Adult vertebrates require a continuous supply of osteoblasts for both bone remodeling and regeneration during fracture repair. This implies the existence of a reservoir of cells in the body capable of osteogenesis. One source of these osteoprogenitors is the mesenchymal stem cells within the fibroblastic component of bon marrow stroma. Evidence for this derives from in vivo studies showing that stromal cells placed in diffusion chambers and implanted in a host give rise to cartilage and bone. In vitro, cultured bone marrow stem cells have been shown to yield adipocytic, fibroblastic and reticular cells as well as osteoblastic lines. We have recently developed a culture system in which rat bone marrow stromal cells give rise to cultures containing a high proportion of osteoblasts. This system involves brief culture of adherent stromal cells with dexamethasone in the presence of non-adherent marrow cells, followed by removal of non-adherent cells and culture with dexamethasone and/or bone morphogenetic proteins. The stromal cells, when treated with inducers, express high alkaline phosphatase, osteopontin and bone sialoprotein mRNA levels, and l,25 dihydroxyvitamin D3 induces osteocalcin mRNA. We propose to utilize this stromal cell culture system to examine the time course of changes in cultured marrow stem cells as they differentiate into osteoblasts, to identify changes in gene expression characteristic of early osteoprogenitor cells, and to identify transcriptional control mechanisms involved in this differentiation. Using the powerful techniques of flow cytometry and mRNA analyses, we will follow the progressive acquisition of markers associated with recruitment of stem cells and their subsequent differentiation into osteoblasts. We will examine the hypothesis that bone morphogenetic proteins promote bone formation by inducing osteogenesis from stromal stem cells. We will define progenitor cell subpopulations based on their expression of early markers, sort them by flow cytometry, and assess their capacity to form osteoblasts, chondrocytes and adipocytes. Finally, we will test the hypothesis that helix-loop-helix transcription factors related to those responsible for myogenesis are involved in osteogenesis. Our objective is to gain an understanding of the pathway by which stem cells give rise to osteoblasts as well as identifying factors controlling activation of this pathway, with the goal of identifying therapies for accelerated bone remodelling and increased bone formation.
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