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  1. Growth inhibition of mouse embryonic stem (ES) cells on the feeders ...

    African Journals Online (AJOL)

    Mouse embryonic stem cells (mESCs) can be propagated in vitro on the feeders of mouse embryonic fibroblasts. In this study, we found growth inhibition of mESCs cultured on embryonic fibroblast feeders derived from different livestock animals. Under the same condition, mESCs derived from mouse embryonic fibroblast ...

  2. Improved Establishment of Embryonic Stem (ES) Cell Lines from the Chinese Kunming Mice by Hybridization with 129 Mice

    Science.gov (United States)

    Yu, Shumin; Yan, Xingrong; Liu, Huanhuan; Cai, Xin; Cao, Suizhong; Shen, Liuhong; Zuo, Zhicai; Deng, Junliang; Ma, Xiaoping; Wang, Ya; Ren, Zhihua

    2014-01-01

    Chinese Kunming mice (Mus musculus Km), widely used as laboratory animals throughout China, remain very refractory for embryonic stem (ES) cell isolation. The present study was aimed to evaluate the effects of hybridization with 129/Sv mice, and culture media containing fetal bovine serum (FBS) or Knockout serum replacement (KSR) on ES cell isolation from Kunming mice. The results demonstrated that ES cells had been effectively isolated from the hybrid embryos of Kunming and 129/Sv mice using all three media containing 15% FBS, 15% KSR and their mixture of 14% KSR and 1% FBS, individually. These isolated ES cells had maintained in vitro undifferentiated for a long time, exhibiting all features specific for mouse ES cells. In addition, the rates of ES cell isolation in the medium containing 14% KSR and 1% FBS, was 46.67% and significantly higher than those in another two media containing only FBS or KSR (p < 0.05). Contrarily, no ES cell line had been established from Kunming mouse inbred embryos using the same protocols. These results suggested that ES cells with long-term self-renewal ability could be efficiently generated from hybrid embryos of Kunming and 129/Sv mice, and a small volume of FBS was necessary to isolate ES cells in the KSR medium when embryos and early ES cells cultured. PMID:24573251

  3. Improved Establishment of Embryonic Stem (ES Cell Lines from the Chinese Kunming Mice by Hybridization with 129 Mice

    Directory of Open Access Journals (Sweden)

    Shumin Yu

    2014-02-01

    Full Text Available Chinese Kunming mice (Mus musculus Km, widely used as laboratory animals throughout China, remain very refractory for embryonic stem (ES cell isolation. The present study was aimed to evaluate the effects of hybridization with 129/Sv mice, and culture media containing fetal bovine serum (FBS or Knockout serum replacement (KSR on ES cell isolation from Kunming mice. The results demonstrated that ES cells had been effectively isolated from the hybrid embryos of Kunming and 129/Sv mice using all three media containing 15% FBS, 15% KSR and their mixture of 14% KSR and 1% FBS, individually. These isolated ES cells had maintained in vitro undifferentiated for a long time, exhibiting all features specific for mouse ES cells. In addition, the rates of ES cell isolation in the medium containing 14% KSR and 1% FBS, was 46.67% and significantly higher than those in another two media containing only FBS or KSR (p < 0.05. Contrarily, no ES cell line had been established from Kunming mouse inbred embryos using the same protocols. These results suggested that ES cells with long-term self-renewal ability could be efficiently generated from hybrid embryos of Kunming and 129/Sv mice, and a small volume of FBS was necessary to isolate ES cells in the KSR medium when embryos and early ES cells cultured.

  4. High glucose suppresses embryonic stem cell differentiation into cardiomyocytes : High glucose inhibits ES cell cardiogenesis.

    Science.gov (United States)

    Yang, Penghua; Chen, Xi; Kaushal, Sunjay; Reece, E Albert; Yang, Peixin

    2016-12-09

    Babies born to mothers with pregestational diabetes have a high risk for congenital heart defects (CHD). Embryonic stem cells (ESCs) are excellent in vitro models for studying the effect of high glucose on cardiac lineage specification because ESCs can be differentiated into cardiomyocytes. ESC maintenance and differentiation are currently performed under high glucose conditions, whose adverse effects have never been clarified. We investigated the effect of high glucose on cardiomyocyte differentiation from a well-characterized ESC line, E14, derived from mouse blastocysts. E14 cells maintained under high glucose (25 mM) failed to generate any beating cardiomyocytes using the hanging-drop embryonic body method. We created a glucose-responsive E14 cell line (GR-E14) through a graduated low glucose adaptation. The expression of stem cell markers was similar in the parent E14 cells and the GR-E14 cells. Glucose transporter 2 gene was increased in GR-E14 cells. When GR-E14 cells were differentiated into cardiomyocytes under low (5 mM) or high (25 mM) glucose conditions, high glucose significantly delayed the appearance and reduced the number of TNNT2 (Troponin T Type 2)-positive contracting cardiomyocytes. High glucose suppressed the expression of precardiac mesoderm markers, cardiac transcription factors, mature cardiomyocyte markers, and potassium channel proteins. High glucose impaired the functionality of ESC-derived cardiomyocytes by suppressing the frequencies of Ca 2+ wave and contraction. Our findings suggest that high glucose inhibits ESC cardiogenesis by suppressing key developmental genes essential for the cardiac program.

  5. Derivation of normal macrophages from human embryonic stem (hES cells for applications in HIV gene therapy

    Directory of Open Access Journals (Sweden)

    Kaufman Dan S

    2006-04-01

    Full Text Available Abstract Background Many novel studies and therapies are possible with the use of human embryonic stem cells (hES cells and their differentiated cell progeny. The hES cell derived CD34 hematopoietic stem cells can be potentially used for many gene therapy applications. Here we evaluated the capacity of hES cell derived CD34 cells to give rise to normal macrophages as a first step towards using these cells in viral infection studies and in developing novel stem cell based gene therapy strategies for AIDS. Results Undifferentiated normal and lentiviral vector transduced hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34 hematopoietic progenitor cells. The differentiated CD34 cells isolated from cystic bodies were further cultured in cytokine media to derive macrophages. Phenotypic and functional analyses were carried out to compare these with that of fetal liver CD34 cell derived macrophages. As assessed by FACS analysis, the hES-CD34 cell derived macrophages displayed characteristic cell surface markers CD14, CD4, CCR5, CXCR4, and HLA-DR suggesting a normal phenotype. Tests evaluating phagocytosis, upregulation of the costimulatory molecule B7.1, and cytokine secretion in response to LPS stimulation showed that these macrophages are also functionally normal. When infected with HIV-1, the differentiated macrophages supported productive viral infection. Lentiviral vector transduced hES cells expressing the transgene GFP were evaluated similarly like above. The transgenic hES cells also gave rise to macrophages with normal phenotypic and functional characteristics indicating no vector mediated adverse effects during differentiation. Conclusion Phenotypically normal and functionally competent macrophages could be derived from hES-CD34 cells. Since these cells are susceptible to HIV-1 infection, they provide a uniform source of macrophages for viral infection studies. Based on these results, it is also now feasible to

  6. Growth inhibition of mouse embryonic stem (ES) cells on the feeders ...

    African Journals Online (AJOL)

    ajl4

    -specific markers Oct 4 and stage-specific embryonic antigen 1 (SSEA 1). Here, our results indicate that the feeders from goat, porcine and bovine embryonic fibroblasts inhibit the proliferation of mESCs. Key words: Domestic animals, feeders, ...

  7. Wnt/ß-catenin signalling and the dynamics of fate decisions in early mouse embryos and embryonic stem (ES) cells.

    Science.gov (United States)

    Muñoz-Descalzo, Silvia; Hadjantonakis, Anna-Katerina; Arias, Alfonso Martinez

    2015-12-01

    Wnt/ß-catenin signalling is a widespread cell signalling pathway with multiple roles during vertebrate development. In mouse embryonic stem (mES) cells, there is a dual role for ß-catenin: it promotes differentiation when activated as part of the Wnt/ß-catenin signalling pathway, and promotes stable pluripotency independently of signalling. Although mES cells resemble the preimplantation epiblast progenitors, the first requirement for Wnt/ß-catenin signalling during mouse development has been reported at implantation [1,2]. The relationship between ß-catenin and pluripotency and that of mES cells with epiblast progenitors suggests that ß-catenin might have a functional role during preimplantation development. Here we summarize the expression and function of Wnt/ß-catenin signalling elements during the early stages of mouse development and consider the reasons why the requirement in ES cells do not reflect the embryo. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  8. Human embryonic stem cell (hES derived dendritic cells are functionally normal and are susceptible to HIV-1 infection

    Directory of Open Access Journals (Sweden)

    Bandi Sriram

    2008-01-01

    Full Text Available Abstract Background Human embryonic stem (hES cells hold considerable promise for cell replacement and gene therapies. Their remarkable properties of pluripotency, self-renewal, and tractability for genetic modification potentially allows for the production of sizeable quantities of therapeutic cells of the hematopoietic lineage. Dendritic cells (DC arise from CD34+ hematopoietic progenitor cells (HPCs and are important in many innate and adaptive immune functions. With respect to HIV-1 infection, DCs play an important role in the efficient capture and transfer of the virus to susceptible cells. With an aim of generating DCs from a renewable source for HIV-1 studies, here we evaluated the capacity of hES cell derived CD34+ cells to give rise to DCs which can support HIV-1 infection. Results Undifferentiated hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34+ HPCs which were subsequently grown in specific cytokine differentiation media to promote the development of DCs. The hES derived DCs (hES-DC were subjected to phenotypic and functional analyses and compared with DCs derived from fetal liver CD34+ HPC (FL-DC. The mature hES-DCs displayed typical DC morphology consisting of veiled stellate cells. The hES-DCs also displayed characteristic phenotypic surface markers CD1a, HLA-DR, B7.1, B7.2, and DC-SIGN. The hES-DCs were found to be capable of antigen uptake and stimulating naïve allogeneic CD4+ T cells in a mixed leukocyte reaction assay. Furthermore, the hES-DCs supported productive HIV-1 viral infection akin to standard DCs. Conclusion Phenotypically normal and functionally competent DCs that support HIV-1 infection can be derived from hES cells. hES-DCs can now be exploited in applied immunology and HIV-1 infection studies. Using gene therapy approaches, it is now possible to generate HIV-1 resistant DCs from anti-HIV gene transduced hES-CD34+ hematopoietic progenitor cells.

  9. Embryonic Stem Cell Markers

    Directory of Open Access Journals (Sweden)

    Lan Ma

    2012-05-01

    Full Text Available Embryonic stem cell (ESC markers are molecules specifically expressed in ES cells. Understanding of the functions of these markers is critical for characterization and elucidation for the mechanism of ESC pluripotent maintenance and self-renewal, therefore helping to accelerate the clinical application of ES cells. Unfortunately, different cell types can share single or sometimes multiple markers; thus the main obstacle in the clinical application of ESC is to purify ES cells from other types of cells, especially tumor cells. Currently, the marker-based flow cytometry (FCM technique and magnetic cell sorting (MACS are the most effective cell isolating methods, and a detailed maker list will help to initially identify, as well as isolate ESCs using these methods. In the current review, we discuss a wide range of cell surface and generic molecular markers that are indicative of the undifferentiated ESCs. Other types of molecules, such as lectins and peptides, which bind to ESC via affinity and specificity, are also summarized. In addition, we review several markers that overlap with tumor stem cells (TSCs, which suggest that uncertainty still exists regarding the benefits of using these markers alone or in various combinations when identifying and isolating cells.

  10. Porcine embryonic stem cells

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane

    2008-01-01

    The development of porcine embryonic stem cell lines (pESC) has received renewed interest given the advances being made in the production of immunocompatible transgenic pigs. However, difficulties are evident in the production of pESCs in-vitro. This may largely be attributable to differences...

  11. Graphene for enhanced embryonic stem cell photo-transfection efficiency

    CSIR Research Space (South Africa)

    Mthunzi, P

    2013-04-01

    Full Text Available Due to their pluripotency properties, embryonic stem (ES) cells possess great potential in regenerative therapy. Since reported a promising tissue engineering scaffold material, here, graphene is demonstrated to significantly improve the ES cell...

  12. Cytokine signalling in embryonic stem cells

    DEFF Research Database (Denmark)

    Kristensen, David Møbjerg; Kalisz, Mark; Nielsen, Jens Høiriis

    2006-01-01

    Cytokines play a central role in maintaining self-renewal in mouse embryonic stem (ES) cells through a member of the interleukin-6 type cytokine family termed leukemia inhibitory factor (LIF). LIF activates the JAK-STAT3 pathway through the class I cytokine receptor gp130, which forms a trimeric...... pathways seem to converge on c-myc as a common target to promote self-renewal. Whereas LIF does not seem to stimulate self-renewal in human embryonic stem cells it cannot be excluded that other cytokines are involved. The pleiotropic actions of the increasing number of cytokines and receptors signalling...... via JAKs, STATs and SOCS exhibit considerable redundancy, compensation and plasticity in stem cells in accordance with the view that stem cells are governed by quantitative variations in strength and duration of signalling events known from other cell types rather than qualitatively different stem...

  13. Epigenetic control of embryonic stem cell fate

    DEFF Research Database (Denmark)

    Christophersen, Nicolaj Strøyer; Helin, Kristian

    2010-01-01

    Embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and are pluripotent, as they are able to differentiate into all cell types of the adult organism. Once established, the pluripotent ES cells can be maintained under defined culture conditions, but can also...... be induced rapidly to differentiate. Maintaining this balance of stability versus plasticity is a challenge, and extensive studies in recent years have focused on understanding the contributions of transcription factors and epigenetic enzymes to the "stemness" properties of these cells. Identifying...... the molecular switches that regulate ES cell self-renewal versus differentiation can provide insights into the nature of the pluripotent state and enhance the potential use of these cells in therapeutic applications. Here, we review the latest models for how changes in chromatin methylation can modulate ES cell...

  14. Directional differentiation of chicken embryonic stem cells into ...

    African Journals Online (AJOL)

    Chicken embryonic stem (ES) cells are useful for producing transgenic chickens and preserving genetic material in avian species. In this study, the differentiation potential of chicken ES cells was investigated in vitro. Chicken ES cells were differentiated into osteoblasts cultured for 15 to 21 days in the induction media ...

  15. Embryonic stem cells in pig and cattle

    DEFF Research Database (Denmark)

    Maddox-Hyttel, Poul; Wolf, Xenia Asbæk; Rasmussen, Mikkel Aabech

    2007-01-01

    Porcine and bovine cell lines derived from the inner cell mass (ICM) or epiblasts of blastocysts have been maintained over extended periods of time and characterized by morphology, identification of some stem cell markers and, in few cases, by production of chimaeric offspring. However, germ line...... transmission in chimaeras has never been obtained. Due to this incomplete characterization of the cell lines, the expression embryonic stem (ES)-like cells is presently used in pig and cattle. The ICM or epiblast can be isolated from the blastocyst by whole blastocyst culture, mechanical isolation......, or immunosurgery, and they are generally cultured on feeder cells. The resulting ES-like cells may be differentiated in vivo by chimaera and teratoma formation or in vitro by embryoid body formation and monolayer induction. It is likely that more well characterized and stable porcine and bovine ES cell lines...

  16. The liberation of embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Kathryn Blair

    2011-04-01

    Full Text Available Mouse embryonic stem (ES cells are defined by their capacity to self-renew and their ability to differentiate into all adult tissues including the germ line. Along with efficient clonal propagation, these properties have made them an unparalleled tool for manipulation of the mouse genome. Traditionally, mouse ES (mES cells have been isolated and cultured in complex, poorly defined conditions that only permit efficient derivation from the 129 mouse strain; genuine ES cells have not been isolated from another species in these conditions. Recently, use of small molecule inhibitors of glycogen synthase kinase 3 (Gsk3 and the Fgf-MAPK signaling cascade has permitted efficient derivation of ES cells from all tested mouse strains. Subsequently, the first verified ES cells were established from a non-mouse species, Rattus norvegicus. Here, we summarize the advances in our understanding of the signaling pathways regulating mES cell self-renewal that led to the first derivation of rat ES cells and highlight the new opportunities presented for transgenic modeling on diverse genetic backgrounds. We also comment on the implications of this work for our understanding of pluripotent stem cells across mammalian species.

  17. Mapping the stem cell state: eight novel human embryonic stem and embryonal carcinoma cell antibodies

    DEFF Research Database (Denmark)

    Wright, A; Andrews, N; Bardsley, K

    2011-01-01

    The antigenic profile of human embryonic stem (ES) and embryonal carcinoma (EC) cells has served as a key element of their characterization, with a common panel of surface and intracellular markers now widely used. Such markers have been used to identify cells within the 'undifferentiated state...... of reactivity for all antibodies against both ES and EC cells, suggesting that these markers will afford recognition of unique sub-states within the undifferentiated stem cell compartment....... and EC cells, and herein describe their characterization. The reactivity of these antibodies against a range of cell lines is reported, as well as their developmental regulation, basic biochemistry and reactivity in immunohistochemistry of testicular germ cell tumours. Our data reveal a range...

  18. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... Studies have shown that embryonic stem (ES) cells can be successfully differentiated into liver cells, which offer the potential unlimited cell source for a variety of end-stage liver disease. In our study, in order to induce mouse ES cells to differentiate into hepatocyte-like cells under chemically defined.

  19. p53 regulation and activity in mouse embryonic stem cells

    OpenAIRE

    Solozobova, Valeriya

    2010-01-01

    P53 is a tumour development p53. The aim of this work was to study the regulation of p53 in embryonic stem cells and its activation in response to DNA damage. p53 was found that p53 becomes transcriptionally active in ES cells after DNA damage. Embryonic stem cells contain a relatively high amount of p53 protein and p53 RNA. After differentiation p53 level is rapidly downregulated. The high abundance of p53 in undifferentiated ES cells is a result of enhanced translation.

  20. Derivation of human embryonic stem cells in defined conditions.

    Science.gov (United States)

    Ludwig, Tenneille E; Levenstein, Mark E; Jones, Jeffrey M; Berggren, W Travis; Mitchen, Erika R; Frane, Jennifer L; Crandall, Leann J; Daigh, Christine A; Conard, Kevin R; Piekarczyk, Marian S; Llanas, Rachel A; Thomson, James A

    2006-02-01

    We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.

  1. [Progress in the research of germ cell from human embryonic stem cells].

    Science.gov (United States)

    Guo, Xin; Cai, Zhi-Ming; Gui, Yao-Ting

    2006-01-01

    As the development of spontaneous differentiation of germ cells and gametogenesis from mouse embryonic stem cells (mES) in vitro, hES (human embryonic stem cells) also have potential to differentiated into germ cells in theory. This review focuses on the stem cells niches and genes regulating the hES differentiation toward germ cells, as well as the recent advance and application on the reproductive medicine and therapy of infertility.

  2. Assessment of Stem Cell Markers During Long-Term Culture of Mouse Embryonic Stem Cells

    OpenAIRE

    Berrill, A.; Tan, H L; Wuang, S.C.; Fong, W.J.; Choo, Andre B. H.; Oh, Steve K. W.

    2004-01-01

    Embryonic stem (ES) cells have been in the fore front of scientific literature lately as having the potential for regeneration of many tissue types. Two important issues that need to be addressed are the culture conditions for maintaining ES cells and the accuracy of ES cell markers in monitoring the undifferentiated state. Leukaemia inhibitory factor (LIF) is routinely used to sustain mouse ES cells (mES) in a pluripotent fashion. In this paper, we assessed three markers during long-term mai...

  3. Gene targeting in embryonic stem cells, II: conditional technologies

    Science.gov (United States)

    Genome modification via transgenesis has allowed researchers to link genotype and phenotype as an alternative approach to the characterization of random mutations through evolution. The synergy of technologies from the fields of embryonic stem (ES) cells, gene knockouts, and protein-mediated recombi...

  4. Gene expression heterogeneities in embryonic stem cell populations

    DEFF Research Database (Denmark)

    Martinez Arias, Alfonso; Brickman, Joshua M

    2011-01-01

    Stem and progenitor cells are populations of cells that retain the capacity to populate specific lineages and to transit this capacity through cell division. However, attempts to define markers for stem cells have met with limited success. Here we consider whether this limited success reflects...... an intrinsic requirement for heterogeneity with stem cell populations. We focus on Embryonic Stem (ES) cells, in vitro derived cell lines from the early embryo that are considered both pluripotent (able to generate all the lineages of the future embryo) and indefinitely self renewing. We examine the relevance...... of recently reported heterogeneities in ES cells and whether these heterogeneities themselves are inherent requirements of functional potency and self renewal....

  5. Genetic engineering of human embryonic stem cells with lentiviral vectors.

    Science.gov (United States)

    Xiong, Chen; Tang, Dong-Qi; Xie, Chang-Qing; Zhang, Li; Xu, Ke-Feng; Thompson, Winston E; Chou, Wayne; Gibbons, Gary H; Chang, Lung-Ji; Yang, Li-Jun; Chen, Yuqing E

    2005-08-01

    Human embryonic stem (hES) cells present a valuable source of cells with a vast therapeutic potential. However, the low efficiency of directed differentiation of hES cells remains a major obstacle in their uses for regenerative medicine. While differentiation may be controlled by the genetic manipulation, effective and efficient gene transfer into hES cells has been an elusive goal. Here, we show stable and efficient genetic manipulations of hES cells using lentiviral vectors. This method resulted in the establishment of stable gene expression without loss of pluripotency in hES cells. In addition, lentiviral vectors were effective in conveying the expression of an U6 promoter-driven small interfering RNA (siRNA), which was effective in silencing its specific target. Taken together, our results suggest that lentiviral gene delivery holds great promise for hES cell research and application.

  6. Embryonic Stem Cells and their Genetic Modification

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 13; Issue 2. Embryonic Stem Cells and their Genetic Modification - The Nobel Prize in Physiology or Medicine 2007. Mitradas M Panicker. General Article Volume 13 Issue 2 February 2008 pp 172-180 ...

  7. Functional Role of Mst1/Mst2 in Embryonic Stem Cell Differentiation

    OpenAIRE

    Peng Li; Ying Chen; Kinglun Kingston Mak; Chun Kwok Wong; Chi Chiu Wang; Ping Yuan

    2013-01-01

    The Hippo pathway is an evolutionary conserved pathway that involves cell proliferation, differentiation, apoptosis and organ size regulation. Mst1 and Mst2 are central components of this pathway that are essential for embryonic development, though their role in controlling embryonic stem cells (ES cells) has yet to be exploited. To further understand the Mst1/Mst2 function in ES cell pluripotency and differentiation, we derived Mst1/Mst2 double knockout (Mst-/-) ES cells to completely pertur...

  8. Embryoid body formation from embryonic and induced pluripotent stem cells:Benefits of bioreactors

    National Research Council Canada - National Science Library

    Sasitorn Rungarunlert Mongkol Techakumphu Melinda K Pirity Andras Dinnyes

    2009-01-01

    Embryonic stem(ES)cells have the ability to differ-entiate into all germ layers,holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies...

  9. Transcriptome coexpression map of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Mattson Mark P

    2006-05-01

    Full Text Available Abstract Background Human embryonic stem (ES cells hold great promise for medicine and science. The transcriptome of human ES cells has been studied in detail in recent years. However, no systematic analysis has yet addressed whether gene expression in human ES cells may be regulated in chromosomal domains, and no chromosomal domains of coexpression have been identified. Results We report the first transcriptome coexpression map of the human ES cell and the earliest stage of ES differentiation, the embryoid body (EB, for the analysis of how transcriptional regulation interacts with genomic structure during ES self-renewal and differentiation. We determined the gene expression profiles from multiple ES and EB samples and identified chromosomal domains showing coexpression of adjacent genes on the genome. The coexpression domains were not random, with significant enrichment in chromosomes 8, 11, 16, 17, 19, and Y in the ES state, and 6, 11, 17, 19 and 20 in the EB state. The domains were significantly associated with Giemsa-negative bands in EB, yet showed little correlation with known cytogenetic structures in ES cells. Different patterns of coexpression were revealed by comparative transcriptome mapping between ES and EB. Conclusion The findings and methods reported in this investigation advance our understanding of how genome organization affects gene expression in human ES cells and help to identify new mechanisms and pathways controlling ES self-renewal or differentiation.

  10. Autophagy in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Thien Tra

    Full Text Available Autophagy (macroautophagy is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC.

  11. Embryonic Stem Cells: Isolation, Characterization and Culture

    Science.gov (United States)

    Amit, Michal; Itskovitz-Eldor, Joseph

    Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.

  12. Stepwise development of hematopoietic stem cells from embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Kenji Matsumoto

    Full Text Available The cellular ontogeny of hematopoietic stem cells (HSCs remains poorly understood because their isolation from and their identification in early developing small embryos are difficult. We attempted to dissect early developmental stages of HSCs using an in vitro mouse embryonic stem cell (ESC differentiation system combined with inducible HOXB4 expression. Here we report the identification of pre-HSCs and an embryonic type of HSCs (embryonic HSCs as intermediate cells between ESCs and HSCs. Both pre-HSCs and embryonic HSCs were isolated by their c-Kit(+CD41(+CD45(- phenotype. Pre-HSCs did not engraft in irradiated adult mice. After co-culture with OP9 stromal cells and conditional expression of HOXB4, pre-HSCs gave rise to embryonic HSCs capable of engraftment and long-term reconstitution in irradiated adult mice. Blast colony assays revealed that most hemangioblast activity was detected apart from the pre-HSC population, implying the early divergence of pre-HSCs from hemangioblasts. Gene expression profiling suggests that a particular set of transcripts closely associated with adult HSCs is involved in the transition of pre-HSC to embryonic HSCs. We propose an HSC developmental model in which pre-HSCs and embryonic HSCs sequentially give rise to adult types of HSCs in a stepwise manner.

  13. Chromatin regulation landscape of embryonic stem cell identity.

    Science.gov (United States)

    Lee, Yun Hwa; Wu, Qiang

    2011-04-01

    ES cells (embryonic stem cells) derived from the ICM (inner cell mass) of blastocysts are pluripotent and are capable of giving rise to most cell types. The ES cell identity is mainly maintained by the Oct4 (octamer-binding transcription factor 4) and Nanog transcriptional networks. Recently, a tremendous amount of work has focused on deciphering how ES cell identity is regulated epigenetically. It has been shown that histone methylation/demethylation, histone acetylation/deacetylation, histone variants and chromatin remodelling play crucial roles in ES cell maintenance and differentiation. Moreover, perturbation of those chromatin regulators results in loss of ES cell identity or aberrant differentiation. Therefore, it is important to fully understand the chromatin regulation landscape of ES cells. The knowledge gained will help us to harness the unique characteristics of ES cells for stem cell-related therapy and regenerative medicine. In the present review, we will discuss recent proceedings that provide novel insights into chromatin regulation of ES cell identity.

  14. Embryonic stem cells and property rights.

    Science.gov (United States)

    Andersson, Anna-Karin M

    2011-06-01

    This article contributes to the current debate on human embryonic stem cell researchers' possible complicity in the destruction of human embryos and the relevance of such complicity for the issue of commodification of human embryos. I will discuss if, and to what extent, researchers who destroy human embryos, and researchers who merely use human embryos destroyed by others, have moral use rights, and/or moral property rights, in these embryos. I argue that the moral status of the human embryo, however justified, places few restrictions on the latter researchers' use of it, and property rights in it, once it is destroyed. I argue that the former researchers have no property rights in the destroyed embryo but use rights in it to the extent allowed by the legitimate owners of the destroyed embryo. I discuss the implications of this account for previous and current US federal law regulating human embryonic stem cell research.

  15. Will embryonic stem cells change health policy?

    Science.gov (United States)

    Sage, William M

    2010-01-01

    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.

  16. Embryonic stem cell biology: insights from molecular imaging.

    Science.gov (United States)

    Sallam, Karim; Wu, Joseph C

    2010-01-01

    Embryonic stem (ES) cells have therapeutic potential in disorders of cellular loss such as myocardial infarction, type I diabetes and neurodegenerative disorders. ES cell biology in living subjects was largely poorly understood until incorporation of molecular imaging into the field. Reporter gene imaging works by integrating a reporter gene into ES cells and using a reporter probe to induce a signal detectable by normal imaging modalities. Reporter gene imaging allows for longitudinal tracking of ES cells within the same host for a prolonged period of time. This has advantages over postmortem immunohistochemistry and traditional imaging modalities. The advantages include expression of reporter gene is limited to viable cells, expression is conserved between generations of dividing cells, and expression can be linked to a specific population of cells. These advantages were especially useful in studying a dynamic cell population such as ES cells and proved useful in elucidating the biology of ES cells. Reporter gene imaging identified poor integration of differentiated ES cells transplanted into host tissue as well as delayed donor cell death as reasons for poor long-term survival in vivo. This imaging technology also confirmed that ES cells indeed have immunogenic properties that factor into cell survival and differentiation. Finally, reporter gene imaging improved our understanding of the neoplastic risk of undifferentiated ES cells in forming teratomas. Despite such advances, much remains to be understood about ES cell biology to translate this technology to the bedside, and reporter gene imaging will certainly play a key role in formulating this understanding.

  17. Comparison of American mink embryonic stem and induced pluripotent stem cell transcriptomes

    DEFF Research Database (Denmark)

    Menzorov, Aleksei G; Matveeva, Natalia M.; Markakis, Marios Nektarios

    2015-01-01

    BACKGROUND: Recently fibroblasts of many mammalian species have been reprogrammed to pluripotent state using overexpression of several transcription factors. This technology allows production of induced pluripotent stem (iPS) cells with properties similar to embryonic stem (ES) cells....... The completeness of reprogramming process is well studied in such species as mouse and human but there is not enough data on other species. We produced American mink (Neovison vison) ES and iPS cells and compared these cells using transcriptome analysis. RESULTS: We report the generation of 10 mink ES and 22 i......PS cell lines. The majority of the analyzed cell lines had normal diploid chromosome number. The only ES cell line with XX chromosome set had both X-chromosomes in active state that is characteristic of pluripotent cells. The pluripotency of ES and iPS cell lines was confirmed by formation of teratomas...

  18. The polycomb group protein Suz12 is required for embryonic stem cell differentiation

    DEFF Research Database (Denmark)

    Pasini, Diego; Bracken, Adrian P; Hansen, Jacob Bo Højberg

    2007-01-01

    results in early lethality of mouse embryos. Here, we demonstrate that Suz12(-/-) mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12(-/-) ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation...

  19. Electrophysiological properties of embryonic stem cell-derived neurons.

    Directory of Open Access Journals (Sweden)

    Jessica R Risner-Janiczek

    Full Text Available In vitro generation of functional neurons from embryonic stem (ES cells and induced pluripotent stem cells offers exciting opportunities for dissecting gene function, disease modelling, and therapeutic drug screening. To realize the potential of stem cells in these biomedical applications, a complete understanding of the cell models of interest is required. While rapid advances have been made in developing the technologies for directed induction of defined neuronal subtypes, most published works focus on the molecular characterization of the derived neural cultures. To characterize the functional properties of these neural cultures, we utilized an ES cell model that gave rise to neurons expressing the green fluorescent protein (GFP and conducted targeted whole-cell electrophysiological recordings from ES cell-derived neurons. Current-clamp recordings revealed that most neurons could fire single overshooting action potentials; in some cases multiple action potentials could be evoked by depolarization, or occurred spontaneously. Voltage-clamp recordings revealed that neurons exhibited neuronal-like currents, including an outward current typical of a delayed rectifier potassium conductance and a fast-activating, fast-inactivating inward current, typical of a sodium conductance. Taken together, these results indicate that ES cell-derived GFP(+ neurons in culture display functional neuronal properties even at early stages of differentiation.

  20. Intestinal lineage commitment of embryonic stem cells.

    Science.gov (United States)

    Cao, Li; Gibson, Jason D; Miyamoto, Shingo; Sail, Vibhavari; Verma, Rajeev; Rosenberg, Daniel W; Nelson, Craig E; Giardina, Charles

    2011-01-01

    Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue. Copyright © 2010 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  1. Human embryonic stem cells and patent protection

    Directory of Open Access Journals (Sweden)

    Radovanović Sanja M.

    2015-01-01

    Full Text Available Given the importance of biotechnological research in modern diagnostics and therapeutics, on the one hand, and stimulative function of a patent, on the other hand, this work deals with the question of the possibility of pa-tent protection of human embryonic stem cells. Taking into account that this is a biotechnological invention, the key question that this paper highlights is the interpretation of the provisions of their patentability. Namely, thanks to the advanced methods of isolation, purification and preparation for implementation, modern patent systems do not exclude a priori living organisms from patent protection. Therefore, the analysis of representative administrative decisions or court rulings sought to define the criteria that would be applied in order to give patent protection to a certain biotechnological invention (stem cells while others do not.

  2. LIF signal in mouse embryonic stem cells

    Science.gov (United States)

    Ohtsuka, Satoshi; Nakai-Futatsugi, Yoko; Niwa, Hitoshi

    2015-01-01

    Since the establishment of mouse embryonic stem cells (mESCs) in the 1980s, a number of important notions on the self-renewal of pluripotent stem cells in vitro have been found. In serum containing conventional culture, an exogenous cytokine, leukemia inhibitory factor (LIF), is absolutely essential for the maintenance of pluripotency. In contrast, in serum-free culture with simultaneous inhibition of Map-kinase and Gsk3 (so called 2i-culture), LIF is no longer required. However, recent findings also suggest that LIF may have a role not covered by the 2i for the maintenance of naïve pluripotency. These suggest that LIF functions for the maintenance of naïve pluripotency in a context dependent manner. We summarize how LIF-signal pathway is converged to maintain the naïve state of pluripotency. PMID:27127728

  3. TET2 deficiency inhibits mesoderm and hematopoietic differentiation in human embryonic stem cells

    DEFF Research Database (Denmark)

    Langlois, Thierry; da Costa Reis Monte Mor, Barbara; Lenglet, Gaëlle

    2014-01-01

    . Here, we show that TET2 expression is low in human embryonic stem (ES) cell lines and increases during hematopoietic differentiation. ShRNA-mediated TET2 knockdown had no effect on the pluripotency of various ES cells. However, it skewed their differentiation into neuroectoderm at the expense...... profile, including abnormal expression of neuronal genes. Intriguingly, when TET2 was knockdown in hematopoietic cells, it increased hematopoietic development. In conclusion, our work suggests that TET2 is involved in different stages of human embryonic development, including induction of the mesoderm...... and hematopoietic differentiation. Stem Cells 2014....

  4. The cell cycle as a brake for ?-cell regeneration from embryonic stem cells

    OpenAIRE

    El-Badawy, Ahmed; El-Badri, Nagwa

    2016-01-01

    The generation of insulin-producing ? cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic ? cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle ...

  5. Embryonic stem cells: testing the germ-cell theory.

    Science.gov (United States)

    Hochedlinger, Konrad

    2011-10-25

    The exact cellular origin of embryonic stem cells remains elusive. Now a new study provides compelling evidence that embryonic stem cells, established under conventional culture conditions, originate from a transient germ-cell state. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Human embryonic stem cells: preclinical perspectives

    Directory of Open Access Journals (Sweden)

    Sarda Kanchan

    2008-01-01

    Full Text Available Abstract Human embryonic stem cells (hESCs have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic.

  7. No shortcuts to pig embryonic stem cells.

    Science.gov (United States)

    Brevini, T A L; Pennarossa, G; Gandolfi, F

    2010-09-01

    The establishment of embryonic stem cell (ESC) lines in domestic species could have great impact in the agricultural as well as in the biomedical field. In particular, derivation of pig ESC would find important applications aimed at improving health and production traits of this species through genetic engineering. Similarly, the immunological, morphological, physiological, and functional similarities to the human make the pig a very effective and suitable animal model for biomedical studies and pre-clinical trials. While proven blastocyst-derived mouse and human ESC lines have been established, no validated porcine ESC (pESC) lines are available. In the present manuscript we briefly discuss some of the factors that make the establishment of ESC lines in the pig, and in animal species other than mouse and human, a very slow process. The paucity of information related to morphology, pluripotency markers, differentiation capability hampers a thorough evaluation of the validity of putative lines. These difficulties are further increased by the lack of reliable antibodies, reagents, and in vitro culture systems that could ensure reliable results in the pig and allow for the screening and long-term maintenance of pESC. Data from the literature suggest that similar regulatory pathways are likely to exist among different species. Coupling of these pathways with their distinct expression patterns, the relative concentrations of pluripotency-related molecules, and timing of embryo development, along with supportive micro-environmental conditions, would appear to vary in a species-specific manner. We feel that the understanding of these subtle but meaningful diversities may provide beneficial information about the isolation of genuine porcine embryonic stem cells. Copyright 2010 Elsevier Inc. All rights reserved.

  8. Tracking the mechanical dynamics of human embryonic stem cell chromatin

    Directory of Open Access Journals (Sweden)

    Hinde Elizabeth

    2012-12-01

    Full Text Available Abstract Background A plastic chromatin structure has emerged as fundamental to the self-renewal and pluripotent capacity of embryonic stem (ES cells. Direct measurement of chromatin dynamics in vivo is, however, challenging as high spatiotemporal resolution is required. Here, we present a new tracking-based method which can detect high frequency chromatin movement and quantify the mechanical dynamics of chromatin in live cells. Results We use this method to study how the mechanical properties of chromatin movement in human embryonic stem cells (hESCs are modulated spatiotemporally during differentiation into cardiomyocytes (CM. Notably, we find that pluripotency is associated with a highly discrete, energy-dependent frequency of chromatin movement that we refer to as a ‘breathing’ state. We find that this ‘breathing’ state is strictly dependent on the metabolic state of the cell and is progressively silenced during differentiation. Conclusions We thus propose that the measured chromatin high frequency movements in hESCs may represent a hallmark of pluripotency and serve as a mechanism to maintain the genome in a transcriptionally accessible state. This is a result that could not have been observed without the high spatial and temporal resolution provided by this novel tracking method.

  9. Therapeutic approaches for treating hemophilia A using embryonic stem cells.

    Science.gov (United States)

    Kasuda, Shogo; Tatsumi, Kohei; Sakurai, Yoshihiko; Shima, Midori; Hatake, Katsuhiko

    2016-06-01

    Hemophilia A is an X-linked rescessive bleeding disorder that results from F8 gene aberrations. Previously, we established embryonic stem (ES) cells (tet-226aa/N6-Ainv18) that secrete human factor VIII (hFVIII) by introducing the human F8 gene in mouse Ainv18 ES cells. Here, we explored the potential of cell transplantation therapy for hemophilia A using the ES cells. Transplant tet-226aa/N6-Ainv18 ES cells were injected into the spleens of severe combined immunodeficiency (SCID) mice, carbon tetrachloride (CCl4)-pretreated wild-type mice, and CCl4-pretreated hemophilia A mice. F8 expression was induced by doxycycline in drinking water, and hFVIII-antigen production was assessed in all cell transplantation experiments. Injecting the ES cells into SCID mice resulted in an enhanced expression of the hFVIII antigen; however, teratoma generation was confirmed in the spleen. Transplantation of ES cells into wild-type mice after CCl4-induced liver injury facilitated survival and engraftment of transplanted cells without teratoma formation, resulting in hFVIII production in the plasma. Although CCl4 was lethal to most hemophilia A mice, therapeutic levels of FVIII activity, as well as the hFVIII antigen, were detected in surviving hemophilia A mice after cell transplantation. Immunolocalization results for hFVIII suggested that transplanted ES cells might be engrafted at the periportal area in the liver. Although the development of a safer induction method for liver regeneration is required, our results suggested the potential for developing an effective ES-cell transplantation therapeutic model for treating hemophilia A in the future. Copyright © 2016 King Faisal Specialist Hospital & Research Centre. Published by Elsevier Ltd. All rights reserved.

  10. EDA-Containing Fibronectin Increases Proliferation of Embryonic Stem Cells

    Science.gov (United States)

    Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F.; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

    2013-01-01

    Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA+). Here, we investigated if the FN EDA+ isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA-), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC’s proliferation rate. Here we showed for the first time that this FN isoform enhances ESC’s proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy. PMID:24244705

  11. Contrasting expression of keratins in mouse and human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Jochen Maurer

    Full Text Available RNA expression data reveals that human embryonic stem (hES cells differ from mouse ES (mES cells in the expression of RNAs for keratin intermediate filament proteins. These differences were confirmed at the cellular and protein level and may reflect a fundamental difference in the epithelial nature of embryonic stem cells derived from mouse and human blastocysts. Mouse ES cells express very low levels of the simple epithelial keratins K8, K18 and K19. By contrast hES cells express moderate levels of the RNAs for these intermediate filament proteins as do mouse stem cells derived from the mouse epiblast. Expression of K8 and K18 RNAs are correlated with increased c-Jun RNA expression in both mouse and human ES cell cultures. However, decreasing K8 and K18 expression associated with differentiation to neuronal progenitor cells is correlated with increasing expression of the Snai2 (Slug transcriptional repression and not decreased Jun expression. Increasing K7 expression is correlated with increased CDX2 and decreased Oct4 RNA expression associated with the formation of trophoblast derivatives by hES cells. Our study supports the view that hES cells are more similar to mouse epiblast cells than mouse ES cells and is consistent with the epithelial nature of hES cells. Keratin intermediate filament expression in hES cells may modulate sensitivity to death receptor mediated apoptosis and stress.

  12. Embryonic Stem Cell Proteins and MicroRNAs in the Etiology of Germ Cell Cancer

    NARCIS (Netherlands)

    R. Eini (Ronak)

    2013-01-01

    textabstractIn the early 1980s, a population of unique cells was isolated from the inner cell mass (ICM) of the mouse pre-implantation embryo named embryonic stem (ES) cells. These cells were generated by removing the ICM from pre-implantation blastocysts. The resulting cells were found to be

  13. Human embryonic stem cells and microenvironment

    Directory of Open Access Journals (Sweden)

    Banu İskender

    2014-09-01

    Full Text Available Human embryonic stem cells (hESCs possess a great potential in the field of regenerative medicine by their virtue of pluripotent potential with indefinite proliferation capabilities. They can self renew themselves and differentiate into three embryonic germ layers. Although they are conventionally grown on mitotically inactivated mouse feeder cells, there are in vitro culture systems utilizing feeder cells of human origin in order to prevent cross-species contamination. Recently established in vitro culture systems suggested that direct interaction with feeder cells is not necessary but rather attachment to a substrate is required to ensure long-term, efficient hESC culture in vitro. This substrate is usually composed of a mixture of extracellular matrix components representing in vivo natural niche. In hESC biology, the mechanism of interaction of hESCs with extracellular matrix molecules remained insufficiently explored area of research due to their transient nature of interaction with the in vivo niche. However, an in vitro culture system established using extracellular matrix molecules may provide a safer alternative to culture systems with feeder cells while paving the way to Good Manufacturing Practice-GMP production of hESCs for therapeutic purposes. Therefore, it is essential to study the interaction of extracellular matrix molecules with hESCs in order to standardize in vitro culture systems for large-scale production of hESCs in a less labor-intensive way. This would not only provide valuable information regarding the mechanisms that control pluripotency but also serve to dissect the molecular signaling pathways of directed differentiation for prospective therapeutic applications in the future. J Clin Exp Invest 2014; 5 (3: 486-495

  14. Transforming pluripotency: an exon-level study of malignancy-specific transcripts in human embryonal carcinoma and embryonic stem cells.

    Science.gov (United States)

    Alagaratnam, Sharmini; Harrison, Neil; Bakken, Anne Cathrine; Hoff, Andreas M; Jones, Mark; Sveen, Anita; Moore, Harry D; Andrews, Peter W; Lothe, Ragnhild A; Skotheim, Rolf I

    2013-04-01

    To circumvent difficulties of isolating pure populations of cancer stem cells (CSCs) for the purpose of identifying malignancy-specific gene expression, we have compared exon-resolution transcriptomic profiles of 5 embryonal carcinoma (EC) cell lines, a histological subtype of germ cell tumor (GCT), to their nonmalignant caricature, specifically 6 human embryonic stem (ES) cell lines. Both cell types are readily accessible, and were purified for undifferentiated cells only. We identified a set of 28 differentially expressed genes, many of which had cancer and stemness roles. Overexpression of the recently discovered pluripotency gene NR5A2 in malignant EC cells revealed an intriguing indication of how WNT-mediated dysregulation of pluripotency is involved with malignancy. Expression of these 28 genes was further explored within 2 publically available data sets of primary EC tumors and normal testis. At the exon-level, alternative splicing events were detected in ZNF195, DNMT3B, and PMF1, and alternative promoters were detected for ASH2L and ETV5. These events were validated by reverse transcriptase-polymerase chain reaction-based methods in EC and ES lines, where the alternative splicing event in the de novo DNA methyltransferase DNMT3B may have functional consequences. In conclusion, we have identified malignancy-specific gene expression differences within a rigorous pluripotent stem cell context. These findings are of particular interest for both GCT and ES cell biology, and, in general, to the concept of CSCs.

  15. Human Embryonic Stem Cell Research Debates: A Confucian Argument

    National Research Council Canada - National Science Library

    D. F.-C. Tsai

    2005-01-01

    Human embryonic stem cell research can bring about major biomedical breakthroughs and thus contribute enormously to human welfare, yet it raises serious moral problems because it involves using human...

  16. Nucleosome Organization in Human Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Puya G Yazdi

    Full Text Available The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a "ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this "ground state" by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational

  17. A Focused Microarray for Screening Rat Embryonic Stem Cell Lines

    OpenAIRE

    Hong, James; He, Hong; Bui, Phuoc; Ryba-White, Ben; Rumi, Mohammad A.K.; Soares, Michael J.; Dutta, Debasree; Paul, Soumen; Kawamata, Masaki; Ochiya, Takahiro; Ying, Qi-Long; Rajanahalli, Pavan; Mark L. Weiss

    2012-01-01

    Here, we describe a focused microarray for screening rat embryonic stem cells (ESCs) and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem (TS) cells, rat extraembryonic endoderm cells, mouse embryonic fibroblast feeder cells, and differentiated rat ESCs. Using this tool, genuine rat ESC lines, which have been expanded in a conventional rat ESC medium containing two inhibitors (2i), for example, glycogen synthase kinase 3 (GSK3) and mi...

  18. Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Morizane, Ryuji [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Monkawa, Toshiaki, E-mail: monkawa@sc.itc.keio.ac.jp [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Itoh, Hiroshi [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan)

    2009-12-25

    Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

  19. Primordial germ cell specification from embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Wei Wei

    Full Text Available BACKGROUND: Primordial germ cell (PGC specification is the first crucial step in germ line development. However, owing to significant challenges regarding the in vivo system, such as the complex cellular environment and potential problems with embryo manipulation, it is desirable to generate embryonic stem (ES cells that are capable of overcoming these aforementioned limitations in order to provide a potential in vitro model to recapitulate the developmental processes in vivo. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we studied the detailed process of PGC specification from stella-GFP ES cells. We first observed the heterogeneous expression of stella in ES cells. However, neither Stella-positive ES cells nor Stella-negative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors. Second, we derived PGCs from ES cells using two differentiation methods, namely the attachment culture technique and the embryoid body (EB method. Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method. Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation. CONCLUSIONS AND SIGNIFICANCE: The in vitro model we have established can recapitulate the developmental processes in vivo and provides new insights into the mechanism of PGC specification.

  20. Retroviral transcriptional regulation and embryonic stem cells: war and peace.

    Science.gov (United States)

    Schlesinger, Sharon; Goff, Stephen P

    2015-03-01

    Retroviruses have evolved complex transcriptional enhancers and promoters that allow their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES cell pluripotency. This overlap is not coincidental; retrovirus-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps "noisy" control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    DEFF Research Database (Denmark)

    Van Hoof, Dennis; Muñoz, Javier; Braam, Stefan R

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during...

  2. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    NARCIS (Netherlands)

    van Hoof, D.; Munoz, J.; Braam, S.R.; Pinkse, M.W.H.; Linding, R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during

  3. Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics

    Science.gov (United States)

    Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultur...

  4. Plasma treatment of biomaterials to direct the differentiation of embryonic stem cells

    Science.gov (United States)

    Hanley, Erik

    In this work, we explore how embryonic stem (ES) cell differentiation patterns are affected by surface interactions with plasma-processed materials. We hypothesize that mouse embryonic stem-cell exposure to certain plasma-polymerized tetraglyme surfaces will direct their differentiation into endothelial cells. R1 mouse embryonic stem (ES) cells were plated on surfaces onto which tetraglyme was deposited by plasma polymerization. In addition, tissue-treated polystyrene and control glass cover slips were also examined. Some samples were fixed three days after plating and immunofluorescence stained with platelet endothelial-cell adhesion molecule, while the others were fixed seven days after plating and immunofluorescence stained with von Willebrand Factor. Positive results seen by ES cell derivatives precociously expressing the vWF and PECAM genetic markers on the plasma-polymerized tetraglyme treated surfaces suggest that the plasma-polymerized surfaces direct differentiation of ES cells into endothelial cells. Research goals of this dissertation include: characterization of the material properties of the plasma-polymerized tetraglyme surfaces that induce directed differentiation of ES cells into endothelial cells, optimization of the plasma-polymerization process to maximize the number of endothelial cells derived from R1 ES cells, and biological experimentation to characterize properties of the mechanism of directed differentiation. A potential application of this work is in the design and construction of an artificial blood vessel. Current small-scale arterial substitutes have proved inadequate because of thrombogenicity and infection. Moreover, the lower blood flow velocities of smaller vessels pose a different set of design criteria and introduce new problems not encountered in large arterial substitutes. By utilizing a tissue engineering approach that incorporates embryonic stem cell-derived endothelial cells, the longevity of the prosthesis can be ensured.

  5. Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

    Science.gov (United States)

    Wang, Zhongde

    2011-01-01

    Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

  6. Hematopoietic cell differentiation from embryonic and induced pluripotent stem cells

    Science.gov (United States)

    2013-01-01

    Pluripotent stem cells, both embryonic stem cells and induced pluripotent stem cells, are undifferentiated cells that can self-renew and potentially differentiate into all hematopoietic lineages, such as hematopoietic stem cells (HSCs), hematopoietic progenitor cells and mature hematopoietic cells in the presence of a suitable culture system. Establishment of pluripotent stem cells provides a comprehensive model to study early hematopoietic development and has emerged as a powerful research tool to explore regenerative medicine. Nowadays, HSC transplantation and hematopoietic cell transfusion have successfully cured some patients, especially in malignant hematological diseases. Owing to a shortage of donors and a limited number of the cells, hematopoietic cell induction from pluripotent stem cells has been regarded as an alternative source of HSCs and mature hematopoietic cells for intended therapeutic purposes. Pluripotent stem cells are therefore extensively utilized to facilitate better understanding in hematopoietic development by recapitulating embryonic development in vivo, in which efficient strategies can be easily designed and deployed for the generation of hematopoietic lineages in vitro. We hereby review the current progress of hematopoietic cell induction from embryonic stem/induced pluripotent stem cells. PMID:23796405

  7. Assessment of stem cell markers during long-term culture of mouse embryonic stem cells.

    Science.gov (United States)

    Berrill, A; Tan, H L; Wuang, S C; Fong, W J; Choo, Andre B H; Oh, Steve K W

    2004-01-01

    Embryonic stem (ES) cells have been in the fore front of scientific literature lately as having the potential for regeneration of many tissue types. Two important issues that need to be addressed are the culture conditions for maintaining ES cells and the accuracy of ES cell markers in monitoring the undifferentiated state. Leukaemia inhibitory factor (LIF) is routinely used to sustain mouse ES cells (mES) in a pluripotent fashion. In this paper, we assessed three markers during long-term maintenance of ES cells with various concentrations of LIF to see if decreasing concentration would lead to changes in marker expressions and growth behavior. Common markers of pluripotency such as alkaline phosphatase enzyme activity (ALP), surface staining for stage specific embryonic antigen 1 (SSEA-1), Oct-4 transcription factor, cell doubling time, as well as visual observations of cell morphology were analyzed during long-term maintenance of mES cells with LIF concentrations ranging from 0 to 500 pM. The morphology of the cells at LIF concentrations of 0 25 pM changed from being tight clusters to more flattened shapes while cells in 50-500 pM retained the clustered shape but growth rates remained essentially identical at between 10 and 16 h. ES cells at all concentrations of LIF continued expressing ALP, SSEA-1 and Oct-4 markers over a period of 6 weeks, which indicate that mES cells are capable of either producing autocrine LIF or are able to proliferate at very low levels of LIF. Pluripotency markers such as Oct-4 and SSEA-1 are only moderately reduced after 5-6 weeks. Oct-4 mRNA expression levels were partially diminished in LIF free conditions only at weeks 5 and 6 compared to controls with LIF at 500 pM. Changes in morphology of cells by visual observation seemed to be a faster indication of the onset of differentiation in mES cells, although other reliable means also include decreased levels of Oct-4, SSEA-1 and ALP markers. It is preferable to maintain long

  8. STELLA facilitates differentiation of germ cell and endodermal lineages of human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Patompon Wongtrakoongate

    Full Text Available Stella is a developmentally regulated gene highly expressed in mouse embryonic stem (ES cells and in primordial germ cells (PGCs. In human, the gene encoding the STELLA homologue lies on chromosome 12p, which is frequently amplified in long-term cultured human ES cells. However, the role played by STELLA in human ES cells has not been reported. In the present study, we show that during retinoic acid (RA-induced differentiation of human ES cells, expression of STELLA follows that of VASA, a marker of germline differentiation. By contrast, human embryonal carcinoma cells express STELLA at a higher level compared with both karyotypically normal and abnormal human ES cell lines. We found that over-expression of STELLA does not interfere with maintenance of the stem cell state of human ES cells, but following retinoic acid induction it leads to up-regulation of germline- and endodermal-associated genes, whereas neural markers PAX6 and NEUROD1 are down-regulated. Further, STELLA over-expression facilitates the differentiation of human ES cells into BE12-positive cells, in which the expression of germline- and endodermal-associated genes is enriched, and suppresses differentiation of the neural lineage. Taken together, this finding suggests a role for STELLA in facilitating germline and endodermal differentiation of human ES cells.

  9. The ethics of patenting human embryonic stem cells.

    Science.gov (United States)

    Chapman, Audrey R

    2009-09-01

    Just as human embryonic stem cell research has generated controversy about the uses of human embryos for research and therapeutic applications, human embryonic stem cell patents raise fundamental ethical issues. The United States Patent and Trademark Office has granted foundational patents, including a composition of matter (or product) patent to the Wisconsin Alumni Research Foundation (WARF), the University of Wisconsin-Madison's intellectual property office. In contrast, the European Patent Office rejected the same WARF patent application for ethical reasons. This article assesses the appropriateness of these patents placing the discussion in the context of the deontological and consequentialist ethical issues related to human embryonic stem cell patenting. It advocates for a patent system that explicitly takes ethical factors into account and explores options for new types of intellectual property arrangements consistent with ethical concerns.

  10. The cell cycle as a brake for β-cell regeneration from embryonic stem cells.

    Science.gov (United States)

    El-Badawy, Ahmed; El-Badri, Nagwa

    2016-01-13

    The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.

  11. Differentiation of Adipocytes in Monolayer from Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Cuaranta-Monroy, Ixchelt; Simandi, Zoltan; Nagy, Laszlo

    2016-01-01

    Obesity and its comorbidity incidence have increased worldwide during the past 10 years. In consequence, researchers have drawn their attention to the understanding of adipocyte differentiation. Several cellular model systems have been established; however no efficient protocol could be developed so far to differentiate the pluripotent embryonic stem cells to adipocytes. In this chapter, we describe a detailed protocol that is optimized for mouse embryonic stem cells. The result of this differentiation is a homogenous adipocyte monolayer culture that can be used for several applications including developmental and pharmacological research.

  12. Development of buffalo (Bubalus bubalis embryonic stem cell lines from somatic cell nuclear transferred blastocysts

    Directory of Open Access Journals (Sweden)

    Syed Mohmad Shah

    2015-11-01

    Full Text Available We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

  13. Reconstitution of mammary epithelial morphogenesis by murine embryonic stem cells undergoing hematopoietic stem cell differentiation.

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    Shuxian Jiang

    2010-03-01

    Full Text Available Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo.To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo.Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.

  14. Functional Role of Mst1/Mst2 in Embryonic Stem Cell Differentiation

    Science.gov (United States)

    Li, Peng; Chen, Ying; Mak, Kinglun Kingston; Wong, Chun Kwok; Wang, Chi Chiu; Yuan, Ping

    2013-01-01

    The Hippo pathway is an evolutionary conserved pathway that involves cell proliferation, differentiation, apoptosis and organ size regulation. Mst1 and Mst2 are central components of this pathway that are essential for embryonic development, though their role in controlling embryonic stem cells (ES cells) has yet to be exploited. To further understand the Mst1/Mst2 function in ES cell pluripotency and differentiation, we derived Mst1/Mst2 double knockout (Mst-/-) ES cells to completely perturb Hippo signaling. We found that Mst-/- ES cells express higher level of Nanog than wild type ES cells and show differentiation resistance after LIF withdrawal. They also proliferate faster than wild type ES cells. Although Mst-/- ES cells can form embryoid bodies (EBs), their differentiation into tissues of three germ layers is distorted. Intriguingly, Mst-/- ES cells are unable to form teratoma. Mst-/- ES cells can differentiate into mesoderm lineage, but further differentiation to cardiac lineage cells is significantly affected. Microarray analysis revealed that ligands of non-canonical Wnt signaling, which is critical for cardiac progenitor specification, are significantly repressed in Mst-/- EBs. Taken together our results showed that Mst1/Mst2 are required for proper cardiac lineage cell development and teratoma formation. PMID:24224013

  15. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    In vitro differentiation of mouse embryonic stem cells into functional hepatocytes by sodium butyrate, hepatocyte growth factor and dexamethasone under ... under chemically defined conditions, which might be useful as an in vitro system for hepatocyte transplantation therapy and toxicity screening in drug discovery.

  16. Twenty years of embryonic stem cell research in farm animals

    Science.gov (United States)

    Notable distinctions between an embryonic stem cell (ESC) and somatic cell are that the ESC can maintain an undifferentiated state indefinitely, self renew, and is pluripotent, meaning that the ESC can potentially generate cells representing all the three primordial germ layers and contribute to the...

  17. Improved genetic manipulation of human embryonic stem cells.

    NARCIS (Netherlands)

    Braam, S.R.; Denning, C.; van den Brink, S.; Kats, P.; Hochstenbach, R.; Passier, R.; Mummery, C.L.

    2008-01-01

    Low efficiency of transfection limits the ability to genetically manipulate human embryonic stem cells (hESCs), and differences in cell derivation and culture methods require optimization of transfection protocols. We transiently transferred multiple independent hESC lines with different growth

  18. Impact of 2-bromopropane on mouse embryonic stem cells and ...

    African Journals Online (AJOL)

    This study shows that 2-BP (5 to 10 μM) induces apoptotic processes in mouse embryonic stem cells (ESC-B5), but exerts no effects at treatment dosages below 5 μM. In ESC-B5 cells, 2-BP directly increased the content of reactive oxygen species (ROS), significantly increased the cytoplasmic free calcium and nitric oxide ...

  19. Human embryonic stem cell transplantation to repair the infarcted myocardium

    National Research Council Canada - National Science Library

    Leor, Jonathan; Gerecht, Sharon; Cohen, Smadar; Miller, Liron; Holbova, Radka; Ziskind, Anna; Shachar, Michal; Feinberg, Micha S; Guetta, Esther; Itskovitz-Eldor, Joseph

    2007-01-01

    To test the hypothesis that human embryonic stem cells (hESCs) can be guided to form new myocardium by transplantation into the normal or infarcted heart, and to assess the influence of hESC-derived cardiomyocytes (hESCMs...

  20. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... Key words: Embryonic stem cells, hepatic-like cells, in vitro differentiation, sodium butyrate, hepatocyte growth factor, dexamethason. INTRODUCTION. The liver is the major organ that provides multiple metabolic functions critical for the maintenance of homeostasis. One of the major causes of morbidity and.

  1. Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines.

    Directory of Open Access Journals (Sweden)

    Olivier Féraud

    Full Text Available Hematopoiesis generated from human embryonic stem cells (ES and induced pluripotent stem cells (iPS are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process.

  2. The p53 inhibitor, pifithrin-{alpha}, suppresses self-renewal of embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Abdelalim, Essam Mohamed, E-mail: essam_abdelalim@yahoo.com [Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan); Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522 (Egypt); Tooyama, Ikuo [Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan)

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer We determine the role of p53 in ES cells under unstressful conditions. Black-Right-Pointing-Pointer PFT-{alpha} suppresses ES cell proliferation. Black-Right-Pointing-Pointer PFT-{alpha} induces ES cell cycle arrest. Black-Right-Pointing-Pointer PFT-{alpha} downregulates Nanog and cyclin D1. -- Abstract: Recent studies have reported the role of p53 in suppressing the pluripotency of embryonic stem (ES) cells after DNA damage and blocking the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. However, to date no evidence has been presented to support the function of p53 in unstressed ES cells. In this study, we investigated the effect of pifithrin (PFT)-{alpha}, an inhibitor of p53-dependent transcriptional activation, on self-renewal of ES cells. Our results revealed that treatment of ES cells with PFT-{alpha} resulted in the inhibition of ES cell propagation in a dose-dependent manner, as indicated by a marked reduction in the cell number and colony size. Also, PFT-{alpha} caused a cell cycle arrest and significant reduction in DNA synthesis. In addition, inhibition of p53 activity reduced the expression levels of cyclin D1 and Nanog. These findings indicate that p53 pathway in ES cells rather than acting as an inactive gene, is required for ES cell proliferation and self-renewal under unstressful conditions.

  3. Mouse Embryonic Stem Cells Inhibit Murine Cytomegalovirus Infection through a Multi-Step Process

    Science.gov (United States)

    Kawasaki, Hideya; Kosugi, Isao; Arai, Yoshifumi; Iwashita, Toshihide; Tsutsui, Yoshihiro

    2011-01-01

    In humans, cytomegalovirus (CMV) is the most significant infectious cause of intrauterine infections that cause congenital anomalies of the central nervous system. Currently, it is not known how this process is affected by the timing of infection and the susceptibility of early-gestational-period cells. Embryonic stem (ES) cells are more resistant to CMV than most other cell types, although the mechanism responsible for this resistance is not well understood. Using a plaque assay and evaluation of immediate-early 1 mRNA and protein expression, we found that mouse ES cells were resistant to murine CMV (MCMV) at the point of transcription. In ES cells infected with MCMV, treatment with forskolin and trichostatin A did not confer full permissiveness to MCMV. In ES cultures infected with elongation factor-1α (EF-1α) promoter-green fluorescent protein (GFP) recombinant MCMV at a multiplicity of infection of 10, less than 5% of cells were GFP-positive, despite the fact that ES cells have relatively high EF-1α promoter activity. Quantitative PCR analysis of the MCMV genome showed that ES cells allow approximately 20-fold less MCMV DNA to enter the nucleus than mouse embryonic fibroblasts (MEFs) do, and that this inhibition occurs in a multi-step manner. In situ hybridization revealed that ES cell nuclei have significantly less MCMV DNA than MEF nuclei. This appears to be facilitated by the fact that ES cells express less heparan sulfate, β1 integrin, and vimentin, and have fewer nuclear pores, than MEF. This may reduce the ability of MCMV to attach to and enter through the cellular membrane, translocate to the nucleus, and cross the nuclear membrane in pluripotent stem cells (ES/induced pluripotent stem cells). The results presented here provide perspective on the relationship between CMV susceptibility and cell differentiation. PMID:21407806

  4. DNA damage responses in human induced pluripotent stem cells and embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Olga Momcilovic

    2010-10-01

    Full Text Available Induced pluripotent stem (iPS cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming, which uses a defined set of transcription factors, iPS cells represent important sources of patient-specific cells for clinical applications. However, before these cells can be used in therapeutic designs, it is essential to understand their genetic stability.Here, we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation, iPS cells activate checkpoint signaling, evidenced by phosphorylation of ATM, NBS1, CHEK2, and TP53, localization of ATM to the double strand breaks (DSB, and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2 phase of the cell cycle, displaying a lack of the G(1/S cell cycle arrest similar to human embryonic stem (ES cells. Furthermore, both cell types remove DSB within six hours of γ-irradiation, form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally, we report elevated expression of genes involved in DNA damage signaling, checkpoint function, and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts.High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage, dramatic changes in cell cycle structure, including a high percentage of cells in the S phase, increased radiosensitivity and loss of DNA damage-induced G(1/S cell cycle arrest, were observed in stem cells generated by induced pluripotency.

  5. A comparative study of protocols for mouse embryonic stem cell culturing.

    Directory of Open Access Journals (Sweden)

    Christoffer Tamm

    Full Text Available Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem (ES cells. These involve growing cells on mouse embryonic fibroblast feeder cells or on gelatin in media supplemented with fetal bovine serum and leukemia inhibitory factor (LIF. However, these techniques have several drawbacks including the need for feeder-cells and/or use of undefined media containing animal derived components. Culture of stem cells under undefined conditions can induce spontaneous differentiation and reduce reproducibility of experiments. In recent years several new ES cell culture protocols, using more well-defined conditions, have been published and we have compared the standard culture protocols with two of the newly described ones: 1 growing cells in semi-adherence in a medium containing two small molecule inhibitors (CHIR99021, PD0325901 and; 2 growing cells in a spheroid suspension culture in a defined medium containing LIF and bFGF. Two feeder-dependent mouse ES (mES cell lines and two cell lines adapted to feeder-independent growth were used in the study. The overall aim has not only been to compare self-renewal and differentiation capacity, but also ease-of-use and cost efficiency. We show that mES cells when grown adherently proliferate much faster than when grown in suspension as free-floating spheres, independent of media used. Although all the tested culture protocols could maintain sustained pluripotency after prolonged culturing, our data confirm previous reports showing that the media containing two chemical inhibitors generate more pure stem cell cultures with negligible signs of spontaneous differentiation as compared to standard mES media. Furthermore, we show that this medium effectively rescues and cleans up cultures that have started to deteriorate, as well as allow for effective adaption of feeder-dependent mES cell lines to be maintained in feeder-free conditions.

  6. Properties and uses of embryonic stem cells: prospects for application to human biology and gene therapy.

    Science.gov (United States)

    Rathjen, P D; Lake, J; Whyatt, L M; Bettess, M D; Rathjen, J

    1998-01-01

    Embryonic stem cells are pluripotent cells derived from the early mouse embryo that can be propagated stably in the undifferentiated state in vitro. They retain the ability to differentiate into all cell types found in an embryonic and adult mouse in vivo, and can be induced to differentiate into many cell types in vitro. Exploitation of ES cell technology for the creation of mice bearing predetermined genetic alterations has received widespread attention because of the sophistication that it brings to the study of gene function in mammals. Analysis of cell differentiation in vitro has also been of value, leading to the identification of novel bioactive factors and the elucidation of cell specification mechanisms. In this paper, we summarise the features of pluripotent cell lines and their applications, foreshadowing the impact that these systems may have on human biology. While the isolation of definitive human pluripotent cell lines has not yet been achieved, potential applications for these cells in the study of human biology, particularly cell specification, can be envisaged. Of particular interest is the possibility that human embryonic stem cells with properties similar to mouse embryonic stem cells might provide a generic system for gene therapy.

  7. Transcription factor network in embryonic stem cells: heterogeneity under the stringency.

    Science.gov (United States)

    Nakai-Futatsugi, Yoko; Niwa, Hitoshi

    2013-01-01

    Leukemia inhibitory factor (LIF) signaling regulates transcription factors to maintain the self-renewability and pluripotency of embryonic stem (ES) cells. Recently, we have proposed a network model that consists of transcription factors such as, Klf4, Sox2, Tbx3, Nanog, and Oct3/4, which form a parallel pathway downstream from LIF signaling (Nature, 460, 2009, Niwa et al.). In this parallel pathway, the transcription factors maintain the pluripotency of ES cells through mutual balance with some degree of redundancy and compensation. While self-renewability and pluripotency are maintained well under such seemingly stringent regulation, studies of single cells revealed heterogeneity among individual ES cells. This heterogeneity may underlie the mechanism that allows ES cells to exit self-renewal and enter into differentiation to exert pluripotency. Here we focus on recent studies on the heterogeneity of ES cells and discuss their inherent metastability.

  8. Abortion, embryonic stem cell research, and waste.

    Science.gov (United States)

    Jensen, David A

    2008-01-01

    Can one consistently deny the permissibility of abortion while endorsing the killing of human embryos for the sake of stem cell research? The question is not trivial; for even if one accepts that abortion is prima facie wrong in all cases, there are significant differences with many of the embryos used for stem cell research from those involved in abortion--most prominently, many have been abandoned in vitro, and appear to have no reasonably likely meaningful future. On these grounds one might think to maintain a strong position against abortion but endorse killing human embryos for the sake of stem cell research and its promising benefits. I will argue, however, that these differences are not decisive. Thus, one who accepts a strong view against abortion is committed to the moral impermissibility of killing human embryos for the sake of stem cell research. I do not argue for the moral standing of either abortion or the killing of embryos for stem cell research; I only argue for the relation between the two. Thus the conclusion is relevant to those with a strong view in favor of the permissibility of killing embryos for the sake of research as much as for those who may strongly oppose abortion; neither can consider their position in isolation from the other.

  9. Oct-4 controls cell-cycle progression of embryonic stem cells

    Science.gov (United States)

    Lee, Jungwoon; Go, Yeorim; Kang, Inyoung; Han, Yong-Mahn; Kim, Jungho

    2009-01-01

    Mouse and human ES (embryonic stem) cells display unusual proliferative properties and can produce pluripotent stem cells indefinitely. Both processes might be important for maintaining the ‘stemness’ of ES cells; however, little is known about how the cell-cycle fate is regulated in ES cells. Oct-4, a master switch of pluripotency, plays an important role in maintaining the pluripotent state of ES cells and may prevent the expression of genes activated during differentiation. Using ZHBTc4 ES cells, we have investigated the effect of Oct-4 on ES cell-cycle control, and we found that Oct-4 down-regulation in ES cells inhibits proliferation by blocking cell-cycle progression in G0/G1. Deletion analysis of the functional domains of Oct-4 indicates that the overall integrity of the Oct-4 functional domains is important for the stimulation of S-phase entry. We also show in the present study that the p21 gene is a target for Oct-4 repression. Furthermore, p21 protein levels were repressed by Oct-4 and were induced by the down-regulation of Oct-4 in ZHBTc4 ES cells. Therefore the down-regulation of p21 by Oct-4 may contribute to the maintenance of ES cell proliferation. PMID:19968627

  10. Differentiation of Mouse Embryonic Stem Cells into Ventral Foregut Precursors

    DEFF Research Database (Denmark)

    Rothová, Michaela; Hölzenspies, Jurriaan J; Livigni, Alessandra

    2016-01-01

    Anterior definitive endoderm (ADE), the ventral foregut precursor, is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other organs. Here, a method is described for the differentiation of mouse embryonic stem cells (mESCs) to definitive...... endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (embryoid body) culture or in a serum-free adherent monolayer culture. ESC-derived ADE cells are committed to endodermal fates and can undergo further differentiation in vitro towards ventral foregut...

  11. Dissecting the heterogeneity of gene expressions in mouse embryonic stem cells

    Science.gov (United States)

    Zou, Ling-Nan; Thomson, Matt; Liu, S. John; Ramanathan, Sharad

    2011-03-01

    A population of genetically identical cells, of the same nominal cell type, and cultured in the same petri dish, will nevertheless often exhibit varying patterns of gene expression. Taking mouse embryonic stem (ES) cells as a model system, we use immunofluorescence and flow cytometry to examine in detail the distribution of expression levels for various transcription factors key to the maintenance of the ES cell identity. We find the population-level distribution of many proteins, once rescaled by the average expression level, have very similar shapes. This suggest the largest component of observed heterogeneity comes from a single source. More subtly, we find the expression many of genes appears to modulate with the cell cycle. This may suggest that the program for maintaining ES cell identity is tightly coupled to the cell cycle machinery. This work is supported by the Harvard Stem Cell Institute and the Jane Coffin Childs Memorial Fund for Medical Research.

  12. Evaluation of biological effects of intermediate frequency magnetic field on differentiation of embryonic stem cell

    Directory of Open Access Journals (Sweden)

    Sachiko Yoshie

    2016-01-01

    Full Text Available The embryotoxic effect of intermediate frequency (IF magnetic field (MF was evaluated using murine embryonic stem (ES cells and fibroblast cells based on the embryonic stem cell test (EST. The cells were exposed to 21 kHz IF–MF up to magnetic flux density of 3.9 mT during the cell proliferation process (7 days or the cell differentiation process (10 days during which an embryonic body differentiated into myocardial cells. As a result, there was no significant difference in the cell proliferation between sham- and IF–MF-exposed cells for both ES and fibroblast cells. Similarly, the ratio of the number of ES-derived cell aggregates differentiated to myocardial cells to total number of cell aggregates was not changed by IF–MF exposure. In addition, the expressions of a cardiomyocytes-specific gene, Myl2, and an early developmental gene, Hba-x, in the exposed cell aggregate were not altered. Since the magnetic flux density adopted in this study is much higher than that generated by an inverter of the electrical railway, an induction heating (IH cooktop, etc. in our daily lives, these results suggested that IF–MF in which the public is exposed to in general living environment would not have embryotoxic effect.

  13. The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimerism.

    NARCIS (Netherlands)

    L. Longo; A. Bygrave; F.G. Grosveld (Frank); P.P. Pandolfi

    1997-01-01

    textabstractMouse pluripotent embryonic stem (ES) cells, once reintroduced into a mouse blastocyst, can contribute to the formation of all tissues, including the germline, of an organism referred to as a chimaeric. However, the reasons why this contribution often appears erratic are poorly

  14. β-Cell Replacement in Mice Using Human Type 1 Diabetes Nuclear Transfer Embryonic Stem Cells.

    Science.gov (United States)

    Sui, Lina; Danzl, Nichole; Campbell, Sean R; Viola, Ryan; Williams, Damian; Xing, Yuan; Wang, Yong; Phillips, Neil; Poffenberger, Greg; Johannesson, Bjarki; Oberholzer, Jose; Powers, Alvin C; Leibel, Rudolph L; Chen, Xiaojuan; Sykes, Megan; Egli, Dieter

    2018-01-01

    β-Cells derived from stem cells hold great promise for cell replacement therapy for diabetes. Here we examine the ability of nuclear transfer embryonic stem cells (NT-ESs) derived from a patient with type 1 diabetes to differentiate into β-cells and provide a source of autologous islets for cell replacement. NT-ESs differentiate in vitro with an average efficiency of 55% into C-peptide-positive cells, expressing markers of mature β-cells, including MAFA and NKX6.1. Upon transplantation in immunodeficient mice, grafted cells form vascularized islet-like structures containing MAFA/C-peptide-positive cells. These β-cells adapt insulin secretion to ambient metabolite status and show normal insulin processing. Importantly, NT-ES-β-cells maintain normal blood glucose levels after ablation of the mouse endogenous β-cells. Cystic structures, but no teratomas, were observed in NT-ES-β-cell grafts. Isogenic induced pluripotent stem cell lines showed greater variability in β-cell differentiation. Even though different methods of somatic cell reprogramming result in stem cell lines that are molecularly indistinguishable, full differentiation competence is more common in ES cell lines than in induced pluripotent stem cell lines. These results demonstrate the suitability of NT-ES-β-cells for cell replacement for type 1 diabetes and provide proof of principle for therapeutic cloning combined with cell therapy. © 2017 by the American Diabetes Association.

  15. Germline transmission of a novel rat embryonic stem cell line derived from transgenic rats.

    Science.gov (United States)

    Men, Hongsheng; Bauer, Beth A; Bryda, Elizabeth C

    2012-09-20

    Germline-competent rat embryonic stem (ES) cell lines are important resources for the creation of mutant rat models using ES-cell-based gene targeting technology. The ability to isolate germline-competent ES cell lines from any rat strain, including genetically modified strains, would allow for more sophisticated genetic manipulations without extensive breeding. Sprague Dawley (SD) males carrying an enhanced green fluorescent protein (EGFP) transgene were used as the founder animals for the derivation of ES cell lines. A number of ES cell lines were established and subjected to rigorous quality control testing that included assessment of pluripotency factor expression, karyotype analysis, and pathogen/sterility testing. Two male ES cell lines, SD-Tg.EC1/Rrrc and SD-Tg.EC8/Rrrc, were injected into blastocysts recovered from a cross of Dark Agouti (DA) males with SD females. Resulting chimeric animals were bred with wild-type SD mates to verify the germline transmissibility of the ES cell lines by identifying pups carrying the ES cell line-derived EGFP transgene. While both ES cell lines gave rise to chimeric animals, only SD-Tg.EC1 was germline competent. This confirms the feasibility of deriving germline-competent ES cell lines from transgenic rat strains and provides a novel ES cell line with a stable green fluorescent protein (GFP) reporter for future genetic manipulations to create new rat models.

  16. Dynamic Proteomic Profiling of Extra-Embryonic Endoderm Differentiation in Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Mulvey, Claire M; Schröter, Christian; Gatto, Laurent; Dikicioglu, Duygu; Fidaner, Isik Baris; Christoforou, Andy; Deery, Michael J; Cho, Lily T Y; Niakan, Kathy K; Martinez-Arias, Alfonso; Lilley, Kathryn S

    2015-09-01

    During mammalian preimplantation development, the cells of the blastocyst's inner cell mass differentiate into the epiblast and primitive endoderm lineages, which give rise to the fetus and extra-embryonic tissues, respectively. Extra-embryonic endoderm (XEN) differentiation can be modeled in vitro by induced expression of GATA transcription factors in mouse embryonic stem cells. Here, we use this GATA-inducible system to quantitatively monitor the dynamics of global proteomic changes during the early stages of this differentiation event and also investigate the fully differentiated phenotype, as represented by embryo-derived XEN cells. Using mass spectrometry-based quantitative proteomic profiling with multivariate data analysis tools, we reproducibly quantified 2,336 proteins across three biological replicates and have identified clusters of proteins characterized by distinct, dynamic temporal abundance profiles. We first used this approach to highlight novel marker candidates of the pluripotent state and XEN differentiation. Through functional annotation enrichment analysis, we have shown that the downregulation of chromatin-modifying enzymes, the reorganization of membrane trafficking machinery, and the breakdown of cell-cell adhesion are successive steps of the extra-embryonic differentiation process. Thus, applying a range of sophisticated clustering approaches to a time-resolved proteomic dataset has allowed the elucidation of complex biological processes which characterize stem cell differentiation and could establish a general paradigm for the investigation of these processes. © 2015 AlphaMed Press.

  17. Porphyrin Homeostasis Maintained by ABCG2 Regulates Self-Renewal of Embryonic Stem Cells

    Science.gov (United States)

    Chen, Yun-Nan; Shen, Chia-Rui; Yan, Yu-Ting; Tsai, Sheng-Ta; Chen, Chung-Hsuan; Shen, Chia-Ning

    2008-01-01

    Background Under appropriate culture conditions, undifferentiated embryonic stem (ES) cells can undergo multiple self-renewal cycles without loss of pluripotency suggesting they must be equipped with specific defense mechanisms to ensure sufficient genetic stability during self-renewal expansion. The ATP binding cassette transporter ABCG2 is expressed in a wide variety of somatic and embryonic stem cells. However, whether it plays an important role in stem cell maintenance remains to be defined. Methodology/Principal Findings Here we provide evidence to show that an increase in the level of ABCG2 was observed accompanied by ES colony expansion and then were followed by decreases in the level of protoporphyrin IX (PPIX) indicating that ABCG2 plays a role in maintaining porphyrin homoeostasis. RNA-interference mediated inhibition of ABCG2 as well as functional blockage of ABCG2 transporter with fumitremorgin C (FTC), a specific and potent inhibitor of ABCG2, not only elevated the cellular level of PPIX, but also arrest the cell cycle and reduced expression of the pluripotent gene Nanog. Overexpression of ABCG2 in ES cells was able to counteract the increase of endogenous PPIX induced by treatment with 5-Aminolevulinic acid suggesting ABCG2 played a direct role in removal of PPIX from ES cells. We also found that excess PPIX in ES cells led to elevated levels of reactive oxygen species which in turn triggered DNA damage signals as indicated by increased levels of γH2AX and phosphorylated p53. The increased level of p53 reduced Nanog expression because RNA- interference mediated inhibition of p53 was able to prevent the downregulation of Nanog induced by FTC treatment. Conclusions/Significance The present work demonstrated that ABCG2 protects ES cells from PPIX accumulation during colony expansion, and that p53 and γH2AX acts as a downstream checkpoint of ABCG2-dependent defense machinery in order to maintain the self-renewal of ES cells. PMID:19107196

  18. Femtosecond laser pulses for chemical-free embryonic and mesenchymal stem cell differentiation

    Science.gov (United States)

    Mthunzi, Patience; Dholakia, Kishan; Gunn-Moore, Frank

    2011-10-01

    Owing to their self renewal and pluripotency properties, stem cells can efficiently advance current therapies in tissue regeneration and/or engineering. Under appropriate culture conditions in vitro, pluripotent stem cells can be primed to differentiate into any cell type some examples including neural, cardiac and blood cells. However, there still remains a pressing necessity to answer the biological questions concerning how stem cell renewal and how differentiation programs are operated and regulated at the genetic level. In stem cell research, an urgent requirement on experimental procedures allowing non-invasive, marker-free observation of growth, proliferation and stability of living stem cells under physiological conditions exists. Femtosecond (fs) laser pulses have been reported to non-invasively deliver exogenous materials, including foreign genetic species into both multipotent and pluripotent stem cells successfully. Through this multi-photon facilitated technique, directly administering fs laser pulses onto the cell plasma membrane induces transient submicrometer holes, thereby promoting cytosolic uptake of the surrounding extracellular matter. To display a chemical-free cell transfection procedure that utilises micro-litre scale volumes of reagents, we report for the first time on 70 % transfection efficiency in ES-E14TG2a cells using the enhanced green fluorescing protein (EGFP) DNA plasmid. We also show how varying the average power output during optical transfection influences cell viability, proliferation and cytotoxicity in embryonic stem cells. The impact of utilizing objective lenses of different numerical aperture (NA) on the optical transfection efficiency in ES-E14TG2a cells is presented. Finally, we report on embryonic and mesenchymal stem cell differentiation. The produced specialized cell types could thereafter be characterized and used for cell based therapies.

  19. Efficient and Fast Differentiation of Human Neural Stem Cells from Human Embryonic Stem Cells for Cell Therapy

    Directory of Open Access Journals (Sweden)

    Xinxin Han

    2017-01-01

    Full Text Available Stem cell-based therapies have been used for repairing damaged brain tissue and helping functional recovery after brain injury. Aberrance neurogenesis is related with brain injury, and multipotential neural stem cells from human embryonic stem (hES cells provide a great promise for cell replacement therapies. Optimized protocols for neural differentiation are necessary to produce functional human neural stem cells (hNSCs for cell therapy. However, the qualified procedure is scarce and detailed features of hNSCs originated from hES cells are still unclear. In this study, we developed a method to obtain hNSCs from hES cells, by which we could harvest abundant hNSCs in a relatively short time. Then, we examined the expression of pluripotent and multipotent marker genes through immunostaining and confirmed differentiation potential of the differentiated hNSCs. Furthermore, we analyzed the mitotic activity of these hNSCs. In this report, we provided comprehensive features of hNSCs and delivered the knowledge about how to obtain more high-quality hNSCs from hES cells which may help to accelerate the NSC-based therapies in brain injury treatment.

  20. Induction of murine embryonic stem cell differentiation by medicinal plant extracts

    Energy Technology Data Exchange (ETDEWEB)

    Reynertson, Kurt A. [Center for Complementary and Integrative Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States); Department of Pharmacology, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States); Charlson, Mary E. [Center for Complementary and Integrative Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States); Department of Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States); Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu [Center for Complementary and Integrative Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States); Department of Pharmacology, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States); Department of Medicine, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065 (United States)

    2011-01-01

    Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extracts for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from 12 species of ethnomedically utilized plants, we found fractions from 3 species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells.

  1. Endothelial cells derived from human embryonic stem cells

    Science.gov (United States)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  2. Genomic and proteomic analyses of Prdm5 reveal interactions with insulator binding proteins in embryonic stem cells

    DEFF Research Database (Denmark)

    Galli, Giorgio Giacomo; Carrara, Matteo; Francavilla, Chiara

    2013-01-01

    PRDM proteins belong to the SET- domain protein family involved in the regulation of gene expression. Although few PRDM members possess histone methyltransferase activity, the molecular mechanisms by which the other members exert transcriptional regulation remain to be delineated. In this study, ......-occupies genomic loci. In summary, our data indicate how Prdm5 may modulate transcription by interacting with factors involved in genome organization in mouse embryonic stem cells....... find that Prdm5 is highly expressed in mouse embryonic stem cells (mES) and exploit this cellular system to characterize molecular functions of Prdm5. By combining proteomics and next generation sequencing technologies we identify Prdm5 interaction partners and genomic occupancy. We demonstrate that......, despite Prdm5 is dispensable for mES cell maintenance, it directly targets genomic regions involved in early embryonic development and affects the expression of a subset of developmental regulators during cell differentiation. Importantly, Prdm5 interacts with Ctcf, Cohesin and TFIIIC and co...

  3. Fabrication of synthetic polymer coatings and their use in feeder-free culture of human embryonic stem cells

    OpenAIRE

    Nandivada, Himabindu; Villa-Diaz, Luis G; O'Shea, K. Sue; Smith, Gary D.; Krebsbach, Paul H.; Lahann, Joerg

    2011-01-01

    The culture of human embryonic stem (hES) cells in defined and xenogeneic-free conditions will contribute substantially to future biotechnological and medical applications. To achieve this goal, we developed the first fully defined synthetic polymer coating poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH) that sustains long-term growth of hES cells in different culture media. Here we describe a detailed protocol for the reproducible fabrication of PMEDSAH co...

  4. Unique Differentiation Profile of Mouse Embryonic Stem Cells in Rotary and Stirred Tank Bioreactors

    Science.gov (United States)

    Fridley, Krista M.; Fernandez, Irina; Li, Mon-Tzu Alice; Kettlewell, Robert B.

    2010-01-01

    Embryonic stem (ES)-cell-derived lineage-specific stem cells, for example, hematopoietic stem cells, could provide a potentially unlimited source for transplantable cells, especially for cell-based therapies. However, reproducible methods must be developed to maximize and scale-up ES cell differentiation to produce clinically relevant numbers of therapeutic cells. Bioreactor-based dynamic culture conditions are amenable to large-scale cell production, but few studies have evaluated how various bioreactor types and culture parameters influence ES cell differentiation, especially hematopoiesis. Our results indicate that cell seeding density and bioreactor speed significantly affect embryoid body formation and subsequent generation of hematopoietic stem and progenitor cells in both stirred tank (spinner flask) and rotary microgravity (Synthecon™) type bioreactors. In general, high percentages of hematopoietic stem and progenitor cells were generated in both bioreactors, especially at high cell densities. In addition, Synthecon bioreactors produced more sca-1+ progenitors and spinner flasks generated more c-Kit+ progenitors, demonstrating their unique differentiation profiles. cDNA microarray analysis of genes involved in pluripotency, germ layer formation, and hematopoietic differentiation showed that on day 7 of differentiation, embryoid bodies from both bioreactors consisted of all three germ layers of embryonic development. However, unique gene expression profiles were observed in the two bioreactors; for example, expression of specific hematopoietic genes were significantly more upregulated in the Synthecon cultures than in spinner flasks. We conclude that bioreactor type and culture parameters can be used to control ES cell differentiation, enhance unique progenitor cell populations, and provide means for large-scale production of transplantable therapeutic cells. PMID:20528675

  5. Neurotrophin receptor-mediated death of misspecified neurons generated from embryonic stem cells lacking Pax6.

    Science.gov (United States)

    Nikoletopoulou, Vassiliki; Plachta, Nicolas; Allen, Nicolas D; Pinto, Luisa; Götz, Magdalena; Barde, Yves-Alain

    2007-11-01

    Pax6-positive radial glial (RG) cells are the progenitors of most glutamatergic neurons in the cortex, a lineage that can be recapitulated in vitro using embryonic stem (ES) cells. We show here that ES cells lacking Pax6, a transcription factor long known to be essential for cortical development, generate Mash1-positive RG cells that differentiate in GABAergic neurons. These neurons express high levels of the neurotrophin receptor p75NTR causing their rapid death. Pax6 function was also investigated following transplantation of ES cells in the developing chick telencephalon and in mice lacking both Pax6 and p75NTR. Taken together, our results indicate that reliable predictions can be made with cultured ES cells when used to explore the role of genes impacting early aspects of mammalian neurogenesis. They also provide a novel opportunity to compare the molecular constituents of glutamatergic with those of GABA-ergic neurons and to explore the mechanisms of their generation.

  6. Polycomb enables primitive endoderm lineage priming in embryonic stem cells

    DEFF Research Database (Denmark)

    Illingworth, Robert S; Hölzenspies, Jurriaan J; Roske, Fabian V

    2016-01-01

    Mouse embryonic stem cells (ESCs), like the blastocyst from which they are derived, contain precursors of the epiblast (Epi) and primitive endoderm (PrEn) lineages. While transient in vivo, these precursor populations readily interconvert in vitro. We show that altered transcription is the driver...... polycomb with dynamic changes in transcription and stalled lineage commitment, allowing cells to explore alternative choices prior to a definitive decision....

  7. Epigenetic stability, adaptability, and reversibility in human embryonic stem cells

    OpenAIRE

    Tompkins, Joshua D.; Hall, Christine; Chen, Vincent Chang-yi; Li, Arthur Xuejun; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

    2012-01-01

    The stability of human embryonic stem cells (hESCs) is of critical importance for both experimental and clinical applications. We find that as an initial response to altered culture conditions, hESCs change their transcription profile for hundreds of genes and their DNA methylation profiles for several genes outside the core pluripotency network. After adaption to conditions of feeder-free defined and/or xeno-free culture systems, expression and DNA methylation profiles are quite stable for a...

  8. Viscoelastic and dynamic properties of embryonic stem cells

    DEFF Research Database (Denmark)

    Ritter, Christine

    ofthe cells themselves. In this thesis, the viscoelastic properties of mouse embryonic stem cells primedeither toward the epiblast (Epi) or the primitive endoderm (PrE) lineage were investigated.Optical tweezers were used to measure the fluctuations of endogenous lipid granules and therebydraw...... diffusive process was determined to becontinuous time random walk (CTRW).Upon exciting pluripotency, changes occur in the nucleus of stem cells. Chromatin remodeling,the recruitment of lamin A to the nucleoskeleton and stiffening of the cells were reported changescaused by differentiation...

  9. Generation of stomach tissue from mouse embryonic stem cells.

    Science.gov (United States)

    Noguchi, Taka-aki K; Ninomiya, Naoto; Sekine, Mari; Komazaki, Shinji; Wang, Pi-Chao; Asashima, Makoto; Kurisaki, Akira

    2015-08-01

    Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, β-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.

  10. The ethical dilemma of embryonic stem cell research.

    Science.gov (United States)

    Manzar, Nabeel; Manzar, Bushra; Hussain, Nuzhat; Hussain, M Fawwad Ahmed; Raza, Sajjad

    2013-03-01

    To determine the knowledge, attitude, and ethical concerns of medical students and graduates with regard to Embryonic Stem Cell (ESC) research. This questionnaire based descriptive study was conducted at the Civil Hospital Karachi (CHK), Pakistan from February to July 2008. A well structured questionnaire was administered to medical students and graduate doctors, which included their demographic profile as well as questions in line with the study objective. Informed consent was taken and full confidentiality was assured to the participants. Data were entered in a Statistical Package for Social Sciences (SPSS version.12) and analyzed. A total of 204 male and 216 female medical students and doctors were administered questionnaires out of which 105 males (51.4%) and 108 females (50%) were aware of the embryonic stem cell research and its ethical implications. Forty percent males and 47% of females were of the opinion that life begins at conception. Forty-six percent males and 39% females were in favor of stem cell research while only 31% males and 28% females supported the ESC research. Less than 1/3 of students supported using frozen embryos for research purposes while more than 2/3 indicated that they were unlikely to support abortion for stem cell research purposes. The majority of the students were in favor of stem cell research with some reservations regarding ESC research. A sizeable number of students withheld their views, reflecting their poor understanding of medical ethics. The result of the study indicates a need for incorporating bioethics into the medical curriculum.

  11. Mechanisms Regulating Stemness and Differentiation in Embryonal Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Gregory M. Kelly

    2017-01-01

    Full Text Available Just over ten years have passed since the seminal Takahashi-Yamanaka paper, and while most attention nowadays is on induced, embryonic, and cancer stem cells, much of the pioneering work arose from studies with embryonal carcinoma cells (ECCs derived from teratocarcinomas. This original work was broad in scope, but eventually led the way for us to focus on the components involved in the gene regulation of stemness and differentiation. As the name implies, ECCs are malignant in nature, yet maintain the ability to differentiate into the 3 germ layers and extraembryonic tissues, as well as behave normally when reintroduced into a healthy blastocyst. Retinoic acid signaling has been thoroughly interrogated in ECCs, especially in the F9 and P19 murine cell models, and while we have touched on this aspect, this review purposely highlights how some key transcription factors regulate pluripotency and cell stemness prior to this signaling. Another major focus is on the epigenetic regulation of ECCs and stem cells, and, towards that end, this review closes on what we see as a new frontier in combating aging and human disease, namely, how cellular metabolism shapes the epigenetic landscape and hence the pluripotency of all stem cells.

  12. Derivation of a germline competent transgenic Fischer 344 embryonic stem cell line.

    Directory of Open Access Journals (Sweden)

    Hongsheng Men

    Full Text Available Embryonic stem (ES cell-based gene manipulation is an effective method for the generation of mutant animal models in mice and rats. Availability of germline-competent ES cell lines from inbred rat strains would allow for creation of new genetically modified models in the desired genetic background. Fischer344 (F344 males carrying an enhanced green fluorescence protein (EGFP transgene were used as the founder animals for the derivation of ES cell lines. After establishment of ES cell lines, rigorous quality control testing that included assessment of pluripotency factor expression, karyotype analysis, and pathogen/sterility testing was conducted in selected ES cell lines. One male ES cell line, F344-Tg.EC4011, was further evaluated for germline competence by injection into Dark Agouti (DA X Sprague Dawley (SD blastocysts. Resulting chimeric animals were bred with wild-type SD mates and germline transmissibility of the ES cell line was confirmed by identification of pups carrying the ES cell line-derived EGFP transgene. This is the first report of a germline competent F344 ES cell line. The availability of a new germline competent ES cell line with a stable fluorescence reporter from an inbred transgenic rat strain provides an important new resource for genetic manipulations to create new rat models.

  13. Dual Function of Wnt Signaling during Neuronal Differentiation of Mouse Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Hanjun Kim

    2015-01-01

    Full Text Available Activation of Wnt signaling enhances self-renewal of mouse embryonic and neural stem/progenitor cells. In contrast, undifferentiated ES cells show a very low level of endogenous Wnt signaling, and ectopic activation of Wnt signaling has been shown to block neuronal differentiation. Therefore, it remains unclear whether or not endogenous Wnt/β-catenin signaling is necessary for self-renewal or neuronal differentiation of ES cells. To investigate this, we examined the expression profiles of Wnt signaling components. Expression levels of Wnts known to induce β-catenin were very low in undifferentiated ES cells. Stable ES cell lines which can monitor endogenous activity of Wnt/β-catenin signaling suggest that Wnt signaling was very low in undifferentiated ES cells, whereas it increased during embryonic body formation or neuronal differentiation. Interestingly, application of small molecules which can positively (BIO, GSK3β inhibitor or negatively (IWR-1-endo, Axin stabilizer control Wnt/β-catenin signaling suggests that activation of that signaling at different time periods had differential effects on neuronal differentiation of 46C ES cells. Further, ChIP analysis suggested that β-catenin/TCF1 complex directly regulated the expression of Sox1 during neuronal differentiation. Overall, our data suggest that Wnt/β-catenin signaling plays differential roles at different time points of neuronal differentiation.

  14. Differentiating Mouse Embryonic Stem Cells into Embryoid Bodies by Hanging-Drop Cultures.

    Science.gov (United States)

    Behringer, Richard; Gertsenstein, Marina; Nagy, Kristina Vintersten; Nagy, Andras

    2016-12-01

    Embryonic stem (ES) cells can develop into many types of differentiated tissues if they are placed into a differentiating environment. This can occur in vivo when the ES cells are injected into or aggregated with an embryo, or in vitro if their culture conditions are modified to induce differentiation. There are an increasing number of differentiating culture conditions that can bias the differentiation of ES cells into desired cell types. Determining the mechanisms that control ES cell differentiation into therapeutically important cell types is a quickly growing area of research. Knowledge gained from these studies may eventually lead to the use of stem cells to repair specific damaged tissues. Many times ES cell differentiation proceeds through an intermediate stage called the embryoid body (EB). EBs are round structures composed of ES cells that have undergone some of the initial stages of differentiation. EBs can then be manipulated further to generate more specific cell types. This protocol describes a method to differentiate ES cells into EBs. It produces EBs of comparable size. This aspect is important because the differentiation processes taking place inside an EB are influenced by its size. © 2016 Cold Spring Harbor Laboratory Press.

  15. Relative developmental toxicity potencies of retinoids in the embryonic stem cell test compared with their relative potencies in in vivo and two other in vitro assays for developmental toxicity

    NARCIS (Netherlands)

    Louisse, J.; Gönen, S.; Rietjens, I.M.C.M.; Verwei, M.

    2011-01-01

    The present study determines the relative developmental toxicity potencies of retinoids in the embryonic stem (ES)-D3 cell differentiation assay of the embryonic stem cell test, and compares the outcomes with their relative potencies in in vivo and two other in vitro assays for developmental

  16. Human embryonic stem cells and embryonal carcinoma cells have overlapping and distinct metabolic signatures.

    Directory of Open Access Journals (Sweden)

    Raed Abu Dawud

    Full Text Available While human embryonic stem cells (hESCs and human embryonal carcinoma cells (hECCs have been studied extensively at the levels of the genome, transcriptome, proteome and epigenome our knowledge of their corresponding metabolomes is limited. Here, we present the metabolic signatures of hESCs and hESCs obtained by untargeted gas chromatography coupled to mass spectrometry (GC-MS. Whilst some metabolites are common to both cell types, representing the self-renewal and house-keeping signatures, others were either higher (e.g., octadecenoic acid, glycerol-3-phosphate, 4-hydroxyproline or lower (e.g., glutamic acid, mannitol, malic acid, GABA in hESCs (H9 compared to hECCs (NTERA2, these represent cell type specific signatures. Further, our combined results of GC-MS and microarray based gene expression profiling of undifferentiated and OCT4-depleted hESCs are consistent with the Warburg effect which is increased glycolysis in embryonic cells and tumor cells in the presence of O(2 while oxidative phosphorylation (OXPHOS is impaired or even shut down. RNAi-based OCT4 knock down mediated differentiation resulted in the activation of the poised OXPHOS machinery by expressing missing key proteins such as NDUFC1, UQCRB and COX, increase in TCA cycle activity and decreased lactate metabolism. These results shed light on the metabolite layer of pluripotent stem cells and could potentially establish novel metabolic markers of self renewal and pluripotency.

  17. Suppression of Th1-mediated autoimmunity by embryonic stem cell-derived dendritic cells.

    Directory of Open Access Journals (Sweden)

    Tokunori Ikeda

    Full Text Available We herein demonstrate the immune-regulatory effect of embryonic stem cell-derived dendritic cells (ES-DCs using two models of autoimmune disease, namely non-obese diabetic (NOD mice and experimental autoimmune encephalomyelitis (EAE. Treatment of pre-diabetic NOD mice with ES-DCs exerted almost complete suppression of diabetes development during the observation period for more than 40 weeks. The prevention of diabetes by ES-DCs was accompanied with significant reduction of insulitis and decreased number of Th1 and Th17 cells in the spleen. Development of EAE was also inhibited by the treatment with ES-DCs, and the therapeutic effect was obtained even if ES-DCs were administrated after the onset of clinical symptoms. Treatment of EAE-induced mice with ES-DCs reduced the infiltration of inflammatory cells into the spinal cord and suppressed the T cell response to the myelin antigen. Importantly, the ES-DC treatment did not affect T cell response to an exogenous antigen. As the mechanisms underlying the reduction of the number of infiltrating Th1 cells, we observed the inhibition of differentiation and proliferation of Th1 cells by ES-DCs. Furthermore, the expression of VLA-4α on Th1 cells was significantly inhibited by ES-DCs. Considering the recent advances in human induced pluripotent stem cell-related technologies, these results suggest a clinical application for pluripotent stem cell-derived dendritic cells as a therapy for T cell-mediated autoimmune diseases.

  18. Pluripotency factors and Polycomb Group proteins repress aryl hydrocarbon receptor expression in murine embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Chia-I Ko

    2014-01-01

    Full Text Available The aryl hydrocarbon receptor (AHR is a transcription factor and environmental sensor that regulates expression of genes involved in drug-metabolism and cell cycle regulation. Chromatin immunoprecipitation analyses, Ahr ablation in mice and studies with orthologous genes in invertebrates suggest that AHR may also play a significant role in embryonic development. To address this hypothesis, we studied the regulation of Ahr expression in mouse embryonic stem cells and their differentiated progeny. In ES cells, interactions between OCT3/4, NANOG, SOX2 and Polycomb Group proteins at the Ahr promoter repress AHR expression, which can also be repressed by ectopic expression of reprogramming factors in hepatoma cells. In ES cells, unproductive RNA polymerase II binds at the Ahr transcription start site and drives the synthesis of short abortive transcripts. Activation of Ahr expression during differentiation follows from reversal of repressive marks in Ahr promoter chromatin, release of pluripotency factors and PcG proteins, binding of Sp factors, establishment of histone marks of open chromatin, and engagement of active RNAPII to drive full-length RNA transcript elongation. Our results suggest that reversible Ahr repression in ES cells holds the gene poised for expression and allows for a quick switch to activation during embryonic development.

  19. Aire promotes the self-renewal of embryonic stem cells through Lin28.

    Science.gov (United States)

    Bin, Gu; Jiarong, Zhang; Shihao, Wang; Xiuli, Song; Cheng, Xu; Liangbiao, Chen; Ming, Zhang

    2012-10-10

    Abstract Autoimmune regulator (Aire) is one of the most well-characterized molecules in autoimmunity, but its function outside the immune system is largely unknown. The recent discovery of Aire expression in stem cells and early embryonic cells and its function in the self-renewal of embryonic stem (ES) cells highlight the importance of Aire in these cells. In this study, we present evidence that Aire promotes the expression of the pluripotent factor Lin28 and the self-renewal of ES cells. We presented the first evidence that the let-7 microRNA family contributed to the self-renewal promoting effect of Aire on ES cells. Moreover, we showed that Aire and Lin28 are co-expressed in the genital ridge, oocytes, and cleavage-stage embryos, and the expression level of Lin28 is correlated with the expression level of Aire. Although it is widely considered to be a promiscuous gene expression activator, these results indicated that Aire promotes the self-renewal of ES cells through a specific pathway (i.e., the activation of Lin28 and the inhibition of the let-7 microRNA family). The correlation between Aire and Lin28 expression in germ cells and early embryos indicated an in vivo function for Aire in toti- and pluripotent stem cells. This study presents the first molecular pathway that incorporates Aire into the pluripotency network. Moreover, it presents the first evidence that microRNAs contribute to the regulatory function of Aire and highlights a novel function of Aire in stem cell biology and reproduction. These functions reveal novel perspectives for studying the molecular mechanisms behind the establishment and sustenance of pluripotent identity.

  20. A Sweet Potion to Put Embryonic Stem Cells to Sleep

    Directory of Open Access Journals (Sweden)

    Kaushik D. Deb

    2009-01-01

    Full Text Available Human embryonic stem cells (hESCs are rapidly revolutionizing the areas of drug screening and therapy. In view of their applications and high operational costs at global multicentric setups, the ability to store and transport hESCs and derivatives under ambient temperatures, and their cryopreservation without compromising the stemness, function, and viability, is becoming imperative. Here we discuss the need for a natural cryoprotectant and biopreservative with a potential to improve cryopreservation, ambient temperature storage, and shipping of hESCs and derivatives. Trehalose, a naturally occurring disaccharide with therapeutic properties, protects the integrity of cells against desiccation, dehydration, and extreme heat or cold, and has been successfully tested for some somatic stem cell types. However, the biggest setback is the inability of mammalian cells to internalize trehalose. Here we review the methods being developed at different laboratories to facilitate its intercellular transport and advocate the need for similar advances in hESCs.

  1. Measurement of human embryonic stem cell in the growing cycle

    Science.gov (United States)

    Li, X.; Zhao, L.; Oh, Steve K. W.; Chong, W. K.; Ong, J. K.; Chen, Allen K.; Choo, Andre B. H.

    2008-09-01

    A measurement and imaging system has been developed for in-line continuous measurement of live, unmodified, human embryonic stem cells (hESC). The measurement will not affect cell growth, structure, sterility and suitability for clinical use. The stem cell imaging system (SCIS) can be used to support the optimization of automated stem cell growth for invitro study and for high-volume bio-manufacture. This paper present the experimental and analysis for the optimization of system parameters. A non-linear lighting is developed to obtain a clear images. The individual cluster can be traced from day one to day two. The whole system is calibrated with measurement microscope and haemacytometer. The measurement accuracy is better than 90%.

  2. Embryonic stem cell research and the argument of complicity.

    Science.gov (United States)

    Birnbacher, Dieter

    2009-01-01

    While the argument of complicity is only rarely discussed in bioethics, it is of obvious relevance to the issue of imported embryonic stem cells in countries in which the derivation of stem cells from early human embryos is legally prohibited and/or morally rejected. Complicity means that making use of the results or products of an illegal or morally problematic activity is itself morally problematic, although generally to a lesser degree than the original activity. The question arises as to which conditions make the argument of complicity plausible, thus supporting attacks against legislation that aims to promote research based on 'fruits of a forbidden tree'. This paper distinguishes a number of different variants of complicity, proposes that they deserve different kinds of moral valuation and applies the results to the ongoing debate about the German stem cell research law.

  3. AIRE is a critical spindle-associated protein in embryonic stem cells.

    Science.gov (United States)

    Gu, Bin; Lambert, Jean-Philippe; Cockburn, Katie; Gingras, Anne-Claude; Rossant, Janet

    2017-07-25

    Embryonic stem (ES) cells go though embryo-like cell cycles regulated by specialized molecular mechanisms. However, it is not known whether there are ES cell-specific mechanisms regulating mitotic fidelity. Here we showed that Autoimmune Regulator ( Aire ), a transcription coordinator involved in immune tolerance processes, is a critical spindle-associated protein in mouse ES(mES) cells. BioID analysis showed that AIRE associates with spindle-associated proteins in mES cells. Loss of function analysis revealed that Aire was important for centrosome number regulation and spindle pole integrity specifically in mES cells. We also identified the c-terminal LESLL motif as a critical motif for AIRE's mitotic function. Combined maternal and zygotic knockout further revealed Aire's critical functions for spindle assembly in preimplantation embryos. These results uncovered a previously unappreciated function for Aire and provide new insights into the biology of stem cell proliferation and potential new angles to understand fertility defects in humans carrying Air e mutations.

  4. Cryopreservation of human embryonic stem cells without the use of a programmable freezer.

    Science.gov (United States)

    Ha, Sung Yun; Jee, Byung Chul; Suh, Chang Suk; Kim, Hee Sun; Oh, Sun Kyung; Kim, Seok Hyun; Moon, Shin Yong

    2005-07-01

    An effective freezing-thawing technique is crucial for the clinical application of human embryonic stem (ES) cells. The aim of this study was to find an optimal cryopreservation protocol for human ES cells using slow freezing-rapid thawing without a programmable freezer. The human ES cell line, SNUhES-3, was cultured on an STO feeder layer in gelatin-coated tissue culture dishes. All cryopreservation steps were performed using a simple commercial freezing container. The survival rate of cryopreserved-thawed human ES cells was estimated by counting colony numbers under a stereomicroscope. Initially, we compared the survival rates of cryopreserved human ES cells using three cryoprotectants: dimethylsulphoxide (DMSO), ethylene glycol (EG) and glycerol. In this experiment, 5% DMSO/95% fetal bovine serum (FBS) (vol/vol) showed the highest survival rate. We next tested the impact of various concentrations of FBS (95, 50 and 5%) with 5% DMSO, and then examined the effects of adding EG or glycerol to 5% DMSO + optimal FBS. No significant difference in survival rate was observed between 95 and 50% FBS in the presence of 5% DMSO. A significant improvement in survival rate was obtained by adding 10% EG to 5% DMSO+50% FBS. After thawing, surviving cells were found to maintain the inherent characteristics of human ES cells. 5% DMSO+50% FBS+10% EG may be an optimal cryoprotectant for the slow freezing-rapid thawing of human ES cells.

  5. p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Moscatelli, Ilana; Pierantozzi, Enrico; Camaioni, Antonella; Siracusa, Gregorio [Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome (Italy); Campagnolo, Luisa, E-mail: campagno@med.uniroma2.it [Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome (Italy)

    2009-11-01

    Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75{sup NTR}), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75{sup NTR} and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75{sup NTR} and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75{sup NTR}/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75{sup NTR} or TrkA. Interestingly, immunoreactivity to anti-p75{sup NTR} antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75{sup NTR}, when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75{sup NTR} is turned on.

  6. Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

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    Mandy Y M Lo

    Full Text Available Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

  7. Global transcriptional profiles of beating clusters derived from human induced pluripotent stem cells and embryonic stem cells are highly similar

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    Gupta Manoj K

    2010-09-01

    Full Text Available Abstract Background Functional and molecular integrity of cardiomyocytes (CMs derived from induced pluripotent stem (iPS cells is essential for their use in tissue repair, disease modelling and drug screening. In this study we compared global transcriptomes of beating clusters (BCs microdissected from differentiating human iPS cells and embryonic stem (ES cells. Results Hierarchical clustering and principal component analysis revealed that iPS-BCs and ES-BCs cluster together, are similarly enriched for cardiospecific genes and differ in expression of only 1.9% of present transcripts. Similarly, sarcomeric organization, electrophysiological properties and calcium handling of iPS-CMs were indistinguishable from those of ES-CMs. Gene ontology analysis revealed that among 204 genes that were upregulated in iPS-BCs vs ES-BCs the processes related to extracellular matrix, cell adhesion and tissue development were overrepresented. Interestingly, 47 of 106 genes that were upregulated in undifferentiated iPS vs ES cells remained enriched in iPS-BCs vs ES-BCs. Most of these genes were found to be highly expressed in fibroblasts used for reprogramming and 34% overlapped with the recently reported iPS cell-enriched genes. Conclusions These data suggest that iPS-BCs are transcriptionally highly similar to ES-BCs. However, iPS-BCs appear to share some somatic cell signature with undifferentiated iPS cells. Thus, iPS-BCs may not be perfectly identical to ES-BCs. These minor differences in the expression profiles may occur due to differential cellular composition of iPS-BCs and ES-BCs, due to retention of some genetic profile of somatic cells in differentiated iPS cell-derivatives, or both.

  8. Early gene regulation of osteogenesis in embryonic stem cells

    KAUST Repository

    Kirkham, Glen R.

    2012-01-01

    The early gene regulatory networks (GRNs) that mediate stem cell differentiation are complex, and the underlying regulatory associations can be difficult to map accurately. In this study, the expression profiles of the genes Dlx5, Msx2 and Runx2 in mouse embryonic stem cells were monitored over a 48 hour period after exposure to the growth factors BMP2 and TGFβ1. Candidate GRNs of early osteogenesis were constructed based on published experimental findings and simulation results of Boolean and ordinary differential equation models were compared with our experimental data in order to test the validity of these models. Three gene regulatory networks were found to be consistent with the data, one of these networks exhibited sustained oscillation, a behaviour which is consistent with the general view of embryonic stem cell plasticity. The work cycle presented in this paper illustrates how mathematical modelling can be used to elucidate from gene expression profiles GRNs that are consistent with experimental data. © 2012 The Royal Society of Chemistry.

  9. Raman microscopy of individual living human embryonic stem cells

    DEFF Research Database (Denmark)

    Novikov, Sergey M.; Beermann, Jonas; Bozhevolnyi, Sergey I.

    2010-01-01

    We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing...... cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal...

  10. Embryoid body formation from embryonic and induced pluripotent stem cells: Benefits of bioreactors.

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    Rungarunlert, Sasitorn; Techakumphu, Mongkol; Pirity, Melinda K; Dinnyes, Andras

    2009-12-31

    Embryonic stem (ES) cells have the ability to differentiate into all germ layers, holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies and for drug screening. Embryoid body (EB) formation from ES cells is a common method for producing different cell lineages for further applications. However, conventional techniques such as hanging drop or static suspension culture are either inherently incapable of large scale production or exhibit limited control over cell aggregation during EB formation and subsequent EB aggregation. For standardized mass EB production, a well defined scale-up platform is necessary. Recently, novel scenario methods of EB formation in hydrodynamic conditions created by bioreactor culture systems using stirred suspension systems (spinner flasks), rotating cell culture system and rotary orbital culture have allowed large-scale EB formation. Their use allows for continuous monitoring and control of the physical and chemical environment which is difficult to achieve by traditional methods. This review summarizes the current state of production of EBs derived from pluripotent cells in various culture systems. Furthermore, an overview of high quality EB formation strategies coupled with systems for in vitro differentiation into various cell types to be applied in cell replacement therapy is provided in this review. Recently, new insights in induced pluripotent stem (iPS) cell technology showed that differentiation and lineage commitment are not irreversible processes and this has opened new avenues in stem cell research. These cells are equivalent to ES cells in terms of both self-renewal and differentiation capacity. Hence, culture systems for expansion and differentiation of iPS cells can also apply methodologies developed with ES cells, although direct evidence of their use for iPS cells is still limited.

  11. Neural Progenitor Cells Derived from Human Embryonic Stem Cells as an Origin of Dopaminergic Neurons

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    Parinya Noisa

    2015-01-01

    Full Text Available Human embryonic stem cells (hESCs are able to proliferate in vitro indefinitely without losing their ability to differentiate into multiple cell types upon exposure to appropriate signals. Particularly, the ability of hESCs to differentiate into neuronal subtypes is fundamental to develop cell-based therapies for several neurodegenerative disorders, such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. In this study, we differentiated hESCs to dopaminergic neurons via an intermediate stage, neural progenitor cells (NPCs. hESCs were induced to neural progenitor cells by Dorsomorphin, a small molecule that inhibits BMP signalling. The resulting neural progenitor cells exhibited neural bipolarity with high expression of neural progenitor genes and possessed multipotential differentiation ability. CBF1 and bFGF responsiveness of these hES-NP cells suggested their similarity to embryonic neural progenitor cells. A substantial number of dopaminergic neurons were derived from hES-NP cells upon supplementation of FGF8 and SHH, key dopaminergic neuron inducers. Importantly, multiple markers of midbrain neurons were detected, including NURR1, PITX3, and EN1, suggesting that hESC-derived dopaminergic neurons attained the midbrain identity. Altogether, this work underscored the generation of neural progenitor cells that retain the properties of embryonic neural progenitor cells. These cells will serve as an unlimited source for the derivation of dopaminergic neurons, which might be applicable for treating patients with Parkinson’s disease.

  12. CrxOS maintains the self-renewal capacity of murine embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Saito, Ryota; Yamasaki, Tokiwa; Nagai, Yoko; Wu, Jinzhan [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan); Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan); Kajiho, Hiroaki [Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan); Yokoi, Tadashi; Noda, Eiichiro [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan); Department of Ophthalmology, National Center for Child Health and Development, Tokyo 157-8535 (Japan); Nishina, Sachiko [Department of Ophthalmology, National Center for Child Health and Development, Tokyo 157-8535 (Japan); Niwa, Hitoshi [Laboratory for Pluripotent Cell Studies, RIKEN Center for Developmental Biology, Hyogo 650-0047 (Japan); Azuma, Noriyuki [Department of Ophthalmology, National Center for Child Health and Development, Tokyo 157-8535 (Japan); Katada, Toshiaki [Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan); Nishina, Hiroshi, E-mail: nishina.dbio@mri.tmd.ac.jp [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan)

    2009-12-25

    Embryonic stem (ES) cells maintain pluripotency by self-renewal. Several homeoproteins, including Oct3/4 and Nanog, are known to be key factors in maintaining the self-renewal capacity of ES cells. However, other genes required for the mechanisms underlying this process are still unclear. Here we report the identification by in silico analysis of a homeobox-containing gene, CrxOS, that is specifically expressed in murine ES cells and is essential for their self-renewal. ES cells mainly express the short isoform of endogenous CrxOS. Using a polyoma-based episomal expression system, we demonstrate that overexpression of the CrxOS short isoform is sufficient for maintaining the undifferentiated morphology of ES cells and stimulating their proliferation. Finally, using RNA interference, we show that CrxOS is essential for the self-renewal of ES cells, and provisionally identify foxD3 as a downstream target gene of CrxOS. To our knowledge, ours is the first delineation of the physiological role of CrxOS in ES cells.

  13. Improvement of embryonic stem cell line derivation efficiency with novel medium, glucose concentration, and epigenetic modifications.

    Science.gov (United States)

    Kim, Chul; Amano, Tomokazu; Park, Joonghoon; Carter, Mark G; Tian, Xiuchun; Yang, Xiangzhong

    2009-03-01

    Although the first mouse embryonic stem (ES) cell lines were derived 2 decades ago, and standard protocols for ES cell derivation are widely used today, the technical difficulty of these protocols still pose a challenge for many investigators attempting to produce large numbers of ES cell lines, and are limited to only a few mouse strains. Recently, glucose concentration was shown to have a significant effect on the efficiency of ES cell derivation, but the mechanism(s) mediating this effect are still the subject of debate. In this report, we investigated the effect of glucose concentration on ES cell derivation efficiency from blastocysts in the context of a new medium, Minimum Essential Medium alpha (MEMalpha). Furthermore, we propose novel methods to improve mouse ES cell derivation efficiency using in vitro epigenetic modifications during early passages, combined with detection of Oct4-expressing cells. Based on the results reported here, modified MEMalpha containing high glucose improves the efficiency of ES cell derivation remarkably, compared with Knockout Dulbecco's-Modified Eagle Media (KDMEM). Epigenetic modifications are able to improve the efficiency even further.

  14. In vitro germ cell differentiation from cynomolgus monkey embryonic stem cells.

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    Kaori Yamauchi

    Full Text Available BACKGROUND: Mouse embryonic stem (ES cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. METHODS AND FINDINGS: To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis. VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. CONCLUSION: VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the

  15. Gene function in early mouse embryonic stem cell differentiation

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    Campbell Pearl A

    2007-03-01

    Full Text Available Abstract Background Little is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5 undergoing undirected differentiation into embryoid bodies (EBs over a period of two weeks. Results We identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1, our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2 that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set. Conclusion Our analysis profiles for the first time gene expression at a very early stage of m

  16. The N-glycome of human embryonic stem cells

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    Olonen Anne

    2009-06-01

    Full Text Available Abstract Background Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC, the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses. Results The data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Lex and H type 2 antennae in sialylated complex-type N-glycans. Conclusion The N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.

  17. Transplantation of neural stem cells clonally derived from embryonic stem cells promotes recovery after murine spinal cord injury.

    Science.gov (United States)

    Salewski, Ryan P; Mitchell, Robert A; Shen, Carl; Fehlings, Michael G

    2015-01-01

    The pathology of spinal cord injury (SCI) makes it appropriate for cell-based therapies. Treatments using neural stem cells (NSCs) in animal models of SCI have shown positive outcomes, although uncertainty remains regarding the optimal cell source. Pluripotent cell sources such as embryonic stem cells (ESCs) provide a limitless supply of therapeutic cells. NSCs derived using embryoid bodies (EB) from ESCs have shown tumorigenic potential. Clonal neurosphere generation is an alternative method to generate safer and more clinically relevant NSCs without the use of an EB stage for use in cell-based therapies. We generated clonally derived definitive NSCs (dNSCs) from ESC. These cells were transplanted into a mouse thoracic SCI model. Embryonic stem cell-derived definitive neural stem cell (ES-dNSC)-transplanted mice were compared with controls using behavioral measures and histopathological analysis of tissue. In addition, the role of remyelination in injury recovery was investigated using transmission electron microscopy. The SCI group that received ES-dNSC transplantation showed significant improvements in locomotor function compared with controls in open field and gait analysis. The cell treatment group had a significant enhancement of spared neural tissue. Immunohistological assessments showed that dNSCs differentiated primarily to oligodendrocytes. These cells were shown to express myelin basic protein, associate with axons, and support nodal architecture as well as display proper compact, multilayer myelination in electron microscopic analysis. This study provides strong evidence that dNSCs clonally derived from pluripotent cells using the default pathway of neuralization improve motor function after SCI and enhance sparing of neural tissue, while remaining safe and clinically relevant.

  18. Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos.

    Science.gov (United States)

    Behboodi, E; Bondareva, A; Begin, I; Rao, K; Neveu, N; Pierson, J T; Wylie, C; Piero, F D; Huang, Y J; Zeng, W; Tanco, V; Baldassarre, H; Karatzas, C N; Dobrinski, I

    2011-03-01

    Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos. In vivo-derived embryos were cultured on goat fetal fibroblast feeders. Embryos either attached to the feeder layer or remained floating and expanded in culture. Embryos that attached showed a prominent inner cell mass (ICM) and those that remained floating formed structures resembling ICM disks surrounded by trophectodermal cells. ICM cells and embryonic disks were isolated mechanically, cultured on feeder cells in the presence of hLIF, and outgrown into ES-like colonies. Two cell lines were cultured for 25 passages and stained positive for alkaline phosphatase, POU5F1, NANOG, SOX2, SSEA-1, and SSEA-4. Embryoid bodies formed in suspension culture without hLIF. One cell line was cultured for 2 years (over 120 passages). This cell line differentiated in vitro into epithelia and neuronal cells, and could be stably transfected and selected for expression of a fluorescent marker. When cells were injected into SCID mice, teratomas were identified 5-6 weeks after transplantation. Expression of known ES cell markers, maintenance in vitro for 2 years in an undifferentiated state, differentiation in vitro, and formation of teratomas in immunodeficient mice provide evidence that the established cell line represents goat ES cells. This also is the first report of teratoma formation from large animal ES cells. Copyright © 2011 Wiley-Liss, Inc.

  19. A Focused Microarray for Screening Rat Embryonic Stem Cell Lines

    Science.gov (United States)

    Hong, James; He, Hong; Bui, Phuoc; Ryba-White, Ben; Rumi, Mohammad A.K.; Soares, Michael J.; Dutta, Debasree; Paul, Soumen; Kawamata, Masaki; Ochiya, Takahiro; Ying, Qi-Long; Rajanahalli, Pavan

    2013-01-01

    Here, we describe a focused microarray for screening rat embryonic stem cells (ESCs) and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem (TS) cells, rat extraembryonic endoderm cells, mouse embryonic fibroblast feeder cells, and differentiated rat ESCs. Using this tool, genuine rat ESC lines, which have been expanded in a conventional rat ESC medium containing two inhibitors (2i), for example, glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (MEK) inhibitors, and leukemia inhibitory factor, and genuine rat ESCs, which have been expanded in rat ESC medium containing four inhibitors (4i), for example, GSK3, MEK, Alk5, and Rho-associated kinase inhibitors were compared; as were genuine rat ESCs from 4 different strains of rats. Expression of Cdx2, a gene associated with trophoblast determination, was observed in genuine, undifferentiated rat ESCs from 4 strains and from both 2i and 4i ESC derivation medium. This finding is in contrast to undifferentiated mouse ESCs that do not express Cdx2. The rat ESC focused microarray described in this report has utility for rapid screening of rat ESCs. This tool will enable optimization of culture conditions in the future. PMID:22889370

  20. Lipofectamine RNAiMAX: an efficient siRNA transfection reagent in human embryonic stem cells.

    Science.gov (United States)

    Zhao, Ming; Yang, Hong; Jiang, Xingjun; Zhou, Wen; Zhu, Bin; Zeng, Ying; Yao, Kaitai; Ren, Caiping

    2008-09-01

    RNA interference methodology suppresses specific gene expression, thus mimicking loss-of-function mutation and enabling in vitro and in vivo gene function analysis. Lipofectamine RNAiMAX, a new transfection reagent, has been confirmed high efficiency in delivering small interfering RNA (siRNA) into mesenchymal stem cells and neural stem cells. In this study, we used three transfection reagents (Lipofectamine RNAiMAX, Oligofectamine and Lipofectamine 2000) to deliver siRNA into human embryonic stem (hES) cells and compared the silencing efficiency of enhanced green fluorescent protein transgene and Oct4, a key regulator of pluripotency. As a result, siRNA can be delivered into hES cells more efficiently by Lipofectamine RNAiMAX compared with the other two transfection reagents and high efficient knockdown of target genes was obtained by Lipofectamine RNAiMAX even at a low concentration of siRNA. Quantitative real-time PCR showed approximately 90% knockdown of Oct4 transcript with cognate Oct4 siRNA transfection by Lipofectamine RNAiMAX compared to control siRNA. These results demonstrated that Lipofectamine RNAiMAX is an efficient and excellent transfection reagent for delivering siRNA into hES cells.

  1. Reprogramming Malignant Cancer Cells toward a Benign Phenotype following Exposure to Human Embryonic Stem Cell Microenvironment

    Science.gov (United States)

    Arena, Vincenzo; Arena, Manuel; Arena, Goffredo Orazio

    2017-01-01

    The embryonic microenvironment is well known to be non-permissive for tumor development because early developmental signals naturally suppress the expression of proto-oncogenes. In an analogous manner, mimicking an early embryonic environment during embryonic stem cell culture has been shown to suppress oncogenic phenotypes of cancer cells. Exosomes derived from human embryonic stem cells harbor substances that mirror the content of the cells of origin and have been reported to reprogram hematopoietic stem/progenitor cells via horizontal transfer of mRNA and proteins. However, the possibility that these embryonic stem cells-derived exosomes might be the main effectors of the anti-tumor effect mediated by the embryonic stem cells has not been explored yet. The present study aims to investigate whether exosomes derived from human embryonic stem cells can reprogram malignant cancer cells to a benign stage and reduce their tumorigenicity. We show that the embryonic stem cell-conditioned medium contains factors that inhibit cancer cell growth and tumorigenicity in vitro and in vivo. Moreover, we demonstrate that exosomes derived from human embryonic stem cells display anti-proliferation and pro-apoptotic effects, and decrease tumor size in a xenograft model. These exosomes are also able to transfer their cargo into target cancer cells, inducing a dose-dependent increase in SOX2, OCT4 and Nanog proteins, leading to a dose-dependent decrease of cancer cell growth and tumorigenicity. This study shows for the first time that human embryonic stem cell-derived exosomes play an important role in the tumor suppressive activity displayed by human embryonic stem cells. PMID:28068409

  2. Rho-associated kinase inhibitors promote the cardiac differentiation of embryonic and induced pluripotent stem cells.

    Science.gov (United States)

    Cheng, Ya-Ting; Yeih, Dong-Feng; Liang, Shu-Man; Chien, Chia-Ying; Yu, Yen-Ling; Ko, Bor-Sheng; Jan, Yee-Jee; Kuo, Cheng-Chin; Sung, Li-Ying; Shyue, Song-Kun; Chen, Ming-Fong; Yet, Shaw-Fang; Wu, Kenneth K; Liou, Jun-Yang

    2015-12-15

    Rho-associated kinase (ROCK) plays an important role in maintaining embryonic stem (ES) cell pluripotency. To determine whether ROCK is involved in ES cell differentiation into cardiac and hematopoietic lineages, we evaluated the effect of ROCK inhibitors, Y-27632 and fasudil on murine ES and induced pluripotent stem (iPS) cell differentiation. Gene expression levels were determined by real-time PCR, Western blot analysis and immunofluorescent confocal microscopy. Cell transplantation of induced differentiated cells were assessed in vivo in a mouse model (three groups, n=8/group) of acute myocardial infarction (MI). The cell engraftment was examined by immunohistochemical staining and the outcome was analyzed by echocardiography. Cells were cultured in hematopoietic differentiation medium in the presence or absence of ROCK inhibitor and colony formation as well as markers of ES, hematopoietic stem cells (HSC) and cells of cardiac lineages were analyzed. ROCK inhibition resulted in a drastic change in colony morphology accompanied by loss of hematopoietic markers (GATA-1, CD41 and β-Major) and expressed markers of cardiac lineages (GATA-4, Isl-1, Tbx-5, Tbx-20, MLC-2a, MLC-2v, α-MHC, cTnI and cTnT) in murine ES and iPS cells. Fasudil-induced cardiac progenitor (Mesp-1 expressing) cells were infused into a murine MI model. They engrafted into the peri-infarct and infarct regions and preserved left ventricular function. These findings provide new insights into the signaling required for ES cell differentiation into hematopoietic as well as cardiac lineages and suggest that ROCK inhibitors are useful in directing iPS cell differentiation into cardiac progenitor cells for cell therapy of cardiovascular diseases. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  3. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

    Science.gov (United States)

    The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

  4. Functional network integration of embryonic stem cell-derived astrocytes in hippocampal slice cultures.

    Science.gov (United States)

    Scheffler, Björn; Schmandt, Tanja; Schröder, Wolfgang; Steinfarz, Barbara; Husseini, Leila; Wellmer, Jörg; Seifert, Gerald; Karram, Khalad; Beck, Heinz; Blümcke, Ingmar; Wiestler, Otmar D; Steinhäuser, Christian; Brüstle, Oliver

    2003-11-01

    Embryonic stem (ES) cells provide attractive prospects for neural transplantation. So far, grafting strategies in the CNS have focused mainly on neuronal replacement. Employing a slice culture model, we found that ES cell-derived glial precursors (ESGPs) possess a remarkable capacity to integrate into the host glial network. Following deposition on the surface of hippocampal slices, ESGPs actively migrate into the recipient tissue and establish extensive cell-cell contacts with recipient glia. Gap junction-mediated coupling between donor and host astrocytes permits widespread delivery of dye from single donor cells. During maturation, engrafted donor cells display morphological, immunochemical and electrophysiological properties that are characteristic of differentiating native glia. Our findings provide the first evidence of functional integration of grafted astrocytes, and depict glial network integration as a potential route for widespread transcellular delivery of small molecules to the CNS.

  5. The phenotype of FancB-mutant mouse embryonic stem cells

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    Kim, Tae Moon; Ko, Jun Ho; Choi, Yong Jun; Hu Lingchuan [Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245 (United States); Hasty, Paul, E-mail: hastye@uthscsa.edu [Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245 (United States)

    2011-07-01

    Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, developmental defects and cancer. There are multiple FA genes that enable the repair of interstrand crosslinks (ICLs) in coordination with a variety of other DNA repair pathways in a way that is poorly understood. Here we present the phenotype of mouse embryonic stem (ES) cells mutated for FancB. We found FancB-mutant cells exhibited reduced cellular proliferation, hypersensitivity to the crosslinking agent mitomycin C (MMC), increased spontaneous and MMC-induced chromosomal abnormalities, reduced spontaneous sister chromatid exchanges (SCEs), reduced gene targeting, reduced MMC-induced Rad51 foci and absent MMC-induced FancD2 foci. Since FancB is on the X chromosome and since ES cells are typically XY, FancB is an excellent target for an epistatic analysis to elucidate FA's role in ICL repair.

  6. Generation of genetically modified embryonic stem cells for the development of knockout mouse animal model systems.

    Science.gov (United States)

    Robinson, Stephen D; Wilson, Stephen; Hodivala-Dilke, Kairbaan M

    2006-01-01

    The aim of our lab is to understand the contributions made by cell adhesion molecules in the processes of disease. Much of our recent work has focused on the role played by beta3-integrin in mediating pathological angiogenesis. It is fair to state that without the ability to manipulate the mouse genome, and specifically to create knockout mice, the advances we have made in this field would not be nearly as significant as they are. The ability to generate knockout mice depends on the two technological breakthroughs of the ability to isolate and culture mouse embryonic stem (ES) cells and the methods employed for achieving targeted gene replacement in these cells by homologous recombination. Here, we present the methods we have found to be successful, and that we routinely employ to grow and manipulate ES cells, as well as those to screen and identify homologous recombinants.

  7. Effects of Feeder Cell Types on Culture of Mouse Embryonic Stem Cell In Vitro.

    Science.gov (United States)

    Park, Yun-Gwi; Lee, Seung-Eun; Kim, Eun-Young; Hyun, Hyuk; Shin, Min-Young; Son, Yeo-Jin; Kim, Su-Young; Park, Se-Pill

    2015-09-01

    The suitable feeder cell layer is important for culture of embryonic stem (ES) cells. In this study, we investigated the effect of two kinds of the feeder cell, MEF cells and STO cells, layer to mouse ES (mES) cell culture for maintenance of stemness. We compare the colony formations, alkaline phosphatase (AP) activities, expression of pluripotency marker genes and proteins of D3 cell colonies cultured on MEF feeder cell layer (D3/MEF) or STO cell layers (D3/STO) compared to feeder free condition (D3/-) as a control group. Although there were no differences to colony formations and AP activities, interestingly, the transcripts level of pluripotency marker genes, Pou5f1 and Nanog were highly expressed in D3/MEF (79 and 93) than D3/STO (61and 77) or D3/- (65 and 81). Also, pluripotency marker proteins, NANOG and SOX-2, were more synthesized in D3/MEF (72.8±7.69 and 81.2±3.56) than D3/STO (32.0±4.30 and 56.0±4.90) or D3/- (55.0±4.64 and 62.0±6.20). These results suggest that MEF feeder cell layer is more suitable to mES cell culture.

  8. Derivation and characterization of hepatic progenitor cells from human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Dongxin Zhao

    Full Text Available The derivation of hepatic progenitor cells from human embryonic stem (hES cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential hepatic progenitor cells from hES cells. These hES cell-derived hepatic progenitor cells could be effectively used as an in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.

  9. Puerarin Facilitates T-Tubule Development of Murine Embryonic Stem Cell-Derived Cardiomyocytes

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    Lu Wang

    2014-07-01

    Full Text Available Aims: The embryonic stem cell-derived cardiomyocytes (ES-CM is one of the promising cell sources for repopulation of damaged myocardium. However, ES-CMs present immature structure, which impairs their integration with host tissue and functional regeneration. This study used murine ES-CMs as an in vitro model of cardiomyogenesis to elucidate the effect of puerarin, the main compound found in the traditional Chinese medicine the herb Radix puerariae, on t-tubule development of murine ES-CMs. Methods: Electron microscope was employed to examine the ultrastructure. The investigation of transverse-tubules (t-tubules was performed by Di-8-ANEPPS staining. Quantitative real-time PCR was utilized to study the transcript level of genes related to t-tubule development. Results: We found that long-term application of puerarin throughout cardiac differentiation improved myofibril array and sarcomeres formation, and significantly facilitated t-tubules development of ES-CMs. The transcript levels of caveolin-3, amphiphysin-2 and junctophinlin-2, which are crucial for the formation and development of t-tubules, were significantly upregulated by puerarin treatment. Furthermore, puerarin repressed the expression of miR-22, which targets to caveolin-3. Conclusion: Our data showed that puerarin facilitates t-tubule development of murine ES-CMs. This might be related to the repression of miR-22 by puerarin and upregulation of Cav3, Bin1 and JP2 transcripts.

  10. High-Efficient Transfection of Human Embryonic Stem Cells by Single-Cell Plating and Starvation.

    Science.gov (United States)

    Liu, Hui; Ren, Caiping; Zhu, Bin; Wang, Lei; Liu, Weidong; Shi, Jia; Lin, Jianxing; Xia, Xiaomeng; Zeng, Fei; Chen, Jiawen; Jiang, Xingjun

    2016-03-15

    Nowadays, the low efficiency of small interfering RNA (siRNA) or plasmid DNA (pDNA) transfection is a critical issue in genetic manipulation of human embryonic stem (hES) cells. Development of an efficient transfection method for delivery of siRNAs and plasmids into hES cells becomes more and more imperative. In this study, we tried to modify the traditional transfection protocol by introducing two crucial processes, single-cell plating and starvation, to increase the transfection efficiency in hES cells. Furthermore, we comparatively examined the transfection efficiency of some commercially available siRNA or pDNA transfection reagents in hES cells. Our results showed that the new developed method markedly enhanced the transfection efficiency without influencing the proliferation and pluripotency of hES cells. Lipofectamine RNAiMAX exhibited much higher siRNA transfection efficiency than the other reagents, and FuGENE HD was identified as the best suitable reagent for efficient pDNA transfection of hES cells among the tested reagents.

  11. Zinc Chloride Transiently Maintains Mouse Embryonic Stem Cell Pluripotency by Activating Stat3 Signaling.

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    Jing Hu

    Full Text Available An improved understanding of the pluripotency maintenance of embryonic stem (ES cells is important for investigations of early embryo development and for cell replacement therapy, but the mechanism behind pluripotency is still incompletely understood. Recent findings show that zinc, an essential trace element in humans, is critically involved in regulating various signaling pathways and genes expression. However, its role in ES cell fate determination remains to be further explored. Here we showed that 2μM zinc chloride (ZnCl2 transiently maintained mouse ES cell pluripotency in vitro. The cultured mouse ES cells remained undifferentiated under 2μM ZnCl2 treatment in leukemia inhibitory factor (LIF withdrawal, retinoic acid (RA or embryoid bodies (EBs differentiation assays. In addition, ZnCl2 increased pluripotency genes expression and inhibited differentiation genes expression. Further mechanistic studies revealed that ZnCl2 transiently activated signal transducers and activators of transcription 3 (Stat3 signaling through promoting Stat3 phosphorylation. Inhibition of Stat3 signaling abrogated the effects of ZnCl2 on mouse ES cell pluripotency. Taken together, this study demonstrated a critical role of zinc in the pluripotency maintenance of mouse ES cells, as well as an important regulator of Stat3 signaling.

  12. Embryonic stem-like cells derived from in vitro produced bovine blastocysts

    Directory of Open Access Journals (Sweden)

    Erika Regina Leal de Freitas

    2011-06-01

    Full Text Available The aim of this work was to study the derivation of bovine embryonic stem-like (ES-like cells from the inner cell mass (ICM of in vitro produced blastocysts. The ICMs were mechanically isolated and six out of seventeen (35% ICMs could attach to a monolayer of murine embryonic fibroblasts (MEF. Ten days after, primary outgrowths were mechanically dissected into several small clumps and transferred to a new MEF layer. Cells were further propagated and passaged by physical dissociation over a 60 days period. The pluripotency of the bovine ES-like cells was confirmed by RT-PCR of Oct-4 and STAT-3 gene markers. The colonies were weakly stained for alkaline phosphatase and the mesoderm and endoderm differentiation gene markers such as GATA-4 and Flk-1, respectively, were not expressed. Embryoid bodies were spontaneously formed at the seventh passage. Results showed that bovine ES-like cells could be obtained and passaged by mechanical procedures from the fresh in vitro produced blastocysts.

  13. Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells.

    Science.gov (United States)

    Bibel, Miriam; Richter, Jens; Lacroix, Emmanuel; Barde, Yves-Alain

    2007-01-01

    A detailed protocol is described allowing the generation of essentially pure populations of glutamatergic neurons from mouse embryonic stem (ES) cells. It is based on the culture of ES cells that are kept undifferentiated by repeated splitting and subsequently amplified as non-adherent cell aggregates. Treatment with retinoic acid causes these ES cells to essentially become neural progenitors with the characteristics of Pax6-positive radial glial cells. As they do in vivo, these progenitors differentiate in glutamatergic pyramidal neurons that form functional synaptic contacts and can be kept in culture for long periods of time. This protocol does not require the use of ES lines expressing resistance or fluorescent markers and can thus be applied in principle to any wild-type or mutant ES line of interest. At least 2 weeks are required from starting ES cell culture until plating progenitors and differentiating neurons establish synaptic transmission within about 10 days.

  14. Embryonic stem cells in science and medicine, part II: law, ethics, and the continuing need for dialogue.

    Science.gov (United States)

    Solomon, Louis M; Brockman-Lee, Sandra A

    2008-03-01

    Just as our first article, "Embryonic Stem Cells in Science and Medicine: An Invitation for Dialogue," in the December 2007 issue of Gender Medicine went to press, two groups of researchers had just announced that adult human somatic cells had been reprogrammed to behave like pluripotent stem cells, and that the reprogrammed cells were able to differentiate into cell types of the 3 germ layers in vitro and in a mouse model. A third group has since done so. Because the reprogrammed cells were not embryonic in origin, the announcements were heralded as "stunning" and "leaps forward," because, it was argued, the ability to generate stem cells, without destroying embryos in the process, would avoid the difficult ethical questions raised by human embryonic stem (hES) cell research. This article addresses the most recent announcements and briefly retraces the relevant history so that we may consider whether the moral, ethical, and social issues do in fact disappear as a result of these new advancements. We conclude that, despite the hoopla, little has changed. If indeed there were ethical issues surrounding hES cell research, they remain-and remain as urgent to address and resolve as they had been previously. Lastly, we argue that the medical and scientific communities continue to do themselves a disservice by failing to create a cohesive governing body to address and make concrete recommendations concerning the moral, ethical, and related social issues affecting their communities.

  15. Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Varga, Nora [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Vereb, Zoltan; Rajnavoelgyi, Eva [Department of Immunology, Medical and Health Science Centre, University of Debrecen, Debrecen (Hungary); Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Apati, Agota, E-mail: apati@kkk.org.hu [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  16. A novel SALL4/OCT4 transcriptional feedback network for pluripotency of embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Jianchang Yang

    Full Text Available BACKGROUND: SALL4 is a member of the SALL gene family that encodes a group of putative developmental transcription factors. Murine Sall4 plays a critical role in maintaining embryonic stem cell (ES cell pluripotency and self-renewal. We have shown that Sall4 activates Oct4 and is a master regulator in murine ES cells. Other SALL gene members, especially Sall1 and Sall3 are expressed in both murine and human ES cells, and deletions of these two genes in mice lead to perinatal death due to developmental defects. To date, little is known about the molecular mechanisms controlling the regulation of expressions of SALL4 or other SALL gene family members. METHODOLOGY/PRINCIPAL FINDINGS: This report describes a novel SALL4/OCT4 regulator feedback loop in ES cells in balancing the proper expression dosage of SALL4 and OCT4 for the maintenance of ESC stem cell properties. While we have observed that a positive feedback relationship is present between SALL4 and OCT4, the strong self-repression of SALL4 seems to be the "break" for this loop. In addition, we have shown that SALL4 can repress the promoters of other SALL family members, such as SALL1 and SALL3, which competes with the activation of these two genes by OCT4. CONCLUSIONS/SIGNIFICANCE: Our findings, when taken together, indicate that SALL4 is a master regulator that controls its own expression and the expression of OCT4. SALL4 and OCT4 work antagonistically to balance the expressions of other SALL gene family members. This novel SALL4/OCT4 transcription regulation feedback loop should provide more insight into the mechanism of governing the "stemness" of ES cells.

  17. 78 FR 25091 - Submission for OMB Review; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To...

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    2013-04-29

    ... for Human Embryonic Stem Cell Line To Be Approved for Use in NIH-Funded Research SUMMARY: Under the... be requested in writing. Proposed Collection: Request for Human Embryonic Stem Cell Line to be... used by applicants to request that human embryonic stem cell lines be approved for use in NIH-funded...

  18. 78 FR 13688 - Proposed Collection; 60-Day Comment Request: Request for Human Embryonic Stem Cell Line To Be...

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    2013-02-28

    ... Human Embryonic Stem Cell Line To Be Approved for Use in NIH Funded Research SUMMARY: In compliance with... Information Collection: The form is used by applicants to request that human embryonic stem cell lines be... within 60 days of the date of this publication. Proposed Collection: Request for Human Embryonic Stem...

  19. EXAMINATION OF THE GERM CELL CHIMERA FORMING POTENTIAL OF MOUSE EMBRYONIC STEM CELLS

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    CÂRSTEA V. B

    2007-01-01

    Full Text Available The aim of this study was to examine the factors, which influence the chimeraforming potential of mouse embryonic stem cells (ES cells. In our work, we examinethe chimera producing ability of R1 and R1/E mouse ES cell lines. We found that thepassage number affects chimera-forming capability of the ES cells. With theincreasing of the passage number, it could be getting less chimera animal, and onlythe R1/E ES cell line derived cells could contribute to the germ cells. At first, wecompared the marker of pluripotency using immunostaining and RT PCR, but wecould not find any difference between the R1 and R1/E cell in this way. Atchromosome analysis, we found, that the number of aneuploid cells, in R1 ES cellline, dramatically increased after 10 passages. We thought that the reason is thatduring the cell division Y chromosome could not arrange correctly between the twonewly derived progeny cells. To prove our conception, we made X and YchromosomeFISH analyses. We found, that the aneuploid R1 and R1/E ES cellscontain only one X and one Y chromosome, so not the loss of Y chromosome causethe problem at the germ cell formation. At last, we made the karyotypeanalysis of R1 and R1/E ES cells at different passages. The karyotype analysisdemonstrated that in the case of R1 ES cell line, the 41 and 42-chromosomecontaining cells hold trisomy. With the increasing of the passages number, thenumber of trisomy containing aneuploid cells increased. The aneuploid ES cells cancontribute to the different tissuses of chimera animals, but cannot form viable germcells.

  20. Human embryonic stem cell registries: value, challenges and opportunities.

    Science.gov (United States)

    Luong, Mai X; Smith, Kelly P; Stein, Gary S

    2008-10-15

    The accelerating pace of human embryonic stem cell (hESC) research has created an urgent need for the development of hESC registries, information repositories intended to gather, organize and disseminate hESC information. Although of enormous value to this evolving field, registries face significant challenges to their development. These challenges include addressing the legal and ethical issues surrounding hESC derivation as well as complex intellectual property concerns. In addition to these issues, registries must develop tools to efficiently gather, validate and present many different types of hESC information from a variety of sources. Given the pace and regulatory complexities of this field, it is important that registries develop cooperative mechanisms to avoid duplication and more efficiently support hESC research. (c) 2008 Wiley-Liss, Inc.

  1. Dynamic heterogeneity and DNA methylation in embryonic stem cells.

    KAUST Repository

    Singer, Zakary S

    2014-07-01

    Cell populations can be strikingly heterogeneous, composed of multiple cellular states, each exhibiting stochastic noise in its gene expression. A major challenge is to disentangle these two types of variability and to understand the dynamic processes and mechanisms that control them. Embryonic stem cells (ESCs) provide an ideal model system to address this issue because they exhibit heterogeneous and dynamic expression of functionally important regulatory factors. We analyzed gene expression in individual ESCs using single-molecule RNA-FISH and quantitative time-lapse movies. These data discriminated stochastic switching between two coherent (correlated) gene expression states and burst-like transcriptional noise. We further showed that the "2i" signaling pathway inhibitors modulate both types of variation. Finally, we found that DNA methylation plays a key role in maintaining these metastable states. Together, these results show how ESC gene expression states and dynamics arise from a combination of intrinsic noise, coherent cellular states, and epigenetic regulation.

  2. Extracellular Matrix and Integrins in Embryonic Stem Cell Differentiation.

    Science.gov (United States)

    Wang, Han; Luo, Xie; Leighton, Jake

    2015-01-01

    Embryonic stem cells (ESCs) are pluripotent cells with great therapeutic potentials. The in vitro differentiation of ESC was designed by recapitulating embryogenesis. Significant progress has been made to improve the in vitro differentiation protocols by toning soluble maintenance factors. However, more robust methods for lineage-specific differentiation and maturation are still under development. Considering the complexity of in vivo embryogenesis environment, extracellular matrix (ECM) cues should be considered besides growth factor cues. ECM proteins bind to cells and act as ligands of integrin receptors on cell surfaces. Here, we summarize the role of the ECM and integrins in the formation of three germ layer progenies. Various ECM-integrin interactions were found, facilitating differentiation toward definitive endoderm, hepatocyte-like cells, pancreatic beta cells, early mesodermal progenitors, cardiomyocytes, neuroectoderm lineages, and epidermal cells, such as keratinocytes and melanocytes. In the future, ECM combinations for the optimal ESC differentiation environment will require substantial study.

  3. GATA-1 directly regulates Nanog in mouse embryonic stem cells

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    Li, Wen-Zhong; Ai, Zhi-Ying [College of Life Sciences, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Wang, Zhi-Wei [School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui 230027 (China); Chen, Lin-Lin [College of Life Sciences, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Guo, Ze-Kun, E-mail: gzknwaf@126.com [College of Veterinary Medicine, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Zhang, Yong, E-mail: zylabnwaf@126.com [College of Veterinary Medicine, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China)

    2015-09-25

    Nanog safeguards pluripotency in mouse embryonic stem cells (mESCs). Insight into the regulation of Nanog is important for a better understanding of the molecular mechanisms that control pluripotency of mESCs. In a silico analysis, we identify four GATA-1 putative binding sites in Nanog proximal promoter. The Nanog promoter activity can be significantly repressed by ectopic expression of GATA-1 evidenced by a promoter reporter assay. Mutation studies reveal that one of the four putative binding sites counts for GATA-1 repressing Nanog promoter activity. Direct binding of GATA-1 on Nanog proximal promoter is confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Our data provide new insights into the expanded regulatory circuitry that coordinates Nanog expression. - Highlights: • The Nanog proximal promoter conceives functional element for GATA-1. • GATA-1 occupies the Nanog proximal promoter in vitro and in vivo. • GATA-1 transcriptionally suppresses Nanog.

  4. Isolation and culture of embryonic stem-like cells from pig nuclear transfer blastocysts of different days.

    Science.gov (United States)

    Tan, Guangyun; Ren, Linzhu; Huang, Yongye; Tang, Xiaochun; Zhou, Yang; Zhou, Yan; Li, Dong; Song, Hongxiao; Ouyang, Hongsheng; Pang, Daxin

    2012-11-01

    This study was conducted to establish pig embryonic stem (ES)-like cell lines from nuclear transfer blastocysts. A green fluorescent protein (GFP)-expressing cell line was used as the source of donor cells injected into the enucleated oocytes. Blastocysts were collected at D5 (the fifth day), D7 (the seventh day) and D9 (the ninth day). Differential staining was used to assay the viability and development of blastocysts from the 3 days. The number of inner cell mass (ICM) cells increased from 1.83 ± 0.8 (D5) to 5.37 ± 1.2 (D7) to 7.56 ± 1.5 (D9). The expression profiles of embryonic stem (ES) cell factors (OCT4, SOX2, KLF4 and c-MYC) correlated best with the undifferentiated ES state and were identified by qPCR. The expression of the four factors was increased from D5 to D7, whereas the expression decreased from D7 to D9. We tried to isolate ES-like cells from these embryos. However, ES-like cells from the D7 blastocysts grew slowly and expressed alkaline phosphatase. The cells from the D9 blastocysts grew rapidly but did not express alkaline phosphatase. ES-like cells were not isolated from the D5 blastocysts. These results show that the cells from the D7 embryos are pluripotent but grow slowly. The cells from the D9 embryos grow rapidly but start to lose pluripotency.

  5. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    Directory of Open Access Journals (Sweden)

    Rajvi H Mehta

    2014-01-01

    Full Text Available The ability to successfully derive human embryonic stem cells (hESC lines from human embryos following in vitro fertilization (IVF opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ′discarded′ or ′spare′ fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. In case a couple does not desire to ′cryopreserve′ their embryos then all the embryos remaining following embryo transfer can be considered ′spare′ or if a couple is no longer in need of the ′cryopreserved′ embryos then these also can be considered as ′spare′. But, the question raised by the ethicists is, "what about ′slightly′ over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to ′discarded′ embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of ′discarding′ embryos. What would be the criteria for discarding embryos and the potential ′use′ of ESC derived from the ′abnormal appearing′ embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.

  6. Genetic selection system allowing monitoring of myofibrillogenesis in living cardiomyocytes derived from mouse embryonic stem cells

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    R Bugorsky

    2009-08-01

    Full Text Available Embryonic stem (ES cell-derived cardiomyocytes recapitulate cardiomyogenesis in vitro and are a potential source of cells for cardiac repair. However, this requires enrichment of mixed populations of differentiating ES cells into cardiomyocytes. Toward this goal, we have generated bicistronic vectors that express both the blasticidin S deaminase (bsd gene and a fusion protein consisting of either myosin light chain (MLC-3f or human a-actinin 2A and enhanced green fluorescent protein (EGFP under the transcriptional control of the a-cardiac myosin heavy chain (a-MHC promoter. Insertion of the DNase I-hypersensitive site (HS-2 element from the b-globin locus control region, which has been shown to reduce transgene silencing in other cell systems, upstream of the transgene promoter enhanced MLC3f-EGFP gene expression levels in mouse ES cell lines. The a-MHC-a- actinin-EGFP, but not the a-MHC-MLC3f-EGFP, construct resulted in the correct incorporation of the newly synthesized fusion protein at the Z-band of the sarcomeres in ES cellderived cardiomyocytes. Exposure of embryoid bodies to blasticidin S selected for a relatively pure population of cardiomyocytes within 3 days. Myofibrillogenesis could be monitored by fluorescence microscopy in living cells due to sarcomeric epitope tagging. Therefore, this genetic system permits the rapid selection of a relatively pure population of developing cardiomyocytes from a heterogeneous population of differentiating ES cells, simultaneously allowing monitoring of early myofibrillogenesis in the selected myocytes.

  7. Profiling TRA-1-81 antigen distribution on a human embryonic stem cell.

    Science.gov (United States)

    Qiu, Dengli; Xiang, Jialing; Li, Zhaoxia; Krishnamoorthy, Aparna; Chen, Liaohai; Wang, Rong

    2008-05-02

    Human embryonic stem (hES) cells hold great promise in regenerative medicine. Although hES cells have unlimited self-renewal potential, they tend to differentiate spontaneously in culture. TRA-1-81 is a biomarker of undifferentiated hES cells. Quantitative characterization of TRA-1-81 expression level in a single cell helps capture the "turn-on" signal and understand the mechanism of early differentiation. Here, we report on our examination of TRA-1-81 distribution and association on a hES cell membrane using an atomic force microscope (AFM). Our results suggest that aggregated distribution of TRA-1-81 antigen is characteristic for undifferentiated hES cells. We also evaluated the TRA-1-81 expression level at approximately 17,800 epitopes and approximately 700 epitopes per cell on an undifferentiated cell and a spontaneously differentiated cell, respectively. The method in this study can be adapted in examining other surface proteins on various cell types, thus providing a general tool for investigating protein distribution and association at the single cell level.

  8. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells.

    Science.gov (United States)

    Zheng, Li-Wei; Linthicum, Logan; DenBesten, Pamela K; Zhang, Yan

    2013-03-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.

  9. Characterization of an Atlantic cod (Gadus morhua embryonic stem cell cDNA library

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    Holen Elisabeth

    2009-05-01

    Full Text Available Abstract Background The Atlantic cod is an ecologically and economically important North Atlantic fish species and also an emerging aquaculture species. To study gene expression in Atlantic cod embryonic stem (ES cells, our goal was to generate and analyze expressed sequence tags (ESTs from an ES cell cDNA library of mRNA consisting of approximately 3,900 ESTs. Results We sequenced 3,935 EST clones using a directional cDNA library made from pooled ES cells harvested at the blastula stage. Quality filtering of these ESTs allowed identification of 2,719 high-quality sequences with an average length of 442 bp containing 368 contigs and 1,276 singletons (1,644 unique sequences. BLASTX searches produced 889 significant (E-value -3 hits, of which 698 (42.5% were annotated with Gene Ontology terms (E-value -6. The number of unknown unique sequences was 946 (57.5%. All the high-quality EST sequences have been deposited in GenBank (GenBank: 2,719 sequences in UniGene library dbEST id: 22,021. Gene discovery and annotations are presented and discussed. Conclusion This set of ESTs represents one of the first attempts to describe mRNA in ES cells from a marine cold-water fish species, and provides a basis for gene expression studies of Atlantic cod ES cells.

  10. Differentiation of neurons from neural precursors generated in floating spheres from embryonic stem cells

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    Forrester Jeff

    2009-09-01

    Full Text Available Abstract Background Neural differentiation of embryonic stem (ES cells is usually achieved by induction of ectoderm in embryoid bodies followed by the enrichment of neuronal progenitors using a variety of factors. Obtaining reproducible percentages of neural cells is difficult and the methods are time consuming. Results Neural progenitors were produced from murine ES cells by a combination of nonadherent conditions and serum starvation. Conversion to neural progenitors was accompanied by downregulation of Oct4 and NANOG and increased expression of nestin. ES cells containing a GFP gene under the control of the Sox1 regulatory regions became fluorescent upon differentiation to neural progenitors, and ES cells with a tau-GFP fusion protein became fluorescent upon further differentiation to neurons. Neurons produced from these cells upregulated mature neuronal markers, or differentiated to glial and oligodendrocyte fates. The neurons gave rise to action potentials that could be recorded after application of fixed currents. Conclusion Neural progenitors were produced from murine ES cells by a novel method that induced neuroectoderm cells by a combination of nonadherent conditions and serum starvation, in contrast to the embryoid body method in which neuroectoderm cells must be selected after formation of all three germ layers.

  11. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells

    Science.gov (United States)

    Zheng, Li-Wei; Linthicum, Logan; DenBesten, Pamela K; Zhang, Yan

    2013-01-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel. PMID:23538640

  12. Brown adipogenesis of mouse embryonic stem cells in alginate microstrands

    Science.gov (United States)

    Unser, Andrea Mannarino

    The ability of brown adipocytes (fat cells) to dissipate energy as heat shows great promise for the treatment of obesity and other metabolic disorders. Employing pluripotent stem cells, with an emphasis on directed differentiation, may overcome many issues currently associated with primary fat cell cultures. However, brown adipocytes are difficult to transplant in vivo due to the instability of fat, in terms of necrosis and neovascularization, once injected. Thus, 3D cell culture systems that have the potential to mimic adipogenic microenvironments are needed, not only to advance brown fat implantation, but also to better understand the role of brown adipocytes in treating obesity. To address this need, we created 3D "Brown-Fat-in-Microstrands" by microfluidic synthesis of alginate hydrogel microstrands that encapsulated cells and directly induced cell differentiation into brown adipocytes, using mouse embryonic stem cells (ESCs) as a model of pluripotent stem cells and brown preadipocytes as a positive control. The effect of hydrogel formation parameters on brown adipogenesis was studied, leading to the establishment of "Brown-Fat-in-Microstrands". Brown adipocyte differentiation within microstrands was confirmed by lipid droplet accumulation, immunocytochemistry and qPCR analysis of gene expression of brown adipocyte marker uncoupling protein 1 (UCP1) in addition to adipocyte marker expression. Compared to a 2D approach, 3D differentiated "Brown-Fat-in-Microstrands" exhibited higher level of brown adipocyte marker expression. The functional analysis of "Brown-Fat-in-Microstrands" was attempted by measuring the mitochondrial activity of ESC-differentiated brown adipocytes in 3D using Seahorse XF24 3 Extracellular Flux Analyzer. The ability to create "Brown-Fat-in-Microstrands" from pluripotent stem cells opens up a new arena to understanding brown adipogenesis and its implications in obesity and metabolic disorders.

  13. ERG is required for the differentiation of embryonic stem cells along the endothelial lineage

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    Le Bras Alexandra

    2009-12-01

    Full Text Available Abstract Background The molecular mechanisms that govern stem cell differentiation along the endothelial lineage remain largely unknown. Ets related gene (ERG has recently been shown to participate in the transcriptional regulation of a number of endothelial specific genes including VE-cadherin (CD144, endoglin, and von Willebrand's Factor (vWF. The specific role of the ETS factor ERG during endothelial differentiation has not been evaluated. Results ERG expression and function were evaluated during the differentiation of embryonic stem cells into embryoid bodies (EB. The results of our study demonstrate that ERG is first expressed in a subpopulation of vascular endothelial growth factor receptor 2 (VEGF-R2 expressing cells that also express VE-cadherin. During ES cell differentiation, ERG expression remains restricted to cells of the endothelial lineage that eventually coalesce into primitive vascular structures within embryoid bodies. ERG also exhibits an endothelial cell (EC-restricted pattern during embryogenesis. To further define the role of ERG during ES cell differentiation, we used a knockdown strategy to inhibit ERG expression. Delivery of three independent shRNA led to 70-85% reductions in ERG expression during ES cell differentiation compared to no change with control shRNA. ERG knockdown was associated with a marked reduction in the number of ECs, the expression of EC-restricted genes, and the formation of vascular structures. Conclusion The ETS factor ERG appears to be a critical regulator of EC differentiation.

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

    Science.gov (United States)

    Plachta, Nicolas; Bibel, Miriam; Tucker, Kerry Lee; Barde, Yves-Alain

    2004-11-01

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

  15. Chromosomal transposition of PiggyBac in mouse embryonic stem cells.

    Science.gov (United States)

    Wang, Wei; Lin, Chengyi; Lu, Dong; Ning, Zeming; Cox, Tony; Melvin, David; Wang, Xiaozhong; Bradley, Allan; Liu, Pentao

    2008-07-08

    Transposon systems are widely used for generating mutations in various model organisms. PiggyBac (PB) has recently been shown to transpose efficiently in the mouse germ line and other mammalian cell lines. To facilitate PB's application in mammalian genetics, we characterized the properties of the PB transposon in mouse embryonic stem (ES) cells. We first measured the transposition efficiencies of PB transposon in mouse embryonic stem cells. We next constructed a PB/SB hybrid transposon to compare PB and Sleeping Beauty (SB) transposon systems and demonstrated that PB transposition was inhibited by DNA methylation. The excision and reintegration rates of a single PB from two independent genomic loci were measured and its ability to mutate genes with gene trap cassettes was tested. We examined PB's integration site distribution in the mouse genome and found that PB transposition exhibited local hopping. The comprehensive information from this study should facilitate further exploration of the potential of PB and SB DNA transposons in mammalian genetics.

  16. Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Jiafei Xi

    Full Text Available In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs we isolated human fetal liver stromal cells (hFLSCs from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days. Basic fibroblast growth factor (bFGF is known to play an important role in promoting self-renewal of human embryonic stem (hES cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2, and transforming growth factor β (TGF-β, thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.

  17. An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

    DEFF Research Database (Denmark)

    Massumi, Mohammad; Pourasgari, Farzaneh; Nalla, Amarnadh

    2016-01-01

    developed an abbreviated five-stage protocol (25-30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems......The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have...... positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux...

  18. Leukemia inhibitory factor (LIF) enhances MAP2 + and HUC/D + neurons and influences neurite extension during differentiation of neural progenitors derived from human embryonic stem cells.

    Science.gov (United States)

    Leukemia Inhibitory Factor (L1F), a member of the Interleukin 6 cytokine family, has a role in differentiation of Human Neural Progenitor (hNP) cells in vitro. hNP cells, derived from Human Embryonic Stem (hES) cells, have an unlimited capacity for self-renewal in monolayer cultu...

  19. Investigation of a redox-sensitive predictive model of mouse embryonic stem cells differentiation using quantitative nuclease protection assays and glutathione redox status

    Science.gov (United States)

    Investigation of a redox-sensitive predictive model of mouse embryonic stem cell differentiation via quantitative nuclease protection assays and glutathione redox status Chandler KJ,Hansen JM, Knudsen T,and Hunter ES 1. U.S. Environmental Protection Agency, Research Triangl...

  20. Src-family tyrosine kinase activities are essential for differentiation of human embryonic stem cells

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    Xiong Zhang

    2014-11-01

    Full Text Available Embryonic stem (ES cells are characterized by pluripotency, defined as the developmental potential to generate cell lineages derived from all three primary germ layers. In the past decade, great progress has been made on the cell culture conditions, transcription factor programs and intracellular signaling pathways that control both murine and human ES cell fates. ES cells of mouse vs. human origin have distinct culture conditions, responding to some tyrosine kinase signaling pathways in opposite ways. Previous work has implicated the Src family of non-receptor protein–tyrosine kinases in mouse ES cell self-renewal and differentiation. Seven members of the Src kinase family are expressed in mouse ES cells, and individual family members appear to play distinct roles in regulating their developmental fate. Both Hck and c-Yes are important in self-renewal, while c-Src activity alone is sufficient to induce differentiation. While these findings implicate Src-family kinase signaling in mouse ES cell renewal and differentiation, the role of this kinase family in human ES cells is largely unknown. Here, we explored Src-family kinase expression patterns and signaling in human ES cells during self-renewal and differentiation. Of the eleven Src-related kinases in the human genome, Fyn, c-Yes, c-Src, Lyn, Lck and Hck were expressed in H1, H7 and H9 hES cells, while Fgr, Blk, Srm, Brk, and Frk transcripts were not detected. Of these, c-Yes, Lyn, and Hck transcript levels remained constant in self-renewing human ES cells vs. differentiated EBs, while c-Src and Fyn showed a modest increase in expression as a function of differentiation. In contrast, Lck expression levels dropped dramatically as a function of EB differentiation. To assess the role of overall Src-family kinase activity in human ES cell differentiation, cultures were treated with inhibitors specific for the Src kinase family. Remarkably, human ES cells maintained in the presence of the potent

  1. Human embryonic stem cells as models for aneuploid chromosomal syndromes.

    Science.gov (United States)

    Biancotti, Juan-Carlos; Narwani, Kavita; Buehler, Nicole; Mandefro, Berhan; Golan-Lev, Tamar; Yanuka, Ofra; Clark, Amander; Hill, David; Benvenisty, Nissim; Lavon, Neta

    2010-09-01

    Syndromes caused by chromosomal aneuploidies are widely recognized genetic disorders in humans and often lead to spontaneous miscarriage. Preimplantation genetic screening is used to detect chromosomal aneuploidies in early embryos. Our aim was to derive aneuploid human embryonic stem cell (hESC) lines that may serve as models for human syndromes caused by aneuploidies. We have established 25 hESC lines from blastocysts diagnosed as aneuploid on day 3 of their in vitro development. The hESC lines exhibited morphology and expressed markers typical of hESCs. They demonstrated long-term proliferation capacity and pluripotent differentiation. Karyotype analysis revealed that two-third of the cell lines carry a normal euploid karyotype, while one-third remained aneuploid throughout the derivation, resulting in eight hESC lines carrying either trisomy 13 (Patau syndrome), 16, 17, 21 (Down syndrome), X (Triple X syndrome), or monosomy X (Turner syndrome). On the basis of the level of single nucleotide polymorphism heterozygosity in the aneuploid chromosomes, we determined whether the aneuploidy originated from meiotic or mitotic chromosomal nondisjunction. Gene expression profiles of the trisomic cell lines suggested that all three chromosomes are actively transcribed. Our analysis allowed us to determine which tissues are most affected by the presence of a third copy of either chromosome 13, 16, 17 or 21 and highlighted the effects of trisomies on embryonic development. The results presented here suggest that aneuploid embryos can serve as an alternative source for either normal euploid or aneuploid hESC lines, which represent an invaluable tool to study developmental aspects of chromosomal abnormalities in humans.

  2. Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Taru Sharma, G., E-mail: gts553@gmail.com [Reproductive Physiology Laboratory, Division of Physiology and Climatology, Indian Veterinary Research Institute, Izatnagar-243 122, Bareilly, U.P. (India); Dubey, Pawan K.; Verma, Om Prakash; Pratheesh, M.D.; Nath, Amar; Sai Kumar, G. [Reproductive Physiology Laboratory, Division of Physiology and Climatology, Indian Veterinary Research Institute, Izatnagar-243 122, Bareilly, U.P. (India)

    2012-08-03

    Graphical abstract: EBs formation, characterization and expression of germinal layers marker genes of in vivo developed teratoma using four different types of extracellular matrices. Highlights: Black-Right-Pointing-Pointer Collagen-IV matrix is found cytocompatible for EBs formation and differentiation. Black-Right-Pointing-Pointer Established 3D microenvironment for ES cells development and differentiation into three germ layers. Black-Right-Pointing-Pointer Collagen-IV may be useful as promising candidate for ES cells based therapeutic applications. -- Abstract: Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it

  3. Mouse embryonic stem cells with a multi-integrase mouse artificial chromosome for transchromosomic mouse generation.

    Science.gov (United States)

    Yoshimura, Yuki; Nakamura, Kazuomi; Endo, Takeshi; Kajitani, Naoyo; Kazuki, Kanako; Kazuki, Yasuhiro; Kugoh, Hiroyuki; Oshimura, Mitsuo; Ohbayashi, Tetsuya

    2015-08-01

    The mouse artificial chromosome (MAC) has several advantages as a gene delivery vector, including stable episomal maintenance of the exogenous genetic material and the ability to carry large and/or multiple gene inserts including their regulatory elements. Previously, a MAC containing multi-integration site (MI-MAC) was generated to facilitate transfer of multiple genes into desired cells. To generate transchromosomic (Tc) mice containing a MI-MAC with genes of interest, the desired genes were inserted into MI-MAC in CHO cells, and then the MI-MAC was transferred to mouse embryonic stem (mES) cells via microcell-mediated chromosome transfer (MMCT). However, the efficiency of MMCT from CHO to mES cells is very low (lines containing a MI-MAC vector to directly insert a gene of interest into the MI-MAC in mES cells via a simple transfection method for Tc mouse generation. The recombination rate of the GFP gene at each attachment site (FRT, PhiC31attP, R4attP, TP901-1attP and Bxb1attP) on MI-MAC was greater than 50% in MI-MAC mES cells. Chimeric mice with high coat colour chimerism were generated from the MI-MAC mES cell lines and germline transmission from the chimera was observed. As an example for the generation of Tc mice with a desired gene by the MI-MAC mES approach, a Tc mouse strain ubiquitously expressing Emerald luciferase was efficiently established. Thus, the findings suggest that this new Tc strategy employing mES cells and a MI-MAC vector is efficient and useful for animal transgenesis.

  4. Quality Assurance in Stem Cell Banking: Emphasis on Embryonic and Induced Pluripotent Stem Cell Banking.

    Science.gov (United States)

    Kallur, Therése; Blomberg, Pontus; Stenfelt, Sonya; Tryggvason, Kristian; Hovatta, Outi

    2017-01-01

    For quality assurance (QA) in stem cell banking, a planned system is needed to ensure that the banked products, stem cells, meet the standards required for research, clinical use, and commercial biotechnological applications. QA is process oriented, avoids, or minimizes unacceptable product defects, and particularly encompasses the management and operational systems of the bank, as well as the ethical and legal frameworks. Quality control (QC ) is product oriented and therefore ensures the stem cells of a bank are what they are expected to be. Testing is for controlling, not assuring, product quality, and is therefore a part of QC , not QA. Like QA, QC is essential for banking cells for quality research and translational application (Schwartz et al., Lancet 379:713-720, 2012). Human embryonic stem cells (hESCs), as cells derived from donated supernumerary embryos from in vitro fertilization (IVF) therapy, are different from other stem cell types in resulting from an embryo that has had two donors . This imposes important ethical and legal constraints on the utility of the cells, which, together with quite specific culture conditions, require special attention in the QA system. Importantly, although the origin and derivation of induced pluripotent stem cells (iPSCs ) differ from that of hESCs, many of the principles of QA for hESC banking are applicable to iPSC banking (Stacey et al., Cell Stem Cell 13:385-388, 2013). Furthermore, despite differences between the legal and regulatory frameworks for hESC and iPSC banking between different countries, the requirements for QA are being harmonized (Stacey et al., Cell Stem Cell 13:385-388, 2013; International Stem Cell Banking Initiative, Stem Cell Rev 5:301-314, 2009).

  5. Transplantation of porcine embryonic stem cells and their derived neuronal progenitors in a spinal cord injury rat model.

    Science.gov (United States)

    Yang, Jenn-Rong; Liao, Chia-Hsin; Pang, Cheng-Yoong; Huang, Lynn Ling-Huei; Chen, Yi-Ling; Shiue, Yow-Ling; Chen, Lih-Ren

    2013-02-01

    The purpose of this study was to investigate therapeutic potential of green fluorescent protein expressing porcine embryonic stem (pES/GFP(+)) cells in A rat model of spinal cord injury (SCI). Undifferentiated pES/GFP(+) cells and their neuronal differentiation derivatives were transplanted into the contused spinal cord of the Long Evans rat, and in situ development of the cells was determined by using a live animal fluorescence optical imaging system every 15 days. After pES/GFP(+) cell transplantation, the behavior functional recovery of the SCI rats was assessed with the Basso, Beattie, and Bresnahan Locomotor Rating Scale (BBB scale), and the growth and differentiation of the grafted pES/GFP(+) cells in the SCI rats were analyzed by immunohistochemical staining. The relative green fluorescent protein expression level was decreased for 3 months after transplantation. The pES/GFP(+)-derived cells positively stained with neural specific antibodies of anti-NFL, anti-MBP, anti-SYP and anti-Tuj 1 were detected at the transplanted position. The SCI rats grafted with the D18 neuronal progenitors showed a significant functional recovery of hindlimbs and exhibited the highest BBB scale score of 15.20 ± 1.43 at week 24. The SCI rats treated with pES/GFP(+)-derived neural progenitors demonstrated a better functional recovery. Transplantation of porcine embryonic stem (pES)-derived D18 neuronal progenitors has treatment potential for SCI, and functional behavior improvement of grafted pES-derived cells in SCI model rats suggests the potential for further application of pES cells in the study of replacement medicine and functionally degenerative pathologies. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  6. Transcriptional profiling of Sox17-positive, endoderm-committed mouse embryonic stem cells

    OpenAIRE

    Nolden, Tobias

    2010-01-01

    The major first step in embryonic development of vertebrates is the formation of three germ layers during the process of gastrulation - the ectoderm, the mesoderm and the endoderm. During gastrulation, embryonic cells of the epiblast loose their pluripotency and adopt different developmental fates. However, the underlying molecular mechanisms have not been fully elucidated. Embryonic stem cells (ESCs) derived from the epiblast largely express the same genes as the pluripotent epiblast cells ...

  7. Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology.

    Science.gov (United States)

    Moralli, Daniela; Monaco, Zoia L

    2015-02-01

    De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.

  8. Embryonic stem cells improve skeletal muscle recovery after extreme atrophy in mice.

    Science.gov (United States)

    Artioli, Guilherme Giannini; De Oliveira Silvestre, João Guilherme; Guilherme, João Paulo Limongi França; Baptista, Igor Luchini; Ramos, Gracielle Vieira; Da Silva, Willian José; Miyabara, Elen Haruka; Moriscot, Anselmo Sigari

    2015-03-01

    We injected embryonic stem cells into mouse tibialis anterior muscles subjected to botulinum toxin injections as a model for reversible neurogenic atrophy. Muscles were exposed to botulinum toxin for 4 weeks and allowed to recover for up to 6 weeks. At the onset of recovery, a single muscle injection of embryonic stem cells was administered. The myofiber cross-sectional area, single twitch force, peak tetanic force, time-to-peak force, and half-relaxation time were determined. Although the stem cell injection did not affect the myofiber cross-sectional area gain in recovering muscles, most functional parameters improved significantly compared with those of recovering muscles that did not receive the stem cell injection. Muscle function recovery was accelerated by embryonic stem cell delivery in this durable neurogenic atrophy model. We conclude that stem cells should be considered a potential therapeutic tool for recovery after extreme skeletal muscle atrophy. © 2014 Wiley Periodicals, Inc.

  9. Gro/TLE enables embryonic stem cell differentiation by repressing pluripotent gene expression

    DEFF Research Database (Denmark)

    Laing, Adam F; Lowell, Sally; Brickman, Joshua M

    2015-01-01

    Gro/TLE proteins (TLE1-4) are a family of transcriptional corepressors acting downstream of multiple signalling pathways. Several TLEs are expressed in a dynamic manner throughout embryonic development and at high levels in embryonic stem cells (ESCs). Here we find that Gro/TLE is not required...

  10. Human embryonic stem cells have enhanced repair of multiple forms of DNA damage

    DEFF Research Database (Denmark)

    Maynard, Scott; Swistowska, Anna Maria; Lee, Jae Wan

    2008-01-01

    Embryonic stem cells need to maintain genomic integrity so that they can retain the ability to differentiate into multiple cell types without propagating DNA errors. Previous studies have suggested that mechanisms of genome surveillance, including DNA repair, are superior in mouse embryonic stem...... cells compared with various differentiated murine cells. Using single-cell gel electrophoresis (comet assay) we found that human embryonic stem cells (BG01, I6) have more efficient repair of different types of DNA damage (generated from H2O2, UV-C, ionizing radiation, or psoralen) than human primary...... fibroblasts (WI-38, hs27) and, with the exception of UV-C damage, HeLa cells. Microarray gene expression analysis showed that mRNA levels of several DNA repair genes are elevated in human embryonic stem cells compared with their differentiated forms (embryoid bodies). These data suggest that genomic...

  11. Restricted intra-embryonic origin of bona fide hematopoietic stem cells in the chicken

    NARCIS (Netherlands)

    Yvernogeau, Laurent; Robin, Catherine

    2017-01-01

    Hematopoietic stem cells (HSCs), which are responsible for blood cell production, are generated during embryonic development. Human and chicken embryos share features that position the chicken as a reliable and accessible alternative model to study developmental hematopoiesis. However, the existence

  12. Femtosecond optical transfection as a tool for genetic manipulation of human embryonic stem cells

    Science.gov (United States)

    Torres-Mapa, M. L.; Gardner, J.; Bradburn, H.; King, J.; Dholakia, K.; Gunn-Moore, F.

    2013-03-01

    We demonstrate the use of femtosecond optical transfection for the genetic manipulation of human embryonic stem cells. Using a system with an SLM combined with a scanning mirror allows poration of both single-cell and colony-formed human embryonic stem cells in a rapid and targeted manner. In this work, we show successful transfection of plasmid DNA tagged with fluorescent reporters into human embryonic stem cells using three doses of focused femtosecond laser. A significant number of transfected cells retained their undifferentiated morphological feature of large nucleus with high nucleus to cytoplasmic ratio, 48h after photoporation. Furthermore, DNA constructs driven by different types of promoters were also successfully transfected into human embryonic stem cells using this technique.

  13. Discovery of pluripotency-associated microRNAs in rabbit preimplantation embryos and embryonic stem-like cells.

    Science.gov (United States)

    Maraghechi, Pouneh; Hiripi, László; Tóth, Gábor; Bontovics, Babett; Bősze, Zsuzsanna; Gócza, Elen

    2013-04-01

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate multiple biological processes. Increasing experimental evidence implies an important regulatory role of miRNAs during embryonic development and in embryonic stem (ES) cell biology. In the current study, we have described and analyzed the expression profile of pluripotency-associated miRNAs in rabbit embryos and ES-like cells. The rabbit specific ocu-miR-302 and ocu-miR-290 clusters, and three homologs of the human C19MC cluster (ocu-miR-512, ocu-miR-520e, and ocu-miR-498) were identified in rabbit preimplantation embryos and ES-like cells. The ocu-miR-302 cluster was highly similar to its human homolog, while ocu-miR-290 revealed a low level of evolutionary conservation with its mouse homologous cluster. The expression of the ocu-miR-302 cluster began at the 3.5 days post-coitum early blastocyst stage and they stayed highly expressed in rabbit ES-like cells. In contrast, a high expression level of the ocu-miR-290 cluster was detected during preimplantation embryonic development, but a low level of expression was found in rabbit ES-like cells. Differential expression of the ocu-miR-302 cluster and ocu-miR-512 miRNA was detected in rabbit trophoblast and embryoblast. We also found that Lefty has two potential target sites in its 3'UTR for ocu-miR-302a and its expression level increased upon ocu-miR-302a inhibition. We suggest that the expression of the ocu-miR-302 cluster is characteristic of the rabbit ES-like cell, while the ocu-miR-290 cluster may play a crucial role during early embryonic development. This study presents the first identification, to our knowledge, of pluripotency-associated miRNAs in rabbit preimplantation embryos and ES-like cells, which can open up new avenues to investigate the regulatory function of ocu-miRNAs in embryonic development and stem cell biology.

  14. Data for whole and mitochondrial proteome of human embryonic stem cells

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    Faezeh Shekari

    2017-08-01

    Full Text Available The data presented here pertain to the research article entitled “Proteome Analysis of Human Embryonic Stem Cell Organelles” (Shekariet al., 2017 [1]. In the present article we endeavour to locate new proteins and pathways in human embryonic stem cells (hESCs by mass spectrometry and bioinformatics analysis. We have analyzed total and mitochondrial proteins extracted from three biological replicates of the hESC H9 cell line according to mass spectrometry proteomics and bioinformatics investigations.

  15. Progressing a human embryonic stem-cell-based regenerative medicine therapy towards the clinic

    OpenAIRE

    Whiting, Paul; Kerby, Julie; Coffey, Peter; da Cruz, Lyndon; McKernan, Ruth

    2015-01-01

    Since the first publication of the derivation of human embryonic stem cells in 1998, there has been hope and expectation that this technology will lead to a wave of regenerative medicine therapies with the potential to revolutionize our approach to managing certain diseases. Despite significant resources in this direction, the path to the clinic for an embryonic stem-cell-based regenerative medicine therapy has not proven straightforward, though in the past few years progress has been made. H...

  16. Functional effect of mouse embryonic stem cell implantation after spinal cord injury

    OpenAIRE

    Lee, Tae-Hoon

    2013-01-01

    We transplanted mouse embryonic stem cells (mESCs) to improve functional loss in a rat model of clip-compression spinal cord injury (SCI). The mouse embryonic stem cells were transplanted to injured cord 7 days after injury. We include minimizing the progression of secondary injury, manipulating the neuroinhibitory environment of the spinal cord, replacing lost tissue with transplanted cells and substantial improvement of motor. A number of potential approaches optimize functional recovery af...

  17. Computational analysis of expression of human embryonic stem cell-associated signatures in tumors

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    Wang Xiaosheng

    2011-10-01

    Full Text Available Abstract Background The cancer stem cell model has been proposed based on the linkage between human embryonic stem cells and human cancer cells. However, the evidences supporting the cancer stem cell model remain to be collected. In this study, we extensively examined the expression of human embryonic stem cell-associated signatures including core genes, transcription factors, pathways and microRNAs in various cancers using the computational biology approach. Results We used the class comparison analysis and survival analysis algorithms to identify differentially expressed genes and their associated transcription factors, pathways and microRNAs among normal vs. tumor or good prognosis vs. poor prognosis phenotypes classes based on numerous human cancer gene expression data. We found that most of the human embryonic stem cell- associated signatures were frequently identified in the analysis, suggesting a strong linkage between human embryonic stem cells and cancer cells. Conclusions The present study revealed the close linkage between the human embryonic stem cell associated gene expression profiles and cancer-associated gene expression profiles, and therefore offered an indirect support for the cancer stem cell theory. However, many interest issues remain to be addressed further.

  18. Differences in chimera formation and germline transmission between E14 and C2J embryonic stem cells in mice.

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    Zhu, Yan; Li, Dun-Gao; Sun, Zhao-Gui; Chen, Xue-Jin; Jiang, Man-Xi

    2014-05-01

    Summary The goal of this project was to determine whether the originating strain of mouse embryonic stem (ES) cells affects the maintenance of their pluripotency under uniform culture conditions. ES cells from two strains of mice, E14 and C2J, were tested. Both ES cell lines were cultured in KOSR + 2i medium and then injected into C57BL/6J blastocysts. Our results demonstrate that this medium could support both E14 and C2J ES cells to keep their pluripotency, though E14 ES cells were found to have a higher chimeric rate than C2J ES cells. However, analysis by backcrossing revealed that C2J and E14 ES cells have the same ability for germline transmission. Our results demonstrate that ES cells derived from E14 and C2J cells have the same capacity for germline transmission when injected into C57BL/6J blastocysts; however, due to the limitation of mixed genetic background between E14 cells and host C57BL/6J embryos, C2J ES cells are preferable to E14 ES cells for use in gene-targeting and should become the cell line of choice for the generation of genetically engineered mutant mouse lines.

  19. Potential applications of keratinocytes derived from human embryonic stem cells.

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    Movahednia, Mohammad M; Kidwai, Fahad K; Jokhun, Doorgesh S; Squier, Christopher A; Toh, Wei Seong; Cao, Tong

    2016-01-01

    Although skin grafting is one of the most advanced cell therapy technique, wide application of skin substitutes is hampered by the difficulty in securing sufficient amount of epidermal substitute. Additionally, in understanding the progression of skin aging and disease, and in screening the cosmetic and pharmaceutical products, there is lack of a satisfactory human skin-specific in vitro model. Recently, human embryonic stem cells (hESCs) have been proposed as an unlimited and reliable cell source to obtain almost all cell types present in the human body. This review focuses on the potential off-the-shelf use of hESC-derived keratinocytes for future clinical applications as well as a powerful in vitro skin model to study skin function and integrity, host-pathogen interactions and disease pathogenesis. Furthermore, we discuss the industrial applications of hESC-derived keratinized multi-layer epithelium which provides a human-like test platform for understanding disease pathogenesis, evaluation of new therapeutic modalities and assessment of the safety and efficacy of skin cosmetics and therapeutics. Overall, we conclude that the hESC-derived keratinocytes have great potential for clinical, research and industrial applications. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Generative models: Human embryonic stem cells and multiple modeling relations.

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    Fagan, Melinda Bonnie

    2016-04-01

    Model organisms are at once scientific models and concrete living things. It is widely assumed by philosophers of science that (1) model organisms function much like other kinds of models, and (2) that insofar as their scientific role is distinctive, it is in virtue of representing a wide range of biological species and providing a basis for generalizations about those targets. This paper uses the case of human embryonic stem cells (hESC) to challenge both assumptions. I first argue that hESC can be considered model organisms, analogous to classic examples such as Escherichia coli and Drosophila melanogaster. I then discuss four contrasts between the epistemic role of hESC in practice, and the assumptions about model organisms noted above. These contrasts motivate an alternative view of model organisms as a network of systems related constructively and developmentally to one another. I conclude by relating this result to other accounts of model organisms in recent philosophy of science. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Selenite benefits embryonic stem cells therapy in Parkinson's disease.

    Science.gov (United States)

    Tian, L-P; Zhang, S; Xu, L; Li, W; Wang, Y; Chen, S-D; Ding, J-Q

    2012-09-01

    Embryonic stem cells (ESC) transplantation is a potential therapeutic approach for Parkinson's disease (PD). However, one of the main challenges to this therapy is the post-transplantation survival of dopaminergic (DA) neurons. In this study, mouse ESC were differentiated into DA neurons by a modified serum free protocol. These ESC-derived neurons were then transplanted into striatum of 6-OHDA lesioned rat. The viability of grafted DA neurons was decreased, accompanied by activated microglia and high levels of proinflammatory factors, such as TNF-α and iNOS, in the graft niche. This suggested that the local neuroinflammation might be involved in the reduced cells viability. Selenite, the source of essential micronutrient selenium, could inhibit NF-κB p65 nuclear translocation and subsequently reduce iNOS, COX-2 and TNF-α expression in LPS-treated BV2 cells in a dose dependant manner. Before the transplantation of ESC-derived DA neurons, 6-OHDA lesioned rats were intraperitoneally injected with selenite. The expression levels of TNF-α and iNOS were decreased by 30% and 50%, respectively, in selenite treated group. The survival of implanted DA neurons and the rotational behavior of transplanted rats were also remarkably improved by selenite treatment. To sum up, selenite might benefit ESCs transplantation therapy in PD through anti-inflammation effects.

  2. Epigenetic stability, adaptability, and reversibility in human embryonic stem cells.

    Science.gov (United States)

    Tompkins, Joshua D; Hall, Christine; Chen, Vincent Chang-yi; Li, Arthur Xuejun; Wu, Xiwei; Hsu, David; Couture, Larry A; Riggs, Arthur D

    2012-07-31

    The stability of human embryonic stem cells (hESCs) is of critical importance for both experimental and clinical applications. We find that as an initial response to altered culture conditions, hESCs change their transcription profile for hundreds of genes and their DNA methylation profiles for several genes outside the core pluripotency network. After adaption to conditions of feeder-free defined and/or xeno-free culture systems, expression and DNA methylation profiles are quite stable for additional passaging. However, upon reversion to the original feeder-based culture conditions, numerous transcription changes are not reversible. Similarly, although the majority of DNA methylation changes are reversible, highlighting the plasticity of DNA methylation, a few are persistent. Collectively, this indicates these cells harbor a memory of culture history. For culture-induced DNA methylation changes, we also note an intriguing correlation: hypomethylation of regions 500-2440 bp upstream of promoters correlates with decreased expression, opposite to that commonly seen at promoter-proximal regions. Lastly, changes in regulation of G-coupled protein receptor pathways provide a partial explanation for many of the unique transcriptional changes observed during hESC adaptation and reverse adaptation.

  3. Gene expression analysis of embryonic stem cells expressing VE-cadherin (CD144 during endothelial differentiation

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    Libermann Towia

    2008-05-01

    Full Text Available Abstract Background Endothelial differentiation occurs during normal vascular development in the developing embryo. This process is recapitulated in the adult when endothelial progenitor cells are generated in the bone marrow and can contribute to vascular repair or angiogenesis at sites of vascular injury or ischemia. The molecular mechanisms of endothelial differentiation remain incompletely understood. Novel approaches are needed to identify the factors that regulate endothelial differentiation. Methods Mouse embryonic stem (ES cells were used to further define the molecular mechanisms of endothelial differentiation. By flow cytometry a population of VEGF-R2 positive cells was identified as early as 2.5 days after differentiation of ES cells, and a subset of VEGF-R2+ cells, that were CD41 positive at 3.5 days. A separate population of VEGF-R2+ stem cells expressing the endothelial-specific marker CD144 (VE-cadherin was also identified at this same time point. Channels lined by VE-cadherin positive cells developed within the embryoid bodies (EBs formed by differentiating ES cells. VE-cadherin and CD41 expressing cells differentiate in close proximity to each other within the EBs, supporting the concept of a common origin for cells of hematopoietic and endothelial lineages. Results Microarray analysis of >45,000 transcripts was performed on RNA obtained from cells expressing VEGF-R2+, CD41+, and CD144+ and VEGF-R2-, CD41-, and CD144-. All microarray experiments were performed in duplicate using RNA obtained from independent experiments, for each subset of cells. Expression profiling confirmed the role of several genes involved in hematopoiesis, and identified several putative genes involved in endothelial differentiation. Conclusion The isolation of CD144+ cells during ES cell differentiation from embryoid bodies provides an excellent model system and method for identifying genes that are expressed during endothelial differentiation and that

  4. Efficient differentiation of embryonic stem cells into mesodermal precursors by BMP, retinoic acid and Notch signalling.

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    Josema Torres

    Full Text Available The ability to direct differentiation of mouse embryonic stem (ES cells into specific lineages not only provides new insights into the pathways that regulate lineage selection but also has translational applications, for example in drug discovery. We set out to develop a method of differentiating ES cells into mesodermal cells at high efficiency without first having to induce embryoid body formation. ES cells were plated on a feeder layer of PA6 cells, which have membrane-associated stromal-derived inducing activity (SDIA, the molecular basis of which is currently unknown. Stimulation of ES/PA6 co-cultures with Bone Morphogenetic Protein 4 (BMP4 both favoured self-renewal of ES cells and induced differentiation into a Desmin and Nestin double positive cell population. Combined stimulation with BMP4 and all-trans Retinoic Acid (RA inhibited self-renewal and resulted in 90% of cells expressing Desmin and Nestin. Quantitative reverse transcription-polymerase chain reaction (qPCR analysis confirmed that the cells were of mesodermal origin and expressed markers of mesenchymal and smooth muscle cells. BMP4 activation of a MAD-homolog (Smad-dependent reporter in undifferentiated ES cells was attenuated by co-stimulation with RA and co-culture with PA6 cells. The Notch ligand Jag1 was expressed in PA6 cells and inhibition of Notch signalling blocked the differentiation inducing activity of PA6 cells. Our data suggest that mesodermal differentiation is regulated by the level of Smad activity as a result of inputs from BMP4, RA and the Notch pathway.

  5. Derivation of rat embryonic stem cells and generation of protease-activated receptor-2 knockout rats.

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    Yamamoto, Satoshi; Nakata, Mitsugu; Sasada, Reiko; Ooshima, Yuki; Yano, Takashi; Shinozawa, Tadahiro; Tsukimi, Yasuhiro; Takeyama, Michiyasu; Matsumoto, Yoshio; Hashimoto, Tadatoshi

    2012-08-01

    One of the remarkable achievements in knockout (KO) rat production reported during the period 2008-2010 is the derivation of authentic embryonic stem (ES) cells from rat blastocysts using a novel culture medium containing glycogen synthase kinase 3 and mitogen-activated protein kinase kinase inhibitors (2i medium). Here, we report gene-targeting technology via homologous recombination in rat ES cells, demonstrating its use through production of a protease-activated receptor-2 gene (Par-2) KO rat. We began by generating germline-competent ES cells from Dark Agouti rats using 2i medium. These ES cells, which differentiate into cardiomyocytes in vitro, can produce chimeras with high ES cell contribution when injected into blastocysts. We then introduced a targeting vector with a neomycin-resistant gene driven by the CAG promoter to disrupt Par-2. After a 7-day drug selection, 489 neomycin-resistant colonies were obtained. Following screening by polymerase chain reaction (PCR) genotyping and quantitative PCR analysis, we confirmed three homologous recombinant clones, resulting in chimeras that transmitted the Par-2 targeted allele to offspring. Par-2 KO rats showed a loss of Par-2 messenger RNA expression in their stomach cells and a lack of PAR-2 mediated smooth muscle relaxation in the aorta as indicated by pharmacological testing. Compared with mice, rats offer many advantages in biomedical research, including a larger body size; consequently, they are widely used in scientific investigation. Thus, the establishment of a gene-targeting technology using rat ES cells will be a valuable tool in human disease model production and drug discovery.

  6. Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells

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    Horiuchi, Rie [Division of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan); Akimoto, Takayuki, E-mail: akimoto@m.u-tokyo.ac.jp [Division of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan); Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Waseda-tsurumaki, Shinjuku, Tokyo 162-0041 (Japan); Hong, Zhang [Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Waseda-tsurumaki, Shinjuku, Tokyo 162-0041 (Japan); Ushida, Takashi [Division of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan)

    2012-08-15

    Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17 Hz. The expression of Nanog was reduced during ES cell differentiation in response to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling. -- Highlights: Black-Right-Pointing-Pointer The expression of Nanog, which is an essential regulator of 'stemness' was reduced during embryonic stem (ES) cell differentiation. Black-Right-Pointing-Pointer Cyclic mechanical strain attenuated the reduction of Nanog expression. Black-Right-Pointing-Pointer Cyclic mechanical strain promoted PI3K-Akt signaling and mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor and an inhibitor of actin polymerization.

  7. Alginate encapsulation technology supports embryonic stem cells differentiation into insulin-producing cells.

    Science.gov (United States)

    Wang, Nan; Adams, Gary; Buttery, Lee; Falcone, Franco H; Stolnik, Snow

    2009-12-01

    This work investigates an application of the alginate encapsulation technology to the differentiation of embryonic stem (ES) cells into insulin-producing cells. It shows that the ES cells can efficiently be encapsulated within the alginate beads, retaining a high level of cell viability. The alginate encapsulation achieves approximately 10-fold increase in the cell density in the culture, in comparison to the two-dimensional conditions, opening a potential benefit of the technology in large-scale cell culture applications. Manipulations of encapsulation conditions, particularly of the initial alginate concentration, allow the control over both the diffusion of molecules into the alginate matrix (e.g. differentiation factors) as well as control over the matrix porosity/flexibility to permit the proliferation and growth of encapsulated ES aggregates within the bead. Post-differentiation analysis confirms the presence of insulin-positive cells, as judged from immunostaining, insulin ELISA and RT-PCR analysis. The functionality of the encapsulated and differentiated cells was confirmed by their insulin production capability, whereby on glucose challenge the insulin production by the cells differentiated within alginate beads was found to be statistically significantly higher than for the cells from conventional two-dimensional differentiation system.

  8. Mineralocorticoid receptor overexpression facilitates differentiation and promotes survival of embryonic stem cell-derived neurons.

    Science.gov (United States)

    Munier, Mathilde; Law, Frédéric; Meduri, Geri; Le Menuet, Damien; Lombès, Marc

    2012-03-01

    Mineralocorticoid receptor (MR), highly expressed in the hippocampus, binds corticosteroid hormones and coordinately participates, with the glucocorticoid receptor, to the control of stress responses, memorization, and behavior. To investigate the impact of MR in neuronal survival, we generated murine embryonic stem (ES) cells that overexpress human MR (hMR) (P1-hMR) and are induced to differentiate into mature neurons. We showed that recombinant MR expression increased throughout differentiation and is 2-fold higher in P1-hMR ES-derived neurons compared with wild-type controls, whereas glucocorticoid receptor expression was unaffected. Although proliferation and early neuronal differentiation were comparable in P1-hMR and wild-type ES cells, MR overexpression was associated with higher late neuronal marker expression (microtubule-associated protein 2 and β-tubulin III). This was accompanied by a shift towards neuron survival with an increased ratio of anti- vs. proapoptotic molecules and 50% decreased caspase 3 activity. Knocking down MR overexpression by small interfering RNA drastically reversed neuroprotective effects with reduced Bcl(2)/Bax ratio and decreased microtubule-associated protein 2 expression. P1-hMR neurons were protected against oxidative stress-induced apoptosis through reduced caspase 3 activation and drastically increased Bcl(2)/Bax ratio and β-tubulin III expression. We demonstrated the involvement of MR in neuronal differentiation and survival and identify MR as an important neuroprotective mediator opening potential pharmacological strategies.

  9. Characterization of Bovine 5′-flanking Region during Differentiation of Mouse Embryonic Stem Cells

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    Hye-Jeong Jang

    2015-12-01

    Full Text Available Embryonic stem cells (ESCs have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (−420/+181 bovine NANOG 5′-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (−420/+181 promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells.

  10. Arrayed mutant haploid embryonic stem cell libraries facilitate phenotype-driven genetic screens.

    Science.gov (United States)

    Liu, Guang; Wang, Xue; Liu, Yufang; Zhang, Meili; Cai, Tao; Shen, Zhirong; Jia, Yuyan; Huang, Yue

    2017-12-15

    Forward genetic screens using mammalian embryonic stem (ES) cells have identified genes required for numerous cellular processes. However, loss-of-function screens are more difficult to conduct in diploid cells because, in most cases, both alleles of a gene must be mutated to exhibit a phenotype. Recently, mammalian haploid ES cell lines were successfully established and applied to several recessive genetic screens. However, all these screens were performed in mixed pools of mutant cells and were mainly based on positive selection. In general, negative screening is not easy to apply to these mixed pools, although quantitative deep sequencing of mutagen insertions can help to identify some 'missing' mutants. Moreover, the interplay between different mutant cells in the mixed pools would interfere with the readout of the screens. Here, we developed a method for rapidly generating arrayed haploid mutant libraries in which the proportion of homozygous mutant clones can reach 85%. After screening thousands of individual mutant clones, we identified a number of novel factors required for the onset of differentiation in ES cells. A negative screen was also conducted to discover mutations conferring cells with increased sensitivity to DNA double-strand breaks induced by the drug doxorubicin. Both of these screens illustrate the value of this system. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Three-dimensional epithelial tissues generated from human embryonic stem cells.

    Science.gov (United States)

    Hewitt, Kyle J; Shamis, Yulia; Carlson, Mark W; Aberdam, Edith; Aberdam, Daniel; Garlick, Jonathan A

    2009-11-01

    The use of pluripotent human embryonic stem (hES) cells for tissue engineering may provide advantages over traditional sources of progenitor cells because of their ability to give rise to multiple cell types and their unlimited expansion potential. We derived cell populations with properties of ectodermal and mesenchymal cells in two-dimensional culture and incorporated these divergent cell populations into three-dimensional (3D) epithelial tissues. When grown in specific media and substrate conditions, two-dimensional cultures were enriched in cells (EDK1) with mesenchymal morphology and surface markers. Cells with a distinct epithelial morphology (HDE1) that expressed cytokeratin 12 and beta-catenin at cell junctions became the predominant cell type when EDK1 were grown on surfaces enriched in keratinocyte-derived extracellular matrix proteins. When these cells were incorporated into the stromal and epithelial tissue compartments of 3D tissues, they generated multilayer epithelia similar to those generated with foreskin-derived epithelium and fibroblasts. Three-dimensional tissues demonstrated stromal cells with morphologic features of mature fibroblasts, type IV collagen deposition in the basement membrane, and a stratified epithelium that expressed cytokeratin 12. By deriving two distinct cell lineages from a common hES cell source to fabricate complex tissues, it is possible to explore environmental cues that will direct hES-derived cells toward optimal tissue form and function.

  12. ESSA1 embryonic stem like cells from gilthead seabream: a new tool to study mesenchymal cell lineage differentiation in fish.

    Science.gov (United States)

    Parameswaran, Vijayakumar; Laizé, Vincent; Gavaia, Paulo J; Leonor Cancela, M

    2012-10-01

    Embryonic stem (ES) cells are a promising tool for generation of transgenic animals and an ideal experimental model for in vitro studies of embryonic cell development, differentiation and gene manipulation. Here we report the development and initial characterization of a pluripotent embryonic stem like cell line, designated as ESSA1, derived from blastula stage embryos of the gilthead seabream (Sparus aurata, L). ESSA1 cells are cultured in Leibovitz's L-15 medium supplemented with 5% fetal bovine serum and, unlike other ES cells, without a feeder layer. They have a round or polygonal morphology, grow exponentially in culture and form dense colonies. ESSA1 cells also exhibit intense alkaline phosphatase activity, normal karyotype and are positive for stage-specific embryonic antigen-1 (SSEA1) and octamer-binding transcription factor 4 (Oct4) markers for up to 30 passages. Upon treatment with all-trans retinoic acid, ESSA1 cells differentiate into neuron-like, oligodendritic, myocyte and melanocyte cells; they can also form embryoid bodies when seeded in bacteriological plates, a characteristic usually associated with pluripotency. The capacity of ESSA1 cells to differentiate into osteoblastic, chondroblastic or osteoclastic cell lineages and to produce a mineralized extracellular matrix in vitro was demonstrated through histochemical techniques and further confirmed by immunocytochemistry using lineage-specific markers. Furthermore, ESSA1 cells can be used to produce chimera, where they contribute to the development of a variety of tissues including the trunk and gut of zebrafish embryos and fry. Thus, ESSA1 cells represent a promising model for investigating bone-lineage cell differentiation in fish and also highlight the potential of piscine stem cell research. Copyright © 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  13. Skeletogenic phenotype of human Marfan embryonic stem cells faithfully phenocopied by patient-specific induced-pluripotent stem cells

    Science.gov (United States)

    Quarto, Natalina; Leonard, Brian; Li, Shuli; Marchand, Melanie; Anderson, Erica; Behr, Barry; Francke, Uta; Reijo-Pera, Renee; Chiao, Eric; Longaker, Michael T.

    2012-01-01

    Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the gene coding for FIBRILLIN-1 (FBN1), an extracellular matrix protein. MFS is inherited as an autosomal dominant trait and displays major manifestations in the ocular, skeletal, and cardiovascular systems. Here we report molecular and phenotypic profiles of skeletogenesis in tissues differentiated from human embryonic stem cells and induced pluripotent stem cells that carry a heritable mutation in FBN1. We demonstrate that, as a biological consequence of the activation of TGF-β signaling, osteogenic differentiation of embryonic stem cells with a FBN1 mutation is inhibited; osteogenesis is rescued by inhibition of TGF-β signaling. In contrast, chondrogenesis is not perturbated and occurs in a TGF-β cell-autonomous fashion. Importantly, skeletal phenotypes observed in human embryonic stem cells carrying the monogenic FBN1 mutation (MFS cells) are faithfully phenocopied by cells differentiated from induced pluripotent-stem cells derived independently from MFS patient fibroblasts. Results indicate a unique phenotype uncovered by examination of mutant pluripotent stem cells and further demonstrate the faithful alignment of phenotypes in differentiated cells obtained from both human embryonic stem cells and induced pluripotent-stem cells, providing complementary and powerful tools to gain further insights into human molecular pathogenesis, especially of MFS. PMID:22178754

  14. Derivation of Stromal (Skeletal, Mesenchymal) Stem-like cells from Human Embryonic Stem Cells

    DEFF Research Database (Denmark)

    Mahmood, Amer; Harkness, Linda; Abdallah, Basem

    2012-01-01

    Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESC) is a pre-requisite for their use in clinical applications. However, there is no standard protocol for differentiating hESC into osteoblastic cells. The aim of this study was to identify the emergence of a human...... stromal (mesenchymal, skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESC in a feeder-free environment using serum replacement and as suspension aggregates (embryoid...... bodies; hEBs). Over a 20 day developmental period, the hEBs demonstrated increasing enrichment for cells expressing hMSC markers: CD29, CD44, CD63, CD56, CD71, CD73, CD105, CD106 and CD166 as revealed by immunohistochemical staining and flow cytometry (FACS) analysis. Ex vivo differentiation of h...

  15. RNA polymerase III transcriptomes in human embryonic stem cells and induced pluripotent stem cells, and relationships with pluripotency transcription factors.

    Science.gov (United States)

    Alla, Ravi K; Cairns, Bradley R

    2014-01-01

    Recent genomic approaches have revealed that the repertoire of RNA Pol III-transcribed genes varies in different human cell types, and that this variation is likely determined by a combination of the chromatin landscape, cell-specific DNA-binding transcription factors, and collaboration with RNA Pol II. Although much is known about this regulation in differentiated human cells, there is presently little understanding of this aspect of the Pol III system in human ES cells. Here, we determine the occupancy profiles of Pol III components in human H1 ES cells, and also induced pluripotent cells, and compare to known profiles of chromatin, transcription factors, and RNA expression. We find a relatively large fraction of the Pol III repertoire occupied in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In ES cells we find clear correlations between Pol III occupancy and active chromatin. Interestingly, we find a highly significant fraction of Pol III-occupied genes with adjacent binding events by pluripotency factors in ES cells, especially NANOG. Notably, in human ES cells we find H3K27me3 adjacent to but not overlapping many active Pol III loci. We observe in all such cases, a peak of H3K4me3 and/or RNA Pol II, between the H3K27me3 and Pol III binding peaks, suggesting that H3K4me3 and Pol II activity may "insulate" Pol III from neighboring repressive H3K27me3. Further, we find iPSCs have a larger Pol III repertoire than their precursors. Finally, the active Pol III genome in iPSCs is not completely reprogrammed to a hESC like state and partially retains the transcriptional repertoire of the precursor. Together, our correlative results are consistent with Pol III binding and activity in human ES cells being enabled by active/permissive chromatin that is shaped in part by the pluripotency network of transcription factors and RNA Pol II activity.

  16. Which bank? A guardian model for regulation of embryonic stem cell research in Australia.

    Science.gov (United States)

    McLennan, A

    2007-08-01

    In late 2005 the Legislation Review: Prohibition of Human Cloning Act 2002 (Cth) and the Research Involving Human Embryos Act 2002 (Cth) recommended the establishment of an Australian stem cell bank. This article aims to address a lack of discussion of issues surrounding stem cell banking by suggesting possible answers to the questions of whether Australia should establish a stem cell bank and what its underlying philosophy and functions should be. Answers are developed through an analysis of regulatory, scientific and intellectual property issues relating to embryonic stem cell research in the United Kingdom, United States and Australia. This includes a detailed analysis of the United Kingdom Stem Cell Bank. It is argued that a "guardian" model stem cell bank should be established in Australia. This bank would aim to promote the maximum public benefit from human embryonic stem cell research by providing careful regulatory oversight and addressing ethical issues, while also facilitating research by addressing practical scientific concerns and intellectual property issues.

  17. Identification of insulin-producing cells derived from embryonic stem cells by zinc-chelating dithizone.

    Science.gov (United States)

    Shiroi, Akira; Yoshikawa, Masahide; Yokota, Hiroshi; Fukui, Hiroshi; Ishizaka, Shigeaki; Tatsumi, Kouko; Takahashi, Yoshiko

    2002-01-01

    Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently identified the emergence of cellular clusters within differentiated ES cell cultures by staining with dithizone (DTZ). DTZ is a zinc-chelating agent known to selectively stain pancreatic beta cells because of their high zinc content. The aim of the present study was to investigate the characteristics of DTZ-stained cellular clusters originating from ES cells. Embryoid bodies (EBs), formed by a 5-day hanging drop culture of ES cells, were allowed to form outgrowths in the culture. The outgrowths were incubated in DTZ solution (final concentration, 100 microg/ml ) for 15 minutes before being examined microscopically. The gene expression of endocrine pancreatic markers was also analyzed by reverse transcriptase-polymerase chain reaction. In addition, insulin production was examined immunohistochemically, and its secretion was examined using enzyme-linked immunosorbent assay. DTZ-stained cellular clusters appeared after approximately 16 days in the EB culture and became more apparent by day 23. They were found to be immunoreactive to insulin and expressed pancreatic-duodenal homeobox 1 (PDX1), proinsulin 1, proinsulin 2, glucagon, pancreatic polypeptide, glucose transporter-2 (GLUT2), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) mRNA. They were also able to secrete detectable amounts of insulin. ES cell-derived DTZ-positive cellular clusters possess characteristics of the endocrine pancreas, including insulin secretion. Further, DTZ staining is a useful method for the identification of differentiated pancreatic islets developed from EBs in vitro.

  18. Control of leucocyte differentiation from embryonic stem cells upon vasculogenesis and confrontation with tumour tissue.

    Science.gov (United States)

    Hannig, Madeleine; Figulla, Hans-Reiner; Sauer, Heinrich; Wartenberg, Maria

    2010-01-01

    Embryonic stem (ES) cells spontaneously differentiate capillary-like structures as well as leucocytes such as monocytes/macrophages, neutrophils, natural killer (NK) cells and cytototoxic T lymphocytes. The interplay between vasculogenesis and leucocyte differentiation as well as the population of tumour tissues with ES cell-derived leucocytes and endothelial cells is, however, not sufficiently specified. In the present study, gene expression of the cell surface markers CD68 and CD14 (expressed on monocytes and macrophages), Mac-1 (CD11b) (expressed on granulocytes, monocytes and NK cells) and CD16 (expressed on neutrophils) was investigated in murine CGR8 ES cells in relation to the endothelial cell markers CD31 and vascular endothelial (VE)-cadherin. Expression of leucocyte markers increased from day 7-8 of cell culture on. Furthermore, addition of macrophage colony-stimulating factor to the cell culture medium resulted in a threefold increase in the number of CD68(+) monocytes/macrophages. Treatment of embryoid bodies with lipopolysaccharide (LPS) up-regulated CD14 thus suggesting functionality of the CD14 LPS receptor. Differentiation of vascular structures positive for CD31 and VE-cadherin preceded leucocyte differentiation by 2 days (i.e. from day 5-6 on) suggesting that vasculogenesis may be a determinant of leucocyte differentiation. Consequently the Flk-1 antagonist SU5416 which inhibits vasculogenesis of ES cells significantly blunted leucocyte differentiation. Confrontation culture of embryoid bodies with multicellular breast tumour spheroids initiated significant increase of leucocyte cell numbers and invasion of leucocytes into the tumour tissue. In summary our data demonstrate that during ES cell differentiation vasculogenesis precedes leucocyte differentiation, and point towards the direction that leucocyte cell invasion into tumour tissue may initiate the pro-inflammatory microenvironment necessary for tumour vascularization.

  19. Searching ChIP-seq genomic islands for combinatorial regulatory codes in mouse embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Chen Gong

    2011-10-01

    Full Text Available Abstract Background To facilitate deciphering underlying transcriptional regulatory circuits in mouse embryonic stem (ES cells, recent ChIP-seq data provided genome-wide binding locations of several key transcription factors (TFs; meanwhile, existing efforts profiled gene expression in ES cells and in their early differentiated state. It has been shown that the gene expression profiles are correlated with the binding of these TFs. However, it remains unclear whether other TFs, referred to as cofactors, participate the gene regulation by collaborating with the ChIP-seq TFs. Results Based on our analyses of the ES gene expression profiles and binding sites of potential cofactors in vicinity of the ChIP-seq TF binding locations, we identified a list of co-binding features that show significantly different characteristics between different gene expression patterns (activated or repressed gene expression in ES cells at a false discovery rate of 10%. Gene classification with a subset of the identified features achieved up to 20% improvement over classification only based on the ChIP-seq TFs. More than 1/3 of reasoned regulatory roles of cofactor candidates involved in these features are supported by existing literatures. Finally, the predicted target genes of the majority candidates present expected expression change in another independent data set, which serves as a supplementary validation of these candidates. Conclusions Our results revealed a list of combinatorial genomic features that are significantly associated with gene expression in ES cells, suggesting potential cofactors of the ChIP-seq TFs for gene regulation.

  20. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

    Directory of Open Access Journals (Sweden)

    Alberto Miranda

    2011-04-01

    Full Text Available Cellular prion protein (PRNP is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs. Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB differentiation in mouse Prnp-null (KO and WT embryonic stem cell (ESC lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5 in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel and SPRN (Shadoo, whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  1. Mechanism of arsenite toxicity in embryonic stem cells.

    Science.gov (United States)

    Beeravolu, Naimisha; McKee, Christina; Chaudhry, G Rasul

    2017-10-01

    Environmental arsenite exposure has been linked to cancer as well as other diseases, presenting an important and serious public health problem. Toxicity of inorganic arsenite (iAs) has been investigated using animal models and cell culture, yet its developmental effects are poorly understood. This study investigated the molecular mechanism of iAs toxicity to ascertain insight into development and differentiation processes using mouse embryonic stem cells (ESCs). The results showed that iAs exposure affected morphology and integrity of ESC colonies as well as inhibited cell growth in a concentration-dependent manner, excluding concentrations <1 μM iAs which stimulated ESC growth. ESCs self-renewal and pluripotency was also affected as evident from the downregulation of transcription circuitry, Oct4, Nanog, Sox2 and Klf4 resulting in non-specific differentiation. ESCs exposed to iAs randomly differentiated into three germ layers, mesoderm, endoderm and ectoderm, as judged by transcriptional expression of Brachyury, Gata4 and FGF2, as well as translational expression of BRACHYURY, GATA4 and TUJ1 respectively. The differentiated cells represented osteogenic, chondrogenic, myogenic and neurogenic lineages as evident from upregulation of Col1, Sox9, Col2, Myog, Notch, Nes and Nef. Although iAs caused slight apoptosis with a concomitant increase in ROS levels, the exposed ESCs had significant Bcl2 expression, which could be involved in the protection against apoptosis. Further analysis revealed upregulation of Jun and P38 in ESCs with an increase in iAs concentration. These observations indicated that iAs stress caused random differentiation of ESCs via JNK/P38 pathways. These findings suggest that iAs exposure may cause teratogenicity during early fetal development. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  2. High throughput sequencing identifies an imprinted gene, Grb10, associated with the pluripotency state in nuclear transfer embryonic stem cells.

    Science.gov (United States)

    Li, Hui; Gao, Shuai; Huang, Hua; Liu, Wenqiang; Huang, Huanwei; Liu, Xiaoyu; Gao, Yawei; Le, Rongrong; Kou, Xiaochen; Zhao, Yanhong; Kou, Zhaohui; Li, Jia; Wang, Hong; Zhang, Yu; Wang, Hailin; Cai, Tao; Sun, Qingyuan; Gao, Shaorong; Han, Zhiming

    2017-07-18

    Somatic cell nuclear transfer and transcription factor mediated reprogramming are two widely used techniques for somatic cell reprogramming. Both fully reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells hold potential for regenerative medicine, and evaluation of the stem cell pluripotency state is crucial for these applications. Previous reports have shown that the Dlk1-Dio3 region is associated with pluripotency in induced pluripotent stem cells and the incomplete somatic cell reprogramming causes abnormally elevated levels of genomic 5-methylcytosine in induced pluripotent stem cells compared to nuclear transfer embryonic stem cells and embryonic stem cells. In this study, we compared pluripotency associated genes Rian and Gtl2 in the Dlk1-Dio3 region in exactly syngeneic nuclear transfer embryonic stem cells and induced pluripotent stem cells with same genomic insertion. We also assessed 5-methylcytosine and 5-hydroxymethylcytosine levels and performed high-throughput sequencing in these cells. Our results showed that Rian and Gtl2 in the Dlk1-Dio3 region related to pluripotency in induced pluripotent stem cells did not correlate with the genes in nuclear transfer embryonic stem cells, and no significant difference in 5-methylcytosine and 5-hydroxymethylcytosine levels were observed between fully and partially reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells. Through syngeneic comparison, our study identifies for the first time that Grb10 is associated with the pluripotency state in nuclear transfer embryonic stem cells.

  3. Efficient differentiation of embryonic stem cells into hepatic cells in vitro using a feeder-free basement membrane substratum.

    Directory of Open Access Journals (Sweden)

    Nobuaki Shiraki

    Full Text Available The endoderm-inducing effect of the mesoderm-derived supportive cell line M15 on embryonic stem (ES cells is partly mediated through the extracellular matrix, of which laminin α5 is a crucial component. Mouse ES or induced pluripotent stem cells cultured on a synthesized basement membrane (sBM substratum, using an HEK293 cell line (rLN10-293 cell stably expressing laminin-511, could differentiate into definitive endoderm and subsequently into pancreatic lineages. In this study, we investigated the differentiation on sBM of mouse and human ES cells into hepatic lineages. The results indicated that the BM components played an important role in supporting the regional-specific differentiation of ES cells into hepatic endoderm. We show here that knockdown of integrin β1 (Itgb1 in ES cells reduced their differentiation into hepatic lineages and that this is mediated through Akt signaling activation. Moreover, under optimal conditions, human ES cells differentiated to express mature hepatocyte markers and secreted high levels of albumin. This novel procedure for inducing hepatic differentiation will be useful for elucidating the molecular mechanisms controlling lineage-specific fates during gut regionalization. It could also represent an attractive approach to providing a surrogate cell source, not only for regenerative medicine, but also for pharmaceutical and toxicologic studies.

  4. Sox2 in Embryonic Stem Cells and Lung Development

    NARCIS (Netherlands)

    C.G. Pardo (Cristina Gontan)

    2009-01-01

    markdownabstract__Abstract__ Sox2 is a fascinating transcription factor with multiple roles during embryonic development. In early embryonic development, Sox2 is one of the key transcription factors in the maintenance of the pluripotent status of the cells of the inner cell mass (ICM). Sox2 is

  5. In vitro generation of motor neuron precursors from mouse embryonic stem cells using mesoporous nanoparticles

    DEFF Research Database (Denmark)

    Garcia-Bennett, Alfonso E; König, Niclas; Abrahamsson, Ninnie

    2014-01-01

    Aim: Stem cell-derived motor neurons (MNs) are utilized to develop replacement strategies for spinal cord disorders. Differentiation of embryonic stem cells into MN precursors involves factors and their repeated administration. We investigated if delivery of factors loaded into mesoporous...

  6. Procedures for Derivation and Characterisation of Human Embryonic Stem Cells from Odense, Denmark

    DEFF Research Database (Denmark)

    Harkness, Linda; Kassem, Moustapha

    2012-01-01

    In 1998, a development occurred in stem cell biology with the fi rst report of the derivation of a human embryonic stem cell (hESC) line. Since then a number of techniques have been used to derive and characterise hESCs. Here, we describe the derivation methods used by our laboratory for isolation...

  7. CXXC Finger Protein 1 Contains Redundant Functional Domains That Support Embryonic Stem Cell Cytosine Methylation, Histone Methylation, and Differentiation▿

    OpenAIRE

    Tate, Courtney M.; Lee, Jeong-Heon; Skalnik, David G.

    2009-01-01

    CXXC finger protein 1 (Cfp1) is a regulator of both cytosine methylation and histone methylation. Murine embryonic stem (ES) cells lacking Cfp1 exhibit a decreased plating efficiency, decreased cytosine methylation, elevated global levels of histone H3-Lys4 trimethylation, and a failure to differentiate in vitro. Remarkably, transfection studies reveal that expression of either the amino half of Cfp1 (amino acids 1 to 367 [Cfp11-367]) or the carboxyl half of Cfp1 (Cfp1361-656) is sufficient t...

  8. DNA Methyltransferase Protein Synthesis Is Reduced in CXXC Finger Protein 1–Deficient Embryonic Stem Cells

    OpenAIRE

    Butler, Jill S; Palam, Lakshmi R.; Tate, Courtney M.; Sanford, Jeremy R; Wek, Ronald C.; Skalnik, David G.

    2009-01-01

    CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is required for embryogenesis. CFP1 is also a component of the Setd1A and Setd1B histone H3K4 methyltransferase complexes. Murine embryonic stem (ES) cells lacking CFP1 fail to differentiate, and exhibit a 70% reduction in global genomic cytosine methylation and a 50% reduction in DNA methyltransferase (DNMT1) protein and activity. This study investigated the underlying mechanism for reduced DNMT1 expression in CFP1-defi...

  9. Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage

    Directory of Open Access Journals (Sweden)

    Rolletschek Alexandra

    2009-06-01

    Full Text Available Abstract Background P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. Results In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells activates transcription of the target genes mdm2, p21, puma and noxa. We observed bi-phasic kinetics for nuclear accumulation of p53 after ionizing radiation. During the first wave of nuclear accumulation, p53 levels were increased and the p53 target genes mdm2, p21 and puma were transcribed. Transcription of noxa correlated with the second wave of nuclear accumulation. Transcriptional activation of p53 target genes resulted in an increased amount of proteins with the exception of p21. While p21 transcripts were efficiently translated in 3T3 cells, we failed to see an increase in p21 protein levels after IR in embryonal stem cells. Conclusion In embryonic stem cells where (anti-proliferative p53 activity is not necessary, or even unfavorable, p53 is retained in the cytoplasm and prevented from activating its target genes. However, if its activity is beneficial or required, p53 is allowed to accumulate in the nucleus and activates its target genes, even in embryonic stem cells.

  10. Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage.

    Science.gov (United States)

    Solozobova, Valeriya; Rolletschek, Alexandra; Blattner, Christine

    2009-06-17

    P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells activates transcription of the target genes mdm2, p21, puma and noxa. We observed bi-phasic kinetics for nuclear accumulation of p53 after ionizing radiation. During the first wave of nuclear accumulation, p53 levels were increased and the p53 target genes mdm2, p21 and puma were transcribed. Transcription of noxa correlated with the second wave of nuclear accumulation. Transcriptional activation of p53 target genes resulted in an increased amount of proteins with the exception of p21. While p21 transcripts were efficiently translated in 3T3 cells, we failed to see an increase in p21 protein levels after IR in embryonal stem cells. In embryonic stem cells where (anti-proliferative) p53 activity is not necessary, or even unfavorable, p53 is retained in the cytoplasm and prevented from activating its target genes. However, if its activity is beneficial or required, p53 is allowed to accumulate in the nucleus and activates its target genes, even in embryonic stem cells.

  11. The TRA-1-60 and TRA-1-81 human pluripotent stem cell markers are expressed on podocalyxin in embryonal carcinoma.

    Science.gov (United States)

    Schopperle, William M; DeWolf, William C

    2007-03-01

    We have previously identified the cell adhesion protein podocalyxin expressed in a human pluripotent stem cell, embryonal carcinoma (EC), which is a malignant germ cell. Podocalyxin is a heavily glycosylated membrane protein with amino acid sequence homology to the hematopoietic stem cell marker CD34. Since the initial discovery of podocalyxin in a cancerous stem cell, numerous new studies have identified podocalyxin in many different human cancers and in embryonic stem cells lines (ES) derived from human embryos. Embryonal carcinoma, as do all human pluripotent stem cells, expresses TRA-1-60 and TRA-1-81 antigens, and although their molecular identities are unknown, they are commonly used as markers of undifferentiated pluripotent human stem cells. We report here that purified podocalyxin from embryonal carcinoma has binding activity with the TRA-1-60 and TRA-1-81 antibodies. Embryonal carcinoma cells treated with retinoic acid undergo differentiation and lose the TRA-1-60/TRA-1-81 markers from their plasma membrane surface. We show that podocalyxin is modified in the retinoic acid-treated cells and has an apparent molecular mass of 170 kDa on protein blots as compared with the apparent 200-kDa molecular weight form of podocalyxin expressed in untreated cells. Furthermore, the modified form of podocalyxin no longer reacts with the TRA-1-60/TRA-1-81 antibodies. Thus, embryonal carcinoma expresses two distinct forms of podocalyxin, and the larger version is a molecular carrier of the human stem cell-defining antigens TRA-1-60 and TRA-1-81.

  12. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

    Energy Technology Data Exchange (ETDEWEB)

    Yue, Xiao-shan [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan); Fujishiro, Masako [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Toyoda, Masashi [Department of Reproductive Biology, National Institute for Child Health and Development, 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535 (Japan); Akaike, Toshihiro [Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan); Ito, Yoshihiro, E-mail: y-ito@riken.jp [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan)

    2010-04-16

    In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.

  13. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E).

    Science.gov (United States)

    Yue, Xiao-shan; Fujishiro, Masako; Toyoda, Masashi; Akaike, Toshihiro; Ito, Yoshihiro

    2010-04-16

    In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells. Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.

  14. Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Kim, Jong Hyun; Kim, Hyung Woo; Cha, Kyoung Je; Han, Jiyou; Jang, Yu Jin; Kim, Dong Sung; Kim, Jong-Hoon

    2016-03-22

    Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells.

  15. High frequency electromagnetic fields (GSM signals) affect gene expression levels in tumor suppressor p53-deficient embryonic stem cells.

    Science.gov (United States)

    Czyz, Jaroslaw; Guan, Kaomei; Zeng, Qinghua; Nikolova, Teodora; Meister, Armin; Schönborn, Frank; Schuderer, Jürgen; Kuster, Niels; Wobus, Anna M

    2004-05-01

    Effects of electromagnetic fields (EMF) simulating exposure to the Global System for Mobile Communications (GSM) signals were studied using pluripotent embryonic stem (ES) cells in vitro. Wild-type ES cells and ES cells deficient for the tumor suppressor p53 were exposed to pulse modulated EMF at 1.71 GHz, lower end of the uplink band of GSM 1800, under standardized and controlled conditions, and transcripts of regulatory genes were analyzed during in vitro differentiation. Two dominant GSM modulation schemes (GSM-217 and GSM-Talk), which generate temporal changes between GSM-Basic (active during talking phases) and GSM-DTX (active during listening phases thus simulating a typical conversation), were applied to the cells at and below the basic safety limits for local exposures as defined for the general public by the International Commission on Nonionizing Radiation Protection (ICNIRP). GSM-217 EMF induced a significant upregulation of mRNA levels of the heat shock protein, hsp70 of p53-deficient ES cells differentiating in vitro, paralleled by a low and transient increase of c-jun, c-myc, and p21 levels in p53-deficient, but not in wild-type cells. No responses were observed in either cell type after EMF exposure to GSM-Talk applied at similar slot-averaged specific absorption rates (SAR), but at lower time-averaged SAR values. Cardiac differentiation and cell cycle characteristics were not affected in embryonic stem and embryonic carcinoma cells after exposure to GSM-217 EMF signals. Our data indicate that the genetic background determines cellular responses to GSM modulated EMF. Bioelectromagnetics 25:296-307, 2004. Copyright 2004 Wiley-Liss, Inc.

  16. Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations

    Directory of Open Access Journals (Sweden)

    Siebler Mario

    2009-08-01

    Full Text Available Abstract Background The present work was performed to investigate the ability of two different embryonic stem (ES cell-derived neural precursor populations to generate functional neuronal networks in vitro. The first ES cell-derived neural precursor population was cultivated as free-floating neural aggregates which are known to form a developmental niche comprising different types of neural cells, including neural precursor cells (NPCs, progenitor cells and even further matured cells. This niche provides by itself a variety of different growth factors and extracellular matrix proteins that influence the proliferation and differentiation of neural precursor and progenitor cells. The second population was cultivated adherently in monolayer cultures to control most stringently the extracellular environment. This population comprises highly homogeneous NPCs which are supposed to represent an attractive way to provide well-defined neuronal progeny. However, the ability of these different ES cell-derived immature neural cell populations to generate functional neuronal networks has not been assessed so far. Results While both precursor populations were shown to differentiate into sufficient quantities of mature NeuN+ neurons that also express GABA or vesicular-glutamate-transporter-2 (vGlut2, only aggregate-derived neuronal populations exhibited a synchronously oscillating network activity 2–4 weeks after initiating the differentiation as detected by the microelectrode array technology. Neurons derived from homogeneous NPCs within monolayer cultures did merely show uncorrelated spiking activity even when differentiated for up to 12 weeks. We demonstrated that these neurons exhibited sparsely ramified neurites and an embryonic vGlut2 distribution suggesting an inhibited terminal neuronal maturation. In comparison, neurons derived from heterogeneous populations within neural aggregates appeared as fully mature with a dense neurite network and punctuated

  17. Chemical and physical factors influencing the dynamics of differentiation in embryonic stem cells.

    Science.gov (United States)

    Jyoti, Saras; Tandon, Simran

    2015-01-01

    Differentiating embryonic stem cells into a specific lineage or cell type is one of the most investigated areas of stem cell research; however it is wrought with hurdles. The differentiation process during embryogenesis is greatly influenced by physiochemical factors. They direct key genes for lineage commitment, which in turn are responsible for patterning into highly organized tissues resulting in organ formation. In this review, we have focused on the influence of physiochemical factors on the differentiation process in embryonic stem cells, which mimics, embryogenesis in vivo.

  18. Efficient femtosecond driven SOX 17 delivery into mouse embryonic stem cells: Differentiation study

    CSIR Research Space (South Africa)

    Thobakgale, Setumo L

    2017-01-01

    Full Text Available Efficient femtosecond laser driven SOX 17 delivery into mouse embryonic stem cells: Differentiation study S.L Thobakgale1,2, S.L Manoto1, S. Ombinda-Lemboumba1, M. Maaza2, P. Mthunzi-Kufa1,2* 1Council for Scientific and Industrial Research (CSIR) National... and definitive endoderm in vitro. Development Growth and Differentiation, 50(7), pp.585–593 7. Berrill, A. et al., 2004. Assessment of stem cell markers during long-term culture of mouse embryonic stem cells. Cytotechnology, 44(1-2), pp.77–91. 8. Matthew J...

  19. The role of nanotechnology in induced pluripotent and embryonic stem cells research.

    Science.gov (United States)

    Chen, Lukui; Qiu, Rong; Li, Lushen

    2014-12-01

    This paper reviews the recent studies on development of nanotechnology in the field of induced pluripotent and embryonic stem cells. Stem cell therapy is a promising therapy that can improve the quality of life for patients with refractory diseases. However, this option is limited by the scarcity of tissues, ethical problem, and tumorigenicity. Nanotechnology is another promising therapy that can be used to mimic the extracellular matrix, label the implanted cells, and also can be applied in the tissue engineering. In this review, we briefly introduce implementation of nanotechnology in induced pluripotent and embryonic stem cells research. Finally, the potential application of nanotechnology in tissue engineering and regenerative medicine is also discussed.

  20. A micro-channel-well system for culture and differentiation of embryonic stem cells on different types of substrate.

    Science.gov (United States)

    Liu, Li; Luo, Chunxiong; Ni, Xiaofeng; Wang, Li; Yamauchi, Kaori; Nomura, Shinichi M; Nakatsuji, Norio; Chen, Yong

    2010-06-01

    We have developed a combined micro-channel and micro-well system for easy cell loading, culture and post-culture operation on a chip. To demonstrate the reliability of the system, on chip cell culture and differentiation were performed with different types of substrates made of culture dish, glass cover slide and polydimethylsiloaxe (PDMS). As expected, mouse embryo fibroblasts (MEF) showed different adhesion and growth rate on different substrates. When embryonic stem (ES) cells were co-cultured with MEFs, the formation of ES colonies is efficient on both glass and Petri dish, although PDMS could also be used. Finally, ES cell differentiation with neuron growth factors was performed on different substrates, showing clear advantages of using culture Petri dish over both glass and PDMS.

  1. Cooperative transcription activation by Nurr1 and Pitx3 induces embryonic stem cell maturation to the midbrain dopamine neuron phenotype

    DEFF Research Database (Denmark)

    Martinat, Cecile; Bacci, Jean-Jacques; Leete, Thomas

    2006-01-01

    Midbrain dopamine (DA) neurons play a central role in the regulation of voluntary movement, and their degeneration is associated with Parkinson's disease. Cell replacement therapies, and in particular embryonic stem (ES) cell-derived DA neurons, offer a potential therapeutic venue for Parkinson......'s disease. We sought to identify genes that can potentiate maturation of ES cell cultures to the midbrain DA neuron phenotype. A number of transcription factors have been implicated in the development of midbrain DA neurons by expression analyses and loss-of-function knockout mouse studies, including Nurr1......, Pitx3, Lmx1b, Engrailed-1, and Engrailed-2. However, none of these factors appear sufficient alone to induce the mature midbrain DA neuron phenotype in ES cell cultures in vitro, suggesting a more complex regulatory network. Here we show that Nurr1 and Pitx3 cooperatively promote terminal maturation...

  2. Enhancement of polysialic acid expression improves function of embryonic stem-derived dopamine neuron grafts in Parkinsonian mice.

    Science.gov (United States)

    Battista, Daniela; Ganat, Yosif; El Maarouf, Abderrahman; Studer, Lorenz; Rutishauser, Urs

    2014-01-01

    There has been considerable progress in obtaining engraftable embryonic stem (ES) cell-derived midbrain dopamine neurons for cell replacement therapy in models of Parkinson's disease; however, limited integration and striatal reinnervation of ES-derived grafts remain a major challenge for future clinical translation. In this paper, we show that enhanced expression of polysialic acid results in improved graft efficiency in correcting behavioral deficits in Parkinsonian mice. This result is accompanied by two potentially relevant cellular changes: greater survival of transplanted ES-derived dopamine neurons and robust sprouting of tyrosine hydroxylase-positive processes into host tissue. Because the procedures used to enhance polysialic acid are easily translated to other cell types and species, this approach may represent a general strategy to improve graft integration in cell-based therapies.

  3. The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

    Science.gov (United States)

    Araki, Ryoko; Mizutani, Eiji; Hoki, Yuko; Sunayama, Misato; Wakayama, Sayaka; Nagatomo, Hiroaki; Kasama, Yasuji; Nakamura, Miki; Wakayama, Teruhiko; Abe, Masumi

    2017-05-01

    Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196. © 2017 AlphaMed Press.

  4. Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm

    DEFF Research Database (Denmark)

    Rasmussen, Camilla Holzmann; Petersen, Dorthe Roenn; Møller, Jonas Bech

    2015-01-01

    Human embryonic stem cells have the ability to generate all cell types in the body and can potentially provide an unlimited source of cells for cell replacement therapy to treat degenerative diseases such as diabetes. Current differentiation protocols of human embryonic stem cells towards insulin...... embryonic stem cells to the definitive endoderm lineage. The percentage of definitive endoderm cells after differentiation on collagen I and fibronectin was >85% and 65%, respectively. The cells on collagen I substrates displayed different morphology and gene expression during differentiation as assessed...... and consistent differentiation of stem cells to definitive endoderm. The results shed light on the importance of extracellular matrix proteins for differentiation and also points to a cost effective and easy method to improve differentiation....

  5. Regulation of APC/C (Cdh1) ubiquitin ligase in differentiation of human embryonic stem cells.

    Science.gov (United States)

    Bar-On, Ortal; Shapira, Ma'anit; Skorecki, Karl; Hershko, Avram; Hershko, Dan D

    2010-05-15

    We have recently shown that Skp2 levels are high in undifferentiated human embryonic stem cells, but decline rapidly following induction of differentiation, thereby leading to accumulation of p27. Changes in Skp2 levels were found to be caused mainly by its rate of degradation. Here we show that the activity of APC/C (Cdh1), the ubiquitin ligase that targets Skp2 for degradation, increases markedly during the differentiation process of human embryonic stem cells. APC/C (Cdh1) is present but inactive in undifferentiated embryonic stem cells and becomes active in the differentiated state. The rise in APC/C (Cdh1) activity with differentiation appears to be due, at least in part, to a dramatic decline in the levels of its inhibitor Emi1. In addition, protein kinase activity also appears to contribute to the suppression of APC/C (Cdh1) activity in undifferentiated stem cells, possibly by inhibitory phosphorylation of Cdh1.

  6. Improvement of mouse cloning using nuclear transfer-derived embryonic stem cells and/or histone deacetylase inhibitor.

    Science.gov (United States)

    Wakayama, Sayaka; Wakayama, Teruhiko

    2010-01-01

    Nuclear transfer-derived ES (ntES) cell lines can be established from somatic cell nuclei with a relatively high success rate. Although ntES cells have been shown to be equivalent to ES cells, there are ethical objections concerning human cells, such as the use of fresh oocyte donation from young healthy woman. In contrast, the use of induced pluripotent stem (iPS) cells for cloning poses few ethical problems and is a relatively easy technique compared with nuclear transfer. Therefore, although there are several reports proposing the use of ntES cells as a model of regenerative medicine, the use of these cells in preliminary medical research is waning. However, in theory, 5 to 10 donor cells can establish one ntES cell line and, once established, these cells will propagate indefinitely. These cells can be used to generate cloned animals from ntES cell lines using a second round of NT. Even in infertile and "unclonable" strains of mice, we can generate offspring from somatic cells by combining cloning with ntES technology. Moreover, cloned offspring can be generated potentially even from the nuclei of dead bodies or freeze-dried cells via ntES cells, such as from an extinct frozen animal. Currently, only the ntES technology is available for this purpose, because all other techniques, including iPS cell derivation, require significant numbers of living donor cells. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.

  7. Polarized Secretion of PEDF from Human Embryonic Stem Cell–Derived RPE Promotes Retinal Progenitor Cell Survival

    Science.gov (United States)

    Zhu, Danhong; Deng, Xuemei; Spee, Christine; Sonoda, Shozo; Hsieh, Chih-Lin; Barron, Ernesto; Pera, Martin

    2011-01-01

    Purpose. Human embryonic stem cell–derived RPE (hES-RPE) transplantation is a promising therapy for atrophic age-related macular degeneration (AMD); however, future therapeutic approaches may consider co-transplantation of hES-RPE with retinal progenitor cells (RPCs) as a replacement source for lost photoreceptors. The purpose of this study was to determine the effect of polarization of hES-RPE monolayers on their ability to promote survival of RPCs. Methods. The hES-3 cell line was used for derivation of RPE. Polarization of hES-RPE was achieved by prolonged growth on permeable inserts. RPCs were isolated from 16- to 18-week-gestation human fetal eyes. ELISA was performed to measure pigment epithelium–derived factor (PEDF) levels from conditioned media. Results. Pigmented RPE-like cells appeared as early as 4 weeks in culture and were subcultured at 8 weeks. Differentiated hES-RPE had a normal chromosomal karyotype. Phenotypically polarized hES-RPE cells showed expression of RPE-specific genes. Polarized hES-RPE showed prominent expression of PEDF in apical cytoplasm and a marked increase in secretion of PEDF into the medium compared with nonpolarized culture. RPCs grown in the presence of supernatants from polarized hES-RPE showed enhanced survival, which was ablated by the presence of anti-PEDF antibody. Conclusions. hES-3 cells can be differentiated into functionally polarized hES-RPE cells that exhibit characteristics similar to those of native RPE. On polarization, hES-RPE cells secrete high levels of PEDF that can support RPC survival. These experiments suggest that polarization of hES-RPE would be an important feature for promotion of RPC survival in future cell therapy for atrophic AMD. PMID:21087957

  8. Prospects and Ethical Concerns of Embryonic Stem Cells Research-A Review

    Directory of Open Access Journals (Sweden)

    Jayanti Tokas and P. D. Mathur

    2011-12-01

    Full Text Available Stem cell research has appeared as a silver lining of hope over the dark cloud of some untreatable diseases like cancer and certain neurological disorders. Embryonic stem cells, the tabula rasa, holds much promise in this regard owing to its totipotency, howbeit, it has whirled a severe tempest all over the world on the point of humanity. The present review article includes the chronology of stem cell research with special reference to the techniques that were evolved in due course of research, the controversy over the application of embryonic stem cells for therapeutics and present status of stem cell research under Indian context. India is being increasingly alluring the foreign companies to invest in this project since a huge prospect in stem cell marketing business is foreseen in this country. [Vet. World 2011; 4(6.000: 281-286

  9. YAP/TAZ enhance mammalian embryonic neural stem cell characteristics in a Tead-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Han, Dasol; Byun, Sung-Hyun; Park, Soojeong; Kim, Juwan; Kim, Inhee; Ha, Soobong; Kwon, Mookwang; Yoon, Keejung, E-mail: keejung@skku.edu

    2015-02-27

    Mammalian brain development is regulated by multiple signaling pathways controlling cell proliferation, migration and differentiation. Here we show that YAP/TAZ enhance embryonic neural stem cell characteristics in a cell autonomous fashion using diverse experimental approaches. Introduction of retroviral vectors expressing YAP or TAZ into the mouse embryonic brain induced cell localization in the ventricular zone (VZ), which is the embryonic neural stem cell niche. This change in cell distribution in the cortical layer is due to the increased stemness of infected cells; YAP-expressing cells were colabeled with Sox2, a neural stem cell marker, and YAP/TAZ increased the frequency and size of neurospheres, indicating enhanced self-renewal- and proliferative ability of neural stem cells. These effects appear to be TEA domain family transcription factor (Tead)–dependent; a Tead binding-defective YAP mutant lost the ability to promote neural stem cell characteristics. Consistently, in utero gene transfer of a constitutively active form of Tead2 (Tead2-VP16) recapitulated all the features of YAP/TAZ overexpression, and dominant negative Tead2-EnR resulted in marked cell exit from the VZ toward outer cortical layers. Taken together, these results indicate that the Tead-dependent YAP/TAZ signaling pathway plays important roles in neural stem cell maintenance by enhancing stemness of neural stem cells during mammalian brain development. - Highlights: • Roles of YAP and Tead in vivo during mammalian brain development are clarified. • Expression of YAP promotes embryonic neural stem cell characteristics in vivo in a cell autonomous fashion. • Enhancement of neural stem cell characteristics by YAP depends on Tead. • Transcriptionally active form of Tead alone can recapitulate the effects of YAP. • Transcriptionally repressive form of Tead severely reduces stem cell characteristics.

  10. Evaluation of 309 environmental chemicals using a mouse embryonic stem cell adherent cell differentiation and cytotoxicity assay.

    Science.gov (United States)

    Chandler, Kelly J; Barrier, Marianne; Jeffay, Susan; Nichols, Harriette P; Kleinstreuer, Nicole C; Singh, Amar V; Reif, David M; Sipes, Nisha S; Judson, Richard S; Dix, David J; Kavlock, Robert; Hunter, Edward S; Knudsen, Thomas B

    2011-01-01

    The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7) and cytotoxicity (DRAQ5™/Sapphire700™) were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC₅₀) values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500) revealed significant associations for a subset of chemicals (26) that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A) were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation.

  11. Evaluation of 309 environmental chemicals using a mouse embryonic stem cell adherent cell differentiation and cytotoxicity assay.

    Directory of Open Access Journals (Sweden)

    Kelly J Chandler

    Full Text Available The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7 and cytotoxicity (DRAQ5™/Sapphire700™ were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC₅₀ values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500 revealed significant associations for a subset of chemicals (26 that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation.

  12. Embryonic Stem Cell-Like Subpopulations in Venous Malformation

    Directory of Open Access Journals (Sweden)

    Elysia M. S. Tan

    2017-10-01

    Full Text Available BackgroundVenous malformation (VM consists of a network of ectatic anomalous thin-walled venous channels. A role for an activating TIE2 mutation in the development of the dilated luminal vessels in VM, and its proposed involvement of embryonic stem cells (ESCs, led us to investigate the expression of ESC markers in subcutaneous VM (SCVM and intramuscular VM (IMVM.MethodsFormalin-fixed paraffin-embedded sections of SCVM from seven patients and IMVM samples from seven patients were analyzed for the expression of Nanog, pSTAT3, OCT4, SOX2, SALL4, and CD44, using 3,3′-diaminobenzidine (DAB immunohistochemical (IHC staining. All these samples did not express lymphatic marker D2-40. NanoString mRNA analysis and RT-PCR were performed on snap-frozen samples of SCVM (n = 3 and IMVM (n = 3 from the respective original cohorts of patients included in DAB IHC staining. To confirm co-expression of two proteins, immunofluorescent (IF IHC staining on two representative samples of IMVM and SCVM samples from the original cohorts of patients included for DAB IHC staining was performed.ResultsDAB IHC staining demonstrated expression of all of the above ESC markers in both SCVM and IMVM samples. IF IHC staining showed that these markers were localized to the endothelium within these lesions and that Nanog, pSTAT3, SOX2, and CD44 were also expressed by cells outside of the endothelium. NanoString mRNA analysis confirmed transcription activation of pSTAT3, OCT4, and CD44. RT-qPCR confirmed transcription activation of Nanog, SOX2, and SALL4.ConclusionOur findings support the presence of two ESC-like subpopulations, one within and one outside of the endothelium, of both SCVM and IMVM. Given that the endothelial ESC-like subpopulation expresses the more primitive marker, OCT4, it is exciting to speculate that they give rise to the non-endothelial subpopulation.

  13. Monitoring embryonic stem cell transplantation into rat corpus cavernosum using optical imaging system

    Energy Technology Data Exchange (ETDEWEB)

    Min, Jung Joon; Moon, Sung Min; Le, Uyenchi N.; Park, Kwang Sung; Lee, Hyun Suk; Song, Ho Cheon; Bom, Hee Seung [Chonnam National University Medical School, Gwangju (Korea, Republic of); Han, Ha Jae [Chonnam National University College of Veterinary Medicine, Gwangju (Korea, Republic of)

    2005-07-01

    The conventional method for the analysis of stem cell transplantation depends on postmortem histology. Here, we have sought to demonstrate the feasibility of a longitudinal monitoring of transplanted cell survival in living animals, by employing optical imaging techniques. Mouse embryonic stem cells (ESC) were obtained from American Type Culture Collection (ES-E14TG2a). Mouse ES cells were cultured in the DMEM (Gibco-BRL, Gaithersburg, MD) supplemented with 3.7 g/L sodium bicarbonate, 1 % penicillin and streptomycin, 1.7 mM L-glutamine, 0.1mM {beta}-mercaptoethanol 5 ng/mL mouse leukemia inhibitory factor (LIF), and 15% fetal bovine serum (FBS) with or without a feeder layer and cultured for five days in standard medium plus LIF. ESCs were then transfected (MOI=100) overnight with Ad-CMV-Fluc. Our experimental Sprague-Dawley rats (n=7) were given with different numbers of ESCs 6) expressing Fluc into corpus cavernosum. In cell cultures, firefly luciferase activity correlated linearly with cell numbers from 10 5 to 5x10 6 (r2=0.95). In living animal imaging, imaging signal activity correlated linearly with cell numbers injected from 10 5 to 5x10 6 at each time point (r2=0.62 {approx} 0.98), In all three groups of rats, imaging signal was detected in rat genital area from the 2nd day to the 47th day after cellular injection. Adenovirus mediated transient expression of firefly luciferase reporter gene in ESCs was feasible to monitor cell survival over a month after transplantation. The locations, magnitude, and survival duration of the ESCs were noninvasively monitored with a bioluminescence optical imaging system.

  14. Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions

    DEFF Research Database (Denmark)

    Morgani, Sophie M; Canham, Maurice A; Nichols, Jennifer

    2013-01-01

    Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought...... not directly support Nanog-positive epiblast-like ESCs. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants....... to represent an embryonically restricted ground state. However, we observed heterogeneous expression of the extraembryonic endoderm marker Hex in 2i-cultured embryos, suggesting that 2i blocked development prior to epiblast commitment. Similarly, 2i ESC cultures were heterogeneous and contained a Hex...

  15. Embryonic stem cells and prospects for their use in regenerative medicine approaches to motor neurone disease.

    Science.gov (United States)

    Christou, Y A; Moore, H D; Shaw, P J; Monk, P N

    2007-10-01

    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.

  16. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal

    Directory of Open Access Journals (Sweden)

    Macmaster Suzanne

    2002-06-01

    Full Text Available Abstract Background Non-invasive autofluorescent reporters have revolutionized lineage labeling in an array of different organisms. In recent years green fluorescent protein (GFP from the bioluminescent jellyfish Aequoria Victoria has gained popularity in mouse transgenic and gene targeting regimes 1. It offers several advantages over conventional gene-based reporters, such as lacZ and alkaline phosphatase, in that its visualization does not require a chromogenic substrate and can be realized in vivo. We have previously demonstrated the utility and developmental neutrality of enhanced green fluorescent protein (EGFP in embryonic stem (ES cells and mice 2. Results In this study we have used embryonic stem (ES cell-mediated transgenesis to test the enhanced cyan fluorescent protein (ECFP and enhanced yellow fluorescent protein (EYFP, two mutant and spectrally distinct color variants of wild type (wt GFP. We have also tested DsRed1, the novel red fluorescent protein reporter recently cloned from the Discostoma coral by virtue of its homology to GFP. To this end, we have established lines of ES cells together with viable and fertile mice having widespread expression of either the ECFP or EYFP GFP-variant reporters. However, we were unable to generate equivalent DsRed1 lines, suggesting that DsRed1 is not developmentally neutral or that transgene expression cannot be sustained constitutively. Balanced (diploid diploid and polarized (tetraploid diploid chimeras comprising combinations of the ECFP and EYFP ES cells and/or embryos, demonstrate that populations of cells expressing each individual reporter can be distinguished within a single animal. Conclusions GFP variant reporters are unique in allowing non-invasive multi-spectral visualization in live samples. The ECFP and EYFP-expressing transgenic ES cells and mice that we have generated provide sources of cells and tissues for combinatorial, double-tagged recombination experiments, chimeras or

  17. Rotary orbital suspension culture of embryonic stem cell-derived neural stem/progenitor cells: impact of hydrodynamic culture on aggregate yield, morphology and cell phenotype.

    Science.gov (United States)

    Laundos, Tiago L; Silva, Joana; Assunção, Marisa; Quelhas, Pedro; Monteiro, Cátia; Oliveira, Carla; Oliveira, Maria J; Pêgo, Ana P; Amaral, Isabel F

    2017-08-01

    Embryonic stem (ES)-derived neural stem/progenitor cells (ES-NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single-cell suspensions of ES-NSPCs, to obtain homogeneously-sized ES-NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES-NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller-sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES-NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N-cadherin and β-catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase-2 (MMP-2) activity and percentage of neuronal cells. In line with the increased MMP-2 activity levels found, hydrodynamically-cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single-cell suspensions of ES-NSPCs, besides allowing the formation of homogeneously-sized neurospheres, promoted ES-NSPC differentiation and outward migration, possibly by influencing the expression of cell-cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly-sized ES-NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or

  18. A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Mummery, C.L.; Krijgsveld, J.; Heck, A.

    2008-01-01

    The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological

  19. Propagation of Human Embryonic Stem Cells on Human Amniotic Fluid Cells as Feeder Cells in Xeno-Free Culture Conditions.

    Science.gov (United States)

    Jung, Juwon; Baek, Jin Ah; Seol, Hye Won; Choi, Young Min

    2016-03-01

    Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feederlayers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KOSR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xenofree conditions for clinical grade hESCs culture will be useful data in future clinical studies.

  20. Contested embryonic culture in Japan--public discussion, and human embryonic stem cell research in an aging welfare society.

    Science.gov (United States)

    Sleeboom-Faulkner, Margaret

    2010-01-01

    This article explores the reasons for the lack of a broad discussion on bioethical regulation of human embryonic stem cell research (hESR) in Japan and asks why scientists experience difficulties accessing resources for hESR despite the acclaimed indifference of dominant Japanese culture to embryo research. The article shows how various social actors express their views on the embryo and oocyte donation in terms of dominant Japanese culture, foiled against what is regarded as Western culture. Second, it shows how the lack of concern with hESR should be understood in the context of public health policies and communications and bioethics decision making in Japan. Finally, it interprets the meaning of the embryo in the context of Japan as an aging modern welfare society, explaining how policymakers have come to emphasize the urgency of infertility problems over issues around abortion and embryonic life.

  1. Comparison of the glycosphingolipids of human-induced pluripotent stem cells and human embryonic stem cells.

    Science.gov (United States)

    Säljö, Karin; Barone, Angela; Vizlin-Hodzic, Dzeneta; Johansson, Bengt R; Breimer, Michael E; Funa, Keiko; Teneberg, Susann

    2017-04-01

    High expectations are held for human-induced pluripotent stem cells (hiPSC) since they are established from autologous tissues thus overcoming the risk of allogeneic immune rejection when used in regenerative medicine. However, little is known regarding the cell-surface carbohydrate antigen profile of hiPSC compared with human embryonic stem cells (hESC). Here, glycosphingolipids were isolated from an adipocyte-derived hiPSC line, and hiPSC and hESC glycosphingolipids were compared by concurrent characterization by binding assays with carbohydrate-recognizing ligands and mass spectrometry. A high similarity between the nonacid glycosphingolipids of hiPSC and hESC was found. The nonacid glycosphingolipids P1 pentaosylceramide, x2 pentaosylceramide and H type 1 heptaosylceramide, not previously described in human pluripotent stem cells (hPSC), were characterized in both hiPSC and hESC. The composition of acid glycosphingolipids differed, with increased levels of GM3 ganglioside, and reduced levels of GD1a/GD1b in hiPSC when compared with hESC. In addition, the hESC glycosphingolipids sulf-globopentaosylceramide and sialyl-globotetraosylceramide were lacking in hiPSC. Neural stem cells differentiating from hiPSC had a reduced expression of sialyl-lactotetra, whereas expression of the GD1a ganglioside was significantly increased. Thus, while sialyl-lactotetra is a marker of undifferentiated hPSC, GD1a is a novel marker of neural differentiation. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Trace metals alter DNA repair and histone modification pathways concurrently in mouse embryonic stem cells.

    Science.gov (United States)

    Gadhia, Sanket R; Calabro, Anthony R; Barile, Frank A

    2012-07-20

    Exposure to metals alters gene expression, changes transcription rates or interferes with DNA repair mechanisms. We tested a hypothesis to determine whether in vitro acute metal exposure, with or without recovery, alters epigenetic pathways in mouse embryonic stem (mES) cells. We measured cell viability, total and histone protein production, changes in gene expression for differentiation and DNA repair, and histone lysine mono-methylation (H3K27me1), in differentiated cells. Confluent differentiated cultures of mES cells were exposed to arsenic (As), cadmium (Cd), copper (Cu), lead (Pb), lithium (Li), mercury (Hg), and nickel (Ni), for 1-h and 24-h, followed by a recovery period. The data demonstrate that maximum cell death occurred during the first few hours of exposure at 24-h IC₅₀ concentrations for all metals. Prolonged in vitro exposure to metals at low concentrations also inhibited protein production and cell proliferation. Subsequently, we determined that metals alter cell differentiation (Oct-4 and egfr) and DNA repair mechanisms (Rad-18, Top-3a and Ogg-1). Interestingly, As, Cd, Hg, and Ni decreased cell proliferation to a greater extent than total histone protein production. Yet, at equivalent concentrations, As and Hg significantly decreased total histone protein production per cell compared to respective controls, suggesting suppression of repair or compensatory mechanisms involving histone pathways. And, acute exposure to As, Cd, Hg and Ni decreased H3K27me1 residue, when compared to control cells. Because activation of cellular differentiation, histone modification, and DNA repair are linked by common transcriptional pathways, and the data propose that metals alter these conduits, then it is reasonable to conclude that trace quantities of metals are capable of suppressing regulation of chromatin structure, cellular differentiation, and controlled cell proliferation in mES cells. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  3. H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells.

    OpenAIRE

    Karmodiya Krishanpal; Krebs Arnaud R; Oulad-Abdelghani Mustapha; Kimura Hiroshi; Tora Laszlo

    2012-01-01

    Abstract Background Transcription regulation in pluripotent embryonic stem (ES) cells is a complex process that involves multitude of regulatory layers, one of which is post-translational modification of histones. Acetylation of specific lysine residues of histones plays a key role in regulating gene expression. Results Here we have investigated the genome-wide occurrence of two histone marks, acetylation of histone H3K9 and K14 (H3K9ac and H3K14ac), in mouse embryonic stem (mES) cells. Genom...

  4. Diabetes-induced effects on cardiomyocytes in chick embryonic heart micromass and mouse embryonic D3 differentiated stem cells.

    Science.gov (United States)

    Mohammed, Omar J; Latif, Muhammad Liaque; Pratten, Margaret K

    2017-04-01

    Diabetes mellitus during pregnancy is a considerable medical challenge, since it is related to ‎augmented morbidity and mortality concerns for both the fetus ‎and the pregnant woman. Records show that the etiology of diabetic ‎embryopathy is complicated, as many teratological factors might be involved ‎in the mechanisms of diabetes mellitus-induced congenital malformation. ‎In this study, the potential cardiotoxic effect of hyperglycemia with hyperketonemia was investigated by using two in vitro models; primary chick embryonic cardiomyocytes and stem cell derived cardiomyocytes, where adverse effects were recorded in both systems. The cells were evaluated by changes in beating activity, cell activity, protein content, ROS production, DNA damage and differentiating stem cell migration. The diabetic formulae used produced an increase in DNA damage and a decline in cell migration in mouse embryonic stem cells. These results provide an additional insight into adverse effects during gestational diabetes mellitus and a recommendation for expectant mothers and maternity staff to monitor glycaemic levels months ahead of conception. This study also supports the recommendation of using antioxidants during pregnancy to prevent DNA damage by the production of ROS, which might result in heart defects as well as other developmental anomalies. Copyright © 2017. Published by Elsevier Inc.

  5. Embryonic Stem Cell Culture Conditions Support Distinct States Associated with Different Developmental Stages and Potency

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    Martin Gonzalez, Javier; Morgani, Sophie M; Bone, Robert A

    2016-01-01

    Embryonic stem cells (ESCs) are cell lines derived from the mammalian pre-implantation embryo. Here we assess the impact of derivation and culture conditions on both functional potency and ESC transcriptional identity. Individual ESCs cultured in either two small-molecule inhibitors (2i) or with ......Embryonic stem cells (ESCs) are cell lines derived from the mammalian pre-implantation embryo. Here we assess the impact of derivation and culture conditions on both functional potency and ESC transcriptional identity. Individual ESCs cultured in either two small-molecule inhibitors (2i...

  6. Transcriptomic changes in mouse embryonic stem cells exposed to thalidomide during spontaneous differentiation

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    Xiugong Gao

    2015-09-01

    Full Text Available Thalidomide is a potent developmental toxicant that induces a range of birth defects, notably severe limb malformations. To unravel the molecular mechanisms underpinning the teratogenic effects of thalidomide, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on the differentiation of mouse embryonic stem cells (mESCs, and published the major findings in a research article entitled “Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells” [1]. The data presented herein contains complementary information related to the aforementioned research article.

  7. Maturation of spinal motor neurons derived from human embryonic stem cells.

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    Tomonori Takazawa

    Full Text Available Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases.

  8. Maturation of Spinal Motor Neurons Derived from Human Embryonic Stem Cells

    Science.gov (United States)

    Takazawa, Tomonori; Croft, Gist F.; Amoroso, Mackenzie W.; Studer, Lorenz; Wichterle, Hynek; MacDermott, Amy B.

    2012-01-01

    Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP) in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases. PMID:22802953

  9. Criteria that optimize the potential of murine embryonic stem cells for in vitro and in vivo developmental studies.

    Science.gov (United States)

    Brown, D G; Willington, M A; Findlay, I; Muggleton-Harris, A L

    1992-01-01

    Cultured mouse embryonic stem (ES) cells are used for both in vitro and in vivo studies. The uncommitted pluripotent cells provide a model system with which to study cellular differentiation and development; they can also be used as vectors to carry specific mutations into the mouse genome by homologous recombination. To ensure successful integration into the germ line, competent totipotent diploid ES cell lines are selected using a cell injection bioassay that is both time consuming and technically demanding. The prolonged in vitro culture of rapidly dividing ES cells can lead to accumulated changes and chromosomal abnormalities that will compromise the biological function and abrogate germ line transmission of chimeric mice carrying novel genetic mutations. Such in vitro conditions will vary between individual laboratories; for example, differences in the serums used for maintenance. Using a number of different criteria we attempt in this paper to define the parameters that we found to be key factors for optimization of the biological potential of established ES cell lines. The successful integration into the germ line is dependant on acquiring or deriving a competent totipotent mouse ES diploid cell line. In this paper parameters and criteria are defined which we found to be key factors for the optimization of the biological potential of established ES cell lines.

  10. Oct3/4 directly regulates expression of E2F3a in mouse embryonic stem cells

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    Kanai, Dai; Ueda, Atsushi; Akagi, Tadayuki; Yokota, Takashi; Koide, Hiroshi, E-mail: hkoide@med.kanazawa-u.ac.jp

    2015-04-10

    Embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, have a characteristic cell cycle with truncated G1 and G2 phases. Recent findings that suppression of Oct3/4 expression results in a reduced proliferation rate of ES cells suggest the involvement of Oct3/4 in the regulation of ES cell growth, although the underlying molecular mechanism remains unclear. In the present study, we identified E2F3a as a direct target gene of Oct3/4 in ES cells. Oct3/4 directly bound to the promoter region of the E2F3a gene and positively regulated expression of E2F3a in mouse ES cells. Suppression of E2F3a activity by E2F6 overexpression led to the reduced proliferation in ES cells, which was relieved by co-expression of E2F3a. Furthermore, cell growth retardation caused by loss of Oct3/4 was rescued by E2F3a expression. These results suggest that Oct3/4 upregulates E2F3a expression to promote ES cell growth. - Highlights: • Oct3/4 positively regulates E2F3a expression in ES cells. • Oct3/4 binds to the promoter region of the E2F3a gene. • Overexpression of E2F6, an inhibitor of E2F3a, reduces ES cell growth. • E2F3a recovers growth retardation of ES cells caused by Oct3/4 reduction.

  11. Estrogen receptor beta-selective agonists stimulate calcium oscillations in human and mouse embryonic stem cell-derived neurons.

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    Lili Zhang

    2010-07-01

    Full Text Available Estrogens are used extensively to treat hot flashes in menopausal women. Some of the beneficial effects of estrogens in hormone therapy on the brain might be due to nongenomic effects in neurons such as the rapid stimulation of calcium oscillations. Most studies have examined the nongenomic effects of estrogen receptors (ER in primary neurons or brain slices from the rodent brain. However, these cells can not be maintained continuously in culture because neurons are post-mitotic. Neurons derived from embryonic stem cells could be a potential continuous, cell-based model to study nongenomic actions of estrogens in neurons if they are responsive to estrogens after differentiation. In this study ER-subtype specific estrogens were used to examine the role of ERalpha and ERbeta on calcium oscillations in neurons derived from human (hES and mouse embryonic stem cells. Unlike the undifferentiated hES cells the differentiated cells expressed neuronal markers, ERbeta, but not ERalpha. The non-selective ER agonist 17beta-estradiol (E(2 rapidly increased [Ca2+]i oscillations and synchronizations within a few minutes. No change in calcium oscillations was observed with the selective ERalpha agonist 4,4',4''-(4-Propyl-[1H]-pyrazole-1,3,5-triyltrisphenol (PPT. In contrast, the selective ERbeta agonists, 2,3-bis(4-Hydroxyphenyl-propionitrile (DPN, MF101, and 2-(3-fluoro-4-hydroxyphenyl-7-vinyl-1,3 benzoxazol-5-ol (ERB-041; WAY-202041 stimulated calcium oscillations similar to E(2. The ERbeta agonists also increased calcium oscillations and phosphorylated PKC, AKT and ERK1/2 in neurons derived from mouse ES cells, which was inhibited by nifedipine demonstrating that ERbeta activates L-type voltage gated calcium channels to regulate neuronal activity. Our results demonstrate that ERbeta signaling regulates nongenomic pathways in neurons derived from ES cells, and suggest that these cells might be useful to study the nongenomic mechanisms of estrogenic compounds.

  12. Human dental follicle cells express embryonic, mesenchymal and neural stem cells markers.

    Science.gov (United States)

    Lima, Rodrigo Lopes; Holanda-Afonso, Rosenilde Carvalho; Moura-Neto, Vivaldo; Bolognese, Ana Maria; DosSantos, Marcos Fabio; Souza, Margareth Maria

    2017-01-01

    This study was conducted to identify and characterize dental follicle stem cells (DFSCs) by analyzing expression of embryonic, mesenchymal and neural stem cells surface markers. Design Dental follicle cells (DFCs) were evaluated by immunocytochemistry using embryonic stem cells markers (OCT4 and SOX2), mesenchmal stem cells (MSCs) markers (Notch1, active Notch1, STRO, CD44, HLA-ABC, CD90), neural stem cells markers (Nestin and β-III-tubulin), neural crest stem cells (NCSCs) markers (p75 and HNK1) and a glial cells marker (GFAP). RT-PCR was performed to identify the expression of OCT4 and NANOG in DFCs and dental follicle tissue. Immunocytochemistry and RT-PCR analysis revealed that a significant proportion of the DFCs evaluated expressed human embryonic stem cells marker OCT4 (75%) whereas NANOG was weakly expressed. A considerable amount of MSCs (90%) expressed Notch1, STRO, CD44 and HLA-ABC. However, they were weakly positive for CD90. Moreover, it was possible to demonstrate that dental follicle contains a significant proportion of neural stem/progenitors cells, expressing β-III-tubulin (90%) and nestin (70%). Interestingly, immunocytochemistry showed DFCs positive for p75 (50%), HNK1 (cells. This is the first study reporting the presence of NCSCs and glial-like cells in the dental follicle. The results of the present study suggest the occurrence of heterogeneous populations of stem cells, particularly neural stem/progenitor cells, in the dental follicle, Therefore, the human dental follicle might be a promising source of adult stem cells for regenerative purposes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Equine induced pluripotent stem cells have a reduced tendon differentiation capacity compared to embryonic stem cells

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    Emma Patricia Bavin

    2015-11-01

    Full Text Available Tendon injuries occur commonly in horses and their repair through scar tissue formation predisposes horses to a high rate of re-injury. Pluripotent stem cells may provide a cell replacement therapy to improve tendon tissue regeneration and lower the frequency of re-injury. We have previously demonstrated that equine embryonic stem cells (ESCs differentiate into the tendon cell lineage upon injection into the damaged horse tendon and can differentiate into functional tendon cells in vitro to generate artificial tendons. Induced pluripotent stem cells (iPSCs have now been derived from horses but, to date, there are no reports on their ability to differentiate into tendon cells. As iPSCs can be produced from adult cell types, they provide a more accessible source of cells than ESCs, which require the use of horse embryos. The aim of this study was to compare tendon differentiation by ESCs and iPSCs produced through two independent methods. In 2-dimensional differentiation assays the iPSCs expressed tendon associated genes and proteins, which were enhanced by the presence of transforming growth factor-β3. However, in 3-dimensional differentiation assays the iPSCs failed to differentiate into functional tendon cells and generate artificial tendons. These results demonstrate the utility of the 3-dimensional in vitro tendon assay for measuring tendon differentiation and the need for more detailed studies to be performed on equine iPSCs to identify and understand their epigenetic differences from pluripotent ESCs prior to their clinical application.

  14. Embryonic stem cell-derived neural stem cells fuse with microglia and mature neurons.

    Science.gov (United States)

    Cusulin, Carlo; Monni, Emanuela; Ahlenius, Henrik; Wood, James; Brune, Jan Claas; Lindvall, Olle; Kokaia, Zaal

    2012-12-01

    Transplantation of neural stem cells (NSCs) is a novel strategy to restore function in the diseased brain, acting through multiple mechanisms, for example, neuronal replacement, neuroprotection, and modulation of inflammation. Whether transplanted NSCs can operate by fusing with microglial cells or mature neurons is largely unknown. Here, we have studied the interaction of a mouse embryonic stem cell-derived neural stem (NS) cell line with rat and mouse microglia and neurons in vitro and in vivo. We show that NS cells spontaneously fuse with cocultured cortical neurons, and that this process requires the presence of microglia. Our in vitro data indicate that the NS cells can first fuse with microglia and then with neurons. The fused NS/microglial cells express markers and retain genetic and functional characteristics of both parental cell types, being able to respond to microglia-specific stimuli (LPS and IL-4/IL-13) and to differentiate to neurons and astrocytes. The NS cells fuse with microglia, at least partly, through interaction between phosphatidylserine exposed on the surface of NS cells and CD36 receptor on microglia. Transplantation of NS cells into rodent cortex results in fusion with mature pyramidal neurons, which often carry two nuclei, a process probably mediated by microglia. The fusogenic role of microglia could be even more important after NSC transplantation into brains affected by neurodegenerative diseases associated with microglia activation. It remains to be elucidated how the occurrence of the fused cells will influence the functional outcome after NSC transplantation in the diseased brain. Copyright © 2012 AlphaMed Press.

  15. Maintenance of human embryonic stem cells in mesenchymal stem cell-conditioned media augments hematopoietic specification.

    Science.gov (United States)

    Ramos-Mejía, Verónica; Fernández, Agustín F; Ayllón, Verónica; Real, Pedro J; Bueno, Clara; Anderson, Per; Martín, Francisco; Fraga, Mario F; Menendez, Pablo

    2012-06-10

    The realization of human embryonic stem cells (hESC) as a model for human developmental hematopoiesis and in potential cell replacement strategies relies on an improved understanding of the extrinsic and intrinsic factors regulating hematopoietic-specific hESC differentiation. Human mesenchymal stem cells (hMSCs) are multipotent cells of mesodermal origin that form a part of hematopoietic stem cell niches and have an important role in the regulation of hematopoiesis through production of secreted factors and/or cell-to-cell interactions. We have previously shown that hESCs may be successfully maintained feeder free using hMSC-conditioned media (MSC-CM). Here, we hypothesized that hESCs maintained in MSC-CM may be more prone to differentiation toward hematopoietic lineage than hESCs grown in standard human foreskin fibroblast-conditioned media. We report that specification into hemogenic progenitors and subsequent hematopoietic differentiation and clonogenic progenitor capacity is robustly enhanced in hESC lines maintained in MSC-CM. Interestingly, co-culture of hESCs on hMSCs fully abrogates hematopoietic specification of hESCs, thus suggesting that the improved hematopoietic differentiation is mediated by MSC-secreted factors rather than by MSC-hESC physical interactions. To investigate the molecular mechanism involved in this process, we analyzed global (LINE-1) methylation and genome-wide promoter DNA methylation. hESCs grown in MSC-CM showed a decrease of 17% in global DNA methylation and a promoter DNA methylation signature consisting of 45 genes commonly hypomethylated and 102 genes frequently hypermethylated. Our data indicate that maintenance of hESCs in MSC-CM robustly augments hematopoietic specification and that the process seems mediated by MSC-secreted factors conferring a DNA methylation signature to undifferentiated hESCs which may influence further predisposition toward hematopoietic specification.

  16. Putative embryonic stem cells derived from porcine cloned blastocysts using induced pluripotent stem cells as donors.

    Science.gov (United States)

    Kim, Eunhye; Hwang, Seon-Ung; Yoo, Hyunju; Yoon, Junchul David; Jeon, Yubyeol; Kim, Hyunggee; Jeung, Eui-Bae; Lee, Chang-Kyu; Hyun, Sang-Hwan

    2016-03-01

    The establishment of porcine embryonic stem cells (ESCs) would have great impact in biomedical studies and preclinical trials through their use in genetic engineering. However, authentic porcine ESCs have not been established until now. In this study, a total of seven putative ESC lines were derived from porcine embryos of various origins, including in vitro fertilization, parthenogenetic activation, and, in particular, induced pluripotent stem (iPS) nuclear transfer (NT) from a donor cell with induced pluripotent stem cells (iPSCs). To characterize these cell lines, several assays including an assessment of intensive alkaline phosphatase activity, karyotyping, embryoid body formation, expression analysis of the pluripotency-associated markers, and the three germ layerassociated markers were performed. Based on quantitative polymerase chain reaction, the expression levels of REX1 and FGFR2 in iPS-NT lines were higher than those of cells of other origins. Additionally, only iPS-NT lines showed multiple aberrant patterns of nuclear foci elucidated by immunofluorescence staining of H3K27me3 as a marker of the state of X chromosome inactivation and a less mature form of mitochondria like naive ESCs, by transmission electron microscopy. Together, these data suggested that established putative porcine ESC lines generally exhibited a primed pluripotent state, like human ESCs. However, iPS-NT lines have especially unique characteristics distinct from other origins because they have more epigenetic instability and naive-like mitochondrial morphology than other putative ESC lines. This is the first study to establish and characterize the iPSC-derived putative ESC lines and compare them with other lines derived from different origins in pigs. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Induced pluripotent stem cells from pigs and other ungulate species: an alternative to embryonic stem cells?

    Science.gov (United States)

    Ezashi, T; Telugu, B P V L; Roberts, R M

    2012-08-01

    Robust embryonic stem cell (ESC) lines from livestock species have been difficult to derive and maintain, and unlike mouse ESC, have not contributed to our ability to understand directed differentiation in vitro. Nor have such cells yet provided a simpler means than pronuclear injection to manipulate the genomes of agriculturally important species, such as cattle, sheep and pigs. Induced pluripotent stem cells (iPSC) generated by reprogramming somatic cells, such as fibroblasts, with a set of stemness genes, most usually but not exclusively POU5F1, SOX2, KLF4 and c-MYC, offer an alternative to ESC in these regards, as they exhibit a pluripotent phenotype resembling that of ESC, yet are readily generated in the laboratory. Accordingly, such cells, in association with cloning technologies, may be useful for introducing complex genetic changes into livestock, although this potential has yet to be demonstrated. Porcine iPSC may be especially valuable because the pig is a prime biomedical model for tissue transplantation. In general, iPSC from livestock, like those from humans, are of the epiblast type and depend upon FGF2 and activin/nodal signalling systems to maintain their pluripotency and growth. Recent experiments, in which newly reprogrammed porcine and bovine cells were selected on a LIF-based medium in presence of specific protein kinase inhibitors, have allowed iPSC cells of the naïve type, resembling the more amenable blastocyst-derived mouse ESC and iPSC to be isolated. However, hurdles still remain if such cells are to achieve their biotechnological promise. © 2012 Blackwell Verlag GmbH.

  18. Effects of Pulsed Electromagnetic Field on Differentiation of HUES-17 Human Embryonic Stem Cell Line

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    Yi-Lin Wu

    2014-08-01

    Full Text Available Electromagnetic fields are considered to potentially affect embryonic development, but the mechanism is still unknown. In this study, human embryonic stem cell (hESC line HUES-17 was applied to explore the mechanism of exposure on embryonic development to pulsed electromagnetic field (PEMF for 400 pulses at different electric field intensities and the differentiation of HUES-17 cells was observed after PEMF exposure. The expression of alkaline phosphatase (AP, stage-specific embryonic antigen-3 (SSEA-3, SSEA-4 and the mRNA level and protein level of Oct4, Sox2 and Nanog in HUES-17 cells remained unchanged after PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m. Four hundred pulses PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m did not affect the differentiation of HUES-17 cells. The reason why electromagnetic fields affect embryonic development may be due to other mechanisms rather than affecting the differentiation of embryonic stem cells.

  19. Co-incident insertion enables high efficiency genome engineering in mouse embryonic stem cells.

    Science.gov (United States)

    Shy, Brian R; MacDougall, Matthew S; Clarke, Ryan; Merrill, Bradley J

    2016-09-19

    CRISPR/Cas9 nucleases have enabled powerful, new genome editing capabilities; however, the preponderance of non-homologous end joining (NHEJ) mediated repair events over homology directed repair (HDR) in most cell types limits the ability to engineer precise changes in mammalian genomes. Here, we increase the efficiency of isolating precise HDR-mediated events in mouse embryonic stem (ES) cells by more than 20-fold through the use of co-incidental insertion (COIN) of independent donor DNA sequences. Analysis of on:off-target frequencies at the Lef1 gene revealed that bi-allelic insertion of a PGK-Neo cassette occurred more frequently than expected. Using various selection cassettes targeting multiple loci, we show that the insertion of a selectable marker at one control site frequently coincided with an insertion at an unlinked, independently targeted site, suggesting enrichment of a sub-population of HDR-proficient cells. When individual cell events were tracked using flow cytometry and fluorescent protein markers, individual cells frequently performed either a homology-dependent insertion event or a homology-independent event, but rarely both types of insertions in a single cell. Thus, when HDR-dependent selection donors are used, COIN enriches for HDR-proficient cells among heterogeneous cell populations. When combined with a self-excising selection cassette, COIN provides highly efficient and scarless genome editing. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Science.gov (United States)

    Bibel, Miriam; Richter, Jens; Schrenk, Katrin; Tucker, Kerry Lee; Staiger, Volker; Korte, Martin; Goetz, Magdalena; Barde, Yves-Alain

    2004-09-01

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

  1. Human IL-12 p40 as a reporter gene for high-throughput screening of engineered mouse embryonic stem cells

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    Shaffer Benjamin

    2008-06-01

    Full Text Available Abstract Background Establishing a suitable level of exogenous gene expression in mammalian cells in general, and embryonic stem (ES cells in particular, is an important aspect of understanding pathways of cell differentiation, signal transduction and cell physiology. Despite its importance, this process remains challenging because of the poor correlation between the presence of introduced exogenous DNA and its transcription. Consequently, many transfected cells must be screened to identify those with an appropriate level of expression. To improve the screening process, we investigated the utility of the human interleukin 12 (IL-12 p40 cDNA as a reporter gene for studies of mammalian gene expression and for high-throughput screening of engineered mouse embryonic stem cells. Results A series of expression plasmids were used to study the utility of IL-12 p40 as an accurate reporter of gene activity. These studies included a characterization of the IL-12 p40 expression system in terms of: (i a time course of IL-12 p40 accumulation in the medium of transfected cells; (ii the dose-response relationship between the input DNA and IL-12 p40 mRNA levels and IL-12 p40 protein secretion; (iii the utility of IL-12 p40 as a reporter gene for analyzing the activity of cis-acting genetic elements; (iv expression of the IL-12 p40 reporter protein driven by an IRES element in a bicistronic mRNA; (v utility of IL-12 p40 as a reporter gene in a high-throughput screening strategy to identify successful transformed mouse embryonic stem cells; (vi demonstration of pluripotency of IL-12 p40 expressing ES cells in vitro and in vivo; and (vii germline transmission of the IL-12 p40 reporter gene. Conclusion IL-12 p40 showed several advantages as a reporter gene in terms of sensitivity and ease of the detection procedure. The IL-12 p40 assay was rapid and simple, in as much as the reporter protein secreted from the transfected cells was accurately measured by ELISA using

  2. Ambivalent journeys of hope: embryonic stem cell therapy in a clinic in India.

    Science.gov (United States)

    Prasad, Amit

    2015-03-01

    Stem cell therapy in non-Western countries such as India has received a lot of attention. Apart from media reports, there are a number of social science analyses of stem cell policy, therapy, and research, their ethical implications, and impact of advertising on patients. Nevertheless, in the media reports as well as in academic studies, experiences of patients, who undertake overseas journeys for stem cell therapy, have largely been either ignored or presented reductively, often as a "false hope." In this article, I analyze the experiences of patients and their "journeys of hope" to NuTech Mediworld, an embryonic stem cell therapy clinic in New Delhi, India. My analysis, which draws on my observations in the clinic and patients' experiences, instead of seeking to adjudicate whether embryonic stem cell therapy in clinics such as NuTech is right or wrong, true or false, focuses on how patients navigate and contest these concerns. I utilize Gilles Deleuze and Felix Guattari's "concepts," lines of flight and deterritorialization, to highlight how embryonic stem cell therapy's "political economy of hope" embodies deterritorialization of several "regimes of truth" and how these deterritorializations impact patients' experiences. © The Author(s) 2014.

  3. Asynchronous replication and autosome-pair non-equivalence in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Devkanya Dutta

    Full Text Available A number of mammalian genes exhibit the unusual properties of random monoallelic expression and random asynchronous replication. Such exceptional genes include genes subject to X inactivation and autosomal genes including odorant receptors, immunoglobulins, interleukins, pheromone receptors, and p120 catenin. In differentiated cells, random asynchronous replication of interspersed autosomal genes is coordinated at the whole chromosome level, indicative of chromosome-pair non-equivalence. Here we have investigated the replication pattern of the random asynchronously replicating genes in undifferentiated human embryonic stem cells, using fluorescence in situ hybridization based assay. We show that allele-specific replication of X-linked genes and random monoallelic autosomal genes occur in human embryonic stem cells. The direction of replication is coordinated at the whole chromosome level and can cross the centromere, indicating the existence of autosome-pair non-equivalence in human embryonic stem cells. These results suggest that epigenetic mechanism(s that randomly distinguish between two parental alleles are emerging in the cells of the inner cell mass, the source of human embryonic stem cells.

  4. Fingerprinting of neurotoxic compounds using a mouse embryonic stem cell dual luminescence reporter assay

    NARCIS (Netherlands)

    Colaianna, M.; Ilmjärv, S.; Peterson, H.; Ilse Kern, I.; Julien, S.; Baquié, M.; allocca, G.; Bosgra, S.; Sachinidis, A.; Hengstler, J.G.; Leist, M.; Krause, K.H.

    2017-01-01

    Identification of neurotoxic drugs and environmental chemicals is an important challenge. However, only few tools to address this topic are available. The aim of this study was to develop a neurotoxicity/developmental neurotoxicity (DNT) test system, using the pluripotent mouse embryonic stem cell

  5. Redox Disrupting Potential of ToxCast™Chemicals Ranked by Activity in Mouse Embryonic Stem Cells

    Science.gov (United States)

    Little is known regarding the adverse outcome pathways responsible for developmental toxicity following exposure to chemicals. An evaluation of Toxoast™ Phase I chemicals in an adherent mouse embryonic stem cell (mESC) assay revealed a redox sensitive pathway that correlated with...

  6. Global phosphoproteome profiling reveals unanticipated networks responsive to cisplatin treatment of embryonic stem cells

    DEFF Research Database (Denmark)

    Pines, Alex; Kelstrup, Christian D; Vrouwe, Mischa G

    2011-01-01

    (stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia...

  7. Generation of a homozygous GBA deletion human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Anna Lisa Gündner

    2017-08-01

    Full Text Available We describe the generation of a biallelic GBA deletion human embryonic stem cell line using zinc finger nuclease-mediated gene targeting. The homozygous targeting of exon 4 of the GBA locus leads to a complete loss of glucocerebrosidase (GCase protein expression.

  8. Partial promoter substitutions generating transcriptional sentinels of diverse signaling pathways in embryonic stem cells and mice

    DEFF Research Database (Denmark)

    Serup, Palle; Gustavsen, Carsten; Klein, Tino

    2012-01-01

    extracellular signals. We thereby created an allelic series of embryonic stem cells and mice, each containing a signal-responsive sentinel with different fluorescent reporters that respond with sensitivity and specificity to retinoic acids, bone morphogenic proteins, activin A, Wnts or Notch, and that can...

  9. NANOG reporter cell lines generated by gene targeting in human embryonic stem cells

    DEFF Research Database (Denmark)

    Fischer, Yvonne; Ganic, Elvira; Ameri, Jacqueline

    2010-01-01

    Pluripotency and self-renewal of human embryonic stem cells (hESCs) is mediated by a complex interplay between extra- and intracellular signaling pathways, which regulate the expression of pluripotency-specific transcription factors. The homeodomain transcription factor NANOG plays a central role...

  10. REDOX DISRUPTING POTENTIAL OF TOXCAST CHEMICALS RANKED BY ACTIVITY IN MOUSE EMBRYONIC STEM CELLS

    Science.gov (United States)

    To gain insight regarding the adverse outcome pathways leading to developmental toxicity following exposure to chemicals, we evaluated ToxCast™ Phase I chemicals in an adherent mouse embryonic stem cell (mESC) assay and identified a redox sensitive pathway that correlated with al...

  11. Efficient femtosecond driven SOX 17 delivery into mouse embryonic stem cells: differentiation studies

    Science.gov (United States)

    Thobakgale, Lebogang; Manoto, Sello Lebohang; Lemboumba, Satuurnin Ombinda; Maaza, Malik; Mthunzi-Kufa, Patience

    2017-02-01

    Embryonic stem cells have great promise in regenerative medicine because of their ability to self-renew and differentiate into various cell types. Delivery of therapeutic genes into cells has already been achieved using of chemical agents and viral vectors with high transfection efficiencies. However, these methods have also been documented as toxic and in the latter case they can cause latent cell infections. In this study we use femtosecond laser pulses to optically deliver genetic material in mouse embryonic stem cells. Femtosecond laser pulses in contrast to the conventional approach, minimises the risk of unwanted side effects because photons are used to create transient pores on the membrane which allow free entry of molecules with no need for delivery agents. Using an Olympus microscope, fluorescence imaging of the samples post irradiation was performed and decreased expression of stage specific embryonic antigen one (SSEA-1) consistent with on-going cellular differentiation was observed. Our results also show that femtosecond laser pulses were effective in delivering SOX 17 plasmid DNA (pSOX17) which resulted in the differentiation of mouse embryonic stem cells into endoderm cells. We thus concluded that laser transfection of stem cells for the purpose of differentiation, holds potential for applications in tissue engineering as a method of generating new cell lines.

  12. Erk signaling suppresses embryonic stem cell self-renewal to specify endoderm

    DEFF Research Database (Denmark)

    Hamilton, William B; Brickman, Joshua M

    2014-01-01

    Fgf signaling via Erk activation has been associated with both neural induction and the generation of a primed state for the differentiation of embryonic stem cells (ESCs) to all somatic lineages. To dissect the role of Erk in both ESC self-renewal and lineage specification, we explored...

  13. Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line

    Science.gov (United States)

    The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...

  14. Functionally deficient neuronal differentiation of mouse embryonic neural stem cells in vitro

    NARCIS (Netherlands)

    Balasubramaniyan, [No Value; de Haas, AH; Bakels, R; Koper, A; Boddeke, HWGM; Copray, JM

    Embryonic mouse neural stem cells (NSCs) were isolated from E14 mice, multiplied in medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and plated in laminin-coated wells in basic serum-free neurobasal medium. After 7 days in vitro, approximately 20% of the

  15. Toward Development of Pluripotent Porcine Stem Cells by Road Mapping Early Embryonic Development

    DEFF Research Database (Denmark)

    Petkov, Stoyan; Freude, Kristine; Mashayekhi-Nezamabadi, Kaveh

    2017-01-01

    The lack in production of bona fide porcine pluripotent stem cells has definitely been hampered by a lack of research into porcine embryo development. Embryonic development in mammals is the extraordinary transition of a single-celled fertilized zygote into a complex fetus, which occurs in the ut...

  16. Regional differentiation of retinoic acid-induced human pluripotent embryonic carcinoma stem cell neurons.

    Directory of Open Access Journals (Sweden)

    Dennis E Coyle

    Full Text Available The NTERA2 cl D1 (NT2 cell line, derived from human teratocarcinoma, exhibits similar properties as embryonic stem (ES cells or very early neuroepithelial progenitors. NT2 cells can be induced to become postmitotic central nervous system neurons (NT2N with retinoic acid. Although neurons derived from pluripotent cells, such as NT2N, have been characterized for their neurotransmitter phenotypes, their potential suitability as a donor source for neural transplantation also depends on their ability to respond to localized environmental cues from a specific region of the CNS. Therefore, our study aimed to characterize the regional transcription factors that define the rostocaudal and dorsoventral identity of NT2N derived from a monolayer differentiation paradigm using quantitative PCR (qPCR. Purified NT2N mainly expressed both GABAergic and glutamatergic phenotypes and were electrically active but did not form functional synapses. The presence of immature astrocytes and possible radial glial cells was noted. The NT2N expressed a regional transcription factor code consistent with forebrain, hindbrain and spinal cord neural progenitors but showed minimal expression of midbrain phenotypes. In the dorsoventral plane NT2N expressed both dorsal and ventral neural progenitors. Of major interest was that even under the influence of retinoic acid, a known caudalization factor, the NT2N population maintained a rostral phenotype subpopulation which expressed cortical regional transcription factors. It is proposed that understanding the regional differentiation bias of neurons derived from pluripotent stem cells will facilitate their successful integration into existing neuronal networks within the CNS.

  17. Generation of hematopoietic stem cells from human embryonic stem cells using a defined, stepwise, serum-free, and serum replacement-free monolayer culture method.

    Science.gov (United States)

    Kim, So-Jung; Jung, Ji-Won; Ha, Hye-Yeong; Koo, Soo Kyung; Kim, Eung-Gook; Kim, Jung-Hyun

    2017-03-01

    Embryonic stem cells (ESCs) can be expanded infinitely in vitro and have the potential to differentiate into hematopoietic stem cells (HSCs); thus, they are considered a useful source of cells for HSC production. Although several technical in vitro methods for engineering HSCs from pluripotent stem cells have been developed, clinical application of HSCs engineered from pluripotent stem cells is restricted because of the possibility of xenogeneic contamination resulting from the use of murine materials. Human ESCs (CHA-hES15) were cultured on growth factor-reduced Matrigel-coated dishes in the mTeSR1 serum-free medium. When the cells were 70% confluent, we initiated HSC differentiation by three methods involving (1) knockout serum replacement (KSR), cytokines, TGFb1, EPO, and FLT3L; (2) KSR, cytokines, and bFGF; or (3) cytokines and bFGF. Among the three differentiation methods, the minimal number of cytokines without KSR resulted in the greatest production of HSCs. The optimized method resulted in a higher proportion of CD34+CD43+ hematopoietic progenitor cells (HPCs) and CD34+CD45+ HPCs compared to the other methods. In addition, the HSCs showed the potential to differentiate into multiple lineages of hematopoietic cells in vitro. In this study, we optimized a two-step, serum-free, animal protein-free, KSR-free, feeder-free, chemically defined monolayer culture method for generation of HSCs and hematopoietic stem and progenitor cells (HSPCs) from human ESCs.

  18. Hyaluronan esters drive Smad gene expression and signaling enhancing cardiogenesis in mouse embryonic and human mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Margherita Maioli

    Full Text Available BACKGROUND: Development of molecules chemically modifying the expression of crucial orchestrator(s of stem cell commitment may have significant biomedical impact. We have recently developed hyaluronan mixed esters of butyric and retinoic acids (HBR, turning cardiovascular stem cell fate into a high-yield process. The HBR mechanism(s remain still largely undefined. METHODOLOGY/PRINCIPAL FINDINGS: We show that in both mouse embryonic stem (ES cells and human mesenchymal stem cells from fetal membranes of term placenta (FMhMSCs, HBR differentially affected the patterning of Smad proteins, one of the major conductors of stem cell cardiogenesis. Real-time RT-PCR and Western blot analyses revealed that in both cell types HBR enhanced gene and protein expression of Smad1,3, and 4, while down-regulating Smad7. HBR acted at the transcriptional level, as shown by nuclear run-off experiments in isolated nuclei. Immunofluorescence analysis indicated that HBR increased the fluorescent staining for Smad1,3, and 4, confirming that the transcriptional action of HBR encompassed the upregulation of the encoded Smad proteins. Chromatin immune precipitation and transcriptional analyses showed that HBR increased the transcription of the cardiogenic gene Nkx-2.5 through Smad4 binding to its own consensus Smad site. Treatment of mouse ES cells and FMhMSCs with HBR led to the concomitant overexpression of both Smad4 and α-sarcomeric actinin. Smad4 silencing by the aid of lentiviral-mediated Smad4 shRNA confirmed a dominant role of Smad4 in HBR-induced cardiogenesis. CONCLUSIONS/SIGNIFICANCE: The use of HBR may pave the way to novel combinatorial strategies of molecular and stem cell therapy based on fine tuning of targeted Smad transciption and signaling leading to a high-throughput of cardiogenesis without the needs of gene transfer technologies.

  19. Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

    Science.gov (United States)

    Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

    2015-02-25

    Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Two sides of the same coin? Unraveling subtle differences between human embryonic and induced pluripotent stem cells by Raman spectroscopy.

    Science.gov (United States)

    Parrotta, Elvira; De Angelis, Maria Teresa; Scalise, Stefania; Candeloro, Patrizio; Santamaria, Gianluca; Paonessa, Mariagrazia; Coluccio, Maria Laura; Perozziello, Gerardo; De Vitis, Stefania; Sgura, Antonella; Coluzzi, Elisa; Mollace, Vincenzo; Di Fabrizio, Enzo Mario; Cuda, Giovanni

    2017-11-28

    Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, hold enormous promise for many biomedical applications, such as regenerative medicine, drug testing, and disease modeling. Although induced pluripotent stem cells resemble embryonic stem cells both morphologically and functionally, the extent to which these cell lines are truly equivalent, from a molecular point of view, remains controversial. Principal component analysis and K-means cluster analysis of collected Raman spectroscopy data were used for a comparative study of the biochemical fingerprint of human induced pluripotent stem cells and human embryonic stem cells. The Raman spectra analysis results were further validated by conventional biological assays. Raman spectra analysis revealed that the major difference between human embryonic stem cells and induced pluripotent stem cells is due to the nucleic acid content, as shown by the strong positive peaks at 785, 1098, 1334, 1371, 1484, and 1575 cm-1, which is enriched in human induced pluripotent stem cells. Here, we report a nonbiological approach to discriminate human induced pluripotent stem cells from their native embryonic stem cell counterparts.

  1. Two sides of the same coin? Unraveling subtle differences between human embryonic and induced pluripotent stem cells by Raman spectroscopy

    KAUST Repository

    Parrotta, Elvira

    2017-11-28

    Background: Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, hold enormous promise for many biomedical applications, such as regenerative medicine, drug testing, and disease modeling. Although induced pluripotent stem cells resemble embryonic stem cells both morphologically and functionally, the extent to which these cell lines are truly equivalent, from a molecular point of view, remains controversial. Methods: Principal component analysis and K-means cluster analysis of collected Raman spectroscopy data were used for a comparative study of the biochemical fingerprint of human induced pluripotent stem cells and human embryonic stem cells. The Raman spectra analysis results were further validated by conventional biological assays. Results: Raman spectra analysis revealed that the major difference between human embryonic stem cells and induced pluripotent stem cells is due to the nucleic acid content, as shown by the strong positive peaks at 785, 1098, 1334, 1371, 1484, and 1575 cm–1, which is enriched in human induced pluripotent stem cells. Conclusions: Here, we report a nonbiological approach to discriminate human induced pluripotent stem cells from their native embryonic stem cell counterparts.

  2. Up-scaling single cell-inoculated suspension culture of human embryonic stem cells.

    Science.gov (United States)

    Singh, Harmeet; Mok, Pamela; Balakrishnan, Thavamalar; Rahmat, Siti Norfiza Binte; Zweigerdt, Robert

    2010-05-01

    We have systematically developed single cell-inoculated suspension cultures of human embryonic stem cells (hESC) in defined media. Cell survival was dependent on hESC re-aggregation. In the presence of the Rho kinase inhibitor Y-27632 (Ri) only approximately 44% of the seeded cells were rescued, but an optimized heat shock treatment combined with Ri significantly increased cell survival to approximately 60%. Mechanistically, our data suggest that E-cadherin plays a role in hESC aggregation and that dissociation and re-aggregation upon passaging functions as a purification step towards a pluripotency markers-enriched population. Mass expansion of hESC was readily achieved by up-scaling 2 ml cultures to serial passaging in 50 ml spinner flasks. A media comparison revealed that mTeSR was superior to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency markers was achieved for two lines (hES2, hES3) that were used at higher passages (>86). In contrast, rapid down regulation of Oct4, Tra-1-60, and SSEA4 was observed for ESI049, a clinically compliant line, used at passages 20-36. The up-scaling strategy has significant potential to provide pluripotent cells on a clinical scale. Nevertheless, our data also highlights a significant line-to-line variability and the need for a critical assessment of novel methods with numerous relevant cell lines. Copyright 2010 Elsevier B.V. All rights reserved.

  3. Regulation of mineralocorticoid receptor expression during neuronal differentiation of murine embryonic stem cells.

    Science.gov (United States)

    Munier, Mathilde; Meduri, Geri; Viengchareun, Say; Leclerc, Philippe; Le Menuet, Damien; Lombès, Marc

    2010-05-01

    Mineralocorticoid receptor (MR) plays a critical role in brain function. However, the regulatory mechanisms controlling neuronal MR expression that constitutes a key element of the hormonal response are currently unknown. Two alternative P1 and P2 promoters drive human MR gene transcription. To examine promoter activities and their regulation during neuronal differentiation and in mature neurons, we generated stably transfected recombinant murine embryonic stem cell (ES) lines, namely P1-GFP and P2-GFP, in which each promoter drove the expression of the reporter gene green fluorescent protein (GFP). An optimized protocol, using embryoid bodies and retinoic acid, permitted us to obtain a reproducible neuronal differentiation as revealed by the decrease in phosphatase alkaline activity, the concomitant appearance of morphological changes (neurites), and the increase in the expression of neuronal markers (nestin, beta-tubulin III, and microtubule-associated protein-2) as demonstrated by immunocytochemistry and quantitative PCR. Using these cell-based models, we showed that MR expression increased by 5-fold during neuronal differentiation, MR being preferentially if not exclusively expressed in mature neurons. Although the P2 promoter was always weaker than the P1 promoter during neuronal differentiation, their activities increased by 7- and 5-fold, respectively, and correlated with MR expression. Finally, although progesterone and dexamethasone were ineffective, aldosterone stimulated both P1 and P2 activity and MR expression, an effect that was abrogated by knockdown of MR by small interfering RNA. In conclusion, we provide evidence for a tight transcriptional control of MR expression during neuronal differentiation. Given the neuroprotective and antiapoptotic role proposed for MR, the neuronal differentiation of ES cell lines opens potential therapeutic perspectives in neurological and psychiatric diseases.

  4. Evidence for a critical role of catecholamines for cardiomyocyte lineage commitment in murine embryonic stem cells.

    Science.gov (United States)

    Lehmann, Martin; Nguemo, Filomain; Wagh, Vilas; Pfannkuche, Kurt; Hescheler, Jürgen; Reppel, Michael

    2013-01-01

    Catecholamine release is known to modulate cardiac output by increasing heart rate. Although much is known about catecholamine function and regulation in adults, little is known about the presence and role of catecholamines during heart development. The present study aimed therefore to evaluate the effects of different catecholamines on early heart development in an in vitro setting using embryonic stem (ES) cell-derived cardiomyocytes. Effects of catecholamine depletion induced by reserpine were examined in murine ES cells (line D3, αPIG44) during differentiation. Cardiac differentiation was assessed by immunocytochemistry, qRT-PCR, quantification of beating clusters, flow cytometry and pharmacological approaches. Proliferation was analyzed by EB cross-section measurements, while functionality of cardiomyocytes was studied by extracellular field potential (FP) measurements using microelectrode arrays (MEAs). To further differentiate between substance-specific effects of reserpine and catecholamine action via α- and β-receptors we proved the involvement of adrenergic receptors by application of unspecific α- and β-receptor antagonists. Reserpine treatment led to remarkable down-regulation of cardiac-specific genes, proteins and mesodermal marker genes. In more detail, the average ratio of ∼40% spontaneously beating control clusters was significantly reduced by 100%, 91.1% and 20.0% on days 10, 12, and 14, respectively. Flow cytometry revealed a significant reduction (by 71.6%, n = 11) of eGFP positive CMs after reserpine treatment. By contrast, reserpine did not reduce EB growth while number of neuronal cells in reserpine-treated EBs was significantly increased. MEA measurements of reserpine-treated EBs showed lower FP frequencies and weak responsiveness to adrenergic and muscarinic stimulation. Interestingly we found that developmental inhibition after α- and β-adrenergic blocker application mimicked developmental changes with reserpine. Using several

  5. ERK2 suppresses self-renewal capacity of embryonic stem cells, but is not required for multi-lineage commitment.

    Directory of Open Access Journals (Sweden)

    William B Hamilton

    Full Text Available Activation of the FGF-ERK pathway is necessary for naïve mouse embryonic stem (ES cells to exit self-renewal and commit to early differentiated lineages. Here we show that genetic ablation of Erk2, the predominant ERK isozyme expressed in ES cells, results in hyper-phosphorylation of ERK1, but an overall decrease in total ERK activity as judged by substrate phosphorylation and immediate-early gene (IEG induction. Normal induction of this subset of canonical ERK targets, as well as p90RSK phosphorylation, was rescued by transgenic expression of either ERK1 or ERK2 indicating a degree of functional redundancy. In contrast to previously published work, Erk2-null ES cells exhibited no detectable defect in lineage specification to any of the three germ layers when induced to differentiate in either embryoid bodies or in defined neural induction conditions. However, under self-renewing conditions Erk2-null ES cells express increased levels of the pluripotency-associated transcripts, Nanog and Tbx3, a decrease in Nanog-GFP heterogeneity, and exhibit enhanced self-renewal in colony forming assays. Transgenic add-back of ERK2 is capable of restoring normal pluripotent gene expression and self-renewal capacity. We show that ERK2 contributes to the destabilization of ES cell self-renewal by reducing expression of pluripotency genes, such as Nanog, but is not specifically required for the early stages of germ layer specification.

  6. Lovastatin Decreases the Expression of CD133 and Influences the Differentiation Potential of Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Ade Kallas-Kivi

    2016-01-01

    Full Text Available The lipophilic statin lovastatin decreases cholesterol synthesis and is a safe and effective treatment for the prevention of cardiovascular diseases. Growing evidence points at antitumor potential of lovastatin. Therefore, understanding the molecular mechanism of lovastatin function in different cell types is critical to effective therapy design. In this study, we investigated the effects of lovastatin on the differentiation potential of human embryonic stem (hES cells (H9 cell line. Multiparameter flow cytometric assay was used to detect changes in the expression of transcription factors characteristic of hES cells. We found that lovastatin treatment delayed NANOG downregulation during ectodermal and endodermal differentiation. Likewise, expression of ectodermal (SOX1 and OTX2 and endodermal (GATA4 and FOXA2 markers was higher in treated cells. Exposure of hES cells to lovastatin led to a minor decrease in the expression of SSEA-3 and a significant reduction in CD133 expression. Treated cells also formed fewer embryoid bodies than control cells. By analyzing hES with and without CD133, we discovered that CD133 expression is required for proper formation of embryoid bodies. In conclusion, lovastatin reduced the heterogeneity of hES cells and impaired their differentiation potential.

  7. Collagen scaffolds with or without the addition of RGD peptides support cardiomyogenesis after aggregation of mouse embryonic stem cells.

    Science.gov (United States)

    Dawson, Jennifer; Schussler, Olivier; Al-Madhoun, Ashraf; Menard, Claudine; Ruel, Marc; Skerjanc, Ilona S

    2011-10-01

    Embryonic stem (ES) cell-based cardiac muscle repair using tissue-engineered scaffolds is an attractive prospective treatment option for patients suffering from heart disease. In this study, our aim was to characterize mouse ES cell-derived cardiomyocytes growing on collagen I/III scaffolds, modified with the adhesion peptides arginine-glycine-aspartic acid (RGD). Mouse ES-derived embryoid bodies (EBs) differentiated efficiently into beating cardiomyocytes on the collagen scaffolds. QPCR analysis and immunofluorescent staining showed that cardiomyocytes expressed cardiac muscle-related transcripts and proteins. Analysis of cardiomyocytes by electron microscopy identified muscle fiber bundles and Z bands, typical of ES-derived cardiomyocytes. No differences were detected between the collagen + RGD and collagen control scaffolds. ES cells that were not differentiated as EBs prior to seeding on the scaffold, did not differentiate into cardiomyocytes. These results indicate that a collagen I/III scaffold supports cardiac muscle development and function after EB formation, and that this scaffold appears suitable for future in vivo testing. The addition of the RGD domain to the collagen scaffold did not improve cardiomyocyte development or viability, indicating that RGD signaling to integrins was not a rate-limiting event for cardiomyogenesis from EBs seeded on a collagen scaffold.

  8. Cloning of embryonal stem cell-specific genes: characterization of the transcriptionally controlled gene esg-1.

    Science.gov (United States)

    Bierbaum, P; MacLean-Hunter, S; Ehlert, F; Möröy, T; Müller, R

    1994-01-01

    We have isolated, by differential library screening, eight cDNAs representing genes that are specifically expressed in the embryonal stem cell line IMT-11, when compared to the parietal endoderm-like cell line PYS-2 or to NIH3T3 fibroblasts. One of these genes, embryonal stem cell gene 1 (esg-1), was analyzed in detail. esg-1 mRNA is found at high levels in both IMT-11 and F9 embryonal carcinoma cells and disappears during the differentiation of the stem cells. Furthermore, expression of the gene was found to be extremely low in, or absent from, oocytes and fertilized eggs, but it is strongly induced at the 2-cell stage, reaching maximum levels at the 4-cell stage. In contrast, esg-1 expression is detectable neither in midgestation embryos nor in neonatal tissues. These results strongly suggest that esg-1 is expressed specifically or at least predominantly in embryonal stem cells. Antibodies directed against a glutathione S-transferase-esg-1 fusion product detect a protein of M(r) approximately 14,000 in F9 embryonal carcinoma cells, but not in differentiated cells. Apart from the esg-1 gene, which contains two introns, there are at least seven esg-1-related pseudogenes in the mouse genome that differ from the esg-1 gene by the presence of multiple point mutations, by the lack of intervening sequences, and/or by the presence of a polyadenylated stretch at the 3' end. The esg-1 gene is under stringent transcriptional control in differentiating and differentiated cells, as shown by both nuclear run-on assays and the transient F9 stem cell-specific expression of constructs consisting of esg-1 upstream sequences fused to a luciferase reporter gene.

  9. A porous membrane-mediated isolation of mesenchymal stem cells from human embryonic stem cells.

    Science.gov (United States)

    Hong, Ki-Sung; Bae, Daekyeong; Choi, Youngsok; Kang, Sun-Woong; Moon, Sung-Hwan; Lee, Hoon Taek; Chung, Hyung-Min

    2015-03-01

    Pluripotent human embryonic stem cells (hESCs) acquire mesenchymal characteristics during the epithelial-mesenchymal transition (EMT) process. Here, we report a simple and an efficient isolation method for mesenchymal stem cells (MSCs) from hESCs undergoing EMT using a commercialized porous membrane transwell culture insert. Suspension culture of hESC colonies results in the formation of embryoid bodies, which adhered on the upper compartment of 8 μm porous membrane in the presence of EMG2-MV media. The population migrating through the permeable membrane to the lower compartment not only exhibited EMT markers but also expressed high levels of a panel of typical MSC surface antigen markers, and demonstrated multipotent differentiation capability. In addition, they have a prolonged proliferation capacity without characteristics and chromosomal changes. Furthermore, the isolated MSCs significantly enhanced cardiac functions in a rat model of myocardial infarction (MI) as measured by the left ventricle wall thickness (MI control, 32.9%±3.2% vs. hESCs-MSCs, 38.7%±2.4%), scar length (MI control, 46.1%±2.5% vs. hESCs-MSCs, 41.8%±1.3%), fibrosis area (MI control, 34.3%±1.6% vs. hESCs-MSCs, 28.9%±3.5%), and capillary density. Our findings demonstrate an ease with which hESCs-MSCs can be effectively isolated using the porous membrane, which overcomes the lack of availability of MSCs for therapeutic applications in various diseased animal models.

  10. Photo-transfection and selective optical differentiation of embryonic stem cells

    CSIR Research Space (South Africa)

    Mthunzi, P

    2010-09-01

    Full Text Available and differentiation programs are operated and regulated at the genetic level. Genetic manipulation such as delivery of exogenous gene expression or knockout with small interfering RNA (siRNA) is relatively rare in mouse embryonic (ES) cells. However, genetic species...

  11. [Generation of high proliferative potential hematopoietic progenitor cells from embryonic stem cell-derived BL-CFC].

    Science.gov (United States)

    Yao, Hui-Yu; Liu, Bing; Yuan, Ye; Mao, Ning

    2003-08-01

    The blast colony-forming cells (BL-CFC), which are detected within embryoid bodies derived from embryonic stem cells (ES cells) differentiated for 2.5-3.5 days, have dual-potential of differentiation to hematopoietic and endothelial cells. In this investigation the culture method of BL-CFC was established and colony forming assay, immunofluorescent technique as well as nested RT-PCR was employed to identify the differentiation capacity of adherent and nonadherent cells derived from individual blast colony. The results showed that the adherent cells could intake DiI-Ac-LDL and expressed the endothelium-specific surface markers including CD31, UEA-I and VE-cadherin. In addition, nonadherent cells were capable of developing primitive or/and definitive hematopoiesis potential. High proliferative potential colony-forming cells (HPP-CFC) bearing self-renewal capacity was found in 20% of BL-CFC. It is concluded that BL-CFC derived from embryonic stem cells can generate high proliferative potential hematopoietic progenitor cells. However, the whether BL-CFC can reconstitute the adult bone marrow hematopoiesis in long-term remains to be further determined.

  12. Notch-1 mediated cardiac protection following embryonic and induced pluripotent stem cell transplantation in doxorubicin-induced heart failure.

    Directory of Open Access Journals (Sweden)

    Hilda Merino

    Full Text Available Doxorubicin (DOX, an effective chemotherapeutic drug used in the treatment of various cancers, is limited in its clinical applications due to cardiotoxicity. Recent studies suggest that transplanted adult stem cells inhibit DOX-induced cardiotoxicity. However, the effects of transplanted embryonic stem (ES and induced pluripotent stem (iPS cells are completely unknown in DOX-induced left ventricular dysfunction following myocardial infarction (MI. In brief, C57BL/6 mice were divided into five groups: Sham, DOX-MI, DOX-MI+cell culture (CC media, DOX-MI+ES cells, and DOX-MI+iPS cells. Mice were injected with cumulative dose of 12 mg/kg of DOX and 2 weeks later, MI was induced by coronary artery ligation. Following ligation, 5×10(4 ES or iPS cells were delivered into the peri-infarct region. At day 14 post-MI, echocardiography was performed, mice were sacrificed, and hearts were harvested for further analyses. Our data reveal apoptosis was significantly inhibited in ES and iPS cell transplanted hearts compared with respective controls (DOX-MI+ES: 0.48±0.06% and DOX-MI+iPS: 0.33±0.05% vs.1.04±0.07% and DOX-MI+CC: 0.96±0.21%; p<0.05. Furthermore, a significant increase in levels of Notch-1 (p<0.05, Hes1 (p<0.05, and pAkt (p<0.05 were observed whereas a decrease in the levels of PTEN (p<0.05, a negative regulator of Akt, was evident following stem cell transplantation. Moreover, hearts transplanted with stem cells demonstrated decreased vascular and interstitial fibrosis (p<0.05 as well as MMP-9 expression (p<0.01 compared with controls. Additionally, heart function was significantly improved (p<0.05 in both cell-transplanted groups. In conclusion, our data show that transplantation of ES and iPS cells blunt DOX-induced adverse cardiac remodeling, which is associated with improved cardiac function, and these effects are mediated by the Notch pathway.

  13. From embryonic stem cells to testicular germ cell cancer-- should we be concerned?

    DEFF Research Database (Denmark)

    Almstrup, Kristian; Sonne, Si Brask; Hoei-Hansen, Christina E

    2006-01-01

    that initial hypothesis but also indicating that CIS cells have a striking phenotypic similarity to embryonic stem cells (ESC). Many cancers have been proposed to originate from tissue-specific stem cells [so-called 'cancer stem cells' (CSC)] and we argue that CIS may be a very good example of a CSC......, but with exceptional features due to the retention of embryonic pluripotency. In addition, considering the fact that pre-invasive CIS cells are transformed from early fetal cells, possibly due to environmentally induced alterations of the niche, we discuss potential risks linked to the uncontrolled therapeutic use......Since the discovery of testicular carcinoma in situ (CIS) -- the precursor cell for the vast majority of germ cell tumours -- it has been proposed that CIS cells could be derived from transformed primordial germ cells or gonocytes. Here, we review recent discoveries not only substantiating...

  14. Differential effects on cell motility, embryonic stem cell self-renewal and senescence by diverse Src kinase family inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Tamm, Christoffer, E-mail: christoffer.tamm@imbim.uu.se; Galito, Sara Pijuan, E-mail: sara.pijuan@imbim.uu.se; Anneren, Cecilia, E-mail: cecilia.anneren@imbim.uu.se

    2012-02-15

    The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt in proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2. -- Highlights: Black-Right-Pointing-Pointer SFK inhibitor SU6656 induces senescence in mouse ES cells. Black-Right-Pointing-Pointer SU6656 inhibits mitosis in a SFK-independent manner via cross-selectivity for Aurora kinases. Black-Right-Pointing-Pointer SFK inhibitor PP2 impairs cell motility in various cell lines, including mouse ES cells. Black-Right-Pointing-Pointer Ensuing impeded motility, PP2 inhibits proliferation of various cells lines except for mouse ES cells. Black-Right-Pointing-Pointer SFK inhibitors PP2 and PD173952 impede spontaneous differentiation in standard mouse ES culture maintenance.

  15. Homozygous mutation of focal adhesion kinase in embryonic stem cell derived neurons: normal electrophysiological and morphological properties in vitro

    Directory of Open Access Journals (Sweden)

    Komiyama NH

    2006-06-01

    Full Text Available Abstract Background Genetically manipulated embryonic stem (ES cell derived neurons (ESNs provide a powerful system with which to study the consequences of gene manipulation in mature, synaptically connected neurons in vitro. Here we report a study of focal adhesion kinase (FAK, which has been implicated in synapse formation and regulation of ion channels, using the ESN system to circumvent the embryonic lethality of homozygous FAK mutant mice. Results Mouse ES cells carrying homozygous null mutations (FAK-/- were generated and differentiated in vitro into neurons. FAK-/- ESNs extended axons and dendrites and formed morphologically and electrophysiologically intact synapses. A detailed study of NMDA receptor gated currents and voltage sensitive calcium currents revealed no difference in their magnitude, or modulation by tyrosine kinases. Conclusion FAK does not have an obligatory role in neuronal differentiation, synapse formation or the expression of NMDA receptor or voltage-gated calcium currents under the conditions used in this study. The use of genetically modified ESNs has great potential for rapidly and effectively examining the consequences of neuronal gene manipulation and is complementary to mouse studies.

  16. Effects of Synthetic Neural Adhesion Molecule Mimetic Peptides and Related Proteins on the Cardiomyogenic Differentiation of Mouse Embryonic Stem Cells

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    Ruodan Xu

    2015-04-01

    Full Text Available Background/Aims: Pluripotent stem cells differentiating into cardiomyocyte-like cells in an appropriate cellular environment have attracted significant attention, given the potential use of such cells for regenerative medicine. However, the precise mechanisms of lineage specification of pluripotent stem cells are still largely to be explored. Identifying the role of various small synthetic peptides involved in cardiomyogenesis may provide new insights into pathways promoting cardiomyogenesis. Methods: In the present study, using a transgenic murine embryonic stem (ES cell lineage expressing enhanced green fluorescent protein (EGFP under the control of α-myosin heavy chain (α-MHC promoter (pαMHC-EGFP, we investigated the cardiomyogenic effects of 7 synthetic peptides (Betrofin3, FGLs, FGLL, hNgf_C2, EnkaminE, Plannexin and C3 on cardiac differentiation. The expression of several cardiac-specific markers was determined by RT-PCR whereas the structural and functional properties of derived cardiomyocytes were examined by immunofluorescence and electrophysiology, respectively. Results: The results revealed that Betrofin3, an agonist of brain derived neurotrophic factor (BDNF peptide exerted the most striking pro-cardiomyogenic effect on ES cells. We found that BDNF receptor, TrkB expression was up-regulated during differentiation. Treatment of differentiating cells with Betrofin3 between days 3 and 5 enhanced the expression of cardiac-specific markers and improved cardiomyocyte differentiation and functionality as revealed by genes regulation, flow cytometry and patch clamp analysis. Thus Betrofin3 may exert its cardiomyogenic effects on ES cells via TrkB receptor. Conclusion: Taken together, the results suggest that Betrofin3 modulates BDNF signaling with positive cardiomyogenic effect in stage and dose-dependent manner providing an effective strategy to increase ES cell-based generation of cardiomyocytes and offer a novel therapeutic approach to

  17. Utx Is Required for Proper Induction of Ectoderm and Mesoderm during Differentiation of Embryonic Stem Cells

    DEFF Research Database (Denmark)

    Morales Torres, Cristina; Laugesen, Anne; Helin, Kristian

    2013-01-01

    for the activation of lineage choice genes in response to developmental signals. To further understand the function of Utx in pluripotency and differentiation we generated Utx knockout embryonic stem cells (ESCs). Here we show that Utx is not required for the proliferation of ESCs, however, Utx contributes......Embryonic development requires chromatin remodeling for dynamic regulation of gene expression patterns to ensure silencing of pluripotent transcription factors and activation of developmental regulators. Demethylation of H3K27me3 by the histone demethylases Utx and Jmjd3 is important...

  18. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Xiugong, E-mail: xiugong.gao@fda.hhs.gov; Sprando, Robert L.; Yourick, Jeffrey J.

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposure to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds.

  19. Photo-transfection of mouse embryonic stem cells with plasmid DNA using femtosecond laser pulses

    CSIR Research Space (South Africa)

    Thobakgale, Lebogang

    2017-01-01

    Full Text Available - blastocyst, germ layers Multipotent- related cell types, adult stem cells, red and white blood cells Oligopotent- Adult, lymphoid, myeloid Unipotent- Only one type, muscle Induced pluripotent- Adult ES genetically reprogrammed to pluripotency . Karla K et... al. Stem Cell: Basics, Classification and Applications. American Journal of Phytomedicine and Clinical Therapeutics.2014,27, pg 919-930 Therapeutic applications of ES Regeneration therapy • Diabetes- pancreatic β cells Fibroblasts, DiPS • Parkinson’s...

  20. Impact of homeopathic remedies on the expression of lineage differentiation genes: an in vitro approach using embryonic stem cells.

    Science.gov (United States)

    Jyoti, Saras; Tandon, Simran

    2016-05-01

    Well-documented studies of the potential effects and safety of homeopathic medicines in pregnancy are required. In this study, specific genes were studied which could serve as biomarkers for specification of three lineages to predict the safety of homeopathic remedies using mouse embryonic stem (ES) cells. Thus, the present work was to study the effects of homeopathic remedies taken during pregnancy using ES cells as the model. Mouse ES cells were exposed to 30C potency of Nux Vomica and Sepia, which are homeopathic medicines prescribed for the management of pregnancy related symptoms. Cytotoxicity studies were done using a modified Embryonic Stem cell test (EST). The expression levels of key genes and proteins were analyzed using real time polymerase chain reaction and immunocytochemistry, respectively. Homeopathic treatment led to modulations in the expression of certain lineage specific genes but this difference was not significant with respect to solvent control and showed normal differentiation as demonstrated by the expression of α/β MHC and α-actinin proteins in the differentiated ES cells. Our study for the first time has shown the feasibility of using ES cells in the developmental toxicity testing of remedies. The results suggest that they are not associated with developmental toxicity. Copyright © 2015 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.

  1. Use of "MGE enhancers" for labeling and selection of embryonic stem cell-derived medial ganglionic eminence (MGE progenitors and neurons.

    Directory of Open Access Journals (Sweden)

    Ying-Jiun J Chen

    Full Text Available The medial ganglionic eminence (MGE is an embryonic forebrain structure that generates the majority of cortical interneurons. MGE transplantation into specific regions of the postnatal central nervous system modifies circuit function and improves deficits in mouse models of epilepsy, Parkinson's disease, pain, and phencyclidine-induced cognitive deficits. Herein, we describe approaches to generate MGE-like progenitor cells from mouse embryonic stem (ES cells. Using a modified embryoid body method, we provided gene expression evidence that mouse ES-derived Lhx6(+ cells closely resemble immature interneurons generated from authentic MGE-derived Lhx6(+ cells. We hypothesized that enhancers that are active in the mouse MGE would be useful tools in detecting when ES cells differentiate into MGE cells. Here we demonstrate the utility of enhancer elements [422 (DlxI12b, Lhx6, 692, 1056, and 1538] as tools to mark MGE-like cells in ES cell differentiation experiments. We found that enhancers DlxI12b, 692, and 1538 are active in Lhx6-GFP(+ cells, while enhancer 1056 is active in Olig2(+ cells. These data demonstrate unique techniques to follow and purify MGE-like derivatives from ES cells, including GABAergic cortical interneurons and oligodendrocytes, for use in stem cell-based therapeutic assays and treatments.

  2. Differences in the epigenetic and reprogramming properties of pluripotent and extra-embryonic stem cells implicate chromatin remodelling as an important early event in the developing mouse embryo

    Directory of Open Access Journals (Sweden)

    Santos Joana

    2010-01-01

    Full Text Available Abstract Background During early mouse development, two extra-embryonic lineages form alongside the future embryo: the trophectoderm (TE and the primitive endoderm (PrE. Epigenetic changes known to take place during these early stages include changes in DNA methylation and modified histones, as well as dynamic changes in gene expression. Results In order to understand the role and extent of chromatin-based changes for lineage commitment within the embryo, we examined the epigenetic profiles of mouse embryonic stem (ES, trophectoderm stem (TS and extra-embryonic endoderm (XEN stem cell lines that were derived from the inner cell mass (ICM, TE and PrE, respectively. As an initial indicator of the chromatin state, we assessed the replication timing of a cohort of genes in each cell type, based on data that expressed genes and acetylated chromatin domains, generally, replicate early in S-phase, whereas some silent genes, hypoacetylated or condensed chromatin tend to replicate later. We found that many lineage-specific genes replicate early in ES, TS and XEN cells, which was consistent with a broadly 'accessible' chromatin that was reported previously for multiple ES cell lines. Close inspection of these profiles revealed differences between ES, TS and XEN cells that were consistent with their differing lineage affiliations and developmental potential. A comparative analysis of modified histones at the promoters of individual genes showed that in TS and ES cells many lineage-specific regulator genes are co-marked with modifications associated with active (H4ac, H3K4me2, H3K9ac and repressive (H3K27me3 chromatin. However, in XEN cells several of these genes were marked solely by repressive modifications (such as H3K27me3, H4K20me3. Consistent with TS and XEN having a restricted developmental potential, we show that these cells selectively reprogramme somatic cells to induce the de novo expression of genes associated with extraembryonic differentiation

  3. The "future like ours" argument and human embryonic stem cell research.

    Science.gov (United States)

    Kuflik, A

    2008-06-01

    The most closely argued and widely discussed case against abortion in the philosophical literature today is Don Marquis's "future like ours" argument. The argument moves from an analysis of why there is a serious presumption against killing someone "like us" to the conclusion that most abortions are seriously wrong for the same reason: they deprive "an individual" of a future of valuable experiences and activities, a "future like ours". Julian Savulescu has objected that "preventing" such a future could not be as seriously presumptively wrong as Marquis contends for if it were, even contraception and failure to engage in reproductive cloning would be seriously presumptively wrong. Savulescu maintains that there is only a modest presumption against preventing a "future of value like ours" and that in the case of human embryonic stem cell research, it is clearly outweighed by "the enormous potential to save people's lives and to improve their quality of life". Marquis defends his strong anti-abortion stance against Savulescu's "contraception" and "failure to clone" objections but surprisingly says nothing about the implications of the "future like ours" argument for the controversy surrounding human embryonic stem cell research. I argue that key features of Marquis's response actually support the view that embryos used in stem cell research are not included within the protective scope of the "future like ours" argument. It is significant that the most philosophically rigorous anti-abortion case thus far presented does not entail that human embryonic stem cell research is even presumptively wrong.

  4. Human embryonic stem cells differentiated to lung lineage-specific cells ameliorate pulmonary fibrosis in a xenograft transplant mouse model.

    Directory of Open Access Journals (Sweden)

    Ena Ray Banerjee

    Full Text Available Our aim was to differentiate human (h embryonic stem (ES cells into lung epithelial lineage-specific cells [i.e., alveolar epithelial type I (AEI and type II (AEII cells and Clara cells] as the first step in the development of cell-based strategies to repair lung injury in the bleomycin mouse model of idiopathic pulmonary fibrosis (IPF. A heterogeneous population of non-ciliated lung lineage-specific cells was derived by a novel method of embryoid body (EB differentiation. This differentiated human cell population was used to modulate the profibrotic phenotype in transplanted animals.Omission or inclusion of one or more components in the differentiation medium skewed differentiation of H7 hES cells into varying proportions of AEI, AEII, and Clara cells. ICG-001, a small molecule inhibitor of Wnt/β-catenin/Creb-binding protein (CBP transcription, changed marker expression of the differentiated ES cells from an AEII-like phenotype to a predominantly AEI-like phenotype. The differentiated cells were used in xenograft transplantation studies in bleomycin-treated Rag2γC(-/- mice. Human cells were detected in lungs of the transplanted groups receiving differentiated ES cells treated with or without ICG-001. The increased lung collagen content found in bleomycin-treated mice receiving saline was significantly reduced by transplantation with the lung-lineage specific epithelial cells differentiated from ES cells. A significant increase in progenitor number was observed in the airways of bleomycin-treated mice after transplantation of differentiated hES cells.This study indicates that ES cell-based therapy may be a powerful novel approach to ameliorate lung fibrosis.

  5. Generation of gene-targeted mice using embryonic stem cells derived from a transgenic mouse model of Alzheimer's disease.

    Science.gov (United States)

    Yamamoto, Satoshi; Ooshima, Yuki; Nakata, Mitsugu; Yano, Takashi; Matsuoka, Kunio; Watanabe, Sayuri; Maeda, Ryouta; Takahashi, Hideki; Takeyama, Michiyasu; Matsumoto, Yoshio; Hashimoto, Tadatoshi

    2013-06-01

    Gene-targeting technology using mouse embryonic stem (ES) cells has become the "gold standard" for analyzing gene functions and producing disease models. Recently, genetically modified mice with multiple mutations have increasingly been produced to study the interaction between proteins and polygenic diseases. However, introduction of an additional mutation into mice already harboring several mutations by conventional natural crossbreeding is an extremely time- and labor-intensive process. Moreover, to do so in mice with a complex genetic background, several years may be required if the genetic background is to be retained. Establishing ES cells from multiple-mutant mice, or disease-model mice with a complex genetic background, would offer a possible solution. Here, we report the establishment and characterization of novel ES cell lines from a mouse model of Alzheimer's disease (3xTg-AD mouse, Oddo et al. in Neuron 39:409-421, 2003) harboring 3 mutated genes (APPswe, TauP301L, and PS1M146V) and a complex genetic background. Thirty blastocysts were cultured and 15 stable ES cell lines (male: 11; female: 4) obtained. By injecting these ES cells into diploid or tetraploid blastocysts, we generated germline-competent chimeras. Subsequently, we confirmed that F1 mice derived from these animals showed similar biochemical and behavioral characteristics to the original 3xTg-AD mice. Furthermore, we introduced a gene-targeting vector into the ES cells and successfully obtained gene-targeted ES cells, which were then used to generate knockout mice for the targeted gene. These results suggest that the present methodology is effective for introducing an additional mutation into mice already harboring multiple mutated genes and/or a complex genetic background.

  6. Human embryonic stem cells differentiate into functional renal proximal tubular-like cells.

    Science.gov (United States)

    Narayanan, Karthikeyan; Schumacher, Karl M; Tasnim, Farah; Kandasamy, Karthikeyan; Schumacher, Annegret; Ni, Ming; Gao, Shujun; Gopalan, Began; Zink, Daniele; Ying, Jackie Y

    2013-04-01

    Renal cells are used in basic research, disease models, tissue engineering, drug screening, and in vitro toxicology. In order to provide a reliable source of human renal cells, we developed a protocol for the differentiation of human embryonic stem cells into renal epithelial cells. The differentiated stem cells expressed markers characteristic of renal proximal tubular cells and their precursors, whereas markers of other renal cell types were not expressed or expressed at low levels. Marker expression patterns of these differentiated stem cells and in vitro cultivated primary human renal proximal tubular cells were comparable. The differentiated stem cells showed morphological and functional characteristics of renal proximal tubular cells, and generated tubular structures in vitro and in vivo. In addition, the differentiated stem cells contributed in organ cultures for the formation of simple epithelia in the kidney cortex. Bioreactor experiments showed that these cells retained their functional characteristics under conditions as applied in bioartificial kidneys. Thus, our results show that human embryonic stem cells can differentiate into renal proximal tubular-like cells. Our approach would provide a source for human renal proximal tubular cells that are not affected by problems associated with immortalized cell lines or primary cells.

  7. Generation and Characterization of Erythroid Cells from Human Embryonic Stem Cells and Induced Pluripotent Stem Cells: An Overview

    Directory of Open Access Journals (Sweden)

    Kai-Hsin Chang

    2011-01-01

    Full Text Available Because of the imbalance in the supply and demand of red blood cells (RBCs, especially for alloimmunized patients or patients with rare blood phenotypes, extensive research has been done to generate therapeutic quantities of mature RBCs from hematopoietic stem cells of various sources, such as bone marrow, peripheral blood, and cord blood. Since human embryonic stem cells (hESCs and induced pluripotent stem cells (iPSCs can be maintained indefinitely in vitro, they represent potentially inexhaustible sources of donor-free RBCs. In contrast to other ex vivo stem-cell-derived cellular therapeutics, tumorigenesis is not a concern, as RBCs can be irradiated without marked adverse effects on in vivo function. Here, we provide a comprehensive review of the recent publications relevant to the generation and characterization of hESC- and iPSC-derived erythroid cells and discuss challenges to be met before the eventual realization of clinical usage of these cells.

  8. Serial in vivo positive contrast MRI of iron oxide-labeled embryonic stem cell-derived cardiac precursor cells in a mouse model of myocardial infarction.

    Science.gov (United States)

    Mani, Venkatesh; Adler, Eric; Briley-Saebo, Karen C; Bystrup, Anne; Fuster, Valentin; Keller, Gordon; Fayad, Zahi A

    2008-07-01

    Myocardial regeneration with stem-cell transplantation is a possible treatment option to reverse deleterious effects that occur after myocardial infarction. Since little is known about stem cell survival after transplantation, developing techniques for "tracking" cells would be desirable. Iron-oxide-labeled stem cells have been used for in vivo tracking using MRI but produce negative contrast images that are difficult to interpret. The aim of the current study was to test a positive contrast MR technique using reduced z-gradient rephasing (GRASP) to aid in dynamically tracking stem cells in an in vivo model of mouse myocardial infraction. Ferumoxides and protamine sulfate were complexed and used to magnetically label embryonic stem cell-derived cardiac-precursor-cells (ES-CPCs). A total of 500,000 ES-CPCs were injected in the border zone of infarcted mice and MR imaging was performed on a 9.4T scanner using T(2)*-GRE sequences (negative contrast) and positive contrast GRASP technique before, 24 hours, and 1 week after ES-CPC implantation. Following imaging, mice were sacrificed for histology and Perl's staining was used to confirm iron within myocardium. Good correlation was observed between signal loss seen on conventional T(2)* images, bright areas on GRASP, and the presence of iron on histology. This demonstrated the feasibility of in vivo stem cell imaging with positive contrast MRI. (c) 2008 Wiley-Liss, Inc.

  9. The class I-specific HDAC inhibitor MS-275 modulates the differentiation potential of mouse embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Gianluigi Franci

    2013-08-01

    Exploitation of embryonic stem cells (ESC for therapeutic use and biomedical applications is severely hampered by the risk of teratocarcinoma formation. Here, we performed a screen of selected epi-modulating compounds and demonstrate that a transient exposure of mouse ESC to MS-275 (Entinostat, a class I histone deacetylase inhibitor (HDAC, modulates differentiation and prevents teratocarcinoma formation. Morphological and molecular data indicate that MS-275-primed ESCs are committed towards neural differentiation, which is supported by transcriptome analyses. Interestingly, in vitro withdrawal of MS-275 reverses the primed cells to the pluripotent state. In vivo, MS275-primed ES cells injected into recipient mice give only rise to benign teratomas but not teratocarcinomas with prevalence of neural-derived structures. In agreement, MS-275-primed ESC are unable to colonize blastocysts. These findings provide evidence that a transient alteration of acetylation alters the ESC fate.

  10. Kidney specific protein-positive cells derived from embryonic stem cells reproduce tubular structures in vitro and differentiate into renal tubular cells.

    Science.gov (United States)

    Morizane, Ryuji; Monkawa, Toshiaki; Fujii, Shizuka; Yamaguchi, Shintaro; Homma, Koichiro; Matsuzaki, Yumi; Okano, Hideyuki; Itoh, Hiroshi

    2014-01-01

    Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP)-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.

  11. Linking incomplete reprogramming to the improved pluripotency of murine embryonal carcinoma cell-derived pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Gang Chang

    Full Text Available Somatic cell nuclear transfer (SCNT has been proved capable of reprogramming various differentiated somatic cells into pluripotent stem cells. Recently, induced pluripotent stem cells (iPS have been successfully derived from mouse and human somatic cells by the over-expression of a combination of transcription factors. However, the molecular mechanisms underlying the reprogramming mediated by either the SCNT or iPS approach are poorly understood. Increasing evidence indicates that many tumor pathways play roles in the derivation of iPS cells. Embryonal carcinoma (EC cells have the characteristics of both stem cells and cancer cells and thus they might be the better candidates for elucidating the details of the reprogramming process. Although previous studies indicate that EC cells cannot be reprogrammed into real pluripotent stem cells, the reasons for this remain unclear. Here, nuclei from mouse EC cells (P19 were transplanted into enucleated oocytes and pluripotent stem cells (P19 NTES cells were subsequently established. Interestingly, P19 NTES cells prolonged the development of tetraploid aggregated embryos compared to EC cells alone. More importantly, we found that the expression recovery of the imprinted H19 gene was dependent on the methylation state in the differential methylation region (DMR. The induction of Nanog expression, however, was independent of the promoter region DNA methylation state in P19 NTES cells. A whole-genome transcriptome analysis further demonstrated that P19 NTES cells were indeed the intermediates between P19 cells and ES cells and many interesting genes were uncovered that may be responsible for the failed reprogramming of P19 cells. To our knowledge, for the first time, we linked incomplete reprogramming to the improved pluripotency of EC cell-derived pluripotent stem cells. The candidate genes we discovered may be useful not only for understanding the mechanisms of reprogramming, but also for deciphering the

  12. The dynamic changes of X chromosome inactivation during early culture of human embryonic stem cells.

    Science.gov (United States)

    Xie, Pingyuan; Ouyang, Qi; Leng, Lizhi; Hu, Liang; Cheng, Dehua; Tan, Yueqiu; Lu, Guangxiu; Lin, Ge

    2016-07-01

    X chromosome inactivation (XCI) is required for dosage compensation of X-linked genes in human female cells. Several previous reports have described the promiscuous XCI status in long-term cultured female human embryonic stem cells (hESCs), and the majority of them exhibit non-random XCI. However, when and how such female hESCs acquire the aberrant XCI states during culture is unknown. Herein, through comparing the XCI states in 18 paired hES cell lines throughout early culture, we revealed a uniform dynamic change during this culture period under a widely used culture condition. The female initial hESCs (ihESCs, P4-P9) expressed XIST RNA, H3K27me3 punctate enrichment and displayed random XCI pattern. By further culturing, the female early hESCs (ehESCs, P20-P30) lost the expression of XIST RNA, H3K27me3 punctate enrichment and exhibited a completely skewed XCI pattern. Importantly, a subset of X-linked genes was up-regulated in ehESCs, including some cancer-related genes. At last, we found 5% physiological oxygen was beneficial for the expression of XIST and H3K27me3 punctate enrichment, but not for the XCI pattern. We conclude that the XCI dynamic change is a frequent epigenetic instability event during early culture, which is accompanied by the up-regulation of some X-linked genes. Furthermore, we emphasize that physiological oxygen is beneficial for XCI fidelity. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Chan-Jung Chang

    Full Text Available We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.

  14. "Harvesting" and Use of Human (Embryonic) Stem Cells: An Islamic Evaluation.

    Science.gov (United States)

    Bouzenita, Anke I

    2017-03-01

    This paper gives insight into the Islamic bioethical discussion on harvesting and using human embryonic (hESC) and adult stem cells. It describes some of the Islamic legal mechanisms involved in the bioethical discourse among Muslims. As the contemporary Islamic bioethical discourse is very diverse, the paper focuses on the critical discussion of related resolutions of the Saudi-based Islamic Fiqh Academy due to the esteem in which the IFA is held in the Islamic world and the pertinence of their rulings on this issue. This study discusses the different sources of human adult and embryonic stem cells and their use from an Islamic perspective, while questioning some directions the Islamic bioethical discourse has taken. The paper invites interested parties to deliberate the use of some of the legal means resorted to in the ongoing Islamic bioethical discourse.

  15. New frontiers in gene targeting and cloning: success, application and challenges in domestic animals and human embryonic stem cells

    National Research Council Canada - National Science Library

    C Denning; H Priddle

    2003-01-01

    Until recently, precise modification of the animal genome by gene targeting was restricted to the mouse because germline competent embryonic stem cells are not available in any other mammalian species. Nuclear transfer (NT...

  16. Development of heart muscle-cell diversity: a help or a hindrance for phenotyping embryonic stem cell-derived cardiomyocytes

    NARCIS (Netherlands)

    Fijnvandraat, Arnoud C.; Lekanne Deprez, Ronald H.; Moorman, Antoon F. M.

    2003-01-01

    Despite the advances in cardiovascular treatment, cardiac disease remains a major cause of morbidity in all industrialized countries. The extraordinary potential of (embryonic) stem cells for therapeutic purposes has revolutionized ideas about cardiac repair of diseased cardiac muscle to exciting

  17. Dynamic MicroRNA Expression Programs During Cardiac Differentiation of Human Embryonic Stem Cells: Role for miR-499

    National Research Council Canada - National Science Library

    Wilson, Kitchener D; Hu, Shijun; Venkatasubrahmanyam, Shivkumar; Fu, Ji-Dong; Sun, Ning; Abilez, Oscar J; Baugh, Joshua J.A; Jia, Fangjun; Ghosh, Zhumur; Li, Ronald A; Butte, Atul J; Wu, Joseph C

    2010-01-01

    .... Human embryonic stem cells are known to express miRNAs that are often undetectable in adult organs, and a growing body of evidence has implicated miRNAs as important arbiters of heart development and disease...

  18. Transcriptomic concentration-response evaluation of valproic acid, cyproconazole, and hexaconazole in the neural embryonic stem cell test (ESTn)

    NARCIS (Netherlands)

    Theunissen, P.; Robinson, J.F.; Pennings, J.L.A.; de Jong, E.; Claessen, S.M.H.; Kleinjans, J.C.S.; Piersma, A.H.

    2012-01-01

    Alternative developmental toxicity assays are urgently needed to reduce animal use in regulatory developmental toxicology. We previously designed an in vitro murine neural embryonic stem cell test (ESTn) as a model for neurodevelopmental toxicity testing (Theunissen et al., 2010). Toxicogenomic

  19. Β-amyloid 1-42 oligomers impair function of human embryonic stem cell-derived forebrain cholinergic neurons.

    Directory of Open Access Journals (Sweden)

    Linn Wicklund

    Full Text Available Cognitive impairment in Alzheimer's disease (AD patients is associated with a decline in the levels of growth factors, impairment of axonal transport and marked degeneration of basal forebrain cholinergic neurons (BFCNs. Neurogenesis persists in the adult human brain, and the stimulation of regenerative processes in the CNS is an attractive prospect for neuroreplacement therapy in neurodegenerative diseases such as AD. Currently, it is still not clear how the pathophysiological environment in the AD brain affects stem cell biology. Previous studies investigating the effects of the β-amyloid (Aβ peptide on neurogenesis have been inconclusive, since both neurogenic and neurotoxic effects on progenitor cell populations have been reported. In this study, we treated pluripotent human embryonic stem (hES cells with nerve growth factor (NGF as well as with fibrillar and oligomeric Aβ1-40 and Aβ1-42 (nM-µM concentrations and thereafter studied the differentiation in vitro during 28-35 days. The process applied real time quantitative PCR, immunocytochemistry as well as functional studies of intracellular calcium signaling. Treatment with NGF promoted the differentiation into functionally mature BFCNs. In comparison to untreated cells, oligomeric Aβ1-40 increased the number of functional neurons, whereas oligomeric Aβ1-42 suppressed the number of functional neurons. Interestingly, oligomeric Aβ exposure did not influence the number of hES cell-derived neurons compared with untreated cells, while in contrast fibrillar Aβ1-40 and Aβ1-42 induced gliogenesis. These findings indicate that Aβ1-42 oligomers may impair the function of stem cell-derived neurons. We propose that it may be possible for future AD therapies to promote the maturation of functional stem cell-derived neurons by altering the brain microenvironment with trophic support and by targeting different aggregation forms of Aβ.

  20. Alginate Encapsulation Parameters Influence the Differentiation of Microencapsulated Embryonic Stem Cell Aggregates

    OpenAIRE

    Wilson, Jenna L.; Najia, Mohamad Ali; Saeed, Rabbia; McDevitt, Todd C.

    2013-01-01

    Pluripotent embryonic stem cells (ESCs) have tremendous potential as tools for regenerative medicine and drug discovery, yet the lack of processes to manufacture viable and homogenous cell populations of sufficient numbers limits the clinical translation of current and future cell therapies. Microencapsulation of ESCs within microbeads can shield cells from hydrodynamic shear forces found in bioreactor environments while allowing for sufficient diffusion of nutrients and oxygen through the en...

  1. Highly efficient differentiation of embryonic stem cells into adipocytes by ascorbic acid

    OpenAIRE

    Cuaranta-Monroy, Ixchelt; Simandi, Zoltan; Kolostyak, Zsuzsanna; Doan-Xuan, Quang-Minh (1986-) (biofizikus); Poliska, Szilard; Horvath, Attila; Nagy, Gergely; Bacso, Zsolt; Nagy, Laszlo

    2014-01-01

    Adipocyte differentiation and function have become the major research targets due to the increasing interest in obesity and related metabolic conditions. Although, late stages of adipogenesis have been extensively studied, the early phases remain poorly understood. Here we present that supplementing ascorbic acid (AsA) to the adipogenic differentiation cocktail enables the robust and efficient differentiation of mouse embryonic stem cells (mESCs) to mature adipocytes. Such ESC-derived adipocy...

  2. Microencapsulation Technology: A Powerful Tool for Integrating Expansion and Cryopreservation of Human Embryonic Stem Cells

    OpenAIRE

    Margarida Serra; Cláudia Correia; Rita Malpique; Catarina Brito; Janne Jensen; Petter Bjorquist; Carrondo, Manuel J. T.; Alves, Paula M

    2011-01-01

    The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) ag...

  3. Microencapsulation technology: a powerful tool for human embryonic stem cells expansion and cryopreservation

    OpenAIRE

    Correia, Cláudia Susana Pedreira

    2010-01-01

    Dissertation presented to obtain a Master degree in Biotechnology at the Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia Human embryonic stem cells (hESCs) are known by their ability to either self-renewal and differentiate into any adult cell type. These properties confer to hESCs a huge applicability for cell therapy, tissue engineering and drug screening. However, successful implementation of hESCs-based technologies requires the production of large numbers of well chara...

  4. Adapted cytokinesis-block micronucleus assay (CBMn) for mouse embryonic stem cells

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Hamid Kalantari, Hamid Gourabi & Hossein Baharvand ### Abstract Our observation showed the addition of cytochalasin-B to mouse embryonic stem cells (mESC) culture for CBMn analysis led to the induction of apoptosis in these cells. On the other hand, addition of cyt-B is the most critical part of the cytokinesis-block micronucleus assay (CBMn) technique that cannot be omitted. Thus, modification of the traditional CBMn assay seems to be necessary. In this paper, we attempt to ...

  5. Radial glial cells defined and major intermediates between embryonic stem cells and CNS neurons.

    Science.gov (United States)

    Götz, Magdalena; Barde, Yves-Alain

    2005-05-05

    Radial glial cells have been identified as a major source of neurons during development. Here, we review the evidence for the distinct "glial" nature of radial glial cells and contrast these cells with their progenitors, the neuroepithelial cells. Recent results also suggest that not only during neurogenesis in vivo, but also during the differentiation of cultured embryonic stem cells toward neurons, progenitors with clear glial antigenic characteristics act as cellular intermediates.

  6. Derivation of human embryonic stem cell from spinal muscular atrophy patient

    Directory of Open Access Journals (Sweden)

    Pingyuan Xie

    2016-03-01

    Full Text Available We established a human embryonic stem cell (hESC line chHES-427 from the abnormal embryo carrying homozygous deletion of exon 7 of survival motor neuron gene (SMN. This cell line maintained a normal karyotype 46, XX during long-term culture. Further characteristic analysis suggested that the cells expressed the pluripotency-related markers and had the capacity to differentiate into the derivatives from the three germ layers in vitro.

  7. A protocol for embryonic stem cell derivation by somatic cell nuclear transfer into human oocytes

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Dieter Egli & Gloryn Chia ### Abstract Here we describe detailed methods that allowed us to derive embryonic stem cell lines by nuclear transfer of fibroblasts from a newborn and from a type 1 diabetic adult. The protocol is based on the insight that 1) agents for cell fusion can act as potent mediators of oocyte activation by compromising maintaining plasma membrane integrity; minimizing the concentration at which they are used, and at least transiently remove calcium from ...

  8. Phenotypic and Functional Characterization of Peripheral Sensory Neurons derived from Human Embryonic Stem Cells

    OpenAIRE

    Alshawaf, Abdullah Jawad; Viventi, Serena; Qiu, Wanzhi; D’Abaco, Giovanna; Nayagam, Bryony; Erlichster, Michael; Chana, Gursharan; Everall, Ian; Ivanusic, Jason; Skafidas, Efstratios; Dottori, Mirella

    2018-01-01

    The dorsal root ganglia (DRG) consist of a multitude of sensory neuronal subtypes that function to relay sensory stimuli, including temperature, pressure, pain and position to the central nervous system. Our knowledge of DRG sensory neurons have been predominantly driven by animal studies and considerably less is known about the human DRG. Human embryonic stem cells (hESC) are valuable resource to help close this gap. Our previous studies reported an efficient system for deriving neural crest...

  9. The effects of silver nanoparticles on mouse embryonic stem cell self-renewal and proliferation

    OpenAIRE

    Rajanahalli, Pavan; Stucke, Christopher J.; Hong, Yiling

    2015-01-01

    Silver nanoparticles (AgNPs) are gaining rapid popularity in many commonly used medical and commercial products for their unique anti-bacterial properties. The molecular mechanisms of effects of AgNPs on stem cell self-renewal and proliferation have not yet been well understood. The aim of the work is to use mouse embryonic stem cells (mESCs) as a cellular model to evaluate the toxicity of AgNPs. mESC is a very special cell type which has self-renewal and differentiation properties. The objec...

  10. The postischemic environment differentially impacts teratoma or tumor formation after transplantation of human embryonic stem cell-derived neural progenitors

    DEFF Research Database (Denmark)

    Seminatore, Christine; Polentes, Jerome; Ellman, Ditte

    2010-01-01

    Risk of tumorigenesis is a major obstacle to human embryonic and induced pluripotent stem cell therapy. Likely linked to the stage of differentiation of the cells at the time of implantation, formation of teratoma/tumors can also be influenced by factors released by the host tissue. We have...... analyzed the relative effects of the stage of differentiation and the postischemic environment on the formation of adverse structures by transplanted human embryonic stem cell-derived neural progenitors....

  11. Novel Method To Differentiate Human Embryonic Stem Cells Into Dopaminergic Nerve Cells | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Institute on Drug Abuse's Development and Plasticity Section is seeking statements of capability or interest from parties interested in licensing opportunities to further develop, evaluate, or commercialize novel methods to differentiate human embryonic stem cells into dopaminergic nerve cells. The invention described here is a novel method of differentiating human embryonic stem cells (hESCs) into dopaminergic nerve cells, which is preferable to the currently available dopaminergic differentiation techniques.

  12. Nuclear Magnetic Resonance Detects Phosphoinositide 3-Kinase/Akt-Independent Traits Common to Pluripotent Murine Embryonic Stem Cells and Their Malignant Counterparts

    Directory of Open Access Journals (Sweden)

    Hanna M. Romanska

    2009-12-01

    Full Text Available Pluripotent embryonic stem (ES cells, a potential source of somatic precursors for cell therapies, cause tumors after transplantation. Studies of mammalian carcinogenesis using nuclear magnetic resonance (NMR spectroscopy have revealed changes in the choline region, particularly increased phosphocholine (PCho content. High PCho levels in murine ES (mES cells have recently been attributed to cell pluripotency. The phosphoinositide 3-kinase (PI3K/Akt pathway has been implicated in tumor-like properties of mES cells. This study aimed to examine a potential link between the metabolic profile associated with choline metabolism of pluripotent mES cells and PI3K/Akt signaling. We used mES (ES-D3 and murine embryonal carcinoma cells (EC-F9 and compared the metabolic profiles of 1 pluripotent mES (ESD0, 2 differentiated mES (ESD14, and 3 pluripotent F9 cells. Involvement of the PI3K/Akt pathway was assessed using LY294002, a selective PI3K inhibitor. Metabolic profiles were characterized in the extracted polar fraction by 1H NMR spectroscopy. Similarities were found between the levels of choline phospholipid metabolites (PCho/total choline and PCho/glycerophosphocholine [GPCho] in ESD0 and F9 cell spectra and a greater-than five-fold decrease of the PCho/GPCho ratio associated with mES cell differentiation. LY294002 caused no significant change in relative PCho levels but led to a greater-than two-fold increase in PCho/GPCho ratios. These results suggest that the PCho/GPCho ratio is a metabolic trait shared by pluripotent and malignant cells and that PI3K does not underlie its development. It is likely that the signature identified here in a mouse model may be relevant for safe therapeutic applications of human ES cells.

  13. Changes in glycosaminoglycan structure on differentiation of human embryonic stem cells towards mesoderm and endoderm lineages.

    Science.gov (United States)

    Gasimli, Leyla; Hickey, Anne Marie; Yang, Bo; Li, Guoyun; dela Rosa, Mitche; Nairn, Alison V; Kulik, Michael J; Dordick, Jonathan S; Moremen, Kelley W; Dalton, Stephen; Linhardt, Robert J

    2014-06-01

    Proteoglycans are found on the cell surface and in the extracellular matrix, and serve as prime sites for interaction with signaling molecules. Proteoglycans help regulate pathways that control stem cell fate, and therefore represent an excellent tool to manipulate these pathways. Despite their importance, there is a dearth of data linking glycosaminoglycan structure within proteoglycans with stem cell differentiation. Human embryonic stem cell line WA09 (H9) was differentiated into early mesoderm and endoderm lineages, and the glycosaminoglycanomic changes accompanying these transitions were studied using transcript analysis, immunoblotting, immunofluorescence and disaccharide analysis. Pluripotent H9 cell lumican had no glycosaminoglycan chains whereas in splanchnic mesoderm lumican was glycosaminoglycanated. H9 cells have primarily non-sulfated heparan sulfate chains. On differentiation towards splanchnic mesoderm and hepatic lineages N-sulfo group content increases. Differences in transcript expression of NDST1, HS6ST2 and HS6ST3, three heparan sulfate biosynthetic enzymes, within splanchnic mesoderm cells compared to H9 cells correlate to changes in glycosaminoglycan structure. Differentiation of embryonic stem cells markedly changes the proteoglycanome. The glycosaminoglycan biosynthetic pathway is complex and highly regulated, and therefore, understanding the details of this pathway should enable better control with the aim of directing stem cell differentiation. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Fibroblasts derived from human embryonic stem cells direct development and repair of 3D human skin equivalents.

    Science.gov (United States)

    Shamis, Yulia; Hewitt, Kyle J; Carlson, Mark W; Margvelashvilli, Mariam; Dong, Shumin; Kuo, Catherine K; Daheron, Laurence; Egles, Christophe; Garlick, Jonathan A

    2011-02-21

    Pluripotent, human stem cells hold tremendous promise as a source of progenitor and terminally differentiated cells for application in future regenerative therapies. However, such therapies will be dependent upon the development of novel approaches that can best assess tissue outcomes of pluripotent stem cell-derived cells and will be essential to better predict their safety and stability following in vivo transplantation. In this study we used engineered, human skin equivalents (HSEs) as a platform to characterize fibroblasts that have been derived from human embryonic stem (hES) cell. We characterized the phenotype and the secretion profile of two distinct hES-derived cell lines with properties of mesenchymal cells (EDK and H9-MSC) and compared their biological potential upon induction of differentiation to bone and fat and following their incorporation into the stromal compartment of engineered, HSEs. While both EDK and H9-MSC cell lines exhibited similar morphology and mesenchymal cell marker expression, they demonstrated distinct functional properties when incorporated into the stromal compartment of HSEs. EDK cells displayed characteristics of dermal fibroblasts that could support epithelial tissue development and enable re-epithelialization of wounds generated using a 3D tissue model of cutaneous wound healing, which was linked to elevated production of hepatocyte growth factor (HGF). Lentiviral shRNA-mediated knockdown of HGF resulted in a dramatic decrease of HGF secretion from EDK cells that led to a marked reduction in their ability to promote keratinocyte proliferation and re-epithelialization of cutaneous wounds. In contrast, H9-MSCs demonstrated features of mesenchymal stem cells (MSC) but not those of dermal fibroblasts, as they underwent multilineage differentiation in monolayer culture, but were unable to support epithelial tissue development and repair and produced significantly lower levels of HGF. Our findings demonstrate that hES-derived cells

  15. Human embryonic stem cell-derived oligodendrocyte progenitors aid in functional recovery of sensory pathways following contusive spinal cord injury.

    Directory of Open Access Journals (Sweden)

    Angelo H All

    Full Text Available Transplantations of human stem cell derivatives have been widely investigated in rodent models for the potential restoration of function of neural pathways after spinal cord injury (SCI. Studies have already demonstrated cells survival following transplantation in SCI. We sought to evaluate survival and potential therapeutic effects of transplanted human embryonic stem (hES cell-derived oligodendrocyte progenitor cells (OPCs in a contusive injury in rats. Bioluminescence imaging was utilized to verify survivability of cells up to 4 weeks, and somatosensory evoked potential (SSEPs were recorded at the cortex to monitor function of sensory pathways throughout the 6-week recovery period.hES cells were transduced with the firefly luciferase gene and differentiated into OPCs. OPCs were transplanted into the lesion epicenter of rat spinal cords 2 hours after inducing a moderate contusive SCI. The hES-treatment group showed improved SSEPs, including increased amplitude and decreased latencies, compared to the control group. The bioluminescence of transplanted OPCs decreased by 97% in the injured spinal cord compared to only 80% when injected into an uninjured spinal cord. Bioluminescence increased in both experimental groups such that by week 3, no statistical difference was detected, signifying that the cells survived and proliferated independent of injury. Post-mortem histology of the spinal cords showed integration of human cells expressing mature oligodendrocyte markers and myelin basic protein without the expression of markers for astrocytes (GFAP or pluripotent cells (OCT4.hES-derived OPCs transplanted 2 hours after contusive SCI survive and differentiate into OLs that produce MBP. Treated rats demonstrated functional improvements in SSEP amplitudes and latencies compared to controls as early as 1 week post-injury. Finally, the hostile injury microenvironment at 2 hours post-injury initially caused increased cell death but did not affect the long

  16. Derivation of keratinocytes from chicken embryonic stem cells: Establishment and characterization of differentiated proliferative cell populations

    Directory of Open Access Journals (Sweden)

    Mathilde Couteaudier

    2015-03-01

    Full Text Available A common challenge in avian cell biology is the generation of differentiated cell-lines, especially in the keratinocyte lineage. Only a few avian cell-lines are available and very few of them show an interesting differentiation profile. During the last decade, mammalian embryonic stem cell-lines were shown to differentiate into almost all lineages, including keratinocytes. Although chicken embryonic stem cells had been obtained in the 1990s, few differentiation studies toward the ectodermal lineage were reported. Consequently, we explored the differentiation of chicken embryonic stem cells toward the keratinocyte lineage by using a combination of stromal induction, ascorbic acid, BMP4 and chicken serum. During the induction period, we observed a downregulation of pluripotency markers and an upregulation of epidermal markers. Three homogenous cell populations were derived, which were morphologically similar to chicken primary keratinocytes, displaying intracellular lipid droplets in almost every pavimentous cell. These cells could be serially passaged without alteration of their morphology and showed gene and protein expression profiles of epidermal markers similar to chicken primary keratinocytes. These cells represent an alternative to the isolation of chicken primary keratinocytes, being less cumbersome to handle and reducing the number of experimental animals used for the preparation of primary cells.

  17. Confrontation cultures of embryonic stem cells with multicellular tumor spheroids to study tumor-induced angiogenesis.

    Science.gov (United States)

    Wartenberg, Maria; Finkensieper, Andreas; Hescheler, Jürgen; Sauer, Heinrich

    2006-01-01

    Human embryonic stem cells efficiently differentiate blood vessels, which allows using this in vitro model to study the interaction of blood vessels with adjacent tissues. Herein, we introduce confrontation cultures of human embryonic stem cells with multicellular tumor spheroids to investigate molecular mechanisms of tumor-induced angiogenesis. Vascularization of tumor tissue by the host is a prerequisite for tumor growth, which has led to the development of antiangiogenic therapy. This promising anti-cancer therapy intends to reduce, halt, or even regress tumor growth by deprivation from blood, oxygen, and nutrient supply. Confrontation cultures of human embryonic stem cells with multicellular tumor spheroids allow the investigation of the time course of endothelial cell invasion into the tumor tissue, the concomitant analysis of changes in angiogenesis-related gene expression, and analysis of the cellular microenvironment (i.e., pericellular oxygen pressure, tissue pH, and levels of tissue reactive oxygen species). The in vitro model of confrontation cultures is suitable for routine screening of antiangiogenic agents in pre-clinical trials and may be used to replace animal experiments applied in antiangiogenesis research.

  18. Induction of corneal epithelium-like cells from cynomolgus monkey embryonic stem cells and their experimental transplantation to damaged cornea.

    Science.gov (United States)

    Kumagai, Yuta; Kurokawa, Manae S; Ueno, Hiroki; Kayama, Maki; Tsubota, Kazuo; Nakatsuji, Norio; Kondo, Yasushi; Ueno, Satoki; Suzuki, Noboru

    2010-04-01

    We previously reported the successful transplantation of corneal epithelium-like cells derived from mouse embryonic stem (ES) cells onto injured mouse cornea. Here, we tested whether nonhuman primate ES cells have ability to differentiate into corneal epithelial cells and whether monkey ES cell-derived corneal epithelium-like cells were applicable for the experimental transplantation to damaged cornea. Monkey ES cells were cultivated on type IV collagen-coated dishes for various days to induce differentiation into corneal epithelium-like cells. The differentiation was evaluated by reverse transcription-polymerase chain reaction and immunostaining. The corneal epithelium-like cells were transplanted to the injured mouse cornea. Reconstitution of the corneal epithelium was evaluated by immunostaining. The cells cultured on type IV collagen showed cobblestone-like appearance resembling epithelial cells. They expressed messenger RNA of pax6, p63, E-cadherin, CD44, proliferating cell nuclear antigen, keratin 3, and keratin 12. Protein expressions of pax6, keratin 3/12, p63, proliferating cell nuclear antigen, E-cadherin, and CD44 were confirmed by immunostaining. When the corneal epithelium-like cells were transplanted, they adhered to the corneal stroma, leading to formation of multiple cell layers. The grafted cells were stained with anti-human nuclear protein antibody, which cross-reacted with nuclei of monkey cells but not with those of mouse cells. They retained the expressions of keratin 3/12, E-cadherin, and CD44. We induced corneal epithelium-like cells from monkey ES cells with moderate efficiency. The cells were successfully transplanted onto the injured mouse cornea. This is the first demonstration that nonhuman primate ES cells were induced to differentiate into corneal epithelium-like cells, which were applicable for transplantation to an animal model of corneal injury.

  19. Inactivation of SAG E3 ubiquitin ligase blocks embryonic stem cell differentiation and sensitizes leukemia cells to retinoid acid.

    Directory of Open Access Journals (Sweden)

    Mingjia Tan

    Full Text Available Sensitive to Apoptosis Gene (SAG, also known as RBX2 (RING box protein-2, is the RING component of SCF (SKP1, Cullin, and F-box protein E3 ubiquitin ligase. Our previous studies have demonstrated that SAG is an anti-apoptotic protein and an attractive anti-cancer target. We also found recently that Sag knockout sensitized mouse embryonic stem cells (mES to radiation and blocked mES cells to undergo endothelial differentiation. Here, we reported that compared to wild-type mES cells, the Sag(-/- mES cells were much more sensitive to all-trans retinoic acid (RA-induced suppression of cell proliferation and survival. While wild-type mES cells underwent differentiation upon exposure to RA, Sag(-/- mES cells were induced to death via apoptosis instead. The cell fate change, reflected by cellular stiffness, can be detected as early as 12 hrs post RA exposure by AFM (Atomic Force Microscopy. We then extended this novel finding to RA differentiation therapy of leukemia, in which the resistance often develops, by testing our hypothesis that SAG inhibition would sensitize leukemia to RA. Indeed, we found a direct correlation between SAG overexpression and RA resistance in multiple leukemia lines. By using MLN4924, a small molecule inhibitor of NEDD8-Activating Enzyme (NAE, that inactivates SAG-SCF E3 ligase by blocking cullin neddylation, we were able to sensitize two otherwise resistant leukemia cell lines, HL-60 and KG-1 to RA. Mechanistically, RA sensitization by MLN4924 was mediated via enhanced apoptosis, likely through accumulation of pro-apoptotic proteins NOXA and c-JUN, two well-known substrates of SAG-SCF E3 ligase. Taken together, our study provides the proof-of-concept evidence for effective treatment of leukemia patients by RA-MLN4924 combination.

  20. Abrogation of E-cadherin-mediated cellular aggregation allows proliferation of pluripotent mouse embryonic stem cells in shake flask bioreactors.

    Directory of Open Access Journals (Sweden)

    Lisa Mohamet

    2010-09-01

    Full Text Available A fundamental requirement for the exploitation of embryonic stem (ES cells in regenerative medicine is the ability to reproducibly derive sufficient numbers of cells of a consistent quality in a cost-effective manner. However, undifferentiated ES cells are not ideally suited to suspension culture due to the formation of cellular aggregates, ultimately limiting scalability. Significant advances have been made in recent years in the culture of ES cells, including automated adherent culture and suspension microcarrier or embryoid body bioreactor culture. However, each of these methods exhibits specific disadvantages, such as high cost, additional downstream processes or reduced cell doubling times.Here we show that abrogation of the cell surface protein E-cadherin, using either gene knockout (Ecad-/- or the neutralising antibody DECMA-1 (EcadAb, allows culture of mouse ES cells as a near-single cell suspension in scalable shake flask culture over prolonged periods without additional media supplements. Both Ecad-/- and EcadAb ES cells exhibited adaptation phases in suspension culture, with optimal doubling times of 7.3 h±0.9 and 15.6 h±4.7 respectively and mean-fold increase in viable cell number of 95.1±2.0 and 16±0.9-fold over 48 h. EcadAb ES cells propagated as a dispersed cell suspension for 15 d maintained expression of pluripotent markers, exhibited a normal karyotype and high viability. Subsequent differentiation of EcadAb ES cells resulted in expression of transcripts and proteins associated with the three primary germ layers.This is the first demonstration of the culture of pluripotent ES cells as a near-single cell suspension in a manual fed-batch shake flask bioreactor and represents a significant improvement on current ES cell culture techniques. Whilst this proof-of-principle method would be useful for the culture of human ES and iPS cells, further steps are necessary to increase cell viability of hES cells in suspension.

  1. E-cadherin acts as a regulator of transcripts associated with a wide range of cellular processes in mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Francesca Soncin

    Full Text Available We have recently shown that expression of the cell adhesion molecule E-cadherin is required for LIF-dependent pluripotency of mouse embryonic stem (ES cells.In this study, we have assessed global transcript expression in E-cadherin null (Ecad-/- ES cells cultured in either the presence or absence of LIF and compared these to the parental cell line wtD3.We show that LIF has little effect on the transcript profile of Ecad-/- ES cells, with statistically significant transcript alterations observed only for Sp8 and Stat3. Comparison of Ecad-/- and wtD3 ES cells cultured in LIF demonstrated significant alterations in the transcript profile, with effects not only confined to cell adhesion and motility but also affecting, for example, primary metabolic processes, catabolism and genes associated with apoptosis. Ecad-/- ES cells share similar, although not identical, gene expression profiles to epiblast-derived pluripotent stem cells, suggesting that E-cadherin expression may inhibit inner cell mass to epiblast transition. We further show that Ecad-/- ES cells maintain a functional β-catenin pool that is able to induce β-catenin/TCF-mediated transactivation but, contrary to previous findings, do not display endogenous β-catenin/TCF-mediated transactivation. We conclude that loss of E-cadherin in mouse ES cells leads to significant transcript alterations independently of β-catenin/TCF transactivation.

  2. Radiolabeling and in vivo imaging of transplanted renal lineages differentiated from human embryonic stem cells in fetal rhesus monkeys.

    Science.gov (United States)

    Tarantal, Alice F; Lee, C Chang I; Batchelder, Cynthia A; Christensen, Jared E; Prater, Daniel; Cherry, Simon R

    2012-04-01

    The goals of this study were to optimize radiolabeling of renal lineages differentiated from human embryonic stem (hES) cells and use noninvasive imaging (positron emission tomography (PET) and bioluminescence imaging (BLI)) to detect the cells in fetal monkeys post-transplant. hES cells expressing firefly luciferase (5 × 10(6)) were radiolabeled with the optimized concentration of 10 μCi/ml (64)Cu-PTSM then transplanted under ultrasound guidance into early second trimester fetal monkey kidneys. Fetuses were imaged in utero with PET and tissues collected for analysis 3 days post-transplant. Fetal kidneys were imaged ex vivo (PET and BLI) post-tissue harvest, and serial kidney sections were assessed by PCR for human-specific DNA sequences, fluorescent in situ hybridization (FISH) for human-specific centromere probes, and immunohistochemistry (IHC) to assess engrafted cells. Transplanted cells were readily imaged in vivo and identified at the site of injection; tissue analyses confirmed the imaging findings. Using a semi-quantitative method, one in approximately 650 cells in the kidney was shown to be of human origin by PCR and FISH. These studies suggest that hES cells differentiated toward renal lineages can be effectively radiolabeled, transplanted into fetal monkey kidneys under ultrasound guidance, monitored with PET post-transplant, and identified by PET, BLI, PCR, FISH, and IHC post-tissue harvest.

  3. Human embryonic stem cells and good manufacturing practice: Report of a 1- day workshop held at Stem Cell Biology Research Center, Yazd, 27th April 2017.

    Science.gov (United States)

    Akyash, Fatemeh; Sadeghian-Nodoushan, Fatemeh; Tahajjodi, Somayyeh Sadat; Nikukar, Habib; Farashahi Yazd, Ehsan; Azimzadeh, Mostafa; D Moore, Harry; Aflatoonian, Behrouz

    2017-05-01

    This report explains briefly the minutes of a 1-day workshop entitled; "human embryonic stem cells (hESCs) and good manufacturing practice (GMP)" held by Stem Cell Biology Research Center based in Yazd Reproductive Sciences Institute at Shahid Sadoughi University of Medical Sciences, Yazd, Iran on 27th April 2017. In this workshop, in addition to the practical sessions, Prof. Harry D. Moore from Centre for Stem Cell Biology, University of Sheffield, UK presented the challenges and the importance of the biotechnology of clinical-grade human embryonic stem cells from first derivation to robust defined culture for therapeutic applications.

  4. Pathways in pluripotency and differentiation of embryonic cells

    NARCIS (Netherlands)

    du Puy, L.

    2010-01-01

    Pluripotency - the potential to differentiate into derivatives of the three embryonic germ layers endoderm, ectoderm and mesoderm - is the main characteristic of embryonic stem (ES) cells. ES cells are derived from the inner cell mass (ICM) of a pre-implantation blastocyst and can self-renew

  5. Comparison of FRPE and Human Embryonic Stem Cell–Derived RPE Behavior on Aged Human Bruch's Membrane

    Science.gov (United States)

    Sugino, Ilene K.; Sun, Qian; Wang, Jianqiu; Nunes, Celia F.; Cheewatrakoolpong, Noounanong; Rapista, Aprille; Johnson, Adam C.; Malcuit, Christopher; Klimanskaya, Irina; Lanza, Robert

    2011-01-01

    Purpose. To compare RPE derived from human embryonic stem cells (hES-RPE) and fetal RPE (fRPE) behavior on human Bruch's membrane (BM) from aged and AMD donors. Methods. hES-RPE of 3 degrees of pigmentation and fRPE were cultured on BM explants. Explants were assessed by light, confocal, and scanning electron microscopy. Integrin mRNA levels were determined by real-time polymerase chain reaction studies. Secreted proteins in media were analyzed by multiplex protein analysis after 48-hour exposure at culture day 21. Results. hES-RPE showed impaired initial attachment compared to fRPE; pigmented hES-RPE showed nuclear densities similar to fRPE at day 21. At days 3 and 7, hES-RPE resurfaced BM to a limited degree, showed little proliferation (Ki-67), and partial retention of RPE markers (MITF, cytokeratin, and CRALBP). TUNEL-positive nuclei were abundant at day 3. fRPE exhibited substantial BM resurfacing at day 3 with decreased resurfacing at later times. Most fRPE retained RPE markers. Ki-67–positive nuclei decreased with time in culture. TUNEL staining was variable. Increased integrin mRNA expression did not appear to affect cell survival at day 21. hES-RPE and fRPE protein secretion was similar on equatorial BM except for higher levels of nerve growth factor and thrombospondin-2 (TSP2) by hES-RPE. On submacular BM, fRPE secreted more vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor, and platelet-derived growth factor; hES-RPE secreted more TSP2. Conclusions. Although pigmented hES-RPE and fRPE resurfaced aged and AMD BM to a similar, limited degree at day 21, cell behavior at earlier times was markedly dissimilar. Differences in protein secretion may indicate that hES-RPE may not function identically to native RPE after seeding on aged or AMD BM. PMID:21460262

  6. Gelatin–PMVE/MA composite scaffold promotes expansion of embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chhabra, Hemlata [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); Gupta, Priyanka [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); IITB-Monash Research Academy, Mumbai (India); Department of Chemical Engineering, Monash University, Melbourne (Australia); Verma, Paul J. [Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, South Australia (Australia); Jadhav, Sameer; Bellare, Jayesh R. [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India)

    2014-04-01

    We introduce a new composite scaffold of gelatin and polymethyl vinyl ether-alt-maleic anhydride (PMVE/MA) for expansion of embryonic stem cells (ESCs) in an in vitro environment. To optimize the scaffold, we prepared a gelatin scaffold (G) and three composite scaffolds namely GP-1, GP-2, and GP-3 with varying PMVE/MA concentrations (0.2–1%) and characterized them by scanning electron microscopy (SEM), swelling study, compression testing and FTIR. SEM micrographs revealed interconnected porous structure in all the scaffolds. The permissible hemolysis ratio and activation of platelets by scaffolds confirmed the hemocompatibility of scaffolds. Initial biocompatibility assessment of scaffolds was conducted using hepatocarcinoma (Hep G2) cells and adhesion, proliferation and infiltration of Hep G2 cells in depth of scaffolds were observed, proving the scaffold's biocompatibility. Further Oct4B2 mouse embryonic stem cells (mESCs), which harbor a green fluorescence protein transgene under regulatory control of the Oct4 promotor, were examined for expansion on scaffolds with MTT assay. The GP-2 scaffold demonstrated the best cell proliferation and was further explored for ESC adherence and infiltration in depth (SEM and confocal), and pluripotent state of mESCs was assessed with the expression of Oct4-GFP and stage-specific embryonic antigen-1 (SSEA-1). This study reports the first demonstration of biocompatibility of gelatin–PMVE/MA composite scaffold and presents this scaffold as a promising candidate for embryonic stem cell based tissue engineering. - Highlights: • Composite scaffolds of gelatin and PMVE/MA were prepared by freeze-drying method. • SEM micrographs showed porous structure in all scaffolds of varying pore dimension. • GP-2 composite exhibited better cellular response in comparison to other scaffolds. • mESCs proliferated and expressed Oct-4 and SSEA-1, when cultured on GP-2 scaffold.

  7. Deciphering the Epigenetic Code in Embryonic and Dental Pulp Stem Cells.

    Science.gov (United States)

    Bayarsaihan, Dashzeveg

    2016-12-01

    A close cooperation between chromatin states, transcriptional modulation, and epigenetic modifications is required for establishing appropriate regulatory circuits underlying self-renewal and differentiation of adult and embryonic stem cells. A growing body of research has established that the epigenome topology provides a structural framework for engaging genes in the non-random chromosomal interactions to orchestrate complex processes such as cell-matrix interactions, cell adhesion and cell migration during lineage commitment. Over the past few years, the functional dissection of the epigenetic landscape has become increasingly important for understanding gene expression dynamics in stem cells naturally found in most tissues. Adult stem cells of the human dental pulp hold great promise for tissue engineering, particularly in the skeletal and tooth regenerative medicine. It is therefore likely that progress towards pulp regeneration will have a substantial impact on the clinical research. This review summarizes the current state of knowledge regarding epigenetic cues that have evolved to regulate the pluripotent differentiation potential of embryonic stem cells and the lineage determination of developing dental pulp progenitors.

  8. Quantitative glycomics monitoring of induced pluripotent- and embryonic stem cells during neuronal differentiation

    Directory of Open Access Journals (Sweden)

    Michiyo Terashima

    2014-11-01

    Full Text Available Alterations in the structure of cell surface glycoforms occurring during the stages of stem cell differentiation remain unclear. We describe a rapid glycoblotting-based cellular glycomics method for quantitatively evaluating changes in glycoform expression and structure during neuronal differentiation of murine induced pluripotent stem cells (iPSCs and embryonic stem cells (ESCs. Our results show that changes in the expression of cellular N-glycans are comparable during the differentiation of iPSCs and ESCs. The expression of bisect-type N-glycans was significantly up-regulated in neurons that differentiated from both iPSCs and ESCs. From a glycobiological standpoint, iPSCs are an alternative neural cell source in addition to ESCs.

  9. Molecular Biomarkers for Embryonic and Adult Neural Stem Cell and Neurogenesis

    Science.gov (United States)

    2015-01-01

    The procedure of neurogenesis has made numerous achievements in the past decades, during which various molecular biomarkers have been emerging and have been broadly utilized for the investigation of embryonic and adult neural stem cell (NSC). Nevertheless, there is not a consistent and systematic illustration to depict the functional characteristics of the specific markers expressed in distinct cell types during the different stages of neurogenesis. Here we gathered and generalized a series of NSC biomarkers emerging during the procedures of embryonic and adult neural stem cell, which may be used to identify the subpopulation cells with distinguishing characters in different timeframes of neurogenesis. The identifications of cell patterns will provide applications to the detailed investigations of diverse developmental cell stages and the extents of cell differentiation, which will facilitate the tracing of cell time-course and fate determination of specific cell types and promote the further and literal discoveries of embryonic and adult neurogenesis. Meanwhile, via the utilization of comprehensive applications under the aiding of the systematic knowledge framework, researchers may broaden their insights into the derivation and establishment of novel technologies to analyze the more detailed process of embryogenesis and adult neurogenesis. PMID:26421301

  10. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells.

    Science.gov (United States)

    Gao, Xiugong; Sprando, Robert L; Yourick, Jeffrey J

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72h after exposure to 0.25mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. Published by Elsevier Inc.

  11. Culture conditions have an impact on the maturation of traceable, transplantable mouse embryonic stem cell-derived otic progenitor cells.

    Science.gov (United States)

    Abboud, Nesrine; Fontbonne, Arnaud; Watabe, Isabelle; Tonetto, Alain; Brezun, Jean Michel; Feron, François; Zine, Azel

    2017-09-01

    The generation of replacement inner ear hair cells (HCs) remains a challenge and stem cell therapy holds the potential for developing therapeutic solutions to hearing and balance disorders. Recent developments have made significant strides in producing mouse otic progenitors using cell culture techniques to initiate HC differentiation. However, no consensus has been reached as to efficiency and therefore current methods remain unsatisfactory. In order to address these issues, we compare the generation of otic and HC progenitors from embryonic stem (ES) cells in two cell culture systems: suspension vs. adherent conditions. In the present study, an ES cell line derived from an Atoh1-green fluorescent protein (GFP) transgenic mouse was used to track the generation of otic progenitors, initial HCs and to compare these two differentiation systems. We used a two-step short-term differentiation method involving an induction period of 5 days during which ES cells were cultured in the presence of Wnt/transforming growth factor TGF-β inhibitors and insulin-like growth factor IGF-1 to suppress mesoderm and reinforce presumptive ectoderm and otic lineages. The generated embryoid bodies were then differentiated in medium containing basic fibroblast growth factor (bFGF) for an additional 5 days using either suspension or adherent culture methods. Upon completion of differentiation, quantitative polymerase chain reaction analysis and immunostaining monitored the expression of otic/HC progenitor lineage markers. The results indicate that cells differentiated in suspension cultures produced cells expressing otic progenitor/HC markers at a higher efficiency compared with the production of these cell types within adherent cultures. Furthermore, we demonstrated that a fraction of these cells can incorporate into ototoxin-injured mouse postnatal cochlea explants and express MYO7A after transplantation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons

  12. Fish Stem Cell Cultures

    OpenAIRE

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-01-01

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is th...

  13. Usefulness of field potential as a marker of embryonic stem cell-derived cardiomyocytes, and endpoint analysis of embryonic stem cell test.

    Science.gov (United States)

    Koseki, Naoteru; Deguchi, Jiro; Yamada, Toru; Funabashi, Hitoshi; Seki, Takaki

    2010-12-01

    The embryonic stem cell test (EST) is a validated method and a useful screening tool for drug discovery. EST requires microscopic observation of beating cells to be considered cardiomyocytes as an endpoint assay. However, this procedure is time-consuming and limits the throughput performance. Instead of microscopic observation, we previously established a novel assay method based on cardiac field potential as an endpoint. However, cardiac specificity of this field potential is not yet clarified, because beating cells have not been rigorously evaluated as skeletal or cardiomyocyte. Here, we investigated the relationships between field potential, beating, and cardiac troponin T (cTnT) expression, selected as a cardiomyocyte-specific marker, and evaluated suitability of the field potential as a marker for cardiomyocyte in vehicle or 5-fluorouracil treated embryo bodies. Embryoid bodies of mouse embryonic stem cells (D3) were differentiated in a chamber with multi-electrode array for 5 days, and field potential and beating were measured at the end of differentiation. In addition, these chambers were immunohistochemically stained with anti-cTnT antibody, and the correlation between field potential, beating, and cTnT expression was examined. These results indicated the area of field potential or beating mainly coincided with that of cTnT expression. 5-fluorouracil treatment decreased not only the number of field potential detecting electrodes and beating area, but also cTnT expression, and the area of these parameters was also nearly identical. These results indicate that field potential can be used as a suitable cardiac differentiation marker, and can be a promising parameter of EST.

  14. Hypocapnia leads to enhanced expression of pluripotency and meso-endodermal differentiation genes in mouse embryonic stem cells.

    Science.gov (United States)

    Jyoti, Saras; Tandon, Simran

    2014-04-01

    The efficient utilization of embryonic stem cells for applications like cell based therapy, transplantation and drug discovery largely depends upon the culturing conditions of these cells. In this report, we have analyzed gene, protein expression and morphological changes of embryonic stem cells when subjected to lowered CO2 levels i.e. hypocapnia. We studied the quantitative expression of pluripotent genes, Oct3/4, Nanog and Sox2 and genes involved in the differentiation to the three lineages, under varying CO2 levels. Enhanced expression of these genes was seen at cultures maintained at 1.5% CO2 as compared to those maintained at 5% CO2. The cells exposed to hypocapnic conditions when subjected to immunocytochemical analysis stained positive for Oct-3/4, Nanog and Sox2 transcription factors. Flow cytometry and western blot further showed that the pluripotent proteins in the 1.5% CO2 maintained cultures have higher levels of expression as compared to the ES cells at 5% CO2. In addition, there was enhanced differentiation particularly towards the mesodermal and endodermal lineages at cultures maintained and differentiated at 1.5% CO2 at all the time periods analyzed i.e. day 10 (5+5d), 12 (5+7d) and day 15 (5+10d). These results, which we feel are the first of their kind, indicate that lowered CO2 levels seem to be preferred for the maintenance of pluripotency and the subsequent differentiation. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Stemness-related Factor Sall4 Interacts with Transcription Factors Oct-3/4 and Sox2 and Occupies Oct-Sox Elements in Mouse Embryonic Stem Cells*

    Science.gov (United States)

    Tanimura, Nobuyuki; Saito, Motoki; Ebisuya, Miki; Nishida, Eisuke; Ishikawa, Fuyuki

    2013-01-01

    A small number of transcription factors, including Oct-3/4 and Sox2, constitute the transcriptional network that maintains pluripotency in embryonic stem (ES) cells. Previous reports suggested that some of these factors form a complex that binds the Oct-Sox element, a composite sequence consisting of closely juxtaposed Oct-3/4 binding and Sox2 binding sites. However, little is known regarding the components of the complex. In this study we show that Sall4, a member of the Spalt-like family of proteins, directly interacts with Sox2 and Oct-3/4. Sall4 in combination with Sox2 or Oct-3/4 simultaneously occupies the Oct-Sox elements in mouse ES cells. Overexpression of Sall4 in ES cells increased reporter activities in a luciferase assay when the Pou5f1- or Nanog-derived Oct-Sox element was included in the reporter. Microarray analyses revealed that Sall4 and Sox2 bound to the same genes in ES cells significantly more frequently than expected from random coincidence. These factors appeared to bind the promoter regions of a subset of the Sall4 and Sox2 double-positive genes in precisely similar distribution patterns along the promoter regions, suggesting that Sall4 and Sox2 associate with such Sall4/Sox2-overlapping genes as a complex. Importantly, gene ontology analyses indicated that the Sall4/Sox2-overlapping gene set is enriched for genes involved in maintaining pluripotency. Sall4/Sox2/Oct-3/4 triple-positive genes identified by referring to a previous study identifying Oct-3/4-bound genes in ES cells were further enriched for pluripotency genes than Sall4/Sox2 double-positive genes. These results demonstrate that Sall4 contributes to the transcriptional network operating in pluripotent cells together with Oct-3/4 and Sox2. PMID:23269686

  16. Mouse Embryonic Fibroblasts (MEF) Exhibit a Similar but not Identical Phenotype to Bone Marrow Stromal Stem Cells (BMSC)

    DEFF Research Database (Denmark)

    Saeed, Hamid; Taipaleenmäki, Hanna; Aldahmash, Abdullah M

    2012-01-01

    Mouse embryonic fibroblasts have been utilized as a surrogate stem cell model for the postnatal bone marrow-derived stromal stem cells (BMSC) to study mesoderm-type cell differentiation e.g. osteoblasts, adipocytes and chondrocytes. However, no formal characterization of MEF phenotype has been re...

  17. Establishment of a congenital tooth agenesis related gene MSX1 knockout human embryonic stem cell lines by CRISPR-Cas9 technology

    Directory of Open Access Journals (Sweden)

    Yanting Xue

    2017-10-01

    Full Text Available Human MSX1 gene is mapped to chromosome 4 and encodes a 303aa homeobox protein MSX1. MSX1 expression appears during early tooth development of vertebrate embryogenesis. Mutations in this protein are related to human tooth anomalie, cleft lip and palate and congenital ectodermal dysplasia syndrome. Most of the confirmed pathogenic mutations are located in exon2 encoded homeobox domain. Here, we report the establishment of MSX1 gene knockout human embryonic stem (hES cell lines by CRISPR-Cas9 technology. These cell lines provide good materials for further studies of the roles MSX1 plays in human tooth development and congenital tooth agenesis.

  18. Vitrification by Ultra-fast Cooling at a Low Concentration of Cryoprotectants in a Quartz Microcapillary: A Study Using Murine Embryonic Stem Cells

    Science.gov (United States)

    He, Xiaoming; Park, Eric Y.H.; Fowler, Alex; Yarmush, Martin L.; Toner, Mehmet

    2009-01-01

    Conventional cryopreservation protocols for slow-freezing or vitrification involve cell injury due to ice formation/cell dehydration or toxicity of high cryoprotectant (CPA) concentrations, respectively. In this study, we developed a novel cryopreservation technique to achieve ultra-fast cooling rates using a quartz microcapillary (QMC). The QMC enabled vitrification of murine embryonic stem (ES) cells using an intracellular cryoprotectant concentration in the range used for slowing freezing (1–2 M). The cryoprotectants used included 2 M 1,2-propanediol (PROH, cell membrane permeable) and 0.5 M extracellular trehalose (cell membrane impermeable). More than 70% of the murine ES cells post-vitrification attached with respect to non-frozen control cells, and the proliferation rates of the two groups were similar. Preservation of undifferentiated properties of the pluripotent murine ES cells post vitrification cryopreservation was verified using three different types of assays: the expression of transcription factor Oct-4, the presentation of the membrane surface glycoprotein SSEA-1, and the elevated expression of the intracellular enzyme alkaline phosphatase. These results indicate that vitrification at a low concentration (2 M) of intracellular cryoprotectants is a viable and effective approach for the cryopreservation of murine embryonic stem cells. PMID:18462712

  19. Artificial niche substrates for embryonic and induced pluripotent stem cell cultures.

    Science.gov (United States)

    Joddar, Binata; Ito, Yoshihiro

    2013-10-20

    Stem cells possess the ability to self-renew and differentiate into other cell types. In vivo, stem cells reside in their own anatomic niches in a defined physiological environment, from which they are released to differentiate into a required cell type when deemed appropriate. While a resident within the niche, the stem cell receives signals that in turn maintain the cell in a pluripotent state. In addition, the niche also provides nourishment to the cell. Physically, the niche also serves to anchor the cell via various ECM components and cell-adhesion molecules. Therefore, in vitro models that replicate the in vivo niche will lead to a better understanding of stem cell fate and turnover. In turn, this will help inform attempts to culture stem cells in vitro on artificial niche-like substrates. In this review, we have highlighted recent studies describing artificial niche-like substrates used to culture embryonic and induced pluripotent stem cells in vitro. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  20. Electrophysiological properties of neurosensory progenitors derived from human embryonic stem cells.

    Science.gov (United States)

    Needham, Karina; Hyakumura, Tomoko; Gunewardene, Niliksha; Dottori, Mirella; Nayagam, Bryony A

    2014-01-01

    In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, stem cell-derived neurons may provide a potential source of replacement cells. The success of such a therapy relies upon producing a population of functional neurons from stem cells, to enable precise encoding of sound information to the brainstem. Using our established differentiation assay to produce sensory neurons from human stem cells, patch-clamp recordings indicated that all neurons examined generated action potentials and displayed both transient sodium and sustained potassium currents. Stem cell-derived neurons reliably entrained to stimuli up to 20 pulses per second (pps), with 50% entrainment at 50 pps. A comparison with cultured primary auditory neurons indicated similar firing precision during low-frequency stimuli, but significant differences after 50 pps due to differences in action potential latency and width. The firing properties of stem cell-derived neurons were also considered relative to time in culture (31-56 days) and revealed no change in resting membrane potential, threshold or firing latency over time. Thus, while stem cell-derived neurons did not entrain to high frequency stimulation as effectively as mammalian auditory neurons, their electrical phenotype was stable in culture and consistent with that reported for embryonic auditory neurons. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Sorting and expansion of murine embryonic stem cell colonies using micropallet arrays.

    Science.gov (United States)

    Shadpour, Hamed; Sims, Christopher E; Thresher, Randy J; Allbritton, Nancy L

    2009-02-01

    Isolation of cell colonies is an essential task in most stem cell studies. Conventional techniques for colony selection and isolation require significant time, labor, and consumption of expensive reagents. New microengineered technologies hold the promise for improving colony manipulation by reducing the required manpower and reagent consumption. Murine embryonic stem cells were cultured on arrays composed of releasable elements termed micropallets created from a biocompatible photoresist. Micropallets containing undifferentiated colonies were released using a laser-based technique followed by cell collection and expansion in culture. The micropallet arrays provided a biocompatible substrate for maintaining undifferentiated murine stem cells in culture. A surface coating of 0.025% gelatin was shown to be optimal for cell culture and collection. Arrays composed of surface-roughened micropallets provided further improvements in culture and isolation. Colonies of viable stem cells were efficiently isolated and collected. Colonies sorted in this manner were shown to remain undifferentiated even after collection and further expansion in culture. Qualitative and quantitative analyses of sorting, collection efficiency, and cell viability after release and expansion of stem cell colonies demonstrated that the micropallet array technology is a promising alternative to conventional sorting methods for stem cell applications. Copyright 2008 International Society for Advancement of Cytometry

  2. Electrophysiological properties of neurosensory progenitors derived from human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Karina Needham

    2014-01-01

    Full Text Available In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, stem cell-derived neurons may provide a potential source of replacement cells. The success of such a therapy relies upon producing a population of functional neurons from stem cells, to enable precise encoding of sound information to the brainstem. Using our established differentiation assay to produce sensory neurons from human stem cells, patch-clamp recordings indicated that all neurons examined generated action potentials and displayed both transient sodium and sustained potassium currents. Stem cell-derived neurons reliably entrained to stimuli up to 20 pulses per second (pps, with 50% entrainment at 50 pps. A comparison with cultured primary auditory neurons indicated similar firing precision during low-frequency stimuli, but significant differences after 50 pps due to differences in action potential latency and width. The firing properties of stem cell-derived neurons were also considered relative to time in culture (31–56 days and revealed no change in resting membrane potential, threshold or firing latency over time. Thus, while stem cell-derived neurons did not entrain to high frequency stimulation as effectively as mammalian auditory neurons, their electrical phenotype was stable in culture and consistent with that reported for embryonic auditory neurons.

  3. Undifferentiated Embryonic Cell Transcription Factor 1 Regulates ESC Chromatin Organization and Gene Expression

    NARCIS (Netherlands)

    Kooistra, Susanne M.; van den Boom, Vincent; Thummer, Rajkumar P.; Johannes, Frank; Wardenaar, Rene; Tesson, Bruno M.; Veenhoff, Liesbeth M.; Fusetti, Fabrizia; O'Neill, Laura P.; Turner, Bryan M.; de Haan, Gerald; Eggen, Bart J. L.; O’Neill, Laura P.

    2010-01-01

    Previous reports showed that embryonic stem (ES) cells contain hyperdynamic and globally transcribed chromatin-properties that are important for ES cell pluripotency and differentiation. Here, we demonstrate a role for undifferentiated embryonic cell transcription factor 1 (UTF1) in regulating ES

  4. Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

    Energy Technology Data Exchange (ETDEWEB)

    Yamano, Noriko [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Kimura, Tohru, E-mail: tkimura@patho.med.osaka-u.ac.jp [Department of Pathology, Medical School, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Watanabe-Kushima, Shoko [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Shinohara, Takashi [Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501 (Japan); Nakano, Toru, E-mail: tnakano@patho.med.osaka-u.ac.jp [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Department of Pathology, Medical School, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan)

    2010-02-12

    Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.

  5. Differential bone-forming capacity of osteogenic cells from either embryonic stem cells or bone marrow-derived mesenchymal stem cells

    NARCIS (Netherlands)

    Both, Sanne Karijn; van Apeldoorn, Aart A.; Jukes, J.M.; Englund, Mikael C.O.; Hyllner, Johan; van Blitterswijk, Clemens; de Boer, Jan

    2011-01-01

    For more than a decade, human mesenchymal stem cells (hMSCs) have been used in bone tissue-engineering research. More recently some of the focus in this field has shifted towards the use of embryonic stem cells. While it is well known that hMSCs are able to form bone when implanted subcutaneously in

  6. Sel Kumulus Sebagai Feeder Layer pada Kultur Stem Cells Embrionic Mencit (CUMULUS CELLS AS FEEDER LAYER IN CULTURE OF MOUSE EMBRYONIC STEM CELLS

    Directory of Open Access Journals (Sweden)

    Thomas Mata Hine

    2014-08-01

    Full Text Available The purpose of this study was to verify the effectiveness of cumulus cells as a feeder layer in supportingthe growth of mouse embryonic stem cells. Embryos at blastocyst stage were exposed in pronase solution,and then incubated in rabbit anti-mouse antibody and guinea pig complement to lyse and separate thetrophoblast cells from the inner cell mass. Inner cell mass subsequently cultured in a petri dish containinga cumulus feeder layer, mouse embryonic fibroblasts, or without a feeder layer, in stem cells medium. Theresulting stem cells colony passaged in trypsin solution, pipetted repeatedly to produce subcolonies orsingle cells, and cultured as before in some new petri dishes. Characterization of stem cells was identifiedby using alkaline phosphatase staining. The results showed the effectiveness of cumulus cells as feederlayer for culture of mouse embryonic stem cells was comparable with mouse embryonic fibroblasts, andboth of them were better than without a feeder layer method. The number of primary colony, cell lines, andcolony growth rate increased 41.30, 8.70, and 54.20%, respectively, while doubling time was shorter 10:21hours compared to the growth without feeder layer method. These results prove that the cumulus feederlayer effectively supports the growth of mouse embryonic stem cells.

  7. Transcriptional and functional profiling of human embryonic stem cell-derived cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Feng Cao

    Full Text Available Human embryonic stem cells (hESCs can serve as a potentially limitless source of cells that may enable regeneration of diseased tissue and organs. Here we investigate the use of human embryonic stem cell-derived cardiomyocytes (hESC-CMs in promoting recovery from cardiac ischemia reperfusion injury in a mouse model. Using microarrays, we have described the hESC-CM transcriptome within the spectrum of changes that occur between undifferentiated hESCs and fetal heart cells. The hESC-CMs expressed cardiomyocyte genes at levels similar to those found in 20-week fetal heart cells, making this population a good source of potential replacement cells in vivo. Echocardiographic studies showed significant improvement in heart function by 8 weeks after transplantation. Finally, we demonstrate long-term engraftment of hESC-CMs by using molecular imaging to track cellular localization, survival, and proliferation in vivo. Taken together, global gene expression profiling of hESC differentiation enables a systems-based analysis of the biological processes, networks, and genes that drive hESC fate decisions, and studies such as this will serve as the foundation for future clinical applications of stem cell therapies.

  8. Embryonic stem-cell research and the moral status of embryos.

    Science.gov (United States)

    Chu, G

    2003-11-01

    Stem-cell research has the potential to significantly advance our knowledge of cell differentiation with the promise of exciting and innovative therapeutic applications for otherwise incurable genetic and degenerative disorders. The issue has been the subject of debate in federal and state parliament as research of embryonic stem cells and their application have come under intense scrutiny. Scientists and medical practitioners are highly skilled in the technical aspects of biomedicine but are decidedly less comfortable with ethics. The place of novel and controversial biomedical technology is commonly left to an ad hoc and complex process whereby social acceptability eventually becomes the final arbiter. Utilitarianism is a popular ethical approach that attempts to weigh all the known and anticipated merits and pitfalls. The scientific background of biomedical professionals, the vast explosion of information, and specialization and subspecialization, have all contributed to a reductionistic view of life and ethics. The 'sanctity of life' doctrine is altogether quite different, as secular and religious advocates appear inflexible and unyielding to the logical propositions of utilitarianism and reductionism or to the consensus of democracy. These four major ethical approaches are discussed with reference to the embryonic stem-cell research debate.

  9. Striking a balance: regulation of transposable elements by Zfp281 and Mll2 in mouse embryonic stem cells.

    Science.gov (United States)

    Dai, Qian; Shen, Yang; Wang, Yan; Wang, Xin; Francisco, Joel Celio; Luo, Zhuojuan; Lin, Chengqi

    2017-12-01

    Transposable elements (TEs) compose about 40% of the murine genome. Retrotransposition of active TEs such as LINE-1 (L1) tremendously impacts genetic diversification and genome stability. Therefore, transcription and transposition activities of retrotransposons are tightly controlled. Here, we show that the Krüppel-like zinc finger protein Zfp281 directly binds and suppresses a subset of retrotransposons, including the active young L1 repeat elements, in mouse embryonic stem (ES) cells. In addition, we find that Zfp281-regulated L1s are highly enriched for 5-hydroxymethylcytosine (5hmC) and H3K4me3. The COMPASS-like H3K4 methyltransferase Mll2 is the major H3K4me3 methylase at the Zfp281-regulated L1s and required for their proper expression. Our studies also reveal that Zfp281 functions partially through recruiting the L1 regulators DNA hydroxymethylase Tet1 and Sin3A, and restricting Mll2 at these active L1s, leading to their balanced expression. In summary, our data indicate an instrumental role of Zfp281 in suppressing the young active L1s in mouse ES cells. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Ethanol diverts early neuronal differentiation trajectory of embryonic stem cells by disrupting the balance of lineage specifiers.

    Directory of Open Access Journals (Sweden)

    Rosa Sánchez-Alvarez

    Full Text Available Ethanol is a toxin responsible for the neurodevelopmental deficits of Fetal Alcohol Spectrum Disorders (FASD. Recent evidence suggests that ethanol modulates the protein expression of lineage specifier transcription factors Oct4 (Pou5f1 and Sox2 in early stages of mouse embryonic stem (ES cell differentiation. We hypothesized that ethanol induced an imbalance in the expression of Oct4 and Sox2 in early differentiation, that dysregulated the expression of associated and target genes and signaling molecules and diverted cells from neuroectodermal (NE formation.We showed modulation by ethanol of 33 genes during ES cell differentiation, using high throughput microfluidic dynamic array chips measuring 2,304 real time quantitative PCR assays. Based on the overall gene expression dynamics, ethanol drove cells along a differentiation trajectory away from NE fate. These ethanol-induced gene expression changes were observed as early as within 2 days of differentiation, and were independent of cell proliferation or apoptosis. Gene expression changes were correlated with fewer βIII-tubulin positive cells of an immature neural progenitor phenotype, as well as a disrupted actin cytoskeleton were observed. Moreover, Tuba1a and Gapdh housekeeping genes were modulated by ethanol during differentiation and were replaced by a set of ribosomal genes with stable expression.These findings provided an ethanol-response gene signature and pointed to the transcriptional dynamics underlying lineage imbalance that may be relevant to FASD phenotype.

  11. Astrocytes derived from trisomic human embryonic stem cells express markers of astrocytic cancer cells and premalignant stem-like progenitors

    Directory of Open Access Journals (Sweden)

    Iverson Linda E

    2010-04-01

    Full Text Available Abstract Background Trisomic variants of human embryonic stem cells (hESCs arise spontaneously in culture. Although trisomic hESCs share many properties with diploid hESCs, they also exhibit features of cancer stem cells. Since most hESC-based therapies will utilize differentiated derivatives, it is imperative to investigate the potential of trisomic hESCs to undergo malignant transformation during differentiation prior to their use in the clinical setting. Methods Diploid and trisomic hESCs were differentiated into astrocytic progenitors cells (APCs, RNA extracted and hybridized to human exon-specific microarrays. Global gene expression profiles of diploid and trisomic APCs were compared to that of an astrocytoma cell line and glioblastoma samples, analyzed by others, using the same microarray platform. Results Bioinformatic analysis of microarray data indicates that differentiated trisomic APCs exhibit global expression profiles with similarities to the malignant astrocytoma cell line. An analogous trend is observed in comparison to glioblastoma samples indicating that trisomic APCs express markers of astrocytic cancer cells. The analysis also allowed identification of transcripts predicted to be differentially expressed in brain tumor stem cells. These data indicate that in vitro differentiation of trisomic hESCs along astrocytic pathways give rise to cells exhibiting properties of premalignant astrocytic stem/progenitor cells. Conclusions Given their occult nature, opportunities to study premalignant stem/progenitor cells in human have been few. The ability to propagate and direct the differentiation of aneuploid hESCs provides a powerful in vitro system for investigating biological properties of human cells exhibiting features of premalignant stem cells. This in vitro culture system can be used to elucidate changes in gene expression occurring enroute to malignant transformation and to identify molecular markers of cancer stem

  12. Induced Pluripotent Stem Cells Show Metabolomic Differences to Embryonic Stem Cells in Polyunsaturated Phosphatidylcholines and Primary Metabolism

    Science.gov (United States)

    Meissen, John K.; Yuen, Benjamin T. K.; Kind, Tobias; Riggs, John W.; Barupal, Dinesh K.; Knoepfler, Paul S.; Fiehn, Oliver

    2012-01-01

    Induced pluripotent stem cells are different from embryonic stem cells as shown by epigenetic and genomics analyses. Depending on cell types and culture conditions, such genetic alterations can lead to different metabolic phenotypes which may impact replication rates, membrane properties and cell differentiation. We here applied a comprehensive metabolomics strategy incorporating nanoelectrospray ion trap mass spectrometry (MS), gas chromatography-time of flight MS, and hydrophilic interaction- and reversed phase-liquid chromatography-quadrupole time-of-flight MS to examine the metabolome of induced pluripotent stem cells (iPSCs) compared to parental fibroblasts as well as to reference embryonic stem cells (ESCs). With over 250 identified metabolites and a range of structurally unknown compounds, quantitative and statistical metabolome data were mapped onto a metabolite networks describing the metabolic state of iPSCs relative to other cell types. Overall iPSCs exhibited a striking shift metabolically away from parental fibroblasts and toward ESCs, suggestive of near complete metabolic reprogramming. Differences between pluripotent cell types were not observed in carbohydrate or hydroxyl acid metabolism, pentose phosphate pathway metabolites, or free fatty acids. However, significant differences between iPSCs and ESCs were evident in phosphatidylcholine and phosphatidylethanolamine lipid structures, essential and non-essential amino acids, and metabolites involved in polyamine biosynthesis. Together our findings demonstrate that during cellular reprogramming, the metabolome of fibroblasts is also reprogrammed to take on an ESC-like profile, but there are select unique differences apparent in iPSCs. The identified metabolomics signatures of iPSCs and ESCs may have important implications for functional regulation of maintenance and induction of pluripotency. PMID:23077522

  13. Persistent donor cell gene expression among human induced pluripotent stem cells contributes to differences with human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Zhumur Ghosh

    Full Text Available Human induced pluripotent stem cells (hiPSCs generated by de-differentiation of adult somatic cells offer potential solutions for the ethical issues surrounding human embryonic stem cells (hESCs, as well as their immunologic rejection after cellular transplantation. However, although hiPSCs have been described as "embryonic stem cell-like", these cells have a distinct gene expression pattern compared to hESCs, making incomplete reprogramming a potential pitfall. It is unclear to what degree the difference in tissue of origin may contribute to these gene expression differences. To answer these important questions, a careful transcriptional profiling analysis is necessary to investigate the exact reprogramming state of hiPSCs, as well as analysis of the impression, if any, of the tissue of origin on the resulting hiPSCs. In this study, we compare the gene profiles of hiPSCs derived from fetal fibroblasts, neonatal fibroblasts, adipose stem cells, and keratinocytes to their corresponding donor cells and hESCs. Our analysis elucidates the overall degree of reprogramming within each hiPSC line, as well as the "distance" between each hiPSC line and its donor cell. We further identify genes that have a similar mode of regulation in hiPSCs and their corresponding donor cells compared to hESCs, allowing us to specify core sets of donor genes that continue to be expressed in each hiPSC line. We report that residual gene expression of the donor cell type contributes significantly to the differences among hiPSCs and hESCs, and adds to the incompleteness in reprogramming. Specifically, our analysis reveals that fetal fibroblast-derived hiPSCs are closer to hESCs, followed by adipose, neonatal fibroblast, and keratinocyte-derived hiPSCs.

  14. Induced pluripotent stem cells show metabolomic differences to embryonic stem cells in polyunsaturated phosphatidylcholines and primary metabolism.

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    John K Meissen

    Full Text Available Induced pluripotent stem cells are different from embryonic stem cells as shown by epigenetic and genomics analyses. Depending on cell types and culture conditions, such genetic alterations can lead to different metabolic phenotypes which may impact replication rates, membrane properties and cell differentiation. We here applied a comprehensive metabolomics strategy incorporating nanoelectrospray ion trap mass spectrometry (MS, gas chromatography-time of flight MS, and hydrophilic interaction- and reversed phase-liquid chromatography-quadrupole time-of-flight MS to examine the metabolome of induced pluripotent stem cells (iPSCs compared to parental fibroblasts as well as to reference embryonic stem cells (ESCs. With over 250 identified metabolites and a range of structurally unknown compounds, quantitative and statistical metabolome data were mapped onto a metabolite networks describing the metabolic state of iPSCs relative to other cell types. Overall iPSCs exhibited a striking shift metabolically away from parental fibroblasts and toward ESCs, suggestive of near complete metabolic reprogramming. Differences between pluripotent cell types were not observed in carbohydrate or hydroxyl acid metabolism, pentose phosphate pathway metabolites, or free fatty acids. However, significant differences between iPSCs and ESCs were evident in phosphatidylcholine and phosphatidylethanolamine lipid structures, essential and non-essential amino acids, and metabolites involved in polyamine biosynthesis. Together our findings demonstrate that during cellular reprogramming, the metabolome of fibroblasts is also reprogrammed to take on an ESC-like profile, but there are select unique differences apparent in iPSCs. The identified metabolomics signatures of iPSCs and ESCs may have important implications for functional regulation of maintenance and induction of pluripotency.

  15. Induced pluripotent stem cells show metabolomic differences to embryonic stem cells in polyunsaturated phosphatidylcholines and primary metabolism.

    Science.gov (United States)

    Meissen, John K; Yuen, Benjamin T K; Kind, Tobias; Riggs, John W; Barupal, Dinesh K; Knoepfler, Paul S; Fiehn, Oliver

    2012-01-01

    Induced pluripotent stem cells are different from embryonic stem cells as shown by epigenetic and genomics analyses. Depending on cell types and culture conditions, such genetic alterations can lead to different metabolic phenotypes which may impact replication rates, membrane properties and cell differentiation. We here applied a comprehensive metabolomics strategy incorporating nanoelectrospray ion trap mass spectrometry (MS), gas chromatography-time of flight MS, and hydrophilic interaction- and reversed phase-liquid chromatography-quadrupole time-of-flight MS to examine the metabolome of induced pluripotent stem cells (iPSCs) compared to parental fibroblasts as well as to reference embryonic stem cells (ESCs). With over 250 identified metabolites and a range of structurally unknown compounds, quantitative and statistical metabolome data were mapped onto a metabolite networks describing the metabolic state of iPSCs relative to other cell types. Overall iPSCs exhibited a striking shift metabolically away from parental fibroblasts and toward ESCs, suggestive of near complete metabolic reprogramming. Differences between pluripotent cell types were not observed in carbohydrate or hydroxyl acid metabolism, pentose phosphate pathway metabolites, or free fatty acids. However, significant differences between iPSCs and ESCs were evident in phosphatidylcholine and phosphatidylethanolamine lipid structures, essential and non-essential amino acids, and metabolites involved in polyamine biosynthesis. Together our findings demonstrate that during cellular reprogramming, the metabolome of fibroblasts is also reprogrammed to take on an ESC-like profile, but there are select unique differences apparent in iPSCs. The identified metabolomics signatures of iPSCs and ESCs may have important implications for functional regulation of maintenance and induction of pluripotency.

  16. Derivation of Huntington Disease affected Genea046 human embryonic stem cell line

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    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea046 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying HTT gene CAG expansion of 45 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 85% of cells expressed Nanog, 92% Oct4, 75% Tra1–60 and 99% SSEA4 and demonstrated Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and visible contamination.

  17. Generation of human embryonic stem cells from abnormal blastocyst diagnosed with adrenoleukodystrophy.

    Science.gov (United States)

    Ouyang, Qi; Zhou, Xiaoying; Chen, Jing; Du, Juan; Lu, Guangxiu; Lin, Ge; Sun, Yi

    2016-11-01

    Human embryonic stem cell (hESC) line chHES-480 was derived from abnormal blastocyst diagnosed with adrenoleukodystrophy (ALD) after preimplantation genetic diagnosis (PGD) treatment. DNA sequencing analysis confirmed that chHES-480 cell line carried a hemizygous missense mutation c.1825G>A(p.Glu609Lys) of ABCD1 gene. Characteristic tests proved that the chHES-480 cell line presented typical markers of pluripotency and had the capability to form the three germ layers both in vitro and in vivo. Copyright © 2016 Michael Boutros, German Cancer Research Center, Heidelberg, Germany. Published by Elsevier B.V. All rights reserved.

  18. Laser-induced fusion of human embryonic stem cells with optical tweezers

    Energy Technology Data Exchange (ETDEWEB)

    Chen Shuxun; Wang Xiaolin; Sun Dong [Department of Mechanical and Biomedical Engineering, City University of Hong Kong (Hong Kong); Cheng Jinping; Han Cheng, Shuk [Department of Biology and Chemistry, City University of Hong Kong (Hong Kong); Kong, Chi-Wing [Stem Cell and Regenerative Medicine Consortium, and Departments of Medicine and Physiology, LKS Faculty of Medicine, University of Hong Kong (Hong Kong); Li, Ronald A. [Stem Cell and Regenerative Medicine Consortium, and Departments of Medicine and Physiology, LKS Faculty of Medicine, University of Hong Kong (Hong Kong); Center of Cardiovascular Research, Mount Sinai School of Medicine, New York, New York 10029 (United States)

    2013-07-15

    We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

  19. Derivation of the human embryonic stem cell line RCe010-A (RC-6

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    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe010-A (RC-6 was derived from a frozen and thawed blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  20. Derivation of Trisomy 21 affected human embryonic stem cell line Genea021

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    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea021 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Trisomy 21, indicative of Down Syndrome. Following ICM outgrowth on inactivated human feeders, CGH and STR analyses demonstrated a 47, XY, +21 karyotype and male allele pattern. The hESC line had pluripotent cell morphology, 71% of cells expressed Nanog, 84% Oct4, 23% Tra1–60 and 95% SSEA4, gave a Pluritest Pluripotency score of 21.85, Novelty of 1.42, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  1. Human Embryonic Stem Cells: A Model for the Study of Neural Development and Neurological Diseases

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    Piya Prajumwongs

    2016-01-01

    Full Text Available Although the mechanism of neurogenesis has been well documented in other organisms, there might be fundamental differences between human and those species referring to species-specific context. Based on principles learned from other systems, it is found that the signaling pathways required for neural induction and specification of human embryonic stem cells (hESCs recapitulated those in the early embryo development in vivo at certain degree. This underscores the usefulness of hESCs in understanding early human neural development and reinforces the need to integrate the principles of developmental biology and hESC biology for an efficient neural differentiation.

  2. Derivation of the human embryonic stem cell line RCe006-A (RC-2

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    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe006-A (RC-2 was derived from a frozen and thawed blastocyst voluntarily donated as surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line exhibits expression of expected pluripotency markers and in vitro differentiation potential to three germinal lineage representative cell populations. It has a male trisomy 12 karyotype (47XY, +12. Microsatellite DNA marker identity and HLA and blood group typing data are available.

  3. Derivation of the human embryonic stem cell line RCe014-A (RC-10

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    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe014-A (RC-10 was derived from a fresh oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XY and 47XY +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  4. Derivation of the human embryonic stem cell line RCe012-A (RC-8

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe012-A (RC-8 was derived from a frozen and thawed day 5 embryo cultivated to the blastocyst stage. The embryo was voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  5. Derivation of the clinical grade human embryonic stem cell line RCe013-A (RC-9

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    P.A. De Sousa

    2016-07-01

    Full Text Available The human embryonic stem cell line RCe013-A (RC-9 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a failed to fertilise oocyte voluntarily donated as unsuitable and surplus to fertility requirements following informed consent. RCe013-A (RC-9 shows normal pluripotency marker expression and differentiation to the three germ layers in vitro and in vivo. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  6. Derivation of the clinical grade human embryonic stem cell line RCe016-A (RC-12

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    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe016-A (RC-12 was derived under quality assured compliance with UK regulations, EU Directives and International guidance for tissue procurement, processing and storage according to good manufacturing practice (GMP standards. The cell line was derived from a cryopreserved blastocyst stage embryo voluntarily donated as surplus to fertility requirements following informed consent. RCe016-A (RC-12 shows normal pluripotency marker expression and differentiation to three germ layers in vitro. Karyology revealed a mixed male karyotype at early passage (P15, which resolved as normal 46XY by passage 33. Microsatellite PCR identity, HLA and blood group typing data is available.

  7. Derivation of the clinical grade human embryonic stem cell line RCe019-A (RC-15

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    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe019-A (RC-15 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe019-A (RC-15 shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XX/47XX, +8 female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  8. Derivation of the clinical grade human embryonic stem cell line RCe018-A (RC-14

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe018-A (RC-14 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a blastocyst stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe018-A (RC-14 shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a male karyotype with an extra copy of chromosome 8 (47XY, +8. Microsatellite PCR identity, HLA and blood group typing data are available.

  9. Derivation of the clinical grade human embryonic stem cell line RCe015-A (RC-11

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    P.A. De Sousa

    2016-07-01

    Full Text Available The human embryonic stem cell line RCe015-A (RC-11 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a fragmented cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe015-A (RC-11 shows normal pluripotency marker expression and differentiation to the three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  10. Derivation of the clinical grade human embryonic stem cell line RCe017-A (RC-13

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    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe017-A (RC-13 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a frozen and thawed blastocyst stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe017-A (RC-13 shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 47XY, +12/48XY, +1, +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  11. Derivation of the clinical grade human embryonic stem cell line RCe020-a (RC-16

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    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe020-A (RC-16 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a failed to fertilise oocyte voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe020-A (RC-16 shows normal pluripotency marker expression and differentiates to mesoderm and potentially ectoderm in vitro. It has an abnormal 47XX, +14, i(20(q10 female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  12. Derivation of the clinical grade human embryonic stem cell line RCe021-A (RC-17

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    P.A. De Sousa

    2016-07-01

    Full Text Available The human embryonic stem cell line RCe021-A (RC-17 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a day 3 embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe021-A (RC-17 shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  13. Stable isotope labelling with amino acids in cell culture for human embryonic stem cell proteomic analysis

    DEFF Research Database (Denmark)

    Harkness, Linda; Prokhorova, Tatyana A; Kassem, Moustapha

    2012-01-01

    The identification and quantitative measurements of proteins in human embryonic stem cells (hESC) is a fast growing interdisciplinary area with an enormous impact on understanding the biology of hESC and the mechanism controlling self-renewal and differentiation. Using a quantitative mass...... spectroscopic method of stable isotope labelling with amino acids during cell culture (SILAC), we are able to analyse differential expression of proteins from different cellular compartments and to identify intracellular signalling pathways involved in self-renewal and differentiation. In this chapter, we...

  14. Patenting, morality and human embryonic stem cell science: bioethics and cultural politics in Europe.

    Science.gov (United States)

    Salter, Brian

    2007-05-01

    As the recent experience of the European Patent Office graphically demonstrates, there is an inherent political tension between the individual ownership rights necessary for the operation of an international market in human embryonic stem cell science and the communal values of the many cultures in which such markets operate. This report examines the basis of the conflict between patenting and morality at national and international levels, the manifestation of those tensions in European patenting policy, and the contribution of bioethics to the attempt by European institutions to develop a governance response.

  15. Functional neuromuscular junctions formed by embryonic stem cell-derived motor neurons.

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    Joy A Umbach

    Full Text Available A key objective of stem cell biology is to create physiologically relevant cells suitable for modeling disease pathologies in vitro. Much progress towards this goal has been made in the area of motor neuron (MN disease through the development of methods to direct spinal MN formation from both embryonic and induced pluripotent stem cells. Previous studies have characterized these neurons with respect to their molecular and intrinsic functional properties. However, the synaptic activity of stem cell-derived MNs remains less well defined. In this study, we report the development of low-density co-culture conditions that encourage the formation of active neuromuscular synapses between stem cell-derived MNs and muscle cells in vitro. Fluorescence microscopy reveals the expression of numerous synaptic proteins at these contacts, while dual patch clamp recording detects both spontaneous and multi-quantal evoked synaptic responses similar to those observed in vivo. Together, these findings demonstrate that stem cell-derived MNs innervate muscle cells in a functionally relevant manner. This dual recording approach further offers a sensitive and quantitative assay platform to probe disorders of synaptic dysfunction associated with MN disease.

  16. PROPOSALS FOR EMBRYONIC STEM CELL PRODUCTION WITHOUT DESTROYING HUMAN EMBRYOS: SCIENTIFIC AND BIOETHICAL CHALLENGES PROPUESTAS DE PRODUCCIÓN DE CÉLULAS MADRE EMBRIONARIAS SIN DESTRUIR EMBRIONES HUMANOS: RETOS CIENTÍFICOS Y BIOÉTICOS PROPOSTAS DE PRODUÇÃO DE CÉLULAS-TRONCO EMBRIONÁRIAS SEM DESTRUIR EMBRIÕES HUMANOS: DESAFIOS CIENTÍFICOS E BIOÉTICOS

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    Patricio Ventura-Juncá

    2009-11-01

    Full Text Available The possibility of using human embryonic stem cells (ESC for therapeutic purposes raises serious ethical objections, the most fundamental one being that until recently the only way to obtain ESC was with procedures that necessarily destroyed living human embryos. Due to this, research in this field has been rejected by many scientists, bioeticists, and has been banned by law in several countries. Efforts have been made to find procedures to obtain ESC without destroying embyros or putting them at risk. This paper reviews the scientific, technical and ethical aspects of the different strategies developed for this purpose. Embryo biopsy, ESC obtained from "dead" embryos, ESC produced by "parthenogenetic embryos", ESC obtained by Altered Nuclear Transfer and induced pluripotent cells (iPSC obtained by direct epigenetic reprogramming of somatic cells are the main five alternative reported in recent studies.La posibilidad de usar células madre embrionarias humanas (ESC para finalidades terapéuticas plantea graves objeciones éticas; la más fundamental es que, hasta hace poco, la única manera de obtener ESC era mediante procedimientos que destruían necesariamente embriones humanos vivos. Debido a esto, la investigación en este campo ha sido rechazada por muchos científicos, bioeticistas y ha sido prohibida por ley en varios países. Se han realizado esfuerzos para encontrar procedimientos que permitan obtener ESC sin destruir embriones o sin ponerlos en riesgo. En este documento examinamos los aspectos científicos, técnicos y éticos de las diferentes estrategias elaboradas para esta finalidad. La biopsia de embriones, ESC obtenidas de embriones "muertos"; ESC producidas por partenogénesis de embriones; ESC obtenidas mediante Transferencia Nuclear Alterada y células pluripotentes inducidas (iPSC, obtenidas mediante la reprogramación epigenética directa de las células somáticas, son las principales cinco opciones informadas en estudios

  17. Art and human embryonic stem cells: from the bench to the high street.

    Science.gov (United States)

    Duprat, Sebastien

    2009-03-01

    ESTOOLS, a project funded by the European Commission (FP6), gathers expertise on human embryonic stem cells in 10 countries of the European Research Area. The ESTOOLS outreach program uses Art extensively as the only universal cross-cultural and cross-religion means of communication. The Smile of a Stem Cell photo exhibition, a major component of this program, aims to fill a missing link between public dissemination of science and science-illiterate citizens. Scientists are also engaged to stand at a distance from their work and observe it with an outsider's perspective, which enhances their competency to communicate science. The photo exhibition, by its situation upstream of scientific education, makes itself open to interest and enthusiasm among a public with no prerequired scientific knowledge or abilities.

  18. Variations in Humanized and Defined Culture Conditions Supporting Derivation of New Human Embryonic Stem Cell Lines

    DEFF Research Database (Denmark)

    Fletcher, Judy M; Ferrier, Patricia M; Gardner, John O

    2006-01-01

    The evolution of "humanized" (i.e., free of animal sourced reagents) and ultimately chemically defined culture systems for human embryo stem cell (hESC) isolation and culture is of importance to improving their efficacy and safety in research and therapeutic applications. This can be achieved...... serum-free medium (SFM) containing only human sourced and recombinant protein. Further, outgrowth of embryonic cells from whole blastocysts in both media could be achieved for up to 1 week without reliance on feeder cells. All variant conditions sustained undifferentiated cell status, a stable karyotype......, with a transitional requirement for human feeder cells. This represents another sequential step in the generation of therapeutic grade stem cells with reduced risk of zoonotic pathogen transmission....

  19. Loss of pluripotency in human embryonic stem cells directly correlates with an increase in nuclear zinc.

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    Janet L Wolford

    Full Text Available The pluripotency of human embryonic stem cells (hESCs is important to investigations of early development and to cell replacement therapy, but the mechanism behind pluripotency is incompletely understood. Zinc has been shown to play a key role in differentiation of non-pluripotent cell types, but here its role in hESCs is directly examined. By mapping the distribution of metals in hESCs at high resolution by x-ray fluorescence microprobe (XFM and by analyzing subcellular metal content, we have found evidence that loss of pluripotency is directly correlated with an increase in nuclear zinc. Zinc elevation not only redefines our understanding of the mechanisms that support pluripotency, but also may act as a biomarker and an intervention point for stem cell differentiation.

  20. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells

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    Jared Carlson-Stevermer

    2016-01-01

    Full Text Available CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.

  1. Electrophysiological properties and calcium handling of embryonic stem cell-derived cardiomyocytes

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    Jae Boum Youm

    2016-03-01

    Full Text Available Embryonic stem cell-derived cardiomyocytes (ESC-CMs hold great interest in many fields of research including clinical applications such as stem cell and gene therapy for cardiac repair or regeneration. ESC-CMs are also used as a platform tool for pharmacological tests or for investigations of cardiac remodeling. ESC-CMs have many different aspects of morphology, electrophysiology, calcium handling, and bioenergetics compared with adult cardiomyocytes. They are immature in morphology, similar to sinus nodal-like in the electrophysiology, higher contribution of trans-sarcolemmal Ca2+ influx to Ca2+ handling, and higher dependence on anaerobic glycolysis. Here, I review a detailed electrophysiology and Ca2+ handling features of ESC-CMs during differentiation into adult cardiomyocytes to gain insights into how all the developmental changes are related to each other to display cardinal features of developing cardiomyocytes.

  2. Loss of pluripotency in human embryonic stem cells directly correlates with an increase in nuclear zinc.

    Energy Technology Data Exchange (ETDEWEB)

    Finney, L.; Vogt, S.; Wolford, J. L.; Chishti, Y.; Jin, Q.; Ward, J.; Chen, L. (Biosciences Division); ( XSD)

    2010-01-01

    The pluripotency of human embryonic stem cells (hESCs) is important to investigations of early development and to cell replacement therapy, but the mechanism behind pluripotency is incompletely understood. Zinc has been shown to play a key role in differentiation of non-pluripotent cell types, but here its role in hESCs is directly examined. By mapping the distribution of metals in hESCs at high resolution by x-ray fluorescence microprobe (XFM) and by analyzing subcellular metal content, we have found evidence that loss of pluripotency is directly correlated with an increase in nuclear zinc. Zinc elevation not only redefines our understanding of the mechanisms that support pluripotency, but also may act as a biomarker and an intervention point for stem cell differentiation.

  3. Layered double hydroxide nanoparticles promote self-renewal of mouse embryonic stem cells through the PI3K signaling pathway

    Science.gov (United States)

    Wu, Youjun; Zhu, Rongrong; Zhou, Yang; Zhang, Jun; Wang, Wenrui; Sun, Xiaoyu; Wu, Xianzheng; Cheng, Liming; Zhang, Jing; Wang, Shilong

    2015-06-01

    Embryonic stem cells (ESCs) hold great potential for regenerative medicine due to their two unique characteristics: self-renewal and pluripotency. Several groups of nanoparticles have shown promising applications in directing the stem cell fate. Herein, we investigated the cellular effects of layered double hydroxide nanoparticles (LDH NPs) on mouse ESCs (mESCs) and the associated molecular mechanisms. Mg-Al-LDH NPs with an average diameter of ~100 nm were prepared by hydrothermal methods. To determine the influences of LDH NPs on mESCs, cellular cytotoxicity, self-renewal, differentiation potential, and the possible signaling pathways were explored. Evaluation of cell viability, lactate dehydrogenase release, ROS generation and apoptosis demonstrated the low cytotoxicity of LDH NPs. The alkaline phosphatase activity and the expression of pluripotency genes in mESCs were examined, which indicated that exposure to LDH NPs could support self-renewal and inhibit spontaneous differentiation of mESCs under feeder-free culture conditions. The self-renewal promotion was further proved to be independent of the leukemia inhibitory factor (LIF). Furthermore, cells treated with LDH NPs maintained the potential to differentiate into all three germ layers both in vitro and in vivo through formation of embryoid bodies and teratomas. In addition, we observed that LDH NPs initiated the activation of the PI3K/Akt pathway, while treatment with the PI3K inhibitor LY294002 could block the effects of LDH NPs on mESCs. The results confirmed that the promotion of self-renewal by LDH NPs was associated with activation of the PI3K/Akt signaling pathway. Altogether, our studies identified a new role of LDH NPs in maintaining self-renewal of mouse ES cells which could potentially be applied in stem cell research.Embryonic stem cells (ESCs) hold great potential for regenerative medicine due to their two unique characteristics: self-renewal and pluripotency. Several groups of nanoparticles

  4. Identification and characterization of secondary neural tube-derived embryonic neural stem cells in vitro.

    Science.gov (United States)

    Shaker, Mohammed R; Kim, Joo Yeon; Kim, Hyun; Sun, Woong

    2015-05-15

    Secondary neurulation is an embryonic progress that gives rise to the secondary neural tube, the precursor of the lower spinal cord region. The secondary neural tube is derived from aggregated Sox2-expressing neural cells at the dorsal region of the tail bud, which eventually forms rosette or tube-like structures to give rise to neural tissues in the tail bud. We addressed whether the embryonic tail contains neural stem cells (NSCs), namely secondary NSCs (sNSCs), with the potential for self-renewal in vitro. Using in vitro neurosphere assays, neurospheres readily formed at the rosette and neural-tube levels, but less frequently at the tail bud tip level. Furthermore, we identified that sNSC-generated neurospheres were significantly smaller in size compared with cortical neurospheres. Interestingly, various cell cycle analyses revealed that this difference was not due to a reduction in the proliferation rate of NSCs, but rather the neuronal commitment of sNSCs, as sNSC-derived neurospheres contain more committed neuronal progenitor cells, even in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). These results suggest that the higher tendency for sNSCs to spontaneously differentiate into progenitor cells may explain the limited expansion of the secondary neural tube during embryonic development.

  5. Identification of Thalidomide-Specific Transcriptomics and Proteomics Signatures during Differentiation of Human Embryonic Stem Cells

    Science.gov (United States)

    Meganathan, Kesavan; Jagtap, Smita; Wagh, Vilas; Winkler, Johannes; Gaspar, John Antonydas; Hildebrand, Diana; Trusch, Maria; Lehmann, Karola; Hescheler, Jürgen; Schlüter, Hartmut; Sachinidis, Agapios

    2012-01-01

    Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2), that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide. PMID:22952932

  6. β-Adrenergic receptor antagonists inhibit vasculogenesis of embryonic stem cells by downregulation of nitric oxide generation and interference with VEGF signalling.

    Science.gov (United States)

    Sharifpanah, Fatemeh; Saliu, Fatjon; Bekhite, Mohamed M; Wartenberg, Maria; Sauer, Heinrich

    2014-11-01

    The β-adrenoceptor antagonist Propranolol has been successfully used to treat infantile hemangioma. However, its mechanism of action is so far unknown. The hypothesis of this research was that β-adrenoceptor antagonists may interfere with endothelial cell differentiation of stem cells. Specifically, the effects of the non-specific β-adrenergic receptor (β-adrenoceptor) antagonist Propranolol, the β1-adrenoceptor-specific antagonist Atenolol and the β2-adrenoceptor-specific antagonist ICI118,551 on vasculogenesis of mouse embryonic stem (ES) cells were investigated. All three β-blockers dose-dependently downregulated formation of capillary structures in ES cell-derived embryoid bodies and decreased the expression of the vascular cell markers CD31 and VE-cadherin. Furthermore, β-blockers downregulated the expression of fibroblast growth factor-2 (FGF-2), hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor 165 (VEGF165), VEGF receptor 2 (VEGF-R2) and phospho VEGF-R2, as well as neuropilin 1 (NRP1) and plexin-B1 which are essential modulators of embryonic angiogenesis with additional roles in vessel remodelling and arteriogenesis. Under conditions of β-adrenoceptor inhibition, the endogenous generation of nitric oxide (NO) as well as the phosphorylation of endothelial nitric oxide synthase (eNOS) was decreased in embryoid bodies, whereas an increase in NO generation was observed with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP). Consequently, vasculogenesis of ES cells was restored upon treatment of differentiating ES cells with β-adrenoceptor antagonists in the presence of NO donor. In summary, our data suggest that β-blockers impair vasculogenesis of ES cells by interfering with NO generation which could be the explanation for their anti-angiogenic effects in infantile hemangioma.

  7. Selective microRNA-Offset RNA expression in human embryonic stem cells.

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    Suvi Asikainen

    Full Text Available Small RNA molecules, including microRNAs (miRNAs, play critical roles in regulating pluripotency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs are similar in length to miRNAs, align to miRNA precursor (pre-miRNA loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence. Recent next generation sequencing (NGS studies have reported the presence of moRNAs in human neurons and cancer cells and in several tissues in mouse, including pluripotent stem cells. In order to gain additional knowledge about human moRNAs and their putative development-related expression, we applied NGS of small RNAs in human embryonic stem cells (hESCs and fibroblasts. We found that certain moRNA isoforms are notably expressed in hESCs from loci coding for stem cell-selective or cancer-related miRNA clusters. In contrast, we observed only sparse moRNAs in fibroblasts. Consistent with earlier findings, most of the observed moRNAs derived from conserved loci and their expression did not appear to correlate with the expression of the adjacent miRNAs. We provide here the first report of moRNAs in hESCs, and their expression profile in comparison to fibroblasts. Moreover, we expand the repertoire of hESC miRNAs. These findings provide an expansion on the known repertoire of small non-coding RNA contents in hESCs.

  8. Mapping dynamic histone acetylation patterns to gene expression in nanog-depleted murine embryonic stem cells.

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    Florian Markowetz

    2010-12-01

    Full Text Available Embryonic stem cells (ESC have the potential to self-renew indefinitely and to differentiate into any of the three germ layers. The molecular mechanisms for self-renewal, maintenance of pluripotency and lineage specification are poorly understood, but recent results point to a key role for epigenetic mechanisms. In this study, we focus on quantifying the impact of histone 3 acetylation (H3K9,14ac on gene expression in murine embryonic stem cells. We analyze genome-wide histone acetylation patterns and gene expression profiles measured over the first five days of cell differentiation triggered by silencing Nanog, a key transcription factor in ESC regulation. We explore the temporal and spatial dynamics of histone acetylation data and its correlation with gene expression using supervised and unsupervised statistical models. On a genome-wide scale, changes in acetylation are significantly correlated to changes in mRNA expression and, surprisingly, this coherence increases over time. We quantify the predictive power of histone acetylation for gene expression changes in a balanced cross-validation procedure. In an in-depth study we focus on genes central to the regulatory network of Mouse ESC, including those identified in a recent genome-wide RNAi screen and in the PluriNet, a computationally derived stem cell signature. We find that compared to the rest of the genome, ESC-specific genes show significantly more acetylation signal and a much stronger decrease in acetylation over time, which is often not reflected in a concordant expression change. These results shed light on the complexity of the relationship between histone acetylation and gene expression and are a step forward to dissect the multilayer regulatory mechanisms that determine stem cell fate.

  9. Generation of a constitutively expressing Tetracycline repressor (TetR human embryonic stem cell line BJNhem20-TetR

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    Ronak Shetty

    2016-03-01

    Full Text Available Human embryonic stem cell line BJNhem20-TetR was generated using non-viral method. The construct pCAG-TetRnls was transfected using microporation procedure. BJNhem20-TetR can subsequently be transfected with any vector harbouring a TetO (Tet operator sequence to generate doxycycline based inducible line. For example, in human embryonic stem cells, the pSuperior based TetO system has been transfected into a TetR containing line to generate OCT4 knockdown cell line (Zafarana et al., 2009. Thus BJNhem20-TetR can be used as a tool to perturb gene expression in human embryonic stem cells.

  10. Activation of retinoic acid receptor signaling coordinates lineage commitment of spontaneously differentiating mouse embryonic stem cells in embryoid bodies.

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    Simandi, Zoltan; Balint, Balint Laszlo; Poliska, Szilard; Ruhl, Ralph; Nagy, Laszlo

    2010-07-16

    Retinoid signaling has been implicated in embryonic stem cell differentiation. Here we present a systematic analysis of gene expression changes in mouse embryonic stem cells (mESCs), during their spontaneous differentiation into embryoid bodies and the effect of all-trans retinoic acid (ATRA) on this process. We show that retinoic acid is present in the serum and is sufficient to activate retinoid signaling at a basal level in undifferentiated mESCs. This signal disappears during embryoid body formation. However exogenously added ATRA resets the spontaneous differentiation programs in embryoid bodies and initiates a distinct genetic program. These data suggest that retinoid signaling not only promotes a particular pathway but also acts as a context dependent general coordinator of the differentiation states in embryonic stem cells. Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  11. Cryopreservation of murine embryos, human spermatozoa and embryonic stem cells using a liquid nitrogen-free, controlled rate freezer.

    Science.gov (United States)

    Morris, G J; Acton, E; Faszer, K; Franklin, A; Yin, H; Bodine, R; Pareja, J; Zaninovic, N; Gosden, R

    2006-09-01

    A Stirling Cycle Cryocooler has been developed as an alternative to conventional liquid nitrogen controlled rate freezers. Unlike liquid nitrogen systems, the Stirling Cycle freezer does not pose a contamination risk, can be used in sterile conditions and has no need for a constant supply of cryogen. Three types of samples from two species (murine embryos, human spermatozoa and embryonic stem cells), each requiring different cooling protocols, were cryopreserved in the Stirling Cycle freezer. For comparison, cells were also frozen in a conventional liquid nitrogen controlled rate freezer. Upon thawing, the rates of survival of viable cells were generally greater than 50% for mouse embryos and human embryonic stem cells, based on morphology (mouse embryos) and staining and colony formation (human embryonic stem cells). Survival rates of human spermatozoa frozen in the Stirling Cycle freezer, based on motility and dead cell staining, were similar to those of samples frozen in a conventional controlled rate freezer using liquid nitrogen.

  12. Porcine induced pluripotent stem cells analogous to naïve and primed embryonic stem cells of the mouse.

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    Telugu, Bhanu Prakash V L; Ezashi, Toshihiko; Roberts, R Michael

    2010-01-01

    Authentic or naïve embryonic stem cells (ESC) have probably never been derived from the inner cell mass (ICM) of pig blastocysts, despite over 25 years of effort. Recently, several groups, including ours, have reported induced pluripotent stem cells (iPSC) from swine by reprogramming somatic cells with a combination of four factors, OCT4 (POU5F1)/SOX2/KLF4/c-MYC delivered by retroviral transduction. The porcine (p) iPSC resembled human (h) ESC and the mouse "Epiblast stem cells" (EpiSC) in their colony morphology and expression of pluripotent genes, and are likely dependent on FGF2/ACTIVIN/NODAL signaling, therefore representing a primed ESC state. These cells are likely to advance swine as a model in biomedical research, since grafts could potentially be matched to the animal that donated the cells for re-programming. The objective of the present work has been to develop naïve piPSC. Employing a combination of seven reprogramming factors assembled on episomal vectors, we successfully reprogrammed porcine embryonic fibroblasts on a modified LIF-medium supplemented with two kinase inhibitors; CHIR99021, which inhibits GSK-3beta, and PD0325901, a MEK inhibitor. The derived piPSC bear a striking resemblance to naïve mESC in colony morphology, are dependent on LIF to maintain an undifferentiated phenotype, and express markers consistent with pluripotency. They exhibit high telomerase activity, a short cell cycle interval, and a normal karyotype, and are able to generate teratomas. Currently, the competence of these lines for contributing to germ-line chimeras is being tested.

  13. Requirement of B-Raf, C-Raf, and A-Raf for the growth and survival of mouse embryonic stem cells

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    Guo, Wenjing; Hao, Baixia; Wang, Qian; Lu, Yingying; Yue, Jianbo, E-mail: jbyue@me.com

    2013-11-01

    Extracellular signal-regulated kinases (ERKs) have been implicated to be dispensable for self-renewal of mouse embryonic stem (ES) cells, and simultaneous inhibition of both ERK signaling and glycogen synthase kinase 3 (GSK3) not only allows mouse ES cells to self-renew independent of extracellular stimuli but also enables more efficient derivation of naïve ES cells from mouse and rat strains. Interestingly, some ERKs stay active in mouse ES cells which are maintained in regular medium containing leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP). Yet, the upstream signaling for ERK activation and their roles in mouse ES cells, other than promoting or priming differentiation, have not been determined. Here we found that mouse ES cells express three forms of Raf kinases, A-Raf, B-Raf, and C-Raf. Knocking-down each single Raf member failed to affect the sustained ERK activity, neither did A-Raf and B-Raf double knockdown or B-Raf and C-Raf double knockdown change it in ES cells. Interestingly, B-Raf and C-Raf double knockdown, not A-Raf and B-Raf knockdown, inhibited the maximal ERK activation induced by LIF, concomitant with the slower growth of ES cells. On the other hand, A-Raf, B-Raf, and C-Raf triple knockdown markedly inhibited both the maximal and sustained ERK activity in ES cells. Moreover, Raf triple knockdown, similar to the treatment of U-0126, an MEK inhibitor, significantly inhibited the survival and proliferation of ES cells, thereby compromising the colony propagation of mouse ES cells. In summary, our data demonstrate that all three Raf members are required for ERK activation in mouse ES cells and are involved in growth and survival of mouse ES cells. - Highlights: ●Mouse ES (mES) cells express all three Raf members, A-Raf, B-Raf, and C-Raf. ●Leukemia inhibitory factor (LIF) temporally activates ERKs in mES cells. ●B-Raf and C-Raf are required for LIF-induced maximal ERKs activity in mES cells. ●All Raf members are

  14. Generation of Two Noradrenergic-Specific Dopamine-Beta-Hydroxylase-FLPo Knock-In Mice Using CRISPR/Cas9-Mediated Targeting in Embryonic Stem Cells.

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    Jenny J Sun

    Full Text Available CRISPR/Cas9 mediated DNA double strand cutting is emerging as a powerful approach to increase rates of homologous recombination of large targeting vectors, but the optimization of parameters, equipment and expertise required remain barriers to successful mouse generation by single-step zygote injection. Here, we sought to apply CRISPR/Cas9 methods to traditional embryonic stem (ES cell targeting followed by blastocyst injection to overcome the common issues of difficult vector construction and low targeting efficiency. To facilitate the study of noradrenergic function, which is implicated in myriad behavioral and physiological processes, we generated two different mouse lines that express FLPo recombinase under control of the noradrenergic-specific Dopamine-Beta-Hydroxylase (DBH gene. We found that by co-electroporating a circular vector expressing Cas9 and a locus-specific sgRNA, we could target FLPo to the DBH locus in ES cells with shortened 1 kb homology arms. Two different sites in the DBH gene were targeted; the translational start codon with 6-8% targeting efficiency, and the translational stop codon with 75% targeting efficiency. Using this approach, we established two mouse lines with DBH-specific expression of FLPo in brainstem catecholaminergic populations that are publically available on MMRRC (MMRRC_041575-UCD and MMRRC_041577-UCD. Altogether, this study supports simplified, high-efficiency Cas9/CRISPR-mediated targeting in embryonic stem cells for production of knock-in mouse lines in a wider variety of contexts than zygote injection alone.

  15. Differentiation of mouse embryonic stem cells into endoderm without embryoid body formation.

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    Peter T W Kim

    Full Text Available Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes. Herein, we describe a protocol using all-trans-retinoic acid, basic fibroblast growth factor and dibutyryl cAMP (DBcAMP in the absence of embryoid body formation, for differentiation of murine embryonic stem cells into definitive endoderm that may serve as pancreatic precursors. The produced cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, and pancreas. Differentiated cells displayed increased Sox17 and Foxa2 expression consistent with definitive endoderm production. There was minimal production of Sox7, an extraembryonic endoderm marker, and Oct4, a marker of pluripotency. There was minimal mesoderm or neuroectoderm formation based on expression levels of the markers brachyury and Sox1, respectively. Various assays revealed that the cell clusters generated by this protocol express markers of the pancreatic lineage including insulin I, insulin II, C-peptide, PDX-1, carboxypeptidase E, pan-cytokeratin, amylase, glucagon, PAX6, Ngn3 and Nkx6.1. This protocol using all-trans-retinoic acid, DBcAMP, in the absence of embryoid bodies, generated cells that have features of definitive endoderm that may serve as pancreatic endocrine precursors.

  16. Progesterone increases dopamine neurone number in differentiating mouse embryonic stem cells.

    Science.gov (United States)

    Díaz, N F; Díaz-Martínez, N E; Velasco, I; Camacho-Arroyo, I

    2009-08-01

    Progesterone participates in the regulation of several functions in mammals, including brain differentiation and dopaminergic transmission, but the role of progesterone in dopaminergic cell differentiation is unknown. We investigated the effects of progesterone on dopaminergic differentiation of embryonic stem cells using a five-stage protocol. Cells were incubated with different progesterone concentrations during the proliferation (stage 4) or differentiation (stage 5) phases. Progesterone added at 1, 10 and 100 nm during stage 4 increased the number of dopamine neurones at stage 5 by 72%, 80% and 62%, respectively, compared to the control group. The administration of progesterone at stage 5 did not induce significant changes in the number of dopamine neurones. These actions were not mediated by the activation of intracellular progesterone receptors because RU 486 did not block the positive effects of progesterone on differentiation to dopaminergic neurones. The results obtained suggest that progesterone should prove useful with respect to producing higher proportions of dopamine neurones from embryonic stem cells in the treatment of Parkinson's disease.

  17. DNA context represents transcription regulation of the gene in mouse embryonic stem cells

    Science.gov (United States)

    Ha, Misook; Hong, Soondo

    2016-04-01

    Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac.

  18. Human embryonic stem cell-derived neuronal cells form spontaneously active neuronal networks in vitro.

    Science.gov (United States)

    Heikkilä, Teemu J; Ylä-Outinen, Laura; Tanskanen, Jarno M A; Lappalainen, Riikka S; Skottman, Heli; Suuronen, Riitta; Mikkonen, Jarno E; Hyttinen, Jari A K; Narkilahti, Susanna

    2009-07-01

    The production of functional human embryonic stem cell (hESC)-derived neuronal cells is critical for the application of hESCs in treating neurodegenerative disorders. To study the potential functionality of hESC-derived neurons, we cultured and monitored the development of hESC-derived neuronal networks on microelectrode arrays. Immunocytochemical studies revealed that these networks were positive for the neuronal marker proteins beta-tubulin(III) and microtubule-associated protein 2 (MAP-2). The hESC-derived neuronal networks were spontaneously active and exhibited a multitude of electrical impulse firing patterns. Synchronous bursts of electrical activity similar to those reported for hippocampal neurons and rodent embryonic stem cell-derived neuronal networks were recorded from the differentiated cultures until up to 4 months. The dependence of the observed neuronal network activity on sodium ion channels was examined using tetrodotoxin (TTX). Antagonists for the glutamate receptors NMDA [D(-)-2-amino-5-phosphonopentanoic acid] and AMPA/kainate [6-cyano-7-nitroquinoxaline-2,3-dione], and for GABAA receptors [(-)-bicuculline methiodide] modulated the spontaneous electrical activity, indicating that pharmacologically susceptible neuronal networks with functional synapses had been generated. The findings indicate that hESC-derived neuronal cells can generate spontaneously active networks with synchronous communication in vitro, and are therefore suitable for use in developmental and drug screening studies, as well as for regenerative medicine.

  19. Embryonic origin of adult stem cells required for tissue homeostasis and regeneration

    Science.gov (United States)

    Davies, Erin L; Lei, Kai; Seidel, Christopher W; Kroesen, Amanda E; McKinney, Sean A; Guo, Longhua; Robb, Sofia MC; Ross, Eric J; Gotting, Kirsten; Alvarado, Alejandro Sánchez

    2017-01-01

    Planarian neoblasts are pluripotent, adult somatic stem cells and lineage-primed progenitors that are required for the production and maintenance of all differentiated cell types, including the germline. Neoblasts, originally defined as undifferentiated cells residing in the adult parenchyma, are frequently compared to embryonic stem cells yet their developmental origin remains obscure. We investigated the provenance of neoblasts during Schmidtea mediterranea embryogenesis, and report that neoblasts arise from an anarchic, cycling piwi-1+ population wholly responsible for production of all temporary and definitive organs during embryogenesis. Early embryonic piwi-1+ cells are molecularly and functionally distinct from neoblasts: they express unique cohorts of early embryo enriched transcripts and behave differently than neoblasts in cell transplantation assays. Neoblast lineages arise as organogenesis begins and are required for construction of all major organ systems during embryogenesis. These subpopulations are continuously generated during adulthood, where they act as agents of tissue homeostasis and regeneration. DOI: http://dx.doi.org/10.7554/eLife.21052.001 PMID:28072387

  20. Impact of transient down-regulation of DREAM in human embryonic stem cell pluripotency

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    A. Fontán-Lozano

    2016-05-01

    Full Text Available Little is known about the functions of downstream regulatory element antagonist modulator (DREAM in embryonic stem cells (ESCs. However, DREAM interacts with cAMP response element-binding protein (CREB in a Ca2+-dependent manner, preventing CREB binding protein (CBP recruitment. Furthermore, CREB and CBP are involved in maintaining ESC self-renewal and pluripotency. However, a previous knockout study revealed the protective function of DREAM depletion in brain aging degeneration and that aging is accompanied by a progressive decline in stem cells (SCs function. Interestingly, we found that DREAM is expressed in different cell types, including human ESCs (hESCs, human adipose-derived stromal cells (hASCs, human bone marrow-derived stromal cells (hBMSCs, and human newborn foreskin fibroblasts (hFFs, and that transitory inhibition of DREAM in hESCs reduces their pluripotency, increasing differentiation. We stipulate that these changes are partly mediated by increased CREB transcriptional activity. Overall, our data indicates that DREAM acts in the regulation of hESC pluripotency and could be a target to promote or prevent differentiation in embryonic cells.

  1. Sensitivity of human embryonic stem cells to different conditions during cryopreservation.

    Science.gov (United States)

    Xu, Yanqing; Zhang, Liang; Xu, Jiandong; Wei, Yuping; Xu, Xia

    2015-12-01

    Low cell recovery rate of human embryonic stem cells (hESCs) resulting from cryopreservation damages leads to the difficulty in their successful commercialization of clinical applications. Hence in this study, sensitivity of human embryonic stem cells (hESCs) to different cooling rates, ice seeding and cryoprotective agent (CPA) types was compared and cell viability and recovery after cryopreservation under different cooling conditions were assessed. Both extracellular and intracellular ice formation were observed. Reactive oxidative species (ROS) accumulation of hESCs was determined. Cryopreservation of hESCs at 1 °C/min with the ice seeding and at the theoretically predicted optimal cooling rate (TPOCR) led to lower level of intracellular ROS, and prevented irregular and big ice clump formation compared with cryopreservation at 1 °C/min. This strategy further resulted in a significant increase in the hESC recovery when glycerol and 1,2-propanediol were used as the CPAs, but no increase for Me2SO. hESCs after cryopreservation under all the tested conditions still maintained their pluripotency. Our results provide guidance for improving the hESC cryopreservation recovery through the combination of CPA type, cooling rate and ice seeding. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Expression and function of cannabinoid receptors CB1 and CB2 and their cognate cannabinoid ligands in murine embryonic stem cells.

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    Shuxian Jiang

    2007-07-01

    Full Text Available Characterization of intrinsic and extrinsic factors regulating the self-renewal/division and differentiation of stem cells is crucial in determining embryonic stem (ES cell fate. ES cells differentiate into multiple hematopoietic lineages during embryoid body (EB formation in vitro, which provides an experimental platform to define the molecular mechanisms controlling germ layer fate determination and tissue formation.The cannabinoid receptor type 1 (CB1 and cannabinoid receptor type 2 (CB2 are members of the G-protein coupled receptor (GPCR family, that are activated by endogenous ligands, the endocannabinoids. CB1 receptor expression is abundant in brain while CB2 receptors are mostly expressed in hematopoietic cells. However, the expression and the precise roles of CB1 and CB2 and their cognate ligands in ES cells are not known. We observed significant induction of CB1 and CB2 cannabinoid receptors during the hematopoietic differentiation of murine ES (mES-derived embryoid bodies. Furthermore, mES cells as well as ES-derived embryoid bodies at days 7 and 14, expressed endocannabinoids, the ligands for both CB1 and CB2. The CB1 and CB2 antagonists (AM251 and AM630, respectively induced mES cell death, strongly suggesting that endocannabinoids are involved in the survival of mES cells. Treatment of mES cells with the exogenous cannabinoid ligand Delta(9-THC resulted in the increased hematopoietic differentiation of mES cells, while addition of AM251 or AM630 blocked embryoid body formation derived from the mES cells. In addition, cannabinoid agonists induced the chemotaxis of ES-derived embryoid bodies, which was specifically inhibited by the CB1 and CB2 antagonists.This work has not been addressed previously and yields new information on the function of cannabinoid receptors, CB1 and CB2, as components of a novel pathway regulating murine ES cell differentiation. This study provides insights into cannabinoid system involvement in ES cell

  3. Expression and function of cannabinoid receptors CB1 and CB2 and their cognate cannabinoid ligands in murine embryonic stem cells.

    Science.gov (United States)

    Jiang, Shuxian; Fu, Yigong; Williams, John; Wood, Jodianne; Pandarinathan, Lakshmipathi; Avraham, Shiri; Makriyannis, Alexandros; Avraham, Shalom; Avraham, Hava Karsenty

    2007-07-25

    Characterization of intrinsic and extrinsic factors regulating the self-renewal/division and differentiation of stem cells is crucial in determining embryonic stem (ES) cell fate. ES cells differentiate into multiple hematopoietic lineages during embryoid body (EB) formation in vitro, which provides an experimental platform to define the molecular mechanisms controlling germ layer fate determination and tissue formation. The cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2) are members of the G-protein coupled receptor (GPCR) family, that are activated by endogenous ligands, the endocannabinoids. CB1 receptor expression is abundant in brain while CB2 receptors are mostly expressed in hematopoietic cells. However, the expression and the precise roles of CB1 and CB2 and their cognate ligands in ES cells are not known. We observed significant induction of CB1 and CB2 cannabinoid receptors during the hematopoietic differentiation of murine ES (mES)-derived embryoid bodies. Furthermore, mES cells as well as ES-derived embryoid bodies at days 7 and 14, expressed endocannabinoids, the ligands for both CB1 and CB2. The CB1 and CB2 antagonists (AM251 and AM630, respectively) induced mES cell death, strongly suggesting that endocannabinoids are involved in the survival of mES cells. Treatment of mES cells with the exogenous cannabinoid ligand Delta(9)-THC resulted in the increased hematopoietic differentiation of mES cells, while addition of AM251 or AM630 blocked embryoid body formation derived from the mES cells. In addition, cannabinoid agonists induced the chemotaxis of ES-derived embryoid bodies, which was specifically inhibited by the CB1 and CB2 antagonists. This work has not been addressed previously and yields new information on the function of cannabinoid receptors, CB1 and CB2, as components of a novel pathway regulating murine ES cell differentiation. This study provides insights into cannabinoid system involvement in ES cell survival and

  4. Neurons derived from human embryonic stem cells extend long–distance axonal projections through growth along host white matter tracts after intra-cerebral transplantation.

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    Mark eDenham

    2012-03-01

    Full Text Available Human pluripotent stem cells have the capacity for directed differentiation into a wide variety of neuronal subtypes that may be useful for brain repair. While a substantial body of research has lead to a detailed understanding of the ability of neurons in fetal tissue grafts to structurally and functionally integrate after intra-cerebral transplantation, we are only just beginning to understand the in vivo properties of neurons derived from human pluripotent stem cells. Here we have utilised the human embryonic stem (ES cell line Envy, which constitutively expresses green fluorescent protein (GFP, in order to study the in vivo properties of neurons derived from human ES cells. Rapid and efficient neural induction, followed by differentiation as neurospheres resulted in a GFP+ neural precursor population with traits of neuroepithelial and dorsal forebrain identity. Ten weeks after transplantation into neonatal rats, GFP+ fibre patterns revealed extensive axonal growth in the host brain, particularly along host white matter tracts, although innervation of adjacent nuclei was limited. The grafts were composed of a mix of neural cell types including differentiated neurons and glia, but also dividing neural progenitors and migrating neuroblasts, indicating an incomplete state of maturation at 10 weeks. This was reflected in patch-clamp recordings showing stereotypical properties appropriate for mature functional neurons, including the ability to generate action potentials, as well profiles consistent for more immature neurons. These findings illustrate the intrinsic capacity for neurons derived from human ES cells to integrate at a structural and functional level following transplantation.

  5. Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics

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    Kleinstreuer, N.C., E-mail: kleinstreuer.nicole@epa.gov [NCCT, US EPA, RTP, NC 27711 (United States); Smith, A.M.; West, P.R.; Conard, K.R.; Fontaine, B.R. [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States); Weir-Hauptman, A.M. [Covance, Inc., Madison, WI 53704 (United States); Palmer, J.A. [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States); Knudsen, T.B.; Dix, D.J. [NCCT, US EPA, RTP, NC 27711 (United States); Donley, E.L.R. [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States); Cezar, G.G. [Stemina Biomarker Discovery, Inc., Madison, WI 53719 (United States); University of Wisconsin-Madison, Madison, WI 53706 (United States)

    2011-11-15

    Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast Trade-Mark-Sign chemical screening and prioritization research project. Metabolites from hES cultures were evaluated for known and novel signatures that may be indicative of developmental toxicity. Significant fold changes in endogenous metabolites were detected for 83 putatively annotated mass features in response to the subset of ToxCast chemicals. The annotations were mapped to specific human metabolic pathways. This revealed strong effects on pathways for nicotinate and nicotinamide metabolism, pantothenate and CoA biosynthesis, glutathione metabolism, and arginine and proline metabolism pathways. Predictivity for adverse outcomes in mammalian prenatal developmental toxicity studies used ToxRefDB and other sources of information, including Stemina Biomarker Discovery's predictive DevTox Registered-Sign model trained on 23 pharmaceutical agents of known developmental toxicity and differing potency. The model initially predicted developmental toxicity from the blinded ToxCast compounds in concordance with animal data with 73% accuracy. Retraining the model with data from the unblinded test compounds at one concentration level increased the predictive accuracy for the remaining concentrations to 83%. These preliminary results on a 11-chemical subset of the ToxCast chemical library indicate that metabolomics analysis of the hES secretome provides information valuable for predictive modeling and mechanistic understanding of mammalian developmental toxicity. -- Highlights: Black-Right-Pointing-Pointer We tested 11 environmental compounds in a hESC metabolomics platform. Black-Right-Pointing-Pointer Significant changes in secreted small molecule metabolites were observed. Black-Right-Pointing-Pointer Perturbed mass features map to pathways critical for normal

  6. Very Small Embryonic-Like Stem Cells: Implications in Reproductive Biology

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    Deepa Bhartiya

    2013-01-01

    Full Text Available The most primitive germ cells in adult mammalian testis are the spermatogonial stem cells (SSCs whereas primordial follicles (PFs are considered the fundamental functional unit in ovary. However, this central dogma has recently been modified with the identification of a novel population of very small embryonic-like stem cells (VSELs in the adult mammalian gonads. These stem cells are more primitive to SSCs and are also implicated during postnatal ovarian neo-oogenesis and primordial follicle assembly. VSELs are pluripotent in nature and characterized by nuclear Oct-4A, cell surface SSEA-4, and other pluripotent markers like Nanog, Sox2, and TERT. VSELs are considered to be the descendants of epiblast stem cells and possibly the primordial germ cells that persist into adulthood and undergo asymmetric cell division to replenish the gonadal germ cells throughout life. Elucidation of their role during infertility, endometrial repair, superovulation, and pathogenesis of various reproductive diseases like PCOS, endometriosis, cancer, and so on needs to be addressed. Hence, a detailed review of current understanding of VSEL biology is pertinent, which will hopefully open up new avenues for research to better understand various reproductive processes and cancers. It will also be relevant for future regenerative medicine, translational research, and clinical applications in human reproduction.

  7. Synthetic niches for differentiation of human embryonic stem cells bypassing embryoid body formation.

    Science.gov (United States)

    Liu, Yarong; Fox, Victoria; Lei, Yuning; Hu, Biliang; Joo, Kye-Il; Wang, Pin

    2014-07-01

    The unique self-renewal and pluripotency features of human embryonic stem cells (hESCs) offer the potential for unlimited development of novel cell therapies. Currently, hESCs are cultured and differentiated using methods, such as monolayer culture and embryoid body (EB) formation. As such, achieving efficient differentiation into higher order structures remains a challenge, as well as maintaining cell viability during differentiation into homogeneous cell populations. Here, we describe the application of highly porous polymer scaffolds as synthetic stem cell niches. Bypassing the EB formation step, these scaffolds are capable of three-dimensional culture of undifferentiated hESCs and subsequent directed differentiation into three primary germ layers. H9 hESCs were successfully maintained and proliferated in biodegradable polymer scaffolds based on poly (lactic-co-glycolic acid) (PLGA). The results showed that cells within PLGA scaffolds retained characteristics of undifferentiated pluripotent stem cells. Moreover, the scaffolds allowed differentiation towards the lineage of interest by the addition of growth factors to the culture system. The in vivo transplantation study revealed that the scaffolds could provide a microenvironment that enabled hESCs to interact with their surroundings, thereby promoting cell differentiation. Therefore, this approach, which provides a unique culture/differentiation system for hESCs, will find its utility in various stem cell-based tissue-engineering applications. © 2013 Wiley Periodicals, Inc.

  8. Manipulation of the extracellular microenvironment by micro- and nanotechnology approaches to improve the generation of pancreatic endocrine cells from human embryonic stem cells

    DEFF Research Database (Denmark)

    Rasmussen, Camilla Holzmann; Rønn Petersen, Dorthe

    ) alteration of substrate topography and elasticity (paper 2) and 3) initial seeding and cell distribution (paper 3). With the first strategy an array screen was performed to systematically identify ECM protein coatings, which induced or inhibited the differentiation of hES cells towards DE. Almost 500...... 2 induced DE differentiation to a higher degree than the control fibronectin. Especially, all the analyses pointed to collagen 1 having unique properties. Cultures on collagen 1 had distinct morphology, proliferated faster and most importantly resulted in purer DE cultures with very few...... previously not been reported with embryonic stem cells, indicating that additional dimensions, such as the physical environment, should be taken into account when directing stem cell differentiation. With the last strategy, the cell seeding density and cell distribution across a well was investigated...

  9. Retinoic acid signaling and mouse embryonic stem cell differentiation: Cross talk between genomic and non-genomic effects of RA.

    Science.gov (United States)

    Rochette-Egly, Cécile

    2015-01-01

    Retinoic acid (RA), the active derivative of vitamin A, a fat-soluble vitamin, plays key roles in cell growth and differentiation by activating nuclear receptors, RARs (α, β and γ), which are ligand dependent regulators of transcription. The past years highlighted several novelties in the field that increased the complexity of RA effects. Indeed, in addition to its classical genomic effects, RA also has extranuclear and non-transcriptional effects. RA induces the rapid and transient activation of kinase cascades, which are integrated in the nucleus via the phosphorylation of RARs at a conserved serine residue located in the N-terminal domain and their coregulators. In order to investigate the relevance of RARs' phosphorylation in cell differentiation, mouse embryonic stem (mES) cells were used as a model. When treated with RA, these pluripotent cells give rise to neuronal cells. Cells invalidated for each RAR were generated as well as stable rescue lines expressing RARs mutated in phosphor acceptor sites. Such a strategy revealed that RA-induced neuronal differentiation involves the RARγ2 subtype and requires RARγ2 phosphorylation. Moreover, in gene expression profiling experiments, the phosphorylated form of RARγ2 was found to regulate a small subset of genes through binding a novel RA response element consisting of two direct repeats with a 7 base pair spacer. These new findings suggest an important role for RAR phosphorylation during cell differentiation, and pave the way for further investigations with other cell types and during embryonic development. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Gene expression signatures of extracellular matrix and growth factors during embryonic stem cell differentiation.

    Science.gov (United States)

    Nair, Rekha; Ngangan, Alyssa V; Kemp, Melissa L; McDevitt, Todd C

    2012-01-01

    Pluripotent stem cells are uniquely capable of differentiating into somatic cell derivatives of all three germ lineages, therefore holding tremendous promise for developmental biology studies and regenerative medicine therapies. Although temporal patterns of phenotypic gene expression have been relatively well characterized during the course of differentiation, coincident patterns of endogenous extracellular matrix (ECM) and growth factor expression that accompany pluripotent stem cell differentiation remain much less well-defined. Thus, the objective of this study was to examine the global dynamic profiles of ECM and growth factor genes associated with early stages of pluripotent mouse embryonic stem cell (ESC) differentiation. Gene expression analysis of ECM and growth factors by ESCs differentiating as embryoid bodies for up to 14 days was assessed using PCR arrays (172 unique genes total), and the results were examined using a variety of data mining methods. As expected, decreases in the expression of genes regulating pluripotent stem cell fate preceded subsequent increases in morphogen expression associated with differentiation. Pathway analysis generated solely from ECM and growth factor gene expression highlighted morphogenic cell processes within the embryoid bodies, such as cell growth, migration, and intercellular signaling, that are required for primitive tissue and organ developmental events. In addition, systems analysis of ECM and growth factor gene expression alone identified intracellular molecules and signaling pathways involved in the progression of pluripotent stem cell differentiation that were not contained within the array data set. Overall, these studies represent a novel framework to dissect the complex, dynamic nature of the extracellular biochemical milieu of stem cell microenvironments that regulate pluripotent cell fate decisions and morphogenesis.

  11. Method of derivation and differentiation of mouse embryonic stem cells generating synchronous neuronal networks.

    Science.gov (United States)

    Gazina, Elena V; Morrisroe, Emma; Mendis, Gunarathna D C; Michalska, Anna E; Chen, Joseph; Nefzger, Christian M; Rollo, Benjamin N; Reid, Christopher A; Pera, Martin F; Petrou, Steven

    2018-01-01

    Stem cells-derived neuronal cultures hold great promise for in vitro disease modelling and drug screening. However, currently stem cells-derived neuronal cultures do not recapitulate the functional properties of primary neurons, such as network properties. Cultured primary murine neurons develop networks which are synchronised over large fractions of the culture, whereas neurons derived from mouse embryonic stem cells (ESCs) display only partly synchronised network activity and human pluripotent stem cells-derived neurons have mostly asynchronous network properties. Therefore, strategies to improve correspondence of derived neuronal cultures with primary neurons need to be developed to validate the use of stem cell-derived neuronal cultures as in vitro models. By combining serum-free derivation of ESCs from mouse blastocysts with neuronal differentiation of ESCs in morphogen-free adherent culture we generated neuronal networks with properties recapitulating those of mature primary cortical cultures. After 35days of differentiation ESC-derived neurons developed network activity very similar to that of mature primary cortical neurons. Importantly, ESC plating density was critical for network development. Compared to the previously published methods this protocol generated more synchronous neuronal networks, with high similarity to the networks formed in mature primary cortical culture. We have demonstrated that ESC-derived neuronal networks recapitulating key properties of mature primary cortical networks can be generated by optimising both stem cell derivation and differentiation. This validates the approach of using ESC-derived neuronal cultures for disease modelling and in vitro drug screening. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Fresh or frozen? Classifying 'spare' embryos for donation to human embryonic stem cell research.

    Science.gov (United States)

    Ehrich, Kathryn; Williams, Clare; Farsides, Bobbie

    2010-12-01

    United Kingdom (UK) funding to build human embryonic stem cell (hESC) derivation labs within assisted conception units (ACU) was intended to facilitate the 'In-vitro fertilisation (IVF)-stem cell interface', including the flow of fresh 'spare' embryos to stem cell labs. However, in the three sites reported on here, which received this funding, most of the embryos used for hESC research came from long term cryopreservation storage and/or outside clinics. In this paper we explore some of the clinical, technical, social and ethical factors that might help to explain this situation. We report from our qualitative study of the ethical frameworks for approaching women/couples for donation of embryos to stem cell research. Members of staff took part in 44 interviews and six ethics discussion groups held at our study sites between February 2008 and October 2009. We focus here on their articulations of social and ethical, as well as scientific, dimensions in the contingent classification of 'spare' embryos, entailing uncertainty, fluidity and naturalisation in classifying work. Social and ethical factors include acknowledging and responding to uncertainty in classifying embryos; retaining 'fluidity' in the grading system to give embryos 'every chance'; tensions between standardisation and variation in enacting a 'fair' grading system; enhancement of patient choice and control, and prevention of regret; and incorporation of patients' values in construction of ethically acceptable embryo 'spareness' ('frozen' embryos, and embryos determined through preimplantation genetic diagnosis (PGD) to be genetically 'affected'). We argue that the success of the 'built moral environment' of ACU with adjoining stem cell laboratories building projects intended to facilitate the 'IVF-stem cell interface' may depend not only on architecture, but also on the part such social and ethical factors play in configuration of embryos as particular kinds of moral work objects. Copyright © 2010

  13. High expression of hTERT and stemness genes in BORIS/CTCFL positive cells isolated from embryonic cancer cells.

    Directory of Open Access Journals (Sweden)

    Loredana Alberti

    Full Text Available BORIS/CTCFL is a member of cancer testis antigen family normally expressed in germ cells. In tumors, it is aberrantly expressed although its functions are not completely well-defined. To better understand the functions of BORIS in cancer, we selected the embryonic cancer cells as a model. Using a molecular beacon, which specifically targets BORIS mRNA, we demonstrated that BORIS positive cells are a small subpopulation of tumor cells (3-5% of total. The BORIS-positive cells isolated using BORIS-molecular beacon, expressed higher telomerase hTERT, stem cell (NANOG, OCT4, SOX2 and cancer stem cell marker genes (CD44 and ALDH1 compared to the BORIS-negative tumor cells. In order to define the functional role of BORIS, stable BORIS-depleted embryonic cancer cells were generated. BORIS silencing strongly down-regulated the expression of hTERT, stem cell and cancer stem cell marker genes. Moreover, the BORIS knockdown increased cellular senescence in embryonic cancer cells, revealing a putative role of BORIS in the senescence biological program. Our data indicate an association of BORIS expressing cells subpopulation with the expression of stemness genes, highlighting the critical role played by BORIS in embryonic neoplastic disease.

  14. Applying a "double-feature" promoter to identify cardiomyocytes differentiated from human embryonic stem cells following transposon-based gene delivery.

    Science.gov (United States)

    Orbán, Tamás I; Apáti, Agota; Németh, Andrea; Varga, Nóra; Krizsik, Virág; Schamberger, Anita; Szebényi, Kornélia; Erdei, Zsuzsa; Várady, György; Karászi, Eva; Homolya, László; Német, Katalin; Gócza, Elen; Miskey, Csaba; Mátés, Lajos; Ivics, Zoltán; Izsvák, Zsuzsanna; Sarkadi, Balázs

    2009-05-01

    Human embryonic stem (HuES) cells represent a new potential tool for cell-therapy and gene-therapy applications. However, these approaches require the development of efficient, stable gene delivery, and proper progenitor cell and tissue separation methods. In HuES cell lines, we have generated stable, enhanced green fluorescent protein (EGFP)-expressing clones using a transposon-based (Sleeping Beauty) system. This method yielded high percentage of transgene integration and expression. Similarly to a lentiviral expression system, both the undifferentiated state and the differentiation pattern of the HuES cells were preserved. By using the CAG promoter, in contrast to several other constitutive promoter sequences (such as CMV, elongation factor 1alpha, or phosphoglycerate kinase), an exceptionally high EGFP expression was observed in differentiated cardiomyocytes. This phenomenon was independent of the transgene sequence, methods of gene delivery, copy number, and the integration sites. This "double-feature" promoter behavior, that is providing a selectable marker for transgene expressing undifferentiated stem cells, and also specifically labeling differentiated cardiomyocytes, was assessed by transcriptional profiling. We found a positive correlation between CAG promoter-driven EGFP transcription and expression of cardiomyocyte-specific genes. Our experiments indicate an efficient applicability of transposon-based gene delivery into HuES cells and provide a novel approach to identify differentiated tissues by exploiting a nontypical behavior of a constitutively active promoter, thereby avoiding invasive drug selection methods.

  15. Canonical Wnt signaling promotes early hematopoietic progenitor formation and erythroid specification during embryonic stem cell differentiation.

    Science.gov (United States)

    Tarafdar, Anuradha; Dobbin, Edwina; Corrigan, Pamela; Freeburn, Robin; Wheadon, Helen

    2013-01-01

    The generation of hematopoietic stem cells (HSCs) during development is a complex process linked to morphogenic signals. Understanding this process is important for regenerative medicine applications that require in vitro production of HSC. In this study we investigated the effects of canonical Wnt/β-catenin signaling during early embryonic differentiation and hematopoietic specification using an embryonic stem cell system. Our data clearly demonstrates that following early differentiation induction, canonical Wnt signaling induces a strong mesodermal program whilst maintaining a degree of stemness potential. This involved a complex interplay between β-catenin/TCF/LEF/Brachyury/Nanog. β-catenin mediated up-regulation of TCF/LEF resulted in enhanced brachyury levels, which in-turn lead to Nanog up-regulation. During differentiation, active canonical Wnt signaling also up-regulated key transcription factors and cell specific markers essential for hematopoietic specification, in particular genes involved in establishing primitive erythropoiesis. This led to a significant increase in primitive erythroid colony formation. β-catenin signaling also augmented early hematopoietic and multipotent progenitor (MPP) formation. Following culture in a MPP specific cytokine cocktail, activation of β-catenin suppressed differentiation of the early hematopoietic progenitor population, with cells displaying a higher replating capacity and a propensity to form megakaryocytic erythroid progenitors. This bias towards erythroid lineage commitment was also observed when hematopoietic progenitors were directed to undergo myeloid colony formation. Overall this study underscores the importance of canonical Wnt/β-catenin signaling in mesodermal specification, primitive erythropoiesis and early hematopietic progenitor formation during hematopoietic induction.

  16. Canonical Wnt signaling promotes early hematopoietic progenitor formation and erythroid specification during embryonic stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Anuradha Tarafdar

    Full Text Available The generation of hematopoietic stem cells (HSCs during development is a complex process linked to morphogenic signals. Understanding this process is important for regenerative medicine applications that require in vitro production of HSC. In this study we investigated the effects of canonical Wnt/β-catenin signaling during early embryonic differentiation and hematopoietic specification using an embryonic stem cell system. Our data clearly demonstrates that following early differentiation induction, canonical Wnt signaling induces a strong mesodermal program whilst maintaining a degree of stemness potential. This involved a complex interplay between β-catenin/TCF/LEF/Brachyury/Nanog. β-catenin mediated up-regulation of TCF/LEF resulted in enhanced brachyury levels, which in-turn lead to Nanog up-regulation. During differentiation, active canonical Wnt signaling also up-regulated key transcription factors and cell specific markers essential for hematopoietic specification, in particular genes involved in establishing primitive erythropoiesis. This led to a significant increase in primitive erythroid colony formation. β-catenin signaling also augmented early hematopoietic and multipotent progenitor (MPP formation. Following culture in a MPP specific cytokine cocktail, activation of β-catenin suppressed differentiation of the early hematopoietic progenitor population, with cells displaying a higher replating capacity and a propensity to form megakaryocytic erythroid progenitors. This bias towards erythroid lineage commitment was also observed when hematopoietic progenitors were directed to undergo myeloid colony formation. Overall this study underscores the importance of canonical Wnt/β-catenin signaling in mesodermal specification, primitive erythropoiesis and early hematopietic progenitor formation during hematopoietic induction.

  17. Screen for abnormal mitochondrial phenotypes in mouse embryonic stem cells identifies a model for succinyl-CoA ligase deficiency and mtDNA depletion

    Directory of Open Access Journals (Sweden)

    Taraka R. Donti

    2014-02-01

    Full Text Available Mutations in subunits of succinyl-CoA synthetase/ligase (SCS, a component of the citric acid cycle, are associated with mitochondrial encephalomyopathy, elevation of methylmalonic acid (MMA, and mitochondrial DNA (mtDNA depletion. A FACS-based retroviral-mediated gene trap mutagenesis screen in mouse embryonic stem (ES cells for abnormal mitochondrial phenotypes identified a gene trap allele of Sucla2 (Sucla2SAβgeo, which was used to generate transgenic mice. Sucla2 encodes the ADP-specific β-subunit isoform of SCS. Sucla2SAβgeo homozygotes exhibited recessive lethality, with most mutants dying late in gestation (e18.5. Mutant placenta and embryonic (e17.5 brain, heart and muscle showed varying degrees of mtDNA depletion (20–60%. However, there was no mtDNA depletion in mutant liver, where the gene is not normally expressed. Elevated levels of MMA were observed in embryonic brain. SCS-deficient mouse embryonic fibroblasts (MEFs demonstrated a 50% reduction in mtDNA content compared with wild-type MEFs. The mtDNA depletion resulted in reduced steady state levels of mtDNA encoded proteins and multiple respiratory chain deficiencies. mtDNA content could be restored by reintroduction of Sucla2. This mouse model of SCS deficiency and mtDNA depletion promises to provide insights into the pathogenesis of mitochondrial diseases with mtDNA depletion and into the biology of mtDNA maintenance. In addition, this report demonstrates the power of a genetic screen that combines gene trap mutagenesis and FACS analysis in mouse ES cells to identify mitochondrial phenotypes and to develop animal models of mitochondrial dysfunction.

  18. Transplantation of Embryonic and Induced Pluripotent Stem Cell-Derived 3D Retinal Sheets into Retinal Degenerative Mice

    Directory of Open Access Journals (Sweden)

    Juthaporn Assawachananont

    2014-05-01

    Full Text Available In this article, we show that mouse embryonic stem cell- or induced pluripotent stem cell-derived 3D retinal tissue developed a structured outer nuclear layer (ONL with complete inner and outer segments even in an advanced retinal degeneration model (rd1 that lacked ONL. We also observed host-graft synaptic connections by immunohistochemistry. This study provides a “proof of concept” for retinal sheet transplantation therapy for advanced retinal degenerative diseases.

  19. Expression and loss of alleles in cultured mouse embryonic fibroblasts and stem cells carrying allelic fluorescent protein genes

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    Stringer Saundra L

    2006-10-01

    Full Text Available Abstract Background Loss of heterozygosity (LOH contributes to many cancers, but the rate at which these events occur in normal cells of the body is not clear. LOH would be detectable in diverse cell types in the body if this event were to confer an obvious cellular phenotype. Mice that carry two different fluorescent protein genes as alleles of a locus would seem to be a useful tool for addressing this issue because LOH would change a cell's phenotype from dichromatic to monochromatic. In addition, LOH caused by mitotic crossing over might be discernable in tissues because this event produces a pair of neighboring monochromatic cells that are different colors. Results As a step in assessing the utility of this approach, we derived primary embryonic fibroblast populations and embryonic stem cell lines from mice that carried two different fluorescent protein genes as alleles at the chromosome 6 locus, ROSA26. Fluorescence activated cell sorting (FACS showed that the vast majority of cells in each line expressed the two marker proteins at similar levels, and that populations exhibited expression noise similar to that seen in bacteria and yeast. Cells with a monochromatic phenotype were present at frequencies on the order of 10-4 and appeared to be produced at a rate of approximately 10-5 variant cells per mitosis. 45 of 45 stably monochromatic ES cell clones exhibited loss of the expected allele at the ROSA26 locus. More than half of these clones retained heterozygosity at a locus between ROSA26 and the centromere. Other clones exhibited LOH near the centromere, but were disomic for chromosome 6. Conclusion Allelic fluorescent markers allowed LOH at the ROSA26 locus to be detected by FACS. LOH at this locus was usually not accompanied by LOH near the centromere, suggesting that mitotic recombination was the major cause of ROSA26 LOH. Dichromatic mouse embryonic cells provide a novel system for studying genetic/karyotypic stability and factors

  20. Efficient derivation of multipotent neural stem/progenitor cells from non-human primate embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Hiroko Shimada

    Full Text Available The common marmoset (Callithrix jacchus is a small New World primate that has been used as a non-human primate model for various biomedical studies. We previously demonstrated that transplantation of neural stem/progenitor cells (NS/PCs derived from mouse and human embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs promote functional locomotor recovery of mouse spinal cord injury models. However, for the clinical application of such a therapeutic approach, we need to evaluate the efficacy and safety of pluripotent stem cell-derived NS/PCs not only by xenotransplantation, but also allotransplantation using non-human primate models to assess immunological rejection and tumorigenicity. In the present study, we established a culture method to efficiently derive NS/PCs as neurospheres from common marmoset ESCs. Marmoset ESC-derived neurospheres could be passaged repeatedly and showed sequential generation of neurons and astrocytes, similar to that of mouse ESC-derived NS/PCs, and gave rise to functional neurons as indicated by calcium imaging. Although marmoset ESC-derived NS/PCs could not differentiate into oligodendrocytes under default culture conditions, these cells could abundantly generate oligodendrocytes by incorporating additional signals that recapitulate in vivo neural development. Moreover, principal component analysis of microarray data demonstrated that marmoset ESC-derived NS/PCs acquired similar gene expression profiles to those of fetal brain-derived NS/PCs by repeated passaging. Therefore, marmoset ESC-derived NS/PCs may be useful not only for accurate evaluation by allotransplantation of NS/PCs into non-human primate models, but are also applicable to analysis of iPSCs established from transgenic disease model marmosets.

  1. A single-cell and feeder-free culture system for monkey embryonic stem cells.

    Science.gov (United States)

    Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

    2014-01-01

    Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

  2. Human Embryonic and Induced Pluripotent Stem Cell Research Trends: Complementation and Diversification of the Field

    Directory of Open Access Journals (Sweden)

    Sabine Kobold

    2015-05-01

    Full Text Available Research in human induced pluripotent stem cells (hiPSCs is rapidly developing and there are expectations that this research may obviate the need to use human embryonic stem cells (hESCs, the ethics of which has been a subject of controversy for more than 15 years. In this study, we investigated approximately 3,400 original research papers that reported an experimental use of these types of human pluripotent stem cells (hPSCs and were published from 2008 to 2013. We found that research into both cell types was conducted independently and further expanded, accompanied by a growing intersection of both research fields. Moreover, an in-depth analysis of papers that reported the use of both cell types indicates that hESCs are still being used as a “gold standard,” but in a declining proportion of publications. Instead, the expanding research field is diversifying and hESC and hiPSC lines are increasingly being used in more independent research and application areas.

  3. Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells.

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    Amy Sebeson

    Full Text Available The binding sequence for any transcription factor can be found millions of times within a genome, yet only a small fraction of these sequences encode functional transcription factor binding sites. One of the reasons for this dichotomy is that many other factors, such as nucleosomes, compete for binding. To study how the competition between nucleosomes and transcription factors helps determine a functional transcription factor site from a predicted transcription factor site, we compared experimentally-generated in vitro nucleosome occupancy with in vivo nucleosome occupancy and transcription factor binding in murine embryonic stem cells. Using a solution hybridization enrichment technique, we generated a high-resolution nucleosome map from targeted regions of the genome containing predicted sites and functional sites of Oct4/Sox2 regulation. We found that at Pax6 and Nes, which are bivalently poised in stem cells, functional Oct4 and Sox2 sites show high amounts of in vivo nucleosome displacement compared to in vitro. Oct4 and Sox2, which are active, show no significant displacement of in vivo nucleosomes at functional sites, similar to nonfunctional Oct4/Sox2 binding. This study highlights a complex interplay between Oct4 and Sox2 transcription factors and nucleosomes among different target genes, which may result in distinct patterns of stem cell gene regulation.

  4. ZFX controls the self-renewal of human embryonic stem cells.

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    Sivan Harel

    Full Text Available Embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs offer great promise in regenerative medicine and disease modeling due to their unlimited self-renewal and broad differentiation capacity. There is evidence that the growth properties and critical signaling pathways differ between murine and human ESCs; therefore, it is essential to perform functional studies to test the putatively conserved mechanisms of pluripotent stem cell self-renewal between species. Previously, we identified the transcription factor Zfx as a key regulator of self-renewal in murine ESCs. Here we extend those findings to human ESCs. ZFX knockdown in hESCs hindered clonal growth and decreased colony size after serial replating. ZFX overexpression enhanced clone formation in the presence of Y-27632, increased colony size at low density and decreased expression of differentiation-related genes in human ESCs. ZFX-overexpressing hESCs resisted spontaneous differentiation but could be directed to differentiate into endodermal and neural cell fates when provided with the appropriate cues. Thus, ZFX acts as a molecular rheostat regulating the balance between self-renewal and differentiation in hESCs, revealing the close evolutionary conservation of the self-renewal mechanisms in murine and human ESCs.

  5. A large-scale proteomic analysis of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Sherrer Eric

    2007-12-01

    Full Text Available Abstract Background Much of our current knowledge of the molecular expression profile of human embryonic stem cells (hESCs is based on transcriptional approaches. These analyses are only partly predictive of protein expression however, and do not shed light on post-translational regulation, leaving a large gap in our knowledge of the biology of pluripotent stem cells. Results Here we describe the use of two large-scale western blot assays to identify over 600 proteins expressed in undifferentiated hESCs, and highlight over 40 examples of multiple gel mobility variants, which are suspected protein isoforms and/or post-translational modifications. Twenty-two phosphorylation events in cell signaling molecules, as well as potential new markers of undifferentiated hESCs were also identified. We confirmed the expression of a subset of the identified proteins by immunofluorescence and correlated the expression of transcript and protein for key molecules in active signaling pathways in hESCs. These analyses also indicated that hESCs exhibit several features of polarized epithelia, including expression of tight junction proteins. Conclusion Our approach complements proteomic and transcriptional analysis to provide unique information on human pluripotent stem cells, and is a framework for the continued analyses of self-renewal.

  6. Effects of ß-TCP scaffolds on neurogenic and osteogenic differentiation of human embryonic stem cells.

    Science.gov (United States)

    Arpornmaeklong, Premjit; Pressler, Michael J

    2018-01-01

    Extracellular matrix (ECM) and adhesion molecules play crucial roles in regulating growth and differentiation of stem cells. The current study aimed to investigate the effects of beta-tricalcium phosphate (ß-TCP) scaffolds on differentiation and expression of ECM and adhesion molecules of human embryonic stem cells (hESCs). Undifferentiated hESCs were seeded on ß-TCP scaffolds and cell culture plates and cultured in growth and osteogenic medium for 21 days. Scanning electron microscopy (SEM) displayed adhesion and growth of hESCs on the porous ß-TCP scaffolds. Histological analysis, immunohistochemical staining and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) demonstrated that the scaffolds supported growth and differentiation of hESCs. Expression levels of neural crest related genes (AP2a, FoxD3, HNK1, P75, Sox1, Sox10) and osteoblast-related genes (Runx2, SPP1 and BGLA) on the scaffolds in osteogenic medium were significantly higher than on the scaffolds in growth and cell culture plates in osteogenic medium, respectively (pTCP scaffolds promoted differentiation of hESCs, particularly expression of genes related to neural crest stem cell and osteoblastic differentiations. Beta-TCP scaffolds could be an alternative cell culture substrate for neural crest and osteogenic differentiation of hESCs. Optimization of culture medium may be necessary to enhance lineage restriction of hESCs on the ß-TCP scaffolds. Copyright © 2017 Elsevier GmbH. All rights reserved.

  7. Restoration of heart functions using human embryonic stem cells derived heart muscle cells.

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    Gepstein, Lior; Kehat, Izhak

    2005-02-01

    Extract: Recent advances in molecular and cellular biology and specifically in the areas of stem cell biology and tissue engineering have paved the way for the development of a new field in biomedicine, regenerative medicine. This exciting approach seeks to develop new biological solutions, using the mobilization of endogenous stem cells or delivery of exogenous cells to replace or modify the function of diseased, absent, or malfunctioning tissue. The adult heart represents an attractive candidate for these emerging technologies, since adult cardiomyocytes have limited regenerative capacity. Thus, any significant heart cell loss or dysfunction, such as occurs during heart attack, is mostly irreversible and may lead to the development of progressive heart failure, one of the leading causes of world-wide morbidity and mortality. Similarly, dysfunction of the specialized electrical conduction system within the heart may result in inefficient rhythm initiation or impulse conduction, leading to significant slowing of the heart rate, usually requiring the implantation of a permanent electronic pacemaker. Replacement of the dysfunctional myocardium (heart muscle) by implantation of external heart muscle cells is emerging as a novel paradigm for restoration of the myocardial electromechanical properties, but has been significantly hampered by the paucity of cell sources for human heart cells and by the relatively limited evidence for functional integration between grafted and host cells. The recently described human embryonic stem cell (hESC) lines may provide a possible solution for the aforementioned cell sourcing problem.

  8. Effects of the EVCAM chemical validation library on differentiation using marker gene expression in lmouse embryonic stem cells

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    The adherent cell differentiation and cytotoxicity (ACDC) assay was used to profile the effects of the ECVAM EST validation chemical library (19 compounds) on J1 mouse embryonic stem cells (mESC). PCR-based TaqMan Low Density Arrays (TLDA) provided a high-content assessment of al...

  9. Tet Proteins Connect the O-Linked N-acetylglucosamine Transferase Ogt to Chromatin in Embryonic Stem Cells

    DEFF Research Database (Denmark)

    Vella, Pietro; Scelfo, Andrea; Jammula, Sriganesh

    2013-01-01

    O-linked N-acetylglucosamine (O-GlcNAc) transferase (Ogt) activity is essential for embryonic stem cell (ESC) viability and mouse development. Ogt is present both in the cytoplasm and the nucleus of different cell types and catalyzes serine and threonine glycosylation. We have characterized...

  10. Effects of oxidative stress on human embryonic stem cells; global gene expression, advanced glycation end products and NEDD1 levels

    NARCIS (Netherlands)

    Barandalla Sobrados, M.

    2017-01-01

    A number of unfavorable conditions can affect the development of the early embryo inducing oxidative stress both in vivo, for instance in gestational diabetes, and in vitro, when embryos are derived from Assisted Reproductive Technologies (ART). Human Embryonic Stem Cells (hESCs) potentially offer a

  11. An optimized gene set for transcriptomics based neurodevelopmental toxicity prediction in the neural embryonic stem cell test

    NARCIS (Netherlands)

    Pennings, J.L.A.; Theunissen, P.T.; Piersma, A.H.|info:eu-repo/dai/nl/071276947

    2012-01-01

    The murine neural embryonic stem cell test (ESTn) is an in vitro model for neurodevelopmental toxicity testing. Recent studies have shown that application of transcriptomics analyses in the ESTn is useful for obtaining more accurate predictions as well as mechanistic insights. Gene expression

  12. A vector-based system for the differentiation of mouse embryonic stem cells toward germ-line cells

    Directory of Open Access Journals (Sweden)

    Reza Ebrahimzadeh-Vesal

    2014-08-01

    Conclusion: In this study, we demonstrated the in vitro generation of mouse embryonic stem cells to germ cells by using a backbone vector containing the fusion gene Stra8-EGFP. The Stra8 gene is a retinoic acid-responsive protein and is able to regulate meiotic initiation.

  13. Immunoflourescence and mRNA analysis of human embryonic stem cells (hESCs) grown under feeder-free conditions

    DEFF Research Database (Denmark)

    Awan, Aashir; Oliveri, Roberto S; Jensen, Pernille L

    2010-01-01

    This chapter describes the procedures in order to do immunofluorescence (IF) microscopy and quantitative PCR (qPCR) analysis of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol outlining the steps from initially growing the cells, passaging...

  14. High School Students Debate the Use of Embryonic Stem Cells: The Influence of Context on Decision-Making

    Science.gov (United States)

    Molinatti, Gregoire; Girault, Yves; Hammond, Constance

    2010-01-01

    The present study analyzes decision-making and argumentation by high school students in a debate situation on a socioscientific issue, the use of embryonic stem cells in research and therapy. We tested the influence on the debates of two different contexts. Adolescent students at the high school level in the same grade (mean age 16.4 years) from…

  15. Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells

    NARCIS (Netherlands)

    Sorkio, A.; Haimi, Suvi; Verdoold, Vincent; Juuti-Uusitalo, K.; Grijpma, Dirk W.; Skottman, H.

    2017-01-01

    Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great

  16. Developing predictions of in vivo developmental toxicity of ToxCast chemicals using mouse embryonic stem cells.

    Science.gov (United States)

    Developing predictions of in vivo developmental toxicity of ToxCast chemicals using mouse embryonic stem cells S. Hunter, M. Rosen, M. Hoopes, H. Nichols, S. Jeffay, K. Chandler1, Integrated Systems Toxicology Division, National Health and Environmental Effects Research Labor...

  17. The transcriptomes of novel marmoset monkey embryonic stem cell lines reflect distinct genomic features.

    Science.gov (United States)

    Debowski, Katharina; Drummer, Charis; Lentes, Jana; Cors, Maren; Dressel, Ralf; Lingner, Thomas; Salinas-Riester, Gabriela; Fuchs, Sigrid; Sasaki, Erika; Behr, Rüdiger

    2016-07-07

    Embryonic stem cells (ESCs) are useful for the study of embryonic development. However, since research on naturally conceived human embryos is limited, non-human primate (NHP) embryos and NHP ESCs represent an excellent alternative to the corresponding human entities. Though, ESC lines derived from naturally conceived NHP embryos are still very rare. Here, we report the generation and characterization of four novel ESC lines derived from natural preimplantation embryos of the common marmoset monkey (Callithrix jacchus). For the first time we document derivation of NHP ESCs derived from morula stages. We show that quantitative chromosome-wise transcriptome analyses precisely reflect trisomies present in both morula-derived ESC lines. We also demonstrate that the female ESC lines exhibit different states of X-inactivation which is impressively reflected by the abundance of the lncRNA X inactive-specific transcript (XIST). The novel marmoset ESC lines will promote basic primate embryo and ESC studies as well as preclinical testing of ESC-based regenerative approaches in NHP.

  18. Role of ALKBH1 in the Core Transcriptional Network of Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Rune Ougland

    2016-01-01

    Full Text Available Background/Aims: ALKBH1, an AlkB homologue in the 2-oxoglutarate and Fe2+ dependent hydroxylase family, is a histone dioxygenase that removes methyl groups from histone H2A. Studies of transgenic mice lacking Alkbh1 reveal that most Alkbh1-/- embryos die during embryonic development. Embryonic stem cells (ESCs derived from these mice have prolonged expression of pluripotency markers and delayed induction of genes involved in neural differentiation, indicating that ALKBH1 is involved in regulation of pluripotency and differentiation. The aim of this study was to further investigate the role ALKBH1 in early de