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Nick Di Girolamo, Yas Eghtedari, Alexander Richardson, Kelly Mai, Benjamin Heng, Gilles Guillemin, Denis Wakefield; Characterizing Human Fetal Corneal Epithelial Stem Cells for Ocular Surface Reconstruction. Invest. Ophthalmol. Vis. Sci. 2016;57(12):882.
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© ARVO (1962-2015); The Authors (2016-present)
Limbal stem cell deficiency (LSCD) is a challenging disease to treat. A myriad of ocular and non-ocular cells have been transplanted to replenish the patient’s depleted, damaged or diseased stem cell (SC) pool; however graft failure is still unacceptably high. We postulated that the fetal cornea harbors SCs with inherent properties comparable to their adult counterparts.
Immunofluorescence was performed on corneal sections and flat-mounts. Cells were expanded from fetal and adult corneal explants under several culture conditions, and qRT-PCR and colony forming efficiency assays were employed to establish their phenotypic and functional properties. In total, >60 different corneas were used in these analyses.
Standard histological staining with H&E was used to identify the rudimentary SC niche (otherwise referred to as the limbal dome) in fetal corneas. Keratin-(K)-14, a putative adult limbal epithelial SC marker, was used to map the spatiotemporal distribution of precursor cell activity across the developing cornea, divulging a restricted pattern of vertical and horizontal expression. K14 was initially widespread in superficial epithelia across the entire cornea, with staining confined to basal limbal epithelium with increasing gestational age. K14 was co-expressed with αv-integrin in both fetal and adult corneas, and in 4% of cultured corneolimbal epithelia. qRT-PCR disclosed the enhanced expression of K14 mRNA over αv-integrin in both fetal and adult cells, and colony forming efficiency was not statistically different in cells from the developing and mature cornea. Finally, both cell types were adherent, grew exceptionally well, and maintained a K14 phenotype on therapeutic contact lenses, a substrate we previously used to deliver corneal cells to patients with LSCD.
This study provides valuable insights into the development of the human cornea, including the formation of the SC repository, the distribution of these cells across the ocular surface, and a preliminary attempt at harnessing, phenotyping and functionally characterizing these cells for potential clinical applications.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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