iPSCs were manually dissected from MEF, then grown in suspension culture for 4 days in EB medium supplemented with 2 μM dorsomorphin (R&D Systems, 3096; Minneapolis, MN, USA) and 5 μM SB431542 (R&D Systems, 1614). Medium was then changed to neural induction medium consisting of DMEM/F12, 1 × N2 supplement (Invitrogen, 17502-048), 1 × NEAA, 2 mM glutamax, 0.1 mM β-mercaptoethanol, 2 μM dorsomorphin, 5 μM SB431542 and 20 ng ml−1 FGF2 for an additional 3 days in suspension. Neurospheres were then planted on laminin (Sigma-Aldrich, L2020)-coated culture dishes for rosette formation. Rosettes were manually picked 2–3 times, dissociated in Accutase then re-plated on poly-D-ornithine-laminin-coated substrates for neuronal differentiation in medium composed of neural basal medium (Invitrogen, 21103–049), 1 × B27 supplement (Invitrogen, 17504–044), 1 mM glutamine, 1% NEAA and 1 × Penicillin/Streptomycin for an additional 4, 8 or 12 weeks. Medium was changed every other day. To determine whether dorsal–ventral fate could be respecified, cells were grown with the Smoothened agonist (Hedgehog pathway activator) purmorphamine (1 μm; Cayman Chemical, Ann Arbor, MI, USA, 10009634), or with the dorsalizing agent lithium chloride (LiCl; 1 mM; Sigma-Aldrich). Controls were exposed to DMSO (carrier) alone.42,43
