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Organoids derived from mouse and human stem cells have recently emerged as a powerful tool to study organ development and disease. We here established a three-dimensional (3D) murine bronchioalveolar lung organoid (BALO) model that allows clonal expansion and self-organization of FACS-sorted bronchioalveolar stem cells (BASCs) upon co-culture with lung-resident mesenchymal cells. BALOs yield a highly branched 3D structure within 21 days of culture, mimicking the cellular composition of the bronchioalveolar compartment as defined by single-cell RNA sequencing and fluorescence as well as electron microscopic phenotyping. Additionally, BALOs support engraftment and maintenance of the cellular phenotype of injected tissue-resident macrophages. We also demonstrate that BALOs recapitulate lung developmental defects after knockdown of a critical regulatory gene, and permit modeling of viral infection. We conclude that the BALO model enables reconstruction of the epithelial-mesenchymal-myeloid unit of the distal lung, thereby opening numerous new avenues to study lung development, infection, and regenerative processes in vitro., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Bronchioalveolar stem cells (BASCs) are a potential source for lung regeneration, but direct in vivo evidence for a multipotential lineage contribution during homeostasis and disease is critically missing, since specific genetic labeling of BASCs has not been possible. We developed a novel cell tracing approach based on intein‐mediated assembly of newly engineered split‐effectors, allowing selective targeting of dual‐marker expressing BASCs in the mouse lung. RNA sequencing of isolated BASCs demonstrates that BASCs show a distinct transcriptional profile, characterized by co‐expression of bronchiolar and alveolar epithelial genes. We found that BASCs generate the majority of distal lung airway cells after bronchiolar damage but only moderately contribute to cellular turnover under homeostatic conditions. Importantly, DTA‐mediated ablation of BASCs compromised proper regeneration of distal airways. The study defines BASCs as crucial components of the lung repair machinery and provides a paradigmatic example for the detection and manipulation of stem cells that cannot be recognized by a single marker alone. Synopsis: Contribution of lung progenitors or stem cells to replenishment of mature cell types after injury has been debated. Here, a newly‐developed genetic complementation approach identifies bronchioalveolar stem cells (BASCs) located at the duct junction as a dominant component of the mouse distal airway repair. Genetic targeting based on intein‐mediated assembly of split‐effectors allows for selective visualization, tracing and manipulation of dual‐marker expressing BASCs in the lung.BASCs show a distinct transcriptional profile characterized by co‐expression of bronchiolar and alveolar epithelial genes.BASCs emerge postnatally and contribute modestly to cellular turnover in the undamaged lung.BASCs contribute substantially to bronchioalveolar regeneration and differentiate into alveolar type 2, club‐ and ciliated cells.Cellular ablation of BASCs impairs efficient regeneration of distal airways. [ABSTRACT FROM AUTHOR]