Fig 1.
Generation of hFIBROs and hKERAs.
(A) Differentiation protocol of hFIBROs. (B) The morphology of hFIBROs at each day (Day 4, 16 and 30). Left scale bar: 300 µm and right scale bar: 100 µm. (C) IF staining for fibronectin and vimentin. Scale bar: 100 µm. (D) FACS for Vimentin+ cells in hFIBROs. (E) Differentiation protocol of hKERAs. (F) The morphology of hKERAs at each day (Day 4, 17 and 30). Left scale bar: 300 µm and right scale bar: 100 µm. (G) IF staining for KRT14 and ΔNp63. Scale bar: 100 µm. (H) FACS for KRT14+ cells in hKERAs.
Fig 2.
(A) Protocol for generation of hiPSC-SKE. (B) Images of hiPSC-SKE in the inserts at each differentiation stage. (C) Comparison of epidermis formation according to ALI culture duration (10 and 20 days) through H&E staining. SC: stratum corneum. SG: stratum granulosum. SB: stratum basale. Scale bar: 50 µm. (D) Comparison of epidermis thickness according to ALI culture duration (10 and 20 days) (n = 4). (E) Morphology between native skin (human and mouse) and hiPSC-SKE through H&E staining. Scale bar: 25 and 50 µm. Yellow dotted boxes: enlarged images. Data are presented as mean values ± SEM. p value: *p < 0.05.
Fig 3.
Verification of skin related markers in hiPSC-SKE.
IF staining for (A) KRT14 and (B) ΔNp63 identifying SB distribution in hiPSC-SKE according to ALI culture duration (10 and 20 days). IF staining for (C) KRT10 to identify SS distribution and (D) Loricrin to identify SC distribution. IF staining for (E) vimentin, (F) PDGFRα, and (G) fibronectin to identify hFIBROs in hiPSC-SKE at D30 (ALI culture for 20 days). Scale bar: 50 µm. Yellow dotted boxes: enlarged images.
Fig 4.
Skin irritation testing using hiPSC-SKEs.
(A) Schematic diagram of skin irritation induced chemical stimulation. (B) Protocol of the skin irritation testing by CCK-8 assay. Arrow: O.D value measurement time. (C) Comparison of color changes before (Pre-treat) and after treatment (Post-treat) with the control group and 3% Triton using CCK-8 solution. (D) Changes in O.D value of CTL and 3% Triton-treated groups (n = 4). Data are presented as mean values ± SEM. n.s: not significant. p value: ***p < 0.0001. (E) Comparison of morphology between the control and 3% Triton using H&E staining. Scale bar: 100 µm. Yellow dotted boxes: enlarged images.