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Directed Differentiation of Human Embryonic Stem Cells Into Haematopoietic and Definitive Endodermal Lineages

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Directed Differentiation of Human Embryonic Stem Cells Into Haematopoietic and Definitive Endodermal Lineages DIRECTED DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS INTO HAEMATOPOIETIC AND DEFINITIVE ENDODERMAL LINEAGES ABRAHAM SUMAN MARY (M.Sc MICROBIOLOGY, UNIV. OF MUMBAI, INDIA) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS In all things I give YOU glory! You have always led me through amazing paths and given me gifts that I don’t deserve. I thank you Lord for all the blessings you constantly shower on me. Everything is possible with God! I thank Dr. Alan Colman for being my guide and helping me to initiate the work contained in this dissertation. His support and encouragement have been invaluable. Thank you, Alan for your support through the years. Dr. Norris Ray Dunn took me under his wing and guided me through this endeavour. He has been a true mentor, always willing to teach, and I have learned a lot from him. Ray, thank you for showing me the way and helping me reach this juncture. A significant part of the research was conducted at ES Cell International Pte Ltd to whom I would like to express my sincere gratitude. Triona, Jacqui, Robert, Michael, Bruce, Chirag, Suzan and so many others have played important roles and encouraged me at all times. Critical portions of this work were done at the Institute of Medical Biology, A*Star. I would like to register my appreciation for the support and help provided by many people in IMB especially, members of the Ray Dunn lab, Mike Jones lab and Alan Colman lab. Kee Yew, thank you for giving me your precious time and helping me with some of the most important data in this dissertation. I would like to thank the staff at the Department of Biochemistry and Yong Loo Lin School of Medicine at the National University of Singapore for providing administrative support. Through the years many colleagues and friends have expressed their concern, spoken encouraging words and pushed me to get to the finish. I thank you all. Varsha, Akila, Raj, Suzan and Thava, Chaaya, Ajith and Surinder– you have seen my struggles, listened to my problems and never hesitated to help. Thank you. My teachers through the years have influenced me in so many fantastic ways. I remember you all and thank God that you were in my life. Ms. Susan, Mrs. Chinnamma Kovoor, Mrs. Phadnis, Mrs. Shantini Nair, Mrs. Vaidya, Mrs. V.P.Kale and George– thank you. None of this would have been possible but for the support system my family has been. Thank you, Appa and Mummy for all the love and laughter and for inspiring me to follow my heart. Sumitha, Aji and Tamanna, and Susan, thank you for the joy you bring to my life and my world. Pappa and Mamma, thank you for your love and unconditional support. My grandparents and the rest of my extended family have been encouraging me whole-heartedly and I sincerely thank all of them. Ajit, you are my strength and joy. You have been waiting patiently, working so hard not to distract me and making me smile even when things were tough. Thank you for being by my side, egging me on and never losing faith in me. You’re simply the best! i TABLE OF CONTENTS Summary…………………………………………………………………………..….v List of tables………………………………………………………………………....vii List of figures…………………………………………………………………….....viii List of symbols………………………………………………………………………..x Chapter 1: General Introduction 1.1. Embryonic stem cells..............................................................................................1 1.2. Gastrulation– formation of mesoderm and endoderm in the embryo.....................8 1.3. Regenerative medicine and embryonic stem cells................................................10 1.3.1. Diabetes– a candidate disease for cell therapy .........................................10 1.3.2. Transplantation tolerance of hESC-derived cell therapy ..........................10 1.4. Haematopoiesis.....................................................................................................18 1.4.1. Haematopietic development in the mouse..................................................18 1.4.2. Haematopoiesis in the human embryo.......................................................25 1.4.3. Haematopoietic differentiation from mESCs.............................................26 1.4.4. Haematopoietic differentiation from hESCs..............................................29 1.5. Definitive endoderm formation in the vertebrate embryo ....................................34 1.5.1. Differentiation of mESCs to endodermal derivatives ................................40 1.5.2. Endodermal differentiation from hESCs....................................................45 Chapter 2: Materials and Methods 2.1. Cell culture............................................................................................................52 2.1.1. Human embryonic stem cell culture ..........................................................52 2.1.2. Stromal feeder cells....................................................................................53 2.2. Differentiation protocols.......................................................................................53 2.2.1. Haematopoietic differentiation: Co-culture with stromal cell lines..........53 2.2.2. Haematopoietic differentiation: Use of cytokines......................................54 2.2.3. Endodermal differentiation: 3D Matrigel protocol ...................................55 2.3. CFU assay .............................................................................................................57 2.4. Flow Cytometry ....................................................................................................58 2.5. Immunocytochemistry ..........................................................................................58 2.6. Differential staining ..............................................................................................59 ii 2.7. RNA extraction .....................................................................................................60 2.8. Quantitative RT-PCR............................................................................................61 2.9. Western blotting....................................................................................................61 2.10. Microarray...........................................................................................................63 2.11. Whole Mount In Situ Hybridisation (WISH)......................................................63 2.11.1. Cloning of genes ......................................................................................63 2.11.2. Riboprobe synthesis .................................................................................64 2.11.3 Whole-mount In Situ Hybridisation (WISH).............................................65 Chapter 3: Haematopoietic Differentiation Introduction.………………………………………………………………………….67 Results………………………………………………………………………………..70 3.1. hESC-derived embryoid bodies (EBs) give rise to haematopoietic-like cells when co-cultured with stromal cell lines………………………………………..70 3.2. hESCs form haematopoietic-like cells when differentiated in presence of pro- haematopoietic- cytokines ....................................................................................76 3.3. hESCs maintained on human feeder cells are amenable to haematopoietic differentiation........................................................................................................80 3.3.1. hESCs maintained on CCD919 cells .........................................................82 3.3.2. hESCs maintained on Ortec143 cells ........................................................85 Conclusion and Discussion…………………………...…….......………………........92 Chapter 4: Endodermal Differentiation Introduction.………………………………………………………………………….96 Results………………………………………………………………………………..98 4.1. Formation of definitive endoderm within embryoid bodies derived from hESCs ……………………………………………………………………………98 4.1.1. Bmp4 enhances the endoderm-inducing potential of Activin A in Matrigel.......................................................................................................99 4.1.2. Matrigel affects the extent, not the outcome of differentiation ................102 4.1.3. Cells expressing FOXA2 and SOX17 are of definitive endodermal origin as visceral endoderm is suppressed during differentiation .....................107 4.2. Detailed analysis of the role played by Bmp4 in the formation of definitive iii endoderm in vitro...............................................................................................110 4.2.1. Activin A signaling leads to the expression of known target genes during differentiation............................................................................................112 4.2.3. Bmp4 signaling and its downstream effects.............................................118 4.2.4. Bmp4 does not generate cell types associated with its pleiotropic activities ............................................................................................................................123 4.2.5. No evidence of formation of alternate lineages during DE differentiation ............................................................................................................................128 4.2.6. Gene Expression Analysis of Differentiation Using Microarray Technology................................................................................................131 Conclusion and Discussion…………………………………………………………153 Chapter 5– Future Direction Introduction...……………………………………………………………………….159
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