Fig. 2

Sheet formation and characterization of keratinocytes derived from T21- and D21-iPSC. a Schematic diagram of the experimental design. Keratinocytes derived from iPSCs were cultivated under culture condition A followed by condition B for the purpose of propagation of the lifespan. As the growth rate decreased, the culture conditions were changed from A to B. In the culture condition A, keratinocytes were maintained in DKSFM supplemented with 20 ng/mL EGF and 10 μM Y-27632 at a plate coated with type I collagen and fibronectin. In culture condition B, keratinocytes were cultured in the ESTEM-EP medium supplemented with 10 μM Y-27632 on irradiated mouse embryo fibroblasts (MEF). b Phase-contrast photomicrographs of keratinocytes derived from T21-KC at passage 4, D21-KC#1 at passage 5, and D21-KC#2 at passage 4. c Thin section of T21-KC at day 42 in iPGell. Hematoxylin and eosin (HE) stain. d Thin section of D21-KC#1 at day 50 and D21-KC#2 at day 48 in iPGell. HE stain. e Fabrication of D21-KC#1 epithelial sheet after dispase treatment. (Left panel) Histology of the sheet. HE stain. Scale bar, 100 μm. (Right panel) Macroscopic view of the sheet. Scale bar, 1 cm. f Immunohistochemical analysis of skin, HDK1-K4DT, T21-KCs at day 42, D21-KC#1 at day 50, and D21-KC#2 at day 48 with antibodies to pan-cytokeratins (Pan-CK), KRT14, P63, KRT10, integrin β4, involucrin, loricrin, filaggrin, and OCT3/4. D21-KC exhibited terminal differentiation and formed structured cell sheets. The expression patterns of these markers in intact skin are shown for reference. g Real-time qPCR analysis of epithelial markers (KRT14, TP63, Involucrin) and pluripotent markers (OCT3/4, NANOG) in T21-KC at day 47, D21-KC#1 at day 49, and undifferentiated iPSCs. Values are shown as means ± SD from two or three independent experiments. *p < 0.05