Neural induction was performed as previously reported33. Briefly, cells were dissociated to single cells using Accutase and plated on gelatin for 10 minutes to remove MEFs. Non-adherent cells were collected and plated on Geltrex-treated dishes at a density of 150-200k cells per well of a 24-well plate in the presence of MEF-conditioned hESC media containing 10 ng/ml of FGF-2 (Life Tech) and 10 uM of Y-27632 (Tocris). Neural differentiation was initiated when cells were confluent using KSR media containing 820 ml of Knockout DMEM (Life Tech), 150 ml Knockout Serum Replacement (Life Tech), 1 mM L-glutamine (Life Tech), 100 uM MEM non-essential amino acids (Life Tech), and 0.1 mM beta- mercaptoethanol (Life Tech) to inhibit SMAD signaling, 100 nM of LDN-193189 (Cat. no. ab142186, Abcam) and 5 uM of SB431542 (Cat. No. 13031, Cayman Chemical) were added on Days 0 through 9. Cells were fed daily, and N2 media (Life Tech) was added in increasing 25% increments every other day starting on Day 4 (100% N2 on Day 10).
