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    • br Experimental Procedures Briefly hiPSCs were generated fro

    2018-10-22


    Experimental Procedures Briefly, hiPSCs were generated from IMR90, CCD-1097SK, BJ1, and NPC cells derived from 11-week-old fetal brain using retroviral expression of OCT4, SOX2, KLF4, and c-MYC or OCT4, NANOG, KLF4, and LIN-28. This study of hESCs and hiPSCs was approved by the UCLA Embryonic Stem Cell Research Oversight Committee. We used the HumanMethylation27 DNA Analysis BeadChip from Illumina to interrogate 26,837 CpG sites over 14,152 genes. Full experimental procedures and data analysis are available in the Supplemental Experimental Procedures.
    Acknowledgments
    Introduction A safe and efficient method for generating patient-specific pluripotent cells from easily accessible tissues via expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC (collectively referred to as the four factors or 4F) (Takahashi et al., 2007), or some other combination of defined factors (Yu et al., 2007), would have important applications in cell-replacement therapy and disease modeling. A key concern with reprogramming somatic cells into induced pluripotent stem cells (iPSCs) is the increased load of genomic aberrations. We and others have shown that human iPSCs harbor genomic aberrations and point coding mutations that are absent in the parental cells (Gore et al., 2011; Hussein et al., 2011; Ji et al., 2012; Martins-Taylor et al., 2011; Pasi et al., 2011; Taapken et al., 2011). These mutations occur despite the exclusion of c-MYC as the reprogramming factor and the use of nonintegrating methods for transgene delivery (Gore et al., 2011; Young et al., 2012). The causes of these mutations remain largely unknown. Evidence indicates that preferential reprogramming of mutated cells in the starting somatic cell population (Gore et al., 2011; Ji et al., 2012) and CB-5083 to growth in culture (Hussein et al., 2011; Laurent et al., 2011) may contribute to the somatic mutations found in iPSCs. However, a significant proportion of the mutations in iPSCs cannot be exclusively attributed to preexisting rare mutations in the parental cells or to acquisition of mutations during the passaging of iPSCs (Ji et al., 2012), suggesting that the mutation rate might be elevated during reprogramming. This finding is consistent with the oncogenic potential of at least some of the reprogramming factors, and with the fact that reprogramming-factor-transduced mouse cells develop genome instability (González et al., 2013; Marión et al., 2009). It is known that the P53 pathway is activated in cells transduced with reprogramming factors and results in apoptosis, cell-cycle arrest, and senescence (Banito et al., 2009; Hong et al., 2009; Kawamura et al., 2009; Krizhanovsky and Lowe, 2009; Marión et al., 2009; Utikal et al., 2009). However, in a previous study, we found that the TP53 gene was not mutated in any of the iPSCs that harbored mutations (Ji et al., 2012). Thus, an important challenge now is to identify the aspects of reprogramming that cause mutations to optimize reprogramming, in order to minimize genome instability during the derivation of iPSCs. The acceleration of growth rate following the induction of reprogramming factors (Ruiz et al., 2011) is expected to impose greater metabolic demands for energy and precursors for biosynthesis on the cells. During reprogramming, mitochondria get progressively smaller and less active (Prigione et al., 2010; Suhr et al., 2010), and metabolism shifts from oxidative respiration to oxidative glycolysis (Varum et al., 2011). Such a metabolic shift can lead to a buildup of electrons in the electron transport chain, increasing their leakage into the cytoplasm as reactive oxygen species (ROS) that will cause oxidative stress if the radical-scavenging systems in the cell are not sufficiently upregulated. High ROS levels can result in the modification of individual nucleotide bases (such as the mutagenic 7,8 dihydro-8-oxoguanine), single- and double-strand breaks (Vafa et al., 2002), and telomere attrition (von Zglinicki, 2002). Indeed, reprogramming-factor-transduced fibroblasts have elevated levels of oxidative DNA damage and ROS (Banito et al., 2009; Esteban et al., 2010). One way to prevent this damage is to supplement the reprogramming cells with ROS scavengers such as N-acetyl-cysteine (NAC) or vitamin C (Vc). Here, we show that supplementation of NAC and Vc during reprogramming and early passaging of iPSCs generated from human fibroblasts results in a significant reduction in de novo copy number variations (CNVs).