Paper pools were established by

Paper pools were established by searches of the PubMed database, which is accessible through the NIH National Library of Medicine (NIH/NLM). Data bank searches were performed separately to identify hESC- and hiPSC-related publications using the search strings described earlier (Guhr et al., 2006; Müller et al., 2010) and modified as indicated in Supplemental Experimental Procedures. The complete procedures used to identify research papers with relevance for our analysis are described in the Supplemental Information and outlined schematically in Figure S1. Briefly, initial searches of the database resulted in about 17,400 hits (11,137 for hESC-related papers and 6,291 for hiPSC-related papers). We excluded articles that were categorized by PubMed as non-research papers, as well as studies that appeared in journals that do not publish original experimental research. Abstracts and/or full texts of the remaining ∼11,000 papers were inspected manually for the use of hESCs or hiPSCs before they were added to the paper repositories. Therefore, our paper pools only contain original research papers in which hESCs and/or hiPSCs were used experimentally. Allocation of a paper to a country was done according to the corresponding author’s affiliation. Citation analysis was performed as described previously (Löser et al., 2012) using the Scopus database. Details are given in Supplemental Experimental Procedures.
Author Contributions
Introduction The molecular basis for the self-renewal and differentiation of human embryonic stem (ES) metabotropic receptors is not fully understood. In the mouse, self-renewal depends on the maintenance of a core regulatory network of three transcription factors, Nanog, Oct4, and Sox2, which function as a unit to block differentiation (reviewed in Jaenisch and Young, 2008). Mouse embryos null for any of these factors are incapable of maintaining a pluripotent inner cell mass, and cells destined to become epiblast instead develop into extraembryonic lineages (Avilion et al., 2003; Nichols et al., 1998; Chambers et al., 2003; Mitsui et al., 2003). The involvement of these transcription factors has been more recently extended to human ES cells, as they occupy the promoters of a number of genes shown to be differentially upregulated or repressed in human ES cells versus differentiated cells (Boyer et al., 2005). Unlike mouse ES cells, these three factors do not function as a unit to regulate self-renewal of human ES cells and each represses the differentiation of different cell fates (Wang et al., 2012). Little is known about the factors working with either NANOG, OCT4, or SOX2 to block lineage specific differentiation in human ES cells. Co-immunoprecipitation experiments have been successfully utilized to detect proteins binding to and cooperating with Nanog, Oct4, and Sox2 in mouse ES cells (Wang et al., 2006; Liang et al., 2008; van den Berg et al., 2010; Mallanna et al., 2010; Pardo et al., 2010). We would like to develop a complementary forward genetic approach to identify genes that cooperate with a factor such as NANOG in regulating important biological processes in human ES cells. A forward genetic approach not only has the power to interrogate the genome in an unbiased fashion, but also has the potential to identify cooperating genes that are either not in the same protein complex or have low transcript or protein abundance. We have previously shown that the piggyBac (PB) transposon modified from moth can efficiently transpose in the mouse and human genomes (Ding et al., 2005). Here we present a gain of function screen in human ES cells using PB transposon mutagenesis. The transposon is specially designed for identifying genes that cooperate with NANOG to block differentiation and support human ES cell self-renewal. As proof of principle, we show that the screen identified DENND2C, whose overexpression is capable of genetic cooperation with NANOG to block retinoic acid (RA)-induced differentiation. Further characterization revealed that DENND2C negatively regulates RHOA, affecting the localization, activity, and DNA association of nuclear RHOA.