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Results for “Mouse Embryonic Stem Cells”

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PubMed1998

Disruption of genes regulated during hematopoietic differentiation of mouse embryonic stem cells.

A retroviral gene trap vector (U3Tkneo) that selects for integrations in or near expressed 5' exons has been used to identify genes that are repressed during hematopoietic differentiation of mouse totipotent embryonic stem cells. The vector contains coding sequences for an HSV-thymidine kinase/neomycin phosphotransferase fusion protein in the U3 region of a Moloney murine leukemia virus LTR and allows selection for (G418) and against (Ganciclovir; GC) U3 gene expression. A total of 208 neomycin-resistant clones were isolated following infection with U3tkneo and screened for integrations into regulated genes by using a two-step, semisolid culture system that supports hematopoietic differentiation. Two clones contained U3Tkneo integrations in genes that were repressed selectively in hematopoietic cells. Analysis of upstream proviral flanking sequences indicated that both integrations occurred into unknown genes. One up-stream sequence identified a cellular transcript that was expressed differentially in the kidneys and liver of adult mice. When this fusion gene was passaged to the germ line, homozygous offspring with nearly null mutations were obtained. However, mutant mice were normal, suggesting that potential loss of function phenotypes are subtle and may be restricted to the kidneys and the liver.

Animals↗

PubMed2004

Complementation of replication origin function in mouse embryonic stem cells by human DNA sequences.

A functional origin of replication was mapped to the transcriptional promoter and exon 1 of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in the mouse and human genomes. This origin was lost in mouse embryonic stem (ES) cells with a spontaneous deletion (approximately 36 kb) at the 5' end of the HPRT locus. Restoration of HPRT activity by homologous recombination with human/mouse chimeric sequences reconstituted replication origin activity in two independent ES cell lines. Quantitative PCR analyses of abundance of genetic markers in size-fractionated nascent DNA indicated that initiation of DNA replication coincided with the site of insertion in the mouse genome of the 335 bp of human DNA containing the HPRT exon 1 and a truncated promoter. The genetic information contained in the human sequence and surrounding mouse DNA was analyzed for cis-acting elements that might contribute to selection and functional activation of a mammalian origin of DNA replication.

Animals↗

PubMed1989

Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes.

A strategy was devised for identifying regions of the mouse genome that are transcriptionally active in a temporally and spatially restricted manner during development. The approach is based on the introduction into embryonic stem cells of two types of lacZ reporter constructs that can be activated by flanking mouse genomic sequences. Embryonic stem cells containing the lacZ constructs were used to produce chimaeric mice. Developmental regulation of lacZ expression occurred at a high frequency. Molecular cloning of the flanking endogenous genes and introduction of these potential insertional mutations into the mouse germ line should provide an efficient means of identifying and mutating novel genes important for the control of mammalian development.

Animals↗

PubMed1994

Gene replacement of the p53 gene with the lacZ gene in mouse embryonic stem cells and mice by using two steps of homologous recombination.

Two steps of gene targeting were used to replace the p53 gene with the E. coli beta-galactosidase (lacZ) gene in mouse embryonic stem (ES) cells. The first targeting vector consisted of neo and herpes simplex virus thymidine kinase (HSV-tk) genes as a neo-tk cassette in the middle of the targeting vector. At the first targeting, the homologous recombinants became G418 resistant and ganciclovir (GANC) sensitive and were selected by G418 alone. At the second targeting, homologous recombination reciprocally exchanged the neo-tk casette in the ES cell chromosome with the lacZ fragment in the second targeting vector and thus made the ES cells GANC resistant. We obtained two ES cell clones, in which the p53 gene for both had been replaced with a totally non-homologous sequence of the lacZ gene. The germ-line transmission of the manipulated ES cells also demonstrated that the entire procedure had no detrimental effects on ES cells at all.

Animals↗

PubMed2004

Differences between human and mouse embryonic stem cells.

We compared gene expression profiles of mouse and human ES cells by immunocytochemistry, RT-PCR, and membrane-based focused cDNA array analysis. Several markers that in concert could distinguish undifferentiated ES cells from their differentiated progeny were identified. These included known markers such as SSEA antigens, OCT3/4, SOX-2, REX-1 and TERT, as well as additional markers such as UTF-1, TRF1, TRF2, connexin43, and connexin45, FGFR-4, ABCG-2, and Glut-1. A set of negative markers that confirm the absence of differentiation was also developed. These include genes characteristic of trophoectoderm, markers of germ layers, and of more specialized progenitor cells. While the expression of many of the markers was similar in mouse and human cells, significant differences were found in the expression of vimentin, beta-III tubulin, alpha-fetoprotein, eomesodermin, HEB, ARNT, and FoxD3 as well as in the expression of the LIF receptor complex LIFR/IL6ST (gp130). Profound differences in cell cycle regulation, control of apoptosis, and cytokine expression were uncovered using focused microarrays. The profile of gene expression observed in H1 cells was similar to that of two other human ES cell lines tested (line I-6 and clonal line-H9.2) and to feeder-free subclones of H1, H7, and H9, indicating that the observed differences between human and mouse ES cells were species-specific rather than arising from differences in culture conditions.

Animals↗

PubMed1992

Rescue of erythroid development in gene targeted GATA-1- mouse embryonic stem cells.

Development of definitive (fetal liver-derived) red cells is blocked by a targeted mutation in the gene encoding the transcription factor GATA-1. We used in vitro differentiation of GATA-1- mouse embryonic stem (ES) cells to reveal a requirement for GATA-1 during primitive (yolk sac-derived) erythropoiesis and to establish a rescue assay. We show that the block to development includes primitive, as well as definitive, erythroid cells and is complete at the level of globin RNA expression; that the introduction of a normal GATA-1 gene restores developmental potential both in vivo and in vitro; and that efficient rescue is dependent on a putative autoregulatory GATA-motif in the distal promoter. Use of in vitro differentiated ES cells bridges a gap between conventional approaches to gene function in cell lines and analysis of loss of function mutations in the whole animal.

Animals↗

PubMed2006

Effects of oxytocin on cardiomyocyte differentiation from mouse embryonic stem cells.

This study sought to investigate the presence of oxytocin receptors and the possible biological role of oxytocin as an effective factor in the differentiation of embryonic stem cells (ESCs) into cardiomyocytes. Mouse ESCs were cultivated in hanging drops to form embryoid bodies (EBs). The EBs were then treated with and without oxytocin (experimental and control groups). Up to 30 days after plating, contraction and beating frequency were monitored and evaluated daily. The growth characteristics of the ESC-derived cardiomyocytes were assessed by cardioactive drugs, immunocytochemistry, transmission electron microscopy (TEM) and reverse transcription-polymerase chain reaction (RT-PCR). In the experimental group, the percentage of the EBs with spontaneous contraction was significantly increased from 17th day onward. The spontaneous beating frequency of each EB in both groups was also changed with cardioactive drugs such as Bay K, carbachol, isopernaline and phenylephrine. However, in the experimental group, changes with isopernaline were more pronounced at the early and intermediate stages of cardiomyocyte development. The beating cells of both groups, stained positive with anti alpha-actinin, desmin, cardiac troponin I and connexin antibodies, and revealed similar ultrastructural features. Oxytocin receptors were detected on the ESCs and derived-differentiated cells. In addition, cardiac-specific genes such as cardiac alpha- and beta-myosin heavy chain, myosin light chain-2v, and atrial natriuretic factor were also detected in the ESC-derived differentiated cells of both groups. In the experimental group, all the specific genes, with the exception of alpha-myosin heavy chain, were more pronounced at the early stage of cardiomyocyte development. In conclusion, oxytocin has receptors on undifferentiated ESCs and derived differentiated cells, and in spite of better improvement of the EBs with spontaneous contraction, it can only promote the early maturation of ESC-derived cardiomyocytes in terms of chronotropic responses and expression of cardiac-specific genes, and have no effect on ultrastructural characteristics of cardiomyocytes in any stage of development.

Animals↗

PubMedDate not supplied

Effect of nicotine on Oct-4 and Rex-1 expression of mouse embryonic stem cells.

To examine the influence that nicotine exposure has on early embryo development, the present study has applied real-time RT-PCR to investigate changes in Oct-4 and Rex-1 expression in mouse embryonic stem (ES) cells exposed to nicotine alone or in the presence of tubocurarine. Oct-4 is regarded as a candidate master regulator gene for the initiation, maintenance, and differentiation of pluripotent cells. Zfp42/Rex-1, another specific gene of pluripotent cells, also plays a critical role in maintaining stem cell character and pluripotency. Results indicated that nicotine (10-1000 nM) enhanced Oct-4 and Rex-1 expression without altering beta-actin expression. This up-regulation with nicotine (100-1000 nM) was prevented with tubocurarine (10 microM), a nicotinic acetylcholine receptor (nAChRs) antagonist. We conclude that nicotine may influence Oct-4 and Rex-1 expression in ES cells through a mechanism involving the nAChRs.

Animals↗

PubMed2003

Mutator phenotype of MUTYH-null mouse embryonic stem cells.

To evaluate the antimutagenic role of a mammalian mutY homolog, namely the Mutyh gene, which encodes adenine DNA glycosylase excising adenine misincorporated opposite 8-oxoguanine in the template DNA, we generated MUTYH-null mouse embryonic stem (ES) cells. In the MUTYH-null cells carrying no adenine DNA glycosylase activity, the spontaneous mutation rate increased 2-fold in comparison with wild type cells. The expression of wild type mMUTYH or mutant mMUTYH protein with amino acid substitutions at the proliferating cell nuclear antigen binding motif restored the increased spontaneous mutation rates of the MUTYH-null ES cells to the wild type level. The expression of a mutant mMUTYH protein with an amino acid substitution (G365D) that corresponds to a germ-line mutation (G382D) found in patients with multiple colorectal adenomas could not suppress the elevated spontaneous mutation rate of the MUTYH-null ES cells. Although the recombinant mMUTYH(G365D) purified from Escherichia coli cells had a substantial level of adenine DNA glycosylase activity as did wild type MUTYH, no adenine DNA glycosylase activity was detected in the MUTYH-null ES cells expressing the mMUTYH(G365D) mutant protein. The germ-line mutation (G382D) of the human MUTYH gene is therefore likely to be responsible for the occurrence of a mutator phenotype in these patients.

Amino Acid Motifs↗

PubMed1998

Impact of tumor necrosis factor alpha on mouse embryonic stem cells.

Previous studies have shown the adverse impact of the cytokine tumor necrosis factor alpha (TNFalpha) on the development of the inner cell mass in mouse blastocysts. In the present study, two embryonic stem (ES) cell lines were used to further investigate the action of TNFalpha. The expression of TNFalpha receptors in ES cells was tested by reverse transcription-polymerase chain reaction and Northern blot analysis. Transcripts encoding the two distinct receptor isoforms were detected in these cells. Using different approaches, our data showed a TNFalpha dose-dependent decrease in the number of ES cells after 24 h of exposure. Simultaneous blocking of the two receptors with antagonist antibodies was needed to completely abrogate the inhibitory effect of the cytokine. Extensive DNA nicks (visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling [TUNEL] method), but not nuclear fragmentation, was found with a higher incidence in ES cells exposed to TNFalpha. The possibility that TNFalpha may stimulate ES cell differentiation was investigated with a test based on the expression of alkaline phosphatase. The results indicated that TNFalpha cannot over-ride the negative control exerted by leukemia inhibitory factor on differentiation. The opposite possibility, that TNFalpha blocks differentiation, was tested in suspended medium drops. In this system, TNFalpha was found to decrease the ability of ES cells to differentiate into embryoid bodies. In addition, expression of Rex-1, a marker gene for undifferentiated ES cells, was increased in ES cells exposed to TNFalpha. Thus our data support the hypothesis that TNFalpha is a significant (negative) effector of proliferation and differentiation in inner cell mass-derived ES cells.

Animals↗

PubMed1998

Selective disruption of genes transiently induced in differentiating mouse embryonic stem cells by using gene trap mutagenesis and site-specific recombination.

A strategy employing gene trap mutagenesis and site-specific recombination (Cre/loxP) has been used to identify genes that are transiently expressed during early mouse development. Embryonic stem cells expressing a reporter plasmid that codes for neomycin phosphotransferase and Escherichia coli LacZ were infected with a retroviral gene trap vector (U3Cre) carrying coding sequences for Cre recombinase (Cre) in the U3 region. Activation of Cre expression from integrations into active genes resulted in a permanent switching between the two selectable marker genes and consequently the expression of beta-galactosidase (beta-Gal). As a result, clones in which U3Cre had disrupted genes that were only transiently expressed could be selected. Moreover, U3Cre-activating cells acquired a cell autonomous marker that could be traced to cells and tissues of the developing embryo. Thus, when two of the clones with inducible U3Cre integrations were passaged in the germ line, they generated spatial patterns of beta-Gal expression.

Animals↗

PubMed2003

Stable expression of hrGFP by mouse embryonic stem cells: promoter activity in the undifferentiated state and during dopaminergic neural differentiation.

Three promoters, cellular polypeptide chain elongation factor 1 alpha (EF1), cytomegalovirus (CMV), and Rous sarcoma virus (RSV) were examined for stable transgene expression in mouse embryonic stem (ES) cells and their progeny during dopaminergic neural differentiation. In undifferentiated ES cells the EF1 promoter was highly effective, while CMV had moderate activity. After 3 months in culture, expression of humanized renilla green fluorescent protein (hrGFP) was unchanged for the EF1 promoter and decreased for CMV. At the nestin-positive stage of differentiation, hrGFP and nestin were colocalized in about 20% of cells for EF1, in contrast to 80% of cells for the CMV promoter. In tyrosine hydroxylase (TH)-positive neurons neither the EF1 nor CMV promoter were effective. The RSV promoter was inactive in undifferentiated, nestin-positive, and TH-positive cells. Thus, EF1 and CMV are effective promoters for transgene expression in undifferentiated ES cells and nestin-positive neural precursors.

Animals↗

PubMed2001

Efficient Cre-loxP-induced mitotic recombination in mouse embryonic stem cells.

FLP/FRT-induced mitotic recombination provides a powerful method for creating genetic mosaics in Drosophila and for discerning the function of recessive genes in a heterozygous individual. Here we show that mitotic recombination can be reproducibly induced in mouse embryonic stem (ES) cells, by Cre/loxP technology, at frequencies ranging from 4.2 x 10(-5) (Snrpn) to 7.0 x 10(-3) (D7Mit178) for single allelic loxP sites, and to 5.0 x 10(-2) (D7Mit178) for multiple allelic lox sites, after transient Cre expression. Notably, much of the recombination occurs in G2 and is followed by X segregation, where the recombinant chromatids segregate away from each other during mitosis. It is X segregation that is useful for genetic mosaic analysis because it produces clones of homozygous mutant daughter cells from heterozygous mothers. Our studies confirm the predictions made from studies in Drosophila that suggest that X segregation will not be limited to organisms with strong mitotic pairing, because the forces (sister-chromatid cohesion) responsible for X segregation are an elemental feature of mitosis in all eukaryotes. Our studies also show that genetic mosaic analysis in mice is feasible, at least for certain chromosomal regions.

Animals↗

PubMed2003

The SHB adapter protein is required for efficient multilineage differentiation of mouse embryonic stem cells.

The SH2 domain-containing adapter protein SHB transmits signals from receptor tyrosine kinases regulating diverse processes such as apoptosis and differentiation. To elucidate a role for SHB in cell differentiation, wild-type and R522K (inactive SH2 domain-mutant) SHB were transfected and expressed in mouse embryonic stem (ES) cells. Microarray analysis using Affymetrix U74A chips on undifferentiated ES cells and expression of selected differentiation markers after generation of embryoid bodies were subsequently assessed. Wild-type SHB altered the expression of 16 genes in undifferentiated ES cells, many of which have been found to relate to neural cell function. R522K-SHB altered the expression of 128 genes in undifferentiated ES cells, the majority of which were decreased, including several transcription factors related to development. When grown as embryoid bodies, after 4 days R522K-SHB ES cells were already found to display a different morphological appearance, with an impaired cavity formation that occurred in the absence of altered OCT4 expression. This impairment was reversed by exogenous addition of Matrigel. In addition, R522K-SHB embryoid bodies displayed reduced mRNA contents of the liver protein albumin, the pancreatic proteins amylase, glucagon and insulin after 20 days of differentiation. Matrigel did not restore the impaired expression of albumin in the R522K-SHB cells. Expression of the mesodermal marker cardiac actin and the neural marker neurofilament heavy chain alpha was not affected by wild-type or R522K-SHB overexpression. It is concluded that SHB is required for efficient differentiation of ES cells into embryoid bodies with normal cavities and cells belonging to endodermal lineages.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Syncytiotrophoblastic giant cells in teratocarcinoma-like tumors derived from Parp-disrupted mouse embryonic stem cells.

The enzyme poly(ADP-ribose) polymerase (Parp) catalyzes poly(ADP-ribosyl)ation reaction and is involved in DNA repair and cell death induction upon DNA damages. Meanwhile, poly(ADP-ribosyl)ation of chromosome-associated proteins is suggested to be implicated in the regulation of gene expression and cellular differentiation, both of which are important in tumorigenesis. To investigate directly the role of Parp deficiency in tumorigenicity and differentiation of embryonic stem (ES) cells during tumor formation, studies were conducted by using wild-type J1 (Parp(+/+)) ES cells and Parp(+/-) and Parp(-/-) ES clones generated by disrupting Parp exon 1. These ES cells, irrespective of the Parp genotype, produced tumors phenotypically similar to teratocarcinoma when injected s.c. into nude mice. Remarkably, all tumors derived from Parp(-/-) clones contained syncytiotrophoblastic giant cells (STGCs), which possess single or multiple megalo-nuclei. The STGCs were present within large areas of intratumoral hemorrhage. In contrast, neither STGC nor hemorrhage was observed in tumors of both wild-type J1 cells and Parp(+/-) clones. Electron microscopic examination showed that the STGCs possess microvilli on the cell surface and contained secretory granules in the cytoplasm. Furthermore, the cytoplasms of STGCs were strongly stained with antibody against mouse prolactin, which could similarly stain trophoblasts in placenta. These morphological and histochemical features indicate that the STGCs in teratocarcinoma-like tumors derived from Parp(-/-) clones belong to the trophoblast cell lineage. Our findings thus suggest that differentiation of ES cells into STGCs was possibly induced by the lack of Parp during the development of teratocarcinoma.

Animals↗

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

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↗