br Conclusions br Acknowledgements br

Conclusions:
Acknowledgements
Background Much of the recent attention paid to human bone marrow stromal erk pathway (hBMSCs) is a consequence of their promise in cell-based therapy (Yi and Song, 2012). Features of BMSCs claimed that beneficial may include the capacity to home to sites of damage, differentiate into mature cells of the mesenchymal lineages, and secrete or stimulate the production of bioactive molecules. However, numerous studies have shown that extensive engraftment of BMSCs occurs rarely and thus they may act primarily through the release of soluble factors or through cell–cell interactions (Bernardo et al., 2012; Devine et al., 2003). Following their initial discovery in bone marrow, cells with similar surface characteristics have been isolated from many postnatal tissues (Otto and Wright, 2011), and these cells share common properties such as the capacity to adhere to plastic in cell culture and expression of common marker proteins (e.g., CD73, CD90, and CD105) or lack of other markers (e.g., CD11b, CD14, CD19, CD45, and HLA-DR) (Dominici et al., 2006; Keating, 2012). In bone marrow, they are a rare population (at times, from adult bone marrow only as few as 2000 BMSCs could be recovered) (Ksiazek, 2009). And yet, considerably higher cell numbers are often required for effective laboratory research or for clinical applications (106–109 cells per kg body weight (Wang et al., 2011; Yi and Song, 2012)). Hence, to obtain BMSCs in sufficient quantity, extensive in vitro culturing of primary cells is often necessary. In regard to long-term culturing, the optimization of protocols continues to be a work in progress. One key challenge is the decline in cell fitness with in vitro passaging and this may impact the therapeutic benefit of BMSCs. For example, while early passage BMSCs support the expansion and differentiation of hematopoietic cells, the late passages (passage nine and beyond) did not exhibit this property (Briquet et al., 2010). Studies show that late passage BMSCs exhibit various indices of aging including altered growth kinetics with subsequent proliferation arrest, altered morphology and clonogenicity, diminished differentiation capacity, increased levels of reactive oxygen species (ROS), p21 and p53 (Bruder et al., 1997; Lo Surdo and Bauer, 2012; Lo Surdo et al., 2013; Wagner et al., 2008). These changes could be the result of reorganization at genomic, epigenetic, transcriptomic, and proteomic levels. BMSCs acquire chromosomal aberrations, albeit at a lower frequency, during passaging (4% in human BMSCs vs 9% in human pluripotent stem cells (hPSCs)) (Ben-David et al., 2011). Some transcriptome profiling experiments revealed that genes associated with cell death and chromatin assembly were up-regulated while those related to cell cycle, DNA repair, and DNA metabolism were largely down-regulated with in vitro aging (Bin Noh et al., 2010; Wagner et al., 2008). Meanwhile, epigenetic studies suggest that DNA-methylation changes better correlate with the in vitro cell aging than other genetic changes (Redaelli et al., 2012; Schellenberg et al., 2011). Traditional 2D-gel based approaches have been applied to study various aspects of the BMSC proteome including differentiation-associated changes (Kasper et al., 2009; Sun et al., 2006), alterations in BMSCs as a result of disease (Seshi, 2006), changes initiated by mechanical stress (Yi et al., 2010), and the changes associated with in vitro cell passaging of BMSCs (Celebi and Elcin, 2009; Madeira et al., 2012). Taken together with other related studies, proteomic analyses undoubtedly provide important insights into the proteomic composition of BMSCs and help evolve our understanding about their biology. Nonetheless, more studies are needed, especially when many questions regarding the determinants of cell growth and development in culture media remain unanswered. In addition, molecular markers that correlate with developmental stages of cells, which may be utilized to monitor quality attributes of BMSCs, are yet to be revealed. Contemporary proteomic techniques are better placed to satisfy higher demands of characterizing complex biological systems than what could be achieved a few years ago. Such techniques combine the use of multidimensional chromatographic separation, high resolution mass spectrometry (MS), and improved bioinformatic data handling systems. The present work utilizes a workflow, which we recently optimized to survey the proteomics of human BMSCs and investigate the impact of long-term in vitro cultivation on protein expression in BMSCs. Based on multiple replicates; we performed an in-depth proteomic survey on hBMSCs at three passage stages (P3, P5 and P7). We used a label-free protein quantification technique to determine protein abundance and changes in response to long-term passaging. The technique uses the average intensity of the three most intense tryptic peptides per mole of a protein as surrogate to estimate protein abundance, a trend described by Silva et al. (2006). Our results provide detailed molecular insights of those changes that follow long-term in vitro cell passaging and offer potentially valuable protein targets for further studies to identify molecular markers that may correlate with hBMSC function.