ACKNOWLEDGEMENTS It is a pleasure to thank the many people who made this thesis possible. First and foremost, I would like to extend my most sincere thanks to my supervisor, Assistant Professor Cao Tong for his patience, encouragement and sound advice throughout my candidature. Thank you for giving me the opportunity to immerse myself in this intriguing field of stem cell research. I am also especially grateful to my co-supervisor, Associate Professor Manoor Prakash Hande for his unfailing guidance and direction especially during the many challenges that arose during the course of study. The cytogenetic work done for this thesis would have been impossible without his support. I would like to specially thank Dr. Yang Zheng with whom I have shared many insightful conversations during the development of ideas during the course of work. I am indebted to Dr. Alexis Heng Boon Chin for his endless ideas and suggestions that provided many new insights to my project. Heartfelt thanks to my fellow Stem cell laboratory mates, Mr. Lu Kai, Ms. Sui Lin, and Dr. Ge Zigang with whom I have shared many cherished moments. Special thanks to Dr. Liu Hua, Ms. Rufaihah bte Abdul Jalil and Dr. Tian Xian Feng, who have generously extended their support and friendship on many occasions. My sincere appreciation to Toh Wei Seong for his invaluable help with RT-PCR. Last but not least, I would also like to thank my friends at Genome Stability Laboratory for their cooperation and comradeship. Special thanks to Dr.Anuradha Poonepalli, Dr. Birendranath Banerjee and Ms. Lakshmi Devi for their constant support and encouragement which was invaluable in solving many challenges which arose. i TABLE OF CONTENTS ACKNOWLEDGMENTS………………………………………………….i TABLE OF CONTENTS…………………………………………………..ii LIST OF FIGURES……………………………………………………….vi LIST OF TABLES…………………………………………………….....viii SUMMARY………………………………………………………………..ix CHAPTER 1 1. INTRODUCTION……………………………………………………1 1.1 OBJECTIVES………………………………………………………...3 CHAPTER 2 2. LITERATURE REVIEW…………………………………………….4 2.1 Human Embryonic Stem Cells (hESCs): Derivation, Isolation and Maintenance of hESCs……………………………………………………………………………………...4 2.2 ESCs Characterization…………………………………………………………….6 2.3 Effect of temperature on hESC viability…………………………………………..7 2.4 Genotoxicity Testing ……………………………………………………………...8 2.5 Molecular cytogenetics…………………………………………………………..11 2.6 Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH)………...12 2.7 Multi-color Fluorescence In Situ Hybridization (mFISH)……………………….14 ii CHAPTER 3 3. MATERIALS AND METHODS……………………………………15 3.1 Cell Culture………………………………………………………………………15 3.11 Human Lung fibroblast culture…………………………………………………..15 3.12 Human embryonic stem cell (hESCs) culture……………………………............15 3.13 Differentiation of hESCs…………………………………………………………16 3.2 Characterization of Human Embryonic stem cells ……………………………...17 3.21 Real –time reverse transcriptase Polymerase Chain Reaction (rtPCR)………….17 3.22 Immunohistochemistry…………………………………………………………..18 3.3 Exposure of hESC to reduced temperature………………………………………20 3.31 MTT assay……………………………………………………………………….21 3.4 Metaphase preparation…………………………………………………………...22 3.5 Peptide Nucleic Acid- Fluorescence In Situ Hybridization (PNA-FISH)……….23 3.6 Multi-color Fluorescence In Situ Hybridization (mFISH)………………………24 3.7 Determination of Genotoxicity using Mitomycin C……………………………..26 iii CHAPTER 4 4. RESULTS………………………………………………………..27 4.1 Characterization of human embryonic stem cells………………………..27 4.2 Temperature tolerance of human embryonic stem cells………………...30 4.21 Survival rate of hESC after exposure to low temperature……………….30 4.22 Spontaneous differentiation of hESC after exposure to low temperature..30 4.23 Chromosomal Analysis of hESC after exposure to low temperature……31 4.3 Evaluation of genotoxic effect of Mitomycin C on human embryonic stem cells using PNA-FISH and mFISH…………………………………………………..41 CHAPTER 5 5. DISCUSSION………………………………………………………52 CHAPTER 6 6. CONCLUSION……………………………………………………...56 CHAPTER 7 7. APPENDIX………………………………………………………….57 iv CHAPTER 8 8. REFERENCES…………………………………………………59 LIST OF PUBLICATIONS.....................................................................68 v LIST OF FIGURES Title Page 28 Figure 1 Bright field and Phase contrast microscopy to visualize morphology of hESCs with different grades of differentiation. Immunohistochemistry was undertaken to assess the presence of the pluripotent markers: SSEA-3 and TRA-1-181. Figure 2 Evaluation of pluripotency by Real –time reverse transcriptase polymerase Chain Reaction (rtPCR). 29 Figure 3 hESCs maintained at 37oC (physiological control) by PNA FISH 33 Figure 4 Figure 5 hESCs maintained at 4 oC for 24h by PNA FISH hESCs maintained at 25 oC for 24h by PNA FISH 33 34 Figure 6 hESCs maintained at 4 oC for 48h by PNA FISH 34 Figure 7 hESCs maintained at 25 oC for 48h by PNA FISH 35 Figure 8 Metaphase spread of hESCs maintained at 37 oC (physiological control) by mFISH 36 Figure 9 Metaphase spread of hESCs maintained at 37 oC (physiological control) by mFISH 36 Figure 10 Metaphase spread of hESCs maintained at 4 oC for 24h by mFISH 37 Figure 11 Karyotype of hESCs maintained at 4 oC for 24h by mFISH 37 Figure 12 Metaphase spread of hESCs maintained at 25 oC for 24h by mFISH 38 Figure 13 Metaphase spread of hESCs maintained at 4 oC for 48h by mFISH 38 Figure 14 Metaphase spread of hESCs maintained at 4 oC for 48h by mFISH 39 Figure 15 Karyotype of hESCs maintained at 4 oC for 48h by mFISH 39 Figure 16 Metaphase spread of hESCs maintained at 25 oC for 48h by mFISH 40 Figure 17 Karyotype of hESCs maintained at 25 oC for 48h by mFISH 40 Figure 18 Percentage of chromosomal aberrations detected by PNA-FISH 42 Figure 19 hESCs control metaphase spreads as observed by PNA-FISH 43 vi Figure 20 MMC-treated hESC with extra-telomeric signals by PNA-FISH 43 Figure 21 MMC-treated hESCs with break by PNA-FISH 44 Figure 22 MMC-treated hESCs with sister chromatid splits by PNA FISH 44 Figure 23 IMR90 control metaphases spreads as observed by PNA-FISH 45 Figure 24 MMC-treated IMR90 metaphase spreads with break by PNA FISH 45 Figure 25 MMC-treated IMR90 metaphase spreads with break by PNA FISH 46 Figure 26 MMC-treated IMR90 metaphase spreads with loss of telomeric end and sister chromatid split by PNA FISH 46 Figure 27 MMC-treated IMR90 metaphase spreads with break by PNA FISH 47 Figure 28 MMC-treated IMR90 metaphase spreads with breaks by PNA FISH 47 Figure 29 hESCs control metaphase as observed by mFISH 48 Figure 30 hESC control karyotype as observed by mFISH 48 Figure 31 MMC-treated hESCs metaphase as observed by mFISH 49 Figure 32 MMC-treated hESCs karyotype as observed by mFISH 49 Figure 33 IMR90 control metaphase spread as observed by mFISH 50 Figure 34 IMR90 control karyotype as observed by mFISH 50 Figure 35 MMC-treated IMR90 metaphase as observed by mFISH 51 Figure 36 MMC-treated IMR90 metaphase as observed by mFISH 51 vii LIST OF TABLES Title Table 1 Cell viability by MTT assay following exposure of hESC to reduced temperature (4oC and 25oC) for 24h and 48h. Table 2 Percentage of aberrant cells in untreated controls and in MMCtreated IMR90 and hESCs by PNA-FISH. Page 32 42 viii SUMMARY Introduction: Established mammalian cell lines and primary explanted cells from mammals are commonly used in vitro to analyze the genotoxic potential of environmental factors, drugs, biomaterials as well as chemical, physical and biological agents. However, established cell lines poorly reflect human physiology. Lack of standardization is also a limitation with the use of primary explanted cells. Human embryonic stem cells (hESCs) have been validated as a permanently stable and healthy human cell source. Theoretically, hESCs are an ideal cell source for in vitro toxicology studies. However, such studies are yet to be reported. Aim: This study aimed to investigate the utility of hESCs as a cellular model for genotoxicity testing. Method: hESCs were characterized by RT- PCR and immunohistochemistry to confirm their pluripotency. Genotoxic effects of DNA cross-linking agent, Mitomycin C, were evaluated in hESCs and normal human lung primary fibroblast cells (IMR-90). Peptide Nucleic Acid Fluorescence in-situ hybridization (PNA-FISH) was performed to determine if any chromosomal alterations were produced by Mitomycin C. Multi-color FISH (mFISH) allowed for further elucidation of the specific type of chromosomal aberrations. Result: Pluripotent markers, Oct4, SSEA-3 and TRA-1-81 were present only in hESCs, confirming pluripotency. Chromosomal analysis by PNA-FISH and mFISH following ix Mitomycin C treatment revealed that hESCs are significantly more resistant to genotoxic damage than IMR-90 as evidenced by the lower levels of chromosomal aberration in hESCs compared to IMR-90. The resistance of hESCs to genotoxic damage highlights the possibility of efficient repair mechanisms in these cells. It also emphasizes, how, established cell lines used for genotoxicity testing may not reflect the in vivo conditions. Hence, this study highlights the versatility and usefulness of hESCs for application in genotoxicity testing. x  ... of Genotoxicity using Mitomycin C…………………………… 26 iii CHAPTER 4 RESULTS……………………………………………………… 27 4.1 Characterization of human embryonic stem cells …………………… 27 4.2 Temperature tolerance of human embryonic. .. established cell lines poorly reflect human physiology Lack of standardization is also a limitation with the use of primary explanted cells Human embryonic stem cells (hESCs) have been validated... culture…………………………… 15 3.13 Differentiation of hESCs…………………………………………………………16 3.2 Characterization of Human Embryonic stem cells …………………………… 17 3.21 Real –time reverse transcriptase Polymerase Chain Reaction