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(478) CHARACTERIZATION AND TRANSPLANTATION OF CULTURED SPERMATOGONIAL STEM CELLS. Jeong, DongKee1, McLean, Derek1, Griswold, Michael1, 1 Center for Reproductive Biology, Washington State University, Pullman, WA ABSTRACT- The objectives of this study were to develop an in vitro culture system and characterize factors necessary to optimize stem cell proliferation and finally, produce mature spermatozoa in recipient testes by transplanting cultured donor stem cells. Donor stem cells isolated from Rosa-26 male mice were cultured with STO feeder cells in DMEM supplemented with fetal bovine serum (FBS), SCF, LIF, bFGF, IGF-1, IL-11, L-glutamine, Sodium pyrubate, 2-Mercaptoethanol, mOSM and PDGF. To determine the presence of undifferentiated stem cells, cultured germ cells were stained by alkaline phosphatase (AP). Proliferation was assessed using BrdU, which incorporates into S-phase nuclei. Cultured cells were stained with antibodies for germ cell nuclear antigen (GCNA1) and c-kit and transplanted into testes of azoospermic busulfan treated recipient males. Donor cells formed colonies in the primary cultures (8-21 days). These cultured colonies were maintained for 4 weeks or longer without subculture and proliferated for up to eight passages over a period of 3 months. These colonies had AP activity and BrdU incorporation indicating proliferation activity. Also, these colonies were positively stained with antibody for GCNA1 and c-kit. Cultured cells transplanted into testes were identified by X-gal staining and histological screening. Germ cells cultured with this system showed enhanced colonization versus controls indicating these cells were proliferating in culture. This system provides an improved method for culturing germ cells and may be useful in gene therapy and the production of transgenic animals. (Grant number HD35494 from NICHD) KEY WORDS: spermatogonial stem cells, in vitro culture, transplantation, spermatozoa |
