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Background <p>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.</p> Methods <p>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.</p> Results <p>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<sup>low</sup> Tp63<sup>low</sup> Krt17<sup>+</sup> ABCs_1, found in patients with idiopathic pulmonary fibrosis (IPF), and Krt5<sup>hi</sup> Tp63<sup>hi</sup> Krt17<sup>+</sup> 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.</p> Conclusions <p>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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年10月30日

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  • Bin Wu,
  • Shigeyuki Shichino,
  • Satoshi Ueha,
  • Rina Matsukiyo,
  • Yu Ishimura,
  • Haru Ogiwara,
  • Masaki Takasu,
  • Shotaro Yamano,
  • Yumi Umeda,
  • Kouji Matsushima

摘要

Background

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.

Methods

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.

Results

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: Krt5low Tp63low Krt17+ ABCs_1, found in patients with idiopathic pulmonary fibrosis (IPF), and Krt5hi Tp63hi 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.

Conclusions

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.