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

项目摘要 在骨髓中，造血依赖于周围微环境的支持，或 利基骨髓微环境复杂，有造血细胞和间充质细胞群 相互作用以影响造血细胞、血管和骨骼的形成。为了解 多个细胞群支持造血的分子和细胞机制， 有助于同时分析大量细胞类型。质谱细胞仪（CyTOF）是一种新的技术 其中使用与稀土金属同位素偶联的抗体进行流式细胞术， 荧光染料，然后质谱。不受光谱重叠的阻碍，CyTOF允许分析 >40个同步参数。使用CyTOF，我们最近发表了主要的参考地图 小鼠和人骨髓中的造血细胞群。我们现建议将这些措施扩展至 包括间充质细胞群。我们的中心假设是，分布和 间充质细胞群的功能直接影响骨髓造血。作为推论， 我们认为，影响间充质细胞的骨病变，如骨质疏松症， 造血细胞发育和功能。甲状旁腺激素（PTH）受体（PTH 1 R）是一种G蛋白 偶联受体，其在骨中的信号传导对骨形成和造血有深远影响。到 研究PTH信号如何改变骨髓微环境，我们提出以下目标： 目的1我们将扩大小鼠骨髓造血细胞的参考图谱， 间充质细胞群，通过掺入抗体来鉴定内皮细胞、间充质细胞 干/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转导 PTH刺激的通路。在目标2中，我们将研究如何通过破坏改变骨髓群体 PTH/PTH 1 R/Gsα信号通路。我们已经证明，缺乏Gsα亚基的小鼠， 骨中的许多PTH依赖性作用，在成骨细胞祖细胞（Gsα-OsxKO小鼠）中表现出严重的 骨质疏松症、B淋巴细胞前体的损失和不能响应PTH而增加骨量。通过 比较Gsα-OsxKO骨髓中造血和间充质细胞群的频率， 目标1中生成的参考图，我们将揭示成骨细胞中Gsα信号的缺失如何影响成骨细胞的生长。 骨髓生态系统我们还将检查细胞因子产生和PTH依赖性 Gsα-OsxKO小鼠骨髓内的信号传导。在目标3中，我们将生成一个参考图， 人骨髓微环境了解正常间充质细胞群的结构 人类骨髓是检查疾病变化的基础，这样的参考图谱将是 在研究骨髓微环境在人类疾病、衰老和应对 针对骨骼和血液的药物

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.