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