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  • Epigenetic regulation of Nanog expression by Ezh2 in pluripotent stem cells. 21490431

    Nanog levels in pluripotent stem cells are heterogeneous and this is thought to reflect two different and interchangeable cell states, respectively poised to self-renew (Nanog-high subpopulation) or to differentiate (Nanog-low subpopulation). However, little is known about the mechanisms responsible for this pattern of Nanog expression. Here, we have examined the impact of the histone methyltransferase Ezh2 on pluripotent stem cells and on Nanog expression. Interestingly, induced pluripotent stem (iPS) cells lacking Ezh2 presented higher levels of Nanog due to a relative expansion of the Nanog-high subpopulation, and this was associated to severe defects in differentiation. Moreover, we found that the Nanog promoter in embryonic stem (ES) cells and iPS cells coexists in two alternative univalent chromatin configurations, either H3K4me3 or H3K27me3, the latter being dependent on the presence of functional Ezh2. Finally, the levels of expression of Ezh2, as well as the amount of H3K27me3 present at the Nanog promoter, were higher in the Nanog-low subpopulation of ES/iPS cells. Together, these data indicate that Ezh2 directly regulates the epigenetic status of the Nanog promoter affecting the balance of Nanog expression in pluripotent stem cells and, therefore, the equilibrium between self-renewal and differentiation.
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  • Radical acceleration of nuclear reprogramming by chromatin remodeling with the transactivation domain of MyoD. 21732495

    Induced pluripotent stem cells (iPSCs) can be created by reprogramming differentiated cells through introduction of defined genes, most commonly Oct4, Sox2, Klf4, and c-Myc (OSKM). However, this process is slow and extremely inefficient. Here, we demonstrate radical acceleration of iPSC creation with a fusion gene between Oct4 and the powerful transactivation domain (TAD) of MyoD (M(3)O). Transduction of M(3) O as well as Sox2, Klf4, and c-Myc into fibroblasts effectively remodeled patterns of DNA methylation, chromatin accessibility, histone modifications, and protein binding at pluripotency genes, raising the efficiency of making mouse and human iPSCs more than 50-fold in comparison to OSKM. These results identified that one of the most critical barriers to iPSC creation is poor chromatin accessibility and protein recruitment to pluripotency genes. The MyoD TAD has a capability of overcoming this problem. Our approach of fusing TADs to unrelated transcription factors has far-reaching implications as a powerful tool for transcriptional reprogramming beyond application to iPSC technology.
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    Reference
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  • Using small molecules to improve generation of induced pluripotent stem cells from somatic cells 20336525

    Induction of pluripotent stem cells from somatic cells by defined factors was shown to be possible only recently, but already several laboratories have made tremendous strive toward improving and understanding the process. Originally, Oct4, Sox2, Klf4, and cMyc were identified as being the combination of genes necessary to induce reprogramming. It was later shown that cMyc was dispensable; however, in its absence the process was less efficient and took a considerably longer period of time to occur. Furthermore, others have shown that the combination of Oct4, Sox2, Nanog, and Lin28 could also induce reprogramming. One major caveat associated with these techniques remains the need for overexpression of several genes using viral systems. Until very recently, most studies were done using integrating viruses such as retroviruses and lentiviruses. This method ensured that the protein of interested would be expressed at a high concentration and for an adequate period of time necessary to induce reprogramming. Up to date, others have now been able to use different nonintegrative method such as adenovirus and plasmid transfection to induce reprogramming. Furthermore, piggyBac transposition was successfully used to induce reprogramming of murine cells. Most importantly, it was recently published that reprogramming can be induced in the absence of virus, with proteins and small molecules. All of the later methods are appealing since they do not require the integration of the virus or plasmid to exert its effect. However, one avenue that would be all the more therapeutically appealing would be to induce reprogramming in the absence of gene overexpression systems, using small molecules to modulate signaling pathways in the somatic cells. A few molecules have already been identified with the ability to either improve the process or replace one or two of the genes deemed necessary for reprogramming. We have screened successfully for compounds that can replace some of these factors, and share the methods developed following these screens.
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    Reference
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  • Large scale phosphoproteome profiles comprehensive features of mouse embryonic stem cells. 21149613

    Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple