Abstract
Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.
Highlights
Autologous epidermal cultures restore a functional epidermis on burned patients
An important step toward the molecular identification of human keratinocyte clonal types came from the discovery of p63 as a key transcription factor underpinning the proliferative potential of epithelial stem cells[12,13,14,15]
Autologous epidermal cultures have been used for over three decades to produce grafts that restore a functional epidermis on severely burned patients[10,11]
Summary
Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so in genetic skin diseases such as Junctional Epidermolysis Bullosa. Formal evidence of holoclone-forming cells being stem cells able to permanently sustain the human epidermis came from the lifesaving use of transgenic epidermal cultures for combined cell and gene therapy of junctional epidermolysis bullosa (JEB), a severe genetic skin disease. Using proviral integrations as clonal genetic marks, clonal tracing performed on the regenerated transgenic epidermis has formally shown that the human epidermis is sustained solely by self-renewing holoclone-forming cells They continuously generate pools of meroclones and paraclones, which behave as TA progenitors, persist for various periods of time, replenish differentiated cells, and play a crucial role both in the engraftment of epidermal cultures and in epidermal regeneration during wound healing[5]. It has recently been shown that nuclear YAP is a key determinant of human holoclones and its interplay with p63 is essential for sustaining their self-renewal and proliferative/ regenerative capacity[16]
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