Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Stem cell competition orchestrates skin homeostasis and ageing

Abstract

Stem cells underlie tissue homeostasis, but their dynamics during ageing—and the relevance of these dynamics to organ ageing—remain unknown. Here we report that the expression of the hemidesmosome component collagen XVII (COL17A1) by epidermal stem cells fluctuates physiologically through genomic/oxidative stress-induced proteolysis, and that the resulting differential expression of COL17A1 in individual stem cells generates a driving force for cell competition. In vivo clonal analysis in mice and in vitro 3D modelling show that clones that express high levels of COL17A1, which divide symmetrically, outcompete and eliminate adjacent stressed clones that express low levels of COL17A1, which divide asymmetrically. Stem cells with higher potential or quality are thus selected for homeostasis, but their eventual loss of COL17A1 limits their competition, thereby causing ageing. The resultant hemidesmosome fragility and stem cell delamination deplete adjacent melanocytes and fibroblasts to promote skin ageing. Conversely, the forced maintenance of COL17A1 rescues skin organ ageing, thereby indicating potential angles for anti-ageing therapeutic intervention.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Chronological ageing causes epidermal atrophy with hemidesmosomal instability.
Fig. 2: Age-associated differential expression of COL17A1 provokes epidermal stem cell competition.
Fig. 3: Epidermal homeostasis is maintained by COL17A1-mediated symmetric cell divisions.
Fig. 4: Heterotypic lineage maintenance in the skin by COL17A1+ epidermal stem cells.

Data availability

Microarray data were deposited in Gene Expression Omnibus under series identifier GSE111825. All other data that support the conclusions are available from the authors on request.

References

  1. 1.

    Medawar, P. B. An Unsolved Problem of Biology (Published for the College by H. K. Lewis, 1952).

  2. 2.

    Kirkwood, T. Ageing: too fast by mistake. Nature 444, 1015–1017 (2006).

    CAS  ADS  Article  Google Scholar 

  3. 3.

    Martincorena, I. & Campbell, P. J. Somatic mutation in cancer and normal cells. Science 349, 1483–1489 (2015).

    CAS  ADS  Article  Google Scholar 

  4. 4.

    van Deursen, J. M. The role of senescent cells in ageing. Nature 509, 439–446 (2014).

    ADS  Article  Google Scholar 

  5. 5.

    Goodell, M. A. & Rando, T. A. Stem cells and healthy aging. Science 350, 1199–1204 (2015).

    CAS  ADS  Article  Google Scholar 

  6. 6.

    Matsumura, H. et al. Hair follicle aging is driven by transepidermal elimination of stem cells via COL17A1 proteolysis. Science 351, aad4395 (2016).

    Article  Google Scholar 

  7. 7.

    Nishimura, E. K., Granter, S. R. & Fisher, D. E. Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche. Science 307, 720–724 (2005).

    CAS  ADS  Article  Google Scholar 

  8. 8.

    Tanimura, S. et al. Hair follicle stem cells provide a functional niche for melanocyte stem cells. Cell Stem Cell 8, 177–187 (2011).

    CAS  Article  Google Scholar 

  9. 9.

    Snippert, H. J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).

    CAS  Article  Google Scholar 

  10. 10.

    Stine, R. R. & Matunis, E. L. Stem cell competition: finding balance in the niche. Trends Cell Biol. 23, 357–364 (2013).

    CAS  Article  Google Scholar 

  11. 11.

    Clayton, E. et al. A single type of progenitor cell maintains normal epidermis. Nature 446, 185–189 (2007).

    CAS  ADS  Article  Google Scholar 

  12. 12.

    Morata, G. & Ripoll, P. Minutes: mutants of Drosophila autonomously affecting cell division rate. Dev. Biol. 42, 211–221 (1975).

    CAS  Article  Google Scholar 

  13. 13.

    Vincent, J. P., Fletcher, A. G. & Baena-Lopez, L. A. Mechanisms and mechanics of cell competition in epithelia. Nat. Rev. Mol. Cell Biol. 14, 581–591 (2013).

    CAS  Article  Google Scholar 

  14. 14.

    Merino, M. M., Levayer, R. & Moreno, E. Survival of the fittest: essential roles of cell competition in development, aging, and cancer. Trends Cell Biol. 26, 776–788 (2016).

    Article  Google Scholar 

  15. 15.

    Clavería, C. & Torres, M. Cell competition: mechanisms and physiological roles. Annu. Rev. Cell Dev. Biol. 32, 411–439 (2016).

    Article  Google Scholar 

  16. 16.

    Maruyama, T. & Fujita, Y. Cell competition in mammals—novel homeostatic machinery for embryonic development and cancer prevention. Curr. Opin. Cell Biol. 48, 106–112 (2017).

    CAS  Article  Google Scholar 

  17. 17.

    Williams, S. E., Beronja, S., Pasolli, H. A. & Fuchs, E. Asymmetric cell divisions promote Notch-dependent epidermal differentiation. Nature 470, 353–358 (2011).

    CAS  ADS  Article  Google Scholar 

  18. 18.

    Poulson, N. D. & Lechler, T. Asymmetric cell divisions in the epidermis. Int. Rev. Cell Mol. Biol. 295, 199–232 (2012).

    CAS  Article  Google Scholar 

  19. 19.

    Kurban, R. S. & Bhawan, J. Histologic changes in skin associated with aging. J. Dermatol. Surg. Oncol. 16, 908–914 (1990).

    CAS  Article  Google Scholar 

  20. 20.

    Farage, M. A., Miller, K. W. & Maibach, H. I. Textbook of Aging Skin (Springer, 2010).

  21. 21.

    Gosain, A. & DiPietro, L. A. Aging and wound healing. World J. Surg. 28, 321–326 (2004).

    Article  Google Scholar 

  22. 22.

    Giangreco, A., Goldie, S. J., Failla, V., Saintigny, G. & Watt, F. M. Human skin aging is associated with reduced expression of the stem cell markers β1 integrin and MCSP. J. Invest. Dermatol. 130, 604–608 (2010).

    CAS  Article  Google Scholar 

  23. 23.

    Mine, S., Fortunel, N. O., Pageon, H. & Asselineau, D. Aging alters functionally human dermal papillary fibroblasts but not reticular fibroblasts: a new view of skin morphogenesis and aging. PLoS ONE 3, e4066 (2008).

    ADS  Article  Google Scholar 

  24. 24.

    Langton, A. K., Halai, P., Griffiths, C. E., Sherratt, M. J. & Watson, R. E. The impact of intrinsic ageing on the protein composition of the dermal-epidermal junction. Mech. Ageing Dev. 156, 14–16 (2016).

    CAS  Article  Google Scholar 

  25. 25.

    Watanabe, M. et al. Type XVII collagen coordinates proliferation in the interfollicular epidermis. eLife 6, e26635 (2017).

    Article  Google Scholar 

  26. 26.

    McGrath, J. A. et al. Mutations in the 180-kD bullous pemphigoid antigen (BPAG2), a hemidesmosomal transmembrane collagen (COL17A1), in generalized atrophic benign epidermolysis bullosa. Nat. Genet. 11, 83–86 (1995).

    CAS  Article  Google Scholar 

  27. 27.

    Jonkman, M. F. et al. 180-kD bullous pemphigoid antigen (BP180) is deficient in generalized atrophic benign epidermolysis bullosa. J. Clin. Invest. 95, 1345–1352 (1995).

    CAS  Article  Google Scholar 

  28. 28.

    Solanas, G. et al. Aged stem cells reprogram their daily rhythmic functions to adapt to stress. Cell 170, 678–692.e20 (2017).

    CAS  Article  Google Scholar 

  29. 29.

    Amano, S. Characterization and mechanisms of photoageing-related changes in skin. Damages of basement membrane and dermal structures. Exp. Dermatol. 25 (Suppl. 3), 14–19 (2016).

    CAS  Article  Google Scholar 

  30. 30.

    Zeman, M. K. & Cimprich, K. A. Causes and consequences of replication stress. Nat. Cell Biol. 16, 2–9 (2014).

    CAS  Article  Google Scholar 

  31. 31.

    Bartkova, J. et al. Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas. Oncogene 29, 5095–5102 (2010).

    CAS  Article  Google Scholar 

  32. 32.

    Mascré, G. et al. Distinct contribution of stem and progenitor cells to epidermal maintenance. Nature 489, 257–262 (2012).

    ADS  Article  Google Scholar 

  33. 33.

    Olasz, E. B. et al. Human bullous pemphigoid antigen 2 transgenic skin elicits specific IgG in wild-type mice. J. Invest. Dermatol. 127, 2807–2817 (2007).

    CAS  Article  Google Scholar 

  34. 34.

    Langton, A. K., Herrick, S. E. & Headon, D. J. An extended epidermal response heals cutaneous wounds in the absence of a hair follicle stem cell contribution. J. Invest. Dermatol. 128, 1311–1318 (2008).

    CAS  Article  Google Scholar 

  35. 35.

    Ueno, M., Aoto, T., Mohri, Y., Yokozeki, H. & Nishimura, E. K. Coupling of the radiosensitivity of melanocyte stem cells to their dormancy during the hair cycle. Pigment Cell Melanoma Res. 27, 540–551 (2014).

    CAS  Article  Google Scholar 

  36. 36.

    Amano, S. Possible involvement of basement membrane damage in skin photoaging. J. Investig. Dermatol. Symp. Proc. 14, 2–7 (2009).

    CAS  Article  Google Scholar 

  37. 37.

    Inomata, S. et al. Possible involvement of gelatinases in basement membrane damage and wrinkle formation in chronically ultraviolet B-exposed hairless mouse. J. Invest. Dermatol. 120, 128–134 (2003).

    CAS  Article  Google Scholar 

  38. 38.

    Aragona, M. et al. Defining stem cell dynamics and migration during wound healing in mouse skin epidermis. Nat. Commun. 8, 14684 (2017).

    ADS  Article  Google Scholar 

  39. 39.

    Falanga, V. et al. Full-thickness wounding of the mouse tail as a model for delayed wound healing: accelerated wound closure in Smad3 knock-out mice. Wound Repair Regen. 12, 320–326 (2004).

    Article  Google Scholar 

  40. 40.

    Rheinwald, J. G. & Green, H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331–343 (1975).

    CAS  Article  Google Scholar 

  41. 41.

    Nanba, D. et al. Cell motion predicts human epidermal stemness. J. Cell Biol. 209, 305–315 (2015).

    CAS  Article  Google Scholar 

  42. 42.

    Inomata, K. et al. Genotoxic stress abrogates renewal of melanocyte stem cells by triggering their differentiation. Cell 137, 1088–1099 (2009).

    CAS  Article  Google Scholar 

  43. 43.

    Jonkman, M. F. et al. Revertant mosaicism in epidermolysis bullosa caused by mitotic gene conversion. Cell 88, 543–551 (1997).

    CAS  Article  Google Scholar 

  44. 44.

    Pasmooij, A. M., Nijenhuis, M., Brander, R. & Jonkman, M. F. Natural gene therapy may occur in all patients with generalized non-Herlitz junctional epidermolysis bullosa with COL17A1 mutations. J. Invest. Dermatol. 132, 1374–1383 (2012).

    CAS  Article  Google Scholar 

  45. 45.

    Kowalewski, C. et al. Amelioration of junctional epidermolysis bullosa due to exon skipping. Br. J. Dermatol. 174, 1375–1379 (2016).

    CAS  Article  Google Scholar 

  46. 46.

    Vasioukhin, V., Degenstein, L., Wise, B. & Fuchs, E. The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin. Proc. Natl Acad. Sci. USA 96, 8551–8556 (1999).

    CAS  ADS  Article  Google Scholar 

  47. 47.

    Niculescu, C. et al. Conditional ablation of integrin alpha-6 in mouse epidermis leads to skin fragility and inflammation. Eur. J. Cell Biol. 90, 270–277 (2011).

    CAS  Article  Google Scholar 

  48. 48.

    Braun, K. M. et al. Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis. Development 130, 5241–5255 (2003).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank H. Yokozeki, Y. Nishimori, F. Toki, Y. Kato, M. Fukuda, R. Yajima, A. Tsuda, Y. Muroyama, H. Shimizu, W. Nishie, K. Yancey, T. Usami, F. Ishino and M. Kanai for support, and DASS Manuscript for editing. E.K.N. is supported by an AMED Project for Elucidating and Controlling Mechanisms of Ageing and Longevity (JP17gm5010002 JP18gm5010002) and a JSPS Grants-in-Aid for Scientific Research S (26221303) and Scientific Research on Innovative Areas “Stem Cell Aging and Disease” (26115003). H. Matsumura is supported by a JSPS Grant-in-Aid for Scientific Research on Innovative Areas “Cell Competition” (26114001).

Reviewer information

Nature thanks Salvador Aznar Benitah, James DeGregori and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Affiliations

Authors

Contributions

E.K.N. and H. Matsumura conceived the study and wrote the manuscript. N.L. performed the majority of experiments, analysed the data and wrote the manuscript. T.K., D.N. and K.A. analysed human keratinocytes. S.I. performed TEM experiments. A.T. and Y.M. prepared Epi-Confetti mice. H. Morinaga performed 8-OHdG staining. T.N. provided human skin specimens. E.G.-L. and A.D.A. provided the Itga6 floxed mice.

Corresponding authors

Correspondence to Hiroyuki Matsumura or Emi K. Nishimura.

Ethics declarations

Competing interests

E.K.N. is an inventor on a patent application (in preparation) related to this manuscript, which will be filed by Tokyo Medical and Dental University.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Chronological ageing induces the instability of hemidesmosome components.

a, Left, schematic of a scale (red) and interscale (yellow) area of mouse tail epidermis. Right, schematic of a magnified view of the scale, which consists of well-stratified keratinocyte layers that contain epidermal melanocytes or melanoblasts (green) in the basal layer and dermal fibroblasts (orange) in the dermis. b, c, Representative HE images (b) and quantification of epidermal thickness (c) of facial skin from young (22–33-year-old, n = 3) and aged (74–80-year-old, n = 4) humans. Human epidermal thickness was significantly decreased by ageing. d, Representative immunofluorescence images of KRT31 in tail scale IFE in young (8 wo) and aged (22 mo) mice. Dashed lines, basement membrane. e, Quantification of the number of KRT31+ spinous layers in tail scale IFE from young (7–8 wo, n = 30 scales) and aged (22 mo, n = 30) wild-type mice. KRT31+ spinous cell layers in the tail scale IFE were decreased by ageing. f, Representative immunofluorescence images of MCM2+ (non-G0) cells in tail scale IFE from 8 wo and 22 mo wild-type mice. Basal cells in young tail skin show a perpendicularly polarized structure against the basement membrane (arrows), whereas basal cells in aged tail skin usually show a flattened structure (arrowheads). Small insets, magnified views of dashed boxes. g, h, Numbers of DAPI+ basal cells (g) and MCM2+ basal cells at the non-G0 state (h) in tail scale IFE from young (7–8 wo, n = 4 in f, n = 5 in g) and aged (22–25 mo, n = 5) wild-type mice. i, j, Representative immunohistochemical images (i) and quantification of COL17A1+ DAB signals (j) in facial skin from young (22–33-year-old, n = 3) and aged (74–80-year-old, n = 3) humans. COL17A1 expression was significantly decreased by ageing. k, l, Representative immunofluorescence images and fluorescent intensities of hemidesmosome and basement membrane components, ITGA6, plectin, ITGB4, COL7A1 and ITGB1 in tail scale areas from young (7–8 wo, n = 50 scales) and aged (22–25 mo, n = 50) mice. m, Representative ultrastructural images of COL17A1 immunogold signals at the basement membrane in young (11 wo) and aged (22 mo) mice. n, Quantification of COL17A1+ immunogold particles on the basement membrane in young (11 wo, n = 5 hemidesmosomes) and aged (22 mo, n = 5) mice. COL17A1+ immunogold signals at the basement membrane were significantly decreased by ageing. o, Representative whole-mount immunofluorescence images of COL17A1/ITGA6, COL17A1/plectin, COL17A1/ITGB4 and COL17A1/ITGB1 expression in tail skin from young (7–8 wo) and aged (22–28 mo) mice. Similar results were obtained in at least two independent experiments. Arrows, intact expression; arrowheads, decreased expression. Heterogeneous destabilization of COL17A1 (arrowheads) was stochastically observed in the aged IFE. Mean ± s.e.m.; two-tailed Mann–Whitney U-test (e, l) or two-tailed t-test (c, g, h, j, n). Source data

Extended Data Fig. 2 COL17A1 is the most unstable hemidesmosome component under genotoxic stress.

a, d, Experimental design of the CHX chase assay. Lysates of NHEKs were collected 0, 1, 3, 6, 12 or 24 h after treatment with or without 10 µM CHX (a) or 0, 7 or 24 h after treatment with 3 µM marimastat (MM) with or without CHX (d). b, Western blot analysis of COL17A1, plectin, ITGA6, ITGB4, ITGB1 and GAPDH in NHEKs after CHX treatment for 0, 1, 3, 6, 12 or 24 h. Only COL17A1 showed protein degradation from 6 h; other hemidesmosome components were more stable. Data are representative of at least two independent experiments. c, Quantification of band intensities of COL17A1 relative to GAPDH (n = 5). e, Western blot analysis of COL17A1 and GAPDH in NHEKs after marimastat treatment with or without CHX. Marimastat partially rescued the CHX-induced decrease in COL17A1 expression. Data are representative of at least three independent experiments. f, Experimental design for western blot analysis of hemidesmosome components in HaCaT keratinocytes. Cells were seeded 24 h before stress treatments. Cell lysates were collected 24 h after UVB or H2O2 treatment and 72 h after ionizing radiation. g, i, k, Western blot analysis of COL17A1, plectin, ITGA6, ITGB4, ITGB1, γ-H2AX and β-actin (ACTB) in HaCaT cells with or without 20 or 40 mJ cm−2 UVB irradiation (g), 20 or 30 Gy ionizing radiation (i), and 250 µM H2O2 (k). h, j, l, Band intensities of hemidesmosome components and ITGB1 relative to ACTB (n = 2). m, Cell lysates of NHEKs were collected 12 h after 0, 40 or 80 mJ cm−2 UVB with or without 3 µM marimastat. Marimastat treatment partially rescued UVB-induced COL17A1 destabilization. n, Western blot analysis of COL17A1 and ITGA6 in NHEKs after transfection with scrambled siRNA (siCont), COL17A1 siRNA (siCOL17A1) or ITGA6 siRNA (siITGA6) for 72 h. Data are representative of at least two independent experiments. o, Band intensities of COL17A1 or ITGA6 relative to tubulin in n (n = 2). COL17A1 knockdown destabilizes ITGA6, whereas ITGA6 knockdown destabilizes COL17A1. Mean ± s.d. (c, h, j, l, o). Source data

Extended Data Fig. 3 The DNA damage response underlies age-associated proteolysis of COL17A1 in vivo.

a, Experimental design. Wild-type mice were exposed to UVB at a dose of 200 mJ cm−2, once daily for 5 consecutive days every week from 7 wo to 11 wo, and skin samples were collected 4 days after the last UVB irradiation. b, Representative immunofluorescence images of COL17A1 in the scale IFE in control and UVB-irradiated mice. UVB irradiation led to a linearized loss of the COL17A1 expression pattern. Data are representative of at least two independent experiments. c, Representative ultrastructural images of epidermal basal cells from control (11 wo, n = 3) and UVB-irradiated (11 wo, n = 3) mice assessed by TEM. Bottom, enlarged views of the dashed boxed areas in top panels; white lines show regions across the hemidesmosome, lamina lucida and lamina densa. d, Intensity histogram (above lines) for hemidesmosomes. UVB irradiation led to the loss of hemidesmosome density. e, Representative ultrastructural images of COL17A1+ immunogold signals at the basement membrane in control (11 wo) and UVB-irradiated (11 wo) mice. f, Numbers of COL17A1+ immunogold particles at the basement membrane in control (11 wo, n = 5 hemidesmosomes) and UVB-irradiated (11 wo, n = 7 hemidesmosomes) mice. COL17A1+ immunogold signals at the basement membrane were significantly decreased by UVB irradiation. g, l, Experimental designs. Seven-week-old wild-type mice were intraperitoneally injected with 100 µl HU (25 mg ml−1) for 4 consecutive days (g) or for 3 consecutive days per week for 3 weeks (l). Samples were collected 2 h (g, l) after the final HU treatment. h, i, Representative immunofluorescence images of COL17A1 and γ-H2AX (h) or p-RPA2/32 (i) 4 days after HU treatment. γ-H2AX foci and p-RPA2/32 foci (arrowheads) were both occasionally found in young tail scale basal cells and were significantly increased by HU treatment. Similar results were obtained in at least two independent experiments. j, Representative immunofluorescence images of COL17A1 and 8-OHdG in young (8 wo) and aged (22 mo) scale IFE. Accumulation of 8-OHdG-retaining basal cells and loss of COL17A1 expression were observed in the aged tail skin. k, 8-OHdG intensity in tail basal scale cells from young (8 wo, n = 3) and aged (22 wo, n = 3) mice. m, Representative immunofluorescence images of COL17A1 three weeks after HU treatment. Reduced COL17A1 expression was observed three weeks after HU treatment. Similar results were obtained in at least two independent experiments. n, Representative immunofluorescence images of COL17A1 and γ-H2AX in aged (30 mo) scale IFE. o, COL17A1 intensity in γ-H2AXhigh (arrowhead in n) and γ-H2AXmean (average level) cells from tail basal scale IFE from aged (22–30 mo, n = 19 scales) mice. γ-H2AXhigh cells contain significantly less COL17A1 than γ-H2AXmean cells. Mean ± s.e.m. (f, k, o); two-tailed Mann–Whitney U-test (o) or two-tailed t-test (f, k). Source data

Extended Data Fig. 4 There is no significant induction of apoptosis or senescence of epidermal stem cells during physiological skin ageing.

a, Representative immunofluorescence images of CASP3 in the tail scale area from 8-wo and 29-mo wild-type mice. CASP3+ cells were found 1 day after 10 Gy irradiation of dorsal skin (positive control). b, Number of CASP3+ cells per basal scale area in positive control, 7–8-wo (n = 4) and 25–29-mo (n = 3) wild-type mice. The number of CASP3+ cells was not significantly increased in epidermal basal cells from young or aged mice. c, Representative immunofluorescence images of p16+ signals in the tail scale area from 7-wo and 29-mo wild-type mice. p16 signals were found in DMBA/TPA-induced mouse papilloma (positive control), but no specific induction of p16 was found in the basal cells of aged mice even at 29 mo. d, Number of p16+ cells per 10 μm in skin from positive control, 7-wo (n = 3) and 25–29-mo (n = 3) wild-type mice. p16+ signals were not significantly increased in epidermal basal cells in aged mice compared to young mice. Mean ± s.e.m.; two-tailed t-test (b, d). Source data

Extended Data Fig. 5 Elimination of Col17a1-deficient cells through differentiation-coupled delamination.

a, Experimental design for analysis of stem and progenitor cell fate after deletion of COL17A1. In brief, 7-wo Col17a1 cKO and control (Epi-Confetti) mice were treated with TAM for 1 day (low dose) to label basal keratinocytes at clonal density. Skin samples were collected at D2, D14, D28 and 24–30 weeks (24–30 w). b, Representative immunofluorescence images of multicolour-labelled scale basal keratinocytes expressing KRT31 at D28 from control and Col17a1 cKO mice. Single-cell (arrowhead) or multi-cell floating clones (open arrowhead) were observed. c, Frequency of floating clones from control (n = 5) and Col17a1 cKO (n = 5) mice in b. Multi-cell floating clones were significantly increased during delamination of Col17a1-deficient cells. d, Representative immunofluorescence images of CASP3 in the tail scale area at 2 and 28 days from TAM-treated control and Col17a1 cKO mice and at 1 day from 10-Gy-irradiated mice (positive control). Dashed lines, basement membrane. e, Number of CASP3+ cells per basal scale area from positive control, control (n = 3) and Col17a1 cKO (n = 3) mice. CASP3+ cells were found 1 day after 10 Gy irradiation of dorsal skin but were not found in Col17a1-deficient cells during differentiation and delamination. f, Representative immunofluorescence images of MCM2 in the tail scale area at 28 days from TAM-treated control and Col17a1 cKO mice. g, Intensity of MCM2+ cells in single-colour clone area from control (n = 3 scales) and Col17a1 cKO (n = 12) mice. MCM2+ cells at the non-G0 state did not show any significant difference in Col17a1-deficient cells in cKO mice during differentiation or delamination. h, i, Areas (h) and numbers (i) of single-colour (red) clones in whole-mount images of tail epidermis at 24–25 w from TAM-treated control (n = 10 images) and Col17a1 cKO (n = 15) mice. j, Representative immunofluorescence images of multicolour-labelled scale basal keratinocytes at 24–25 w from TAM-treated control and Col17a1 cKO mice. Bottom, enlarged views of dashed boxed areas shown in top panels. k, Number of basal clones per scale in control (n = 55 scales) and Col17a1 cKO (n = 72) mice at 24–25 w after TAM administration showing progressive and selective elimination of Col17a1 cells from the skin. l, Experimental design for clonal fate analysis of Col17a1fl/+ basal keratinocyte stem and progenitor cells. In brief, 7-wo Epi-Confetti Col17a1fl/+ and control Col17a1+/+ mice were treated with TAM (low dose) to trace the individual stem and progenitor cell-derived clones. Skin samples were collected at D28. m, Representative immunofluorescence images of COL17A1 in RFP-expressing basal cells from control and Col17a1 fl/+ mice. At D28, the Col17a1+/+ RFP clones replaced the Col17a1fl/+ RFP+ basal clones and occupied the basal layer. n, Quantification of basal clones in control (n = 5) and Col17a1 fl/+ (n = 4) mice at D28 following TAM administration. At D28, basal clones were significantly decreased in number by Col17a1 heterozygous deficiency. Means ± s.e.m.; two-tailed Mann–Whitney U-test (k) and two-tailed t-test (c, e, g, h, i, n). Source data

Extended Data Fig. 6 COL17A1+ basal cells outcompete COL17A1low basal cells in the 3D self-organizing epidermis model.

a, Experimental design for in vitro cell competition assay. Immortalized human HaCaT keratinocytes with inhibitory shRNA (scrambled (shSCR) or shCOL17A1) and EmGFP were mixed with EmGFP parental HaCaT keratinocytes at 1:10, 1:3, 1:0 and 0:1 ratios and were then applied to the 3D culture system for 14 days, after which the competitive contribution of EmGFP+ cells in the basal cell layer was analysed. b, g, i, Representative immunofluorescence images of COL17A1, KRT10 and EmGFP and HE images of 3D cultured HaCaT keratinocytes. Three-dimensional cultures with HaCaT or shSCR-expressing cells or shCOL17A1-expressing cells alone (b) and shSCR- or shCOL17A1-expressing EmGFP+ cells co-cultured with HaCaT keratinocytes at a 1:10 ratio (g) or a 1:3 ratio (i) are shown. Dashed lines, boundary of basal layer. In 3D cultures, basal cells express COL17A1 and suprabasal cells express KRT10. Knockdown of COL17A1 efficiently inhibited COL17A1 expression, but did not affect epidermal stratification and structure (b). Arrow, shSCR EmGFP+ cells in the basal layer; arrowhead, delaminated shCOL17A1 EmGFP+ cells from the basal layer. c, e, Representative immunofluorescence images of phospho-histone H3 (PH3) (c) or CASP3 (e) after 3D culturing alone with HaCaT cells or with shSCR or shCOL17A1. d, f, Quantification of PH3+ cells (HaCaT alone, n = 9; shSCR, n = 7; shCOL17A1, n = 9 images) (d) or CASP3+ cells (HaCaT alone, n = 9; shSCR, n = 7; shCOL17A1, n = 9 images) (f) at the basal layer; there were no significant differences in their frequency among these groups. h, j, Number of shRNA-expressing EmGFP+ cells in the basal cell layer after 3D co-culture with shSCR- or shCOL17A1-expressing cells and wild-type HaCaT cells at 1:10 (h, shSCR, n = 10 images; shCOL17A1, n = 14 images) or 1:3 (j, shSCR, n = 10 images; shCOL17A1, n = 8 images) ratios. k, Ratio of shCOL17A1 or shSCR EmGFP+ cells in the basal layer after 3D co-culture with shSCR or shCOL17A1 EmGFP+ and wild-type HaCaT cells at a 1:3 or a 1:10 ratio (1:3, n = 8; 1:10, n = 14 images). COL17A1 KD cells were significantly eliminated from the basal layer, depending on the ratio of surrounding wild-type HaCaT cells. Mean ± s.e.m.; one-way ANOVA with Tukey–Kramer post hoc test (d), Kruskal–Wallis test with Dunn’s post hoc test (f) or two-tailed t-test (h, j, k). Source data

Extended Data Fig. 7 Maintenance of COL17A1 enhances epidermal stem cell potential.

ad, Colony-forming analysis of tail epidermal keratinocytes from young (7 wo, n = 3) and aged (25 mo, n = 3) mice (a, b) or from wild-type (8 wo, n = 3) and hCOL17A1 tg (8 wo, n = 3) mice (c, d). Colony number and size were significantly decreased by ageing (a, b). Expression of the hCOL17A1 transgene significantly increased the number and size of colonies in primary tail epidermal keratinocytes (c, d). e, Representative immunofluorescence images of DAPI+ nuclei in tail scale IFE from young (7 wo) and aged (22 mo) wild-type mice and from aged (26 mo) hCOL17A1 tg mice. Young and aged hCOL17A1 tg tail basal cells show a rectangular and perpendicularly polarized structure against the basement membrane (arrows), whereas many aged tail basal cells show a flattened structure (arrowheads). Insets, magnified views of dashed boxed areas. Similar results were obtained in at least five independent experiments. f, Representative ultrastructural images of epidermal basal cells from wild-type (22 mo) and hCOL17A1 tg (25 mo) mice assessed by TEM. Bottom, enlarged views of dashed boxed areas. Asterisks, micro-delaminations. g, Numbers of micro-delaminations at the basement membrane in tail scale basal cells from wild-type (22 mo, n = 3) and hCOL17A1 tg (22–30 mo, n = 3) mice. Expression of the hCOL17A1 transgene significantly decreased age-associated micro-delaminations. h, Representative FACS histogram of COL17A1 expression by NHEKs. P4 (COL17A1−/low) and P6 (COL17A1+) fractions were collected by a cell sorter. Data are representative of at least two independent experiments. i, Colony-forming analysis of COL17A1low/− or COL17A1+ NHEKs with or without serial passage. Data are representative of three independent experiments. Mean ± s.e.m.; two-tailed t-test (ad, g). Source data

Extended Data Fig. 8 Hemidesmosome instability in basal keratinocytes causes epidermal dyspigmentation.

a, Schematic of whole-mount views of scale (orange) and interscale (green) areas in mouse tail epidermis. b, Representative whole-mount images of pigment distribution in the tail epidermis from young (8 wo) and aged (22 mo) mice; bottom, enlarged views of dashed outlines. Similar results were obtained in at least three independent experiments. c, GO analysis between total epidermal cells from young (7–8 wo, n = 3) and aged (22–25 mo, n = 3) mice for ≥twofold-downregulated genes in aged total epidermal cells. The GO terms for melanocyte-related genes (asterisks) were significantly enriched in young epidermal cells. d, Heat map showing the fold change (expressed as a log) between total epidermis from young (7–8 wo, n = 3) and aged (22–25 mo, n = 3) mice for melanocyte-related genes such as Mitf, Tyrp1, Sox10 and Dct. e, Representative immunofluorescence image of KIT+ melanocytes in Dct–H2B–GFP+ tg mice. Dct–H2B–GFP+ was merged with KIT+ in epidermal melanocytes and melanoblasts. Data are representative of at least ten independent experiments. f, Representative tail whole-mount immunofluorescence images of melanocyte distribution and KRT31 expression in skin from 7-wo and 22-mo Dct-H2B-GFP tg mice. Dashed line, margin of scale area. g, Numbers of GFP-marked melanocytes per scale area from young (7 wo, n = 29 scales) and aged (22 mo, n = 24) Dct-H2B-GFP tg mice. Dct–H2B–GFP+ melanocytes were significantly decreased during ageing. h, Representative images of tail skin after deletion of Col17a1 and/or Itga6. We treated 7-wo wild-type (control), Col17a1 cKO, Itga6fl/fl;K14-creERT2 (Itga6 cKO) or Col17a1fl/fl;Itga6fl/fl;K14-creERT2 (Col17a1, Itga6 dcKO) mice with TAM to delete each gene in epidermal basal keratinocytes. Arrows, hyperpigmentation; arrowheads, hypopigmentation. i, Representative immunofluorescence images of COL17A1 and ITGA6 in the tail scale area from cont, Col17a1 cKO, Itga6 cKO and Col17a1, Itga6 dcKO (11 wo) mice. Data are representative of at least three independent experiments. j, Representative combined immunofluorescence and bright field (melaninhigh/low) images of GFP at 16 wo from control and Col17a1 or Itga6 cKO mice. Arrows indicate epidermal melanocytes among tail scale basal keratinocytes. k, Numbers of GFP-marked melanocytes per tail scale area (melaninhigh/low) from control (n = 5), Col17a1 cKO (n = 4) and Itga6 cKO (n = 6) combined with Dct-H2B-GFP tg mice. Melaninhigh scale areas from Col17a1 or Itga6 cKO mice showed no significant difference in the number of GFP-marked melanocytes, whereas melaninlow scale areas from Col17a1 or Itga6 cKO mice showed a significant decrease in the number of GFP-marked melanocytes. l, o, Representative images of tail skin from control and UVB-exposed mice at 7 wo, 11 wo and 15 wo (l) or wild-type mice and hCOL17A1 tg mice at 8 wo, 18 mo and 25 mo (o). Repetitive UVB exposure induced hyper-pigmentation (arrows) in a month at 11 wo followed by appearance of depigmented spots upon the cessation of UVB irradiation (arrowheads) at 15 wo. Expression of the hCOL17A1 transgene rescued the age-associated epidermal dyspigmentation. m, Representative ultrastructural images of scale basal cells from control (11 wo) and Col17a1 cKO (11 wo) mice assessed by TEM. Bottom, enlarged views of dashed boxed areas. White lines, show regions across the hemidesmosome, lamina lucida and lamina densa. n, Intensity histograms (above lines) for hemidesmosomes in m. Similar results were obtained in at least three independent experiments. Images are representative of at least five independent experiments (h, l, o). Mean ± s.e.m.; modified Fisher’s exact test (c), two-tailed Mann–Whitney U-test (g) or one-way ANOVA with Dunnett’s post hoc test (k). Source data

Extended Data Fig. 9 Hemidesmosome-mediated lateral stem cell expansion facilitates skin wound healing.

a, d, q, Experimental designs. Full-thickness wounds were created by skin excision on the tails of young (7–8 wo) and aged (20–24 mo) wild-type mice or of 8-wo wild-type and hCOL17A1 tg mice (a); and of 11-wo wild-type (control), TAM-treated Col17a1 cKO and Itga6 cKO mice (d). Wound repair processes were observed over time. q, Vehicle, apocynin or Y-27632 were repeatedly administered during full-thickness wound healing in wild-type mice (9–11 wo). b, e, g, r, Representative images of tail skin wounds from young (n = 7) and aged wild-type (n = 5) mice (b) or from control (n = 13), Col17a1 cKO (n = 10) and Itga6 cKO (n = 4) mice (e) or from wild-type (n = 8) and hCOL17A1 tg (n = 6) mice (g) or from mice treated with vehicle (n = 9), apocynin (n = 4) or Y-27632 (n = 4) (r) at D0, D14 and D21. Arrowheads, delayed wound repair areas in aged, Col17a1 cKO or Itga6 cKO mice. Arrows, facilitated wound healing areas in hCOL17A1 tg mice or apocynin- or Y-27632-treated mice. c, f, h, s, Unrepaired wound area at D0, D14 and D21. Physiological ageing (c) or Col17a1 or Itga6 deficiency (f) significantly delayed wound repair. Expression of the hCOL17A1 transgene (h), or treatment with apocynin or Y-27632 (s) significantly facilitated wound repair. i, j, Western blot analysis of COL17A1 in HaCaT keratinocytes after treatment with vehicle (control), apocynin (i) or Y-27632 (j). k, l, Band intensity of COL17A1 relative to GADPH after treatment with apocynin (k) or Y-27632 (l). Both chemicals significantly induced COL17A1 expression. Similar results were obtained in at least two independent experiments. m, Colony-forming analysis of NHEKs after treatment with vehicle (control), apocynin or Y-27632. Data are representative of at least two independent experiments. np, Colony number (n = 3 wells) was significantly increased by Y-27632 treatment (n), and colony size (n = 3 wells) (diameter >2 mm) (o) or average size (n = 3 wells) of the top five colonies (p) was significantly increased by treatment with Y-27632 or apocynin. Means ± s.e.m.; two-tailed t-test (c, h) or one-way ANOVA with Dunnett’s post hoc test (f, np, s). Source data

Extended Data Fig. 10 A schematic model of epidermal stem cell competition for skin homeostasis and ageing.

Top, homeostatic epidermis. COL17A1+ stem cells undergo parallel cell divisions that spread horizontally on the basement membrane and naturally generate the mechanical driving force for cell competition to eliminate COL17A1low/− stressed stem cells in the basal layer because of spatial constraints. Bottom, aged epidermis. Thin, atrophic and fragile skin with basal cells of COL17A1MCM2 exhausted/quiescent state resulting from repetitive stem cell competition and clonal expansion processes in homeostatic epidermis.

Supplementary information

Supplementary Figures

This file contains uncropped scans with size maker indications

Reporting Summary

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liu, N., Matsumura, H., Kato, T. et al. Stem cell competition orchestrates skin homeostasis and ageing. Nature 568, 344–350 (2019). https://doi.org/10.1038/s41586-019-1085-7

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing