小鼠胚胎干细胞体外诱导分化为精原干细胞的研究

目的:初步探讨在体外条件下诱导小鼠胚胎干细胞(EGFP-mESC)分化为精原干细胞(SSC)的条件,建立有效的技术平台。方法:采用部分模拟胚胎早期发育的方法,将EGFP-mESC(XY)经体外悬滴培养,制备拟胚体(EB);使用免疫磁珠分选法从EB中分离出表达原始生殖细胞(PGC)表面标志SSEA-1的细胞;获得的细胞经2μmol/L维甲酸(RA)诱导增殖,使之向雄性生殖细胞分化。对诱导的细胞采用RT-PCR及免疫荧光检测特异性基因及蛋白的碱性磷酸酶。结果:在诱导EGFP-mESC向雄性生殖细胞分化的过程中,采用RT-PCR方法检测到了雄性生殖细胞特异表达基因Sry、fragilis、stella、Tex14等mRNA的表达;利用激光共聚焦方法检测到SSC表面标志蛋白Stra8、integrin-α6及Hsp90α的表达。结论:采用RA体外诱导EGFP-mESC定向分化为SSC是可行的。

Differentiation of Spermatogonial Stem Cells from Mouse Embryonic Stem Cells in vitro

Objective: To set up a feasible method of differentiation of spermatogonial stem cell(SSC) from mouse embryonic stem cell in vitro. Methods: Mouse embryonic stem cells(EGFP-mESC)was cultured in mode of embryoid bodies(EB) which consist of tissue lineages typical of the early mouse embryo, aiming to partially imitate the process of embryonic development; immunomagnetic beads were used to separate the SSEA-1 expressing cells from EB. Then the EB-derived cells were cultured in 2 μmol/L retinoic acid (RA) inducing them to proliferate and differentiate into SSCs in vitro. RT-PCR and immunofluorescence were used to prove that the obtained cells were indeed SSCs, alkaline phosphatase stain. Results: A set of genes that was exclusively expressed in the male germ line was detected by RT-PCR, including Sry, fragilis, Rnh2, stella and Tex14; some spermatogonial stem cell markers, such as Stra8, integrin-α6, and Hsp90α were detected using confocal microscopy. Conclusion: Derivation of spermatogenic stem cells from ESC induced by RA in vitro isf easible.