Transcriptome analysis of cells of different cycling speed during Yamanaka reprogramming
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE53074 | GEO | 2014/02/12
SECONDARY ACCESSION(S): PRJNA230800
REPOSITORIES: GEO
Similar Datasets
Project description:Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient, and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a non-stochastic manner. Subsets of murine hematopoietic progenitors are privileged, whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after 4-5 divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of ~8 hours. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression, and is increased by p53-knockdown. This ultrafast-cycling population accounts for >99% of the bulk reprogramming activity in wildtype or p53-knockdown fibroblasts. We compared the transcriptomes of the fast cycling cells with those of slower hematopoietic progenitors, bulk fibroblasts and established iPS cells. 3-5 replicates for each of the six cell types were included: 4 replicates for established iPS cells, 4 replicates for bulk mouse embryonic fibroblasts (MEF), 4 replicates for fast cycling MEF, 4 replicates for slow cycling MEF, 5 replicates for fast cycling granulocyte monocyte progenitors (GMP) and 3 replicates for slow cycling GMP.
2014-02-12 | E-GEOD-53074 | biostudies-arrayexpress
Project description:Oct4 is widely considered the most important among the four Yamanaka reprogramming factors. Here we show that the combination of Sox2, Klf4, and cMyc (SKM) suffices for reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs). Simultaneous induction of Sox2 and cMyc in fibroblasts triggers immediate retroviral silencing, which explains the discrepancy with previous studies that attempted but failed to generate iPSCs without Oct4 using retroviral vectors. SKM induction could partially activate the pluripotency network even in Oct4-knockout fibroblasts. Importantly, reprogramming in the absence of exogenous Oct4 results in greatly improved developmental potential of iPSCs, determined by their ability to give rise to all-iPSC mice in the tetraploid complementation assay. Our data suggest that overexpression of Oct4 during reprogramming leads to off-target gene activation during reprogramming and epigenetic aberrations in resulting iPSCs, and thereby bear major implications for further development and application of iPSC technology.
2019-11-06 | GSE137001 | GEO
Project description:Yamanaka reprogramming is revolutionary but inefficient, slow and stochastic. The underlying molecular events for these mixed outcomes of iPSC reprogramming is still unclear. Previous studies about transcriptional responses to reprogramming overlooked human reprogramming, and are compromised by the fact that only a rare population proceeds towards pluripotency and a significant amount of the collected transcriptional data may not represent the positive reprogramming. We recently developed a concept of reprogramome, which allows one to study the early transcriptional responses to the Yamanaka factors in the perspective of reprogramming legitimacy of a gene response to reprogramming. Using RNA-seq, this study scored 579 genes successfully reprogrammed within 48 hours, indicating the potency of the reprogramming factors. This report also tallied 438 genes reprogrammed significantly but insufficiently up to 72 hours, indicating a positive drive with some inadequacy of the Yamanaka factors. In addition, 953 member genes within the reprogramome were transcriptionally irresponsive to reprogramming, showing the inability of the reprogramming factors to directly act on these genes. Furthermore, there were 305 genes undergoing six types of aberrant reprogramming: over, wrong, and unwanted up- or down- reprogramming, revealing significant negative impacts of the Yamanaka factors. The mixed findings about the initial transcriptional responses to the reprogramming factors shed new insights into the robustness as well as limitations of the Yamanaka factors.
2020-06-07 | GSE148158 | GEO
Project description:In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of nonproductive and staggered productive intermediates arise at different reprogramming time points1-11. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells12,13 prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that during reprogramming the cells progressively lose donor cell identity and gradually acquire iPS cell properties1,2,7,8,10. Here, we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200 that are absent in fibroblasts and iPS cells. Single cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic reprogramming phase2. Expression profiling revealed that the transcriptional regulators Nr0b1 and Etv5 are specifically expressed in this early reprogramming state, preceding activation of key pluripotency regulators such as Rex1, Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus mark some of the earliest known regulators of iPS cell induction. Our study shows an ordered sequence of transitions during the earliest steps of iPS cell reprogramming that deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition. Samples for poised (CD73+ or CD49d+) and non-poised (CD73-) reprogramming samples were FACS sorted 6 and 9 days after induction of Klf4, Oct4, Sox2 and cMyc in Rosa-rtTA +/- mouse embryonic fibroblasts (MEFs). 'Total' populations are expression analyses for unsorted populations analyzed at the same time points. Control populations were also sampled: mouse embryonic fibroblasts (MEFs), partially reprogrammed cells (SC4) and mouse embryonic stem cell (ESC).
2015-01-31 | E-GEOD-62957 | biostudies-arrayexpress
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.
2010-04-28 | E-GEOD-21361 | biostudies-arrayexpress
Project description:Somatic cell reprogramming into pluripotent stem cells induced by Oct4, Sox2, Klf4 and Myc (OSKM) occurs at low frequencies and with a considerable delay involving a stochastic phase. In contrast, transdifferentiation of B cells into macrophages induced by C/EBPα is fully efficient and initiated almost immediately. We now discovered that a pulse of C/EBPα in B cell precursors followed by OSKM expression dramatically enhances reprogramming to pluripotency, overcoming the stochastic phase. The cells simultaneously activate mesenchymal-epithelial transition and pluripotency genes to high levels within 2 days, correlating with promoter demethylation and accessibility to Oct4 binding. The conditioning effect of C/EBPα is shared with C/EBPβ but not with transcription factors that induce erythroid, neuronal and muscle fates, which are innocuous or inhibitory. Surprisingly, OSKM induce the transient upregulation of macrophage genes in B cells, including Cebpa and Cebpb, as well as trophectoderm genes. Since both C/EBPs are functionally expressed in trophoblasts their unique pluripotency-conditioning capacity might reflect an early embryonic function. Our findings have removed a major hurdle in elucidating mechanisms that establish pluripotency. Change in gene expression during reprogramming of B cells into iPS cells, comparing the time 0 B cells to with cells induced with OKSM at different time points pre-treated or not with estradiol to induce C/EBPa.
2013-11-17 | E-GEOD-46321 | biostudies-arrayexpress
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.
2010-04-16 | GSE21361 | GEO
Project description:Reactivation of the pluripotency network during somatic cell reprogramming by exogenous transcription factors involves chromatin remodeling and the recruitment of RNA polymerase II (Pol II) to target loci. Here, we report that Pol II is engaged at pluripotency promoters in reprogramming but remains paused and inefficiently released. We also show that bromodomain-containing protein 4 (BRD4) stimulates productive transcriptional elongation of pluripotency genes by dissociating the pause release factor P-TEFb from an inactive complex containing HEXIM1. Consequently, BRD4 overexpression enhances reprogramming efficiency and HEXIM1 suppresses it, whereas Brd4 and Hexim1 knockdown do the opposite. We further demonstrate that the reprogramming factor KLF4 helps recruit P-TEFb to pluripotency promoters. Our work thus provides a mechanism for explaining the reactivation of pluripotency genes in reprogramming and unveils an unanticipated role for KLF4 in transcriptional pause release. Pol II ChIP-seq for MEFs, ESCs and bulk populations of OSKM reprogramming intermediates at two time points.
2014-10-07 | E-GEOD-59828 | biostudies-arrayexpress
Project description:Somatic cells can be directly reprogrammed to pluripotency by exogenous expression of transcription factors, classically Oct4, Sox2, Klf4 and c-Myc. While distinct types of somatic cells can be reprogramed with varying efficiencies and by different modified reprogramming protocols, induced pluripotent stem cell (iPSC) induction remains inefficient and stochastic where a fraction of the cells converts into iPSCs. The nature of rate limiting barrier(s) preventing majority of cells to convert into iPSCs remains elusive. Here we show that neutralizing Mbd3, a core member of the Mbd3/NURD co-repressor and chromatin-remodeling complex, results in deterministic and synchronized reprogramming of multiple differentiated cell types to pluripotency. 100% of Mbd3 depleted mouse and human somatic cells convert into iPSCs after seven days of reprogramming factor induction. Our findings delineate a critical pathway blocking the reestablishment of pluripotency, and offer a novel platform for future dissection of epigenetic dynamics leading to iPSC formation at high resolution. Samples include Mbd3+/+, Mbd3flox/- and Mbd3-/- cells from mouse ES cells and mouse embryonic fibroblast (MEF) before and after DOX induction (initiating reprogramming by OSKM factors).
2013-09-12 | E-GEOD-45352 | biostudies-arrayexpress
Project description:The generation of induced Pluripotent Stem Cells (iPSCs) from somatic cells provides an excellent example to study mechanisms of transcription factor-induced global alterations of the genome and the epigenome. Here, we have investigated the early transcriptional events of cellular reprogramming triggered by the co-expression of OSKM (Oct4, Sox2, Klf4 and c-Myc) in Mouse Embryonic Fibroblasts (MEFs) and mouse Hepatocytes (mHeps) and identified a novel gene regulatory network composed of 9 Transcriptional Regulators (TRs), which are directly targeted by OSKM. Functional studies using single and double shRNA knock-downs of any of these TRs caused disruption of the network and dramatic reductions in reprogramming efficiency, demonstrating that this novel gene regulatory network is essential for the induction and establishment of pluripotency. We demonstrate that that the stochastic co-expression of the 9TRs network components occurs in a remarkably small number of cells approximating the percentage of reprogrammed cells as the result of dynamic molecular events. Thus, the early binding patterns of OSKM and the subsequent stochastic co-expression of pivotal TRs in subpopulations of cells steer the reconstruction of a gene regulatory network crucial for the generation of iPSCs. In this study, we show that OSKM trigger the gradual establishment of the stemness phenotype by inducing the stochastic reconstruction of a novel 9TRs gene regulatory network that guides the acquisition to pluripotency.
2019-02-14 | GSE114581 | GEO