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Mouse Embryonic Stem Cells (mouse + embryonic_stem_cell)
Selected AbstractsEffects of Ethanol on Mouse Embryonic Stem CellsALCOHOLISM, Issue 12 2009Alla Arzumanyan Background:, Fetal alcohol syndrome (FAS) reflects a constellation of congenital abnormalities caused by excess maternal consumption of alcohol. It is likely that interference with embryonic development plays a role in the pathogenesis of the disorder. Ethanol-induced apoptosis has been suggested as a causal factor in the genesis of FAS. Mouse embryonic stem (mES) cells are pluripotent cells that differentiate in vitro to cell aggregates termed embryoid bodies (EBs), wherein differentiation capacity and gene expression profile are similar to those of the early embryo. Methods:, To investigate the effects of ethanol during differentiation, mES cells were cultured on a gelatin surface in the presence of leukemia inhibitory factor which maintains adherent undifferentiated cells or in suspension to promote formation of EBs. All cells were treated (1,6 days) with 80 mM ethanol. The pluripotency and differentiation of mES cells were evaluated by western blotting of stage-specific embryonic antigen (SSEA-1), transcription factors Oct-3/4, Sox-2, and Nanog, using alkaline phosphatase staining. Apoptosis (early to late stages) was assessed by fluorescence-activated cell sorting using TdT-mediated biotin,dUTP nick-end labelling assay and fluorescein isothiocyanate-Annexin V/propidium iodide staining. Results:, Ethanol increased apoptosis during in vitro differentiation of mES cells to EBs, whereas undifferentiated cells were not affected. Ethanol exposure also interfered with pluripotency marker patterns causing an upregulation of SSEA-1 under self-renewal conditions. In EBs, ethanol delayed the downregulation of SSEA-1 and affected the regulation of transcription factors during differentiation. Conclusion:, Our findings suggest that ethanol may contribute to the pathogenesis of FAS by triggering apoptotic pathways during differentiation of embryonic stem cells and deregulating early stages of embryogenesis. [source] Chemically Induced Cardiomyogenesis of Mouse Embryonic Stem CellsCHEMBIOCHEM, Issue 2 2010Albrecht Berkessel Prof. Dr. Abstract A transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of ,-myosine heavy chain (,-MHC) promoter (p,-MHC-EGFP) was used to investigate the effects of (thio)urea and cinchona alkaloid derivatives on cardiomyogenesis. The screening of the compounds yielded cardiomyogenesis inducing substances with good (IV-5, V-4) to very good activities (II-16, IV-8), as determined by a 50 to 80,% increase in the EGFP fluorescence compared to untreated cells. Time-dependent screening approaches in which compounds were added at different developmental stages of the ES cells appeared to be of limited suitability for the identification of potential cellular targets. [source] Three-dimensional culture systems for the expansion of pluripotent embryonic stem cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Michael P. Storm Abstract Mouse embryonic stem cell (ESC) lines, and more recently human ESC lines, have become valuable tools for studying early mammalian development. Increasing interest in ESCs and their differentiated progeny in drug discovery and as potential therapeutic agents has highlighted the fact that current two-dimensional (2D) static culturing techniques are inadequate for large-scale production. The culture of mammalian cells in three-dimensional (3D) agitated systems has been shown to overcome many of the restrictions of 2D and is therefore likely to be effective for ESC proliferation. Using murine ESCs as our initial model, we investigated the effectiveness of different 3D culture environments for the expansion of pluripotent ESCs. Solohill Collagen, Solohill FACT, and Cultispher-S microcarriers were employed and used in conjunction with stirred bioreactors. Initial seeding parameters, including cell number and agitation conditions, were found to be critical in promoting attachment to microcarriers and minimizing the size of aggregates formed. While all microcarriers supported the growth of undifferentiated mESCs, Cultispher-S out-performed the Solohill microcarriers. When cultured for successive passages on Cultispher-S microcarriers, mESCs maintained their pluripotency, demonstrated by self-renewal, expression of pluripotency markers and the ability to undergo multi-lineage differentiation. When these optimized conditions were applied to unweaned human ESCs, Cultispher-S microcarriers supported the growth of hESCs that retained expression of pluripotency markers including SSEA4, Tra-1,60, NANOG, and OCT-4. Our study highlights the importance of optimization of initial seeding parameters and provides proof-of-concept data demonstrating the utility of microcarriers and bioreactors for the expansion of hESCs. Biotechnol. Bioeng. 2010;107:683,695. © 2010 Wiley Periodicals, Inc. [source] Efficient generation of mature cerebellar Purkinje cells from mouse embryonic stem cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2010Osamu Tao Abstract Mouse embryonic stem cells (ESCs) can generate cerebellar neurons, including Purkinje cells (PCs) and their precursor cells, in a floating culture system called serum-free culture of embryoid body-like aggregates (SFEB) treated with BMP4, Fgf8b, and Wnt3a. Here we successfully established a coculture system that induced the maturation of PCs in ESC-derived Purkinje cell (EDPC) precursors in SFEB, using as a feeder layer a cerebellum dissociation culture prepared from mice at postnatal day (P) 6,8. PC maturation was incomplete or abnormal when the adherent culture did not include feeder cells or when the feeder layer was from neonatal cerebellum. In contrast, EDPCs exhibited the morphology of mature PCs and synaptogenesis with other cerebellar neurons when grown for 4 weeks in coculture system with the postnatal cerebellar feeder. Furthermore, the electrophysiological properties of these EDPCs were compatible with those of native mature PCs in vitro, such as Na+ or Ca2+ spikes elicited by current injections and excitatory or inhibitory postsynaptic currents, which were assessed by whole-cell patch-clamp recordings. Thus, EDPC precursors in SFEB can mature into PCs whose properties are comparable with those of native PCs in vitro. © 2009 Wiley-Liss, Inc. [source] Influence of hormones and hormone metabolites on the growth of schwann cells derived from embryonic stem cells and on tumor cell lines expressing variable levels of neurofibromin,DEVELOPMENTAL DYNAMICS, Issue 2 2008Therese M. Roth Abstract Loss of neurofibromin, the protein product of the tumor suppressor gene neurofibromatosis type 1 (NF1), is associated with neurofibromas, composed largely of Schwann cells. The number and size of neurofibromas in NF1 patients have been shown to increase during pregnancy. A mouse embryonic stem cell (mESC) model was used, in which mESCs with varying levels of neurofibromin were differentiated into Schwann-like cells. NF1 cell lines derived from a malignant and a benign human tumor were used to study proliferation in response to hormones. Estrogen and androgen receptors were not expressed or expressed at very low levels in the NF1+/+ cells, at low levels in NF1+/,cells, and robust levels in NF1,/,cells. A 17,-estradiol (E2) metabolite, 2-methoxy estradiol (2ME2) is cytotoxic to the NF1,/, malignant tumor cell line, and inhibits proliferation in the other cell lines. 2ME2 or its derivatives could provide new treatment avenues for NF1 hormone-sensitive tumors at times of greatet hormonal influence. Developmental Dynamics 237:513,524, 2008. © 2008 Wiley-Liss, Inc. [source] Temporal expression changes during differentiation of neural stem cells derived from mouse embryonic stem cellJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2004Joon-Ik Ahn Abstract Temporal analysis in gene expression during differentiation of neural stem cells (NSCs) was performed by using in-house microarrays composed of 10,368 genes. The changes in mRNA level were measured during differentiation day 1, 2, 3, 6, 12, and 15. Out of 10,368 genes analyzed, 259 genes were up-regulated or down-regulated by 2-fold or more at least at one time-point during differentiation, and were classified into six clusters based on their expression patterns by K-means clustering. Clusters characterized by gradual increase have large numbers of genes involved in transport and cell adhesion; those which showed gradual decrease have much of genes in nucleic acid metabolism, cell cycle, transcription factor, and RNA processing. In situ hybridization (ISH) validated microarray data and it also showed that Fox M1, cyclin D2, and CDK4 were highly expressed in CNS germinal zones and ectonucleotide pyrophosphatase/phosphodiesterase 2 (Enpp2) was highly expressed in choroid plexus where stem/progenitor cells are possibly located. Together, this clustering analysis of expression patterns of functionally classified genes may give insight into understanding of CNS development and mechanisms of NSCs proliferation and differentiation. © 2004 Wiley-Liss, Inc. [source] Extrinsic factors derived from mouse embryonal carcinoma cell lines maintain pluripotency of mouse embryonic stem cells through a novel signal pathwayDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2009Shinjirou Kawazoe Embryonic carcinoma (EC) cells, which are malignant stem cells of teratocarcinoma, have numerous morphological and biochemical properties in common with pluripotent stem cells such as embryonic stem (ES) cells. However, three EC cell lines (F9, P19 and PCC3) show different developmental potential and self-renewal capacity from those of ES cells. All three EC cell lines maintain self-renewal capacity in serum containing medium without Leukemia Inhibitory factor (LIF) or feeder layer, and show limited differentiation capacity into restricted lineage and cell types. To reveal the underlying mechanism of these characteristics, we took the approach of characterizing extrinsic factors derived from EC cells on the self-renewal capacity and pluripotency of mouse ES cells. Here we demonstrate that EC cell lines F9 and P19 produce factor(s) maintaining the undifferentiated state of mouse ES cells via an unidentified signal pathway, while P19 and PCC3 cells produce self-renewal factors of ES cells other than LIF that were able to activate the STAT3 signal; however, inhibition of STAT3 activation with Janus kinase inhibitor shows only partial impairment on the maintenance of the undifferentiated state of ES cells. Thus, these factors present in EC cells-derived conditioned medium may be responsible for the self-renewal capacity of EC and ES cells independently of LIF signaling. [source] IFN-, induces apoptosis in mouse embryonic stem cells, a putative mechanism of its embryotoxicityDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2000Gang-Ming Zou It has been reported that interferon (IFN)-, should inhibit in vitro mouse embryo growth by direct cell toxicity. However, the mechanism involved has not been clearly established. In the present study, this question was addressed using the embryonic stem (ES) cell model. It was found that IFN-, induces a dose-dependent apoptosis in ES cells, as assessed by trypan-blue staining, by Annexin-V labeling and DNA analysis. Moreover, IFN-, treatment cooperates with Fas-mediated apoptosis, a phenomenon that has been recently reported. As Bcl-2 oncoprotein functions as a death repressor molecule in an evolutionarily conserved cell death pathway, its expression was analyzed by flow cytometry. It was demonstrated that Bcl-2 is expressed in ES cells. When compared to untreated ES cells, IFN-,-treated, apoptotic cells expressed a lower Bcl-2 level and a normal level of Fas, whereas surviving cells expressed a normal level of Bcl-2 but a lower Fas expression. Altogether, these data suggest that IFN-, may influence early mouse embryo development by promoting apoptosis, which may constitute a novel mechanism of IFN-, embryotoxicity. [source] Changes in gene expression and morphology of mouse embryonic stem cells on differentiation into insulin-producing cells in vitro and in vivoDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2009Ortwin Naujok Abstract Background Embryonic stem (ES) cells have the potential to produce unlimited numbers of surrogate insulin-producing cells for cell replacement therapy of type 1 diabetes mellitus. The impact of the in vivo environment on mouse ES cell differentiation towards insulin-producing cells was analysed morphologically after implantation. Methods ES cells differentiated in vitro into insulin-producing cells according to the Lumelsky protocol or a new four-stage differentiation protocol were analysed morphologically before and after implantation for gene expression by in situ reverse transcription polymerase chain reaction and protein expression by immunohistochemistry and ultrastructural analysis. Results In comparison with nestin positive ES cells developed according to the reference protocol, the number of ES cells differentiated with the four-stage protocol increased under in vivo conditions upon morphological analysis. The cells exhibited, in comparison to the in vitro situation, increased gene and protein expression of Pdx1, insulin, islet amyloid polypeptide (IAPP), the GLUT2 glucose transporter and glucokinase, which are functional markers for glucose-induced insulin secretion of pancreatic beta cells. Renal sub-capsular implantation of ES cells with a higher degree of differentiation achieved by in vitro differentiation with a four-stage protocol enabled further significant maturation for the beta-cell-specific markers, insulin and the co-stored IAPP as well as the glucose recognition structures. In contrast, further in vivo differentiation was not achieved with cells differentiated in vitro by the reference protocol. Conclusions A sufficient degree of in vitro differentiation is an essential prerequisite for further substantial maturation in a beta-cell-specific way in vivo, supported by cell-cell contacts and vascularisation. Copyright © 2009 John Wiley & Sons, Ltd. [source] Cover Picture: Electrophoresis 9'09ELECTROPHORESIS, Issue 9 2009Article first published online: 7 MAY 200 Issue no. 9 is an Emphasis Issue with 7 articles on various aspects of "Microfluidics and Miniaturization" while the remaining articles are grouped into sections on "Detection Sensitivity Enhancement and Stacking", "Binding Studies" and "Other Methodologies". In addition, issue no. 9 has two Fast Track articles. The first on proteome alteration of early-stage differentiation of mouse embryonic stem cells into hepatocyte-like cells, and the second on dielectrophoretic separation of small particles in a sawtooth channel. [source] Activation of JNK and PAK2 is essential for citrinin-induced apoptosis in a human osteoblast cell lineENVIRONMENTAL TOXICOLOGY, Issue 4 2009Yu-Ting Huang Abstract The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells. CTN causes cell injury, including apoptosis. Previous studies by our group showed that CTN triggers apoptosis in mouse embryonic stem cells, as well as embryonic developmental injury. Here, we investigated the precise mechanisms governing this apoptotic effect in osteoblasts. CTN induced apoptotic biochemical changes in a human osteoblast cell line, including activation of c-Jun N-terminal kinase (JNK), loss of mitochondrial membrane potential, and caspase-3 and p21-activated protein kinase 2 (PAK2) activation. Experiments using a JNK-specific inhibitor, SP600125, and antisense oligonucleotides against JNK reduced CTN-induced activation of both JNK and caspase-3 in osteoblasts, indicating that JNK is required for caspase activation in this apoptotic pathway. Experiments using caspase-3 inhibitors and antisense oligonucleotides against PAK2 revealed that active caspase-3 is essential for PAK2 activation. Moreover, both caspase-3 and PAK2 require activation for CTN-induced apoptosis of osteoblasts. Interestingly, CTN stimulates two-stage activation of JNK in human osteoblasts. Early-stage JNK activation is solely ROS-dependent, whereas late-stage activation is dependent on ROS-mediated caspase activity, and regulated by caspase-induced activation of PAK2. On the basis of these results, we propose a signaling cascade model for CTN-induced apoptosis in human osteoblasts involving ROS, JNK, caspases, and PAK2. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009. [source] Electrical and neurotransmitter activity of mature neurons derived from mouse embryonic stem cells by Sox-1 lineage selection and directed differentiationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2004R. J. Lang Abstract Sx1TV2/16C is a mouse embryonic stem (ES) cell line in which one copy of the Sox1 gene, an early neuroectodermal marker, has been targeted with a neomycin (G418) selection cassette. A combination of directed differentiation with retinoic acid and G418 selection results in an enriched neural stem cell population that can be further differentiated into neurons. After 6,7 days post-plating (D6,7PP) most neurons readily fired tetrodotoxin (TTX)-sensitive action potentials due to the expression of TTX-sensitive Na+ and tetraethylammonium (TEA)-sensitive K+ channels. Neurons reached their maximal cell capacitance after D6,7PP; however, ion channel expression continued until at least D21PP. The percentage of cells receiving spontaneous synaptic currents (s.s.c.) increased with days in culture until 100% of cells received a synaptic input by D20PP. Spontaneous synaptic currents were reduced in amplitude and frequency by TTX, or upon exposure to a Ca2+ -free, 2.5 mm Mg2+ saline. S.s.c. of rapid decay time constants were preferentially blocked by the nonNMDA glutamatergic receptor antagonists CNQX or NBQX. Ca2+ levels within ES cell-derived neurons increased in response to glutamate receptor agonists l -glutamate, AMPA, N -methyl- d -aspartate (NMDA) and kainic acid and to acetylcholine, ATP and dopamine. ES cell-derived neurons also generated cationic and Cl, -selective currents in response to NMDA and glycine or GABA, respectively. It was concluded that ES-derived neurons fire action potentials, receive excitatory and inhibitory synaptic input and respond to various neurotransmitters in a manner akin to primary central neurons. [source] Antibiotic-Loaded PLGA Nanofibers for Wound Healing Applications,ADVANCED ENGINEERING MATERIALS, Issue 4 2010David A. Soscia Incorporating antibiotics into biocompatible nanoscale non-woven fibrous mats could provide utility for wound healing applications and for incorporation into wound dressing materials. In this study, the antibiotic chloramphenicol (Cm) was incorporated into electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers, which were then tested for inhibition of bacterial growth for multiple bacterial species (Escherichia coli, Staphylococcus aureus, Bacillus cereus, Salmonella typhimurium, and Pseudomonas aeruginosa). In addition, the cytotoxicity of Cm-PLGA nanofibers was examined for two types of mammalian cells including mouse embryonic stem cells and fibroblasts. Electrospun PLGA nanofibers containing Cm were able to reduce bacterial growth on solid agar plates for all species except for P. aeruginosa. In liquid culture, Cm-loaded nanofibers inhibited growth for E. coli, B. cereus and S. typhimurium by 93% or greater, while P. aeruginosa and S. aureus growth was inhibited by 42% and 56%, respectively. Cm-loaded nanofibers showed limited cytoxicity on fibroblasts and embryonic stem cells, with viability greater than 96% for all conditions tested. These results suggest that Cm can be successfully incorporated into electrospun nanofibers and that these fibers could be used for wound healing applications with minimal cytotoxicity to the surrounding tissue. [source] Genetic engineering of mouse embryonic stem cells by Nurr1 enhances differentiation and maturation into dopaminergic neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2002Sangmi Chung Abstract Nurr1 is a transcription factor critical for the development of midbrain dopaminergic (DA) neurons. This study modified mouse embryonic stem (ES) cells to constitutively express Nurr1 under the elongation factor-1, promoter. The Nurr1-expression in ES cells lead to up-regulation of all DA neuronal markers tested, resulting in about a 4- to 5-fold increase in the proportion of DA neurons. In contrast, other neuronal and glial markers were not significantly changed by Nurr1 expression. It was also observed that there was an additional 4-fold increase in the number of DA neurons in Nurr1-expressing clones following treatment with Shh, FGF8 and ascorbic acid. Several lines of evidence suggest that these neurons may represent midbrain DA neuronal phenotypes; firstly, they coexpress midbrain DA markers such as aromatic l -amino acid decarboxylase, calretinin, and dopamine transporter, in addition to tyrosine hydroxylase and secondly, they do not coexpress other neurotransmitters such as GABA or serotonin. Finally, consistent with an increased number of DA neurons, the Nurr1 transduction enhanced the ability of these neurons to produce and release DA in response to membrane depolarization. This study demonstrates an efficient genetic manipulation of ES cells that facilitates differentiation to midbrain DA neurons, and it will serve as a framework of genetic engineering of ES cells by key transcription factor to regulate their cell fate. [source] Maternal-effect gene Ces5/Ooep/Moep19/Floped is essential for oocyte cytoplasmic lattice formation and embryonic development at the maternal-zygotic stage transitionGENES TO CELLS, Issue 8 2010Fumi Tashiro In a search for genes specifically expressed in mouse embryonic stem cells, we identified one we called Ces5. We found that it corresponded to the Ooep gene, which was recently reported to be expressed specifically in oocytes. Mouse Ces5/Ooep, also called Moep19 or Floped, encoded a 164-amino acid protein, which was detected in the cytoplasm of developing and mature oocytes and in embryos throughout the preimplantation period. To examine its function, we carried out targeted disruption of this gene. The Ces5/Ooep -null mice were grossly normal, but the females were infertile. Although the ovaries and ovulation appeared normal, the embryos from Ces5/Ooep -null females mated with wild-type males showed developmental arrest at the two- or four-cell stage. In addition, their first cleavage was considerably delayed and often asymmetrical. Thus, Ces5/Ooep is a maternal-effect gene. By electron microscopy, we found that the eggs from Ces5/Ooep -null females lacked oocyte cytoplasmic lattices (CPLs), which have long been predicted to function as a storage form for components that are maternally contributed to the early embryo. Further analysis showed that CES5/OOEP was directly associated with the CPLs. These results indicate that CES5/OOEP is an essential component of the CPLs and is required for embryonic development at the maternal-zygotic stage transition. [source] DPPA4 modulates chromatin structure via association with DNA and core histone H3 in mouse embryonic stem cellsGENES TO CELLS, Issue 4 2010Hisaharu Masaki Developmental pluripotency associated 4 (DPPA4) is one of the uncharacterized genes that is highly expressed in embryonic stem (ES) cells. DPPA4 is associated with active chromatin and involved in the pluripotency of mouse ES cells. However, the biological function of DPPA4 remains poorly understood. In this study, we performed fluorescence recovery after photobleaching (FRAP) analysis to examine the dynamics of DPPA4 in ES cells. FRAP analysis showed that the mobility of DPPA4 is similar to that of histone H1. In addition, biochemical analysis with purified proteins and immunoprecipitation analysis showed that DPPA4 directly binds to both DNA and core histone H3. The analysis using truncated proteins indicated that DPPA4 is associated with DNA via the N-terminal region and histone H3 via the C-terminal region. In vitro assembled chromatin showed resistance to micrococcal nuclease (MNase) digestion in the presence of DPPA4. Moreover, MNase assay and FRAP analysis with the truncated proteins implies that DPPA4 binding to both DNA and histone H3 is necessary for the chromatin structure resistant to MNase and for the proper localization of DPPA4 in ES cell nuclei. These results suggest that DPPA4 modulates the chromatin structure in association with DNA and histone H3 in ES cells. [source] Dynamic changes in the epigenomic state and nuclear organization of differentiating mouse embryonic stem cellsGENES TO CELLS, Issue 4 2007Satoru Kobayakawa Changes in nuclear organization and the epigenetic state of the genome are important driving forces for developmental gene expression. However, a strategy that allows simultaneous visualization of the dynamics of the epigenomic state and nuclear structure has been lacking to date. We established an experimental system to observe global DNA methylation in living mouse embryonic stem (ES) cells. The methylated DNA binding domain (MBD) and the nuclear localization signal (nls) sequence coding for human methyl CpG-binding domain protein 1 (MBD1) were fused to the enhanced green fluorescent protein (EGFP) reporter gene, and ES cell lines carrying the construct (EGFP-MBD-nls) were established. The EGFP-MBD-nls protein was used to follow DNA methylation in situ under physiological conditions. We also monitored the formation and rearrangement of methylated heterochromatin using EGFP-MBD-nls. Pluripotent mouse ES cells showed unique nuclear organization in that methylated centromeric heterochromatin coalesced to form large clusters around the nucleoli. Upon differentiation, the organization of these heterochromatin clusters changed dramatically. Time-lapse microscopy successfully captured a moment of dramatic change in chromosome positioning during the transition between two differentiation stages. Thus, this experimental system should facilitate studies focusing on relationships between nuclear organization, epigenetic status and cell differentiation. [source] Genome-wide and locus-specific DNA hypomethylation in G9a deficient mouse embryonic stem cellsGENES TO CELLS, Issue 1 2007Kohta Ikegami In the mammalian genome, numerous CpG-rich loci define tissue-dependent and differentially methylated regions (T-DMRs). Euchromatin from different cell types differs in terms of its tissue-specific DNA methylation profile as defined by these T-DMRs. G9a is a euchromatin-localized histone methyltransferase (HMT) and catalyzes methylation of histone H3 at lysines 9 and 27 (H3-K9 and -K27). To test whether HMT activity influences euchromatic cytosine methylation, we analyzed the DNA methylation status of approximately 2000 CpG-rich loci, which are predicted in silico, in G9a,/, embryonic stem cells by restriction landmark genomic scanning (RLGS). While the RLGS profile of wild-type cells contained about 1300 spots, 32 new spots indicating DNA demethylation were seen in the profile of G9a,/, cells. Virtual-image RLGS (Vi-RLGS) allowed us to identify the genomic source of ten of these spots. These were confirmed to be cytosine demethylated, not just at the Not I site detected by the RLGS but extending over several kilobase pairs in cis. Chromatin immunoprecipitation (ChIP) confirmed these loci to be targets of G9a, with decreased H3-K9 and/or -K27 dimethylation in the G9a,/, cells. These data indicate that G9a site-selectively contributes to DNA methylation. [source] Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3bGENES TO CELLS, Issue 7 2006Akiko Tsumura DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing and maintenance of genome integrity. Proper CpG methylation is required for the normal growth of various somatic cell types, indicating its essential role in the basic cellular function of mammalian cells. Previous studies using Dnmt1,/, or Dnmt3a,/,Dnmt3b,/, ES cells, however, have shown that undifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation. In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function, we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite the absence of CpG methylation, as demonstrated by genome-wide methylation analysis, these triple knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics. TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at Lys9 of histone H3 and heterochromatin protein-1, and maintained their normal chromosome numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA methyltransferases. [source] A mouse embryonic stem cell model of Schwann cell differentiation for studies of the role of neurofibromatosis type 1 in Schwann cell development and tumor formationGLIA, Issue 11 2007Therese M. Roth Abstract The neurofibromatosis Type 1 (NF1) gene functions as a tumor suppressor gene. One known function of neurofibromin, the NF1 protein product, is to accelerate the slow intrinsic GTPase activity of Ras to increase the production of inactive rasGDP, with wide-ranging effects on p21ras pathways. Loss of neurofibromin in the autosomal dominant disorder NF1 is associated with tumors of the peripheral nervous system, particularly neurofibromas, benign lesions in which the major affected cell type is the Schwann cell (SC). NF1 is the most common cancer predisposition syndrome affecting the nervous system. We have developed an in vitro system for differentiating mouse embryonic stem cells (mESC) that are NF1 wild type (+/+), heterozygous (+/,), or null (,/,) into SC-like cells to study the role of NF1 in SC development and tumor formation. These mES-generated SC-like cells, regardless of their NF1 status, express SC markers correlated with their stage of maturation, including myelin proteins. They also support and preferentially direct neurite outgrowth from primary neurons. NF1 null and heterozygous SC-like cells proliferate at an accelerated rate compared to NF1 wild type; this growth advantage can be reverted to wild type levels using an inhibitor of MAP kinase kinase (Mek). The mESC of all NF1 types can also be differentiated into neuron-like cells. This novel model system provides an ideal paradigm for studies of the role of NF1 in cell growth and differentiation of the different cell types affected by NF1 in cells with differing levels of neurofibromin that are neither transformed nor malignant. © 2007 Wiley-Liss, Inc. [source] Stem cell generation and choice of fate: role of cytokines and cellular microenvironmentJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 4 2000S.N. Constantinescu Hematopoietic stem cells (HSC) have provided a model for the isolation, enrichment and transplantation of stem cells. Gene targeting studies in mice have shown that expression of the thrombopoietin receptor (TpoR) is linked to the accumulation of HSCs capable to generate long-term blood repopulation when injected into irradiated mice. The powerful increase in vivo in HSC numbers by retrovirally transduced HOX4B, a homeotic gene, along with the role of the TpoR, suggested that stem cell fate, renewal, differentiation and number can be controlled. The discovery of the precise region of the mouse embryo where HSCs originate and the isolation of supporting stromal cell lines open the possibility of identifying the precise signals required for HSC choice of fate. The completion of human genome sequencing coupled with advances in gene expression profiling using DNA microarrays will enable the identification of key genes deciding the fate of stem cells. Downstream from HSCs, multipotent hematopoietic progenitor cells appear to co-express a multiplicity of genes characteristic of different blood lineages. Genomic approaches will permit the identification of the select group of genes consolidated by the commitment of these multipotent progenitors towards one or the other of the blood lineages. Studies with neural stem cells pointed to the unexpected plastic nature of these cells. Isolation of stem cells from multiple tissues may suggest that, providing the appropriate environment/signal, tissues could be regenerated in the laboratory and used for transplantation. A spectacular example of influence of the environment on cell fate was revealed decades ago by using mouse embryonic stem cells (ES). Injected into blastocysts, ES cells contribute to the formation of all adult tissues. Injected into adult mice, ES cells become cancer cells. After multiple passages as ascites, when injected back into the blastocyst environment, ES- derived cancer cells behaved again as ES cells. More recently, the successful cloning of mammals and reprogramming of transferred nuclei by factors in the cytoplasm of oocytes turned back the clock by showing that differentiated nuclei can be "re-booted" to generate again the stem cells for different tissues. [source] High glucose increase cell cycle regulatory proteins level of mouse embryonic stem cells via PI3-K/Akt and MAPKs signal pathwaysJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2006Yun Hee Kim This study examined the effects of high glucose on cell proliferation and its related signal pathways using mouse embryonic stem (ES) cells. Here, we showed that high glucose level significantly increased [3H]thymidine incorporation, BrdU incorporation, the number of cells, [3H]leucine, and [3H]proline incorporation in a time-(>3 hr) and dose-(>25 mM) dependent manner. Moreover, high glucose level increased the cellular reactive oxygen species (ROS), Akt, and mitogen-activated protein kinases (MAPKs) phosphorylation. Subsequently, these signaling molecules involved in high glucose-induced increase of [3H]thymidine incorporation. High glucose level also increased cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4 protein levels, which is cell cycle regulatory proteins acting in G1,S phase of cell cycle. Inhibition of phosphatidylinositol 3-kinase (PI3-K) (LY 294002: PI3-kinase inhibitor, 10,6 M), Akt (Akt inhibitor, 10,5 M), and p44/42 MAPKs (PD 98059: MEK inhibitor, 10,5 M) decreased these proteins. High glucose level phosphorylated the RB protein, which was decreased by inhibition of PI3-K and Akt. In conclusion, high glucose level stimulates mouse ES cell proliferation via the PI3-K/Akt and MAPKs pathways. J. Cell. Physiol. 209: 94,102, 2006. © 2006 Wiley-Liss, Inc. [source] Efficient generation of mature cerebellar Purkinje cells from mouse embryonic stem cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2010Osamu Tao Abstract Mouse embryonic stem cells (ESCs) can generate cerebellar neurons, including Purkinje cells (PCs) and their precursor cells, in a floating culture system called serum-free culture of embryoid body-like aggregates (SFEB) treated with BMP4, Fgf8b, and Wnt3a. Here we successfully established a coculture system that induced the maturation of PCs in ESC-derived Purkinje cell (EDPC) precursors in SFEB, using as a feeder layer a cerebellum dissociation culture prepared from mice at postnatal day (P) 6,8. PC maturation was incomplete or abnormal when the adherent culture did not include feeder cells or when the feeder layer was from neonatal cerebellum. In contrast, EDPCs exhibited the morphology of mature PCs and synaptogenesis with other cerebellar neurons when grown for 4 weeks in coculture system with the postnatal cerebellar feeder. Furthermore, the electrophysiological properties of these EDPCs were compatible with those of native mature PCs in vitro, such as Na+ or Ca2+ spikes elicited by current injections and excitatory or inhibitory postsynaptic currents, which were assessed by whole-cell patch-clamp recordings. Thus, EDPC precursors in SFEB can mature into PCs whose properties are comparable with those of native PCs in vitro. © 2009 Wiley-Liss, Inc. [source] Generation of hepatocytes from cultured mouse embryonic stem cellsLIVER TRANSPLANTATION, Issue 10 2003Xiao Ling Kuai Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of fertilized blastocysts in vitro. ES cells can be induced to undergo differentiation into potentially all cell types. The aim of this study is to examine the differentiating potential of mouse ES cells into hepatocytes in the presence of retinoic acid (RA), hepatocyte growth factor (HGF), and ,-nerve growth factor (,-NGF). RA, HGF, and ,-NGF were added to the cell culture. Hepatocyte induction was confirmed morphologically, as well as biochemically, through immunohistochemical assays of ,1 -antitrypsin (,1 -AT) and alfafetaprotein (AFP) expression and reverse-transcriptase polymerase chain reaction tests for the presence of albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4, and SAPK/ERK kinase-1 (SEK1) messenger RNA, produced only by functioning hepatocytes. Fifteen days after the addition of HGF and ,-NGF to the cell culture, many epithelioid cells were noticed. ,1 -AT, AFP, albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4, and SEK1 messenger RNA expression also was detected, indicating successful ES cell differentiation into functioning hepatocytes. However, in the presence of RA alone, only transthyretin messenger RNA was positive, whereas no other expression pertaining to functioning hepatocytes could be detected. In the presence of HGF and ,-NGF, mouse ES cells can differentiate into functioning hepatocytes, whereas RA function is limited. [source] Hyperpolarization-activated cyclic nucleotide-modulated ,HCN' channels confer regular and faster rhythmicity to beating mouse embryonic stem cellsTHE JOURNAL OF PHYSIOLOGY, Issue 3 2008Yang Qu The hyperpolarization-activated cation current (If), and the hyperpolarization-activated cyclic nucleotide-modulated ,HCN' subunits that underlie it, are important components of spontaneous activity in the embryonic mouse heart, but whether they contribute to this activity in mouse embryonic stem cell-derived cardiomyocytes has not been investigated. We address this issue in spontaneously beating cells derived from mouse embryonic stem cells (mESCs) over the course of development in culture. If and action potentials were recorded from single beating cells at early, intermediate and late development stages using perforated whole-cell voltage- and current-clamp techniques. Our data show that the proportion of cells expressing If, and the density of If in these cells, increased during development and correlated with action potential frequency and the rate of diastolic depolarization. The If blocker ZD7288 (0.3 ,m) reduced If and the beating rate of embryoid bodies. Taken together, the activation kinetics of If and results from Western blots are consistent with the presence of the HCN2 and HCN3 isoforms. At all stages of development, isoproterenol (isoprenaline) and acetylcholine shifted the voltage dependence of If to more positive and negative voltages, respectively, and they also increased and decreased the beating rate of embryonic cell bodies, respectively. Together, the data suggest that current through HCN2 and HCN3 channels confers regular and faster rhythmicity to mESCs, which mirrors the developing embryonic mouse heart, and contributes to modulation of rhythmicity by autonomic stimulation. [source] An embryonic story: Analysis of the gene regulative network controlling Xist expression in mouse embryonic stem cellsBIOESSAYS, Issue 7 2010Pablo Navarro Abstract In mice, dosage compensation of X-linked gene expression is achieved through the inactivation of one of the two X-chromosomes in XX female cells. The complex epigenetic process leading to X-inactivation is largely controlled by Xist and Tsix, two non-coding genes of opposing function. Xist RNA triggers X-inactivation by coating the inactive X, while Tsix is critical for the designation of the active X-chromosome through cis -repression of Xist RNA accumulation. Recently, a plethora of trans -acting factors and cis -regulating elements have been suggested to act as key regulators of either Xist, Tsix or both; these include ubiquitous factors such as Yy1 and Ctcf, developmental proteins such as Nanog, Oct4 and Sox2, and X-linked regulators such as Rnf12. In this paper we summarise recent advances in our knowledge of the regulation of Xist and Tsix in embryonic stem (ES) and differentiating ES cells. [source] An embryonic story: analysis of the gene regulative network controlling Xist expression in mouse embryonic stem cellsBIOESSAYS, Issue 7 2010Pablo Navarro No abstract is available for this article. [source] Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process controlBIOTECHNOLOGY & BIOENGINEERING, Issue 7 2005Magnus Schroeder Abstract It is well established that embryonic stem (ES) cells can differentiate into functional cardiomyocytes in vitro. ES-derived cardiomyocytes could be used for pharmaceutical and therapeutic applications, provided that they can be generated in sufficient quantity and with sufficient purity. To enable large-scale culture of ES-derived cells, we have developed a robust and scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, stirred 2 L bioreactor following inoculation with a single cell suspension of mouse ES cells. Utilizing a pitched-blade-turbine, parameters for optimal cell expansion as well as efficient ES cell differentiation were established. Optimization of stirring conditions resulted in the generation of high-density suspension cultures containing 12.5,×,106 cells/mL after 9 days of differentiation. Approximately 30%,40% of the EBs formed in this process vigorously contracted, indicating robust cardiomyogenic induction. An ES cell clone carrying a recombinant DNA molecule comprised of the cardiomyocyte-restricted alpha myosin heavy chain (,MHC) promoter and a neomycin resistance gene was used to establish the utility of this bioprocess to efficiently generate ES-derived cardiomyocytes. The genetically engineered ES cells were cultured directly in the stirred bioreactor for 9 days, followed by antibiotic treatment for another 9 days. The protocol resulted in the generation of essentially pure cardiomyocyte cultures, with a total yield of 1.28,×,109 cells in a single 2 L bioreactor run. This study thus provides an important step towards the large-scale generation of ES-derived cells for therapeutic and industrial applications. © 2005 Wiley Periodicals, Inc. [source] Accurate control of oxygen level in cells during culture on silicone rubber membranes with application to stem cell differentiationBIOTECHNOLOGY PROGRESS, Issue 3 2010Daryl E. Powers Abstract Oxygen level in mammalian cell culture is often controlled by placing culture vessels in humidified incubators with a defined gas phase partial pressure of oxygen (pO2gas). Because the cells are consuming oxygen supplied by diffusion, a difference between pO2gas and that experienced by the cells (pO2cell) arises, which is maximal when cells are cultured in vessels with little or no oxygen permeability. Here, we demonstrate theoretically that highly oxygen-permeable silicone rubber membranes can be used to control pO2cell during culture of cells in monolayers and aggregates much more accurately and can achieve more rapid transient response following a disturbance than on polystyrene and fluorinated ethylene-propylene copolymer membranes. Cell attachment on silicone rubber was achieved by physical adsorption of fibronectin or Matrigel. We use these membranes for the differentiation of mouse embryonic stem cells to cardiomyocytes and compare the results with culture on polystyrene or on silicone rubber on top of polystyrene. The fraction of cells that are cardiomyocyte-like increases with decreasing pO2 only when using oxygen-permeable silicone membrane-based dishs, which contract on silicone rubber but not polystyrene. The high permeability of silicone rubber results in pO2cell being equal to pO2gas at the tissue-membrane interface. This, together with geometric information from histological sections, facilitates development of a model from which the pO2 distribution within the resulting aggregates is computed. Silicone rubber membranes have significant advantages over polystyrene in controlling pO2cell, and these results suggest they are a valuable tool for investigating pO2 effects in many applications, such as stem cell differentiation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Maintenance of pluripotency in mouse embryonic stem cells cultivated in stirred microcarrier culturesBIOTECHNOLOGY PROGRESS, Issue 2 2010Paulo A. N. Marinho Abstract The development of efficient and reproducible culture systems for embryonic stem (ES) cells is an essential pre-requisite for regenerative medicine. Culture scale-up ensuring maintenance of cell pluripotency is a central issue, because large amounts of pluripotent cells must be generated to warrant that differentiated cells deriving thereof are transplanted in great amounts and survive the procedure. This study aimed to develop a robust scalable cell expansion system, using a murine embryonic stem cell line that is feeder-dependent and adapted to serum-free medium, thus representing a more realistic model for human ES cells. We showed that high concentrations of murine ES cells can be obtained in stirred microcarrier-based spinner cultures, with a 10-fold concentration of cells per volume of medium and a 5-fold greater cell concentration per surface area, as compared to static cultures. No differences in terms of pluripotency and differentiation capability were observed between cells grown in traditional static systems and cells that were replated onto the traditional system after being expanded on microcarriers in the stirred system. This was verified by morphological analyses, quantification of cells expressing important pluripotency markers (Oct-4, SSEA-1, and SOX2), karyotype profile, and the ability to form embryoid bodies with similar sizes, and maintaining their intrinsic ability to differentiate into all three germ layers. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] | ||||||||||||||||||||||||||||||||||