胚胎干细胞来源的饲养细胞支持自体胚胎干细胞生长  

Mouse Embryonic Stem Cells Derived Feeder Cells Support the Growth of Themselves

作　　者：施英唐[1] 唐帆[1] 褚建新[1] 

机构地区：[1]上海交通大学医学院新华医院发育生物学研究中心,上海200092

出　　处：《上海交通大学学报（医学版）》2006年第5期470-475,共6页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家重点基础研究发展规划("973")项目(2001CB509904)

摘　　要：目的探讨小鼠胚胎干细胞(mESCs)来源的饲养细胞是否具备支持自体mESCs未分化生长的能力。方法先诱导mESCs形成类胚体(EB),进一步诱导其分化为成纤维细胞(mEB-dFE),以用作饲养细胞。采用形态学、集落形成率、细胞分化率、免疫细胞化学、碱性磷酸酶染色和RT-PCR对生长在mEB-dF上的mESCs的未分化特性进行鉴定。采用RT-PCR和诱导mESCs体内外分化方法鉴定mESCs多分化潜能特性。结果从EB三个发育阶段(EB贴壁10、15、20 d)分离出48个mEB-dF系,其中5个(来自15 d的EB)能维持mESCs未分化生长和多潜能性达10代以上。生长在这种饲养层上的mESCs与生长在小鼠胚胎成纤维细胞(MEF)上一样,其特性无明显差异,均表达碱性磷酸酶和特殊的mESCs标记,包括SSEA-1、OCT-4、NANOG,在体外形成EB和体内形成畸胎瘤;其他43个mEB-dF系则不具有这种能力。结论研究不仅为mESCs体外培养提供了一种新的饲养细胞,避免了MEF作饲养细胞的许多不足;而且还提示mESCs可诱导出不同功能的成纤维样细胞,其差异的分子机制值得进一步研究。Objective To test whether feeder cells derived from mouse embryonic stem cells（mESCs） could support the growth of mESCs themselves. Methods mESCs were induced to form mouse embryoid bodies（EB）, and then fibroblast-like cells were derived from further differentiated mEB （mEB-dF）, which served as feeder cells. The undifferentiation of mESCs grown on mEB-dF was confirmed by morphological analysis, colony efficiency and cell differentiation rate of mESCs, immunocytochemistry, alkaline phosphatase staining and RT-PCR. The pluripotency of mESCs grown on mEB-dF was examined by RT-PCR, inducing their differentiation in vivo and in vitro. Results Forty-eight fibroblast-like cells lines were derived from the same EB at three periods （d 10, d 15 and d 20）, and five of them, mostly derived from d15 EB, were able to maintain mESCs in undifferentiated status and pluripotential ability over 10 passages, mESCs cultured on these feeder cell lines expressed alkaline phosphatase and specific mESCs markers, including SSEA-1, OCT-4, NANOG, and formed EB in vitro and teratomas in vivo. However, the majority of mEB-dF lines（43/48） has no such ability. Conclusion This study not only provides a novel feeder system for mESCs culture, avoiding lot of disadvantages of mouse embryo fibroblasts used as the feeder, but also indicates that fibroblast-like cells derived from mESCs take on different functions. The molecular mechanism of different function of these fibroblast cells is worthy of further investigations.

关 键 词：小鼠 胚胎干细胞 类胚体 饲养细胞 

分 类 号：Q813.11[生物学—生物工程]