人原始生殖细胞的分离和体外培养

从4～10周龄药物流产胚胎的生殖嵴和肠系膜组织中分离原始生殖细胞(primordial germ cells,PGCs),培养在添加人重组白血病抑制因子(rh LIF)、人重组碱性成纤维细胞生长因子(rh bFGF)和Forskolin的小鼠饲养层细胞上.经过4～7 d培养,PGCs形成典型的鸟巢状集落.集落在传代过程中保持碱性磷酸酶活性,且胚胎阶段性特异抗原1(SSEA-1)、胚胎阶段性特异抗原3(SSEA-3)免疫荧光染色呈阳性.具有分化潜能的PGCs能在体外连续传代培养12代.结果表明从药物流产胚胎中分离的人类PGCs可以在体外培养成为具有分化潜能的多能性干细胞.

Isolation and long term culture of human primordial germ cells

Human primordial germ cells(PGCs) collected from gonadal ridges and mesenteries were cultured on mouse feeder layers in DMEM/15%FBS with human recombinant leukemia inhibitory factor (LIF), human recombinant basic fibroblast growth factor, and forskolin. Initially, PGCs were visualized by alkaline phosphatase activity staining. Over a period of 4-7 days, PGCs gave rise to large multicellular colonies resembling those of mouse embryonic stem cells. Throughout the culture period, most cells within the colonies maintaine their alkaline phosphatase active and positive staining of Specific Stage Embryonic Antigen (SSEA-1, SSEA-3) which has been routinely used to characterize embryonic stem and PGCs cells. Among all PGC-derived cultures, it was found that one derivation can under go 12 passages. Taken together, these results suggest that human PGC-derived cultures reserve differentiation potential to proliferated pluripotent stem cells.