ln vitro culture of CD9- and α6-integrin-expressing cells enriched by magnetic cell sorting from cryptorchid adult and pup testes in mice

Recently, studies on cell surface markers of spermatogonia in combination with germ cell transplantation technique have made possible their functional analysis and the germline stem cells(GS cell)have established. GS cells are downstream of the stem cells such as ES cells and embryonic germ cells, namely EG cells, which are derived from PGCs. Therefore, GS cells are expected to be useful for the production of genetically-modified animals. In this study, we examined enrichment of GS cells expressing CD9 or α6-integrin from C57BL/6J cryptorchid adult and ICR pup(6-8 dpp)testes by magnetic cell sorting(MACS)and cultivation of those cells in vitro. Flow cytometric analyses demonstrated that MACS effectively enriched CD9-positive(CD9)and α6-integrin-positive (α6-integrin)cells most effectively from pup testis. Therefore, CD9 and α6-integrin cells from pup testes were used for the following cultivation. Cells proliferated in the first few days in suspension, subsequently attached to the culture plates and formed colonies after 3-5 days of culture. Those GS-like cells were passaged on the feeder layers and showed continuous proliferation for more than 2 months. Immunocytochemical and FCM analyses demonstrated that those cells maintained the expression of CD9, α6-integrin and Oct4. These findings indicate that the CD9 and α6-integrin cells collected from mouse pup testes have GS cell properties. Now, transplantation of GS-like cells into testis of recipient W mice which lack spermatogenesis is under way to evaluate their ability of spermatogenesis.