We have made considerable progress towards understanding the cellular and molecular mechanisms that regulate the proliferation, differentiation and survival of neural progenitor cells in the developing and adult central nervous system. We have found that nitric oxide and BDNF function in a positive feedback loop to promote neurogenesis. In other studies we found that SDFalpha, activates CXCR4 in glial progenitor cells resulting in increased migration and differentation of those cells. Our recent research has revealed a new molecular signaling system that regulates the fate of neural stem cells in the cerebral cortex. We used antibody-blocking and genetic experiments to reveal an requirement for laminin/integrin interactions in apical process adhesion and neural stem cell regulation. Transient abrogation of integrin binding and signalling using blocking antibodies to specifically target the ventricular region in utero results in abnormal cerebral cortex development. Using a multidisciplinary approach to analyse stem cell behaviour by expression of fluorescent transgenes and multiphoton time-lapse imaging revealed that the transient embryonic disruption of laminin/integrin signalling resulted in substantial layering defects in the postnatal neocortex. Additional studies of the """"""""Bush approved"""""""" lines of human embryonic stem cells (ESC) have been performed. We made a side-by-side comparison of the following features of 3 different human ESC lines: 1) the ability to maintain an undifferentiated state and to self-renew under standard conditions;2) the ability to spontaneously differentiate into three primary embryonic germ lineages in differentiating embryoid bodies;and 3) the responses to directed neural differentiation Using immunocytochemistry, flow cytometry, qRT-PCR and MPSS, we found that all three cell lines actively proliferated and expressed similar """"""""stemness"""""""" markers including transcription factors POU5F1/Oct3/4 and NANOG, glycolipids SSEA4 and TRA-1-81, and alkaline phosphatase activity. All cell lines differentiated into three embryonic germ lineages in embryoid bodies and into neural cell lineages when cultured in neural differentiation medium. However, a profound variation in colony morphology, growth rate, BrdU incorporation, and relative abundance of gene expression in undifferentiated and differentiated states of the cell lines was observed. Undifferentiated I3 cells grew significantly slower but their differentiation potential was greater than I6 and BG01V. Under the same neural differentiation-promoting conditions, the ability of each cell line to differentiate into neural progenitors varied. The differences among the 3 human ESC lines may be associated with inherited variation in the sex, stage, quality and genetic background of embryos used for hESC line derivation, and/or changes acquired during passaging in culture. We have developed a new research tool that promises to advance an understanding of the mechanisms that control the formation of neurons and oligodendrocytes, the cells that wrap around and insulate axons. Using human embryonic stem cells (ESC) we targeted Olig2, a basic helix-loop-helix transcription factor that plays an important role in motoneuron and oligodendrocyte development. One allele of Olig2 locus was replaced by a green fluorescent protein (GFP) cassette and clones were selected that retained pluripotency, typical hESC morphology, and a normal parental karyotype. GFP expression recapitulated endogenous Olig2 expression when R-Olig2 was induced by sonic hedgehog and retinoic acid, and GFP-positive cells could be purified by fluorescence-activated cell sorting. Consistent with previous reports on rodents, early GFP-expressing cells appeared biased to a neuronal fate, whereas late GFP-expressing cells appeared biased to an oligodendrocytic fate. This was corroborated by myoblast coculture, transplantation into the rat spinal cords, and whole genome expression profiling. We have also investigated the roles of glial progenitor cells in the response of the nervous system to injury in models of traumatic brain injury and multiple sclerosis. This research is contributing to the development of novel approaches for treating neurological disorders based on treatments that stimulate stem cells to form new neurons that integrate into functional circuitry thereby reversing the damage caused by injury or disease.
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