Porcine Spermatogonial Stem Cell Culture

The cell line used in this study was the immortalized porcine SSC line established by us [31 (link)]. The cell line was characterized by immunofluorescence staining of different SSC markers. The cells were cultured in DMEM/high glucose (Gibco, Grand Island, NY, USA) containing 5% fetal bovine serum (Gibco, Mesenchymal Stem Cell FBS Qualified), 5% knockout serum replacement (KSR; Gibco), 100 unit/mL penicillin and streptomycin (Hyclone, Logan, Utah), 1 × MEM vitamin solution (Gibco), 1× non-essential amino acid (NEAA; Gibco), 2 mmol/L Glutamax (Gibco), 40 ng/mL recombinant human GFRA1 (BioLegend, San Diego, CA, USA), 20 ng/mL recombinant human GDNF (Peprotech, Rocky Hill, NJ, USA) and 10 ng/mL recombinant human bFGF (Peprotech). The cells were maintained at 37 ℃ in a humidified incubator containing 5% CO₂.