The Spatial Transcriptomic Atlas of Human Limbus and Vital Niche Microenvironment Regulating the Fate of Limbal Epithelial Stem Cells

Purpose: This study aimed to generate the spatial atlas of the human limbus using spatial transcriptomic technology and reveal the deep interaction among the niche microenvironment. Methods: The spatial transcriptomic atlas of human limbus was performed using 10× Genomics Space Ranger software platform. Single-cell RNA sequencing data of human limbal epithelial stem cells (LESCs) were downloaded for integrating analysis. Results: We profiled more than 400 spots within each sample and spatially located major cell types within the limbus area. LESCs were localized mainly in the basement membrane, and limbal niche cells were situated predominantly within the stromal area. Next, the limbus was divided into four regions based on histological structure, and the differential expressed genes among the four regions were analyzed. Notably, GPHB5 was highly expressed in the epithelium of the middle region and co-staining with deltaNp63 suggested it might be a novel potential biomarker of LESCs. Subsequently, limbal mesenchymal stem cells were found to exhibit the greatest amounts of ligands associated with LESCs. The widespread activity of COL6A2/CD44 signaling among limbal mesenchymal stem cells, melanocytes, immune cells, and LESCs indicate its essential role in mediating bidirectional communication via the collagen pathway. Conclusions: This research mapped the spatial positioning of key cells within the limbal niche and detailed interactions between major cell types. These findings provide a foundation for further LESC research and enhance our understanding of corneal biology.