Role of mesenchymal stem cells in the blood stem cell niche

描述（由申请人提供）：造血干细胞居住在特定的小生境中，调节其承诺、存活、增殖和分化。然而，HSC生态位的细胞成分尚不清楚。一些证据表明，成骨细胞在提供造血干细胞保持静止状态的特定位点方面发挥了作用。其他数据表明造血干细胞主要位于骨髓和脾脏的窦状血管附近。我们最近发现，贩运，无论是强制的还是稳态的，是由交感神经系统（SNS）的信号调节的。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）的小鼠中，表达绿色荧光蛋白的细胞形成HSC生态位。我们的初步数据表明，绝大多数CD150+CD48-造血干细胞和sns纤维都位于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。经过培养，已分类的Nestin+细胞迅速分化为间充质细胞系。此外，我们发现使用新的培养方法，当巢蛋白+细胞在体外或体内作为球体（称为“中间球体”）生长在非贴壁培养皿中或附着在皮下植入的陶瓷立方体上时，巢蛋白+细胞可以自我更新。因此，我们假设Nestin+细胞是真正的间充质干细胞，它们受到SNS的严格调节，并在髓质和髓外部位形成HSC生态位。我们将在三个具体目标中与我们的合作者探讨这一假设。在Specific Aim I中，我们将评估hsc与Nestin+细胞在主动脉-性腺-中肾（AGM）区域和胎儿肝脏中的空间位置及其关系。我们将评估Nestin+细胞耗竭对这些阶段造血的影响。我们将研究Nestin+小生境细胞亚群，使用启动子驱动强力霉素诱导表达早期成骨细胞基因Osterix。我们将评估分选的胎儿肝脏和骨髓来源的Nestin+细胞的差异基因表达，以确定新的小生境调节HSC增殖。在Specific Aim II中，我们将使用表达Nestin+细胞的mCherry红和保留GFP标记的hsc的新型成像模型来表征静止hsc在骨髓中的空间定位和动员行为。在Specific Aim III中，我们将评估Nestin+生态位细胞在骨髓纤维化（骨髓损耗）和镰状细胞病（骨髓扩增）病理模型中的数量和功能。在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学或发育的限制。

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.