| Bone morphogenetic proteins (BMPs) are key morphogenetic regulators of skeletal tissue development and potent inducers of post-developmental mesenchymal stem cell differentiation. Previous studies highlight the complex, temporal expression pattern of BMPs during fracture repair and pre-natal bone development, suggesting that these processes are regulated through the coordinated actions of multiple BMPs. Murine bone marrow stromal cells (MSCs) are a recognized ex vivo system of mesenchymal stem cell differentiation in which the effects of BMPs can be examined. Initial experiments focused on characterizing the culture system and evaluating the conditions necessary to drive MSC osteogenic differentiation. Subsequent studies were performed to determine if MSC differentiation is dependent on the endogenous expression of multiple BMPs, characterize the interactions of the BMPs within this network, and determine if the network is autonomously regulated by the expression of specific BMPs. Stem cell recruitment was measured by monitoring the development of mineralizing nodules, osteogenic differentiation was quantified by AlkPase activity and assessment of mRNA expression levels of specific transcription factors, matrix proteins, and several BMPs by ribonuclease protection assay. Osteogenic differentiation of MSCs in culture was accompanied by increased mRNA levels of Dlx5 and Runx2 and temporal increases in Col1alpha1, osteopontin, bone sialoprotein and osteocalcin expression. The progression of osteogenic differentiation was temporally concurrent and proportional to increases in the expression of BMPs 2, 4, 5, 6, 8A and 3. Noggin, a BMP antagonist, inhibited nodule formation, reduced the expression levels of osteogenic associated transcription factors, extracellular matrix genes, and BMPs 8A, 2, 5, and 3. In contrast, noggin treatment induced the expression of BMP-4 and 6. The addition of rhBMP-7 or 2 increased nodule formation, and the expression of osteogenic extracellular matrix genes. Interestingly, BMP treatment inhibited the endogenous expression of BMPs 4, 6 and 3b, while enhancing the expression of BMP-8a and BMP-3. Treatment with a neutralizing BMP-2 antibody also inhibited MSC differentiation and its effects were rescued by the addition of rhBMP-2. In summary, these data suggest that MSCs express BMP-2 which controls osteogenic differentiation through the dynamic regulation of other BMPs and factors critical to potentiation of stem cell lineage commitment. |
