小鼠胚胎干细胞来源的神经干细胞的稳定传代体系探索

Stable maintaining and propagation of mouse embryonic stem cells-derived neural stem cells in vitro

目的探讨体外诱导、获取均一的神经干细胞群(NSCs)的有效方法,并建立神经干细胞体外稳定传代扩增体系。方法首先采用无血清的诱导培养基贴壁诱导mESCs形成神经上皮祖细胞(NPCs)。然后将经添加表皮生长因子(EGF)和成纤维细胞生长因子-2(FGF2)的无血清培养基短暂悬浮培养后的NPCs再贴壁培养,诱导形成NSCs。通过细胞系46C监测NPCs的形成,同时对分化细胞进行定量PCR和免疫荧光染色,在不同水平检测细胞分化效果。结果 mESCs神经诱导5 d出现大量Sox1+的NPCs;NPCs悬浮培养后,进一步诱导可得到形态均一的NSCs。第2代和第6代NSCs的神经干细胞标志定量PCR检测结果为:Pax6、Nestin、Mash1、BLBP高表达。第8代NSCs免疫荧光染色显示90%以上的细胞均为Nestin、RC2和Pax6阳性。结论成功诱导mESC生成神经干细胞群,并且可以在体外连续稳定的传代。

Objective To establish an efficient way to induce the mouse embryonic stem cells to differentiate into homogeneous neural stem cells and to develop the culture conditions that support the viability and propagation of stable NSCs in vitro.Methods In the serum-free medium,firstly,we induced ESCs to differentiate into neuroepithelial progenitor cells（NPCs）.Then,NPCs were cultured in a suspension medium supplemented with EGF and FGF2 and aggregates were collected and replated to differentiate into neural stem cells（NSCs） in monolayer.We used 46C for monitoring and quantitating the transition from ESCs to NPCs.Expression of NSCs marker was determined by quantitative PCR and immunoflurescence.Results After 5 days of induction,mESCs differentiated into NPCs with high expression of Sox1.NPCs can further differentiate into NSCs after culture in suspension.As determined by quantitative PCR,Pax6,Nestin,Mash1,BLBP genes expressed highly in NSCs.The immunoflurescence demonstrated that 90% of NSCs expressed Nestin,RC2 and Pax6.Conclusions ESCs can be successfully induced to NSCs,and NSCs maintain self-renewal status and propagate in vitro after several passages.