September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Stem cells of the human corneal endothelium – In the peripheral zone or beyond?
Author Affiliations & Notes
  • Gary S L Peh
    Singapore Eye Research Institute, Singapore, Singapore
    Duke-NUS, Singapore, Singapore
  • Padmapriya Sathiyanathan
    Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
  • Benjamin L George
    Singapore Eye Research Institute, Singapore, Singapore
  • Khadijah Adnan
    Singapore Eye Research Institute, Singapore, Singapore
  • Lawrence W Stanton
    Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
  • Jodhbir S Mehta
    Singapore National Eye Centre, Singapore, Singapore
    Singapore Eye Research Institute, Singapore, Singapore
  • Footnotes
    Commercial Relationships   Gary Peh, None; Padmapriya Sathiyanathan, None; Benjamin George, None; Khadijah Adnan, None; Lawrence Stanton, None; Jodhbir Mehta, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 5279. doi:
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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)

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Abstract

Purpose : 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).

Methods : 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.

Results : 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.

Conclusions : 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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