Genetically modified keratinocytes transplanted to wounds reconstitute the epidermis.

Abstract

At 60-801% confluence keratinocyte cultures were assigned as controls or for gene transfer by infection with one or the other of two recombinant retrovirus vectors: (i) an amphotropic helper-free murine leukemia virus vector, MFG-lacZ, containing the (-galactosidase gene, lacZ (R.C.M., unpublished), or (ii) a vector, a-SGC-hGH, containing the gene sequence from hGH (L. Cohen and R.C.M., unpublished). Subconfluent keratinocyte cultures were incubated on 3 consecutive days with the retrovirus in Polybrene (8 pg/ml) and 10% fetal bovine serum containing Dulbecco's modified Eagle's medium (DMEM; GIBCO).During transfection the modified Waymouth medium was removed and replaced daily by an equal amount of DMEM containing the retrovirus at a concentration of 1-2 x 107 plaque-forming units/ml. After 6 hr the virus-containing medium was removed and replaced by Waymouth medium. This procedure was repeated on 3 consecutive days. At confluence, keratinocytes were released from the flasks with 0.01% trypsin and resuspended in normal unbuffered saline (0.9%O) containing 100 international units ofpenicillin and 100 pg of streptomycin per ml, at a concentration of 3 x 106 cells per ml. Keratinocytes resuspended in saline were found to be >90% viable, as determined by trypan blue staining.The procedure for making 170 reproducible standardized f-thickness skin wounds on the backs of 13 anesthetized pigs for these experiments was as follows. Under aseptic conditions using a 2.25-cm2 square template the skin was excised with a scalpel to the level ofthe panniculus carnosus muscle with careful hemostasis. Wound margins were tattooed with India ink, allowing for photoplanimetric evaluation of the surface area on sequential standardized photographs. The chambers, serving as an in vivo cell culture device, were applied to each wound (1, 4). The chamber (P. A. Medical, Columbia, TN) consists of a flexible transparent vinyl top bonded to an adhesive base. The base has a central opening fitting the wound margins. Chambers were filled with 1.2 ml of isotonic saline containing 100 pg of streptomycin and 100 international units penicillin per ml (1, 4). Twenty-two wounds were treated with lacZ transfected Abbreviations: hGH, human growth hormone; H&E, hematoxylin/ eosin; X-Gal, 5-bromo-4-chloro-3-indolyl -D-galactoside. t~o whom reprint requests should be addressed. 9307The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Liposomal IGF-1 gene transfer modulates pro- and anti-inflammatory cytokine mRNA expression in the burn wound

“…18,19 Other gene transfer technologies rely on particle-mediated transfer ('gene gun'), microseeding techniques, or liposomes. [19][20][21] Cholesterol containing cationic liposomes have been efficiently and safely used to deliver a CMV promoterdriven IGF-1-cDNA construct into the burn wound to stimulate re-epithelialization. 22,23 The healing effects of IGF-1 have been attributed to mitogenic effects on fibroblasts and keratinocytes, anti-apoptotic effects via phosphatidylinositol-3 kinase signaling pathway, 24,25 and the stimulation of local collagen formation.…”

Nonviral transfer of genes to pig primary keratinocytes. Induction of angiogenesis by composite grafts of modified keratinocytes overexpressing VEGF driven by a keratin promoter

“…28,44 Using these cells, we have been able to introduce stably genetic material by lipid-mediated transfection. A low concentration G418 selection step for 15-20 days gave rise to a G418-resistant population, with 40-70% of these cells expressing high EGFP and VEGF levels, as determined by FACS and ELISA analysis, respectively.…”

The Production and Applications of Genetically Modified Skin Cells

“…The use of reporter gene transfer to monitor the fate of ill vitro cultured keratinocytes returned to an ill vitro or ill vivo wound has been studied by many groups (Garlick and Taichman, 1992;Garlick and Taichman, 1993;Setoguchi et (/1., 1994;Stockschlader et al, 1994;Vogt et al, 1994;Jensen et al, 1994;Ng el al., 1997;Mackenzie, 1997;Kolodka et a/., 1998). This has been seen as particularly important since the keratinocyte 'stem cell' has yet to be isolated and its ill vivo position has not been convincingly mapped.…”