In bone marrow cell transplantation, the phase of engraftment of donor cells is preceded by a phase of their firm anchoring within the extravascular spaces of bone marrow. Emerging evidence has recently emphasized that not only adequate retention but donor cell partitioning to particular bone marrow zones (""""""""niches"""""""") is necessary for long term hematopoietic reconstitution. Such """"""""niches"""""""" support distinct functions of stem cells, i.e. survival, quiescence and self-renewal (endosteal) vs. proliferation and downstream differentiation (vascular spaces). However, how donor cell partitioning is accomplished is not clear and the assays for partitioning have not been rigorously formulated. In this proposal, using defined criteria, we plan to quantitate donor cell compartmentalization within bone marrow, and ascertain whether there is a selective partitioning to certain bone marrow zones and whether this selectivity is cell context dependent (SA #1). Furthermore, although some of the molecular pathways that govern interactions of donor cells with their particular niches have been described, additional pathways and interactive networks remain to be delineated. We have previously described the role of VLA4/VCAM-1 pathway and interaction with CXCR4/SDF-1 pathway in homing and trafficking of hematopoietic cells. However, VLA4 (1421) is only one of several 21 heterodimers, and the contribution of the latter to the above functions has not been adequately explored. Furthermore, studies from 21-conditionally deficient mice have generated conflicting data. We have procured 21-conditionally deficient mice and using this important model, plan to fully characterize the coordinate contribution of several 21 heterodimers that contribute to phenotypic and functional aspects of hematopoiesis (SA #2-A and -B) in these mice with a particular focus on 1-integrins using alternate 2 partners (i.e., 14 and 16, SA#2-C). Finally, using G protein-coupled receptor kinase 6 (GRK-6) ko mice that lack the ability to down regulate CXCR4 after agonist-induced activation and have prolonged SDF-1 signaling, we will explore functional consequences in hematopoiesis and cell trafficking at steady state hematopoiesis and post stress (SA#3). Independent studies in mice reconstituted with (WHIM) mutated CXCR4 will provide complementary information on perturbed CXCR4/SDF-1 signaling. We believe that the proposed studies will greatly expand our current understanding of donor cell compartmentalization within bone marrow and the molecular pathways that dictate their interactions with microenvironmental cells and extracellular matrix. Transplantation of stem cells from bone marrow is an established practice for treatment of hematopoietic malignancies, non-malignant autoimmune conditions, or marrow failure syndromes. Successful outcomes depend on adequate number of donor cells use or treatment of donor cells, or of recipients to increase the engraftment efficiency. Our grant explores molecular pathways that are important for the efficient homing of donor cells, and therefore has the potential of finding ways to improve homing/engraftment. ? ? ?
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