Periodic Reporting for period 4 - Niche Fibrosis (Identification of regulatory signals from vascular niche in alveolar regeneration and pulmonary fibrosis)
Reporting period: 2020-11-01 to 2021-06-30
Pulmonary fibrosis is a multifaceted and fatal disease that includes damaged alveolar epithelial cells and disorganization of multiple stromal cells. Dysregulation of multicellular crosstalk between epithelial and stromal cells is likely to contribute to fibrosis. However, the precise way this tissue damage occurs is unknown. In this study, we aimed to define functional interactions of epithelial stem cells and their niches that contribute to lung regeneration after fibrotic injury and its implication in pulmonary fibrosis by establishing ex vivo human lung organoid models as well as in vivo animal model systems. Specifically, we have defined a step-wise trajectory of stem cell differentiation during alveolar regeneration after fibrotic injury. Critical niche cells including interstitial macrophages secreting IL-1b, mesenchymal cells expressing Lgr5 and Lgr6, and mature ciliated cells have been demonstrated to govern stem cell differentiation. Furthermore, we have created for the first time the long-term expanding human lung alveolar organdies derived from primary human AT2 cells and airway secretory cells which provide the invaluable ex vivo platform to study human lung diseases including pulmonary fibrosis. 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.
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.
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.
The proposed project was particularly focused on understanding the process of alveolar regeneration, and how dysregulation of this process contributes to pulmonary fibrosis, by identifying dynamic stem cell – niche interactions. We defined, for the first time, the detailed differentiation trajectory of alveolar and airway stem cells during alveolar regeneration after fibrotic damage by analyzing scRNA-seq data of in vivo lineage-tracing and in vitro organoid models that we have newly developed. Critically, we discovered a previously unseen DATPs that are extremely rare at steady-state, yet significantly emerged during lung regeneration after injury. Further lineage-tracing and single-cell profiling of DATPs reveal its unique identity and feature in lung regeneration, as well as lung diseases. Notably, a series of recent studies has highlighted the emergence of DATP-like cells during acute injury repair and disease progression in lung tissue, including pulmonary fibrosis, lung cancer, and acute respiratory distress syndrome (ARDS) caused by SARS-CoV-2 infection. The works from us and others provide an extensive evidence showing the implication of DATPs in human lung diseases including pulmonary fibrosis. We believe that our studies made a significant step forward in the field by identification and characterization of DATPs at cellular and molecular levels in mouse and human lungs. Further demonstration of signaling networks and genetic programs governing their state and fate behaviors during disease development would be our next focus to develop therapeutic strategies targeting them in damaged lungs. Moreover, the model systems that we have developed for human lung biology will beneficial to researchers who study lung development and various lung diseases including idiopathic pulmonary fibrosis. Finally, our project was extremely timely at a time when many researchers, including the respiratory community, are trying to understand the pathogenesis of SARS-CoV-2 infection in human lung tissues where are the main target for COVID-19. In particular, it has been suggested that people infected with SARS-CoV-2 may be left with permanent lung damage such as pulmonary fibrosis, which needs long-term cares. Our works focusing on human lung damage repair and organoid models provide fundamental insights into mechanisms of stem cell-based lung therapies, as well as allow the community to utilize our model systems for their COVID research.