| Objective: To establish the methods of in vitro culttre and characterization of neural stem cells isolated from neonatal rats. To examine the proliferation and differentiation of neural stem cells in vitro. To investigate the mechanisms of how microenvironmental elements support the proliferation and differentiation of stem cells, as a fundamental research for neural stem cell transplantation. Methods:1) Neural stem cells from neonatal rats were isolated, cultured and expanded by neurosphere formation using serum-free medium containing growth factors, bFGF and/or EGF. The cells were passaged continuously by dissociating mechanically in order to harvest them in bulk. The cell morphology was observed under phase-contrast microscope. The cell proliferaticn was confirmed by cell growth curve and flow cytometry. Immunocytochemistry was performed to detect the expression of Nestin, neuron specific enolase (NSE), glial fibril lary acidic protein (GFAP), and cyclic nucleotide phosphohydrolase (CNP).2) The effects of bFGF and EGF on the proliferation of neural stem cells were examined by cell counting. Immunofluorescence and flow cytometry were performed to detect the effects of bFGF, EGF and the different concentrations of fetal bovine serum on the differentiation of neural stem cells into neurons, iistrocytes and oligodendrocytes.3) Astrocytes were isolated and purified by a standiird shaking method and the differential adhesion technique. With immunocytochemical labeling, the purity of astrocytes was determined by the expression of GFAP. For co-culture experiments, astrocytes were cultured on the bottom of 12-well plates, while poly-L-lysine-coated coverslips were placed in the same well without contact. Asirocytes were incubated in co-culturemedium overnight before neural stem cells were plated on the coverslips. Cultures werefed every other day. Immunofluorescence examination was used to detect the effects of astrocyte-derived factors on the expression of NSE, GFAP, and tyrosine hydroxylase (TH) of the differentiated cells. Results:1) The proliferating cells isolated from neonatal rat brain grew into floating neurospheres in the serum-free medium with the presence of bFGF or EOF. The results of cell growth curve and flow cytometry showed that these neurospheres could proliferate and passage in vitro. Irnmunocytochemistry study indicated that the neurospheres were Nestin-positive and could differentiat with multi-directions. They expressed specific antigens of neuron, astrocytes, and oligodendrocy;es.2) Neural stem cells grew best in basal culture with 10~20ng/ml bFGF or 20~30ng/ml EGF. The results of immunofluorescence and flow cytometry suggested that bFGF tend to induce the cells to differentiate into NSE-positive cells (P<0.05 vs control), EGF more likely induce GFAP-positive cells (P<0.0f vs control) , and fetal bovine serum help the differentiation of GFAP-positive cells (P(0.05 vs control).3) Irnmunocytochemistry examination showed thai the purified astrocytes were 98% of GFAP-positive. Co-culturing the neurospheres v/ith astrocytes without contact rapidly promoted the differentiation of neural stem cells into NSE-positive cells (P<0.05 vs control) including more TH-positive cells (P<0.0.5 vs control).Conclusions:1) The cells isolated from neonatal rat brain can only be characterized as stem cells of the central nervous system according to their uncifferentiated features, the capacity of self-renewing, proliferation and pluripotentiality, and the expression of Nestin antigen. The isolation and culture of neural stem cells nethod established here is simple and feasible. It is a great model for studies on induction of differentiation.2) Neural stem cells were best cultured in basal m;dium containing 10~20ng/ml of bFGF or 20~30ng/ml of EGF. bFGF exerted a preferential effect on neuronal development and to a less extent, glial development; EGF may induce more stem cells differentiation into glial cells. While fetal bovine serum tended to...
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