Long-Read Single-Cell RNA Sequencing Reveals Dynamic Isoform Changes During Corneal Epithelial Wound Healing in Cynomolgus Monkeys

Purpose: Corneal epithelial wound healing is essential for visual recovery after injury, yet the molecular mechanisms remain incompletely understood. Different RNA isoforms from the same gene may have distinct functions not captured by gene-level analyses. This study characterized isoform-level transcriptional changes during corneal epithelial wound healing in cynomolgus monkeys using long-read single-cell RNA sequencing (RNA-seq). Methods: We profiled corneal limbal tissues from cynomolgus monkeys under uninjured conditions and at 1 day and 3 days post-injury using HIT-scISOseq, a long-read single-cell approach detecting full-length transcripts. Bioinformatic analyses examined differential isoform expression, usage, diversity, and switching across epithelial cell populations. Key isoforms were validated by RT-qPCR in scratch-wounded HCE-T cells and RNA-FISH in monkey corneal tissue. Results: Analysis revealed widespread isoform-level changes during corneal epithelial repair that were not evident from gene expression alone. Cytoskeletal remodeling genes (TPM1 and DST) and stress-response genes (ERICH5 and CTSL) showed altered isoform usage despite relatively stable total expression. ARID5B and GSN exhibited cell-type-specific isoform switching across repair stages. We identified 27 genes with dynamic isoform diversity changes and 62 genes with significant isoform switching. RT-qPCR and RNA-FISH validation confirmed injury-induced expression dynamics and cell-type-specific localization of key isoforms. Conclusions: Isoform-level regulation represents an important and independent layer of transcriptional control during corneal epithelial wound healing. This single-cell isoform atlas provides a resource for identifying injury-associated transcripts and candidate regulatory pathways, offering a framework for future functional studies and isoform-focused therapeutic strategies.