人诱导性多能干细胞的无饲养层培养及鉴定

Feeder- free culture and identification of human induced pluripotent stem cells

目的探索人诱导性多能干细胞(induced pluripotent stem cells,i PSCs)的无饲养层培养方法,并对此方法培养的i PSCs进行鉴定。方法将人i PSCs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i PSCs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i PSCs多能性基因SSEA-1、Nanog、Sox2的表达情况。结果倒置显微镜下可见i PSCs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i PSCs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i PSCs,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（i PSCs） and identify i PSCs cultured through feeder- free system. Methods i PSCs were plated on the dishwhich was coated with Vitronectin XF, and passaged by EDTA solution. Morphology of i PSCs 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 i PSCs formed typical cell cloneswith 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 i P-SCs culture system provides suitable condition for keeping i PSCs proliferation and pluripotency.