Maintenance and Differentiation of Human iPSCs and Neural Cells

The hiPSC line 1210B2 was maintained as described previously^{51 (link),52 (link)}. The hiPSCs were dissociated using 0.5× TrypLE Select (Thermo Fisher) and plated on iMatrix511 (Nippi)-coated dishes in StemFit (AK03N) medium at feeder-free condition. The hiPSC-derived NECs were established and maintained as described previously^{53 (link),54 (link)}. The hNECs were passaged every 4 days and plated at a ratio of 1:5. The cells were dissociated using TrypLE Select (Thermo Fisher) and plated on Matrigel (Corning)-coated dishes in RHB-A medium (Takara) supplemented with 10 ng/mL EGF (Peprotech) and 10 ng/mL FGF2 (Peprotech). The hiPSC-derived NCCs were established previously and passaged less than three times for analysis. The hNCC medium consisted of 1:1 neurobasal medium (Thermo Fisher Scientific) and D-MEM/Ham’s F-12 (Wako) medium containing 1× GlutaMax (Thermo Fisher Scientific), 0.5× GEM 21 NeuroPlex serum-free supplement (Gemini Bio Products), 0.5× N2 supplement (Thermo Fisher Scientific), 5 mg/mL insulin (Sigma-Aldrich), 0.5% penicillin and streptomycin (Nacalai tesque), 20 ng/mL FGF2 and 20 ng/mL EGF. The hNCCs were dissociated using TrypLE Select and plated on fibronectin (Sigma-Aldrich)-coated dishes.