Recently, studies on cell surface markers of spermatogonia in combination with germ cell transplantation techniques have made possible the functional analysis of germline stem cells (GS cells). The GS cells are downstream of the stem cells such as ES cells and embryonic germ cells (EG cells), which are derived from primordial germ cells (PGCs). Therefore, GS cells are expected to be useful in the production of genetically modified animals. In this study, we examined the enrichment and cultivation of mouse GS cells by magnetic cell sorting (MACS). Testicular cell suspensions were collected from C57BL/6J cryptorchid adult testes at 2 to 3 months after surgery and ICR pup (6 to 8 dpp) testes. They were digested by 0.1% collagenase followed by 0.25% trypsin with gentle shaking. Dissociated cell suspensions were filtrated through a glass-wool column followed by a Falcon cell strainer (40-�m mesh). They were then treated with biotin-conjugated anti-mouse CD9 antibody, whose antigen, CD9, is localized on the GS cell surface, followed by the streptavidin-microbeads treatment. The cell suspension was passed through a MACS-separation column. In Experiment I, MACS-treated fractions were analyzed by flow cytometry (FCM) on the rates of recovery and enrichment and their cellular characteristics. In Experiment II, CD9-positive (CD9+) cells were cultured on gelatin-coated MultiDish (176740, Nunc) with 4-5 � 105 cells/well in StemPro34-SFM supplemented with 1% fetal bovine serum, leukemia inhibitory factor, GDNF, bFGF, EGF, insulin, transferrin, putrescine, MEM vitamin solution, MEM-NEAA and some other reagents at 32�C or 37�C under 5% CO2 in air. They were examined for their proliferation and cytological changes such as CD9, �6-integrin and Oct-1 expression by immunohistochemistry. In Experiment I, MACS selection effectively enriched CD9+ cells from mouse testes. However, FCM analysis revealed that the CD9-negative (CD9-) cells partially remained in MACS-selected fraction from cryptorchid adult testes. In contrast, the CD9+ subpopulation could be successfully separated from CD9- subpopulation from pup testes. Therefore CD9+ subpopulation from pup testes was used for the following cultivation. In Experiment II, the cells proliferated in the first few days in suspension. Then they attached to the dish and formed colonies after 5 days or 3 days of culture at 32�C or 37�C, respectively. Immunohistochemical analysis showed that the cells maintained the expression of CD9 for at least 14 days, but their expression of �6-integrin gradually diminished. It was demonstrated by immunohistochemistry and FCM analysis that the cells in colonies expressed Oct-1, and its expression level was stronger in culture at 37�C than at 32�C. These findings indicate that the CD9+ cells collected from mouse pup testes have stem cell properties.
This work was supported by the Wakayama Prefecture Collaboration of Regional Entities for the Advanced Technological Excellence, JST; by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan; and by a Grant-in Aid for Scientific Research from the Japan Society for the Promotion of Science.
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