Glycobiology of adipose-derived stem cell differentiation

Human adipose tissue is a major source of mesenchymal stem cells, with numbers exceeding those in bone marrow and blood. These stem cells are referred to as adipose-derived stem cells (ADSCs) and have the ability to differentiate into different cell types. In vitro, ADSCs can be made to differentiate into the mesenchymal lineages of adipocytes, osteoblasts and chondrocytes, while in vivo, ADSCs become pre-adipocytes before further differentiating into mature adipocytes. Protein glycosylation has been shown to change in stem cell differentiation, and while ADSCs have been acknowledged for their therapeutic potential, the investigation of protein glycosylation changes during ADSC adipogenic differentiation has not been reported. First, adipogenic differentiation of ADSCs in vitro was optimised and evaluated by measuring the expression levels of established gene-markers and staining of the accumulated lipid in differentiated cells using Oil Red O. These results confirmed the cells had entered the adipogenic lineage and shared some characteristics with, but did not entirely mirror native, in vivo produced adipocytes. ADSCs, in vitro differentiated ADSCs and in vivo native mature adipocytes harvested from the same patients were used to investigate the membrane protein N- and O-linked glycosylation of adipogenesis. Using porous graphitised carbon LC coupled with negative ion ESI-MS/MS, a total of 138 glycan structures carried by the membrane proteins were characterised across the three cell types. Bisecting GlcNAc type N-linked structures were detected at a high level (32.1 % of total glycans) in mature adipocytes with some appearing in the differentiating ADSCs (1.9 %), while overall, ADSCs and their in vitro differentiated progeny showed similar glycosylation expression profiles. This finding was further correlated by increased mRNA expression of the MGAT3 gene responsible for the enzyme synthesis of this glycan structure type in both the in vivo and in vitro differentiated ADSCs. The same cell types (ADSCs, differentiated ADSCs and mature adipocytes) were characterised for their membrane proteome using label-free quantitative shotgun proteomics analysis. Many protein differences were identified between the three cell types, with, importantly, adipocyte-specific proteins found to be up-regulated in both mature adipocytes and differentiating ADSCs. Mitochondrial and lipid-related processes were also up-regulated in both adipogenic cell products, whereas epigenetic-related proteins and processes were found to be up-regulated in in vitro differentiated ADSCs alone. Analysis of the N-linked glycans from SDS-PAGE separated membrane proteins of the three cell types revealed that expression of bisecting GlcNAc structures was present on the majority of adipocyteglyco proteins. A more targeted methodology of carrying out proteomic analysis of de-N-glycosylated peptides of the gel-separated proteins unearthed new glycoproteins not detected previously in the shotgun proteomic analysis without de-glycosylation. This approach identified the adipogenic marker, CD36, as the dominant glycoprotein in the adipocyte membrane proteome that was also upregulated at the mRNA transcript level in both the in vitro differentiated ADSCs (7.1-fold increase) and mature adipocytes (102.9-fold increase). This work highlighted the importance of de-Nglycosylation of proteins in proteomics experiments for increased identification of glycoproteins. The systems glycobiology approach by the integration of glycomics, proteomics and transcriptomics analyses in this thesis extended the investigation of membrane protein glycosylation changes in adipose-derived stem cell differentiation. The work provides a framework for future glycoproteomics-based investigations into the differentiation of stem cells into adipocytes, and will allow their related pathologies and potential therapeutic applications to be discovered.