The spatial transcriptomic atlas of human limbus and vital niche microenvironment regulating the fate of limbal epithelial stem cells

We profiled over 400 spots within each sample and spatially located major cell types through the integration with reference the single-cell RNA sequencing data. Based on Robust Cell Type Decomposition (RCTD) technology, limbal epithelial stem cells (LESCs) were mainly localized in the basement membrane, and limbal niche cells were predominantly situated within the stromal area. Following, the limbus was divided into 4 regions based on histological structure, and the differential expressed genes among the four regions were analyzed. Notably, our investigation revealed significantly higher levels of GPHB5 and PERP within the epithelium of the middle region, precisely where LESCs predominantly reside. Concurrently, the activation of critical pathways such as "Protein Binding" and "ECM-receptor interaction" were discovered in this same region, underscoring its pivotal role in maintaining the limbal niche. Subsequently, cell-cell communication analysis was conducted on essential cells inside the niche microenvironment. Remarkably, we observed that limbal mesenchymal stem cells exhibit the largest amounts of ligands associated with LESCs, with melanocytes closely trailing behind. 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. Additionally, a myriad of ligand-receptor interactions and pathways regulatory crosstalk among diverse cell types were exhibited. The single-cell transcriptomic atlas of two human limbus was performed using 10X Genomics' Space Ranger software platform, followed by visualizing in two-dimensional space by UMAP and unbiased computational informatic analysis. Immunofluorescent staining assays were performed to further validate the analysis results.