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Results for “Mouse Embryonic Stem Cells”

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PubMed2003

The in vitro differentiation of mouse embryonic stem cells into neutrophils.

A reliable and effective in vitro differentiation system is described for the production of functional neutrophils from mouse embryonic stem (ES) cells. A three-step culture method was developed that enables abundant production and effective harvesting of mature neutrophils at high purity without sorting. Utilization of the OP9 stromal cell line, which does not produce macrophage colony stimulating factor (M-CSF) was found to enhance the number, percentage and duration of neutrophils produced. Based on a number of criteria, morphologically and functionally mature neutrophils can be produced using this method in approximately 16 days. This differentiation system provides a useful model system for studying neutrophil development and maturation in vitro and the many factors that regulate this process. Morphologically mature ES-derived neutrophils can be grown in culture that produce superoxide, flux calcium and directionally respond to the chemoattractant MIP-2. In addition, they express the granulocyte markers Gr-1 and the neutrophil specific antigen, as well as specific chloroacetate esterase. Interestingly, during their development in culture, regional areas of apparent neutrophil production can be identified that recapitulate certain aspects of the marrow environment. As ES cells can be genetically modified, this system enables evaluation of the effects of specific genetic alterations on neutrophil differentiation and function.

Animals↗

PubMed2000

Telomere maintenance in telomerase-deficient mouse embryonic stem cells: characterization of an amplified telomeric DNA.

Telomere dynamics, chromosomal instability, and cellular viability were studied in serial passages of mouse embryonic stem (ES) cells in which the telomerase RNA (mTER) gene was deleted. These cells lack detectable telomerase activity, and their growth rate was reduced after more than 300 divisions and almost zero after 450 cell divisions. After this growth crisis, survivor cells with a rapid growth rate did emerge. Such survivors were found to maintain functional telomeres in a telomerase-independent fashion. Although telomerase-independent telomere maintenance has been reported for some immortalized mammalian cells, its molecular mechanism has not been elucidated. Characterization of the telomeric structures in one of the survivor mTER(-/-) cell lines showed amplification of the same tandem arrays of telomeric and nontelomeric sequences at most of the chromosome ends. This evidence implicates cis/trans amplification as one mechanism for the telomerase-independent maintenance of telomeres in mammalian cells.

Animals↗

PubMed2003

The kinase MSK1 is required for induction of c-fos by lysophosphatidic acid in mouse embryonic stem cells.

BACKGROUND: The regulation of the immediate-early gene c-fos serves as a paradigm for signal-activated gene induction. Lysophosphatidic acid is a potent serum-borne mitogen able to induce c-fos. RESULTS: Analysing the signalling events following stimulation of mouse embryonic stem cells with serum and lysophosphatidic acid, we show that the extracellular signal-regulated kinase (ERK) pathway is involved in mediating c-fos induction. We demonstrate that the ERK-activated kinase MSK1 is required for full c-fos promoter activation, as well as for the phosphorylation of cAMP-responsive element (CRE) binding proteins. We propose that MSK1 contributes to ERK-mediated c-fos promoter activation by targeting CRE binding proteins. CONCLUSION: These results show that MSK1 is an important ERK-activated mediator of mitogen-stimulated c-fos induction. In addition, they indicate that MSK1 could act through CRE binding proteins to achieve c-fos promoter activation. Thus, they further our understanding of the complex regulation of the model immediate-early gene c-fos.

Animals↗

PubMed2003

Enhanced genomic instability and defective postreplication repair in RAD18 knockout mouse embryonic stem cells.

In lower eukaryotes, Rad18 plays a crucial role in postreplication repair. Previously, we isolated a human homologue of RAD18 (hRAD18) and showed that human cells overexpressing hRad18 protein with a mutation in the RING finger motif are defective in postreplication repair. Here, we report the construction of RAD18-knockout mouse embryonic stem cells by gene targeting. These cells had almost the same growth rate as wild-type cells and manifested phenotypes similar to those of human cells expressing mutant Rad18 protein: hypersensitivity to multiple DNA damaging agents and a defect in postreplication repair. Mutation was not induced in the knockout cells with any higher frequencies than in wild-type cells, as shown by ouabain resistance. In the knockout cells, spontaneous sister chromatid exchange (SCE) occurred with twice the frequency observed in normal cells. After mild DNA damage, SCE was threefold higher in the knockout cells, while no increase was observed in normal cells. Stable transformation efficiencies were approximately 20-fold higher in knockout cells, and gene targeting occurred with approximately 40-fold-higher frequency than in wild-type cells at the Oct3/4 locus. These results indicate that dysfunction of Rad18 greatly increases both the frequency of homologous as well as illegitimate recombination, and that RAD18 contributes to maintenance of genomic stability through postreplication repair.

Amino Acid Motifs↗

PubMed1994

Hematopoietic development of vav-/- mouse embryonic stem cells.

The vav protooncogene product is expressed nearly exclusively in hematopoietic lineages and contains several structural motifs (SH2/SH3 domains and a dbl-oncogene homology region) typical of proteins functioning in signaling pathways. To ascertain if vav expression is required for hematopoiesis we generated vav-negative mouse embryonic stem cells by gene targeting and examined the consequences of loss of vav function on erythroid and myeloid development in vitro and in vivo. In conflict with the conclusions drawn from expression of antisense vav RNA in embryonic stem cells [Wulf, G. M., Adra, C. N. & Lim, B. (1993) EMBO J. 12, 5065-5074], we observed erythroid and myeloid development in the absence of vav. These experiments demonstrate that vav expression is not absolutely required for hematopoietic development.

Animals↗

PubMed2006

Altered cell adhesion and cell viability in a p38alpha mitogen-activated protein kinase-deficient mouse embryonic stem cell line.

p38 mitogen-activated protein (MAP) kinase alpha (p38alpha) is a broadly expressed protein kinase that regulates growth and development. Most studies of p38alpha have been in somatic cells. Little is known about its function in embryonic stem (ES) cells. Using a ES cell line isolated from p38alpha knockout mouse embryos (p38alpha (-/-) ES cells), we investigated roles of p38alpha in the regulation of ES cell activities. p38alpha (-/-) ES cells displayed several altered features different from wild-type cells. The major findings are that p38alpha (-/-) ES cells have significantly increased cell adhesion to several extracelluar matrix proteins, correlating with elevated phosphorylation of focal adhesion kinase and paxillin. p38alpha (-/-) ES cells also showed increased cell viability, correlating with increased expression of survivin and activation of AKT (protein kinase B), two molecules that are known to improve cell viability. p38alpha (-/-) ES cells reach confluence faster than wild-type cells in routine cell culture. However, this is not due to a higher cell proliferation rate in p38alpha (-/-) ES cells, but rather is likely a result of improved cell adhesion and/or cell viability. Together our results indicated that p38alpha may negatively regulate mouse ES cell adhesion and viability.

Animals↗

PubMed2002

Chromosome instability as a result of double-strand breaks near telomeres in mouse embryonic stem cells.

Telomeres are essential for protecting the ends of chromosomes and preventing chromosome fusion. Telomere loss has been proposed to play an important role in the chromosomal rearrangements associated with tumorigenesis. To determine the relationship between telomere loss and chromosome instability in mammalian cells, we investigated the events resulting from the introduction of a double-strand break near a telomere with I-SceI endonuclease in mouse embryonic stem cells. The inactivation of a selectable marker gene adjacent to a telomere as a result of the I-SceI-induced double-strand break involved either the addition of a telomere at the site of the break or the formation of inverted repeats and large tandem duplications on the end of the chromosome. Nucleotide sequence analysis demonstrated large deletions and little or no complementarity at the recombination sites involved in the formation of the inverted repeats. The formation of inverted repeats was followed by a period of chromosome instability, characterized by amplification of the subtelomeric region, translocation of chromosomal fragments onto the end of the chromosome, and the formation of dicentric chromosomes. Despite this heterogeneity, the rearranged chromosomes eventually acquired telomeres and were stable in most of the cells in the population at the time of analysis. Our observations are consistent with a model in which broken chromosomes that do not regain a telomere undergo sister chromatid fusion involving nonhomologous end joining. Sister chromatid fusion is followed by chromosome instability resulting from breakage-fusion-bridge cycles involving the sister chromatids and rearrangements with other chromosomes. This process results in highly rearranged chromosomes that eventually become stable through the addition of a telomere onto the broken end. We have observed similar events after spontaneous telomere loss in a human tumor cell line, suggesting that chromosome instability resulting from telomere loss plays a role in chromosomal rearrangements associated with tumor cell progression.

Animals↗

PubMed2002

Deficiency of Retinoblastoma gene in mouse embryonic stem cells leads to genetic instability.

Genetic instability has been recognized as a hallmark of human cancers. Retinoblastoma (Rb) tumor suppressor protein has an essential role in modulating cell cycle progression. However, there is no direct evidence supporting its role in maintaining genetic stability. Here, we developed a sensitive method to examine the level of chromosome instability by using retrovirus carrying both positive and negative selectable markers that integrated randomly into individual chromosomes, and the frequency of loss of this selectable chromosomal marker (LOM) in normal mammalian cells was measured. Our results showed that normal mouse embryonic stem (ES) cells had a very low frequency of LOMs, which was less than 10(-8)/cell/generation. In Rb-/- mouse ES cells, the frequency was increased to approximately 10(-5)/cell/generation, whereas in Rb+/- ES cells, the frequency was approximately 10(-7)/cell/generation. LOMs was mediated mainly through chromosomal mechanisms and not through point mutations. These results, therefore, revealed that Rb, with a haploinsufficiency, plays a critical role in the maintenance of chromosome stability. The mystery of why Rb heterozygous carriers have early-onset tumor formation with high penetrance can be, at least, partially explained by this novel activity.

Animals↗

PubMed1999

Chromosome healing in mouse embryonic stem cells.

The addition of new telomeres to the ends of broken chromosomes, termed chromosome healing, has been extensively studied in unicellular organisms; however, its role in the mammalian cell response to double-strand breaks is unknown. A system for analysis of chromosome healing, which involves the integration of plasmid sequences immediately adjacent to a telomere, has been established in mouse embryonic stem cells. This "marked" telomere contains a neo gene for positive selection in G418, an I-SceI endonuclease recognition sequence for introducing double-strand breaks, and a herpes simplex virus thymidine kinase gene for negative selection with ganciclovir for cells that have lost the telomere. Transient expression of the I-SceI endonuclease results in terminal deletions involving telomeric repeat sequences added directly onto the end of the broken chromosome. The sites of addition of the new telomeres contain short regions of complementarity to telomeric repeat sequences. The most common site of addition is the last A of the ATAA 3' overhang generated by the I-SceI endonuclease, without the loss of a single nucleotide from the end of the chromosome. The next most frequent site involved 5 bp of complementarity, which occurred after the loss of four nucleotides from the end of the chromosome. The new telomeres are generally much shorter than in the parental cell line, and most increase in size with time in culture. These results demonstrate that chromosome healing is a mechanism for repair of chromosome breaks in mammalian cells.

Animals↗

PubMed2006

Immunogenicity and engraftment of mouse embryonic stem cells in allogeneic recipients.

Embryonic stem cells (ESCs) are pluripotent and therefore able to differentiate both in vitro and in vivo into specialized tissues under appropriate conditions, a property that could be exploited for cellular therapies. However, the immunological nature of these cells in vivo has not been well understood. In vitro, mouse-derived ESCs fail to stimulate T cells, but they abrogate ongoing alloresponses by a process that requires cell-cell contact. We further show that despite a high expression of the NKG2D ligand retinoic acid early inducible-1 by mouse ESCs, they remain resistant to natural killer cell lysis. In vivo, allogeneic mouse ESCs populate the thymus, spleen, and liver of sublethally irradiated allogeneic host mice, inducing apoptosis to T cells and establishing multilineage mixed chimerism that significantly inhibits alloresponses to donor major histocompatibility complex antigens. Immunohistochemical imaging revealed a significant percentage of ESC-derived cells in the splenic marginal zones, but not in the follicles. Taken together, the data presented here reveal that nondifferentiated mouse embryonic stem cells are non-immunogenic and appear to populate lymphoid tissues in vivo, leading to T-cell deletion by apoptosis.

Animals↗

PubMed1996

Recycling selectable markers in mouse embryonic stem cells.

As a result of gene targeting, selectable markers are usually permanently introduced into the mammalian genome. Multiple gene targeting events in the same cell line can therefore exhaust the pool of markers available and limit subsequent manipulations or genetic analysis. In this study, we describe the combined use of homologous and CRE-loxP-mediated recombination to generate mouse embryonic stem cell lines carrying up to four targeted mutations and devoid of exogenous selectable markers. A cassette that contains both positive and negative selectable markers flanked by loxP sites, rendering it excisable by the CRE protein, was constructed. Homologous recombination and positive selection were used to disrupt the Rep-3 locus, a gene homologous to members of the mutS family of DNA mismatch repair genes. CRE-loxP-mediated recombination and negative selection were then used to recover clones in which the cassette had been excised. The remaining allele of Rep-3 was then subjected to a second round of targeting and excision with the same construct to generate homozygous, marker-free cell lines. Subsequently, both alleles of mMsh2, another mutS homolog, were disrupted in the same fashion to obtain cell lines homozygous for targeted mutations at both the Rep-3 and mMsh2 loci and devoid of selectable markers. Thus, embryonic stem cell lines obtained in this fashion are suitable for further manipulation and analysis involving the use of selectable markers.

Animals↗

PubMed1996

De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells.

It has been a controversial issue as to how many DNA cytosine methyltransferase mammalian cells have and whether de novo methylation and maintenance methylation activities are encoded by a single gene or two different genes. To address these questions, we have generated a null mutation of the only known mammalian DNA methyltransferase gene through homologous recombination in mouse embryonic stem cells and found that the development of the homozygous embryos is arrested prior to the 8-somite stage. Surprisingly, the null mutant embryonic stem cells are viable and contain low but stable levels of methyl cytosine and methyltransferase activity, suggesting the existence of a second DNA methyltransferase in mammalian cells. Further studies indicate that de novo methylation activity is not impaired by the mutation as integrated provirus DNA in MoMuLV-infected homozygous embryonic stem cells become methylated at a similar rate as in wild-type cells. Differentiation of mutant cells results in further reduction of methyl cytosine levels, consistent with the de novo methylation activity being down regulated in differentiated cells. These results provide the first evidence that an independently encoded DNA methyltransferase is present in mammalian cells which is capable of de novo methylating cellular and viral DNA in vivo.

Animals↗

PubMed1996

In vitro development of hematopoietic system from mouse embryonic stem cells: a new approach for embryonic hematopoiesis.

We developed an efficient differentiation induction system (OP9 system) from mouse embryonic stem (ES) cell to blood cells by coculture on a novel stromal cell line, OP9. This stromal cell line does not produce functional macrophage colony-stimulating factor (M-CSF) and presumably the deficiency of M-CSF production of OP9 cells might induce preferential differentiation of ES cells into hematopoietic cells other than the monocyte-macrophage lineage. ES cells gave rise to embryonic primitive erythrocytes, and then adult type definitive erythrocytes, myeloid and B lineage cells. The OP9 system should facilitate to elucidate the molecular mechanisms of hematopoietic system development and hematopoietic cell differentiation.

Animals↗

PubMed2003

In vitro development of mouse embryonic stem cells lacking JNK/stress-activated protein kinase-associated protein 1 (JSAP1) scaffold protein revealed its requirement during early embryonic neurogenesis.

The Jsap1 gene encodes a scaffold protein for c-Jun N-terminal kinase cascades. We established c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1)-null mouse embryonic stem cell lines by homologous recombination. The JSAP1-null embryonic stem cells were viable, however, exhibited hyperplasia of the ectoderm during embryoid body formation, and spontaneously differentiated into neurons more efficiently than did wild type. The expression of components of c-Jun N-terminal kinase cascades and a subset of marker mRNAs during early embryogenesis was altered in the JSAP1-null mutants. Retinoic acid dramatically increased the expression of JSAP1 and JNK3, which were co-precipitated with anti-JNK3 in the neuroectoderm of wild type but not JSAP1-null embryoid bodies. In the neurons differentiated from the wild type embryoid bodies, JSAP1 was localized in the soma, neurites, and growth cone-like structure of the neurites, and neurite outgrowth from the JSAP1-null embryoid bodies was apparently less efficient than from wild type. JSAP1 and c-Jun N-terminal kinase 3 were coexpressed in the embryonic ectoderm of E7.5 mouse embryo, whereas Wnt1 and Pax2 were coexpressed with JSAP1 at the midbrain-hindbrain junction in E12.5 mouse embryo, thus suggesting that JSAP1 is required for early embryonic neurogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed2006

The transcription factor ZBP-89 controls generation of the hematopoietic lineage in zebrafish and mouse embryonic stem cells.

Hematopoietic development is closely linked to that of blood vessels and the two processes are regulated in large part by transcription factors that control cell fate decisions and cellular differentiation. Both blood and blood vessels derive from a common progenitor, termed the hemangioblast, but the factor(s) specifying the development and differentiation of this stem cell population into the hematopoietic and vascular lineages remain ill defined. Here, we report that knockdown of the Krüppel-like transcription factor ZBP-89 in zebrafish embryos results in a bloodless phenotype, caused by disruption of both primitive and definitive hematopoiesis, while leaving primary blood vessel formation intact. Injection of ZBP-89 mRNA into cloche zebrafish embryos, which lack both the hematopoietic and endothelial lineages, rescues hematopoiesis but not vasculogenesis. Injection of mRNA for Stem Cell Leukemia (SCL), a transcription factor that directs hemangioblast development into blood cell precursors, rescues the bloodless phenotype in ZBP-89 zebrafish morphants. Forced expression of ZBP-89 induces the expansion of hematopoietic progenitors in wild-type zebrafish and in mouse embryonic stem cell cultures but inhibits angiogenesis in vivo and in vitro. These findings establish a unique regulatory role for ZBP-89, positioned at the interface between early blood and blood vessel development.

Animals↗

PubMed2001

Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.

In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein-tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.

Animals↗

PubMed2005

Differential roles for Sox15 and Sox2 in transcriptional control in mouse embryonic stem cells.

Sox family transcription factors play essential roles in cell differentiation, development, and sex determination. Sox2 was previously thought to be the sole Sox protein expressed in mouse embryonic stem (ES) cells. Sox2 associates with Oct3/4 to maintain self-renewal of ES cells. In the current study, digital differential display identified transcripts for an additional Sox family member, Sox15, enriched in mouse ES cells. Reverse transcription-PCR confirmed that Sox15 expression is highest in undifferentiated ES cells and repressed upon differentiation. Sox15 is expressed at low levels in several tissues, including testis and muscle. In vitro studies showed that Sox15, like Sox2, associated with Oct3/4 on DNA sequences containing the octamer motif and Sox-binding site. Gel mobility shift assays and SELEX analyses showed that Sox15 binds similar DNA sequences as Sox2 but with weaker affinity. In contrast to the early embryonic lethality observed in Sox2-null mice, Sox15-null ES cells and mice were grossly normal. DNA microarray analyses revealed that Otx2, Ctgf, Ebaf, and Hrc are dysregulated in Sox15-null ES cells, however. Chromatin immunoprecipitation showed that Sox15, but not Sox2, bound to a Sox consensus binding site within the Hrc gene. Taken together, these data demonstrate differential roles for Sox15 and Sox2 in transcriptional control in mouse ES cells.

Amino Acid Motifs↗

PubMed2004

Dnmt1 deficiency leads to enhanced microsatellite instability in mouse embryonic stem cells.

DNA hypomethylation is frequently seen in cancer and imparts genomic instability in mouse models and some tissue culture systems. However, the effects of genomic DNA hypomethylation on mutation rates are still elusive. We have developed a model system to analyze the effects of DNA methyltransferase 1 (Dnmt1) deficiency on DNA mismatch repair (MMR) in mouse embryonic stem (ES) cells. We generated sibling ES cell clones with and without functional Dnmt1 expression, containing a stable reporter gene that allowed us to measure the slippage rate at a mononucleotide repeat. We found that Dnmt1 deficiency led to a 7-fold increase in the microsatellite slippage rate. Interestingly, the region flanking the mononucleotide repeat was unmethylated regardless of Dnmt1 status, suggesting that it is not the local levels of DNA methylation that direct the increase in microsatellite instability (MSI). The enhanced MSI was associated with higher levels of histone H3 acetylation and lower MeCP2 binding at regions near the assayed microsatellite, suggesting that Dnmt1 loss may decrease MMR efficiency by modifying chromatin structure.

Animals↗