Single-cell RNA sequencing analysis of murine lung epithelial cells and fibroblasts derived from bleomycin-injured alveolospheres

Pulmonary fibrosis is a devatating lung disease with limited effective treatment. Because fibrotic responses are considered to be regulated by complex cellular and molecular circuits, it is difficult to demonstrate a causal relationship of cells, molecules, and cell-cell interactions with diseases in vivo. To overcome this issue, we established bleomycin (BLM)-stimulated alveolosphere model as an ex vivo culture model. To clarify whether our ex vivo model recapitulates the cellular and molecular response to BLM-induced lung injury, we performed single-cell RNA-seq (scRNA-seq) analysis of the BLM-stimulated alveolospheres generated by co-culturing murine lung epithelial cells and lung fibroblasts. Alveolopheres were generated by co-culturing primary mouse CD326 positive lung epithelial cells and Cd140a positive lung fibroblasts. To clarify transcriptomic changes of BLM-injured alveolospheres at single-cell level, we stimulated alveolospheres with bleomycin (0.1 ug/ml) in a culture media from day 8 to day 10 post co-cultured. After that, bleomycin was removed from the culture media and the alveolospheres were further cultured until day 14 post co-cultured. scRNA-seq analysis was performed at day 10 and day 14 post co-cultured.