This proposal plans to exploit the human long term marrow culture system in a large group of patients with severe aplastic anemia to determine the functional and biosynthetic properties of cultured marrow stromal cells in this disorder. When marrow buffy coat cells are placed in long term flask culture, and adherent stromal layer forms which consists of cells which synthesize collagen types I, III, and IV, synthesize proteoglycans, variably express Factor VIII associated antigen, but do not express the T-200, an antigen found on hematopoietic cells. Surprisingly, studies have shown that following allogeneic marrow transplantation, these stromal cells become donor-derived. In addition, stromal cells and/or their synthetic products are essential for proliferation and maturation of hematopoietic stem cells in vitro. We propose to examine the ability of stromal cells derived from aplastic anemia marrows to promote the differentiation of anti-Ia antibody plus complement treated normal, HLA-identical marrow cells to CFU-C detectable in semi-solid culture. In addition, biosynthetic patterns of collagen and proteoglycans will be measured directly and correlated with the functional integrity of the stromal cells. The derivation of marrow stromal cells after marrow transplantation of siblings of the opposite sex will be determined by Y-body analysis of cultured stromal cells from long term marrow cultures. It is of considerable interest to determine whether microenvironmental cells are transplanted in aplastic anemia as well as in acute leukemia. The third component of this project involves use of long term marrow culture as an indirect measure of stem cells with high proliferative potential not detectable in cloning assays. The effect of in vitro T-cell depletion and in vivo treatment with antithymocyte globulin on the generation of CFU-C in long term marrow cultures will be assessed to determine whether this system might prove useful in determining which patients are likely to respond to immunosuppression before it is given, and whether it could prove useful in determining response prior to the 2 to 3 months needed for evaluation of clinical responsiveness.
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