In Vitro Induced Differentiatio Into Neural Cells Using Rat Fetal Neural Stem Cells And Child Bone Marrow Stromal Cells

Objective: (1)To explore the methods of purification, expanding, marking, conservation, induced differentiation and identification of neural stem cells (NSCs) in vitro for research and treatment animal model of tethered cord syndrome and other neural system diseases further. (2)To investigate the feasibility of differentiation of children marrow stromal stem cells (MSCs) into NSCs and neural cells in vitro induced by butylatedhydroxyanisole (BHA). Methods: (D The cells derived from the cerebral cortex of frontal lobe in 14.5d rat fetal 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 purified by the limited dilution, then expanded numerously with sub-colon in consecutive generations .The cells of the purified and expanded NSCs were frozen, recovered and incubated in BrdU, and then the NSCs were induced to differentiate in serum or feeder layer. ?The MSCs donated voluntarily by a fourteen girl were originally cultured in oc-MEM supplemented with 20% FBS, 2mM L-glutamine, lOOU/ml penicillin, lOOmg/ml streptomycin, and 25ng/ml amphotericin B. The cells that were expanded as undifferentiated cells in culture for several passages were applied to be introduced into neural stem cells (NSCs) and neural cells. The induction media is composed of DMEM, 2%DMSO and 150 uM BHA. Results: ? The NSCs of the purified and expanded express Nestin antigen by Immunohistochemistry techniques. These anabiotic NSCs from frozen cells could express Nestin antigen and be induced in serum or feeder layer to differentiate into neurons and glias express Tubulin-III and GFAP respectively. ?After induction by BHA for 7d, about 35% of the MSCs extended long processes terminating in typical growth cones and filopodia and processes formed extensive networks. The induced cells expressed both beta-tublin III and Glial fibrillary acidic protein. In addition, the differentiating cells expressed nestin, characteristic of neuronal precursor stem cells after 30 min, but the trait was undetectable after 6 days. In contrast, expression of trkB, the nerve growth factor receptor, persisted from 5.5 hr to 6 days. Conclusions: ? It is a good and simple way for purification and proliferation 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 beinduced on feeder layer to differentiate into neural cells numerously. BrdU can mark and trace NSCs for research and treatment of neural system diseases in animal model. It is a potential to find a new way for research of biologic specificity and treatment of the disease in nervous system further with the NSCs of the purification, proliferation and induced differentiation in vitro. ?It is capable that the MSCs of the Child differentiate into neurons and glial cells with an optimal differentiation protocol. It also is possible to provide abundant the cells which differentiated from the stem cell for treatment of tethered cord syndrome and other neurological diseases in children.