Ectopic expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the fibroblast genome, generating induced pluripotent stem (iPS) cells. It remains unknown if nuclear reprogramming induced by these four factors globally resets epigenetic differences between differentiated and pluripotent cells. Here, using novel selection approaches, we have generated iPS cells from fibroblasts to characterize their epigenetic state. Female iPS cells showed reactivation of a somatically silenced X chromosome and underwent random X inactivation upon differentiation. Genome-wide analysis of two key histone modifications indicated that iPS cells are highly similar to ES cells. Consistent with these observations, iPS cells gave rise to viable high-degree chimeras with contribution to the germline. These data show that transcription factor-induced reprogramming leads to the global reversion of the somatic epigenome into an ES-like state. Our results provide a paradigm for studying the epigenetic modifications that accompany nuclear reprogramming and suggest that abnormal epigenetic reprogramming does not pose a problem for the potential therapeutic applications of iPS cells.

Summary Induced pluripotent stem (iPS) cells can be obtained from fibroblasts upon expression of Oct4, Sox2, Klf4 and c-Myc. To understand how these factors induce pluripotency, we carried out genome-wide analyses of their promoter binding and expression in iPS and partially reprogrammed cells. We find that target genes of the four factors strongly overlap in iPS and embryonic stem (ES) cells. In partially reprogrammed cells, many genes co-occupied by c-Myc and any of the other three factors already show an ES-like binding and expression pattern. In contrast, genes that are specifically co-bound by Oct4, Sox2 and Klf4 in ES cells and encode pluripotency regulators severely lack binding and transcriptional activation. Among the four factors, c-Myc promotes the most ES cell-like transcription pattern when expressed individually in fibroblasts. These data uncover temporal and separable contributions of the four factors during the reprogramming process and indicate that ectopic c-Myc predominantly acts before pluripotency regulators are activated.

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Abstract-Neuropilin-1 (Npn-1) is a cell surface receptor that binds vascular endothelial growth factor (VEGF), a potent mediator of endothelial permeability, chemotaxis, and proliferation. In vitro, Npn-1 can complex with VEGF receptor-2 (VEGFR2) to enhance VEGFR2-mediated endothelial cell chemotaxis and proliferation. To determine the role of Npn-1/VEGFR2 complexes in VEGF-induced endothelial barrier dysfunction, endothelial cells were stably transfected with Npn1 or VEGFR2 alone (PAE/Npn and PAE/KDR, respectively), or VEGFR2 and Npn-1 (PAE/KDR/Npn-1). Permeability, estimated by measurement of transendothelial electrical resistance (TER), of PAE/Npn and PAE/KDR cell lines was not altered by VEGF 165 . In contrast, TER of PAE/KDR/Npn-1 cells decreased in dose-dependent fashion following VEGF 165 (10 to 200 ng/mL). Activation of VEGFR2, and 2 downstream signaling intermediates (p38 and ERK1/2 MAPK) involved in VEGF-mediated permeability, also increased in PAE/KDR/Npn-1. Consistent with these data, inhibition of Npn-1, but not VEGFR2, attenuated VEGF 165 -mediated permeability of human pulmonary artery endothelial cells (HPAE), and VEGF 121 (which cannot ligate Npn-1) did not alter TER of HPAE. Npn-1 inhibition also attenuated both VEGF 165 -mediated pulmonary vascular leak and activation of VEGFR2, p38, and ERK1/2 MAPK, in inducible lung-specific VEGF transgenic mice. These data support a critical role for Npn-1 in regulating endothelial barrier dysfunction in response to VEGF and suggest that activation of distinct receptor complexes may determine specificity of cellular response to VEGF. Key Words: vascular endothelial growth factor receptor-2 Ⅲ transendothelial electrical resistance Ⅲ chemotaxis Ⅲ mitogen-activated protein kinase Ⅲ acute lung injury V ascular endothelial growth factor (VEGF) was first isolated and purified from tumor cells because of its ability to markedly increase vascular permeability to plasma proteins. 1 VEGF is expressed normally in most tissues, including lung, 2,3 with abundant expression of VEGF in alveolar cells of normal rats. 3,4 Published data support a significant role for VEGF in the development of acute lung injury and acute respiratory distress syndrome (ARDS) after various insults. [5][6][7][8][9][10] In the adult lung, VEGF may promote vascular injury 6 -9,11 via effects on increased endothelial permeability. On the other hand, VEGF stimulates endothelial cell proliferation 12 and chemotaxis, 13 potential reparative responses to pulmonary vascular injury. Because cellular responses to VEGF may be either injurious or reparative, understanding how the endothelium transduces specific VEGF-mediated signals is critical.Vascular endothelial cells express 2 tyrosine kinase receptors for VEGF, termed VEGF receptor 1 (VEGFR1) 14 and VEGF receptor 2 (VEGFR2). 15 Intermolecular cross-talk between VEGFR1 and VEGFR2 has been reported, 16 -18 and represents one possible pathway for differential regulation of VEGF signals. Endothelial cells also express neuropilin-1 (Npn-1), a class 3 s...