Abstract
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.
Highlights
The limbus forms the outer rim of the cornea at the corneoscleral junction and harbours a population of stem cells for corneal maintenance
In-vitro sphere characterization Peripheral corneal spheres were initially characterized in vitro to confirm they were stem-cell enriched and possessed the ability to respond to a collagen matrix with cell migration and division
Spheres immobilized on poly-L-lysine-coated dishes stained positively for putative limbal stem cell markers ΔNp63α, ABCG2 and the recently proposed limbal stem cell marker ATP-binding cassette sub-family B member 5 (ABCB5) [32] (Fig. 1a–c) when compared with the background fluorescence emitted by the secondary antibody only (Fig. 1g), primary antibody only and no antibody controls
Summary
The limbus forms the outer rim of the cornea at the corneoscleral junction and harbours a population of stem cells for corneal maintenance. The limbal epithelial crypts (long extensions of the interpalisadal rete ridges) described relatively recently have been shown to be potential reservoirs of stem cells [6]. Mathan et al Stem Cell Research & Therapy (2016)7:8 diseases, chemical injuries and thermal burns [7] can damage the limbal environment and deplete the stem cell population therein, impairing the regenerative capacity of the cornea leading to redness and pain, persisting epithelial defects, corneal vascularization, conjunctivalization and severe visual impairment or blindness. Severe depletion of stem cells within the limbal environment can lead to a condition known as limbal stem cell deficiency (LSCD)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
