Fig. 2

Generation and characterization of iPSC-derived EpSCs. a Schematic of the EpSC differentiation showing the presence of retinoic acid, BMP-4, and EGF at different times. b Morphology of cells at different stages of differentiation: iPSCs, high-density polygonal morphology of cells differentiated for 11 days, and paving stone morphology of cells differentiated for 18 days. Scale bar, 50 μm. c Percentage of positive cells were 20.73 ± 7.26%, 20.05 ± 6.02%, 18.57 ± 4.59%, 17.71 ± 6.19%, 17.90 ± 4.42%, 22.94 ± 6.37%, and 0.01% ± 0.01% for ITGA6, CD200, Krt14, Krt15, ITGB1, Krt19, and NANOG respectively (n = 5, mean ± SD). d Immunofluorescence analysis showing positive expression of CD200, Krt15, Krt19, ITGB1, and ITGA6 and negative expression of NANOG in differentiated cells in comparison with iPSCs. Scale bar, 30 μm. e–g RT-PCR analyses of epithelial stem cell-related genes (LGR5, LGR6, TCF4, FZD2, DDK3, CTNNB1, Krt14, LEF1, and LHX2) (e), hair follicle stem cell-related genes (CD200, Krt15, Krt19, and ITGA6) (f), and pluripotent genes (NANOG, OCT4, and REOX1) (g) in cells induced for 11 days compared with control hair follicle stem cells (hHFSCs) and iPSCs. The housekeeping gene GAPDH was used as an internal reference. Error bars represent the S.D. (n = 3). h Flow cytometric analysis of Krt14 in iPSC-derived EpSCs in comparison with iPSCs. i Flow cytometric analysis of CD200 and ITGA6 in iPSC-derived EpSCs in comparison with iPSCs