Rationale:The respiratory tract constitutes an elaborated line of defense based on a unique cellular ecosystem. Single-cell profiling methods enable the investigation of cell population distributions and transcriptional changes along the airways. Methods:We have explored cellular heterogeneity of the human airway epithelium in 10 healthy living volunteers by single-cell RNA profiling. 77,969 cells were collected by bronchoscopy at 35 distinct locations, from the nose to the 12 th division of the airway tree. Results:The resulting atlas is composed of a high percentage of epithelial cells (89.1%), but also immune (6.2%) and stromal (4.7%) cells with peculiar cellular proportions in different sites of the airways. It reveals differential gene expression between identical cell types (suprabasal, secretory, and multiciliated cells) from the nose (MUC4, PI3, SIX3) and tracheobronchial (SCGB1A1, TFF3) airways. By contrast, cell-type specific gene expression was stable across all tracheobronchial samples. Our atlas improves the description of ionocytes, pulmonary neuroendocrine (PNEC) and brush cells, which are likely derived from a common population of precursor cells. We also report a population of KRT13 positive cells with a high percentage of dividing cells which are reminiscent of "hillock" cells previously described in mouse.Conclusions: Robust characterization of this unprecedented large single-cell cohort establishes an important resource for future investigations. The precise description of the continuum existing from nasal epithelium to successive divisions of lung airways and the stable gene expression profile of these regions better defines conditions under which relevant tracheobronchial proxies of human respiratory diseases can be developed.

The upper airway epithelium, which is mainly composed of multiciliated, goblet, club and basal cells, ensures proper mucociliary function and can regenerate in response to assaults. In chronic airway diseases, defective repair leads to tissue remodeling. Delineating key drivers of differentiation dynamics can help understand how normal or pathological regeneration occurs. Using single-cell transcriptomics and lineage inference, we have unraveled trajectories from basal to luminal cells, providing novel markers for specific populations. We report that: (1) a precursor subgroup of multiciliated cells, which we have entitled deuterosomal cells, is defined by specific markers, such as DEUP1, FOXN4, YPEL1, HES6 and CDC20B; (2) goblet cells can be precursors of multiciliated cells, thus explaining the presence of hybrid cells that co-express markers of goblet and multiciliated cells; and (3) a repertoire of molecules involved in the regeneration process, such as keratins or components of the Notch, Wnt or BMP/TGFβ pathways, can be identified. Confirmation of our results on fresh human and pig airway samples, and on mouse tracheal cells, extend and confirm our conclusions regarding the molecular and cellular choreography at work during mucociliary epithelial differentiation.