The different binding properties of cultured human corneal endothelial cell subpopulations to Descemet’s membrane components

Abstract

Purpose: Human corneal endothelial cells (HCECs) have poor proliferative ability under in vitro culture conditions. The tendency to enter into cell senescence or phase transition (epithelial-mesenchymal transition (EMT), cell senescence, and fibrosis) during cultivation produces different subpopulations from HCECs. To date, we have developed definitive subpopulations based on their cell surface markers. To clarify the adherent properties of these subpopulations, we compared the binding ability of cultured HCEC subpopulations to major Descemet’s membrane components that distribute to the endothelial face; i.e., laminin-511, -411, Type IV collagen, and proteoglycans.

Methods: Each subpopulation was prepared by controlling the culture conditions or by using magnetic cell separation, and then confirmed by staining with several cell surface markers. To examine the binding abilities of HCEC subpopulations, the cells were added to 96-well culture plates immobilized with collagens, laminins, or proteoglycans, and the plates were then centrifuged. The attached cells were then evaluated under a phase contrast microscope.

Results: Both the fully differentiated mature HCEC subpopulation and the EMT-phenotype subpopulation were found to be attached to laminin or collagen coated plates. Among the examined laminins, HCEC subpopulations were most strongly bound to laminin-511 and weakly bound to Perlecan, Agrin, and TSP-1. Interestingly, the binding properties to laminins were different among these subpopulations. Although the level of attached cells to the laminin-411 coated plate was the same among the HCEC subpopulations, the fully differentiated mature HCEC subpopulation was significantly more tightly bound to laminin-511 than was the EMT-phenotype subpopulation.

Conclusions: Our results suggest that the binding ability of HCECs to the major Descemet’s membrane components is distinct among subpopulations of cultivated HCECs. Moreover, the simple methods used in this study are effective for evaluating the interaction between HCECs and extracellular matrix components.