Retroviral marking identifies grafted autologous keratinocytes in porcine wounds receiving cultured epithelium

Abstract

Cultured epithelial autografts are often applied to wounds with a capacity for regeneration from dermal appendages. It is unclear in these circumstances whether the cultured autografts act merely as a biologic dressing or whether they become incorporated into the new epithelium. We have used retroviral gene transfer techniques to identify autologous keratinocytes in an established porcine model of cultured epidermal (CE) grafting. Porcine keratinocytes were transduced with an MFG-lacZ nls vector produced by the amphotropic packaging line GP+EnvAm12. Transduction rates of 15.1%, in the absence of selection, were achieved by a single passage on γ-irradiated retroviral producers as a feeder layer. Full-thickness wounds were created on Large White pigs and isolated from the surrounding skin by a polytetrafluoroethylene chamber. Wounds were grafted initially with autologous de-epidermized dermis (DED), followed 7 d later by sheets of retrovirally marked or unmarked CE autografts. Two weeks after grafting, the mean area of epithelium was 48.4% in wounds that received CE grafts and 32.3% in wounds that were left as DED alone. The epithelium on DED represents regeneration from dermal appendages. The contribution made by the autograft cells to the new epidermis was demonstrated unequivocally, however, by lacZ-positive areas visible macroscopically on the surface of the excised wound. In cryostat sections through the lacZ-positive areas, retrovirally marked cells were present at both superficial and basal positions in the new epithelium.