Live Imaging of Intact Airway

气道衬里的完整性依赖于其上皮干细胞的多种功能。 我们假设气道干细胞的行为是由细胞和力量调节的， 周围，包括其他上皮细胞，上皮下间充质细胞，和外部 施加在上皮上的机械力。这些不同的成分如何调节 气道祖细胞的行为在很大程度上是未知的。此外，基底干细胞龛 这种相互作用可以解释这种祖细胞内越来越多的异质性， 群体，包括区域和组织水平的异质性基础干细胞的行为。 虽然在其他研究中已经用活体成像描述了祖细胞-小生境相互作用， 系统，呼吸系统的不断运动和具有挑战性的访问阻碍了 开发高分辨率气道实时成像平台。 我们将新兴的干细胞生物学和活体成像技术相结合， 气道外植活体成像平台。我们想象了 小鼠气道外植体中的气道上皮，被其所有主要成分包围， 3D多组分微环境，包括ECM和间充质。我们提出 用双光子激光跟踪再生过程中单个干细胞的初步数据 扫描显微镜，包括用荧光显微镜观察干细胞分化 报道者和纤毛细胞功能的分析与新的视频率成像方式。到 定义气道干细胞微环境并测试其在祖细胞功能中的作用，我们 可视化和推定的细胞生态位成分，并通过激光和毒素介导的 消融术我们建议使用细胞移植，然后施加机械力， 检查微环境在建立平面极性中的作用，并研究 气道集体细胞迁移中增殖、迁移和分化的关系 上皮

The integrity of the lining of the airways relies on multiple functions of its epithelial stem cells. We hypothesize that the airway stem cell behavior is regulated by the cells and the forces that surrounds them, including other epithelial cells, subepithelial mesenchymal cells, and external mechanical forces exerted on the epithelium. How these varied components regulate the behavior of airway progenitor cells is largely unknown. Furthermore, basal stem cell – niche interactions may explain the increasingly recognized heterogeneity within this progenitor cell population, including regional and tissue-level heterogeneity in basal stem cell behavior. Although progenitor cell – niche interactions have been described with live imaging in other systems, the constant motion and challenging access of the respiratory system has hampered the development of platforms for live imaging of the airways at high resolution. We combined emerging stem cell biology and live imaging technologies to develop a novel airway explant live imaging platform. We imaged the maintenance and regeneration of the airway epithelium in a mouse airway explant, surrounded by all of the major components of its 3D multicomponent microenvironment, including the ECM and the mesenchyme. We present preliminary data on tracking individual stem cells during regeneration with two photon laser scanning microscopy, including visualization of stem cell differentiation with fluorescent reporters and analysis of ciliated cell function with novel video-rate imaging modalities. To define the airway stem cell microenvironment and to test its role in progenitor cells function, we visualize and putative cellular niche components and test their role by laser and toxin-mediated ablation. We propose to use cellular engraftment followed by application of mechanical forces to check the role of the microenvironment in establishing planar polarity, and to study the relationship of proliferation, migration and differentiation in collective cell migration in the airway epithelium.