3D mapping of potential markers for human corneal endothelial cell

Abstract

Purpose: At present there is no unique marker for normal human corneal endothelial cell (hCEC). Nevertheless, a perfect characterization of hCEC is a prerequisite for future works of endothelial bioengineering, especially in order to define the best quality controls to validate the identity and purity of cells obtained after in vitro expansion or differentiation. Considering that the correct subcellular localization of each protein is the true hallmark of hCEC, the present study aimed at establishing a clear 3D mapping of the main proteins expressed by hCEC

Methods: Using optimized immunolocalization techniques that we previously developed for the endothelium of flat mounted whole corneas, we tested more than hundred antibodies. Cytoskeleton, intercellular junctions, organelles, ionic pumps, and adhesion complexes were targeted (previously known to be expressed by hCEC or not). We studied both fresh (unstored corneas procured within 4-12 hours after death, body donation to Science) and organ cultured corneas with normal endothelium and repeated the staining at least in triplicate. Stacks of high magnification images (x60 objective) were acquired using a confocal microscope (Fluoview, Olympus) with high resolution and a with a 150nm pitch. We selected only markers with highly specific expression patterns.

Results: A 3D mapping of numerous proteins was clearly established and modelled. Tight junction (ZO-1) and contractile acto-myosin complex together form the typical hexagonal shape at the apical pole. NCAM (CD56), N-cadherin, Na/K ATPase and Aquaporin 1 localized at lateral plasma membrane expansions forming numerous interdigitations with neighbour cells. Vimentin, Tubulin and COXIV (mitochondriae) filled the cytoplasm. Intergrin α3β1 labelled only the basal membrane also forming interdigitations. By comparison with epithelial cells, keratocytes and trabecular meshwork endothelial cells, the 3D subcellular locations of this markers set were specific to hCEC

Conclusions: Normal hCEC have a unique 3D structure highlighted by a combination of several markers in relation with specific endothelial functions. We propose to use this 3D mapping to assess the quality of cultured endothelial cells during bioengineering processes.<br /> Grant: IFRESIS, St ETIENNE METROPOLE<br />