Role of CD44 in hematopoietic stem cell homing

描述(申请人提供)：移植造血干细胞(HSC)是接受高剂量化疗和放射治疗的患者的必备程序，其效率取决于HSC的归巢能力，例如细胞进入受者骨髓并建立稳定造血的能力。HSC归巢是一个复杂的过程，包括多个步骤，受到黏附分子、可溶性因子和细胞外基质的严格调控。我们先前已经证明CD44黏附分子是骨髓归巢受体之一。然而，CD44介导的人HSC归巢的细胞和分子机制仍然知之甚少。虽然现有的研究HSC归巢的技术提供了大量的重要信息，但它们不允许我们区分血管内皮细胞上的渗出细胞和滞留细胞，也不允许我们评估生理流动条件下造血微环境对内皮细胞功能的影响。因此，需要新的体外方法来研究切应力条件下造血微环境对HSC-内皮细胞和HSC-基质细胞相互作用的影响。作为第一个具体目标，我们建议利用一种新的技术来监测造血微环境对人HSC运输的影响，包括滚动、黏附和跨内皮层迁移，以及利用体外两细胞平行流室在这些生理流动条件下种植骨髓支持造血的基质。结合印迹实验，该技术将有助于我们寻找CD44的新的配体和反受体，并阐明CD44介导的人HSC体外归巢的细胞和分子机制。因此，所提出的研究将为研究人HSC归巢提供一种新的方法，这可能为开发化疗或全身照射后干细胞移植的新策略提供基础。

DESCRIPTION (provided by applicant): Transplantation of hematopoietic stem cells (HSC) is a required procedure for patients who have undergone high-dose chemotherapy and irradiation, and its efficiency depends on the homing ability of HSC, e.g., capacity of the cells to enter the recipient's bone marrow and establish steady hematopoiesis. HSC homing is a complex process that includes multiple steps and is strictly regulated by adhesion molecules as well as soluble factors and the extracellular matrix. We have previously demonstrated that the CD44 adhesion molecule is one of the bone marrow homing receptors. However, the cellular and molecular mechanisms of CD44-mediated human HSC homing are still poorly understood. While existing techniques for studying HSC homing provide a large amount of important information, they do not allow us to distinguish between extravasated cells and cells arrested on the vascular endothelium, nor do they allow us to assess the influence of the hematopoietic microenvironment on endothelial cell function under conditions of physiological flow. Therefore, new in vitro methods are required to investigate the effects of the hematopoietic microenvironment on both HSC - endothelial cell and HSC - stromal cell interactions under shear stress conditions. As the first specific aim, we propose to utilize a new technique that would allow us to monitor the effect of the hematopoietic microenvironment on trafficking of human HSC, including rolling on, adhesion to, and transmigration across the endothelial layer, and the seeding of the bone marrow hematopoietic-supportive stroma under these conditions of physiological flow by utilizing a two-cell parallel flow chamber in vitro. In combination with a blot-rolling assay, this technique will help us to identify novel ligands and counter-receptors for CD44 and delineate the cellular and molecular mechanisms of CD44 - mediated homing of human HSC in vitro. The proposed studies will therefore define a new approach for studying homing of human HSC that can potentially provide a basis for the development of new strategies for stem cell transplantation following chemotherapy or total body irradiation.