This grant will develop a cell culture model to identify, isolate and characterize cells as they traverse stages of increasing differentiation within the osteoblast lineage. It is based on the observation of bone nodule formation by marrow stromal fibroblasts (MSF) which are believed to be a primary source for the most primitive osteoprogenitor cell. In contrast to other strategies that use cell surface markers of lineage development in rat or human, this approach uses promoter-driven transgenes obtained from transgenic mice which activate at different stages of bone cell differentiation. The marker transgenes are subfragments of the Col1A1 promoter which activate either prior to or at the time of easy bone differentiation, BSP which activates at the time of bone mineralization and OC which is expressed well after mineralization has formed. The temporal expression of two promoter constructs driving different marker genes (CAT or beta-gal) will be used to optimize the system for the time, number and size of bone nodules that express the markers in culture. Subsequently, markers and substrates will be utilized which allow identifying cells expressing the transgenes while maintaining the vitality of the cell (green fluorescent protein and lipophilic beta-gal substrates). This is one of the primary advantages of the strategy because it will permit isolation of a relatively homogeneous population of living cells along the lineage pathway by FACS based on the pattern of expression of the transgenes. Thus, a subpopulation of cells at a known stage of development will be tested for its ability to be repassaged and still maintain the potential to form bone in vitro and in vivo when cocultured with freshly isolated MSF from non-transgenic mice. Because the model permits transgene identification in ongoing cultured cells, a retroviral gene trap strategy will be developed for new genes that are activate either before or after a clonal population of cells begin early bone differentiation. The model has the potential for identifying and expanding osteoprogenitor cells that would be optimal candidates for corrective somatic gene therapy of heritable diseases of bone. It will have many applications to questions of stem cell number and progression in murine models of osteoporosis because the existing and new markers of differentiation can be used in intact animals.
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