Characterisation of small leucine rich proteins gene and protein expression in mesenchymal stem cell differentiation into osteoblasts, adipocytes and chondrocytes

Abstract

This thesis is directed to understanding the role of Small Leucine Rich Proteins (SLRPs) in the cell biology of mesenchymal tissue in particular bone and cartilage. SLRPs are a family of 17 biologically active macromolecules which form the extracellular matrix in a variety of tissues and may play a role in bone and cartilage biology and diseases, in particular osteoporosis. It was hypothesised that: 1) The gene and protein expression of specific SLRPs will be up-regulated during the development of bone and cartilage. 2) During osteogenesis, the location of these SLRPs shows a pattern of distribution within the extracellular matrix. 3) Osteogenesis related SLRPs are specific to the cell development of that tissue. To investigate the first hypothesis, a bioinformatics study of a human osteosarcoma cell was initially used to determine the gene expression on all 17 SLRP members. The six highest expressed members Lumican, Epiphycan, Tskushi, Biglycan Decorin, and Osteomodulin (OMD) were selected for further analysis. To investigate the second hypothesis, the gene expression of these six selected members were analysed using real time quantitative reverse transcriptase polymerase chain reaction in both long term (up to 28 days) and short term (up to 7 days) osteogenesis of donor matched human adipose and bone marrow mesenchymal stem cells. These results showed the increase in expression of OMD in osteogenic stimulated media. As a result of these studies OMD was selected for further study, as a potential biomarker of osteoblasts. The gene expression of OMD was only increased significantly in osteoblast-like cells compared to other mesenchymal stem cell lineages including cartilage and adipose tissue. Protein expression of OMD was further investigated by western blotting. This was followed by confocal microscopy to further understand the expression of this protein. It was found through both methods that the protein expression of OMD was increased during osteogenesis, reflecting the gene expression previously observed. In conclusion, it was shown that the gene and protein expression of OMD was increased specifically during osteogenesis, and therefore could be used as a marker of osteogenesis of mesenchymal stem cells. Furthermore, its role in osteogenic development should be further studied to understand its role in osteogenesis