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      A method to recapitulate early embryonic spatial patterning in human embryonic stem cells

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          Abstract

          Embryos allocate cells to the three germ layers in a spatially ordered sequence. Human embryonic stem cells (hESCs) can generate the three germ layers in culture, however, differentiation is typically heterogeneous and spatially disordered. Here we show that geometric confinement is sufficient to trigger self-organized patterning in hESCs. In response to BMP4, these colonies reproducibly differentiate to an outer trophectoderm-like ring, an inner ectodermal circle and a ring of mesendoderm expressing primitive-streak markers in between. Fates are defined relative to the boundary with a fixed length scale: small colonies correspond to the outer layers of larger ones. Inhibitory signals limit the range of BMP4 signaling to the colony edge and induce a gradient of Activin/Nodal signaling that patterns mesendodermal fates. These results demonstrate that the intrinsic tendency of stem cells to make patterns can be harnessed by controlling colony geometries, and provide a quantitative assay for studying paracrine signaling.

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          Most cited references 26

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          TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells.

          Human embryonic stem cells (hESCs) self-renew indefinitely and give rise to derivatives of all three primary germ layers, yet little is known about the signaling cascades that govern their pluripotent character. Because it plays a prominent role in the early cell fate decisions of embryonic development, we have examined the role of TGFbeta superfamily signaling in hESCs. We found that, in undifferentiated cells, the TGFbeta/activin/nodal branch is activated (through the signal transducer SMAD2/3) while the BMP/GDF branch (SMAD1/5) is only active in isolated mitotic cells. Upon early differentiation, SMAD2/3 signaling is decreased while SMAD1/5 signaling is activated. We next tested the functional role of TGFbeta/activin/nodal signaling in hESCs and found that it is required for the maintenance of markers of the undifferentiated state. We extend these findings to show that SMAD2/3 activation is required downstream of WNT signaling, which we have previously shown to be sufficient to maintain the undifferentiated state of hESCs. Strikingly, we show that in ex vivo mouse blastocyst cultures, SMAD2/3 signaling is also required to maintain the inner cell mass (from which stem cells are derived). These data reveal a crucial role for TGFbeta signaling in the earliest stages of cell fate determination and demonstrate an interconnection between TGFbeta and WNT signaling in these contexts.
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            Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells.

            Nanog, Oct4, and Sox2 are the core regulators of mouse (m)ESC pluripotency. Although their basic importance in human (h)ESCs has been demonstrated, the mechanistic functions are not well defined. Here, we identify general and cell-line-specific requirements for NANOG, OCT4, and SOX2 in hESCs. We show that OCT4 regulates, and interacts with, the BMP4 pathway to specify four developmental fates. High levels of OCT4 enable self-renewal in the absence of BMP4 but specify mesendoderm in the presence of BMP4. Low levels of OCT4 induce embryonic ectoderm differentiation in the absence of BMP4 but specify extraembryonic lineages in the presence of BMP4. NANOG represses embryonic ectoderm differentiation but has little effect on other lineages, whereas SOX2 and SOX3 are redundant and repress mesendoderm differentiation. Thus, instead of being panrepressors of differentiation, each factor controls specific cell fates. Our study revises the view of how self-renewal is orchestrated in hESCs. Copyright © 2012 Elsevier Inc. All rights reserved.
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              Pluripotency factors in embryonic stem cells regulate differentiation into germ layers.

              Cell fate decisions are fundamental for development, but we do not know how transcriptional networks reorganize during the transition from a pluripotent to a differentiated cell state. Here, we asked how mouse embryonic stem cells (ESCs) leave the pluripotent state and choose between germ layer fates. By analyzing the dynamics of the transcriptional circuit that maintains pluripotency, we found that Oct4 and Sox2, proteins that maintain ESC identity, also orchestrate germ layer fate selection. Oct4 suppresses neural ectodermal differentiation and promotes mesendodermal differentiation; Sox2 inhibits mesendodermal differentiation and promotes neural ectodermal differentiation. Differentiation signals continuously and asymmetrically modulate Oct4 and Sox2 protein levels, altering their binding pattern in the genome, and leading to cell fate choice. The same factors that maintain pluripotency thus also integrate external signals and control lineage selection. Our study provides a framework for understanding how complex transcription factor networks control cell fate decisions in progenitor cells. Copyright © 2011 Elsevier Inc. All rights reserved.
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                Author and article information

                Journal
                101215604
                32338
                Nat Methods
                Nat. Methods
                Nature methods
                1548-7091
                1548-7105
                18 June 2014
                29 June 2014
                August 2014
                26 February 2015
                : 11
                : 8
                : 847-854
                Affiliations
                [1 ]Center for Studies in Physics and Biology, The Rockefeller University, New York, NY
                [2 ]Laboratory of Molecular Vertebrate Embryology, The Rockefeller University, New York, NY 10065
                Author notes
                Correspondence should be addressed to: E.D.S ( siggiae@ 123456rockefeller.edu ) or A.H.B ( brvnlou@ 123456rockefeller.edu )
                [3]

                These authors contributed equally.

                Article
                NIHMS603221
                10.1038/nmeth.3016
                4341966
                24973948
                Categories
                Article

                Life sciences

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