Perivascular Cell Characterization and Contribution to the Hematopoietic Niche

描述(由申请人提供)：摘要了解造血细胞的生态环境对我们理解造血学很重要。目前，间充质基质细胞(MSC)已被认为建立了造血细胞的生态位，但其体内鉴定和鉴定尚未完成。目前的研究认为，MSC来源于血管周围细胞。已知骨髓间充质干细胞分泌大量的基质衍生因子-1(SDF1)，这是一种参与造血细胞招募和维持的趋化因子。我们已经在斑马鱼中创造了第一个转SDF1报告基因的脊椎动物，之前的研究表明，高SDF1分泌细胞实际上是血管周围细胞，并在过继转移后有助于SDF1-造血细胞的募集。这个模型第一次允许先验地分离造血细胞。我们假设血管周围细胞在体外培养扩增环境中能分化为间充质干细胞，并在体外和体内建立了造血细胞的生态位环境。这项工作的主要目的是表征血管周围细胞的生化和功能。在特定的AIM 1中，我们将检测从SDF1：DsRed转基因斑马鱼中分离的血管周围细胞的体外特性，在特定的AIM 2中，我们将确定血管周围细胞在体外支持造血细胞增殖的潜力。在此之前，MSC的特征主要集中在中胚层标志物的检测和中胚层分化(成骨、血管生成和脂肪生成)。利用分离和培养的血管周围细胞，我们将验证细胞表面标志，并将它们分化为中胚层细胞系。此外，我们还将展示血管周围细胞在体外可以支持造血细胞的生长/维持。利用转基因斑马鱼的好处是体内血管周围细胞的可视化，在特定的AIM 3中，我们将确定血管周围细胞在体内维持造血细胞生态位中的作用。为了确定血管周围细胞的功能意义，我们将构建一种SDF启动子驱动的白喉毒素受体转基因FISH，它将允许靶向消融血管周围的壁龛细胞。随后将采用标记的供者骨髓的过继转移来量化血管周围壁龛消融对归巢的影响，使用视觉显微镜分析和定量归巢分析。这里概述的实验方法将使我们更好地了解造血微环境。 公共卫生相关性：骨髓移植是一些儿科癌症的唯一治疗措施，但由于准备方案导致的中性粒细胞减少症持续时间较长，因此具有显著的风险。我们将学习造血细胞是如何归宿并与提供支持的骨髓细胞相互作用的。了解这一过程将使我们能够开发新的药物和治疗方法，以提高造血细胞植入的效率，从而减少中性粒细胞减少的时间，并最终提高患者的存活率。此外，从这项工作中获得的知识可能被用来更好地理解骨髓衰竭领域更广泛的疾病机制，包括再生障碍性贫血、范可尼贫血和辐射暴露。

DESCRIPTION (provided by applicant): Abstract Comprehending the hemopoietic cell niche environment is important in our understanding of hematopoiesis. It has been hypothesized that mesenchymal stromal cells (MSC) establish the hematopoietic niche, but their identification and characterization in vivo has not been accomplished. Current thinking suggests that MSC are derived from perivascular cells. MSC are known to secrete high amounts of stroma- derived factor-1 (SDF1), a chemokine involved in the recruitment and maintenance of hematopoietic cells. We have created the first SDF1 reporter transgenic vertebrate in a zebrafish and previously shown that high-SDF1 secreting cells were, in fact, perivascular cells and contribute to SDF1-recruitment of hematopoietic cells after adoptive transfer. This model allows, for the first time, the isolation of hematopoietic niche cells a priori. We hypothesize that perivascular cells give rise to MSC in the ex vivo culture expansion setting and establish the hematopoietic cell niche environment in vitro and in vivo. The primary aim of this work is to characterize perivascular cells both biochemically and functionally. In specific AIM 1, we will determine the in vitro properties of perivascular cells isolated from the sdf1:DsRed transgenic zebrafish and in specific AIM 2 we will determine the potential of perivascular cells to support hematopoietic cell expansion in vitro. Prior characterization of MSC has been focused on detection of mesodermal markers and mesodermal differentiation (osteogenesis, vasculogenesis, and adipogenesis). Using isolated and cultured perivascular cells we will verify cell surface markers and differentiate them into mesodermal lineages. Furthermore, we will show that perivascular cells can support the growth/maintenance of hematopoietic cells in vitro. The benefit of utilizing a transgenic zebrafish is the visualization of the perivascular cells in vivo and in specific AIM 3, we will determine the role of perivascular cells in the maintenance of the hematopoietic cell niche in vivo. To determine the functional significance of perivascular cells, we will construct an sdf promoter driven diphtheria toxin receptor transgenic fish that will allow targeted ablation of the perivascular niche cells. This will be followed by adoptive transfer of labeled donor marrow to quantify the effects of perivascular niche ablation on homing using both visual microscopic assays and quantitative homing assays. The experimental approach outlined here wills us to better understand the hematopoietic microenvironment. PUBLIC HEALTH RELEVANCE: Bone marrow transplant is the only curative measure for some pediatric cancers, but carries with it significant risk due to prolonged neutropenia associated with preparative regimen. We will learn how the hematopoietic cells home and interact with the marrow niche cells which provide their support. Understanding this process will allow us to develop new drugs and therapies to allow hematopoietic cell engraftment to occur with increased efficiency thereby decreasing time of neutropenia and ultimately increasing patient survival. Additionally, knowledge form this work may be used to better understand broader disease mechanisms in the field on bone marrow failure including aplastic anemia, Fanconi's anemia, and radiation exposure.