A human embryonic stem cell based model of trophoblast formation

A Human Embryonic Stem Cell–based Model of Trophoblast Formation LUO WENLONG National University of Singapore 2008 A Human Embryonic Stem Cell–based Model of Trophoblast Formation LUO WENLONG (BSc., China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2008 i ACKNOWLEDGMENTS I would like to thank my supervisor Dr Paul Robson for his guidance and help Paul is a responsible supervisor, training me to develop my scientific critical thinking ability; he also sets an excellent example of a passionate scientist with integrity, sincerity, understanding and compassion, greatly influencing my personal philosophy I would like to thank colleagues who were involved in this project and helped me to accomplish it From our lab, Jameelah Sheik Mohamed and Sun Lili for the human embryonic stem cell (hESC) culture work and Woon Chow Thai for the microarray experiments and RNA quality control From Dr Neil Clarke’s lab at GIS, Mikael Huss and Yeo Zhen Xuan have been involved in the microarray data analysis This project could only have been successfully accomplished with intimate cooperation between the above team members In addition, I would like to thank other members of our lab - Dr David Rodda, Dr Chu Lee Thean, Dr Wang Chao Yang, Lim Leng Hiong, Guo Guoji and Teo Tang Yi Roy for their suggestions and encouragement I would like to thank Li Pin and Soh Boon Seng from Dr Lim Bing’s lab, both of whom kindly shared their cell culture experience with me I also would like to thank Andrew Hutchins and Mathia Lee Yu Chun for helping to proof read my thesis and to provide many constructive comments Last but not least, I would like to thank my mother for her love and support in all my ii life Especially during my Ph.D study period, during which she has had to bear with my extended absence far away over the South China Sea and its accompanying loneliness, waiting for my Ph.D accomplishment She must be more proud of this thesis than I am iii TABLE OF CONTENTS ACKNOWLEDGMENTS i TABLE OF CONTENTS iii SUMMARY ix LIST OF FIGURES x LIST OF ABBREVIATIONS xii LIST OF PUBLICATIONS xix CHAPTER I INTRODUCTION 1.1 Introductory statement 1.2 The early embryo and placenta development 1.2.1 Human preimplantation development: from zygote to implanting blastocyst 1.2.2 Early trophoblast morphology and function 1.2.3 Key factors modulating early trophoblast development 1.2.3.1 Hormone related genes 1.2.3.2 Growth factors 11 1.2.3.3 Transcription Factors 13 1.2.3.4 Immune related factors in placenta 18 2.3.4.1 Immunosuppression in placenta 18 2.3.4.2 Non-immune function of immune cells 20 1.2.3.5 Other placenta related factors 21 iv 1.2.4 Clinical relevance of placenta development 1.3 Embryonic stem cells 1.3.1 Mouse embryonic stem cells 22 23 23 1.3.1.1 Derivation of mESC 23 1.3.1.2 Culture condition of mESC 24 1.3.1.3 Core signaling pathway of mESC 24 1.3.1.3.1 LIF/STAT3 24 1.3.1.3.2 Transcription factors 25 1.3.1.4 Mouse trophoblast Stem cells 27 1.3.2 Human embryonic stem cell (hESC) 27 1.3.2.1 hESC derivation 27 1.3.2.2 hESC culture condition 30 1.3.2.3 Core signaling pathways of hESC 32 1.3.2.4 hESC-derived trophoblast cells 33 1.4 FGF and TGF-β Signaling pathway 1.4.1 Fibroblast growth factor (FGF) signaling 35 35 1.4.1.1 Signaling cascade 36 1.4.1.2 FGF receptor isoforms 39 1.4.1.3 FGF signaling involved in mouse early embryo development 39 1.4.1.4 FGF signaling involved in hESC 42 1.4.1.5 Down-stream pathway of FGF signaling in hESCs 44 v 1.4.2 TGF-β Superfamily of signaling molecules 1.4.1.1 BMP signaling 44 45 1.4.1.1.1 Effectors of BMP signaling 45 1.4.1.1.2 Transcriptional targets of BMP signaling 48 1.4.1.1.3 BMPs in mESC 50 1.4.1.1.4 BMPs in hESC 52 1.5 Specific aims of this study CHAPTER II MATERIALS AND METHODS 2.1 Cell culture 2.1.1 Mouse embryonic fibroblasts (MEFs) 54 56 57 57 2.1.1.1 Derivation of MEFs 57 2.1.1.2 Culture of primary MEF 58 2.1.2 Mouse ES cell culture 58 2.1.3 Human ES cell culture 59 2.1.3.1 Culture of hESC on MEF 66 2.1.3.2 Culture of feeder-free hESC 67 2.1.3.3 Cryopreservation of hESC 69 2.1.3.4 Karyotyping 69 2.1.4 Human embryoid body (EB) culture 2.2 Bioactive molecule treatments 69 70 vi 2.3 RNA analysis 70 2.3.1 RNA isolation 70 2.3.2 RNA integrity analysis 71 2.3.3 Reverse transcriptase (RT) 72 2.3.4 Polymerase chain reaction (PCR) 72 2.3.4.1 Conventional PCR 72 2.3.4.2 Taqman real time PCR 73 2.3.5 Microarray analysis 2.4 Protein analysis 73 74 2.4.1 Protein extraction and quantitation 74 2.4.2 Western blot 74 2.4.3 Immunostaining 75 2.4.4 Fluorescence activated cell sorting (FACs) analysis 76 CHAPTER III RESULTS 78 3.1 Characterization of FGF pathway genes in hESCs 79 3.2 Inhibition of FGF signaling pathway in hESC 83 3.2.1 Morphology of H1 hESC treated with SU5402 83 3.2.2 mRNA expression profiling of H1 hESC treated with SU5402 84 3.2.3 Protein expression of H1 hESC treated with SU5402 3.3 BMP4 treatment on H1 89 90 vii 3.3.1 Morphology of H1 hESC treated with BMP4 90 3.3.2 mRNA expression profiling of H1 hESC treated with BMP4 91 3.3.3 Comparison between hESCs treated with BMP4 versus SU5402 92 3.4 Combined treatment of FGFR inhibitor and BMP4 on hESC 94 3.4.1 Morphology of H1 hESC treated with BS 95 3.4.2 Global gene expression profiling of H1 hESC treated with BS 95 3.4.3 Protein expression of H1 hESC treated with BS 100 3.4.3.1 Immunostaining of H1 hESC treated with BS 3.4.3.1.1 Immunostaining of trophoblast related transcription factors 100 100 3.4.3.1.2 Immunostaining of C-Terminal-Phosphorylated-SMAD5 101 3.4.3.2 Western blot of H1 hESC treated with BS 102 3.4.3.3 BS-treated hESC secrete hCG 102 3.4.4 BMP signaling is involved in the differentiation induced by FGF inhibition 104 3.4.4.1 Morphology 105 3.4.4.2 mRNA expression profiling 105 3.5 Short time treatment of BMP4, SU5402 and BMP4+SU5402 on H1 hESC 3.5.1 Early Response Genes 108 109 3.5.2 Response of BMP direct target genes in SU5402 treatment 113 3.5.3 SU5402 Does Stabilize SMAD5 115 3.5.4 BMP4-independent role of FGF 117 viii 3.5.5 BS effect is not specific to the H1 hESC line 120 3.6 Cell fate commitment time point of H1 hESC treated with BS 124 3.7 Inhibition of FGF signaling in mESC 129 3.7.1 Combination effect of LIF pathway activation and FGF receptor inhibition on mESCs 129 3.7.1.1 Morphology of treated mESC 129 3.7.1.2 mRNA expression profiling of treated mESC 131 3.7.1.3 Protein expression of treated mESC detected by Western Blot 132 CHAPTER IV DISCUSSION 134 4.1 How does this model compare with others? 135 4.2 Mechanism of trophoblast induction by BMP4 and SU5402 136 4.3 Regulatory Networks in Human Placental Development 139 4.4 Totipotency of Human ES Cells? 144 CHAPTER V CONCLUSIONS AND FUTURE STUDY 147 5.1 Conclusion 148 5.2 Future work 149 REFERENCES 150 170 Morasso, M.I., Grinberg, A., Robinson, G., Sargent, T.D., and Mahon, K.A (1999) Placental failure in mice lacking the homeobox gene Dlx3 Proc 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Stem Cell Research (ISSCR) Conference 2007, Cairns Australia) A Human Embryonic Stem Cell? ? ?based Model of Trophoblast Formation (manuscript in preparation, 2009) Inhibition of methylation releases... (Carosella et al., 200 8a; Carosella et al., 2008b) Trophoblast cells are also able to avoid maternal natural killer cell monitoring and attacking independent of HLA class I expression (Avril et al., 1999)... the maternal-fetal interface (Auman et al., 2002) Redundancies in the Tcfap2 family may mask an even earlier role of Tcfap 2a in the mouse trophoblast lineage (Winger et al., 2006) as a number of