Mesenchymal stem cells (MSC) are self-renewing multipotent cells which hold great potential in reconstructive medicine and tissue engineering. They have the ability to differentiate into cells of the mesoderm lineage and have been shown to be beneficial for the treatment of a variety of diseases. MSC can be derived from multiple adult tissues but have only limited expansion capacity in cell culture. Highly proliferative ESC-derived MSC can be an alternative source for MSC but currently no standardized protocol exists which meets clinical standards. We further developed and improved a protocol (Raynaud et al., 2013) to differentiate human embryonic stem cells (ESC) into highly-proliferative MSC. ESC-derived MSCs were tested for their characteristic surface markers by flow cytometry and the differentiation capabilities typical for MSCs (bone, fat) were verified. To characterize the differentiation process in-depth we performed comprehensive large-scale proteomic and phosphoproteomic profiling experiments using quantitative high resolution mass spectrometric analysis based on reductive dimethylation (Boersema et al., 2009). Experiments were designed as triplex comprising an internal standard, labeled with light isotopes, and time points, labeled either with medium or heavy isotopes. Differentiation was followed over a time course of 30 days including sampling days 0, 1, 2, 5, 15, and 30. Samples for proteomic analysis were fractionated by in-solution isoelectric focusing prior to mass spectrometry. Phosphoproteomic profiling was performed according to the TiSH protocol (Engholm-Keller et al., 2012) including phosphopeptide enrichment by titanium dioxide combined with sequential immobilized metal affinity chromatography. ESC-derived MSC were compared to adult tissue-derived MSC (bone marrow MSC) as well as to their origin (ESC). In total, 8615 proteins were identified with 5800 proteins on average quantified per sample. A total of 4064 proteins were quantified in all samples at all stages and were subjected to stringent statistical analysis. For phosphoproteomics, we identified and quantified more than 8000 phosphosites on 4000 proteins with around 4600 phosphosites on 1800 proteins per sample. A large overlap (75%) was observed for quantified proteins between proteomic and phosphoproteomic workflows. To further enhance the analytical depth, data was integrated with transctriptome profiling derived from next-generation RNA sequencing which enabled us to quantify the expression of over 14000 genes. To identify important regulators of differentiation, we performed clustering according to expression patterns. The established differentiation protocol is highly reproducible and robust, may be adapted for clinical use and thus be of great value for the stem cell research community. The comprehensive analysis of the differentiation process will improve understanding of MSC biology and therefore directly benefit MSC-based therapies. References Boersema, P.J., et al. (2009). Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc. 4, 484-494. Engholm-Keller, K., et al. (2012). TiSH--a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC. J. Proteomics 75, 5749-5761. Raynaud, C.M., et al. (2013). Human Embryonic Stem Cell Derived Mesenchymal Progenitors Express Cardiac Markers but Do Not Form Contractile Cardiomyocytes. PLoS ONE 8.


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