Comparison of mRNA expression in primary cultures of human anterior lens epithelium and human corneal endothelium.

Abstract

Purpose : Corneal endothelial dysfunctions are the main cause of corneal blindness in developed countries and to date, the shortage of endothelial tissue for its treatment represents an emerging problem. Therefore, finding substitutes to corneal endothelium in other tissues should be a priority in order to treat more patients. In this study we compare the gene expression from primary xeno-free cultures of human lens capsule epithelium and human corneal endothelium in order to evaluate the potential use of lens capsule epithelial cells as a surrogate of corneal endothelium.

Methods : Four corneal endothelia deemed inappropriate for surgery and four lenses were obtained from Asturias tissue bank. Corneal endothelium was dissected following the Schwalbe line and anterior lens capsule was dissected from the stroma. Both tissues were digested with TrypLE for 90 min at 37 °C and seeded on a culture plate and cultured until confluence. Subsequently, we performed bulk RNASeq in both cell types and analyzed their gene expression profiles. RNA was isolated using the Arcturus^TM PicoPure^TM kit followed by NEBNext Ultra II RNA library preparation. Samples were sequenced with Illumina NextSeq550. Additionally, cultured cells were fixed using ice-cold methanol for 10 min and immunocytochemistry of major corneal endothelial markers (Na⁺/K⁺ ATPase, ZO-1) were performed.

Results : We identified 19.677 similarly expressed genes out of 22.641 lens capsule epithelium and corneal endothelium genes. Essential genes for the correct function of the corneal endothelium such as, those responsible for the barrier function and ion pump (Na⁺/K⁺ ATPase, ZO-1) present a similar level of expression. Moreover, protein expression of major functional markers was confirmed by immunocytochemistry. The 2964 differentially expressed genes (DEGs) were mainly clustered in the gene ontology of epithelial cell proliferation, extracellular matrix organization and eye development.

Conclusions : Corneal endothelium and lens epithelium share substantial similarities in functional gene and protein expression, supporting the proposal to use lens capsule epithelial cells as a possible surrogate for corneal endothelium

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.