scholarly journals An Inducible Expression System of the Calcium-Activated Potassium Channel 4 to Study the Differential Impact on Embryonic Stem Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Stefan Liebau ◽  
Michael Tischendorf ◽  
Daniel Ansorge ◽  
Leonhard Linta ◽  
Marianne Stockmann ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
Cell Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Pacemaker Cell ◽  
Es Cell ◽  
Sk Channel ◽  
Mouse Es Cell

Rationale. The family of calcium-activated potassium channels consists of four members with varying biological functions and conductances. Besides membrane potential modulation, SK channels have been found to be involved in cardiac pacemaker cell development from ES cells and morphological shaping of neural stem cells.Objective. Distinct SK channel subtype expression in ES cells might elucidate their precise impact during cardiac development. We chose SK channel subtype 4 as a potential candidate influencing embryonic stem cell differentiation.Methods. We generated a doxycycline inducible mouse ES cell line via targeted homologous recombination of a cassette expressing a bicistronic construct encoding SK4 and a fluorophore from the murine HPRT locus.Conclusion. We characterized the mouse ES cell line iSK4-AcGFP. The cassette is readily expressed under the control of doxycycline, and the overexpression of SK4 led to an increase in cardiac and pacemaker cell differentiation thereby serving as a unique tool to characterize the cell biological variances due to specific SK channel overexpression.

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 MicroRNA-1 transfected embryonic stem cells enhance cardiac myocyte differentiation and inhibit apoptosis by modulating the PTEN/Akt pathway in the infarcted heart

2011 ◽  
Vol 301 (5) ◽  
pp. H2038-H2049 ◽  
Author(s):  
Carley Glass ◽  
Dinender K. Singla
Keyword(s):  
Cell Differentiation ◽  
Cardiac Myocyte ◽  
Heart Function ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Border Zone ◽  
Es Cell ◽  
Infarcted Heart ◽  
Myocyte Differentiation

microRNAs (miRs) have emerged as critical modulators of various physiological processes including stem cell differentiation. Indeed, miR-1 has been reported to play an integral role in the regulation of cardiac muscle progenitor cell differentiation. However, whether overexpression of miR-1 in embryonic stem (ES) cells (miR-1-ES cells) will enhance cardiac myocyte differentiation following transplantation into the infarcted myocardium is unknown. In the present study, myocardial infarction (MI) was produced in C57BL/6 mice by left anterior descending artery ligation. miR-1-ES cells, ES cells, or culture medium (control) was transplanted into the border zone of the infarcted heart, and 2 wk post-MI, cardiac myocyte differentiation, adverse ventricular remodeling, and cardiac function were assessed. We provide evidence demonstrating enhanced cardiac myocyte commitment of transplanted miR-1-ES cells in the mouse infarcted heart as compared with ES cells. Assessment of apoptosis revealed that overexpression of miR-1 in transplanted ES cells protected host myocardium from MI-induced apoptosis through activation of p-AKT and inhibition of caspase-3, phosphatase and tensin homolog, and superoxide production. A significant reduction in interstitial and vascular fibrosis was quantified in miR-1-ES cell and ES cell transplanted groups compared with control MI. However, no statistical significance between miR-1-ES cell and ES cell groups was observed. Finally, mice receiving miR-1-ES cell transplantation post-MI had significantly improved heart function compared with respective controls ( P < 0.05). Our data suggest miR-1 drives cardiac myocyte differentiation from transplanted ES cells and inhibits apoptosis post-MI, ultimately giving rise to enhanced cardiac repair, regeneration, and function.

scholarly journals Asf1a resolves bivalent chromatin domains for the induction of lineage-specific genes during mouse embryonic stem cell differentiation

2018 ◽  
Vol 115 (27) ◽  
pp. E6162-E6171 ◽  
Author(s):  
Yuan Gao ◽  
Haiyun Gan ◽  
Zhenkun Lou ◽  
Zhiguo Zhang
Keyword(s):  
Cell Differentiation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation ◽  
Es Cell ◽  
Nucleosome Assembly ◽  
Chromatin Domains ◽  
Nucleosome Disassembly ◽  
Lineage Specific Genes

Bivalent chromatin domains containing repressive H3K27me3 and active H3K4me3 modifications are barriers for the expression of lineage-specific genes in ES cells and must be resolved for the transcription induction of these genes during differentiation, a process that remains largely unknown. Here, we show that Asf1a, a histone chaperone involved in nucleosome assembly and disassembly, regulates the resolution of bivalent domains and activation of lineage-specific genes during mouse ES cell differentiation. Deletion of Asf1a does not affect the silencing of pluripotent genes, but compromises the expression of lineage-specific genes during ES cell differentiation. Mechanistically, the Asf1a–histone interaction, but not the role of Asf1a in nucleosome assembly, is required for gene transcription. Asf1a is recruited to several bivalent promoters, partially through association with transcription factors, and mediates nucleosome disassembly during differentiation. We suggest that Asf1a-mediated nucleosome disassembly provides a means for resolution of bivalent domain barriers for induction of lineage-specific genes during differentiation.

Filamin A Controls Embryonic Stem Cell Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4599-4599
Author(s):  
Taisuke Kanaji ◽  
Takashi Okamura ◽  
Peter J. Newman
Keyword(s):  
Cell Differentiation ◽  
Signaling Pathways ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryoid Bodies ◽  
Actin Binding ◽  
Embryonic Stem Cell Differentiation ◽  
Filamin A ◽  
Es Cell

Abstract Abstract 4599 Filamin A is a major non-muscle actin binding protein that plays an important role in cross-linking cortical actin filaments into three-dimensional networks. In addition to its role as a cytoskeletal scaffolding molecule, Filamin A is also known to bind more than 30 other proteins, regulating their subcellular location and coordinating their ability to signal. To analyze the role of filamin A in mouse embryonic stem (ES) cell maturation, we generated filamin ALow ES cells by introducing a micro-RNA that specifically downregulates filamin A expression under the control of a cytomegalovirus promoter. Filamin ALow ES cells exhibited a more rounded morphology than did their wild-type filamin ANormal counterparts, and expressed increased levels of the ES cell transcription factor Nanog. In contrast, non-transfected cells in the same culture dish retained normal expression of filamin A, expressed low levels of Nanog, and exhibited a more elongated and spread phenotype characteristic of differentiating cells. Further evidence for a role for filamin A in ES cell differentiation was provided by the observation that withdrawing leukemia inhibitory factor (LIF) to induce ES cell differentiation was accompanied by increased expression of filamin A, a concomitant loss of Nanog expression, and acquisition of a differentiated morphology. Filamin ALow ES cells were able to retain their undifferentiated phenotype, as evaluated by alkaline phosphatase (Alp) activity, in the presence of a 10-fold lower concentration of LIF than was permissive for filamin ANormal ES cells, or following exposure to the differentiating agent, bone morphogenic protein 4 (BMP4). LIF-induced phosphorylation of ERK was decreased in filamin ALow relative to filamin ANormal ES cells, as was BMP-induced phosphorylation of Smad1/5 - two signaling pathways that initiate ES cell differentiation. Finally, embryoid bodies comprised of filamin ALow ES cells were unable to differentiate into CD41+ hematopoietic progenitor cells. Taken together, these data demonstrate that filamin A plays a previously unrecognized, but critical, scaffolding function that support both the LIF - ERK and BMP4 - Smad1/5 signaling pathways leading to ES and hematopoietic cell differentiation. Manipulation of filamin levels might be useful in the future to modulate the differentiation requirements for a variety of clinically-and therapeutically-useful stem cells. Disclosures: Newman: Novo Nordisk: Consultancy; New York Blood Center: Membership on an entity's Board of Directors or advisory committees.

scholarly journals The Polycomb Group Protein Suz12 Is Required for Embryonic Stem Cell Differentiation

2007 ◽  
Vol 27 (10) ◽  
pp. 3769-3779 ◽  
Author(s):  
Diego Pasini ◽  
Adrian P. Bracken ◽  
Jacob B. Hansen ◽  
Manuela Capillo ◽  
Kristian Helin
Keyword(s):  
Cell Differentiation ◽  
Transcriptional Activation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Polycomb Group ◽  
Embryonic Stem Cell Differentiation ◽  
Es Cell ◽  
Polycomb Group Protein ◽  
Specific Expression

ABSTRACT Polycomb group (PcG) proteins form multiprotein complexes, called Polycomb repressive complexes (PRCs). PRC2 contains the PcG proteins EZH2, SUZ12, and EED and represses transcription through methylation of lysine (K) 27 of histone H3 (H3). Suz12 is essential for PRC2 activity and its inactivation results in early lethality of mouse embryos. Here, we demonstrate that Suz12 −/− mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12 −/− ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation-specific genes. Moreover, Suz12 −/− ES cells are impaired in proper differentiation, resulting in a lack of repression of ES cell markers as well as activation of differentiation-specific genes. Finally, we demonstrate that the PcGs are actively recruited to several genes during ES cell differentiation, which despite an increase in H3K27me3 levels is not always sufficient to prevent transcriptional activation. In summary, we demonstrate that Suz12 is required for the establishment of specific expression programs required for ES cell differentiation. Furthermore, we provide evidence that PcGs have different mechanisms to regulate transcription during cellular differentiation.

scholarly journals The topographical regulation of embryonic stem cell differentiation

2004 ◽  
Vol 359 (1446) ◽  
pp. 1009-1020 ◽  
Author(s):  
Patricia Murray ◽  
David Edgar
Keyword(s):  
Cell Differentiation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
The Body ◽  
Embryonic Stem Cell Differentiation ◽  
Es Cell ◽  
Mouse Es Cells ◽  
Intercellular Signalling ◽  
Potential Use

The potential use of pluripotent stem cells for tissue repair or replacement is now well recognized. While the ability of embryonic stem (ES) cells to differentiate into all cells of the body is undisputed, their use is currently restricted by our limited knowledge of the mechanisms controlling their differentiation. This review discusses recent work by ourselves and others investigating the intercellular signalling events that occur within aggregates of mouse ES cells. The work illustrates that the processes of ES cell differentiation, epithelialization and programmed cell death are dependent upon their location within the aggregates and coordinated by the extracellular matrix. Establishment of the mechanisms involved in these events is not only of use for the manipulation of ES cells themselves, but it also throws light on the ways in which differentiation is coordinated during embryogenesis.

scholarly journals RNF219 interacts with CCR4-NOT in regulating stem cell differentiation

2020 ◽  
Author(s):  
Hao Du ◽  
Chen Chen ◽  
Yan Wang ◽  
Yang Yang ◽  
Zhuanzhuan Che ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Es Cells ◽  
Rna Stability ◽  
Embryonic Stem ◽  
Ring Finger ◽  
Stem Cell Differentiation ◽  
Es Cell ◽  
Gene Expression Control ◽  
Neuronal Genes

ABSTRACTRegulation of RNA stability plays a crucial role in gene expression control. Deadenylation is the initial rate-limiting step for the majority of RNA decay events. Here, we show that RING Finger Protein 219 (RNF219) interacts with the CCR4-NOT deadenylase complex. RNF219-CCR4-NOT exhibits deadenylation activity in vitro. RNA-seq analyses identify some of the 2-cell specific genes and the neuronal genes significantly down-regulated upon RNF219 knockdown, while up-regulated after depletion of the CCR4-NOT subunit CNOT10 in mouse embryonic stem (ES) cells. RNF219 depletion leads to impaired neuronal lineage commitment during ES cell differentiation. Our study suggests that RNF219 is a novel interacting partner of CCR4-NOT, and required for maintenance of ES cell pluripotency.

An in situ assessment of the routes and extents of colonisation of the mouse embryo by embryonic stem cells and their descendants

Development ◽  
1990 ◽  
Vol 110 (4) ◽  
pp. 1241-1248 ◽  
Author(s):  
Y. Lallemand ◽  
P. Brulet
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Line ◽  
Mouse Embryo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Low Degree ◽  
Morula Stage

An embryonic stem (ES) cell line stably expressing lacZ under the control of an endogenous promoter has been isolated and used as a marker to follow the fate of ES cells injected into blastocysts and morulae, before midgestation. The results show a multisite pattern of blastocyst colonization by ES cells deposited into the blastocoel cavity and a low degree of mingling between ES cells and ICM cells. Furthermore, analysis of dispersal of ES cell descendants in postimplantation chimaeric embryos showed that colonization can be highly variable from one region of the embryo to another. In contrast, a high and reproducible degree of chimaerism was obtained when the ES cells were injected at the morula stage prior to ICM formation.

scholarly journals Changes in the Distributions and Dynamics of Polycomb Repressive Complexes during Embryonic Stem Cell Differentiation

2008 ◽  
Vol 28 (9) ◽  
pp. 2884-2895 ◽  
Author(s):  
Xiaojun Ren ◽  
Claudius Vincenz ◽  
Tom K. Kerppola
Keyword(s):  
Cell Differentiation ◽  
Protein Localization ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Bimolecular Fluorescence Complementation ◽  
Global Changes ◽  
Embryonic Stem Cell Differentiation ◽  
Es Cell ◽  
A Genome

ABSTRACT Polycomb group (PcG) transcription regulatory proteins maintain cell identity by sustained repression of numerous genes. The differentiation of embryonic stem (ES) cells induces a genome-wide shift in PcG target gene expression. We investigated the effects of differentiation and protein interactions on CBX family PcG protein localization and dynamics by using fluorescence imaging. In mouse ES cells, different CBX proteins exhibited distinct distributions and mobilities. Most CBX proteins were enriched in foci known as Polycomb bodies. Focus formation did not affect CBX protein mobilities, and the foci dispersed during ES cell differentiation. The mobilities of CBX proteins increased upon the induction of differentiation and decreased as differentiation progressed. The deletion of the chromobox, which mediates interactions with RING1B, prevented the immobilization of CBX proteins. In contrast, the deletion of the chromodomain, which can bind trimethylated lysine 27 of histone H3, had little effect on CBX protein dynamics. The distributions and mobilities of most CBX proteins corresponded to those of CBX-RING1B complexes detected by using bimolecular fluorescence complementation analysis. Epigenetic reprogramming during ES cell differentiation is therefore associated with global changes in the subnuclear distributions and dynamics of CBX protein complexes.

scholarly journals Isolation and Initial Application of a Novel Peptide That Specifically Recognizes the Neural Stem Cells Derived from Rhesus Monkey Embryonic Stem Cells

2010 ◽  
Vol 15 (6) ◽  
pp. 687-694 ◽  
Author(s):  
Wenxiu Zhao ◽  
Hang Yuan ◽  
Xing Xu ◽  
Lan Ma
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
Rhesus Monkey ◽  
Neural Stem Cells ◽  
Phage Display ◽  
Es Cells ◽  
Embryonic Stem ◽  
Peptide Sequence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Es Cell

The search for new receptor ligands is important in the study of embryonic stem (ES) cell differentiation processes. In this study, a novel peptide (HGE VPRFHAVHL) with a specific ability to bind with neural stem cells derived from rhesus monkey ES cells was successfully screened out using a Ph.D-12 peptide phage display library. High affinity and specificity of the HGE phage were shown in an enzyme-linked immunosorbent assay. The binding ability of the phage could be matched with that of a chemically synthesized peptide with a sequence identical to that displayed by the phage, indicating that this binding capability manifests a peptide sequence. Combined with quantum dots, the HGE peptide can be used as a direct tool to show optical imaging of specific binding on a single cell membrane. Further results of Western blot showed that the HGE peptide interacted with 48/34-kDa proteins on the membrane of neural stem cells. This work is the first time that a phage display technique has been applied in ES cell differentiation studies. The findings extend the utilization of a targeting agent for neural stem cells and can also be used as a research tool in studying other cell lineages derived from ES cells.

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