Supplementary Materials01. 1981). ES cells are pluripotent, meaning they are able to expand indefinitely while retaining the capacity to generate derivatives of all three germ layers both and em in vivo /em . The discovery of murine ES (mES) cells was a major breakthrough in developmental biology, since it enabled the study of mammalian gene function in vivo, using transgenic and knockout technologies. The subsequent derivation of human ES (hES) cells raised the expectation that these cells would similarly revolutionize our insights into human development and disease. Unfortunately, human pluripotent stem cells are remarkably resilient to non-viral genetic manipulation and to date only a handful of human knock-in or knock-out cell lines exist. As a result, the application of human pluripotent stem cells has been more limited than previously anticipated. While both human and murine ES cells are derived from blastocyst-stage embryos, they demonstrate profound differences (Thomson et al., 1998). Murine ES cells grow in three-dimensional, tightly packed colonies with a population doubling time of approximately 16 hours and their maintenance is dependent on LIF and BMP4 growth factor signaling (Smith et al., 1988; Xu et al., 2005; Ying et al., 2003). In contrast, human ES cells form flattened two-dimensional colonies and are maintained in a bFGF and Activin A/TGFbeta signaling dependent manner (Thomson et al., 1998). HES cells proliferate slowly, with a population doubling time averaging 36 hours. Epigenetically, human and murine ES cells display a different X-chromosome inactivation pattern and promoter occupancy by pluripotency transcription factors (Boyer et al., 2005; Silva et al., 2008; Tesar et al., 2007). In addition, hES cells are passaged as small clumps of cells, and most hES cell lines cannot be passaged as single cells by trypsin digest. The inability of hES cell lines to grow from single cells greatly impedes genetic modification of these cells, since the introduction of transgenes is followed Pozanicline by clonal selection. Two reports for the derivation of murine epiblast stem cells (EpiSCs) lately provided a fresh perspective on the type of human being Sera cells (Brons et al., 2007; Tesar et al., 2007). EpiSCs derive from post-implantation murine epiblast RLC embryos under tradition conditions just like hES cell tradition conditions. EpiSCs screen lots Pozanicline of the features of human being Sera cells including their reliance on bFGF/Activin A signaling, their flattened colony morphology, their slower proliferation price in comparison to murine Sera cells, their X-inactivation position and their necessity to become passaged as little clumps of cells (Brons et al., 2007; Tesar et al., 2007). The tradition dynamics and the precise characteristics of murine ES cells and EpiSCs appear to be largely determined by the growth factor conditions under which these cell types are derived and maintained. Indeed, recent work from our group demonstrates Pozanicline that culture growth factor conditions play a critical role in defining the pluripotent stem cell state (Chou et al., 2008). Intriguingly, while pluripotent stem cells can be stably derived and propagated from multiple species in an epiblast-like state, including the rat and non-permissive mouse strains, the LIF-dependent pluripotent state appears to be unstable in these species. (Buehr et al., 2008; Hanna et al., 2009; Li et al., 2009; Pozanicline Liao et al., 2009). However the LIF-dependent pluripotent state can be stabilized through the constitutive ectopic expression of one or more of the reprogramming factors (Oct4, Sox2, Klf4, cMyc), which induce the generation of induced pluripotent stem cells (iPS cells) from somatic cells (Takahashi et al., 2007; Takahashi and Yamanaka, 2006). In the non-permissive NOD mouse strain for example, the constitutive ectopic expression of either Klf4 or cMyc is sufficient to allow the derivation of ES-like cells from blastocyst embryos (Hanna et.