Abstract
Purpose :
Balanced interplay of cell-type specific transcriptomic regulation is critical for nerve regeneration. We use cornea to study cell-type specific transcriptomic regulation. Single nucleus-RNA-sequencing (snRNASeq) and spatial transcriptomics were used to study cell populations and gene expression in central cornea/limbus from male, 3-month, C57BL/6 wild-type (WT) and injured mice 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 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, corneas excised, formalin-fixed paraffin-embedded, and 5µm sections prepared for Visium CytAssist mediated probe hybridization, library construction, sequencing, data analysis using Spaceranger, data visualization, cell cluster annotation using Loupe Browser. Cell populations distinguished by t-distributed stochastic neighbor embedding (t-SNE). Using RNAscope® assay data from Visium CytAssist was validated for genes (ALDH3A1, SPARC) associated wound healing.
Results :
Unbiassed cell clustering recovered 25039 corneal and 24183 limbal single nuclei, dimensionality reduction detected specific clusters: 8 (cornea), 5 (limbus); 5 (normal cornea) and 1 (damaged cornea) based on unique molecular signatures. Cell clusters identified in cornea included transient amplifying cells, epithelial cells, stromal keratocytes, lymphocytes, and dendritic cells. Limbal progenitor, limbal stem cells and lymphatic endothelium in limbus cluster. Central cornea and ocular surface epithelium transcription profiles were different and varying. Spatial clusters showed varying gene expression in WT and injured mice cornea. Gene expression of ALDH3A1 and SPARC decreased in central cornea after injury, validated using RNAscope® assay.
Conclusions :
Cell clusters associated with central cornea and limbal populations were identified and impact of spatial gene clusters identified from Visium CytAssist in WT and injured cornea/limbus on nerve regeneration were studied and validated using RNAscope® assay.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.