Engineered alveolar organoids to understand ECM signaling

项目摘要 特发性肺纤维化(IPF)远端肺的特点是空间不均匀的区域。 成纤维细胞/肌成纤维细胞和过多的细胞外基质(ECM)积聚破坏肺泡 建筑。现在认为肺泡上皮细胞直接参与了肺泡上皮细胞的发育和进展。 纤维化，但新的ECM沉积如何影响肺泡上皮细胞功能和反馈以加强纤维化 改建是未知的。这在一定程度上是由于缺乏研究ECM动力学或直接测量细胞的工具 命运对体内改变的细胞外基质的反应。有机培养物可在体内产生类似于肺泡的补体 组织；然而，当前的体外模型依赖于矩阵的使用，其特征是可变的 构成，不利于受控操纵。 拟议工作的总体重点是将分泌的肺泡ECM信号的变化与 上皮细胞功能(K99时相)和上皮细胞间充质信号(R00)。在K99阶段，定义 将开发水凝胶基质，以在特定的微环境中形成肺泡有机物质，并确定 新分泌的ECM成分和机制如何指导上皮细胞的功能。使用这个平台， 分泌的细胞外基质将被外部交联，以检测细胞外基质硬化是否促进上皮细胞 功能障碍。随着对分泌型细胞外基质如何启动上皮细胞功能障碍的了解，第二个目标将 确定分泌的ECM如何改变上皮细胞对间质信号的反应 R00阶段。我们将确定细胞外基质的积累是否改变了AT2细胞和 间充质细胞，以及这是否会加强纤维化重塑。接下来，将使用微结构水凝胶 为了控制空间关系，并检查上皮细胞和细胞的物理分离的效果 间充质细胞对上皮细胞功能的影响。 为了了解细胞及其持续变化的环境中的双向信号 肺泡利基，这项研究将使用有机培养和工程方法来操纵和 解构细胞与细胞外基质的相互作用。结果将包括确定新的ECM介导的机制 参与牙槽化和修复过程，并为测试治疗方法提供新的途径 参与IPF的旁分泌信号通路的调控。重要的是，这项建议包含了严格的培训 该计划将为促进申请者在生物医学研究方面的事业奠定基础。具体地说，K99 训练将包括学习小鼠模型、谱系追踪和原代细胞分离，以建立 利用工程水凝胶研究细胞-细胞信号转导的基础 调查员R00阶段。

PROJECT ABSTRACT Idiopathic pulmonary fibrosis (IPF) of the distal lung is characterized by spatially heterogeneous areas of fibroblasts/myofibroblasts and accumulation of excess extracellular matrix (ECM) that disrupts the alveolar architecture. Alveolar epithelial cells are now thought to directly contribute to the development and progression of fibrosis, but how new ECM deposition impacts alveolar epithelia function and feedbacks to reinforce fibrotic remodeling is unknown. This is due in part to the lack of tools to study ECM dynamics or to directly measure cell fate in response to altered ECM in vivo. Organoid cultures can generate an in vivo-like complement of alveolar tissue; however, current in vitro models depend on the use of Matrigel matrices, which feature variable compositions and is not conducive to controlled manipulations. The overall focus of the proposed work is to connect changes in secreted alveolar ECM cues and epithelial cell function (K99 phase) and epithelia to mesenchyme signaling (R00). During the K99 phase, defined hydrogel matrices will be developed to form alveolar organoids in a defined microenvironment and determine how newly secreted ECM composition and mechanics guide epithelial cell function. Using this platform, the secreted ECM will be externally crosslinked to examine whether ECM stiffening promotes epithelial cells dysfunction. With an understanding of how secreted ECM initiates epithelial cell dysfunction, the second aim will determine how the secreted ECM alters the response of epithelial cells to signals from the mesenchyme during the R00 phase. We will determine if the accumulation of ECM changes the interaction between AT2 cells and mesenchymal cells, and whether this reinforces fibrotic remodeling. Next, microstructured hydrogels will be used to control for spatial relationships and examine the effect of the physical separation of epithelial and mesenchymal cells on epithelial cell function. To understand the bidirectional signaling of cells and their continuously changing surroundings within the alveolar niche, this research will use organoid cultures and engineering approaches to manipulate and deconstruct cell-ECM interactions. The outcomes will comprise identification of new ECM mediated mechanisms involved in alveolarization and reparative processes, and provide new avenues for testing therapeutics modulating paracrine signaling pathways involved in IPF. Importantly, this proposal comprises a rigorous training plan that will build the foundation to advance the applicant’s career in biomedical research. Specifically, the K99 training will consist of learning mouse models, lineage tracing, and primary cell isolation to establish the foundation towards investigating cell-cell signaling using engineered hydrogels during the independent investigator R00 phase.