PARENT SESSION

(73) ISOLATION AND TRANSPLANTATION OF ADULT MAMMALIAN FEMALE GERMLINE STEM CELLS AND PROGENITORS.

Johnson, Joshua¹, Bagley, Jessamyn¹, Skaznik-Wikiel, Malgorzata¹, Lee, Ho-Joon¹, Adams, Gregor¹, Canning Tilly, Jacqueline¹, Cortes, Maria¹, Forkert, Randolf¹, Spitzer, Thomas¹, Iacomini, John¹, Scadden, David¹, Tilly, Jonathan¹, ¹ Massachusetts General Hospital, Harvard Medical School, Boston, MA

ABSTRACT- Recent studies have reported that oocyte production is sustained in postnatal mouse ovaries by germline stem cells (GSC). We have since confirmed that adult mouse ovaries can generate or regenerate hundreds of oocytes within 24-26 hours. Our current objective is to more fully understand the mechanism by which GSC produce new oocytes. Accordingly, multiple approaches to isolate populations of cells containing GSC and/or their progenitors from adult female mice were explored. Transplantation studies were initiated to test whether fractions of cells containing GSC could generate oocytes in chemotherapy-sterilized recipients. In initial experiments, adult C57BL/6 female mice were conditioned with busulfan and cyclophosphamide (BuCy). Oocyte and follicle production were rescued within 60 days of transplanting GSC-containing fractions to BuCy-treated recipients, while mock-transplanted BuCy-treated animals completely lacked immature oocytes at 60 days post-treatment. Delivery of GSC-containing fractions to BuCy-conditioned Ataxia-telangiectasia mutated gene deficient (AtmKO) female mice, which are incapable of making a single oocyte, resulted in the development of follicle-enclosed oocytes. Importantly, oocyte production was shown to last for up to 11 months post-transplant in both wild-type and AtmKO mice treated with BuCy, suggesting that the cell fractions contain self-renewing GSC. More recent experiments have yielded what we believe represents a post-GSC progenitor germ cell (GPC) fraction. Wild-type and AtmKO recipient female mice were conditioned with BuCy, and received GPC preparations from transgenic donors that express green fluorescent protein (GFP) specifically in the germline. Within 30 hours post-transplant, both groups of recipients exhibited GFP-positive oocytes. In summary, these data support the existence of mammalian female GSC and their progenitors, and validate the ability of these cells to contribute to postnatal oocyte production in the mouse. Current and future studies are directed at more fully characterizing this novel adult stem cell niche and its potential for therapeutic manipulation. This work was supported by NIH R01-AG12279, The MGH Fund for Medical Discovery, and Vincent Memorial Research Funds (http://www.vcrb.org).

KEY WORDS: oocyte, germ, stem, transplantation