Objective: To improve the efficiency of induced differentiation of primitive neural epithelial cells derived from human induced pluripotent stem cells (hiPSCs-NECs) into functional midbrain dopaminergic progenitor cells (DAPs).
Methods: HiPSCs were cultured in mTeSRTM medium containing DMH1 (10 μmol/L), SB431542 (10 μmol/L), SHH (200 ng/mL), FGF8 (100 ng/mL), purmorphamine (2 μmol/L), CHIR99021 (3 μmol/L), and N2 (1%) for 12 days to induce their differentiation into primitive neuroepithelial cells (NECs). The hiPSCs-NECs were digested with collagenase Ⅳ and then cultured in neurobasal medium supplemented with 1% N2, 2% B27-A, BDNF (10 ng/mL), GDNF (10 ng/mL), AA, TGF-β, cAMP, and 1% GlutaMax in the presence of different concentrations of Rho kinase inhibitor Y27632, and the culture medium was changed the next day to remove Y27632. Continuous induction was performed until day 28 to obtain DAPs.
Results: Human iPSCs expressed the pluripotency markers OCT4, SOX2, Nanog, and SSEA1 and were positive for alkaline phosphatase staining. The hiPSCs-NECs were obtained on day 13 in the form of neural rosettes expressing neuroepithelial markers SOX2, nestin, and PAX6. In digested hiPSCs-NECs, the addition of 5 μmol/L Y27632 significantly promoted survival of the adherent cells, increased cell viability and the proportion of S-phase cells (P < 0.01), and reduced the rate of apoptotic cells (P < 0.05). On day 28 of induction, the obtained cells highly expressed the specific markers of DAPS (TH, FOXA2, NURR1, and Tuj1).
Conclusion: Treatment with Y27632 (5 μmol/L) for 24 h significantly promotes the survival of human iPSCs-NECs during their differentiation into DPAs without affecting the cell differentiation, which indirectly enhances the efficiency of cell differentiation.
目的: 提高人诱导多能干细胞来源原始神经上皮细胞（hiPSCs-NECs）定向诱导中脑多巴胺能神经前体细胞（DAPs）的效率。
方法: 将人iPSCs在含有小分子组合[DMH1（10 μmol/L）、SB431542（10 μmol/L）、音猬因子（SHH 200 ng/mL）、成纤维细胞生长因8（FGF8 100 ng/mL）、嘌吗啡胺（2 μmol/L）、CHIR99021（3 μmol/L）、N2（1%）]的mTeSRTM培养基中诱导至12 d，细胞分化为原始神经上皮细胞（NECs）。特异性诱导DAPs，将诱导的hiPSCs-NECs用胶原酶Ⅳ消化后，在神经分化培养基[添加1%N2、2%B27-A、BDNF（10 ng/mL）、GDNF（10 ng/mL）、AA、TGF-β、cAMP、1% GlutaMax]中加入不同浓度的Rho激酶抑制剂Y27632重新接种，并于次日更换培养基去除Y27632，持续诱导至第28天获得DAPs。
结果: 人iPSCs可在基质胶上稳定传代，表达多潜能性标记物OCT4、SOX2、Nanog和SSEA1，且碱性磷酸酶染色呈阳性。诱导分化至第13天获得hiPSCs-NECs，形成玫瑰花结样结构，能够表达特征性标记物（SOX2、Nestin和PAX6）。将hiPSCs-NECs消化后，添加5 μmol/L Y27632的hiPSCs-NECs相比较未加Y27632的对照组，细胞解离后贴壁存活率明显增加，处于S期的细胞（P＜0.01）与细胞活力显著提高（P＜0.01），且凋亡率显著降低（P＜0.05），持续诱导至第28天，细胞高表达DAPs特异性的标记物（TH、FOXA2、NURR1和Tuj1）。
结论: 添加Y27632（5 μmol/L）24 h可显著提高人iPSCs-NECs细胞向iPSCs-DA神经前体转化时的细胞存活率，并且Y27632去除后，不会影响后续向DA神经前体细胞分化，间接的提高了细胞的分化效率
Keywords: Y27632; dopaminergic neural progenitor cells; human induced pluripotent stem cells; primitive neuroepithelial cells.