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.
