Identification of differentiated mature cultured human corneal endothelial cells and their distinct cell propensity from other immature subpopulations

Abstract

Purpose: Human corneal endothelium cells (HCECs) have poor proliferative ability under in vitro culture conditions. Tendency to enter into cell senescence during cultivation hampers detailed analysis of their differentiation potential. To detail molecular mechanisms underlying the impaired proliferation of HCECs, we attempted to clarify the presence of functionally heterogeneous subpopulations in cultured HCECs based on their cell surface markers.

Methods: Each subpopulation was stained with several cell surface markers selected by global analysis and to characterize candidate markers for the fully differentiated HCECs. Expression of LGR5, Endoglin, CD166, and 5 other CDAgs were analyzed in HCECs subpopulations, either fully differentiated or partly phase transitioned (EMT, cell senescence, fibrosis), by flow cytometry and immunostaining. Proliferative tendency and mitochondrial contents of each population were evaluated by BrdU assay and carboxyfluorescein succinimidyl ester (CFSE) dye dilution assay, and by MitoTracker Red staining.

Results: Two subpopulations with different proliferative properties were detected in cultured HCECs by CFSE dye dilution assay. One divided at most 7 times in 8 days cultivation, while the other stopped cell division at 3 times (the latter thought to enter cell senescence at the early stage of cultivation, resulting in cell cycle arrest). Cell surface marker based analysis revealed the presence of at least 5 distinct subpopulations in cultivated HCECs under our culture condition. During the course of extended passages of the culture, the ratio of the population X, identified as the fully differentiated mature HCECs, tended to decrease, indicating the plasticity dictated by culture microenvironments. The other two populations with EMT-phenotype-like stem cells exhibited far higher proliferation propensity than population X. The ratio of population X in the cultured HCECs well correlated inversely with the age of the donors of the corneas, consistent with thus-far reported corneal transplantation clinical findings.

Conclusions: Our results suggest the existence of a distinct subpopulation in cultured HCECs. Definition of the fully matured HCECs based on cell surface markers will provide a novel regenerative medicine intervention for patients with impaired HCEC functions.

Keywords: 481 cornea: endothelium