Proliferation Propensity of Cultured Human Corneal Endothelial Cells and Their Plasticity Dictated by Culture Microenvironments

Abstract

Purpose: It is well known that human corneal endothelium cells (HCECs) have poor proliferative ability under in vitro culture conditions. The difficulty of cultivating HCECs hampers a detailed analysis of their proliferation propensity. To detail molecular mechanisms underlying this impaired HCEC proliferation, we attempted to clarify the presence and proliferation propensity of functionally heterogeneous subpopulations in cultured HCECs.

Methods: The proliferative properties of cultured HCECs were evaluated by BrdU assay and carboxyfluorescein succinimidyl ester (CFSE) dye dilution assay. To investigate if cultured HCECs contain subpopulations with distinct metabolic requirements, HCECs were stained with MitoTracker® Red (Life Technologies Corp., Carlsbad, CA) to evaluate their mitochondria content. Flow cytometry (FCM) using several surface markers was performed to characterize the subpopulations.

Results: Cell percentages in the G1, S, and G2/M phase of the cell cycle were determined by FCM using BrdU and 7-AAD. The percentage of cells in the S- and G2/M-phase was about 40%, and about 60% of the HCECs were arrested in the G1-phase. CFSE assay detected 2 subpopulations with different proliferative properties. One divided 7 times in 8 days of cultivation, while the other stopped cell division at 3 times. We theorize that the latter entered premature cell senescence at the early stage of cultivation, resulting in cell-cycle arrest. These results suggest that each subpopulation has unique metabolic turnover rates and energy requirements, as it is theorized that poor proliferation is tied to energy from mitochondria, not from glucose metabolism. Moreover, FMC using MitoTracker® Red detected different mitochondrial content in the cultured HCECs. Each subpopulation was stained with several cell surface markers selected by global analysis, and we tried to characterize candidate markers for the high-proliferative population.

Conclusions: These findings show that cultured HCECs have different subpopulations and provide the possibility of establishing an effective method for culturing HCECs containing an enriched subpopulation with high proliferation ability.

Keywords: 481 cornea: endothelium