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

描述(申请人提供)：造血是一种生理过程，少数多能造血干细胞(HSCs)存在于骨髓(BM)增殖和分化，以产生完整的血液和免疫细胞。造血干细胞和祖细胞(HSPC)的自我更新和谱系分化依赖于不同的微环境，称为生态位，由细胞成分、可溶性调节因子和细胞外基质定义。由于常规成像(共聚焦、荧光)技术的限制，尚未完成对骨髓中HSPC壁龛的位置以及细胞和细胞外特征的最终鉴定。在这项研究计划中，我们使用新的成像技术，激光扫描细胞术(LSC)和多光子活体显微镜(MP-IVM)，来确定骨髓微环境中HSPC与其生态位(S)之间细胞-细胞相互作用的定位和动态行为。我们将这项赠款提案组织成两个目标。目标1将量化HSPC在血管外骨髓间隔内不同解剖位置的分布。LSC将被用来量化和定位HSPC在骨髓的骨干(骨内膜与髓质)和干骺端区域的形态位置(S)。将使用BMI-1基因靶向报告(GFP)小鼠模型来鉴定HSPC，其中HSPC的GFP水平最高，并对HSPC群体特有的表面抗原(c-Kit、SCA-1、flk2、CD150、CD34)进行荧光免疫染色。LSC允许扫描股骨BM腔的整个纵向切片，并以单细胞级别的精度映射BM腔的全部细胞内容(2.5x105个/图像)，从而使客观量化和统计比较即使是极其罕见的BM群体的定位也成为可能。目标2将描述BM内HSPC生态位的细胞成分(S)。多光子活体显微镜(MP-IVM)将用于体内分析HSPC行为的动力学，特别是HSPC与三种不同的潜在HSPC生态位细胞之间的相互作用：成骨细胞、血管内皮细胞和CXCL12丰富的网状细胞(CAR)。体内免疫染色将被用来识别BM内每个拟议的HSPC利基细胞。LSC结合HSPC和壁龛细胞表面抗原的荧光免疫染色，也将被用来客观地量化HSPC与整个骨髓腔内每个壁龛细胞密切相关的相对频率。BMI-1靶向(GFP)报告鼠模型将再次被用作识别HSPC的基础。S在骨髓中对HSPC生态位的定义的改进可能对临床相关HSPC群体的体外扩增具有重要意义，并将有助于理解白血病发生、骨髓增生异常和免疫缺陷等病理疾病的病因。公共卫生相关性：这项研究计划将使用新的成像技术来定义骨髓微环境中造血干细胞和祖细胞及其生态位之间细胞-细胞相互作用的定位和动态行为(S)。这些分析应该为调节造血细胞发育的骨髓环境信号提供重要的洞察力。这些信息可能被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，也将有助于理解白血病发生、骨髓发育不良和免疫缺陷等病理疾病的病因。

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.