Although hematopoietic stem cells and progenitor cells (HPC) circulate, they only develop in hematopoietic stroma. This has led to the hypothesis that HPC must recognize and adhere to appropriate tissue of hematopoietic organs where specific cellular interactions can influence their growth. This application proposes to develop an assay that can be used to identify the cells and molecules that mediate these interactions. The methodology proposed is based on in vitro binding of target cells to tissue sections. This adhesion assay has been developed by others to identify lymphocyte surface molecules or """"""""homing"""""""" receptors responsible for organ-specific binding of lymphocytes. The possible relevance of this assay to HPC-stromal cell interactions has been suggested by preliminary studies showing differential binding patterns of bone marrow cells to sections of normal and genetically defective hematopoietic tissue. Physiologically relevant adhesion assays will be developed for normal HPC on sections of marrow and spleen obtained from normal donors and patients with hematological diseases. Initial studies will require the preparation of relatively pure populations of HPC. Current pilot studies using a combination of selection techniques have yielded cell populations with a 30% plating efficiency for CFU-GM, which satisfies this requirement. Once specific binding patterns for normal HPC have been defined, cell lines that mimic these specific binding patterns, as well as auto- logous variant cell lines that do not bind will be established. Paired autologous cell lines that are positive and negative (+/-) for binding will be used to generate monoclonal antibodies that can distinguish the +/- variants. These monoclonal antibodies will then be tested for their ability to block binding and/or distinguish distinct classes of HPC. The +/- variants will also be used to provide mRNA for differential cDNA hybridization techniques that can identify gene sequences expressed in one variant but not the other. Differentially expressed sequences will then be cloned. The proposed studies should provide a panel of antibodies and gene sequences that can be used as tools to define cellular interactions involved in normal hematopoietic regulation and identify mechanisms associated with disease.
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