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Kishore Reddy Katikireddy, Thore Schmedt, Yuming Chen, Ula Jurkunas; Stem cell characteristics in distinct subpopulation of human corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2056.
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© ARVO (1962-2015); The Authors (2016-present)
Recently, we identified the existence of morphologically distinct subpopulation of human corneal endothelial cells (HCEnCs) that exhibit enhanced self-renewal competence and lack of phenotypic signs of cellular senescence. The purpose of the study was to identify and compare stem cell (SC) characteristics between distinct subpopulations of HCEnCs.
Three primary HCEnCs were isolated from 21, 56, and 70 year-old male donors by EDTA treatment. Each donor showed a distinct subpopulation of highly proliferative cells (called HCEnC-21, -56, -70) within surrounding slowly proliferating cells. After isolation, distinct subpopulation cells grew more than passage 75, while surrounding cells expanded to passage 5 and senesced. Cell morphology was monitored using phase-contrast microscopy. Gene expression of ABCG2, p63, Oct4, SOX2, Nanog, Rex1, and LGR5 was compared between distinct subpopulation of cells at early (3-10) and late (60-70) passages and slowly proliferating cells using TaqMan® real-time PCR.
HCEnC-21, -56, and -70 exhibited a greater level of regular and hexagonal morphology as compared to surrounding primary cells which were more fibroblast like. Higher expression of ABCG2, p63, Oct4, SOX2, Nanog, Rex1, and LGR5 were observed in HCEnC-21, -56, and -70 as compared to slow-proliferating HCEnCs (p<0.001). There was in increase in p63 (1.4 and 1.9 fold), Oct4 (1.2 and 2.0 fold), and SOX2 (1.4 and 3.8 fold) in early compared to late passages in both HCEnC-21 and -70 respectively (p<0.001). Oct4 expression was 1.5-fold decreased in HCEnC-56, and HCEnC-70 compared to HCEnC-21 of early passages (p<0.001). No significant difference of Rex1 was observed at early, late passages and at different ages. Early passage of HCEnC-21 showed significantly higher expression of LGR5 as compared to HCEnC-56 (3.1 fold, p<0.001) HCEnC-70 (3.6 fold, p<0.001) at early passages (p<0.001).
Morphologically distinct subpopulations of HCEnC are detected in both younger and older donors and demonstrate elevated expression of SC markers when compared to slow proliferating cells. Expression of Oct4, p63, and LGR5 was highest in the early passage of HCEnC-21 cells. Appearance of SC markers at early and late passages signifies the importance of these markers in corneal endothelial cell proliferation and survival.
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