In situ spatial transcriptomics reveals novel markers of the limbal stem cell niche and ocular surface epithelia.

The mammalian cornea is endowed with stem cells (SCs) that have lifelong regenerative activity. The niche for these cells is the limbus, and damage to it or its SCs results in limbal stem cell deficiency (LSCD). Despite the numerous studies that employ single-cell RNA sequencing, the identity of these cells remains an enigma principally because their spatial positioning is lost upon dissociation. These adversities were avoided via on-tissue spatial transcriptomics where Krt16 and Nkiras1 were differentially expressed. Krt16 was dynamically expressed in the developing limbus, correlated with slow-cycling label-retaining limbal epithelial SCs and was induced during corneal injury, observations consistent with marking functional SCs. Additionally, we established Nkiras1 as a novel maker of limbal neutrophils. Because current gold-standard treatments for LSCD include SC transplantation, our data will inform future studies in delivering a more reliable standard therapy that incorporates an identifiable SC population to improve clinical outcomes.