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

## TL;DR

This study uses spatial transcriptomics to identify new markers for limbal stem cells and neutrophils in the eye, which could improve treatments for corneal stem cell deficiency.

## Contribution

The study identifies Krt16 and Nkiras1 as novel markers for limbal stem cells and neutrophils using spatial transcriptomics.

## Key findings

- Krt16 is dynamically expressed in the developing limbus and during corneal injury, marking functional stem cells.
- Nkiras1 identifies a population of limbal neutrophils.
- Limbal span forms in mice postnatally after eyelid opening.

## Abstract

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.

•Spatial transcriptomics on ocular surface tissue identified K16 as a limbal stem cell marker•K16+ epithelial cells partake in corneolimbal development and wound healing•NKIRAS1+ cells identify a population of limbal neutrophils•Limbal span forms in mice postnatally after eyelid open

Spatial transcriptomics on ocular surface tissue identified K16 as a limbal stem cell marker

K16+ epithelial cells partake in corneolimbal development and wound healing

NKIRAS1+ cells identify a population of limbal neutrophils

Limbal span forms in mice postnatally after eyelid open

In this article, Di Girolamo and colleagues report on the tissue-specific spatial transcriptomic profiles of healthy corneal, stem cell-containing limbal and conjunctival epithelia using the mouse as a model. Unique genes and networks are identified and validated that may improve cell isolation for therapeutic use and assist the ongoing search for reliable stem cell biomarkers to enhance clinical outcomes.