Human epidermis is naturally endowed with an efficient capacity for long-term renewal and regeneration, due to the presence of stem cells within its basal layer. The high regenerative potential of native epidermal keratinocyte stem cells has enabled the development of skin replacement strategies, historically exemplified by the treatment of massive burn wounds, and recently by whole epidermis combined cell and gene therapy. Bioengineering of large surfaces of skin substitute requires massive ex vivo cellular expansion, during which maintenance of functional stem cells constitutes a critical endpoint to ensure long-term graft success. In this context, further understanding the cellular and molecular regulators involved in keratinocyte stem cell maintenance may impact the design of next-generation skin graft bioengineering principles. Alternatively, lineage-oriented differentiation of human pluripotent stem cells, either hESCs or iPSCs, into keratinocytes has been proposed as a complementary approach to generate epidermis substitutes. Effectiveness of this potential keratinocyte source will be greatly improved by robust differentiation procedures as a critical aspect of this technology is the generation of keratinocytes corresponding to an immature ‘stem-like’ cell status.

In the work we just published in Nature Biomedical Engineering (https://doi.org/10.1038/s41551-019-0464-6), we have explored the regulatory function of the transcription factor KLF4 in native keratinocytes from adult human skin (Ker-Ads), and in human embryonic stem cell-derived keratinocytes (Ker-ESCs). Integrating these two cellular systems was rendered possible by setting a collaboration between our ‘Laboratory of Genomics and Radiobiology of Keratinopoiesis’ (CEA - LGRK) and the ‘Institute for Stem cell Therapy and Exploration of Monogenic diseases’ (I-Stem), both located in Evry, France.

Firstly, a stable lentiviral-based KLF4 knock-down (KD) approach was developed and used as a proof-of-concept to study the properties of KLF4^KD versus KLF4 wild-type Ker-Ads. Using long-term cultures and clonal assays, we found that reduced KLF4 expression in Ker-Ads increased ‘stemness’ and ex vivo amplification. Importantly, KLF4^KD cells exhibited an improved grafting capacity in a serial skin grafting model (a). To explore the mechanisms responsible for these gains-of-function, the interplay between KLF4 and TGF-β1 was investigated. Treatment of KLF4^WT keratinocytes with TGF-β1 induced differentiation, but this process was impaired in KLF4^KD, notably through down-regulation of TGF-β1 receptor expression, leading to maintenance of a more immature cellular and molecular phenotype. As a consequence, expression of stemness transcripts and immaturity-associated markers were up-modulated in KLF4^KD keratinocytes, whereas keratinocyte differentiation program was shut-down (b-c).

In a translational perspective, we next analyzed pharmacological repression of KLF4, obtained using the small molecule kenpaullone, and showed its efficiency to promote beneficial effects. Notably, kenpaullone promoted stemness both in culture systems using serum and feeder layers of growth-arrested fibroblasts, and in serum- and feeder-free conditions, which anticipates the expected evolution towards more defined conditions in skin substitute bioengineering. Considering the safety issues, we determined that KLF4 down-modulation did not promote DNA mutations, genomic deletions or duplications, supporting absence of impact on genomic integrity.

To widen the perspectives of our work, we finally extended the anti-KLF4 strategy to ESC derivatives. Keratinocytes derived from ESCs can be obtained in large numbers, but they currently do not entirely reproduce adult stem cell phenotype, and notably their growth potential. We show that KLF4 constitutes a relevant molecular target to improve the properties of Ker-ESCs. Notably, kenpaullone treatment improved the growth capacity and epidermis regeneration potential of Ker‑ESCs.

In conclusion, the present publication documents an original function of KLF4, which we identify as a gate-keeper of stemness in human keratinocytes. The presented results pinpoint its pharmaceutical down-modulation as a promising approach for promoting ex vivo cell amplification for two different types of cells of interest for skin cell therapy, native and ESC-derived keratinocytes.

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Written by Nicolas O. FORTUNEL & Michèle T. MARTIN

Correspondence: nicolas.fortunel@cea.fr (+33 6 78 49 35 30); michele.martin@cea.fr (+33 6 76 81 76 09)

Laboratoire de Génomique et Radiobiologie de la Kératinopoïèse, CEA/DRF/IBFJ/IRCM, 91000 Evry, France; INSERM U967, Fontenay-aux-Roses; Université Paris-Diderot, Paris 7; Université Paris-Saclay, Paris 11, France.