2种诱导iPSC向神经干细胞分化方法的比较

目的:整体比较2种促进诱导性多能干细胞(induced pluripotent stem cells,i PSC)向神经干细胞(neural stem cells,NSC)分化的方法,确定一种稳定、高效的获得NSC的方法,并对NSC进行系统鉴定。方法:方法A:SB431542和drosomophorin的浓度均为5μmmol/L,诱导初始密度100%;方法 B:SB431542的浓度为5 mmol/L,drosomophorin的浓度为1 mmol/L,诱导初始密度为40%。比较及鉴定方法:镜下观察诱导获得NSC的状态;realtime PCR比较神经干细胞相关基因Pax6、nestin、Sox1、Sox2等表达量;流式细胞术分析诱导第16天Pax6阳性率;免疫荧光定性分析神经干细胞相关蛋白的表达及其自发分化的能力。结果:方法 A获得的NSC悬起后成球趋势明显,圆形,透明;方法 B诱导获得NSC形状不规则,色灰暗。Real-time PCR结果证明方法 A诱导获得的细胞神经干细胞相关基因的表达量高于方法 B。流式细胞术分析证明第16天,PAX6的阳性率,方法 A高于方法 B。经鉴定,方法 A获得的神经干细胞高表达Pax6、nestin、Sox2等基因自发分化30 d,形成明显的神经纤维束,表达TUJ-1、MAP2及GFAP等神经元和胶质细胞的特异性标志物。结论:方法 A整体优于方法 B,我们推荐方法 A作为诱导i PSC向神经干细胞分化的方法。

Comparison of 2 methods for inducing iPSC to differentiate into neural stem cells

AIM: To select an efficient way of promoting induced pluripotent stem cells (iPSC) to differentiate into neural stem cells (NSC) by comparing 2 methods. METHODS: The culture system in method A contained SB431542 (5 mmol/L) and drosomophorin (5 mmol/L) with 100% initial cell density, while that in method B contained SB431542 (5 mmol/L) and drosomophorin (1 mmol/L) with 30%~50% initial cell density. For comparison and identification of the 2 methods, the growth state was observed under microscope, and the expression of Pax6, nestin, Sox1 and Sox2 was quantitatively detected by real-time PCR and flow cytometry. The related protein expression and the ability of spontaneous differentiation were determined by immunofluorescence analysis. RESULTS: The cells derived from method A with 5 mmol/L of SB431542 and drosomophorin and 100% initial cell density achieved the higher expression of Pax6, nestin, Sox1 and Sox2. The growth state was better and the cells differentiated into neurons and astrocytes normally. CONCLUSION: The method A was superior to method B, and we recommend the method A with 5 mmol/L of SB431542 and drosomophorin and 100% initial cell density as the method for differentiating NSC.