Leung, Sze Lee Cecilia. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 144-153). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.iii / Acknowledgements --- p.v / Table of Content --- p.vi / Abbreviations --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Stem cells --- p.1 / Chapter 1.1.1 --- Adult stem cells --- p.2 / Chapter 1.1.2 --- Embryonic stem cells --- p.2 / Chapter 1.1.3 --- Pros and cons of embryonic and adult stem cells --- p.5 / Chapter 1.1.4 --- Human embryonic stem cells (hESCs) --- p.6 / Chapter 1.1.5 --- Mouse embryonic stem cells (mESCs) --- p.7 / Chapter 1.1.6 --- Characteristics of ESC-derived cardiomyocytes --- p.7 / Chapter 1.2 --- Cardiovascular Diseases (CVD) --- p.9 / Chapter 1.2.1 --- Causes and statistics of CVD --- p.9 / Chapter 1.2.2 --- Current treatment for CVD --- p.10 / Chapter 1.2.3 --- Current hurdles of putting hESC-CMs into clinical use --- p.11 / Chapter 1.3 --- Myosin light chain2v --- p.13 / Chapter 1.4 --- Genetic-engineering of hESCs & their cardiac derivatives by lentiviral-mediate gene transfer --- p.14 / Chapter 1.5 --- Cytokines secretion during myocardial infarction --- p.15 / Chapter 1.6 --- Aims of the Project --- p.19 / Chapter 1.7 --- Significance of the Project --- p.19 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Subcloning --- p.20 / Chapter 2.1.1 --- Amplification of MLC-2v --- p.20 / Chapter 2.1.2 --- Purification of DNA product --- p.21 / Chapter 2.1.3 --- Restriction enzyme digestion --- p.21 / Chapter 2.1.4 --- Ligation of MLC-2v promoter with DuetO 11 vector --- p.22 / Chapter 2.1.5 --- Transformation of ligation product into competent cells --- p.22 / Chapter 2.1.6 --- PCR confirmation of successful ligation --- p.23 / Chapter 2.1.7 --- Small-scale preparation of bacterial plasmid DNA --- p.23 / Chapter 2.1.8 --- Restriction enzyme digestions to reconfirm positive clones --- p.24 / Chapter 2.1.9 --- DNA sequencing of the cloned plasmid DNA --- p.25 / Chapter 2.1.10 --- Large-scale preparation of target recombinant expression vector --- p.25 / Chapter 2.2 --- Mouse Embryonic Fibroblast (MEF) Culture --- p.26 / Chapter 2.2.1 --- Derivation of MEF --- p.26 / Chapter 2.2.2 --- Mouse embryonic fibroblast cells culture --- p.27 / Chapter 2.2.3 --- Irradiation of mouse embryonic fibroblast --- p.28 / Chapter 2.3 --- HESC culture --- p.29 / Chapter 2.3.1 --- Thawing and Plating hESCs --- p.29 / Chapter 2.3.2 --- Splitting hESCs --- p.30 / Chapter 2.3.3 --- "Culture maintainence, selection and colony removal" --- p.31 / Chapter a) --- Distinguish differentiated and undifferentiated cells and colonies / Chapter b) --- "Remove differentiated cells by ""Picking to Remove""" / Chapter c) --- "Remove undifferentiated cells by ""Picking to Keep""" / Chapter 2.3.4 --- Freezing hESCs --- p.31 / Chapter 2.3.5 --- Differentiation of hESCs --- p.32 / Chapter 2.3.6 --- "HESC culture on feeder free system, mTeSR TM1" --- p.34 / Chapter a) --- Preparation of mTeSRTMl / Chapter b) --- Preparation of BD MatrigelTM hESC-qualified Matrix aliquots / Chapter c) --- Coating plates with BD MatrigelTM hESC-qualified Matrix / Chapter d) --- Human Embryonic stem cells culture in mTeSRTMl / Chapter 2.4 --- ES Cell Characterization (Chemicon Cat# SCR001) --- p.36 / Chapter 2.4.1 --- Alkaline Phosphatase Staining --- p.36 / Chapter 2.4.2 --- Immunofluorescence staining --- p.37 / Chapter 2.5 --- MESC culture --- p.38 / Chapter 2.5.1 --- Thawing and Plating mESCs --- p.38 / Chapter 2.5.2 --- Splitting mESCs --- p.38 / Chapter 2.5.3 --- Differentiation of mESCs --- p.39 / Chapter 2.5.4 --- To study the effects of cytokines on mESC differentiation --- p.40 / Chapter 2.6 --- Lentivirus (LV) Packaging --- p.41 / Chapter 2.6.1 --- Transfection of lentiviral vectors into HEK293FT cells --- p.41 / Chapter 2.6.2 --- LV titering --- p.42 / Chapter 2.7 --- MultipleTransduction --- p.43 / Chapter 2.8 --- Selection of transduced cells by hygromycin --- p.43 / Chapter 2.8.1 --- Determination of hygromycin selection dosage --- p.43 / Chapter 2.8.2 --- Selection of stable clones --- p.44 / Chapter 2.9 --- Isolation of green fluorescent cardiomyocytes derived from differentiated hESCs --- p.45 / Chapter 2.9.1 --- Collagenase digestion of embryoid bodies into single cells --- p.45 / Chapter 2.9.2 --- FACS --- p.46 / Chapter 2.10 --- Gene expression study / Chapter 2.10.1 --- Primer design --- p.46 / Chapter 2.10.2 --- RNA extraction --- p.46 / Chapter 2.10.3 --- DNase Treatment --- p.47 / Chapter 2.10.4 --- Synthesis of Double-stranded cDNA from Total RNA --- p.47 / Chapter 2.10.5 --- Quantitative real-time PCR --- p.48 / Chapter 2.10.6 --- Quantification of mRNA expression --- p.49 / Chapter 2.11 --- Protein Expression study --- p.49 / Chapter 2.11.1 --- Crude protein extraction --- p.49 / Chapter 2.11.2 --- Quantitation of protein samples --- p.50 / Chapter 2.11.3 --- SDS-PAGE --- p.50 / Chapter 2.11.4 --- Western Blot --- p.51 / Chapter 2.11.5 --- Western blot luminal detection --- p.52 / Chapter 2.11.6 --- Quantification of protein expression --- p.52 / Chapter CHAPTER 3 --- PURIFICATION OF CARDIOMYOCYTES DERIVED FROM DIFFERENTIATED HESCs / Chapter 3.1 --- Subcloning --- p.57 / Chapter 3.1.1 --- Linearization of DuetO11 and excision of UBC promoter --- p.58 / Chapter 3.1.2 --- PCR cloning of MLC-2V --- p.59 / Chapter 3.1.3 --- Ligation of MLC-2v promoter to linearized DuetO11 --- p.60 / Chapter 3.1.3.1 --- Colony PCR to screen for positive clones --- p.61 / Chapter 3.1.3.2 --- Restriction digestion to confirm the success of ligation --- p.61 / Chapter 3.2 --- Lentivirus (LV) packaging --- p.62 / Chapter 3.2.1 --- Transfection --- p.63 / Chapter 3.2.2 --- LV titering --- p.64 / Chapter 3.3 --- HESC culture --- p.66 / Chapter 3.4 --- Multi-transduction of hESCs with LVs --- p.67 / Chapter 3.5 --- Differentiation after transduction --- p.69 / Chapter 3.6 --- Antibiotic selection --- p.71 / Chapter 3.6.1 --- Characterization of hESCs on feeder free system --- p.72 / Chapter 3.6.1.1 --- Alkaline Phosphatase (AP) staining --- p.72 / Chapter 3.6.1.2 --- Immunostaining with pluripotency marker --- p.73 / Chapter 3.6.2 --- Determination of hygromycin dosage by MTT assay --- p.74 / Chapter 3.6.3 --- HESCs after selection in feeder free system --- p.75 / Chapter 3.7 --- Differentiation of hESCs after selection --- p.76 / Chapter 3.8 --- FACS --- p.77 / Chapter 3.9 --- QPCR of cells after FACS --- p.80 / Chapter 3.9.1 --- Gene expression of Nkx2.5 --- p.81 / Chapter 3.9.2 --- Gene expression of c-Tnl --- p.82 / Chapter 3.9.3 --- Gene expression of c-TnT --- p.83 / Chapter 3.9.3 --- Gene expression of MLC-2v --- p.84 / Chapter CHAPTER 4 --- THE STUDY OF CYTOKINES' EFFECT ON MESC DIFFERENTIATION / Chapter 4.1 --- mESC culture --- p.85 / Chapter 4.2 --- The effect of cytokines on the differentiation of mESCs --- p.86 / Chapter 4.2.1 --- Beating curves of mESCs treated with different concentrations of cytokines at differentiation day 2 to 6 before attachment --- p.88 / Chapter 4.2.2 --- qPCR to determine the cytokines' effect on the differentiation of mESCs --- p.94 / Chapter 4.2.2.1 --- The effect of IL-1α on the expression of cardiac specific genes --- p.95 / Chapter 4.2.2.2 --- The effect of IL-1β on the expression of cardiac specific genes --- p.98 / Chapter 4.2.2.3 --- The effect of IL-6 on the expression of cardiac specific genes --- p.101 / Chapter 4.2.2.4 --- The effect of IL-10 on the expression of cardiac specific genes --- p.104 / Chapter 4.2.2.5 --- The effect of IL-18 on the expression of cardiac specific genes --- p.107 / Chapter 4.2.2.6 --- The effect of TNF-α on the expression of cardiac specific genes --- p.110 / Chapter 4.2.3 --- Western blot analysis of the cytokines' effect on the differentiation of mESCs --- p.113 / Chapter 4.2.3.1 --- The effect of IL-lα on the abundance of cardiac specific proteins --- p.114 / Chapter 4.2.3.2 --- The effect of IL-1β on the abundance of cardiac specific proteins --- p.116 / Chapter 4.2.3.3 --- The effect of IL-6 on the abundance of cardiac specific proteins --- p.118 / Chapter 4.2.3.4 --- The effect of IL-10 on the abundance of cardiac specific proteins --- p.120 / Chapter 4.2.3.5 --- The effect of IL-18 on the abundance of cardiac specific proteins --- p.122 / Chapter 4.2.3.6 --- The effect of TNF-α on the abundance of cardiac specific proteins --- p.124 / Chapter CHAPTER 5 --- DISCUSSION / Chapter 5.1 --- Purification of cardiomyocytes derived from differentiated hESCs --- p.127 / Chapter 5.2 --- Study on the effect of cytokines on mESC differentiation --- p.135 / Chapter 5.3 --- Conclusion --- p.142 / REFERENCES --- p.144

Ng, Wing Chi Linda. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 120-142). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.iv / Publications --- p.v / Abbreviations --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xiii / List of Figures --- p.xiv / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Stem Cells --- p.1 / Chapter 1.1.1 --- Definition of Stem Cells --- p.1 / Chapter 1.1.2 --- Different Origins of Stem Cells --- p.2 / Chapter 1.1.3 --- Challenges and Importance of Stem Cell Research --- p.5 / Chapter 1.2 --- Adult Mesenchymal Stem Cells --- p.7 / Chapter 1.2.1 --- Characteristics of Adult Mesenchymal Stem Cells --- p.7 / Chapter 1.2.2 --- Adipose Tissue as an Alternate Source of MSCs --- p.8 / Chapter 1.2.3 --- Adipose Tissue Versus Bone Marrow as a Source of MSCs --- p.10 / Chapter 1.3 --- Adipose Tissue-derived Stem Cells (ATSCs) --- p.11 / Chapter 1.3.1 --- Cell Surface Marker Characteristic of ATSCs --- p.11 / Chapter 1.3.2 --- Global Gene Expression Profile of ATSCs --- p.14 / Chapter 1.3.3 --- Immunomodulatory Effect of ATSCs --- p.15 / Chapter 1.3.4 --- Proliferation Capacity of ATSCs --- p.17 / Chapter 1.3.5 --- Multilineage Differentiation of ATSCs --- p.18 / Chapter 1.3.5.1 --- Differentiation Capability of ATSCs : Adipogenesis --- p.18 / Chapter 1.3.5.2 --- Osteogenesis --- p.19 / Chapter 1.3.5.3 --- Skeletal and Smooth Muscle Myogenesis --- p.21 / Chapter 1.3.5.4 --- Cardiomyogenesis --- p.23 / Chapter 1.3.5.5 --- Chondrogenesis --- p.24 / Chapter 1.3.5.6 --- Neurogenesis --- p.27 / Chapter 1.4 --- Signaling Pathways in Stem Cells --- p.31 / Chapter 1.4.1 --- Wnt Signaling --- p.31 / Chapter 1.4.2 --- Notch Signaling --- p.33 / Chapter 1.4.3 --- Signaling Pathway of the TGF-β Superfamily --- p.34 / Chapter 1.5 --- Pathways Controlling Chondrogenesis --- p.36 / Chapter 1.6 --- MicroRNA --- p.39 / Chapter 1.6.1 --- MicroRNA - A Novel Gene Regulator --- p.39 / Chapter 1.6.2 --- Biogenesis of MicroRNAs --- p.40 / Chapter 1.6.3 --- Post-transcriptional Repression by MicroRNAs --- p.43 / Chapter 1.6.4 --- Role of MicroRNAs in Development --- p.45 / Chapter 1.6.5 --- MicroRNAs in Stem Cell Differentiation --- p.46 / Chapter 1.6.5.1 --- MicroRNA Expression Profile in ESCs --- p.46 / Chapter 1.6.5.2 --- Lineage Differentiation --- p.47 / Chapter 1.7 --- Project Aims --- p.52 / Chapter 1.8 --- Significance of Study --- p.53 / Chapter Chapter 2 --- Materials and Methods --- p.54 / Chapter 2.1 --- Sample Collection --- p.54 / Chapter 2.2 --- Isolation and Culture of ATSCs --- p.54 / Chapter 2.3 --- Measurement of Cell Growth --- p.55 / Chapter 2.4 --- Effect of Estrogen Treatment on ATSC Proliferation --- p.55 / Chapter 2.5 --- Multilineage Differentiation of ATSCs --- p.55 / Chapter 2.5.1 --- Chondrogenic Differentiation --- p.56 / Chapter 2.5.2 --- Neural Differentiation --- p.56 / Chapter 2.6 --- Immunocytochemical Analysis of Surface Markers and Lineage Specific Markers --- p.57 / Chapter 2.7 --- Alcian Blue Staining --- p.58 / Chapter 2.8 --- RNA Extraction --- p.58 / Chapter 2.9 --- Reverse Transcription --- p.59 / Chapter 2.10 --- Quantitative Real-time Polymerase Chain Reaction --- p.59 / Chapter 2.11 --- Statistical Analysis of Real-time PCR Data --- p.61 / Chapter 2.12 --- MicroRNA Profiling --- p.61 / Chapter 2.12.1 --- Reverse Transcription --- p.62 / Chapter 2.12.2 --- Quantitative Real-time Polymerase Chain Reaction --- p.62 / Chapter 2.13 --- mRNA Target Prediction of MicroRNA --- p.63 / Chapter 2.14 --- MicroRNA Knockdown Assay --- p.63 / Chapter 2.15 --- MicroRNA Over-expression Assay --- p.64 / Chapter 2.15.1 --- Vector Amplification --- p.64 / Chapter 2.15.1.1 --- Transformation --- p.64 / Chapter 2.15.1.2 --- Purification of Plasmid DNA --- p.65 / Chapter 2.15.1.3 --- Confirmation of Construct Insertion --- p.66 / Chapter 2.15.2 --- Transfection of Plasmid and Establishment of MicroRNA Precursor Expressing Cell Lines --- p.66 / Chapter 2.16 --- Gene Expression Microarry --- p.67 / Chapter 2.16.1 --- Preparation of Amplification and Labeling Reaction --- p.67 / Chapter 2.16.2 --- Purification of the Labeled/Amplified RNA --- p.68 / Chapter 2.16.3 --- RNA Fragmentation --- p.68 / Chapter 2.16.4 --- Hybridization --- p.69 / Chapter 2.16.5 --- Array Washing and Scanning --- p.69 / Chapter 2.16.6 --- Statistical Analysis of Microarray Data --- p.69 / Chapter CHAPTER 3 --- RESULTS --- p.71 / Chapter 3.1 --- Isolation and Characterization of ATSCs --- p.71 / Chapter 3.2 --- ATSCs Exhibited Multilineage Differentiation --- p.75 / Chapter 3.2.1 --- Chondrogenic Differentiation --- p.75 / Chapter 3.2.2 --- Expression of Chondrogenic Markers --- p.76 / Chapter 3.2.3 --- Neural Differentiation --- p.80 / Chapter 3.2.4 --- Expression of Neural Markers --- p.83 / Chapter 3.3 --- Effect of Donor's Reproductive Status on the Proliferation and Differentiation Capacity of ATSCs --- p.83 / Chapter 3.3.1 --- Expression of Stem Cell Makers --- p.86 / Chapter 3.3.2 --- Cell Proliferation Assay --- p.86 / Chapter 3.3.3 --- Differentiation Capacity of ATSCs --- p.89 / Chapter 3.4 --- Effect of E2 Treatment on the Proliferation Rate of ATSCs --- p.89 / Chapter 3.5 --- MicroRNA --- p.91 / Chapter 3.5.1 --- MicroRNA Expression Profile of Undifferentiated and Chondrogenic Differentiated ATSCs --- p.91 / Chapter 3.5.2 --- Clustering Analysis Identified MicroRNAs Segregate with ATSCs --- p.91 / Chapter 3.5.3 --- Identification of Differentially Expressed MicroRNAs in Chondrogenic-induced ATSCs --- p.95 / Chapter 3.5.4 --- mRNA Target Prediction for miR-199a --- p.97 / Chapter 3.6 --- Correlating MicroRNA Expression and mRNA Levels: Clues to MicroRNA Function --- p.97 / Chapter 3.6.1 --- Effect ofmiR-199a RNAi in Phenotypic Changes of Chondrogenic-induced ATSCs --- p.97 / Chapter 3.6.2 --- Identification of Potential Target Genes by Microarray Analysis of ATSCs with miR-199a Over-expression and Knockdown --- p.102 / Chapter CHAPTER 4 --- DISCUSSION --- p.104 / Chapter CHAPTER 5 --- CONCLUSIONS --- p.115 / APPENDICES --- p.117 / REFERENCES --- p.120

Type 1 Diabetes (T1D) is an autoimmune disorder in which the pancreatic β-cells are destroyed by the body's immune system. The reduced number of β-cells leads to inadequate insulin secretion and high glucose levels in the body. The requirement of insulin injection throughout life and lack of donors for islet transplantations has prompted a search for more accessible and available sources of insulin producing cells that can be transplanted in T1D patients. To that end, the discovery of induced pluripotent stem (iPS) cells has provided a potential source of precursors for cell therapy for T1D. iPS cells are reprogrammed somatic cells which can be transplanted back into the patient from whom the somatic cells were initially derived, thus potentially avoiding immune rejection when transplanted. As a potential therapy for T1D, we aim to derive insulin producing cells (IPCs) from human iPS cells. In contrast to the conventional two dimensional (2D) cell culture systems used in many iPS derived IPC studies, the inner cell mass (ICM) from which various organs differentiate during embryogenesis is a cluster of cells that enables signaling crosstalk between cells of different types. Three dimensional (3D) cell culture systems allows cells to form cell clusters that promote cell - cell signaling. Hence, we hypothesized that 3D cell culture systems will yield better efficiency of differentiation to functional IPCs in vitro than 2D cultures. Initially, the synthetic polymers sodium alginate and matrigel were analyzed for their ability to enable cell clustering to establish 3D cell culture systems. The 3D cell environment established using matrigel was used for the differentiation of human iPS cells to Insulin Producing Cells (IPC). The cells were first converted to endodermal cells. A mixture of growth factors then induced the differentiation of endodermal cells to pancreatic cells. The pancreatic cells were converted to IPCs that resemble pancreatic β-cells. Our 3D differentiated IPCs strongly expressed pancreatic endocrine transcription factors and pancreatic hormones. The IPCs also produced insulin when exposed to a high glucose environment. But the number of IPCs obtained at the end of the differentiation was low. Hence, our results demonstrate that 3D differentiation generates functional IPCs in vitro unlike 2D differentiation. In the future we aim to improve the percentage of IPCs that we generate from the 3D differentiation. Our expectation is that these cells will be able to cure hyperglycemia in diabetic mice more rapidly compared to the 2D differentiated cells owing to their proven insulin production in the presence of a high glucose environment in vitro.

Thesis (doctoral)--Humboldt-Universiẗat, Berlin, 2005. / Includes bibliographical references (p. 231-260) and index.

Colony-stimulating factor 1 receptor (CSF1R, Fms) is an integral transmembrane glycoprotein with tyrosine specific protein kinase activity that it is found on the mononuclear phagocytes to promote their survival, proliferation and differentiation. Colony-stimulating factor 1 (CSF-1), also known as M-CSF, is a protein ligand that acts on the CSF1R. There is a variable association of Fms with the stem cell marker CD34 on acute myeloid leukemia (AML) cells and this suggests different structures of the AML hierarchy in different patients. Mouse stromal cells (MS-5) were transduced with a plasmid containing human CSF-1 because mouse CSF-1 is inactive on human CSF1R. Results show that AML cells cultured with CSF-1-expressing stroma had a much better growth and survival than the control stroma, suggesting that CSF-1 might be a stimulating factor for the growth of leukemic stem cells.

Construction of the vertebrate nervous system begins with the decision of a group of ectoderm cells to take on a neural fate. Studies using Xenopus ectodermal explants, or with mouse ectoderm cells or embryonic stem (ES) cells, demonstrated that this process of neural determination occurred by default – the ectoderm cells became neural after the removal of inhibitory signals. Whether ectoderm or ES cells directly differentiate into bona fide neural stem cells is not clear. One model suggests that there is an intermediate stage where “primitive” neural stem cells (pNSC) emerge harbouring properties of both ES cells and neural stem cells. The goal of my research was to address this question by evaluating the role of growth factor signaling pathways and their impact on the function of the zinc-finger homeobox transcription factor, Zfhx1b, during mouse neural stem cell development. I tested whether FGF and Wnt signaling pathways could regulate Zfhx1b expression to control early neural stem cell development. Inhibition of FGF signaling at a time when the ectoderm is acquiring a neural fate resulted in the accumulation of too many pNSCs, at the expense of the definitive neural stem cells. Interestingly, over-expression of Zfhx1b was sufficient to rescue the transition from a pNSC to definitive NSC. These data suggested that definitive NSC fate specification in the mouse ectoderm was facilitated by FGF activation of Zfhx1b, whereas canonical Wnt signaling repressed Zfhx1b expression. Next I sought to determine whether Zfhx1b was similarly required during neural lineage development in ES cells. FGF and Wnt signaling modulated expression of Zfhx1b in ES cell cultures in manner resembling my observations from similar experiments using mouse ectoderm. Knockdown of Zfhx1b in ES cells using siRNA did not affect the initial transition of ES cells to pNSC fate, but did limit the ability of these neural cells to further develop into definitive NSCs. Thus, my findings using ES cells were congruent with evidence from mouse embryos and supported a model whereby intercellular signaling induced Zfhx1b, required for the development of definitive NSCs, subsequent to an initial neural specification event that was independent of this pathway.

Construction of the vertebrate nervous system begins with the decision of a group of ectoderm cells to take on a neural fate. Studies using Xenopus ectodermal explants, or with mouse ectoderm cells or embryonic stem (ES) cells, demonstrated that this process of neural determination occurred by default – the ectoderm cells became neural after the removal of inhibitory signals. Whether ectoderm or ES cells directly differentiate into bona fide neural stem cells is not clear. One model suggests that there is an intermediate stage where “primitive” neural stem cells (pNSC) emerge harbouring properties of both ES cells and neural stem cells. The goal of my research was to address this question by evaluating the role of growth factor signaling pathways and their impact on the function of the zinc-finger homeobox transcription factor, Zfhx1b, during mouse neural stem cell development. I tested whether FGF and Wnt signaling pathways could regulate Zfhx1b expression to control early neural stem cell development. Inhibition of FGF signaling at a time when the ectoderm is acquiring a neural fate resulted in the accumulation of too many pNSCs, at the expense of the definitive neural stem cells. Interestingly, over-expression of Zfhx1b was sufficient to rescue the transition from a pNSC to definitive NSC. These data suggested that definitive NSC fate specification in the mouse ectoderm was facilitated by FGF activation of Zfhx1b, whereas canonical Wnt signaling repressed Zfhx1b expression. Next I sought to determine whether Zfhx1b was similarly required during neural lineage development in ES cells. FGF and Wnt signaling modulated expression of Zfhx1b in ES cell cultures in manner resembling my observations from similar experiments using mouse ectoderm. Knockdown of Zfhx1b in ES cells using siRNA did not affect the initial transition of ES cells to pNSC fate, but did limit the ability of these neural cells to further develop into definitive NSCs. Thus, my findings using ES cells were congruent with evidence from mouse embryos and supported a model whereby intercellular signaling induced Zfhx1b, required for the development of definitive NSCs, subsequent to an initial neural specification event that was independent of this pathway.

Source: Masters Abstracts International, Volume: 47-02, page: 0981. Adviser: Luisa Franzini. Includes bibliographical references.

Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009. / Committee Chair: Alfred H. Merrill, Jr; Committee Member: John Cairney; Committee Member: M. Cameron Sullards; Committee Member: Marion B. Sewer; Committee Member: Yuhong Fan. Part of the SMARTech Electronic Thesis and Dissertation Collection.

published_or_final_version / Anatomy / Master / Master of Philosophy