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Alena Bartakova, Karen Alvarez-Delfin, Enrique Salero, Alejandra D Weisman, Noelia J Kunzevitzky, Jeffrey L Goldberg; Human Corneal Endothelial Cell Characterization For Cell Therapy. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2038.
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The corneal endothelium regulates corneal hydration and transparency. Decrease in human corneal endothelial cell (HCEC) density and function occurs with aging, and is accelerated after ocular surgery or trauma, leading to loss of function, corneal edema, decreased vision, and pain. Surgical keratoplasty is limited by expense, availability of donor corneas, and access to trained specialists. Here we characterize human corneal endothelial cells (HCECs) in vitro in search of a subpopulation optimized towards cell therapy.
HCECs were isolated from cadaveric donor corneas, cultured and further characterized for morphology and expression of cell surface markers by RT-PCR, immunofluorescence and flow cytometry. Cell function was assessed by measuring transepithelial electric resistance (TEER).
In vitro, HCECs exhibited polygonal, canonical morphology in early passages; later they became more fibroblastic. This endothelial-mesenchymal transition was related to the quality of donor tissue: corneas from healthy donors remained canonical for a higher number of passages compared to corneas from unhealthy donors. Immunofluorescence showed common markers such as zonula occludens-1 (ZO1) and Na+/K+ ATPase expressed in canonical-looking HCECs; in fibroblastic-looking cells, their expression disappeared, with increase in α-smooth muscle actin (α-SMA). Other markers, selected after microarray analysis and tested by flow cytometry, differed between canonical and fibroblastic HCECs, permitting identification and sorting of these two cell populations. To assess functional ability to form an endothelial barrier, TEER was measured and showed a marked difference between the two morphologies: canonical cells demonstrated high resistance compared to fibroblastic-looking cells.
We have identified a subset of HCEC markers that allow for selection of high-functioning HCECs, based on morphology and ability to form a tight barrier. Such markers may enhance efficacy in future pre-clinical and clinical cell therapy studies.
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