Fig. 1

Keratinocytic differentiation of Down syndrome-iPSCs and autonomously rescued disomy 21-iPSCs. a Protocol for keratinocytic differentiation from amniotic fluid-iPSCs. DKSFM, defined keratinocyte serum-free medium; RA, retinoic acid; BMP4, bone morphogenetic protein 4; VTN, vitronectin; E8, Essential 8 medium; EGF, epidermal growth factor. b Phase-contrast photomicrograph of normal human epidermal keratinocytes (NHEK) at passage 3, and keratinocytes derived from T21-iPSCs (T21-KC), D21-iPSC#1 (D21-KC#1) and D21-iPSC#2 (D21-KC#2) at passage 2 (day 25). T21-KC, D21-KC#1, and D21-KC#2 exhibited keratinocyte-like morphology. Scale bars, 200 μm. c Immunocytochemical analysis of T21-KC at day 28 and D21-KC#1 at day 34 with the antibodies to epithelial markers, i.e., KRT14, KRT10, involucrin and loricrin. Scale bars, 100 μm. d Immunocytochemistry with the epithelial stem cell marker KRT14. The percentage of KRT-positive cells is shown for HDK1-K4DT (control), T21-KCs at day 56, D21-KC#1 at day 55, and D21-KC#2 at day 57. Scale bars, 100 μm. **p < 0.01, ***p < 0.001, one-way ANOVA with Bonferroni correction. e Real-time qPCR analysis of OCT3/4 in T21-KCs at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. Values are shown as means ± SD from three independent experiments. f Real-time qPCR analysis of NANOG in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. Values are shown as means ± SD from two or three independent experiments. g Real-time qPCR analysis of KRT14 in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. The expression level of KRT14 was relatively high in D21-KCs, compared with T21-KCs (p-value, 0.42). Values are shown as means ± SD from three independent experiments. h Real-time qPCR analysis of TP63 in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. The expression level of TP63 was relatively high in D21-KCs, compared with T21-KCs (p-value, 0.31). Values are shown as means ± SD from three independent experiments. i Real-time qPCR analysis of Filaggrin in T21-KC at day 28, D21-KC#1 at day 28, and undifferentiated iPSCs. The expression level of Filaggrin was relatively high in T21-KCs, compared with D21-KCs (p-value, 0.34). Values are shown as means ± SD from three independent experiments. j Real-time qPCR analysis of KRT14 in D21-KC#1 at passages 2 and 3. Values are shown as means ± SD from three independent experiments. k Real-time qPCR analysis of TP63 in D21-KC#1 at passages 2 and 3. Values are shown as means ± SD from three independent experiments. l Growth of iPSC-derived keratinocytes. The total number of population doublings (PDs) was calculated using the formula [log10 (total number of harvested cells/number of plated cells)]/log10 (2). Phase-contrast photomicrograph of D21-KC#1 at senescence is shown