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Over the last decade, several organoid models have evolved to acquire increasing cellular, structural, and functional complexity. Advanced lung organoid platforms derived from various sources, including adult, fetal, and induced pluripotent stem cells, have now been generated, which more closely mimic the cellular architecture found within the airways and alveoli. In this regard, the establishment of novel protocols with optimized stem cell isolation and culture conditions has given rise to an array of models able to study key cellular and molecular players involved in lung injury and repair. In addition, introduction of other nonepithelial cellular components, such as immune, mesenchymal, and endothelial cells, and employment of novel precision gene editing tools have further broadened the range of applications for these systems by providing a microenvironment and/or phenotype closer to the desired in vivo scenario. Thus, these developments in organoid technology have enhanced our ability to model various aspects of lung biology, including pathogenesis of diseases such as chronic obstructive pulmonary disease, pulmonary fibrosis, cystic fibrosis, and infectious disease and host-microbe interactions, in ways that are often difficult to undertake using only in vivo models. In this Review, we summarize the latest developments in lung organoid technology and their applicability for disease modeling and outline their strengths, drawbacks, and potential avenues for future development.

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 .

Influenza A virus (IAV) infection mobilizes bone marrow-derived macrophages (BMDM) that gradually undergo transition to tissue-resident alveolar macrophages (TR-AM) in the inflamed lung. Combining high-dimensional single-cell transcriptomics with complex lung organoid modeling, in vivo adoptive cell transfer, and BMDM-specific gene targeting, we found that transitioning (“regenerative”) BMDM and TR-AM highly express Placenta-expressed transcript 1 (Plet1). We reveal that Plet1 is released from alveolar macrophages, and acts as important mediator of macrophage-epithelial cross-talk during lung repair by inducing proliferation of alveolar epithelial cells and re-sealing of the epithelial barrier. Intratracheal administration of recombinant Plet1 early in the disease course attenuated viral lung injury and rescued mice from otherwise fatal disease, highlighting its therapeutic potential.

Alveolar type 2 (AT2) cells are heterogeneous cells, with specialised AT2 subpopulations within this lineage exhibiting stem cell properties. However, the existence of quiescent, immature cells within the AT2 lineage that are activated during lung regeneration is unknown.Sftpc^CreERT2/+;tdTomato^flox/floxmice were used for the labelling of AT2 cells and labelled subpopulations were analysed by flow cytometry, quantitative PCR, assay for transposase-accessible chromatin using sequencing (ATAC-seq), gene arrays, pneumonectomy and culture of precision-cut lung slices. Single-cell RNA-sequencing (scRNA-seq) data from human lungs were analysed.In mice, we detected two distinct AT2 subpopulations, with low tdTomato level (Tom^Low) and high tdTomato level (Tom^High). Tom^Lowcells express lower levels of the AT2 differentiation markersFgfr2bandEtv5, while Tom^High, asbona fidemature AT2 cells, show higher levels ofSftpc,Sftpb,Sftpa1,Fgfr2bandEtv5expression. ATAC-seq analysis indicates that Tom^Lowand Tom^Highcells constitute two distinct cell populations, with specific silencing ofSftpc,Rosa26and cell cycle gene loci in the Tom^Lowpopulation. Upon pneumonectomy, the number of Tom^Lowbut not Tom^Highcells increases and Tom^Lowcells show upregulated expression ofFgfr2b,Etv5,Sftpc,Ccnd1andCcnd2compared to Sham. Tom^Lowcells overexpress programmed cell death 1 ligand 1 (PD-L1), an immune inhibitory membrane receptor ligand, which is used by flow cytometry to differentially isolate these two subpopulations. In the human lung, data mining of a recent scRNA-seq AT2 data set demonstrates the existence of aPD-L1^Pospopulation. Therefore, we have identified a novel population of AT2 quiescent, immature progenitor cells in mouse that expand upon pneumonectomy and we have provided evidence for the existence of such cells in human.

Background: Fibrosis is often associated with aberrant repair mechanisms that ultimately lead to organ failure. In the lung, idiopathic pulmonary fibrosis (IPF) is a fatal form of interstitial lung disease for which there is currently no curative therapy. From the cell biology point of view, the cell of origin and eventual fate of activated myofibroblasts (aMYFs) have taken centre stage, as these cells are believed to drive structural remodelling and lung function impairment. While aMYFs are now widely believed to originate from alveolar fibroblasts, the heterogeneity and ultimate fate of aMYFs during fibrosis resolution remain elusive. We have shown previously that aMYF dedifferentiation and acquisition of a lipofibroblast (LIF)-like phenotype represent a route of fibrosis resolution.Methods: In this study, we combined genetic lineage tracing and single-cell transcriptomics in mice, and data mining of human IPF datasets to decipher the heterogeneity of aMYFs and investigate differentiation trajectories during fibrosis resolution. Furthermore, organoid cultures were utilised as a functional readout for the alveolar mesenchymal niche activity during various phases of injury and repair in mice.Results: Our data demonstrate that aMYFs consist of four subclusters displaying unique pro-alveologenicversuspro-fibrotic profiles. Alveolar fibroblasts displaying a high LIF-like signature largely constitute both the origin and fate of aMYFs during fibrogenesis and resolution, respectively. The heterogeneity of aMYFs is conserved in humans and a significant proportion of human aMYFs displays a high LIF signature.Conclusion: Our work identifies a subcluster of aMYFs that is potentially relevant for future management of IPF.