hESCs (WA-09, HES-3 (NKX2.1::GFP) and iPSCs (C72, SeV6) were maintained on mouse embryonic fibroblasts (MEFs) and dissociated with Accutase (Innovative Cell Technologies) for differentiation or dispase for passaging (Chambers et al., 2009). Differentiation media were described previously (Kriks et al., 2011): KSR- and N2 medium for neural induction, and Neurobasal medium + B27 (Gibco) and N2 supplements (Invitrogen) for neuronal differentiation. Small molecule compounds: XAV939 (2 μM; Stemgent;), LDN193189 (100 nM; Stemgent), SB431542 (10 μM; Tocris), and Purmorphamine (1–2 μM; Calbiochem), ascorbic acid (200 μM) and dibutyryl-cyclic AMP (dbcAMP, 200 μM; both from Sigma). Recombinant growth factors: SHH (C25II; 50–500 ng/ml), Noggin (125 ng/ml), DKK1 (250 ng/ml), BDNF (10 ng/ml; all R&D Systems) and FGF2 (5–10 ng/ml; Promega).
