Javidjam, D.; Vattulainen, M.; Lagali, N.; Moustardas, P.

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.