The Culture In Vitro And The Characterization Of Differentiation And Proliferation Of Cortex-originated Neural Stem Cells From Embryonic Rat Brain

Objective To established the culture and identification methods of cortex-originated neural stem cells from embryonic rat brain,and study their characterization of differentiation and proliferation , in order to provide a cell source for transplantation therapy of neurodegenerative disorders such as Parkinson's disease.Methods The cortical cells from E14.5d rat embryonic were isolated and cultured in suspended condition with serum-free medium containing 20 ng/ml mitogen epidermal growth factor and 1% B27. The characterization of the neural stem cells was identified by nestin immunocytochemistry.The self-renew capacity was demonstrated by Single-cell clonal analyses and BrdU immunocytochemistry.The multipotency of differentiation was identified by MAP-2 and GFAP immunocytochemistry after the cells were induced to differentiate into neuronal-like cells .Results (1) The cultured cells formed typical neurospheres and could be subcultured continously in the medium. Over 90% of the cells are positive for neural stem cells marker Nestin when plated on the poly-1-lysine precoated coverlip for 4 hours.Over 85% of the cells were BrdU positive; Single-cell clonal analyses showed that single cell could be propagated into neurosphere in a week.While induced to differentiate into neuronal-like cells, they were showed to express neuronal cell markers MAP- 2 or GFAP. (2) The cells cultured in suspended condition without EGF propagated slower and tended to differentiate,compared with the cells incubated with EGF. The cells cultured in this condition showed...