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Results for “Mouse Embryonic Stem Cells”

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PubMed1999

Human XIST yeast artificial chromosome transgenes show partial X inactivation center function in mouse embryonic stem cells.

Initiation of X chromosome inactivation requires the presence, in cis, of the X inactivation center (XIC). The Xist gene, which lies within the XIC region in both human and mouse and has the unique property of being expressed only from the inactive X chromosome in female somatic cells, is known to be essential for X inactivation based on targeted deletions in the mouse. Although our understanding of the developmental regulation and function of the mouse Xist gene has progressed rapidly, less is known about its human homolog. To address this and to assess the cross-species conservation of X inactivation, a 480-kb yeast artificial chromosome containing the human XIST gene was introduced into mouse embryonic stem (ES) cells. The human XIST transcript was expressed and could coat the mouse autosome from which it was transcribed, indicating that the factors required for cis association are conserved in mouse ES cells. Cis inactivation as a result of human XIST expression was found in only a proportion of differentiated cells, suggesting that the events downstream of XIST RNA coating that culminate in stable inactivation may require species-specific factors. Human XIST RNA appears to coat mouse autosomes in ES cells before in vitro differentiation, in contrast to the behavior of the mouse Xist gene in undifferentiated ES cells, where an unstable transcript and no chromosome coating are found. This may not only reflect important species differences in Xist regulation but also provides evidence that factors implicated in Xist RNA chromosome coating may already be present in undifferentiated ES cells.

Animals↗

PubMed2005

Mouse embryonic stem cell colonisation of carbonated apatite surfaces.

Apatites play a crucial role in the body and have been used extensively in biomedical implants. The influence on stem cell behaviour is not known and so this study will explore whether sintered carbonated apatites are favourable for propagation of stem cells. Different weight substitutions of carbonated apatite, specifically 2.5 wt% (2.5 wt%CAP) and 5 wt% (5 wt%CAP), were sintered and characterised prior to the investigation of their potential as a matrix for the support of mouse embryonic stem (ES) cells. Characterisation of the apatites included elemental analysis, X-ray diffraction, surface roughness, specific surface area, density, and solubility. The ability of carbonated apatite to support mouse ES cell colonisation and maintenance in the presence of leukaemia inhibitory factor was determined by an enumeration of live versus dead cells within a population, and immunoreactivity to Oct4, a transcription factor and stem cell marker, following growth on each matrix. It was found that while both compositions allowed for the colonisation of mouse ES cells, the cells were not maintained in an undifferentiated state, as evidenced by a reduction in the number of cells staining positive for Oct4 expression. This study shows that an increase in carbonate content within sintered apatites leads to a higher cell number, a desired aspect for stem cells to populate scaffolds intended for tissue engineering. This study presents carbonated apatites as a suitable matrix for the initial colonisation and differentiation of ES cells for tissue engineering applications.

Animals↗

PubMed2004

Msh2 deficiency does not contribute to cisplatin resistance in mouse embryonic stem cells.

Several reports have suggested that a defect in the DNA mismatch repair (MMR) system not only causes resistance to methylating agents but also confers low-level resistance to the chemotherapeutic drug cisplatin. Here we report that in a clonogenic assay, mouse embryonic stem (ES) cells deficient for the MMR protein MSH2 respond similarly as wild-type cells to cisplatin. Furthermore, restoring MSH2 expression in a cisplatin-resistant subclone selected from an Msh2(-/-) cell population did not sensitize cells to cisplatin. To ascertain that our observations were not the result of a mutation in the Msh2(-/-) cells that obscured the contribution of a defective MMR machinery to cisplatin resistance, we made use of the Cre-lox system to create a cell line in which the Msh2 gene can be conditionally inactivated. However, while de novo inactivation of Msh2 rendered cells tolerant to the methylating drug N-methyl-N'-nitro-N-nitrosoguanidine as expected, it did not alter the sensitivity to cisplatin. In addition, we were not able to derive cisplatin-resistant subclones from this freshly generated MMR-deficient cell line. Thus, in ES cells we did not find evidence for direct involvement of MMR deficiency in cisplatin resistance.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Inhibition of the ATR/Chk1 pathway induces a p38-dependent S-phase delay in mouse embryonic stem cells.

Ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases, family members of the PI-3 kinase related proteins, play a key role in checkpoint activation and maintenance of genomic stability following DNA damage. We have used wild type (WT) and p38alpha-deficient mouse embryonic stem (ES) cells to investigate the role of ATR and ATM kinases during embryonic cell cycle. We have found that inhibition of ATR and ATM kinases with caffeine or Chk1 with UCN-01, results in activation of a p38-dependent intra-S-phase checkpoint and activation of apoptosis in ES cells. However, wortmannin at a concentration, that inhibits ATM kinase but not ATR kinase, did not affect cell cycle progression. Furthermore, the presence of caffeine results in activation of p38 kinase, accumulation of p21/Waf1 in a complex with Cdk2 and decrease of Cdk2 kinase activity. In contrast, caffeine-treated p38alpha-/- ES cells show less apoptosis, and fail to trigger an effective S-phase checkpoint and accumulation of p21/Waf1. We conclude that ATR kinase activity is essential for normal cell cycle progression of exponentially proliferating mouse ES cells even in the absence of exogenous DNA damage, and ATR deregulation triggers p38alpha-dependent cell-cycle checkpoint and apoptotic responses.

Animals↗

PubMed1992

Targeted integration of the Ren-1D locus in mouse embryonic stem cells.

We have introduced a Ren-1D targeting vector into embryonic stem cells containing the two highly homologous mouse renin genes Ren-1D and Ren-2. Using a polymerase chain reaction (PCR) screen designed to detect targeted integration at Ren-1D and Ren-2, we isolated 15 targeted embryonic stem cell clones, all of which had undergone a gene conversion event at the Ren-1D locus. We did not isolate any clones in which the incoming DNA had recombined with Ren-2. Over the region encompassed by our transgene, Ren-1D and Ren-2 display greater than 95% homology. Our results suggest that the machinery driving gene targeting by means of homologous recombination in mammalian cells is capable of distinguishing between these two sequences. Construction of transgenic mice with the embryonic stem cells reported here carrying a mutated renin gene will permit a greater understanding of the functions of the Ren-1D and Ren-2 gene products and their relative contribution to cardiovascular homeostasis.

Animals↗

PubMed2025

Analysis of a BCOR internal tandem duplication in mouse embryonic stem cell to neuronal precursor differentiation.

BCOR (BCL-6 corepressor) is a component of variant Polycomb Repressive Complex 1.1 (vPRC1.1), one of several vPRC1 complexes that catalyze histone H2A ubiquitination thought to play an important role in PRC2 binding and the deposition of H3K27me3 to silence genes. The PCGF Ub-like fold discriminator (PUFD) domain at the C-terminus of BCOR forms a heterodimer with PCGF1, serving as a critical interface for its polycomb-dependent functions. Internal tandem duplication (ITD) mutations in BCOR, causing in-frame duplications of 20 to 40 amino acids in the PUFD domain, are observed in heterogeneous tumors including sarcomas (kidney, bone, and endometrium) and neuroepithelial tumors in the brain. To dissect the molecular mechanisms underlying aberrant function of BCOR-ITD mutants, we employed mouse embryonic stem (mES) cells expressing either transgenic or endogenous BCOR-ITD. Our results indicate that the BCOR-ITD mutation does not disrupt the BCOR-PCGF1 interaction, instead maintaining the integrity of the vPRC1.1 complex. While displaying subtle changes in imprinted gene expression during differentiation toward a neural lineage, BCOR-ITD mutants also had no growth phenotype in culture. Furthermore, we found that CD24+ cells were enriched, as expected, during neural progenitor differentiation in both wildtype and mutant cells. However, sensitization of BCOR-ITD mES cells with EZH2 inhibitor during differentiation resulted in an unexpected enrichment of a CD24+CD26+ subpopulation, indicating aberrant cell fate that was also prevalent in a BCOR truncation mutant. Together, our results suggest that BCOR-ITD may largely retain wildtype function, but with increased susceptibility to synergistic stress on the Polycomb pathway.

Animals↗

PubMed2005

Stromal cell-derived inducing activity, Nurr1, and signaling molecules synergistically induce dopaminergic neurons from mouse embryonic stem cells.

To induce differentiation of embryonic stem cells (ESCs) into specialized cell types for therapeutic purposes, it may be desirable to combine genetic manipulation and appropriate differentiation signals. We studied the induction of dopaminergic (DA) neurons from mouse ESCs by overexpressing the transcription factor Nurr1 and coculturing with PA6 stromal cells. Nurr1-expressing ESCs (N2 and N5) differentiated into a higher number of neurons (approximately twofold) than the naïve ESCs (D3). In addition, N2/N5-derived cells contained a significantly higher proportion (>50%) of tyrosine hydroxylase (TH)+ neurons than D3 (<30%) and an even greater proportion of TH+ neurons (approximately 90%) when treated with the signaling molecules sonic hedgehog, fibroblast growth factor 8, and ascorbic acid. N2/N5-derived cells express much higher levels of DA markers (e.g., TH, dopamine transporter, aromatic amino acid decarboxylase, and G protein-regulated inwardly rectifying K+ channel 2) and produce and release a higher level of dopamine, compared with D3-derived cells. Furthermore, the majority of generated neurons exhibited electrophysiological properties characteristic of midbrain DA neurons. Finally, transplantation experiments showed efficient in vivo integration/generation of TH+ neurons after implantation into mouse striatum. Taken together, our results show that the combination of genetic manipulation(s) and in vitro cell differentiation conditions offers a reliable and effective induction of DA neurons from ESCs and may pave the way for future cell transplantation therapy in Parkinson's disease.

Animals↗

PubMed1995

Targeted mutagenesis of the transcription factor GATA-4 gene in mouse embryonic stem cells disrupts visceral endoderm differentiation in vitro.

Transcription factor GATA-4 belongs to a family of zinc finger proteins involved in lineage determination. GATA-4 is first expressed in yolk sac endoderm of the developing mouse and later in cardiac tissue, gut epithelium and gonads. To delineate the role of this transcription factor in differentiation and early development, we studied embryoid bodies derived from mouse embryonic stem (ES) cells in which both copies of the Gata-4 gene were disrupted. Light and electron microscopy demonstrated that embryoid bodies formed from wild-type and heterozygous deficient ES cells were covered with a layer of visceral yolk sac endoderm, whereas no yolk sac endoderm was evident on the surface of the homozygous deficient embryoid bodies. Independently selected homozygous deficient cell lines displayed this distinctive phenotype, suggesting that it was not an artifact of clonal variation. Biochemical markers of visceral endoderm formation, such as alpha-feto-protein, hepatocyte nuclear factor-4 and binding sites for Dolichos biflorus agglutinin, were absent from the homozygous deficient embryoid bodies. Examination of other differentiation markers in the mutant embryoid bodies, studies of ES cell-derived teratocarcinomas and chimeric mouse analysis demonstrated that GATA-4-deficient ES cells have the capacity to differentiate along other lineages. We conclude that, under in vitro conditions, disruption of the Gata-4 gene results in a specific block in visceral endoderm formation. These homozygous deficient cells should yield insights into the regulation of yolk sac endoderm development and the factors expressed by visceral endoderm that influence differentiation of adjoining ectoderm/mesoderm.

Animals↗

PubMed1996

A novel strategy for the negative selection in mouse embryonic stem cells operated with immunotoxin-mediated cell targeting.

Immunotoxoin-mediated cell targeting (IMCT) is a technique for conditionally ablating specific cell types based on the cytotoxic activity of a recombinant immunotoxin anti-Tac (Fv)-PE40. To examine the feasibility of this technique for the negative selection in mouse embryonic stem (ES) cells, we investigated the responsiveness of cells expressing human interleukin-2 receptor alpha subunit to anti-Tac(Fv)-PE40. The immunotoxin treatment efficiently eliminated only ES cells bearing the receptor as a consequence of the target specificity of anti-Tac(Fv)-PE40, indicating that IMCT can be used as a novel strategy for positive and negative selection to enrich ES cell clones with a targeted mutation.

ADP Ribose Transferases↗

PubMed2005

Phenotype-specific cells with proliferative potential are produced by polyethylene glycol-induced fusion of mouse embryonic stem cells with fetal cardiomyocytes.

Because cardiomyocytes lose the ability to divide upon differentiation, myocardial failure is assumed to be generally irreversible. For terminal cardiac insufficiency, the potential for regenerative treatment by stem cells, especially embryonic stem (ES) cells, offers hope for the future. Recent studies showed that stem cells fuse spontaneously with cells remaining in damaged tissues, and restore tissue function. To imitate spontaneous fusion in vivo, we used polyethylene glycol (PEG) in vitro to fuse mouse ES cells and fetal cardiomyocytes and analyzed the cytochemical properties of the fused cells. Confocal laser scanning microscopy coupled with lipophilic dye labeling of the living cell membranes showed that there were fused cells of ES cells and cardiomyocytes after PEG treatment. By flow cytometry, the fusion efficiency between ES cells and cardiomyocytes was estimated to be about 45% of the total resulting cells. When green fluorescent protein (GFP)-expressing ES cells were fused with cardiomyocytes, the fused cells had immunoreactivity for GFP in their cytoplasm and cardiac troponin I in their myofibrils. Some of these cells also expressed proliferating cell nuclear antigen up to 11 days after fusion, the last time point examined. This study shows that PEG-induced fusions of mouse ES cells and cardiomyocytes have the cardiomyocyte phenotype and proliferation potential.

Animals↗

PubMed2004

Requirement of MKK4 and MKK7 for CdCl2- or HgCl2-induced activation of c-Jun NH2-terminal kinase in mouse embryonic stem cells.

c-Jun NH(2)-terminal kinase (JNK), also known as stress-activated protein kinase (SAPK), is activated primarily by inflammatory cytokines and environmental stresses including toxic metal exposure. To reveal the upstream kinase responsible for JNK activation by toxic metals, the phosphorylation status and the activity of JNK were examined in mouse embryonic stem (ES) cells lacking MKK4 or MKK7 following exposure to CdCl(2) or HgCl(2). Treatment with CdCl(2) or HgCl(2) induced the phosphorylation of JNK in a dose- and time-dependent manner in wild-type ES cells. In both mkk4(-/-) and mkk7(-/-) ES cells, CdCl(2)- or HgCl(2)-induced phosphorylation and activation of JNK were suppressed significantly. However, in mkk7(-/-) ES cells treated with CdCl(2) and HgCl(2), JNK activation was not abolished (suppressed by 56% and 78%, respectively). These findings suggest that the full activation of JNK by toxic metal exposure requires both MKK4 and MKK7, and these upstream kinases might contribute differentially in JNK activation between mouse ES cells exposed to CdCl(2) and HgCl(2).

Animals↗

PubMed2006

Enrichment of cardiomyocytes derived from mouse embryonic stem cells.

BACKGROUND: Embryonic stem (ES) cell-derived cardiomyocytes transplantation and tissue engineering together represent a promising approach for the treatment of myocardial infarction, despite the limited supply of cardiac myocytes. This study examines whether functional cardiomyocytes can be efficiently enriched from mouse embryonic stem (mES) cells. METHODS: mES cells were induced by ascorbic acid to differentiate into cardiomyocytes. Beating cells were observed after 1 week and increased in number with time while under differentiation conditions. Furthermore, the differentiated cultures could be dissociated and enriched by Percoll gradient density centrifugation. RESULTS: The beating cells expressed markers characteristic of cardiomyocytes, such as cardiac troponin T (cTnT). The enriched population contained 88.7% cardiomyocytes and showed expression of cardiomyocyte markers of troponin T and cardiac genes, including alpha-MHC, beta-MHC, ANF and Nkx2.5. However, Oct-4, a marker of early-stage ES cells, was not expressed in the mES cell-derived cardiac cell clusters. Moreover, the mES cell-derived and Percoll-enriched cardiomyocytes responded appropriately to cardioactive drugs, as did normal neonatal rat cardiomyocytes. CONCLUSIONS: mES cell-derived functional cardiomyocytes can be enriched by the method of discontinuous Percoll gradient centrifugation. The ability to differentiate and enrich for functional mouse cardiomyocytes makes it possible for further development of these cells as a model of myocardial repair through cell transplantation or tissue engineering.

Animals↗

PubMed2005

Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing.

Dicer is the enzyme that cleaves double-stranded RNA (dsRNA) into 21-25-nt-long species responsible for sequence-specific RNA-induced gene silencing at the transcriptional, post-transcriptional, or translational level. We disrupted the dicer-1 (dcr-1) gene in mouse embryonic stem (ES) cells by conditional gene targeting and generated Dicer-null ES cells. These cells were viable, despite being completely defective in RNA interference (RNAi) and the generation of microRNAs (miRNAs). However, the mutant ES cells displayed severe defects in differentiation both in vitro and in vivo. Epigenetic silencing of centromeric repeat sequences and the expression of homologous small dsRNAs were markedly reduced. Re-expression of Dicer in the knockout cells rescued these phenotypes. Our data suggest that Dicer participates in multiple, fundamental biological processes in a mammalian organism, ranging from stem cell differentiation to the maintenance of centromeric heterochromatin structure and centromeric silencing.

Animals↗

PubMedDate not supplied

Approaching the multifaceted nature of energy metabolism: inactivation of the cytosolic creatine kinases via homologous recombination in mouse embryonic stem cells.

To study the physiological role of the creatine kinase/phosphocreatine (CK/PCr) system in cells and tissues with a high and fluctuating energy demand we have concentrated on the site-directed inactivation of the B- and M-CK genes encoding the cytosolic CK protein subunits. In our approach we used homologous recombination in mouse embryonic stem (ES) cells from strain 129/Sv. Using targeting constructs based on strain 129/Sv isogenic DNA we managed to ablate the essential exons of the B-CK and M-CK genes at reasonably high frequencies. ES clones with fully disrupted B-CK and two types of M-CK gene mutations, a null (M-CK-) and leaky (M-CK1) mutation, were used to generate chimaeric mutant mice via injection in strain C57BL/6 derived blastocysts. Chimaeras with the B-CK null mutation have no overt abnormalities but failed to transmit the mutation to their offspring. For the M-CK- and M-CK1 mutations successful transmission was achieved and heterozygous and homozygous mutant mice were bred. Animals deficient in MM-CK are phenotypically normal but lack muscular burst activity. Fluxes through the CK reaction in skeletal muscle are highly impaired and fast fibres show adaptation in cellular architecture and storage of glycogen. Mice homozygous for the leaky M-CK allele, which have 3-fold reduced MM-CK activity, show normal fast fibres but CK fluxes and burst activity are still not restored to wildtype levels.

Animals↗

PubMed2004

[Establishing mouse embryonic stem cell line carrying a fluorescent undifferentiated marker].

To label mouse ES cells,a cell line derived from the inner cell mass of 3.5-day blastocysts,with enhanced green fluorescent protein (EGFP), the vector of pRex-1-EGFP was transferred into mouse ES cells by electroporation. The expressions of Rex-1 in undifferentiated and differentiated ES cells were detected by the microscopic observation of EGFP and by RT-PCR. The results showed that the EGFP gene was transferred into the mouse ES cell line, and the transfected cells in undifferentiated state showed high levels of EGFP expression. When the cells began to differentiate, the EGFP expressions were gradually reduced. A mouse ES cell line expressing EGFP under the control of Rex-1 gene promoter was generated. The cell line provides a powerful approach for the research of the process of mammalian development and for the screening of small molecules that can regulate this process.

Animals↗

PubMed1999

Factors affecting de novo methylation of foreign DNA in mouse embryonic stem cells.

Integration of foreign DNA into an established host genome can lead to changes in methylation in both the inserted DNA and in host sequences and potentially alters transgene and cellular transcription patterns. This work addresses the questions of what factors influence de novo methylation, and whether the integration site or inserted DNA can affect de novo methylation. Homologous recombination was used to integrate foreign DNA into a specific gene, B lymphocyte kinase (BLK), in mouse embryonic stem (ES) cells. Two plasmids were chosen for integration; one contained the adenovirus type 2 E2AL promoter upstream of the luciferase reporter gene, and the second carried the early SV40 promoter. The methylation patterns were analyzed using HpaII and MspI restriction endonucleases for both homologously recombined and randomly integrated foreign DNA in the ES cell clones. Upon homologous reinsertion of the BLK gene into the genome of mouse ES cells, methylation patterns in this gene were reestablished. In DNA segments adjoined to the BLK gene, the de novo patterns of DNA methylation depended on the viral sequences in these clones and on the locations of the inserts, i.e. on whether the insertions resulted from homologously recombined or randomly integrated foreign DNA. In homologously recombined DNA, sequences carrying the adenovirus type 2 promoter were heavily methylated, and those with an SV40 promoter and an SV40 enhancer element remained unmethylated or hypomethylated. Upon removal of the enhancer element, these inserted constructs also became heavily methylated. In addition, all randomly integrated constructs were heavily methylated independently of the promoter and enhancer element present in the construct. These results indicate that modes and sites of integration as well as the inserted nucleotide sequence, possibly promoter strength, are factors affecting de novo methylation.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Mouse ubiquitous mitochondrial creatine kinase: gene organization and consequences from inactivation in mouse embryonic stem cells.

Individual members of the creatine kinase isoenzyme family (CK; EC 2.7.3.2), which play a prominent role in energy homeostasis, are encoded by four separate nuclear genes. We have isolated and characterized the complete mouse UbCKmit gene, the product of which is ubiquitously expressed and is located in the intermembrane space of mitochondria. Transcription of this gene is initiated at multiple adjacent positions and the region immediately upstream of these sites shares many features with genes encoding housekeeping proteins. These include a high G/C content, absence of TATA and CCAAT motifs, and presence of SP1 and AP2 recognition sequences. In addition, a binding site for HIP1, hormone-responsive elements, and three Mt-motifs, known as boxes shared between nuclear genes encoding mitochondrial proteins, were identified. To study the functional role of the UbCKmit protein, we have inactivated both UbCKmit alleles in mouse embryonic stem (ES) cells. UbCKmit-deficient cells, obtained by consecutive rounds of gene targeting using homologous recombination and drug selection-driven gene conversion events, show no obvious growth disadvantage or abnormal differentiation potential. Activities of mitochondrial cytochrome c oxidase and citrate synthase, as well as the rate of pyruvate oxidation, showed values equal to wild-type cells, indicating a normal aerobic metabolism. Mitochondria of in vivo differentiated knock-out cells were structurally intact, as demonstrated by electron microscopy. Approaches to study the role of the UbCKmit gene further are discussed.

Animals↗

PubMed1990

Mouse embryonic stem cells express the cardiac myosin heavy chain genes during development in vitro.

In the mouse embryo, early organogenesis is characterized by the formation of a functional cardiac muscle, such that 9-day embryos exhibit beating, although not fully developed hearts. In light of this observation, we found it intriguing that mouse embryoid bodies (EB), which can develop in vitro from totipotential embryonic stem cells, undergo spontaneous contractile activity. To determine if these cells are capable of recapitulating aspects of cardiogenesis, a cDNA library was prepared from beating EB and screened with a chicken skeletal myosin heavy chain cDNA. We found that the predominant myosin transcripts in the library encode the alpha- and beta-cardiac isoforms. In addition, an embryonic skeletal myosin cDNA was isolated. The myosin heavy chain transcripts in both EB and 9-day embryonic hearts were found to be the same. Transcript-specific primers were prepared, and polymerase chain reaction analyses on single EB were carried out. The data show that a single EB is capable of expressing both the alpha- and beta-isoforms as well as very low amounts of the embryonic skeletal transcript. These data indicate that EB transcribe the appropriate tissue- and developmental stage-specific myosin heavy chain genes and therefore serve as a model system for studying early cardiogenic processes at the molecular level.

Animals↗