Cutaneous wound-healing disorders are a major health problem that requires the development of innovative treatments. Whithin this context, the search for reliable human wound-healing models that allow us to address both mechanistic and therapeutic matters is warranted. In this study, we have developed a novel invivo wound-healing model in a genetically modified human context. Our model is based on the regeneration of human skin on the back of nude mice by transplantation of a cultured bioengineered skin equivalent previously designed in our laboratory. In this setting, human keratinocytes in the epidermal compartment were genetically modified with a retroviral vector encoding the enhanced green fluorescent protein (EGFP). After stable engraftment of the EGFP skin was achieved (9-12 wk after grafting), a small circular full thickness wound was performed on this mature human skin. A wide variety of parameters involved in wound healing were monitored, including tissue architecture, cell proliferation, epidermal differentiation, dermal remodelling, and basement membrane regeneration. Wounded gene-targeted skin-humanized mice re-capitulated native skin wound-healing features. In addition, when keratinocyte growth factor (KGF), a growth factor that has been shown to improve wound healing, was added to wounds during 3 d, the re-epithelialization was significantly accelerated. The present wound-healing model system provides a suitable in vivo tool to test gene transfer strategies for human skin repair. It also serves as a complementary platform for studies in genetically modified mice and as a model to evaluate pharmaceutical therapeutic approaches for impaired wound healing.

Killer immunoglobulin-like receptors (KIRs) on natural killer cells (NKs) recognize groups of human leukocyte antigen (HLA) class I alleles. Cells without an inhibitory HLA ligand may trigger NK activation. Reduced risk of relapse has been reported in malignant hematologic diseases after haploidentical transplantation when HLA ligands against the inhibitory KIRs present in the donor were absent in the recipient. We performed haploidentical transplant in three children with refractory solid tumors. Our results showed that beneficial antitumor effects could be observed in the presence of inhibitory KIR-HLA mismatch. These preliminary results suggest a possible association between disease control and NK cell alloreactivity.

Although skin is perhaps the most accessible of all somatic tissues for therapeutic gene transfer, it is a challenging site when attempting gene delivery. In addition to the transience of gene expression, important obstacles to cutaneous gene therapy have included the inability to sustain gene expression in a large proportion of keratinocytes within a given skin compartment. In this study, we have developed a novel experimental strategy that allows long-term regeneration of entirely genetically engineered human skin on the backs of NOD/SCID mice. Primary human keratinocytes were infected with a retroviral vector encoding the enhanced green fluorescent protein (EGFP) produced by transient transfection of 293T cells. EGFP expression allowed cell-sorting selection of a polyclonal population of productively transduced keratinocytes that were assembled in a live fibroblast-containing fibrin dermal matrix and orthotopically grafted onto mice. Epifluorescent illumination of the transplanted zone allowed in vivo monitoring of the genetically modified graft. EGFP-positive human skin was present on mice for 22 weeks after grafting. In addition, frozen sections prepared from the grafts displayed consistently strong EGFP-based fluorescence in all epidermal strata at every time point examined. Persistence of transgene expression was further confirmed through EGFP protein immunodetection. Purified EGFP-positive keratinocytes grafted as part of the fibrin-based artificial skin were capable of generating multilayer human epidermis on mice, with well-developed granulosum and corneum strata, and clearly defined rete ridges. Finally, the large proportion of transduced keratinocytes in our grafts allowed us to study, for the first time, the long-term in vivo clonal reconstitution pattern of the regenerated skin. Analysis of the provirus insertion sites indicates that a discrete number of epidermal stem cell clones was responsible for the maintenance of human skin regenerated in NOD/SCID recipients.

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