Verbatim): Hematopoietic stem cells undergo a development stage-specific translocation during ontogeny and ultimately reside in the adult bone marrow. Maintenance of this highly regenerative cell pool through adult life is dependent upon their relative quiescence. We generated a cDNA library from quiescent human hematopoietic stem-like cells derived from bone marrow and identified by subtractive cloning a seven transmembrane molecule with a signature motif of the chemokine receptor family. Antiserum raised against this gene product identified cells from human fetal bone marrow, but not other fetal hematopoietic organs and very rare cells from adult bone marrow. These cells were enriched for quiescent cells with the ability to sustain mature blood cell generation for prolonged periods on stromal feeder layers. Calcium flux was induced upon exposure of transfected cells to bone marrow stroma conditioned medium, but not medium from other hematopoietic tissue stromal sources or from a panel of recombinant chemokines. Transduction of the receptor into hematopoietic cell lines resulted in enhanced transmigration toward bone marrow stroma in vitro and to bone marrow in irradiated mice. Transduced primary CD34+ progenitors had reduced proliferative potential, but sustained LTC-IC capability. Stem cell-G protein-coupled receptor-i (SC-GPR1) is a chemokine receptor that identifies bone marrow-derived hematopoietic stem cells and mediates growth regulatory and cell localization signals. This proposal builds upon these observations to address the following specific aims: 1. Define the functional role of SC-GPR1 in development using a mouse engineered to be deficient in the gene and its role in adult hematopoiesis using transplantation of cells identified by a monoclonal antibody specific for SC-GPR1. 2. Purify and clone the ligand for SC-GPR1. 3. Determine the mechanism by which ectopic expression of SC-GPR1 induces or maintains a primitive phenotype.
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