Moustardas, P. | Search bioRxiv

bioRxiv2026

Decoding murine corneal epithelial specification and homeostasis by single-cell spatial transcriptomics with scRNA-seq enrichment

Investigation of gene regulatory programs underlying corneal epithelial cell specification and homeostasis is essential for understanding how the cornea maintains vision. Here, we describe the use of true single-cell resolution spatial transcriptomics (ST), enriched with full-tissue single-cell RNAseq (SC), to improve spatial resolution and enhance cell cluster size up to 65-fold and per-cell transcriptomic depth up to 17-fold. This enabled cell type specification across the full differentiation trajectory from limbal stem cells (LSC) to superficial corneal epithelium and identification of an activated signature (Atf3, Zfp36, Gsta4 and Dapl1) marking differentiation-primed states across multiple cell types, including a major activated intermediate epithelium (AIE) population. Validation using ST data from murine corneas at different postnatal ages and multiple human SC datasets confirms a large AIE population, which spatial localization and transcriptomic profiling suggest is an active intermediate state distinct from quiescent wing cells. Sub-clustering further revealed early (Sox9, Hes1), proliferative (Mki67, Top2a) and mature (Ccdn1, Dapl1) transient amplifying cell subpopulations and four LSC subpopulations, including putative active (Atf3, Socs3, Zfp36), quiescent (Gpha2, Ifitm3, Cd63) and Apoe-specific. Direct ST-to-SC comparison revealed enhanced axonal processes and genes (Sema3f, Sema 4d, Pax6) and cell-cell adhesion and cell-matrix markers (Itgb4, Tns4, Tjp3) in ST data, suggesting cell dissociation from tissue in SC masks epithelial innervation, adhesion and barrier functions. Our findings identify and localize key transcriptional programs in situ, prompting a re-evaluation of epithelial states in scRNA-seq data.

cell biology↗

bioRxiv2025

Perturbation of epithelial and limbal stem cell identity in a mouse model of pathologic corneal neovascularization

The epithelial layer of the cornea is a critical physical and ocular immune barrier for maintaining tissue integrity, homeostasis, and transparency for proper vision. Corneal injury can trigger inflammation, impair wound healing and compromise immune privilege and avascularity, leading to vision loss. Moreover, injury to the cornea can disrupt the engine of epithelial repair and restoration, the limbal stem cell (LSC) niche. Here we used a corneal suture model to induce epithelial damage, sustained inflammation and neovascularization, to examine the impact on LSCs. Using single-cell transcriptomics, we analyzed corneal cell state changes and additionally evaluated the potential of duloxetine, an FDA-approved medicine, to promote wound healing and corneal homeostasis. Single-cell RNA-seq analysis revealed loss of homeostatic limbal stem cells, basal and differentiated epithelial cells and an increase in distinct limbal-like, conjunctival, inflammatory, and vascular cell states, suggesting a coordinated wound healing response in different tissue layers. Importantly, duloxetine treatment promoted epithelial homeostasis, enhanced stem cell-like and stromal repair processes, and suppressed immune and vascular responses. Examination of corneal cell perturbation and transformations at the single-cell level thorough marker profile annotations can improve the understanding of LSC plasticity and function while yielding potential biomarkers of corneal repair processes.

molecular biology↗