Rho/SMAD/mTOR triple inhibition enables long-term expansion of human neonatal tracheal aspirate-derived airway basal cell-like cells

Background: Bronchopulmonary dysplasia remains one of the most common complications of prematurity, despite significant improvements in perinatal care. Functional modeling of human lung development and disease, like BPD, is limited by our ability to access the lung and to maintain relevant progenitor cell populations in culture. Methods: We supplemented Rho/SMAD signaling inhibition with mTOR inhibition to generate epithelial basal cell-like cell lines from tracheal aspirates of neonates. Results: Single-cell RNA-sequencing confirmed the presence of epithelial cells in tracheal aspirates obtained from intubated neonates. Using Rho/SMAD/mTOR triple signaling inhibition, neonatal tracheal aspirate-derived (nTAD) basal cell-like cells can be expanded long term and retain the ability to differentiate into pseudostratified airway epithelium. Conclusions: Our data demonstrate that neonatal tracheal aspirate-derived epithelial cells can provide a novel ex vivo human cellular model to study neonatal lung development and disease. Impact: Airway epithelial basal cell-like cell lines were derived from human neonatal tracheal aspirates.mTOR inhibition significantly extends in vitro proliferation of neonatal tracheal aspirate-derived basal cell-like cells (nTAD BCCs).nTAD BCCs can be differentiated into functional airway epithelium.nTAD BCCs provide a novel model to investigate perinatal lung development and diseases.