Investigation on cultural conditions of neural stem cells from embryonic midbrain

Abstract:

Objective：To investigate the cultural condition of neural stem cells（NSCs） from embryonic midbrain. Methods：Midrains of embryonic SD rice（12.5 d） were used. Cells were maintained and expanded in the medium with serum-free supplement containing basic fibroblast growth factor（bFGF） and epidermal growth factor（EGF）. The cultured neurospheres were identified by neuroepithelial stem cell protein（nestin）. 5-bromodeoxyuridine（Brdu） was added into the medium to see the proliferative ability. Differentiation of cultured NSC was checked by immunocytochemistry to see the expression of glial fibrillary acidic protein（GFAP），and β-Ⅲ tublin. Clone forming of different cell densities was checked. The effects of separated and combined use of FGF and EGF on cell growth were investigated. Effect of different inoculation density（0.5-16.0）×105/ml and leukemia inhibitory factor（LIF） on cell growth was also taken into consideration. Results：The cultured neurospheres positively expressed nestin. Brdu tests showed that most NSC in neuro－spheres had proliferative capacity. After differentiation，NSCs differentiated into cells expressing GFAP（68.32±5.70）% or β-Ⅲ tublin（21.65±3.40）%. Cell clone numbers were higher in EGF and bFGF combined use group than in separated use group（P＜0.05，compared with those of EGF group；P＜0.01，compared with those of bFGF group）. Number of cells was significantly increased in LIF group than in control group（F=2 630.114，P=0.000）. The results of different inoculation density showed that cell density had signifi－cant impact on the number of neural spheres（F=155.1００，P=0.001）. The number of neural spheres increased within the inoculation density from 0.5×105/ml to 16.0×105/ml. Conclusion：Cytokines bFGF and EGF is necessary for the growth of NSCs. LIF promotes the growth of NSCs during long-term culture. In a suitable range，NSCs clone forming shows density-dependent.