scholarly journals Hypoxia Downregulates MAPK/ERK but Not STAT3 Signaling in ROS-Dependent and HIF-1-Independent Manners in Mouse Embryonic Stem Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-16 ◽  
Author(s):  
Jan Kučera ◽  
Julie Netušilová ◽  
Stanislava Sladeček ◽  
Martina Lánová ◽  
Ondřej Vašíček ◽  
...  
Keyword(s):  
Cell Fate ◽  
Intracellular Signaling ◽  
Es Cells ◽  
Hypoxia Inducible Factor ◽  
Embryonic Stem ◽  
Erk Signaling ◽  
Independent Manner ◽  
Hypoxic Response ◽  
Stat3 Phosphorylation ◽  
Phosphorylation Pattern

Hypoxia is involved in the regulation of stem cell fate, and hypoxia-inducible factor 1 (HIF-1) is the master regulator of hypoxic response. Here, we focus on the effect of hypoxia on intracellular signaling pathways responsible for mouse embryonic stem (ES) cell maintenance. We employed wild-type and HIF-1α-deficient ES cells to investigate hypoxic response in the ERK, Akt, and STAT3 pathways. Cultivation in 1% O2 for 24 h resulted in the strong dephosphorylation of ERK and its upstream kinases and to a lesser extent of Akt in an HIF-1-independent manner, while STAT3 phosphorylation remained unaffected. Downregulation of ERK could not be mimicked either by pharmacologically induced hypoxia or by the overexpression. Dual-specificity phosphatases (DUSP) 1, 5, and 6 are hypoxia-sensitive MAPK-specific phosphatases involved in ERK downregulation, and protein phosphatase 2A (PP2A) regulates both ERK and Akt. However, combining multiple approaches, we revealed the limited significance of DUSPs and PP2A in the hypoxia-mediated attenuation of ERK signaling. Interestingly, we observed a decreased reactive oxygen species (ROS) level in hypoxia and a similar phosphorylation pattern for ERK when the cells were supplemented with glutathione. Therefore, we suggest a potential role for the ROS-dependent attenuation of ERK signaling in hypoxia, without the involvement of HIF-1.

scholarly journals Placental cell fates are regulated in vivo by HIF-mediated hypoxia responses

2000 ◽  
Vol 14 (24) ◽  
pp. 3191-3203
Author(s):  
David M. Adelman ◽  
Marina Gertsenstein ◽  
Andras Nagy ◽  
M. Celeste Simon ◽  
Emin Maltepe
Keyword(s):  
Cell Fate ◽  
Es Cells ◽  
Hypoxia Inducible Factor ◽  
Embryonic Stem ◽  
Giant Cells ◽  
Cell Fate Determination ◽  
Placental Development ◽  
Fate Determination

Placental development is profoundly influenced by oxygen (O2) tension. Human cytotrophoblasts proliferate in vitro under low O2 conditions but differentiate at higher O2 levels, mimicking the developmental transition they undergo as they invade the placental bed to establish the maternal–fetal circulation in vivo. Hypoxia-inducible factor-1 (HIF-1), consisting of HIF-1α and ARNT subunits, activates many genes involved in the cellular and organismal response to O2deprivation. Analysis of Arnt−/− placentas reveals an aberrant cellular architecture due to altered cell fate determination of Arnt−/− trophoblasts. Specifically, Arnt−/− placentas show greatly reduced labyrinthine and spongiotrophoblast layers, and increased numbers of giant cells. We further show that hypoxia promotes the in vitro differentiation of trophoblast stem cells into spongiotrophoblasts as opposed to giant cells. Our results clearly establish that O2 levels regulate cell fate determination in vivo and that HIF is essential for mammalian placentation. The unique placental phenotype of Arnt−/− animals also provides an important tool for studying the disease of preeclampsia. Interestingly, aggregation of Arnt−/− embryonic stem (ES) cells with tetraploid wild-type embryos rescues their placental defects; however, these embryos still die from yolk sac vascular and cardiac defects.

scholarly journals Embryonal Carcinoma P19 Cells Produce Erythropoietin Constitutively But Express Lactate Dehydrogenase in an Oxygen-Dependent Manner

Blood ◽  
1998 ◽  
Vol 91 (4) ◽  
pp. 1185-1195 ◽  
Author(s):  
Taiho Kambe ◽  
Junko Tada ◽  
Mariko Chikuma ◽  
Seiji Masuda ◽  
Masaya Nagao ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Binding Site ◽  
Embryonal Carcinoma ◽  
Hypoxia Inducible Factor ◽  
Embryonic Stem ◽  
Gene Promoter ◽  
Dependent Manner ◽  
P19 Cells ◽  
Independent Manner ◽  
Hep3b Cells

Abstract Embryonic stem cells and embryonal carcinoma P19 cells produce erythropoietin (Epo) in an oxygen-independent manner, although lactate dehydrogenase A (LDHA) is hypoxia-inducible. To explore this paradox, we studied the operation of cis-acting sequences from these genes in P19 and Hep3B cells. The Epo gene promoter and 3′ enhancer from P19 cells conveyed hypoxia-inducible responses in Hep3B cells but not in P19 cells. Together with DNA sequencing and the normal transcription start site of P19 Epo gene, this excluded the possibility that the noninducibility of Epo gene in P19 cells was due to mutation in these sequences or unusual initiation of transcription. In contrast, reporter constructs containing LDHA enhancer and promoter were hypoxia inducible in P19 and Hep3B cells, and mutation of a hypoxia- inducible factor 1 (HIF-1) binding site abolished the hypoxic inducibility in both cells, indicating that HIF-1 activation operates normally in P19 cells. Neither forced expression of hepatocyte nuclear factor 4 in P19 cells nor deletion of its binding site from the Epo enhancer was effective in restoring Epo enhancer function. P19 cells may lack an unidentified regulator(s) required for interaction of the Epo enhancer with Epo and LDHA promoters.

Human embryonic stem cells: challenges and opportunities

2006 ◽  
Vol 18 (8) ◽  
pp. 839 ◽  
Author(s):  
Steven L. Stice ◽  
Nolan L. Boyd ◽  
Sujoy K. Dhara ◽  
Brian A. Gerwe ◽  
David W. Machacek ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Cell Line ◽  
Cell Fate ◽  
Neural Development ◽  
Es Cells ◽  
Embryonic Stem ◽  
Normal Karyotype ◽  
Es Cell ◽  
Cell Therapies

Human and non-human primate embryonic stem (ES) cells are invaluable resources for developmental studies, pharmaceutical research and a better understanding of human disease and replacement therapies. In 1998, subsequent to the establishment of the first monkey ES cell line in 1995, the first human ES cell line was developed. Later, three of the National Institute of Health (NIH) lines (BG01, BG02 and BG03) were derived from embryos that would have been discarded because of their poor quality. A major challenge to research in this area is maintaining the unique characteristics and a normal karyotype in the NIH-registered human ES cell lines. A normal karyotype can be maintained under certain culture conditions. In addition, a major goal in stem cell research is to direct ES cells towards a limited cell fate, with research progressing towards the derivation of a variety of cell types. We and others have built on findings in vertebrate (frog, chicken and mouse) neural development and from mouse ES cell research to derive neural stem cells from human ES cells. We have directed these derived human neural stem cells to differentiate into motoneurons using a combination of developmental cues (growth factors) that are spatially and temporally defined. These and other human ES cell derivatives will be used to screen new compounds and develop innovative cell therapies for degenerative diseases.

scholarly journals Histone demethylase KDM6A directly senses oxygen to control chromatin and cell fate

Science ◽  
2019 ◽  
Vol 363 (6432) ◽  
pp. 1217-1222 ◽  
Author(s):  
Abhishek A. Chakraborty ◽  
Tuomas Laukka ◽  
Matti Myllykoski ◽  
Alison E. Ringel ◽  
Matthew A. Booker ◽  
...  
Keyword(s):  
Cell Fate ◽  
Histone Methylation ◽  
Mammalian Cells ◽  
Control Cell ◽  
Hypoxia Inducible Factor ◽  
Histone Demethylase ◽  
Histone Demethylases ◽  
Independent Manner ◽  
H3k27 Methylation ◽  
Oxygen Sensitive

Oxygen sensing is central to metazoan biology and has implications for human disease. Mammalian cells express multiple oxygen-dependent enzymes called 2-oxoglutarate (OG)-dependent dioxygenases (2-OGDDs), but they vary in their oxygen affinities and hence their ability to sense oxygen. The 2-OGDD histone demethylases control histone methylation. Hypoxia increases histone methylation, but whether this reflects direct effects on histone demethylases or indirect effects caused by the hypoxic induction of the HIF (hypoxia-inducible factor) transcription factor or the 2-OG antagonist 2-hydroxyglutarate (2-HG) is unclear. Here, we report that hypoxia promotes histone methylation in a HIF- and 2-HG–independent manner. We found that the H3K27 histone demethylase KDM6A/UTX, but not its paralog KDM6B, is oxygen sensitive. KDM6A loss, like hypoxia, prevented H3K27 demethylation and blocked cellular differentiation. Restoring H3K27 methylation homeostasis in hypoxic cells reversed these effects. Thus, oxygen directly affects chromatin regulators to control cell fate.

scholarly journals Stem cell culture on polyvinyl alcohol hydrogels having different elasticity and immobilized with ECM-derived oligopeptides

2017 ◽  
Vol 37 (7) ◽  
pp. 647-660 ◽  
Author(s):  
Saradaprasan Muduli ◽  
Li-Hua Chen ◽  
Meng-Pei Li ◽  
Zhao-wen Heish ◽  
Cheng-Hui Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Extracellular Matrix ◽  
Stem Cell ◽  
Cell Fate ◽  
Es Cells ◽  
Embryonic Stem ◽  
Poly Vinyl Alcohol ◽  
Hematopoietic Stem ◽  
Stem Cell Culture

Abstract The physical characteristics of cell culture materials, such as their elasticity, affect stem cell fate with respect to cell proliferation and differentiation. We systematically investigated the morphologies and characteristics of several stem cell types, including human amniotic-derived stem cells, human hematopoietic stem cells, human induced pluripotent stem (iPS) cells, and embryonic stem (ES) cells on poly(vinyl alcohol) (PVA) hydrogels immobilized with and without extracellular matrix-derived oligopeptide. Human ES cells did not adhere well to soft PVA hydrogels immobilized with oligovitronectin, whereas they did adhere well to PVA hydrogel dishes with elasticities greater than 15 kPa. These results indicate that biomaterials such as PVA hydrogels should be designed to possess minimum elasticity to facilitate human ES cell attachment. PVA hydrogels immobilized with and without extracellular matrix-derived oligopeptides are excellent candidates of cell culture biomaterials for investigations into how cell culture biomaterial elasticity affects stem cell culture and differentiation.

scholarly journals Dichotomous role of Shp2 for naïve and primed pluripotency maintenance in embryonic stem cells

2021 ◽  
Author(s):  
Seong Min Kim ◽  
Eun-Ji Kwon ◽  
Yun-Jeong Kim ◽  
Young-Hyun Go ◽  
Ji-Young Oh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Erk Signaling ◽  
Differentiation Potential ◽  
Stat3 Signaling ◽  
Stat3 Phosphorylation ◽  
Pluripotency Maintenance

Abstract The requirement of the Mek1 inhibitor (iMek1) during naïve pluripotency maintenance results from the activation of the Mek1-Erk1/2 (Mek/Erk) signaling pathway upon leukemia inhibitory factor (LIF) stimulation. Through a meta-analysis of previous genome-wide screening for negative regulators of naïve pluripotency, Ptpn11 (encoding the Shp2 protein, which serves both as a tyrosine phosphatase and putative adapter), was predicted as one of the key factors for the negative modulation of naïve pluripotency through LIF-dependent Jak/Stat3 signaling. Using an isogenic pair of naïve and primed mouse embryonic stem cells (mESCs), we demonstrated the differential role of Shp2 in naïve and primed pluripotency. Loss of Shp2 increased naive pluripotency by promoting Jak/Stat3 signaling and disturbed in vivo differentiation potential. In sharp contrast, Shp2 depletion significantly impeded the self-renewal of ESCs under primed culture conditions, which was concurrent with a reduction in Mek/Erk signaling. Similarly, upon treatment with an allosteric Shp2 inhibitor (iShp2), the cells sustained Stat3 phosphorylation and decoupled Mek/Erk signaling, thus replacing the use of iMek1 not only for maintenance but also for the establishment of naïve ESCs through reprogramming. Taken together, our findings highlight the differential roles of Shp2 in naïve and primed pluripotency and propose the usage of iShp2 instead of iMek1 for the efficient maintenance and establishment of naïve pluripotency.

scholarly journals Transcriptional repression by FACT is linked to regulation of chromatin accessibility at the promoter of ES cells

2018 ◽  
Author(s):  
Constantine Mylonas ◽  
Peter Tessarz
Keyword(s):  
Rna Polymerase Ii ◽  
Cell Fate ◽  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Antisense Transcription ◽  
Chromatin Accessibility ◽  
Promoter Architecture ◽  
Neuronal Lineage

The conserved and essential histone chaperone FACT (Facilitates Chromatin Transcription) reorganizes nucleosomes during DNA transcription, replication and repair and ensures both, efficient elongation of RNA Pol II and nucleosome integrity. In mammalian cells, FACT is a heterodimer, consisting of SSRP1 and SUPT16. Here, we show that in contrast to yeast, FACT accumulates at the transcription start site of genes reminiscent of RNA Polymerase II profile. Depletion of FACT in mouse embryonic stem cells leads to up-regulation of pro-proliferative genes and key pluripotency factors concomitant with hyper-proliferation of mES cells. Using MNase-, ATAC-, and Nascent Elongating Transcript Sequencing (NET-seq) we show that up-regulation of genes coincides with loss of nucleosomes upstream of the TSS and concomitant increase in antisense transcription, indicating that FACT impacts the promoter architecture to regulate expression of these genes. Finally, we demonstrate a role for FACT in cell fate determination and show that FACT depletion primes ES cells for the neuronal lineage.

scholarly journals Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions

2018 ◽  
Author(s):  
Daniel Strebinger ◽  
Cédric Deluz ◽  
Elias T. Friman ◽  
Subashika Govindan ◽  
Andrea B. Alber ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Fate ◽  
Es Cells ◽  
Embryonic Stem ◽  
Chromatin Accessibility ◽  
Directed Differentiation ◽  
Es Cell ◽  
Endogenous Fluctuations ◽  
Cell Fate Decisions ◽  
Oct4 Protein

AbstractSOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. However, how temporal fluctuations in their expression levels bias lineage commitment is unknown. Here we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cell towards both neuroectodermal and mesendodermal fates. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers.

scholarly journals Nucleus-cytoskeleton communication impacts on OCT4-chromatin interactions in embryonic stem cells

BMC Biology ◽  
2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Juan José Romero ◽  
María Cecilia De Rossi ◽  
Camila Oses ◽  
Camila Vázquez Echegaray ◽  
Paula Verneri ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Fate ◽  
Opposite Effect ◽  
Es Cells ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Cytoskeleton Organization ◽  
Chromatin Interactions ◽  
Cellular Environment ◽  
Dimensional Distribution

Abstract Background The cytoskeleton is a key component of the system responsible for transmitting mechanical cues from the cellular environment to the nucleus, where they trigger downstream responses. This communication is particularly relevant in embryonic stem (ES) cells since forces can regulate cell fate and guide developmental processes. However, little is known regarding cytoskeleton organization in ES cells, and thus, relevant aspects of nuclear-cytoskeletal interactions remain elusive. Results We explored the three-dimensional distribution of the cytoskeleton in live ES cells and show that these filaments affect the shape of the nucleus. Next, we evaluated if cytoskeletal components indirectly modulate the binding of the pluripotency transcription factor OCT4 to chromatin targets. We show that actin depolymerization triggers OCT4 binding to chromatin sites whereas vimentin disruption produces the opposite effect. In contrast to actin, vimentin contributes to the preservation of OCT4-chromatin interactions and, consequently, may have a pro-stemness role. Conclusions Our results suggest roles of components of the cytoskeleton in shaping the nucleus of ES cells, influencing the interactions of the transcription factor OCT4 with the chromatin and potentially affecting pluripotency and cell fate.

scholarly journals Transcriptional repression by FACT is linked to regulation of chromatin accessibility at the promoter of ES cells

2018 ◽  
Vol 1 (3) ◽  
pp. e201800085 ◽  
Author(s):  
Constantine Mylonas ◽  
Peter Tessarz
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Rna Polymerase Ii ◽  
Cell Fate ◽  
Transcription Start Site ◽  
Mammalian Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Start Site ◽  
Transcription Start

The conserved and essential histone chaperone, facilitates chromatin transcription (FACT), reorganizes nucleosomes during DNA transcription, replication, and repair and ensures both efficient elongation of RNA Pol II and nucleosome integrity. In mammalian cells, FACT is a heterodimer, consisting of SSRP1 and SUPT16. Here, we show that in contrast to yeast, FACT accumulates at the transcription start site of genes reminiscent of RNA polymerase II profile. Depletion of FACT in mouse embryonic stem cells leads to deregulation of developmental and pro-proliferative genes concomitant with hyper-proliferation of mES cells. Using MNase-seq, Assay for Transposase-Accessible Chromatin sequencing, and nascent elongating transcript sequencing, we show that up-regulation of genes coincides with loss of nucleosomes upstream of the transcription start site and concomitant increase in antisense transcription, indicating that FACT impacts the promoter architecture to regulate the expression of these genes. Finally, we demonstrate a role for FACT in cell fate determination and show that FACT depletion primes embryonic stem cells for the neuronal lineage.

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