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The spermatogonial-enriched cell fraction was seeded on laminin-coated plates and three different base media were evaluated after 7 days in culture, namely, MEMα, DMEM/F-12, and StemPro™-34 SFM. StemPro™-34 SFM medium had the highest colony formation, increased number of cells, and was chosen as the medium for continued use in the current study (data not shown). The medium was supplemented with human forms of growth factors: human glial cell line-derived neurotrophic factor (hGDNF) (20 ng/ml; R&D Systems), human fibroblast growth factor 2 (hFGF2) (1 μg/ml; BD Biosciences), human leukemia inhibiting factor (hLIF) (10 ng/ml; R&D Systems), and human stromal-derived factor 1 (hSDF-1) (10 ng/ml; PeproTech, Inc.). The cells at a concentration of 0.02 × 10⁶ cells per well in the StemPro™-34 SFM culture medium (Table 1) were seeded on 96-well laminin-coated plates in the incubator with an air atmosphere containing 5% CO₂ at 37°C and the media changed every other day. The serum-free medium was conditioned by incubating goat fetal fibroblast monolayer of cells overnight and then passing through a 0.2-μm filter Oatley and Oatley (16 ). The cells were passaged after 7 days. The colony growth was monitored for up to 45 days and images were taken using the ZEISS Axio Vert.A1 inverted microscope.

Mouse SSC cultures were established as described previously^{10 (link),31 (link),57 (link)} with minor modifications. In brief, murine testicular cells were prepared from 5-day-old GFP mice (C57BL/6-Tg (CAG-EGFP), Japan SLC, Shizuoka, Japan) by enzymatic digestion using trypsin-EDTA, and cultured on SNL 76/7 STO cell (a kind gift from Dr. A. Bradley, Baylor College of Medicine, USA) feeders in SFM with 10 ng/mL hGDNF (R&D Systems, Minneapolis, MN, USA) and 0.5 ng/mL human FGF2 (Corning). In some experiments, 30 μL/mL of pGDNF were used instead of hGDNF. One day after seeding, weakly attached spermatogonia were collected and cultured on fresh feeders. Medium was changed every other day, and the cells were subcultured every 5–7 days as described previously^{31 (link)}.