Fig 2: Keratinocyte derivation procedure in defined conditions. See also Figure S4. A. Schematic drawing of the optimized keratinocyte differentiation protocol. B. Cell morphology changes in the differentiation process. Scale bar,100µm. C. Stagewise keratinocyte gene expression by RT-qPCR. The results were normalized to hESC (D0); Data are representative of 3 independent experiments. D. Immunostaining of TP63 (red), KRT14 (green), KRT1 (green) and KRT10 (green) on day 24 and day 32 of differentiation. Scale bar, 50µm. E. Flow cytometry analysis of KRT14 (left panel) and KRT1 (right panel) on Day 28 of differentiation. Blue peak represents undifferentiated hESCs; pink peak represents keratinocyte. F. Keratinocyte differentiation from multiple hESC (H1, H9) and hiPSC (ND1-4, NL-1, NL-4) lines. Gene expression was analyzed on day 20 of differentiation and compared with HaCaT keratinocyte cell line, primary human foreskin keratinocytes (HFK-1, HFK-2) and undifferentiated hESCs (H1). The results were normalized to hESC (H1); Data are representative of 3 independent experiments.

Fig 3: Generation of air-liquid interface (ALI)-skin organoids(A) Schematic overview and timeline of the culture protocol for generating ALI-skin organoids from hiPSCs. Day 0 refers to when hiPSC colonies were detached to form EBs and an overview of the ALI-skin organoid culture model.(B) Bright-field images of ALI-skin organoids after 0, 2, 4, and 6 days of culture in dry conditions. Scale bars, 50 µm, and H&E staining of ALI-skin organoids after 0, 2, 4, and 6 days of culture in dry conditions. Scale bars, 100 µm.(C) Immunostaining for epidermal barrier markers in ALI-skin organoids after 0, 2, 4, and 6 days of culture in dry conditions and in human adult skin. Cornified (Filaggrin; green) and basal (KRT5; red) epidermal layer markers are shown on the top, and spinous (KRT10; green) and granular (Loricrin; red) epidermal layer protein markers are characterized on the bottom panel. Scale bars, 50 µm.(D) Immunostaining for dermal layer markers (Vimentin and Collagen 3; green) and basal epithelial layer markers (KRT5; red) in ALI-skin organoids after 6 days in dry conditions (top panel) and in human adult skin (bottom panel). Scale bars, 50 µm.(E–J) Hair follicle characterization in ALI-skin organoids. Shown are immunostaining images for Sox2+ dermal papilla (arrows; left and right) (E), melanocytes (melanA) found in elongated hair follicles (arrow; left) and epidermal layer (arrow; right) (F), KRT15+KRT5+ bulge stem cells (arrow; left) and NFATc1+KRT5+ bulge stem cells (arrow; right) (G), KRT17+KRT5+ outer sheaths of hair shafts; dashed box, magnified region (arrow; right) (H), KRT71+ inner sheaths of hair shafts (arrowhead; left), AE13+ hair shaft cortex (asterisk; left), and ?SMA+KRT5+ dermal sheath (arrow; right) (I), and ki67+p63+ hair matrix cells, dashed box, magnified region (arrow; right) (J). Scale bars, 50 µm.

Fig 4: The effect of Cutibacterium acnes (C. acnes) on ALI-skin organoids infected with S. aureus(A) Schematic outline for C. acnes pre-treatment on S. aureus-infected ALI-skin organoids.(B–D) Epidermal barrier markers were analyzed in 0 CFU (control; PBS only), S. aureus (SA) 106 only, C. acnes (CA) 106CFU + SA 106 CFU, and CA 107CFU + SA 106 CFU infected ALI-skin organoids. Immunostaining images for KRT5+ basal and FLG+ cornified epidermal layer marker (top panel) and KRT10+ spinous and LOR+ granular epithelial layer markers (bottom panel) are shown (B). Scale bars, 50 µm. Quantifications of Filaggrin (C) and Loricrin (D) expression levels are shown. One-way ANOVA with Bonferroni post-hoc test; *p < 0.05, **p < 0.01, ***p < 0.001, compared to S. aureus (SA) 106; independent replicates = 5. The results are presented as the means ± SEM.

Fig 5: Isolated CDC20 KO epithelial cells undergo apoptosis.a Apoptotic sub-G1 fraction as measured by PI, in control cells (CDC20+/+; Cre), upon vehicle only (48 h), Plk1 inhibitor BI2536 (48 h), lethal UV irradiation (48 h) or TAM for 3 days, as indicated; black bar for CDC20 KO; Cre cells upon TAM treatment for 3 days. Percent of cells 4C (G2/M + tetraploids) and >4C (polyploids) 48 h after BI2536 or UV irradiation, or 4 days after TAM. Fraction of cells positive for squamous differentiation markers K1 and K10 48 h after BI2536 or UV irradiation, or 3 days after TAM. c Frame from a representative time-lapse video by phase-contrast microscopy and DNA labelling by NucBlue, 31 h after addition of TAM to delete CDC20. Scale bar: 50 µm. b Flow-cytometry analyses for detection of apoptotic early marker Annexin V after BI2536 (48 h), UV irradiation (48 h) or TAM (4 days). Bar histogram displays quantification of Annexin positive cells. **P < 0.01, *P < 0.05, ns not significant. Data are mean ± SD of duplicate or triplicate samples (n = 2 or n = 3) of at least two mice per group (N = 2) and five independent experiments. See also Supplementary Fig. 8.