A map of signaling responses in human airway epithelium

Receptor-mediated signaling plays a central role in tissue regeneration, and it is dysregulated in disease. Here, we build a signaling–response map for a model regenerative human tissue: the airway epithelium. We analyzed the effect of 17 receptor-mediated signaling pathways on organotypic cultures to determine changes in abundance and phenotype of all epithelial cell types. This map recapitulates the gamut of known airway epithelial signaling responses to these pathways. It defines convergent states induced by multiple ligands and diverse, ligand-specific responses in basal-cell and secretory-cell metaplasia. We show that loss of canonical differentiation induced by multiple pathways is associated with cell cycle arrest, but that arrest is not sufficient to block differentiation. Using the signaling-response map, we show that a TGFB1-mediated response underlies specific aberrant cells found in multiple lung diseases and identify interferon responses in COVID-19 patient samples. Thus, we offer a framework enabling systematic evaluation of tissue signaling responses. Overall design: Human bronchial epithelial cells were cultured at air-liquid interface (ALI) without treatment to drive differentiation into a pseudo- stratified epithelium that recapitulates the physiological cell types of the airway. The differentiated luminal cells were then stripped through calcium depletion, leaving the remaining basal cells to regenerate the tissue. This was done in the presence of a signaling agonist added to each well at a dosage 10- to 100-fold greater than the IC50. One condition was kept unperturbed for control. After 2 weeks of differentiation, the final composition of the tissue was analyzed by scRNA-Seq and imaging. Cells were run in two separate experiments: donor #429581 was collected individually; donors #221175 and #323353 were pooled and captured simultaneously, except for one condition (ActA) for which we only collected donor #323353. We call these two experiments as ali12 and ali13 respectively in the metadata files.