Differentiation of iPSCs to Neural Crest Cells

Differentiation protocol was adapted from [16 (link)]. iPSC colonies were dissociated into small clusters (50–200 cells) with 0.5 mM EDTA (Sigma Aldrich, Darmstadt, Germany #E6758). The cell clusters were transferred with neural induction media made of 1:1 DMEM/F-12 Glutamax (Gibco #10565-018, Waltham, MA USA) media supplemented with N2 (Gibco #17502048) and Neurobasal media (Gibco #21103-049) supplemented with B27 (Gibco #17504044), 1% Penicillin/Streptomycin, 5 μg/mL Insulin (Sigma Aldrich #I6634), 20 ng/mL bFGF (PeproTech #100-18B, Rocky Hill, NJ, USA) and 20 ng/mL EGF (Sigma Aldrich #E9644) into an ultra-low attachment culture dish (Corning #CLS3261, Corning, NY, USA). Media were changed every day for 12 days till the neuroepithelial rosettes formed. The mature neuroectodermal spheres were then plated on a 1 μg/mL fibronectin (Sigma Aldrich #F1141) coated dish where the spheres attached, and migratory neural crest-like cells emerged.