Background: Despite compelling data describing pro-regenerative effects of all-trans retinoic acid (ATRA) in pre-clinical models of chronic obstructive pulmonary disease (COPD), clinical trials using retinoids for emphysema patients have failed. Crucial information about the specific role of RA signaling in adult rodent and human lung epithelial progenitor cells is largely missing.Methods: Adult lung organoid cultures were generated from isolated primary mouse and human lung epithelial cells, and incubated with pharmacological pathway modulators and recombinant proteins. Organoid number and size were measured, and differentiation was assessed with quantitative imrnunofluorescence and gene expression analyses.Findings: We unexpectedly found that ATRA decreased lung organoid size, whereas RA pathway inhibition increased mouse and human lung organoid size. RA pathway inhibition stimulated mouse lung epithelial proliferation via YAP pathway activation and epithelial-mesenchymal FGF signaling, while concomitantly suppressing alveolar and airway differentiation. HDAC inhibition rescued differentiation in growth-augmented lung organoids.Interpretation: In contrast to prevailing notions, our study suggests that regenerative pharmacology using transient RA pathway inhibition followed by HDAC inhibition might hold promise to promote lung epithelial regeneration in diseased adult lung tissue. (C) 2018 The Authors. Published by Elsevier B.V.