The aim of the present study was to produce a reconstructed human cornea in vitro by tissue engineering and to characterize the expression of integrins and basement membrane proteins in this reconstructed cornea. Epithelial cells and fibroblasts were isolated from human corneas (limbus or centre) and cultured on plastic substrates in vitro. Reconstructed human corneas were obtained by culturing epithelial cells on collagen gels containing fibroblasts. Histological (Masson’s trichrome staining) and immunohistological (laminin, type VII collagen, fibronectin as well as β₁, α₃, α₄, α₅, and α₆ integrin subunits) studies were performed. Human corneal epithelial cells from the limbus yielded colonies of small fast-growing cells when cultured on plastic substrates. They could be subcultured for several passages in contrast to central corneal cells. In reconstructed cornea, the epithelium had 4–5 cell layers by the third day of culture; basal cells were cuboidal. The basement membrane components were already detected after 3 days of culture. Integrin stainings, except for the α₄ integrin, were also positive after 3 days. They were mostly detected at the epithelium-stroma junction. Such in vitro tissue-engineered human cornea, which shows appropriate histology and expression of basement membrane components and integrins, provides tools for further physiological, toxicological and pharmacological studies as well as being an attractive model for gene expression studies.

We have developed an improved method for determining CAT activity directed by stably (transgenic mice) or transiently (tissue culture cell lines) introduced CAT reporter gene constructs. The procedure is based on the use of a new buffer system which considerably increases the stability of the CAT enzyme during the preparation of the crude cell extracts. When compared to other procedures, our method enables an increase of up to 100-fold in the sensitivity of the assay, depending on the transgenic tissue tested. Furthermore, a strong increase (up to 23-fold) was also observed with various promoter/CAT constructs transiently transfected in established tissue culture cell lines. This increase in sensitivity provides a significant reduction in the time required to perform the CAT assay when strong promoters are studied (from 18 to 1 hr) and is also very useful for the analysis of CAT gene expression driven by weak promoters.

The similarity observed with the in vivo wound healing process supports the use of this tissue-engineered model for investigating the basic mechanisms involved in corneal reepithelialization. Moreover, this model may also be used as a tool to screen agents that affect reepithelialization or to evaluate the effect of growth factors before animal testing.

The accumulation of fibronectin (FN) in response to corneal epithelium injury has been postulated to turn on expression of the FN-binding integrin ␣ 5 ␤ 1 . In this work, we determined whether the activity directed by the ␣ 5 gene promoter can be modulated by FN in rabbit corneal epithelial cells (RCEC). The activity driven by chloramphenicol acetyltransferase/␣ 5 promoter-bearing plasmids was drastically increased when transfected into RCEC grown on FN-coated culture dishes. The promoter sequence mediating FN responsiveness was shown to bear a perfect inverted repeat that we designated the fibronectin-responsive element (FRE). Analyses in electrophoretic mobility shift assays provided evidence that Sp1 is the predominant transcription factor binding the FRE. Its DNA binding affinity was found to be increased when RCEC are grown on FN-coated dishes. The addition of the MEK kinase inhibitor PD98059 abolished FN responsiveness suggesting that alteration in the state of phosphorylation of Sp1 likely accounts for its increased binding to the ␣ 5 FRE. The FRE also proved sufficient to confer FN responsiveness to an otherwise unresponsive heterologous promoter. However, site-directed mutagenesis indicated that only the 3 half-site of the FRE was required to direct FN responsiveness. Collectively, binding of FN to its ␣ 5 ␤ 1 integrin activates a signal transduction pathway that results in the transcriptional activation of the ␣ 5 gene likely through altering the phosphorylation state of Sp1.Corneal wounds account for a substantial proportion of all visual disabilities and medical consultations for ocular problems in North America. They can be superficial with damage limited to the epithelium or associated with a deeper involvement of the epithelial basement membrane and of the stromal lamella. Severe recurrent and persistent corneal wounds are most commonly secondary to ocular diseases and damage such as recurrent erosion, mild chemical burns, superficial herpetic infections, neuroparalytic cornea, autoimmune diseases, and stromal ulcerations due to viral or bacterial infections or to severe burns (1). Despite currently available treatments, many of these corneal wounds persist for weeks and months or else recur frequently and can progress to corneal perforation.Tissue repair requires cell migration, proliferation, and adhesion. Cell adhesion and migration in turn require extracellular matrix (ECM) 1 synthesis and assembly. ECM is a complex, cross-linked structure of proteins and polysaccharides. It organizes the geometry of normal tissues. Fibronectin (FN) is an ECM adhesion protein identified as a potential wound healing agent because of its cell attachment, migration, differentiation, and orientation properties (for a review see Refs. 2-4). In the unwounded rat eye, FN is observed by immunohistological staining at the level of the corneal epithelium basement membrane (5-7). Shortly after corneal injury, the basal cells that border the injured area and stromal keratocytes start producing massive amounts of FN (5,[8]...

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A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3) can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes.

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