Figure 3
Evaluation of the proliferative activity of recombinant pGDNF on murine SSCs. (A,B) Measurement of GDNF concentration in the culture media of pGDNF-293 and hGDNF-293 cells by western blot analysis. The culture media (10 μL) of pGDNF-293, hGDNF-293 and non-transfected 293 cells and purified recombinant hGDNF (1, 3 and 5 ng) for preparing a calibration curve were electrophoresed. A calibration curve was prepared by the gray values of the purified hGDNF. The amounts of GDNF in the culture media of pGDNF-293 and hGDNF-293 were calculated to be 4.3 and 4.9 ng, respectively. (C) Proliferation of mouse clump-forming germ cells enriched for SSCs by pGDNF. Cells were seeded at 2 × 105 cells/mL/well in a 24-well plate and cultured with pGDNF (supernatant), hGDNF (supernatant) or purified recombinant hGDNF (standard, 1, 3, 10 and 30 ng/mL) for 7 days. The supernatants (30 μL) of pGDNF-293, hGDNF-293 or non-transfected 293 cells were added to 1 mL of SFM supplemented with FGF2. Data are shown as mean ± SEM (n = 3). Different letters indicate significant differences (P < 0.01 except for a vs. b = P < 0.05). (D) Phase contrast images of mouse SSCs cultured with pGDNF or purified recombinant hGDNF. Mouse SSCs formed cellular clumps and proliferated in the presence of GDNF, while no cell proliferation was observed in the absence of GDNF. Bars = 100 µm. (E) Initiation and continuous proliferation of germ cell clumps by pGDNF. The cell numbers of clump-forming germ cells cultured from freshly isolated testis cells with pGDNF or purified recombinant hGDNF are indicated. (F) Antigenic profile of clump-forming germ cells cultured with pGDNF or hGDNF. The surface phenotypes of cells cultured in either condition were THY1lo ITGA6+ ITGAVlo KIT−.
