Role of mesenchymal stem cells in the blood stem cell niche

间充质干细胞在血液干细胞生态位中的作用

• 批准号: 7928911
• 负责人: Paul S Frenette
• 金额: $ 41.5万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-06 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7928911
• 关键词: Ablation; Adipocytes; Adult; Aorta; Behavior; Blood; Blood Vessels; Bone Marrow; Bone Marrow Cells; CD34 gene; Cell Maintenance; Cell Proliferation; Cells; Ceramics; Characteristics; Chondrocytes; Collaborations; Data; Development; Disease; Doxycycline; Embryo; Fetal Liver; Fiber; Fluorescence; Gene Expression; Genes; Gonadal structure; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Human; Image; Implant; In Vitro; Intermediate Filament Proteins; Label; Laboratories; Location; Maps; Marrow; Mesenchymal; Mesenchymal Stem Cells; Mesonephric structure; Methods; Modeling; Mus; Myelofibrosis; Nuclear; Oklahoma; Osteoblasts; Proteins; Regulatory Element; Role; Sickle Cell Anemia; Signal Transduction; Site; Sorting - Cell Movement; Spleen; Staging; Staining method; Stains; Stem cells; Sympathetic Nervous System; Techniques; Tissue-Specific Gene Expression; Toxin; Transgenic Mice; cell type; in vivo; mouse model; nerve supply; nestin protein; novel; postnatal; progenitor; promoter; public health relevance; self-renewal; spatial relationship; stem cell niche; tissue culture; trafficking

DESCRIPTION (provided by applicant): HSCs inhabit specific niches that regulate their commitment, survival, proliferation and differentiation. However the cellular constituents of the HSC niche remain unclear. Several lines of evidence have suggested a role for osteoblasts in providing a specific site where HSCs are maintained in a quiescent state. Other data have suggested that HSCs are predominantly localized near sinusoidal vessels in the bone marrow and spleen. We have recently found that the trafficking, both enforced and homeostatic, is regulated by signals from the sympathetic nervous system (SNS). Searching for the cellular stromal target of the SNS, we have found using a mouse expressing the green fluorescence protein (GFP) under the Nestin gene promoter, that GFP expressing cells formed the HSC niche. Our preliminary data suggest that the vast majority of CD150+CD48- HSCs and SNS-fibers are localized near Nestin+ cells, and that these cells express high levels of key genes involved in stem cell maintenance. Upon culturing, sorted Nestin+ cells rapidly differentiate into mesenchymal lineages. Moreover, we show using novel culture methods that sorted Nestin+ cells can self-renew in vitro or in vivo when grown as spheres (termed "mesenspheres") in non-adherent dishes or attached to ceramic cubes implanted subcutaneously. We thus hypothesize that Nestin+ cells are bona fide mesenchymal stem cells which are tightly regulated by the SNS, and which form the HSC niche in both medullary and extra-medullary sites. We will explore this hypothesis with our collaborators in three Specific Aims. In Specific Aim I, we will evaluate the spatial location and relationships of HSCs with Nestin+ cells in the aorta-gonad-mesonephros (AGM) region and the fetal liver. We will assess the impact of Nestin+ cell depletion on hematopoiesis at these stages. We will study a subset of Nestin+ niche cells expressing the early osteoblast gene Osterix using promoter-driven doxycycline-induced expression. We will evaluate differential gene expression in sorted fetal liver and bone marrow-derived Nestin+ cells to identify novel niche regulators of HSC proliferation. In Specific Aim II, we will characterize the spatial localization and mobilization behavior of quiescent HSCs within the bone marrow using novel imaging models using mCherry red expressing Nestin+ cells and GFP label-retaining HSCs. In Specific Aim III, we will assess the number and function of Nestin+ niche cells in pathological models of myelofibrosis (bone marrow attrition) and sickle cell disease (bone marrow expansion). The analyses proposed in these pathologically relevant models will lay the groundwork for studies to define core universal niche genes whose function extends beyond anatomical or developmental confines. PUBLIC HEALTH RELEVANCE: The spatial localization and cellular constituents forming the hematopoietic stem cell (HSC) niche are unclear, with studies suggesting that HSC localizes either near osteoblasts or the vasculature. Preliminary studies supporting this proposal suggest that bona fide mesenchymal stem cells, peri-vascular and isolatable by Nestin expression, form a unique HSC niche in the bone marrow. This proposal explores the function of this candidate niche cell during the normal development and pathological states of bone marrow attrition and expansion.

描述（由申请人提供）：HSC 存在于调节其承诺、生存、增殖和分化的特定生态位中。然而，HSC 生态位的细胞成分仍不清楚。多项证据表明，成骨细胞在提供 HSC 保持静止状态的特定位点方面发挥着重要作用。其他数据表明，HSC 主要位于骨髓和脾脏中的窦状血管附近。我们最近发现，无论是强制贩运还是稳态贩运，都受到交感神经系统（SNS）信号的调节。在寻找 SNS 的细胞基质靶标时，我们发现使用在 Nestin 基因启动子下表达绿色荧光蛋白（GFP）的小鼠，表达 GFP 的细胞形成了 HSC 生态位。我们的初步数据表明，绝大多数 CD150+CD48- HSC 和 SNS 纤维位于 Nestin+ 细胞附近，并且这些细胞表达高水平的参与干细胞维持的关键基因。培养后，分选的 Nestin+ 细胞迅速分化为间充质谱系。此外，我们展示了使用新的培养方法，当在非贴壁培养皿中生长为球体（称为“中间球”）或附着在皮下植入的陶瓷立方体上时，分选的巢蛋白+细胞可以在体外或体内自我更新。因此，我们假设 Nestin+ 细胞是真正的间充质干细胞，受到 SNS 的严格调控，并在髓质和髓外位点形成 HSC 生态位。我们将与我们的合作者在三个具体目标中探讨这一假设。在具体目标 I 中，我们将评估主动脉-性腺-中肾 (AGM) 区域和胎儿肝脏中 HSC 与巢蛋白 + 细胞的空间位置和关系。我们将评估巢蛋白+细胞耗竭对这些阶段造血的影响。我们将使用启动子驱动的多西环素诱导表达来研究表达早期成骨细胞基因 Osterix 的 Nestin+ 微环境细胞子集。我们将评估分选的胎儿肝脏和骨髓来源的 Nestin+ 细胞中的差异基因表达，以确定 HSC 增殖的新生态位调节因子。在特定目标 II 中，我们将使用表达 mCherry red Nestin+ 细胞和保留 GFP 标记的 HSC 的新型成像模型来表征骨髓内静止 HSC 的空间定位和动员行为。在特定目标 III 中，我们将评估骨髓纤维化（骨髓消耗）和镰状细胞病（骨髓扩张）病理模型中 Nestin+ 微环境细胞的数量和功能。这些病理相关模型中提出的分析将为定义核心通用生态位基因的研究奠定基础，这些基因的功能超出了解剖或发育的范围。 公共健康相关性：造血干细胞 (HSC) 生态位的空间定位和细胞成分尚不清楚，研究表明 HSC 位于成骨细胞或脉管系统附近。支持这一提议的初步研究表明，血管周围的、可通过巢蛋白表达分离的真正的间充质干细胞在骨髓中形成独特的 HSC 生态位。该提案探讨了该候选微环境细胞在骨髓磨损和扩张的正常发育和病理状态下的功能。