Identifying a Lung Stem Cell Subpopulation by Combining Single-Cell Morphometrics, Organoid Culture, and Transcriptomics.

Single-cell RNA sequencing is a valuable tool for dissecting cellular heterogeneity in complex systems. However, it is still challenging to estimate the proliferation and differentiation potentials of subpopulations within dormant tissue stem cells. Here, we established a new single-cell analysis method for profiling the organoid-forming capacity and differentiation potential of tissue stem cells to disclose stem cell subpopulations by integrating single-cell morphometrics, organoid-forming assay, and RNA sequencing, a method named scMORN. To explore lung epithelial stem cells, we initially developed feeder-free culture system, which could expand all major lung stem cells, including basal, club, and alveolar type 2 (AT2) cells, and found that club cells contained a subpopulation, which showed better survival rate and high proliferation capacity and could differentiate into alveolar cells. Using the scMORN method, we discovered a club cell subpopulation named Muc5b+ and large club (ML-club) cells that efficiently formed organoids than other club or AT2 cells in our feeder-free organoid culture and differentiated into alveolar cells in vitro. Single-cell transcriptome profiling and immunohistochemical analysis revealed that ML-club cells localized at the intrapulmonary proximal airway and distinct from known subpopulations of club cells such as BASCs. Furthermore, we identified CD14 as a cell surface antigen of ML-club cells and showed that purified CD14+ club cells engrafted into injured mouse lungs had better engraftment rate and expansion than other major lung stem cells, reflecting the observations in organoid culture systems. The scMORN method could be adapted to different stem cell tissues to discover useful stem-cell subpopulations.

Autocrine TGF-ß-positive feedback in profibrotic AT2-lineage cells plays a crucial role in non-inflammatory lung fibrogenesis.

The molecular etiology of idiopathic pulmonary fibrosis (IPF) has been extensively investigated to identify new therapeutic targets. Although anti-inflammatory treatments are not effective for patients with IPF, damaged alveolar epithelial cells play a critical role in lung fibrogenesis. Here, we establish an organoid-based lung fibrosis model using mouse and human lung tissues to assess the direct communication between damaged alveolar type II (AT2)-lineage cells and lung fibroblasts by excluding immune cells. Using this in vitro model and mouse genetics, we demonstrate that bleomycin causes DNA damage and activates p53 signaling in AT2-lineage cells, leading to AT2-to-AT1 transition-like state with a senescence-associated secretory phenotype (SASP). Among SASP-related factors, TGF-ß plays an exclusive role in promoting lung fibroblast-to-myofibroblast differentiation. Moreover, the autocrine TGF-ß-positive feedback loop in AT2-lineage cells is a critical cellular system in non-inflammatory lung fibrogenesis. These findings provide insights into the mechanism of IPF and potential therapeutic targets.