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Munetoyo Toda, Morio Ueno, Asako Hiraga, Kazuko Asada, Monty Montoya, Chie Sotozono, Junji Hamuro, Shigeru Kinoshita; The cell homogeneity is indispensable for regenerative medicine by cultivated human corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5281. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Cultivated human corneal endothelial cells (cHCECs) have an inclination towards cell-state transition (CST) into a senescence phenotype, epithelial-mesenchymal transition (EMT), and transformed fibroblastic cell morphology, thereby hampering their use in the clinical setting. The aim of this study was to identify the subpopulations (SPs) among heterogeneous cHCECs that are devoid of CST and are adaptable for cell-based therapy in the clinical setting.
The presence of SPs in cHCECs was confirmed based on surface cluster determinant (CD) marker expression levels by flow cytometry. CD markers effective to distinguish distinct SPs were selected by analyzing those on established cHCECs with a small cell area and exhibiting a high cell density. ZO-1 and Na+/K+ ATPase, CD200, and human leukocyte antigen expression was then compared among those heterogeneous SPs.
Flow cytometric analyses identified the existence of at least 5 SPs in cHCECs. One of those SPs was found to be nearly identical to fully matured HCECs present in fresh tissues in regard to the expression pattern of CD markers. That SP had a hexagonal-cobblestone shape, expressed ZO-1 and Na+/K+ ATPase, and was around 200 ~ 220 μm2 in size with a cell density of over 2500 cells/mm2. The proportion of the SPs (E-ratio) was inversely paralleled with donor age and decreased during prolonged culture passages.
Fully-matured cHCEC SPs, as defined by flow cytometry, may possibly serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunctions.
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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