Mechanisms underlying the self-renewal characteristic and cardiac differentiation of mouse embryonic stem cells.

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.iAcknowledgements --- p.iiContents --- p.iiiAbstract --- p.vii論文摘要 --- p.xAbbreviations --- p.xiList of Figures --- p.xiiiList of Tables --- p.xviiChapter CHAPTER ONE --- INTRODUCTION --- p.1Chapter 1.1 --- Embryonic Stem Cells (ESCs) --- p.1Chapter 1.1.1 --- What are ESCs and the characteristics of ESCs --- p.1Chapter 1.1.1.1 --- Pluripotent markers --- p.2Chapter 1.1.1.2 --- Germ layers' markers --- p.3Chapter 1.1.2 --- Mouse ESCs (mESCs) --- p.4Chapter 1.1.2.1 --- mESCs co-culture with mitotically inactivated mouse embryonic fibroblast (MEF) feeder layers --- p.4Chapter 1.1.2.2 --- Feeder free mESCs --- p.4Chapter 1.1.3 --- Promising uses of ESCs and their shortcomings --- p.5Chapter 1.1.4 --- Characteristics of ESC-derived cardiomyocytes (ESC-CMs) --- p.6Chapter 1.2 --- Cardiovascular diseases (CVD) --- p.7Chapter 1.2.1 --- Background --- p.7Chapter 1.2.2 --- Current treatments --- p.8Chapter 1.2.3 --- Potential uses of ESC-CMs for basic science research and therapeutic purposes --- p.9Chapter 1.2.4 --- Current hurdles in application of ESC-CMs for clinical uses --- p.10Chapter 1.3 --- Cardiac gene markers --- p.13Chapter 1.3.1 --- Atrial-specific --- p.13Chapter 1.3.2 --- Ventricular-specific --- p.19Chapter 1.4 --- Lentiviral vector-mediated gene transfer --- p.27Chapter 1.5 --- Cell cycle in ESCs --- p.29Chapter 1.5.1 --- Cell cycle --- p.29Chapter 1.5.2 --- Characteristics of cell cycle in ESCs --- p.30Chapter 1.6 --- Potassium (K+) channels --- p.31Chapter 1.6.1 --- Voltage gated potassium (Kv) channels --- p.32Chapter 1.6.2 --- Role of Kv channels in maintenance of membrane potential --- p.32Chapter 1.7 --- Objectives and significances --- p.33Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.35Chapter 2.1 --- Mouse embryonic fibroblast (MEF) culture --- p.35Chapter 2.1.1 --- Derivation of MEF --- p.3 5Chapter 2.1.2 --- MEF culture --- p.37Chapter 2.1.3 --- Irradiation of MEF --- p.37Chapter 2.2 --- mESC culture and their differentiation --- p.38Chapter 2.2.1 --- mESC culture --- p.38Chapter 2.2.2 --- Differentiation of mESCs --- p.39Chapter 2.3 --- Subcloning --- p.40Chapter 2.3.1 --- Amplification of Irx4 --- p.40Chapter 2.3.2 --- Purification of DNA products --- p.41Chapter 2.3.3 --- Restriction enzyme digestion --- p.42Chapter 2.3.4 --- Ligation of Irx4 with iDuet101A vector --- p.43Chapter 2.3.5 --- Transformation of ligation product into competent cells --- p.43Chapter 2.3.6 --- Small scale preparation of bacterial plasmid DNA --- p.44Chapter 2.3.7 --- Confirmation of positive clones by restriction enzyme digestion --- p.45Chapter 2.3.8 --- DNA sequencing of the cloned plasmid DNA --- p.45Chapter 2.3.9 --- Large scale preparation of target recombinant expression vector --- p.45Chapter 2.4 --- Lentiviral vector-mediated gene transfer to mESCs --- p.47Chapter 2.4.1 --- Lentivirus packaging --- p.47Chapter 2.4.2 --- Lentivirus titering --- p.48Chapter 2.4.3 --- Multiple transduction to mESCs --- p.48Chapter 2.4.4 --- Hygromycin selection on mESCs --- p.49Chapter 2.5 --- Selection of stable clone --- p.49Chapter 2.5.1 --- Monoclonal establishment and clone selection --- p.49Chapter 2.6 --- Differentiation of cell lines after selection --- p.50Chapter 2.7 --- Gene expression study on control and Irx4-overexpressed mESC lines --- p.50Chapter 2.8 --- Analysis of mESCs at different phases of the cell cycle --- p.55Chapter 2.8.1 --- Go/Gi and S phase synchronization --- p.55Chapter 2.8.2 --- Cell cycle analysis by propidium iodide (PI) staining followed by flow cytometric analysis --- p.55Chapter 2.8.3 --- Gene expression study by qPCR of Kv channel isoforms --- p.56Chapter 2.8.4 --- Membrane potential measurement by membrane potential-sensitive dye followed by flow cytometry --- p.57Chapter 2.9 --- Apoptotic study --- p.58Chapter 2.10 --- Determination of pluripotent characteristic of mESCs --- p.59Chapter 2.10.1 --- Expression of germ layers' markers by qPCR --- p.59Chapter 2.10.2 --- Differentiation by hanging drop method and suspension method --- p.61Chapter CHAPTER THREE --- RESULTS --- p.62Chapter 3.1 --- mESC culture --- p.62Chapter 3.1.1 --- Cell colony morphology of feeder free mESCs --- p.62Chapter 3.2 --- Subcloning --- p.63Chapter 3.2.1 --- PCR cloning of Irx4 --- p.63Chapter 3.2.2 --- Restriction digestion on iDuet101A --- p.64Chapter 3.2.3 --- Ligation of Irx4 to iDuet101A backbone --- p.66Chapter 3.2.4 --- Confirmation of successful ligation --- p.67Chapter 3.3 --- Lentivirus packaging --- p.68Chapter 3.3.1 --- Transfection --- p.68Chapter 3.4 --- Multiple transduction of mESCs and hygromycin selection of positively-transduced cells --- p.69Chapter 3.5 --- FACS --- p.70Chapter 3.6 --- Irx4 and iduet clone selection --- p.71Chapter 3.7 --- Characte rization of mESCs after clone selection --- p.74Chapter 3.7.1 --- Immunostaining of pluripotent and differentiation markers --- p.74Chapter 3.8 --- Differentiation of cell lines after selection --- p.77Chapter 3.8.1 --- Size of EBs of the cell lines during differentiation --- p.77Chapter 3.9 --- Gene expression study by qPCR --- p.79Chapter 3.10 --- Kv channel expression and membrane potential of mESCs at Go/Gi phase and S phases --- p.84Chapter 3.10.1 --- Expression of Kv channels subunits at G0/Gi phase and S phase --- p.86Chapter 3.10.2 --- Membrane potential at Go/Gi phase and S phase --- p.87Chapter 3.11 --- Effects of TEA+ on feeder free mESCs --- p.89Chapter 3.11.1 --- Apoptotic study --- p.89Chapter 3.11.2 --- Expression of germ layers´ة markers --- p.91Chapter 3.11.3 --- Embryo id bodies (EBs) measurement after differentiation --- p.92Chapter CHAPTER FOUR --- DISCUSSION --- p.95Chapter 4.1 --- Effect of overexpression of Irx4 on the cardiogenic potential of mESCs --- p.95Chapter 4.2 --- Role of Kv channels in maintaining the chacteristics of mESCs --- p.99Chapter 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.99Chapter 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.102Chapter 4.3 --- Insights from the present investigation on the future uses of ESCs --- p.105Conclusions --- p.108References --- p.11