Modelling metabolomic changes in human bone marrow derived mesenchymal stem cells during oesteogenic differentiation via genome scale metabolic models to further their use in regenerative medicine

Abstract

In recent years the fields of regenerative and translational medicine have become the subjects of significantly growing interest due to their offer of previously unimaginable therapeutics. Within these fields are several novel tools believed to hold the keys to furthering existing and new developments and one of those tools is human mesenchymal stem cells. One of the applications hMSCs have been studied for is enhanced osteogenic regeneration or reconstruction of new bone tissue. Although various studies have been performed and some strides been made towards a plausible clinical application there is still lot left to be discovered. A methodical studying and relatively detailed in silico genome scale metabolic modelling occurring changes and the accompanying metabolic phenotypes could provide a means to fill in the existing knowledge gaps (from the protein level all the way to the genomic level) and, additionally, a means to perform hypotheses testing with a significant reduction when it comes to the accompanying cost. The objective of this thesis was to study the metabolomic changes in hMSCs during osteogenic differentiation using original transcriptomic, intracellular and extracellular metabolomic data in order try and define possible metabolic stages over the course of the differentiation and use genome scale network reconstruction to create in silico models. In the first part of this work extracellular and intracellular data were used to define possible stages to osteogenic differentiation and hypothesise which pathways may characterise the different metabolic phenotypes. Three stages were suggested based on the data and labelled intracellular metabolomics indicated a decrease in glycolytic dependencies throughout the differentiation period with an increase in mitochondria related energy producing functions as the osteogenesis progressed. This will help focus specific time points of interest and relevance when it comes to mapping the significant metabolomic changes. In the second part of this work extracellular metabolomic data collected from BM-hMSCs during proliferation, adipogenic and osteogenic differentiation was used along with experimentally specific data to create three directly comparable genome scale metabolic models, two of which have no comparable predecessors, for those cell lineages during the first 7 days of cell culture. Models were biologically feasible and showed all lineage specific characteristic reactions as active. By analysis and comparison, the various enriched subsystems and pathways most significant for each lineage were found. Results were varied for proliferating cells, which matches that they have to synthesise various metabolites and substances to expand, whilst fatty acid oxidation and fatty acid synthesis was most prominent in adipogenesis with fatty acid oxidation as well as transport reactions characteristic for osteogenesis. These models can be used for model-driven experimental design to engineer osteogenesis and, with modifications, to create disease model for osteoporosis. In the third part presented we summarized the various characteristics and possibilities that lie in using MSCs as a tool in tissue engineering and regenerative medicine and how, via implementation of genome scale metabolic model reconstruction, their possibilities could possibly be taken much further in a faster, more methodical manner while reducing the related experimental cost. To summarise, this work has provided real strides when it comes to closing the existing gap regarding metabolomic changes during differentiation of hMSCs as well as providing novel tools that can be used to make further studies more efficient and cost effective.