Vincent Center for Reproductive Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Nat Protoc. 2013 May;8(5):966-88. doi: 10.1038/nprot.2013.047. Epub 2013 Apr 18.
Accruing evidence indicates that production of new oocytes (oogenesis) and their enclosure by somatic cells (folliculogenesis) are processes not limited to the perinatal period in mammals. Endpoints ranging from oocyte counts to genetic lineage tracing and transplantation experiments support a paradigm shift in reproductive biology involving active renewal of oocyte-containing follicles during postnatal life. The recent purification of mitotically active oocyte progenitor cells, termed female germline stem cells (fGSCs) or oogonial stem cells (OSCs), from mouse and human ovaries opens up new avenues for research into the biology and clinical utility of these cells. Here we detail methods for the isolation of mouse and human OSCs from adult ovarian tissue, cultivation of the cells after purification, and characterization of the cells before and after ex vivo expansion. The latter methods include analysis of germ cell-specific markers and in vitro oogenesis, as well as the use of intraovarian transplantation to test the oocyte-forming potential of OSCs in vivo.
越来越多的证据表明，哺乳动物中新卵母细胞（卵子发生）的产生及其被体细胞包裹（卵泡发生）的过程并不局限于围产期。从卵母细胞计数到遗传谱系追踪和移植实验的终点支持生殖生物学中的范式转变，即在产后生活中卵母细胞包含的卵泡发生积极更新。最近从老鼠和人类卵巢中纯化出有丝分裂活性的卵母细胞祖细胞，称为雌性生殖干细胞（fGSCs）或卵原干细胞（OSCs），为研究这些细胞的生物学和临床应用开辟了新途径。在这里，我们详细介绍了从成年卵巢组织中分离小鼠和人类 OSCs 的方法、纯化后细胞的培养以及细胞在体外扩增前后的特性。后一种方法包括分析生殖细胞特异性标记物和体外卵子发生，以及利用卵巢内移植在体内测试 OSCs 的卵母细胞形成潜能。
