After developing and selecting the iPSC colonies, those colonies were cultured on Geltrex matrix-coated plates with E8 medium (Invitrogen). For differentiation into 2D neurons, the medium was replaced every other day by neural induction medium (Invitrogen) for seven days. On day seven, the neural stem cells were exposed to accutase (Invitrogen) for ~5 min and plated on Geltrex matrix-coated 10 cm plates with a Rock inhibitor (Thiazovivin; 1 uM) (Miltenyi Biotec, San Diego, CA). The following day the medium was replaced by neural expansion medium without Rock inhibitor for 5 days. After 5 passages in neural expansion medium, neural stem cells were plated in neural expansion medium on 6-well plates (2.5–5 x 105 cells) or 8-chamber slides (2.5–5 x104 cells) coated with poly-L-ornithine (Sigma) and Laminin (Life Technology). After two days, the medium was replaced by neuronal differentiation medium (Neurobasal medium with B27 and GlutaMAX) and changed every 3–4 days thereafter.
