Marco Fraccaro Lecture - Life-saving regeneration of the entire human epidermis by transgenic stem cells

Laminin beta3-deficient generalized Junctional Epidermolyis Bullosa is the first genetic disease targeted by transplantation of epidermal cultures originated from transgenic epidermal stem cells. Three patients were treated with autologous epidermal cultures transduced with a MLV-derived retroviral vector carrying the LAMB3 cDNA under the control of the viral LTR. Several skin biopsies were taken from each patients to perform histological analysis, immunofluorescence, in situ hybridization and genome-wide analysis of the retroviral integration sites. The regenerated epidermis was normal-looking, remained mechanically stable throughout the entire follow-up period and did not form blisters, even upon shear force. We observed a proper expression and location of laminin 332 in the basal lamina. In situ hybridization performed using vector-specific LAMB3 probes showed homogenous expression of LAMB3 mRNA in all epidermal layers, confirming that the regenerated epidermis consists only of transgenic keratinocytes. Histological analysis showed a normal and fully differentiated epidermis with a normal dermal-epidermal junction. Electron Microscopy confirmed the presence of well-defined, organized hemidesmosomes comparable to those of healthy controls. In particular, we report life-saving regeneration of the entire epidermis on a seven-year-old JEB child. 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.

Michele De Luca, MD is Director of the Centre for Regenerative Medicine “Stefano Ferrari”, University of Modena and Reggio Emilia, and Scientific Director of Holostem Terapie Avanzate S.r.l. Michele De Luca is internationally recognized as a leading scientist in epithelial stem cell biology aimed at clinical application in regenerative medicine and played a pivotal role in epithelial stem cell-mediated cell therapy and gene therapy. Beside his work on the use of human epidermal stem cell cultures in life-saving treatment of massive full-thickness burns, he, and his principal collaborator Graziella Pellegrini, were first in establishing human urethral epithelia stem cell cultures aimed at urethra regeneration in posterior hypospadias and human limbal stem cell culture aimed at corneal regeneration in patients with severe limbal stem cell deficiency. This latter treatment leads to recovery of vision in patients with poor alternative therapy and was recently approved as advanced therapy medicinal product by the European Medicine Agency with the name of Holoclar®. He is currently coordinating several ex-vivo epidermal stem cell-mediated combined cell and gene therapy clinical trials for genetic skin diseases as Epidermolysis Bullosa. During these studies, Michele De Luca and Graziella Pellegrini reported life-saving regeneration of the entire human epidermis of a Junctional EB patient by means of transgenic epidermal stem cells.