scholarly journals TET2 Inhibits Differentiation of Embryonic Stem Cells but Does Not Overcome Methylation-Induced Gene Silencing

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Louis Norman ◽  
Paul Tarrant ◽  
Timothy Chevassut
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Genomic Dna ◽  
Es Cells ◽  
Embryonic Stem ◽  
Gene Promoters ◽  
Loss Of Function ◽  
Promoter Sequences ◽  
Maintenance Methylation

TET2 is a methylcytosine dioxygenase that is frequently mutated in myeloid malignancies, notably myelodysplasia and acute myeloid leukemia. TET2 catalyses the conversion of 5′-methylcytosine to 5′-hydroxymethylcytosine within DNA and has been implicated in the process of genomic demethylation. However, the mechanism by which TET2 loss of function results in hematopoietic dysplasia and leukemogenesis is poorly understood. Here, we show that TET2 is expressed in undifferentiated embryonic stem cells and that its knockdown results in reduction of 5′-hydroxymethylcytosine in genomic DNA. We also present DNA methylation data from bone marrow samples obtained from patients with TET2-mutated myelodysplasia. Based on these findings, we sought to identify the role of TET2 in regulating pluripotency and differentiation. We show that overexpression of TET2 in a stably integrated transgene leads to increased alkaline phosphatase expression in differentiating ES cells and impaired differentiation in methylcellulose culture. We speculate that this effect is due to TET2-mediated expression of stem cell genes in ES cells via hydroxylation of 5′-methylcytosines at key promoter sequences within genomic DNA. This leads to relative hypomethylation of gene promoters as 5′-hydroxymethylcytosine is not a substrate for DNMT1-mediated maintenance methylation. We sought to test this hypothesis by cotransfecting the TET2 gene with methylated reporter genes. The results of these experiments are presented.

scholarly journals Role of TGFβ1 and WNT6 in FGF2 and BMP4-driven endothelial differentiation of murine embryonic stem cells

Angiogenesis ◽  
2021 ◽  
Author(s):  
Anna Gualandris ◽  
Alessio Noghero ◽  
Davide Cora’ ◽  
Elena Astanina ◽  
Marco Arese ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Culture Medium ◽  
Expression Profiles ◽  
Es Cells ◽  
Embryonic Stem ◽  
Endothelial Differentiation ◽  
Loss Of Function ◽  
Pure Cultures

AbstractEmbryonic stem cells (ES) are a valuable source of endothelial cells. By co-culturing ES cells with the stromal PA6 cells, the endothelial commitment can be achieved by adding exogenous FGF2 or BMP4. In this work, the molecular pathways that direct the differentiation of ES cells toward endothelium in response to FGF2 are evaluated and compared to those activated by BMP4. To this purpose the genes expression profiles of both ES/PA6 co-cultures and of pure cultures of PA6 cells were obtained by microarray technique at different time points. The bioinformatics processing of the data indicated TGFβ1 as the most represented upstream regulator in FGF2-induced endothelial commitment while WNT pathway as the most represented in BMP4-activated endothelial differentiation. Loss of function experiments were performed to validate the importance of TGFβ1 and WNT6 respectively in FGF2 and BMP4-induced endothelial differentiation. The loss of TGFβ1 expression significantly impaired the accomplishment of the endothelial commitment unless exogenous recombinant TGFβ1 was added to the culture medium. Similarly, silencing WNT6 expression partially affected the endothelial differentiation of the ES cells upon BMP4 stimulation. Such dysfunction was recovered by the addition of recombinant WNT6 to the culture medium. The ES/PA6 co-culture system recreates an in vitro complete microenvironment in which endothelial commitment is accomplished in response to alternative signals through different mechanisms. Given the importance of WNT and TGFβ1 in mediating the crosstalk between tumor and stromal cells this work adds new insights in the mechanism of tumor angiogenesis and of its possible inhibition.

Thrombopoietin (TPO) Enhances Heamatopoietic Differentiation of Human Embryonic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4157-4157
Author(s):  
Anand S. Srivastava ◽  
Rangrath Mishra ◽  
Ewa Carrier
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cytoplasmic Domain ◽  
Embryoid Bodies ◽  
Janus Kinase ◽  
Human Es Cells

Abstract Recently, it was demonstrated that TPO enhances hematopoietic differentiation of primate ES cells, but its role in differentiating human ES cells is unknown. We sought to investigate the regulatory mechanism of TPO induced signals mediated by the c-mpl cytoplasmic domain during human embryonic stem (hES) cells hematopoietic commitment. We hypothesize that in human embryonic stem cells, binding of TPO to its c-mpl receptor causes three-dimensional alterations which bring the c-mpl cytoplasmic domain and Janus Kinase into close-proximity and thus induces the phosphorylation and dimerization of STAT5 molecule. Dimerized STAT5 molecules detach from the receptors and migrate to the nucleus where they bind GAS site and induce transcription of a set of target, hematopoiesis-related genes. NIH human ES cell lines (WI01) were used in this experiment. In brief, to induce EB formation, cells were incubated in differentiation medium, which consisted of knockout DMEM medium (GIBCO/BRL, Carlsbad, USA), supplemented with 20% non-heat-inactivated fetal bovine serum (FBS, Hyclone, USA), 1% nonessential amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Subsequently, DMEM was replaced by IMDM (GIBCO/BRL, USA) with the same supplements and additional two cytokines (100 ng/mL SCF and 100 ng/mL Flt-3 ligand (Flt-3L)) (control group). To investigate the role of TPO and VEGF, cells were additionally treated with 100 ng/mL TPO alone or in combination with 100 ng/mL rhVEGF. All cytokines were from the R&D systems (USA). Significant increase in the numbers of embryoid bodies (EBs) formation in TPO (125/105), TPO/VEGF (150/105 cells) when compared to controls (10/105 planted ES cells) was documented. This corresponded with the increase in CFU-C and the number of CD31/CD34 positive and CD34-positive progenitors. Analysis of gene expression during hematopoietic development demonstrated that TPO/VEGF combination increased mRNA expression of the TPO receptor (TPO-R) and VEGF (VEGF-R) receptors in hematopoietic progenitors obtained from human ES cells. We are in the process of determining the role of JAK/STAT pathway in this process; functional studies involve blocking of TPO/c-mpl using TPO-R-specific antibodies and determining its impact on human ES-derived hematopoiesis.

Generation of embryos directly from embryonic stem cells by tetraploid embryo complementation reveals a role for GATA factors in organogenesis

2005 ◽  
Vol 33 (6) ◽  
pp. 1534-1536 ◽  
Author(s):  
S.A. Duncan
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Recipient Mouse ◽  
Gata Factors ◽  
Embryonic Tissues ◽  
Tetraploid Complementation ◽  
Traditional Procedure

Gene targeting in ES (embryonic stem) cells has been used extensively to study the role of proteins during embryonic development. In the traditional procedure, this requires the generation of chimaeric mice by introducing ES cells into blastocysts and allowing them to develop to term. Once chimaeric mice are produced, they are bred into a recipient mouse strain to establish germline transmission of the allele of interest. Although this approach has been used very successfully, the breeding cycles involved are time consuming. In addition, genes that are essential for organogenesis often have roles in the formation of extra-embryonic tissues that are essential for early stages of post-implantation development. For example, mice lacking the GATA transcription factors, GATA4 or GATA6, arrest during gastrulation due to an essential role for these factors in differentiation of extra-embryonic endoderm. This lethality has frustrated the study of these factors during the development of organs such as the liver and heart. Extraembryonic defects can, however, be circumvented by generating clonal mouse embryos directly from ES cells by tetraploid complementation. Here, we describe the usefulness and efficacy of this approach using GATA factors as an example.

scholarly journals LncRNA Mrhl orchestrates differentiation programs in mouse embryonic stem cells through chromatin mediated regulation

2019 ◽  
Author(s):  
Debosree Pal ◽  
C V Neha ◽  
Utsa Bhaduri ◽  
Zenia ◽  
Subbulakshmi Chidambaram ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Fate ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Loss Of Function ◽  
Genome Wide ◽  
Development And Differentiation ◽  
Lineage Specific Genes

AbstractLong non-coding RNAs (lncRNAs) have been well-established to act as regulators and mediators of development and cell fate specification programs. LncRNA Mrhl (meiotic recombination hotspot locus) has been shown to act in a negative feedback loop with WNT signaling to regulate male germ cell meiotic commitment. In our current study, we have addressed the role of Mrhl in development and differentiation using mouse embryonic stem cells (mESCs) as our model system of study. We found Mrhl to be a nuclear-localized, chromatin-bound lncRNA with moderately stable expression in mESCs. Transcriptome analyses and loss-of-function phenotype studies revealed dysregulation of developmental processes and lineage-specific genes along with aberrance in specification of early lineages during differentiation of mESCs. Genome-wide chromatin occupancy studies suggest regulation of chromatin architecture at key target loci through triplex formation. Our studies thus reveal a role for lncRNA Mrhl in regulating differentiation programs in mESCs in the context of appropriate cues through chromatin-mediated responses.

scholarly journals The role of nucleotide excision repair in protecting embryonic stem cells from genotoxic effects of UV-induced DNA damage

1999 ◽  
Vol 27 (16) ◽  
pp. 3276-3282 ◽  
Author(s):  
P. P. H. Van Sloun ◽  
J. G. Jansen ◽  
G. Weeda ◽  
L. H. F. Mullenders ◽  
A. A. van Zeeland ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Embryonic Stem Cells ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Embryonic Stem ◽  
Genotoxic Effects ◽  
Nucleotide Excision

scholarly journals Modulation of Differentiation of Embryonic Stem Cells by Polypyrrole: The Impact on Neurogenesis

2021 ◽  
Vol 22 (2) ◽  
pp. 501
Author(s):  
Kateřina Skopalová ◽  
Katarzyna Anna Radaszkiewicz ◽  
Věra Kašpárková ◽  
Jaroslav Stejskal ◽  
Patrycja Bober ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Active Role ◽  
Low Molecular Weight ◽  
Conducting Materials ◽  
The Impact ◽  
Erk Kinase

The active role of biomaterials in the regeneration of tissues and their ability to modulate the behavior of stem cells in terms of their differentiation is highly advantageous. Here, polypyrrole, as a representantive of electro-conducting materials, is found to modulate the behavior of embryonic stem cells. Concretely, the aqueous extracts of polypyrrole induce neurogenesis within embryonic bodies formed from embryonic stem cells. This finding ledto an effort to determine the physiological cascade which is responsible for this effect. The polypyrrole modulates signaling pathways of Akt and ERK kinase through their phosphorylation. These effects are related to the presence of low-molecular-weight compounds present in aqueous polypyrrole extracts, determined by mass spectroscopy. The results show that consequences related to the modulation of stem cell differentiation must also be taken into account when polypyrrole is considered as a biomaterial.

The role of microRNAs in embryonic stem cells: A proteomics approach

2014 ◽  
Vol 237 (2) ◽  
pp. e8
Author(s):  
P. Gyambibi-Barnett ◽  
X. Yin ◽  
Y. Chung ◽  
A. Zampetaki ◽  
M. Mayr
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Proteomics Approach

scholarly journals Cell Adhesion and Spreading Affect Adipogenesis from Embryonic Stem Cells: The Role of Calreticulin

Stem Cells ◽  
2009 ◽  
Vol 27 (9) ◽  
pp. 2092-2102 ◽  
Author(s):  
Eva Szabo ◽  
Tianshu Feng ◽  
Ewa Dziak ◽  
Michal Opas
Keyword(s):  
Stem Cells ◽  
Cell Adhesion ◽  
Embryonic Stem Cells ◽  
Embryonic Stem
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