Oct4 and Nanog are transcription factors required to maintain the pluripotency and self-renewal of embryonic stem (ES) cells. Using the chromatin immunoprecipitation paired-end ditags method, we mapped the binding sites of these factors in the mouse ES cell genome. We identified 1,083 and 3,006 high-confidence binding sites for Oct4 and Nanog, respectively. Comparative location analyses indicated that Oct4 and Nanog overlap substantially in their targets, and they are bound to genes in different configurations. Using de novo motif discovery algorithms, we defined the cis-acting elements mediating their respective binding to genomic sites. By integrating RNA interference-mediated depletion of Oct4 and Nanog with microarray expression profiling, we demonstrated that these factors can activate or suppress transcription. We further showed that common core downstream targets are important to keep ES cells from differentiating. The emerging picture is one in which Oct4 and Nanog control a cascade of pathways that are intricately connected to govern pluripotency, self-renewal, genome surveillance and cell fate determination.

The maintenance of pluripotency and specification of cellular lineages during embryonic development are controlled by transcriptional regulatory networks, which coordinate specific sets of genes through both activation and repression. The transcriptional repressor RE1-silencing transcription factor (REST) plays important but distinct regulatory roles in embryonic (ESC) and neural (NSC) stem cells. We investigated how these distinct biological roles are effected at a genomic level. We present integrated, comparative genome- and transcriptome-wide analyses of transcriptional networks governed by REST in mouse ESC and NSC. The REST recruitment profile has dual components: a developmentally independent core that is common to ESC, NSC, and differentiated cells; and a large, ESC-specific set of target genes. In ESC, the REST regulatory network is highly integrated into that of pluripotency factors Oct4-Sox2-Nanog. We propose that an extensive, pluripotency-specific recruitment profile lends REST a key role in the maintenance of the ESC phenotype.

Human embryonic stem cells (hESCs) herald tremendous promise for the production of clinically useful cell types for the treatment of injury and disease. Numerous reports demonstrate their differentiation into definitive endoderm (DE) cells, the germ layer from which pancreatic b cells and hepatocytes arise, solely from exposure to a high dose of recombinant Activin/Nodal. We show that combining a second related ligand, BMP4, in combination with Activin A yields 15%-20% more DE as compared with Activin A alone. The addition of recombinant BMP4 accelerates the downregulation of pluripotency genes, particularly SOX2, and results in upregulation of endogenous BMP2 and BMP4, which in turn leads to elevated levels of phospho-SMAD1/5/8. Combined Activin A and BMP4 treatment also leads to an increase in the expression of DE genes CXCR4, SOX17, and FOXA2 when compared with Activin A addition alone. Comparative microarray studies between DE cells harvested on day 3 of differentiation further reveal a novel set of genes upregulated in response to initial BMP4 exposure. Several of these, including APLNR, LRIG3, MCC, LEPREL1, ROR2, and LZTS1, are expressed in the mouse primitive streak, the site of DE formation. Thus, this synergism between Activin A and BMP4 during the in vitro differentiation of hESC into DE suggests a complex interplay between BMP and Activin/Nodal signaling during the in vivo allocation and expansion of the endoderm lineage.

Pluripotent embryonic stem cells (ESCs) are capable of differentiating into cell types belonging to all three germ layers within the body, which makes them an interesting and intense field of research. Inefficient specific differentiation and contamination with unwanted cell types are the major issues in the use of ESCs in regenerative medicine. Lineage-specific progenitors generated from ESCs could be utilized to circumvent the issue. We demonstrate here that sustained activation of the Wnt pathway (using Wnt3A or an inhibitor of glycogen synthase kinase 3␤) in multiple mouse and human ESCs results in meso/endodermspecific differentiation. Using monolayer culture conditions, we have generated multipotential "mesendodermal progenitor clones" (MPC) from mouse ESCs by sustained Wnt pathway activation. MPCs express increased levels of meso/endodermal and mesendodermal markers and exhibit a stable phenotype in culture over a year. The MPCs have enhanced potential to differentiate along endothelial, cardiac, vascular smooth muscle, and skeletal lineages than undifferentiated ESCs. In conclusion, we demonstrate that the Wnt pathway activation can be utilized to generate lineage-specific progenitors from ESCs, which can be further differentiated into desired organ-specific cells.The unique property of pluripotent ESCs 2 to differentiate into three germ layers derivatives makes ESCs an ideal source of cells for regenerative therapy (1). One of the most pressing problems in developing ESC-based applications has been the inefficient differentiation of ESCs into the specific therapeutic cell type of choice and the presence of unwanted differentiated cells of other germ layers. A strategy to overcome this problem is to derive lineage-specific progenitor stem cells from ESCs.The advantages of lineage-specific progenitor cells over ESCs are that they differentiate into a limited number of cell types of a particular lineage and, therefore, the differentiation will be robust and more efficient. In addition, they can self-renew, and thus, can be maintained as a renewable source of cells.ESCs can aggregate to form embryoid bodies (EBs) (2, 3), which resemble an intact embryo, and thus, many protocols for in vitro differentiation of the ESCs utilize formation of EB as the first step. However, there can be a mixture of differentiating cells in using an EB approach, whereas differentiation using monolayer culture can yield more uniform and homogenous results. A successful strategy for the use of ESCs in regenerative medicine could involve formation of lineage-restricted progenitor cell using a monolayer culture system as a first step.Gene expression analysis and fate maps together indicate that endoderm and mesoderm are derived, at least in part, from bipotent mesendodermal cells that separate during gastrulation (4, 5) Active canonical Wnt signaling is detected in pregastrulating embryo, through primitive streak (PS) formation, and during gastrulation (6). The evolutionary conserved Wnt signaling pathway is absolutely essential for...