Genetically Modified Human Epidermis Overexpressing PDGF-A Directs the Development of a Cellular and Vascular Connective Tissue Stroma When Transplanted to Athymic Mice–Implications for the Use of Genetically Modified Keratinocytes to Modulate Dermal Regeneration

Abstract

We investigated the hypothesis that keratinocyte-produced platelet-derived growth factor-AA (PDGF-AA) is involved in epidermal-dermal interactions and that PDGF-AA is an important mediator of the temporal and spatial events of tissue repair. Retroviral-mediated gene transfer was used to introduce the gene encoding human PDGF-A into cultures of human diploid keratinocytes. Genetic modification boosted the endogenous in vitro level of PDGF-AA secretion by over 300 fold. When PDGF-secreting cells were transplanted as epithelial sheets to athymic mice, modified keratinocytes underwent terminal differentiation and generated a stratified epithelium comparable to unmodified cells. Seven days after grafting the newly synthesized connective tissue layer subjacent to the PDGF-A-modified grafts was significantly thicker, was rich in mononuclear cells and fibroblasts, and had increased numbers of blood vessels when compared to control grafts of unmodified cells. These results suggest that PDGF-AA secreted by the epidermis is an important mediator of epithelial-mesenchymal interactions and helps to promote growth and vascularization of the underlying dermal tissue. Further, these data demonstrate the feasibility of using genetically modified cells to modulate tissue regeneration.

Evaluation of Transplanted Tissue-Engineered Oral Mucosa Equivalents in Severe Combined Immunodeficient Mice

“…[35][36][37] Viac and others 37 showed that VEGF expression in the epithelium could be modified according to the stage of cell differentiation and during rapid growth or activation of keratinocytes. In cultured keratinocytes, the amount of cell-associated and secreted VEGF increased with time, and was released extracellularly, after its production (data not shown).…”

Alternative Proteolytic Processing of Hepatocyte Growth Factor during Wound Repair

“…34 Briefly, to initiate primary cultures, keratinocytes were cocultivated with 3T3-J2 mouse fibroblasts, pretreated with mitomycin C (Sigma, Germany). Keratinocyte growth medium was changed every 3-4 days with a 3:1 mixture of Dulbecco's modified Eagle's medium (Gibco, Karlsruhe, Germany) and Ham's F12 (Gibco, Karlsruhe, Germany), supplements were added as previously described.…”

“…Keratinocyte growth medium was changed every 3-4 days with a 3:1 mixture of Dulbecco's modified Eagle's medium (Gibco, Karlsruhe, Germany) and Ham's F12 (Gibco, Karlsruhe, Germany), supplements were added as previously described. 34 Cells were subcultured by first removing the feeder layer cells with a brief EDTA wash and then treating keratinocytes with trypsin-EDTA. Madin-Darby canine kidney cells were cultured in Dulbecco's modified Eagle's medium (low glucose) (Gibco, Karlsruhe, Germany) containing 10% fetal calf serum (Perbio, Bonn, Germany) and penicillin/streptomycin (Biochrom AG, Berlin, Germany).…”

RETRACTED: Gene therapy and wound healing

“…Genetically modified keratinocytes were shown to synthesize and secrete high levels of transgenes in vitro. When modified cells were transplanted as an epithelial sheet to athymic mice, PDGF-A expressing cells promoted the development of a granulation tissue in the adjacent dermal tissue (105). IGF-1 expressing keratinocytes increased the proliferation of modified cells, demonstrating that genetic modification can be used to modify the autocrine control of keratinocyte proliferation (106).…”