Single-cell transcriptomics of the murine cornea reveals novel age driven changes in gene expression, cell identity composition, and limbal trajectory

Abstract

Purpose : Single-cell RNA-sequencing (scRNA-seq) was utilized to investigate the aging wild-type murine single cell transcriptome of the corneal epithelium (CEp), endothelium (CEn) and limbal proliferating cells (LPC). The purpose of this study was to elucidate the cell identity changes, and the global and cell type specific transcriptome changes attributable to corneal aging.

Methods : Corneal buttons were harvested from 3 wildtype 129/Sv mice from 3 age groups - 2 months (2MO, immature), 6 months (6MO, mature) and 11 months old (11MO, mature and aged). Pooled single cell suspensions were generated with papain digestion, enriching for isolation of CEp and CEn, from each age group were submitted to 10X Chromium Single Cell 3’ library preparation and NovaSeq 4000 sequencing. scRNA-seq was performed utilizing Seurat and Monocle3.

Results : Unbiased clustering across all three ages revealed 18 cell populations of the cornea based on known molecular markers. A great percentage of less differentiated cell types were contributed by the 2MO sample followed by the 6MO and the 11MO. Globally, the 2MO sample demonstrated an upregulation of heat shock proteins (Hspa1a, Hspa1b, Hspa8) while the older samples demonstrated an upregulation of mitochondrial genes (Lars2, mt-Nd2, mt-Nd4l) and cytokine-like protein Isg15 and Histone protein Hist1h1e. Older LPCs and CEn exhibited an upregulation of markers of keratinization (Krt6a, Krt6b, Krt16) and bicellular adhesion (Cldn4, Cldn7, Cdh1), pointing to the role of these genes. Additionally, older CEn were found to have a downregulation of Hippo pathway constituents including Wwtr1, which encodes TAZ, and Ctgf whilst noting an upregulation of TGF-Beta receptor III , potentially linked with increased TGF-Beta signaling leading to a reduced proliferative capacity. Limbal changes with age were also characterized by a decreased LPC population, increased percentage of Krt6b+ LPCs (most differentiated LPCs) and a change in trajectory from CEp to conjunctival epithelium, suggesting a transcriptomic lineage shift towards conjunctival epithelium.

Conclusions : We established an updated cell atlas with scRNA-seq of the murine cornea with age. It revealed a discrete change in the transcriptome, limbal trajectory changes from corneal to conjunctival epithelium, and implication of the Hippo pathway in the aging endothelium.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.