Divergence Between Marker Expression and Phenotype During Human Corneal Endothelial Cell Expansion

The purpose of this study was to compare whether conventional marker-based assessments adequately reflect phenotypic stability during serial passaging of two established culture protocols for primary human corneal endothelial cells (CEnCs).

Primary CEnCs were isolated from paired donor corneas from 25 individuals (18-44 years of age), and were cultured using 2 established methods. Cells were evaluated at passages 0 to 2 using quantitative morphology (cell area, form factor, nucleus-to-cytoplasm ratio, and cell density), RT-qPCR of endothelial-associated genes, flow cytometry, immunofluorescence, and ELISA quantification of secreted thrombospondin-2 (TSP2). Statistical comparisons were performed using 1- or 2-way ANOVA with multiple comparisons (P < 0.05).

CEnCs cultured with method 1 maintained higher cell density, smaller cell area, and greater hexagonality across passages compared with method 2 (P < 0.01 to P < 0.0001). In contrast, transcript levels of selected endothelial-associated markers (ATP1A1 and TJP1) and ZO-1 protein expression were higher in method 2 at later passages (P < 0.01), despite progressive morphological deterioration and loss of membrane localization. Proteins associated with loss of the CEnC phenotype (CD44 and CD10) were significantly elevated in method 2 (P < 0.05 to P < 0.0001), and TSP2 secretion was increased at passage 2 (P < 0.001). Across both methods, most canonical endothelial genes were ≥75% downregulated compared with native tissue.

Culture-induced phenotypic drift can occur in CEnCs despite preserved canonical endothelial marker expression. Divergence among marker expression, morphology, localization, and endothelial-to-mesenchymal transition (EndMT) readouts highlights the limits of marker-based evaluation alone. Integrated, multidimensional phenotyping should guide assessment of cultured CEnCs and optimization of expansion protocols, although functional validation is needed to link these readouts to endothelial performance.