Efficient Neural Progenitor Cell Generation

The procedure to generate neural progenitor cells (NPCs) was adapted from Reinhardt et al. [49 (link)] and is described in detail in Grochowska et al. [11 (link)]. Briefly, iPSCs were detached and cultured as embryoid bodies in suspension in a shaker. Neural induction was initiated by promoting Wnt and Shh signalling with the addition of SB431542 (Tocris, 1614), dorsomorphin (Abcam, ab120843), CHIR (Sigma-Aldrich, SML1046), and Purmorphamine (Stem Cell Technologies, 72,202). Embryoid bodies showing neuroepithelial development were selected, dissociated, and plated on Matrigel® (Corning, 356238)-coated plates as NPCs. From the sixth passage, NPCs were kept in NPC medium, consisting of N2B27 medium (DMEM/F-12—Neurobasal in 1:1 ratio, 1% B27 w/o Vitamin A, 0.5% N2, 1% Penicillin–Streptomycin, all from Thermo Fisher Scientific) supplemented with 3 μM CHIR, 200 μM ascorbic acid (AA, Sigma-Aldrich, A92902), and 0.5 μM Smoothened Agonist (SAG, Abcam, ab142160). Cells were kept on Matrigel-coated plates, refreshed every other day, and split with accutase (Sigma-Aldrich) in a 1:10–1:20 ratio.