Fgf 2

hiPSCs (414C2 line) were cultured for 12 days in adhesion cultures with mouse embryonic fibroblasts and then allowed to form embryonic bodies in floating culture for 30 days. Aggregated cells were differentiated into NS/PCs derived from hiPSC-NS/PCs using various factors during each day of the incubation period (Okada et al., 2008 (link)).
hiPSCs (201B7 line) were pretreated for 6 days with 3 μM SB431542 (Tocris, 301836-41-9) and 150 nM LDN193189 (StemRD, 1062368-24-4). The cells were then dissociated and seeded at a density of 1×10⁵ cells per milliliter in ultra-low-attachment culture dishes (Corning) in neuronal induction medium consisting of medium hormone mix (MHM) (Okada et al., 2008 (link)) supplemented with 2% B27 supplement without vitamin A (Thermo Fisher, 17504-044), 20 ng/mL FGF-2, 10 μM Y27632 (Nacalai Tesque, 08945-71), 1 μM retinoic acid (RA; Sigma, R2625-1G), 3 μM CHIR 99021 (Reprocell, 04-0004) and 10 μM SB431542 (Calbiochem, 301836-41-9) in a hypoxic and humidified atmosphere (4% O₂, 5% CO₂) for 6 days. The formed neurospheres were passaged by dissociation into single cells and then cultured in slightly modified neuronal induction medium, MHM supplemented with 2% B27 without vitamin A, 20 ng/mL FGF-2, 10 μM Y27632, and 1 μM RA for 6 days under 4% O₂ (hypoxic) conditions.

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.