New Advanced Engineering Tools for Investigating Lung Injury and Repair

PROJECT SUMMARY Idiopathic pulmonary fibrosis (IPF) is characterized by scarring and distortion of the alveolar architecture and a reduction in alveolar function. Under normal conditions, alveolar type II (AT2) cell turnover is low. However, extensive lineage tracing studies have demonstrated that, upon injury, AT2 cells can function as stem cells, replacing both themselves and alveolar type I (AT1) cells. Despite the critical role of AT2 cells in alveolar regeneration, virtually nothing is known about their dynamic behavior during lung injury and repair. Understanding these processes is critical to determining how the alveolar niche balances self-renewal with differentiation in order to maintain and repair lung tissue. Unfortunately, there is a distinct lack of technologies that can track these cells over extended periods, at single cell resolution, and in real time, and quantify their behaviours in response to defined perturbations. To address this need, we have brought together two teams with extensive expertise in bioengineering, optics, in vivo lung imaging, IPF, and molecular and cellular biology in order to develop two new bioengineering approaches capable of visualizing AT2 cell behavior in the alveolar niche. The first, called the Lung Explant Imaging System (LEIS), consists of a custom imaging chamber designed to maintain lung explanted, precision cut lung slices (PCLS) for extended periods of time while simultaneously performing single cell 3D imaging. Using LEIS, we will directly visualize AT2 cell proliferation, division, migration, and differentiation in real time during progression of fibrosis post-bleomycin- induced lung injury. The second, called Single Cell Ablation through the Window for High-Resolution Intravital imaging of the Lung (SCA-WHRIL), is an intravital imaging-based system capable of simultaneously performing single cell-ablation and intravital imaging of the intact, living, breathing mouse lung. We will use SCA-WHRIL to ablate individual cell types from within the alveolar niche and formally test their requirement for alveolar repair. We will use different mouse strains bearing fluorescent labels of different cell types (AT1 cells, AT2 cells) to track and quantify the dynamic cell behaviors of different niche components during injury and regeneration. Taken together, these two approaches will transform our ability to address fundamental questions concerning the molecular and cellular mechanisms of alveolar repair in real time during a physiological regeneration process. Through application of our approaches, we will specify for the first time the spatio-temporal dynamics of alveolar stem cell-mediated repair and will identify the mechanism of action and target cells of key regeneration signals in the alveolar niche in real time. Our findings will have important implications for understanding stem cell biology and regeneration of the alveolar niche during lung injury, and may help to inform targeted strategies for therapeutic intervention of IPF.