Human alveolar progenitors generate dual lineage bronchioalveolar organoids

The GSK3 inhibitor CHIR99021 is required to maintain the alveolar lineage in alveolar progenitors grown in airway media or AT2 cells become plastic, with the observed effects acting outside of its role as a GSK3B inhibitor. Mechanisms of epithelial renewal in the alveolar compartment remain incompletely understood. To this end, we aimed to characterize alveolar progenitors. Single-cell RNA-sequencing (scRNA-seq) analysis of the HTII-280(+)/EpCAM(+) population from adult human lung revealed subclusters enriched for adult stem cell signature (ASCS) genes. We found that alveolar progenitors in organoid culture in vitro show phenotypic lineage plasticity as they can yield alveolar or bronchial cell-type progeny. The direction of the differentiation is dependent on the presence of the GSK-3 beta inhibitor, CHIR99021. By RNA-seq profiling of GSK-3 beta knockdown organoids we identified additional candidate target genes of the inhibitor, among others FOXM1 and EGF. This gives evidence of Wnt pathway independent regulatory mechanisms of alveolar specification. Following influenza A virus (IAV) infection organoids showed a similar response as lung tissue explants which confirms their suitability for studies of sequelae of pathogen-host interaction.

Automated summary

The GSK3 inhibitor CHIR99021 is necessary for the maintenance of the alveolar lineage and regulates the differentiation of alveolar progenitors. Single-cell RNA-sequencing analysis identified subclusters enriched for adult stem cell signature genes in alveolar progenitors. The presence of the GSK-3 beta inhibitor, CHIR99021, determines the direction of differentiation, and RNA-seq profiling revealed additional target genes of the inhibitor, suggesting independent regulatory mechanisms of alveolar specification.