Sphere-forming cells from peripheral cornea demonstrate the ability to repopulate the ocular surface

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.

Limbal stem cells: identity, developmental origin, and therapeutic potential

“…This original report and additional studies by independent laboratories have confirmed the presence of ABCB5+ LSC in human and mouse limbal epithelium. [48][49][50][51][52][53] Moreover, our most recent studies utilizing genetic lineage tracing in Abcb5/Cre reporter mice crossed with tdTomato [B6;129S6-Gt(ROSA)26Sor tm14(CAG-tdTo mato)Hze /J] mice, 54 aimed at further dissecting the origin of LSC and their progeny cell fate, identified tdTomato-positive ABCB5-derived progeny within the entire adult mouse corneal epithelium (Figure 2), identifying at the level of genetic lineage tracing an ABCB5-expressing precursor cell that gives rise to self-renewing corneal epithelium during development and regeneration, consistent with the LSC phenotype.…”

Limbal niche cells are a potent resource of adult mesenchymal progenitors

“…24,62 Limbal ESC obtained from young donors may produce better stem cells for clinical therapies. 63 Sphere forming cells are also a promising cell type for stem cell repopulations 30,64 and native LNC may promote the expansion of LEPC. 19 Human corneal stromal SC may survive in stored organ or culture, 26 although biomechanics exhibit a powerful effect on LNC and their growth and differentiation.…”

Defining the Limbal Stem Cell Niche

“…Even if the limbal cells themselves were abated, cells from the central cornea would move towards the limbal area to fill the void that had been created. Taking this data in conjunction with the data above, we then decided to examine the migration properties of the peripheral corneal spheres when transplanted back onto a denuded corneal stroma and adjacent limbus [15].…”