Transcriptomic Analysis of Cultured Corneal Endothelial Cells as a Validation for Their Use in Cell Replacement Therapy

Abstract

The corneal endothelium plays a primary role in maintaining corneal homeostasis and clarity, and must be surgically replaced with allogenic donor corneal endothelium in the event of visually significant dysfunction. However, a worldwide shortage of donor corneal tissue has led to a search for alternative sources of transplantable tissue. Cultured human corneal endothelial cells (HCEnC) have been shown to restore corneal clarity in experimental models of corneal endothelial dysfunction in animal models, but characterization of cultured HCEnC remains incomplete. To this end, we utilized next-generation RNA sequencing technology to compare the transcriptomic profile of ex vivo human corneal endothelial cells (evHCEnC) with that of primary HCEnC (pHCEnC) and HCEnC lines, and to determine the utility of cultured and immortalized corneal endothelial cells as models of in vivo corneal endothelium. Multidimensional analyses of the transcriptome datasets demonstrated that primary HCEnC have a closer relationship to evHCEnC than do immortalized HCEnC. Subsequent analyses showed that the majority of the genes specifically expressed in HCEnC (not expressed in ex vivo corneal epithelium or fibroblasts) demonstrated a marked variability of expression in cultured cells compared with evHCEnC. In addition, genes associated with either corneal endothelial cell function or corneal endothelial dystrophies were investigated. Significant differences in gene expression and protein levels were observed in the cultured cells compared with evHCEnC for each of the genes tested except for AGBL1 and LOXHD1, which were not detected by RNA-seq or qPCR. Our transcriptomic analysis suggests that at a molecular level pHCEnC most closely resemble evHCEnC and thus represent the most viable cell culture based therapeutic option for managing corneal endothelial cell dysfunction. Our findings also suggest that investigators should perform an assessment of the entire transcriptome of cultured HCEnC prior to determination of their potential clinical utility for the management of corneal endothelial cell failure.

Transcriptomic Profiling of Posterior Polymorphous Corneal Dystrophy

“…32 Using a threshold of expression of RPKM ≥ 0.1, 83.7% (87/104) and 53.8% (56/104) of the 104 protein-coding evHCEnC-specific genes were expressed in P5 and P6, respectively, as compared to 97.1% (101/104) and 95.2% (99/104) in the respective controls, C5 and C6. A total of 49 evHCEnC-specific genes were expressed in each of the two PPCD and two control HCEnC samples, while six evHCEnC-specific genes ( CA10 , CRB2 , EPHB1 , GPR158 , MGAT3, and PPP1R1B ) were not expressed in either P5 or P6, but were expressed in the controls (Supplementary Table S4).…”

“…32 Therefore it is likely that the loss of expression of evHCEnC-specific genes is contributing to the alterations in corneal endothelial morphology and function that characterize PPCD. In addition to demonstrating a loss of evHCEnC-specific genes, both individuals with PPCD exhibited ectopic expression of the classic epithelial marker CDH1 while showing a greater than 6-fold decrease in ZEB1 expression (Table 3), supporting the hypothesis that PPCD is caused by MET as a result of ZEB1 inactivation.…”

“…32 Cells were plated in a 12-well tissue culture–treated, nonpyrogenic polystyrene plastic plate, and experiments were performed when the cells achieved an intact and confluent monolayer, prior to the first passage.…”

Cultivation and characterisation of the surface markers and carbohydrate profile of human corneal endothelial cells

“…The immortalised, transformed nature of the cell line as well as the ex vivo cultivation of HCEnCs may have contributed to such differences. According to another transcriptome analysis of the cell line versus primary and ex vivo cultured corneal endothelial cells, B4G12 represents a distinct type of cell, thus not being an optimal model for corneal endothelial studies . Future clinically oriented experiments should therefore focus on using primary HCEnCs, possibly obtained from cadavers or other sources such as embryonic or induced pluripotent stem cells.…”

“…Commercially available SV40 immortalised cell lines such as HCEnC‐B4G12 or HCEnC‐H9C1 have also been used in research for their expression of functional proteins resembling primary HCEnC . Although other clonal cell lines have been produced in an attempt to set up ex vivo models for corneal endothelial diseases, recent findings report a better resemblance of primary isolated cells to the in vivo ones compared with any cell line . Corneal endothelial cells have been successfully generated from human embryonic stem cells (ESCs) as well, the latter expressing pump function‐related transcripts as well as other tight junction‐related proteins, such as zonula occludens‐1 (ZO‐1) and Na/K ATPase .…”