| Research Abstract |
In the present study, we have first examined the possibility of neuronal differentiation of human teratocartinoma cell line (NT-2) with a purpose of alternative source for neuronal transplantation. By means of treating the teratocarcinoma cells (NT-2) in vitro with 10 mM of retinoic acid for five weeks, neuronal like cells in morphology (NT-2N) were successfully obtained. NT-2N cells as well as NT-2 cells were then studied electrophysiologicaly for their property as neuronal cells. We have found that voltage sensitive Na+ currents were observed in NT-2N cells but not in the NT-2 cells and that in addition to the glutamate receptors, the NT-2N cells expressed GABA-A receptors, suggesting that those receptors have a crucial role in neurotansmission from the earlier stage of the brain development.
Second, neural stem cells isolated from rat fetus striatum were cultured in serum free epidermal growth factor containing medium as spheres for two months without dissociation. Each nonpassaged s
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phere maintained the potential to differentiate to neurons and glia cells when replated in serum containing medium. The electronmicroscopical study revealed immature cells with scant dark cytoplasm or in proliferation stage. The cells differentiated heterogeneously in the sphere ; some had rough endoplasmic reticula and others had long processes with intermediate filaments. Cells undergoing apoptosis were also observed in the sphere. Those were dominant in the core of spheres where abundant cellular debris including apoptotic bodies were observed in the large extracellular spaces. These neural spheres with ongoing differentiation of the cells could fit as an alternative source for neuonal transplantation.
Third, a human astrocyte cell line was generated from a tissue of white matter containing reactive astrocytes in a routine neurosurgical procedure. Those astrocytes were cryopreserved and grafted into normal rat striatum with immunosurpression by cycrosporine. Immunohistochemical study with human specific glial fibrillary acidic protein antigen revealed that grafted astrocytes formed dense cell clusters in the host brain and that part of the cells migrated away into the host striatum. Inside the grafts, there were vessels which were positive with rat specific endothelial barrier antigen (EBA), suggesting that the angiogenesis of the grafts was performed by the host-derived endothelial cells. The expression of EBA also suggests that the blood-brain-barrier was likely formed in the xenograft of astrocytes. In the animals in which the immunosurpression discontinued at two weeks postgrafting, the grafts were able to survive for three additional weeks, however, rejected at eight weeks after discontinuation of the immunosurpression. This indicated that the barrier formation as an immunological privilege was incomplete. Our study indicates that although the formation of blood-brain-barrier is incomplete in the xenografts of astrocytes, the grafted astrocytes themselves were remarkablely integrated with host circumstances and that the autograft of the astrocytes might be feasible for the therapeutic purpose. Less
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