Epigenetic and Environmental Determinants of Undifferentiated Human Embryonic Stem Cell Renewal

I Declaration IV Acknowledgements V List of Figures VIII List of Tables XVII List of Abbreviations XX Chapter 1: General Introduction 1 1.1 Human Embryonic Stem Cells 2 1.1.1 Derivation of Human Embryonic Stem Cells 2 1.1.2 Culture of Human Embryonic Stem Cells 4 1.1.3 Characterization of Human Embryonic Stem Cells 5 1.1.4 Applications of Human Embryonic Stem Cells 6 1.2 Embryonic Stem Cell Pluripotency 8 1.3 Induced Pluripotency 10 1.4 Epigenetic Modifications 13 1.4.1 DNA Methylation 13 1.4.2 Histone Modifications 15 1.4.3 5-Hydroxymethylcytosine 17 1.4.3.1 Historic Overview of 5-hydroxymethylcytosine 17 1.4.3.2 Ten-Eleven-Translocation (TET) enzyme family 19 1.4.3.3 5-Hydroxymethylcytosine can serve as a biomarker of toxicity in predictive epigenetic toxicology 21 1.5 Oxygen Tension and Development 23 1.5.1 Hypoxia-inducible factors (HIFs) 24 1.5.2 Oxygen Tension Influence in Stem Cell Biology 29 1.5.2.1 The Role of Oxygen in the Regulation of Neural Stem Cells 30 1.5.2.2 Hypoxia and Hematopoietic Stem Cells 32 1.5.2.3 Low Oxygen Tension and Mesenchymal Stem Cells 33 1.5.3 Oxygen Tensions and Embryonic Stem Cell Pluripotency 34 1.5.4 Low Oxygen Tensions and Induced Pluripotency 39 1.5.5 Low Oxygen Tensions and “De-differentiation” 42 1.6 Oxidative Stress 43 1.6.1 Generation of Reactive Oxygen Species (ROS) 44 1.6.2 Determination and Quantification of ROS 53 1.6.3 Antioxidant Cellular Systems and Antioxidant-related Enzymes 56 1.6.3.1 Glutathione (GSH) System 56 1.6.3.2 Endogenous Antioxidant Defence Enzymes 57 1.6.3.3 Exogenous Administration of Antioxidants 59 1.7 Epigenetic Responses to Hypoxia 60 1.7.1 Hypoxia and DNA Methylation 61 1.7.2 Hypoxia and Histone Modifications 62 1.7.3 Hypoxia and Jumonji-Domain Containing Histone Demethylases 63 1.8 Environmental Epigenetics 66 1.8.1 Cadmium 67 1.8.2 Arsenic 68 1.8.3 5-Azacytidine 71 1.8.4 Valproic Acid 72 1.9 Objectives of the thesis 74 Chapter 2: Materials and Methods 75 2.1 Mammalian Cell Culture 76 2.1.1 Culture of human embryonic stem cells 76 2.1.2 Microscopy of the morphology of human embryonic stem cells 76 2.1.3 Passaging of human embryonic stem cells 76 2.1.4 Freezing of human embryonic stem cells 77 2.1.5 Thawing of human embryonic stem cells 77 2.1.6 Hypoxic induction of human embryonic stem cells 78 2.1.7 Culture and passaging of Human Dermal-Fibroblasts 78 2.2 Study Test Compounds 79 2.2.1 Cytotoxicity range finder study 79 2.2.2 Cell viability assay 80 2.2.3 Subcytotoxic treatment of compounds on undifferentiated human ES cells 81 2.3 Determination of reactive oxygen species (ROS) production 81 2.4 Determination of mitochondrial membrane potential (MMP) 82 2.5 Immunocytochemistry 83 2.5.1 OCT4 and NANOG 83 2.5.2 5-Hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC) 84 2.6 Molecular Techniques 85 2.6.1 RNA Isolation and Quantification 85 2.6.2 Complementary DNA (cDNA) Synthesis 86 2.6.3 Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) 86 2.6.4 DNA Purification 90 2.7 Enzyme-linked immunosorbent assay (ELISA) of global DNA 5-hmC content 91 2.8 Small Interfering RNA (siRNA) knockdown 94 2.9 Statistical Analysis 95 Chapter 3: The Functional Significance of Epigenetically-Defined Biomarkers of the Undifferentiated Human Embryonic Stem Cell Phenotype 99 3.