ESTABLISHMENT OF AUTOLOGOUS CULTURE SYSTEMS FOR HUMAN EMBRYONIC STEM CELLS FU XIN (B.Sci (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ORAL AND MAXILLOFACIAL SURGERY FACULTY OF DENTISTRY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements Acknowledgements With great pleasure in the completion of my project I would like to express my deepest appreciation and gratitude to all those who provided their kind support and motivation First and the foremost, I would like to express my sincere thanks to my supervisor, Associate Professor Cao Tong, Vice Dean of Research, Faculty of Dentistry, National University of Singapore, for giving me the opportunity to join as a graduate student I would like to thank my supervisor for his constant encouragement, invaluable guidance and infinite patience throughout the course of this study He opened a new door in my life in the year 2006 and molded me into a better human being filled with energy and exuberance to go further in the road of academics I am deeply thankful to my Head of the Department, Associate Professor Yeo Jin Fei, for his constant support towards the completion of my work on time Without the excellent facilities, this work would not have been accomplished The help and support provided by Associate Professor Grace Ong Hui Lian, Dean, Faculty of Dentistry, in my studies, conference visits and overseas studies is greatly appreciated I wish to express my warm and sincere thanks to Professor Yu Guang Yan, Department of Oral and Maxillofacial Surgery, Peking University School of Stomotology, for his advices and helps during my stay in Beijing, China when I was conducting my joint project I also wish to thank my thesis committee members, Dr Liu Hua and Dr i Acknowledgements Toh Wei Seong for their invaluable helps, comments and guidance Working with them was a privilege Their helps extending from the work to the minute details during my thesis writing contributed a lot towards the timely completion of my project The sincere help from all my group members, Mr Lu Kai, Mr Li Ming Ming, Dr Vinoth, Dr Fahad and Dr Sriram helped me a lot in working and gaining knowledge I wish to acknowledge their support and friendly working environment I am also thankful to Mr Chan Sweeheng and Miss Lina for their help The main backbone for my achievement is contributed to my beloved family and precious friends Their faith, encouragement and help pushed me to become better by day in whatever I Without them, my life in Singapore and the pursuit of my doctorate degree would not have been the same This work was supported by grants from the Ministry of Education of Singapore (R223000014112 and R223000018112) ii Table of Contents Table of Contents Acknowledgements i Table of contents iii Summary ix List of Tables xii List of Figures xiii List of Abbreviations xvi List of Publications xx Chapter I: Literature Review Literature Review 1.0 Introduction to Literature Review 1.1 Human embryonic stem cells (hESCs) 1.1.1 Derivation of hESCs 1.1.2 Characterization of hESCs 1.1.2.1 Cell surface and molecular markers 1.1.2.2 Pluripotency of hESCs 11 1.1.3 Signaling pathways involved in the self-renewal and pluripotency of hESCs 12 1.1.3.1 FGF signaling pathway 12 1.1.3.2 Crosstalk between FGF-2 signaling and other signaling pathways 14 1.1.4 Transcription factors controlling self-renewal and pluripotency of hESCs 15 1.2 Long-term maintenance of hESCs in culture 17 1.2.1 Maintenance of hESCs on MEF feeder 17 1.2.2 Maintenance of hESCs on human feeder layers 20 1.2.3 Maintenance of hESCs on autologous feeder layers derived iii Table of Contents from hESCs 22 1.2.4 Maintenance of hESCs in feeder-free system 24 1.2.4.1 Components and functions of ECM derived from feeder cells 24 1.2.4.2 ECM components from animal origin 25 1.2.4.3 Substrate derived from human origin 27 1.2.4.4 Growth of hESCs in suspension 30 1.3 Applications of hESCs 32 1.3.1 Regenerative medicine and cell therapy 32 1.3.2 Cytotoxicity testing, embryotoxicity screening and drug discovery 35 1.3.3 Cellular model for basic science study 36 1.4 Towards clinical-grade hESCs 37 Chapter II: Hypotheses and Objectives 39 Chapter III: Materials and Methods 42 3.0 Introduction to materials and methods 43 3.1 Derivation of autologous feeder cells (H9-F) from H9 hESCs 43 3.1.1 Cultivation of H9 hESCs 44 3.1.1.1 Preparation of MEF 44 3.1.1.2 Expansion of H9 hESCs 46 3.1.2 Derivation of autologous fibroblast cells from H9 hESCs 47 3.1.2.1 Differentiation of H9 ebF from H9 hESCs 47 3.1.2.2 Derivation of H9 dF from H9 hESCs 48 3.2 Characterization and comparison of H9-F 48 3.2.1 Growth curve analysis 49 3.2.2 Identity analysis of H9-F by flow cytometry 49 3.2.3 Gene expression analysis by reverse transcription-polymerase chain reaction (RT-PCR) 51 iv Table of Contents 3.2.4 Evaluation of FGF-2 secretion in H9-F conditioned medium by enzyme-linked immunosorbent assay (ELISA) 54 3.3 Characterization and comparison of H9 hESCs cultured on autologous H9-F, MEF and feeder-free MatrigelTM 55 3.3.1 Cultivation of H9 hESCs in various culture systems 55 3.3.2 Cryopreservation assay 57 3.3.3 Immunofluorescence staining 58 3.3.4 Alkaline phosphatase (ALP) staining 60 3.3.5 Proliferation analysis 60 3.3.6 Pluripotency analysis by EB formation and teratoma formation 62 3.3.7 Undifferentiated states analysis of H9 hESCs cultured in various systems 64 3.3.7.1 Immunofluorescence staining and flow cytometry analysis for Oct4 and SSEA-3/4 expression in hESCs 64 3.3.7.2 Conventional RT-PCR and real-time RT-PCR 65 3.3.8 Karyotype analysis 67 3.4 Cultivation of hESCs on H9 ebF-derived ECM in xeno-free, serum-free and feeder-free conditions 68 3.4.1 Extraction of ECM from H9 ebF 68 3.4.2 Characterization of ECM by fluorescence confocal microscopy 69 3.4.3 Cultivation of H9 and H1 hESC lines on ECM in xeno-free, serum-free and feeder-free conditions 70 3.4.3.1 Expansion of hESCs 70 3.4.3.2 Characterization of hESCs by immunofluorescence staining and flow cytometry 71 3.4.3.2 Proliferation analysis 71 3.4.3.3 Pluripotency analysis by EB formation and teratoma formation 72 v Table of Contents 3.4.3.4 Undifferentiated state analysis by RT-PCR and real-time RT-PCR 72 3.4.4 Application of ECM-supported hESCs 73 3.4.4.1 In vitro osteogenic differentiation of ECM-supported hESCs 73 3.4.4.2 Cytotoxicity testing of NaF on ECM-supported H9 hESCs, H9 ebF and CRL1486 by MTS assay 74 3.5 Statistical Analysis 75 Chapter IV: Results 76 4.1 Derivation of autologous fibroblast cells from H9 hESCs 77 4.1.1 Morphology of H9 ebF 77 4.1.2 Morphology of H9 dF 77 4.2 Characterization and comparison of H9 ebF and H9 dF 80 4.2.1 Growth kinetics 80 4.2.2 Purity 81 4.2.3 Identity 83 4.2.4 Characterization 87 4.2.5 Synthesis and secretion of FGF-2 89 4.2.6 Karyotypes 91 4.2.7 Adherence of freeze-thawed H9-F for H9 hESCs culture 92 4.3 Characterization and comparison of H9 hESCs on H9-F, MEF and feeder-free MatrigelTM 93 4.3.1 Morphologies 93 4.3.2 Viability after cryopreservation 96 4.3.3 Expressions of undifferentiated markers 96 4.3.4 ALP activities 98 4.3.5 Comparisons of H9 hESCs on H9-F, MEF and feeder-free MatrigelTM after long-term culture 99 4.3.5.1 Proliferation 99 vi Table of Contents 4.3.5.2 Maintenance of undifferentiated states 103 4.3.5.3 Percentages of undifferentiated H9 hESCs 108 4.3.5.4 Maintenance of pluripotency 111 4.3.6 Karyotypes 116 4.4 Characterization of hESCs on H9 ebF-derived ECM in xeno-free, serum-free and feeder-free conditions 117 4.4.1 Establishment of ECM stratum with H9 ebF 117 4.4.2 Characterization of H9 ebF-derived ECM 118 4.4.3 Morphologies of hESCs 120 4.4.4 Maintenance of undifferentiated states of hESCs 121 4.4.5 Percentage of undifferentiated hESCs 124 4.4.6 Proliferation of hESCs 126 4.4.7 Pluripotency of hESCs 127 4.5 Applications of ECM-supported hESCs 130 4.5.1 In vitro differentiation to osteogenic progenitors 130 4.5.2 Cytotoxicity testing of NaF on ECM-supported H9 hESCs by MTS assay 131 Chapter V: Discussion 133 5.1 Feasibility of H9-F derivation 134 5.2 Similarities and differences in the properties of H9 ebF and H9 dF 135 5.3 Comparison of supportive effect on the growth of H9 hESCs by H9 ebF, H9 dF, MEF and feeder-free MatrigelTM 138 5.3.1 The characteristics of H9 hESCs on H9-F 138 5.3.2 Better effect on the maintenance of undifferentiated states of hESCs by H9 ebF in comparison with H9 dF 139 5.3.3 Better effect on the maintenance of pluripotency of hESCs by H9 ebF in comparison with H9 dF 141 5.3.4 Higher proliferation activities and ectoderm differentiation potentials of H9 hESCs in feeder-free MatrigelTM-mTeSRTM1 system vii Table of Contents 143 5.4 Supportive effect of the xeno-free, feeder-free and serum-free system for the growth of hESCs 145 5.4.1 Establishment of autologous feeder-derived ECM 145 5.4.2 Characterization of H9 ebF-derived ECM 146 5.4.3 Cultivation of H9 and H1 hESCs on ECM 148 5.4.4 Characterization of ECM-supported hESCs 149 5.5 ECM-supported hESCs are applicable for regenerative medicine and cytotoxicity testing 150 5.6 Animal Model 153 Chapter VI: Conclusion and Recommendation of Future Study 155 Chapter VII: Bibliography 159 Appendix 189 viii Summary Summary Background：Human embryonic stem cells (hESCs) hold great promise for regenerative medicine due to their unlimited differentiation potential and proliferation capacity Currently, potential clinical applications of hESCs are hampered by the use of mouse embryonic fibroblasts (MEF) and animal-derived components during culture Experimental modifications and manipulations of hESCs require a feeder-free culture system to exclude the confounding effects of feeder cells Recent literature have demonstrated the possibilities of culturing hESCs on autologous fibroblast, while others have also demonstrated an alternative of culturing hESCs on a feeder-free system with the aid of MatrigelTM But none of them has systematically compared 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P12 P13 P14 P15 P16 P17 P18 P19 P20 H9 ebF (x106) 0.81 1.4 1.74 1.51 1.86 0.97 1.29 1.15 1.22 1.04 1.68 1.48 2.46 2.09 2.46 1.63 1.85 1.61 1.88 H9 dF (x106) 0.91 1.66 1.74 1.7 2.1 1.87 1.28 1.09 1.24 1.8 0.89 0.83 0.65 0.63 0.86 0.6 0.71 0.81 0.89 190 Appendix 2) Table showing the doubling time of H9 ebF and H9 dF from P2 to P20 Passage (P) number P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 H9 ebF doubling time (h) 103.4492 48.47092 40.02029 45.15385 37.98866 75.30934 52.65579 59.91842 55.94922 68.14416 41.17907 45.98884 31.32262 34.89212 31.32262 42.23191 38.14518 42.67778 37.68188 H9 dF doubling time (h) 83.33927 41.59005 40.02029 40.78085 34.77607 37.83424 53.09171 64.03824 54.94757 38.9611 86.55123 98.47053 190.2187 215.9414 92.02353 273.7284 142.3232 103.4492 86.55123 191 Appendix 3) Table showing the normalized concentration of secreted FGF-2 in H9 ebF- and H9 dF-conditioned medium after ELISA analysis Cells H9 dF P2 H9 dF P3 H9 dF P4 H9 dF P5 H9 dF P6 H9 dF P7 H9 dF P8 H9 dF P9 H9 dF P10 H9 dF P11 H9 dF P12 H9 dF P13 H9 dF P14 H9 dF P15 H9 dF P16 H9 dF P17 H9 dF P18 H9 dF P19 H9 dF P20 Mean 139.3342 102.3904 169.4413 115.5462 187.5388 193.8588 100.5027 111.0286 90.60627 137.6712 208.8197 135.169 89.17351 96.62433 223.7503 132.0488 130.1573 195.4701 167.2992 STD 26.55755 47.572 34.25128 82.1093 94.94901 64.5081 68.49873 57.04656 30.51268 31.74526 28.20905 24.31945 26.63748 26.40968 127.4883 43.58178 64.62667 12.98138 23.29959 Cells H9 ebF P2 H9 ebF P3 H9 ebF P4 H9 ebF P5 H9 ebF P6 H9 ebF P7 H9 ebF P8 H9 ebF P9 H9 ebF P10 H9 ebF P11 H9 ebF P12 H9 ebF P13 H9 ebF P14 H9 ebF P15 H9 ebF P16 H9 ebF P17 H9 ebF P18 H9 ebF P19 H9 ebF P20 Mean 84.82689 110.8197 275.6208 442.2026 370.7733 332.3182 270.5126 288.0978 323.9589 472.5619 513.9416 488.4698 553.4046 590.0273 495.8254 341.1012 311.7736 641.3067 357.0067 STD 41.53262 37.06342 20.75693 43.7563 35.59377 35.64507 30.97087 8.109131 42.91505 51.80737 110.7961 4.85699 6.481853 1.776509 42.4272 24.09215 48.74971 15.3471 13.50515 192 Appendix 4) Teratoma of H9 hESCs after cultured in various culture systems A B C D Appendix Teratoma formation of H9 hESCs (A) Teratoma specimen isolated from SCID mice after weeks of injection with MEF-supported H9 hESCs (B) Teratoma specimen isolated from SCID mice after weeks of injection with Matrigel-supported H9 hESCs (C) Teratoma specimen isolated from SCID mice after 11 weeks of injection with H9 ebF-supported H9 hESCs (D) Teratoma specimen isolated from SCID mice after weeks of injection with H9 dF-supported H9 hESCs 193 ... sources, named embryonic stem cells Literature review (ESCs), embryonic germ cells, fetal stem cells, umbilical cord stem cells, adult stem cells and induced pluripotent stem (iPS) cells (Ariff... cell source for future clinical applications Literature review Table Characterization of stem cells by derivation source Category Embryonic stem cells Embryonic germ cells Fetal stem cells Umbilical... Xiao R, and Cao T Establishment of Autologous Feeders for Human Embryonic Stem Cells Propagation 2nd Meeting of IADR Pan Asian Pacific Federation (PAPF) and the 1st Meeting of IADR Asia/Pacific