人胚胎干细胞无血清无饲养层培养体系的建立

摘要

Objective To establish a serum-free and feeder-free culture system for human embryonic stem cells (HESc).Methods The HESX-01 cells were cultured in Knockout medium (the first group),self-made serumfree medium (the second group) and improved serum-free medium with bFGF and heparin (the third group),respectively.The morphology,clone counting and clone size were observed after twenty-fifth generation.The surface labeling immunocytochemical analysis,Fluorescence-activated cell sorter analysis (FACS) and embryoid bodies formation were utilized to determine the character of HESc.Results After twenty-fifth passages the clones of the second group were gradually differentiated,and the HESX-01 cells in the first and third group showed typical human embryonic stem cell morphology,they all expressed Nanog,Oct-4,Tra-1-60 and SSEA-4,but did not express SSEA-1,the same results were observed from FACS analysis.The HESc formed embryoid body.Besides,the significant differences were oberserved in the second group as compared with the first and third group in clone counting and size per field.Conclusions The self-made serum-free and feeder-free culture system for HESX-01 cells has been established,and the efficacy is not significantly different to commercially availabe Knockout medium.%目的 拟建立人胚胎干细胞无血清无饲养层的培养体系.方法 将干细胞分别置于Knockout培养基(第1组)、自制无血清培养基(第2组)和添加bFGF及肝素的改良无血清培养基(第3组)中培养25代后观察比较3组干细胞形态学变化、克隆数目及大小.并借助细胞免疫组化、流式细胞术和体外拟胚体形成等方法检测干细胞的未分化状态和全能性.结果 传代25次后,第2组克隆逐渐分化.第1组和第3组的干细胞均呈典型的人胚胎干细胞形态特征；细胞表达Nanog,Oct-4,Tra-1-60,SSEA-4,但不表达SSEA-1；流式检测也显示SSEA-4高表达,SSEA-1低表达；体外分化能形成拟胚体.通过单位视野下克隆个数及大小的比较,结果显示第2组与第1组和第3组间的差异显著.结论 本研究通过改良本实验室已获得国家专利的无血清培养基,初步建立了人胚胎干细胞无血清无饲养层培养体系,培养效果与公认培养体系无明显差异.