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Gary S L Peh, Padmapriya Sathiyanathan, Benjamin L George, Khadijah Adnan, Lawrence W Stanton, Jodhbir S Mehta; Stem cells of the human corneal endothelium – In the peripheral zone or beyond?. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5279.
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© ARVO (1962-2015); The Authors (2016-present)
It has been reported that human corneal endothelial cells (CEnCs) in the periphery showed increased expression of stem cell-associated genes as compared to CEnCs from central cornea1,2. The aim of this study is to determine (1) if CEnCs of the periphery cornea are more proliferative than donor-matched cells from the central of the cornea and (2) to identify potential stem cells of the human corneal endothelium (CE).
Pairs of donor corneas deemed unsuitable for transplantation were procured for this study. Each cornea was lightly marked with the appropriate trephines to form three concentric zones: (1) Zone A – central 6.0mm; (2) Zone B – between 6.0mm to 8.5mm; and (3) Zone C – all areas from 8.5mm and beyond, up to the trabecular meshwork (Figure 1). A brief wash in a solution containing 0.1% Trypan blue demarcated the borders and the Descemet’s membrane (DM) / CEnCs were carefully peeled and pooled accordingly to their concentric zones within each donor-matched pairs. Subsequent isolation and cultures of CEnCs were achieved using a dual media approach3,4. Cultured CEnCs were assessed for cell morphology and cell proliferation via Click-iT EdU. Concurrently, trabecular meshwork mesenchymal stem cells (TM-MSCs) were isolated from cornealoscerla rims following removal of central cornea storma button, established and characterized as described5. Three combinatory factors were used in the differentiation of TM-MSCs towards a CEnC-like phenotype, and further characterized for their expression of CEnCs genes via quantitative real-time PCR.
Proliferation of CEnCs was found to be comparable between all 3 concentric zones (n=3). Interestingly CEnCs isolated from Zone C, which included areas beyond the Schwalbe’s line generated heterogeneous cells that were significantly larger and non-polygonal (p<0.05, n=5). Compared to control, TM-MSCs treated with a combination of factors for 14 days were found to upreguate known CEnCs genes such as COL8 and SLC4A11.
Although CEnCs in the periphery had been previously shown to express stem cell-associated genes as compared to CEnCs from central cornea, proliferation rates remained similar between concentric zones. Cells beyond Schwalbe’s line contained a small subpopulation of cells that forms TM-MSCs, which possess the capacity to differentiate towards a polygonal morphology and express gene transcript indicative of CEnC-like cells.
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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