Sphere-forming cells from peripheral cornea demonstrate a wound-healing response to injury.

The cornea is the initial refractive interface of the eye. Its transparency is critical for clear vision and is maintained by stem cells which also act to repair injury inflicted by external insults, such as chemical and thermal burns. Damage to the epithelium compromises its clarity and can reduce or eliminate the stem cell population, diminishing the ability for self-repair. This condition has been termed "limbal stem cell deficiency"; severe cases can lead to corneal blindness. Sphere-forming cells isolated from peripheral cornea are a potential source of stem and progenitor cells for corneal repair. When provided with appropriate substrate, these spheres have the ability to adhere and for cells to migrate outwards akin to that of their natural environment. Direct compression injury and remote scratch injury experiments were conducted on the sphere cells to gauge their wound healing capacity. Measures of proliferation, differentiation, and migration were assessed by immunohistochemical detection of EdU incorporation, α-smooth muscle actin expression and confocal image analysis, respectively. Both modes of injury were observed to draw responses from the spheres indicating wound healing processes. Direct wounding induced a rapid, but transient increase in expression of α-SMA, a marker of corneal myofibroblasts, followed by a proliferative and increasing migratory response. The spheres were observed to respond to remote injury as entire units, with no directional response seen for targeted repair over the scratch injury area. These results give strength to the future use of these peripheral corneal spheres as transplantable units for the regeneration of corneal tissue.

Sphere-forming cells from peripheral cornea demonstrate polarity and directed cell migration.

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.

Efficacy and safety assessment of a novel ultraviolet C device for treating corneal bacterial infections.

BACKGROUND: A prototype solid-state Ultraviolet-C (UVC) LED device may be useful in the treatment of corneal microbial infections, as UVC is commonly used for eradicating bacteria, fungi and viruses in other settings. This study assessed the efficacy of 265 nm UVC from this LED, on four different bacterial strains, and investigated the consequences of corresponding exposures on human corneal epithelial cells in vitro. METHODS: Agar plate lawns of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Streptococcus pyogenes were exposed to a 4.5 mm diameter 265 nm UVC beam at a fixed intensity and distance, for 30, 5, 4, 2 and 1 seconds. Growth inhibition was assessed with a BioRad Gel imager, and the diameter of lucent areas of bacterial inhibition recorded. Human corneal epithelial cells cultured on glass cover-slips were exposed to corresponding doses of UVC from the same device. Live/dead staining was performed and the results quantified. RESULTS: There was 100% inhibition of growth for all bacteria tested, at all exposure times. A 30-second exposure of human corneal epithelium to UVC gave no statistically significant decrease (P = 0.877) in the ratio of live to dead cells when compared to control cultures. CONCLUSION: 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.

Sphere-forming corneal cells repopulate dystrophic keratoconic stroma: Implications for potential therapy.

BACKGROUND: Keratoconus is a degenerative corneal disease characterised by aberrant cell behaviour and loss of matrix that can result in vision loss. Cells extracted from peripheral corneas can form stem cell-enriched spheres, which have shown the potential to repopulate the normal peripheral corneal stroma in vitro upon sphere implantation but have not been previously studied in keratoconic tissue. AIM: To investigate the therapeutic potential of stem cell-enriched spheres formed from extracted peripheral human corneal cells when introduced to keratoconic tissue. METHODS: Stem cell-enriched spheres were formed from extracts of normal cadaveric human peripheral corneal cells. These spheres were implanted into incisions created in full thickness and onto the surface of 10 µm thin sections of keratoconic and normal stromal tissues in vitro. Tissue sections were used to maximise use of limited keratoconic tissue available for research. Living cells were stained with Calcein-AM and visualised with stereo and fluorescence microscopy to assess survival and behaviours between the time of implantation day 0 and 14 d (D14) from implantation. Sphere cells in implanted tissues were characterised for stem cell and differentiation markers using immunohistochemistry and droplet digital PCR to assess the potential implications of these characteristics in the use of spheres in keratoconus treatment. RESULTS: Spheres were successfully implanted into full-thickness central corneal tissue and onto the surface of 10 µm thin en face tissue sections. No observable differences were seen in sphere migration, proliferation or differentiation in keratoconic tissue compared to normal between day 0 and D14. Spheres stained positively with Calcein-AM up to D14. Cell migration increased from day 0 to D14, occurring radially in three dimensions from the sphere and in alignment with tissue edges. Cell proliferation marker, EdU, was detected at day 10. Implanted spheres stained positively for putative stem cell markers ∆Np63α and ABCB5, while ABCG2, ABCB5, ∆Np63 and p63α were detectable by droplet digital PCR up to D14. Double immunolabelling revealed absence of ABCB5 staining in migrated cells but positive staining of alpha smooth muscle actin (myofibroblast marker) in some migrated cells. Droplet digital PCR showed similar expression patterns of differentiation markers but a reduction in stem cell markers between normal and keratoconic tissue with an increase in stromal cell markers and a reduction in epithelial cell markers, indicating an appropriate response to repopulating diseased tissue. CONCLUSION: Cells from implanted stem cell-enriched spheres can repopulate a keratoconic corneal stromal surface in a directed manner and exhibit migratory stromal cell phenotypes.