Penicillin-Streptomycin, 100 nM LDN193189 (Reagents Direct), 10 μM SB431542 (StemRD), 2 μM XAV939 (TOCRIS bioscience). Medium was changed daily. On D5, the medium was replaced with 75% NIMX media, 25% N2 medium (DMEM/F12 (1:1), N-2 supplement, 25% (w/v) dextrose (Sigma Aldrich), 55 μM 2-Mercaptoethanol, Penicillin-Streptomycin). On D7, cells were fed with a 50/50 NIMX/N2 medium, on D9, cells were fed with 25% NIMX medium. On D10, cells were dissociated into a single cell suspension with Accutase and plated onto matrigel-coated 24-well plates in 25% NIMX supplemented with 2 μM thiasovivin at a density of 1.3 × 106/cm2. On D11 the medium was replaced with N2/B27 medium (N2 medium (above) + B-27 supplement) containing Purmorphamine (Stemgent) and Sonic Hedgehog (R&D Systems). On D19-23, cells were fed daily with N2/B27 medium without Shh and purmorphamine.
