Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. Little is currently known about what specific factors stimulate osteoblast differentiation from human mesenchymal stem cells (hMSCs). Therefore, the aim for this project is to determine novel factors and mechanisms involved in human bone production which can be targeted to treat osteoporosis, using gene expression profiling and bioinformatic analyses, including the Connectivity Map, as an in silico approach.
Gene expression profiling was performed on hMSCs differentiated towards osteoblasts using Illumina microarrays. Osteogenic hMSC differentiation was assessed by analyses of alkaline phosphatase activity (ALP) and mineralization by calcium assay and alizarin red staining. Gene expression was determined by qPCR. Immunofluorescent analysis was performed to examine changes in the cytoskeleton. Kegg analysis was performed to determine enriched pathways. The gene signature of osteogenic hMSCs (top significantly regulated genes 6 h after induction by dexamethasone) was uploaded into Connectivity Map (www.broadinstitute.org/cmap/). This identified parbendazole as a compound with a statistically significant correlating gene signature to osteogenic hMSCs. Parbendazole stimulated osteogenic hMSC differentiation as indicated by increased ALP and mineralization, which interestingly occurs independent of the presence of glucocorticoids. Moreover, strong upregulation of glucocorticoid receptor target genes by glucocorticoids, is absent in parbendazole-treated cells. Parbendazole caused profound cell morphological and cytosketetal changes including strong inhibition of microtubules. Kegg analysis of the gene signature indicated TGF-β signalling, mineral absorption, and MAPK signalling pathways were enriched. By combining genomic and bioinfomatic tools against the backdrop of highly characterized human osteogenic differentiating hMSCs we have identified a novel bone anabolic candidate that induces osteoblast differentiation independent of glucocorticoid stimulation. In combination with the Kegg analysis we will identify important cellular processes and signalling cascades that can be manipulated to stimulate bone formation.
18 May 2013 - 22 May 2013