br Materials and methods br Results br Discussion The

Materials and methods
Results
Discussion The current work showed that M3O-SKM allows for the establishment and maintenance of iPSCs in the absence of exogenous LIF. We also showed that miR-205-5p synergizes with M3O-SKM. More than 20 individuals or families of miRNAs play critical roles in self-renewal and pluripotency in ESCs and iPSCs by regulating numerous target genes (Moradi et al., 2014; Greve et al., 2013); however, their connections with LIF/Stat3 remains ill defined, and miR-205-5p is no exception. Like mouse miR-205-5p, human miR-205-5p, whose sequence is identical to its mouse counterpart, is more highly expressed in human ESCs and iPSCs than in fibroblasts (Wilson et al., 2009). In addition, Oct4, Sox2, and Nanog proteins bind to the genomic locus of miR-205 in ESCs, suggesting direct regulation by the pluripotency master proteins (Marson et al., 2008). However, miR-205-5p is not essential for pluripotency of early embryos, because mice lacking this miRNA can survive until around 10days after birth, when they die with severe skin defects (Wang et al., 2013). Li et al. reported that threefold overexpression of miR-205-5p in mouse ESCs leads to differentiation into extraembryonic endoderm in the presence of LIF (Li et al., 2013). Somewhat consistently, our result indicates that the positive effect of miR-205-5p in iPSC formation becomes obvious only under a specific condition—used at 1nM with M3O to potentiate Oct4 in the absence of LIF. We showed the capability of miR-205-5p to downregulate Src family kinases and Gsk3β in M3O-lenti-iPSCs-LIF(−) but knockout of the miRNA did not upregulate the target genes. More detailed study of these effects would require a genome-wide gene expression analysis in M3O-lenti-iPSCs-LIF(−) after up- and downregulation of miR-205-5p, which is beyond the scope of the current study. Perhaps a more fundamental message about the wider impact of the current study would be that amplification of Oct4 activity with the MyoD domain can eliminate the necessity of LIF. This could be due to higher expression of the genuine Oct4 target genes and/or expanded target genes. Our microarray analysis of mRNAs excluded the possibility that genes already known to substitute for LIF were upregulated by M3O-SKM, suggesting the existence of other genes that can compensate for the lack of LIF. Future studies on the differentially expressed mRNAs could potentially reveal a novel connection between Oct4 target genes and the LIF signaling pathway. <br> Acknowledgments We thank Toshio Kitamura for Plat-E purchase Regorafenib and pMXs-IP retroviral vectors, Wei-Shou Hu and Andrew Yongky for microarray analyses, and Peter Karian for mouse experiments. mRNA microarray analyses were done at the University of Minnesota Genomics Center. Histology was performed at the University of Minnesota Masonic Cancer Center Comparative Pathology Shared Resource with support from the NIH Grant P30 CA077598. M.F. and N.K were supported by Richard M. Schulze Family Foundation. N.K. was supported by the NIH (R01 GM098294 and R21 CA187232); Engdahl Family Foundation; Grain-in-Aid of Research, Artistry and Scholarship, University of Minnesota (22802); and the University of Minnesota Foundation (4160-9227-13).
Introduction ES cells are pluripotent cells derived from the inner cell mass (ICM) of the blastocyst at least an early post-implantation stage (Evans and Kaufman 1981) and are capable of dividing and self-renewing for extended periods (Loebel et al. 2003). These properties made ES cells can be used for treating Alzheimer\'s disease, Parkinson\'s disease, and other degenerative diseases (Thomson et al. 1998). However, application of ES cells to treatment has major hurdles, such as ethical issue and rejection problem after transplantation (Colman and Burley 2001). One solution to circumvent these problems is the generation of pluripotent cells by reprogramming somatic cells. In 2006, Yamanaka has generated ES cell-like cells, which are named iPS cells, from mouse fibroblast with four transcription factors (Oct3/4, Sox2, C-myc and Klf4) (Takahashi and Yamanaka 2006). Nonetheless, there are still considerations, such as cost, safety, and efficiency in generating iPS cells. Therefore, to resolve these issues, new methods are investigated, such as reducing the number of defined factors (Kim et al. 2008) and using non-viral inducers (Huangfu et al. 2008; Shi et al. 2008; Xu et al. 2008) or proteins (Cho et al. 2010; Zhou et al. 2009) or direct conversions (Han et al. 2014). In our previous study, iPS cells were generated from fibroblast by treating them with mES cells protein extract (Cho et al. 2010). Notably, only cell extracts from C57 mES cells were able to generate iPS cells from fibroblasts, but extracts from E14 mES cells were not (Cho et al. 2010). Through proteomic analysis of two different mES cell lines to find the key proteins associated with reprogramming, we noticed proteomic contrast between C57 and E14 mES cells. The expression of E-Ras in C57 mES cells was significantly higher than in E14 mES cells (Jin et al. 2011). Therefore, we hypothesized that E-Ras influences the reprogramming efficiency. Knowledge about molecular mechanisms of reprogramming will make reprogramming efficiency better. Recent studies have identified signaling pathways which play important roles in the reprogramming, including MAPK/ERK (Silva et al. 2008), p53-p21 (Li et al. 2009; Hong et al. 2009; Kawamura et al. 2009) and Wnt/β-catenin (Marson et al. 2008). However, the efficiency of iPSC generation is still low and the underlying mechanisms are largely unknown. E-Ras is expressed in undifferentiated mES cells, but not in adult mouse tissues and differentiated ES cells (Takahashi et al. 2003). Around 80–95% of E-Ras protein binds to GTP regardless of its membrane localization (Takahashi et al. 2003; Zhang et al. 1993). Therefore, E-Ras can activate such signaling pathways constitutively, not like other Ras proteins (Yu et al. 2014). E-Ras activates phosphatidylinositol-3-OH kinase (PI3K) and promotes cell growth, but the cell cycle was not affected by E-Ras–PI3K pathway (Takahashi et al. 2003; Takahashi et al. 2005). In other words, there is other pathway that E-Ras affects the cell proliferation. In the previous studies, a high proliferation rate is required for reprogramming efficiency and maintenance of stem cell identity (Ruiz et al. 2011; Xu et al. 2013). Even though E-Ras was in the top 24-list of Yamanaka factor candidates (Takahashi and Yamanaka 2006), it has not been well studied in the context of reprogramming.