The Isolation, Culture And Identification Of Neural Stem Cells From The Goat And Rat Fetus

Since Reynolds et al. found the neural stem cells exist in mammalian brain in 1992, there was very much work focused on neural stem cells. The neural stem cell capacities of proliferation and differentiation to every type of neural cell (neurons, astrocytes, and oligodendrocytes) changed the old view which neurocytes in neural system could not proliferate. Researches on neural stem cells have not only remarkable science significance, but also possess the huge potential values which the neural stem cells as substituted cells are used to transplant to therapy neural system diseases. Now, although it has been identified that neural stem cells exist in the neural system in the fetus of human, rat, and mouse, also investigated a little in their suitable fetal age of isolation, cultural conditions, growth characteristic, and induced differentiation in vitro, and so on. But the reports on NSCs from the goat fetal pallium have not been found, and also there are a few researches on them from the fetal spinal cord of rat, not refereed to their freezing conservation. However all these further researches on the NSCs are the key steps to the clinic applications:1．The pallium tissue isolated from six E24~35 d goat fetus by microanatomy technique was made into single-cell suspension and cultured in D-MEM/F12 supplemented with 10%NBS,10ng/mL EGF,10ng/mL bFGF,100 IU/mL penicillin, and 100 IU/mL streptomycin. Though four of six fetus only acquired NSCs global colonies, passaged to third generation by mechanical method.2．The spinal tissue isolated from 128 E12~19 d rat fetus, which acquired from 12 pregnant Wistar rats, by microanatomy technique, was made into single-cell suspension and cultured in D-MEM/F12 supplemented with B27,20ng/mL EGF,20ng/mL bFGF,10ng/mL LIF, 10ng/mL ITS, 100 IU/mL penicillin, and 100 IU/mL streptomycin. The NSCs were derived from nine Wistar rat and passaged to sixth generation by 0.125% tripsin with mechanical method.3．The spinal tissue isolated from 69 E12-19 d rat fetus, which acquired from 6 pregnant SD rats, by microanatomy technique, was made into single-cell suspension and cultured in D-MEM/F12 supplemented with B27,20ng/mL EGF,20ng/mL bFGF,10ng/mL LIF,10ng/mL ITS,100 IU/mL penicillin, and 100 IU/mL streptomycin. In order to investigate the NSCs characters after freezing and thaw, the log growth stage NSCs were frozen through digesting by 0.125% tripsin and making into single-cell suspension by D-MEM/F12 supplemented with 20%NBS and 10% DMSO. The thawed cells still possess NSCs characters.4．The results showed the goat and rat fetus NSCs possess NSCs characters by cellular morphology, monoclonal experiments, induced differentiation experiments and immunochemistry analysis.It is the first time identified there are NSCs in goat brain. The rat NSCs derived from spinal cord were isolated successfully and the study proved NSCs still possess its' intrinsic characters after frozen and thawed, which will establish base for future "spinal-cord-hurted transplant model" experimentation.