Efficient Generation of Neural Stem Cells

For the generation of NPCs, we followed a previously published protocol^{19 (link)}. Briefly, hiPSC colonies were gently disaggregated from the culture plate and plated for 6 hours in non-adherent conditions in DMEM/F12 (Gibco/Invitrogen), 2% B27, 1% N2, 10 μM Y-27632 (Miltenyl-Biotech), 100 nM LDN-193189 (120-10 C; PeproTech), 10 μM SB431542 (S4317-5MG; Sigma-Aldrich) and 2 ng/ml bFGF. Cells were then plated for 10 days on Matrigel-coated dishes in this medium before being detached with accutase and re-plated on Matrigel-coated dishes and cultured in neural induction medium: 1:1 DMEM/F12:neurobasal medium supplemented with 2% B27, 1% N2, 1% ultraglutamine, 10 ng/ml Epidermal Growth Factor (EGF; AF-100-15 Peprotech) and 10 ng/ml bFGF. Culturing cells in this neural induction medium generates homogenous cultures of NSCs (>95% of the cells).