Murine Stem Cell Differentiation

Cells were isolated from 20 CP-treated mice for experiment. Cells were seeded in plates of 24 wells. Each well contained 200,000 cells/500 µl suspended in media-containing StemPro (33%) (Gibco, Waltham, MA, USA), KnockOut serum replacement-KSR (25%) (Gibco, USA), growth factors such as human rEGF (recombinant epidermal growth factor) (20 ng/mL) (Biolegend, San Diego, CA, USA), human rGDNF (glial cell line derived nerve growth factor) (10 ng/mL) (Biolegend), human rLIF (leukemia inhibitory factor) (10 ng/mL) (Biolegend), and human r-bFGF (basic fibroblast growth factor) (10 ng/mL) (Biolegend), penicillin-streptomycin (pen-strep, 1%), and methylcellulose (MC; 42%) (R&D, McKinley Place NE, Minneapolis, MN, USA) as described by AbuMadighem et al., 2018 [46 (link)]. From the beginning of the cultures we added follicle stimulating hormone (FSH) (7.5 IU/mL) (Serono, Geneva, Switzerland) or interleukin 1 alpha (IL-1α) (20 pg/mL) (BioLegend) or tumor necrosis factor alpha (TNF-α) (20 pg/mL) (BioLegend). However, testosterone (T) (10⁻⁷ M) (Bayer, Hod Hasharon, Israel) alone or in combination with other factors (FSH + T, IL-1α + T or TNFα + T) was added at the last week of the culture. The above-mentioned growth factors were just added every 7–10 days in a ×10 concentration (50 μL/well) to the cultures. The cells were usually cultured in MCS for 4–5 weeks.