Genetic manipulation by overexpression of exogenous factors

Genetic manipulation by overexpression of exogenous factors such as Nanog, Klf2, and Prdm14 enables conversion of mouse EpiSCs to ESC-like cells (rESCs) (Gillich et al., 2012; Silva et al., 2009). Furthermore, transition of mouse EpiSCs to rESCs rarely occurs even after stimulation with LIF-STAT3 signaling (Bao et al., 2009). However, the cellular mechanisms that limit reprogramming efficiency remain unclear. Pluripotency in nonrodent PSCs is more like that in rodent primed-PSCs (Nichols and Smith, 2009), so that chimeric animals derived from PSCs are reported only in work with rodents (Nichols and Smith, 2009). Nonrodent PSCs thus are expected not to contribute to chimeras (one reason why knockout or transgenic studies have not been done using nonrodent mammals). We investigated the conditions for efficient conversion of primed PSCs to naive-like PSCs as part of generation of nonrodent naive PSCs. Forced expression of E-cadherin in mouse EpiSCs under primed-PSC culture conditions promotes ICM development after kit inhibitor injection and results in generation of chimeric mice without reprogramming to the naive state (Ohtsuka et al., 2012). E-cadherin is a functional factor that can cooperate with reprogramming factors to promote generation of induced pluripotent stem cells (iPSCs) from somatic cells under naive-PSC culture conditions (Chen et al., 2010). These findings raised the possibility that E-cadherin upregulation under appropriate culture conditions might enhance reprogramming of primed PSCs. We therefore investigated the effects of E-cadherin upregulation in mouse EpiSCs under various culture conditions. We found that combining E-cadherin upregulation with LIF treatment dramatically improves rates of conversion of mouse EpiSCs to naive-like PSCs. E-CADHERIN specifically binds β-CATENIN and regulates its nuclear translocation (Conacci-Sorrell et al., 2003; Sasaki et al., 2000; Stockinger et al., 2001). We found that nuclear translocation of β-CATENIN is negatively regulated by E-cadherin overexpression in mouse EpiSCs. Instead of upregulating E-cadherin expression, we used small-molecule inhibitors of Wnt signaling to study the role of such signaling in conversion of primed PSCs to naive-like PSCs. Interestingly, as did overexpression of E-cadherin, blocking nuclear localization of β-CATENIN significantly enhanced the efficiency of mouse EpiSCs conversion to naive-like PSCs in response to LIF. Our investigations thus provide insight into the significance of E-cadherin and β-CATENIN as well as into approaches for increasing efficiency of conversion of primed PSCs to naive-like PSCs.
Results
Discussion Conversion of mouse EpiSCs to ESC-like cells in response to LIF-STAT3 signaling rarely occurs under ordinary cell culture conditions (Bao et al., 2009). Indeed, a recent study has shown that upregulation of E-cadherin expression does not induce conversion from primed to naive state under culture in media containing bFGF and activin A (Ohtsuka et al., 2012). However, we demonstrated that overexpression of E-cadherin in combination with the cytokine LIF yields highly efficient derivation of cells that express naive-PSC markers and that can contribute to chimeras. We then extended these data by confirming E-cadherin-dependent induction of naive PSC markers and repression of primed PSC markers in the presence of LIF. These results suggest that overexpression of E-cadherin induces conversion of mouse EpiSCs toward the ESC-like naive state. Ohtsuka et al. (2012) showed that 2 days of E-cadherin overexpression enabled chimera formation by EpiSCs without any evident conversion to ESC-like cell status. However, the EpiSC lines that we used did not acquire the ability to form chimeras after short-term (2 days) overexpression of E-cadherin. To explain this discrepancy, two possibilities can be considered. One is differences in culture conditions. Ohtsuka et al. maintained EpiSCs with activin and FGF2 under feeder-free conditions, whereas we maintained EpiSCs in a different medium supplemented with bFGF and on feeder cells. Different culture conditions might set EpiSCs at slightly different stages within the primed pluripotent state and might result in different outcomes with respect to chimera formation after E-cadherin overexpression. The other is use of different vector systems to induce E-cadherin overexpression. We used the all-in-one tet-on lentiviral vector system (Yamaguchi et al., 2012), whereas Ohtsuka et al. used the piggyBack transposon system. Of importance may be that they described E-cadherin expression levels in EpiSC-line cells into which the tet-on system had introduced E-cadherin as “slightly higher” than those in ESCs, whereas cells in our line showed much higher expression than did ESCs after Dox treatment in a “standard” mouse EpiSC line. This may suggest that E-cadherin expression levels must be similar to those in ESCs to permit integration into the ICM.