Role of CD44 in hematopoietic stem cell homing

描述（由申请人提供）：造血干细胞（HSC）移植是接受高剂量化疗和放疗的患者的必要手术，其效率取决于HSC的归家能力，例如，细胞进入受体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，受粘附分子、可溶性因子和细胞外基质的严格调控。我们之前已经证明CD44粘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重要的信息，但它们不能让我们区分外渗细胞和停留在血管内皮上的细胞，也不能让我们评估在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造血微环境在剪切应力条件下对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技术，使我们能够监测造血微环境对人造血干细胞运输的影响，包括在这些生理流动条件下，利用双细胞平行流室监测内皮层的滚动、粘附和转运，以及骨髓造血支持基质的播种。结合印迹滚动试验，该技术将帮助我们识别CD44的新配体和对抗受体，并描述CD44介导的体外人HSC归巢的细胞和分子机制。因此，拟议的研究将为研究人类HSC的归巢提供一种新的方法，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.