A robust human airway organoid platform enables scalable expansion and trajectory mapping of pulmonary neuroendocrine cells

ABSTRACT Pulmonary neuroendocrine cells (PNECs) are rare chemosensory epithelial cells, facultative stem cells, and a cell-of-origin for neuroendocrine lung cancers, yet the mechanisms governing their differentiation and heterogeneity are poorly understood. Here we establish NEr-fAOs, a human fetal airway organoid platform that robustly enriches PNECs, and identify a cooperative requirement for dual GSK3 and NOTCH inhibition to drive directed PNEC differentiation. This strategy yields stable cultures with up to 60-fold expansion of PNECs whose transcriptomes closely match fetal and adult PNECs. In addition to PNEC-enrichment, NEr-fAOs retain diverse airway epithelial cell types, preserving epithelial complexity. Time-resolved single-cell transcriptomics maps PNEC trajectories in NEr-fAOs, resolving precursor and mature states. Comparative analyses further reveal a distal airway bias in NEr-fAOs and enrichment for lower-airway progenitors. NEr-fAOs thus provide a scalable, tractable platform to dissect human PNEC biology and distal airway progenitor hierarchies relevant to lung development, cancer, and disease. Competing Interest Statement The authors have declared no competing interest. Footnotes ↵11 co-last authors ↵12 Lead contact This version of the manuscript has been updated to: 1) correct mistakes in author name spelling 2) add reference and url to ShinyApp for data exploration 3) refined discussion section 4) include data availability information 5) corrections to figures 1, figures 2, and figures 3 6) additional data added to figures S3 and S5