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.

  • Article
  • Published:

KLF4 inhibition promotes the expansion of keratinocyte precursors from adult human skin and of embryonic-stem-cell-derived keratinocytes

Nature Biomedical Engineering volume 3, pages 985–997 (2019)Cite this article

Subjects

Abstract

Expanded autologous skin keratinocytes are currently used in cutaneous cell therapy, and embryonic-stem-cell-derived keratinocytes could become a complementary alternative. Regardless of keratinocyte provenance, for efficient therapy it is necessary to preserve immature keratinocyte precursors during cell expansion and graft processing. Here, we show that stable and transient downregulation of the transcription factor Krüppel-like factor 4 (KLF4) in keratinocyte precursors from adult skin, using anti-KLF4 RNA interference or kenpaullone, promotes keratinocyte immaturity and keratinocyte self-renewal in vitro, and enhances the capacity for epidermal regeneration in mice. Both stable and transient KLF4 downregulation had no impact on the genomic integrity of adult keratinocytes. Moreover, transient KLF4 downregulation in human-embryonic-stem-cell-derived keratinocytes increased the efficiency of skin-orientated differentiation and of keratinocyte immaturity, and was associated with improved generation of epidermis. As a regulator of the cell fate of keratinocyte precursors, KLF4 could be used for promoting the ex vivo expansion and maintenance of functional immature keratinocyte precursors.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: KLF4 expression level controls skin keratinocyte precursor growth and immature status.
Fig. 2: Stable KLF4 knockdown in skin keratinocyte precursors results in improved epidermis regeneration capacity.
Fig. 3: Transient downmodulation of KLF4 increases the clonogenic capacity and immature character of skin keratinocyte precursors.
Fig. 4: Drug-induced KLF4 downmodulation improves ex vivo expansion of skin keratinocyte precursors under feeder- and serum-free culture conditions.
Fig. 5: Drug-induced KLF4 downmodulation promotes an immature precursor status in ESC-derived keratinocytes.
Fig. 6: Drug-induced KLF4 downmodulation improves epidermis reconstruction capacity in ESC-derived keratinocytes.

Similar content being viewed by others

Data availability

The main data supporting the results of this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors. The transcriptome microarray and RNA-Seq datasets that were generated have been deposited in the GEO database (www.ncbi.nlm.nih.gov/geo) and are accessible using the accession codes GSE68583 and GSE111786, respectively.

References

  1. Gallico, G. G. 3rd, O’Connor, N. E., Compton, C. C., Kehinde, O. & Green, H. Permanent coverage of large burn wounds with autologous cultured human epithelium. N. Engl. J. Med. 311, 448–451 (1984).

    Article  PubMed  Google Scholar 

  2. Thivolet, J., Faure, M., Demidem, A. & Mauduit, G. Long-term survival and immunological tolerance of human epidermal allografts produced in culture. Transplantation 42, 274–280 (1986).

    Article  CAS  PubMed  Google Scholar 

  3. 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).

    Article  CAS  PubMed  Google Scholar 

  4. Barrandon, Y. & Green, H. Three clonal types of keratinocyte with different capacities for multiplication. Proc. Natl Acad. Sci. USA 84, 2302–2306 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ronfard, V., Rives, J.-M., Neveux, Y., Carsin, H. & Barrandon, Y. Long-term regeneration of human epidermis on third degree burns transplanted with autologous cultured epithelium grown on a fibrin matrix. Transplantation 70, 1588–1598 (2000).

    Article  CAS  PubMed  Google Scholar 

  6. Fuchs, E. The impact of cell culture on stem cell research. Cell Stem Cell 10, 640–641 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Guenou, H. et al. Human embryonic stem-cell derivatives for full reconstruction of the pluristratified epidermis: a preclinical study. Lancet 374, 1745–1753 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Lemaître, G., Nissan, X., Baldeschi, C. & Peschanski, M. Concise review: epidermal grafting: the case for pluripotent stem cells. Stem Cells 29, 895–899 (2011).

    Article  PubMed  Google Scholar 

  9. Hirsch, T. et al. Regeneration of the entire human epidermis using transgenic stem cells. Nature 551, 327–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Blanpain, C., Lowry, W. E., Geoghegan, A., Polak, L. & Fuchs, E. Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 118, 635–648 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Oshimori, N. & Fuchs, E. The harmonies played by TGF-β in stem cell biology. Cell Stem Cell 11, 751–764 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lien, W. H. & Fuchs, E. Wnt some lose some: transcriptional governance of stem cells by Wnt/β-catenin signaling. Genes Dev. 28, 1517–1532 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Katz, J. P. et al. The zinc-finger transcription factor Klf4 is required for terminal differentiation of goblet cells in the colon. Development 129, 2619–2628 (2002).

    CAS  PubMed  Google Scholar 

  14. Cordes, K. R. et al. miR-145 and miR-143 regulate smooth muscle cell fate and plasticity. Nature 460, 705–710 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Qin, S. & Zhang, C. L. Role of Kruppel-like factor 4 in neurogenesis and radial neuronal migration in the developing cerebral cortex. Mol. Cell. Biol. 32, 4297–4305 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Segre, J. A., Bauer, C. & Fuchs, E. Klf4 is a transcription factor required for establishing the barrier function of the skin. Nat. Genet. 22, 356–360 (1999).

    Article  CAS  PubMed  Google Scholar 

  18. Sen, G. L. et al. ZNF750 is a p63 target gene that induces KLF4 to drive terminal epidermal differentiation. Dev. Cell 22, 669–677 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Fortunel, N. O. et al. Exploration of the functional hierarchy of the basal layer of human epidermis at the single-cell level using parallel clonal microcultures of keratinocytes. Exp. Dermatol. 19, 387–392 (2010).

    Article  PubMed  Google Scholar 

  20. Barrandon, Y. & Green, H. Cell size as a determinant of the clone-forming ability of human keratinocytes. Proc. Natl Acad. Sci. USA 82, 5390–5394 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Candi, E., Amelio, I., Agostini, M. & Melino, G. MicroRNAs and p63 in epithelial stemness. Cell Death Differ. 22, 12–21 (2015).

    Article  CAS  PubMed  Google Scholar 

  22. Lujan, E. et al. Early reprogramming regulators identified by prospective isolation and mass cytometry. Nature 521, 352–356 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lieu, Y. K. & Reddy, E. P. Conditional c-myb knockout in adult hematopoietic stem cells leads to loss of self-renewal due to impaired proliferation and accelerated differentiation. Proc. Natl Acad. Sci. USA 106, 21689–21694 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Delaunay, D., Cortay, V., Patti, D., Knoblauch, K. & Dehay, C. Mitotic spindle asymmetry: a Wnt/PCP-regulated mechanism generating asymmetrical division in cortical precursors. Cell Rep. 6, 400–414 (2014).

    Article  CAS  PubMed  Google Scholar 

  25. Gao, Y. et al. Replacement of Oct4 by Tet1 during iPSC induction reveals an important role of DNA methylation and hydroxymethylation in reprogramming. Cell Stem Cell 12, 453–469 (2013).

    Article  CAS  PubMed  Google Scholar 

  26. Yu, F. et al. Kruppel-like factor 4 (KLF4) is required for maintenance of breast cancer stem cells and for cell migration and invasion. Oncogene 30, 2161–2172 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pellegrini, G. et al. The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin. Transplantation 68, 868–879 (1999).

    Article  CAS  PubMed  Google Scholar 

  28. Yue, J., Gou, X., Li, Y., Wicksteed, B. & Wu, X. Engineered epidermal progenitor cells can correct diet-induced obesity and diabetes. Cell Stem Cell 21, 256–263 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Fortunel, N. O. et al. Cellular adhesion on collagen: a simple method to select human basal keratinocytes which preserves their high growth capacity. Eur. J. Dermatol. 21, 12–20 (2011).

    CAS  PubMed  Google Scholar 

  30. Boxer, L. D., Barajas, B., Tao, S., Zhang, J. & Khavari, P. A. ZNF750 interacts with KLF4 and RCOR1, KDM1A, and CTBP1/2 chromatin regulators to repress epidermal progenitor genes and induce differentiation genes. Genes Dev. 28, 2013–2026 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bao, X. et al. ACTL6a enforces the epidermal progenitor state by suppressing SWI/SNF-dependent induction of KLF4. Cell Stem Cell 12, 193–203 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lopez-Pajares, V. et al. A LncRNA-MAF:MAFB transcription factor network regulates epidermal differentiation. Dev. Cell 32, 693–706 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Patel, S., Xi, Z. F., Seo, E. Y., McGaughey, D. & Segre, J. A. Klf4 and corticosteroids activate an overlapping set of transcriptional targets to accelerate in utero epidermal barrier acquisition. Proc. Natl Acad. Sci. USA 103, 18668–18673 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Tetreault, M. P., Weinblatt, D., Shaverdashvili, K., Yang, Y. & Katz, J. P. KLF4 transcriptionally activates non-canonical WNT5A to control epithelial stratification. Sci. Rep. 6, 26130 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Fortunel, N. O. et al. Comment on “‘Stemness’: transcriptional profiling of embryonic and adult stem cells” and “A stem cell molecular signature”. Science 302, 393 (2003).

    Article  CAS  PubMed  Google Scholar 

  36. Vogel, G. Stem cells. ‘Stemness’ genes still elusive. Science 302, 371 (2003).

    Article  CAS  PubMed  Google Scholar 

  37. Alexaline, M. M. et al. Bioengineering a human plasma-based epidermal substitute with efficient grafting capacity and high content in clonogenic cells. Stem Cells Transl. Med. 4, 643–654 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Lange, C. et al. Small molecule GSK-3 inhibitors increase neurogenesis of human neural progenitor cells. Neurosci. Lett. 488, 36–40 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. Neveux, Y. et al. Clinical interest of cutaneous models reproduced in vitro for severe burn treatment: histopathological and ultrastructural study. Cell Biol. Toxicol. 11, 173–178 (1995).

    Article  CAS  PubMed  Google Scholar 

  40. Bisson, F. et al. Irradiated human dermal fibroblasts are as efficient as mouse fibroblasts as a feeder layer to improve human epidermal cell culture lifespan. Int. J. Mol. Sci. 14, 4684–4704 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Germain, L. et al. Autologous bilayered self-assembled skin substitutes (SASSs) as permanent grafts: a case series of 14 severely burned patients indicating clinical effectiveness. Eur. Cells Mater. 36, 128–141 (2018).

    Article  CAS  Google Scholar 

  42. Gardien, K. L. et al. Outcome of burns treated with autologous cultured proliferating epidermal cells: a prospective randomized multicenter intrapatient comparative trial. Cell Transplant. 25, 437–448 (2016).

    Article  PubMed  Google Scholar 

  43. Zhang, C. et al. Long-term in vitro expansion of epithelial stem cells enabled by pharmacological inhibition of PAK1–ROCK–myosin II and TGF-β signaling. Cell Rep. 25, 598–610 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Ghaleb, A. M. & Yang, V. W. Krüppel-like factor 4 (KLF4): what we currently know. Gene 611, 27–37 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Li, J. et al. Deficiency of the Kruppel-like factor KLF4 correlates with increased cell proliferation and enhanced skin tumorigenesis. Carcinogenesis 33, 1239–1246 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Coraux, C. et al. Reconstituted skin from murine embryonic stem cells. Curr. Biol. 13, 849–853 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Dabelsteen, S. et al. Epithelial cells derived from human embryonic stem cells display p16INK4A senescence, hypermotility, and differentiation properties shared by many P63+ somatic cell types. Stem Cells 27, 1388–1399 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Iuchi, S., Dabelsteen, S., Easley, K., Rheinwald, J. G. & Green, H. Immortalized keratinocyte lines derived from human embryonic stem cells. Proc. Natl Acad. Sci. USA 103, 1792–1797 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Tien, Y. T. et al. Downregulation of the KLF4 transcription factor inhibits the proliferation and migration of canine mammary tumor cells. Vet. J. 205, 244–253 (2015).

    Article  CAS  PubMed  Google Scholar 

  50. Rachidi, W. et al. Sensing radiosensitivity of human epidermal stem cells. Radiother. Oncol. 83, 267–276 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank O. Alibert, P. Soularue and Y. Mesloub for assistance with genomic data management, L. Guibbal (CEA-LGRK), S. Bouet and A. Boukadiri (histology platform, UMR 1313 GABI, INRA/CEA) for technical assistance, and D. Stockholm (imaging platform, Genethon), C. Fauter and H. Gharbi (Mauna Kea Technologies) for technological support. We thank H. Serhal and Y. Diaw (Clinique de l’Essonne) for providing human skin samples from healthy donors. We thank J.-J. Lataillade and M. Trouillas (IRBA, INSERM U1197) for helpful discussions on clinical settings. We also thank Genopole for providing equipment and infrastructures. This work was supported by grants from: CEA and INSERM (UMR967); Délégation Générale de l’Armement; FUI-AAP13 and the ‘Conseil Général de l’Essonne’ within the STEMSAFE grant; and EURATOM (RISK-IR; FP7; grant 323267).

Author information

Author notes

  1. These authors contributed equally: Loubna Chadli, Julien Coutier.

Authors and Affiliations

  1. Laboratoire de Génomique et Radiobiologie de la Kératinopoïèse, CEA/DRF/IBFJ/IRCM, Evry, France

    Nicolas O. Fortunel, Loubna Chadli, Julien Coutier, Frédéric Auvré, Emmanuelle Bouissou-Cadio, Pierre Vaigot, Sophie Cavallero & Michèle T. Martin

  2. INSERM U967, Université Paris-Diderot, Paris, France

    Nicolas O. Fortunel, Loubna Chadli, Julien Coutier, Frédéric Auvré, Emmanuelle Bouissou-Cadio, Pierre Vaigot, Sophie Cavallero, Paul-Henri Roméo & Michèle T. Martin

  3. Université Paris-Saclay, Paris, France

    Nicolas O. Fortunel, Loubna Chadli, Julien Coutier, Frédéric Auvré, Emmanuelle Bouissou-Cadio, Pierre Vaigot, Sophie Cavallero, Paul-Henri Roméo & Michèle T. Martin

  4. Université d’Evry Val d’Essonne, Université Paris-Saclay, INSERM U861, Institut des Cellules Souches pour le Traitement et l’Etude des Maladies Monogéniques, Corbeil Essonne, France

    Gilles Lemaître

  5. Centre d’Etude des Cellules Souches, Institut des Cellules Souches pour le Traitement et l’Etude des Maladies Monogéniques, Corbeil Essonne, France

    Sophie Domingues

  6. Centre National de Recherche en Génomique Humaine, CEA/DRF/IBFJ, Paris, France

    Jean-François Deleuze

  7. Laboratoire de Recherche sur la Réparation et la Transcription dans les Cellules Souches, CEA/DRF/IBFJ/IRCM, Fontenay-aux-Roses, France

    Paul-Henri Roméo

Authors
  1. Nicolas O. Fortunel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Loubna Chadli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Julien Coutier
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gilles Lemaître
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Frédéric Auvré
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sophie Domingues
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Emmanuelle Bouissou-Cadio
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Pierre Vaigot
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sophie Cavallero
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jean-François Deleuze
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Paul-Henri Roméo
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Michèle T. Martin
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.O.F. designed the study, planned and performed the experiments, analysed and interpreted the data and wrote the manuscript. L.C. and J.C. contributed to experimental design, performed the experiments, analysed the data and participated in manuscript writing. F.A., S.D., E.B.-C., P.V. and S.C. performed the experimental work and contributed to the data analysis. J.-F.D. produced the genomic data. G.L. contributed to experimental design, analysed the data and participated in manuscript writing. P.-H.R. assisted with analysis and interpretation of the data, as well as manuscript writing. M.T.M. conceived and initiated the project, designed the study, planned the experiments, analysed and interpreted the data and wrote the manuscript.

Corresponding authors

Correspondence to Nicolas O. Fortunel or Michèle T. Martin.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Supplementary information

Supplementary Information

Supplementary methods, Figs. 1–16, Tables 1–4 and references.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fortunel, N.O., Chadli, L., Coutier, J. et al. KLF4 inhibition promotes the expansion of keratinocyte precursors from adult human skin and of embryonic-stem-cell-derived keratinocytes. Nat Biomed Eng 3, 985–997 (2019). https://doi.org/10.1038/s41551-019-0464-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41551-019-0464-6

This article is cited by

Search

Advanced 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