Endothelialized Bronchioalveolar Lung Organoids Model Endothelial Cell Responses to Injury.

Organoid 3D systems are powerful platforms to study development and disease. Recently, the complexity of lung organoid models derived from adult mouse and human stem cells has increased substantially in terms of cellular composition and structural complexity. However, a murine lung organoid system with a clear integrated endothelial compartment is still missing. Here, we describe a novel method that adds another level of intricacy to our published bronchioalveolar lung organoid (BALO) model by microinjection of FACS-sorted lung endothelial cells (ECs) into differentiated organoid cultures. Before microinjection, ECs obtained from the lung homogenate (LH) of young mice expressed typical ECs markers such as CD31 and vascular endothelial (VE)-Cadherin and showed tube formation capacity. Following microinjection, ECs surrounded BALO´s alveolar-like compartment aligning with both alveolar epithelial cells type I (AECI) and type II (AECII), as demonstrated by confocal and electron microscopy. Notably, expression of Car4 and Aplnr was as well detected, suggesting presence of EC microvascular phenotypes in the cultured ECs. Moreover, upon epithelial cell injury by lipopolysaccharides (LPS) and influenza A virus (IV), endothelialized BALO (eBALO) released proinflammatory cytokines leading to the upregulation of the intercellular adhesion molecule 1 (ICAM-1) in ECs. In summary, we characterized for the first time a organoid model that incorporates ECs into the alveolar structures of lung organoids, not only increasing our previous model ́s cellular and structural complexity but also providing a suitable niche to model lung endothelium responses to injury ex vivo.

Multilineage murine stem cells generate complex organoids to model distal lung development and disease.

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.