In situ spatial transcriptomics reveal 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 for its stratified epithelium. The niche for these cells is the limbus, and damage to this microstructure or its SCs results in a blinding disease known as 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 from their repository, and their gene expression is altered upon exposure to physical and chemical stressors during procurement. Herein, these adversities were avoided by conducting spatial transcriptomic screening directly on corneal, limbal and conjunctival tissues. Differentially expressed genes in the limbus included Krt16 and Nkiras1 among 6 others, which play a role in maintaining stemness, epithelial organization, immune surveillance, and tumor suppression. Krt16 was dynamically expressed in the developing limbus, correlated with slow-cycling label-retaining limbal epithelia and was induced during corneal injury; observations consistent with marking functional SCs. Additionally, we established Nkiras1 as a novel maker of neutrophils in the limbal SC niche, and Alox12e as a specific marker of conjunctival epithelia. Because current gold standard treatments for LSCD include SC transplantation, our data will inform future studies in delivering a more reliable standard therapy which incorporates an identifiable SC population to improve clinical outcomes. Overall design: C57BL/6 male and female wild-type mice (n = 81) were obtained from Australian BioResources (Moss Vale, Sydney, Australia). They were housed under pathogen-free conditions in temperature-controlled rooms and given ad libitum access to standard chow and water. Eyes (n=3) were fixed in 4% paraformaldehyde (PFA) overnight. The superior nasal (SN) quadrant was dissected and paraffin-embedded. Sections (5µm) were mounted onto glass slides within a 35.3mm x 14.1mm area, dried at 58°C for 3 hrs, then subjected to the GeoMx DSP workflow (Bruker Spatial Biology, San Jose, CA). Prior to selecting ROIs, serial sections were stained with hematoxylin or double immunostained for K8 and K12 to identify tissue boundaries. ROIs included corneal, transitional (containing some limbus and cornea), limbal and bulbar conjunctival epithelia.