npg Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) are highly expressed in embryonic stem (ES) cells, and their overexpression can induce pluripotency in both mouse and human somatic cells, indicating that these factors regulate the developmental signaling network necessary for ES cell pluripotency. However, systemic analysis of the signaling pathways regulated by Yamanaka factors has not yet been fully described. In this study, we identified the target promoters of endogenous Yamanaka factors on a whole genome scale using ChIP (chromatin immunoprecipitation)-on-chip in E14.1 mouse ES cells, and we found that these four factors co-occupied 58 promoters. Interestingly, when Oct4 and Sox2 were analyzed as core factors, Klf4 functioned to enhance the core factors for development regulation, whereas c-Myc seemed to play a distinct role in regulating metabolism. The pathway analysis revealed that Yamanaka factors collectively regulate a developmental signaling network composed of 16 developmental signaling pathways, nine of which represent earlier unknown pathways in ES cells, including apoptosis and cellcycle pathways. We further analyzed data from a recent study examining Yamanaka factors in mouse ES cells. Interestingly, this analysis also revealed 16 developmental signaling pathways, of which 14 pathways overlap with the ones revealed by this study, despite that the target genes and the signaling pathways regulated by each individual Yamanaka factor differ significantly between these two datasets. We suggest that Yamanaka factors critically regulate a developmental signaling network composed of approximately a dozen crucial developmental signaling pathways to maintain the pluripotency of ES cells and probably also to induce pluripotent stem cells.

Human embryonic stem (ES) cells possess the potential to differentiate into all the cell types of the human body and provide potential applications in regenerative medicine [1]. However, the concerns of immune rejection hamper transplantation therapies using human ES cells. To avoid the complications of immune rejection, diverse methods, such as somatic nuclear transfer (also called therapeutic cloning) and fusion of somatic cells with human ES cells [2], have been attempted to produce patient-specific pluripotent stem cells. Most of these approaches have resulted in little success. The generation of human iPS cells (induced pluripotent stem cells) from somatic cells with defined transcription factors makes it possible to produce patientspecific ES-like stem cells for therapeutic purposes [3,4]. Two sets of four-factors, OCT4, SOX2, C-MYC, KLF4 reported by Yamanaka's laboratory and OCT4, SOX2, NANOG, LIN28 reported by Thomson's laboratory, have been shown to reprogram human somatic cells to pluripotency with similar efficiency (10-20 iPS cell colonies from 0.1 million initial fibroblasts) [3,4]. We speculated that C-MYC and KLF4 might synergize with Thomson's 4 factors (OCT4, SOX2, NANOG, LIN28) to reprogram the human somatic cells. Our present study shows that a combination of 6 transcription factors, OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4, significantly increases the efficiency of generating iPS cells from human somatic cells.The human genes encoding the transcription factors, OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4, were cloned into a lentiviral vector to produce lentivirus.Half million human newborn foreskin fibroblasts were transduced with the lentivirus carrying GFP (serving as negative control), or a cocktail of lentivirus carrying 4 factors (OCT4, NANOG, SOX2 and LIN28) or 6 factors (OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4). Twenty-four hours later, the cells were dissociated with trypsin, transferred to five flasks (0.1 million initial cells per flask) coated with murine embryonic fibroblast (MEF) feeder, and cultured in human embryonic stem cell media. Colonies with a human ES cell-like morphology (iPS cell colonies) first became visible 12 days after transduction with 4 factors, whereas iPS cell colonies became visible 7 days after transduction with 6 factors. In order to quantify the efficiency of the reprogramming, the cells in one flask for each combination of factors were fixed on day 17 to analyze the alkaline phosphatase expression. A total of 16±3 colonies from 0.1 million initial fibroblast cells transduced with 4 factors were alkaline phosphatase-positive, 166±6 colonies from 0.1 million initial fibroblast cells transduced with 6 factors were alkaline phosphatase-positive, and no colonies were observed in GFP-lentivirus transduced controls ( Figure 1A). The efficiency of 6 factors is 10.4-fold more than that of 4 factors. The iPS colonies generated by transduction of 4 factors were picked on day 26. As reported by Yu et al. [4], these cells expressed alkaline phosphatase and undifferentia...