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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. Synopsis: This resource reports generation of bronchioalveolar lung organoids (BALO) derived from bronchioalveolar stem cells (BASC) and lung‐resident mesenchymal cells (rMC), offering new opportunities to investigate lung development, stromal cell interactions, infection, and regeneration in vitro. BALO are established from adult mouse lung rMC and BASC cells.BALO reproduce 3D structure and proximo‐distal patterning of the bronchioalveolar compartment.BALO allow engraftment of tissue‐resident macrophages to study epithelial‐immune cell cross‐talk.BALO can be used for cell‐specific genetic manipulation and modeling of lung viral infection. [ABSTRACT FROM AUTHOR]

Summary: Accurate estimation of the absolute number of a particular cell‐type in whole organs is increasingly important in studies on organogenesis, and the remodelling and repair of diseased tissues. The unbiased estimation of the absolute number of cells in an organ is complicated, and design‐based stereology remains the method of choice. This has led investigators to explore alternative approaches – such as flow cytometry – as a faster and less labour‐intensive replacement for stereology. To address whether flow cytometry might substitute stereology, design‐based stereology was compared with microfluorosphere‐controlled flow cytometry, for estimation of the absolute number of alveolar epithelial type 2 cells (AEC2) in the lungs of two mouse strains: wild‐type C57BL/6J mice and Sftpc‐YFP mice. Using design‐based stereology, ≈10.7 million and ≈9.0 million AEC2 were estimated in the lungs of wild‐type C57BL/6J mice and Sftpc‐YFP mice, respectively. Substantially fewer AEC2 were estimated using flow cytometry. In wild‐type C57/BL6J mouse lungs, 59% of the AEC2 estimated by design‐based stereology were estimated by flow cytometry (≈6.3 million), using intracellular staining for pro‐surfactant protein C. Similarly, in Sftpc‐YFP mouse lungs, 23% of the AEC2 estimated by design‐based stereology were estimated by flow cytometry (≈2.1 million), using yellow fluorescent protein fluorescence. Our data suggest that flow cytometry underestimates AEC2 number, possibly due to impaired recoverability of AEC2 from dissociated lung tissue. These data suggest design‐based stereology as the method of choice for the unbiased estimation of the absolute number of cells in an organ. Lay Description: There is much interest in studies on the pathological changes that accompany disease, to be able to count or estimate the number of a particular cell‐type in solid tissue, such as an organ. The easiest way to do this is to make liquid suspensions of single cells from solid tissue, and then to count the number of cells of interest, using either a microscope, or automated cell counting (for example, a flow cytometer). Alternatively, solid tissue may be examined microscopically, where the cell‐type of interest might also be counted 'by eye' or in an automated manner using software (called planimetry). All of these approaches to counting cells in solid organs come with serious drawbacks, and estimation of the cell number may thus be inaccurate. To overcome this, we have employed a combination of mathematical tools and statistical principles together with microscopy (called 'design‐based stereology') that permits the unbiased counting of cells in microscopic fields, which can then be extrapolated to the entire solid tissue volume, to accurately estimate the number of a cell‐type of interest in the solid tissue. We have compared this method with the estimation of cell number using a flow cytometer. Our data reveal that flow cytometry appreciably underestimates the total number of cells in solid tissue, where we used the lung as an example of solid tissue, and estimated the number of a unique cell‐type in the lung: the alveolar epithelial type 2 cell, to compare stereology with flow cytometry. We believe that flow cytometry underestimates the cell number due to the difficulty of breaking up solid tissue into single cells, and being able to recover all of those single cells for analysis. Our data supports the recommendation to use stereology, not flow cytometry, to accurately estimate the number of a particular cell‐type in solid tissue. Accurate estimation of the absolute number of a particular cell‐type in whole organs is increasingly important in studies on organogenesis, and the remodelling and repair of diseased tissues. Although estimation of the relative number of cells might be straightforward, unbiased estimation of the absolute number of cells in an organ is complicated, and design‐based stereology remains the method of choice. This has led investigators to explore alternative approaches – such as flow cytometry – as a faster and less labour‐intensive replacement for stereology. To address whether flow cytometry might substitute stereology, design‐based stereology was compared with microfluorosphere‐controlled flow cytometry, for estimation of the absolute number of alveolar epithelial type 2 cells (AEC2) in the lungs of two mouse strains: wild‐type C57BL/6J mice and Sftpc‐YFP mice. Using design‐based stereology, ≈10.7 million and ≈9.0 million AEC2 were estimated in the lungs of wild‐type C57BL/6J mice and Sftpc‐YFP mice, respectively. Substantially fewer AEC2 were estimated using flow cytometry. In wild‐type C57/BL6J mouse lungs, 59% of the AEC2 estimated by design‐based stereology were estimated by flow cytometry (≈6.3 million), using intracellular staining for pro‐surfactant protein C. Similarly, in Sftpc‐YFP mouse lungs, 23% of the AEC2 estimated by design‐based stereology were estimated by flow cytometry (≈2.1 million), using yellow fluorescent protein fluorescence. Our data suggest that flow cytometry underestimates AEC2 number, possibly due to impaired recoverability of AEC2 from dissociated lung tissue. These data suggest design‐based stereology as the method of choice for the unbiased estimation of the absolute number of cells in an organ. [ABSTRACT FROM AUTHOR]

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]