Fig 2: Comparison of cell proliferation between the young and aged groups. a Comparison of Ki67-positive cells with K15 positivity in the two groups (scale bar = 200 µm), image of separately staining of Ki67 in the upper left corner. c Ki67-positive cell count results; the young group was higher than the aged group (n = 58, p = 0.0005). b, d Comparison of Ki67-positive cells with K14 positivity in the two groups and Ki67-positive cell count results (scale bar = 200 µm), image of separately staining of Ki67 in the upper left corner. Similar results were obtained for both K14 and K15 staining (n = 58, p = 0.002). **P < 0.01 ***P < 0.001

Fig 3: K5Tta × TRE-H2BGFP+ slow cycling cells co-localize with corneal stem cell markers. (A) Low power haemotoxylin and eosin staining of the adult cornea, limbus and conjunctiva tissue (left), magnified images of the central cornea (middle) and limbal cornea (right). (B–G) Corneal immunofluorescence staining of K5Tta × TRE-H2BGFP sections, co-stained with putative stem cell markers in the central (upper panels) and limbal cornea (lower panels). (B) At 35 day chase (n = 3) few lowly expressed GFP+ cells were found in the central cornea while Krt15 (red) antibody staining is not expressed. At the limbus, there is high GFP+ LRC staining at the limbal and periphery basal layer, including to one cell layer above in some areas with weaker expression. Krt15 and few GFP+ cells co-localize in limbal cells as shown in the merged panel. (C) In the central cornea, GFP+ LRCs at 53 d chase (n = 3) and ?Np63 (red) labeled staining was absent. At the limbus, GFP+ LRCs were localized to 2–3 cells at the limbus and ?Np63 expression was limbal and basal specific (nuclear expression). The merged image shows the co-localization of GFP and ?Np63. (D) GFP+ LRCs at 53 d chase localized to a few cells in the central cornea (boxed) while Sox9 (red) marked only basal epithelial cells (n = 6). The merged image co-localizes them together. At the limbus, GFP+ LRCs co-localized with Sox9. (E) GFP+ LRCs from 53 d chase were not detected and Actn1 was devoid of staining in the central cornea. At the limbus, few GFP+ cells were visible and co-localized to cytoplasmic Actn1 staining (n = 4). (F) GFP+ LRCs cells at 35 d chase are strongly expressed in the basal and some suprabasal layers of the central cornea, while some weak staining was also observed in cells of the suprabasal layer. Fzd7 staining in the central cornea is devoid of any expression, while at the limbus, the expression is basal and co-localized to GFP+ LRCs (n = 2) (G) GFP+ LRCs are strongly expressed in the basal and some suprabasal layers of the central cornea, while some weak staining was also observed in cells of the suprabasal layer. Krt17 staining was absent in the central cornea, but co-localized with GFP+ LRCs at the limbus (n = 3). Scale bar in A = 200 µm for (A–C,E,F); in D scale bar = 50 µm.

Fig 4: 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 5: Gold particle-labeled secondary antibodies against K15 (10 nm) and CD105 (20 nm) in equine laminar progenitor cells from normal hooves sorted by co-expression of K14 and CD105 antigens. Gold labels (black arrows, a–c) localized throughout the cell cytoplasm (a, b) and in close proximity to the cell membrane (c), both singly and together. There was no labeling of cells treated only with gold particle-labeled secondary antibodies (d).