Dynamic single-cell imaging of direct reprogramming reveals an early specifying event
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE21361 | GEO | 2010/04/16
SECONDARY ACCESSION(S): PRJNA126123
REPOSITORIES: GEO
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Project description:The study of induced pluripotency often relies on experimental approaches that average measurements across a large population of cells, the majority of which do not become pluripotent. Here we used high-resolution, time-lapse imaging to trace the reprogramming process over 2 weeks from single mouse embryonic fibroblasts (MEFs) to pluripotency factor-positive colonies. This enabled us to calculate a normalized cell-of-origin reprogramming efficiency that takes into account only the initial MEFs that respond to form reprogrammed colonies rather than the larger number of final colonies. Furthermore, this retrospective analysis revealed that successfully reprogramming cells undergo a rapid shift in their proliferative rate that corresponds to a reduction in cellular area. This event occurs as early as the first cell division and with similar kinetics in all cells that form induced pluripotent stem (iPS) cell colonies. These data contribute to the theoretical modeling of reprogramming and suggest that certain parts of the reprogramming process follow defined rather than stochastic steps. Gene expression profiling: Mouse E13.5 fibroblasts were generated by blastocyst injection with doxycycline inducible Oct4, Sox2, Klf4, and c-Myc primary iPS cells as previously described. Cells were serum starved with 0.5% FBS containing medium, followed by either continued serum starvation for 96 hours, or by 96 hours of factor induction through medium supplemented with 2 ug/ml doxycycline (Sigma). Two biological replicates of each sample (SerumStarveControl and 96hrInduction) were collected and RNA was isolated in preparation for analysis on Affymetrix Mouse Genome 430 2.0 Arrays as previously described.
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Project description:During the reprogramming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells, the activation of pluripotency genes such as Nanog occurs after the mesenchymal to epithelial transition (MET). Here we report that both adult stem cells (neural stem cells- NSCs) and differentiated cells (astrocytes) of the neural lineage can activate Nanog in the absence of cadherin expression during reprogramming. Gene expression analysis revealed that only the Nanog+Ecadherin+ populations expressed stabilization markers and had upregulated several cell cycle genes; and were transgene independent. Inhibition of Dot1L activity, which has previously been shown to increase MET, enhanced both the numbers of Nanog+ and Nanog+E-cadherin+ colonies, suggesting a MET independent pathway for activation of Nanog in NSCs. Expressing Sox2 in MEFs prior to reprogramming does not alter the ratio of Nanog colonies that express E-cadherin obtained. Taken together these results provide a novel pathway for reprogramming taken by cells of the neural lineage.
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Project description:CYLD is a tumor suppressor gene coding for a deubiquitinating enzyme that has a critical regu-latory function in a variety of signaling pathways and biological processes involved in cancer development and progression, many of which are also key modulators of somatic cell repro-gramming. Nevertheless, the potential role of CYLD in this process has not been studied. With the dual aim of investigating the involvement of CYLD in reprogramming and better under-standing the intricate regulatory system governing this process, we reprogrammed control (CYLDWT/WT ) and CYLD-deficient (CYLDΔ9/Δ9 ) Mouse Embryonic Fibroblasts (MEFs) into induced Pluripotent Stem Cells (iPSCs) through ectopic overexpression of the Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) . CYLD deficiency led to significantly reduced reprogramming efficiency and slower early reprogramming kinetics. Nevertheless, CYLD-deficient cells were capable of estab-lishing pluripotent colonies with full spontaneous differentiation potential. The introduction of WT CYLD to CYLDΔ9/Δ9 MEFs rescued the phenotype. Whole proteome analysis revealed that the Mesenchymal to Epithelial transition (MET) during the early stages of reprogramming was dis-rupted in CYLDΔ9/Δ9 MEFs. Interestingly, pathway analysis revealed that the primary processes affected by CYLD deficiency were associated with the extracellular matrix and several metabolic pathways. Our findings not only establish CYLD's significance as a regulatory component of early reprogramming but also highlight its role as an extracellular matrix regulator, which has profound implications in cancer research.
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Project description:Transfer of somatic cell nuclei to enucleated eggs or ectopic expression of specific transcription factors are two different reprogramming strategies used to generate pluripotent cells from differentiated cells. However, they are poorly efficient and other unknown factors might be required to increase their success rate. Here, we show that Xenopus egg extracts at the metaphase stage (M phase) have a strong reprogramming activity on mouse embryonic fibroblasts (MEFs). First, they reset replication properties of MEF nuclei toward a replication profile characteristic of early development and they erase several epigenetic marks, such as trimethylation of H3K9, H3K4 and H4K20. Second, when MEFs are reversibly permeabilized in the presence of M phase Xenopus egg extracts, they show a transient increase in cell proliferation, form colonies and start to express specific pluripotency markers. Finally, transient exposure of MEF nuclei to M phase Xenopus egg extracts increases the success of nuclear transfer to enucleated mouse oocytes and strongly synergize with the production of pluripotent stem cells by ectopic expression of transcription factors. The mitotic stage of the egg extract is crucial as neither of these effects is detected when using interphasic Xenopus egg extracts. Our data demonstrate that mitosis is essential to make mammalian somatic nuclei prone to reprogramming and that, surprisingly, the heterologous Xenopus system has features that are conserved enough to remodel mammalian nuclei.
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Project description:Transfer of somatic cell nuclei to enucleated eggs or ectopic expression of specific transcription factors are two different reprogramming strategies used to generate pluripotent cells from differentiated cells. However, they are poorly efficient and other unknown factors might be required to increase their success rate. Here, we show that Xenopus egg extracts at the metaphase stage (M phase) have a strong reprogramming activity on mouse embryonic fibroblasts (MEFs). First, they reset replication properties of MEF nuclei toward a replication profile characteristic of early development and they erase several epigenetic marks, such as trimethylation of H3K9, H3K4 and H4K20. Second, when MEFs are reversibly permeabilized in the presence of M phase Xenopus egg extracts, they show a transient increase in cell proliferation, form colonies and start to express specific pluripotency markers. Finally, transient exposure of MEF nuclei to M phase Xenopus egg extracts increases the success of nuclear transfer to enucleated mouse oocytes and strongly synergize with the production of pluripotent stem cells by ectopic expression of transcription factors. The mitotic stage of the egg extract is crucial as neither of these effects is detected when using interphasic Xenopus egg extracts. Our data demonstrate that mitosis is essential to make mammalian somatic nuclei prone to reprogramming and that, surprisingly, the heterologous Xenopus system has features that are conserved enough to remodel mammalian nuclei. MEF cells were infected by retroviruses encoding for Oct4, Sox2, Klf4 and c-Myc and, then reversibly permeabilized and treated with Xenopus M-phase extract. All samples of each cell type (e.g. untreated MEF, ES cells and M-iPS cells) were made in triplicates. Total RNAs were extracted with RNeasy kit and hybridized on Nimblegen MM9 microarrays.
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Project description:Post-translational modification by SUMO is a key regulator of cell identity. In mouse embryonic fibroblasts (MEFs), SUMO impedes reprogramming to pluripotency, while in embryonic stem cells (ESCs), it represses the emergence of totipotent-like cells, suggesting that SUMO targets distinct substrates to preserve somatic and pluripotent states. Using MS-based proteomics, we show that the composition of endogenous SUMOylomes differs dramatically between MEFs and ESCs. In MEFs, SUMO2/3 targets proteins associated with canonical SUMO functions, such as splicing, and transcriptional regulators driving somatic enhancer selection. In contrast, in ESCs, SUMO2/3 primarily modifies highly interconnected repressive chromatin complexes, thereby preventing chromatin opening and transitioning to totipotent-like states. We also characterize several SUMO-modified pluripotency factors and show that SUMOylation of Dppa2 and Dppa4 impedes the conversion to 2-cell-embryo-like states. Altogether, we propose that rewiring the repertoire of SUMO target networks is a major driver of cell fate decision during embryonic development.
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