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Results for “Mouse Embryonic Stem Cells”

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At least 433 records · Page 24Linked to original sources
PubMed2003

Differentiation of mouse embryonic stem cells into pancreatic and hepatic cells.

Here, we present efficient strategies to differentiate ES cells either into pancreatic or into hepatic cell types. We recommend a strategy to select nestin+ cells, an early progenitor cell type with high developmental plasticity, followed by differentiation induction with specific growth and extracellular matrix factors into pancreatic and hepatic cell types. Cells differentiating via nestin+ cells into the pancreatic and hepatic lineage expressed tissue-specific genes. Proteins characteristic for mature endocrine pancreatic or hepatic cells were synthesized and released. Further, a histotypic "spinner" culture system was introduced to generate mature insulin- and albumin-producing cells at high efficiency.

Albumins↗
PubMed1998

Beta1 integrin deficiency impairs migration and differentiation of mouse embryonic stem cell derived neurons.

Cell-matrix interaction plays an important role during neuronal development, which is demonstrated by comparing wild type (D3)- and beta1 integrin-deficient (G201) embryonic stem cell derived neurons. In D3 preparations complex networks of functionally coupled neurons with bi- and multipolar morphologies develop. In contrast, neuronal differentiation is retarded in G201 derived neurons, recognised by limited migration and restricted morphological differentiation. Furthermore, beta1 integrin deficiency causes a delay in expression of major neurotransmitters like GABA and glutamate as well as of synaptophysin. These findings indicate a prominent role of beta1 integrin for both morphological and chemical differentiation.

Animals↗
PubMed1998

Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells.

The dramatic increase in sequence information in the form of expressed sequence tags (ESTs) and genomic sequence has created a 'gene function gap' with the identification of new genes far outpacing the rate at which their function can be identified. The ability to create mutations in embryonic stem (ES) cells on a large scale by tagged random mutagenesis provides a powerful approach for determining gene function in a mammalian system; this approach is well established in lower organisms. Here we describe a high-throughput mutagenesis method based on gene trapping that allows the automated identification of sequence tags from the mutated genes. This method traps and mutates genes regardless of their expression status in ES cells. To facilitate the study of gene function on a large scale, we are using these techniques to create a library of ES cells called Omnibank, from which sequence-tagged mutations in 2,000 genes are described.

Animals↗
PubMed2004

Differentiation of mouse embryonic stem cells into a defined neuronal lineage.

Although it has long been known that cultured embryonic stem cells can generate neurons, the lineage relationships with their immediate precursors remain unclear. We report here that selection of highly proliferative stem cells followed by treatment with retinoic acid generated essentially pure precursors that markers identified as Pax-6-positive radial glial cells. As they do in vivo, these cells went on to generate neurons with remarkably uniform biochemical and electrophysiological characteristics.

Animals↗
PubMed2006

LIGHT induces differentiation of mouse embryonic stem cells associated with activation of ERK5.

LT-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT) is a recently cloned type II transmembrane protein belonging to the TNF family that was originally identified as a weak inducer of apoptosis. This cytokine has been extensively defined in its role on T-cell regulation and dendritic cell maturation. However, whether this cytokine regulates stem cell proliferation and/or differentiation remains unknown. In this study, we transduced exogenous LIGHT into embryonic stem cells (ES cells) and found it induced their differentiation. The expression of phospho-STAT3, Nanog and Oct-4 was reduced in LIGHT-transduced ES cells compared with wild-type ES cells. LIGHT-transduced ES cells exhibit a low level of SSEA-1 surface antigen and alkaline phosphatase staining compared with wild-type cells. Introduction of LIGHT into ES cells results in the dephosphorylation of MKP-3 and activation of extracellular signal-regulated kinase (ERK)5. When ERK5 was inhibited by the specific inhibitor PD184352 or knocked down by ERK5 siRNA, reduction of Oct-4 and SSEA-1 expression was rescued. We conclude that LIGHT overrides Leukemia inhibitory factor to induce ES cell differentiation associated with activation of ERK5.

Animals↗
PubMed2003

Neural differentiation of mouse embryonic stem cells.

Pluripotent embryonic stem cells can give rise to neuroectodermal derivatives in culture. This potential could be harnessed to generate neurons and glia for cell-replacement therapies in the central nervous system and for use in drug discovery. However, current methods of neural differentiation are empirical and relatively inefficient. Here, we review these methodologies and present new tools for quantification, analysis and manipulation of embryonic stem cell neural determination.

Animals↗
PubMed1990

Introduction of specific point mutations into RNA polymerase II by gene targeting in mouse embryonic stem cells: evidence for a DNA mismatch repair mechanism.

We have introduced two specific point mutations, located 20 base pairs apart, into the endogenous murine gene that encodes the largest subunit of RNA polymerase II (RPII215). The first mutation conferred resistance to the mushroom toxin alpha-amanitin (amar), and the second mutation generated a restriction fragment length polymorphism without altering the protein sequence. Targeted amar clones were generated at a frequency of 1 in 30 totipotent embryonic stem cells that expressed stably integrated DNA vectors after electroporation. Thirty to 40% of these clones had acquired both mutations, whereas, surprisingly, the remaining clones had acquired the specific amar point mutation but lacked the restriction fragment length polymorphism. We suggest that the latter clones were generated by independent DNA mismatch repair rather than by double crossover or gene conversion. These results demonstrate that it is possible to introduce specific point mutations into an endogenous gene in embryonic stem cells. Thus it should be possible to introduce single base substitutions into other cellular genes, including nonselectable genes, by optimizing the efficiency of gene transfer and/or the sensitivity of screening for targeted clones.

Amino Acid Sequence↗
PubMed2006

Establishment of new mouse embryonic stem cell lines is improved by physiological glucose and oxygen.

Embryonic stem cell lines are routinely selected and cultured in glucose and oxygen concentrations that are well above those of the intrauterine environment. Supraphysiological glucose and hyperoxia each increase oxidative stress, which could be detrimental to survival in vitro by inhibiting proliferation and/or inducing cell death. The aim of this study was to test whether isolation of new embryonic stem cell lines from murine blastocysts is improved by culture in physiological (5%) oxygen instead of approximately 20%, the concentration of oxygen in room air, or in media containing physiological (100 mg/dL) instead of 450 mg/dL glucose. We found that culturing in either physiological oxygen or physiological glucose improved the success of establishing new murine embryonic stem cell lines, and that culture when concentrations of both oxygen and glucose were physiological improved the success of establishing new lines more than culture in either alone. Physiological oxygen and glucose reduce oxidative stress, as determined by 2',7'-dichloro-dihydrofluorescein fluorescence. BrdU incorporation suggests that physiological oxygen and glucose increase the pool of proliferating cells. Cells isolated in physiological oxygen and glucose are capable of self-renewal and differentiation into all three germ layers in vitro. However, none of the culture conditions prevents cytogenetic instability with prolonged passage. These results suggest that culture of cells derived from murine blastocysts in physiological oxygen and glucose reduces oxidant stress, which increases the success of establishing new embryonic stem cell lines.

Animals↗
PubMed2006

Transfection of CCE mouse embryonic stem cells with EGFP and BDNF genes by the electroporation method.

Embryonic stem (ES) cells are pluripotent cells that can differentiate into a wide variety of cell types. This has made them an attractive source of donor cells for developmental studies and cell therapy. Blocking the differentiation of ES cells in culture and using them in clinical applications requires genetic manipulation of the cells. The aim of the present study was to transfect CCE ES cells with EGFP and BDNF genes, in which pcDNA3-hBDNF-v5 and pIRES2-EGFP plasmids were used, respectively. Transformation of DH5alpha competent bacteria by the plasmids was done. Then the plasmids were purified and transfected into ES cells using the electroporation method. The expression of the EGFP gene was confirmed using invert fluorescent microscopy; accordingly, RT-PCR was used for the BDNF gene. The latter was evaluated by extracting the total RNA from the transfected cells. cDNA was obtained by using reverse transcriptase and was amplified by specific primers. The products of the PCR were separated and visualized by agarose gel electrophoresis. Both techniques revealed a successful transfection of CCE ES cells by both plasmids. The obtained data indicated that electroporation is an efficient method for transfection of CCE ES cells.

Animals↗
PubMed2006

Embryoid body-mediated differentiation of mouse embryonic stem cells along a hepatocyte lineage: insights from gene expression profiles.

Pluripotent embryonic stem (ES) cells represent a promising renewable cell source for the generation of functional differentiated cells. Previous studies incorporating embryoid body (EB)-mediated stem cell differentiation have, either spontaneously or after growth factor and extracellular matrix protein supplementation, yielded populations of hepatocyte lineage cells expressing mature hepatocyte markers such as albumin (ALB). In an effort to promote ES cell commitment to the hepatocyte lineage, we have evaluated the effects of four culture conditions on albumin and gene expression in differentiating ES cells. Quantitative in situ immunofluorescence and cDNA microarray analyses were used to describe not only lineage specificity but also to provide insights into the effects of disparate culture environments on the mechanisms of differentiation. The results of these studies suggest that spontaneous and collagen-mediated differentiation induce cells with the highest levels of ALB expression but mature liver specific genes were only expressed in the spontaneous condition. Further analysis of gene expression profiles indicated that two distinct mechanisms may govern spontaneous and collagen-mediated differentiation.

Acute-Phase Proteins↗
PubMed1999

STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells.

Embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). LIF acts through a receptor complex composed of a low affinity LIF receptor (LIFRbeta) and gp130. We reported that the intracellular domain of gp130 plays an important role in self-renewal of ES cells. In the present study, we examined the signaling pathway through which gp130 contributes to the self-renewal of ES cells. Mutational analysis of the cytoplasmic domain of gp130 revealed that the tyrosine residue of gp130 responsible for STAT3 activation is necessary for self-renewal of ES cells, while that required for SHP2 and MAP kinase activation was dispensable. Next, we constructed a fusion protein composed of the entire coding region of STAT3 and the ligand binding domain of the estrogen receptor. This construction (STAT3ER) induced expression of junB (one of the targets of STAT3) in ES cells in the presence of the synthetic ligand 4-hydroxytamoxifen (4HT), thereby indicating that STAT3ER is a conditionally active form. ES cells transfected with STAT3ER cultured in the presence of 4HT maintained an undifferentiated state. Taken together, these results strongly suggest that STAT3 activation is required and sufficient to maintain the undifferentiated state of ES cells.

Animals↗