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    • Main Text br Acknowledgments I have

    2018-10-20

    Main Text
    Acknowledgments I have been fortunate to work with tremendous mentors, colleagues, and students throughout my career and I am grateful for their past and current support. Research in my laboratory is supported by the NIH, the New York Stem Cell Foundation as a New York Stem Cell Foundation–Robertson Investigator, and private philanthropy.
    Introduction Mesenchymal stromal GSK256066 (MSCs) are currently tested for a wide range of clinical applications (Squillaro et al., 2015), but there are no precise measures for their quality control. Molecular markers to clearly discern MSCs and fibroblasts remain elusive. The major difference between these two cell types is that particularly MSCs comprise a multipotent subset often referred to as “mesenchymal stem cells” (Dominici et al., 2006). Several surface markers have been suggested for enrichment of MSCs, such as CD106, CD146, and CD271 (Buhring et al., 2007; Halfon et al., 2011; Sorrentino et al., 2008), but none of them seems to be exclusively expressed on MSCs. Proteomics and gene-expression profiles can discern cells that have been obtained from different tissues or under different culture conditions (Holley et al., 2015; Ishii et al., 2005), and high-content screening assays based on microRNA or RNAi can elucidate cell type-specific responses (Bae et al., 2009; Erdmann et al., 2015). However, all these profiling and high-throughput techniques are relatively time and labor consuming, require complex computational analysis, and can hardly be standardized for quality control of MSC preparations. Cellular differentiation is reflected by specific epigenetic patterns. DNA methylation (DNAm) is the best characterized epigenetic modification, where cytosine guanine dinucleotides (CpGs) are covalently methylated at the cytosine residue (Jaenisch and Bird, 2003). DNAm has several advantages as a biomarker for classification of cell preparations: (1) it is rather stable; (2) it facilitates quantitative analysis at single-nucleotide resolution, and (3) it is directly coupled to cellular differentiation (Karnik and Meissner, 2013). We have recently described that DNAm levels at two CpGs can reliably discern between pluripotent and non-pluripotent cells (Lenz et al., 2015). In this study, we followed the hypothesis that the DNAm profile of MSCs might also reflect specific modifications that are indicative for the cell type and/or the tissue of origin. Small epigenetic signatures based on site-specific analysis of DNAm in a few CpG sites might therefore be particularly appealing for the classification of MSCs.
    Results
    Discussion Despite extensive efforts, it remains a challenge to distinguish between fibroblasts and MSCs. This definition is usually based on the in vitro differentiation potential of MSCs, although these surrogate assays hardly facilitate quantitative comparison, particularly not between different laboratories (Bortolotti et al., 2015; Dominici et al., 2006; Hematti, 2012). In our comparative study, we had to rely on the classification provided by the authors who deposited the DNAm profiles. Hence, they are not based on common standards in cell culture and quality control. At least for the cell preparations that we analyzed by pyrosequencing, we consistently observed higher differentiation potential in MSCs compared with fibroblasts (Koch et al., 2011), and these were all correctly classified by the Epi-MSC-Score. On the other hand, our clonal analysis indicated that this signature is not directly associated with the subset in MSCs that reveals higher in vitro differentiation potential. The epigenome reflects the tissue of origin even after long-term culture (Reinisch et al., 2015; Schellenberg et al., 2012). MSCs can be isolated from a multitude of different tissues (Crisan et al., 2008), but the vast majority of studies utilize MSCs from BM and AT. In fact, cell preparations derived from other tissues are often rather referred to as fibroblasts, and therefore classification of the Epi-MSC-Score may partly be also attributed to the different tissue sources. Either way, classifications with the Epi-MSC-Score are generally in line with those provided by the corresponding publications. Furthermore, the Epi-Tissue-Score can very reliably distinguish between MSCs from BM and AT. The remarkable difference in the epigenetic makeup of MSCs from different tissues, which are cell intrinsic and not due to cellular heterogeneity, may reflect the stark tissue-specific differences in gene-expression profiles (Wagner et al., 2005), proteome (Wagner et al., 2006), and functional readouts (Reinisch et al., 2015). All the more, such analysis is relevant for quality control.