• 2018-07
  • 2018-10
  • 2018-11
  • Similar to our observation that Nanog


    Similar to our observation that Nanog−/− iPSCs could give rise to chimeric animals, Nanog null ESCs have been shown to contribute to the three germ layers (Chambers et al., 2007). However, in the case of ESC chimeras, Nanog−/− GFP+ nicotinic receptor agonist were not detected in the germline after E12.5. Because the CAGs::GFP construct that marks Nanog−/− cells was introduced by random integration into ESCs, we reasoned that perhaps the GFP transgene might, by chance, not be expressed in cells of the germline that could hamper the ability to detect germline contribution of these cells. We therefore performed crosses using chimeras produced from Nanog null iPSCs and found that they could produce offspring carrying the GFP+ transgene originating from the injected iPSCs. Thus, these results indicate, in contrast to previous results, that Nanog−/− iPSCs can give rise to functional, mature germ cells. Here, we provide global transcriptional analysis of both our Nanog iPSCs as well as Nanog−/− mESCs (Figure 3). Although these cells have been shown to robustly colonize chimeric embryos, we show that there are still many differences in global expression profiles between Nanog-deficient and wild-type (WT) pluripotent stem cells (Figure 3) (Chambers et al., 2007). A number of genes are differentially expressed between pluripotent cells of these two genotypes, and thus, it would be interesting to further investigate both the mechanism of activation of the core pluripotency network as well as the transcriptional circuit involved in pluripotency maintenance in this context. Although we have done this work exclusively in nicotinic receptor agonist murine cells, interactions between members of the core pluripotency network are highly conserved between mouse and human. While the relative inefficiency of iPSC reprogramming in human cells may make rare reprogramming events difficult to detect, it is of great interest to perform similar experiments in human cells. These studies may provide insights between the so-called naive and primed ESC states in the NANOG-deficient context (Gafni et al., 2013). Based on our results, we conclude that although reprogramming is indeed less efficient in the absence of Nanog, Nanog is not required for the establishment of a pluripotent state, as has been previously suggested. Instead, we conclude that even under standard conditions, there are Nanog-independent routes to pluripotency.
    Experimental Procedures
    Acknowledgments We thank I. Chambers for generously sharing Nanog ESC lines, L. Rubin for providing piPSC lines, and members of the Eggan Laboratory for helpful discussion. This work was supported by R01 and P01 grants from NIGMS to K.E. B.N.D.-D. is a Milton Safenowitz Post Doctoral Fellow of the ALS association.
    Introduction Intestinal crypts house self-renewing stem cells and transit-amplifying progenitors that depend on Wnt signaling. Expression of endogenous pathway antagonists such as Dickkopf-1 reduces Wnt signaling, arrests stem and progenitor cell proliferation, and impairs secretory cell differentiation (Kuhnert et al., 2004; Pinto et al., 2003). Additionally, although it is unclear exactly when Wnt signaling begins in the developing intestine (Kim et al., 2007), mice lacking Tcf4, a transcriptional effector of the Wnt pathway, show marked epithelial defects (Korinek et al., 1998; van Es et al., 2012). Conversely, constitutive Wnt activity drives excessive cell replication and tumors, including human colorectal cancer (Korinek et al., 1997; Morin et al., 1997). Intestinal stem cells (ISCs) located at the base of mouse small-intestine crypts express the Wnt-responsive gene Lgr5 (Barker et al., 2007), which encodes a coreceptor for Wnt-agonist R-spondins (Carmon et al., 2011; de Lau et al., 2011; Glinka et al., 2011) and are the origin for epithelial tumors spurred by constitutive Wnt activity (Barker et al., 2009). Although Wnt signaling is thus an imperative aspect of intestinal homeostasis, the bona fide physiologic Wnt source within the ISC niche in vivo is unknown.