Single Cell Transcriptomics Define Cell Heterogeneity in Limbal/Corneal Cells Relevant to Nerve Regeneration

Abstract

Balanced interplay of cell-type specific transcriptomic regulation is critical for nerve regeneration in the central and peripheral nervous system. As a model, we use the cornea, where we recently discovered the RvD6-isomer and its function as a regulator of nerve regeneration, critical for dry eye in aging. 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. Isolated nuclei from cornea/limbus of WT mice (N=12) were processed by 10×Genomics pipeline Cellranger. Central mice corneas were damaged with 2mm trephine, and epithelium and anterior stroma removed with corneal rust ring remover (Algerbrush II). Two days post-injury, corneas were excised, formalin-fixed paraffin-embedded, and 5µm sections prepared for 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). RNAscope® assay was used to validate two genes, ALDH3A1 and SPARC.