Human iPSC/iPSC‐derived NSCs were provided by IxCell Biotechnology, Ltd. The hiPSCs were cultured under feeder‐free culture conditions on Matrigel (Corning)‐coated dishes in mTeSR1 Medium (Stem cell technologies). Passaging was performed enzymatically using Accutase (Gibco) by splitting colonies in clumps every 6‐7 days followed by replating on Matrigel‐coated dishes. Medium were changed every day.
The human iPSC‐derived NSC cells were maintained as adherent culture in 50% DMEM‐F12 and 50% Neurobasal‐A, containing 1× N2 supplement, 1× B27 supplement (Minus Vitamin A), 1× NEAA, 1× Glutamax, 10 ng/mL FGF‐Basic (AA10‐155) Recombinant Human Protein (basic fibroblast growth factor—bFGF, Gibco), 10 ng/mL LIF Recombinant Human Protein (hlif, Gibco), 3 μМ CHIR99021 (Selleckchem), 5 μМ SB431542 (Selleckchem), and 200 μМ L‐Ascorbic acid 2‐phosphate sesquimagnesium salt hydrate (Sigma). For neurospheres assays, cells were cultured in DMEM‐F12 with 1× B27 supplement, 20 ng/mL Recombinant Human Protein (EGF, Gibco), 20 ng/mL bFGF, and 10 ng/mL hlif using low‐attachment culture dishes (Corning).
SK‐N‐SH were purchased from ATCC and cultured in Modified Eagle's Medium (MEM) with 10% (wt/vol) heat‐inactivated fetal bovine serum. All cells were maintained at 37°C in humidified air with 5% CO2.
The human iPSC‐derived NSC cells were maintained as adherent culture in 50% DMEM‐F12 and 50% Neurobasal‐A, containing 1× N2 supplement, 1× B27 supplement (Minus Vitamin A), 1× NEAA, 1× Glutamax, 10 ng/mL FGF‐Basic (AA10‐155) Recombinant Human Protein (basic fibroblast growth factor—bFGF, Gibco), 10 ng/mL LIF Recombinant Human Protein (hlif, Gibco), 3 μМ CHIR99021 (Selleckchem), 5 μМ SB431542 (Selleckchem), and 200 μМ L‐Ascorbic acid 2‐phosphate sesquimagnesium salt hydrate (Sigma). For neurospheres assays, cells were cultured in DMEM‐F12 with 1× B27 supplement, 20 ng/mL Recombinant Human Protein (EGF, Gibco), 20 ng/mL bFGF, and 10 ng/mL hlif using low‐attachment culture dishes (Corning).
SK‐N‐SH were purchased from ATCC and cultured in Modified Eagle's Medium (MEM) with 10% (wt/vol) heat‐inactivated fetal bovine serum. All cells were maintained at 37°C in humidified air with 5% CO2.