P3655

Culture plates were pre-covered with poly-L-ornithine (Sigma P3655) and laminin (Thermo Fisher 23017015) for hGSC culture and passage as previously described (Han et al., 2017 (link)). In brief, each 100 mm dish was treated with 10 ml 0.5 μg/ml poly-L-ornithine (10 mg/ml stock concentration dissolved in water) and maintained at room temperature for 16 h on a flat, clean tabletop. The following day, 1 × PBS (Hyclone SH30258.01) was used applied to wash the dishes after the poly-L-ornithine supernatant was discarded. Then, 10 ml 1 × PBS containing 5 μg/ml laminin was added to the dish and was left to keep infiltrating for at least 16 h at room temperature. Finally, the coated dishes were stored at −20°C. Before cell culture, the coated dishes were incubated at 37°C, and the supernatant was discarded.

Motor neurons were derived from iPSC lines following a monolayer-based differentiation protocol^{81 (link)}. iPSCs were plated on Geltrex and cultured in mTeSR1 medium until confluent. Then, we initiated the differentiation induction with neuron differentiation media (DMEM/F12:Neurobasal 1:1 (ThermoFisher, 11330057 and 21103049) supplemented with non-essential amino acids, glutamax (ThermoFisher, 35050038), B27 (ThermoFisher, 12587010), and N2 (ThermoFisher, 17502048). From day 0 to day 6, neuron differentiation media was supplemented with 1 μM retinoic acid (Sigma-Aldrich, R2625), 1 μM smoothened agonist (Sigma-Aldrich, 566661), 0.1 μM LDN-193189 (Miltenyi Biotech, 130-103-925), and 10 μM SB-431542 (Miltenyi Biotech, 130-105-336). From day 7 to day 14, neuron differentiation media was supplemented with 1 μM retinoic acid, 1 μM smoothened agonist, 4 μM SU-5402 (Sigma-Aldrich, SML0443), and 5 μM DAPT (Sigma-Aldrich, D5942). Motor neurons were split and plated on poly-L-ornithine (Sigma-Aldrich, P3655) and laminin-coated (ThermoFisher, 23017015) plates containing neurobasal media supplemented with non-essential amino acids, glutamax, N2, B27, and neurotrophic factors 10 ng ml⁻¹ BDNF (Biozol, 450-02) and 10 ng ml⁻¹ GDNF (Biozol, 450-10).