人胚胎干细胞的自主分化

The Self-differentiation of Human Embryonic Stem Cells

人胚胎干细胞(human embryonic stem cells,h ESCs)具有自我更新的能力及多向分化的潜能。诱导其定向分化一直是近年来的研究热点,但关于其自主分化的研究却不多。本实验使用无血清培养液mTeSR誖1培养hESCs,碱性磷酸酶检测显示其维持了未分化的状态。改用自主分化培养液后,通过形态观察、实时定量PCR(quantitative real-time PCR,q PCR)、免疫荧光及流式细胞术等方法对自主分化的细胞进行鉴定,从而探讨hESCs自主分化的方向和规律。实验结果表明,形态上分化细胞表现出均一的纤维状;基因和蛋白水平上,中胚层的VIMENTIN和SNAIL2显著表达,而外胚层的NESTIN以及内胚层的SOX17则无表达;流式细胞术检测分化细胞的CD29及CD44,表达量分别为92.8%和30.0%。由此可得出结论:hESCs的自主分化倾向于中胚层方向。

Human embryonic stem cells(hESCs) have the self-renewal ability and multilineage differentiation potential. In recent years, the induced differentiation of h ESCs has been a research focus, but there exists few researches on their self-differentiation. In our study, serum-free medium m Te SR 1 was used to culture h ESCs.The alkaline phosphatase staining indicated that the h ESCs we cultured maintained undifferentiated state. Then,we changed m Te SR 1 into self-differentiation medium. Morphologic observation, quantitative Real-time PCR(q PCR), immunofluorescence and flow cytometry were used to identify the direction and principles of the differentiated cells. The results demonstrated that the differentiated cells exhibited a homogenous fibrous morphology. At the level of gene and protein, they significantly expressed mesoderm marker VIMENTIM and SNAIL2, instead of ectoderm marker NESTIN and entoderm marker SOX17. Flow cytometry indicated that CD29 and CD44 were expressed 92.8% and 30.0%, respectively. Therefore, we concluded that h ESCs have the trend to differentiate into mesoderm cells.