Injection of genetically engineered fibroblasts corrects regenerated human epidermolysis bullosa skin tissue

MethodsRDEB cells.Fibroblasts from four unrelated COL7A1 mutant type VII collagen-deficient RDEB patients (3) fulfilling clinical, immunohistological, ultrastructural, and genetic criteria for the disease (11) were grown as described (1).Integrase-based stable integration of the type VII collagen expression plasmid, pCOL7A1, was performed by cotransfecting fibroblasts with a C31 integrase-encoding plasmid and pCOL7A1 as described (3).For selection, 3 days after transfection, cells were subjected to 10 days of blasticidin (4 g/ml) in culture media to yield cells overexpressing type VII collagen (RDEB + cells).Type VII collagen expression was verified by immunofluorescence microscopy and immunoblot analysis using antibodies to human type VII collagen (Calbiochem-Novabiochem Corp., San Diego, California, USA).Animal studies.For fibroblast injection into mouse skin, 6-week-old athymic nude and CB.17 scid/scid mice were injected intradermally with 10 6 fibroblasts resuspended in 100 l PBS using a 30-gauge needle (n = 3 mice/cell group).The injection was performed by first piercing the skin, then directing the needle as superficially as possible back upward toward the surface; this commonly led to formation of a well-demarcated papule in the center of the injected area.Eight to 16 weeks after injection, biopsies and analyses were performed on mouse skin.For human skin studies, skin of RDEB patients and normal controls was generated using either early-passage RDEB keratinocytes