NSC isolation and oligodendrocyte differentiation.

Mouse NSCs were isolated using neural tissue dissociation kits according to the manufacturer’s instructions (Miltenyi Biotec). In brief, whole Nestin-CreER^T2 Qk^fl/fl mouse forebrains from postnatal day 1 pups (both sexes) were dissociated enzymatically and the single-cell suspension was cultured in NeuroCult Basal Medium (Stemcell Technologies), supplemented with 20 ng/mL epidermal growth factor (ProteinTech), 10 ng/mL basic fibroblast growth factor (ProteinTech), NeuroCult Proliferation Supplement (Stemcell Technologies), 50 units/mL penicillin G, and 50 μg/mL streptomycin in a humidified 37°C incubator with an atmosphere containing 5% CO₂. The NSCs were exposed to 100 nM 4-hydroxytamoxifen (Sigma-Aldrich) twice at an interval of 2 days to knockout Qk. To induce oligodendrocyte differentiation, NSCs were cultured on dishes coated with poly-L-ornithine (Sigma-Aldrich) and laminin (Thermo Fisher Scientific) and maintained in neurobasal medium (Thermo Fisher Scientific) containing B-27 (Thermo Fisher Scientific), 2 mM GlutaMAX-I (Thermo Fisher Scientific), 100 ng/mL insulin-like growth factor 1 (IGF-1) (Peprotech), 50 ng/mL 3,3′,5-triiodo-L-thyronine (T3) (Cayman Chemical), 50 units/mL penicillin G, and 50 μg/mL streptomycin. IGF-1 and T3 were added daily, and the differentiation medium was replaced with fresh medium every other day.