Single-cell High-dimensional Characterization of the Bone Marrow Microenvironment in Health and Disease

健康和疾病中骨髓微环境的单细胞高维表征

• 批准号: 9372908
• 负责人: Kara Lynn Davis
• 金额: $ 25.12万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9372908
• 关键词: Ablation; Affect; Aging; Antibodies; B-Lymphocytes; Blood; Blood Cells; Blood Vessels; Bone Marrow; Cell Count; Cell Lineage; Cells; Complex; Coupled; Cytokine Signaling; Cytometry; Development; Disease; Ecosystem; Endothelial Cells; Exhibits; Failure; Flow Cytometry; Fluorochrome; Foundations; Frequencies; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; Health; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Human; Isotopes; Maintenance; Maps; Mass Spectrum Analysis; Mediating; Mesenchymal; Mesenchymal Stem Cells; Metastatic Neoplasm to the Bone; Molecular; Mus; Osteoblasts; Osteogenesis; Osteoporosis; PTH gene; Parathyroid Hormone Receptor; Pathology; Pathway interactions; Pharmaceutical Preparations; Physiological; Play; Population; Production; Publishing; Rare Earth Metals; Receptor Signaling; Role; Signal Pathway; Signal Transduction; Stem cells; Structure; Techniques; Therapeutic Intervention; Tissues; blood vessel development; bone; bone mass; cell type; cytokine; high dimensionality; human disease; leukemia; novel; progenitor; receptor; response; stem; tool

Project Summary In the bone marrow, hematopoiesis is dependent upon support from the surrounding microenvironment, or niche. The bone marrow microenvironment is complex, with hematopoietic and mesenchymal cell populations interacting to influence the formation of hematopoietic cells, blood vessels and bone. In order to understand the molecular and cellular mechanisms by which multiple cell populations support hematopoiesis, it would be helpful to analyze a large number of cell types simultaneously. Mass cytometry (CyTOF) is a novel technique in which flow cytometry is performed using antibodies coupled to rare earth metal isotopes rather than fluorochromes, followed by mass spectrometry. Unimpeded by spectral overlap, CyTOF allows for the analysis of >40 simultaneous parameters. Using CyTOF we have recently published reference maps of the major hematopoietic cell populations in mouse and human bone marrow. We now propose to expand these to include the mesenchymal populations. Our central hypothesis is that alterations in the distribution and function of mesenchymal populations reciprocally influence bone marrow hematopoiesis. As a corollary, we suggest that bone pathologies affecting mesenchymal cells, such as osteoporosis, will necessarily perturb hematopoietic cell development and function. The parathyroid hormone (PTH) receptor (PTH1R) is a G protein coupled receptor whose signaling in bone has profound effects on bone formation and hematopoiesis. To investigate how PTH signaling alters the bone marrow microenvironment we propose the following aims: In Aim 1 we will expand the reference map of hematopoietic cells in murine bone marrow to include mesenchymal populations by incorporating antibodies to identify endothelial cells, mesenchymal stem/progenitor cells, and osteoblasts. We will further identify major cell type-specific cytokines and signaling pathways stimulated by PTH. In Aim 2 we will examine how bone marrow populations are altered by disruption of PTH/PTH1R/Gsα signaling. We have demonstrated that mice lacking the Gsα subunit, which mediates many PTH-dependent actions in bone, in osteoblast progenitors (Gsα-OsxKO mice) exhibit severe osteoporosis, loss of B lymphocyte precursors, and a failure to increase bone mass in response to PTH. By comparing the frequencies of hematopoietic and mesenchymal populations in Gsα-OsxKO bone marrow to the reference map generated in Aim 1, we will reveal how the absence of Gsα signaling in osteoblasts impacts the bone marrow ecosystem. We will also examine the alterations in cytokine production and PTH-dependent signaling within the bone marrow of Gsα-OsxKO mice. In Aim 3 we will generate a reference map of the human bone marrow microenvironment. Understanding the structure of normal mesenchymal populations in human bone marrow is foundational to examining alterations in disease, and such a reference map will be of value in studying the role of bone marrow microenvironment in human disease, aging, and in response to bone- and blood-targeting medications.

项目摘要 在骨髓中，造血依赖于周围微环境的支持，或者 利基市场。骨髓微环境复杂，有造血细胞和间充质细胞群。 相互作用，影响造血细胞、血管和骨骼的形成。为了理解 多个细胞群体支持造血的分子和细胞机制， 有助于同时分析大量的细胞类型。质量细胞分析(CyTOF)是一种新技术 其中流式细胞术是使用与稀土金属同位素偶联的抗体而不是 荧光显色，然后进行质谱分析。不受光谱重叠的阻碍，CyTOF允许进行分析 &gt；40个同步参数。使用CyTOF，我们最近发布了主要 小鼠和人骨髓中的造血细胞群。我们现在建议将这些范围扩大到 包括间充质种群。我们的中心假设是分布的变化和 间充质细胞群的功能相互影响着骨髓的造血。作为推论， 我们认为影响间充质细胞的骨病理，如骨质疏松症，必然会受到干扰。 造血细胞的发育和功能。甲状旁腺激素受体(PTH1R)是一种G蛋白 偶联受体在骨骼中的信号转导对骨形成和造血有深远的影响。至 为了研究甲状旁腺素信号如何改变骨髓微环境，我们提出了以下目标： 目的1我们将扩大小鼠骨髓造血细胞的参考图，以包括 通过掺入抗体识别内皮细胞的间充质细胞群 干细胞/祖细胞和成骨细胞。我们将进一步确定主要的细胞类型特异性细胞因子和信号 甲状旁腺素刺激的通路。在目标2中，我们将研究破坏是如何改变骨髓数量的。 甲状旁腺激素/甲状旁腺素受体/Gsα信号转导通路。我们已经证明，缺乏Gsα亚单位的小鼠 在成骨细胞前体细胞(Gsα-OsxKo小鼠)中，许多甲状旁腺素在骨中的依赖作用表现出严重的 骨质疏松症，B淋巴细胞前体细胞的丧失，以及甲状旁腺激素未能增加骨量。通过 Gs-α-OsxKO骨髓造血祖细胞和间充质细胞的频率比较 Aim 1中生成的参考图谱，我们将揭示成骨细胞中Gsα信号的缺失如何影响 骨髓生态系统。我们还将研究细胞因子产生和甲状旁腺素依赖的变化。 Gsα-OsxKO小鼠骨髓内的信号转导。在目标3中，我们将生成一个参考地图 人骨髓微环境。了解正常骨髓间充质细胞群体的结构 人类骨髓是检查疾病变化的基础，这样的参考图将是 研究骨髓微环境在人类疾病、衰老和应对中的作用 针对骨骼和血液的药物。