This application addresses two of the goals of the RFA: Use """"""""of in vitro methods to assay stem cell function that reflect individual differences among cell or tissue donors"""""""" and """"""""Potential systemic effects of age-related alterations in stem and precursor cell properties in specific lineages."""""""" Skeletal aging is characterized as a gradual loss of bone mass due to an excess of bone resorption that is not balanced by new bone formation. Thus, with age, there is a gradual loss of bone measured by declining bone mineral density (BMD), to the point of osteopenia or osteoporosis, and bone fracture. Bone marrow becomes fatty and ultimately """"""""gelatinized"""""""" with extreme age and osteoporosis. In addition, elderly people commonly have lower circulating levels of 25-dihydroxyvitamin D (25-D). Marrow stromal cells (MSCs) give rise to osteoblasts, but there is some discrepancy in the literature about the effect of age on osteoblast differentiation. We, and others, reported that there is an age-related decline in osteoblastogenesis from human bone marrow. Some others, using colony assays, report no detectable change. We have been applying an innovative automated cell tracking system developed by Dr. Greenberger for hematopoietic cells for research in skeletal aging. We have extensive experience with human marrow obtained from subjects undergoing total hip replacement for non-inflammatory joint disease. We have developed a research plan with 3 specific aims to test the hypotheses 1) that with aging there is a loss of osteoblast potential in human marrow stromal cells from men and women; 2) that vitamin D-deficiency reduces osteoblast potential, and 3) that cells from elders and vitamin D-deficient subjects can be rejuvenated in vitro by vitamin D metabolites or analogs. We will use biochemical and molecular methods to enumerate and characterize MSCs with osteoblast potential. We propose to use an innovative quantitative cell tracking system to measure differentiation as a function of age of human marrow. We will also use monolayer culture methods to assess the effects of age on progress in osteoblast differentiation. We will determine whether vitamin D metabolites or analogs promote osteoblast differentiation of marrow cells and whether the mechanisms involve increasing numbers of progenitors, changes in proliferation and apoptosis, and/or stimulation of osteoblast differentiation.
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