Understanding the Symbiosis Between Megakaryocytes and the Lung Microenvironment

了解巨核细胞与肺微环境之间的共生关系

• 批准号: 10471448
• 负责人: Anthony K Yeung
• 金额: $ 5.18万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10471448
• 关键词: Acute Respiratory Distress Syndrome; Address; Adhesions; Adult; Architecture; Bacterial exotoxin; Biogenesis; Biological Assay; Biology; Blood Platelets; Bone Marrow; Cell model; Cells; Coagulation Process; Coculture Techniques; Cytoplasmic Granules; Data Set; Development; Disease; Electron Microscopy; Epithelial; Fetal Liver; Flow Cytometry; Goals; Hematopoietic; Histologic; Homeostasis; Human; In Vitro; Inflammation; Investigation; Knowledge; Life; Light; Lung; Lung diseases; Maps; Megakaryocytes; Membrane; Methodology; Microscopy; Modeling; Mus; Natural regeneration; Organ; Organoids; Pattern; Phenotype; Ploidies; Population; Process; Production; Pulmonary Fibrosis; Reporter; Resolution; Role; Structure of parenchyma of lung; Surface; Symbiosis; System; Text; Tissues; Work; base; cell type; combinatorial; directed differentiation; fetal; hematopoietic tissue; improved; in vivo; induced pluripotent stem cell; insight; lung development; next generation sequencing; novel therapeutics; pulmonary function; residence; single-cell RNA sequencing; source localization; stem; stem cell model; tool; trafficking; transcriptome; transcriptomics; tumor progression

Abstract Megakaryocytes (MKs) and the platelets they produce are essential for clot formation, but both are also involved in critical processes such as development, inflammation, homeostasis and regeneration. Platelet biogenesis is canonically thought to occur primarily in the adult bone marrow and the developing fetal liver. As such, our current understanding of MK development stems primarily from the investigation of these tissues. However, emerging evidence highlights the lung as a previously underappreciated residence for MKs, with several groups demonstrating that lung-MKs significantly contribute to circulating platelet mass. While a diversity of cells specific to the bone marrow are known to promote the maturation and trafficking of MKs, investigation into how cells of the lung niche influence the development and function of MKs has not been done. This knowledge gap is highlighted by the fact that platelets have demonstrated lung-specific roles in processes such as development, epithelial protection against bacterial exotoxins, and pulmonary fibrosis. Whether lung-MKs are involved in these processes and/or provide a localized source of platelets that carry out lung-specific functions is unknown. This question led us to hypothesize that symbiotic interactions between lung-MKs and the lung microenvironment aid in the maturation and functionalization of both MKs and the lung itself. Here we propose the use of both in-vivo and in-vitro approaches to investigate this adaptive symbiosis. In Aim 1, we will perform single cell RNA sequencing and functional assays on MKs isolated from fetal and adult mouse tissues to define a lung-specific MK phenotype. Investigating MKs at different stages of development will bolster our understanding of the maturational trajectory and functionalization of lung-MKs. We will also perform the histological assessment of primary mouse lung tissue to determine the cellular makeup of the lung-MK microenvironment. In Aim 2, we will develop an in-vitro human induced pluripotent stem cell (iPSC) based coculture system to model the interaction of MKs in the lung microenvironment. Previous work from the Murphy (Sponsor) and Kotton (Co-Sponsor) labs have established methodologies for directing the patterning of iPSCs towards hematopoietic and lung lineages to robustly produce iPSC derived MKs and lung organoids. Harnessing these tools, we will coculture MKs with lung organoids to investigate how reciprocal interactions between each system influences the development and patterning of these tissues. Resultant cells from coculture will then be subjected to functional and transcriptomic studies to investigate how the phenotypic patterning of MKs and lung organoids is influenced by coculture. These studies will improve our understanding of the biology of MK specification and platelet production, and also shed light on the symbiotic relationship between the lung and resident MKs, thus introducing the previously undescribed role of MKs as an integral component of the lung microenvironment.

摘要 巨核细胞（MK）及其产生的血小板对于血栓形成至关重要，但两者也是 参与关键过程，如发育，炎症，稳态和再生。血小板 生物发生通常被认为主要发生在成人骨髓和发育中的胎儿肝脏中。作为 因此，我们目前对MK发育的理解主要来自于对这些组织的研究。 然而，新出现的证据强调了肺作为MK以前未被充分认识的住所， 几个组证明肺-MK显著促进循环血小板质量。而 已知骨髓特异性细胞的多样性促进MK的成熟和运输， 对肺小生境细胞如何影响MK的发育和功能的研究还没有被 完了这一知识差距突出的事实是，血小板已被证明是肺特异性作用， 例如发育、针对细菌外毒素的上皮保护和肺纤维化。 肺MK是否参与这些过程和/或提供血小板的局部来源， 肺特异性功能未知。这个问题使我们假设， 肺-MK和肺微环境有助于MK和肺的成熟和功能化 本身在这里，我们建议使用在体内和体外的方法来调查这种适应性共生。 在目标1中，我们将对从胎儿和胎儿中分离的MK进行单细胞RNA测序和功能测定。 成年小鼠组织以定义肺特异性MK表型。调查不同阶段的MK 发展将加强我们对肺MK成熟轨迹和功能化的理解。我们 还将对原代小鼠肺组织进行组织学评估，以确定 肺-MK微环境。在目标2中，我们将开发体外人诱导多能干细胞 （iPSC）的共培养系统来模拟肺微环境中MK的相互作用。以前的工作 Murphy（申办者）和Kotton（共同申办者）实验室的研究人员已经建立了指导 iPSC向造血和肺谱系的模式化以稳健地产生iPSC衍生的MK和肺 类有机体利用这些工具，我们将共同培养MK与肺类器官，以研究如何相互作用， 每个系统之间的相互作用影响这些组织的发育和模式。得到的细胞 然后进行功能和转录组学研究，以研究表型 MK和肺类器官的模式化受到共培养的影响。这些研究将增进我们对 MK规格和血小板生产的生物学，也揭示了共生关系 之间的肺和居民MK，从而介绍了以前未描述的作用MK作为一个整体 肺微环境的组成部分。