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Ricardo F Frausto, Anthony J Aldave; Comparing the Transcriptome of Ex Vivo Endothelium with Cultured Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3585.
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© ARVO (1962-2015); The Authors (2016-present)
To compare the gene expression profile of ex vivo human corneal endothelium with that of primary and established cultures of human corneal endothelial cells (HCEnC) and to determine the utility of immortalized corneal endothelial cells as a cell culture model of in vivo corneal endothelium.
Descemet membrane with the attached corneal endothelium was separated from 5 eye bank donor corneas. Total RNA was isolated from ex vivo HCEnC and triplicate cultures of primary and immortalized corneal endothelial cells (HCEC-12, HCEnC21T, HCEC-B4G12) and processed for downstream gene expression analysis using RNA-sequencing technology. Hierarchical clustering and principle component analysis (PCA) were performed to determine relationships between the samples. The expression of genes that are considered functional markers of HCEnC were compared and differences in expression were analyzed statistically.
Primary and immortalized corneal endothelial cells demonstrated polygonal and cobblestone morphology characteristic of ex vivo corneal endothelium. Hierarchical clustering and PCA demonstrated underlying variance between the datasets from each RNA source. Primary cells showed the strongest relationship with ex vivo endothelium, while the cell lines were more distantly related to ex vivo samples. Ex vivo and primary cells showed the highest number and percentage of commonly expressed genes, while ex vivo and HCEC-B4G12 cells demonstrated the lowest number and percentage of commonly expressed genes. The mRNA levels of AQP1 and ATP1A1 were reduced in the cell lines, while ZO1 was increased compared to levels in the ex vivo samples. SLC4A11, ZEB1, TCF4 and COL8A2 demonstrated significant differential expression in the cultured cells that was most pronounced in the cell lines, while expression of LOXHD1 and AGBL1 was undetectable in ex vivo, primary and immortalized HCEnCs.
Our findings caution against reaching the conclusion that the expression of a few functional markers defines the parameters for validating the characteristics of immortalized corneal endothelial cells. The cell lines remain a valuable tool in the absence of robust primary cell culturing techniques, but should be used with an understanding of their limitations. Where possible, follow up validation experiments in animal models or humans should be considered.
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