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    • While stem cell based therapies

    2018-11-12

    While stem cell-based therapies hold great potential for the treatment of a wide array of medical conditions they are so novel that product characterization is particularly challenging. Despite considerable progress, the molecular regulatory mechanisms of self-renewal and lineage specification in these cell types are largely unexplored. In recent years a number of “omics” technologies were applied to investigate MSCs (Jansen et al., 2010; Kulterer et al., 2007; Ng et al., 2008; Ren et al., 2011). The majority of earlier proteomic studies were performed by a combination of two dimensional electrophoresis (2DE) and matrix assisted laser desorption ionization mass spectrometry (MALDI MS). The first proteomic investigation reported by Colter et al. resulted in the identification of 40 differentially regulated proteins between rapidly self-renewing and mature human BMSCs (hBMSCs) (Colter et al., 2001). Similar techniques were used to study the effect of transforming growth factor beta (TGF-β) (Wang et al., 2004), shear stress (Yi et al., 2010), and mechanical strain and TGF-β (Kurpinski et al., 2009), or disease conditions such as rheumatoid arthritis (Kastrinaki et al., 2008), beta-lactamase (Rollin et al., 2008), and idiopathic scoliosis (Zhuang et al., 2011) on hBMSCs. Other studies used combinations of 2DE and MALDI MS to compare the proteomic variability between MSCs isolated from various sources such as amniotic fluid, bone marrow, umbilical cord, placenta, adipose tissue, and synovial membrane (Roche et al., 2009). However, the well-documented poor performance of 2DE with regard to membrane, basic, and low abundance proteins limited the exploration of such complex biological samples as MSCs (Chevalier, 2008). Recent trends show that on-line multi-dimensional liquid chromatography (LC) coupled with MS significantly improves proteomic coverage. This approach dramatically increased the number of proteins identified (~900) including hundreds of membrane proteins from hBMSCs (Niehage et al., 2011). On the other hand, off-line 2D-LC fractionation followed by MALDI MS was also applied successfully to study the proteomic architecture of distinct populations of hBMSC (Mareddy et al., 2009). The number of proteins identified to date in hBMSC (<1000) clearly indicates that we have only scratched the surface of the proteome and detected mainly abundantly and moderately expressed proteins. A deeper molecular analysis of the proteome, transcriptome, and protein interactome of hBMSCs would lead to a better understanding of these cells. An additional difficulty is the absence of a unified analytical approach which makes the comparison of data obtained in different laboratories challenging, particularly in combination with the well-documented heterogeneity of hBMSCs.
    Materials and methods
    Results
    Discussion The current consensus is that not many proteins are expressed in a cell-specific manner. In fact, it has been reported that less than 1% of the human proteome is expressed in either single or a few cell types (Gry et al., 2010). This epitomizes the huge challenge that has to be overcome during the identification of protein markers that correlate with specific biological or clinical outcomes. Human BMSCs are poorly characterized in part because of the lack of comprehensive molecular evaluation. Therefore, the data presented in this report, in addition to its correlation with complex biological and molecular networks operating in hBMSCs, serves as a reference proteomic database for comparative work to identify and evaluate possible marker candidates for hBMSCs. The data obtained in this study also provide insight into donor-to-donor MSC proteome heterogeneity. Previous proteomic studies on hBMSCs had limited proteomic coverage and reported only the most abundant proteins. In contrast, this report is based on a total of 7753 proteins, which encompasses 712 transcription and translation regulators including SOX-9, -11, -13, -15, -18, and -30. The expression of SOX-2 and OCT-4, which are factors for pluripotency maintenance, was not detected (Pierantozzi et al., 2011). The expression of Stro-1 could not be detected, which is consistent with previous reports showing that the expression of Stro-1 ceases with cell culture expansion (Kolf et al., 2007). Many members of the STAT family (signal transducer and activator transcription) including STAT-1, -3, -4, 5A, -5B, and -6 have also been identified in this study. STAT proteins are responsible for multiple cellular activities including the regulation of growth, survival, differentiation, motility, and immune response (Akira, 1999). The proteomic data also revealed the expression of 384 kinases (e.g., MAP kinases, PAKs, and CDKs), 248 receptors (e.g., TLR-2, -4, -6, -7, and -9), and 29 cytokines (e.g., chemokines, interleukins, and tumor necrosis factors).