Genetically modified keratinocytes transplanted to wounds reconstitute the epidermis (original) (raw)
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Culture of Keratinocytes for Transplantation Without the Need of Feeder Layer Cells
Cell Transplantation, 2007
Patients with large burn wounds have a limited amount of healthy donor skin. An alternative for the autologous skin graft is transplantation with autologous keratinocytes. Conventionally, the keratinocytes are cultured with mouse feeder layer cells in medium containing fetal calf serum (FCS) to obtain sufficient numbers of cells. These xenobiotic materials can be a potential risk for the patient. The aim of the present study was to investigate if keratinocytes could be expanded in culture without the need of a feeder layer and FCS. Keratinocytes were cultured on tissue culture plastic with or without collagen type IV coating in medium containing Ultroser G (serum substitute) and keratinocyte growth factor (KGF). An in vitro skin equivalent model was used to examine the capacity of these cells to form an epidermis. Keratinocytes in different passages (P2, P4, and P6) and freshly isolated cells were studied. Keratinocytes grown on collagen type IV were able to form an epidermis at higher passage numbers than cells grown in the absence of collagen type IV (P4 and P2, respectively). In both cases the reconstructed epidermis showed an increased expression of Ki-67, SKALP, involucrin, and keratin 17 compared to normal skin. Only 50,000 keratinocytes grown on collagen type IV in P4 were needed to form 1 cm 2 epidermis, whereas 150,000 of freshly isolated keratinocytes were necessary. Using this culture technique sufficient numbers of keratinocytes, isolated from 1 cm 2 skin, were obtained to cover 400 cm 2 of wound surface in 2 weeks. The results show that keratinocytes can be cultured without the need of a fibroblast feeder layer and FCS and that these cells are still able to create a fully differentiated epidermis. This culture technique can be a valuable tool for the treatment of burn wounds and further development of tissue engineered skin.
Use of gene-modified keratinocytes and fibroblasts to enhance regeneration in a full skin defect
Langenbeck's Archives of Surgery, 2011
Background With the development of cell-based gene transfer techniques, genetically modified human keratinocytes (Kc) and fibroblasts (Fb) have been proven to be a better choice in wound repair. Methods This study was designed to construct in one step a gene-modified artificial skin by a genetically engineered Kc expressing PDGF-BB and Fb expressing VEGF 165 and bFGF. The wound healing effect in a full-thickness wound model was then observed. Unmodified artificial skin served as control. On the post-operative days 7, 14, and 21, residual wound area was calculated and skin wound tissues were subjected to biopsy for further investigation. Results Compared with unmodified artificial skin, genemodified artificial skin resulted in a reduced wound contraction and a well-organized human epidermis and better formed dermis.
Transplantation of keratinocytes in the treatment of wounds
The American Journal of Surgery, 1995
Keratinocyte grafting can be used to treat acute traumatic and chronic non-healing wounds. The keratinocyte sheets are fragile and clinical "take" is difficult to assess, especially as activated keratinocytes secrete many growth factors, which have effects on wound healing apart from take. We have developed animal models of grafting that allow us to examine factors influencing autologous keratinocyte graft take. Results show clearly that pretreatment of the wound bed with viable dermis greatly increases the take of keratinocyte grafts. International literature. As a greater understanding of the complex interactions of cell and matrix evolve, so will potential therapeutic maneuvers, not just in the field of cultured keratinocyte grafts, but clearly in that of benign tumors, for example, keloids, and that of oncology. There is now overwhelming evidence of the requirement for a dermal substitute for cultured keratinocyte autografts, and the sheet complexity of the situation demands that this should approximate live human dermis as closely as possible. The stumbling blocks relate to avoiding the risks of viral transmission, tissue matching of host and donor, providing early epithelial cover, and improving delivery systems for fragile keratinocyte grafts.
Journal of Investigative Dermatology, 1993
This study compares two techniques for making cultured skin substitutes: a composite graft made of human fibroblasts and keratinocytes on a collagen-glycosaminoglycan membrane (CG) and a cultured epidermal cell sheet graft (CEG), without a dermal component. The "take" and quality of these cultured skin substitutes were evaluated by placing them on full-thickness, excised wounds of athymic mice. These cultured skin substitutes were placed onto 2-X-2em wounds created on athymic mice. Mice were sacrificed at days 10, 20, and 42 with histologic sections obtained for light, electron, immunofluorescent, a?d immunohistoche~ ical microscopy. "Take" was determmed separately by a dIrect immunofluorescent stain for human leukocyte ABC antigens. There were ten mice of each graft type with at least two animals sacrificed at each time point. Results showed positive "take" for all animals. Grossly, T he use of cultured skin substitutes in burn care and reconstructive surgery is expanding. Two key factors have shaped the design of these cultured grafts: the ability to grow keratinocytes in vitro and the inc~eas ing practice of early wound excision in the extensively burned patient. Medawar reported the first successful isolation of buman keratinocytes in 1941 [1], but it was not until 1975 that Rheinwald and Green reported the growth of human keratinocytes at clonal densities on a feeder layer [2]. This achievement, the culturing of a great number of epidermal cells with dividing times under 24 h, meant that enough keratinocytes could be grown in 3-4 weeks to cover a whole body surface area. Further work in this field over the last 15 years has led to decreased culture times and the formation of serum-free systems [3-6] that can match the growth of the previous serum-based systems. The second major step leading to more experimentation with permanent biologic wound coverings was the increased use of early excision of the burn wound with major burn victims. Several centers Manuscript
Wound Repair and …, 2000
Gene therapy promises the potential for improved treatment of cutaneous wounds. This study evaluated whether genetically modified cultured skin substitutes can act as vehicles for gene therapy in an athymic mouse model of wound healing. Human keratinocytes and fibroblasts were genetically engineered by retroviral transduction to overexpress human platelet-derived growth factor-A chain. Three types of skin substitutes were prepared from collagen-glycosaminoglycan substrates populated with fibroblasts and keratinocytes: HF-/HK-, containing both unmodified fibroblasts and keratinocytes; HF-/HK+, containing unmodified fibroblasts and modified keratinocytes; and HF+/HK-, containing modified fibroblasts and unmodified keratinocytes. Skin substitutes were cultured for two weeks before grafting to full-thickness wounds on athymic mice. The modified skin substitutes secreted significantly elevated levels of platelet-derived growth factor throughout the culture period. Expression of retroviral platelet-derived growth factor-A mRNA was maintained after grafting to mice, and was detected in all HF-/HK+ grafts and one HF+/HK-graft at two weeks after surgery. Although no differences were seen between control and modified grafts, the results suggest that genetically modified cultured skin substitutes can be a feasible mechanism for cutaneous gene therapy. The cultured skin model used for these studies has advantages over other skin analogs containing only epidermal cells; because it contains both fibroblasts and keratinocytes, it therefore offers greater opportunities for genetic modification and potential modulation of wound healing.
Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society
Stable closure of skin wounds with engineered skin substitutes (ESS) requires indefinite mitotic capacity to generate the epidermis. To evaluate whether keratinocytes in ESS exhibit the stem cell phenotype of label retention, ESS (n = 6-9/group) were pulsed with 5-bromo-2'-deoxyuridine (BrdU) in vitro, and after grafting to athymic mice (n = 3-6/group). Pulse and immediate chase in vitro labeled virtually all basal keratinocytes at day 8, with label uptake decreasing until day 22. Label retention in serial chase decreased more rapidly from day 8 to day 22, with a reorganization of BrdU-positive cells into clusters. Similarly, serial chase of labeled basal keratinocytes in vivo decreased sharply from day 20 to day 48 after grafting. Label uptake was assessed by immediate chases of basal keratinocytes, and decreased gradually to day 126, while total labeled cells remained relatively unchanged. These results demonstrate differential rates of label uptake and retention in basal kera...
Differentiation, 1998
Cultured epithelial autografts (CEA) derived from sole skin were transplanted to full-thickness wounds excised to muscle fascia over a variety of diverse body sites in 12 pediatric patients treated for acute burns or giant congenital nevi. The skin regenerated from the grafts was biopsied from 7 days to 6 years after grafting. The resultant epidermal phenotype was analyzed histologically and by immunohistochemical localization of keratin 9 (K9) as objective evidence of sole-type site-specific differentiation. Expression of K9 was also verified by onedimensional gel electrophoresis of epidermal cytoskeletal extracts and K9 immunoblot analysis. Grafts prepared from epidermis of axilla, groin or foreskin and transplanted to wounds of comparable depth in an identical manner in the same patients served as controls of postgrafting differentiation. Biopsies of sole skin from amputation specimens from patients of comparable age served as normal positive controls, and biopsies of nonsole skin from patients of comparable age served as normal negative controls. As early as 2 weeks postgrafting, the histologic appearance of sole-derived CEA differed substantively from that of axilla-or groin-derived CEA controls and displayed a phenotype characteristic of sole skin with a thick compact stratum corneum, a thick stratum granulosum, and a distinct stratum lucidum. In sole-derived grafts rete ridges regenerated within 2 months postgrafting, whereas nonsole-derived grafts required 4-6 months for rete ridge regeneration. Once acquired, the sole skin phenotype was maintained long-term by all sole-derived CEA. In vitro, sole-derived keratinocytes synthesized little, if any, K9. However, within 7 days after grafting, K9 synthesis by multiple suprabasal keratinocytes was seen within the epidermis regenerated from sole-derived CEA. Protein of K9 appeared progressively more diffuse throughout the suprabasal layers, attaining a confluent pattern of expression comparable to normal controls of sole skin by 6 to 12 months postgrafting, and the confluent pattern of suprabasal K9 synthesis was maintained long-term. The results demonstrate that site-specific differentiation is an intrinsic property of postnatal human keratinocytes and can be expressed and maintained in a permissive environment in the absence of dermal tissue.& b d y :
The Keio Journal of Medicine, 1990
An optimum culture condition was established for our keratinocyte cell culture system from human adult skin using 3T3 feeder cells. Calcium ion (Ca++) concentration was found to be critical and cells grew best at the Ca++ concentration of 0.2mM. Keratinocyte proliferation was promoted when 0.4ƒÊg/ml hydrocortisone and 7ƒÊg/ml insulin were added. However, epidermal growth factor, cholera toxin and transferrin did not show stimulating effects on multiplication of human keratinocytes in our culture system. The epidermal sheets grown in vitro under this optimized condition were transplanted onto athymic mice, and a short term of take was observed.
Gene Therapy, 1999
Cultured epithelial grafts have proven to be life-saving in describe a protocol consisting of lipid-mediated transfecthe treatment of large skin losses. It has become apparent tion, G418 selection and an enhanced green fluorescence that one of the main difficulties of this technology is the protein (EGFP)-based enrichment step for obtaining high overall poor take of the grafts as a consequence of sever-levels of transgene-expressing primary keratinocytes. ely damaged dermal beds. Skin substitutes providing both Using this protocol, the cDNA for vascular endothelial cultured keratinocytes, as an epidermal layer, and a dermal growth factor (VEGF), a potent endothelial cell mitogen analogous offer a more suitable material for skin repair. driven by the 5.2 kb bovine keratin K5 promoter, was stably Ex vivo transfer of stroma regeneration-promoting genes transfected into pig primary keratinocytes. Genetically to keratinocytes appears to be an attractive strategy for modified keratinocytes, expanded on live fibroblast-conimproving the therapeutic action of these grafts. The use taining fibrin gels and transplanted to nude mice as a comof epidermal-specific promoters as expression drivers of posite material, elicited a strong angiogenic response in exogenous genes results in both high expression levels the host stroma as determined by fresh tissue examination and stratum specificity, as shown in transgenic mice stud-and CD31 immunostaining. Since the formation of a wellies. Most current gene transfer protocols to primary kera-vascularized wound bed is a crucial step for permanent tinocytes involve transduction of epidermal cells with retro-wound closure, the use of an 'angiogenic' composite viral vectors. However, transfer of gene constructs material may improve wound bed preparation and coverharboring these long DNA fragment promoters cannot be age with cultured keratinocyte grafts. achieved through viral transduction. In this paper, we
Wound Repair and Regeneration, 2007
The concept of using growth factor therapy to induce wound repair has been endorsed in studies that show reduced growth factors in wound fluid from chronic and aged wounds. In this study, we used cell suspensions of allogenic keratinocytes as gene-delivery vehicles for human epidermal growth factor (hEGF) and analyzed their impact on wound repair in a porcine wound-healing model. Full-thickness wounds were created on the backs of six Yorkshire pigs and covered with a wound chamber to create a wet wound-healing environment. First, 5Â10 5 allogenic, autogenic, or mixed keratinocytes were transplanted into wounds and healing parameters were analyzed. Second, we measured long-term reepithelialization and contraction rates from day 8 until day 35. In the third experiment, allogenic keratinocytes were transfected with an hEGF-expressing plasmid pCEP-hEGF and transplanted in full-thickness wounds to improve repair. Wounds treated with autogenic, allogenic, or mixed keratinocytes showed a significantly higher rate of reepithelialization relative to saline-treated control wounds. Repetitive biopsies indicated that the use of allogenic keratinocytes did not lead to long-term wound breakdown. Wounds treated with hEGF-expressing allogenic keratinocytes reepithelialized faster than wounds treated with allogenic keratinocytes or control wounds. With a peak hEGF expression of 920.8 pg/mL, hEGF was detectable until day 5 after transplantation compared with minimal hEGF expression in control wounds. This study shows that allogenic keratinocytes can serve as efficient gene transfer vehicles for ex vivo growth factor delivery to full-thickness wounds and overexpression of hEGF further improves reepithelialization rates.