Ex vivo lung-organoid model for aberrant basaloid cell induction and activation

Pulmonary fibrosis (PF) is a severe lung disease characterized by the destruction of lung architecture resulting from chronic epithelial injury. The PF microenvironment induces PF-specific epithelial cells, such as aberrant basaloid cells (ABCs). However, limited experimental models capable of inducing and activating PF-specific epithelial cells hinder the understanding of their roles.

To address the lack of experimental models, in this study, we developed an ex vivo murine lung-organoid model designed to induce and activate ABCs. The organoids were subjected to bleomycin (BLM) stimulation. Dose-dependent reductions in number and size, structural disorganization, and transcriptomic changes were assessed following stimulation. Single-cell RNA-sequencing (scRNA-seq) analysis was performed to identify ABC subsets. Cell-cell interaction analysis was also conducted.

Following BLM stimulation, the organoids displayed dose-dependent reductions in number and size, along with structural disorganization and transcriptomic changes that were similar to those observed in the in vivo murine fibrosis model. scRNA-seq analysis identified two ABC subsets: Krt5^low Tp63^low Krt17⁺ ABCs_1, found in patients with idiopathic pulmonary fibrosis (IPF), and Krt5^hi Tp63^hi Krt17⁺ ABCs_2, which have been observed in cultured tissues from patients with IPF but not in traditional murine models. BLM stimulation led to the induction of transforming growth factor beta (TGF-β2) expression in ABCs. Cell-cell interaction analysis suggested that BLM-damaged type 2 alveolar epithelial cells (AT2s) enhanced their direct and indirect interactions with ABCs_2 via ephrin-A signaling. In line with this observation, stimulation experiments of BLM-damaged organoids revealed that Ephrin A4 induced ABC cell differentiation-related gene expression changes, whereas Ephrin A3 enhanced epithelial proliferation-related gene expression changes and suppressed fibroblast activation-related gene expression changes.

The developed organoid model serves as a novel platform for studying the roles and responses of PF-specific ABCs. This model may contribute to advancing the understanding of PF pathogenesis and facilitate the development of ABC-targeted therapies.