Spatial transcriptomics define cell heterogeneity in limbal/corneal epithelial stem/early transit amplifying cells in mice cornea after damage

Abstract

Purpose : Functional integrity in wound healing is sustained through renewal of limbal/corneal epithelial stem cells by the balanced interplay of cell-type specific transcriptome. Single-RNA-sequencing (snRNASeq) and spatial transcriptomics were used to interrogate cell populations and gene expression in central cornea/limbus from male, 3-month, C57BL/6 wild-type (WT) and injured mice cornea. We hypothesize that the transcriptome is different in cornea/limbus and between injured and WT cornea.

Methods : Freshly excised cornea was dissected to separate central/limbal tissues, frozen at -80°C, and processed to isolate single nuclei; snRNASeq was conducted on cornea/limbus of WT mice (N=6) using 10xGenomics pipeline Cellranger. Corneal damage was induced in an anesthetized mouse, central portion of right cornea demarcated with 2mm trephine, and epithelium and anterior stroma removed with corneal rust ring remover (Algerbrush II). 2 days post-injury, injured and normal mice were sacrificed and corneas excised and formalin-fixed paraffin embedded (FFPE). 5µm sections prepared, deparaffinized, and H&E stained followed by Visium Cytassist mediated probe hybridization, library construction, sequencing, data analysis using Spaceranger, data visualization and cell cluster annotation using Loupe Browser. Cell populations were distinguished by t-distributed stochastic neighbor embedding (t-SNE).

Results : We recovered 25039 corneal and 24183 limbal unbiassed cell clustering, and dimensionality reduction detected specific clusters: 8 (cornea), 5 (limbus); 5 (WT normal cornea) and 1 (damaged cornea) based on unique molecular signatures. Corneal clusters included transient amplifying and epithelial cells: corneal, basal, superficial, wing, stromal keratocytes, lymphocytes, and dendritic cells. Limbal clusters included basal and wing, epithelial, limbal progenitor, and limbal stem cells and lymphatic endothelium. Transcription profiles were different in central cornea and ocular surface epithelium with varying expression profiles.

Conclusions : Thus, snRNASeq identifies unique cell clusters spatially in mice cornea/limbus activated by cornea injury that enable delineation of differential gene expression critical for repair.

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