The methods of purification, expanding, marking, conservation, induced differentiation and identification of neural stem cells (NSCs) in vitro were explored for further research and treatment of tethered cord syndrome in children and other neural system diseases. The cells derived from the cerebral cortex of frontal lobe in 14.5 d rat embryos were maintained in serum-free DMEM/F12 medium containing 20 ng/ml bFGF, 20 ng/ml EGF and B27 supplement. The NSCs of suspending single-cell-colon were isolated and passaged, were purified by limited dilution, and were expanded numerously with sub-colon in consecutive generations. Then, Nestin antigen expression was detected by immunohistochemistry techniques. The cells of the purified and expanded NSCs were frozen, recovered and incubated in BrdU, and the NSCs were induced to differentiate in serum or feeder layer. These revived NSCs from frozen cells could express Nestin antigen and could be induced in serum or feeder layer to differentiate into neurons and glias expressing tubulin-III and GFAP respectively. It is good and simple for purification and proliferation of NSCs numerously by the limited dilution and consecutive generations suspending single-cell-colon of NSCs from the cerebral cortex of frontal lobe in rat embryos. The NSCs could be induced on feeder layer to differentiate into neural cells numerously. BrdU can mark and trace NSCs for the research and treatment of the animal model of neural system diseases. By good command of the technlogies for the purification proliferation, and induced differentiation of NSCs in vitro, it is possible to find a new way for further research of the biologic specificity and the treatment of the disease in nervous system.