Recently efforts have been made to

Recently, efforts have been made to identify differently expressed proteins during the osteogenic differentiation of hBMSCs (Alves et al., 2010; Foster et al., 2005; Kim et al., 2008, 2010; Ong and Mann, 2006; Zhang et al., 2007). These types of studies will not only increase our understanding of the proteins and signaling pathways involved in the differentiation process, they could also reveal new, reliable markers of hBMSCs undergoing osteogenic differentiation. Surface markers in particular could be an essential tool for monitoring the differentiation process in future applications, such as cell therapy and tissue engineering, as they are accessible using non-disruptive techniques such as flow cytometry. However, to date, there is no single CD marker or panel of markers, similar to that used for defining undifferentiated hBMSCs, which is able clearly to define a population of hBMSCs differentiating towards the osteogenic lineage. A method for quantitatively measuring changes in protein purchase Paclitaxel levels between different cell types or in different culture conditions was described by Mann and co-workers in 2006 (Kratchmarova et al., 2005; Mann, 2006). Stable-isotope labeling in cell culture (SILAC) is based on the metabolic labeling of proteins with different variants of one or two amino acids, resulting in a mass difference between proteins isolated from different culture conditions (Forsman et al., 2008; Ong and Mann, 2006). Mass spectrometric analysis enables the relative quantification of the amounts of individual proteins expressed by the cells. This method has since been used to explore the differences in the proteome between hBMSCs from different sources and under different conditions (Kratchmarova et al., 2005).
Materials and methods The present study was performed in three steps. In the initial exploratory phase, hBMSCs isolated from bone marrow were metabolically labeled, during expansion or osteogenic differentiation, with arginine (Arg) and lysine (Lys) containing either a normal 12C carbon (light isotope) or a 13C carbon (heavy isotope). The membrane proteins were subsequently isolated and their relative expressions, in the two culture conditions, were quantified by mass spectrometry (MS). In this step, one hBMSC donor was used (n=1). In the second step, the difference in expression of a few selected markers, between cells undergoing expansion or osteogenic differentiation, was validated by flow cytometry or ELISA after one, two and three weeks. In a third and final step, the lineage specificity of three candidate markers was investigated. The expression of these candidate markers during chondrogenic and adipogenic differentiation was analyzed by flow cytometry or ELISA after one and two weeks. In steps two and three, three different hBMSC donors were used (n=3).
Results hBMSCs isolated from four different donors, in passages 5–6, positive for CD166 and CD105 (97.3±5.2% and 99.0±1.1% respectively) and negative for CD45 and CD34 (0.3±0.3% and 0.3±0.5% respectively), were used in this study. Using an exploratory approach, proteins differently expressed in osteogenic differentiating hBMSCs were identified using the SILAC method. The quantitative MS analysis was performed on one SILAC sample, which was divided into two technical replicates to reduce the effects of under-sampling in the mass spectrometric analysis. Based on the results of this experiment, 9 proteins were selected for further validation of their differential expression during osteogenesis. The expression levels of several membrane proteins and one intra-cellular protein were measured at several time points in three independent biological replicates by flow cytometry or ELISA. Finally, the lineage specificity of three candidate markers was investigated.
Discussion In the first exploratory part of the present study, several surface proteins were found to be upregulated during the osteogenic differentiation of hBMSCs. After validating the expression of selected surface markers by flow cytometry, new information about their expression patterns during osteogenic differentiation was revealed. CD49e was upregulated upon osteogenic differentiation, with a more pronounced difference after three weeks of differentiation. Hamidouche et al. (2009, 2010) have demonstrated that CD49e, also known as integrin alpha-5, is not only involved in the differentiation process of hBMSCs towards the osteogenic lineage but that the stimulation of CD49e also promoted an increase in the amount of bone matrix in vivo. Further, the gene expression of CD49e has also been shown to be increased during dexamethasone-induced osteogenic differentiation (Baksh et al., 2007), which was also demonstrated in our study but at the level of protein expression.