Spatial analysis of hematopoietic stem and progenitor cells in the bone marrow

描述（由申请人提供）：造血是一种生理过程，其中存在于骨髓（BM）中的少量多能造血干细胞（HSC）增殖并分化以产生血液和免疫细胞的完全补体。造血干细胞和祖细胞（HSPC）的自我更新和谱系分化取决于不同的微环境，称为小生境，由细胞成分，可溶性调节剂和细胞外基质定义。由于常规成像（共聚焦、荧光）技术的限制，尚未完成BM中HSPC小生境的位置以及细胞和细胞外特征的连续鉴定。在这项研究中，我们采用了新的成像技术，激光扫描细胞计数（LSC）和多光子活体显微镜（MP-IVM），以确定骨髓微环境中HSPC及其生态位之间的细胞-细胞相互作用的定位和动态行为。我们把这项拨款建议分为两个目标。目的1将量化HSPC在血管外BM隔室内不同解剖位置的分布。LSC将用于量化和绘制BM骨干（骨内膜与骨髓）和干骺端区域中HSPC的形态学位置。将使用bmi-1基因靶向报告基因（GFP）小鼠模型鉴定HSPC，其中HSPC的GFP水平最强，沿着HSPC群体特征性表面抗原（c-Kit、Sca-1、flk 2、CD 150、CD 34）的荧光免疫染色。LSC允许扫描股骨BM腔的整个纵切面，并以单细胞水平的精度绘制BM腔的整个细胞内容物（约2.5x105个细胞/图像），使得即使是极其罕见的BM群体的定位的客观定量和统计比较成为可能。目的2将表征BM内HSPC小生境的细胞组分。多光子活体显微镜（MP-IVM）将用于HSPC行为动力学的体内分析，特别是HSPC与三种不同的潜在HSPC生态位细胞群体（成骨细胞、血管内皮细胞和富含CXCL 12的网状（CAR）细胞）之间的相互作用。将使用体内免疫染色来鉴定BM内的每个拟定HSPC小生境细胞。LSC与HSPC和小生境细胞的表面抗原的荧光免疫染色结合，也将用于客观地量化HSPC与整个BM腔中的每个小生境细胞密切相关的相对频率。bmi-1靶向（GFP）报告小鼠模型将再次用作鉴定HSPC的基础。改善BM中HSPC生态位的定义可能对临床相关HSPC群体的离体扩增具有重要意义，并将有助于理解病理状况（如白血病发生、骨髓增生异常和免疫缺陷）的病因。公共卫生相关性：这项研究计划将采用新的成像技术来确定造血干细胞和祖细胞及其在骨髓微环境中的小生境之间的细胞-细胞相互作用的定位和动态行为。这些分析应该提供重要的洞察骨髓环境信号调节造血细胞的发展。这些信息可能证明与临床上重要的造血干细胞和祖细胞群体的体外扩增高度相关，并且也将有助于理解病理条件如白血病发生、骨髓增生异常和免疫缺陷的病因。

DESCRIPTION (provided by applicant): Hematopoiesis is a physiological process where a small number of pluripotent hematopoietic stem cells (HSCs) residing in the bone marrow (BM) proliferate and differentiate to generate the full complement of blood and immune cells. Hematopoietic stem and progenitor cell (HSPC) self-renewal and lineage differentiation depend on distinct microenvironments, termed niches, defined by cellular components, soluble regulators, and the extracellular matrix. Definitive identification of the location as well as cellular and extracellular characteristics of HSPC niches in the BM has not been completed due to limitations of conventional imaging (confocal, fluorescent) techniques. In this research proposal, we employ novel imaging technologies, Laser Scanning Cytometry (LSC) and multiphoton intravital microscopy (MP-IVM), to define the localization and dynamic behavior of cell-cell interactions between HSPCs and their niche(s) in the bone marrow microenvironment. We have organized this grant proposal into two aims. Aim 1 will quantify the distribution of HSPCs in distinct anatomical locations within the extravascular BM compartment. LSC will be used to quantify and map the morphological position(s) of HSPCs in the diaphyseal (endosteal versus medullary) and metaphyseal regions of the BM. HSPCs will be identified using the bmi-1 gene targeted reporter (GFP) mouse model, where GFP levels are strongest for HSPCs, along with fluorescent immunostaining of surface antigens (c-Kit, Sca-1, flk2, CD150, CD34) characteristic of HSPC populations. LSC allows whole longitudinal sections of the femoral BM cavity to be scanned and the entire cellular content of the BM cavity (order 2.5x105 cells/image) mapped with single cell level precision, making objective quantification and statistical comparison of the localization of even extremely rare BM populations possible. Aim 2 will characterize the cellular component(s) of the HSPC niche within the BM. Multiphoton intravital microscopy (MP-IVM) will be used for in vivo analysis of the kinetics of HSPC behavior, specifically interactions between HSPCs and three distinct populations of potential HSPC niche cells: osteoblasts, vascular endothelial cells, and CXCL12-abundant reticular (CAR) cells. In vivo immunostaining will be used to identify each proposed HSPC niche cell within the BM. LSC, in conjunction with fluorescent immunostaining of surface antigens for HSPCs and niche cells, also will be used to objectively quantify the relative frequency that HSPCs are found in close association with each niche cell throughout the BM cavity. The bmi-1 targeted (GFP) reporter mouse model will again be used as the basis for identifying HSPCs. Improved definition of HSPC niche(s) in the BM may have important implications for ex vivo expansion of clinically relevant HSPC populations, and will contribute to understanding the etiology of pathological conditions such as leukemogenesis, myelodysplasia and immunodeficiency. PUBLIC HEALTH RELEVANCE: This research proposal will employ novel imaging technologies to define the localization and dynamic behavior of cell-cell interactions between hematopoietic stem and progenitor cells and their niche(s) in the bone marrow microenvironment. Such analyses should provide important insight into bone marrow environmental signals regulating hematopoietic cell development. This information may prove highly relevant to the expansion of clinically important hematopoietic stem and progenitor cell populations ex vivo, and also will contribute to understanding the etiology of pathological conditions such as leukemogenesis, myelodysplasia and immunodeficiency.