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Noelia Kunzevitzky, Karen Alvarez-Delfin, Richard Merkhofer, Alejandra Weisman, Jeffrey Goldberg; The transparency transcriptome: gene expression profile of human corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1698.
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© ARVO (1962-2015); The Authors (2016-present)
The corneal endothelium is a monolayer on the inner cornea whose main function is to maintain corneal transparency. Loss of function due to dystrophy, trauma or genetic abnormalities leads to swelling, pain and loss of vision. Current treatment options include DSAEK or penetrating keratoplasty—both limited by the shortage of donor corneas. Injection of cultured human corneal endothelial cells (HCECs) could provide a less invasive and readily available solution. The protocol for purifying and expanding HCECs in vitro from cadaveric corneas has been extensively described, but the criteria for identifying these cells are not definitive. In order to better define the HCECs’ identity and to ask basic cell biology questions about these cells, we analyzed the gene expression profile of purified HCECs at different numbers of passages in vitro and compared it to the transcriptome of freshly dissected corneal layers.
Human cadaveric corneas preserved in Optisol® were procured by the Lions Eye Institute for Transplant and Research (Tampa, FL). For some corneas, the HCECs were peeled off Descemet’s membrane and cultured. For other corneas, epithelial, stromal and endothelial layers were acutely dissected and minimally processed for RNA extraction. RNA from at least 3 biological replicates was independently collected, amplified and processed for hybridization to Affymetrix GeneChip® arrays. We classified probes by Gene Ontology and compared the gene expression profiles of cultured HCECs and the three corneal layers.
Cultured HCECs expressed ~82% of 28,869 total probes. Further gene ontology analysis revealed the overrepresentation of pathways related to cell survival, cytoskeletal remodeling, oxidative phosphorylation and cell adhesion, among others. We also identified a number of HCEC-specific markers that can be verified via immunostaining and RT-PCR.
We have begun to describe the transcriptome of the cornea, a tissue whose proper function is essential to maintaining visual clarity, yet remains poorly characterized. In doing so we hope to unveil the essential molecular players that regulate cell morphology, survival, and proliferation and to better define HCECs’ identity for further use in cell therapy.
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