Modulation of Wnt and Activin/Nodal supports efficient derivation, cloning and suspension expansion of human pluripotent stem cells
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PROVIDER: PRJNA606248 | ENA |
REPOSITORIES: ENA
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Project description:Various culture systems have been used to derive and maintain human pluripotent stem cells (hPSCs), but they are inefficient in sustaining cloning and suspension expansion of hPSCs. Through systematically modulating Wnt and Activin/Nodal signaling, we developed a defined medium (termed AIC), which enables efficient cloning and long-term expansion of hPSCs (AIC-hPSCs) through single-cell passage on feeders, matrix or in suspension (25-fold expansion in 4 days) and maintains genomic stability of hPSCs over extensive expansion. Moreover, the AIC medium supports efficient derivation of hPSCs from blastocysts or somatic cells under feeder-free conditions. Compared to conventional-culture hPSCs, AIC-hPSCs have similar gene expression profiles but down-regulated differentiation genes and display higher metabolic activity. The AIC medium shows a good compatibility for suspension expansion of different hPSC lines. Our study provides a robust culture system for derivation, cloning and suspension expansion of high-quality hPSCs that benefits GMP production and processing of therapeutic hPSC products
2020-04-23 | GSE145163 | GEO
Project description:Members of the transforming growth factor (TGF)-β superfamily play essential roles in the pluripotency, self-renewal, and differentiation of embryonic stem cells. While bone morphogenic proteins maintain pluripotency of undifferentiated mouse ES cells, the role of Activin/Nodal signaling is less clear. To determine the target genes of Activin/Nodal-Smad2 signaling in undifferentiated embryonic stem cells, changes in gene expression were examined following stimulation with recombinant Activin (2 hours) or after inhibition of Activin/Nodal with SB431542 (24 hours) using defined media culture conditions with LIF and 20 ng/mL BMP4. SB431542 is a specific inhibitor of ALK4/5/7 receptors and antagonizes both Activin and Nodal signaling. Via western analysis, Activin stimulation increased pSmad2 in ES cells after 2 hours, and treatment with SB431542 for 24 hours virtually eliminated pSmad2. Total Smad2 expression remained unchanged through these manipulations. RNA from cells treated with Activin or SB431542 was extracted by standard methods with Qiagen RNeasy columns. The RNA was analyzed with the Mouse Genome 430A Array from Affymetrix. Samples were performed in duplicate, and RNA from cells treated with Activin or SB431542 was compared to untreated embryonic stem cells.
2009-08-31 | GSE17879 | GEO
Project description:Members of the transforming growth factor (TGF)-β superfamily play essential roles in the pluripotency, self-renewal, and differentiation of embryonic stem cells. While bone morphogenic proteins maintain pluripotency of undifferentiated mouse ES cells, the role of Activin/Nodal signaling is less clear. To determine the target genes of Activin/Nodal-Smad2 signaling in undifferentiated embryonic stem cells, changes in gene expression were examined following stimulation with recombinant Activin (2 hours) or after inhibition of Activin/Nodal with SB431542 (24 hours) using defined media culture conditions with LIF and 20 ng/mL BMP4. SB431542 is a specific inhibitor of ALK4/5/7 receptors and antagonizes both Activin and Nodal signaling. Via western analysis, Activin stimulation increased pSmad2 in ES cells after 2 hours, and treatment with SB431542 for 24 hours virtually eliminated pSmad2. Total Smad2 expression remained unchanged through these manipulations. RNA from cells treated with Activin or SB431542 was extracted by standard methods with Qiagen RNeasy columns. The RNA was analyzed with the Mouse Genome 430A Array from Affymetrix. Samples were performed in duplicate, and RNA from cells treated with Activin or SB431542 was compared to untreated embryonic stem cells. Experiment Overall Design: RNA from cells treated with Activin or SB431542 was extracted by standard methods with Qiagen RNeasy columns. The RNA was analyzed with the Mouse Genome 430A Array from Affymetrix. Samples were performed in duplicate, and RNA from cells treated with Activin or SB431542 was compared to untreated embryonic stem cells.
2009-09-12 | E-GEOD-17879 | biostudies-arrayexpress
Project description:Activin/Nodal signalling is necessary to maintain pluripotency of human Embryonic Stem Cells (hESCs) and to induce their differentiation towards endoderm. However, the mechanisms by which Activin/Nodal signalling achieves these opposite functions remain unclear. To unravel these mechanisms, we examined the transcriptional network controlled in hESCs by Smad2 and Smad3 which represent the direct effectors of Activin/Nodal signalling. These analyses reveal that Smad2/3 participate in the control of the core transcriptional network characterising pluripotency which includes Oct-4, Nanog, FoxD3, Dppa4, Tert, Myc and UTF-1. In addition, similar experiments performed on endoderm cells confirm that a broad part of the transcriptional network directing differentiation is downstream of Smad2/3. Therefore, Activin/Nodal signalling appears to control divergent transcriptional networks in hESCs and in endoderm. Importantly, we observed an overlap between the transcriptional network downstream of Nanog and Smad2/3 in hESCs while functional studies showed that both factors cooperate to control the expression of pluripotency genes. Therefore, the effect of Activin/Nodal signalling on pluripotency and differentiation could be dictated by tissue specific Smad2/3 partners such as Nanog, explaining the mechanisms by which signalling pathways can orchestrate divergent cell fate decisions.
2011-11-11 | GSE19461 | GEO
Project description:Activin/Nodal signalling is necessary to maintain pluripotency of human Embryonic Stem Cells (hESCs) and to induce their differentiation towards endoderm. However, the mechanisms by which Activin/Nodal signalling achieves these opposite functions remain unclear. To unravel these mechanisms, we examined the transcriptional network controlled in hESCs by Smad2 and Smad3 which represent the direct effectors of Activin/Nodal signalling. These analyses reveal that Smad2/3 participate in the control of the core transcriptional network characterising pluripotency which includes Oct-4, Nanog, FoxD3, Dppa4, Tert, Myc and UTF-1. In addition, similar experiments performed on endoderm cells confirm that a broad part of the transcriptional network directing differentiation is downstream of Smad2/3. Therefore, Activin/Nodal signalling appears to control divergent transcriptional networks in hESCs and in endoderm. Importantly, we observed an overlap between the transcriptional network downstream of Nanog and Smad2/3 in hESCs while functional studies showed that both factors cooperate to control the expression of pluripotency genes. Therefore, the effect of Activin/Nodal signalling on pluripotency and differentiation could be dictated by tissue specific Smad2/3 partners such as Nanog, explaining the mechanisms by which signalling pathways can orchestrate divergent cell fate decisions. Identification of Smad2/3 binding sites in pluripotent hESCs. 5 ChIP-Seq samples including 1 input control sample and 4 ChIP samples (two conditions x two replicates).
2011-11-11 | E-GEOD-19461 | biostudies-arrayexpress
Project description:This experiment was designed to probe the function of Activin/Nodal signalling in the deposition of m6A in human pluripotent stem cells (hPSCs). hPSCs were cultured in presence of Activin or subjected to short-term inhibition of Activin/Nodal signalling for 2h using the receptor antagonist SB-431542 (IP). The global abundance of m6A was then measured by nuclear-enriched methylated RNA immunoprecipitation followed by deep sequencing (NeMeRIP-seq). Pre-NeMeRIP input RNA was used as control to normalise for the changes in gene expression in the two conditions.
2017-12-26 | E-MTAB-5229 | biostudies-arrayexpress
Project description:To explore the molecular basis of functional differences observed between Nodal versus Activin-derived endoderm, we compared their respective gene expression profiles. Sox17-GFP mouse ES cells were differentiated in the presence of Nodal or Activin for 7 days, after which GFP(+) cells were purified by FACs. Undifferentiated ES cells were also included for comparison as a control. Results indicate that the two endoderm populations are nearly identical at the level of global transcription. Subtle differences suggest a difference in the degree of endoderm progression. Total RNA obtained from SOX17-GFP (+) mouse ES cells, Sox17-GFP treated with Nodal, Sox17-GFP treated with Activin. Two replicates for each sample; Sample data table contains average values of two replicates. The non_normalized.txt file contains individual signal values for each replicate.
2013-01-01 | E-GEOD-41086 | biostudies-arrayexpress
Project description:In differentiated mouse ESCs, most of the nodal/activin responsive genes are dependent on both Smad4 and Trim33, some are solely dependent on Smad4, and some are dependent on Trim33. Ebs at Day2.5 from WT, Smad4 null, and Trim33 knock-down ESCs, were treated with activin or SB 431542 for 2 h.
2012-01-09 | E-GEOD-32903 | biostudies-arrayexpress
Project description:Nodal and Activin are morphogens of the TGFbeta superfamily of signaling molecules that direct differential cell fate decisions in a dose- and distance-dependent manner. During early embryonic development the Nodal/Activin pathway is responsible for the specification of mesoderm, endoderm, node and mesendoderm. In contradiction to this drive towards cellular differentiation, the pathway also plays important roles in the maintenance of self-renewal and pluripotency in embryonic and epiblast stem cells. The molecular basis behind stem cell interpretation of Nodal/Activin signaling gradients and the undertaking of disparate cell fate decisions remains poorly understood. Here, we show that any perturbation of endogenous signaling levels in mouse ES cells leads to their exit from self renewal towards divergent differentiation programs. Increasing Nodal signals above basal levels by direct stimulation with Activin promotes differentiation towards the mesendodermal lineages while repression of signaling with the specific Nodal/Activin receptor inhibitor SB431542 induces trophectodermal differentiation. To address how quantitative Nodal/Activin signals are translated qualitatively into distinct cell fates decisions, we performed chromatin immunoprecipitation of phospho-Smad2 the primary downstream transcriptional factor of the Nodal/Activin pathway followed by massively parallel sequencing and show that phospho-Smad2 binds to and regulates distinct subsets of target genes in a dose-dependent manner. Crucially, Nodal/Activin signaling directly controls the Oct4 master regulator of pluripotency by graded phospho-Smad2 binding in the promoter region. Hence stem cells interpret and carry out differential Nodal/Activin signaling instructions via a corresponding gradient of Smad2 phosphorylation that selectively titrates self-renewal against alternative differentiation programs by direct regulation of distinct target gene subsets and Oct4 expression. Four biological replicates consisting of 4 different passages of E14TG2a ES cells at P20, P21, P23 and P24
2011-06-30 | E-GEOD-23239 | biostudies-arrayexpress
Project description:Nodal and Activin are morphogens of the TGFbeta superfamily of signaling molecules that direct differential cell fate decisions in a dose- and distance-dependent manner. During early embryonic development the Nodal/Activin pathway is responsible for the specification of mesoderm, endoderm, node and mesendoderm. In contradiction to this drive towards cellular differentiation, the pathway also plays important roles in the maintenance of self-renewal and pluripotency in embryonic and epiblast stem cells. The molecular basis behind stem cell interpretation of Nodal/Activin signaling gradients and the undertaking of disparate cell fate decisions remains poorly understood. Here, we show that any perturbation of endogenous signaling levels in mouse ES cells leads to their exit from self renewal towards divergent differentiation programs. Increasing Nodal signals above basal levels by direct stimulation with Activin promotes differentiation towards the mesendodermal lineages while repression of signaling with the specific Nodal/Activin receptor inhibitor SB431542 induces trophectodermal differentiation. To address how quantitative Nodal/Activin signals are translated qualitatively into distinct cell fates decisions, we performed chromatin immunoprecipitation of phospho-Smad2 the primary downstream transcriptional factor of the Nodal/Activin pathway followed by massively parallel sequencing and show that phospho-Smad2 binds to and regulates distinct subsets of target genes in a dose-dependent manner. Crucially, Nodal/Activin signaling directly controls the Oct4 master regulator of pluripotency by graded phospho-Smad2 binding in the promoter region. Hence stem cells interpret and carry out differential Nodal/Activin signaling instructions via a corresponding gradient of Smad2 phosphorylation that selectively titrates self-renewal against alternative differentiation programs by direct regulation of distinct target gene subsets and Oct4 expression.
2011-06-30 | GSE23239 | GEO