Development of long term marrow cultures which form osteoclast-like multinucleated cells (MNC) from mononuclear precursors, has allowed us to characterize the mechanism of action of osteotropic factors which regulate osteoclast formation. Although these culture systems have been extremely useful for these studies, they are limited by the fact that they are composed of heterogeneous cell populations, making it impossible to determine: 1) the molecular mechanisms involved in the differentiation of osteoclast precursors to mature osteoclasts, 2) the identity of the osteoclast precursor(s), or 3) if osteotropic factors act directly on osteoclast precursors. Such studies require purified populations of osteoclast precursors. To overcome these limitations, we have recently developed monoclonal antibodies to these MNC. The monoclonal antibodies react with a subpopulation of marrow mononuclear cells and freshly isolated osteoclasts from fetal baboons as well as MNC, suggesting that they may react with MNC precursors. The antibodies do not cross-react with peripheral blood monocytes or macrophage polykaryons demonstrating that they are not directed against macrophage antigens. With development of these reagents we can no initiate studies to purify and characterize osteoclast precursors. In this proposal we will: 1) further characterize these newly developed anti-MNC monoclonal antibodies and test their ability to purify osteoclast precursors from human bone marrow. If purified populations of MNC precursors are obtained, we will use these purified precursor populations to identify the cell lineage of the osteoclast by determining the antigenic phenotype of the osteoclast precursor(s) and determine the effects of osteotropic factors on purified populations of osteoclasts precursors. 2) extend our culture studies in which we have developed semi-solid culture systems which permit the clonal growth of committed osteoclast (OCL) precursors. These clones will be used to raise monoclonal antibodies against the committed OCL precursor. These antibodies should be useful in purification of OCL precursors and identification of unique differentiation antigens present on early OCL precursors ; 3) transfect human marrow cell populations containing OCL precursors with recombinant adenoviruses to determine if OCL precursors can be transformed, and identify the growth conditions required for their maturation to osteoclasts.
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