Perivascular Cell Characterization and Contribution to the Hematopoietic Niche

摘要了解造血细胞生态位环境对我们理解造血是非常重要的。间充质基质细胞（MSC）建立了造血生态位，但其在体内的鉴定和表征尚未完成。目前认为间充质干细胞来源于血管周围细胞。MSC分泌大量基质衍生因子-1 (SDF1)，这是一种参与造血细胞募集和维持的趋化因子。我们已经在斑马鱼中创造了第一个SDF1报告基因转基因脊椎动物，并且之前已经证明，高SDF1分泌细胞实际上是血管周围细胞，并且在过继移植后有助于造血细胞的SDF1募集。该模型首次允许预先分离造血生态位细胞。我们假设血管周围细胞在离体培养扩增环境中产生MSC，并在体外和体内建立造血细胞生态位环境。这项工作的主要目的是表征血管周围细胞的生化和功能。在特异性AIM 1中，我们将确定从sdf1:DsRed转基因斑马鱼中分离的血管周围细胞的体外特性；在特异性AIM 2中，我们将确定血管周围细胞在体外支持造血细胞扩增的潜力。先前MSC的表征主要集中在检测中胚层标志物和中胚层分化（成骨、血管和脂肪生成）。使用分离和培养的血管周围细胞，我们将验证细胞表面标记并将其分化为中胚层细胞系。此外，我们将证明血管周围细胞可以支持体外造血细胞的生长/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维持体内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，我们将构建一个sdf启动子驱动的白喉毒素受体转基因鱼，它将允许靶向消融血管周围生态位细胞。随后将采用标记供体骨髓的过继性转移，通过视觉显微镜分析和定量归巢分析来量化血管周围生态位消融对归巢的影响。这里概述的实验方法将使我们更好地理解造血微环境。

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.