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.

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.

Cardiomyocyte differentiation by GATA-4-deficient embryonic stem cells

Development ◽  
1997 ◽  
Vol 124 (19) ◽  
pp. 3755-3764 ◽  
Author(s):  
N. Narita ◽  
M. Bielinska ◽  
D.B. Wilson
Keyword(s):  
Transcription Factor ◽  
In Situ Hybridization ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Cardiomyocyte Differentiation ◽  
Wild Type ◽  
Cell Stage

In situ hybridization studies, promoter analyses and antisense RNA experiments have implicated transcription factor GATA-4 in the regulation of cardiomyocyte differentiation. In this study, we utilized Gata4−/− embryonic stem (ES) cells to determine whether this transcription factor is essential for cardiomyocyte lineage commitment. First, we assessed the ability of Gata4−/− ES cells form cardiomyocytes during in vitro differentiation of embryoid bodies. Contracting cardiomyocytes were seen in both wild-type and Gata4−/− embryoid bodies, although cardiomyocytes were observed more often in wild type than in mutant embryoid bodies. Electron microscopy of cardiomyocytes in the Gata4−/− embryoid bodies revealed the presence of sarcomeres and junctional complexes, while immunofluorescence confirmed the presence of cardiac myosin. To assess the capacity of Gata4−/− ES cells to differentiate into cardiomyocytes in vivo, we prepared and analyzed chimeric mice. Gata4−/− ES cells were injected into 8-cell-stage embryos derived from ROSA26 mice, a transgenic line that expresses beta-galactosidase in all cell types. Chimeric embryos were stained with X-gal to discriminate ES cell- and host-derived tissue. Gata4−/− ES cells contributed to endocardium, myocardium and epicardium. In situ hybridization showed that myocardium derived from Gata4−/− ES cells expressed several cardiac-specific transcripts, including cardiac alpha-myosin heavy chain, troponin C, myosin light chain-2v, Nkx-2.5/Csx, dHAND, eHAND and GATA-6. Taken together these results indicate that GATA-4 is not essential for terminal differentiation of cardiomyocytes and suggest that additional GATA-binding proteins known to be in cardiac tissue, such as GATA-5 or GATA-6, may compensate for a lack of GATA-4.

206 COMPARATIVE EVALUATION OF VARIOUS PROTOCOLS FOR NEURAL DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 211
Author(s):  
A. Tas ◽  
S. Arat ◽  
H. Dalcik
Keyword(s):  
Neuronal Differentiation ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Neuronal Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Dependent Manner ◽  
Neuronal Precursor Cells ◽  
Mesenchymal Differentiation

Mouse embryonic stem (ES) cells derived from the inner cell mass of blastocysts can differentiate into neuronal cells by treatment with retinoic acid (RA). ES cells cultured as aggregates and as single cell suspensions were then exposed to RA which induced multiple phenotypes of neuronal cells. Differentiation was dependent on the concentration of RA and the time of exposure. In this study, we cultured ES cells as a suspension in which they formed embryoid bodies (EBs). The EBs were treated with varying concentrations of RA for differing times. We used increasing concentrations of RA (50 nM, 100 nM, 1 �M, and 3 �M) prepared from a stock of 10 mM RA in DMSO. Immunocytochemistry staining was carried out on 2, 5, 7, and 9 days of culture. We formed EBs for 4 days with standard ES cell medium (without LIF) plus an additional 4 days of treatment with 1 �M RA. ES cells were treated with 1 �M RA for 2 days in suspension culture. Two-day-old EBs plated on culture dishes were treated with 1 �M RA for 3 days. To test for the effect of RA concentration on embryonic differentiation, 2-day-old EBs were treated with 50 nM, 100 nM, 1 �M, and 3 �M RA for 3 days. The time-dependent effects of RA on the 4-/4+ RA group were investigated. Results showed that neuronal precursor cells appeared on the second day of culture; they were stained with nestin antibody. On the 5th day of culture, neurons were detected with NCAM antibody. On the 7th day of culture, glial cells were observed with GFAP, and on the 9th day of culture GFAP, expression increased. In EBs that were plated and then treated with RA, the same results were obtained. RA induced neuronal differentiation in a concentration-dependent manner. Low concentrations (50 nM and 100 nM) of RA induced neuronal differentiation besides mesenchymal differentiation; however, higher concentrations (1 �M and 3 �M) of RA did not induce mesenchymal differentiation. The most efficient neuronal differentiation was obtained at 3 mM RA concentration. This study was performed in TUBITAK Research Institute for Genetic Engineering and biotechnology.

Effect of Arsenic Trioxide on E14 Murine Embryonic Stem Cells In Vitro.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4409-4409
Author(s):  
Barbara Graham-Evans ◽  
Hal Broxmeyer
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Es Cells ◽  
Embryonic Stem ◽  
Time Dependent ◽  
Dependent Manner ◽  
Murine Embryonic Stem Cells ◽  
Cell Cycle Status

Abstract The ability of pluripotent murine embryonic stem (muES) cells to differentiate into all cell types makes them a very promising tool for cell therapy. The understanding of the molecular mechanisms underlying the growth and differentiation of the ES cells is a pre-requisite for selecting adequately the cell conditions which will be accessible for future use of ES cells in treating hematological malignancies. Pharmacological agents such as arsenic trioxide (As2O3) have shown to be an effective anticancer agent for acute promyelocytic leukemia but its effect on murine embryonic stem cells prior to differentiation and subsequent target organ toxicity is yet to be determined. This study was done to gain insight into the biological effects of As2O3 on muES cell line, E14, with respect to differentiation, proliferation, cytotoxicity and cell cycle status in vitro. MuEs cells were cultured in complete DMEM in the presence of LIF on gelatin coated plates for 24 hours prior to the addition of varying concentrations of As2O3 (0, 0.5,1, 5, and 10 μmol/L, respectively), and the cells were incubated for an additional 48 hours. The differentiation potential of As2O3 was determined by measuring Oct-4 levels by flow cytometry. Proliferation was measured by trypan blue exclusion. Cytotoxicity of As2O3 was determined by MTT assay. Cell cycle status was measured by Propidium Iodide using flow cytometry. Our results demonstrated that As2O3 at different concentrations did not induce differentiation. However, the proliferation of muES cells treated with As2O3 was inhibited in a concentration and time dependent manner. The cell-killing rate of As2O3 on muES cells was both dose and time dependent with an inhibitory concentration (IC50) of ~ 5±0.2 μM and ~3.75±0.7 μM at 24 and 48 hours respectively. Propidium iodide DNA staining revealed that after 48 hour incubation with arsenic, cells in S phase remained relatively constant (~62–67%) at low doses of As2O3 but this decreased by half (33%) at a high dose of 10uM As2O3. The percent of cells in the G2 phase of the cell cycle increased to 41±9% in 10μM treated cells compared to 6±3% in control. In conclusion, As2O3 does not induce the differentiation of muES cells into different lineages, but does inhibit growth and alter the cell cycle kinetics of these cells in a dose dependent manner.

scholarly journals The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yick W Fong ◽  
Jaclyn J Ho ◽  
Carla Inouye ◽  
Robert Tjian
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
In Vitro Transcription ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Ips Cell ◽  
Ribonucleoprotein Complex ◽  
Transcription System

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.

Abstract P412: Mitochondrial Metabolic Transcriptome Profiles During Cardiomyocyte Differentiation From Mouse And Human Pluripotent Stem Cells

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Sung Woo Cho ◽  
Hyoung Kyu Kim ◽  
Jin Han ◽  
Ji-Hee Sung
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Oxidative Metabolism ◽  
Time Dependent ◽  
Lineage Commitment ◽  
Simultaneous Increase ◽  
Dependent Manner ◽  
Cardiomyocyte Differentiation ◽  
Cardiac Lineage ◽  
Mitochondrial Oxidative Metabolism

Simultaneous increase of myofibrils and mitochondria is a key process of cardiomyocyte differentiation from pluripotent stem cells (PSCs). Specifically, development of mitochondrial oxidative energy metabolism in cardiomyocytes is essential to providing the beating function. Although previous studies reported that mitochondrial oxidative metabolism have some correlation with the differentiation of cardiomyocytes, the mechanism by which mitochondrial oxidative metabolism is regulated and the link between cardiomyogenesis and mitochondrial function are still poorly understood. In the present study, we performed transcriptome analysis on cells at specific stages of cardiomyocyte differentiation from mouse embryonic stem cells (mESCs) and human induced PSCs (hiPSCs). We selected highly upregulated mitochondrial metabolic genes at cardiac lineage commitment and time-dependent manner during cardiomyocyte differentiation and identified the protein-protein interaction network connecting between mitochondrial metabolic and cardiac developmental genes. We found several mitochondrial metabolic regulatory genes at cardiac lineage commitment (Cck, Bdnf, Fabp4, Cebpa, Cdkn2a in mESC-derived cells and CCK, NOS3 in hiPSC-derived cells) and time-dependent manner during cardiomyocyte differentiation (Eno3, Pgam2, Cox6a2, Fabp3 in mESC-derived cells and PGAM2, SLC25A4 in hiPSC-derived cells). Notably, mitochondrial metabolic proteins are highly interacted with cardiac developmental proteins time-dependent manner during cardiomyocyte differentiation rather than cardiac lineage commitment. Furthermore, mitochondrial metabolic proteins are mainly interacted with cardiac muscle contractile proteins rather than cardiac transcription factors in cardiomyocyte. Therefore, mitochondrial metabolism is critical at cardiac maturation rather than cardiac lineage commitment.

Strain-specific transgene methylation occurs early in mouse development and can be recapitulated in embryonic stem cells

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 2853-2859 ◽  
Author(s):  
A. Weng ◽  
T. Magnuson ◽  
U. Storb
Keyword(s):  
De Novo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dependent Manner ◽  
Mouse Development ◽  
Partial Methylation ◽  
Distal Chromosome ◽  
Before And After ◽  
Endogenous Genes ◽  
De Novo Methylation

A murine transgene, HRD, is methylated only when carried in certain inbred strain backgrounds. A locus on distal chromosome 4, Ssm1 (strain-specific modifier), controls this phenomenon. In order to characterize the activity of Ssm1, we have investigated developmental acquisition of methylation over the transgene. Analysis of postimplantation embryos revealed that strain-specific methylation is initiated prior to embryonic day (E) 6.5. Strain-specific transgene methylation is all-or-none in pattern and occurs exclusively in the primitive ectoderm lineage. A strain-independent pattern of partial methylation occurs in the primitive endoderm and trophectoderm lineages. To examine earlier stages, embryonic stem (ES) cells were derived from E3.5 blastocysts and examined for transgene methylation before and after differentiation. Though the transgene had already acquired some methylation in undifferentiated ES cells, differentiation induced further, de novo methylation in a strain-dependent manner. Analysis of methylation in ES cultures suggests that the transgene and endogenous genes (such as immunoglobulin genes) are synchronously methylated during early development. These results are interpreted in the context of a model in which Ssm1-like modifier genes produce alterations in chromatin structure during and/or shortly after implantation, thereby marking target loci for de novo methylation with the rest of the genome during gastrulation.

scholarly journals Deacetylation of Histone H4 Accompanying Cardiomyogenesis is Weakened in HDAC1-Depleted ES Cells

2018 ◽  
Vol 19 (8) ◽  
pp. 2425 ◽  
Author(s):  
Orazio Angelo Arcidiacono ◽  
Jana Krejčí ◽  
Jana Suchánková ◽  
Eva Bártová
Keyword(s):  
Histone Deacetylases ◽  
Trichostatin A ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cardiomyocyte Differentiation ◽  
Histone H4 ◽  
Epigenetic Factors ◽  
Double Knockout ◽  
Physiological Processes

Cell differentiation into cardiomyocytes requires activation of differentiation-specific genes and epigenetic factors that contribute to these physiological processes. This study is focused on the in vitro differentiation of mouse embryonic stem cells (mESCs) induced into cardiomyocytes. The effects of clinically promising inhibitors of histone deacetylases (HDACi) on mESC cardiomyogenesis and on explanted embryonic hearts were also analyzed. HDAC1 depletion caused early beating of cardiomyocytes compared with those of the wild-type (wt) counterpart. Moreover, the adherence of embryonic bodies (EBs) was reduced in HDAC1 double knockout (dn) mESCs. The most important finding was differentiation-specific H4 deacetylation observed during cardiomyocyte differentiation of wt mESCs, while H4 deacetylation was weakened in HDAC1-depleted cells induced to the cardiac pathway. Analysis of the effect of HDACi showed that Trichostatin A (TSA) is a strong hyperacetylating agent, especially in wt mESCs, but only SAHA reduced the size of the beating areas in EBs that originated from HDAC1 dn mESCs. Additionally, explanted embryonic hearts (e15) responded to treatment with HDACi: all of the tested HDACi (TSA, SAHA, VPA) increased the levels of H3K9ac, H4ac, H4K20ac, and pan-acetylated lysines in embryonic hearts. This observation shows that explanted tissue can be maintained in a hyperacetylation state several hours after excision, which appears to be useful information from the view of transplantation strategy and the maintenance of gene upregulation via acetylation in tissue intended for transplantation.

scholarly journals SUMO conjugation susceptibility of Akt/protein kinase B affects the expression of the pluripotency transcription factor Nanog in embryonic stem cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254447
Author(s):  
Marcos Francia ◽  
Martin Stortz ◽  
Camila Vazquez Echegaray ◽  
Camila Oses ◽  
Paula Verneri ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dependent Manner ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Sumo Conjugation ◽  
Heterologous System ◽  
Canonical Pathways ◽  
The Impact

Akt/PKB is a kinase involved in the regulation of a wide variety of cell processes. Its activity is modulated by diverse post-translational modifications (PTMs). Particularly, conjugation of the small ubiquitin-related modifier (SUMO) to this kinase impacts on multiple cellular functions, such as proliferation and splicing. In embryonic stem (ES) cells, this kinase is key for pluripotency maintenance. Among other functions, Akt is known to promote the expression of Nanog, a central pluripotency transcription factor (TF). However, the relevance of this specific PTM of Akt has not been previously analyzed in this context. In this work, we study the effect of Akt1 variants with differential SUMOylation susceptibility on the expression of Nanog. Our results demonstrate that both, the Akt1 capability of being modified by SUMO conjugation and a functional SUMO conjugase activity are required to induce Nanog gene expression. Likewise, we found that the common oncogenic E17K Akt1 mutant affected Nanog expression in ES cells also in a SUMOylatability dependent manner. Interestingly, this outcome takes places in ES cells but not in a non-pluripotent heterologous system, suggesting the presence of a crucial factor for this induction in ES cells. Remarkably, the two major candidate factors to mediate this induction, GSK3-β and Tbx3, are non-essential players of this effect, suggesting a complex mechanism probably involving non-canonical pathways. Furthermore, we found that Akt1 subcellular distribution does not depend on its SUMOylatability, indicating that Akt localization has no influence on the effect on Nanog, and that besides the membrane localization of E17K Akt mutant, SUMOylation is also required for its hyperactivity. Our results highlight the impact of SUMO conjugation in the function of a kinase relevant for a plethora of cellular processes, including the control of a key pluripotency TF.

X chromosome retains the memory of its parental origin in murine embryonic stem cells

Development ◽  
1993 ◽  
Vol 119 (3) ◽  
pp. 813-821 ◽  
Author(s):  
T. Tada ◽  
M. Tada ◽  
N. Takagi
Keyword(s):  
X Chromosome ◽  
Polar Region ◽  
Biochemical Study ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Parental Origin ◽  
Visceral Endoderm ◽  
Es Cell ◽  
Preferential Inactivation

A cytogenetic and biochemical study of balloon-like cystic embryoid bodies, formed by newly established embryonic stem (ES) cell lines having a cytogenetically or genetically marked X chromosome, revealed that the paternally derived X chromosome was inactivated in the majority of cells in the yolk sac-like mural region consisting of the visceral endoderm and mesoderm. The nonrandomness was less evident in the more solid polar region containing the ectodermal vesicle, mesoderm and visceral endoderm. Since the same was true in embryoid bodies derived from ES cells at the 30th subculture generation, it was concluded that the imprinting responsible for the preferential inactivation of the paternal X chromosome that was limited to non-epiblast cells of the female mouse embryos, was stably maintained in undifferentiated ES cells. Differentiating epiblast cells should be able to erase or avoid responding to the imprint.

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