The cytokines essential for human osteociastogenesis, and the mechanism by which estrogen regulates this process, remain conjectural at best, mainly due to the lack of adequate models. This has prompted us to developed a new method for generating human osteoclasts capable of resorbing bone in vitro. This was achieved by culturing peripheral blood stem cells (CD 34+ cells), with GM-CSF, IL-1, IL-3, IL-6, Stem Cell Factor (SCF) and erythropoietin in absence of exogenous stromal cells and 1,25(OH)2D3. Since CD34+ cells can be harvested from normal donors, and induced to produce large numbers of osteoclasts, our method is suitable for determining the factors which regulate the differentiation of human osteoclast precursors into mature, functional osteoclasts, and for assessing the effects of menopause on human osteoclastogenesis. Therefore, in Specific Aim 1 we will determine the essential factors for human osteoclastogenesis. This will be accomplished by culturing blood CD 34+ cells with all possible combinations of the cytokines listed above. We will then determine if these cytokines stimulate osteoclastogenesis by targeting, CD34+ cells which can differentiate into stromal cells (CD34+, HLA-DR- cells) or CD34+ cells which can differentiate into stromal cells (CD34+, HLA-DR- cells) or CD34+ cells which can differentiate only into hemopoietic elements (CD34+, HL-DR+ cells). This will be accomplished by separating total CD34+ cells in CD34+, HLA-DR+ cells by FACS analysis, and assessing the formation of osteoclasts in these populations. Finally, we will determine if in vitro estrogen treatment decreases the differentiation of osteoclast precursors.
In Specific Aim 2, we will investigate the effect of cytokines and steroid hormones on the expression of integrin receptors essential for the fusion of mononucleated precursors into multinucleated cells (alphaMbeta2), and the adherence of these cells to bone (alphavbeta3), in CD34+ cells, CFU-GM derived cells, and more mature osteoclast precursors.
In Specific Aim 3 we will determine the effects of menopause and in vivo estrogen treatment on human osteoclastogenesis. This will be accomplished by harvesting and purifying CD34+ cells from the peripheral blood of premenopausal women, untreated postmenopausal women within 7 years since menopause and age-matched estrogen treated postmenopausal women. The end points of this study will be to determine if menopause increases the production of osteoclast-like cells in cultures of CD34+ cells. The significance of this proposal is high; First it will allow optimization as a new experimental model suitable for general large numbers of human osteoclasts. Second, it will determine the essential cytokines for human osteoclastogenesis. Third, it will elucidate the effects of estrogen and menopause on human osteoclastogenesis.
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