Both limbal and central epithelial cells are capable of forming spheres in cultures that have stem cell properties. Central and limbal epithelial cells cannot be differentiated using FACS, but younger donor tissues give rise to greater numbers of large cells with high dye efflux. Therefore, results indicate that human central corneal epithelium contains cells with stem/progenitor properties, and these stem properties decline with age.

PURPOSE. Gap junctions play a major role in corneal wound healing. This study used reproducible models of corneal wound healing to evaluate the effect of a gap junction channel modulator, connexin43 (Cx43) antisense oligodeoxynucleotides (AsODN), on corneal healing dynamics. METHODS. A mechanical scrape wound model was used to evaluate Cx43 AsODN penetration and initial wound reepithelialization 12 hours postsurgery. Thereafter, detailed analyses of corneal edema, inflammation, and healing were performed in an excimer laser surface ablation model. In vivo confocal microscopy determined clinical parameters (edema, haze) and cellular changes (stromal hypercellularity, reepithelialization), whereas histology and immunohistochemistry were used to quantify stromal edema, inflammation, and reepithelialization. RESULTS. Cx43 AsODN penetrated through the hydrophilic stroma where the epithelium had been removed and accumulated in the basal epithelium close to the wound edge. Twelve hours after scrape wounding, Cx43 AsODN-treated eyes showed a significant reduction in wound area compared with the vehicle alone (1.59 Ϯ 0.37 and 2.29 Ϯ 0.58 mm 2 , respectively, P Ͻ 0.01). After excimer laser ablation, stromal edema and inflammation were reduced, with endothelial structures being clearly visible, and reepithelialization rates were again increased in Cx43 AsODN-treated eyes. Histologic analysis confirmed reduced edema in the central wound site and at the periphery of treated corneas (P Ͻ 0.05), whereas immunohistochemistry showed lower Cx43 levels (P Ͻ 0.05), reduced myofibroblast activation, and improved epithelial basal lamina deposition in antisense-treated wounds (P Ͻ 0.01). CONCLUSIONS. Application of Cx43 AsODN to the cornea reduces stromal edema and inflammation, promoting faster wound closure and a more uniform repair of the epithelial basal lamina after laser ablation. (Invest Ophthalmol Vis Sci.

The results confirmed that a 1 second exposure to germicidal UVC from this LED source was sufficient to inhibit microbial proliferation in the four bacterial strains tested in vitro. The literature suggests UVC at this dose could potentially be beneficial in treating corneal surface infections, without causing significant adverse effects, supported by our findings in human corneal epithelium exposed to UVC.

BackgroundThe limbus forms the outer rim of the cornea at the corneoscleral junction and harbours a population of stem cells for corneal maintenance. Injuries to the limbus, through disease or accidents such as chemical injuries or burns, may lead to significant visual impairment due to depletion of the native stem cells of the tissue.MethodsSphere-forming cells were isolated from peripheral cornea for potential use as transplantable elements for limbal stem cell repopulation and limbal reconstruction. Immunocytochemistry, live cell imaging and quantitative PCR were used to characterize spheres and elucidate activity post implantation into human cadaveric corneal tissue.ResultsSpheres stained positively for stem cell markers ∆NP63α, ABCG2 and ABCB5 as well as the basal limbal marker and putative niche marker, notch 1. In addition, spheres also stained positively for markers of corneal cells, vimentin, keratin 3, keratocan and laminin, indicating a heterogeneous mix of stromal and epithelial-origin cells. Upon implantation into decellularized corneoscleral tissue, 3D, polarized and radially orientated cell migration with cell proliferation was observed. Cells migrated out from the spheres and repopulated the entire corneal surface over 14 days. Post-implantation analysis revealed qualitative evidence of stem, stromal and epithelial cell markers while quantitative PCR showed a quantitative reduction in keratocan and laminin expression indicative of an enhanced progenitor cell response. Proliferation, quantified by PCNA expression, significantly increased at 4 days subsequently followed by a decrease at day 7 post implantation.ConclusionThese observations suggest great promise for the potential of peripheral corneal spheres as transplantable units for corneal repair, targeting ocular surface regeneration and stem cell repopulation.

Sphere-forming cells from peripheral cornea represent a potential source of progenitor cells for treatment of corneal degenerative diseases. Control of cellular repopulation on transplantable substrates is important to prevent uncontrolled growth in unfavourable directions. The coordination of cellular outgrowth may be in response to environmental cues and/or cellular signals from other spheres. To investigate this, cell migration patterns were observed following placement of spheres on an adhesive surface. Human peripheral corneal cells were maintained using a sphere-forming assay and their behaviour on collagen substrate recorded by time-lapse imaging. Immunocytochemistry and proliferation assays were used to detect protein expression and cell division. Proliferation assays showed that spheres formed by a combination of cell division and aggregation. Cell division continued within spheres for up to 4 months and was up-regulated when exposed to differentiation medium and collagen substrate. The spheres expressed both epithelial and stromal cell markers. When exposed to collagen; (1) 25% of the spheres showed spontaneous polarised outgrowth. (2) One sphere initially showed polarised outgrowth followed by collective migration with discrete morphological changes to form leading and trailing compartments. (3) A sphere which did not show polarised outgrowth was also capable of collective migration using cell protrusion and retraction. (4) Active recruitment of cells into spheres was observed. (5) Placement of spheres in close proximity led to production of a cell exclusion area adjacent to spheres. Thus peripheral corneal cell spheres are dynamic entities capable of developing polarity and modifying migration in response to their environment.