Keratinocyte Differentiation of Human iPSCs

Human iPSCs from RTHα patients P1 and P4 and control subjects, generated as described previously (9 (link)), were differentiated into keratinocytes using RA and BMP4 (10 (link)). Briefly, hiPSCs were seeded on dishes coated with Geltrex hESC-qualified Reduced Growth Factor Basement Membrane Matrix (Gibco) and collagen type I, 3 mg/mL solution (Advanced BioMatrix) and grown in N2B27 medium. N2B27 medium was obtained by combining DMEM/F12 and Neurobasal medium (Gibco) in a 1:1 ratio and supplemented with 0.1 mM nonessential amino acids, 1 mM glutamine, 55 μM 2-mercaptoethanol, N2 supplement (100 × ; Life Technologies), B27 supplement (50 × ; Life Technologies), 50 μg/mL ascorbic acid, 0.05% BSA, 50 U/mL penicillin/streptomycin, 100 ng/mL basic FGF (Life Technologies), and 10 μM Y27632 (Sigma-Aldrich). After 1 day, hiPSC colonies were seeded in DKSFM (Gibco) supplemented with DKSFM and 50 U/mL penicillin/streptomycin, containing 25 ng/mL of BMP4 (R&D systems) and 1 μM RA (Sigma Aldrich). On day 14 of differentiation, cells were plated on coated dishes generated by combining collagen type IV powder (Sigma-Aldrich), 0.25% glacial acetic acid, and collagen type I (Advanced BioMatrix) and grown in keratinocyte medium (11 (link)).