Neonatal Rat Hippocampal Neural Stem Cells In Vitro And Induced Differentiation

Objective(1)To establish the methods of culturing and identificating of neural stem cells isolated from the hippocampus of neonatal rat in vitro, to examine the proliferation and differentiation of neural stem cells in vitro.(2)To approach the characteristic of the proliferation and different -iation of neural stem cells cultured in vitro,and investigate the mechanisms that the microenvironment how to affect the proliferation and differentiation of neural stem cells,which as a fundamental rese -arch for neural stem cell transplantation.Methods(1)Neural stem cells were isolated from neonatal rat hippocampus, cult-ured and expanded by DMEM/F12 serum-free medium containing growth factors bFGF,EGF and B₂₇.The cells were passaged continuously by disassociating mechanically in order to purify and obtain the cells bulk.The cells' morphous was observed under inverted phase-contrast microscope,and flow cytometry was applied to estimate the proliferation of neural stem cells.Immunocytochemistry was performed to detect the expression of Nestin,neuron specific enolase(NSE),glial fibrillary acidic protein(GFAP),and cyclic nucleotide phosphohydrolase(CNP).(2)Immunofluorescence and flow cytometry were performed to detect the effects of bFGF,EGF on the differentiation of neural stem cells into neurons,astrocytes and oligodendrocytes.(3)Astrocytes were isolatedand purified by L-leucine-methyl-ester, a standard shaking method and the differential adhesion technique. By immunocytochemical labeling,the purity of astrocytes was determined by the expression of GFAP.For the co-culture experiments, astrocytes were cultured on the bottom of 6-well plates,while poly-L-lysine -coated coverslips were placed in the same well without contact.Astrocytes were incubated in co-culture medium overnight before neural stem cells were plated on the coverslips.They were fed every other day.Immunocytochemistry examination was used to detect the effects of astrocytes on the expression of NSE,GFAP and tyrosine hydroxylase(TH)of the differentiated cells.Results(1)The proliferating cells isolated from neonatal rat hippocampus grew into floating neurospheres in the serum-free medium with the presence of bFGF and/or EGF.The neurospheres could proliferate and passage in vitro.Immunocytochemistry study indicated that the neurospheres were Nestin-positive and could differentiate into multi-directions.Specific antigens of neurons,astrocytes,and oligodendrocytes were expressed also.(2)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＜0.05 vs control).(3)The results of immunocytochemistry showed that the purified astrocytes were 98%of GFAP-positive.Co-culturing the neurospheres with astrocytes without contact rapidly promoted the differentiation of neural stem cells into NSE-positive cells(P＜0.05 vs control), including more TH-positiye cells(P＜0.05 vs control).Conclusions:(1)The cells isolated from neonatal rat hippocampus can only be characterized as stem cells of the central nervous system according to their undifferentiated features,the capacity of self-renewing, proliferation and pluripotentiality,and the expression of Nestin antigen.The method isolate and culture neural stem cells established here is feasible.(2)bFGF expressed a preferential effect on neuronal development and to a less extent glial development;EGF may induce more neural stem cells differentiation into glial cells.(3)Astrocytes induced the differentiaton of neural stem cells into neurons,including dopaminergic neurons.This neurogenesis pathway provided a new direction in the mechanisms of neural cell development and also provided a simple,fast and efficient procedure for the generation of a number of neurons in vitro.