Two reprogramming factors Oct and Sox are core

Two reprogramming factors, Oct4 and Sox2, are core regulatory factors that function cooperatively with Nanog in both human and mouse ESCs (Boyer et al., 2005; Loh et al., 2006). Oct4 has been studied at length and is recognized as being essential for the early development and maintenance of pluripotency. Oct4-deficient embryos fail to form an inner cell mass (Nichols et al., 1998). Reduced Oct4 expression in ESCs causes them to differentiate into the trophectoderm, while doubly high expression induces differentiation into the primitive endoderm and mesoderm (Niwa et al., 2000). Sox2, which is a high-mobility group DNA-binding domain transcription factor, is also essential for early embryogenesis in mice (Avilion et al., 2003). The twenty genes that comprise the mouse Sox-family are classified into ten subgroups, whereby Sox2 is categorized into group B1 (Schepers et al., 2002). Sox2 could be substituted for using Sox1 or Sox3 to generate iPSCs (Nakagawa et al., 2008), even if low expression of these two genes occurs in ESCs. Sox2 is expressed in the developing central nervous system and maintains the properties of neural precursor Etonogestrel (Pevny and Nicolis, 2010). Oct4 and Sox2 cooperatively bind to and activate the transcription of genes that bear Octamer and Sox elements, such as Oct4, Sox2, Nanog, Fgf4, Utf1 and Lefty1 (Okumura-Nakanishi et al., 2005; Tomioka et al., 2002; Kuroda et al., 2005; Johnson et al., 1998; Nishimoto et al., 1999; Nakatake et al., 2006). Similar to Oct4, Sox2 depletion and overexpression promotes differentiation. A two-fold or less increase in Sox2 reduces the transcription of Oct4/Sox2 target genes in ESCs. This results in differentiation into various lineages, including the ectoderm and mesoderm (Boer et al., 2007; Kopp et al., 2008; Adachi et al., 2010), while the knockdown of Sox2 induces differentiation into endoderm and trophectoderm lineages (Adachi et al., 2010; Chew et al., 2005; Fong et al., 2008). Therefore, precisely regulating the expression of the transcription factors is crucial to maintain the pluripotent state of ESCs. However, the effects of quantitative changes in reprogramming factors when inducing pluripotent stem cells remain poorly understood. Here, we examined the suppressive effects of excessive Sox2 on direct reprogramming. Decreased Sox2 virus led to a marked increase in the number of Oct4-GFP-positive cells. However, resulting cell fates were different depending on whether the transduction occurred with or without c-Myc (OSKM and OSK). For OKM and reduced Sox2, the majority of the GFP-positive colonies was trapped in a partially reprogrammed state, while for OK and reduced Sox2, most of the cells turned into fully reprogrammed iPSCs. A significant increase in the number of differentiation markers, which depended on the level of Sox2, was indicative that excessive Sox2 expression promotes differentiation into the mesoderm or ectoderm in parallel with reprogramming. Thus, our data is supportive that the appropriate expression of Sox2 is critical for efficiently generating iPSCs.
Results
Discussion A simple method for decreasing Sox2 virus improved the efficiency of Oct4-GFP-positive cells to emerge for OKM+LS and OK+LS. This is indicative of the attenuating effects on the reprogramming efficiency of overexpressed Sox2 for conventional OKSM and OKS. The increased reprogramming efficiency for LS resulted from the decreased cell differentiation of MEFs into ectoderm and mesoderm lineages as observed by using qPCR analyses. The increased number of GFP-positive cells for OKM+LS was caused by the increase in partially reprogrammed iPSCs. Improved reprogramming efficiency of somatic cells into fully reprogrammed iPSCs was significant for OK+LS. These data are indicative that 1) the reprogramming efficiency of MEFs into iPSCs was significantly increased by low level Sox2 expression in a dose-dependent manner: 2) improved reprogramming efficiency for reprogramming into iPSCs by using OK+LS was associated with impeding cell differentiation of MEFs into the ectoderm and mesoderm lineages: and 3) the generation efficiency of partial iPSCs decreased through reprogramming with OK+LS.