Keratinocyte Differentiation from iPSCs

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.