Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a FACSbased protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically-active cells that exhibit a gene expression profile consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and spontaneously generate 35-50 µm

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... oocytes, as determined by morphology, gene expression and attainment of haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1-2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, like adult mice, possess rare mitotically-active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo. Since the early 1950s, management of ovarian insufficiency and failure, including infertility due to aging or insults, was restricted by the belief that the oocyte-containing pool of follicles set forth at birth is not amenable to renewal 1 . In 2004, studies with mice challenged the idea of a fixed ovarian reserve being endowed during the perinatal period 2 . Based on results from several approaches, it was concluded that ovaries of adult mice possess rare female germline or oogonial stem cells (OSCs) that generate oocytes analogous to spermatogonial stem cell (SSC) support of sperm production in adult testes 3 . Although this Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: