Ldn193189

For neural crest induction, human ESC/iPSC colonies were treated with Accutase cell detachment solution (STEMCELL Technologies) and re-suspended in induction medium plus 3 μM CHIR 99021 and 10 μM ROCK inhibitor, Y-27632 (Tocris Bioscience) plated at the 20,000 cells/cm2 density on Matrigel-coated surfaces. Induction medium contained DMEM/F12 medium supplemented with 2% B27 supplement (Gibco), 1X Glutamax (Gibco) and 0.5% bovine serum albumin (wt/vol) (Sigma-Aldrich). CHIR and ROCK inhibitor Y-27632 were added for the first two days, induction media without CHIR and ROCK inhibitor was used for next 3-days. For non-neural ectodermal differentiation, basal media with 2% B27, 1x Glutamax, and 0.5% BSA was used for 5 days (Leung et al., 2016 (link)). For neuroectodermal differentiation, DMEM/F12 medium with 2% B27 supplement, 1 × Glutamax, 100 nM LDN-193189 (Tocris) and 10 μM SB431542 (Tocris) was used for 5 days (Leung et al., 2019 (link)). For mesoderm induction, DMEM/F12 supplemented with 2.5 ng/ml FGF basic (Tocris Bioscience) with 3 μM CHIR was used for 3 days, while for endoderm induction DMEM/F12 supplemented with 1% FBS and 10 ng/ml Activin A (Tocris Bioscience) with 3 μM CHIR was used for first day and without CHIR next two days. Plating density for all differentiation were maintained at 20,000 cells/cm2. Small molecules and medium were replenished daily.