br Materials and methods br Results br Discussion The
Materials and methods
Discussion The robust expression and purification of stable and biologically active eukaryotic proteins represent a bottleneck of protein transduction technology (Pan et al., 2010; Patsch & Edenhofer, 2007; Yang et al., 2011). In this study, we demonstrate recombinant expression and purification of a cell-permeant version of biologically active Nanog protein. Although several studies have reported purification of recombinant Nanog from different species including mouse (Jauch et al., 2008; Loh et al., 2006), human (Ha et al., 2009; Yang et al., 2009; Yang et al., 2011), Luxi cattle (Hu et al., 2012) and chicken (Yu et al., 2013) the biological functionality, however, remained unclear. Recombinant Nanog proteins have been reported to bind to a consensus DNA sequence using EMSA or EMSA-like technique (Ha et al., 2009; Jauch et al., 2008; Loh et al., 2006; Yang et al., 2009; Yang et al., 2011). One study employing CPP-fused Nanog protein further reports that recombinant Nanog could translocate to the nucleus (Yang et al., 2009); however, no further data was shown to demonstrate the functionality of the protein. Our study shows that our purified recombinant mouse Nanog protein from E. coli is stable at cell culture conditions, translocates to both cytoplasm and nucleus and is biologically active. Proof of functionality is based on i) the DNA-binding ability to a well-described consensus sequence, ii) enhancement of proliferation and iii) supporting self-renewal of mouse ESCs in the absence of LIF but in the presence of LIF inhibitor to abrogate residual LIF signaling. DNA binding ability insufficiently determines the functionality of pluripotency-associated transcription factors. Yang et al. reported that although recombinant Oct4 and Sox2 proteins bind to the consensus DNA sequence, the proteins are not fully biologically active as they fail to regulate most of the target genes investigated (Yang et al., 2011). Further, we did not observe any mixed or undifferentiated colonies on the basis of morphological analysis in the absence of LIF and Nanog-TAT, but we did observe mixed colonies upon treatment with Nanog-TAT and in the absence of LIF which were AP positive. Surprisingly, we did not observe any undifferentiated colonies in the latter condition although the translocation of Nanog into the Bleomycin Sulfate Supplier was high. The reason for this could be that the amount of Nanog protein was not ample enough to generate undifferentiated colonies but was sufficient to enhance proliferation and self-renewal of subpopulations in ESCs. These mixed colonies expressed pluripotency-associated markers and remained pluripotent in the absence of LIF when compared to cells cultured in the absence of LIF and Nanog-TAT. The fact that for technical reasons we had to culture Nanog-TAT-treated cells prior to teratoma as well as blastocyst injection in normal ESC medium raises the possibility of a selective outgrowth of a rare subpopulation of cells that may be responsible for formation of teratoma and chimera. Although we cannot rule out, we consider this a rather unlikely scenario since before that Nanog-TAT-treated cells were cultured for up to 11 passages in the absence of LIF and in the presence of LIF inhibitor. As demonstrated, control cells expanded in the absence of LIF and Nanog and in the presence of LIF inhibitor could not maintain their self-renewal and pluripotency characteristics, ceased to proliferate and differentiated within 2-4 passages only. These results suggest that continuous application of Nanog-TAT kept the pluripotency network active and maintained self-renewal as well as pluripotency of these cells in the absence of LIF. In addition, our data indicates that Nanog suppresses endoderm differentiation indicating that Nanog exerts its intracellular function in a Stat3-independent manner. We found endodermal genes such as GATA6 and transthyretin downregulated upon Nanog gain-of-function by protein transduction. Thus, Nanog protein transduction data presented here is in line with other studies reporting that Nanog suppresses differentiation of ESCs by inhibiting endodermal specification (Chambers et al., 2003; Darr et al., 2006; Hamazaki et al., 2004; Mitsui et al., 2003). Our finding that Nanog-TAT not only increases proliferation but also clonogenicity of ESCs might build a basis for enhanced suspension culture as a prerequisite for biomedical applications (Chen et al., 2012; Singh et al., 2010). In conclusion, our results readily recapitulate previously published reports employing conventional genetic Nanog overexpression (Chambers et al., 2003; Mitsui et al., 2003) to enhance proliferation and self-renewal and maintenance of pluripotency of ESCs. This confirms that our recombinant Nanog protein from E. coli is biologically active and provides an effective model to study the molecular control of pluripotency by modulating stem cell properties in target cells by non-genetic means.