Neural Induction of Human iPSCs

For neural induction, hiPSC colonies were detached with Accutase (Life Technologies), suspended as single cells, and plated on 0.1% gelatin-coated wells (Sigma-Aldrich) for 1 h to allow MEFs to attach. Floating iPSCs were then collected and plated on Matrigel (BD Pharmingen)-coated dishes (50,000 cells/cm²) in MEF preconditioned iPSCM in the presence of 10 μM of the Rho kinase inhibitor Y-27632. When the cell culture reached ≈90% confluence (usually 2 d after plating), the culture medium was replaced with KO serum replacement medium supplemented with 200 ng/ml of rhNOGGIN (R&D) and 10 μM of SB431542 (Sigma-Aldrich). The medium was changed daily for the next 3 d. Thereafter, it was switched every other day to gradually expose the cells to increasing (1:3, 1:1, 3:1) ratios of NSC/KO serum replacement medium. 2 d after the final switch, the cells were detached using Accutase and plated on Matrigel-coated dishes in neural stem cell medium (NSCM) supplemented with 20 ng/ml basic fibroblast growth factor and 20 ng/ml EGF in the presence of 10 μM of the Rho kinase inhibitor Y-27632, according to the published Dual Smad inhibition protocol (Chambers et al., 2009 (link)).