Laminin 332-Dependent YAP Dysregulation Depletes Epidermal Stem Cells in Junctional Epidermolysis Bullosa

Laminin 332-deficient junctional epidermolysis bullosa (JEB) is a severe genetic skin disease. JEB is marked by epidermal stem cell depletion, the origin of which is unknown. We show that dysregulation of the YAP and TAZ pathway underpins such stem cell depletion. Laminin 332-mediated YAP activity sustains human epidermal stem cells, detected as holoclones. Ablation of YAP selectively depletes holoclones, while enforced YAP blocks conversion of stem cells into progenitors and indefinitely extends the keratinocyte lifespan. YAP is dramatically decreased in JEB keratinocytes, which contain only phosphorylated, inactive YAP. In normal keratinocytes, laminin 332 and alpha 6 beta 4 ablation abolish YAP activity and recapitulate the JEB phenotype. In JEB keratinocytes, laminin 332-gene therapy rescues YAP activity and epidermal stem cells in vitro and in vivo. In JEB cells, enforced YAP recapitulates laminin 332-gene therapy, thus uncoupling adhesion from proliferation in epidermal stem cells. This work has important clinical implication for ex vivo gene therapy of JEB

Single-keratinocyte transcriptomic analyses identify different clonal types and proliferative potential mediated by FOXM1 in human epidermal stem cells

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

Regeneration of the entire human epidermis using transgenic stem cells

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, lifethreatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies