Enhanced method to select human oogonial stem cells for fertility research

Alternative methods to obtain mature oocytes are still needed for women with premature ovarian failure (POF). Oogonial stem cells (OSCs), found in adult ovaries, have provided insight into potential paths to treating infertility. Previously, the DDX4 antibody marker alone was utilized to isolate OSCs; however, extensive debate over its location in OSCs versus resulting oocytes (transmembrane or intracytoplasmic) has raised doubt about the identity of these cells. Separate groups, however, have efficiently isolated OSCs using another antibody marker Ifitm3 which is consistently recognized to be transmembrane in location. We hypothesized that by using anti-DDX4 and anti-IFITM3 antibodies, in combination, with MACS, we would improve the yield of isolated OSCs versus using anti-DDX4 antibodies alone. Our study supports earlier findings of OSCs in ovaries during the entire female lifespan: from reproductive age through post-menopausal age. MACS sorting ovarian cells using a the two-marker combination yielded a similar to twofold higher percentage of OSCs from a given mass of ovarian tissue compared to existing single marker methods while minimizing the debate surrounding germline marker selection. During in vitro culture, isolated cells retained the germline phenotype expression of DDX4 and IFITM3 as confirmed by gene expression analysis, demonstrated characteristic germline stem cell self-assembly into embryoid bodies, and formed > 40 mu m "oocyte-like" structures that expressed the early oocyte markers GDF9, DAZL, and ZP1. This enhanced and novel method is clinically significant as it could be utilized in the future to more efficiently produce mature, secondary oocytes, for use with IVF/ICSI to treat POF patients.