Tài liệu hạn chế xem trước, để xem đầy đủ mời bạn chọn Tải xuống
1
/ 214 trang
THÔNG TIN TÀI LIỆU
Thông tin cơ bản
Định dạng
Số trang
214
Dung lượng
7,34 MB
Nội dung
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 the supportive abilities of these various systems for long-term undifferentiated growth and pluripotency of hESC In addition, animal origin of MatrigelTM necessitates the development of a feeder-free system with innovation of a xeno-free matrix Hypotheses:The main hypothesis is that derivation of autologous feeder cells from hESCs can be optimized and the ECM derived from the autologous feeders can further support long-term undifferentiated growth of hESC in xeno-free, serum-free and feeder-free condition ix Bibliography Price PJ GM, Tilkins ML (1998) Embryonic stem cell serum replacement International Patent Application Prowse AB, McQuade LR, Bryant KJ, Van Dyk DD, Tuch BE, Gray PP (2005) A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells Proteomics 5(4):978-89 Puceat M, Ballis A (2007) Embryonic stem cells: from bench to bedside Clin Pharmacol Ther 82(3):337-9 Qiu D, Xiang J, Li Z, Krishnamoorthy A, Chen L, Wang R (2008) Profiling TRA-1-81 antigen distribution on a human embryonic stem cell Biochem Biophys Res Commun 369(2):735-40 Rajala K, Lindroos B, Hussein SM, Lappalainen RS, Pekkanen-Mattila M, Inzunza J, et al (2010) A defined and xeno-free culture method enabling the establishment of clinical-grade human embryonic, induced pluripotent and adipose stem cells PLoS One 5(4):e10246 Rajarajan A, Bloor BK, Desai H, Stokes A, Odell EW (2008) Variant CD44 expression by human fibroblasts Biomarkers 13(3):307-18 Rao RR, Calhoun JD, Qin X, Rekaya R, Clark JK, Stice SL (2004) Comparative transcriptional profiling of two human embryonic stem cell lines Biotechnol Bioeng 88(3):273-86 Ratajczak MZ, Machalinski B, Wojakowski W, Ratajczak J, Kucia M (2007) A hypothesis for an embryonic origin of pluripotent Oct-4(+) stem cells in adult bone marrow and other tissues Leukemia 21(5):860-7 RD L (1965) Histopathologic technic and practical histochemistry 3rd ed New York: McGraw-Hill Rehn A (2008) Regulation and function of mineralized tissue extra-cellular matrix proteins: studies of ADAMTS-1 and osteoadherin Stockholm, Karolinska institute Reiland J, Rapraeger AC (1993) Heparan sulfate proteoglycan and FGF receptor target basic FGF to different intracellular destinations J Cell Sci 105 ( Pt 4): 1085-93 Reim G, Mizoguchi T, Stainier DY, Kikuchi Y, Brand M (2004) The POU domain protein spg (pou2/Oct4) is essential for endoderm formation in 179 Bibliography cooperation with the HMG domain protein casanova Dev Cell 6(1):91-101 Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro Nat Biotechnol 18(4):399-404 Richards M, Fong CY, Chan WK, Wong PC, Bongso A (2002) Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells Nat Biotechnol 20(9):933-6 Richards M, Tan S, Fong CY, Biswas A, Chan WK, Bongso A (2003) Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells Stem Cells 21(5):546-56 Riento K, Ridley AJ (2003) Rocks: multifunctional kinases in cell behaviour Nat Rev Mol Cell Biol 4(6):446-56 Risau W, Sariola H, Zerwes HG, Sasse J, Ekblom P, Kemler R, et al (1988) Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies Development 102(3):471-8 Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, et al (2005) Transcriptional regulation of nanog by OCT4 and SOX2 J Biol Chem 280(26):24731-7 Rohwedel J, Guan K, Hegert C, Wobus AM (2001) Embryonic stem cells as an in vitro model for mutagenicity, cytotoxicity and embryotoxicity studies: present state and future prospects Toxicol In Vitro 15(6):741-53 Romaris M, Bassols A, David G (1995) Effect of transforming growth factor-beta and basic fibroblast growth factor on the expression of cell surface proteoglycans in human lung fibroblasts Enhanced glycanation and fibronectin-binding of CD44 proteoglycan, and down-regulation of glypican Biochem J 310 ( Pt 1): 73-81 Rosler ES, Fisk GJ, Ares X, Irving J, Miura T, Rao MS, et al (2004) Long-term culture of human embryonic stem cells in feeder-free conditions Dev Dyn 229(2):259-74 Rotunda AM, Suzuki H, Moy RL, Kolodney MS (2004) Detergent effects of sodium deoxycholate are a major feature of an injectable phosphatidylcholine formulation used for localized fat dissolution Dermatol Surg 30(7):1001-8 180 Bibliography Salih DA, Tripathi G, Holding C, Szestak TA, Gonzalez MI, Carter EJ, et al (2004) Insulin-like growth factor-binding protein (Igfbp5) compromises survival, growth, muscle development, and fertility in mice Proc Natl Acad Sci U S A 101(12):4314-9 Samadikuchaksaraei A, Cohen S, Isaac K, Rippon HJ, Polak JM, Bielby RC, et al (2006) Derivation of distal airway epithelium from human embryonic stem cells Tissue Eng 12(4):867-75 Sato N, Sanjuan IM, Heke M, Uchida M, Naef F, Brivanlou AH (2003) Molecular signature of human embryonic stem cells and its comparison with the mouse Dev Biol 260(2):404-13 Sausville EA, Burger AM (2006) Contributions of human tumor xenografts to anticancer drug development Cancer Res 66(7):3351-4, discussion 3354 Saxena S, Hanwate M, Deb K, Sharma V, Totey S (2008) FGF2 secreting human fibroblast feeder cells: a novel culture system for human embryonic stem cells Mol Reprod Dev 75(10):1523-32 Saylam C, Ozdemir N, Itil IM, Sendag F, Terek MC (2002) Distribution of fibronectin, laminin and collagen type IV in the materno-fetal boundary zone of the developing mouse placenta Experimental study Arch Gynecol Obstet 266(2):83-5 Scholer HR, Hatzopoulos AK, Balling R, Suzuki N, Gruss P (1989) A family of octamer-specific proteins present during mouse embryogenesis: evidence for germline-specific expression of an Oct factor EMBO J 8(9):2543-50 Schopperle WM, DeWolf WC (2007) The TRA-1-60 and TRA-1-81 human pluripotent stem cell markers are expressed on podocalyxin in embryonal carcinoma Stem Cells 25(3):723-30 Sharp J, Frame J, Siegenthaler M, Nistor G, Keirstead HS (2010) Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants improve recovery after cervical spinal cord injury Stem Cells 28(1):152-63 Shiba Y, Hauch KD, Laflamme MA (2009) Cardiac applications for human pluripotent stem cells Curr Pharm Des 15(24):2791-806 Shimozaki K, Nakashima K, Niwa H, Taga T (2003) Involvement of Oct3/4 in the enhancement of neuronal differentiation of ES cells in neurogenesis-inducing cultures Development 130(11):2505-12 181 Bibliography Silva AK, Yi H, Hayes SH, Seigel GM, Hackam AS (2010) Lithium chloride regulates the proliferation of stem-like cells in retinoblastoma cell lines: a potential role for the canonical Wnt signaling pathway Mol Vis 16: 36-45 Sjogren-Jansson E, Zetterstrom M, Moya K, Lindqvist J, Strehl R, Eriksson PS (2005) Large-scale propagation of four undifferentiated human embryonic stem cell lines in a feeder-free culture system Dev Dyn 233(4):1304-14 Skottman H, Mikkola M, Lundin K, Olsson C, Stromberg AM, Tuuri T, et al (2005) Gene expression signatures of seven individual human embryonic stem cell lines Stem Cells 23(9):1343-56 Skottman H, Hovatta O (2006) Culture conditions for human embryonic stem cells Reproduction 132(5):691-8 Slukvin, II, Vodyanik MA, Thomson JA, Gumenyuk ME, Choi KD (2006) Directed differentiation of human embryonic stem cells into functional dendritic cells through the myeloid pathway J Immunol 176(5):2924-32 Smith AG, Hooper ML (1987) Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells Dev Biol 121(1):1-9 Smith LA, Liu X, Hu J, Ma PX (2010) The enhancement of human embryonic stem cell osteogenic differentiation with nano-fibrous scaffolding Biomaterials 31(21):5526-35 Soldner F, Hockemeyer D, Beard C, Gao Q, Bell GW, Cook EG, et al (2009) Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors Cell 136(5):964-77 Sommer F, Kobuch K, Brandl F, Wild B, Framme C, Weiser B, et al (2007) Ascorbic acid modulates proliferation and extracellular matrix accumulation of hyalocytes Tissue Eng 13(6):1281-9 Sorgner NKDSSL (2007) Humanbiotechnology as social challenge : an interdisciplinary introduction to bioethics Hampshire: Ashgate Publishing, Ltd Sperger JM, Chen X, Draper JS, Antosiewicz JE, Chon CH, Jones SB, et al (2003) Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors Proc Natl Acad Sci U S A 100(23):13350-5 Stacey GN, Cobo F, Nieto A, Talavera P, Healy L, Concha A (2006) The 182 Bibliography development of 'feeder' cells for the preparation of clinical grade hES cell lines: challenges and solutions J Biotechnol 125(4):583-8 Stachowiak MK, Fang X, Myers JM, Dunham SM, Berezney R, Maher PA, et al (2003) Integrative nuclear FGFR1 signaling (INFS) as a part of a universal "feed-forward-and-gate" signaling module that controls cell growth and differentiation J Cell Biochem 90(4):662-91 Stein H, Wilensky M, Tsafrir Y, Rosenthal M, Amir R, Avraham T, et al (2009) Production of bioactive, post-translationally modified, heterotrimeric, human recombinant type-I collagen in transgenic tobacco Biomacromolecules 10(9):2640-5 Steiner D, Khaner H, Cohen M, Even-Ram S, Gil Y, Itsykson P, et al (2010) Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension Nat Biotechnol 28(4):361-4 Stojkovic M, Lako M, Stojkovic P, Stewart R, Przyborski S, Armstrong L, et al (2004) Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture Stem Cells 22(5):790-7 Stojkovic P, Lako M, Przyborski S, Stewart R, Armstrong L, Evans J, et al (2005a) Human-serum matrix supports undifferentiated growth of human embryonic stem cells Stem Cells 23(7):895-902 Stojkovic P, Lako M, Stewart R, Przyborski S, Armstrong L, Evans J, et al (2005b) An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells Stem Cells 23(3):306-14 Strelchenko N, Verlinsky O, Kukharenko V, Verlinsky Y (2004) Morula-derived human embryonic stem cells Reprod Biomed Online 9(6):623-9 Tai MH, Chang CC, Kiupel M, Webster JD, Olson LK, Trosko JE (2005) Oct4 expression in adult human stem cells: evidence in support of the stem cell theory of carcinogenesis Carcinogenesis 26(2):495-502 Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors Cell 126(4):663-76 Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors Cell 131(5):861-72 183 Bibliography Talens-Visconti R, Sanchez-Vera I, Kostic J, Perez-Arago MA, Erceg S, Stojkovic M, et al (2010) Neural differentiation from human embryonic stem cells as a tool to study early brain development and the neuroteratogenic effects of ethanol Stem Cells Dev Epub ahead of print Taori K, Rathod J, Deshmukh A, Sheorain VS, Jawale R, Sanyal R, et al (2006) Primary extragonadal retroperitoneal teratoma in an adult Br J Radiol 79(946):e120-2 Tavakoli T, Xu X, Derby E, Serebryakova Y, Reid Y, Rao MS, et al (2009) Self-renewal and differentiation capabilities are variable between human embryonic stem cell lines I3, I6 and BG01V BMC Cell Biol 10(44 Taylor RA, Cowin PA, Cunha GR, Pera M, Trounson AO, Pedersen J, et al (2006) Formation of human prostate tissue from embryonic stem cells Nat Methods 3(3):179-81 Tecirlioglu RT, Nguyen L, Koh K, Trounson AO, Michalska AE (2010) Derivation and maintenance of human embryonic stem cell line on human adult skin fibroblast feeder cells in serum replacement medium In Vitro Cell Dev Biol Anim 46(3-4):231-5 Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al (1998) Embryonic stem cell lines derived from human blastocysts Science 282(5391):1145-7 Timmermans F, Velghe I, Vanwalleghem L, De Smedt M, Van Coppernolle S, Taghon T, et al (2009) Generation of T cells from human embryonic stem cell-derived hematopoietic zones J Immunol 182(11):6879-88 Toh WS, Lee EH, Guo XM, Chan JK, Yeow CH, Choo AB, et al (2010a) Cartilage repair using hyaluronan hydrogel-encapsulated human embryonic stem cell-derived chondrogenic cells Biomaterials 31(27):6968-80 Toh WS, Lee EH, Richards M, Cao T (2010b) In vitro derivation of chondrogenic cells from human embryonic stem cells Methods Mol Biol 584(317-31 Tombran-Tink J, Johnson LV (1989) Neuronal differentiation of retinoblastoma cells induced by medium conditioned by human RPE cells Invest Ophthalmol Vis Sci 30(8):1700-7 Tomioka M, Nishimoto M, Miyagi S, Katayanagi T, Fukui N, Niwa H, et al (2002) Identification of Sox-2 regulatory region which is under the control of 184 Bibliography Oct-3/4-Sox-2 complex Nucleic Acids Res 30(14):3202-13 Tontonoz P, Hu E, Spiegelman BM (1994) Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor Cell 79(7):1147-56 Touboul T, Hannan NR, Corbineau S, Martinez A, Martinet C, Branchereau S, et al (2010) Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development Hepatology 51(5):1754-65 Tseng SY, Nishimoto KP, Silk KM, Majumdar AS, Dawes GN, Waldmann H, et al (2009) Generation of immunogenic dendritic cells from human embryonic stem cells without serum and feeder cells Regen Med 4(4):513-26 Tuhkanen AL, Agren UM, Tammi MI, Tammi RH (1999) CD44 expression marks the onset of keratinocyte stratification and mesenchymal maturation into fibrous dermis in fetal human skin J Histochem Cytochem 47(12):1617-24 Vallier L, Alexander M, Pedersen RA (2005) Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells J Cell Sci 118(Pt 19):4495-509 Vats A, Bielby RC, Tolley N, Dickinson SC, Boccaccini AR, Hollander AP, et al (2006) Chondrogenic differentiation of human embryonic stem cells: the effect of the micro-environment Tissue Eng 12(6):1687-97 Venable A, Mitalipova M, Lyons I, Jones K, Shin S, Pierce M, et al (2005) Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces BMC Dev Biol 5:15 Vinoth KJ, Heng BC, Poonepalli A, Banerjee B, Balakrishnan L, Lu K, et al (2008) Human embryonic stem cells may display higher resistance to genotoxic stress as compared to primary explanted somatic cells Stem Cells Dev 17(3):599-607 Vo E, Hanjaya-Putra D, Zha Y, Kusuma S, Gerecht S (2010) Smooth-Muscle-Like Cells Derived from Human Embryonic Stem Cells Support and Augment Cord-Like Structures In Vitro Stem Cell Rev 6(2):237-47 Vukicevic S, Kleinman HK, Luyten FP, Roberts AB, Roche NS, Reddi AH (1992) Identification of multiple active growth factors in basement membrane 185 Bibliography Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components Exp Cell Res 202(1):1-8 Wada T, Honda M, Minami I, Tooi N, Amagai Y, Nakatsuji N, et al (2009) Highly efficient differentiation and enrichment of spinal motor neurons derived from human and monkey embryonic stem cells PLoS One 4(8):e6722 Wang DD, Kriegstein AR, Ben-Ari Y (2008) GABA regulates stem cell proliferation before nervous system formation Epilepsy Curr 8(5):137-9 Wang D, Haviland DL, Burns AR, Zsigmond E, Wetsel RA (2007a) A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells Proc Natl Acad Sci U S A 104(11):4449-54 Wang D, Morales JE, Calame DG, Alcorn JL, Wetsel RA (2010) Transplantation of human embryonic stem cell-derived alveolar epithelial type II cells abrogates acute lung injury in mice Mol Ther 18(3):625-34 Wang G, Zhang H, Zhao Y, Li J, Cai J, Wang P, et al (2005a) Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers Biochem Biophys Res Commun 330(3):934-42 Wang L, Schulz TC, Sherrer ES, Dauphin DS, Shin S, Nelson AM, et al (2007b) Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling Blood 110(12):4111-9 Wang Q, Fang ZF, Jin F, Lu Y, Gai H, Sheng HZ (2005b) Derivation and growing human embryonic stem cells on feeders derived from themselves Stem Cells 23(9):1221-7 Wang ZZ, Au P, Chen T, Shao Y, Daheron LM, Bai H, et al (2007c) Endothelial cells derived from human embryonic stem cells form durable blood vessels in vivo Nat Biotechnol 25(3):317-8 Ware CB, Nelson AM, Blau CA (2006) A comparison of NIH-approved human ESC lines Stem Cells 24(12):2677-84 Wei CL, Miura T, Robson P, Lim SK, Xu XQ, Lee MY, et al (2005) Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain the stem cell state Stem Cells 23(2):166-85 Wikipedia Collagen http://en.wikipedia.org/wiki/Collagen 186 Bibliography Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya T, et al (2003) Wnt proteins are lipid-modified and can act as stem cell growth factors Nature 423(6938):448-52 Wobus AM, Boheler KR (2005) Embryonic stem cells: prospects for developmental biology and cell therapy Physiol Rev 85(2):635-78 Woll PS, Martin CH, Miller JS, Kaufman DS (2005) Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity J Immunol 175(8):5095-103 Xiao L, Yuan X, Sharkis SJ (2006) Activin A maintains self-renewal and regulates fibroblast growth factor, Wnt, and bone morphogenic protein pathways in human embryonic stem cells Stem Cells 24(6):1476-86 Xie CQ, Lin G, Luo KL, Luo SW, Lu GX (2004) Newly expressed proteins of mouse embryonic fibroblasts irradiated to be inactive Biochem Biophys Res Commun 315(3):581-8 Xiong C, Xie CQ, Zhang L, Zhang J, Xu K, Fu M, et al (2005) Derivation of adipocytes from human embryonic stem cells Stem Cells Dev 14(6):671-5 Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, et al (2001) Feeder-free growth of undifferentiated human embryonic stem cells Nat Biotechnol 19(10):971-4 Xu C, Rosler E, Jiang J, Lebkowski JS, Gold JD, O'Sullivan C, et al (2005a) Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium Stem Cells 23(3):315-23 Xu RH, Chen X, Li DS, Li R, Addicks GC, Glennon C, et al (2002) BMP4 initiates human embryonic stem cell differentiation to trophoblast Nat Biotechnol 20(12):1261-4 Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA (2005b) Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells Nat Methods 2(3):185-90 Yao S, Chen S, Clark J, Hao E, Beattie GM, Hayek A, et al (2006) Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions Proc Natl Acad Sci U S A 103(18):6907-12 Yen BL, Chang CJ, Liu KJ, Chen YC, Hu HI, Bai CH, et al (2009) Brief report human embryonic stem cell-derived mesenchymal progenitors possess 187 Bibliography strong immunosuppressive effects toward natural killer cells as well as T lymphocytes Stem Cells 27(2):451-6 Yoo SJ, Yoon BS, Kim JM, Song JM, Roh S, You S, et al (2005) Efficient culture system for human embryonic stem cells using autologous human embryonic stem cell-derived feeder cells Exp Mol Med 37(5):399-407 Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin, II, et al (2009) Human induced pluripotent stem cells free of vector and transgene sequences Science 324(5928):797-801 Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al (2007) Induced pluripotent stem cell lines derived from human somatic cells Science 318(5858):1917-20 Zabierowski SE, Herlyn M (2010) Embryonic stem cells as a model for studying melanocyte development Methods Mol Biol 584: 301-16 Zaehres H, Kim JB, Scholer HR (2010) Induced pluripotent stem cells Methods Enzymol 476: 309-25 Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA (2001) In vitro differentiation of transplantable neural precursors from human embryonic stem cells Nat Biotechnol 19(12):1129-33 Zhu GZ, Lin Y, Myles DG, Primakoff P (1999) Identification of four novel ADAMs with potential roles in spermatogenesis and fertilization Gene 234(2):227-37 Zhou J, Su P, Wang L, Chen J, Zimmermann M, Genbacev O, et al (2009) mTOR supports long-term self-renewal and suppresses mesoderm and endoderm activities of human embryonic stem cells Proc Natl Acad Sci U S A 106(19):7840-5 Zimmermann DR, Trueb B, Winterhalter KH, Witmer R, Fischer RW (1986) Type VI collagen is a major component of the human cornea FEBS Lett 197(1-2):55-8 188 Appendix Appendix 189 Appendix Appendix 1) Table showing the details of the number of cells for growth curve analysis 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 (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