An investigation of the early molecular regulation of adipogenesis in human mesenchymal stem cells
Human bone marrow-derived mesenchymal stem cells (hMSCs) can differentiate in vitro into various lineages including adipocytes, osteoblasts, and chondrocytes. Determining the earliest events of hMSC differentiation would provide a greater understanding of stem cell function, leading to novel therapeutic strategies. The molecular regulation of adipogenesis has been studied extensively using preadipocyte models that are committed to the adipocyte lineage. However, the events that occur in uncommitted stem cells such as hMSCs, and thus control the early stages of differentiation, have not been identified. In this thesis, hMSC differentiation to adipocytes was induced using a classic induction cocktail, and transcriptional profiles were obtained at early timepoints using Affymetrix U133 Plus 2.0 microarrays. Adipogenesis was shown to involve the regulation of over 4200 genes, which were clustered into distinct temporal patterns of gene expression. Recognised adipogenic regulators were expressed at the later stages of adipogenesis alongside genes involved in lipid metabolism, thus demonstrating the validity of the system. Global annotation of genes showing early expression changes revealed temporal regulation of signaling pathways known to control adipogenesis, together with molecular processes and pathway regulators not previously implicated in adipogenesis. A further detailed analysis of gene function identified 83 potential candidates for novel adipogenic regulators. Knock-down of gene expression induced by virally-mediated shRNA is an effective method to assess gene function. To investigate whether the candidate genes identified from the transcriptional profiling played a role in hMSC adipogenesis, retroviral shRNA library screens and an additional lentiviral shRNA strategy were developed and implemented. Finally, the effect on hMSC differentiation of the introduction of oncogenic regulators was investigated. Oncogenic transformation of hMSCs may lead to aberrant differentiation and mesenchymal tumour formation. It was found that the progressive addition of transforming genes did not block adipogenic or osteogenic differentiation, but caused significant impairment to the process.