Mouse glial cell line derived neurotrophic factor gdnf

The cells used in this study were an SSC line that we had established previously 13 (link), 20 . The SSC culture system employed a mitotically inactivated STO (SIM mouse embryo-derived thioguanine and ouabainresistant feeder) feeder layer. STO cells were cultured in DMEM supplemented with fetal bovine serum (FBS, Life Technologies), 2 mM glutamine (Amresco), 100 µg/ml penicillin (Amresco), and 100 µg/ml streptomycin (Amresco) (STO culture medium). The culture medium for SSCs was alpha-minimum essential medium supplemented with 1 mM sodium pyruvate (Amresco), 10% fetal bovine serum (FBS, Life Technologies), 10 ng/ml mouse glial cell line‐derived neurotrophic factor (GDNF) (PeproTech), 1 mM non-essential amino acids (Invitrogen), 2 mM L-glutamine (Amresco), 0.1 mM β-mercaptoethanol (Biotech), 10 ng/ml leukemia inhibitory factor (Santa Cruz Biotechnology, CA, USA), 20 µg/ml transferrin (Sigma), 60 µM putrescine, 20 ng/ml mouse epidermal growth factor (PeproTech), 5 µg/ml insulin, 30 mg/l penicillin (Amresco), 75 mg/l streptomycin (Amresco) (SSC medium), and 10 ng/ml human basic fibroblast growth factor (PeproTech). The medium was changed every 2-3 days, and cells were subcultured at 1:2 or 1:3 ratios by enzymatic digestion every 5-7 days. The cells were maintained at 37°C in 5% CO₂20 .