Aim 1. In order to understand the alteration of stem cell differentiation properties post chronic lung injury, we first aimed to determine the step-wise trajectory of stem cell differentiation during alveolar regeneration after bleomycin injury. By utilizing combined lineage-tracing and single-cell RNA sequencing (scRNA-seq) technologies, we for the first time determined the detailed differentiation trajectory of stem cell populations that contribute to alveolar regeneration. Significantly, we newly identified a novel Damage-Associated Transient Progenitors (DATPs) that are derived either from alveolar type II (AT2) or airway secretory cells, and integral to alveolar regeneration after injury. Furthermore, we determined critical niche cells directing the behaviors of stem cell fate and activity during alveolar regeneration post injury. Distinct mesenchymal cell populations expressing Lgr5, Lgr6, and Tnfrsf19 have been identified in the lungs. We also defined the functional interactions of stem cells with immune cells that trigger the alveolar differentiation process of alveolar and airway stem cells upon fibrotic damage. Finally, we uncovered molecular mechanisms driving alveolar differentiation of stem cells during alveolar regeneration post injury. IL-1β-mediated inflammatory signals derived from interstitial macrophages are integral for alveolar regeneration by triggering differentiation of alveolar stem cells. Moreover, we also demonstrated that IL-1β-mediated inflammatory signals modulate the expression of Notch ligands in ciliated cells which then control the fate conversion of airway secretory cells into alveolar lineages during alveolar regeneration post fibrotic injury. Notably, sustained IL-1β signals impair maturation of alveolar lineage differentiation with aberrant accumulation of DATPs, suggesting the implication of spatiotemporal modulation of niche signals in tissue resolution of pulmonary fibrosis. Our study sheds the light on the mechanism how unresolved damage signals alter stem cell properties which can contribute to the initiation and/or progression of chronic lung diseases including pulmonary fibrosis.
Aim 2. We have established, for the first time, the long-term expanding human lung alveolar organoids derived from primary human AT2 cells and airway organoids derived from primary secretory cells in stroma-free culture condition. These organoids retained self-renewing AT2 and airway secretory cells that are capable of differentiation into mature cell types including AT1 cells and ciliated cells, respectively. Importantly, we are able to maintain them over multiple passages up to 1-yr, and establish the protocol for freezing and thawing enabling their robust usage. We have also established an optimized protocol for co-culturing stem cells with various stromal cells including endothelial, mesenchymal and immune cells. Furthermore, we established human lung alveolar and airway organoids derived from primary lung patient tissues of idiopathic pulmonary fibrosis (IPF). Importantly, IPF-AT2 cells showed altered stem cell properties including decreased self-renewal capacity and increased plasticity. Notably, we showed the aberrant accumulation of DATP-like cells in IPF tissues, as well as IPF-organoids, suggesting the functional contribution of altered stem cell properties to IPF. Our study provides important insights which accelerate the development of novel and selective therapeutic approaches that directly target stem cells or their niches in pulmonary fibrosis.
