Role of mesenchymal stem cells in the blood stem cell niche

描述(由申请者提供)：造血干细胞居住在特定的生态环境中，调节其承诺、存活、增殖和分化。然而，HSC生态位的细胞成分仍然不清楚。一些证据表明，成骨细胞在提供一个特定的位置，使HSC保持在静止状态中起到了作用。其他数据表明，HSCs主要分布在骨髓和脾中的血窦血管附近。我们最近发现，无论是强制的还是稳态的贩运，都受到交感神经系统(SNS)信号的调节。在寻找SNS的细胞基质靶点时，我们发现在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-HSCs和SNS-纤维定位于Nestin+细胞附近，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，分离的巢蛋白+细胞迅速分化为间充质细胞系。此外，我们还展示了使用新的培养方法，当分离的巢蛋白+细胞在非粘附性培养皿中生长为球状(称为介质球)或附着于植入皮下的陶瓷立方体时，在体外或体内可以自我更新。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，并在髓内和髓外形成HSC生态位。我们将在三个具体目标上与我们的合作者一起探索这一假设。在特定的目标I中，我们将评估HSCs在腹主动脉-性腺-中肾(AGM)区和胎肝中与Nestin+细胞的空间位置和相互关系。我们将在这些阶段评估Nestin+细胞耗尽对造血的影响。我们将使用启动子驱动的多西环素诱导表达早期成骨细胞基因Osterix的Nestin+NICE细胞亚群进行研究。我们将评估分离的胎肝和骨髓源性Nestin+细胞的差异基因表达，以确定新的HSC增殖的生态位调节因子。在特定的目标II中，我们将使用表达Nestin+细胞的mCherry Red和保留GFP标记的HSCs的新型成像模型来表征静止HSCs在骨髓中的空间定位和动员行为。在特定目的III中，我们将评估在骨髓纤维化(骨髓磨损)和镰状细胞病(骨髓扩张)病理模型中Nestin+NICE细胞的数量和功能。这些病理相关模型中提出的分析将为研究定义其功能超出解剖学或发育范围的核心通用利基基因奠定基础。 公共卫生相关性：形成造血干细胞(HSC)生态位的空间定位和细胞成分尚不清楚，研究表明HSC定位于成骨细胞或血管系统附近。支持这一建议的初步研究表明，真心间充质干细胞位于血管周围，可通过巢蛋白表达分离，在骨髓中形成独特的HSC生态位。这项建议探讨了这种候选的生态位细胞在骨髓磨损和扩张的正常发育和病理状态中的作用。

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.