Previous studies of the hematopoietic microenvironment have centered on the role of cellular elements (Progenitor cells and stromal cells) and soluble factors in hematopoietic differentiation. This proposal will test the hypothesis that, in addition to these two components, extracellular matrix (ECM) plays a crucial role in the hematopoietic microenvironment. A new system in which murine long-term bone marrow cultures (LT- BMC) are established on marrow-derived ECM will be used. In these matrix-based cultures (as opposed to controls), there is a marked proliferation of both hematopoietic progenitor cells and stromal cells. Using this system, the cell biology, biochemistry and functional characteristics of hematopoietic ECM will be addressed. Studies on the biology of ECM:hematopoietic cell interactions will focus on the characterization of cell types responding to ECM. First, the direct and indirect effects of ECM on hematopoietic progenitor cells and/or stromal cells will be determined. Other studies will investigate the organ-specificity and lineage specificity of ECM. These experiments will involve establishing LT-BMC on ECM derived from non-hematopoietic (e.g. lung, kidney) and hematopoietic (e.g. bone marrow and spleen) organs. ECM from hematopoietic organs will also be utilized to examine whether ECM determines organ-related lineage specificity (i.e. marrow is predominantly granulopoietic and spleen erythropoietic). Concomitant with the above studies, biochemical and functional characterization of ECM will be determined. The ability of known ECM proteins to support hematopoiesis will be examined. As well, distinct biochemical differences between various matrices will be studied by SDS- PAGE. Functional studies of these proteins will involve attachment of marrow cells to ECM components resolved on SDS- PAGE and transferred to nitrocellulose. In addition, antibodies against specific marrow ECM attachment proteins will be made and used to determine the distribution/function of these antigens in vivo and in vitro. Finally, hematopoietic cell surface receptors for these ECM proteins will be isolated and characterized. The systems developed in this proposal will show that, in addition to cellular elements and soluble factors, ECM is a crucial third component of the hematopoietic microenvironment. This may have important implications in the understanding of disorders such a aplastic anemia, myelofibrosis, and leukemia.
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