresh, Babu and all my seniors Taruna, Anuradha and Yogesh and lovely juniors- Jaspreet (Jassi), Jagdish, Moloya, Sharabani etc. for

CONTENTS
Chapter
Title Page No.

1. 2.

INTRODUCTION REVIEW OF LITERATURE 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 A brief historical account of stem cells Embryonic stem cells Human vs. mouse embryonic stem cells Sources of embryonic stem cells Embryonic stem cell derivation and maintenance Feeder layers and leukemia inhibitory factor Culture of embryonic stem cells on feeder layer Supplementation of embryonic stem cell media Characterization of embryonic stem cells 2.9.1 2.9.2 2.9.3 2.9.4 2.9.5 2.9.5.1 2.9.5.2 2.9.5.3

.... .... .... .... .... .... .... .... .... .... ....

1-3 4-29 4-5 6 6-7 7 8-9 9-11 12-14 14 14-24 15 15 16 16-18 18-23 18-20 21-22 22-23 23-24 25-27 28 28-29 30-43

Morphology of embryonic stem cell colonies .... Cytogenetic analysis Expression of alkaline phosphatase Expression of stage-specific embryonic antigens .... .... ....

Expression of transcription-based .... markers OCT4 NANOG SOX2 .... .... ....

2.9.6 Importance of transcription-based markers: OCT4, NANOG, SOX2 .... 2.10 In vitro differentiation of embryonic stem cells 2.11 Utility of embryonic stem cells in farm animals 2.12 Status in buffalo embryonic stem cells 3. MATERIALS AND METHODS .... .... .... ....

3.1

Materials 3.1.1 3.1.2 3.1.3 Chemicals, cell supplements Equipment culture media and

.... .... .... .... .... .... …. …. …. .... .... ….

30-33 30 31 31-33 31-32 32 32 32-33 33 33-43 33 33 33-36 33-34 34 35 35 35-36 36 36-38 36-37 37 38 38 39-40 40-43 40 41

Glassware and plasticware

3.1.3.1 Microscopes 3.1.3.2 CO2 incubator 3.1.3.3 Thermal cycler 3.1.3.4 3.1.3.5 3.2 Methods 3.2.1 3.2.2 3.2.3 Preparation of different media Collection of buffalo follicular fluid Electrophoresis unit Gel documentation

In vitro maturation and fertilization of oocytes .... .... .... .... ....

3.2.3.1 Collection and grading of oocytes 3.2.3.2 In vitro maturation of oocytes 3.2.3.3 Sperm preparation and in vitro fertilization 3.2.3.4 In vitro culture

3.2.3.5 Determination of total cell number of blastocyst .... 3.2.4 3.2.5 Production of parthenogenetic embryos ….

Production of buffalo embryonic stem cells ....

3.2.5.1 Isolation of embryonic stem cells and their maintenance in culture .... 3.2.5.2 Development of feeder layers 3.2.5.3 Cryopreservation of feeder layers 3.2.6 3.2.7 3.2.8 Preparation of RNase free plasticware Primer designing .... .... .... ....

Characterization of buffalo embryonic stem cells .... ....

3.2.8.1 Alkaline phosphatase staining

3.2.8.2 Reverse transcription polymerase chain reaction (RT-PCR) ....

3.2.8.3 Semi-quantification of RT-PCR products 3.2.8.4 Karyotyping

.... ....

41 42 43 43 44-65 44-45 45 45-53 46-48 48-53 53-56 55-57 57-65 57-58 58-63 59-61 61-63 63

3.2.8.5 Embryoid body formation and induced differentiation in vitro .... 3.2.9 Statistical analysis .... .... .... .... obtained derived from .... from from .... ….

4.

RESULTS AND DISCUSSION 4.1 4.2 4.3 In vitro production of blastocysts Evaluation of total cell number Comparison of feeder different species 4.3.1 4.3.2 4.4 4.5 4.6 layers

Embryonic stem cells hatched blastocysts

Embryonic stem cells derived early/expanded blastocysts

Comparison of the source material for isolation of inner cell mass …. Morphology of buffalo embryonic stem cells Characterization of buffalo embryonic stem cells 4.6.1 4.6.2 4.6.2.1 4.6.2.2 4.6.3 4.6.4 Expression of alkaline phosphatase Expression markers of transcription-based …. …. …. ….

Expression of OCT4 and NANOG in embryonic stem cells …. Expression of OCT4 and NANOG in embryonic stages …. Karyotyping …. Embryiod body formation and induced differentiation in the presence of all-trans …. retinoic acid .... .... ….

63-65 66-72 i-xviii xix-xxiii

5.

SUMMARY AND CONCLUSIONS BIBLIOGRAPHY ANNEXURE

LIST OF FIGURES
Figure No.
1.

Title

After Page No.

Intracellular LIF/gp130/STAT3 activated by LIF. Structure and function of Oct4.

signaling

pathway:

… 10 … 18 … 20

2. 3. 4.

A schematic structure of Nanog gene.

A venn diagram representing the overlap of Oct4, Nanog and Sox2 promoter bound regions in human ES cells … 23 (Boyer et al., 2005). Usable quality of immature buffalo oocytes.
… 45

5. 6.

A group of hatched blastocysts obtained on day 8 post … 45 insemination. A group of hatched and expanded obtained on day 9 post insemination. blastocysts
… 45

7.

8.

Buffalo blastocyst showing inner cell mass (ICM), trophectodermal (TE) cells, blastocoel and broken zona … 45 pellucida (ZP). A hatched buffalo blastocyst obtained on day 8 (A) and expanded blastocyst on day 9 (C) under normal light. Figures (B) and (D) show respective photographs under fluorescence after staining with Hoechst 33342 for cell … 45 count. Total cell number (Mean ± SEM) of hatched blastocysts obtained on day 8 and expanded blastocysts obtained on … 45 day 9. Derivation of primary culture from tissue explants of (A) Buffalo embryonic fibroblast from buffalo fetus, (B) Goat embryonic fibroblast from goat fetus and (C) Sheep 47 embryonic fibroblast from sheep fetus. ... Morphology of different feeder cells at passage 5. (A) Buffalo embryonic fibroblast, (B) Goat embryonic fibroblast and (C) Sheep embryonic fibroblast. The goat 47 feeder cells appeared comparatively thinner and longer. …

9.

10.

11.

12.

Figure No.
13.

Title

After Page No.

Morphology of intact blastocyst during primary culture. (A) In vitro produced buffalo hatched blastocyst collected at day 8 seeded on feeder layer for ICM isolation (ICM indicated in circle), (B) Proliferated ICM (as indicated in circle) after attachment on feeder layer and (C) Established primary colony of buffalo ES cells. … 47 (A) A pulled Pasture’s pipette and (B) A sharp needle used for isolation of ICMs and cutting of buES cell colonies. … 47 The buES cell colonies cultured on different feeder layers. The buES cells on buffalo feeder at passage 4 and 9 (A, B), buES cells on goat feeder at passage 3 and 5 (C, D). … 49 The buES cell colonies cultured on sheep feeder cells at passage 2 and 4 (E and F). … 49 Morphology of enzyme treated early/expanded blastocysts during primary culture; (A) In vitro produced early/expanded buffalo blastocysts collected at day 9, (B) After removal of zona pellucida and trophectodermal cells by enzyme treatment and (C) Morphology of primary colony. … 49 In vitro produced buffalo (A) Hatched blastocyst and (B) Early/expanded blastocysts used for isolation of ICM cells. … 55 Degenerated colonies of buES cells showing (A) floating appearance and (B) lack of compactness and with scattered cells in culture. … 55 Morphology of buES cell colony on buffalo feeder appears compact and dome shaped (A), on goat feeder appears compact and flat (B), whereas on sheep feeder appears scattered (C). … 57 The buES cell colonies showing positive expression of alkaline phosphatase on (A) Buffalo embryonic fibroblast, (B) Goat embryonic fibroblast and (C) Sheep embryonic fibroblast. … 57

14.

15.

15.

16.

17.

18.

19.

20.

21.

RT-PCR analysis for expression of pluripotency marker OCT4 (341bp) in buES cells on each feeder layer. … 59
Title After Page No.

Figure No.
22.

RT-PCR analysis for expression of pluripotency marker. NANOG (211 bp) in buES cells on each feeder layer. … 59 Expression levels of OCT4 and NANOG in buES cells grown on different feeder layers. … 59 Expression of (A) OCT4 gene and (B) actin (house keeping gene in different embryonic stages of in vitro fertilized buffalo embryos. … 63 Expression of (A) OCT4 gene and (B) actin (house keeping) gene in different embryonic stages of parthenogenetically produced buffalo embryos. … 63 Expression of (A) NANOG gene and (B) 18s-rRNA (house keeping) gene in different embryonic stages of in vitro fertilized buffalo embryos. … 63 Expression of (A) NANOG gene and (B) 18s-rRNA (house keeping) gene in different embryonic stages of parthenogenetically produced buffalo embryos. … 63 Karyotype analysis of buES cells at passage 10.
… 63

23.

24.

25.

26.

27.

28. 29.

The buES cells cultured in suspension in the absence of LIF and feeder cells formed (A) embryoid body and (B) cystic embryoid body. … 65 Induced differentiation of buES cells into neuronal cells at passage 4 (A) and 9 (B) in the presence of all-trans retinoic acid (10-6 M). … 65 Characterization of induced neurons by expression of NF-68 gene. _______
… 65

30.

31.

LIST OF TABLES
Table No. 2.1 Title Page No.

Transcription-based markers of undifferentiated embryonic stem cells.

pluripotent

…. …. …. ….

23

2.2

Directed differentiation of ES cells using different growth factors and antagonists. Analysis of differentiated cells through markers. PCR primers used in the present study.

27 27 42

2.3 3.1 4.1

Buffalo blastocysts produced in vitro on Day 8 and 9 post insemination. …. Effect of feeder layers cells from different species on culture characteristics of hatched buffalo blastocysts. …. Effect of feeder layers cells from different species on characterristics of cultured inner cell masses isolated from early/expanded buffalo blastocysts. …. Comparison of source material for isolation and culture of buES cells on buffalo feeder cells. ….
_______

45

4.2

47

4.3

49

4.4

54

ABSTRACT

The present study was carried out to 1) compare different feeder layers by culturing inner cell mass cells, 2) identify expression of transcription based markers in the buffalo embryonic stem cells and 3) examine the differentiation of cultured embryonic stem cells on progressive subculture. For the production of buffalo embryonic stem (buES) cells, intact seeding of hatched blastocysts on feeder layers offered better results than seeding of ICMs isolated from early/expanded blastocysts through enzymatic treatment. Feeder layers prepared from buffalo embryonic fibroblasts (BEF) were superior to those prepared from goat (GEF) or sheep embryonic fibroblasts (SEF) in terms of attachment rate, time required to attach to feeder layers, primary colony formation rate, time required to form primary colonies, maximum number of passages for which ES cells could survive, colony morphology, chromosomal stability and alkaline phosphatase expression. The expression of OCT4 and NANOG was found to be positive in buES cells on all the three types of feeder layers. Whereas the OCT4 expression level was similar on three types of

feeder layers, that of NANOG was higher (P<0.01) in buES cells grown on BEF feeder layers compared to those grown on GEF or SEF feeder layers. Transcripts of OCT4 could be detected in 2-, 4-, 8 to 16-cell, morula and blastocysts stage embryos generated through IVF or parthenegenetic activation, whereas, NANOG expression was observed to begin only at morula stage. When subjected to spontaneous differentiation by suspension culture in the absence of feeder layer and LIF support, buES cells were able to form embryoid bodies. When subjected to induced differentiation in presence of alltrans retinoic acid, they differentiated to neuron-like cells, which were found to express NF-68 gene, a specific marker for neuronal cells confirming that the cells produced were indeed neurons.

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