In this study, we focus on the localization, specification and function of the newly identified pulmonary ionocyte. We first validated the presence of the pulmonary ionocyte population by immunofluorescence. expressed. The functions of epithelial tissues are dictated by the types, abundance, and distribution of the differentiated cells they contain. Attempts to restore tissue function after damage require knowledge of how physiological tasks are distributed among cell types, and how cell states vary between homeostasis, injury/repair, and disease. In the conducting airway, a heterogeneous basal cell population gives rise to specialized Fluoxymesterone luminal cells that perform mucociliary clearance1. We performed single cell profiling of human bronchial epithelial HSPC150 cells and mouse tracheal epithelial cells to obtain a comprehensive picture of cell types in the conducting airway and their behavior in homeostasis and regeneration. Our analysis reveals cell states that represent known and novel cell populations, delineates their heterogeneity, and identifies distinct differentiation trajectories during homeostasis and tissue repair. Finally, we identified a novel, rare cell type, which we call the pulmonary ionocyte, that co-expresses expression sufficient to drive the production of the pulmonary ionocyte, and that the pulmonary ionocyte is a major source of CFTR activity in the conducting airway epithelium. The conducting airway is lined by a pseudostratified epithelium consisting of basal, secretory and ciliated cells, as well as rare pulmonary neuroendocrine cells (PNECs) and brush cells2. Studies of lineage tracing and regeneration post-injury show that basal cells are a heterogeneous population containing the epithelial Fluoxymesterone stem cells3,4. Basal cells differ in their expression of cytokeratins 14 and 8 (Krt14 and Krt8) and luminal cell fate determinants that are upregulated upon injury2,5. To identify the full repertoire of basal cell molecular states, and to identify candidate gene expression programs that might bias basal cells to self-renew or to adopt differentiated fates, we performed single-cell RNA profiling on airway epithelial cells. We also sought to elucidate the molecular composition of rare PNECs and brush cells, which have fewer lineage markers and are harder to define functionally6,7. Because our approach is unbiased and comprehensive, it could also identify new cell types with a role in mucociliary clearance. We performed single-cell RNA-seq8 (scRNA-seq) on 7,662 mouse tracheal epithelial cells and 2,970 primary human bronchial epithelial cells (HBECs) differentiated at an air-liquid-interface (ALI)9 (Fig. 1a,b). As there are well-documented differences between mouse and human airways10, using these two systems allows comparative analyses and prioritization of common findings between mouse and human. This Fluoxymesterone also provided validation of findings in the culture model, which lacks non-epithelial cells and uses defined culture conditions. A similar analysis of mouse tracheal epithelial cells in a co-submitted paper (Montoro et al., co-submitted) corroborates many of our findings. Open in a separate window Figure 1: Single-cell RNA-seq of proximal airway epithelial cells in mouse and human.a, Mouse tracheal epithelial cells were isolated, dissociated and collected for inDrops scRNA-seq. Human bronchial epithelial cells (HBECs) were cultured for 1 week submerged, followed by 2 weeks at an air-liquid-interface (ALI) and collected for scRNA-seq. b, Mouse tracheal epithelium (n=3 mice) and differentiated HBEC culture (n=3 donors) are pseudostratified, containing basal cells (KRT5) secretory cells (Scgb1a1 in mouse; MUC5B in human), and ciliated cells (AcTub, Acetylated Tubulin). Scale bars, 20m. c,d, SPRING plots of scRNA-seq data for mouse tracheal epithelial cells (n=4 mice, 7,662 cells) (c) and HBECs (n=3 donors, 2,970 cells) (d) colored by inferred cell type, with heat maps of lineage-specific genes by biological replicates (rows). Cell numbers are post quality control. PNEC=pulmonary neuroendocrine cells. Lineage markers for PNECs and brush cells were expressed in rare cells in HBEC cultures, and formed just one human cluster. We visualized the single cell data using a graph-based algorithm (SPRING11) that conserves neighboring relationships of gene expression, facilitating analysis of differentiation trajectories. The resulting graphs revealed a non-uniform continuum structure spanning basal-to-luminal differentiation, with rare gene expression states representing.