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PubMed2005

[Transcription and mRNA splicing of the human lactoferrin gene controlled by the regulatory region of the bovine alphaS1 casein gene in the mammary gland of transgenic mice and in mouse embryonic stem cells].

The regulatory region of the bovine alphaS1 casein gene was used to obtain two genetic constructs for expression of human lactoferrin in the mammary gland of transgenic animals. Several transfected mouse embryonic stem cell (ESC) lines and primary transgenic mice were generated with these constructs. Recombinant lactoferrin was not detected in milk of transgenic mice by Western blotting. However, a recombinant transcript was found in RNAs isolated from mammary glands of transgenic females during lactation and from transfected ESC lines.

Animals↗

PubMed1988

Targeted mutation of the Hprt gene in mouse embryonic stem cells.

The hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene has been mutated in mouse blastocyst-derived embryonic stem cells by site-directed homologous recombination. Embryonic stem cells were electroporated in the presence of a targeting DNA fragment containing two specific features: (i) The targeting DNA contained a promoterless neomycin phosphotransferase (neo) gene that, when located within the endogenous Hprt locus, could be transcribed from the promoter of the target locus. (ii) The targeting fragment had two short regions of homology with the endogenous Hprt gene: one, 132 base pairs long and the other, 1.2 kilobase pairs long. Targeted cells in which the designed homologous recombination event occurred were isolated either by selection with G418 followed by 6-thioguanine or by selection with 6-thioguanine alone. Even though less than 2 kilobases of homology existed between the exogenous and target DNAs, an average of 2.6 embryonic stem cells were successfully targeted for every 10(5) colonies surviving electroporation. Six of the Hprt- cell lines showed homologous recombination. These six lines were further analyzed by nucleotide sequencing a fragment that spans one crossover point after amplification by the polymerase chain reaction. Four lines had the expected sequence, whereas two lines had small deletions abutting the 132-base-pair region of homology.

Amino Acid Sequence↗

PubMed2006

Meta-analysis of differentiating mouse embryonic stem cell gene expression kinetics reveals early change of a small gene set.

Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined, undifferentiated ESC in culture. In each dataset, we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets, despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest, we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728_at, 8430410A17Rik, Klf2, Nr0b1, Sox2, Tcl1, and Zfp42) showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis, this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types.

Adaptation, Physiological↗

PubMed1998

Erythroid 5-aminolevulinate synthase is required for erythroid differentiation in mouse embryonic stem cells.

We have examined the induction of the enzymes of the heme biosynthetic pathway during erythroid differentiation of mouse embryonic stem (ES) cells. Following transfer to appropriate medium all of the pathway enzymes are induced within three days. Unlike differentiating mouse erythroleukemia cells (Lake-Bullock, H. and Dailey, H.A. Mol Cell Biol 13:7122-7132, 1993), all of the enzymes appear to be induced simultaneously and not sequentially in differentiating ES cells. The role of erythroid 5-aminolevulinate synthase (ALAS-2) in this differentiation process was examined by disruption of the ALAS-2 gene. The targeting vector used for disruption replaced all of exons 4 to 6 with a selectable neomycin resistance gene. The resulting genetically modified (ALAS-2 knockout) cells, as well as normal ES cells were used to study induction of heme biosynthesis. Following 10 days of culture in methylcellulose media significant morphological differences between the embryoid bodies (EBs) of the two cell lines were observed. ES cells exhibited morphology of typical EBs with a dark field (blood island) in the center, while ALAS-2 knockout ES cells developed very poorly both in size and shape. At 8 days of differentiation, only 3% of all EBs contained visible erythropoietic cells (i.e., stained positively for hemoglobin) in the ALAS-2 knockout cell line, compared with 50% in ES cells. Most of the genes in the heme synthetic pathway were expressed to a stable level within 3 to 6 days after induction in normal ES cells, while the ALAS-2 knockout cell line failed to significantly increase the level of expression of these genes. Fetal beta-globin mRNA was not detectable in the differentiating ALAS-2 knockout cells, whereas mRNA for this gene was detected in normal ES cells within 3 days of differentiation. These results suggest that ALAS-2 is necessary for ES cell erythroid differentiation and that there is an interrelationship between heme and globin synthesis in differentiating ES cells.

5-Aminolevulinate Synthetase↗

PubMed2002

Genetic manipulation of mouse embryonic stem cells by mutant lambda integrase.

Mutant lambda integrases catalyze site-specific recombination reactions inside mammalian cells. Here we demonstrate that the integrase system can be used to eliminate resistance marker genes from the genome of mouse embryonic stem cells. So-called integrative and excisive recombination pathways led to the precise deletion of the neomycin gene, which was inserted together with a flanking pair of directly repeated recombination sites into the ROSA26 locus by standard targeting techniques. The excision of the resistance gene led to the expression of enhanced green fluorescence protein, which served as a means to sort out cells that had undergone site-specific recombination. Southern analysis and DNA sequencing confirmed that strand exchange reactions had occurred in the genome as expected. Hence, the integrase system may be used in conjunction with other site-specific recombinases as a tool in genome manipulation protocols.

Animals↗

PubMed2002

The human cytomegalovirus immediate-early promoter is transcriptionally active in undifferentiated mouse embryonic stem cells.

It has been reported recently that the cytomegalovirus (CMV) immediate-early promoter is transcriptionally inactive in undifferentiated mouse embryonic stem (ES) cells. This result is surprising, since the CMV promoter is used to express transgenes in a variety of cell lines. We studied the expression of a human CMV-driven enhanced green fluorescent protein (EGFP) reporter gene (pEGFP-N1) in five undifferentiated mouse ES cell lines (BL/6III, D3, E14TG2a, MESC20, and 129) and found EGFP expression in all of these cell lines. Under optimal conditions, between 50%-80% transfection efficiencies could be achieved, and EGFP expression levels were maintained for at least 72 hours. Therefore, the human CMV promoter remains a useful system for transgene expression in undifferentiated ES cells.

Animals↗

PubMed2004

p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression.

The tumour suppressor p53 becomes activated in response to upstream stress signals, such as DNA damage, and causes cell-cycle arrest or apoptosis. Here we report a novel role for p53 in the differentiation of mouse embryonic stem cells (ESCs). p53 binds to the promoter of Nanog, a gene required for ESC self-renewal, and suppresses Nanog expression after DNA damage. The rapid down-regulation of Nanog mRNA during ESC differentiation correlates with the induction of p53 transcriptional activity and Ser 315 phosphorylation. The importance of Ser 315 phosphorylation was revealed by the finding that induction of p53 activity is impaired in p53(S315A) knock-in ESCs during differentiation, leading to inefficient suppression of Nanog expression. The decreased inhibition of Nanog expression in p53(S315A) ESCs during differentiation is due to an impaired recruitment of the co-repressor mSin3a to the Nanog promoter. These findings indicate an alternative mechanism for p53 to maintain genetic stability in ESCs, by inducing the differentiation of ESCs into other cell types that undergo efficient p53-dependent cell-cycle arrest and apoptosis.

Animals↗

PubMed1998

Moesin is not a receptor for measles virus entry into mouse embryonic stem cells.

The involvement of moesin in measles virus (MV) entry was investigated with moesin-positive and -negative mouse embryonic stem (ES) cells. MV infection of these cells was very ineffective and was independent of moesin expression. Furthermore, when these cells were transfected to express human CD46, a 100-fold increase in syncytium formation was observed with these cells and was independent of the expression of moesin. The only obvious difference between moesin-positive and -negative ES cells was the shape of the syncytia formed. Moesin-negative ES cells expressing or not expressing human CD46 formed separate pieces of fragmented syncytia which were torn apart during spreading, whereas ES cells expressing moesin exhibited typical syncytia. In addition, moesin was not detected on the surface of any murine cells or cell lines that we have tested by a flow cytometric assay with moesin-specific antibodies. These findings indicate that murine moesin is neither a receptor nor a CD46 coreceptor for MV entry into mouse ES cells. Moesin is involved in actin filament-plasma membrane interactions as a cross-linker, and it affects only the spreading and shape of MV-mediated syncytia.

Animals↗

PubMed2001

BRCA1 is a selective co-activator of 14-3-3 sigma gene transcription in mouse embryonic stem cells.

BRCA1 gene is a tumor suppressor for breast and ovarian cancers with the putative role in DNA repair and transcription. To characterize the role of BRCA1 in transcriptional regulation, we analyzed gene expression profiles of mouse embryonic stem cells deficient in BRCA1 using microarray technology. We found that loss of BRCA1 correlated with decreased expression of several groups of genes including stress response genes, cytoskeleton genes, and genes involved in protein synthesis and degradation. Previous study showed that BRCA1 is a transcriptional co-activator of p53 protein; however the majority of p53 target genes remained at the same expression levels in BRCA1 knockout cells as in the wild type cells. The only p53 target gene down-regulated with the loss of BRCA1 was 14-3-3 sigma, a major G(2)/M checkpoint control gene. Similar to cells with decreased 14-3-3 sigma activity, BRCA1-deficient cells were unable to sustain G(2)/M growth arrest after exposure to ionizing radiation. We find that BRCA1 induction of 14-3-3 sigma requires the presence of wild type p53 and can be regulated by a minimal p53 response element.

14-3-3 Proteins↗

PubMed2005

Role of hepatocyte-like cells in the differentiation of cardiomyocytes from mouse embryonic stem cells.

Cell replacement therapy, while being a promising approach, is challenged by the limited supply of appropriate cells and incomplete understanding of the cardiac differentiation process. In this manuscript, we show the generation of spontaneously beating cardiomyocytes without using dimethylsulfoxide (DMSO), the most well-known cardiotrophic factor. Here, we employ basic fibroblast growth factor (FGF), a commonly used growth factor for embryonic stem (ES) cell differentiation, to initiate and maintain in vitro cardiac differentiation. Upon differentiation, beating or pulsating cardiac cells grown on tissue culture plates that interspersed with oval-shaped cells appeared after 1 week in culture. The number of beating colonies increased with time, and cells remained contractile for at least 45 days. Within 12 days of differentiation, these cells expressed markers characteristic of cardiomyocytes and hepatocytes such as GATA4, Nkx2.5, beta-myosin heavy chain, myosin light chain 2V, cardiac troponin T, sodium calcium exchanger and HNF-4alpha, alpha-fetoprotein, albumin, transthyretin, and CK-18, respectively. Thus, molecular and cellular characterization of these oval-shaped cells identified them as hepatocyte-like cells. Furthermore, we have identified a candidate set of signaling molecules like bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) and demonstrated their interactive role in in vitro cardiogenesis. To our knowledge, this is the first report elucidating the intrinsic signaling pathway of hepatocyte-like cells in the differentiation of cardiomyocytes from mouse ES cells without employing co-culture techniques. Hence, the study provides a significant insight into the mechanism of in vitro derivation of cardiomyocytes, mediated through interactive signaling with adjoining endodermal derivatives.

Albumins↗

PubMed2004

Developmental potential of defined neural progenitors derived from mouse embryonic stem cells.

The developmental potential of a uniform population of neural progenitors was tested by implanting them into chick embryos. These cells were generated from retinoic acid-treated mouse embryonic stem (ES) cells, and were used to replace a segment of the neural tube. At the time of implantation, the progenitors expressed markers defining them as Pax6-positive radial glial (RG) cells, which have recently been shown to generate most pyramidal neurons in the developing cerebral cortex. Six days after implantation, the progenitors generated large numbers of neurons in the spinal cord, and differentiated into interneurons and motoneurons at appropriate locations. They also colonized the host dorsal root ganglia (DRG) and differentiated into neurons, but, unlike stem cell-derived motoneurons, they failed to elongate axons out of the DRG. In addition, they neither expressed the DRG marker Brn3a nor the Trk neurotrophin receptors. Control experiments with untreated ES cells indicated that when colonizing the DRG, these cells did elongate axons and expressed Brn3a, as well as Trk receptors. Our results thus indicate that ES cell-derived progenitors with RG characteristics generate neurons in the spinal cord and the DRG. They are able to respond appropriately to local cues in the spinal cord, but not in the DRG, indicating that they are restricted in their developmental potential.

Animals↗

PubMed2006

Dual in vivo magnetic resonance evaluation of magnetically labeled mouse embryonic stem cells and cardiac function at 1.5 t.

Cell therapy has demonstrated the potential to restore injured myocardium. A reliable in vivo imaging method to localize transplanted cells and monitor their restorative effects will enable a systematic investigation of this therapeutic modality. The dual MRI capability of imaging both magnetically labeled mouse embryonic stem cells (mESC) and their restorative effects on cardiac function in a murine model of acute myocardial infarction is demonstrated. Serial in vivo MR detection of transplanted mESC and monitoring of the mESC-treated myocardium was conducted over a 4-week period using a 1.5 T clinical scanner. During the 4-week duration, the mESC-treated myocardium demonstrated sustained improvement of the left ventricular (LV) ejection fraction and conservation of LV mass. Furthermore, no significant difference of their restorative effects on the cardiac function was created by the magnetic labeling of mESC. Thus, in vivo MRI enables simultaneous detection of transplanted mESC and their therapeutic effect on the injured myocardium.

Animals↗

PubMed1991

Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers.

A defined cultivation system was developed for the differentiation of pluripotent embryonic stem cells of the mouse into spontaneously beating cardiomyocytes, allowing investigations of chronotropic responses, as well as electrophysiological studies of different cardioactive drugs in vitro. The beta-adrenoceptor agonists (-)isoprenaline and clenbuterol, the mediators of cAMP metabolism, forskolin and isobutylmethylxanthine (IBMX), the alpha 1-adrenoceptor agonist (-)phenylephrine, and the heart glycoside digitoxin induced a positive, the muscarinic cholinoceptor agonist carbachol and L-type Ca2+ channel blockers nisoldipine, gallopamil and diltiazem induced a negative chronotropic response. In early differentiated cardiomyocytes beta 1-, alpha 1-, but not beta 2-adrenoceptors, cholinoceptors, as well as L-type Ca2+ channels participated in the chronotropic response. In terminally differentiated cardiomyocytes beta 2-adrenoceptors and digitoxin responses were also functionally expressed. The contractions of spontaneously beating cardiomyocytes were concomitant with rhythmic action potentials very similar to those described for embryonic cardiomyocytes and sinus-node cells. We conclude that cardiomyocytes differentiating from pluripotent embryonic stem cells are able to develop adrenoceptors and cholinoceptors and signal transduction pathways as well as L-type Ca2+ channels as a consequence of cell-cell interactions during embryoid body formation in vitro, independent of the development in living organisms. The cellular system described may be useful as in vitro assay for toxicological investigations of chronotropic drugs and a model system for studying commitment and cellular differentiation in vitro.

1-Methyl-3-isobutylxanthine↗

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↗

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↗