Dksfm

The induction of differentiation into keratinocytes was carried out as previously described [19 (link)]. We subcultured small clumps of undifferentiated iPSC on VTN-coated 100-mm dish in E8 medium on day 0. iPSCs were cultured in DKSFM (Invitrogen, #10744-019) supplemented with 1 μM all-trans RA (Wako, #182-01111) and 10 ng/mL bone morphogenetic protein 4 (BMP4) (R&D systems, #314-BP-010/CF) from day 1 to day 4. After 4 days, iPSCs were maintained in DKSFM supplemented with 20 ng/mL epidermal growth factor (EGF) (R&D systems, #236-EG-200) for 10 days, then passaged to a 100-mm dish coated with 0.03 mg/mL type I collagen (Advanced Biomatrix, #5005-B) and 0.01 mg/mL fibronectin (Sigma-Aldrich, #F0895-1MG), and maintained in DKSFM supplemented with 20 ng/mL EGF and 10 μM Y-27632 (Wako, #251-00514). Cells were subcultured when approximately 80 to 90% confluence. Cells were rinsed with PBS and incubated with 0.25w/v% trypsin-1mmol/L EDTA (Wako, #209-16941) at 37 °C for 5 min for passaging. Cells were passaged at 0.3 × 10⁵ cells/cm² to a new plate coated with type I collagen and fibronectin and enriched by rapid adherence to fibronectin and type I collagen-coated dished for 15 min at 37 °C. Nonadherent cells were removed and rapidly attached cells were cultured. The medium was changed every 2 or 3 days. All cells were maintained at 37 °C in a humidified incubator with 5% CO₂.

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)).

The induction of differentiation into keratinocytes was carried out as previously described. We subcultured small clumps of undifferentiated iPSC on VTN coated 10-mm dish in E 8 medium on day 1. iPSCs were then cultured for 4 days in DKSFM (Invitrogen, #10744-019) supplemented with 1 mM all-trans RA (Wako, #182-01111) and 10 ng/mL bone morphogenetic protein 4 (BMP4) (R&D systems, #314-BP-010/CF). Subsequently, iPSC was maintained in DKSFM supplemented with 20 ng/mL EGF (R&Dsystems, #236-EG-200) for 10 days, then passaged to a 10-mm dish coated with 0.03 mg/mL type I collagen and 0.01 mg/mL fibronectin, and maintained in DKSFM supplemented with 10 μM Y-27632 (Wako, #251-00514) and 20 ng/mL EGF.