Dmem f12 growth medium

Isolation, culture, and assays of NSCs were carried out as previously described [25 (link)]. Briefly, NSCs were isolated from the mouse SVZ and grown for 10 days in DMEM/F12 growth medium (Sigma) in the presence of EGF (20 ng/mL, Sigma) and FGF-2 (20 ng/mL, Gibco). Primary neurospheres after being counted, were treated with accutase (Sigma) for 5 min, mechanically dissociated to a single-cell suspension and re-plated in growth medium containing EGF and FGF for 10 days (secondary neurospheres, 2^ry). Serial passage experiment was done repeating this methodology up to seven passages (7^ry neurospheres).

Lund human mesencephalic cells were cultured as described previously (Höllerhage et al., 2017 ). Briefly, cells were plated in T75 flasks (EasYFlasks, Nunclon DELTA, Thermo Fisher Scientific, Waltham, MA, United States) coated with 50 μg/mL poly-L-ornithine (Sigma-Aldrich, St. Louis, MO, United States) in DMEM/F12 growth medium (Sigma-Aldrich) with 1% N2-supplement (Life Technologies, Carlsbad, CA, United States) and 0.04 μg/mL human basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill, CT, United States). Multi-well dishes and flasks (Nunc MicroWell plates, Thermo Fisher Scientific, Waltham, MA, United States) were coated with 50 μg/mL poly-L-ornithine (Sigma-Aldrich) at 37°C overnight and washed three times with phosphate-buffered saline (PBS; LifeTechnologies) followed by coating with 5 μg/mL fibronectin (Sigma-Aldrich) for 24 h in the incubator (37°C, 5% CO₂). For experiments, cells were plated at a density of 110,000 cells/cm² in differentiation medium [DMEM/F12 with 1% N2-supplement, 1 μg/mL tetracycline, 0.49 mg/mL dibutyryl cyclic-AMP (Sigma-Aldrich), and 2 ng/mL human glial cell-derived neurotrophic factor (GDNF; R&D Systems, Minneapolis, MN, United States)]. Cells were routinely tested for mycoplasma contamination.