Characterisation of skeletal stem cell populations by western blotting, aptamer selection and mass spectrometry

Abstract

With an aging population, the spectrum of treatable conditions broadens, especially within the orthopoedic field. The burden of bone related diseases has been foreseen to be potentially alleviated by skeletal stem cells (SSCs). However, the clinical potential of these cells is hampered by the absence of specific isolation techniques, due to the lack of specific markers. The aim of this work was to identify specific markers for SSC isolation, thereby allowing the therapeutic potential of SCC to be harnessed. Advanced quantitative mass spectrometry was used to analyse SSC proteome of four patients , 2 males aged 75 and 64 and 2 females aged 42 and 67. Cells were sorted for the Stro-1 marker (a cell surface marker widely used for SSCs), and cultured in osteogenic and basal media. Changes in protein expression were investigated. More than 7,000 proteins were identified and quantified, 6 proteins were significantly upregulated in the osteogenic group. Data suggest that protein modulation was highly governed by the chemical stimuli artificially introduced in the culture condition through the addition of dexamethasone and ascorbic acid (osteogenic media). In addition to the mass spectrometry experiments, characterisation of the Stro-1 antigen was undertaken by Western Blotting. The Stro-1 antibody was observed to react with an epitope of 50 kDa. Furthermore characterisation of the surface markers of SSC was performed adopting a naptamer SELEX process. The results presented here are a further step towards the characterisation of the proteomic profile of SSCs, and identification of unique markersfor the isolation of a homogeneous SSC population.

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ORCID for Richard Oreffo: orcid.org/0000-0001-5995-6726

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