Characterisation of STRO-1 expression on human mesenchymal stem cells and identification of putative cancer stem cells in osteosarcoma: prevention by micronutrients
It is becoming increasingly more common to use culture expanded human mesenchymal stem cells (hMSCs) in regenerative medicine due to their low incidence in vivo. However, their successful application is hampered by a lack of selective markers to positively identify the expanded multipotent cells. This study aimed to characterise STRO-1 antigen as a potential biomarker of multipotency on cultured bone marrow derived hMSCs. In an attempt to identify the nature of this antigen, two techniques were implemented: peptide phage display technology and a microarray based approach. Changes in the expression of STRO-1 were investigated during culture expansion of hMSCs. STRO-1 expression positively correlated with cellular morphology and multilineage potential, whereby senescent cells down-regulated STRO-1 antigen and exhibited decreased adipogenic and osteogenic potential. Furthermore, STRO-1 was found to be heterogeneously expressed on hMSC populations and enrichment followed by lineage specific induction of the STRO-1BRIGHT fraction resulted in enhanced adipogenic and osteogenic differentiation potential. The expression of STRO-1 antigen was further characterised as a marker of differentiation, whereby differentiating cells were found to down-regulate STRO-1. A cellular hierarchy in hMSC population was therefore proposed based on STRO-1 status, with the highest STRO-1 expressive cells representing the multipotent subset. In an attempt to identify the epitope that STRO-1 IgM antibody recognised, peptide phage display technology was used as solid and liquid phase panning systems but the approach yielded no promising peptide candidate. Subsequently, comparative gene expression microarray analysis of osteosarcoma cell lines (143B, CAL72, G-292, HOS, MG-63, Saos-2 and U-2-OS) was implemented and a list of eight potential candidate genes encoding STRO-1 antigen was selected. This work ultimately led to the identification of putative cancer stem cells (CSCs) in seven osteosarcoma cell lines initially based on STRO-1 expression. With MG-63 strongly expressing STRO-1, the maintenance of MSC-like properties by STRO-1 expressing cell lines was investigated. A heterogeneous pattern of osteogenic differentiation was observed between and within the cell lines. Closer inspection revealed a cellular hierarchy comprising of holoclones and paraclones, with the holoclones representing the putative CSCs in osteosarcoma. Overall, this thesis addressed the fields of regenerative medicine as well as oncology by proposing STRO-1 antigen as a marker of multipotency on hMSCs and osteosarcoma holoclonal cells as the putative cancer stem cell targets for anti-cancer drug development.
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