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

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.

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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.

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.

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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).

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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.

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

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

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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

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