Shengqian Dou1, Qun Wang1, Xia Qi1, Bin Zhang1, Hui Jiang1, Shengwen Chen1, Haoyun Duan1, Yao Lu2, Jiaoyang Dong2, Yihai Cao3, Lixin Xie1, Qingjun Zhou4, Weiyun Shi5. 1. State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Qingdao, China; Qingdao Eye Hospital of Shandong First Medical University, Qingdao, China. 2. OE Biotech Co., Ltd, Shanghai, Shanghai, China. 3. Department of Microbiology, Tumor, and Cell Biology, Karolinska Institute, Stockholm, Sweden. 4. State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Qingdao, China; Qingdao Eye Hospital of Shandong First Medical University, Qingdao, China. Electronic address: qjzhou2000@126.com. 5. State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Qingdao, China; Qingdao Eye Hospital of Shandong First Medical University, Qingdao, China; Eye Hospital of Shandong First Medical University, Jinan, China. Electronic address: weiyunshi@163.com.
Abstract
PURPOSE: The corneal limbus maintains the homeostasis, immune and angiogenic privilege of cornea. This study aimed to depict the landscape of human limbal tissues by single-cell RNA sequencing (scRNA-seq). METHODS: Single cells of human limbus collected from donor corneas were subjected to 10x scRNA-seq, followed by clustering cell types through the t-distributed stochastic neighbor embedding (t-SNE) and unbiased computational informatic analysis. Immunofluorescent staining was performed using human corneas to validate the analysis results. RESULTS: 47,627 cells acquired from six human limbal tissues were collected and subjected to scRNA-seq. 14 distinct clusters were identified and 8 cell types were annotated with representative markers. In-depth dissection revealed three limbal epithelial cell subtypes and refined the X-Y-Z hypothesis of corneal epithelial maintenance. We further unveiled two cell states with higher stemness (TP63+ and CCL20+ cells), and two other differentiated cell states (GPHA2+ and KRT6B + cells) in homeostatic limbal stem/progenitor cells (LSPCs) that differ in transcriptional profiles. Cell-cell communication analysis revealed the central role of LSPCs and their bidirectional regulation with various niche cells. Moreover, comparative analysis between limbus and skin deciphered the pivotal contribution of limbal immune cells, vascular and lymphatic endothelial cells to corneal immune and angiogenic privilege. CONCLUSIONS: The human limbus atlas provided valuable resources and foundations for understanding corneal biology, disease and potential interventions.
PURPOSE: The corneal limbus maintains the homeostasis, immune and angiogenic privilege of cornea. This study aimed to depict the landscape of human limbal tissues by single-cell RNA sequencing (scRNA-seq). METHODS: Single cells of human limbus collected from donor corneas were subjected to 10x scRNA-seq, followed by clustering cell types through the t-distributed stochastic neighbor embedding (t-SNE) and unbiased computational informatic analysis. Immunofluorescent staining was performed using human corneas to validate the analysis results. RESULTS: 47,627 cells acquired from six human limbal tissues were collected and subjected to scRNA-seq. 14 distinct clusters were identified and 8 cell types were annotated with representative markers. In-depth dissection revealed three limbal epithelial cell subtypes and refined the X-Y-Z hypothesis of corneal epithelial maintenance. We further unveiled two cell states with higher stemness (TP63+ and CCL20+ cells), and two other differentiated cell states (GPHA2+ and KRT6B + cells) in homeostatic limbal stem/progenitor cells (LSPCs) that differ in transcriptional profiles. Cell-cell communication analysis revealed the central role of LSPCs and their bidirectional regulation with various niche cells. Moreover, comparative analysis between limbus and skin deciphered the pivotal contribution of limbal immune cells, vascular and lymphatic endothelial cells to corneal immune and angiogenic privilege. CONCLUSIONS: The human limbus atlas provided valuable resources and foundations for understanding corneal biology, disease and potential interventions.
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