Epithelial differentiation of human skin equivalents after grafting onto nude mice

Human skin equivalents were developed in vitro with a unidimensionally retracted dermal equivalent made of human type I + III collagen and human MRC5 fibroblasts, and a multilayered epithelium grown in liquid medium from normal human keratinocytes in suspension. We investigated the degree of epidermal differentiation that could be achieved after in vivo grafting onto nude mice by means of light and electronmicroscopy as well as by immunohistochemistry. All transplanted grafts showed a primary take. The grafts formed an epidermis with a stratum corneum and from day 7 to 14 after transplantation a distribution of a 56.5 Kd keratin protein, involucrin, profilaggrin/filaggrin, and MHC-I antigens that was similar to what is noted in normal human epidermis. These data indicate that a full terminal differentiation was only achieved after in vivo transplantation of the cultured epithelium. Pigmentation was present, but no marker of Langerhans cells was seen at 4 weeks. Although there was no evidence of the dermal equivalent after 2 weeks, we noted a strong adherence of the graft to the wound bed, with the presence of type-IV collagen, laminin, and bullous pemphigoid antigen at the dermo-epidermal junction (day 7) and hemidesmosomes, a lamina lucida and a lamina densa (day 30). No epithelial damage was noted in spite of an inflammatory infiltrate in the underlying tissue. This represents a preliminary step in the use of such a skin-equivalent in the treatment of human patients with wounds.