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Embryonic Stem Cells (embryonic + stem_cell)
Kinds of Embryonic Stem Cells Terms modified by Embryonic Stem Cells Selected AbstractsEmbryonic Stem Cells and Gene TargetingEXPERIMENTAL PHYSIOLOGY, Issue 6 2000Birgit Ledermann The development of gene targeting technology, the exchange of an endogenous allele of a target gene for a mutated copy via homologous recombination, and the application of this technique to murine embryonic stem cells has made it possible to alter the germ-line of mice in a predetermined way. Gene targeting has enabled researchers to generate mouse strains with defined mutations in their genome allowing the analysis of gene function in vivo. This review presents the essential tools and methodologies used for gene targeting that have been developed over the past decade. Special emphasis has been laid on the available embryonic stem cell lines and the importance of the genetic background. Also, the state-of-the art of gene targeting approaches in species other than mice will be discussed. [source] Mapping the Interactions among Biomaterials, Adsorbed Proteins, and Human Embryonic Stem CellsADVANCED MATERIALS, Issue 27 2009Ying Mei An integrated high-throughput polymer synthesis and rapid material/protein/cell interaction assays were developed to optimize stem cell microenvironments. Microarrayed polymers were synthesized and studied for the ability to support the growth of partially differentiated human embryonic stem cells. In parallel, a programmed laser scanning cytometry system was developed to allow for rapid quantification of cell material interaction. [source] The Neuropeptide Pituitary Adenylate Cyclase-Activating Polypeptide Exerts Anti-Apoptotic and Differentiating Effects during Neurogenesis: Focus on Cerebellar Granule Neurones and Embryonic Stem CellsJOURNAL OF NEUROENDOCRINOLOGY, Issue 5 2007A. Falluel-Morel Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from ovine hypothalamus on the basis of its hypophysiotrophic activity. It has subsequently been shown that PACAP and its receptors are widely distributed in the central nervous system of adult mammals, indicating that PACAP may act as a neurotransmitter and/or neuromodulator. It has also been found that PACAP and its receptors are expressed in germinative neuroepithelia, suggesting that PACAP could be involved in neurogenesis. There is now compelling evidence that PACAP exerts neurotrophic activities in the developing cerebellum and in embryonic stem (ES) cells. In particular, the presence of PACAP receptors has been demonstrated in the granule layer of the immature cerebellar cortex, and PACAP has been shown to promote survival, inhibit migration and activate neurite outgrowth of granule cell precursors. In cerebellar neuroblasts, PACAP is a potent inhibitor of the mitochondrial apoptotic pathway through activation of the MAPkinase extracellular regulated kinase. ES cells and embryoid bodies (EB) also express PACAP receptors and PACAP facilitates neuronal orientation and induces the appearance of an electrophysiological activity. Taken together, the anti-apoptotic and pro-differentiating effects of PACAP characterised in cerebellar neuroblasts as well as ES and EB cells indicate that PACAP acts not only as a neurohormone and a neurotransmitter, but also as a growth factor. [source] Effects 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] Will Embryonic Stem Cells Change Health Policy?THE JOURNAL OF LAW, MEDICINE & ETHICS, Issue 2 2010William M. Sage Embryonic stem cells are actively debated in political and public policy arenas. However, the connections between stem cell innovation and overall health care policy are seldom elucidated. As with many controversial aspects of medical care, the stem cell debate bridges to a variety of social conversations beyond abortion. Some issues, such as translational medicine, commercialization, patient and public safety, health care spending, physician practice, and access to insurance and health care services, are core health policy concerns. Other issues, such as economic development, technologic progress, fiscal politics, and tort reform, are only indirectly related to the health care system but are frequently seen through a health care lens. These connections will help determine whether the stem cell debate reaches a resolution, and what that resolution might be. [source] Injection of Embryonic Stem Cells Into Scarred Rabbit Vocal Folds Enhances Healing and Improves Viscoelasticity: Short-Term Results,THE LARYNGOSCOPE, Issue 11 2007Jessica Cedervall MSc Abstract Objectives: Scarring caused by trauma, postcancer treatment, or inflammation in the vocal folds is associated with stiffness of the lamina propria and results in severe voice problems. Currently there is no effective treatment. Human embryonic stem cells (hESC) have been recognized as providing a potential resource for cell transplantations, but in the undifferentiated state, they are generally not considered for therapeutic use due to risk of inadvertent development. This study assesses the functional potential of hESC to prevent or diminish scarring and improve viscoelasticity following grafting into scarred rabbit vocal folds. Study Design: hESC were injected into 22 scarred vocal folds of New Zealand rabbits. After 1 month, the vocal folds were dissected and analyzed for persistence of hESC by fluorescence in situ hybridization using a human specific probe, and for differentiation by evaluation in hematoxylin-eosin-stained tissues. Parallel-plate rheometry was used to evaluate the functional effects, i.e., viscoelastic properties, after treatment with hESC. Results: The results revealed significantly improved viscoelasticity in the hESC-treated vs. non-treated vocal folds. An average of 5.1% engraftment of human cells was found 1 month after hESC injection. In the hESC-injected folds, development compatible with cartilage, muscle and epithelia in close proximity or inter-mixed with the appropriate native rabbit tissue was detected in combination with less scarring and improved viscoelasticity. Conclusions: The histology and location of the surviving hESC-derived cells strongly indicate that the functional improvement was caused by the injected cells, which were regenerating scarred tissue. The findings point toward a strong impact from the host microenvironment, resulting in a regional specific in vivo hESC differentiation and regeneration of three types of tissue in scarred vocal folds of adult rabbits. [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] Automated maintenance of embryonic stem cell culturesBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2007Stefanie Terstegge Abstract Embryonic stem cell (ESC) technology provides attractive perspectives for generating unlimited numbers of somatic cells for disease modeling and compound screening. A key prerequisite for these industrial applications are standardized and automated systems suitable for stem cell processing. Here we demonstrate that mouse and human ESC propagated by automated culture maintain their mean specific growth rates, their capacity for multi-germlayer differentiation, and the expression of the pluripotency-associated markers SSEA-1/Oct-4 and Tra-1-60/Tra-1-81/Oct-4, respectively. The feasibility of ESC culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications. Biotechnol. Bioeng. 2007;96: 195,201. © 2006 Wiley Periodicals, Inc. [source] Nuclear proteome analysis of undifferentiated mouse embryonic stem and germ cellsELECTROPHORESIS, Issue 11 2008Nicolas Buhr Abstract Embryonic stem cells (ESCs) and embryonic germ cells (EGCs) provide exciting models for understanding the underlying mechanisms that make a cell pluripotent. Indeed, such understanding would enable dedifferentiation and reprogrammation of any cell type from a patient needing a cell therapy treatment. Proteome analysis has emerged as an important technology for deciphering these biological processes and thereby ESC and EGC proteomes are increasingly studied. Nevertheless, their nuclear proteomes have only been poorly investigated up to now. In order to investigate signaling pathways potentially involved in pluripotency, proteomic analyses have been performed on mouse ESC and EGC nuclear proteins. Nuclei from ESCs and EGCs at undifferentiated stage were purified by subcellular fractionation. After 2-D separation, a subtractive strategy (subtracting culture environment contaminating spots) was applied and a comparison of ESC, (8.5 day post coïtum (dpc))-EGC and (11.5,dpc)-EGC specific nuclear proteomes was performed. A total of 33 ESC, 53 (8.5,dpc)-EGC, and 36 (11.5,dpc)-EGC spots were identified by MALDI-TOF-MS and/or nano-LC-MS/MS. This approach led to the identification of two isoforms (with and without N -terminal acetylation) of a known pluripotency marker, namely developmental pluripotency associated 5 (DPPA5), which has never been identified before in 2-D gel-MS studies of ESCs and EGCs. Furthermore, we demonstrated the efficiency of our subtracting strategy, in association with a nuclear subfractionation by the identification of a new protein (protein arginine N -methyltransferase 7; PRMT7) behaving as proteins involved in pluripotency. [source] Embryonic stem cells reduce liver fibrosis in CCl4 -treated miceINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 6 2008Kei Moriya Summary We transplanted undifferentiated embryonic stem (ES) cells into the spleens of carbon tetrachloride (CCl4)-treated mice to determine their effects on liver fibrosis. Carbon tetrachloride at 0.5 ml/kg of body weight was injected intraperitoneally into C57BL/6 mice twice weekly for up to 20 weeks. Four weeks after the first injection, the mice were divided into two groups and those in group 1 received 1 × 105 ES cells genetically labelled with enhanced green fluorescent protein (GFP) in the spleens, while group 2 mice received 0.1 ml of phosphate-buffered saline. In group 1, GFP-immunopositive cells were retained and found in areas of fibrosis in the liver, and reduced liver fibrosis was observed as compared with group 2. Secondary transplantation of ES cells at 12 weeks after the initial transplantation enhanced the reduction in liver fibrosis. No teratoma formation or uncontrolled growth of ES cells in organs, including the liver and spleen, was observed in any of the mice. In the livers of group 1 mice, metalloproteinase 9-immunopositive cells derived from ES cells as well as those from the recipient were observed. These cells were also found to be immunopositive for the hepatoblast marker Delta-like (DlK-1), a member of the DlK-1 family of transmembrane proteins. These results suggest that ES-based cell therapy is potentially useful for liver fibrosis treatment and that reduction in CCl4 -induced liver fibrosis by transplantation of ES cells may be related closely to the emergence of metalloproteinase-producing hepatoblast-like cells. [source] Programming the genome in embryonic and somatic stem cellsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 4 2007Philippe Collas ,,Introduction ,,Epigenetic makeup of embryonic stem cells: keeping chromatin loose -,DNA methylation and gene expression -,CpG methylation profiles in mouse ESCs -,CpG methylation patterns in human ESCs -,Both active and inactive histone modification marks on developmentally regulated genes in ESCs suggest transcriptional activation potential -,A regulatory role of histone H1 in gene expression in embryonic stem cells? -,Polycomb group proteins impose a transcriptional brake on lineage-priming genes ,,The epigenetic makeup of mesenchymal stem cells reflects restricted differentiation potential -,CpG methylation patterns on lineage-specific promoters in adipose stem cells -,CpG content affects the relationship between promoter DNA methylation and transcriptional activity -,Bivalent histone modifications on potentially active genes? ,,Linking DNA methylation to histone modifications, chromatin packaging and (re)organization of the nuclear compartment ,,Perspectives: towards remodelling the stem cell epigenome? Abstract In opposition to terminally differentiated cells, stem cells can self-renew and give rise to multiple cell types. Embryonic stem cells retain the ability of the inner cell mass of blastocysts to differentiate into all cell types of the body and have acquired in culture unlimited self-renewal capacity. Somatic stem cells are found in many adult tissues, have an extensive but finite lifespan and can differentiate into a more restricted array of cell types. A growing body of evidence indicates that multi-lineage differentiation ability of stem cells can be defined by the potential for expression of lineage-specification genes. Gene expression, or as emphasized here, potential for gene expression, is largely controlled by epigenetic modifications of DNA and chromatin on genomic regulatory and coding regions. These modifications modulate chromatin organization not only on specific genes but also at the level of the whole nucleus; they can also affect timing of DNA replication. This review highlights how mechanisms by which genes are poised for transcription in undifferentiated stem cells are being uncovered through primarily the mapping of DNA methylation, histone modifications and transcription factor binding throughout the genome. The combinatorial association of epigenetic marks on developmentally regulated and lineage-specifying genes in undifferentiated cells seems to define a pluripotent state. [source] Workshop 5: NAAG and NAALADase: Functional Properties in the Central and Peripheral Nervous SystemJOURNAL OF NEUROCHEMISTRY, Issue 2002D. Bacich Glutamate carboxypeptidase II (GCPII, also known as N-acetylated-alpha-linked acidic dipeptidase or NAALADase) knockout (KO) mice were generated by inserting a GCPII targeting cassette containing a PGK-Neo resistance marker and stop codons in exons 1 and 2, and removal of exons 1 and 2 intron/exon boundary sequence. Embryonic stem cells were injected into C57BL6 blastocysts, and chimeric offspring born. Germline transmission was confirmed by mating the chimeras to generate heterozygous KO mice. Crossing heterozygous mice generated F2 generation mice homozygous for the null mutant, as confirmed by loss of GCPII protein. NAAG hydrolyzing activity was minimal (0.07 pmol/mg/min) in KO tissue, with normal levels (4.82 pmol/mg/min) in wild types and intermediate levels (1.73 pmol/mg/min) in heterozygotes. Preliminary neuropathy experiments showed KO mice are less affected by nerve-crush and recover faster from the damage-induced neuropathy, as indicated by EMG recording and nerve morphology. Similarly, GCPII KO mice subjected to high dose vitamin B6 displayed less severe neuropathy than wild types, as indicated by reduced sensory nerve conduction velocity and morphological deficits. Also, in a transient middle cerebral artery occlusion model, GCPII KO mice were significantly more resistant to the effects of cerebral ischemia than their wildtype littermates. Findings support GCPII involvement in stroke and in mediating chronic neuropathic conditions and suggest GCPII inhibitors may be useful in treatment of brain ischemia as well as peripheral neuropathies. [source] Embryonic stem cells produce neurotrophins in response to cerebral tissue extract: Cell line-dependent differencesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2007Kristine Bentz Abstract In the present study, we compare the capacity of two different embryonic stem (ES) cell lines to secrete neurotrophins in response to cerebral tissue extract derived from healthy or injured rat brains. The intrinsic capacity of the embryonic cell lines BAC7 (feeder cell-dependent cultivation) to release brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) exceeded the release of these factors by CGR8 cells (feeder cell-free growth) by factors of 10 and 4, respectively. Nerve growth factor (NGF) was secreted only by BAC7 cells. Conditioning of cell lines with cerebral tissue extract derived from healthy or fluid percussion-injured rat brains resulted in a significant time-dependent increase in BDNF release in both cell lines. The increase in BDNF release by BAC7 cells was more pronounced when cells were incubated with brain extract derived from injured brain. However, differences in neurotrophin release associated with the origin of brain extract were at no time statistically significant. Neutrophin-3 and NGF release was inhibited when cell lines were exposed to cerebral tissue extract. The magnitude of the response to cerebral tissue extract was dependent on the intrinsic capacity of the cell lines to release neurotrophins. Our results clearly demonstrate significant variations in the intrinsic capability of different stem cell lines to produce neurotrophic factors. Furthermore, a significant modulation of neurotrophic factor release was observed following conditioning of cell lines with tissue extract derived from rat brains. A significant modulation of neurotrophin release dependent on the source of cerebral tissue extract used was not observed. © 2007 Wiley-Liss, Inc. [source] Stem cell therapy of the liver, Fusion or fiction?LIVER TRANSPLANTATION, Issue 4 2004Marc H. Dahlke Various stem cell populations have been described in distinct models of liver regeneration. This review provides an overview of these different stem cell populations aimed at unifying diverse views of liver stem cell biology. Embryonic stem cells, hemopoietic stem cells, mesenchymal stem cells, liver-derived hepatic stem cells, bone marrow,derived hepatic stem cells, and mature hepatocytes (as cells with stemlike properties) are considered separately. In so doing, we seek to clarify the nomenclature of putative liver stem cell types. Experiments that address the question of cellular fusion versus transdifferentiation as explanations for observed liver regeneration are highlighted. This review concludes with a series of open questions that should be addressed in the context of clinical liver disease before attempts at human therapeutic interventions. (Liver Transpl 2004;10:471,479.) [source] Embryonic stem cells and prospects for their use in regenerative medicine approaches to motor neurone diseaseNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 5 2007Y. A. Christou Human embryonic stem cells are pluripotent cells with the potential to differentiate into any cell type in the presence of appropriate stimulatory factors and environmental cues. Their broad developmental potential has led to valuable insights into the principles of developmental and cell biology and to the proposed use of human embryonic stem cells or their differentiated progeny in regenerative medicine. This review focuses on the prospects for the use of embryonic stem cells in cell-based therapy for motor neurone disease or amyotrophic lateral sclerosis, a progressive neurodegenerative disease that specifically affects upper and lower motor neurones and leads ultimately to death from respiratory failure. Stem cell-derived motor neurones could conceivably be used to replace the degenerated cells, to provide authentic substrates for drug development and screening and for furthering our understanding of disease mechanisms. However, to reliably and accurately culture motor neurones, the complex pathways by which differentiation occurs in vivo must be understood and reiterated in vitro by embryonic stem cells. Here we discuss the need for new therapeutic strategies in the treatment of motor neurone disease, the developmental processes that result in motor neurone formation in vivo, a number of experimental approaches to motor neurone production in vitro and recent progress in the application of stem cells to the treatment and understanding of motor neurone disease. [source] Proteomic analysis of membrane proteins expressed specifically in pluripotent murine embryonic stem cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2009Atsushi Intoh Abstract Embryonic stem cells (ESCs) are established from the inner cell mass of preimplantation embryos, are capable of self-renewal, and exhibit pluripotency. Given these unique properties, ESCs are expected to have therapeutic potential in regenerative medicine and as a powerful tool for in vitro differentiation studies of stem cells. Various growth factors and extracellular matrix components regulate the pluripotency and differentiation of ESC progenies. Thus, the cell surface receptors that bind these regulatory factors are crucial for the precise regulation of stem cells. To identify membrane proteins that are involved in the regulation of pluripotent stem cells, the membrane proteins of murine ESCs cultured with or without leukemia inhibitory factor (LIF) were purified and analyzed by quantitative proteomics. 2-D PAGE-based analysis using fluorescently labeled proteins and shotgun-based analysis with isotope-labeled peptides identified 338 proteins, including transmembrane, membrane-binding, and extracellular proteins, which were expressed specifically in pluripotent or differentiated murine ESCs. Functions of the identified proteins revealed cell adhesion molecules, channels, and receptors, which are expected to play important roles in the maintenance of murine ESC pluripotency. Membrane proteins that are expressed in pluripotent ESCs but not in differentiated cells such as Slc16a1 and Bsg could be useful for the selection of the stem cells in vitro. [source] Will Embryonic Stem Cells Change Health Policy?THE JOURNAL OF LAW, MEDICINE & ETHICS, Issue 2 2010William M. Sage Embryonic stem cells are actively debated in political and public policy arenas. However, the connections between stem cell innovation and overall health care policy are seldom elucidated. As with many controversial aspects of medical care, the stem cell debate bridges to a variety of social conversations beyond abortion. Some issues, such as translational medicine, commercialization, patient and public safety, health care spending, physician practice, and access to insurance and health care services, are core health policy concerns. Other issues, such as economic development, technologic progress, fiscal politics, and tort reform, are only indirectly related to the health care system but are frequently seen through a health care lens. These connections will help determine whether the stem cell debate reaches a resolution, and what that resolution might be. [source] Lowering oxygen tension enhances the differentiation of mouse embryonic stem cells into neuronal cellsBIOTECHNOLOGY PROGRESS, Issue 5 2009Paul Mondragon-Teran Abstract Embryonic stem cells (ESC) are capable of proliferating indefinitely in vitro whilst retaining their ability to differentiate into cells of every adult lineage. Efficient, high yield processes, which direct differentiation of ESC to specific lineages, will underpin the development of cost-effective drug screening and cell therapy products. The aim of this study was to investigate whether laboratory oxygen tension currently used for the neuronal differentiation of ESC was suboptimal resulting in inefficient process yields. An adherent monolayer protocol for the neuronal differentiation of mouse ESC (mESC) was performed in oxygen controlled chambers using a chemically defined media over an 8 day period of culture. When exposed to oxygen tensions more appropriate to in vivo neuronal development (2% O2), there was a 34-fold increase in the yield of viable cells from the differentiation process. Low oxygen tension inhibited cell death during an early phase (48 to 96 h) and toward the end (120 to 192 h) of the process. The percentage of cells expressing neuronal markers was determined by flow cytometry, revealing a small rise in the ,III tubulin and a threefold increase in the MAP2 populations at 2% O2. The total increase in the yield of viable cells expressing neuronal markers was shown to be 55-fold for ,III tubulin and 114-fold for MAP2. In conclusion, this study revealed that low oxygen tension can be used to enhance the yield of neuronal cells derived from ESCs and has implications for the development of efficient, cost-effective production processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] An efficient experimental strategy for mouse embryonic stem cell differentiation and separation of a cytokeratin-19-positive population of insulin-producing cellsCELL PROLIFERATION, Issue 4 2008O. Naujok Objectives: Embryonic stem cells are a potential source for insulin-producing cells, but existing differentiation protocols are of limited efficiency. Here, the aim has been to develop a new one, which drives development of embryonic stem cells towards insulin-producing cells rather than to neuronal cell types, and to combine this with a strategy for their separation from insulin-negative cells. Materials and methods: The cytokeratin-19 (CK19) promoter was used to control the expression of enhanced yellow fluorescence protein in mouse embryonic stem cells during their differentiation towards insulin-producing cells, using a new optimized four-stage protocol. Two cell populations, CK19+ and CK19, cells, were successfully fluorescence sorted and analysed. Results: The new method reduced neuronal progeny and suppressed differentiation into glucagon- and somatostatin-producing cells. Concomitantly, ,-cell like characteristics of insulin-producing cells were strengthened, as documented by high gene expression of the Glut2 glucose transporter and the transcription factor Pdx1. This novel protocol was combined with a cell-sorting technique. Through the combined procedure, a fraction of glucose-responsive insulin-secreting CK19+ cells was obtained with 40-fold higher insulin gene expression and 50-fold higher insulin content than CK19, cells. CK19+ cells were immunoreactive for C-peptide and had ultrastructural characteristics of an insulin-secretory cell. Conclusion: Differentiated CK19+ cells reflect an endocrine precursor cell type of ductal origin, potentially suitable for insulin replacement therapy in diabetes. [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] Proteome analysis of the culture environment supporting undifferentiated mouse embryonic stem and germ cell growthELECTROPHORESIS, Issue 10 2007Nicolas Buhr Abstract The therapeutical interest of pluripotent cells and ethical issues related to the establishment of human embryonic stem cell (ESC) or embryonic germ cell (EGC) lines raise the understanding of the mechanism underlying pluripotency to a fundamental issue. Establishing a protein pluripotency signature for these cells can be complicated by the presence of unrelated proteins produced by the culture environment. Here, we have analyzed the environment supporting ESC and EGC growth, and established 2-D reference maps for each constituent present in this culture environment: mouse embryonic fibroblast feeder cells, culture medium (CM) and gelatin. The establishment of these reference maps is essential prior to the study of ESC and EGC specific proteomes. Indeed, these maps can be subtracted from ESC or EGC maps to allow focusing on spots specific for ESCs or EGCs. Our study led to the identification of 110 unique proteins from fibroblast feeder cells and 23 unique proteins from the CM, which represent major contaminants of ESC and EGC proteomes. For gelatin, no collagen-specific proteins were identified, most likely due to difficulties in resolution and low quantities. Furthermore, no differences were observed between naive and conditioned CM. Finally, we compared these reference maps to ESC 2-D gels and isolated 17 ESC specific spots. Among these spots, proteins that had already been identified in previous human and mouse ESC proteomes were identified but no apparent ESC-specific pluripotency marker could be identified. This work represents an essential step in furthering the knowledge of environmental factors supporting ESC and EGC growth. [source] A gene trap knockout of the abundant sperm tail protein, outer dense fiber 2, results in preimplantation lethality,GENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 11 2006Nicholas A. Salmon Abstract Outer dense fiber 2 (Odf2) is highly expressed in the testis where it encodes a major component of the outer dense fibers of the sperm flagellum. Furthermore, ODF2 protein has recently been identified as a widespread centrosomal protein. While the expression of Odf2 highlighted a potential role for this gene in male germ cell development and centrosome function, the in vivo function of Odf2 was not known. We have generated Odf2 knockout mice using an Odf2 gene trapped embryonic stem cell (ESC) line. Insertion of a gene trap vector into exon 9 resulted in a gene that encodes a severely truncated protein lacking a large portion of its predicted coil forming domains as well as both leucine zipper motifs that are required for protein,protein interactions with ODF1, another major component of the outer dense fibers. Although wild-type and heterozygous mice were recovered, no mice homozygous for the Odf2 gene trap insertion were recovered in an extended breeding program. Furthermore, no homozygous embryos were found at the blastocyst stage of embryonic development, implying a critical pre-implantation role for Odf2. We show that Odf2 is expressed widely in adults and is also expressed in the blastocyst stage of preimplantation development. These findings are in contrast with early studies reporting Odf2 expression as testis specific and suggest that embryonic Odf2 expression plays a critical role during preimplantation development in mice. genesis 44:515,522, 2006. Published 2006 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] Manganese-guided cellular MRI of human embryonic stem cell and human bone marrow stromal cell viabilityMAGNETIC RESONANCE IN MEDICINE, Issue 4 2009Mayumi Yamada Abstract This study investigated the ability of MnCl2 as a cellular MRI contrast agent to determine the in vitro viability of human embryonic stem cells (hESC) and human bone marrow stromal cells (hBMSC). Basic MRI parameters including T1 and T2 values of MnCl2 -labeled hESC and hBMSC were measured and viability signal of manganese (Mn2+)-labeled cells was validated. Furthermore, the biological activity of Ca2+ -channels was modulated utilizing both Ca2+ -channel agonist and antagonist to evaluate concomitant signal changes. Metabolic effects of MnCl2 -labeling were also assessed using assays for cell viability, proliferation, and apoptosis. Finally, in vivo Mn2+ -guided MRI of the transplanted hESC was successfully achieved and validated by bioluminescence imaging. Magn Reson Med, 2009. © 2009 Wiley-Liss, Inc. [source] THE AMBIGUITY OF THE EMBRYO: ETHICAL INCONSISTENCY IN THE HUMAN EMBRYONIC STEM CELL DEBATEMETAPHILOSOPHY, Issue 2-3 2007KATRIEN DEVOLDER Abstract: We argue in this essay that (1) the embryo is an irredeemably ambiguous entity and its ambiguity casts serious doubt on the arguments claiming its full protection or, at least, protection against its use as a means for stem cell research, (2) those who claim the embryo should be protected as "one of us" are committed to a position even they do not uphold in their practices, (3) views that defend the protection of the embryo in virtue of its potentiality to become a person fail, and (4) the embryo does not have any rights or interests to be protected. Given that many are willing to treat the embryo as a means in other practices, and that human embryonic stem cell (hESC) research holds great potential to benefit many people, one cannot but conclude that hESC research is permissible and, because of its immense promise for alleviating human suffering, even obligatory. [source] Patent Policy for Human Embryonic Stem Cell Research in TaiwanTHE JOURNAL OF WORLD INTELLECTUAL PROPERTY, Issue 4 2010Jerry I.-H. The potential of human embryonic stem cell (ESC) research could prove to provide immense therapeutic value for illnesses not curable under currently existing therapies. However, human ESC research is controversial as it touches the fundamental value of human life. Taiwan has been aiming to become the biotech hub of Asia-Pacific and is becoming a major player in human ESC research. Whether or not the research results from human ESC are patentable could have a profound impact on the progress in this field. In this article, the science of human ESC research is clarified and tested against the existing murky Taiwan patent standards. In particular, this article distinguishes between therapeutic cloning and reproductive cloning techniques, asks questions about the patentability of totipotent human ESCs and explores the meaning of the word embryo. This article draws comparison with the European practice on ethical standards and concludes that patenting human ESC research might not be so controversial, but Taiwan has to make its patent law clearer in this field to fulfill the country's intended goal. [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] Stem cell separation: A bottleneck in stem cell therapyBIOTECHNOLOGY JOURNAL, Issue 1 2010Kornelia Schriebl Dr. Abstract The substantial progress in embryonic stem cell (ESC) research could lead to new possibilities in the treatment of various diseases. Currently, applications of ESC for cell therapy are impeded by the presence of potentially teratoma-forming undifferentiated ESC. Thus, a selective and quantitative removal of undifferentiated ESC from a pool of differentiated and undifferentiated cells is essential before cell therapy. We evaluated the highly selective magnetic activated cell sorting (MACS) method for the quantitative removal of undifferentiated ESC. We found that the clearance rates for undifferentiated ESC decreased with decreasing amount of undifferentiated ESC in the cell pool. Using a simplified model calculation we could predict that, assuming an initial purity of 60%, an estimated 31 steps are required to achieve less than 10,1 cell per 109 cells. Thus, a log clearance rate of 10, which would be necessary for a therapeutically application, is hard to achieve. Our work clearly indicates that the current MACS technology is insufficient to meet the purification needs for cell therapy. [source] Population estimation of human embryonic stem cell culturesBIOTECHNOLOGY PROGRESS, Issue 2 2010Thomas Thurnherr Abstract Traditionally, the population of human embryonic stem cell (hESC) culture is estimated through haemacytometer counts, which include harvesting the cells and manually analyzing a fraction of an entire population. Obviously, through this highly invasive method, it is not possible to preserve any spatial information on the cell population. The goal of this study is to identify a fast and consistent method for in situ automated hESC population estimation to quantitatively estimate the cell growth. Therefore, cell cultures were fixed, stained, and their nuclei imaged through high-resolution microscopy, and the images were processed with different image analysis techniques. The proposed method first identifies signal and background by computing an image specific threshold for image segmentation. By applying a morphological operator (watershed), we split most physically overlapping nuclei, leading to a pixel area distribution of isolated signal areas on the image. On the basis of this distribution, we derive a nucleus area model, describing the distribution of the area of cell debris, single nuclei, and small groups of connected nuclei. Through the model, we can give a quantitative estimation of the population. The focus of this study is on low-density human embryonic stem cell populations; hence cultures were measured at days 2,3 after seeding. Compared with manual cell counts, the automatic method achieved higher accuracy with <6% error. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Noggin maintains pluripotency of human embryonic stem cells grown on MatrigelCELL PROLIFERATION, Issue 4 2009G. Chaturvedi Objective:, Spontaneous differentiation of human embryonic stem cell (hESC) cultures is a major concern in stem cell research. Physical removal of differentiated areas in a stem cell colony is the current approach used to keep the cultures in a pluripotent state for a prolonged period of time. All hESCs available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Under experimental conditions, stem cells are grown on various matrices, the most commonly used being Matrigel. Materials and Methods:, We propose an alternative method to prevent spontaneous differentiation of hESCs grown on Matrigel that uses low amounts of recombinant noggin. We make use of the porosity of Matrigel to serve as a matrix that traps noggin and gradually releases it into the culture to antagonize bone morphogenetic proteins (BMP). BMPs are known to initiate differentiation of hESCs and are either present in the conditioned medium or are secreted by hESCs themselves. Results:, hESCs grown on Matrigel supplemented with noggin in conditioned medium from feeder layers (irradiated mouse embryonic fibroblasts) retained both normal karyotype and markers of hESC pluripotency for 14 days. In addition, these cultures were found to have increased cell proliferation of stem cells as compared to hESCs grown on Matrigel alone. Conclusion:, Noggin can be utilized for short term prevention of spontaneous differentiation of stem cells grown on Matrigel. 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