Wong, Chun Kit. / "September 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 104-118). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Acknowledgements --- p.ii / Contents --- p.iii / Declaration --- p.vi / Abstract --- p.vii / 摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xiii / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- Embryonic Stem Cells (ESCs) / Chapter 1.1.1 --- Characteristics of ESC --- p.1 / Chapter 1.1.2 --- Therapeuticotential of ESCs --- p.2 / Chapter 1.2 --- 17β-estradiol (E2) / Chapter 1.2.1 --- Genomic Actions of E2 --- p.3 / Chapter 1.2.2 --- Non-genomic Actions of E2 --- p.5 / Chapter 1.2.3 --- hysiological Roles of E2 on Early Mammalian Development --- p.9 / Chapter 1.2.4 --- E2 and Cell Proliferation --- p.10 / Chapter 1.3 --- Ca2+ homeostasis / Chapter 1.3.1 --- Overview --- p.11 / Chapter 1.3.2 --- Ca2+ Signaling in mESCs --- p.14 / Chapter 1.4 --- Store-operated Ca2+ Entry (SOCE) / Chapter 1.4.1 --- Overview --- p.15 / Chapter 1.4.2 --- Store Depletion --- p.15 / Chapter 1.4.3 --- Activation of SOCE --- p.16 / Chapter 1.5 --- Molecular Identities of Store-operated Ca2+ Channels (SOCCs) on plasma Membrane / Chapter 1.5.1 --- TRPC Channels --- p.17 / Chapter 1.5.2 --- ORAI Channels --- p.18 / Chapter 1.5.3 --- Regulation of SOCCs at Different Levels --- p.18 / Chapter 1.5.4 --- Regulation of SOCE --- p.19 / Chapter 1.6 --- Nuclear Factor of Activated T-cells (NFAT) / Chapter 1.6.1 --- Overview --- p.20 / Chapter 1.6.2 --- Mechanisms of Action --- p.21 / Chapter 1.6.3 --- Functions --- p.22 / Chapter 1.7 --- Aims of the Study --- p.23 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS / Chapter 2.1 --- Maintenance of mESCs --- p.24 / Chapter 2.2 --- Cell proliferation Assay and Viability Test --- p.24 / Chapter 2.3 --- "RNAreparation, Reverse Transcription (RT) and Quantitative Polymerase Chain Reaction (qPCR)" --- p.25 / Chapter 2.4 --- Totalrotein Extraction --- p.27 / Chapter 2.5 --- Measurement of protein Concentration --- p.27 / Chapter 2.6 --- De-phosphorylation Assay --- p.28 / Chapter 2.7 --- Western Blot --- p.28 / Chapter 2.8 --- Ca2+ Measurement by Confocal Microscopy --- p.30 / Chapter 2.9 --- Ca2+ Measurement by Flow Cytometry --- p.31 / Chapter 2.10 --- siRNA Transfection --- p.31 / Chapter 2.11 --- DNAlasmid Transfection --- p.32 / Chapter 2.12 --- Molecular and Fluorescence Imaging --- p.33 / Chapter 2.13 --- Statistical Analysis --- p.34 / Chapter 2.14 --- Primers used in the Study (Table 1:Primers List) --- p.34 / Chapter 2.15 --- Drugs used in the Study (Table 2: Drugs List) --- p.36 / Chapter 2.16 --- Antibodies used in the Study (Table 3: Antibodies List) --- p.37 / Chapter CHAPTER THREE: --- RESULTS / Chapter 3.1 --- Expression of SOCE in mESCs --- p.38 / Chapter 3.2 --- SOCC Blockers Attenuated mESCroliferation --- p.43 / Chapter 3.3 --- E2 Increased mESCroliferation --- p.48 / Chapter 3.4 --- E2 Increased Intracellular Ca2+ ([Ca2+]i) Level in mESCs --- p.48 / Chapter 3.5 --- E2 Increased the Amplitude of SOCE --- p.51 / Chapter 3.6 --- Increase in mESC proliferation and SOCE Caused by E2 Could be Reversed by SOCC Blocker --- p.51 / Chapter 3.7 --- Relative Expression of SOCC Candidates at mRNA Level Under the Treatment of E2 --- p.56 / Chapter 3.8 --- E2 Down-regulated the Expression of ORAI3 --- p.56 / Chapter 3.9 --- Knockdown of ORAI3 in mESCs --- p.61 / Chapter 3.10 --- Identification of NFATc3 Specific Bands --- p.63 / Chapter 3.11 --- E2 Increased the phosphorylation of NFATc3 --- p.67 / Chapter 3.12 --- Effects of 2-APB on NFATc3 phosphorylation Status --- p.67 / Chapter 3.13 --- Identification of NFATc4 Specific Bands ？ --- p.72 / Chapter 3.14 --- E2 Increased the Translocation of GFP-NFATc4 From the Cytoplasm to the Nucleus and This Effect Could be Reversed by 2-APB --- p.80 / Chapter 3.15 --- CsA Reversed E2-induced Increase in proliferation --- p.82 / Chapter CHAPTER FOUR: --- DISCUSSION / Chapter 4.1 --- Expression of SOCE in mESCs --- p.84 / Chapter 4.2 --- proliferation of mESCs Depends on SOCE --- p.85 / Chapter 4.3 --- E2 Acts an Extrinsic Factor for Stimulatingroliferation of mESCs Via SOCE --- p.87 / Chapter 4.4 --- roposed Mechanism to Show an Increment of SOCE Can be Due to a Down-regulation of ORAI3 --- p.89 / Chapter 4.5 --- Experiments Aiming to Knockdown ORAI3 --- p.92 / Chapter 4.6 --- roposed Mechanism to Show an Increment of SOCE by Other SOCC Candidates Rather than ORAI3 --- p.93 / Chapter 4.7 --- Activation of NFATc3 and NFATc4 by E2 in mESCs --- p.94 / Chapter 4.8 --- possible Downstream Targets of NFAT Responsible for E2-induced mESCs proliferation --- p.96 / Chapter CHAPTER FIVE: --- FUTUREERSPECTIVES --- p.98 / Chapter CHAPTER SIX: --- CONCLUSION --- p.100 / REFERENCES --- p.104

Precise control of stem cell fate is a fundamental issue in the use of human embryonic stem (hES) cells in the context of cell therapy We examined three ways in which the microenvironment can be controlled to alter hES cell behavior, providing insight into the best conditions for maintenance of pluripotency and neural differentiation in developmental and therapeutic studies. We first examined the effects of polydimethylsiloxane (PDMS) growth surfaces on hES cell survival and maintenance of pluripotency. Lightly cured, untreated PDMS was shown to be a poor growth surface for hES cells. Some of the adverse effects caused by PDMS could be mitigated with increased curing or UV treatment of the surface, but neither modification provided a growth surface that supported pluripotent hES cells as well as polystyrene. This work provides a basis for further optimizing PDMS for hES cell culture, moving towards the use of microdevices in establishing precise control over stem cell fate. The second study explored the use of an easily constructed diffusion-based device to grow hES cells in culture on a defined, physiologic oxygen (O₂) gradient. We observed greater hES cell survival and higher levels of pluripotency markers in the lower O₂ regions of the gradient. The greatest benefit was observed at O₂ levels below 5%, narrowing the potential optimal range of O₂ for the maintenance of pluripotent hES cells. Finally, we developed a small molecule-mediated adherent and feeder-free neural differentiation protocol that reduced the cost and time scale for in vitro differentiation of neural precursors and functional neurons from human pluripotent cells. hES cell-derived neural precursors transplanted into a murine model of focal ischemic stroke survived, improved neurogenesis, and differentiated into neurons. Transplant also led to a more consistent and measurable sensory recovery after stroke as compared to untransplanted controls. This protocol represents a potentially translatable method for the generation of CNS progenitors from human pluripotent stem cells.

Stem and progenitor cells are an attractive cell source for the treatment of degenerative diseases due to their potential to differentiate into multiple cell types and provide large cell yields. Thus far, however, clinical applications have been limited due to inefficient differentiation into desired cell types with sufficient yields for adequate tissue repair and regeneration. The ability to spontaneously aggregate in suspension makes embryonic stem cells (ESCs) amenable to large-scale culture techniques for the production of large yields of differentiating cell spheroids (termed embryoid bodies or EBs); however, the introduction of hydrodynamic conditions may alter differentiation profiles within EBs and should be methodically examined. The work presented here employs a novel, laboratory-scale hydrodynamic culture model to systematically interrogate the effects of ESC culture hydrodynamics on cardiomyocyte differentiation through the modulation of a developmentally-relevant signaling pathway. The fluidic environment was defined using computational fluid dynamic modeling, and the effects of hydrodynamic conditions on EB formation, morphology and structure were assessed. Additionally, EB differentiation was examined through gene and protein expression, and indicated that hydrodynamic conditions modulate differentiation patterns, particularly cardiogenic lineage development. This work illustrates that mixing conditions can modulate common signaling pathways active in ESC differentiation and suggests that differentiation may be regulated via bioprocessing parameters and bioreactor design.

Current therapeutic strategies to stimulate endogenous angiogenic processes within injured tissue areas are typically based on introducing exogenous pro-angiogenic molecules or cell populations. Stem cell transplantation for angiogenic therapy aims to deliver populations of cells that secrete angiogenic factors and/or engraft in the new branching vasculature within the damaged tissue. Utilizing stem or progenitor cells has been shown to induce a rather robust angiogenic response despite minimal repopulation of the host vasculature, suggesting that stem cells may provide paracrine factors that transiently induce endogenous angiogenesis of tissues undergoing regeneration. Early differentiating embryonic stem cell (ESC) aggregates, referred to as embryoid bodies (EBs), can undergo vasculogenic differentiation, and also produce extracellular matrix and growth factors that induce proliferation, differentiation, and tissue morphogenesis. Taken together, the ESC extracellular environment may be an effective means by which to manipulate cell behavior. Thus, the objective of this project was to harness morphogens derived from ESCs undergoing differentiation and analyze their bioactive potential. To examine the expression of extracellular factors within EBs, gene expression arrays in conjunction with a variety of analytical tools were utilized to gain an understanding of the importance of extracellular factors in ESC differentiation. Furthermore, the soluble fraction of secreted factors contained within EB-conditioned media was compared to the matrix-associated factors produced by EBs, which led to the development of novel ESC-derived matrices via mechanical acellularization methods. Acellular embryonic stem cell-derived matrices demonstrated the retention of bioactive factors that impacted aspects of angiogenesis. In conclusion, extracellular factors were modulated in response to the progression of EB differentiation and can further be harnessed via acellularization techniques, in order to deliver bioactive ESC-secreted factors in a cell-free manner.

H1 linker histone facilitates the formation of higher order chromatin structure and is essential for mammalian development. Mice have 11 H1 variants which are differentially regulated and conserved in human. Previous research indicates that H1 regulates the expression of specific genes in mouse embryonic stem cells (ESCs). However, whether individual variants have distinct functions and how H1 participates in gene regulation remain elusive. An investigation of the precise localization of individual H1 variants in vivo would facilitate the elucidation of mechanisms underlying chromatin compaction regulated gene expression, while it has been extremely difficult due to the lacking of specific antibodies toward H1 variants. In this dissertation, I have generated a knock-in system in ESCs and shown that the N-terminally tagged H1 proteins are functionally interchangeable to their endogenous counterparts in vivo. H1d and H1c are depleted from GC- and gene-rich regions and active promoters, inversely correlated with H3K4me3, but positively correlated with H3K9me3 and associated with characteristic sequence features. Surprisingly, both H1d and H1c are significantly enriched at major satellites, which display increased nucleosome spacing compared with bulk chromatin. While also depleted at active promoters and enriched at major satellites, overexpressed H10 displays differential binding patterns in specific repetitive sequences compared with H1d and H1c. Depletion of H1c, H1d ,and H1e causes pericentric chromocenter clustering and de-repression of major satellites. Collectively, these results integrate the localization of an understudied type of chromatin proteins, namely the H1 variants, into the epigenome map of mouse ESCs, and demonstrate significant changes at pericentric heterochromatin upon depletion of this epigenetic mark.

Indiana University-Purdue University Indianapolis (IUPUI) / Embryonic stem cells (ESCs) are unique in that they have potential to give rise to every cell type of the body. Little is known about stimuli that promote mouse (m)ESC differentiation and proliferation. Therefore the purpose of this study was to determine the role of Toll Like Receptor (TLR) ligands in mESCs proliferation, survival, and differentiation in the presence of Leukemia Inhibitory Factor (LIF). We hypothesized that TLRs are expressed and functional, and when activated by their ligand will induce survival, proliferation, and prevent differentiation. In this study, mESC line E14 was used to determine the expression of TLRs at the mRNA level and three mESC lines, R1, CGR8, and E14, were used to determine cell surface protein levels. We found expression of TLRs 1, 2, 3, 5, and 6 at the mRNA level, but no expression of TLRs 4, 7, 8, and 9 in the E14 mESC line. We confirmed the presence of TLR-2 but not of TLR-4, protein on the cell surface using flow cytometric analysis for all three cell lines. We focused our studies mainly on TLR-2 using the E14 cell line. Pam3Cys, is a synthetic triacyl lipoprotein and a TLR-2 ligand, which induced a significant increase in mESC proliferation on Days 3, 4, and 5 and enhanced survival of mESC in a dose dependent manner in the context of delayed addition of serum. All the latter experiments were performed in triplicate and student T-test was performed to establish significant differences. Next, we demonstrated functionality of TLR-2 via the MyD88/IKK pathway, where MyD88 was expressed and IKKα/β phosphorylation was enhanced. This was associated with increased NF-κB nuclear translocation upon activation by Pam3Cys. Finally, we showed that there were no changes in expression of mESCs markers Oct-4, KLF-4, Sox-2, and SSEA-1, thus illustrating that the mESCs may have remained in a pluripotent state after activation with the TLR-2 ligand in the presence of LIF. These results demonstrate that mESCs can respond to microbial products, such as Pam3Cys, and can induce proliferation and survival of the mESCs. This finding expands the role of TLRs and has some implications in understanding embryonic stem cell biology.

Master of Science / Department of Anatomy and Physiology / Mark L. Weiss / When mouse embryonic stem cells (ESCs) were grown on mitotically inactivated rat umbilical cord-derived stromal cells (RUCs) in the absence of leukemia inhibitory factor (LIF) and 2-mercaptoethanol (2-MCE), the ESCs showed alkaline phosphatase (AP) staining. ESCs cultured on RUCs maintain expression of the following pluripotency genes, Nanog, Sox2 and Oct4 and grow at a slower rate when compared with ESCs grown on mitotically inactivated mouse embryonic fibroblasts (MEFs). Differences in gene expression for the markers of pluripotency Oct4, Sox2 and Nanog, AP staining and ESC growth rate were also observed after LIF and 2-MCE were removed from the co-cultures. Reverse transcriptase polymerase chain reaction (RT-PCR) suggested differences in Sox2 and Nanog mRNA expression, with both genes being expressed at higher levels in the ESCs cultured on RUCs in the absence of LIF/2-MCE as compared to ESCs cultured on MEFs. Semi-quantitative RT-PCR indicated that Nanog expression was higher when ESCs were grown on RUCs in the absence of LIF and 2-MCE as compared to MEFs in the same treatment conditions. Bisulfite-mediated methylation analysis of the Nanog proximal promoter suggested that the maintenance of Nanog gene expression found in ESCs grown on RUCs after culture for 96 hours in the absence of LIF/2-MCE may be due to prevention of methylation of the CpG dinucleotides in the Nanog proximal promoter as compared to ESCs grown on MEFs. Thus, RUCs may release factors into the medium that maintain the pluripotent state of mouse ESCs in the absence of LIF and 2-MCE.

Ng, Sze Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 110-124). / Abstract also in Chinese. / Thesis Committee --- p.i / Acknowledgements --- p.ii / Contents --- p.iii / Abstract --- p.vii / 論文摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xiii / List of Tables --- p.xvii / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Embryonic Stem Cells (ESCs) --- p.1 / Chapter 1.1.1 --- What are ESCs and the characteristics of ESCs --- p.1 / Chapter 1.1.1.1 --- Pluripotent markers --- p.2 / Chapter 1.1.1.2 --- Germ layers' markers --- p.3 / Chapter 1.1.2 --- Mouse ESCs (mESCs) --- p.4 / Chapter 1.1.2.1 --- mESCs co-culture with mitotically inactivated mouse embryonic fibroblast (MEF) feeder layers --- p.4 / Chapter 1.1.2.2 --- Feeder free mESCs --- p.4 / Chapter 1.1.3 --- Promising uses of ESCs and their shortcomings --- p.5 / Chapter 1.1.4 --- Characteristics of ESC-derived cardiomyocytes (ESC-CMs) --- p.6 / Chapter 1.2 --- Cardiovascular diseases (CVD) --- p.7 / Chapter 1.2.1 --- Background --- p.7 / Chapter 1.2.2 --- Current treatments --- p.8 / Chapter 1.2.3 --- Potential uses of ESC-CMs for basic science research and therapeutic purposes --- p.9 / Chapter 1.2.4 --- Current hurdles in application of ESC-CMs for clinical uses --- p.10 / Chapter 1.3 --- Cardiac gene markers --- p.13 / Chapter 1.3.1 --- Atrial-specific --- p.13 / Chapter 1.3.2 --- Ventricular-specific --- p.19 / Chapter 1.4 --- Lentiviral vector-mediated gene transfer --- p.27 / Chapter 1.5 --- Cell cycle in ESCs --- p.29 / Chapter 1.5.1 --- Cell cycle --- p.29 / Chapter 1.5.2 --- Characteristics of cell cycle in ESCs --- p.30 / Chapter 1.6 --- Potassium (K+) channels --- p.31 / Chapter 1.6.1 --- Voltage gated potassium (Kv) channels --- p.32 / Chapter 1.6.2 --- Role of Kv channels in maintenance of membrane potential --- p.32 / Chapter 1.7 --- Objectives and significances --- p.33 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.35 / Chapter 2.1 --- Mouse embryonic fibroblast (MEF) culture --- p.35 / Chapter 2.1.1 --- Derivation of MEF --- p.3 5 / Chapter 2.1.2 --- MEF culture --- p.37 / Chapter 2.1.3 --- Irradiation of MEF --- p.37 / Chapter 2.2 --- mESC culture and their differentiation --- p.38 / Chapter 2.2.1 --- mESC culture --- p.38 / Chapter 2.2.2 --- Differentiation of mESCs --- p.39 / Chapter 2.3 --- Subcloning --- p.40 / Chapter 2.3.1 --- Amplification of Irx4 --- p.40 / Chapter 2.3.2 --- Purification of DNA products --- p.41 / Chapter 2.3.3 --- Restriction enzyme digestion --- p.42 / Chapter 2.3.4 --- Ligation of Irx4 with iDuet101A vector --- p.43 / Chapter 2.3.5 --- Transformation of ligation product into competent cells --- p.43 / Chapter 2.3.6 --- Small scale preparation of bacterial plasmid DNA --- p.44 / Chapter 2.3.7 --- Confirmation of positive clones by restriction enzyme digestion --- p.45 / Chapter 2.3.8 --- DNA sequencing of the cloned plasmid DNA --- p.45 / Chapter 2.3.9 --- Large scale preparation of target recombinant expression vector --- p.45 / Chapter 2.4 --- Lentiviral vector-mediated gene transfer to mESCs --- p.47 / Chapter 2.4.1 --- Lentivirus packaging --- p.47 / Chapter 2.4.2 --- Lentivirus titering --- p.48 / Chapter 2.4.3 --- Multiple transduction to mESCs --- p.48 / Chapter 2.4.4 --- Hygromycin selection on mESCs --- p.49 / Chapter 2.5 --- Selection of stable clone --- p.49 / Chapter 2.5.1 --- Monoclonal establishment and clone selection --- p.49 / Chapter 2.6 --- Differentiation of cell lines after selection --- p.50 / Chapter 2.7 --- Gene expression study on control and Irx4-overexpressed mESC lines --- p.50 / Chapter 2.8 --- Analysis of mESCs at different phases of the cell cycle --- p.55 / Chapter 2.8.1 --- Go/Gi and S phase synchronization --- p.55 / Chapter 2.8.2 --- Cell cycle analysis by propidium iodide (PI) staining followed by flow cytometric analysis --- p.55 / Chapter 2.8.3 --- Gene expression study by qPCR of Kv channel isoforms --- p.56 / Chapter 2.8.4 --- Membrane potential measurement by membrane potential-sensitive dye followed by flow cytometry --- p.57 / Chapter 2.9 --- Apoptotic study --- p.58 / Chapter 2.10 --- Determination of pluripotent characteristic of mESCs --- p.59 / Chapter 2.10.1 --- Expression of germ layers' markers by qPCR --- p.59 / Chapter 2.10.2 --- Differentiation by hanging drop method and suspension method --- p.61 / Chapter CHAPTER THREE --- RESULTS --- p.62 / Chapter 3.1 --- mESC culture --- p.62 / Chapter 3.1.1 --- Cell colony morphology of feeder free mESCs --- p.62 / Chapter 3.2 --- Subcloning --- p.63 / Chapter 3.2.1 --- PCR cloning of Irx4 --- p.63 / Chapter 3.2.2 --- Restriction digestion on iDuet101A --- p.64 / Chapter 3.2.3 --- Ligation of Irx4 to iDuet101A backbone --- p.66 / Chapter 3.2.4 --- Confirmation of successful ligation --- p.67 / Chapter 3.3 --- Lentivirus packaging --- p.68 / Chapter 3.3.1 --- Transfection --- p.68 / Chapter 3.4 --- Multiple transduction of mESCs and hygromycin selection of positively-transduced cells --- p.69 / Chapter 3.5 --- FACS --- p.70 / Chapter 3.6 --- Irx4 and iduet clone selection --- p.71 / Chapter 3.7 --- Characte rization of mESCs after clone selection --- p.74 / Chapter 3.7.1 --- Immunostaining of pluripotent and differentiation markers --- p.74 / Chapter 3.8 --- Differentiation of cell lines after selection --- p.77 / Chapter 3.8.1 --- Size of EBs of the cell lines during differentiation --- p.77 / Chapter 3.9 --- Gene expression study by qPCR --- p.79 / Chapter 3.10 --- Kv channel expression and membrane potential of mESCs at Go/Gi phase and S phases --- p.84 / Chapter 3.10.1 --- Expression of Kv channels subunits at G0/Gi phase and S phase --- p.86 / Chapter 3.10.2 --- Membrane potential at Go/Gi phase and S phase --- p.87 / Chapter 3.11 --- Effects of TEA+ on feeder free mESCs --- p.89 / Chapter 3.11.1 --- Apoptotic study --- p.89 / Chapter 3.11.2 --- Expression of germ layers´ة markers --- p.91 / Chapter 3.11.3 --- Embryo id bodies (EBs) measurement after differentiation --- p.92 / Chapter CHAPTER FOUR --- DISCUSSION --- p.95 / Chapter 4.1 --- Effect of overexpression of Irx4 on the cardiogenic potential of mESCs --- p.95 / Chapter 4.2 --- Role of Kv channels in maintaining the chacteristics of mESCs --- p.99 / Chapter 4.2.1 --- Inhibition of Kv channels led to a redistribution of the proportion of cells in different phases of the cell cycle: importance of Kv channels in cell cycle progression in native ESCs --- p.99 / Chapter 4.2.2 --- Inhibition of Kv channels led to a loss of pluripotency at molecular and functional levels: importance of Kv channels in the fate determination of mESCs --- p.102 / Chapter 4.3 --- Insights from the present investigation on the future uses of ESCs --- p.105 / Conclusions --- p.108 / References --- p.110

Law, Sau Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 111-117). / Abstract also in Chinese. / Thesis Committee --- p.i / Acknowledgements --- p.ii / Contents --- p.iii / Abstract --- p.vii / 論文摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xiii / List of Tables --- p.xxiii / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1.1 --- Embryonic Stem (ES) Cells / Chapter 1.1.1 --- Characteristics of ES Cells l / Chapter 1.1.2 --- Therapeutic Potential of ES Cells --- p.3 / Chapter 1.1.3 --- Myocardial Infarction and ES cells-derived Cardiomyocytes --- p.4 / Chapter 1.1.4 --- Current Hurdles of Using ES cells-derived Cardiomyocytes for Research and Therapeutic Purposes --- p.6 / Chapter 1.2 --- Transcription Factors for Cardiac Development / Chapter 1.2.1 --- GATA-binding Protein 4 (GATA-4) --- p.8 / Chapter 1.2.2 --- Myocyte Enhancer Factor 2C (MEF2C) --- p.10 / Chapter 1.2.3 --- "NK2 Transcription Factor Related, Locus 5 (Nkx2.5)" --- p.11 / Chapter 1.2.4 --- Heart and Neural Crest Derivatives Expressed 1 /2 (HANDI/2) --- p.11 / Chapter 1.2.5 --- T-box Protein 5 (Tbx5) --- p.13 / Chapter 1.2.6 --- Serum Response Factor (SRF) --- p.14 / Chapter 1.2.7 --- Specificity Protein 1 (Spl) --- p.15 / Chapter 1.2.8 --- Activator Protein 1 (AP-1) --- p.16 / Chapter 1.3 --- Reactive Oxygen Species (ROS) / Chapter 1.3.1 --- Cellular Production of ROS --- p.18 / Chapter 1.3.2 --- Maintenance of Redox balance --- p.18 / Chapter 1.3.3 --- Redox Signaling --- p.19 / Chapter 1.4 --- Nitric Oxide (NO) and NO Signaling --- p.20 / Chapter 1.5 --- Aims of the Study --- p.22 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- Mouse Embryonic Fibroblast (MEF) Culture / Chapter 2.1.1 --- Derivation of MEF --- p.23 / Chapter 2.1.2 --- Maintenance of MEF Culture --- p.24 / Chapter 2.1.3 --- Irradiation of MEF --- p.25 / Chapter 2.2 --- Mouse ES Cell Culture / Chapter 2.2.1 --- Maintenance of Undifferentiated Mouse ES Cell Culture --- p.26 / Chapter 2.2.2 --- Differentiation of Mouse ES Cells --- p.26 / Chapter 2.2.3 --- Exogenous addition of hydrogen peroxide (H2O2) and NO --- p.27 / Chapter 2.3 --- ROS Localization Study / Chapter 2.3.1 --- Frozen Sectioning --- p.28 / Chapter 2.3.2 --- Confocal microscopy for ROS detection --- p.28 / Chapter 2.4 --- Intracellular ROS Measurement / Chapter 2.4.1 --- "Chemistry of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA)" --- p.29 / Chapter 2.4.2 --- Flow Cytometry for ROS Measurement --- p.29 / Chapter 2.5 --- Gene Expression Study / Chapter 2.5.1 --- Primer Design --- p.30 / Chapter 2.5.2 --- RNA Extraction --- p.31 / Chapter 2.5.3 --- DNase Treatment --- p.32 / Chapter 2.5.4 --- Reverse Transcription --- p.32 / Chapter 2.5.5 --- Quantitative Real Time PCR --- p.33 / Chapter 2.5.6 --- Quantification of mRNA Expression --- p.34 / Chapter 2.6 --- Protein Expression Study / Chapter 2.6.1 --- Total Protein Extraction --- p.34 / Chapter 2.6.2 --- Nuclear and Cytosolic Protein Extraction --- p.35 / Chapter 2.6.3 --- Measurement of Protein Concentration --- p.36 / Chapter 2.6.4 --- De-sumoylation Assay --- p.36 / Chapter 2.6.5 --- De-phosphorylation Assay --- p.37 / Chapter 2.6.6 --- De-glycosylation Assay --- p.38 / Chapter 2.6.7 --- Western Blot --- p.39 / Chapter 2.7 --- Statistical Analysis --- p.41 / Chapter CHAPTER THREE --- RESULTS / Chapter 3.1 --- Study of Endogenous ROS / Chapter 3.1.1 --- Level and Distribution of Endogenous ROS --- p.47 / Chapter 3.1.2 --- Quantification of intracellular ROS --- p.48 / Chapter 3.2 --- Effect of Exogenous Addition of Nitric Oxide (NO) on Cardiac Differentiation / Chapter 3.2.1 --- Beating Profile of NO-treated Embryoid Bodies (EBs) --- p.50 / Chapter 3.3 --- Effect of Exogenous Addition of H2O2 on Cardiac Differentiation / Chapter 3.3.1 --- Beating Profile of H2O2-treated EBs --- p.51 / Chapter 3.3.2 --- mRNA Expression of Cardiac Structural Genes --- p.52 / Chapter 3.3.3 --- Protein Expression of Cardiac Structural Genes --- p.54 / Chapter 3.3.4 --- mRNA Expression of Cardiac Transcription Factors --- p.58 / Chapter 3.3.5 --- Protein Expression of Cardiac Transcription Factors --- p.67 / Chapter 3.3.6 --- Post-translational Modifications of Cardiac Transcription Factors --- p.74 / Chapter 3.3.7 --- Translocation of Cardiac Transcription Factors --- p.89 / Chapter CHAPTER FOUR --- DISCUSSION / Chapter 4.1 --- Changes in the Level of Endogenous ROS During Cardiac Differentiation of Mouse ES Cells --- p.96 / Chapter 4.2 --- H2O2 and NO Have Opposite Effects Towards Cardiac Differentiation --- p.97 / Chapter 4.3 --- Exogenous Addition of H2O2 Advances Differentiation of Mouse ES Cells into Cardiac Lineage --- p.99 / Chapter 4.4 --- Possible Role of H2O2 in Mediating Cardiac Differentiation of Mouse ES Cells --- p.103 / Chapter 4.5 --- Future Directions --- p.108 / Conclusions --- p.110 / References --- p.111

Genesis of life begins with the fusion of female and male haploid gametes through a process of fertilization leading to the formation of a diploid cell, the zygote. This undergoes successive cleavage divisions forming 2-, 4- and 8- cell embryos and their individual cells (blastomeres) are totipotent. As development proceeds, there is a gradual restriction in their totipotency, resulting in the generation of two distinct cell lineages i.e., the differentiated trophectoderm (TE) cells and the undifferentiated, inner cell mass (ICM) during blastocyst morphogenesis (Rossant and Tam 2009). During the course of development, the ICM cells can give rise to all cell types of an organism and can also provide embryonic stem (ES)-cells when cultured in vitro (Evan and Kaufman 1981). ES-cells are pluripotent cells, having the ability to self-renew indefinitely and differentiate into all the three primary germ layers (ectoderm, mesoderm and endoderm) derived-cell types. ES-cells are an excellent developmental model system to understand basic mechanisms of self-renewal, cell differentiation and function of various genes in vitro and in vivo (Capecchi 2001). Importantly, their cell derivatives could potentially be used for experimental cell-based therapy for a number of diseases. Although, human ES-cell lines have been successfully derived and differentiated to various cell types (Thomson et al., 1998; Odorico et al., 2001), their cell-therapeutic potential is far from being tested, in view of the lack of our understanding of lineage-specific differentiation, homing and structural-functional integration of differentiated cell types in the host environment. To understand these mechanisms, it is desirable to have fluorescently-marked ES-cells and their differentiated cell-types, which could facilitate experimental cell transplantation studies. In this regard, our laboratory has earlier generated enhanced green fluorescent protein (EGFP)-expressing FVB/N transgenic ‘green’ mouse, under the control of ubiquitous chicken -actin promoter (Devgan et al., 2003). This transgenic mouse has been an excellent source of intrinsically green fluorescent cell types. We have been attempting to derive ES-cell line from this transgenic mouse. Because the derivation of ES-cell line is genetic strain-dependent, with some strains being relatively permissible for ES-cell derivation while others are quite resistant (non permissive), it has been extremely difficult to derive ES-cell line from the FVB/N mouse strain. There is a need to evolve experimental strategies to derive ES-cell line from FVB/N mouse, a strain extensively used for transgenesis. Thus, the aims of the study described in the thesis are to: (1) develop an experimental system to derive EGFP-expressing fluorescently-marked ES-cell line from a non-permissive FVB/N mouse strain; (2) characterize the established ES-cell line; (3) achieve differentiation of various cell types from EGFP-expressing ES-cell line and (4) understand role of FGF signaling in cardiac differentiation from the established ES-cell line. In order to have an appropriate and relevant literature background, the 1st chapter in this thesis describes a comprehensive up-to-date review of literature, pertaining to the early mammalian development and differentiation of blastocyst, followed by origin and properties of ES-cells. Various ES-cell derivation strategies from genetically permissive and non-permissive mouse strains are described and also the ES-cell differentiation potential to various progenitors and differentiated cell types. Subsequently, details on molecular basis of cardiac differentiation and the therapeutic potential of ES-cell derived differentiated cell types to treat disease(s) are described. This chapter is followed by three data chapters (II-IV). Chapter-II describes the issues related to non-permissiveness of FVB/N strain for ES-cell derivation and strategies to overcome this hurdle. This is followed by detailed results pertaining to generation of homozygous EGFP-expressing transgenic mice and development of a two-pronged ES-cell derivation approach to successfully establish a permanent ES-cell line (named ‘GS-2’ ES-cell line) from the EGFP-transgenic ‘green’ mouse. This chapter also provides results pertaining to detailed characterization of the ‘GS-2’ ES-cell line which includes colony morphology, expansion efficiency, alkaline phosphatase staining, expression analysis of pluripotent markers by RT-PCR and immunostaining approaches and karyotyping. Following this, the outcome of results and significance in the context of reported information are discussed in detail. Having successfully derived the ‘GS-2’ ES-cell line, it is necessary to thoroughly assess the differentiation competence of the ‘GS-2’ ES-cell line. Therefore, the Chapter-III describes detailed assessment of the in vitro and in vivo differentiation potential of the ‘GS-2’ ES-cell line. For in vitro differentiation, results pertaining to ES-cell derived embryoid body (EB) formation and their differentiation to ectodermal, mesodermal and endodermal cell types, expressing nestin, BMP-4 and α-fetoprotein, respectively, are described. Besides, the robustness of adaptability of ‘GS-2’ ES-cells to various culture conditions for their maintenance and differentiation are described. Also shown in the chapter is the relatively greater propensity of this cell line to cardiac differentiation. For in vivo differentiation, the ‘GS-2’ ES-cell derived teratoma formation in nude mice and its detailed histological analysis showing three germ layer cell types and their derivatives are described. Last part of the data described in this chapter, pertains to generation of chimeric blastocysts by aggregation method. Because the ‘GS-2’ ES-cell line exhibited a robust differentiation potential, including an efficient cardiomyocyte differentiation, it is of interest to enhance the efficiency of cardiomyocyte differentiation by exogenous addition of one of the key growth factors i.e., FGF8b since this has been implicated to be critical for cardiogenesis in non-mammalian verterbrate species. Therefore, Chapter-IV is focused on assessing the ability of ‘GS-2’ ES-cell line for its cardiomyocyte differentiation property with particular emphasis on the FGF-induced cardiac differentiation. Results pertaining to the expressions of various FGF ligands and their receptors during differentiation of ES-cells are described. Besides, increases in the cardiac efficiency, following FGF8b treatment and the associated up-regulation of cardiac-specific markers such as GATA-4, ISL-1 and α-MHC are shown. At the end of data chapters, separate sections are devoted for ‘Summary and Conclusion’ and for ‘Bibliography’.