人诱导性多能干细胞的无饲养层培养及鉴定

摘要

目的：探索人诱导性多能干细胞（induced pluripotent stem cells，iPSCs）的无饲养层培养方法，并对此方法培养的iPSCs进行鉴定。方法将人iPSCs接种于玻璃粘连蛋白（Vitronectin XF）包被的培养皿上培养，采用EDTA消化传代。倒置显微镜下观察iPSCs的生长状态；碱性磷酸酶（ALP）染色鉴定；采用PCR和免疫荧光检测iPSCs多能性基因SSEA⁃1、Nanog、Sox2的表达情况。结果倒置显微镜下可见iPSCs呈典型的克隆状生长，克隆呈圆形或椭圆形，边界清晰、整齐；ALP染色结果阳性；PCR结果显示人iPSCs强表达多能性基因SSEA⁃1、Nanog、Sox2；免疫荧光结果显示多能干细胞特异性指标SSEA⁃1、Nanog、Sox2均呈阳性。结论无饲养层培养体系培养人iPSCs，细胞能稳定增殖，保持自我更新潜能及多能性。%Objective To explore a stable feeder⁃free culture system of induced pluripotent stem cells (iPSCs) and identify iPSCs cultured through feeder⁃free system. Methods iPSCs were plated on the dish which was coated with Vitronectin XF, and passaged by EDTA solution. Morphology of iPSCs was observed un⁃der a microscope, and the pluripotency marker ALP, was analyzed. Moreover, the pluripotent genes SSEA⁃1, Nanog and Sox2 were detected by PCR and inmunofluorescence. Results iPSCs formed typical cell clones with clear boundary, and the clones were round or oval. The immunohistochemistry of ALP showed positive reac⁃tion. PCR showed that pluripotent genes SSEA⁃1, Nanog and Sox2 were expressed strongly. Moreover, the immu⁃noinfluorescence analysis of SSEA⁃1, Nanog and Sox2 revealed positive results. Conclusion Feeder⁃free iP⁃SCs culture system provides suitable condition for keeping iPSCs proliferation and pluripotency.