Establishing Mouse Germline Stem Cell Culture

Mouse GSCs were cultured as previously reported (25 (link), 27 (link), 29 (link)). The cells were cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs; Gibco, A34962), using a medium (medium I) composed of StemPro-34 SFM medium (Thermo Fisher Scientific), StemPro-34 Supplement (Thermo Fisher Scientific), 1% fetal bovine serum (FBS), recombinant human GDNF (10 ng/ml, 450-10, Peprotech), recombinant human bFGF (10 ng/ml, 100-18B, Peprotech), recombinant human EGF (20 ng/ml, AF-100-15, Peprotech), recombinant human LIF (10 ng/ml, CYT-644, Prospec), as well as other components as previously reported (29 (link)) and described in Supplementary Table 1. The cells were refreshed every 2-3 days, and passaged every 5-7 days at a ratio of 1:4-6 on freshly plated mitotically inactivated mouse embryonic fibroblasts. The cells were maintained at 37°C in 5% CO2 in air.
As a feeder cell to support in vitro meiosis of mouse mGSCs, we used an available immortalized Sertoli cell lines SK49 (30 (link)). The cells were cultured at 37°C and 5% CO2 in Dulbecco’s Eagle’s medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 U/mL).