Injury and disease reconfigure corneal epithelial stem cell differentiation distinct from homeostasis

Abstract

Purpose : The clear corneal epithelium is crucial for vision and regenerates from limbal stem cells (LSCs) whose differentiation in homeostasis has been resolved transcriptionally by recent single cell RNA sequencing (scRNAseq) studies. Yet, it remains poorly understood how LSC differentiation becomes co-opted after injury to promote wound healing or whether differentiation is perturbed in aging, diabetes, and dry eye disease (DED). In the present work, we evaluate--at the single cell level--corneal epithelium in animal models of these conditions that could impact LSC regeneration of the ocular surface.

Methods : Using scRNAseq, we profiled mouse corneal epithelium in homeostasis, in aging, in streptozotocin-induced diabetes, following debridement of the central cornea, and in aqueous tear-deficient DED resulting from lacrimal gland excision.

Results : In homeostasis, we captured the full sequence of corneal epithelial differentiation from LSC to superficial squamous cell, as well as identify candidate genes and gene networks that characterize key stages of differentiation. In models of aging or diabetes, we found that there were only mild transcriptional changes, with ≤10 dysregulated genes in total across all cell types (adjusted p-value < 0.05, |log₂[fold-change]| ≥ 1.5). However, epithelial debridement or DED induced more dramatic changes to aid the regeneration of the injured ocular surface. First, there was expanded expression of some putative LSC markers to other non-LSC cell types, such as Krt14 and Ifitm3 with log₂[fold-change] ranging from 1.5-2.5 (adjusted p-value < 0.05). Second, there was upregulation of epidermal wound healing-associated genes Fabp5 and Rbp1 with log₂[fold-change] ranging from 1.5-2.5 (adjusted p-value < 0.05). Finally, we provide markers for new corneal epithelial cell states that appeared after epithelial debridement or in DED. These wound healing-elicited cell states were not apparent in homeostasis (1.7% of cells in control eye vs. 27% after debridement; 0.5% of cells in control eye vs. 11% in DED).

Conclusions : This transcriptional dissection uncovers dramatic reconfiguration of the corneal epithelial stem cell compartment in injury and DED, providing a framework and atlas for future study of these ocular surface stem cells in health and disease.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.