Developing A Heterotypic Cell Culture Model To Determine Vegf Action Of Neural Stem Cell Fate Choice

Brain development requires tight cell and molecular regulation for proper neural stem cell (NSC) proliferation and differentiation. Increasing interest in NSCs has arisen due to the flexibility inherent within their proliferative capacity and their ability to generate multiple neuronal cell types. Our main focus is on the cortex, the primary integrator for higher order brain function. Cortical NSC proliferation and differentiation occur in concert with blood vessel ingression into the primitive cortex to vascularize the neuroepithelium. One potential influence derived from vascular cells is Vascular endothelial growth factor A (Vegf). Vegf has the ability to regulate both neurogenesis and angiogenesis making it an attractive candidate for a role in regulating NSC fate choice. My hypothesis is that NSCs require vascular cells and Vegf isoforms to regulate their transition from NSC to differentiated neurons and this regulation occurs through epigenetic mechanisms. To address this hypothesis a triculture assay system was used to recapitulate the microenvironment of the early cortical neuroepithelium. I have immortalized neuroepithelium and explored their potential cell fate based on the influence of microvascular cells. The results indicate that NSCs in contact with the microvasculature express neural stem cell (Pax6 & Sox2), neural progenitor (Id1 & Tbr2), and neuron (Tbr1) genes. Also, the presence of microvascular cells leads to a significant increase in NSC proliferation. Furthermore transgenic mice expressing modified Vegf isoform profiles were used to test the hypothesis that Vegf isoforms have distinct regulatory potential in the early cortical microenvironment. In order to determine if vascular cells and Vegf isoforms are influencing NSC fate choice through epigenetic mechanisms I investigated promoter methylation of Pax6 and ID1 as well as global methylation and hydroxymethylation. These investigations are important not only for understanding normal neural developmental, but also for clarifying the potential role of NSC in neural degenerative disorders