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    • At present the strategies for direct generation of

    2018-10-24

    At present, the strategies for direct generation of iNSCs from somatic buy S63845 can be broadly classified into two groups according to whether the procedure passes through intermediate or pluripotent state. Lujen et al. generated iNSCs from fibroblasts by introducing Brn2, Sox2, and FoxG1; but the iNPCs require constant expression of transgenes and generate less mature neurons (Lujan et al., 2012). Our lab and collaborators used pax6, Ngn2, Hes1, Id1, Ascl1, Brn2, c-myc and Klf4 to produce iNSCs from mouse Sertoli cells (Sheng et al., 2012b; Wu et al., 2015), but the same reprogramming strategy did not work well with human cells (data not shown). The above two methods did not involve passing through an intermediate states. Other studies employed part or all of the four Yamanaka reprogramming factors (Lu et al., 2013; Thier et al., 2012; Wang et al., 2013; Han et al., 2012; Kim et al., 2011; Lee et al., 2015) to derive iNSCs which can stably self-renew and differentiate to functional neuronal subtypes and astroglia. Our induction strategy includes two major steps and employs a non-integrative approach: the first two days after infection is for inducing an intermediate state, followed by several days for lineage-specific induction (Fig. 1A). The conversion and maintenance of iNSC phenotype are also dependent on the chemical defined medium. LIF is required for long-term self-renewal of primitive NSCs (Hitoshi et al., 2004), and CHIR99021, the GSK 3β inhibitor can direct human embryonic stem cells to a pre-neuroepithelial state (Denham et al., 2012; Wang et al., 2015). SB431542 as a TGFβ receptor inhibitor can promote neuronal specification in early neural development (Smith et al., 2008). The combination of LIF, GSK3β inhibitor, and TGFβ receptor inhibitor is critical for the generation of neural stem cells from intermediate states.
    Conclusion The following are the supplementary data related to this article.
    Introduction Human iPS cells provide an unprecedented opportunity for the science community to model and study different disorders in vitro (Inoue et al., 2014). These cells can be used for many in vitro applications such as disease modeling, drug screening, genome editing and even in vivo regenerative purposes (Tedesco et al., 2012; Wang et al., 2012; Xie et al., 2014; Zou et al., 2011; Soldner et al., 2009; Dimos et al., 2008; Ebert et al., 2009; Moretti et al., 2010; Raya et al., 2009; Maehr et al., 2009). Therefore, generation of different lineage progenitors and tissues from iPS cells has been considered the cornerstone of iPS technology (Mauritz et al., 2008; Zhang et al., 2009; Choi et al., 2009; Grigoriadis et al., 2010). Thus, generation of lineage-specific reporter ES/iPS cell lines enables prospective identification and isolation of various progenitors from differentiating cells and allows optimization of differentiation protocols. Knock-in reporter cells have been generated through site-specific gene targeting and homologous recombination (HR) mediated inclusion of the reporter cassette in the appropriate genomic locus (Leavitt & Hamlett, 2011; Zwaka & Thomson, 2003). This allowed for in-frame inclusion of the reporter cassette without any undesirable side effects associated with random integration based methods. However, the low efficiency of HR in human iPS cells makes it hard for gene modification in human iPS cells (Zwaka & Thomson, 2003; Xue et al., 2009; Davis et al., 2008; Ruby & Zheng, 2009). Nevertheless, nuclease-mediated introductions of site-specific DNA double-stranded breaks (DSBs) have significantly increased the efficiency of HR. These include site-specific nucleases such as zinc-finger (ZFN) and transcription activator-like effector nucleases (TALENs), which allowed for precise gene editing (Wang et al., 2012; Zou et al., 2011; Kim et al., 1996; Urnov et al., 2010; Carroll et al., 2006; Deng et al., 2012; Ma et al., 2013; Maetzel et al., 2014; Shin et al., 2014). The only limiting factor with these methods is their complex time-consuming design.