New Advanced Engineering Tools for Investigating Lung Injury and Repair

项目摘要 特发性肺纤维化（IPF）的特征是肺泡结构的瘢痕形成和扭曲， 肺泡功能下降在正常情况下，肺泡II型（AT 2）细胞周转率低。然而，在这方面， 广泛的谱系追踪研究已经证明，在损伤时，AT 2细胞可以作为干细胞， 替换自身和肺泡I型（AT 1）细胞。尽管AT 2细胞在肺泡巨噬细胞中起着关键作用， 然而，尽管它们在肺损伤和修复过程中的动态行为几乎一无所知。 了解这些过程对于确定肺泡生态位如何平衡自我更新与 分化，以维持和修复肺组织。不幸的是， 它可以在长时间内以单细胞分辨率和真实的时间跟踪这些细胞，并量化它们的 对确定的扰动的反应。为了满足这一需求，我们召集了两个团队 在生物工程、光学、体内肺成像、IPF以及分子和细胞方面拥有丰富的专业知识， 生物学，以开发两种新的生物工程方法，能够可视化AT 2细胞的行为， 牙槽龛第一种称为肺移植成像系统（LEIS），由自定义成像 设计用于长时间维持肺组织切片（PCLS）的腔室 同时进行单细胞3D成像。使用LEIS，我们将直接可视化AT 2细胞， 博来霉素治疗后纤维化进展过程中真实的时间内的增殖、分裂、迁移和分化， 导致肺损伤。第二个，称为通过窗口进行高分辨率活体内单细胞消融 肺成像（SCA-WHRIL）是一种基于活体成像的系统，能够同时 对完整的、活的、呼吸的小鼠肺进行单细胞消融和活体成像。我们将使用 SCA-WHRIL从肺泡龛内消融单个细胞类型，并正式测试其需求 进行牙槽修复我们将使用携带不同细胞类型（AT 1）的荧光标记的不同小鼠品系 细胞，AT 2细胞）来跟踪和量化损伤期间不同小生境组分的动态细胞行为 和再生。这两种方法结合在一起，将改变我们解决基本问题的能力， 关于肺泡修复的分子和细胞机制的问题，在真实的时间， 生理再生过程通过应用我们的方法，我们将首次指定 肺泡干细胞介导的修复的时空动力学，并将确定作用机制 和关键再生信号的靶细胞在肺泡龛中的真实的时间。我们的发现将对 对于了解肺损伤期间干细胞生物学和肺泡生态位再生的影响，以及 可能有助于为IPF的治疗干预提供有针对性的策略。

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.