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

描述（由申请人提供）：造血是一个生理过程，其中少量的多能造血干细胞（hsc）驻留在骨髓（BM）中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，尚未完成对骨髓中HSPC小生境的位置以及细胞和细胞外特征的明确鉴定。在这项研究计划中，我们采用新的成像技术，激光扫描细胞术（LSC）和多光子活体显微镜（MP-IVM），来定义骨髓微环境中HSPCs及其生态位之间的细胞-细胞相互作用的定位和动态行为。我们将这项拨款提案分为两个目标。目的1将量化HSPCs在骨髓腔室内不同解剖位置的分布。LSC将用于量化和绘制脑脊骨干骺端（髓内与髓内）和干骺端HSPCs的形态位置。HSPCs将使用bmi-1基因靶向报告基因（GFP）小鼠模型进行鉴定，其中GFP水平对HSPCs最强，同时对HSPC群体特征的表面抗原（c-Kit, Sca-1, flk2, CD150, CD34）进行荧光免疫染色。LSC可以扫描股骨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（2.5 × 105个细胞/图像），从而可以对极其罕见的BM种群进行客观量化和定位统计比较。目标2将描述BM中HSPC生态位的细胞成分。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生态位细胞群体之间的相互作用：成骨细胞、血管内皮细胞和富含cxcl12的网状（CAR）细胞。体内免疫染色将用于鉴定BM内每个提出的HSPC小生境细胞。LSC结合HSPCs和生态位细胞表面抗原的荧光免疫染色，也将用于客观量化HSPCs与BM腔中每个生态位细胞密切相关的相对频率。bmi-1靶向（GFP）报告鼠模型将再次作为鉴定HSPCs的基础。改进骨髓中HSPC生态位的定义可能对临床相关HSPC群体的体外扩增具有重要意义，并有助于理解诸如白血病发生、骨髓发育不良和免疫缺陷等病理条件的病因学。公共卫生相关性：本研究计划将采用新的成像技术来定义造血干细胞和祖细胞及其在骨髓微环境中的生态位之间的细胞-细胞相互作用的定位和动态行为。这样的分析应该为骨髓环境信号调节造血细胞发育提供重要的见解。这一信息可能被证明与临床重要的造血干细胞和祖细胞群的体外扩增高度相关，也将有助于理解病理条件的病因，如白血病发生、骨髓发育不良和免疫缺陷。

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.