hiPSCs were differentiated into neurons as described57,58 with modifications. hiPSCs were treated with collagenase IV (Stem Cell Technologies) and grown in suspension as embryoid bodies in hES medium without bFGF. The medium was replaced every day for 5 days. On day 5, embryoid bodies were grown in Neural Induction Medium containing Dulbecco’s modified Eagle’s medium/F12 and Neurobasal Medium (1:1, Thermo Fisher), 1% N2 Supplement (Life Technologies), 1% B27 Supplement (Life Technologies), nonessential amino acids, and 0.5% penicillin/streptomycin (Life Technologies) supplemented with inhibitors of the TGF-β receptor (SB431542, Stemgent; 5 µM) and the bone morphogenetic protein receptor (LDN-193189, Stemgent; 0.25 µM). On day 7, spheres were transferred to wells coated with Matrigel (BD Biosciences) and grown in Neural Progenitor Medium, which consisted of Neural Induction Medium without SB and LDN, but containing 10 ng/ml bFGF (PeproTech), 10 ng/mL epidermal growth factor (EGF) (PeproTech), and 2 µg/ml heparin (Sigma). Attached neuroepithelial cells were fed every other day for 7 days. Neural rosette structures appeared by around day 15, were lifted off the plate, and grown in suspension in Neural Progenitor Medium to
