Single-Cell Insights into Endothelial Cell Heterogeneity in the Thymus 2267370

Abstract Introduction The thymus contains a complex vascular network that supports progenitor entry, thymocyte egress, and blood-thymus barrier integrity. Endothelial cells (ECs) line the luminal surface of blood and lymphatic vessels and mainly regulate these processes. However, their diversity and distinct functions remain unexplored in the thymus. Methods Thymi will be harvested from 6-week-old C57BL/6 mice, with stromal cells isolated from enzymatic digestion and density gradient centrifugation. ECs will be sorted by flow cytometry for single-cell RNA sequencing. Single-cell barcoding and cDNA synthesis will be performed using 10X Genomics Chromium platform and libraries sequenced on an Illumina platform. Data will be processed with Cell Ranger and analyzed in R using Seurat for dimensional reduction, clustering, and differential gene expression. Results We anticipate that this analysis will reveal transcriptionally distinct EC subsets corresponding to structurally distinct vessels. These subsets are expected to contribute to thymic homeostasis and differentially express genes involved in barrier function/regulation, cytokine signaling, and tissue regeneration. Conclusion This study will be the first to provide a single-cell atlas of thymic ECs, revealing specialized roles of thymic EC subtypes. These findings will establish a framework for further studies on how EC diversity contributes to thymic organization, function, and regeneration. Funding Source National Heart, Lung, and Blood Institute of the National Institute of Health (R01HL171971-01A1), Institute of General Medical Sciences of the National Institutes of Health (T32GM141862) Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)