br Acknowledgments We thank all the

Acknowledgments We thank all the members of Wagers laboratory and E. Gussoni and E. Dzierzak for helpful discussions. We also thank A. Wakabayashi, G. Buruzula and J. LaVecchio at Joslin/HSCI Flow Cytometry Core for excellent flow cytometry support, funded in part by NIHP30 DK036836. This work was supported in part by grants from the National Institutes of Health (NIH, OD004345 and HL088582 to AJW and DK67536 and DK103215 to RNK), from the National Health and Medical Research Council of Australia1042934, 1024364 and 1008515 to JEP), and from Leukaemia and Lymphoma Research (LLR) grant 10015 to KO. Content is solely the responsibility of the authors and does not necessarily reflect the official views of the NIH or other funding agencies. The mouse strain used for this research project, B6N.129S5-/Mmcd, identification number 032342-UCD, was obtained from the Mutant Mouse Regional Resource Center, a NCRR–NIH funded strain repository, and was donated to the MMRRC by Genentech, Inc.
Introduction Induced pluripotent stem zolmitriptan (iPSC) are an important research tool and have a potential to become a significant source of autologous cells differentiated from iPSC for therapeutic treatments. However, prior to therapeutic application appropriate characterization of human iPSC (hiPSC) is needed. To date, iPSC have been generated from numerous somatic cell types including dermal fibroblasts (Lowry et al., 2008; Takahashi et al., 2007; Yu et al., 2007), lymphocytes (Staerk et al., 2010; Loh et al., 2010), mesenchymal stem cells (Zou et al., 2011), endogenous kidney tubular renal epithelial cells (Montserrat et al., 2012), and CD34+ hematopoietic stem cells (Loh et al., 2009). It is believed that iPSC of different somatic origins may be predisposed toward re-differentiation to a particular cell lineage via “epigenetic memory” (Bar-Nur et al., 2011; Kim et al., 2010). For instance, it has been reported that hiPSC derived from hematopoietic stem cells (CD34+ cells) are particularly suitable for development of research models and treatments for hematopoietic diseases (Zou et al., 2011; Merling et al., 2013). Another recent study has shown that the hepatic lineage epigenetic memory contributed to the differentiation potential of mouse iPSC (Lee et al., 2012). On the mRNA level hiPSC have been found to be clearly distinguishable from hESC and their expression pattern becomes closer to that of hESC after extended culture (Chin et al., 2009); hiPSC have been shown to bear residual gene expression from the donor cell type (Marchetto et al., 2009; Ghosh et al., 2010). Recent analysis of 12 established hiPSC lines has revealed epigenetic and transcriptional variations among them and has shown that these variations can have a significant impact on a cell line\'s ability to differentiate to a particular cell type (Bock et al., 2011). The molecular characterization of hiPSC has been performed previously on different biological levels, including: gene expression profiling, epigenetic evaluation, the role of miRNAs in pluripotency, and genomic DNA alterations (Muller et al., 2012; Benevento and Munoz, 2012). However, quantitative proteomics has not yet been used to characterize hiPSC systematically (Munoz et al., 2011; Phanstiel et al., 2011; Kim et al., 2012; Yamana et al., 2013), and the molecular differences on the proteome level between hiPSC of different somatic origins have not been addressed. Sample preparation and MS-proteomic approaches reported previously on hiPSC vary significantly (Benevento and Munoz, 2012; Munoz et al., 2011; Phanstiel et al., 2011; Kim et al., 2012; Yamana et al., 2013), which complicates direct comparison of these studies. The focal point of this study was the analysis of proteomes of two hiPSC lines at the earlier and later cell culture passages derived in two different laboratories and of different somatic origins: CD34+ cells circulating in peripheral blood (iNC-01) and fibroblasts of healthy donors (SB5-MP1). Both hiPSC lines were generated using the same reprogramming technique: loxP-flanked excisable polycistronic (human Oct4, Klf4, Sox2, and c-Myc) STEMCCA lentiviral vector, which generates transgene-free hiPSC lines upon Cre-mediated vector excision. iNC-01 cell line was previously used to obtain functional neutrophils (Sweeney et al., 2014) and SB5-MP1 was successfully used in differentiation into motor neurons (Grunseich et al., 2014). In parallel, we performed a quantitative global proteome analysis of H9 hESC line at the earlier and later passages, as well as of the somatic cell types (fibroblasts and peripheral blood mononuclear cells (PBMC), the cell population containing CD34+ hematopoietic stem cells).