Differentiation of Excitatory Neurons from iPSCs

The iPSC cultures were maintained in NutriStem hPSC XF Medium (Biological Industries, 01-0005). When iPSCs reached confluency, the cells were differentiated into excitatory neurons on Geltrex (ThermoFisher, A1413202) coated plates. Cells were maintained in N2/B27 media containing 485 mL Neurobasal medium (Life Technologies), 5 mL N2 (Gibco, 17502001), 5 mL Glutamax (ThermoFisher Scientific), 5 mL penicillin–streptomycin), 10 mL B27 supplement (Gibco, 17504044). For the first 7 days, the medium was supplemented with 10 μM SB431542 (Sigma-Aldrich, S4317), 1 μM dorsomorphin (Sigma-Aldrich, P5499) and 100 nM LDN193189 (Sigma-Aldrich, SML0559). On days 8–29, the N2/B27 media was changed daily, without supplementing the media with SB431542, dorsomorphin or LDN193189. On day 30, cells were transitioned in Brainphys Neuronal Medium (Stemcell Technologies, 05790) supplemented with B-27, with media change twice weekly. Methods for differentiation of cerebral organoids and extraction of RNA had been described in our previous studies [19 (link), 20 (link)].