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A3799 - Identification of Subpopulations of Human Small Airway Epithelial Club Cells
Author Block: M. LeBlanc1, S. Shenoy1, M. Rostami1, W. Zuo1, J. G. Mezey1, R. G. Crystal2; 1Weill Cornell Medical College, New York, NY, United States, 2Genetic Medicine, Weill Cornell Medical College, New York, NY, United States.
RATIONALE: Club cells, a secretory cell that uniquely expresses SCGB1A1 (CC10, CC16), comprise approximately 20% of the human small airway epithelium. Single cell transcriptome analysis suggests that, in the human, club cells play a central role in host defense, lung viral infection and hereditary disorders. Based on data demonstrating that ~50% club cells express MUC5B, a secretory mucin critical for mucociliary clearance, the transcriptome of the SCGCB1A1+KRT5‾ MUC5B+ vs SCGB1A1+KRT5‾ MUC5B‾ cells were compared, with the hypothesis that, at least at the biologic level, there may be subpopulations of SAE MUC6B+ vs MUC5B‾ club cells with unique functions.
METHODS: Two single cell transcriptome methods (Drop-seq and Fluidigm) were used to analyze small airway (10th-12th generation bronchi) epithelium recovered by fiberoptic bronchoscopy and brushing of n=6 healthy nonsmokers. Unbiased clustering was followed by analysis of cell populations that were SCGB1A1+ (club cell) KRT5‾ (basal cell). Immunostaining of SAE in lung sections and cells recovered by brushing was used to confirm the transcriptome data.
RESULTS: Both single cell sequencing datasets demonstrated that 45-55% of SCGB1A1+ club cells were MUC5B+. Immunostaining of brushing samples and lung sections verified that a subpopulation of SAE SCGB1A1+KRT5‾ cells were positive for MUC5B. The Drop-seq single cell sequencing data showed 254 genes were differentially expressed in SCGB1A1+KRT5‾ MUC5B+ compared to SCGB1A1+KRT5‾ MUC5B‾ cells. The MUC5B+ subpopulation highly expressed BPIFB1, TFF3 and SCGB3A1 and KEGG analysis demonstrated increase in expression of genes associated with extracellular exosomes. In contrast, the MUC5B‾ subpopulation highly expressed RPS6, RPL13 and PTMA, and KEGG analysis identified characteristic expression of a large number of ribosomal proteins as well as claudin 1, a major constituent of tight junctions. This data was validated using the Fluidigm system transcriptome data, showing over 50% overlap of the up-regulated genes in the genes identified by Drop-seq as characteristic of MUC5B+/MUC5B ‾ subpopulations.
CONCLUSIONS: These observations suggest two club cell subpopulations in the human SAE. The novel MUC5B+ subtype of SCGB1A1+KRT5‾ club cells have unique expression of secretory genes, suggesting this may be the active club cell population, utilizing secretory components in host defense. In contrast, the MUC5B- subtype of SCGB1A1+KRT5‾ club cells have increased ribosomal protein transcripts, suggesting a second subtype that may function separately from the canonical club cell role in host defense.