Epigenetic and metabolic remodeling during the acquisition of extended pluripotency
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE137208 | GEO | 2020/10/02
REPOSITORIES: GEO
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Project description:The pluripotency of mouse embryonic stem cells (ESC) and induced-pluripotent stem cells (iPSC) can be maintained by feeder cells, which secrete Leukemia Inhibitory Factor (LIF). We found that feeder cells provide a relatively low concentration (25 unit/ml) of LIF, which is insufficient to maintain the ESC/iPSC pluripotency in feeder free conditions. In order to identify additional factors involved in the maintenance of pluripotency, we carried out a global transcript expression profiling of mouse iPSC cultured on feeder cells and in feeder-free (LIF-treated) conditions. This identified 17 significantly differentially expressed genes (adjusted p-value<0.05) including 7 chemokines over-expressed in iPSC grown on feeder cells. Ectopic expression of these chemokines in iPSC revealed that CC chemokine ligand 2 (Ccl2) induced the key transcription factor genes for pluripotency, Klf4, Nanog, Sox2 and Tbx3. Further, addition of recombinant Ccl2 protein drastically increased the number of Nanog-GFP-positive iPSC grown in low LIF feeder free conditions. Interestingly, this effect was not observed in the absence of LIF. We further revealed that pluripotency promotion by Ccl2 is mediated by activating the Stat3-pathway followed by Klf4 up-regulation. We demonstrated that Ccl2 mediated increased pluripotency is independent of PI3K and MAPK pathways and that Tbx3 may be directly up-regulated by Klf4. Overall, Ccl2 cooperatively activates the Stat3-pathway with LIF in feeder free condition to maintain pluripotency for ESC/iPSC. Total RNAs were purified from feeder cells (as a reference sample), iPSC grown on feeder cells and iPSC grown in feeder-free condition in triplicates and applied to the arrays. iPSC grown on feeder cells were separated mechanically from the feeder layer to minimize feeder cell contamination.
2011-06-01 | E-GEOD-28262 | biostudies-arrayexpress
Project description:The pluripotency maintenance of pluripotent stem cells (PSCs) cultured in vitro requires the suitable microenvironment, which is commonly provided by the feeder layer. However, the preparation of feeder layer is time consuming and labor exhaustive. More importantly, the feeder cells treated with mitomycin or gamma-ray irradiation brings heterologous contamination to stem cells. The feeder-free PSC cultures are associated with high costs because of the requirement for additional supplements and special media. In this study, we characterize the pluripotency and metabolic status of bovine ESCs-F7 (classic bESCs lines, abbreviated as F7), which were cultured on methanol fixed mouse embryonic fibroblasts (MT-MEFs) or mitomycin C treated MEFs (1M-MEFs). MT-MEFs could be reused several times and were highly resistant to digestive enzymes. The relative expression levels of pluripotent markers were different between F7 cultured on MT-MEFs (marked as MT-F7) and those cultured on the 1M-MEFs (1M-F7). The long-term cultured MT-F7 cells formed embryoid bodies in vitro, showing the ability to differentiate into endodermal, ectodermal, and mesodermal germ layers like 1M-F7. RNA-sequencing analysis showed that the replacement of the feeder layer from 1M-MEFs to MT-MEFs lead to a novel steady transition of the F7, which included alteration of the expression patterns of genes that regulate pluripotency and metabolism. Further, the long-term cultured bovine expanded pluripotent stem cells (bEPSCs) on MT-MEFs (MT-bEPSCs) formed classical colonies, maintained pluripotency, and demonstrated elevated level of metabolic activity. In conclusion, this study demonstrated that methanol-fixed MEFs were efficient feeder layer that maintain the unique pluripotency and the distinctive metabolic characteristics of the bPSCs cultured in vitro.
2022-05-01 | GSE199731 | GEO
Project description:The pluripotency of mouse embryonic stem cells (ESC) and induced-pluripotent stem cells (iPSC) can be maintained by feeder cells, which secrete Leukemia Inhibitory Factor (LIF). We found that feeder cells provide a relatively low concentration (25 unit/ml) of LIF, which is insufficient to maintain the ESC/iPSC pluripotency in feeder free conditions. In order to identify additional factors involved in the maintenance of pluripotency, we carried out a global transcript expression profiling of mouse iPSC cultured on feeder cells and in feeder-free (LIF-treated) conditions. This identified 17 significantly differentially expressed genes (adjusted p-value<0.05) including 7 chemokines over-expressed in iPSC grown on feeder cells. Ectopic expression of these chemokines in iPSC revealed that CC chemokine ligand 2 (Ccl2) induced the key transcription factor genes for pluripotency, Klf4, Nanog, Sox2 and Tbx3. Further, addition of recombinant Ccl2 protein drastically increased the number of Nanog-GFP-positive iPSC grown in low LIF feeder free conditions. Interestingly, this effect was not observed in the absence of LIF. We further revealed that pluripotency promotion by Ccl2 is mediated by activating the Stat3-pathway followed by Klf4 up-regulation. We demonstrated that Ccl2 mediated increased pluripotency is independent of PI3K and MAPK pathways and that Tbx3 may be directly up-regulated by Klf4. Overall, Ccl2 cooperatively activates the Stat3-pathway with LIF in feeder free condition to maintain pluripotency for ESC/iPSC.
2011-06-01 | GSE28262 | GEO
Project description:Chavez2009 - a core regulatory network of OCT4 in human embryonic stem cells
A core OCT4-regulated network has been identified as a test case, to analyase stem cell characteristics and cellular differentiation.
This model is described in the article:
In silico identification of a core regulatory network of OCT4 in human embryonic stem cells using an integrated approach.
Chavez L, Bais AS, Vingron M, Lehrach H, Adjaye J, Herwig R
BMC Genomics, 2009, 10:314
Abstract:
BACKGROUND: The transcription factor OCT4 is highly expressed in pluripotent embryonic stem cells which are derived from the inner cell mass of mammalian blastocysts. Pluripotency and self renewal are controlled by a transcription regulatory network governed by the transcription factors OCT4, SOX2 and NANOG. Recent studies on reprogramming somatic cells to induced pluripotent stem cells highlight OCT4 as a key regulator of pluripotency.
RESULTS: We have carried out an integrated analysis of high-throughput data (ChIP-on-chip and RNAi experiments along with promoter sequence analysis of putative target genes) and identified a core OCT4 regulatory network in human embryonic stem cells consisting of 33 target genes. Enrichment analysis with these target genes revealed that this integrative analysis increases the functional information content by factors of 1.3 - 4.7 compared to the individual studies. In order to identify potential regulatory co-factors of OCT4, we performed a de novo motif analysis. In addition to known validated OCT4 motifs we obtained binding sites similar to motifs recognized by further regulators of pluripotency and development; e.g. the heterodimer of the transcription factors C-MYC and MAX, a prerequisite for C-MYC transcriptional activity that leads to cell growth and proliferation.
CONCLUSION: Our analysis shows how heterogeneous functional information can be integrated in order to reconstruct gene regulatory networks. As a test case we identified a core OCT4-regulated network that is important for the analysis of stem cell characteristics and cellular differentiation. Functional information is largely enriched using different experimental results. The de novo motif discovery identified well-known regulators closely connected to the OCT4 network as well as potential new regulators of pluripotency and differentiation. These results provide the basis for further targeted functional studies.
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2005-01-01 | MODEL1305010000 | BioModels
Project description:Here, we examined the effect of LDN-193189, an inhibitor of the BMP signaling pathway, on pig embryonic stem cells (ESCs) in the absence of feeder cells to develop a feeder-free culture system. Feeder cells play an important role in supporting the pluripotency of pig ESCs by blocking trophoblast and mesodermal differentiation through inhibition of the BMP pathway. Transcriptome analysis revealed that LDN-193189 treatment instead of coculturing with feeder cells could help maintain the pluripotency and homogenous cell population of pig ESCs by stimulating the secretion of chemokines and suppressing differentiation. We confirmed that LDN-193189-treated pig ESCs cultured without feeder cells maintained pluripotency for an extended period and were capable of differentiating in vivo and in vitro. The pig ESCs represented distinct characteristics of pig epiblasts as an in vivo counterpart at a transcriptomic level. In summary, we developed feeder-free culture systems for pig ESCs, and our findings will improve the understanding of pluripotency gene networks and comparative embryogenesis.
2024-11-17 | GSE189166 | GEO
Project description:The balance between glycolytic and oxidative metabolism shifts during differentiation of human embryonic stem cells (hESCs) and during reprogramming of somatic cells into pluripotent stem cells. However the contribution of glycolytic metabolism to various stages of pluripotency is not well understood. Additionally, few tools have been developed that modulate pluripotent stem cell glycolytic metabolism to influence self-renewal or differentiation. Here we show that the degree of human pluripotency is associated with glycolytic rate, whereby naive hESCs exhibit higher glycolytic flux, increased MYC transcriptional activity, and elevated nuclear N-MYC levels relative to primed hESCs. Consistently, the inner cell mass of human blastocysts also exhibits increased MYC transcriptional activity relative to primed hESCs and elevated nuclear N-MYC levels. Expression of the lactate transporter, monocarboxylate transporter 1 (MCT1), is strongly associated with the pluripotent state, and reduction of glycolysis using a small molecule inhibitor towards MCT1 decreases self-renewal of naïve hESCs and feeder-free cultured primed hESCs, but not that of primed hESCs grown in feeder-supported conditions. Lastly, reduction of glycolytic metabolism via MCT1 inhibition in feeder-free primed hESCs enhances neural lineage specification. These findings validate the association between glycolytic metabolism and pluripotency, reveal differences in the glucose metabolism of feeder- versus feeder-free cultured hESCs, and show that pharmacologic regulation of glycolysis can influence self-renewal and initial cell fate specification of human pluripotent stem cells.
2016-08-16 | GSE83491 | GEO
Project description:Chimeric contribution of human extended pluripotent stem cells to monkey embryos
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Project description:Extended pluripotent stem cells (EPSCs) derived from mice and humans showed an enhanced potential for chimeric formation. By exploiting transcriptomic techniques, we assessed the differences in gene expression profile between extended EPSCs derived from mice and humans, and those newly derived from the common marmoset (marmoset; Callithrix jacchus). Although the marmoset EPSC-like cells displayed a unique colony morphology distinct from murine and human EPSCs, they displayed a pluripotent state akin to embryonic stem cells (ESCs), as confirmed by gene expression and immunocytochemical analyses of pluripotency markers and three-germ-layer differentiation assay. Importantly, the marmoset EPSC-like cells showed interspecies chimeric contribution to mouse embryos, such as E6.5 blastocysts in vitro and E8.5 epiblasts in vivo in mouse development. Also, we discovered that the perturbation of gene expression of the marmoset EPSC-like cells from the original ESCs resembled that of human EPSCs. Thus, we established the efficacy of the method for the derivation of marmoset EPSCs
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