Undifferentiated human embryonic stem cells (hESCs) grow on the extracellular matrix (ECM) substrate fibronectin (FN) in defined feeder-free conditions. The ECM is part of the hESCs pluripotent niche and supports their maintenance, but the contribution to survival remains to be elucidated. Understanding the mechanism of survival is particularly crucial in hESCs, since it affects their expansion in cell culture and ultimately translation of research to the clinic. HESCs bind to FN mainly via alpha5β1- integrin, known to be upstream of important survival cascades in other cell types. However, it is not understood if and how FN/integrin binding supports those molecular pathways in the context of pluripotent hESCs. The aim of this work was to elucidate the survival cascade downstream of the FN/integrin interaction in hESCs. Initially, when hESCs were cultured on a non-integrin activating substrate they initiated an apoptotic response that also occurred when β1-integrin was selectively blocked with antibody, leading the cells to detach from FN. Integrin activation is generally transduced within cells via a complex adhesome of scaffold and kinase proteins, among which the focal adhesion kinase (FAK) plays a key role. Indeed, blocking β1-integrin resulted in dephosphorylation of endogenous FAK in hESCs. When FAK kinase activity was directly inhibited (with small molecule inhibitors), hESCs responded by detaching from FN and activating caspase-3, leading to an increase in apoptosis. Furthermore, flow cytometry analysis showed that the population of hESCs that underwent apoptosis still retained the pluripotency-associated marker NANOG. FAK is a convergent point between growth factor signaling and the PI3K/Akt pathway, with a well-reported role in the maintenance of hESCs. Consistently, FN activated both AKT and its target the ubiquitin ligase MDM2 at the protein levels, while pAkt was reduced after β1-integrin blocking and FAK inhibition. Cell imaging showed that MDM2, which regulates p53 degradation in the nucleus, displayed reduced nuclear localisation after FAK inhibition, opening the possibility for a change in the p53 balance in hESCs. In fact, p53 protein increases after FAK inhibition corresponding also to caspase activation. Further investigation explored if FAK-dependent pathways are also implicated in the maintenance of hESC pluripotency. Inhibition of FAK led the cells that survived apoptosis to lose stem cell morphology, decrease pluripotency-associated markers and change nuclear shape. Moreover, a small pool of FAK was found in the nucleus of hESCs cultured on FN, but decreased after FAK inhibition. FAK was also co- immunoprecipitated with NANOG protein in standard hESC culture while NANOG decreased after sustained FAK inhibition. This data suggests that nuclear roles of FAK could support, together with the cytoplasmic activation of the PI3K cascade, both survival and pluripotency pathways requiring further investigation. In conclusion, the original contribution of this work is to identify in FAK the downstream survival effector of the FN/β1-integrin interaction in hESCs. HESCs survival is maintained by the binding of β1-integrin to FN and activation of FAK kinase and downstream PI3K/Akt, leading to the suppression of p53 and caspase activation. In parallel, promotion of these pathways by FAK is suggested also to support the key pluripotency circuitry, feeding into NANOG. Overall, FAK is proposed here as an important regulator of hESC survival and fate.

Wong, Wai Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 113-130). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.i / Abstract --- p.ii / 中文摘要 --- p.iv / Acknowledgements --- p.vi / List of Figures --- p.vii / List of Tables --- p.ix / Table of Abbreviations --- p.x / Contents --- p.xv / Chapter Chapter I --- Introduction / Chapter 1.1 --- Stem cells --- p.1 / Chapter 1.1.1 --- Embryonic stem cells --- p.2 / Chapter 1.1.2 --- Adult stem cells --- p.2 / Chapter 1.1.3 --- Artificial stem cells --- p.5 / Chapter 1.2 --- Hair bulge stem cells (HBSC) --- p.7 / Chapter 1.2.1 --- Hair follicle --- p.7 / Chapter 1.2.2 --- The discovery of hair bulge stem cells --- p.9 / Chapter 1.2.3 --- The hair bulge niche --- p.10 / Chapter 1.2.4 --- Molecular markers --- p.12 / Chapter 1.2.5 --- Physiological roles of bulge stem cells --- p.13 / Chapter 1.2.6 --- Pathological roles of bulge stem cells --- p.15 / Chapter 1.2.7 --- Developmental plasticity of bulge stem cells --- p.16 / Chapter 1.3 --- "Regulation of adipogenic, osteogenic and cardiogenic differentiation" --- p.17 / Chapter 1.3.1 --- Regulation of adipogenic differentiation --- p.17 / Chapter 1.3.2 --- Regulation of osteogenic differentiation --- p.18 / Chapter 1.3.3 --- Regulation of cardiogenic differentiation --- p.19 / Chapter 1.4 --- Proteomics --- p.23 / Chapter 1.4.1 --- Definition of proteomics --- p.23 / Chapter 1.4.2 --- Two-dimensional gel electrophoresis (2DGE) --- p.25 / Chapter 1.4.3 --- Mass spectrometry and protein identification --- p.29 / Chapter 1.4.4 --- Other techniques associated with proteomics --- p.34 / Chapter 1.4.5 --- Proteomics and stem cells --- p.36 / Chapter 1.5 --- General summary --- p.37 / Chapter 1.6 --- Aims of my study --- p.38 / Chapter Chapter II --- Materials and Methods / Chapter 2.1 --- Animals --- p.39 / Chapter 2.2 --- Immunohistochemistry --- p.39 / Chapter 2.2.1 --- Histology --- p.39 / Chapter 2.2.2 --- Immunohistochemistry --- p.40 / Chapter 2.3 --- Establishment of hair bulge CD34+ stem cell line --- p.41 / Chapter 2.3.1 --- Isolation of hair bulge explants --- p.41 / Chapter 2.3.2 --- Establishing hair bulge primary cultured cells --- p.42 / Chapter 2.3.3 --- Purification of hair bulge stem cells --- p.43 / Chapter 2.4 --- Karyotyping --- p.45 / Chapter 2.5 --- In vitro differentiation --- p.46 / Chapter 2.5.1 --- Adipogenic differentiation --- p.46 / Chapter 2.5.2 --- Osteogenic differentiation --- p.47 / Chapter 2.5.3 --- Cardiogenic differentiation --- p.48 / Chapter 2.6 --- Proteomic analysis --- p.48 / Chapter 2.6.1 --- Sample preparation --- p.48 / Chapter 2.6.2 --- Quantification of proteins --- p.49 / Chapter 2.6.3 --- First-dimensional separation of proteins 226}0ؤ isoelectric focusing (IEF) --- p.50 / Chapter 2.6.4 --- Second-dimensional separation - sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.51 / Chapter 2.6.5 --- "Silver staining, imaging and destaining" --- p.52 / Chapter 2.6.6 --- In-gel digestion and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis --- p.53 / Chapter 2.7 --- Histochemistry --- p.54 / Chapter 2.7.1 --- Oil Red O staining --- p.54 / Chapter 2.7.2 --- Alizarin Red S staining --- p.55 / Chapter 2.8 --- Immunocytochemistry --- p.56 / Chapter 2.9 --- Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) --- p.57 / Chapter 2.9.1 --- Isolation of total cellular RNA --- p.57 / Chapter 2.9.2 --- Complementary DNA (cDNA) synthesis --- p.58 / Chapter 2.9.3 --- Polymerase chain reaction and agarose gel electrophoresis --- p.59 / Chapter 2.10 --- Western blot analysis --- p.62 / Chapter 2.10.1 --- Sample preparation and quantification of proteins --- p.62 / Chapter 2.10.2 --- SDS-PAGE --- p.63 / Chapter 2.10.3 --- Protein transfer --- p.64 / Chapter 2.10.4 --- Immunodetection --- p.65 / Chapter 2.11 --- Cell proliferation assay --- p.66 / Chapter 2.11.1 --- Determination of growth pattern --- p.66 / Chapter 2.11.2 --- MTT assay --- p.66 / Chapter 2.12 --- Ultrastructural analysis --- p.67 / Chapter 2.12.1 --- Scanning electron microscopy (SEM) --- p.67 / Chapter 2.12.2 --- Transmission electron microscopy (TEM) --- p.68 / Chapter 2.13 --- Statistical analysis --- p.68 / Chapter Chapter III --- Results / Chapter 3.1 --- Isolation and characterization hair bulge stem cells --- p.69 / Chapter 3.2 --- Directed adipogenic differentiation --- p.70 / Chapter 3.3 --- Directed osteogenic differentiation --- p.71 / Chapter 3.4 --- Ability of cardiogenol C to induce cardiogenesis in HBSCs --- p.71 / Chapter 3.5 --- Comparative proteomic analysis of HBSC cardiogenic differentiation induced by cardiogenol C --- p.72 / Chapter 3.6 --- Role of Wnt signaling pathway in cardiogenol C-induced cardiogenesis --- p.74 / Chapter 3.7 --- Role of chromatin remodeling in cardiogenol C-induced cardiogenesis --- p.75 / Chapter 3.8 --- Legends and tables --- p.76 / Chapter Chapter IV --- Discussion --- p.98 / References --- p.113 / Appendices --- p.131 / Publication --- p.134

Thesis (Ph.D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 153-173). Also available on the Internet.

Thesis (Ph.D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 153-173). Also available on the Internet.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

abstract / Medicine / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--George Mason University, 2007. / Title from PDF t.p. (viewed Jan. 17, 2008). Thesis director: Richard E. Rubenstein. Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Conflict Analysis and Resolution. Vita: p. 228. Includes bibliographical references (p. 222-227). Also available in print.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2010. / Härtill 4 uppsatser.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

The prognosis for patients diagnosed with glioma has changed little over the past two decades. Many therapies that appeared promising in preclinical studies have been unsuccessful in the clinic. In an attempt to address this problem I developed a method for the efficient derivation of glioma primary cultures from fresh human brain tumours. These cultures are enriched for putative cancer stem-like cells that are thought to be responsible for glioma initiation, therapy resistance and recurrence. This mechanism of tumour development is a departure from the traditional multistep model of cancer. It is hoped that preclinical models incorporating glioma stem-like cells will more effectively recapitulate the biology of human disease and so better predict the likely clinical efficacy of inhibitor compounds tested in vitro and in the preclinical setting. In contrast to the majority of the existing literature, I identified two distinct tumourderived glioma stem-like cell phenotypes in my primary cultures that I have called ‘branched’ and ‘flat.’ The branched cells had similarities to the radial glia-like cells previously described in glioma stem-like cultures. In contrast, the flat cells had mesenchymal-like features. I discuss the implications of these observations for understanding glioma cell biology. I describe the development of high content phenotypic assays that incorporate these putative glioma stem-like cells. I screened inhibitor compounds of the PI3 kinase pathway, which is important in glioma cell behaviour, and identified that PIK75, a drug that targets the p110α catalytic subunit of PI3 kinase, inhibited growth of all the primary cells tested. I examined PIK75 activity in some detail. In vivo models of glioma are used to validate the findings of in vitro compound screening, so I describe my attempt to develop a novel genetically engineered mouse model designed to initiate glioma formation from the glioma stem-like cell. Surprisingly, these mice actually developed malignant peripheral nerve sheath tumours and that gave me a novel insight into the pathogenesis of this rare disease. This also informed future work on my long-term goal of generating a genetic model of glioma that recapitulates human disease.