Directed Differentiation of iPSCs to NSCs

Directed differentiation of patient-derived iPSCs into NSCs was performed according to a previous report [24 (link)] with modifications: Briefly, patient-derived iPSCs were seeded onto Geltrex-coated dishes and were maintained in mTeSR1 medium at 37 °C under humidified atmosphere of 5 % CO₂. After 3 days in adherent culture, culture medium was switched to NSC induction medium [1:1 mixture of DMEM/F12 (Gibco) and Neurobasal medium (Gibco) supplemented with 1 × N2 (Gibco), 1 × B27 minus vitamin A (Gibco), 1 × GlutaMAX (Gibco), 100 units/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco), 10 μM SB431542 (Wako), 100 nM LDN193189 (Wako), 20 ng/mL EGF (Peprotech), 20 ng/mL bFGF (Peprotech)], and patient-derived iPSCs were maintained at 37 °C under humidified atmosphere of 5 % CO₂. Emergent NSCs were expanded in NSC induction medium and were characterized by immunocytochemical staining. Fluorophore-conjugated primary antibody used was 5 μg/mL AlexaFluor 488-conjugated anti-Nestin (Millipore).