无bFGF条件下简便有效建立C57BL/6J小鼠胚胎干细胞系

Efficient method to establish C57BL/6J mouse embryonic stem cell lines with bFGF-free knockout serum replacement

目的:在不含bFGF的Knockout血清替代品(knockout serum replacement,KSR)的培养条件下,简便、有效地建立小鼠胚胎干细胞(embryonic stem cells,ES)系。方法:以C57BL/6J小鼠3.5天囊胚为材料,改良巴氏德管机械法分离内细胞团,于加或不加碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)、含KSR的ES培养基中进行培养和建系,并对建立的小鼠ES系进行鉴定,包括碱性磷酸酶、表面标记分子SSEA-1和多能性相关因子(Oct4、Sox2和Nanog)的检测,核型分析,体内外分化能力等。结果:在无bFGF的ES培养条件,成功从6个小鼠囊胚中分离和建立4株ES系;而添加10 ng/ml bFGF的ES培养条件下,3个优质囊胚未见ES样克隆出现。所建立的小鼠ES可长期传代并维持未分化状态,体内外分化能形成3个胚层结构。结论:辅以改良机械法分离内细胞团,无bFGF的KSR培养条件有助于高效建立C57BL/6J小鼠ES系。

Objective：To establish C57BL/6J mouse embryonic stem（ES） cell lines with bFGF-free knockout serum replacement（KSR）.Methods：C57BL/6J mouse blastocysts 3.5 days post coition（dpc） were collected and cultured in the medium supplemented with KSR and 1 000 U/mL LIF,with or without 10 ng/mL bFGF.The ES cell lines were identificated by morphology,alkaline phosphatase activity,karyotype,cell surface maker SSEA-1 and pluripotent markers（Oct4,Sox2 and Nanog） analysis.The differentiation ability of ES cells was analyzed both in vitro（embryoid bodies） and in vivo（teratoma）.Results：Four cell lines were established out of 6 blastocysts cultured in the medium without bFGF;in contrast,no ES-like colony was observed when bFGF was added to the medium after isolation of 3 high quality blastocysts.The ES cells proliferated well without evidence of differentiation.The ES cells had typical ES-like morphology,expressed alkaline phosphatase,SSEA-1,pluripotent makers such as Oct4,Sox2 and Nanog.They could form embryoid bodies when been cultured in suspension and generate teratomas when been injected subcutaneously into SCID mice,consistent with the the pluripotent abilities of ES cells.Conclusion：KSR containing medium without bFGF could be an efficient culture system for the establishment of mouse ES cell lines.