Divergence Between Marker Expression and Phenotype During Human Corneal Endothelial Cell Expansion.
- 1Department of Cell Biology-Inspired Tissue Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht, The Netherlands.
- 2Department of Pathology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Center (MUMC+), The Netherlands.
- 3Department of Ophthalmology, Utrecht University Medical Center, Utrecht, The Netherlands.
Abstract
Purpose
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).Methods
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).Results
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.Conclusions
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.
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BibTeX
@article{bonelli2026divergence,
title = {{Divergence Between Marker Expression and Phenotype During Human Corneal Endothelial Cell Expansion.}},
author = {Filippo Bonelli and Tom Bailey and Iveta Dzivite and Lieve Temmerman and Mor Dickman and Vanessa LaPointe},
journal = {Investigative ophthalmology \& visual science},
year = {2026},
month = oct,
doi = {10.1167/iovs.67.12.10},
url = {https://europepmc.org/article/MED/42831589},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Divergence Between Marker Expression and Phenotype During Human Corneal Endothelial Cell Expansion.
AU - Filippo Bonelli
AU - Tom Bailey
AU - Iveta Dzivite
AU - Lieve Temmerman
AU - Mor Dickman
AU - Vanessa LaPointe
PY - 2026
DA - 2026/10/01
JO - Investigative ophthalmology & visual science
DO - 10.1167/iovs.67.12.10
UR - https://europepmc.org/article/MED/42831589
AB - Purpose 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).Methods 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).Results 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.Conclusions 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.
SN - 0146-0404
SN - 1552-5783
ER -