278 EFFECT OF XENOESTROGENS ON THE DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS

2009 ◽  
Vol 21 (1) ◽  
pp. 236
Author(s):  
E.-M. Jeung ◽  
K.-C. Choi ◽  
E.-B. Jeung
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Embryoid Bodies ◽  
Time Dependent ◽  
Dependent Manner ◽  
Cardiomyocyte Differentiation ◽  
Differentiation Markers ◽  
Oct4 Expression

Endocrine disruptors (ED) may have adverse impacts on reproductive and immune systems in human and wild animals. It has been shown that octyl-phenol (OP) and nonyl-phenol (NP) have estrogenicity in estrogen-responding cells or tissues. In this study, we further investigated the effect(s) of OP and NP on the expression of undifferentiation and differentiation markers in mouse embryonic stem cells (ESC), which function as an important factor in the differentiation of ESC into cardiomyocytes. Mouse ESC were cultured in hanging drops to form embryoid bodies (EB). The medium was replaced with phenol red-free DMEM/F-12 supplemented with 5% charcoal-dextran-stripped FBS. The ESC were treated with OP, NP (1Ã-10-6 and 1Ã-10-7 M) or 17β-estradiol (E2; 1Ã-10-8 and 1Ã-10-9 M) in a time-dependent manner (1, 2 and 3 days), and EB were treated with identical concentrations for 4 and 8 days, respectively. High increasing doses of OP and NP were employed in this study because a binding affinity of ED to estrogen receptors (ER) is about 1000 less than that of E2. We determined the mRNA expression of undifferentiation markers (Oct4, Sox2 and Zfp206) and cardiomyocyte differentiation markers (cardiac alpha-MHC, beta-MHC and myosin light chain isoform-2V) using real-time PCR. In ESC, undifferentiation markers were identified. It is of interest that treatment with OP, NP or E2 induced a significant increase (1.4 5.5-fold) in Oct4 expression at the transcription levels according to a dose- and time-dependent manner. However, no difference was observed in the expression of Sox2 and Zfp206 genes in ESC, suggesting that OP and NP may play a role as an Oct4 enhancer in ESC. In addition, both undifferentiation and cardiomyocyte differentiation markers were identified in EB. Treatment with OP and NP induced a significant increase in the expression of Oct4, Sox2 and Zfp206 genes at the transcription levels in a dose-dependent manner for 4 days, whereas Oct4 expression was only induced at these doses for 8 days. In contrast, cardiomyocyte differentiation markers were reduced by these ED in EB. Taken together, these results suggest that OP and NP play a role as a positive regulator in the undifferentiation process of ESC and EB, and maintenance and differentiation of mouse ESC.

385 ESTROGENIC EVALUATION OF ALKYPHENOLS IN MOUSE EMBRYONIC STEM CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 349
Author(s):  
E. M. Jung ◽  
E. B. Jeung
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Methylation Pattern ◽  
Embryoid Bodies ◽  
Time Dependent ◽  
Target Cells ◽  
Transcriptional Level ◽  
Dependent Manner ◽  
Cardiomyocyte Differentiation ◽  
Differentiation Markers

Xenoestrogens can have adverse effects on the reproductive and immune systems; for example, 4-tert-octylphenol (OP) and 4-nonylphenol (NP) can have estrogenic effects in target cells. In this study, we investigated the effects of xenoestrogens on the expression of undifferentiation and differentiation markers in mouse embryonic stem (ES) cells, which are important mediators of the differentiation of ES cells into cardiomyocytes. The ES cells were treated with 17β-estradiol or OP and NP in a time-dependent manner (for 1, 2, or 3 days), and embryoid bodies (EB) cells were given the same treatment for 5, 8, 12, or 16 days. The mRNA expressions of undifferentiation markers (Oct-4, Sox2, Zfp206, and Rex-1) and cardiomyocyte differentiation markers (α-MHC, β-MHC, ANF, and MLC-2V) were determined by semi- and quantitative real-time PCR. Treatment with E2 induced an increase (1.3- to 4.6-fold) in Oct-4 expression at the transcriptional level in a dose- and time-dependent manner. However, no difference was observed in the expression of Sox2, Zfp206, or Rex-1 genes in ES cells, suggesting that E2 might be an Oct-4 enhancer in ES cells. However, induction of Oct-4 expression by E2 might result from changes in the Oct-4 promoter methylation pattern rather than from other regulatory mechanisms. We also found that cardiomyocyte differentiation markers were differentially expressed in response to xenoestrogens in EB cells. Taken together, these results suggest that xenoestrogens might play a role as a positive regulator of the undifferentiation process in mouse ES and EB cells and might be involved in the maintenance and differentiation of mouse ES cells.

scholarly journals Transcriptional Silencing of a Novel hTERT Reporter Locus during In Vitro Differentiation of Mouse Embryonic Stem Cells

2007 ◽  
Vol 18 (2) ◽  
pp. 669-677 ◽  
Author(s):  
Shuwen Wang ◽  
Chunguang Hu ◽  
Jiyue Zhu
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Embryoid Bodies ◽  
Histone Deacetylation ◽  
Htert Promoter ◽  
Embryonic Cells ◽  
Human Telomerase Reverse Transcriptase ◽  
Independent Manner

The human telomerase reverse transcriptase hTERT is highly expressed in undifferentiated embryonic cells and silenced in the majority of somatic cells. To investigate the mechanisms of hTERT silencing, we have developed a novel reporter using a bacterial artificial chromosome (BAC) that contained the entire hTERT gene and its neighboring loci, hCRR9 and hXtrp2. Firefly and Renilla luciferases were used to monitor transcription from the hTERT and hCRR9 promoters, respectively. In mouse embryonic stem cells stably integrated with the BAC reporter, both hTERT and hCRR9 promoters were highly expressed. Upon differentiation into embryoid bodies and further into mineral-producing osteogenic cells, the hTERT promoter activity decreased progressively, whereas the hCRR9 promoter remained highly active, both resembling their endogenous counterparts. In fully differentiated cells, the hTERT promoter was completely silenced and adopted a chromatin structure that was similar to its native counterpart in human cells. Inhibition of histone deacetylases led to the opening of the hTERT promoter and partially relieved repression, suggesting that histone deacetylation was necessary but not sufficient for hTERT silencing. Thus, our result demonstrated that developmental silencing of the human TERT locus could be recapitulated in a chromosomal position-independent manner during the differentiation of mouse embryonic stem cells.

scholarly journals Lipid rafts integrity is essential for the prolactin-induced mitogenesis in mouse embryonic stem cells

2021 ◽  
Author(s):  
Sophia Karouzaki ◽  
Charoula Peta ◽  
Emmanouella Tsirimonaki ◽  
George Leondaritis ◽  
Kostas Vougas ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Lipid Rafts ◽  
Membrane Lipids ◽  
Close Association ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Mass Spectrometry Analysis ◽  
Signalling Pathways ◽  
Dependent Manner

Embryonic stem cells, ESCs, retain the capacity to self-renew, yet, the protein machinery essential in maintaining this undifferentiated status remains largely undefined. Signalling interactions are initiated and enhanced at the plasma membrane lipid rafts, within constrains and regulation applied by the actin and tubulin cytoskeleton systems. First, we undertook a comprehensive approach using twodimensional gel electrophoresis and mass spectrometry analysis combined with Western blotting and immunofluorescence analyses at the single cell level to compile the proteome profile of detergentfree preparations of lipid rafts of E14 mouse embryonic stem cells. In comparison with the proteomic profiles of other membrane fractions, recovery of actin and tubulin network proteins, including folding chaperones, was impressively high. At equally high frequency we detected annexins, pleiotropic proteins that may bind membrane lipids and actin filaments to regulate important membrane processes, and we validated their expression in lipid rafts. Next, we tested whether lipid raft integrity is required for completion of mitogenic signalling pathways. Disruption of the rafts with the cholesterol sequestering methyl-β-cyclodextrin (MCD) greatly downregulated the mitotic index of ESCs, in a dose- and time of exposure-dependent manner. Moreover, MCD greatly reduced the mitogenic actions of prolactin, a hormone known to stimulate proliferation in a great variety of stem and progenitor cells. Taken together, our data postulate that lipid rafts in ESCs are in close association with the actin and tubulin cytoskeletons to support signal compartmentalization, especially for signalling pathways pertinent to symmetric divisions for self-renewal.

scholarly journals MOV10 facilitates messenger RNA decay in an N6-methyladenosine (m6A) dependent manner to maintain the mouse embryonic stem cells state

2021 ◽  
Author(s):  
Majid Mehravar ◽  
Yogesh Kumar ◽  
Moshe Olshansky ◽  
Dhiru Bansal ◽  
Craig Dent ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Biology ◽  
Messenger Rna ◽  
Mrna Decay ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Beta Catenin ◽  
Dependent Manner ◽  
Leukaemia Virus

N6-methyladenosine (m6A) is the most predominant internal mRNA modification in eukaryotes, recognised by its reader proteins (so-called m6A-readers) for regulating subsequent mRNA fates, such as splicing, export, localisation, decay, stability, and translation to control several biological processes. Although a few m6A-readers have been identified, yet the list is incomplete. Here, we identify a new m6A-reader protein, Moloney leukaemia virus 10 homologue (MOV10), in mouse embryonic stem cells (mESCs). MOV10 recognises m6A-containing mRNAs with a conserved GGm6ACU motif. Mechanistic studies uncover that MOV10 facilitates mRNA decay of its bound m6A- containing mRNAs in an m6A-dependent manner within the cytoplasmic processing bodies (P-bodies). Furthermore, MOV10 decays the Gsk-3beta mRNA through m6A that stabilises the BETA-CATENIN expression of a WNT/BETA-CATENIN signalling pathway to regulate downstream NANOG expression for maintaining the mESC state. Thus, our findings reveal how a newly identified m6A-reader, MOV10 mediates mRNA decay via m6A that impact embryonic stem cell biology.

Differentiating Mouse Embryonic Stem Cells into Embryoid Bodies by Hanging-Drop Cultures

2016 ◽  
Vol 2016 (12) ◽  
pp. pdb.prot092429 ◽  
Author(s):  
Richard Behringer ◽  
Marina Gertsenstein ◽  
Kristina Vintersten Nagy ◽  
Andras Nagy
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Embryoid Bodies ◽  
Hanging Drop

scholarly journals Involvement of Rictor/mTORC2 in cardiomyocyte differentiation of mouse embryonic stem cells in vitro

2017 ◽  
Vol 13 (1) ◽  
pp. 110-121 ◽  
Author(s):  
Bei Zheng ◽  
Jiadan Wang ◽  
Leilei Tang ◽  
Chao Tan ◽  
Zhe Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Cardiomyocyte Differentiation

scholarly journals Cardiomyocyte differentiation from mouse embryonic stem cells using a simple and defined protocol

2015 ◽  
Vol 245 (2) ◽  
pp. 157-165 ◽  
Author(s):  
Ioannis Kokkinopoulos ◽  
Hidekazu Ishida ◽  
Rie Saba ◽  
Steven Coppen ◽  
Ken Suzuki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Cardiomyocyte Differentiation
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