Fig 2: Expression of stem cell markers and the difference between young and aged participants in human nail stem cells. a Immunohistochemical staining for K14, K15, and K19 in human nails. These three markers were all expressed in the NPF and Mx in both groups. Compared to K14 and K15, the expression of K19 was relatively weak, but there was no significant difference between the two groups (scale bar = 400 µm). b Immunohistochemical staining for CD29, CD34, and Lgr6 in human nails. The markers were expressed in the same area (scale bar = 400 µm). c Proportion of positive cells stained for K14, K15, K19, CD29, CD34, and Lgr6 in analysed slices (n = 58, p > 0.05), illustrating no significant difference between the two groups. d Diagrammatic drawing of the nail root. The area in the blue and black box was used to determine the percentage of positive cells. e Co-immunofluorescence staining for K14 and K15 (scale bar = 400 µm). (Color figure online)

Fig 3: Age-associated hypofunctional in K15+ cells and nail growth rate. a Colony-forming assays of K15+ cells from young and aged nails. b Quantification of colony number (p = 0.02). c Comparison of average fingernail growth rates between the young and aged groups. Nails grew more slowly in the aged group compared to the young group (n = 64, p = 0.03). Average nail growth rates are presented in mm per month (defined as 30 days). Bars represent 95% confidence intervals. *P < 0.05

Fig 4: Localisation of stem cells in human nails. Side view, components of the human nail a Red circles are cells in nail proximal folds (NPFs); green circles are cells in the nail matrix (Mx). We found K15+ label-retaining cells in human nails in both the NPF and Mx. The diagrammatic drawing shows the tangent plane of the slice. b H&E staining of human nail side facing showing the nail bed (NB), nail root at the Mx and the NPF junction (scale bar = 1000 µm). c, d Immunofluorescence staining for K15 (green) and Ki67 (red); cell nuclei were stained blue by DAPI, image of separately staining of Ki67 in the upper left corner. The positive expression of K15 extended from the PF to the proximal Mx (scale bar = 200 µm). e Immumohistochemical staining for K15 (brown) (scale bar = 1000 µm). f Magnified photographs of immumohistochemical staining for K15 (brown) (scale bar = 200 µm). (Color figure online)

Fig 5: 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.