Transcriptome analysis of lens epithelial explants upon vitreous-induced fiber cell differentiation

Abstract

Purpose : Lens epithelial explants provide a useful tool for studying lens cell proliferation and fiber cell differentiation in vitro. Vitreous humor can induce both epithelial cell proliferation and characteristics of fiber cell differentiation including the accumulation of b- and g-crystallins and cell elongation in explants. Here we performed an analysis of transcriptional changes induced by both explanting and by the addition of vitreous. We sought to determine how closely the transcriptome of vitreous-induced fiber cells in explants mirrors that of endogenous lens fiber cells.

Methods : Lens epithelial explants were prepared from 8- day old FVB/N mice. RNA was extracted from fresh lens epithelium, from lens explants after 1 day in culture without vitreous, and from lens, explants after 1, 5, and 10 days of treatment with 50% vitreous and sent for RNA-sequencing. Raw sequence reads were aligned to the mouse transcriptome and gene counts were summarized for differentially expressed genes (DEGs), then used for downstream evaluation such as clustering and gene ontology analysis. The DEG criteria was log2FC >1.5 and p <0.05. Explant RNA-Seq data was compared to that of endogenous FVB/N newborn lens epithelial cells and lens fiber cells.

Results : Gene expression analysis showed that the explants upregulated genes related to immune response and inflammation. Vitreous induced a short-term increase in Cdk1 and reduction in the expression of many crystallin transcripts. However, during the 10 days of vitreous exposure, the explants exhibited an overall loss of transcripts normally enriched in lens epithelial cells (e.g. Sulf1, Flt1, Cdh1, and Gja1) and a gain of transcripts characteristic of fiber cell identity (e.g. Cryba4, Cryg1, Crybb1, Dnase2b, Mip, and Lim2). Gene ontology analysis of the upregulated genes in vitreous-treated explants revealed enrichment for terms relating to developmental process, anatomical structure development, regulation of ERK1 and ERK2 cascade, positive regulation of MAPK cascade.

Conclusions : RNA-Seq analysis revealed that in contrast to endogenous fiber cells, vitreous-exposed explants induced expression of genes related to injury response. However, after 10 days of vitreous exposure, the overall transcriptome of lens epithelial explants matched that of newborn lens fiber cells far more closely than that of endogenous lens epithelium.

This is a 2021 ARVO Annual Meeting abstract.