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  • br Experimental Procedures br Author Contributions br Acknow

    2018-10-20


    Experimental Procedures
    Author Contributions
    Acknowledgments We are grateful for the support from ES/iPSC core facility, Lund University. This work was funded by grants from: The Swedish Research Council, Swedish Cancer Society, Swedish Children\'s Cancer Society, AFA Insurance (Sweden), Lund University Medical Faculty, The HematoLinné Program Grant, and Stem Therapy Program Grant.
    Introduction The reprogramming of somatic cells into pluripotent cells using the classical set of transcription factors, OCT4, KLF4, SOX2, and C-MYC (OKSM) and conventional culture conditions (leukemia inhibitory factor, serum) usually takes several weeks and yields induced pluripotent stem cells (iPSCs) at extremely low frequencies (0.1%–3%) (Takahashi and Yamanaka, 2006). This observation suggests that reprogramming factors need to overcome undefined barriers that have been established by somatic cells to preserve cell identity and resist cell fate change. Identifying roadblocks to iPSC generation thus provides a valuable platform to dissect general principles of cell identity and cell fate change (Apostolou and Hochedlinger, 2013). Previously identified barriers to reprogramming include regulators of kinesin inhibitor progression and senescence (e.g., kinesin inhibitor P53, INK4A/ARF) (Krizhanovsky and Lowe, 2009), histone and DNA modifications (e.g., DNMT1, KDM2B, MBD3) (Mikkelsen et al., 2008; Rais et al., 2013; Wang et al., 2011), as well as signaling pathways and epigenetic processes that can be targeted by small compounds (e.g., ascorbic acid, GSK3 inhibitor, DOT1L inhibitor) (Bar-Nur et al., 2014; Esteban et al., 2010; Onder et al., 2012; Silva et al., 2008). However, suppression of some of these barriers may enhance iPSC formation only under specific culture conditions (e.g., MBD3) (dos Santos et al., 2014; Rais et al., 2013), potentially limiting its usefulness in different cellular contexts. Moreover, manipulation of certain barriers causes permanent aberrations of the epigenome (e.g., DNMT1) (Jackson-Grusby et al., 2001), complicating its applications in a therapeutic setting. More recently, unbiased small hairpin RNA (shRNA) screens have been performed during iPSC formation, leading to the identification of novel roadblocks to reprogramming (Qin et al., 2014; Samavarchi-Tehrani et al., 2010; Yang et al., 2014). Surprisingly, individual suppression of hits that emerged from these screens showed rather modest effects (2- to 4-fold enhancement) compared with the simultaneous suppression of multiple hits (5- to 10-fold enhancement). Furthermore, there was little overlap among independent screening efforts, suggesting that reprogramming may be restrained by additional, yet to be identified barriers. Indeed, our lab recently discovered the histone chaperone CAF-1 as a novel barrier to iPSC generation using a chromatin-focused shRNA screen (Cheloufi et al., 2015).
    Results
    Discussion Here, we identified SUMO2 as a potent roadblock to iPSC generation by combining a well-defined transgenic reprogramming system with a genome-wide shRNA screening approach. In contrast to previous shRNA or siRNA screens conducted during iPSC formation, we employed a serial shRNA enrichment strategy, which may reduce the number of false-positive hits and allow for selection of shRNAs with stronger phenotypes. Indeed, suppression of a top candidate, SUMO2, markedly enhanced and accelerated iPSC formation compared with individual hits that emerged from previous large-scale screens or candidates that were selected based on gene-expression differences between somatic and pluripotent cells. In agreement with the notion that the expression of barrier genes does not necessarily have to be different between MEFs and iPSCs, we found that Sumo2 mRNA levels do not dramatically change during reprogramming. To our knowledge, iPSC formation after 38 hr of OKSM expression represents the shortest time period that has been reported to obtain stably reprogrammed cells from fibroblasts. In addition to SUMO2, our screen uncovered a number of other candidate barriers to iPSC formation, which provide a useful resource for future mechanistic studies of the reprogramming process.