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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.
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