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Identification of regulatory signals from vascular niche in alveolar regeneration and pulmonary fibrosis

Periodic Reporting for period 3 - Niche Fibrosis (Identification of regulatory signals from vascular niche in alveolar regeneration and pulmonary fibrosis)

Reporting period: 2019-05-01 to 2020-10-31

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. Recently, we have identified a crucial interaction between lung endothelial cells and lung stem cells during alveolar injury response, and demonstrated a new regulatory signalling pathway that operates in endothelial cells to support alveolar injury repair by driving alveolar lineage specification of stem cells. Importantly, introduction of endothelial-derived factors into the lung after fibrotic damage enhances alveolar regeneration and prevents pulmonary fibrosis. Given these results, we bring a new concept of stem-niche interactions in alveolar injury repair and pulmonary fibrosis. Using both in vivo murine and organoid culture, as well as human lung organoid culture systems, we aim to identify the key components of the niche that constrain lung alveolar stem cell behaviour in pulmonary fibrosis, and to identify novel mechanisms for stimulating regeneration. Insights gained from these studies will accelerate the development of novel and selective therapeutic approaches that directly target stem cells or their niches in pulmonary fibrosis.

Here we pursue two main aims:
- Define the functional role of vascular niche in stem cell differentiation in alveolar regeneration and pulmonary fibrosis
- Define how endothelial cells and lung epithelial cells are affected in patient samples of pulmonary fibrosis
Aim 1. Define the functional role of vascular niche in stem cell differentiation in alveolar regeneration and pulmonary fibrosis.
We have performed gene expression profiling in individual organoids derived from distal lung stem cells that are co-cultured with endothelial cells. Through comprehensive bioinformatics analysis and validation with qPCR in isolated cells from mouse lungs post fibrotic damage, we found several candidates that are highly expressed in alveolar stem cells (AT2 cells) and their expression levels are dynamically changed during alveolar injury repair. Paired gene sets of receptors and ligands that are expressed in AT2 cells and endothelial cells were selected and validated using CRISPR technologies in organoid co-cultures. We are currently generating mouse lines that specifically target candidates in endothelial cells or AT2 cells using Loxp/CreERT2 system. In addition, we recently identified distinct mesenchymal cell populations expressing Lgr5 in alveolar parenchyma (Lee et al., Cell. 2017). These cells support organoid formation of AT2 cells in vitro and respond to fibrotic damage in vivo, indicating the functional role of these cells in alveolar injury repair. Currently, we investigate the multi-crosstalk among AT2 cells, Lgr5+mesenchymal cells, and endothelial cells during alveolar regeneration and fibrotic progression.

Aim 2. Define how endothelial cells and lung epithelial cells are affected in patient samples of pulmonary fibrosis.
We have defined molecular requirements that allow, for the first time, the long-term expansion of mouse AT2 cells (>I yr) in stroma-free organoid culture. More recently, we have successfully established alveolar organoids derived from distal lung biopsies of human normal tissues. These cultures maintain their ability for self-renewal (expansion) and differentiate towards alveolar lineages. Further functional characterisation is currently carried out at the histological, molecular, and genomic level. More organoids will be established from IPF patients and the impact of niche cells including endothelial cells and fibroblasts will be tested. In addition, we found that endothelial cells secreted various ECM components which have been reported to alter tissue stiffness in fibrotic lungs. We explore the effect of altered tissue stiffness on the AT2 cell behaviour by growing them in hydrogels of varying stiffness in collaboration with Dr Kevin Chalut in our institute.
Lung epithelial cells have emerged as a frequent target of injury, a driver of normal repair, and a key element in the pathobiology of fibrotic lung diseases. Essential to our understanding of the mechanisms involved in injury repair is the identification and characterization of the cells that are potentially capable of repopulating the injured lung. However, the turnover and differentiation of the alveolar epithelium under physiologic conditions and the mechanisms of epithelial regeneration under pathologic situations are poorly understood. An important aspect of epithelial cells is their capacity to respond to microenvironmental cues for proper regeneration and repair. This proposal seeks to evaluate alveolar lineage differentiation of epithelial stem cells in physiologic and pathologic conditions, and define the important role of niche components in providing instructive cues for stem cell differentiation during lung regeneration and injury repair post chronic lung injury.

Despite an increasing incidence in the chronic lung diseases, COPD and IPF, development of new therapies has been held back by a lack of suitable disease models. Current animal models of chronic lung disease do not adequately replicate human pathology. Therefore, there is a growing appreciation of the importance of developing primary human cell-based systems to capture all the features of human disease and streamline research. In this project, we aim to establish and characterize organoids derived from IPF patient tissue samples as models for chronic lung disease. Specifically, we aim to understand the effect of disease-related changes in the niche on stem cell function, and to identify new mechanisms for stimulating regeneration. Developing reliable in vitro models of chronic lung disease will pave the way for developing new therapies and will replace animal models in research.