单层分化法结合Smad信号双抑制剂诱导hiPSCs分化为运动神经元

Monolayer differentiation combined with dual-Smad inhibitors induce motor neuron differentiation from hiPSCs

目的探讨单层细胞分化法结合Smad信号双抑制剂,诱导人诱导多能干细胞(hi PSCs)分化为神经干细胞,并进一步分化为运动神经元的可行性。方法无血清E8培养基培养hi PSCs,当其接近70%融合度时改用神经诱导培养基,并加入SB431542和DMH1。9 d后将贴壁细胞消化,在含b FGF和EGF的培养基中悬浮培养,免疫荧光检测神经干细胞标记物的表达。在神经干细胞培养液中添加视黄酸(RA)和音猬因子(SHH),免疫荧光检测各分化阶段运动神经元相关标记物的表达。结果经9 d神经诱导,hi PSCs分化为表达Nestin、Sox1和Sox2的神经干细胞。神经干细胞在RA和SHH等的作用下进一步分化为运动神经元,免疫荧光检出各不同阶段标志物的表达。结论单层分化法结合Smad信号双抑制剂,可将hi PSCs诱导为神经干细胞,并在RA和SHH等作用下进一步分化为运动神经元。

Objective To investigate the feasibility of induction of neural stem cells differentiation from human induced pluripotent stem cells （hiPSCs） by monolayer differentiation combined with dual-Smad signaling inhibitor, and further differentiated into motor neuron with RA and SHH. Methods hiPSCs were cultured in serum-free E8 medium. When hiPSCs reach -70% confluence, neuralization was initiated by changing medium from Essential 8 medium to Neural Induction Medium combined with SB431542 and DMHI. Upon 9 days neural induction,cells were digested and cultured in medium containing bFGF and EGF. The expression of neural stem cell marker Nestin, Soxl and Sox2 were detected by immunocytochemistry. Motor neuron differentiation was initiated by adding 1 μmol/L RA and 100 ng/ml SHH into the neural stem cell medium; the expression of motor neuron related marker at different stage was detected by immunocytochemistry. Results Upon 9 days induction, hiPSCs were differentiated to neural stem cells positive for Nestin, Soxl, Sox2. The expression of motor neuron related marker Olig2, HB9, βⅢ-tubulin and ChAT were detected at different differentiation stages by immunocytochemistry after adding RA and SHH into neural stem cell medium. Conclusion Neural stem cells can be induced from hiPSCs by monolayer differentiation combined with dual-Smad signaling inhibitor, and then can be further differentiated into motor neuron.