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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE262250 | GEO | 2025/01/30

REPOSITORIES: GEO

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Project description:Limbal stem cells including epithelial and stromal/Mesenchymal stem cells that contribute to sustained corneal homeostasis, maintain their ability to act as self-renewal progenitor cells by virtue of their limbal niche and intercellular communication. Extracellular vehicles (EVs), including exosomes (Exos), are important paracrine mediators through their cargo transfer for intercellular communication in various stem cell niches. Previously we have shown the differential cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos, in limbal epithelial cells (LEC) in normal (N) and diabetic (DM) limbal niche. In the present study, to have a comprehensive knowledge of reciprocal LEC-LSC crosstalk, we investigated the proteomics and miRNA profile of exosomes derived from LEC and their regulatory roles in LSC in N and DM limbus. Our study showed wound healing and proliferation rates in primary N-LSC were significantly enhanced upon treatment by normal LEC-derived Exos (N-Exos), but not by diabetic Exos (DM-Exos). Further, N-Exos treated LSC showed downregulation of keratocyte markers, ALDH3A1 and lumican, but not keratocan, and upregulation of MSC markers, CD105, CD90, and CD73 compared to the DM-Exos treated LSC. Using next generation sequencing (NGS) and proteomics analysis, we revealed some miRNAs and proteins in the Exos that affect the cellular crosstalk and the function of the cornea. We also documented differences in DM vs. normal LEC-derived Exo’s cargos. Overall, DM-Exos have less effect on LSC proliferation, wound healing, and stem cell maintenance than N-Exos, likely by transferring their cargo proteins and/or regulatory miRNAs targeting cell cycle, ERK/MAPK, TGF-β, EMT, PI3K-Akt-mTOR signaling molecules. This suggests that the small RNA and protein cargo differences in DM vs. N LEC-derived Exos could contribute to the disease state. Our study revealed a complex contribution of Exos to health and diabetic state of corneal homeostasis and suggests the potential of EV therapeutics for diabetic cornea regenerative medicine