in Europe and the Swedish Foundation for Strategic Research. The funders had no role in study design, 23388095 data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: [email protected] Introduction Embryonic stem cells have the unique capability to replicate indefinitely while maintaining pluripotency, i.e. the potential to develop into all cell types of the adult organism. In human embryonic stem cells, external ligands like Activin A/ TGFb/Nodal, FGF2 and Insulin/IGF cooperate to activate downstream transcription factors, thereby creating a complex signaling network that ultimately maintains the pluripotent state. One major component of the pluripotency signaling network is the homeodomain transcription factor NANOG, which together with OCT4 and SOX2 constitutes the core transcription factor network in hESCs. Experimental knockdown of NANOG expression leads to hESC differentiation to embryonic and/or extraembryonic lineages, depending on the experimental conditions and on cell line-intrinsic determinants. Conversely, overexpression of NANOG in hESCs promotes self-renewal in the absence of feeders and eliminates the requirement for Activin A in feeder-free systems. Moreover, NANOG expression is required to establish full pluripotency during reprogramming of GSK343 site fibroblasts to induced pluripotent stem cells, as well as for the formation and stabilization of pluripotent epiblast and germ cells in vivo. Thus, it seems that NANOG expression serves both as a determinant and an indicator for bona fide pluripotency, albeit the exact role of NANOG in establishing and maintaining pluripotency remains to be determined. Reporter cell lines, in which a reporter gene is expressed from a specific endogenous promoter, are valuable tools to study gene regulation and function in real-time in living cells, which cannot be achieved by conventional biochemical and immunological methods. Reporter cell lines have been successfully applied in embryonic stem cell research to identify inducers and repressors of specific promoters and to separate subpopulations of differentiated cells. Thus, Nanog reporter lines were created and applied to screen for signaling pathways inducing mouse embryonic stem cell differentiation, to delineate the role of Nanog in pluripotency of mESCs and during embryogenesis, and to monitor iPS cell generation during reprogramming experiments. However, pluripotency and differentiation is regulated through different pathways in murine and human cells, which is reflected by different marker expression and response to signaling molecules of mESCs versus hESCs. This September 2010 | Volume 5 | Issue 9 | e12533 NANOG Reporters from Human ESC restricts the application of principles from mESC biology to hESCs. To enable the study of NANOG expression and NANOGmediated pluripotency in hESCs, we derived NANOG reporter cell lines by gene targeting in hESCs. We chose to pursue a gene targeting strategy rather than random transgenic integration of the reporter construct to avoid uncontrollable position effects on reporter expression, 
and to enable the accurate expression of the reporter gene from the endogenous regulatory sequences of the NANOG locus. These novel NANOG reporter cell lines constitute efficient tools to study the role and regulation of NANOG in human pluripotent cells. Materials and Methods Human embryonic stem cell culture a
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