LAMB3-dependent generalized Junctional Epidermolysis Bullosa (JEB) was targeted by transplantation of epidermal cultures originated from transgenic epidermal stem cells. We report life-saving regeneration of the entire epidermis on a seven-year-old JEB child suffering from a devastating form of JEB. The regenerated transgenic epidermis remained stable throughout the entire follow-up period and did not form blisters, even upon shear force. 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 by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew and produce short-lived progenitors that replenish terminally differentiated keratinocytes. In studying the different behaviour of JEB and COL7A1-dependent generalized Dystrophic EB (RDEB) cultures we discovered a pivotal role of YAP in sustaining human epidermal stem cells, which explains the progressive stem cell loss observed in JEB. Epidermal stem cell depletion of primary JEB keratinocytes is due to perturbation of the YAP/TAZ pathway and consequent alteration of the expression of FOXM1. YAP/TAZ and FOXM1 expression is significantly decreased in JEB keratinocytes, which do not contain nuclear YAP but only phosphorylated, inactive YAP. The JEB phenotype is recapitulaled by Laminin 5 ablation and consequent YAP/TAZ down-regulation in normal cells. Restoration of adhesion properties by Laminin 5-gene therapy rescues normal nuclear levels of YAP/TAZ and clonogenic potential. Both enforced YAP and FOXM1 recapitulate Laminin 5-gene therapy in JEB cells, thus uncoupling adhesion from proliferation in epidermal stem cells. This work has important clinical implication for an efficient ex vivo gene therapy of JEB.
