| Recent development of neural stem cell (NSC) raised hopes for intractable disease such as severe spinal cord injury (SCI) that leaves tens of thousands young people permanently paralyzed every year worldwide. NSC, like other types of stem cells, may be clinically useful in generating cell types that have been damaged in SCI. Animal studies demonstrated that NSCs provided such a sense of optimism ever before for transplantation treatment in SCI than any other cells such as fibroblasts, Schwann cell and olfactory ensheathing cell (OECs) etc. It is possible that manipulating NSC's differentiation prior to transplantation to obtain functionally neurons or glial cells. Thus, the studies of NSCs may bring appealing application prospect on the repair and therapy of SCI. By purification and construction the cell line of NSCs from embryo would make this promising new treatment for SCI possible. Here, We have studied the isolation, cultivation and differentiation of NSCs from mouse and human embryo. Partâ… The purification and characterization of neural stem cell from mouse embryoThe primary cells, isolated from cortex of mouse embryos (14-16 days old),were seeded into serum-free medium containing basic fibroblast growth factor(bFGF) and then passaged continuously. The morphologic and growth of cell were observed by convert microscope. The ultrastructure of NSCs and internal structure of the neurospheres were investigated by transmission electron microscope. The expression of nestin gene, which is specific to NSCs in the CNS, was detected by immunofluorescence technique, reverse transcriptase polymerase chain reaction (RT-PCR) and gene sequencing. The proliferation of cell and the effect of bFGF on it was evaluated by bromodeoxyuridine (BrdU) labeling, 3(4,5dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay, flow cytometry and single-cell cloning technique, and the affect mechanism of bFGF was further assessed. The multipotency of the cells was detected by inducing differentiation. In addition, the effect of serum on differentiation was investigated by comparing with control group. Theresults showed that the purified cells can grow into neurospheres in suspension with typical immatural characteristic and some cells being asymmetric division. Large numbers of nestin-positive cells could be observed in the neurosphere by immunofluorescence technique. RT-PCR and gene sequencing also proved the expression of nestin gene in those cells. BrdU labeling indicated that there were many newly born cells through division and proliferation in the neurosphere. Furthermore, MTT colorimetric assay demonstrated these cells had the remarkable potential of division and proliferation; In other aspect, bFGF could promote them proliferation significantly compared with control group(P<0.01). The result of flow cytometry showed the proportion of cells in S phase and proliferation index(PI) had increased after passaging. Furthermore, the neurosphere can grow into three major types of the CNS: neurons, astrocytes and oligodendrocytes. The astrocyte is preponderant. The majority of GFAP-positive cells in serum-containing medium were protoplasmic-like astrocyte ,but fibrous-like astrocyte was superior in numbers among GFAP-positive cells in serum-free medium. These cells retained their multilineage potential over repeated passages. Present results demonstrate that our cells are self-renewing and multi-potent that can differentiate into neurons, astrocytes and oligodendrocytes under optimal conditions. These cells express nestin gene. Thus, we demonstrated successfully the purification and characterization of NSCs. These cells have the remarkable ability of division and proliferation that they could be maintained and expanded in the presence of bFGF alone. bFGF could preclude differentiation and promote proliferation maybe via a change in cell cycle kinetics of NSCs by decreasing G1 duration. Serum may affect cell differentiation. Without serum, NSCs can generate neurons and glia cells after natural differen... |
