Limbal stem cells: Central concepts of corneal epithelial homeostasis

A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent stud-ies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal

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 10μm 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 10μm 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 markersΔNp63αand ABCB5,while ABCG2,ABCB5,ΔNp63 and p63αwere 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.