Live Imaging of Intact Airway

呼吸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，呼吸道干细胞的行为是由细胞和 围绕它们，包括其他上皮细胞、上皮下间充质细胞和外部 施加在上皮细胞上的机械力。这些不同的组件如何调节 呼吸道祖细胞的行为在很大程度上是未知的。此外，基础干细胞-利基 相互作用可能解释了越来越多人认识到的祖细胞内的异质性 群体，包括基础干细胞行为的区域和组织水平的异质性。 尽管祖细胞-壁龛的相互作用已经用活成像在其他 系统，呼吸系统的持续运动和具有挑战性的通路阻碍了 开发高分辨率的呼吸道实时成像平台。 我们结合了新兴的干细胞生物学和活体成像技术，开发了一种新的 气道植入物活体成像平台。我们描绘了这座建筑的维护和再生 小鼠呼吸道外植体中的呼吸道上皮，被其所有主要成分包围。 三维多组分微环境，包括细胞外基质和间质。我们呈现的是 双光子激光追踪单个干细胞再生过程的初步数据 扫描显微镜，包括用荧光显示干细胞分化 用新的视频率成像模式对纤毛细胞功能进行了报道和分析。至 定义呼吸道干细胞微环境并测试其在祖细胞功能中的作用 利用激光和毒素来可视化和推测细胞生态位组件并测试它们的作用 消融。我们建议使用细胞植入，然后施加机械力来 考察微环境在建立平面极性中的作用，并研究 呼吸道集体细胞迁移中增殖、迁移和分化的关系 上皮组织。

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.