Qing Tingting, Shi Yan, Qin Han, Ye Xin, Wei Wei, Liu Haisong, Ding Mingxiao, Deng Hongkui
Department of Cell Biology and Genetics College of Life Sciences, Peking University, Beijing, China.
Differentiation. 2007 Dec;75(10):902-11. doi: 10.1111/j.1432-0436.2007.00181.x. Epub 2007 May 9.
In vitro derivation of oocytes from embryonic stem (ES) cells has the potential to be an important tool for studying oogenesis as well as advancing the field of therapeutic cloning by providing an alternative source of oocytes. Here, we demonstrate a novel, two-step method for inducing mouse ES cells to differentiate into oocyte-like cells using mouse ovarian granulosa cells. First, primordial germ cells (PGCs) were differentiated within the embryonic body (EB) cells around day 4 as defined by the expression of PGC-specific markers and were distinguished from undifferentiated ES cells. Second, day 4 EB cells were co-cultured with ovarian granulosa cells. After 10 days, these cells formed germ cell colonies as indicated by the expression of the two germ cell markers Mvh and SCP3. These cells also expressed the oocyte-specific genes Figalpha, GDF-9, and ZP1-3 but not any testis-specific genes by RT-PCR analysis. EB cultured alone or cultured in granulosa cell-conditioned medium did not express any of these oocyte-specific markers. In addition, EB co-cultured with Chinese hamster ovary (CHO) cells or cultured in CHO cell-conditioned medium did not express all of these oocyte-specific markers. Immunocytochemistry analysis using Mvh and GDF-9 antibodies confirmed that some Mvh and GDF-9 double-positive oocyte-like cells were generated within the germ cell colonies. Our results demonstrate that granulosa cells were effective in inducing the differentiation of ES cell-derived PGCs into oocyte-like cells through direct cell-to-cell contacts. Our method offers a novel in vitro system for studying oogenesis; in particular, for studying the interactions between PGCs and granulosa cells.
从胚胎干细胞(ES细胞)体外诱导生成卵母细胞,有潜力成为研究卵子发生的重要工具,并且通过提供卵母细胞的替代来源推动治疗性克隆领域的发展。在此,我们展示了一种新颖的两步法,利用小鼠卵巢颗粒细胞诱导小鼠ES细胞分化为卵母细胞样细胞。首先,通过原始生殖细胞(PGC)特异性标志物的表达确定,在第4天左右胚胎体(EB)细胞内PGC分化,并与未分化的ES细胞区分开来。其次,将第4天的EB细胞与卵巢颗粒细胞共培养。10天后,这些细胞形成了生殖细胞集落,这通过两种生殖细胞标志物Mvh和SCP3的表达得以表明。通过逆转录聚合酶链反应(RT-PCR)分析,这些细胞还表达了卵母细胞特异性基因Figalpha、GDF-9和ZP1-3,但未表达任何睾丸特异性基因。单独培养的EB或在颗粒细胞条件培养基中培养的EB均未表达这些卵母细胞特异性标志物中的任何一种。此外,与中国仓鼠卵巢(CHO)细胞共培养或在CHO细胞条件培养基中培养的EB并未表达所有这些卵母细胞特异性标志物。使用Mvh和GDF-9抗体进行的免疫细胞化学分析证实,在生殖细胞集落内产生了一些Mvh和GDF-9双阳性的卵母细胞样细胞。我们的结果表明,颗粒细胞通过直接的细胞间接触有效地诱导ES细胞来源的PGC分化为卵母细胞样细胞。我们的方法提供了一种用于研究卵子发生的新型体外系统;特别是用于研究PGC与颗粒细胞之间的相互作用。