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Glioblastoma is the commonest form of brain neoplasm and among the most malignant forms of cancer. The identification of a subpopulation of self-renewing and multipotent cancer stem cells within glioblastoma has revealed a novel cellular target for the treatment of this disease. The role of developmental cell signaling pathways in these cell populations remains poorly understood. Herein, we examine the role of the Notch signaling pathway in glioblastoma stem cells. In this thesis we have demonstrated that the canonical Notch pathway is active in glioblastoma stem cells and functions to inhibit neuronal lineage commitment in a subset of patient derived glioblastoma stem cells in vitro. Gamma secretase (γ-secretase) small molecule inhibitors or dominant-negative co-activators inhibit glioblastoma stem cell proliferation and induce neuronal lineage commitment in a fashion that synergizes with Wingless pathway activation via GSK-3β blockade. Our data suggest that subsets of patient samples show a Notch gene expression profile that predicts their abilities to undergo neuronal lineage differentiation in response to γ-secretase small molecule inhibitors. Additionally, the data suggests that Notch may perturb the relative fractions of cells undergoing symmetric division, in favour of asymmetric division, limiting clonal expansion from single cells. These data may have important implications for treating human glioblastoma, and suggest that in addition to inhibition of proliferation, influencing lineage choice of the tumor stem cells may be a mechanism by which these tumors may be pharmacologically inhibited.

Glioblastoma is the commonest form of brain neoplasm and among the most malignant forms of cancer. The identification of a subpopulation of self-renewing and multipotent cancer stem cells within glioblastoma has revealed a novel cellular target for the treatment of this disease. The role of developmental cell signaling pathways in these cell populations remains poorly understood. Herein, we examine the role of the Notch signaling pathway in glioblastoma stem cells. In this thesis we have demonstrated that the canonical Notch pathway is active in glioblastoma stem cells and functions to inhibit neuronal lineage commitment in a subset of patient derived glioblastoma stem cells in vitro. Gamma secretase (γ-secretase) small molecule inhibitors or dominant-negative co-activators inhibit glioblastoma stem cell proliferation and induce neuronal lineage commitment in a fashion that synergizes with Wingless pathway activation via GSK-3β blockade. Our data suggest that subsets of patient samples show a Notch gene expression profile that predicts their abilities to undergo neuronal lineage differentiation in response to γ-secretase small molecule inhibitors. Additionally, the data suggests that Notch may perturb the relative fractions of cells undergoing symmetric division, in favour of asymmetric division, limiting clonal expansion from single cells. These data may have important implications for treating human glioblastoma, and suggest that in addition to inhibition of proliferation, influencing lineage choice of the tumor stem cells may be a mechanism by which these tumors may be pharmacologically inhibited.

Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes and chronic liver disease. Various attempts have been made to produce cells that can serve as precursors for pancreas and liver. By using all-trans-retinoic acid, basic fibroblast growth factor, dibutyryl cAMP, and cyclopamine, an attempt has been made to produce definitive endoderm and subsequently cells that can serve as pancreatic and hepatocyte precursors from mouse embryonic stem cells. By using retinoic acid and basic-FGF, in the absence of embryoid body formation, mouse embryonic stem cells were differentiated at different culture periods. Four protocols of varying lengths of culture and reagents and their cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, pancreas and hepatocytes. Inclusion of DBcAMP and extension of culture time resulted in cells that display features of definitive endoderm by expression of Sox 17 and FOXA2 and minimal expression of primitive endoderm and other germ cell layers such as ectoderm and mesoderm. These cells produced insulin and C-peptide and secreted insulin in a glucose responsive manner. However, they seem to lack mature insulin secretion mechanism. There was a production of hepatocyte markers (AFP-2 and transthyretin) but there was insufficient data to assess for convincing production of hepatocytes. In summary, one of the protocols produced cells that displayed characteristics of definitive endoderm and they may serve as pancreatic endocrine precursors.

Telomeres are the repetitive DNA sequence and associated proteins found at the ends of linear chromosomes. They have a role in biological processes including meiosis and aging as well as implications in a number of genomic instability disorders and cancers. Telomeres maintain genomic stability by protecting chromosome ends from terminal fusions and misidentification as DNA damage sites. Their wide range of functions has resulted in an increased interest in developing tools to study the dynamics of telomeres in live cells. To do this, current studies use the ubiquitously expressed protein Telomere Repeat Factor 1 (TRF1) tagged with a fluorescent protein. TRF1 is a negative regulator of telomere length that binds exclusively to telomere repeats. Over-expression of the fluorescent protein fused to TRF1 has been a useful tool to track telomere movement. The foci formed by the tagged TRF1 protein accurately represent the number of telomeres expected in the cells and the localization is maintained throughout the cell cycle. A caveat with this system is that over-expression of TRF1 leads to accelerated telomere shortening, as well as replication defects that can stall telomere replication. These caveats make it difficult to draw conclusions about telomere dynamics based solely on observations of cells over-expressing fluorescently tagged TRF1. To eliminate problems associated with protein over-expression, I have tried to develop knock-in embryonic stem (ES) cells expressing fluorescently tagged TRF1 from the endogenous Trf1 promoter. To do this, I have used a recombineering technique using Bacterial Artificial Chromosomes (BACs). BAC recombineering allows for the direct knock-in of a fluorescent tag into the mouse Trf1gene locus. Genetic constructs with the correct sequence inserts have been obtained and have been used for transfection of ES cells. While no correctly targeted ES cells have been identified so far, the expectation is that ES cell lines with correctly targeted fluorescently tagged TRF1 will be obtained in the near future. Such lines will be used to study telomere dynamics in ES cells, differentiated cells generated from ES cells, as well as to generate mice.