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  • The reprogramming of somatic cells was

    2018-11-02

    The reprogramming of somatic catalase inhibitor was originally demonstrated by producing cloned frogs using somatic cell nuclear transfer (SCNT) into Xenopus oocytes (Gurdon et al., 1958). Reprogramming of mammalian somatic cells has also been achieved using SCNT into oocytes, including those of mice and humans (Noggle et al., 2011; Wakayama et al., 1998). The procedure of SCNT into oocytes is similar to iPSC generation with respect to the time course of extinction of the parental gene-expression profile and activation of pluripotency (Egli et al., 2011). Both somatic cell reprogramming processes involve dynamic rearrangement of the epigenetic profile (Apostolou and Hochedlinger, 2013; Hussein et al., 2014). These findings suggest that the constituents of oocytes include a reprogramming-promoting factor. The linker histone H1 family binds to linker DNA and generates higher-order chromatin structures to control gene expression. Most members of the linker histone family consist of somatic linker histones that condense chromatin; therefore, these structures generally repress global gene-transcription activity (Hebbar and Archer, 2008; Steinbach et al., 1997). Mammalian oocytes contain the maternal-specific linker histone H1foo, a homolog of the Xenopus linker histone B4. H1foo is specifically expressed during the germinal vesicle stage and until the late two-cell or early four-cell stage, coincident with the early wave of zygotic genome reactivation (Gao et al., 2004; Tanaka et al., 2003, 2005). In Xenopus SCNT, somatic linker histones in transplanted nuclei are rapidly exchanged for linker histone B4, and the transplanted nuclei begin to swell and initiate decondensation (Byrne et al., 2003; Jullien et al., 2010); furthermore, in mouse SCNT the same phenomenon is observed with H1foo (Becker et al., 2005). Unlike other somatic linker histones, B4 and H1foo do not restrict the accessibility of the linker DNA, but decondense the chromatin and permit transcriptional activation (Hayakawa et al., 2012; Saeki et al., 2005). Based on these studies, we hypothesized that H1foo has a beneficial effect on iPSC generation. Here, we show that H1foo enhanced the generation of mouse iPSCs when co-expressed with Oct4, Sox2, and Klf4. Furthermore, H1foo promoted several in vitro differentiation characteristics with low heterogeneity in iPSCs that were similar to those of ESCs. Specifically, H1foo enhanced germline-competent chimeric mouse generation. These findings indicate that H1foo contributes to the generation of higher-quality iPSCs.
    Results
    Discussion H1foo promoted the generation of Nanog-GFP-positive colonies when it was co-expressed with OSK. Notably, the proportion of GFP-positive colonies was improved to 90%. OSKH-iPSCs demonstrated differentiation potency more similar to that of ESCs than did OSK-iPSCs, especially with respect to in vitro EB formation, chimerism, and germline transmission in vivo. We also examined the effect of H1foo with Oct4, Sox2, Klf4, and c-Myc, but the number of alkaline phosphatase-positive colonies and Nanog-GFP colonies, and the proportion of GFP-positive colonies were not significantly different to those of OSKM-iPSCs. c-Myc promotes iPSC generation but is not essential for reprogramming. On the other hand, c-Myc lowers the proportion of Nanog-GFP-positive colonies and increases the tumorigenicity of cells (Nakagawa et al., 2008). Therefore, it is preferable to omit c-Myc in iPSC generation, although it is still commonly used to promote reprogramming efficiency. We showed here that H1foo could be substituted for c-Myc in terms of reprogramming efficiency and showed superiority with respect to qualifying iPSCs. Recent studies have demonstrated that oocyte constituents play a key role in somatic cell reprogramming in SCNT. Co-expression of maternal-specific factors in oocytes, such as Glis1 (Maekawa et al., 2011) and TH2A/TH2B (Shinagawa et al., 2014), enhances the reprogramming efficiency of iPSC generation. Investigation of maternal-specific factors in oocytes has great potential for innovating somatic cell reprogramming and deciphering the reprogramming mechanisms (Gurdon and Wilmut, 2011). H1foo is specifically expressed during the germinal vesicle stage and is essential for oocyte maturation (Furuya et al., 2007; Gao et al., 2004). Interestingly, exogenous expression of H1foo in ESCs leads to the prevention of differentiation in vitro due to continuous pluripotency gene activation (Hayakawa et al., 2012). In our study, H1foo was properly silenced in generated iPSCs, which induced successful reprogramming but did not hinder the differentiation potency of OSKH-iPSCs.