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    • br Experimental Procedures Detailed information on the exper

    2018-11-09


    Experimental Procedures Detailed information on the experimental procedures can be found in the Supplemental Information File.
    Author Contributions
    Introduction Although cell-fate decisions are fairly stable in vivo, somatic p38 inhibitor can be reprogrammed back into pluripotency in vitro by ectopic expression of defined transcription factors (Takahashi and Yamanaka, 2006). Successful reprogramming requires complete erasure of somatic cell memory and establishment of a pluripotent stem cell epigenetic landscape (Nashun et al., 2015). Fibroblasts and peripheral blood mononuclear cells (PBMCs) are commonly used for reprogramming (Santostefano et al., 2015). Induced pluripotent stem cells (iPSCs) are known to be epigenetically similar to human embryonic stem cells (hESCs) (Guenther et al., 2010; Maherali et al., 2007), although several reports have suggested retention of epigenetic memory related to the cell of origin (Bar-Nur et al., 2011; Kim et al., 2010, 2011; Ohi et al., 2011; Polo et al., 2010). This phenomenon can have functional consequences by influencing iPSC differentiation propensity and biasing it toward the cell type of origin at the expense of other lineages (Bar-Nur et al., 2011; Kim et al., 2010; Polo et al., 2010). However, conflicting studies have shown that variations in directed differentiation (Kajiwara et al., 2012) and transcriptional heterogeneity (Rouhani et al., 2014) between iPSC lines were ascribed to the genetic background of the donor.
    Results
    Discussion Four major observations emerge from this study. First, we show that source-cell-specific differences are not retained to a significant extent in isogenic iPSC lines. This is in line with a recent report (Rouhani et al., 2014), but contrasts with earlier studies which observed disruptive retention of somatic cell memory in iPSC lines (Bar-Nur et al., 2011; Kim et al., 2010; Polo et al., 2010). Long-term culture has shown to be advantageous in erasing cell-type-specific memory (Kim et al., 2010; Polo et al., 2010), diminishing transcriptional differences between human iPSC and ESC lines (Chin et al., 2010), and eliminating genetic mosaicism (Hussein et al., 2011). In addition, we used the all-in-one type footprint free SeVdp-iPS system for generation of uniform iPSC lines (Nishimura et al., 2011). We performed molecular analyses at later passages and obtained highly similar molecular signatures for genetically matched iPSC lines derived from two different tissues, even though fibroblasts and PBMCs include multiple cell populations with distinct epigenetic and transcriptional landscapes (Sorrell and Caplan, 2004; Zhang and Huang, 2012). Our data show that the majority of transcriptional and epigenetic signatures present in iPSCs are donor determined. This is well in line with recent studies that have suggested the influence of genetic background on transcription (Rouhani et al., 2014; Shao et al., 2013) and differentiation of iPSC lines (Kajiwara et al., 2012; Mills et al., 2013). Also, embryonic stem cells derived from individual donors are shown to maintain line-specific signatures and have distinct differentiation potentials (Bock et al., 2011; Chen et al., 2009; Osafune et al., 2008). Our present data point out that donor-dependent signatures specifically affect gene expressions involved in early embryonic lineage specification, resulting in variability between iPSC lines. Third, we show that iPSC differentiation propensities are significantly biased by donor-dependent variability and not by cell type of origin. Earlier studies have focused on the relationship between genetic variability and molecular signatures of iPSC lines (Kajiwara et al., 2012; Rouhani et al., 2014; Shao et al., 2013). However, only limited information was available on the contribution of donor background to the functional differences of iPSC lines arising from the source-cell-specific differences. This was thoroughly addressed in the present study by combining global transcriptional and epigenetic analyses with spontaneous and targeted differentiation of isogenic iPSC lines. Although we selected iPSC lines from healthy donors showing the highest intra-individual variation in transcriptional and epigenetic analyses, we could not detect major differences in the differentiation potential of isogenic iPSC lines originating from fibroblasts and blood. However, we detected donor-dependent transcriptional differences in spontaneously differentiated EBs and significant variation in erythroid differentiation potential between iPSC lines derived from two healthy donors, regardless of the cell type of origin. Using GSEA we were able to associate the low erythroid-forming potential of the healthy donor to genes previously indicated in DBA. Moreover, the differential expression of these genes in different donors at the iPSC stage was, at least in part, maintained through differentiation to EBs, providing further evidence in support of the donor-related differences affecting the differentiation potential of iPSC lines.