Engineered alveolar organoids to understand ECM signaling

工程化肺泡类器官以了解 ECM 信号传导

• 批准号: 10473808
• 负责人: Claudia Loebel
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10473808
• 关键词: Affect; Alveolar; Alveolar Process; Architecture; Area; Basement membrane; Biochemical; Biological Models; Biomedical Research; Biophysics; Cell Separation; Cell Therapy; Cell physiology; Cells; Chemical Engineering; Cicatrix; Complement; Cues; Deposition; Development; Distal; Engineering; Epithelial; Epithelial Cells; Extracellular Matrix; Feedback; Fibrillar Collagen; Fibroblasts; Fibrosis; Foundations; Functional disorder; Future; Goals; Hydrogels; Impairment; In Situ; In Vitro; Inflammation; Injury; Knowledge; Label; Lead; Learning; Ligands; Lung; Measures; Mechanics; Mediating; Mesenchymal; Mesenchyme; Metabolic; Methods; Modeling; Monitor; Myofibroblast; Natural regeneration; Organ; Organoids; Outcome; Paracrine Communication; Pathway interactions; Phase; Population; Process; Proliferating; Property; Regenerative capacity; Research; Research Personnel; Signal Pathway; Signal Transduction; Source; Structure; Structure of parenchyma of lung; System; Techniques; Technology; Testing; Tissue Model; Tissues; Training; Transforming Growth Factors; Variant; Work; alveolar epithelium; career; cellular engineering; crosslink; epithelial repair; idiopathic pulmonary fibrosis; in vitro Model; in vivo; intercellular communication; matrigel; mouse model; novel strategies; physical separation; profibrotic cytokine; programs; repaired; reparative process; response; spatial relationship; therapeutic evaluation; therapeutic target; tool

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.

项目摘要 远端肺的特发性肺纤维化（IPF）的特征在于肺纤维化的空间异质性区域。 成纤维细胞/肌成纤维细胞和过量细胞外基质（ECM）的积累，破坏肺泡 架构肺泡上皮细胞现在被认为是直接有助于发展和进展 但是新的ECM沉积如何影响肺泡上皮功能和反馈以加强纤维化， 重塑是未知的。这部分是由于缺乏研究ECM动力学或直接测量细胞生长的工具。 在体内响应于改变的ECM的命运。类器官培养物可以产生肺泡上皮细胞的体内样补体， 然而，目前的体外模型依赖于使用Matrigel基质，其特征在于可变的 这是一个复杂的组合物，不利于控制操作。 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期）和上皮至间充质信号传导（R00）。在K99阶段，定义 将开发水凝胶基质以在限定的微环境中形成肺泡类器官， 新分泌的ECM成分和机制如何引导上皮细胞功能。利用这个平台， 分泌的ECM将外部交联以检查ECM硬化是否促进上皮细胞 功能障碍随着对分泌的ECM如何引发上皮细胞功能障碍的理解，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ECM的积累是否改变了AT 2细胞和 间充质细胞，以及这是否加强了纤维化重塑。接下来，将使用微结构水凝胶 以控制空间关系，并检查上皮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。 为了了解细胞的双向信号传导及其在细胞内不断变化的环境， 肺泡龛，这项研究将使用类器官培养和工程方法来操纵和 解构细胞-ECM相互作用。结果将包括新ECM介导机制的鉴定 参与肺泡化和修复过程，并为测试治疗方法提供新的途径 调节参与IPF的旁分泌信号通路。重要的是，这项建议包括严格的培训， 该计划将为推进申请人在生物医学研究领域的职业生涯奠定基础。特别是K99 培训将包括学习小鼠模型，谱系追踪和原代细胞分离，以建立 研究细胞间信号传导的基础，使用工程水凝胶在独立的 研究者R00阶段。