Three-dimensional Skin Equivalents Research Articles

Histidase expression in human epidermal keratinocytes: Regulation by differentiation status and all-trans retinoic acid

Histidase (histidine ammonia lyase) converts histidine into urocanic acid, the main ultraviolet (UV) light absorption factor of the stratum corneum. It is unknown if and how histidase is regulated in the epidermis. We have investigated the transcriptional regulation of histidase expression in epidermal keratinocytes. Human epidermal keratinocytes were cultured in vitro and exposed to UV irradiation, a number of cytokines and all-trans retinoic acid (ATRA) (1 microM). Keratinocyte differentiation was triggered by maintaining confluent cells in monolayer culture and by establishing three-dimensional skin equivalents. The mRNA expression level of histidase in keratinoytes as well as in the epidermis and other tissues was determined by quantitative real-time PCR. Protein expression was determined by Western blot analysis. Human epidermis contained higher levels of histidase transcripts than all other tissues investigated. Expression of histidase strongly increased at the mRNA and protein levels during differentiation of primary keratinocytes in vitro. Treatment of keratinocytes with UVA and UVB did not significantly change the expression level of histidase. By contrast, ATRA suppressed histidase expression almost completely. Our results show that histidase is upregulated during keratinocyte differentiation and that ATRA but not UV irradiation modulates the expression level of histidase. Suppression of histidase-mediated production of urocanic acid may contribute to the increase in UV sensitivity that is caused by treatment with retinoids.

Efficient Gene Transfer to Human Epidermal Keratinocytes on Fibronectin: In Vitro Evidence for Transduction of Epidermal Stem Cells

The epidermis is an attractive target for gene therapy because it is easily accessible and has great potential as an ectopic site for protein delivery in vivo. Genetically modified keratinocytes can be expanded in culture and used to generate three-dimensional skin equivalents, which can deliver therapeutic proteins either locally or systemically for the treatment of wounds or systemic diseases. Here we present an optimum protocol that yields consistently high retroviral gene transfer on a substrate of recombinant fibronectin (rFN). Gene transfer on rFN depends strongly on virus concentration and the density of target cells. Interestingly, the kinetics of gene transfer varies depending upon the origin--mouse or human--of virus-producer cells. Most notably, long-term growth and clonogenic assays show that transduction on rFN promotes gene transfer to epidermal stem cells and prevents loss of clonogenic potential due to exposure of cells to retroviral supernatant. In contrast, the traditional protocol transduces mostly differentiated keratinocytes. We also show that skin equivalents prepared from genetically modified keratinocytes display high levels of transgene expression, mainly in the suprabasal layers. Our results are important for cutaneous gene therapy and for biological studies that require efficient and permanent genetic modification.

Fibroblasts facilitate re-epithelialization in wounded human skin equivalents

The re-epithelialization of the wound involves the migration of keratinocytes from the edges of the wound. During this process, keratinocyte migration and proliferation will depend on the interaction of keratinocytes with dermal fibroblasts and the extracellular matrix. The present study aimed to investigate (1) the role of fibroblasts in the re-epithelialization process and on the reconstitution of the dermal–epidermal junction (DEJ) and (2) differential protein expression during re-epithelialization. For both purposes, three-dimensional human skin equivalents (HSE) were used. A full-thickness wound in HSE was introduced by freezing with liquid nitrogen and a superficial wound by linear incision with a scalpel. The closure of the wound in the absence or presence of exogenous growth factors was followed by monitoring the rate of re-epithelialization and regeneration of the DEJ. The results obtained in this study demonstrate that fibroblasts facilitate wound closure, but they differentially affected the deposition of various basement membrane components. The deposition of laminin 5 at the DEJ was delayed in superficial wounds as compared to the full-thickness wounds. During freeze injury, some basement membrane (BM) components remain associated with the dermal compartment and probably facilitate the BM reconstitution. The re-epithelialization process in full-thickness but not in superficial wounds was accelerated by the presence of keratinocyte growth factor and especially by epidermal growth factor. In addition, we have examined the deposition of various basement membrane components and the differences in protein expression in a laterally expanding epidermis in uninjured HSE. Laminin 5, type IV and VII collagen deposition was decreased in the laterally expanding epidermis, indicating that the presence of these proteins is not required for keratinocyte migration to occur in vitro. Using two-dimensional polyacrylamide gel electrophoresis, we have identified DJ-1, a protein not earlier reported to be differently expressed during the epithelialization process of the skin.

Epithelial-Mesenchymal Interactions Enhance Expression of Collagen VII In Vitro

Expression of collagen VII, the major structural protein of the anchoring fibrils, was assessed in vitro using indirect immunofluorescence staining and immunoblotting of collagen VII isolated from cultures. A very low level of expression was observed in monocultures of normal human fibroblasts or keratinocytes, but the expression was greatly stimulated when fibroblasts and keratinocytes were co-cultured. Primary skin explants under culture conditions supporting growth of both cell types, or mixed co-cultures of purified fibroblasts and keratinocytes, exhibited clearly enhanced synthesis of collagen VII, and the intact tissue form of this collagen could be extracted from small co-cultures. Three-dimensional skin equivalents were constructed with fibroblasts embedded in a contracted gel of collagen I and III, with an overlying stratified keratinocyte epithelium. In these equivalents, expression of collagen VII was observed primarily in the lowest epithelial cells, indicating that these cells are the main manufacturers of collagen VII. Laminin and collagen IV were deposited in a linear fashion onto the epithelial-mesenchymal interface. The results suggest that epithelial-mesenchymal interactions, either through physical interactions and/or through soluble mediators, are necessary for efficient synthesis of collagen VII and biogenesis of the anchoring fibrils.