Deciphering "Immaturity-Stemness" in Human Epidermal Stem Cells at the Levels of Protein-Coding and Non-Coding Genomes: A Prospective Computational Approach.

The epidermis hosts populations of epithelial stem cells endowed with well-documented renewal and regenerative functions. This tissue thus constitutes a model for exploring the molecular characteristics of stem cells, which remain to date partially characterized at the molecular level in human skin. Our group has investigated the regulatory functions of the KLF4/TGFB1 and the MAD4/MAX/MYC signaling pathways in the control of the immaturity-stemness versus differentiation fate of keratinocyte stem and precursor cells from human interfollicular epidermis. We described that down-modulation of either KLF4 or MXD4/MAD4 using RNA interference tools promoted an augmented stemness cellular status; an effect which was associated with significant transcriptional changes, as assessed by RNA-sequencing. Here, we have implemented a computational approach aimed at integrating the level of the coding genome, comprising the transcripts encoding conventional proteins, and the non-coding genome, with a focus on long non-coding RNAs (lncRNAs). In addition, datasets of micro-RNAs (miRNAs) with validated functions were interrogated in view of identifying miRNAs that could make the link between protein-coding and non-coding transcripts. Putative regulons comprising both coding and long non-coding transcripts were built, which are expected to contain original pro-stemness candidate effectors available for functional validation approaches. In summary, interpretation of our basic functional data together with in silico biomodeling gave rise to a prospective picture of the complex constellation of transcripts regulating the keratinocyte stemness status.

MXD4/MAD4 Regulates Human Keratinocyte Precursor Fate.

Deciphering the pathways that regulate human epidermal precursor cell fate is necessary for future developments in skin repair and graft bioengineering. Among them, characterization of pathways regulating the keratinocyte (KC) precursor immaturity versus differentiation balance is required for improving the efficiency of KC precursor ex vivo expansion. In this study, we show that the transcription factor MXD4/MAD4 is expressed at a higher level in quiescent KC stem/progenitor cells located in the basal layer of human epidermis than in cycling progenitors. In holoclone KCs, stable short hairpin-RNA‒mediated decreased expression of MXD4/MAD4 increases MYC expression, whose modulation increases the proliferation of KC precursors and maintenance of their clonogenic potential and preserves the functionality of these precursors in three-dimensional epidermis organoid generation. Altogether, these results characterize MXD4/MAD4 as a major piece of the stemness puzzle in the human epidermis KC lineage and pinpoint an original avenue for ex vivo expansion of human KC precursors.

Immunosuppressive Properties of Epidermal Keratinocytes Differ According to Their Immaturity Status.

Preservation of a functional keratinocyte stem cell pool is essential to ensure the long-term maintenance of epidermis integrity, through continuous physiological renewal and regeneration in case of injury. Protecting stem cells from inflammation and immune reactions is thus a critical issue that needs to be explored. Here, we show that the immature CD49fhigh precursor cell fraction from interfollicular epidermis keratinocytes, comprising stem cells and progenitors, is able to inhibit CD4 + T-cell proliferation. Of note, both the stem cell-enriched CD49fhigh/EGFRlow subpopulation and the less immature CD49fhigh/EGFRhigh progenitors ensure this effect. Moreover, we show that HLA-G and PD-L1 immune checkpoints are overexpressed in CD49fhigh precursors, as compared to CD49flow differentiated keratinocytes. This potency may limit immune reactions against immature precursors including stem cells, and protect them from exacerbated inflammation. Further exploring this correlation between immuno-modulation and immaturity may open perspectives in allogenic cell therapies.

Skin Immunity and Tolerance: Focus on Epidermal Keratinocytes Expressing HLA-G.

Although the role of epidermal cells in skin regeneration has been extensively documented, their functions in immunity and tolerance mechanisms are largely underestimated. The aim of the present review was to outline the state of knowledge on resident immune cells of hematopoietic origin hosted in the epidermis, and then to focus on the involvement of keratinocytes in the complex skin immune networks acting in homeostasis and regeneration conditions. Based on this knowledge, the mechanisms of immune tolerance are reviewed. In particular, strategies based on immunosuppression mediated by HLA-G are highlighted, as recent advances in this field open up perspectives in epidermis-substitute bioengineering for temporary and permanent skin replacement strategies.

Human Keratinocytes Inhibit CD4+ T-Cell Proliferation through TGFB1 Secretion and Surface Expression of HLA-G1 and PD-L1 Immune Checkpoints.

Human skin protects the body against infection and injury. This protection involves immune and epithelial cells, but their interactions remain largely unknown. Here, we show that cultured epidermal keratinocytes inhibit allogenic CD4+ T-cell proliferation under both normal and inflammatory conditions. Inhibition occurs through the secretion of soluble factors, including TGFB1 and the cell-surface expression of HLA-G1 and PD-L1 immune checkpoints. For the first time, we here describe the expression of the HLA-G1 protein in healthy human skin and its role in keratinocyte-driven tissue immunomodulation. The overexpression of HLA-G1 with an inducible vector increased the immunosuppressive properties of keratinocytes, opening up perspectives for their use in allogeneic settings for cell therapy.

When the Search for Stemness Genes Meets the Skin Substitute Bioengineering Field: KLF4 Transcription Factor under the Light.

The transcription factor "Kruppel-like factor 4" (KLF4) is a central player in the field of pluripotent stem cell biology. In particular, it was put under the spotlight as one of the four factors of the cocktail originally described for reprogramming into induced pluripotent stem cells (iPSCs). In contrast, its possible functions in native tissue stem cells remain largely unexplored. We recently published that KLF4 is a regulator of "stemness" in human keratinocytes. We show that reducing the level of expression of this transcription factor by RNA interference or pharmacological repression promotes the ex vivo amplification and regenerative capacity of two types of cells of interest for cutaneous cell therapy: native keratinocyte stem and progenitor cells from adult epidermis, which have been used for more than three decades in skin graft bioengineering, and keratinocytes generated by the lineage-oriented differentiation of embryonic stem cells (ESCs), which have potential for the development of skin bio-bandages. At the mechanistic level, KLF4 repression alters the expression of a large set of genes involved in TGF-ß1 and WNT signaling pathways. Major regulators of TGF-ß bioavailability and different TGF-ß receptors were targeted, notably modulating the ALK1/Smad1/5/9 axis. At a functional level, KLF4 repression produced an antagonist effect on TGFß1-induced keratinocyte differentiation.

Impairment of Base Excision Repair in Dermal Fibroblasts Isolated From Nevoid Basal Cell Carcinoma Patients.

The nevoid basal cell carcinoma syndrome (NBCCS), also called Gorlin syndrome is an autosomal dominant disorder whose incidence is estimated at about 1 per 55,600-256,000 individuals. It is characterized by several developmental abnormalities and an increased predisposition to the development of basal cell carcinomas (BCCs). Cutaneous fibroblasts from Gorlin patients have been shown to exhibit an increased sensitivity to ionizing radiations. Mutations in the tumor suppressor gene PTCH1, which is part of the Sonic Hedgehog (SHH) signaling pathway, are responsible for these clinical manifestations. As several genetic mutations in the DNA repair genes are responsible of photo or radiosensitivity and high predisposition to cancers, we hypothesized that these effects in Gorlin syndrome might be due to a defect in the DNA damage response (DDR) and/or the DNA repair capacities. Therefore, the objective of this work was to investigate the sensitivity of skin fibroblasts from NBCCS patients to different DNA damaging agents and to determine the ability of these agents to modulate the DNA repair capacities. Gorlin fibroblasts showed high radiosensitivity and also less resistance to oxidative stress-inducing agents when compared to control fibroblasts obtained from healthy individuals. Gorlin fibroblasts harboring PTCH1 mutations were more sensitive to the exposure to ionizing radiation and to UVA. However, no difference in cell viability was shown after exposure to UVB or bleomycin. As BER is responsible for the repair of oxidative DNA damage, we decided to assess the BER pathway efficacy in Gorlin fibroblasts. Interestingly, a concomitant decrease of both BER gene expression and BER protein activity was observed in Gorlin fibroblasts when compared to control. Our results suggest that low levels of DNA repair within Gorlin cells may lead to an accumulation of oxidative DNA damage that could participate and partly explain the radiosensitivity and the BCC-prone phenotype in Gorlin syndrome.

Exposure of Human Skin Organoids to Low Genotoxic Stress Can Promote Epithelial-to-Mesenchymal Transition in Regenerating Keratinocyte Precursor Cells.

For the general population, medical diagnosis is a major cause of exposure to low genotoxic stress, as various imaging techniques deliver low doses of ionizing radiation. Our study investigated the consequences of low genotoxic stress on a keratinocyte precursor fraction that includes stem and progenitor cells, which are at risk for carcinoma development. Human skin organoids were bioengineered according to a clinically-relevant model, exposed to a single 50 mGy dose of γ rays, and then xeno-transplanted in nude mice to follow full epidermis generation in an in vivo context. Twenty days post-xenografting, mature skin grafts were sampled and analyzed by semi-quantitative immuno-histochemical methods. Pre-transplantation exposure to 50 mGy of immature human skin organoids did not compromise engraftment, but half of xenografts generated from irradiated precursors exhibited areas displaying focal dysplasia, originating from the basal layer of the epidermis. Characteristics of epithelial-to-mesenchymal transition (EMT) were documented in these dysplastic areas, including loss of basal cell polarity and cohesiveness, epithelial marker decreases, ectopic expression of the mesenchymal marker α-SMA and expression of the EMT promoter ZEB1. Taken together, these data show that a very low level of radiative stress in regenerating keratinocyte stem and precursor cells can induce a micro-environment that may constitute a favorable context for long-term carcinogenesis.

Iterative Three-Dimensional Epidermis Bioengineering and Xenografting to Assess Long-Term Regenerative Potential in Human Keratinocyte Precursor Cells.

The functional definition of somatic adult stem cells is based on their regenerative capacity, which allows tissue regeneration throughout life. Thus, refining methodologies to characterize this capacity is of great importance for progress in the fundamental knowledge of specific keratinocyte subpopulations but also for preclinical and clinical research, considering the high potential of keratinocytes in cell therapy. We present here a methodology which we define as iterative xenografting, which originates in the classical model of human skin substitute xenografts onto immunodeficient recipient mice. The principle of this functional assay is first to perform primary xenografts to assess graft take and the quality of epidermal differentiation. Then, human keratinocytes are extracted from primary graft samples to perform secondary xenografts, to assess the presence and preservation of functional keratinocyte stem cells with long-term regenerative potential. In the example of experiments shown, iterative skin xenografting was used to document the high regenerative potential of epidermal holoclone keratinocytes.