A deep lung cell atlas reveals cytokine-mediated lineage switching of a rare cell progenitor of the human airway epithelium: In-vitro ALI Data
The human airway epithelium contains specialized rare cells, including ionocytes and tuft cells, that have been associated with  respiratory disease. Ionocytes express the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) whereas chemosensory tuft cells express asthma-associated alarmins. Surprisingly, exceedingly few mature tuft cells have been identified in human lung cell atlases.  To enable the identification of rare lung cells and define their lineage specification, we generated a deep lung cell atlas of 311,748 cell profiles with careful anatomical sampling of the large and small human airways from entire lungs declined for transplant. In contrast to prior reports, ionocytes were present in similar numbers in both the large and small airways, suggesting that they may contribute to both large and small airways pathologies in CF, a finding which has important implications for gene therapy. We also identify mature tuft cells in a patient who died from an asthma flare. Computational analysis identified rare, bipotent Tuft-Ionocyte Progenitor cells (TIP cells), which have a comparable/higher fraction of cycling cells in the large airways as basal cells (18% vs 11%)  and are predicted to give rise to either tuft cells or ionocytes based on the airway signaling milieu. Using an optimized in vitro system we show that the default pathway of TIP cell differentiation produces ionocytes, but in the presence of Type 2 and Type 17 cytokines, TIP cells are diverted to differentiate into tuft cells. Using scRNAseq and scATACseq we further predicted transcription factors and intermediate cell states that mark these rare cell lineages. Overall, our findings suggest that the immune signaling pathways that characterize asthma and CF may skew the composition of disease relevant rare cells and show how deep atlases are required to identify disease relevant but scarce cells and link them to molecular mechanisms driving pathology. Overall design: We performed 10x based single-cell RNA sequencing on in-vitro, air-liquid interface cultures of primary bronchus and small airway basal cells, grown in pneumacult ALI media. We applied IL13 treatment and compared it to PBS controls