WorldWideScience

Sample records for monkey ips cells

  1. Non-viral generation of marmoset monkey iPS cells by a six-factor-in-one-vector approach.

    Directory of Open Access Journals (Sweden)

    Katharina Debowski

    Full Text Available Groundbreaking studies showed that differentiated somatic cells of mouse and human origin could be reverted to a stable pluripotent state by the ectopic expression of only four proteins. The resulting pluripotent cells, called induced pluripotent stem (iPS cells, could be an alternative to embryonic stem cells, which are under continuous ethical debate. Hence, iPS cell-derived functional cells such as neurons may become the key for an effective treatment of currently incurable degenerative diseases. However, besides the requirement of efficacy testing of the therapy also its long-term safety needs to be carefully evaluated in settings mirroring the clinical situation in an optimal way. In this context, we chose the long-lived common marmoset monkey (Callithrix jacchus as a non-human primate species to generate iPS cells. The marmoset monkey is frequently used in biomedical research and is gaining more and more preclinical relevance due to the increasing number of disease models. Here, we describe, to our knowledge, the first-time generation of marmoset monkey iPS cells from postnatal skin fibroblasts by non-viral means. We used the transposon-based, fully reversible piggyback system. We cloned the marmoset monkey reprogramming factors and established robust and reproducible reprogramming protocols with a six-factor-in-one-construct approach. We generated six individual iPS cell lines and characterized them in comparison with marmoset monkey embryonic stem cells. The generated iPS cells are morphologically indistinguishable from marmoset ES cells. The iPS cells are fully reprogrammed as demonstrated by differentiation assays, pluripotency marker expression and transcriptome analysis. They are stable for numerous passages (more than 80 and exhibit euploidy. In summary, we have established efficient non-viral reprogramming protocols for the derivation of stable marmoset monkey iPS cells, which can be used to develop and test cell replacement

  2. Non-viral generation of marmoset monkey iPS cells by a six-factor-in-one-vector approach.

    Science.gov (United States)

    Debowski, Katharina; Warthemann, Rita; Lentes, Jana; Salinas-Riester, Gabriela; Dressel, Ralf; Langenstroth, Daniel; Gromoll, Jörg; Sasaki, Erika; Behr, Rüdiger

    2015-01-01

    Groundbreaking studies showed that differentiated somatic cells of mouse and human origin could be reverted to a stable pluripotent state by the ectopic expression of only four proteins. The resulting pluripotent cells, called induced pluripotent stem (iPS) cells, could be an alternative to embryonic stem cells, which are under continuous ethical debate. Hence, iPS cell-derived functional cells such as neurons may become the key for an effective treatment of currently incurable degenerative diseases. However, besides the requirement of efficacy testing of the therapy also its long-term safety needs to be carefully evaluated in settings mirroring the clinical situation in an optimal way. In this context, we chose the long-lived common marmoset monkey (Callithrix jacchus) as a non-human primate species to generate iPS cells. The marmoset monkey is frequently used in biomedical research and is gaining more and more preclinical relevance due to the increasing number of disease models. Here, we describe, to our knowledge, the first-time generation of marmoset monkey iPS cells from postnatal skin fibroblasts by non-viral means. We used the transposon-based, fully reversible piggyback system. We cloned the marmoset monkey reprogramming factors and established robust and reproducible reprogramming protocols with a six-factor-in-one-construct approach. We generated six individual iPS cell lines and characterized them in comparison with marmoset monkey embryonic stem cells. The generated iPS cells are morphologically indistinguishable from marmoset ES cells. The iPS cells are fully reprogrammed as demonstrated by differentiation assays, pluripotency marker expression and transcriptome analysis. They are stable for numerous passages (more than 80) and exhibit euploidy. In summary, we have established efficient non-viral reprogramming protocols for the derivation of stable marmoset monkey iPS cells, which can be used to develop and test cell replacement therapies in

  3. Cynomolgus monkey induced pluripotent stem cells established by using exogenous genes derived from the same monkey species.

    Science.gov (United States)

    Shimozawa, Nobuhiro; Ono, Ryoichi; Shimada, Manami; Shibata, Hiroaki; Takahashi, Ichiro; Inada, Hiroyasu; Takada, Tatsuyuki; Nosaka, Tetsuya; Yasutomi, Yasuhiro

    2013-01-01

    Induced pluripotent stem (iPS) cells established by introduction of the transgenes POU5F1 (also known as Oct3/4), SOX2, KLF4 and c-MYC have competence similar to embryonic stem (ES) cells. iPS cells generated from cynomolgus monkey somatic cells by using genes taken from the same species would be a particularly important resource, since various biomedical investigations, including studies on the safety and efficacy of drugs, medical technology development, and research resource development, have been performed using cynomolgus monkeys. In addition, the use of xenogeneic genes would cause complicating matters such as immune responses when they are expressed. In this study, therefore, we established iPS cells by infecting cells from the fetal liver and newborn skin with amphotropic retroviral vectors containing cDNAs for the cynomolgus monkey genes of POU5F1, SOX2, KLF4 and c-MYC. Flat colonies consisting of cells with large nuclei, similar to those in other primate ES cell lines, appeared and were stably maintained. These cell lines had normal chromosome numbers, expressed pluripotency markers and formed teratomas. We thus generated cynomolgus monkey iPS cell lines without the introduction of ecotropic retroviral receptors or other additional transgenes by using the four allogeneic transgenes. This may enable detailed analysis of the mechanisms underlying the reprogramming. In conclusion, we showed that iPS cells could be derived from cynomolgus monkey somatic cells. To the best of our knowledge, this is the first report on iPS cell lines established from cynomolgus monkey somatic cells by using genes from the same species.

  4. [iPS cells in dentistry].

    Science.gov (United States)

    Egusa, Hiroshi

    2012-01-01

    The discovery of the induced pluripotent stem (iPS) cell technology, which enables us to produce pluripotent stem cells by introducing a few genetic factors, commands considerable attention in the field of dentistry. These iPS cells may be of particular importance for developing innovative technologies to regenerate missing jaw bones and lost teeth, and there are expectations that several types of tissue stem cells and mucosal cells in the oral area can be used as an ideal iPS cell source. We previously reported that the gingiva, which is often resected during general dental treatments and treated as biomedical waste, is a promising source of iPS cells. In this review, the current trends in iPS cell research in dentistry are outlined, and future aspects of potential applications of the iPS cell technologies to dental treatments will be discussed.

  5. Generation of iPS Cells from Granulosa Cells.

    Science.gov (United States)

    Mao, Jian; Liu, Lin

    2016-01-01

    Various types of somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells. Somatic stem cells may generate iPS cells more efficiently than do differentiated cells. We show that granulosa cells exhibit characteristic of somatic stem cells and can be reprogrammed to iPS cells more efficiently or with few factors. Here, we describe generation of mouse and pig iPS cells from granulosa cells with high efficiency.

  6. iPS cells and personalized medicine

    OpenAIRE

    2014-01-01

    iPS cells and personalized medicine Personalized medicine uses patient's genotype and/or level of gene expression for selection of medication, therapy or preventative measures. One of the best approaches for developing patient specific therapy is to use patient specific cells as in vitro drug testing model. Furthermore, patent specific cells in vitro enriched and modified can be used for autologous cell transplantation for treating numerous degenerative conditions.

  7. Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey

    DEFF Research Database (Denmark)

    Hannibal, J; Kankipati, L; Strang, C E

    2014-01-01

    ). The ipRGCs regulate other nonimage-forming visual functions such as the pupillary light reflex, masking behavior, and light-induced melatonin suppression. To evaluate whether PACAP-immunoreactive retinal projections are useful as a marker for central projection of ipRGCs in the monkey brain, we......-expressing cells characterized as inner and outer stratifying melanopsin RGCs. Two macaque monkeys were anesthetized and received a unilateral intravitreal injection of CtB. Bilateral retinal projections containing colocalized CtB and PACAP immunostaining were identified in the SCN, the lateral geniculate complex...... including the pregeniculate nucleus, the pretectal olivary nucleus, the nucleus of the optic tract, the brachium of the superior colliculus, and the superior colliculus. In conclusion, PACAP-immunoreactive projections with colocalized CtB represent retinal projections of ipRGCs in the macaque monkey...

  8. Pluripotent hybrid stem cells from transgenic Huntington's disease monkey.

    Science.gov (United States)

    Laowtammathron, Chuti; Chan, Anthony W S

    2013-01-01

    Huntington's disease (HD) is a devastating disease that currently has no cure. Transgenic HD monkeys have developed key neuropathological and cognitive behavioral impairments similar to HD patients. Thus, pluripotent stem cells derived from transgenic HD monkeys could be a useful comparative model for clarifying HD pathogenesis and developing novel therapeutic approaches, which could be validated in HD monkeys. In order to create personal pluripotent stem cells from HD monkeys, here we present a tetraploid technique for deriving pluripotent hybrid HD monkey stem cells.

  9. Safeguarding nonhuman primate iPS cells with suicide genes.

    Science.gov (United States)

    Zhong, Bonan; Watts, Korashon L; Gori, Jennifer L; Wohlfahrt, Martin E; Enssle, Joerg; Adair, Jennifer E; Kiem, Hans-Peter

    2011-09-01

    The development of technology to generate induced pluripotent stem (iPS) cells constitutes one of the most exciting scientific breakthroughs because of the enormous potential for regenerative medicine. However, the safety of iPS cell-related products is a major concern for clinical translation. Insertional mutagenesis, possible oncogenic transformation of iPS cells or their derivatives, or the contamination of differentiated iPS cells with undifferentiated cells, resulting in the formation of teratomas, have remained considerable obstacles. Here, we demonstrate the utility of suicide genes to safeguard iPS cells and their derivatives. We found suicide genes can control the cell fate of iPS cells in vitro and in vivo without interfering with their pluripotency and self-renewal capacity. This study will be useful to evaluate the safety of iPS cell technology in a clinically highly relevant, large animal model and further benefit the clinical use of human iPS cells.

  10. Cell-Type-Specific Optogenetics in Monkeys.

    Science.gov (United States)

    Namboodiri, Vijay Mohan K; Stuber, Garret D

    2016-09-08

    The recent advent of technologies enabling cell-type-specific recording and manipulation of neuronal activity spurred tremendous progress in neuroscience. However, they have been largely limited to mice, which lack the richness in behavior of primates. Stauffer et al. now present a generalizable method for achieving cell-type specificity in monkeys.

  11. Cell Reprogramming, IPS Limitations, and Overcoming Strategies in Dental Bioengineering

    Directory of Open Access Journals (Sweden)

    Gaskon Ibarretxe

    2012-01-01

    Full Text Available The procurement of induced pluripotent stem cells, or IPS cells, from adult differentiated animal cells has the potential to revolutionize future medicine, where reprogrammed IPS cells may be used to repair disease-affected tissues on demand. The potential of IPS cell technology is tremendous, but it will be essential to improve the methodologies for IPS cell generation and to precisely evaluate each clone and subclone of IPS cells for their safety and efficacy. Additionally, the current state of knowledge on IPS cells advises that research on their regenerative properties is carried out in appropriate tissue and organ systems that permit a safe assessment of the long-term behavior of these reprogrammed cells. In the present paper, we discuss the mechanisms of cell reprogramming, current technical limitations of IPS cells for their use in human tissue engineering, and possibilities to overcome them in the particular case of dental regeneration.

  12. Distinct iPS Cells Show Different Cardiac Differentiation Efficiency.

    Science.gov (United States)

    Ohno, Yohei; Yuasa, Shinsuke; Egashira, Toru; Seki, Tomohisa; Hashimoto, Hisayuki; Tohyama, Shugo; Saito, Yuki; Kunitomi, Akira; Shimoji, Kenichiro; Onizuka, Takeshi; Kageyama, Toshimi; Yae, Kojiro; Tanaka, Tomofumi; Kaneda, Ruri; Hattori, Fumiyuki; Murata, Mitsushige; Kimura, Kensuke; Fukuda, Keiichi

    2013-01-01

    Patient-specific induced pluripotent stem (iPS) cells can be generated by introducing transcription factors that are highly expressed in embryonic stem (ES) cells into somatic cells. This opens up new possibilities for cell transplantation-based regenerative medicine by overcoming the ethical issues and immunological problems associated with ES cells. Despite the development of various methods for the generation of iPS cells that have resulted in increased efficiency, safety, and general versatility, it remains unknown which types of iPS cells are suitable for clinical use. Therefore, the aims of the present study were to assess (1) the differentiation potential, time course, and efficiency of different types of iPS cell lines to differentiate into cardiomyocytes in vitro and (2) the properties of the iPS cell-derived cardiomyocytes. We found that high-quality iPS cells exhibited better cardiomyocyte differentiation in terms of the time course and efficiency of differentiation than low-quality iPS cells, which hardly ever differentiated into cardiomyocytes. Because of the different properties of the various iPS cell lines such as cardiac differentiation efficiency and potential safety hazards, newly established iPS cell lines must be characterized prior to their use in cardiac regenerative medicine.

  13. Distinct iPS Cells Show Different Cardiac Differentiation Efficiency

    Directory of Open Access Journals (Sweden)

    Yohei Ohno

    2013-01-01

    Full Text Available Patient-specific induced pluripotent stem (iPS cells can be generated by introducing transcription factors that are highly expressed in embryonic stem (ES cells into somatic cells. This opens up new possibilities for cell transplantation-based regenerative medicine by overcoming the ethical issues and immunological problems associated with ES cells. Despite the development of various methods for the generation of iPS cells that have resulted in increased efficiency, safety, and general versatility, it remains unknown which types of iPS cells are suitable for clinical use. Therefore, the aims of the present study were to assess (1 the differentiation potential, time course, and efficiency of different types of iPS cell lines to differentiate into cardiomyocytes in vitro and (2 the properties of the iPS cell-derived cardiomyocytes. We found that high-quality iPS cells exhibited better cardiomyocyte differentiation in terms of the time course and efficiency of differentiation than low-quality iPS cells, which hardly ever differentiated into cardiomyocytes. Because of the different properties of the various iPS cell lines such as cardiac differentiation efficiency and potential safety hazards, newly established iPS cell lines must be characterized prior to their use in cardiac regenerative medicine.

  14. Telomere reprogramming and maintenance in porcine iPS cells.

    Science.gov (United States)

    Ji, Guangzhen; Ruan, Weimin; Liu, Kai; Wang, Fang; Sakellariou, Despoina; Chen, Jijun; Yang, Yang; Okuka, Maja; Han, Jianyong; Liu, Zhonghua; Lai, Liangxue; Gagos, Sarantis; Xiao, Lei; Deng, Hongkui; Li, Ning; Liu, Lin

    2013-01-01

    Telomere reprogramming and silencing of exogenous genes have been demonstrated in mouse and human induced pluripotent stem cells (iPS cells). Pigs have the potential to provide xenotransplant for humans, and to model and test human diseases. We investigated the telomere length and maintenance in porcine iPS cells generated and cultured under various conditions. Telomere lengths vary among different porcine iPS cell lines, some with telomere elongation and maintenance, and others telomere shortening. Porcine iPS cells with sufficient telomere length maintenance show the ability to differentiate in vivo by teratoma formation test. IPS cells with short or dysfunctional telomeres exhibit reduced ability to form teratomas. Moreover, insufficient telomerase and incomplete telomere reprogramming and/or maintenance link to sustained activation of exogenous genes in porcine iPS cells. In contrast, porcine iPS cells with reduced expression of exogenous genes or partial exogene silencing exhibit insufficient activation of endogenous pluripotent genes and telomerase genes, accompanied by telomere shortening with increasing passages. Moreover, telomere doublets, telomere sister chromatid exchanges and t-circles that presumably are involved in telomere lengthening by recombination also are found in porcine iPS cells. These data suggest that both telomerase-dependent and telomerase-independent mechanisms are involved in telomere reprogramming during induction and passages of porcine iPS cells, but these are insufficient, resulting in increased telomere damage and shortening, and chromosomal instability. Active exogenes might compensate for insufficient activation of endogenous genes and incomplete telomere reprogramming and maintenance of porcine iPS cells. Further understanding of telomere reprogramming and maintenance may help improve the quality of porcine iPS cells.

  15. [Retinal Cell Therapy Using iPS Cells].

    Science.gov (United States)

    Takahashi, Masayo

    2016-03-01

    Progress in basic research, starting with the work on neural stem cells in the middle 1990's to embryonic stem (ES) cells and induced pluripotent stem (iPS) cells at present, will lead the cell therapy (regenerative medicine) of various organs, including the central nervous system to a big medical field in the future. The author's group transplanted iPS cell-derived retinal pigment epithelial (RPE) cell sheets to the eye of a patient with exudative age-related macular degeneration (AMD) in 2014 as a clinical research. Replacement of the RPE with the patient's own iPS cell-derived young healthy cell sheet will be one new radical treatment of AMD that is caused by cellular senescence of RPE cells. Since it was the first clinical study using iPS cell-derived cells, the primary endpoint was safety judged by the outcome one year after surgery. The safety of the cell sheet has been confirmed by repeated tumorigenisity tests using immunodeficient mice, as well as purity of the cells, karyotype and genetic analysis. It is, however, also necessary to prove the safety by clinical studies. Following this start, a good strategy considering cost and benefit is needed to make regenerative medicine a standard treatment in the future. Scientifically, the best choice is the autologous RPE cell sheet, but autologous cell are expensive and sheet transplantation involves a risky part of surgical procedure. We should consider human leukocyte antigen (HLA) matched allogeneic transplantation using the HLA 6 loci homozyous iPS cell stock that Prof. Yamanaka of Kyoto University is working on. As the required forms of donor cells will be different depending on types and stages of the target diseases, regenerative medicine will be accomplished in a totally different manner from the present small molecule drugs. Proof of concept (POC) of photoreceptor transplantation in mouse is close to being accomplished using iPS cell-derived photoreceptor cells. The shortest possible course for treatment

  16. [Application for Lifestyle disease by iPS cells technologies].

    Science.gov (United States)

    Takashima, Yasuhiro

    2016-03-01

    Currently it is less advanced to understand the pathology of lifestyle disease by using iPS cells because there is partly less direct connection between life style disease and iPS cells. So much more scientists focus on regenerative medicine such as beta cells therapy using iPS cells technologies. It will be indeed a powerful tool to generate beta cells from iPS cells as even in type2 diabetes patients, hyposecretion of insulin from beta cells in pancreas is one of causes. Another reason is complexity of the pathology of life style disease. There are a lot of reasons to cause lifestyle disease. Lifestyle diseases include cancer, chronic liver disease, Type 2 diabetes, heart disease, metabolic syndrome, chronic renal failure, stroke, and obesity. Since obesity is one of major causes of lifestyle diseases, we want to focus on adipogenesis from iPS cells in this review. We analysed and established the differentiation protocol into adipocytes from mouse ES cells and human iPS cells. The other point in this review is the starting pluripotent cells for differentiation. Quality of pluripotent stem cells are one of most critical factors to succeed in getting well-differentiated cells. Recently, we have developed new naive human pluripotent stem cells (PSC),"Reset cells". Naive PSC have more similar to human epibast cells than conventional human PSC. They will be more ideal cells for differentiation because of their hypomethylated status and earlier stage of development.

  17. Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey

    Science.gov (United States)

    Hannibal, J.; Kankipati, L.; Strang, C.E.; Peterson, B.B.; Dacey, D.; Gamlin, P.D.

    2014-01-01

    Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are entrained to the environmental light/dark cycle via intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin and the neuropeptide PACAP. The ipRGCs regulate other non-image-forming visual functions such as the pupillary light reflex, masking behaviour and light induced melatonin suppression. To evaluate whether PACAP immunoreactive retinal projections are useful as a marker for central projection of ipRGCs in the monkey brain, we characterized the occurrence of PACAP in melanopsin expressing ipRGCs and in the retinal target areas in the brain visualized by the anterograde tracer Cholera Toxin subunit B (CtB) in combination with PACAP staining. In the retina, PACAP and melanopsin were found to be co-stored in 99% of melanopsin expressing cells characterized as inner and outer stratifying melanopsin RGCs. Two macaque monkeys were anesthetized and received a unilateral intravitreal injection of CtB. Bilateral retinal projections containing co-localized CtB and PACAP immunostaining were identified in the SCN, the lateral geniculate complex (LGN) including the pregeniculate nucleus (PrGC), the pretectal olivary nucleus (PON), the nucleus of the optic tract (NOT), the brachium of the superior colliculus (BSC), and the superior colliculus (SC). In conclusion, PACAP immunoreactive projections with co-localized CtB represent retinal projections of ipRGCs in the macaque monkey, and support previous retrograde tracer studies demonstrating that melanopsin containing retinal projections reach areas in the primate brain involved in both image and non-image-forming visual processing. PMID:24752373

  18. Central projections of intrinsically photosensitive retinal ganglion cells in the macaque monkey.

    Science.gov (United States)

    Hannibal, J; Kankipati, L; Strang, C E; Peterson, B B; Dacey, D; Gamlin, P D

    2014-07-01

    Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are entrained to the environmental light/dark cycle via intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP). The ipRGCs regulate other nonimage-forming visual functions such as the pupillary light reflex, masking behavior, and light-induced melatonin suppression. To evaluate whether PACAP-immunoreactive retinal projections are useful as a marker for central projection of ipRGCs in the monkey brain, we characterized the occurrence of PACAP in melanopsin-expressing ipRGCs and in the retinal target areas in the brain visualized by the anterograde tracer cholera toxin subunit B (CtB) in combination with PACAP staining. In the retina, PACAP and melanopsin were found to be costored in 99% of melanopsin-expressing cells characterized as inner and outer stratifying melanopsin RGCs. Two macaque monkeys were anesthetized and received a unilateral intravitreal injection of CtB. Bilateral retinal projections containing colocalized CtB and PACAP immunostaining were identified in the SCN, the lateral geniculate complex including the pregeniculate nucleus, the pretectal olivary nucleus, the nucleus of the optic tract, the brachium of the superior colliculus, and the superior colliculus. In conclusion, PACAP-immunoreactive projections with colocalized CtB represent retinal projections of ipRGCs in the macaque monkey, supporting previous retrograde tracer studies demonstrating that melanopsin-containing retinal projections reach areas in the primate brain involved in both image- and nonimage-forming visual processing.

  19. Stem Cells Transplanted in Monkeys without Anti-Rejection Drugs

    Science.gov (United States)

    ... page: https://medlineplus.gov/news/fullstory_160989.html Stem Cells Transplanted in Monkeys Without Anti-Rejection Drugs Scientists say goal is to create banks of stem cells that could be used for any human patient ...

  20. Generation and application of human iPS cells

    Institute of Scientific and Technical Information of China (English)

    CUI Ghun; RAO LingJun; CHENG LinZhao; XIAO Lei

    2009-01-01

    Human embryonic stem (ES) cells are capable of unlimited proliferation and maintenance of pluripo-tency in vitro; these properties may lead to potential applications in regenerative medicine.However,immune rejection hampers the allogenic application of human ES cells.Over-expression of several specific transcription factors has been used to reprogram human adult cells into induced pluripotent stem (iPS) cells,which are similar to hESCs in many aspects.The iPS technique makes it possible to produce patient-specific pluripotent stem cells for transplantation therapy without immune rejection.However,some challenges remain,including viral vector integration into the genome,the existence of exogenous oncogenic factors,and low induction efficiency.Here,we review recent advances in human iPS methodology,as well as remaining challenges and its potential applications.

  1. TRPV-1-mediated elimination of residual iPS cells in bioengineered cardiac cell sheet tissues.

    Science.gov (United States)

    Matsuura, Katsuhisa; Seta, Hiroyoshi; Haraguchi, Yuji; Alsayegh, Khaled; Sekine, Hidekazu; Shimizu, Tatsuya; Hagiwara, Nobuhisa; Yamazaki, Kenji; Okano, Teruo

    2016-02-18

    The development of a suitable strategy for eliminating remaining undifferentiated cells is indispensable for the use of human-induced pluripotent stem (iPS) cell-derived cells in regenerative medicine. Here, we show for the first time that TRPV-1 activation through transient culture at 42 °C in combination with agonists is a simple and useful strategy to eliminate iPS cells from bioengineered cardiac cell sheet tissues. When human iPS cells were cultured at 42 °C, almost all cells disappeared by 48 hours through apoptosis. However, iPS cell-derived cardiomyocytes and fibroblasts maintained transcriptional and protein expression levels, and cardiac cell sheets were fabricated after reducing the temperature. TRPV-1 expression in iPS cells was upregulated at 42 °C, and iPS cell death at 42 °C was TRPV-1-dependent. Furthermore, TRPV-1 activation through thermal or agonist treatment eliminated iPS cells in cardiac tissues for a final concentration of 0.4% iPS cell contamination. These findings suggest that the difference in tolerance to TRPV-1 activation between iPS cells and iPS cell-derived cardiac cells could be exploited to eliminate remaining iPS cells in bioengineered cell sheet tissues, which will further reduce the risk of tumour formation.

  2. Generation of Partially Reprogrammed Cells and Fully Reprogrammed iPS Cells by Plasmid Transfection.

    Science.gov (United States)

    Kim, Jong Soo; Choi, Hyun Woo; Hong, Yean Ju; Do, Jeong Tae

    2016-01-01

    Induced pluripotent stem (iPS) cells can be directly generated from somatic cells by overexpression of defined transcription factors. iPS cells can perpetually self-renew and differentiate into all cell types of an organism. iPS cells were first generated through infection with retroviruses that contain reprogramming factors. However, development of an exogene-free iPS cell generation method is crucial for future therapeutic applications, because integrated exogenes result in the formation of tumors in chimeras and regain pluripotency after differentiation in vitro. Here, we describe a method to generate iPS cells by transfection of plasmid vectors and to convert partially reprogrammed cells into fully reprogrammed iPS cells by switching from mouse ESC culture conditions to KOSR-based media with bFGF. We also describe basic methods used to characterize fully reprogrammed iPS cells.

  3. [The genetic safety of induced pluripotent stem (iPS) cells].

    Science.gov (United States)

    Chen, Qian; Shi, Qing-Hua

    2012-03-01

    Since Takahashi and Yamanaka first generated induced pluripotent stem (iPS) cells from mouse fibroblasts successfully in 2006, iPS cells have rapidly become a new hotspot in the field of stem cells research because of their broad potential application prospects. Meanwhile, more and more attentions are paid to the genetic safety of iPS cells. This article summarizes recent findings on genetic safety of iPS cells and reviews the possible causes leading to genetic instability of iPS cells. Hopefully, this review is helpful to improve the induction of iPS cells and obtain genetically safe iPS cells.

  4. iPS Cells 10 Years Later.

    Science.gov (United States)

    2016-09-08

    In 2006, Takahashi and Yamanaka reported the breakthrough discovery of induction of pluripotent stem cells from fibroblasts by a combination of defined factors. Ten years later, Cell editor João Monteiro brings together Shinya Yamanaka and Hans Schöler, one the original reviewers of the landmark study, to revisit the history behind the paper and its long-lasting legacy.

  5. Oct-4 expression in pluripotent cells of the rhesus monkey.

    Science.gov (United States)

    Mitalipov, Shoukhrat M; Kuo, Hung-Chih; Hennebold, Jon D; Wolf, Don P

    2003-12-01

    The POU (Pit-Oct-Unc)-domain transcription factor, Oct-4, has become a useful marker of pluripotency in the mouse. It is found exclusively in mouse preimplantation-stage embryos after embryonic genome activation and is a characteristic of mouse embryonic stem (ES) cells, and its absence in knockout mice precludes inner cell mass (ICM) formation in blastocysts. Expression of Oct-4 has also been associated with pluripotency in primate cells. Here, we undertook a systematic study of Oct-4 expression in rhesus macaque preimplantation embryos produced by intracytoplasmic sperm injection and in ES cells before and after exposure to differentiating conditions in vitro. We also evaluated Oct-4 expression as a means of monitoring the extent of reprogramming following somatic cell nuclear transfer. Oct-4 was detected by reverse transcription-polymerase chain reaction and immunocytochemistry with a monoclonal antibody. Monkey pronuclear-stage zygotes and cleaving embryos up to the 8-cell stage showed no detectable Oct-4. Nuclear staining for Oct-4 first became obvious at the 16-cell stage, and a strong signal was observed in morula and compact morula stages. Both ICM and trophectodermal cell nuclei of monkey early blastocysts were positive for Oct-4. However, the signal was diminished in trophectodermal cells of expanded blastocysts, whereas expression remained high in ICM nuclei. Similar to the mouse, hatched monkey blastocysts showed strong Oct-4 expression in the ICM, with no detectable signal in the trophectoderm. Undifferentiated monkey ES cells derived from the ICM of in vitro-produced blastocysts expressed Oct-4, consistent with their pluripotent nature, whereas ES cell differentiation was associated with signal loss. Therefore, Oct-4 expression in the monkey, as in the mouse, provides a useful marker for pluripotency after activation of the embryonic genome. Finally, the observed lack or abnormal expression of Oct-4 in monkey nuclear transfer embryos suggests

  6. KSR-Based Medium Improves the Generation of High-Quality Mouse iPS Cells

    OpenAIRE

    2014-01-01

    Induced pluripotent stem (iPS) cells from somatic cells have great potential for regenerative medicine. The efficiency in generation of iPS cells has been significantly improved in recent years. However, the generation of high-quality iPS cells remains of high interest. Consistently, we demonstrate that knockout serum replacement (KSR)-based medium accelerates iPS cell induction and improves the quality of iPS cells, as confirmed by generation of chimeras and all iPS cell-derived offspring wi...

  7. Transcriptional profiling of rhesus monkey embryonic stem cells.

    Science.gov (United States)

    Byrne, James A; Mitalipov, Shoukhrat M; Clepper, Lisa; Wolf, Don P

    2006-12-01

    Embryonic stem cells (ESCs) may be able to cure or alleviate the symptoms of various degenerative diseases. However, unresolved issues regarding survival, functionality, and tumor formation mean a prudent approach should be adopted towards advancing ESCs into human clinical trials. The rhesus monkey provides an ideal model organism for developing strategies to prevent immune rejection and test the feasibility, safety, and efficacy of ESC-based medical treatments. Transcriptional profiling of rhesus monkey ESCs provides a foundation for pre-clinical ESC research in this species. In the present study, we used microarray technology, immunocytochemistry, reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) to characterize and transcriptionally profile rhesus monkey ESCs. We identified 367 stemness gene candidates that were highly (>85%) conserved across five different ESC lines. Rhesus monkey ESC lines maintained a pluripotent undifferentiated state over a wide range of POU5F1 (also known as OCT4) expression levels, and comparisons between rhesus monkey, mouse, and human stemness genes revealed five mammalian stemness genes: CCNB1, GDF3, LEFTB, POU5F1, and NANOG. These five mammalian genes are strongly expressed in rhesus monkey, mouse, and human ESCs, albeit only in the undifferentiated state, and represent the core key mammalian stemness factors.

  8. The elite and stochastic model for iPS cell generation: multilineage-differentiating stress enduring (Muse) cells are readily reprogrammable into iPS cells.

    Science.gov (United States)

    Wakao, Shohei; Kitada, Masaaki; Dezawa, Mari

    2013-01-01

    Induced pluripotent stem (iPS) cells have attracted a great deal of attention, although the mechanism by which they are generated is still not fully understood. Currently, two theories, the stochastic and elite models, have been proposed. Some reports provide theoretical support for the stochastic model. Other reports, however, support the elite model. For example, some human fibroblasts, such as Multilineage-differentiating stress enduring (Muse) cells, are reported to be pluripotent and a primary source of iPS cells. Thus, the mechanism of iPS cell generation continues to be debated. In this review, we discuss the properties of the original cell source, such as the components of the original populations and the potential of each population to become iPS cells, and further discuss the implications of the two theories for iPS cell research.

  9. iPS cells: a source of cardiac regeneration.

    Science.gov (United States)

    Yoshida, Yoshinori; Yamanaka, Shinya

    2011-02-01

    For the treatment of heart failure, a new strategy to improve cardiac function and inhibit cardiac remodeling needs to be established. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are pluripotent cells that can differentiate into cell types from all three germ layers both in vitro and in vivo. The therapeutic effect of ES/iPS cell-derived progeny was reported in animal model. Mouse and human somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by the transduction of four transcription factors, Oct 3/4, Sox2, Klf4, and c-Myc. However, the low induction efficiency hinders the clinical application of iPS technology, and efforts have been made to improve the reprogramming efficiency. There are variations in the characteristics in ES/iPS cell lines, and the further understanding is necessary for the applications of ES/iPS cell technology. Some improvements were also made in the methods to induce cardiomyocytes from ES/iPS cells efficiently. This review article is focused on generation of iPS cells, cardiomyocyte differentiation from ES/iPS cells, and transplantation of derived cardiomyocytes.This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

  10. Monkey hybrid stem cells develop cellular features of Huntington's disease

    Directory of Open Access Journals (Sweden)

    Lorthongpanich Chanchao

    2010-02-01

    Full Text Available Abstract Background Pluripotent stem cells that are capable of differentiating into different cell types and develop robust hallmark cellular features are useful tools for clarifying the impact of developmental events on neurodegenerative diseases such as Huntington's disease. Additionally, a Huntington's cell model that develops robust pathological features of Huntington's disease would be valuable for drug discovery research. Results To test this hypothesis, a pluripotent Huntington's disease monkey hybrid cell line (TrES1 was established from a tetraploid Huntington's disease monkey blastocyst generated by the fusion of transgenic Huntington's monkey skin fibroblast and a wild-type non-transgenic monkey oocyte. The TrES1 developed key Huntington's disease cellular pathological features that paralleled neural development. It expressed mutant huntingtin and stem cell markers, was capable of differentiating to neural cells, and developed teratoma in severely compromised immune deficient (SCID mice. Interestingly, the expression of mutant htt, the accumulation of oligomeric mutant htt and the formation of intranuclear inclusions paralleled neural development in vitro , and even mutant htt was ubiquitously expressed. This suggests the development of Huntington's disease cellular features is influenced by neural developmental events. Conclusions Huntington's disease cellular features is influenced by neural developmental events. These results are the first to demonstrate that a pluripotent stem cell line is able to mimic Huntington's disease progression that parallels neural development, which could be a useful cell model for investigating the developmental impact on Huntington's disease pathogenesis.

  11. [Differentiation of functional cells from iPS cells by efficient gene transfer].

    Science.gov (United States)

    Kawabata, Kenji; Tashiro, Katsuhisa; Mizuguchi, Hiroyuki

    2010-11-01

    Induced pluripotent stem (iPS) cells, which are generated from somatic cells by transducing four genes, are expected to have broad application to regenerative medicine. Although establishment of an efficient gene transfer system for iPS cells is considered to be essential for differentiating them into functional cells, the detailed transduction characteristics of iPS cells have not been examined. By using an adenovirus (Ad) vector containing the cytomegalovirus enhancer/beta-actin (CA) promoters, we have developed an efficient transduction system for mouse mesenchymal stem cells and embryonic stem (ES) cells. Also, we applied our transduction system to mouse iPS cells and investigated whether efficient differentiation could be achieved by Ad vector-mediated transduction of a functional gene. As in the case of ES cells, the Ad vector could efficiently transduce transgenes into mouse iPS cells. We found that the CA promoter had potent transduction ability in iPS cells. Moreover, exogenous expression of a PPARγ gene or a Runx2 gene into mouse iPS cells by an optimized Ad vector enhanced adipocyte or osteoblast differentiation, respectively. These results suggest that Ad vector-mediated transient transduction is sufficient to promote cellular differentiation and that our transduction methods would be useful for therapeutic applications based on iPS cells.

  12. Clinical grade iPS cells: need for versatile small molecules and optimal cell sources.

    Science.gov (United States)

    Wu, Yan-Ling; Pandian, Ganesh N; Ding, Yan-Ping; Zhang, Wen; Tanaka, Yoshimasa; Sugiyama, Hiroshi

    2013-11-21

    Adult mammals possess limited ability to regenerate their lost tissues or organs. The epoch-making strategy of inducing pluripotency in somatic cells incorporates multiple applications in regenerative medicine. However, concerns about the clinical translation of induced pluripotent stem (iPS) cells still exist because of the occurrence of aberrancies, even in genome integration-free methods. As cellular reprogramming is multi-gene-oriented, versatile, bioactive small molecules could concomitantly modulate the transcriptional machinery and aid the generation of clinical grade iPS cells. The availability of optimal cell sources has additional influence on the clinical translation of iPS cells. Herein we provide a critical overview of methods and cell sources available for iPS cell production. We think the review will be a useful resource for researchers who aim to develop small molecules for speeding up the journey of iPS cells from the laboratory to the clinic.

  13. Comparative study of myocytes from normal and mdx mice iPS cells.

    Science.gov (United States)

    Chen, Fei; Cao, Jiqing; Liu, Qiang; Qin, Jie; Kong, Jie; Wang, Yanyun; Li, Yaqin; Geng, Jia; Li, Qiuling; Yang, Liqing; Xiang, Andy Peng; Zhang, Cheng

    2012-02-01

    Recently, induced pluripotent stem cells (iPS cells) have been derived from various techniques and show great potential for therapy of human diseases. Furthermore, the iPS technique can be used to provide cell models to explore pathological mechanisms of many human diseases in vitro, such as Duchenne muscular dystrophy (DMD), which is a severe recessive X-linked form of muscular dystrophy without effective treatment. In this study, we try to determine whether there are different characteristics of myocytes from mdx iPS cells and C57BL/10 iPS cells. Our results showed that both of mdx and C57BL/10 cells could be induced into iPS cells in vitro, whereas colony-forming ability of mdx iPS cells was much weaker than that of C57BL/10 iPS cells. Meanwhile, mdx iPS cells could be induced to differentiate into myocytes, whereas their differentiation efficiency was much lower than that of C57BL/10 iPS cells. And, the number of apoptotic cells in differentiated myocytes from mdx iPS cells was significantly higher than that from C57BL/10 iPS cells. More importantly, treatment of a pan-caspase inhibitor (Z-VAD) produced a significant decrease in apoptotic cells. This study might add some insight to the biology study of dystrophin gene.

  14. Generation of iPS Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors.

    Science.gov (United States)

    Su, Ruijun Jeanna; Neises, Amanda; Zhang, Xiao-Bing

    2016-01-01

    Peripheral blood is the easy-to-access, minimally invasive, and the most abundant cell source to use for cell reprogramming. The episomal vector is among the best approaches for generating integration-free induced pluripotent stem (iPS) cells due to its simplicity and affordability. Here we describe the detailed protocol for the efficient generation of integration-free iPS cells from peripheral blood mononuclear cells. With this optimized protocol, one can readily generate hundreds of iPS cell colonies from 1 ml of peripheral blood.

  15. Genomic imprinting is variably lost during reprogramming of mouse iPS cells

    OpenAIRE

    2013-01-01

    Derivation of induced pluripotent stem (iPS) cells is mainly an epigenetic reprogramming process. It is still quite controversial how genomic imprinting is reprogrammed in iPS cells. Thus, we derived multiple iPS clones from genetically identical mouse somatic cells. We found that parentally inherited imprint was variably lost among these iPS clones. Concurrent with the loss of DNA methylation imprint at the corresponding Snrpn and Peg3 imprinted regions, parental origin-specific expression o...

  16. [Induced pluripotent stem (iPS) cell - issues for clinical application - ].

    Science.gov (United States)

    Aoi, Takashi

    2013-01-01

    Induced pluripotent stem (iPS) cells are generated from somatic cells by introducing small sets of transcription factors. iPS cells demonstrate pluripotency and the ability to self-renew. In addition, iPS cells can be generated from donor individuals with particular characteristics. Based on these features, iPS cells are expected to be applicable in drug discovery, the study of disease mechanisms and cell therapy. From a technical point of view, "diversity" is the key word. At present, iPS cells can be derived using various techniques, resulting in diversity in the quality of iPS cells generated. Therefore, optimization of the derivation technology is one of the most important issues. Another "diversity" is in the propensities amongst iPS cell lines derived using similar techniques. Thus, strategies for selecting good quality lines remain to be established. Considering such technical hurdles, establishment of an iPS cell bank consisting of high quality and versatile iPS lines is a promising idea because of the merits of cost and quality control. Now, we are exploring relevant parameters for the quality control of banked cells. The challenges facing clinical application of iPS cells are new but not unprecedented. To realize clinical applications of iPS cells, we need to make these challenges clear and overcome them through partnership not only with industry, governments and universities, but also patients and society at large.

  17. The first reported generation of human induced pluripotent stem cells (iPS cells) and iPS cell-derived cardiomyocytes in the Netherlands.

    Science.gov (United States)

    Freund, C; Davis, R P; Gkatzis, K; Ward-van Oostwaard, D; Mummery, C L

    2010-01-01

    One of the recent breakthroughs in stem cell research has been the reprogramming of human somatic cells to an embryonic stem cell (ESC)-like state (induced pluripotent stem cells, iPS cells). Similar to ESCs, iPS cells can differentiate into derivatives of the three germ layers, for example cardiomyocytes, pancreatic cells or neurons. This technique offers a new approach to investigating disease pathogenesis and to the development of novel therapies. It may now be possible to generate iPS cells from somatic cells of patients who suffer from vascular genetic diseases, such as hereditary haemorrhagic telangiectasia (HHT). The iPS cells will have a similar genotype to that of the patient and can be differentiated in vitro into the cell type(s) that are affected in the patient. Thus they will serve as excellent models for a better understanding of mechanisms underlying the disease. This, together with the ability to test new drugs, could potentially lead to novel therapeutic concepts in the near future. Here we report the first derivation of three human iPS cell lines from two healthy individuals and one HHT patient in the Netherlands. The iPS cells resembled ESCs in morphology and expressed typical ESC markers. In vitro, iPS cells could be differentiated into cells of the three germ layers, including beating cardiomyocytes and vascular cells. With this technique it will be possible to establish human cardiovascular disease models from patient biopsies provided by the principal hospitals in the Netherlands. (Neth Heart J 2010;18:51-4.).

  18. Genomic imprinting is variably lost during reprogramming of mouse iPS cells.

    Science.gov (United States)

    Takikawa, Sachiko; Ray, Chelsea; Wang, Xin; Shamis, Yulia; Wu, Tien-Yuan; Li, Xiajun

    2013-09-01

    Derivation of induced pluripotent stem (iPS) cells is mainly an epigenetic reprogramming process. It is still quite controversial how genomic imprinting is reprogrammed in iPS cells. Thus, we derived multiple iPS clones from genetically identical mouse somatic cells. We found that parentally inherited imprint was variably lost among these iPS clones. Concurrent with the loss of DNA methylation imprint at the corresponding Snrpn and Peg3 imprinted regions, parental origin-specific expression of the Snrpn and Zim1 imprinted genes was also lost in these iPS clones. This loss of parental genomic imprinting in iPS cells was likely caused by the reprogramming process during iPS cell derivation because extended culture of iPS cells did not lead to significant increase in the loss of genomic imprinting. Intriguingly, one to several paternal chromosomes appeared to have acquired de novo methylation at the Snrpn and Zac1 imprinted regions in a high percentage of iPS clones. These results might have some implications for future therapeutic applications of iPS cells. Since DNA methylation imprint can be completely erased in some iPS clones at multiple imprinted regions, iPS cell reprogramming may also be employed to dissect the underlying mechanisms of erasure, reacquisition and maintenance of genomic imprinting in mammals.

  19. Connexin expression and gap-junctional intercellular communication in ES cells and iPS cells.

    Science.gov (United States)

    Oyamada, Masahito; Takebe, Kumiko; Endo, Aya; Hara, Sachiko; Oyamada, Yumiko

    2013-01-01

    Pluripotent stem cells, i.e., embryonic stem (ES) and induced pluripotent stem (iPS) cells, can indefinitely proliferate without commitment and differentiate into all cell lineages. ES cells are derived from the inner cell mass of the preimplantation blastocyst, whereas iPS cells are generated from somatic cells by overexpression of a few transcription factors. Many studies have demonstrated that mouse and human iPS cells are highly similar but not identical to their respective ES cell counterparts. The potential to generate basically any differentiated cell types from these cells offers the possibility to establish new models of mammalian development and to create new sources of cells for regenerative medicine. ES cells and iPS cells also provide useful models to study connexin expression and gap-junctional intercellular communication (GJIC) during cell differentiation and reprogramming. In 1996, we reported connexin expression and GJIC in mouse ES cells. Because a substantial number of papers on these subjects have been published since our report, this Mini Review summarizes currently available data on connexin expression and GJIC in ES cells and iPS cells during undifferentiated state, differentiation, and reprogramming.

  20. Connexin expression and gap-junctional intercellular communication in ES cells and iPS cells

    Directory of Open Access Journals (Sweden)

    Masahito eOyamada

    2013-07-01

    Full Text Available Pluripotent stem cells, i.e., embryonic stem (ES and induced pluripotent stem (iPS cells, can indefinitely proliferate without commitment and differentiate into all cell lineages. ES cells are derived from the inner cell mass of the preimplantation blastocyst, whereas iPS cells are generated from somatic cells by overexpression of a few transcription factors. Many studies have demonstrated that mouse and human iPS cells are highly similar but not identical to their respective ES cell counterparts. The potential to generate basically any differentiated cell types from these cells offers the possibility to establish new models of mammalian development and to create new sources of cells for regenerative medicine. ES cells and iPS cells also provide useful models to study connexin expression and gap-junctional intercellular communication (GJIC during cell differentiation and reprogramming. In 1996, we reported connexin expression and GJIC in mouse ES cells. Because a substantial number of papers on these subjects have been published since our report, this Mini Review summarizes currently available data on connexin expression and GJIC in ES cells and iPS cells during undifferentiated state, differentiation, and reprogramming.

  1. Derivation and characterization of monkey embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Wolf Don P

    2004-06-01

    Full Text Available Abstract Embryonic stem (ES cell based therapy carries great potential in the treatment of neurodegenerative diseases. However, before clinical application is realized, the safety, efficacy and feasibility of this therapeutic approach must be established in animal models. The rhesus macaque is physiologically and phylogenetically similar to the human, and therefore, is a clinically relevant animal model for biomedical research, especially that focused on neurodegenerative conditions. Undifferentiated monkey ES cells can be maintained in a pluripotent state for many passages, as characterized by a collective repertoire of markers representing embryonic cell surface molecules, enzymes and transcriptional factors. They can also be differentiated into lineage-specific phenotypes of all three embryonic germ layers by epigenetic protocols. For cell-based therapy, however, the quality of ES cells and their progeny must be ensured during the process of ES cell propagation and differentiation. While only a limited number of primate ES cell lines have been studied, it is likely that substantial inter-line variability exists. This implies that diverse ES cell lines may differ in developmental stages, lineage commitment, karyotypic normalcy, gene expression, or differentiation potential. These variables, inherited genetically and/or induced epigenetically, carry obvious complications to therapeutic applications. Our laboratory has characterized and isolated rhesus monkey ES cell lines from in vitro produced blastocysts. All tested cell lines carry the potential to form pluripotent embryoid bodies and nestin-positive progenitor cells. These ES cell progeny can be differentiated into phenotypes representing the endodermal, mesodermal and ectodermal lineages. This review article describes the derivation of monkey ES cell lines, characterization of the undifferentiated phenotype, and their differentiation into lineage-specific, particularly neural, phenotypes

  2. [Stem cell therapy for ischemic stroke using iPS cells].

    Science.gov (United States)

    Oki, Koichi

    2013-01-01

    Recently, several stem cell-based approaches have been considered as a novel treatment for stroke, such as delivery of non neural stem cells, stimulation of endogenous neural stem cells, and transplantation of neural stem cells. Among them, transplantation of neural stem cells is thought to have an advantage for functional recovery through neuronal replacement rather than other mechanisms. We demonstrated that human iPS cells derived neural stem cells form functional neurons and improve recovery after grafting in stroke-damaged rodents without tumor formation. Here we discuss about the potential and clinical application of iPS cells for stroke therapy.

  3. KSR-based medium improves the generation of high-quality mouse iPS cells.

    Science.gov (United States)

    Liu, Kai; Wang, Fang; Ye, Xiaoying; Wang, Lingling; Yang, Jiao; Zhang, Jingzhuo; Liu, Lin

    2014-01-01

    Induced pluripotent stem (iPS) cells from somatic cells have great potential for regenerative medicine. The efficiency in generation of iPS cells has been significantly improved in recent years. However, the generation of high-quality iPS cells remains of high interest. Consistently, we demonstrate that knockout serum replacement (KSR)-based medium accelerates iPS cell induction and improves the quality of iPS cells, as confirmed by generation of chimeras and all iPS cell-derived offspring with germline transmission competency. Both alkaline phosphatase (AP) activity assay and expression of Nanog have been used to evaluate the efficiency of iPS cell induction and formation of ES/iPS cell colonies; however, appropriate expression of Nanog frequently indicates the quality of ES/iPS cells. Interestingly, whereas foetal bovine serum (FBS)-based media increase iPS cell colony formation, as revealed by AP activity, KSR-based media increase the frequency of iPS cell colony formation with Nanog expression. Furthermore, inhibition of MAPK/ERK by a specific inhibitor, PD0325901, in KSR- but not in FBS-based media significantly increases Nanog-GFP+ iPS cells. In contrast, addition of bFGF in KSR-based media decreases proportion of Nanog-GFP+ iPS cells. Remarkably, PD can rescue Nanog-GFP+ deficiency caused by bFGF. These data suggest that MAPK/ERK pathway influences high quality mouse iPS cells and that KSR- and PD-based media could enrich homogeneous authentic pluripotent stem cells.

  4. Cell-mediated responses of immunized vervet monkeys to defined Leishmania T-cell epitopes.

    OpenAIRE

    Curry, A J; Jardim, A; Olobo, J.O.; Olafson, R W

    1994-01-01

    A population of vervet monkeys was immunized with killed parasites and infected with Leishmania major promastigotes either by needle or by infected-fly bite. The responses of recovered monkeys to mitogens, killed parasites, and molecularly defined T-cell epitopes were then compared with those of control animals. Peripheral blood mononuclear cells (PBMC) from both naive and recovered animals proliferated strongly in response to both B- and T-cell mitogens, although the responses of the recover...

  5. 10th anniversary of iPS cells: the challenges that lie ahead.

    Science.gov (United States)

    Aoi, Takashi

    2016-09-01

    In 2006, induced pluripotent stem (iPS) cells were generated by Yamanaka and Takahashi for the first time from a mouse fibroblast culture by introducing four factors. In the 10 years since then, this breakthrough discovery has been making waves in the fields of biology and medical science. For example, various technologies for generating iPS cells have been developed, and we have cultivated a better understanding of the mechanisms involved in reprogramming. In addition, many researchers have explored the applications of iPS cells, such as drug discovery, the study of disease mechanisms and regenerative medicine, and the development of advanced technologies for the differentiation and qualification of the cells. Furthermore, the concept of iPS cell generation has inspired a number of studies that do not use iPS cells. We herein review and discuss the past, present and future of iPS cells and their related issues.

  6. Neural stem cells differentiated from iPS cells spontaneously regain pluripotency.

    Science.gov (United States)

    Choi, Hyun Woo; Kim, Jong Soo; Choi, Sol; Hong, Yean Ju; Kim, Min Jung; Seo, Han Geuk; Do, Jeong Tae

    2014-10-01

    Differentiated somatic cells can be reprogrammed into pluripotent stem cells by transduction of exogenous reprogramming factors. After induced pluripotent stem (iPS) cells are established, exogenous genes are silenced. In the pluripotent state, retroviral genes integrated in the host genome are kept inactive through epigenetic transcriptional regulation. In this study, we tried to determine whether exogenous genes remain silenced or are reactivated upon loss of pluripotency or on differentiation using an in vitro system. We induced differentiation of iPS cells into neural stem cells (NSCs) in vitro; the NSCs appeared morphologically indistinguishable from brain-derived NSCs and stained positive for the NSC markers Nestin and Sox2. These iPS cell-derived NSCs (iPS-NSCs) were also capable of differentiating into all three neural subtypes. Interestingly, iPS-NSCs spontaneously formed aggregates on long-term culture and showed reactivation of the Oct4-GFP marker, which was followed by the formation of embryonic stem cell-like colonies. The spontaneously reverted green fluorescent protein (GFP)-positive (iPS-NSC-GFP(+) ) cells expressed high levels of pluripotency markers (Oct4 and Nanog) and formed germline chimeras, indicating that iPS-NSC-GFP(+) cells had the same pluripotency as the original iPS cells. The reactivation of silenced exogenous genes was tightly correlated with the downregulation of DNA methyltransferases (Dnmts) during differentiation of iPS cells. This phenomenon was not observed in doxycycline-inducible iPS cells, where the reactivation of exogenous genes could be induced only by doxycycline treatment. These results indicate that pluripotency can be regained through reactivation of exogenous genes, which is associated with dynamic change of Dnmt levels during differentiation of iPS cells.

  7. Establishment of human cell type-specific iPS cells with enhanced chondrogenic potential.

    Science.gov (United States)

    Guzzo, Rosa M; Scanlon, Vanessa; Sanjay, Archana; Xu, Ren-He; Drissi, Hicham

    2014-12-01

    The propensity of induced pluripotent stem (iPS) cells to differentiate into specific lineages may be influenced by a number of factors, including the selection of the somatic cell type used for reprogramming. Herein we report the generation of new iPS cells, which we derived from human articular chondrocytes and from cord blood mononucleocytes via lentiviral-mediated delivery of Oct4, Klf4, Sox2, and cMyc. Molecular, cytochemical, and cytogenic analyses confirmed the acquisition of hallmark features of pluripotency, as well as the retention of normal karyotypes following reprogramming of both the human articular chondrocytes (AC) and the cord blood (CB) cells. In vitro and in vivo functional analyses formally established the pluripotent differentiation capacity of all cell lines. Chondrogenic differentiation assays comparing iPS cells derived from AC, CB, and a well established dermal fibroblast cell line (HDFa-Yk26) identified enhanced proteoglycan-rich matrix formation and cartilage-associated gene expression from AC-derived iPS cells. These findings suggest that the tissue of origin may impact the fate potential of iPS cells for differentiating into specialized cell types, such as chondrocytes. Thus, we generated new cellular tools for the identification of inherent features driving high chondrogenic potential of reprogrammed cells.

  8. Efficient generation of iPS cells from skeletal muscle stem cells.

    Directory of Open Access Journals (Sweden)

    Kah Yong Tan

    Full Text Available Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts. However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that "epigenetic memory" significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle.

  9. [Current status and perspective on regenerative medicine for spinal cord injury using iPS cell].

    Science.gov (United States)

    Nakamura, Masaya; Toyama, Yoshiaki; Okano, Hideyuki

    2013-01-01

    Stimulated by the 2012 Nobel Prize in Physiology or Medicine awarded for Shinya Yamanaka and Sir John Gurdon, there is an increasing interest in the iPS cells and reprogramming technologies in medical science. While iPS cells are expected to open new era providing enormous opportunities in the biomedical sciences in terms of cell therapies for regenerative medicine, safety-related concerns for iPS cell-based cell therapy should be resolved prior to the clinical application of iPS cells. In this symposium, the pre-clinical investigations of cell therapy for SCI using neural stem/progenitor cells derived from iPS cells, and their safety issues in vivo are outlined.

  10. Differentiation of monkey embryonic stem cells into neural lineages.

    Science.gov (United States)

    Kuo, Hung-Chih; Pau, K-Y Francis; Yeoman, Richard R; Mitalipov, Shoukhrat M; Okano, Hideyuki; Wolf, Don P

    2003-05-01

    Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.

  11. Preparation of pancreatic β-cells from human iPS cells with small molecules.

    Science.gov (United States)

    Hosoya, Masaki

    2012-01-01

    Human induced pluripotent stem (iPS) cells obtained from patients are expected to be a useful source for cell transplantation therapy, because many patients (including those with type 1 diabetes and severe type 2 diabetes) are on waiting lists for transplantation for a long time due to the shortage of donors. At present, many concerns related to clinical application of human iPS cells have been raised, but rapid development of methods for the establishment, culture, and standardization of iPS cells will lead autologous cell therapy to be realistic sooner or later. However, establishment of a method for preparing some of desired cell types is still challenging. Regarding pancreatic β-cells, there have been many reports about differentiation of these cells from human embryonic stem (ES)/iPS cells, but a protocol for clinical application has still not been established. Since there is clear proof that cell transplantation therapy is effective for diabetes based on the results of clinical islet transplantation, pancreatic β-cells prepared from human iPS cells are considered likely to be effective for reducing the burden on patients. In this article, the current status of procedures for preparing pancreatic β-cells from human ES/iPS cells, including effective use of small molecules, is summarized, and some of the problems that still need to be overcome are discussed.

  12. Mesodermal and hematopoietic differentiation from ES and iPS cells.

    Science.gov (United States)

    Inoue-Yokoo, Tomoko; Tani, Kenzaburo; Sugiyama, Daisuke

    2013-08-01

    Embryonic stem (ES) and induced pluripotent stem (iPS) cells can differentiate into any type of tissue when grown in a suitable culture environment and are considered valuable tools for regenerative medicine. In the field of hematology, generation of hematopoietic stem cells (HSCs) and mature hematopoietic cells (HCs) from ES and iPS cells through mesodermal cells, the ancestors of HCs, can facilitate transplantation and transfusion therapy. Several studies report generation of functional HCs from both mouse and human ES and iPS cells. This approach will likely be applied to individual patient-derived iPS cells for regenerative medicine approaches and drug screening in the future. Here, we summarize current studies of HC-generation from ES and iPS cells.

  13. ERP44 inhibits human lung cancer cell migration mainly via IP3R2.

    Science.gov (United States)

    Huang, Xue; Jin, Meng; Chen, Ying-Xiao; Wang, Jun; Zhai, Kui; Chang, Yan; Yuan, Qi; Yao, Kai-Tai; Ji, Guangju

    2016-06-01

    Cancer cell migration is involved in tumour metastasis. However, the relationship between calcium signalling and cancer migration is not well elucidated. In this study, we used the human lung adenocarcinoma A549 cell line to examine the role of endoplasmic reticulum protein 44 (ERP44), which has been reported to regulate calcium release inside of the endoplasmic reticulum (ER), in cell migration. We found that the inositol 1,4,5-trisphosphate receptors (IP3Rs/ITPRs) inhibitor 2-APB significantly inhibited A549 cell migration by inhibiting cell polarization and pseudopodium protrusion, which suggests that Ca2+ is necessary for A549 cell migration. Similarly, the overexpression of ERP44 reduced intracellular Ca2+ release via IP3Rs, altered cell morphology and significantly inhibited the migration of A549 cells. These phenomena were primarily dependent on IP3R2 because wound healing in A549 cells with IP3R2 rather than IP3R1 or IP3R3 siRNA was markedly inhibited. Moreover, the overexpression of ERP44 did not affect the migration of the human neuroblastoma cell line SH-SY5Y, which mainly expresses IP3R1. Based on the above observations, we conclude that ERP44 regulates A549 cell migration mainly via an IP3R2-dependent pathway.

  14. Induced Pluripotent Stem (iPS) Cell Culture Methods and Induction of Differentiation into Endothelial Cells

    Science.gov (United States)

    Chatterjee, Ishita; Li, Fei; Kohler, Erin E.; Rehman, Jalees; Malik, Asrar B.; Wary, Kishore K.

    2015-01-01

    Summary The studies of stem cell behavior and differentiation in a developmental context is complex, time-consuming and expensive, and for this reason, cell culture remains a method of choice for developmental and regenerative biology and mechanistic studies. Similar to ES cells, iPS cells have the ability to differentiate into endothelial cells (ECs), and the route for differentiation appears to mimic the developmental process that occurs during the formation of an embryo. Traditional EC induction methods from embryonic stem (ES) cells rely mostly on the formation the embryoid body (EB), which employs feeder or feeder-free conditions in the presence or absence of supporting cells. Similar to ES cells, iPS cells can be cultured in feeder-layer or feeder-free conditions. Here, we describe the iPS cell culture methods and induction differentiation of these cells into ECs. We use anti-mouse Flk1 and anti-mouse VE-cadherin to isolate and characterize mouse ECs, because these antibodies are commercially available and their use has been described in the literature, including by our group. The ECs produced by this method have been used by our laboratory, and we have demonstrated their in vivo potential. We also discuss how iPS cells differ in their ability to differentiate into endothelial cells in culture. PMID:25687301

  15. Induced Pluripotent Stem (iPS) Cell Culture Methods and Induction of Differentiation into Endothelial Cells.

    Science.gov (United States)

    Chatterjee, Ishita; Li, Fei; Kohler, Erin E; Rehman, Jalees; Malik, Asrar B; Wary, Kishore K

    2016-01-01

    The study of stem cell behavior and differentiation in a developmental context is complex, time-consuming, and expensive, and for this reason, cell culture remains a method of choice for developmental and regenerative biology and mechanistic studies. Similar to ES cells, iPS cells have the ability to differentiate into endothelial cells (ECs), and the route for differentiation appears to mimic the developmental process that occurs during the formation of an embryo. Traditional EC induction methods from embryonic stem (ES) cells rely mostly on the formation of embryoid body (EB), which employs feeder or feeder-free conditions in the presence or absence of supporting cells. Similar to ES cells, iPS cells can be cultured in feeder layer or feeder-free conditions. Here, we describe the iPS cell culture methods and induction differentiation of these cells into ECs. We use anti-mouse Flk1 and anti-mouse VE-cadherin to isolate and characterize mouse ECs, because these antibodies are commercially available and their use has been described in the literature, including by our group. The ECs produced by this method have been used by our laboratory, and we have demonstrated their in vivo potential. We also discuss how iPS cells differ in their ability to differentiate into endothelial cells in culture.

  16. Cell type list - ChIP-Atlas | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data List Contact us ChIP-Atla...005 Description of data contents A list of all cell type names of data provided on ChIP-Atlas. See details h...License Update History of This Database Site Policy | Contact Us Cell type list - ChIP-Atlas | LSDB Archive ...

  17. Cell tropism and pathogenesis of measles virus in monkeys

    Directory of Open Access Journals (Sweden)

    Sei-ich eKato

    2012-01-01

    Full Text Available Measles virus (MV is an enveloped negative strand RNA virus belonging to the family of Paramyxoviridae, genus Morbillivirus, and causes one of the most contagious diseases in humans. Experimentally infected non-human primates are used as animal models for studies of the pathogenesis of human measles. We established a reverse genetics system based on a highly pathogenic wild-type MV (IC-B strain. Infection of monkeys with recombinant MV strains generated by reverse genetics enabled analysis of the molecular basis of MV pathogenesis. In addition, recombinant wild-type MV strains expressing enhanced green fluorescent protein enable visual tracking of MV-infected cells in vitro and in vivo. To date, 3 different molecules have been identified as receptors for MV. Signaling lymphocyte activation molecule (SLAM, also called CD150, expressed on immune cells, is a major receptor for MV. CD46, ubiquitously expressed in all nucleated cells in humans and monkeys, is a receptor for vaccine and laboratory strains of MV. The newly identified nectin-4 (also called PVRL4 is an epithelial cell receptor for MV. The impact of MV receptor usage in vivo on disease outcomes is now under investigation.

  18. Reprogramming in vivo produces teratomas and iPS cells with totipotency features.

    Science.gov (United States)

    Abad, María; Mosteiro, Lluc; Pantoja, Cristina; Cañamero, Marta; Rayon, Teresa; Ors, Inmaculada; Graña, Osvaldo; Megías, Diego; Domínguez, Orlando; Martínez, Dolores; Manzanares, Miguel; Ortega, Sagrario; Serrano, Manuel

    2013-10-17

    Reprogramming of adult cells to generate induced pluripotent stem cells (iPS cells) has opened new therapeutic opportunities; however, little is known about the possibility of in vivo reprogramming within tissues. Here we show that transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs, implying that full reprogramming can occur in vivo. Analyses of the stomach, intestine, pancreas and kidney reveal groups of dedifferentiated cells that express the pluripotency marker NANOG, indicative of in situ reprogramming. By bone marrow transplantation, we demonstrate that haematopoietic cells can also be reprogrammed in vivo. Notably, reprogrammable mice present circulating iPS cells in the blood and, at the transcriptome level, these in vivo generated iPS cells are closer to embryonic stem cells (ES cells) than standard in vitro generated iPS cells. Moreover, in vivo iPS cells efficiently contribute to the trophectoderm lineage, suggesting that they achieve a more plastic or primitive state than ES cells. Finally, intraperitoneal injection of in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic markers. We conclude that reprogramming in vivo is feasible and confers totipotency features absent in standard iPS or ES cells. These discoveries could be relevant for future applications of reprogramming in regenerative medicine.

  19. Human iPS Cell-Derived Germ Cells: Current Status and Clinical Potential.

    Science.gov (United States)

    Ishii, Tetsuya

    2014-10-13

    Recently, fertile spermatozoa and oocytes were generated from mouse induced pluripotent (iPS) cells using a combined in vitro and in vivo induction system. With regard to germ cell induction from human iPS cells, progress has been made particularly in the male germline, demonstrating in vitro generation of haploid, round spermatids. Although iPS-derived germ cells are expected to be developed to yield a form of assisted reproductive technology (ART) that can address unmet reproductive needs, genetic and/or epigenetic instabilities abound in iPS cell generation and germ cell induction. In addition, there is still room to improve the induction protocol in the female germline. However, rapid advances in stem cell research are likely to make such obstacles surmountable, potentially translating induced germ cells into the clinical setting in the immediate future. This review examines the current status of the induction of germ cells from human iPS cells and discusses the clinical potential, as well as future directions.

  20. Human iPS Cell-Derived Germ Cells: Current Status and Clinical Potential

    Directory of Open Access Journals (Sweden)

    Tetsuya Ishii

    2014-10-01

    Full Text Available Recently, fertile spermatozoa and oocytes were generated from mouse induced pluripotent (iPS cells using a combined in vitro and in vivo induction system. With regard to germ cell induction from human iPS cells, progress has been made particularly in the male germline, demonstrating in vitro generation of haploid, round spermatids. Although iPS-derived germ cells are expected to be developed to yield a form of assisted reproductive technology (ART that can address unmet reproductive needs, genetic and/or epigenetic instabilities abound in iPS cell generation and germ cell induction. In addition, there is still room to improve the induction protocol in the female germline. However, rapid advances in stem cell research are likely to make such obstacles surmountable, potentially translating induced germ cells into the clinical setting in the immediate future. This review examines the current status of the induction of germ cells from human iPS cells and discusses the clinical potential, as well as future directions.

  1. Effects of hypoxia on pluripotency in murine iPS cells.

    Science.gov (United States)

    Sugimoto, Kouji; Yoshizawa, Yuu; Yamada, Shizuka; Igawa, Kazunari; Hayashi, Yoshihiko; Ishizaki, Hidetaka

    2013-10-01

    Retroviral transduction of four transcription factors (Oct4, Sox2, Klf4 and c-Myc) or three factors, excluding c-Myc, has been shown to initiate a reprogramming process that results in the transformation of murine fibroblasts to induced pluripotent stem (iPS) cells, and there has been a rapid increase in the number of iPS cell-based preclinical trials. In this study, the effects of these transcription factors were evaluated regarding the growth and differentiation of murine iPS cells under hypoxia. Based on the results of RT-PCR and alizarin red S staining, there were no statistical differences in the growth and differentiation of iPS cells or the induction of iPS cells to osteoblasts under hypoxia between the transcription factor groups. Furthermore, the function of hypoxia inducible factors (HIFs) in murine iPS cells under hypoxia was investigated in relation to the morphology and expression of transcription factors using RT-PCR and Western blotting. The HIF-2α knockdown group exhibited a decrease in the colony size of the iPS cells. The HIF-2α or -3α knockdown group demonstrated a statistically significant decrease in the transcription factor expression compared to that observed in the control group. These results demonstrate that HIF-2α among HIFs is the most influential candidate for the maintenance of the pluripotency of murine iPS cells.

  2. Generation and In Vitro Expansion of Hepatic Progenitor Cells from Human iPS Cells.

    Science.gov (United States)

    Yanagida, Ayaka; Nakauchi, Hiromitsu; Kamiya, Akihide

    2016-01-01

    Stem cells have the unique properties of self-renewal and multipotency (producing progeny belonging to two or more lineages). Induced pluripotent stem (iPS) cells can be generated from somatic cells by simultaneous expression of pluripotent factors (Oct3/4, Klf4, Sox2, and c-Myc). They share the same properties as embryonic stem (ES) cells and can differentiate into several tissue cells, i.e., neurons, hematopoietic cells, and liver cells. Therefore, iPS cells are suitable candidate cells for regenerative medicine and analyses of disease mechanisms.The liver is the major organ that regulates a multitude of metabolic functions. Hepatocytes are the major cell type populating the liver parenchyma and express several metabolic enzymes that are necessary for liver functions. Although hepatocytes are essential for maintaining homeostasis, it is difficult to alter artificial and transplanted cells because of their multifunctionality, donor shortage, and immunorejection risk. During liver development, hepatic progenitor cells in the fetal liver differentiate into both mature hepatocytes and cholangiocytes. As hepatic progenitor cells have bipotency and high proliferation ability, they could present a potential source for generating transplantable cells or as a liver study model. Here we describe the induction and purification of hepatic progenitor cells derived from human iPS cells. These cells can proliferate for a long term under suitable culture conditions.

  3. Induced Pluripotent Stem (iPS) Cells in Dentistry: A Review

    Science.gov (United States)

    Malhotra, Neeraj

    2016-01-01

    iPS cells are derived from somatic cells via transduction and expression of selective transcription factors. Both viral-integrating (like retroviral) and non-integrating (like, mRNA or protein-based) techniques are available for the production of iPS cells. In the field of dentistry, iPS cells have been derived from stem cells of apical papilla, dental pulp stem cells, and stem cells from exfoliated deciduous teeth, gingival and periodontal ligament fibroblasts, and buccal mucosa fibroblasts. iPS cells have the potential to differentiate into all derivatives of the 3 primary germ layers i.e. ectoderm, endoderm, and mesoderm. They are autogeneically accessible, and can produce patient-specific or disease-specific cell lines without the issue of ethical controversy. They have been successfully tested to produce mesenchymal stem cells-like cells, neural crest-like cells, ameloblasts-like cells, odontoblasts-like cells, and osteoprogenitor cells. These cells can aid in regeneration of periodontal ligament, alveolar bone, cementum, dentin-pulp complex, as well as possible Biotooth formation. However certain key issues like, epigenetic memory of iPS cells, viral-transduction, tumorgenesis and teratoma formation need to be overcome, before they can be successfully used in clinical practice. The article discusses the sources, pros and cons, and current applications of iPS cells in dentistry with an emphasis on encountered challenges and their solutions. PMID:27572712

  4. Smurf1 regulation of DAB2IP controls cell proliferation and migration

    Science.gov (United States)

    Wan, Lixin; Inuzuka, Hiroyuki; Sun, Liankun; North, Brian J.

    2016-01-01

    Tumor cell proliferation, survival and migration are regulated by the deletion of ovarian carcinoma 2/disabled homolog 2 (DOC-2/DAB2) interacting protein (DAB2IP), a tumor suppressor that serves as a scaffold protein for H-Ras and TRAF2. Importantly, the oncogenic histone methyl-transferase EZH2 epigenetically down-regulates DAB2IP in a variety of tumors. Recently, we demonstrated that DAB2IP is negatively regulated by Akt-dependent phosphorylation and SCFFbw7-mediated degradation. Here, we further identify the oncoprotein Smurf1, an E3-ubiquitin ligase, as a novel negative regulator of DAB2IP. Smurf1-mediated cellular proliferation and migration are largely dependent on the presence of DAB2IP, suggesting that DAB2IP is a key effector molecule of Smurf1 oncogenic function. Additionally, we identify that similar to DAB2IP, Smurf1 is also a target of phosphorylation by both Akt1 and Akt2 kinases, which enhances Smurf1 abundance, leading to a reduction in DAB2IP. Given the role of DAB2IP in tumorigenesis and metastasis, our data identify Smurf1 as an upstream oncogenic factor that negatively regulates DAB2IP to govern aberrant cell growth and migration. PMID:27036023

  5. Limitations and possibilities of low cell number ChIP-seq

    Directory of Open Access Journals (Sweden)

    Gilfillan Gregor D

    2012-11-01

    Full Text Available Abstract Background Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq offers high resolution, genome-wide analysis of DNA-protein interactions. However, current standard methods require abundant starting material in the range of 1–20 million cells per immunoprecipitation, and remain a bottleneck to the acquisition of biologically relevant epigenetic data. Using a ChIP-seq protocol optimised for low cell numbers (down to 100,000 cells / IP, we examined the performance of the ChIP-seq technique on a series of decreasing cell numbers. Results We present an enhanced native ChIP-seq method tailored to low cell numbers that represents a 200-fold reduction in input requirements over existing protocols. The protocol was tested over a range of starting cell numbers covering three orders of magnitude, enabling determination of the lower limit of the technique. At low input cell numbers, increased levels of unmapped and duplicate reads reduce the number of unique reads generated, and can drive up sequencing costs and affect sensitivity if ChIP is attempted from too few cells. Conclusions The optimised method presented here considerably reduces the input requirements for performing native ChIP-seq. It extends the applicability of the technique to isolated primary cells and rare cell populations (e.g. biobank samples, stem cells, and in many cases will alleviate the need for cell culture and any associated alteration of epigenetic marks. However, this study highlights a challenge inherent to ChIP-seq from low cell numbers: as cell input numbers fall, levels of unmapped sequence reads and PCR-generated duplicate reads rise. We discuss a number of solutions to overcome the effects of reducing cell number that may aid further improvements to ChIP performance.

  6. Hepatic Differentiation from Murine and Human iPS Cells Using Nanofiber Scaffolds.

    Science.gov (United States)

    Yamazoe, Taiji; Shiraki, Nobuaki; Kume, Shoen

    2016-01-01

    The induced pluripotent stem (iPS) cells of murine and human are capable to differentiate into any cell type of the body through recapitulating normal development, similarly as the embryonic stem (ES) cells. Lines of evidence support that both ES cells and iPS cells are induced to differentiate in vitro by sequential treatment of humoral cues such as growth factors and chemicals, combined with the use of certain microenvironments including extracellular matrices and scaffolds.Here, we describe the procedure to potentiate hepatic lineage cells differentiation from murine and human iPS cells, using growth factor cocktails and nanofiber scaffolds. Nanofiber scaffolds have a three-dimensional surface mimicking the fine structures of the basement membrane in vivo, allow the iPS cells to differentiate into the definitive endoderm and mature hepatocyte-like cells more efficiently than the two-dimensional conventional culture plates.

  7. The Characteristics of Murine iPS Cells and siRNA Transfection Under Hypoxia.

    Science.gov (United States)

    Sugimoto, K; Hayashi, Yoshihiko

    2016-01-01

    iPS cells are attractive for the regenerative medicine. The creation of pluripotent cells from somatic cells has great potential for basic and clinical research and application. Retroviral transduction of four or three transfection factors has been shown to initiate a reprogramming process. Here, we describe the effect of transcription factors regarding the growth and differentiation of mouse iPS cells in normoxia or hypoxia. Furthermore, we introduce the function of hypoxia-inducible factors (HIFs) in mouse iPS cells in hypoxia using RT-PCR and western blotting together with HIFs knockdown techniques.

  8. Inositol Hexakisphosphate Kinase 1 (IP6K1) Regulates Inositol Synthesis in Mammalian Cells.

    Science.gov (United States)

    Yu, Wenxi; Ye, Cunqi; Greenberg, Miriam L

    2016-05-13

    myo-Inositol, the precursor of all inositol compounds, has pivotal roles in cell metabolism and signaling pathways. Although physiological studies indicate a strong correlation between abnormal intracellular inositol levels and neurological disorders, very little is known about the regulation of inositol synthesis in mammalian cells. In this study, we report that IP6K1, an inositol hexakisphosphate kinase that catalyzes the synthesis of inositol pyrophosphate, regulates inositol synthesis in mammalian cells. Ip6k1 ablation led to profound changes in DNA methylation and expression of Isyna1 (designated mIno1), which encodes the rate-limiting enzyme inositol-3-phosphate synthase. Interestingly, IP6K1 preferentially bound to the phospholipid phosphatidic acid, and this binding was required for IP6K1 nuclear localization and the regulation of mIno1 transcription. This is the first demonstration of IP6K1 as a novel negative regulator of inositol synthesis in mammalian cells.

  9. Induced pluripotent stem (iPS) cells repair and regenerate infarcted myocardium.

    Science.gov (United States)

    Singla, Dinender K; Long, Xilin; Glass, Carley; Singla, Reetu D; Yan, Binbin

    2011-10-03

    Cardiac myocyte differentiation reported thus far is from iPS cells generated from mouse and human fibroblasts. However, there is no article on the generation of iPS cells from cardiac ventricular specific cell types such as H9c2 cells. Therefore, whether transduced H9c2 cells, originally isolated from embryonic cardiac ventricular tissue, will be able to generate iPS cells and have the potential to repair and regenerate infarcted myocardium remains completely elusive. We transduced H9c2 cells with four stemness factors, Oct3/4, Sox2, Klf4, and c-Myc, and successfully reprogrammed them into iPS cells. These iPS cells were able to differentiate into beating cardiac myocytes and positively stained for cardiac specific sarcomeric α-actin and myosin heavy chain proteins. Following transplantation in the infarcted myocardium, there were newly differentiated cardiac myocytes and formation of gap junction proteins at 2 weeks post-myocardial infarction (MI), suggesting newly formed cardiac myocytes were integrated into the native myocardium. Furthermore, transplanted iPS cells significantly (p cell groups. Moreover, our iPS cell derived cardiac myocyte differentiation in vitro and in vivo was comparable to embryonic stem cells in the present study. In conclusion we report for the first time that we have H9c2 cell-derived iPS cells which contain the potential to differentiate into cardiac myocytes in the cell culture system and repair and regenerate infarcted myocardium with improved cardiac function in vivo.

  10. The generation of iPS cells using non-viral magnetic nanoparticle based transfection.

    Science.gov (United States)

    Lee, Chang Hyun; Kim, Jung-Hyun; Lee, Hyun Joo; Jeon, Kilsoo; Lim, HyeJin; Choi, Hye yeon; Lee, Eung-Ryoung; Park, Seung Hwa; Park, Jae-Yong; Hong, Sunghoi; Kim, Soonhag; Cho, Ssang-Goo

    2011-10-01

    Induced pluripotent stem (iPS) cells have been generated from various somatic cells; however, a major restriction of the technology is the use of potentially harmful genome-integrating viral DNAs. Here, without a viral vector, we generated iPS cells from fibroblasts using a non-viral magnetic nanoparticle-based transfection method that employs biodegradable cationic polymer PEI-coated super paramagnetic nanoparticles (NP). Our findings support the possible use of transient expression of iPS genes in somatic cells by magnet-based nanofection for efficient generation of iPS cells. Results of dynamic light scattering (DLS) analysis and TEM analyses demonstrated efficient conjugation of NP with iPS genes. After transfection, nanofection-mediated iPS cells showed ES cell-like characteristics, including expression of endogenous pluripotency genes, differentiation of three germ layer lineages, and formation of teratomas. Our results demonstrate that magnet-based nanofection may provide a safe method for use in generation of virus-free and exogenous DNA-free iPS cells, which will be crucial for future clinical applications in the field of regenerative medicine.

  11. Integration-free iPS cells engineered using human artificial chromosome vectors.

    Directory of Open Access Journals (Sweden)

    Masaharu Hiratsuka

    Full Text Available Human artificial chromosomes (HACs have unique characteristics as gene-delivery vectors, including episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs into induced pluripotent stem (iPS cells. Two HAC vectors (iHAC1 and iHAC2 were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown cassette. iHAC1 partially reprogrammed MEFs, and iHAC2 efficiently reprogrammed MEFs. Global gene expression patterns showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. Under non-selecting conditions, we established iHAC-free iPS cells by isolating cells that spontaneously lost iHAC2. Analyses of pluripotent markers, teratomas and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex virus thymidine kinase were eliminated by ganciclovir treatment, indicating that the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a built-in safeguard system.

  12. [Research and Application of iPS Cells in Blood System].

    Science.gov (United States)

    Zhou, Li-Xia; Ye, Jie-Yu; Lian, Qi-Zhou; Yang, Mo

    2015-04-01

    Induced pluripotent stem cells (iPS cells) were first constructed by Takahshi and et al in 2006. They converted the mouse fibroblasts into ES-like cells via viral transduction with four transcription factors (Oct4, Sox2, Klf4 and c-Myc). Since, the significant progress has been made and many researchers have succeeded in inducing iPS cells from other human somatic cells by some novel approaches, such as combining transcriptional factors and small chemicals. IPS cells have significant prospect in clinical application. IPS cells derived from patient somatic cells can be used as a model in studying the pathogenesis of genetic hematological disease and applied in therapeutic screenings. Recent studies suggested that iPS cells can differentiate into red blood cells and platelets in vitro, which may make up a big blood bank for transfusion in future. In this review, current understanding of both recombinant technology of iPS cells and the research progress in hematology are summarized.

  13. Thymic derived iPs cells can be differentiated into cardiomyocytes.

    Science.gov (United States)

    Li, Jian; Cao, Yin-yin; Ma, Xiao-jing; Liu, Fang; Li, Shuo-lin; Zhang, Jing; Gao, Yan; Wang, Hui-jun; Yuan, Yuan; Ma, Duan; Huang, Guo-ying

    2015-06-01

    Ventricular septal defect (VSD) is one of the common congenital heart malformations. Several factors lead to the development of VSD, including familial causes, exposure to certain drugs, infectious agents, and maternal metabolic disturbances. We considered that induced pluripotent stem (iPS) cells derived from VSD patients can be used to study the origin and pathogenesis of the VSD. Here, we show generation and cardiomyocyte differentiation potential of iPS cells from thymic epithelial cells of a patient with VSD (TECs-VSD) by overexpressing the four factors: OCT4, SOX2, NANOG, and LIN28 with lentiviral vectors. The self-renewal and pluripotency of the VSD-iPS cells was verified in iPS cells by in vitro expression of pluripotency markers and formation of teratoma in vivo. iPS cell lines from VSD patients differentiated into functional cardiomyocytes can serve as a model system for studying the pathophysiology and identifying etiology of VSD.

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

    Directory of Open Access Journals (Sweden)

    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. MicroRNA expression profiles of human iPS cells, retinal pigment epithelium derived from iPS, and fetal retinal pigment epithelium.

    Science.gov (United States)

    Greene, Whitney A; Muñiz, Alberto; Plamper, Mark L; Kaini, Ramesh R; Wang, Heuy-Ching

    2014-06-24

    The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

  16. Enhancement of arsenic trioxide-mediated changes in human induced pluripotent stem cells (IPS).

    Science.gov (United States)

    Graham, Barbara; Stevens, Jacqueline; Wells, Phatia; Sims, Jennifer; Rogers, Christian; Leggett, Sophia S; Ekunwe, Stephen; Ndebele, Kenneth

    2014-07-22

    Induced pluripotent stem cells (IPS) are an artificially derived type of pluripotent stem cell, showing many of the same characteristics as natural pluripotent stem cells. IPS are a hopeful therapeutic model; however there is a critical need to determine their response to environmental toxins. Effects of arsenic on cells have been studied extensively; however, its effect on IPS is yet to be elucidated. Arsenic trioxide (ATO) has been shown to inhibit cell proliferation, induce apoptosis and genotoxicity in many cells. Based on ATOs action in other cells, we hypothesize that it will induce alterations in morphology, inhibit cell viability and induce a genotoxic effect on IPS. Cells were treated for 24 hours with ATO (0-9 µg/mL). Cell morphology, viability and DNA damage were documented. Results indicated sufficient changes in morphology of cell colonies mainly in cell ability to maintain grouping and ability to remain adherent. Cell viability decreased in a dose dependent manner. There were significant increases in tail length and moment as well as destruction of intact DNA as concentration increased. Exposure to ATO resulted in a reproducible dose dependent sequence of events marked by changes in morphology, decrease of cell viability, and induction of genotoxicity in IPS.

  17. Induced pluripotent stem (iPS) cells from human fetal stem cells.

    Science.gov (United States)

    Guillot, Pascale V

    2016-02-01

    Pluripotency defines the ability of stem cells to differentiate into all the lineages of the three germ layers and self-renew indefinitely. Somatic cells can regain the developmental potential of embryonic stem cells following ectopic expression of a set of transcription factors or, in certain circumstances, via modulation of culture conditions and supplementation with small molecule, that is, induced pluripotent stem (iPS) cells. Here, we discuss the use of fetal tissues for reprogramming, focusing in particular on stem cells derived from human amniotic fluid, and the development of chemical reprogramming. We next address the advantages and disadvantages of deriving pluripotent cells from fetal tissues and the potential clinical applications.

  18. Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system.

    Science.gov (United States)

    Li, Hongmei Lisa; Gee, Peter; Ishida, Kentaro; Hotta, Akitsu

    2016-05-15

    Precise gene correction using the CRISPR-Cas9 system in human iPS cells holds great promise for various applications, such as the study of gene functions, disease modeling, and gene therapy. In this review article, we summarize methods for effective editing of genomic sequences of iPS cells based on our experiences correcting dystrophin gene mutations with the CRISPR-Cas9 system. Designing specific sgRNAs as well as having efficient transfection methods and proper detection assays to assess genomic cleavage activities are critical for successful genome editing in iPS cells. In addition, because iPS cells are fragile by nature when dissociated into single cells, a step-by-step confirmation during the cell recovery process is recommended to obtain an adequate number of genome-edited iPS cell clones. We hope that the techniques described here will be useful for researchers from diverse backgrounds who would like to perform genome editing in iPS cells.

  19. Inhibition by salmeterol and cilomilast of fluticasone-enhanced IP-10 release in airway epithelial cells.

    Science.gov (United States)

    Reddy, P J; Aksoy, Mark O; Yang, Yi; Li, Xiu Xia; Ji, Rong; Kelsen, Steven G

    2008-02-01

    The CXC chemokines, IP-10/CXCL10 and IL-8/CXCL8, play a role in obstructive lung disease by attracting Th1/Tc1 lymphocytes and neutrophils, respectively. Inhaled corticosteroids (ICS) and long acting beta 2-agonists (LABA) are widely used. However, their effect(s) on the release of IP-10 and IL-8 by airway epithelial cells are poorly understood. This study examined the effects of fluticasone, salmeterol, and agents which raise intracellular cAMP (cilomilast and db-cAMP) on the expression of IP-10 and IL-8 protein and mRNA. Studies were performed in cultured human airway epithelial cells during cytokine-stimulated IP-10 and IL-8 release. Cytokine treatment (TNF-alpha, IL-1beta and IFN-gamma) increased IP-10 and IL-8 protein and mRNA levels. Fluticasone (0.1 nM to 1 microM) increased IP-10 but reduced IL-8 protein release without changing IP-10 mRNA levels assessed by real time RT-PCR. The combination of salmeterol (1 micro M) and cilomilast (1-10 mu M) reduced IP-10 but had no effect on IL-8 protein. Salmeterol alone (1 micro M) and db-cAMP alone (1 mM) antagonised the effects of fluticasone on IP-10 but not IL-8 protein. In human airway epithelial cells, inhibition by salmeterol of fluticasone-enhanced IP-10 release may be an important therapeutic effect of the LABA/ICS combination not present when the two drugs are used separately.

  20. [Retinal regeneration with iPS cells ‒ Clinical trials for retinal degenerative disorders].

    Science.gov (United States)

    Sugita, Sunao

    2015-01-01

    Potential for re-programming cells has become widely accepted as a tool for obtaining transplantation materials. There has been great interest in cell-based therapies, including retinal transplants, because there is a reduced risk of immune rejection. Stem cells have the capacity for self-renewal plus the capacity to generate several differentiated cells. They are derived from many sources including human adult-derived induced pluripotent stem (iPS) cells and have found early application in the context of ocular disease. In results, our established iPS-retinal pigment epithelial (RPE) cells are high-quality RPE cells. iPS cells-derived RPE cells clearly showed polygonal morphology (mostly hexagonal) and contained melanin. Moreover, RPE cells derived from iPS cells had many characteristics of mature RPE cells in vivo, but no characteristics of pluripotent stem cells. Recently, we transplanted RPE cell sheets to treat a patient with wet age-related macular degeneration (September, 2014). In addition, we are now conducting experiments to determine whether allogeneic T cells can recognize iPS-RPE cells from HLA-A, B, DRB1 locus homozygote donors. iPS bank might be useful as allografts in retinal disorders, if the recipient T cells cannot respond to allogeneic RPE cells because of match to some of main HLA antigens.

  1. Nucleus- and cell-specific gene expression in monkey thalamus.

    Science.gov (United States)

    Murray, Karl D; Choudary, Prabhakara V; Jones, Edward G

    2007-02-06

    Nuclei of the mammalian thalamus are aggregations of neurons with unique architectures and input-output connections, yet the molecular determinants of their organizational specificity remain unknown. By comparing expression profiles of thalamus and cerebral cortex in adult rhesus monkeys, we identified transcripts that are unique to dorsal thalamus or to individual nuclei within it. Real-time quantitative PCR and in situ hybridization analyses confirmed the findings. Expression profiling of individual nuclei microdissected from the dorsal thalamus revealed additional subsets of nucleus-specific genes. Functional annotation using Gene Ontology (GO) vocabulary and Ingenuity Pathways Analysis revealed overrepresentation of GO categories related to development, morphogenesis, cell-cell interactions, and extracellular matrix within the thalamus- and nucleus-specific genes, many involved in the Wnt signaling pathway. Examples included the transcription factor TCF7L2, localized exclusively to excitatory neurons; a calmodulin-binding protein PCP4; the bone extracellular matrix molecules SPP1 and SPARC; and other genes involved in axon outgrowth and cell matrix interactions. Other nucleus-specific genes such as CBLN1 are involved in synaptogenesis. The genes identified likely underlie nuclear specification, cell phenotype, and connectivity during development and their maintenance in the adult thalamus.

  2. Rhesus monkey neural stem cell transplantation promotes neural regeneration in rats with hippocampal lesions.

    Science.gov (United States)

    Ye, Li-Juan; Bian, Hui; Fan, Yao-Dong; Wang, Zheng-Bo; Yu, Hua-Lin; Ma, Yuan-Ye; Chen, Feng

    2016-09-01

    Rhesus monkey neural stem cells are capable of differentiating into neurons and glial cells. Therefore, neural stem cell transplantation can be used to promote functional recovery of the nervous system. Rhesus monkey neural stem cells (1 × 10(5) cells/μL) were injected into bilateral hippocampi of rats with hippocampal lesions. Confocal laser scanning microscopy demonstrated that green fluorescent protein-labeled transplanted cells survived and grew well. Transplanted cells were detected at the lesion site, but also in the nerve fiber-rich region of the cerebral cortex and corpus callosum. Some transplanted cells differentiated into neurons and glial cells clustering along the ventricular wall, and integrated into the recipient brain. Behavioral tests revealed that spatial learning and memory ability improved, indicating that rhesus monkey neural stem cells noticeably improve spatial learning and memory abilities in rats with hippocampal lesions.

  3. Induced pluripotent stem (iPS cells offer a powerful new tool for the life sciences

    Directory of Open Access Journals (Sweden)

    Yukio Nakamura

    2010-01-01

    Full Text Available Stem cell biology started with the analysis of somatic stem cells that function to maintain the adult body. We now know that the body is maintained by regeneration of a wide range of cell types, such as skin cells, blood cells and gastrointestinal mucous cells, from somatic stem cells. This regenerative activity is essential for survival. Regenerative medicine was initiated to identify therapies that support and/or accelerate this natural regenerative ability. For example, bone marrow transplantation is a therapy for reconstituting hematopoiesis from the hematopoietic stem cells present in the donor bone marrow. The successful development of a protocol for obtaining human embryonic stem (ES cells prompted medical scientists to utilize human ES cells for regenerative medicine. However, use of these cells raises ethical issues as they are derived from human embryos. An alternative approach using ES-like pluripotent stem cells has the considerable advantage that it does not necessitate use of human embryos. Pluripotent stem cells can be induced from terminally differentiated somatic cells by the introduction of only four defined factors. The products of this method are termed “induced pluripotent stem (iPS" cells. iPS cells have considerable promise as a substitute for ES cells not only for regenerative medicine but also in many other fields. For example, liver and heart cells derived from iPS cells can be used in pharmaceutical research. In addition, iPS cell technology opens new avenues of disease research, for example, by construction of so-called “disease-specific iPS cells” from a patient's somatic cells.

  4. Phenotypic correction of murine hemophilia A using an iPS cell-based therapy.

    Science.gov (United States)

    Xu, Dan; Alipio, Zaida; Fink, Louis M; Adcock, Dorothy M; Yang, Jianchang; Ward, David C; Ma, Yupo

    2009-01-20

    Hemophilia A is caused by mutations within the Factor VIII (FVIII) gene that lead to depleted protein production and inefficient blood clotting. Several attempts at gene therapy have failed for various reasons-including immune rejection. The recent generation of induced pluripotent stem (iPS) cells from somatic cells by the ectopic expression of 3 transcription factors, Oct4, Sox2, and Klf4, provides a means of circumventing the immune rejection barrier. To date, iPS cells appear to be indistinguishable from ES cells and thus provide tremendous therapeutic potential. Here we prepared murine iPS cells from tail-tip fibroblasts and differentiated them to both endothelial cells and endothelial progenitor cells by using the embryoid body differentiation method. These iPS cells express major ES cell markers such as Oct4, Nanog, SSEA-1, alkaline phosphatase, and SALL4. Endothelial/endothelial progenitor cells derived from iPS cells expressed cell-specific markers such as CD31, CD34, and Flk1 and secreted FVIII protein. These iPS-derived cells were injected directly into the liver of irradiated hemophilia A mice. At various times after transplantation (7-90 days) hemophilia A mice and their control mice counterparts were challenged by a tail-clip bleeding assay. Nontransplanted hemophilia A mice died within a few hours, whereas transplanted mice survived for more than 3 months. Plasma FVIII levels increased in transplanted hemophilia A mice during this period to 8% to 12% of wild type and corrected the hemophilia A phenotype. Our studies provide additional evidence that iPS cell therapy may be able to treat human monogenetic disorders in the future.

  5. Inhibition of the integrin signal constitutes a mouse iPS cell niche.

    Science.gov (United States)

    Higuchi, Sayaka; Yoshina, Sawako; Mitani, Shohei

    2016-09-01

    Stem cells are regulated by their surrounding microenvironments, called niche, such as cell-cell interaction and extracellular matrix. Classically, feeder cells as a niche have been used in the culture of iPS cells from both the mouse and the human. However, the regulation mechanism of stem cells by feeder cells as a niche still have been partially unclear. In this study, we used three murine iPS cell lines, iPS-MEF-Ng-20D-17, iPS-MEF-Ng-178B-5 and iPS-MEF-Fb/Ng-440A-3, which were generated by different reprogramming methods. In general, these cell lines commonly need the feeder cells as a niche to culture. Recently, the effect of substrate stiffness is known in stem cell study. First, we focused on the mechanical properties of feeder cells, and then we speculated that feeder-less culture might be made possible by using molecules in place of the mechanical properties of the niche. Finally, we found that the combination of disintegrin (echistatin) and 2i (GSK3 inhibitor and MEK inhibitor) is a sufficient condition for three murine iPS culture. This novel method of mimicking the murine iPS cell niche may be useful to understand signaling pathways to maintain the pluripotency of stem cells.

  6. Induction of iPS cells and of cancer stem cells: the stem cell or reprogramming hypothesis of cancer?

    Science.gov (United States)

    Trosko, James E

    2014-01-01

    This article as designed to examine whether the "stoichiometric" or "elite models" of the origin of the "induced pluripotent stem" (iPS) cells fits some experiment facts from the developmental biology of adult stem cells and from the field of cancer research. In brief, since the evidence presented to support the stoichiometric model failed to recognize the factual existence of adult organ specific stem cells, the model has not been rigorously tested. In addition, the demonstration of a subset of cells (MUSE cells) in normal primary in vitro cultures of human fibroblasts (the usual source of iPS cells) seems to be the origin of the iPS cells. Moreover, from the field of carcinogenesis, the "stem cell" versus "de-differentiation" or "reprogramming" hypotheses were examined. Again, using the role of glycolysis, known to be associated with the Warburg effect in cancer cells, a list of experiments showing that (a) normal stem cells, which have few mitochondria, metabolize via glycolysis; (b) the stem cells are targets for "initiation" or "immortalization" or the blockage of differentiation and apoptosis of the stem cells by "immortalizing viruses"; (c) Lactate dehydrogenase A (LDHA), when expressed, is associated with glycolysis and therefore, must be expressed in normal adult stem cells, as well as in cancer cells; and (d) p53, depleted or rendered dysfunctional by SV40 Large T antigen, is associated with the reduction of mitochondrial function and mass and is associated with the Warburg effect. Together, these observations from the iPS and "cancer stem cell" fields support the idea that both iPS cells and cancer stem cell are derived from adult organ-specific stem cells that do not restore or switch their metabolism of glucose from oxidative metabolism to glycolysis but, rather, in both cases, the adult stem cell, which metabolizes by glycolysis, is prevented from differentiation or from metabolizing by oxidative phosphorylation.

  7. 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.

  8. Modeling Alzheimer's disease with human induced pluripotent stem (iPS) cells.

    Science.gov (United States)

    Mungenast, Alison E; Siegert, Sandra; Tsai, Li-Huei

    2016-06-01

    In the last decade, induced pluripotent stem (iPS) cells have revolutionized the utility of human in vitro models of neurological disease. The iPS-derived and differentiated cells allow researchers to study the impact of a distinct cell type in health and disease as well as performing therapeutic drug screens on a human genetic background. In particular, clinical trials for Alzheimer's disease (AD) have been failing. Two of the potential reasons are first, the species gap involved in proceeding from initial discoveries in rodent models to human studies, and second, an unsatisfying patient stratification, meaning subgrouping patients based on the disease severity due to the lack of phenotypic and genetic markers. iPS cells overcome this obstacles and will improve our understanding of disease subtypes in AD. They allow researchers conducting in depth characterization of neural cells from both familial and sporadic AD patients as well as preclinical screens on human cells. In this review, we briefly outline the status quo of iPS cell research in neurological diseases along with the general advantages and pitfalls of these models. We summarize how genome-editing techniques such as CRISPR/Cas9 will allow researchers to reduce the problem of genomic variability inherent to human studies, followed by recent iPS cell studies relevant to AD. We then focus on current techniques for the differentiation of iPS cells into neural cell types that are relevant to AD research. Finally, we discuss how the generation of three-dimensional cell culture systems will be important for understanding AD phenotypes in a complex cellular milieu, and how both two- and three-dimensional iPS cell models can provide platforms for drug discovery and translational studies into the treatment of AD.

  9. The directed differentiation of human iPS cells into kidney podocytes.

    Science.gov (United States)

    Song, Bi; Smink, Alexandra M; Jones, Christina V; Callaghan, Judy M; Firth, Stephen D; Bernard, Claude A; Laslett, Andrew L; Kerr, Peter G; Ricardo, Sharon D

    2012-01-01

    The loss of glomerular podocytes is a key event in the progression of chronic kidney disease resulting in proteinuria and declining function. Podocytes are slow cycling cells that are considered terminally differentiated. Here we provide the first report of the directed differentiation of induced pluripotent stem (iPS) cells to generate kidney cells with podocyte features. The iPS-derived podocytes share a morphological phenotype analogous with cultured human podocytes. Following 10 days of directed differentiation, iPS podocytes had an up-regulated expression of mRNA and protein localization for podocyte markers including synaptopodin, nephrin and Wilm's tumour protein (WT1), combined with a down-regulation of the stem cell marker OCT3/4. In contrast to human podocytes that become quiescent in culture, iPS-derived cells maintain a proliferative capacity suggestive of a more immature phenotype. The transduction of iPS podocytes with fluorescent labeled-talin that were immunostained with podocin showed a cytoplasmic contractile response to angiotensin II (AII). A permeability assay provided functional evidence of albumin uptake in the cytoplasm of iPS podocytes comparable to human podocytes. Moreover, labeled iPS-derived podocytes were found to integrate into reaggregated metanephric kidney explants where they incorporated into developing glomeruli and co-expressed WT1. This study establishes the differentiation of iPS cells to kidney podocytes that will be useful for screening new treatments, understanding podocyte pathogenesis, and offering possibilities for regenerative medicine.

  10. The directed differentiation of human iPS cells into kidney podocytes.

    Directory of Open Access Journals (Sweden)

    Bi Song

    Full Text Available The loss of glomerular podocytes is a key event in the progression of chronic kidney disease resulting in proteinuria and declining function. Podocytes are slow cycling cells that are considered terminally differentiated. Here we provide the first report of the directed differentiation of induced pluripotent stem (iPS cells to generate kidney cells with podocyte features. The iPS-derived podocytes share a morphological phenotype analogous with cultured human podocytes. Following 10 days of directed differentiation, iPS podocytes had an up-regulated expression of mRNA and protein localization for podocyte markers including synaptopodin, nephrin and Wilm's tumour protein (WT1, combined with a down-regulation of the stem cell marker OCT3/4. In contrast to human podocytes that become quiescent in culture, iPS-derived cells maintain a proliferative capacity suggestive of a more immature phenotype. The transduction of iPS podocytes with fluorescent labeled-talin that were immunostained with podocin showed a cytoplasmic contractile response to angiotensin II (AII. A permeability assay provided functional evidence of albumin uptake in the cytoplasm of iPS podocytes comparable to human podocytes. Moreover, labeled iPS-derived podocytes were found to integrate into reaggregated metanephric kidney explants where they incorporated into developing glomeruli and co-expressed WT1. This study establishes the differentiation of iPS cells to kidney podocytes that will be useful for screening new treatments, understanding podocyte pathogenesis, and offering possibilities for regenerative medicine.

  11. Synthesis of an inositol hexakisphosphate (IP6) affinity probe to study the interactome from a colon cancer cell line.

    Science.gov (United States)

    Yin, Meng-Xin; Catimel, Bruno; Gregory, Mark; Condron, Melanie; Kapp, Eugene; Holmes, Andrew B; Burgess, Antony W

    2016-03-14

    Inositol hexakisphosphate (InsP6 or IP6) is an important signalling molecule in vesicular trafficking, neurotransmission, immune responses, regulation of protein kinases and phosphatases, activation of ion channels, antioxidant functions and anticancer activities. An IP6 probe was synthesised from myo-inositol via a derivatised analogue, which was immobilised through a terminal amino group onto Dynabeads. Systematic analysis of the IP6 interactome has been performed using the IP6 affinity probe using cytosolic extracts from the LIM1215 colonic carcinoma cell line. LC/MS/MS analysis identified 77 proteins or protein complexes that bind to IP6 specifically, including AP-2 complex proteins and β-arrestins as well as a number of novel potential IP6 interacting proteins. Bioinformatic enrichment analysis of the IP6 interactome reinforced the concept that IP6 regulates a number of biological processes including cell cycle and division, signal transduction, intracellular protein transport, vesicle-mediated transport and RNA splicing.

  12. High IP-10 levels decrease T cell function in HIV-1-infected individuals on ART

    Science.gov (United States)

    Ramirez, L. A.; Arango, T. A.; Thompson, E.; Naji, M.; Tebas, P.; Boyer, J. D.

    2014-01-01

    HIV-1-infected subjects, despite control of viral replication with ART, have an altered immune cytokine/chemokine milieu. Changes in systemic cytokines and chemokines can alter immune responses. IP-10, in particular, has been associated with pathogenesis in a number of conditions, and we found that IP-10 is increased in serum in subjects who are HIV-1 infected and on stable ART compared with HIV-1-uninfected individuals. In a series of in vitro studies, we found that PBMCs exposed to IP-10 showed a significant decrease in the number of cells capable of secreting IFN-γ, as well as other cytokines, when stimulated with recall antigens. Furthermore, treatment with IP-10 led to decreased antigen-specific calcium signaling and MAPK38 phosphorylation. Importantly, the cytokines, as well as proliferative responses, could be enhanced with an IP-10 Nab. Our findings suggest that IP-10-modulating drugs may potentially enhance T cell responses to vaccination and HIV-1 in HIV+ subjects on ART. PMID:25157027

  13. Transplantation of adult monkey neural stem cells into a contusion spinal cord injury model in rhesus macaque monkeys

    DEFF Research Database (Denmark)

    Nemati, Shiva Nemati; Jabbari, Reza; Hajinasrollah, Mostafa

    2014-01-01

    OBJECTIVE: Currently, cellular transplantation for spinal cord injuries (SCI) is the subject of numerous preclinical studies. Among the many cell types in the adult brain, there is a unique subpopulation of neural stem cells (NSC) that can self-renew and differentiate into neurons. The study aims......, therefore, to explore the efficacy of adult monkey NSC (mNSC) in a primate SCI model. MATERIALS AND METHODS: In this experimental study, isolated mNSCs were analyzed by flow cytometry, immunocytochemistry, and RT-PCR. Next, BrdU-labeled cells were transplanted into a SCI model. The SCI animal model...... was confirmed by magnetic resonance imaging (MRI) and histological analysis. Animals were clinically observed for 6 months. RESULTS: Analysis confirmed homing of mNSCs into the injury site. Transplanted cells expressed neuronal markers (TubIII). Hind limb performance improved in trans- planted animals based...

  14. Isolation and characterization of liver epithelial progenitor cells from normal adult rhesus monkeys (Macaca mulatta)

    Institute of Scientific and Technical Information of China (English)

    Lifang Jin; Shaohui Ji; Xianghui Tang; Xiangyu Guo; Yongqing Lu; Hongwei Chen; Hongkui Deng; Qi Zhou; Weizhi Ji

    2009-01-01

    @@ Dear Editor, Based on their ability to proliferate and the capacity to differentiate into specific cell types, hepatic progenitor/stem cells (HPCs) from adult human liver may have potential therapeutic effects on end-stage liver failure. In addition, adult HPCs have a reduced risk of teratoma formation and are not subject to the same ethical issues as fetal HPCs or embryonic stem cells [1]. The HPCs from rhesus monkeys are relevant because they may serve as a valuable preclinical model for assessment of cell therapy in humans. To date, there are no reports of HPCs or liver epithelial progenitor cells (LEPCs) isolated from normal adult rhesus monkey although a few studies in other species were reported [2, 3]. We report here for the first time the successful isolation of rhesus monkey LEPCs (mLEPCs) from normal adult livers (n=12).

  15. [A technique of rhesus monkey neural progenitor cells intravitreal transplant to rats].

    Science.gov (United States)

    Bian, Hui; Fan, Yao-Dong; Guo, Li-Yun; Yu, Hua-Lin

    2012-02-01

    To investigate a simple and effective intraocular xenotransplant technique of rhesus monkey neural progenitor cells to rats, mechanical injury was induced in the rat's right retina. And the GFP-labeled rhesus monkey neural progenitor cells suspension was slowly injected into the vitreous space of the right injured and left control eye. Confocal image suggested that the xenografted cells survived in both the injured and control eye, meanwhile the cells integrated in the injured right retina. The results demonstrated that intravitreal xenotransplant could be adopted as a simple and reliable method.

  16. Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines.

    Science.gov (United States)

    Bock, Christoph; Kiskinis, Evangelos; Verstappen, Griet; Gu, Hongcang; Boulting, Gabriella; Smith, Zachary D; Ziller, Michael; Croft, Gist F; Amoroso, Mackenzie W; Oakley, Derek H; Gnirke, Andreas; Eggan, Kevin; Meissner, Alexander

    2011-02-04

    The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines.

  17. Cell-type specificity of ChIP-predicted transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Håndstad Tony

    2012-08-01

    Full Text Available Abstract Background Context-dependent transcription factor (TF binding is one reason for differences in gene expression patterns between different cellular states. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identifies genome-wide TF binding sites for one particular context—the cells used in the experiment. But can such ChIP-seq data predict TF binding in other cellular contexts and is it possible to distinguish context-dependent from ubiquitous TF binding? Results We compared ChIP-seq data on TF binding for multiple TFs in two different cell types and found that on average only a third of ChIP-seq peak regions are common to both cell types. Expectedly, common peaks occur more frequently in certain genomic contexts, such as CpG-rich promoters, whereas chromatin differences characterize cell-type specific TF binding. We also find, however, that genotype differences between the cell types can explain differences in binding. Moreover, ChIP-seq signal intensity and peak clustering are the strongest predictors of common peaks. Compared with strong peaks located in regions containing peaks for multiple transcription factors, weak and isolated peaks are less common between the cell types and are less associated with data that indicate regulatory activity. Conclusions Together, the results suggest that experimental noise is prevalent among weak peaks, whereas strong and clustered peaks represent high-confidence binding events that often occur in other cellular contexts. Nevertheless, 30-40% of the strongest and most clustered peaks show context-dependent regulation. We show that by combining signal intensity with additional data—ranging from context independent information such as binding site conservation and position weight matrix scores to context dependent chromatin structure—we can predict whether a ChIP-seq peak is likely to be present in other cellular contexts.

  18. Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells

    Institute of Scientific and Technical Information of China (English)

    Donghui Zhang; Wei Jiang; Meng Liu; Xin Sui; Xiaolei Yin; Song Chen; Yan Shi; Hongkui Deng

    2009-01-01

    Human pluripotent stem cells represent a potentially unlimited source of functional pancreatic endocrine lineage cells. Here we report a highly efficient approach to induce human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells to differentiate into mature insulin-producing cells in a chemical-defined culture system. The differentiated human ES cells obtained by this approach comprised nearly 25% insulin-positive cells as assayed by flow cytometry analysis, which released insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these insulin-producing cells co-expressed mature β cell-specific markers such as NKX6-1 and PDX1, indicating a similar gene expression pattern to adult islet β cells in vivo. In this study, we also demonstrated that EGF facilitates the expansion of PDX1-positive pancreatic progenitors. Moreover, our protocol also succeeded in efficiently inducing human iPS cells to differentiate into insulin-producing cells. Therefore, this work not only provides a new model to study the mechanism of human pancreatic specialization and maturation in vitro, but also enhances the possibility of utilizing patient-specific iPS cells for the treatment of diabetes.

  19. Novel sequential ChIP and simplified basic ChIP protocols for promoter co-occupancy and target gene identification in human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Elayaperumal Anuratha

    2009-06-01

    Full Text Available Abstract Background The investigation of molecular mechanisms underlying transcriptional regulation, particularly in embryonic stem cells, has received increasing attention and involves the systematic identification of target genes and the analysis of promoter co-occupancy. High-throughput approaches based on chromatin immunoprecipitation (ChIP have been widely used for this purpose. However, these approaches remain time-consuming, expensive, labor-intensive, involve multiple steps, and require complex statistical analysis. Advances in this field will greatly benefit from the development and use of simple, fast, sensitive and straightforward ChIP assay and analysis methodologies. Results We initially developed a simplified, basic ChIP protocol that combines simplicity, speed and sensitivity. ChIP analysis by real-time PCR was compared to analysis by densitometry with the ImageJ software. This protocol allowed the rapid identification of known target genes for SOX2, NANOG, OCT3/4, SOX17, KLF4, RUNX2, OLIG2, SMAD2/3, BMI-1, and c-MYC in a human embryonic stem cell line. We then developed a novel Sequential ChIP protocol to investigate in vivo promoter co-occupancy, which is basically characterized by the absence of antibody-antigen disruption during the assay. It combines centrifugation of agarose beads and magnetic separation. Using this Sequential ChIP protocol we found that c-MYC associates with the SOX2/NANOG/OCT3/4 complex and identified a novel RUNX2/BMI-1/SMAD2/3 complex in BG01V cells. These two TF complexes associate with two distinct sets of target genes. The RUNX2/BMI-1/SMAD2/3 complex is associated predominantly with genes not expressed in undifferentiated BG01V cells, consistent with the reported role of those TFs as transcriptional repressors. Conclusion These simplified basic ChIP and novel Sequential ChIP protocols were successfully tested with a variety of antibodies with human embryonic stem cells, generated a number of novel

  20. The Potential of iPS Cells in Synucleinopathy Research

    Directory of Open Access Journals (Sweden)

    Leonhard Linta

    2012-01-01

    Full Text Available α-synuclein is a protein involved in the pathogenesis of several so-called synucleinopathies including Parkinson's disease. A variety of models have been so far assessed. Human induced pluripotent stem cells provide a patient- and disease-specific model for in vitro studies, pharmacotoxicological screens, and hope for future cell-based therapies. Initial experimental procedures include the harvest of patients’ material for the reprogramming process, the investigation of the patients genetic background in the cultured cells, and the evaluation of disease-relevant factors/proteins under various cell culture conditions.

  1. Expression of neuropeptide receptor mRNA during osteoblastic differentiation of mouse iPS cells.

    Science.gov (United States)

    Nagao, Satomi; Goto, Tetsuya; Kataoka, Shinji; Toyono, Takashi; Joujima, Takaaki; Egusa, Hiroshi; Yatani, Hirofumi; Kobayashi, Shigeru; Maki, Kenshi

    2014-12-01

    Various studies have shown a relationship between nerves and bones. Recent evidence suggests that both sensory and sympathetic nerves affect bone metabolism; however, little is known about how neuropeptides are involved in the differentiation of pluripotent stem cells into osteoblastic (OB) cells. To evaluate the putative effects of neuropeptides during the differentiation of mouse induced pluripotent stem (iPS) cells into calcified tissue-forming OB cells, we investigated the expression patterns of neuropeptide receptors at each differentiation stage. Mouse iPS cells were seeded onto feeder cells and then transferred to low-attachment culture dishes to form embryoid bodies (EBs). EBs were cultured for 4 weeks in osteoblastic differentiation medium. The expression of α1-adrenergic receptor (AR), α2-AR, β2-AR, neuropeptide Y1 receptor (NPY1-R), neuropeptide Y2 receptor (NPY2-R), calcitonin gene-related protein receptor (CGRP-R), and neurokinin 1-R (NK1-R) was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Among these neuropeptide receptors, CGRP-R and β2-AR were expressed at all stages of cell differentiation, including the iPS cell stage, with peak expression occurring at the early osteoblastic differentiation stage. Another sensory nervous system receptor, NK1-R, was expressed mainly in the late osteoblastic differentiation stage. Furthermore, CGRP-R mRNA showed an additional small peak corresponding to EBs cultured for 3 days, suggesting that EBs may be affected by serum CGRP. These data suggest that the sensory nervous system receptor CGRP-R and the sympathetic nervous system receptor β2-AR may be involved in the differentiation of iPS cells into the osteoblastic lineage. It follows from these findings that CGRP and β2-AR may regulate cell differentiation in the iPS and EB stages, and that each neuropeptide has an optimal period of influence during the differentiation process.

  2. Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

    Energy Technology Data Exchange (ETDEWEB)

    Kinoshita, Taisuke [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Nagamatsu, Go, E-mail: gonag@sc.itc.keio.ac.jp [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Kosaka, Takeo [Department of Urology, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Takubo, Keiyo [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Hotta, Akitsu [Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Department of Reprogramming Science, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto (Japan); Ellis, James [Ontario Human iPS Cell Facility, Molecular Genetics, University of Toronto, Developmental and Stem Cell Biology, SickKids, Toronto, Canada MG1L7 (Canada); Suda, Toshio, E-mail: sudato@sc.itc.keio.ac.jp [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan)

    2011-04-08

    Highlights: {yields} iPS cells were induced with a fluorescence monitoring system. {yields} ATM-deficient tail-tip fibroblasts exhibited quite a low reprogramming efficiency. {yields} iPS cells obtained from ATM-deficient cells had pluripotent cell characteristics. {yields} ATM-deficient iPS cells had abnormal chromosomes, which were accumulated in culture. -- Abstract: During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.

  3. Using stem cells and iPS cells to discover new treatments for Parkinson's disease.

    Science.gov (United States)

    Cooper, Oliver; Hallett, Penny; Isacson, Ole

    2012-01-01

    Fetal cell transplantation can improve the symptoms of Parkinson's disease (PD) patients for more than a decade. In some patients, alpha-synuclein aggregates and Lewy bodies have been observed in the transplanted neurons without functional significance. Recently stem cells have emerged as an ethically acceptable source of cells for transplantation but, importantly, the type of stem cell matters. While the lineage restriction of adult neural stem cells limits their clinical applicability for patients with PD, human pluripotent stem cells provide an opportunity to replace specific types of degenerating neurons. Now, cellular reprogramming technology can provide patient-specific neurons for neural transplantation and problems with cell fate specification and safety are resolving. Induced pluripotent stem (iPS) cell-derived neurons are also a unique tool for interpreting the genetic basis for an individual's risk of developing PD into clinically meaningful information. For example, clinical trials for neuroprotective molecules need to be tested in presymptomatic individuals when the neurons can still be protected. Patient-specific neural cells can also be used to identify an individual's responsiveness to drugs and to understand the mechanisms of the disease. Along these avenues of investigation, stem cells are enabling research for new treatments in PD. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study.

    Science.gov (United States)

    Zhu, Yong; Hu, Hong-Liang; Li, Peng; Yang, Shi; Zhang, Wei; Ding, Hui; Tian, Ru-Hui; Ning, Ye; Zhang, Ling-Ling; Guo, Xi-Zhi; Shi, Zhan-Ping; Li, Zheng; He, Zuping

    2012-07-01

    Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.

  5. Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells.

    Science.gov (United States)

    Kinoshita, Taisuke; Nagamatsu, Go; Kosaka, Takeo; Takubo, Keiyo; Hotta, Akitsu; Ellis, James; Suda, Toshio

    2011-04-08

    During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.

  6. Xenotransplantation of microencaps bovine chromaffin cells into hemiparkinsonian monkeys:a analyses of behaviour,biochemistry and pathology

    Institute of Scientific and Technical Information of China (English)

    XUE Y. L.; WANG L. N.; WANG Z. F.; ZHONG D. G.; LI X. J.; CUI X.; MA X. J.; ZHU Ming-wei; SUN A. M.

    2001-01-01

    @@ This study examines the effects of xenografts of microencapsulated bovine chromaffin cells (BCCs) on the rotational behavior of hemiparkinsonian monkey recipients. In addition, it determines the content of monoamine neurotransmitters and their major metabolites in the neostriatum in hemiparkinsonian monkeys. The hemiparkinsonian model in monkeys was induced by a unilateral intracarotid injection of methyl-phenyl-tetrahydropyridine (MPTP). Unencapsulated BCCs (n = 2), BCCs microencapsulated (n= 6) in alginate-polylysine-alginate (APA) membranes as well as empty microencapsules (n = 1) were grafted into the neostriatum of the hemiparkinsonian monkeys.

  7. Post-natal cardiomyocytes can generate iPS cells with an enhanced capacity toward cardiomyogenic re-differentation.

    Science.gov (United States)

    Rizzi, R; Di Pasquale, E; Portararo, P; Papait, R; Cattaneo, P; Latronico, M V G; Altomare, C; Sala, L; Zaza, A; Hirsch, E; Naldini, L; Condorelli, G; Bearzi, C

    2012-07-01

    Adult mammalian cells can be reprogrammed to a pluripotent state by forcing the expression of a few embryonic transcription factors. The resulting induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers. It is well known that post-natal cardiomyocytes (CMs) lack the capacity to proliferate. Here, we report that neonatal CMs can be reprogrammed to generate iPS cells that express embryonic-specific markers and feature gene-expression profiles similar to those of mouse embryonic stem (mES) cell and cardiac fibroblast (CF)-derived iPS cell populations. CM-derived iPS cells are able to generate chimeric mice and, moreover, re-differentiate toward CMs more efficiently then either CF-derived iPS cells or mES cells. The increased differentiation capacity is possibly related to CM-derived iPS cells retaining an epigenetic memory of the phenotype of their founder cell. CM-derived iPS cells may thus lead to new information on differentiation processes underlying cardiac differentiation and proliferation.

  8. The current status of iPS cells in cardiac research and their potential for tissue engineering and regenerative medicine.

    Science.gov (United States)

    Martins, Ana M; Vunjak-Novakovic, Gordana; Reis, Rui L

    2014-04-01

    The recent availability of human cardiomyocytes derived from induced pluripotent stem (iPS) cells opens new opportunities to build in vitro models of cardiac disease, screening for new drugs, and patient-specific cardiac therapy. Notably, the use of iPS cells enables studies in the wide pool of genotypes and phenotypes. We describe progress in reprogramming of induced pluripotent stem (iPS) cells towards the cardiac lineage/differentiation. The focus is on challenges of cardiac disease modeling using iPS cells and their potential to produce safe, effective and affordable therapies/applications with the emphasis of cardiac tissue engineering. We also discuss implications of human iPS cells to biological research and some of the future needs.

  9. Induced pluripotent stem cell (iPS) technology: promises and challenges

    Institute of Scientific and Technical Information of China (English)

    MIGUELA Esteban; GAN Yi; QIN DaJiang; PEI DuanQing

    2009-01-01

    In 2006,an article published in Cell by Shinya Yamanaka took by surprise the stem cell research community.By performing systematic retroviral transduction of factors enriched in embryonic stem (ES) cells,the authors demonstrated the reprogramming of mouse fibroblasts into an ES cell-like state.These cells,baptized iPS (induced pluripotent stem) cells,were immediately recognized as a ground-breaking discovery.Subsequently,the same authors and other groups reported a similar achievement with human fibroblasts.Two years later,the number of top quality papers on iPS is astonishing,and interest in the scientific community has risen to a fever pitch.But although iPS has the potential to revolutionize Regenerative Medicine,important questions still remain unanswered.Work from multiple laboratories worldwide including ours is focused on deciphering the molecular mechanisms of iPS,and trying to improve the technique to make it suitable for the clinic.In this review article we briefly discuss the past,present and future of iPS,with emphasis on urgent issues to be solved.

  10. Long-Term Oocyte-Like Cell Development in Cultures Derived from Neonatal Marmoset Monkey Ovary

    Directory of Open Access Journals (Sweden)

    Bentolhoda Fereydouni

    2016-01-01

    Full Text Available We use the common marmoset monkey (Callithrix jacchus as a preclinical nonhuman primate model to study reproductive and stem cell biology. The neonatal marmoset monkey ovary contains numerous primitive premeiotic germ cells (oogonia expressing pluripotent stem cell markers including OCT4A (POU5F1. This is a peculiarity compared to neonatal human and rodent ovaries. Here, we aimed at culturing marmoset oogonia from neonatal ovaries. We established a culture system being stable for more than 20 passages and 5 months. Importantly, comparative transcriptome analysis of the cultured cells with neonatal ovary, embryonic stem cells, and fibroblasts revealed a lack of germ cell and pluripotency genes indicating the complete loss of oogonia upon initiation of the culture. From passage 4 onwards, however, the cultured cells produced large spherical, free-floating cells resembling oocyte-like cells (OLCs. OLCs strongly expressed several germ cell genes and may derive from the ovarian surface epithelium. In summary, our novel primate ovarian cell culture initially lacked detectable germ cells but then produced OLCs over a long period of time. This culture system may allow a deeper analysis of early phases of female primate germ cell development and—after significant refinement—possibly also the production of monkey oocytes.

  11. Effect of monensin on Mayaro virus replication in monkey kidney and Aedes albopictus cells.

    Science.gov (United States)

    De Campos, R M; Ferreira, D F; Da Veiga, V F; Rebello, M A; Rebello, M C S

    2003-01-01

    The effect of a cationic ionophore, monensin, on the replication of Mayaro virus in monkey kidney TC7 and Aedes albopictus cells has been studied. Treatment of these cells with 1 micromol/l monensin during infection did not affect the virus protein synthesis but inhibited severely the virus replication. Electron microscopy of the cells infected with Mayaro virus and treated with monensin revealed that the morphogenesis of Mayaro virus was impaired in TC7 but not in A. albopictus cells.

  12. Isolation and characterization of novel rhesus monkey embryonic stem cell lines.

    Science.gov (United States)

    Mitalipov, Shoukhrat; Kuo, Hung-Chih; Byrne, James; Clepper, Lisa; Meisner, Lorraine; Johnson, Julie; Zeier, Renee; Wolf, Don

    2006-10-01

    ESCs are important as research subjects since the mechanisms underlying cellular differentiation, expansion, and self-renewal can be studied along with differentiated tissue development and regeneration in vitro. Furthermore, human ESCs hold promise for cell and tissue replacement approaches to treating human diseases. The rhesus monkey is a clinically relevant primate model that will likely be required to bring these clinical applications to fruition. Monkey ESCs share a number of properties with human ESCs, and their derivation and use are not affected by bioethical concerns. Here, we summarize our experience in the establishment of 18 ESC lines from rhesus monkey preimplantation embryos generated by the application of the assisted reproductive technologies. The newly derived monkey ESC lines were maintained in vitro without losing their chromosomal integrity, and they expressed markers previously reported present in human and monkey ESCs. We also describe initial efforts to compare the pluripotency of ESC lines by expression profiling, chimeric embryo formation, and in vitro-directed differentiation into endodermal, mesodermal, and ectodermal lineages.

  13. Differentiation of mouse iPS cells into ameloblast-like cells in cultures using medium conditioned by epithelial cell rests of Malassez and gelatin-coated dishes.

    Science.gov (United States)

    Yoshida, Koki; Sato, Jun; Takai, Rie; Uehara, Osamu; Kurashige, Yoshihito; Nishimura, Michiko; Chiba, Itsuo; Saitoh, Masato; Abiko, Yoshihiro

    2015-09-01

    Induced pluripotent stem (iPS) cells are generated from adult cells and are potentially of great value in regenerative medicine. Recently, it was shown that iPS cells can differentiate into ameloblast-like cells in cultures using feeder cells. In the present study, we sought to induce differentiation of ameloblast-like cells from iPS cells under feeder-free conditions using medium conditioned by cultured epithelial cell rests of Malassez (ERM) cells and gelatin-coated dishes. Two culture conditions were compared: co-cultures of iPS cells and ERM cells; and, culture of iPS cells in ERM cell-conditioned medium. Differentiation of ameloblast-like cells in the cultures was assessed using real-time RT-PCR assays of expression of the marker genes keratin 14, amelogenin, and ameloblastin and by immunocytochemical staining for amelogenin. We found greater evidence of ameloblast-like cell differentiation in the cultures using the conditioned medium. In the latter, the level of amelogenin expression increased daily and was significantly higher than controls on the 7th, 10th, and 14th days. Expression of ameloblastin also increased daily and was significantly higher than controls on the 14th day. The present study demonstrates that mouse iPS cells can be induced to differentiate into ameloblast-like cells in feeder-free cell cultures using ERM cell-conditioned medium and gelatin-coated dishes.

  14. Mouse ES and iPS cells can form similar definitive endoderm despite differences in imprinted genes.

    Science.gov (United States)

    Christodoulou, Constantina; Longmire, Tyler A; Shen, Steven S; Bourdon, Alice; Sommer, Cesar A; Gadue, Paul; Spira, Avrum; Gouon-Evans, Valerie; Murphy, George J; Mostoslavsky, Gustavo; Kotton, Darrell N

    2011-06-01

    The directed differentiation of iPS and ES cells into definitive endoderm (DE) would allow the derivation of otherwise inaccessible progenitors for endodermal tissues. However, a global comparison of the relative equivalency of DE derived from iPS and ES populations has not been performed. Recent reports of molecular differences between iPS and ES cells have raised uncertainty as to whether iPS cells could generate autologous endodermal lineages in vitro. Here, we show that both mouse iPS and parental ES cells exhibited highly similar in vitro capacity to undergo directed differentiation into DE progenitors. With few exceptions, both cell types displayed similar surges in gene expression of specific master transcriptional regulators and global transcriptomes that define the developmental milestones of DE differentiation. Microarray analysis showed considerable overlap between the genetic programs of DE derived from ES/iPS cells in vitro and authentic DE from mouse embryos in vivo. Intriguingly, iPS cells exhibited aberrant silencing of imprinted genes known to participate in endoderm differentiation, yet retained a robust ability to differentiate into DE. Our results show that, despite some molecular differences, iPS cells can be efficiently differentiated into DE precursors, reinforcing their potential for development of cell-based therapies for diseased endoderm-derived tissues.

  15. Comparative study on direction selectivity and functional organization of the primary visual cortical cells in monkeys and cats

    Institute of Scientific and Technical Information of China (English)

    寿天德; 周逸峰; 俞洪波

    2000-01-01

    Although the directionally selective cells in many visual cortical areas are organized in columnar manner, the functional organization of direction selectivity of area VI in the monkey still remains unclear. We quantitatively studied the proportion of directionally selective cells, direction selectivity and the functional organization of the striate cortical cells in the monkey and compared those with the cat. The results show that the direction selectivity and directional organization of striate cortical cells in the monkey are significantly weaker than those in the cat, suggesting that the species difference between the two kinds of animal is related to their different anatomic pathways.

  16. Comparative study on direction selectivity and functional organization of the primary visual cortical cells in monkeys and cats

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Although the directionally selective cells in many visual cortical areas are organized in columnar manner, the functional organization of direction selectivity of area Vl in the monkey still remains unclear. We quantitatively studied the proportion of directionally selective cells, direction selectivity and the functional organization of the striate cortical cells in the monkey and compared those with the cat. The results show that the direction selectivity and directional organization of striate cortical cells in the monkey are significantly weaker than those in the cat, suggesting that the species difference between the two kinds of animal is related to their different anatomic pathways.

  17. Role of protein tyrosine kinase in the effect of IP6 on IL-8 secretion in intestinal epithelial cells.

    Science.gov (United States)

    Wawszczyk, Joanna; Orchel, Arkadiusz; Kapral, Małgorzata; Wéglarz, Ludmiła

    2013-01-01

    Phytic acid (IP6) is a major fiber-associated component of a diet physiologically present in human intestines. Studies showed that this phytochemical can modulate immune functions of intestinal epithelium through regulation of proinflammatory cytokines secretion but mechanisms underlying these cellular response to IP6 have weakly been examined, as yet. The aim of this study was to determine the role of protein tyrosine kinase (PTK) in secretion of IL-8, a central proinflammatory cytokine, by unstimulated and IL-1beta-stimulated intestinal epithelial cells Caco-2 treated with IP6 (1 and 2.5 mM). To study the involvement of PTK signal pathway in IL-8 secretion, inhibitors of phosphotyrosine phosphatase (sodium orthovanadate, OV) and tyrosine kinase (genistein, GEN) were incubated with Caco-2 cells prior to IP6 treatment. IP6 had suppressive effect on basal and IL-1beta-stimulated IL-8 secretion by cells. The effect of OV on IL-8 release by cells treated with IP6 was different under constitutive and stimulated conditions. Secretion of IL-8 was significantly down-regulated in cells with GEN and GEN plus IP6 treatment. In addition, total PTK activity in both unstimulated and IL-1beta stimulated cells was determined in the presence of IP6. The results suggest that physiological intestinal concentrations of IP6 may have an inhibitory effect on IL-8 secretion by Caco-2 cells and one of the mechanisms of its action is the inhibition of PTK signaling cascade. The study revealed for the first time that PTKs could be one of the molecular targets for IP6 effects in the intestinal epithelial cells.

  18. Evaluation of the cytotoxicity of geosmin and 2-methylisoborneol using cultured human, monkey, and dog cells.

    Science.gov (United States)

    Mochida, Kyo

    2009-03-01

    The cytotoxicity of musty odor-emitting substances, geosmin (GM) and 2-methylisoborneol, at a concentration of 10 ng/L - 300 mg/L was investigated using cultured mammalian cells. These two compounds exhibited no cytotoxicity in either the colony-formation of human KB cells or WST-1 assays of human-, monkey-, and dog-derived cells. These results suggest that the maximum concentration (700 ng/L) of GM found in the water of Lake Shinji is not toxic.

  19. Apoptotic effect of IP6 was not enhanced by co-treatment with myo-inositol in prostate carcinoma PC3 cells

    Science.gov (United States)

    Kim, Hyun-Jung; Jang, Yu-mi; Kim, Harriet

    2007-01-01

    Inositol hexaphosphate (IP6) is a major constituent of most cereals, legumes, nuts, oil seeds and soybean. Previous studies reported the anticancer effect of IP6 and suggested that co-treatment of IP6 with inositol may enhance anticancer effect of IP6. Although the anticancer effect of IP6 has been intensively studied, the combinational effect of IP6 and inositol and involved mechanisms are not well understood so far. In the present study, we investigated the effect of IP6 and myo-inositol (MI) on cell cycle regulation and apoptosis using PC3 prostate cancer cell lines. When cells were co-treated with IP6 and MI, the extent of cell growth inhibition was significantly increased than that by IP6 alone. To identify the effect of IP6 and MI on apoptosis, the activity of caspase-3 was measured. The caspase-3 activity was significantly increased when cells were treated with either IP6 alone or both IP6 and MI, with no significant enhancement by co-treatment. To investigate the effect of IP6 and MI of cell cycle arrest, we measured p21 mRNA expression in PC3 cells and observed significant increase in p21 mRNA by IP6. But synergistic regulation by co-treatment with IP6 and MI was not observed. In addition, there was no significant effect by co-treatment compared to IP6 treatment on the regulation of cell cycle progression although IP6 significantly changed cell cycle distribution in the presence of MI or not. Therefore, these findings support that IP6 has anticancer function by induction of apoptosis and regulation of cell cycle. However, synergistic effect by MI on cell cycle regulation and apoptosis was not observed in PC3 prostate cancer cells. PMID:20368938

  20. Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model.

    Science.gov (United States)

    Takagi, Yasushi; Takahashi, Jun; Saiki, Hidemoto; Morizane, Asuka; Hayashi, Takuya; Kishi, Yo; Fukuda, Hitoshi; Okamoto, Yo; Koyanagi, Masaomi; Ideguchi, Makoto; Hayashi, Hideki; Imazato, Takayuki; Kawasaki, Hiroshi; Suemori, Hirofumi; Omachi, Shigeki; Iida, Hidehiko; Itoh, Nobuyuki; Nakatsuji, Norio; Sasai, Yoshiki; Hashimoto, Nobuo

    2005-01-01

    Parkinson disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic (DA) neurons. ES cells are currently the most promising donor cell source for cell-replacement therapy in PD. We previously described a strong neuralizing activity present on the surface of stromal cells, named stromal cell-derived inducing activity (SDIA). In this study, we generated neurospheres composed of neural progenitors from monkey ES cells, which are capable of producing large numbers of DA neurons. We demonstrated that FGF20, preferentially expressed in the substantia nigra, acts synergistically with FGF2 to increase the number of DA neurons in ES cell-derived neurospheres. We also analyzed the effect of transplantation of DA neurons generated from monkey ES cells into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated (MPTP-treated) monkeys, a primate model for PD. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons and attenuated MPTP-induced neurological symptoms.

  1. Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study

    Institute of Scientific and Technical Information of China (English)

    Yong Zhu; Xi-Zhi Guo; Zhan-Ping Shi; Zheng Li; Zuping He; Hong-Liang Hu; Peng Li; Shi Yang; Wei Zhang; Hui Ding; Ru-Hui Tian; Ye Ning; Ling-Ling Zhang

    2012-01-01

    Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells.However,whether iPS cells am capable of producing male germ cells is not known.The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late.stage male germ cells.We used an approach that combines in vitro differentiation and in vivotransplantation.Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium.Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8and Vasa mRNA in the EBs derived from iPS cells.iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs),as evidenced by their expression of VASA,as well as CDH 1 and GFRα 1,which are markers of SSCs.Furthermore,these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan.Notably,iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids,as shown by VASA and SCP3 expression.This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells.The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.

  2. Novel sequential ChIP and simplified basic ChIP protocols for promoter co-occupancy and target gene identification in human embryonic stem cells

    OpenAIRE

    Elayaperumal Anuratha; Goh Saik-Kia; Jadrich Joy; Coley Kristen; Hoium Brian; Papenfuss Kate J; Medeiros Ricardo B; Herrera Julio E; Resnik Ernesto; Ni Hsiao-Tzu

    2009-01-01

    Abstract Background The investigation of molecular mechanisms underlying transcriptional regulation, particularly in embryonic stem cells, has received increasing attention and involves the systematic identification of target genes and the analysis of promoter co-occupancy. High-throughput approaches based on chromatin immunoprecipitation (ChIP) have been widely used for this purpose. However, these approaches remain time-consuming, expensive, labor-intensive, involve multiple steps, and requ...

  3. iPS cells generation: an overview of techniques and methods

    Directory of Open Access Journals (Sweden)

    Telpalo-Carpio SA

    2013-04-01

    Full Text Available Cell therapy is one of the principal and most promising research areas of regenerative medicine. Nevertheless different ethical problems related with the use of embryonic stem cells arise from this technology. Thus the search for a feasible way of stem cells generation is in current investigation. Induced pluripotent stem (iPS cell is one of the most encouraging emerging technologies that represent a solution to this problem. Different methods that change cell protein and gene profiles, as well as its morphology and function, driving the cell to the stem cell state have been developed. These include nuclear transfer, the usage of cell extracts and synthetic molecules, the forced expression of defined genes and cytoplasmatic level modifications. Even though favorable results have been achieved, there are still issues that require special attention. Advances with non-integration methods are now available but dedifferentiation efficiency is yet an area of opportunity. The main objective of this review is to show the most important techniques, their advantages and opportunity areas in the field of iPS cell generation.

  4. Maxadilan prevents apoptosis in iPS cells and shows no effects on the pluripotent state or karyotype.

    Directory of Open Access Journals (Sweden)

    Zhiyi Zhao

    Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP is a structurally endogenous peptide with many biological roles. Maxadilan, a 61-amino acid vasodilatory peptide, specifically activates the PACAP type I receptor (PAC1. Although PAC1 has been identified in embryonic stem cells, little is known about its presence or effects in human induced pluripotent stem (iPS cells. In the present study, we investigated the expression of PAC1 in human iPS cells by reverse transcriptase polymerase chain reaction (RT-PCR and western blot analysis. To study the physiological effects mediated by PAC1, we evaluated the role of maxadilan in preventing apoptotic cell death induced by ultraviolet C (UVC. After exposure to UVC, the iPS cells showed a marked reduction in cell viability and a parallel increase of apoptotic cells, as demonstrated by WST-8 analysis, annexin V/propidium iodide (PI analysis and the terminal transferase dUTP nick end labeling (TUNEL assay. The addition of 30 nM of maxadilan dramatically increased iPS cell viability and reduced the percentage of apoptotic cells. The anti-apoptotic effects of maxadilan were correlated to the downregulation of caspase-3 and caspase-9. Concomitantly, immunofluorescence, western blot analysis, real-time quantitative polymerase chain reaction (RT-qPCR analysis and in vitro differentiation results showed that maxadilan did not affect the pluripotent state of iPS cells. Moreover, karyotype analysis showed that maxadilan did not affect the karyotype of iPS cells. In summary, these results demonstrate that PAC1 is present in iPS cells and that maxadilan effectively protects iPS cells against UVC-induced apoptotic cell death while not affecting the pluripotent state or karyotype.

  5. Maxadilan prevents apoptosis in iPS cells and shows no effects on the pluripotent state or karyotype.

    Science.gov (United States)

    Zhao, Zhiyi; Yu, Rongjie; Yang, Jiayin; Liu, Xiaofei; Tan, Meihua; Li, Hongyang; Chen, Jiansu

    2012-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a structurally endogenous peptide with many biological roles. Maxadilan, a 61-amino acid vasodilatory peptide, specifically activates the PACAP type I receptor (PAC1). Although PAC1 has been identified in embryonic stem cells, little is known about its presence or effects in human induced pluripotent stem (iPS) cells. In the present study, we investigated the expression of PAC1 in human iPS cells by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis. To study the physiological effects mediated by PAC1, we evaluated the role of maxadilan in preventing apoptotic cell death induced by ultraviolet C (UVC). After exposure to UVC, the iPS cells showed a marked reduction in cell viability and a parallel increase of apoptotic cells, as demonstrated by WST-8 analysis, annexin V/propidium iodide (PI) analysis and the terminal transferase dUTP nick end labeling (TUNEL) assay. The addition of 30 nM of maxadilan dramatically increased iPS cell viability and reduced the percentage of apoptotic cells. The anti-apoptotic effects of maxadilan were correlated to the downregulation of caspase-3 and caspase-9. Concomitantly, immunofluorescence, western blot analysis, real-time quantitative polymerase chain reaction (RT-qPCR) analysis and in vitro differentiation results showed that maxadilan did not affect the pluripotent state of iPS cells. Moreover, karyotype analysis showed that maxadilan did not affect the karyotype of iPS cells. In summary, these results demonstrate that PAC1 is present in iPS cells and that maxadilan effectively protects iPS cells against UVC-induced apoptotic cell death while not affecting the pluripotent state or karyotype.

  6. Nuclear-mitochondrial incompatibility in interorder rhesus monkey-cow embryos derived from somatic cell nuclear transfer.

    Science.gov (United States)

    Kwon, Daekee; Koo, Ok-Jae; Kim, Min-Jung; Jang, Goo; Lee, Byeong Chun

    2016-10-01

    Monkey interorder somatic cell nuclear transfer (iSCNT) using enucleated cow oocytes yielded poor blastocysts development and contradictory results among research groups. Determining the reason for this low blastocyst development is a prerequisite for optimizing iSCNT in rhesus monkeys. The aim of this study was to elucidate nuclear-mitochondrial incompatibility of rhesus monkey-cow iSCNT embryos and its relationship to low blastocyst development. Cytochrome b is a protein of complex III of the electron transport chain (ETC). According to meta-analysis of amino acid sequences, the homology of cytochrome b is 75 % between rhesus monkeys and cattle. To maintain the function of ETC after iSCNT, 4n iSCNT embryos were produced by fusion of non-enucleated cow oocytes and rhesus monkey somatic cells. The blastocyst development rate of 4n iSCNT embryos was higher than that of 2n embryos (P monkey iSCNT embryos reconstructed with cow oocytes have nuclear-mitochondrial incompatibility due to fundamental species differences between rhesus monkeys and cattle. Nuclear-mitochondrial incompatibility seems to correlate with low ETC activity and extremely low blastocyst development of rhesus monkey-cow iSCNT embryos.

  7. Reduced cell turnover in lymphocytic monkeys infected by human T-lymphotropic virus type 1.

    Science.gov (United States)

    Debacq, Christophe; Héraud, Jean-Michel; Asquith, Becca; Bangham, Charles; Merien, Fabrice; Moules, Vincent; Mortreux, Franck; Wattel, Eric; Burny, Arsène; Kettmann, Richard; Kazanji, Mirdad; Willems, Luc

    2005-11-17

    Understanding cell dynamics in animal models have implications for therapeutic strategies elaborated against leukemia in human. Quantification of the cell turnover in closely related primate systems is particularly important for rare and aggressive forms of human cancers, such as adult T-cell leukemia. For this purpose, we have measured the death and proliferation rates of the CD4+ T lymphocyte population in squirrel monkeys (Saimiri sciureus) infected by human T-lymphotropic virus type 1 (HTLV-1). The kinetics of in vivo bromodeoxyuridine labeling revealed no modulation of the cell turnover in HTLV-1-infected monkeys with normal CD4 cell counts. In contrast, a substantial decrease in the proliferation rate of the CD4+ T population was observed in lymphocytic monkeys (e.g. characterized by excessive proportions of CD4+ T lymphocytes and by the presence of abnormal flower-like cells). Unexpectedly, onset of HTLV-associated leukemia thus occurs in the absence of increased CD4+ T-cell proliferation. This dynamics significantly differs from the generalized activation of the T-cell turnover induced by other primate lymphotropic viruses like HIV and SIV.

  8. CHCHD2 is down-regulated in neuronal cells differentiated from iPS cells derived from patients with lissencephaly.

    Science.gov (United States)

    Shimojima, Keiko; Okumura, Akihisa; Hayashi, Masaharu; Kondo, Takayuki; Inoue, Haruhisa; Yamamoto, Toshiyuki

    2015-10-01

    The human cerebral cortex is peculiar for a six-layered cellular-sheet structure with convolution, which is a consequence of neuronal migration. Dysfunctions of the pathways contributing to this mechanism typically lead to lissencephaly manifesting smooth brain surfaces. To investigate the unknown mechanism underlying neuronal migration disorders, we generated induced pluripotent stem (iPS) cells from two patients with lissencephaly. Whole gene expression study for iPS cells derived from a patient with a LIS1 deletion showed reduced expression of the coiled-coil-helix-coiled-coil-helix domain containing 2 gene (CHCHD2), which was also confirmed in iPS cells derived from a patient with a TUBA1A mutation. CHCHD2 expression was detected in neuronal cells differentiated from normal iPS cells in a time-dependent manner, as well as in the brain of a fetus at 26-28 week gestational age, suggesting development-dependent expression. Migrating neuronal cells showed CHCHD2 expression, suggesting its functional relevance to neuronal migration.

  9. TAP-deficient human iPS cell-derived myeloid cell lines as unlimited cell source for dendritic cell-like antigen-presenting cells.

    Science.gov (United States)

    Haruta, M; Tomita, Y; Yuno, A; Matsumura, K; Ikeda, T; Takamatsu, K; Haga, E; Koba, C; Nishimura, Y; Senju, S

    2013-05-01

    We previously reported a method to generate dendritic cell (DC)-like antigen-presenting cells (APC) from human induced pluripotent stem (iPS) cells. However, the method is relatively complicated and laborious. In the current study, we attempted to establish a method through which we could obtain a large number of functional APC with a simple procedure. We transduced iPS cell-derived CD11b(+) myeloid cells with genes associated with proliferative or anti-senescence effects, enabling the cells to propagate for more than 4 months in a macrophage colony-stimulating factor (M-CSF)-dependent manner while retaining their capacity to differentiate into functional APC. We named these iPS cell-derived proliferating myeloid cells 'iPS-ML', and the iPS-ML-derived APC 'ML-DC'. In addition, we generated TAP2-deficient iPS cell clones by zinc finger nuclease-aided targeted gene disruption. TAP2-deficient iPS cells and iPS-ML avoided recognition by pre-activated allo-reactive CD8(+) T cells. TAP2-deficient ML-DC expressing exogenously introduced HLA-A2 genes stimulated HLA-A2-restricted MART-1-specific CD8(+) T cells obtained from HLA-A2-positive allogeneic donors, resulting in generation of MART-1-specific cytotoxic T lymphocyte (CTL) lines. TAP-deficient iPS-ML introduced with various HLA class I genes may serve as an unlimited source of APC for vaccination therapy. If administered into allogeneic patients, ML-DC with appropriate genetic modifications may survive long enough to stimulate antigen-specific CTL and, after that, be completely eliminated. Based on the present study, we propose an APC-producing system that is simple, safe and applicable to all patients irrespective of their HLA types.

  10. Interrogation of allelic chromatin states in human cells by high-density ChIP-genotyping.

    Science.gov (United States)

    Light, Nicholas; Adoue, Véronique; Ge, Bing; Chen, Shu-Huang; Kwan, Tony; Pastinen, Tomi

    2014-09-01

    Allele-specific (AS) assessment of chromatin has the potential to elucidate specific cis-regulatory mechanisms, which are predicted to underlie the majority of the known genetic associations to complex disease. However, development of chromatin landscapes at allelic resolution has been challenging since sites of variable signal strength require substantial read depths not commonly applied in sequencing based approaches. In this study, we addressed this by performing parallel analyses of input DNA and chromatin immunoprecipitates (ChIP) on high-density Illumina genotyping arrays. Allele-specificity for the histone modifications H3K4me1, H3K4me3, H3K27ac, H3K27me3, and H3K36me3 was assessed using ChIP samples generated from 14 lymphoblast and 6 fibroblast cell lines. AS-ChIP SNPs were combined into domains and validated using high-confidence ChIP-seq sites. We observed characteristic patterns of allelic-imbalance for each histone-modification around allele-specifically expressed transcripts. Notably, we found H3K4me1 to be significantly anti-correlated with allelic expression (AE) at transcription start sites, indicating H3K4me1 allelic imbalance as a marker of AE. We also found that allelic chromatin domains exhibit population and cell-type specificity as well as heritability within trios. Finally, we observed that a subset of allelic chromatin domains is regulated by DNase I-sensitive quantitative trait loci and that these domains are significantly enriched for genome-wide association studies hits, with autoimmune disease associated SNPs specifically enriched in lymphoblasts. This study provides the first genome-wide maps of allelic-imbalance for five histone marks. Our results provide new insights into the role of chromatin in cis-regulation and highlight the need for high-depth sequencing in ChIP-seq studies along with the need to improve allele-specificity of ChIP-enrichment.

  11. Biological properties of neural progenitor cells isolated from the hippocampus of adult cynomolgus monkeys

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background The existence of neurogenesis in the hippocampus of adult nonhuman primates has been confirmed in recent years, however, the biological properties of adult neural stem cells or neural progenitor cells (NPCs) from this region remain to be extensively explored. The present work was to investigate on the expansion of NSCs/NPCs from the hippocampus of adult cynomolgus monkeys and the examination of their characteristics in vitro.Methods NPCs isolated from the hippocampus of adult cynomolgus monkeys were expanded in vitro in serum-free media containing growth factors, and were then allowed to differentiate by removing mitotic factors. The expansion capacity of NPCs and their differentiation potential were assayed by immunohistochemical and immunocytochemical analysis.Results During primary culture, NPCs underwent cell division, proliferation and aggregation to form neurospheres that were growing in suspension. Without mitotic stimulation, most neurospheres adhered to the culture dish and started to differentiate. Eventually, nearly 12% of the differentiated cells expressed neuron specific marker-βIII-tubulin (Tuj1) and 84% expressed astrocyte specific marker-fibrillary acidic protein (GFAP). In addition, the expression of a neural stem cell marker, nestin, was found both in NPCs and in the subgranular zone of adult monkey hippocampus, where NPCs were originally derived. Conclusions NPCs from the hippocampus of adult cynomolgus monkeys can be expanded to some extent in vitro and are capable of differentiating into neurons and astrocytes. Further experiments to promote the in vitro proliferation capacity of NPCs will be required before adult NPCs can be used as a useful cell model for studying adult neurogenesis and cell replacement therapy using adult stem cells.

  12. Deletion of inositol hexakisphosphate kinase 1 (IP6K1) reduces cell migration and invasion, conferring protection from aerodigestive tract carcinoma in mice

    OpenAIRE

    Jadav, Rathan S.; Kumar, Dharmika; Buwa, Natasha; Ganguli, Shubhra; Thampatty, Sitalakshmi?R.; Balasubramanian, Nagaraj; Bhandari, Rashna

    2016-01-01

    Inositol hexakisphosphate kinases (IP6Ks), a family of enzymes found in all eukaryotes, are responsible for the synthesis of 5-diphosphoinositol pentakisphosphate (5-IP7) from inositol hexakisphosphate (IP6). Three isoforms of IP6Ks are found in mammals, and gene deletions of each isoform lead to diverse, non-overlapping phenotypes in mice. Previous studies show a facilitatory role for IP6K2 in cell migration and invasion, properties that are essential for the early stages of tumorigenesis. H...

  13. Porcine induced pluripotent stem cells may bridge the gap between mouse and human iPS.

    Science.gov (United States)

    Esteban, Miguel A; Peng, Meixiu; Deli, Zhang; Cai, Jie; Yang, Jiayin; Xu, Jianyong; Lai, Liangxue; Pei, Duanqing

    2010-04-01

    Recently, three independent laboratories reported the generation of induced pluripotent stem cells (iPSCs) from pig (Sus scrofa). This finding sums to the growing list of species (mouse, human, monkey, and rat, in this order) for which successful reprogramming using exogenous factors has been achieved, and multiple others are possibly forthcoming. But apart from demonstrating the universality of the network identified by Shinya Yamanaka, what makes the porcine model so special? On one side, pigs are an agricultural commodity and have an easy and affordable maintenance compared with nonhuman primates that normally need to be imported. On the other side, resemblance (for example, size of organs) of porcine and human physiology is striking and because pigs are a regular source of food the ethical concerns that still remain in monkeys are not applicable. Besides, the prolonged lifespan of pigs compared with other domestic species can allow exhaustive follow up of side effects after transplantation. Porcine iPSCs may thus fill the gap between the mouse model, which due to its ease is preferred for mechanistic studies, and the first clinical trials using iPSCs in humans. However, although these studies are relevant and have created significant interest they face analogous problems that we discuss herein together with potential new directions.

  14. Generation of iPS cell lines from schizophrenia patients using a non-integrative method

    Directory of Open Access Journals (Sweden)

    Jaroslaw Sochacki

    2016-07-01

    Full Text Available Skin biopsies were collected from three male patients (age 45, 44 and 44 with clinically diagnosed schizophrenia. The patients were diagnosed according to DSM-5 criteria by a trained psychiatrist. Dermal fibroblast cell lines were established and expanded for subsequent reprogramming procedures. Induced pluripotent stem (iPS cells were derived using the integration-free CytoTune®-iPS 2.0 Sendai Reprogramming Kit, containing Sendai virus particles of the four Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc.

  15. Human amniotic epithelial cell feeder layers maintain human iPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Te, E-mail: liute79@yahoo.com [School of Environmental Science and Engineering, Donghua University, Shanghai 201620 (China); Shanghai Geriatric Institute of Chinese Medicine, Shanghai 200031 (China); Cheng, Weiwei [International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University, Shanghai 200030 (China); Huang, Yongyi [Laboratoire PROTEE, Batiment R, Universite du Sud Toulon-Var, 83957 LA GARDE Cedex (France); Huang, Qin; Jiang, Lizhen [Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Guo, Lihe, E-mail: liute79@yahoo.com [Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)

    2012-02-15

    Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal. Highlights: Black-Right-Pointing-Pointer microRNA-145 inhibits Sox2 expression in human iPS cells. Black-Right-Pointing-Pointer microRNA-145 suppresses the self-renewal and pluripotency of human iPS cells. Black-Right-Pointing-Pointer HuAECs regulate expression of microRNA-145 and Sox2 in human iPS cells. Black-Right-Pointing-Pointer HuAECs feeder

  16. X-chromosome inactivation in monkey embryos and pluripotent stem cells.

    Science.gov (United States)

    Tachibana, Masahito; Ma, Hong; Sparman, Michelle L; Lee, Hyo-Sang; Ramsey, Cathy M; Woodward, Joy S; Sritanaudomchai, Hathaitip; Masterson, Keith R; Wolff, Erin E; Jia, Yibing; Mitalipov, Shoukhrat M

    2012-11-15

    Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.

  17. Tissue Distribution of Memory T and B Cells in Rhesus Monkeys following Influenza A Infection

    Science.gov (United States)

    Yongvanitchit, Kosol; Limsalakpetch, Amporn; Kum-Arb, Utaiwan; Im-Erbsin, Rawiwan; Boonnak, Kobporn; Thitithayanont, Arunee; Jongkaewwattana, Anan; Wiboon-ut, Suwimon; Mongkolsirichaikul, Duangrat; Mahanonda, Rangsini; Spring, Michele; Chuang, Ilin; Mason, Carl J.; Saunders, David L.

    2015-01-01

    Studies of influenza-specific immune responses in humans have largely assessed systemic responses involving serum Ab and peripheral blood T cell responses. However, recent evidence indicates that tissue-resident memory T (TRM) cells play an important role in local murine intrapulmonary immunity. Rhesus monkeys were pulmonary exposed to 2009 pandemic H1N1 virus at days 0 and 28 and immune responses in different tissue compartments were measured. All animals were asymptomatic postinfection. Although only minimal memory immune responses were detected in peripheral blood, a high frequency of influenza nucleoprotein–specific memory T cells was detected in the lung at the “contraction phase,” 49–58 d after second virus inoculation. A substantial proportion of lung nucleoprotein-specific memory CD8+ T cells expressed CD103 and CD69, phenotypic markers of TRM cells. Lung CD103+ and CD103- memory CD8+ T cells expressed similar levels of IFN-γ and IL-2. Unlike memory T cells, spontaneous Ab secreting cells and memory B cells specific to influenza hemagglutinin were primarily observed in the mediastinal lymph nodes. Little difference in systemic and local immune responses against influenza was observed between young adult (6–8 y) and old animals (18–28 y). Using a nonhuman primate model, we revealed substantial induction of local T and B cell responses following 2009 pandemic H1N1 infection. Our study identified a subset of influenza-specific lung memory T cells characterized as TRM cells in rhesus monkeys. The rhesus monkey model may be useful to explore the role of TRM cells in local tissue protective immunity after rechallenge and vaccination. PMID:26408671

  18. Effect of IP3R3 and NPY on age-related declines in olfactory stem cell proliferation.

    Science.gov (United States)

    Jia, Cuihong; Hegg, Colleen C

    2015-02-01

    Losing the sense of smell because of aging compromises health and quality of life. In the mouse olfactory epithelium, aging reduces the capacity for tissue homeostasis and regeneration. The microvillous cell subtype that expresses both inositol trisphosphate receptor type 3 (IP3R3) and the neuroproliferative factor neuropeptide Y (NPY) is critical for regulation of homeostasis, yet its role in aging is undefined. We hypothesized that an age-related decline in IP3R3 expression and NPY signaling underlie age-related homeostatic changes and olfactory dysfunction. We found a decrease in IP3R3(+) and NPY(+) microvillous cell numbers and NPY protein and a reduced sensitivity to NPY-mediated proliferation over 24 months. However, in IP3R3-deficient mice, there was no further age-related reduction in cell numbers, proliferation, or olfactory function compared with wild type. The proliferative response was impaired in aged IP3R3-deficient mice when injury was caused by satratoxin G, which induces IP3R3-mediated NPY release, but not by bulbectomy, which does not evoke NPY release. These data identify IP3R3 and NPY signaling as targets for improving recovery following olfactotoxicant exposure.

  19. G0/G1 arrest and S phase inhibition of human cancer cell lines by inositol hexaphosphate (IP6).

    Science.gov (United States)

    El-Sherbiny, Y M; Cox, M C; Ismail, Z A; Shamsuddin, A M; Vucenik, I

    2001-01-01

    Inositol hexaphosphate (InsP6 or IP6) has shown a striking anti-cancer activity in both in vivo and in vitro models. In an attempt to elucidate the mechanism(s) underlying the anti-neoplastic potential of IP6, we investigated its effect on cell cycle progression of MCF-7 estrogen receptor (ER)-positive and MDA-MB 231 ER-negative human breast cancer cell lines and HT-29 human colon cancer cells. The anti-proliferative effect of IP6 was evaluated using dual-parameter flow cytometric measurements of DNA content, versus the incorporation of 5-bromo-2-deoxyuridine (BrdU) to determine cells actively synthesizing DNA. Combined analysis of the expression of cell cycle-related proteins, proliferation marker Ki-67 and proliferating cell nuclear antigen (PCNA) versus DNA content were used to determine the amount of proliferating cells in each phase, engaged in cell cycle transit. After 3 days of treatment with 5 mM IP6, S-phase, as estimated by BrdU uptake, was significantly decreased in all three cell lines (p = 0.002). MCF-7 and HT-29 cells accumulated in the G0/G1 range of DNA contents (p = 0.002 and p = 0.001, respectively). MDA MB-231 cells transiently accumulated in G0/G1 only after 2 days (p = 0.01). There was a significant decrease in the percentage of Ki-67 expression in IP6-treated cells, from 82.8+/-3.0% to 66.8+/-4.2% in MCF-7 (p = 0.007), from 93.4+/-4.6% to 71.7+/-3.3% in MDA-MB 231 (p = 0.004), and from 95.2+/-1.2% to 73.5+/-2.5% in HT-29 cells (p = 0.002) respectively. PCNA expression levels were also significantly decreased by IP6 in all three cell lines (MCF-7 p = 0.0007; MDA-MB 231 p = 0.0006; HT-29 p = 0.0001). These results show that IP6 controls the progression of cells through the cycle by decreasing S- phase and arresting cells in the G0/G1-phase of the cell cycle. A significant decrease in the expression of proliferation markers indicated that IP6 disengaged cells from actively cycling. Further investigations of cell cycle regulators may lead us to a

  20. Generation and validation of PAX7 reporter lines from human iPS cells using CRISPR/Cas9 technology.

    Science.gov (United States)

    Wu, Jianbo; Hunt, Samuel D; Xue, Haipeng; Liu, Ying; Darabi, Radbod

    2016-03-01

    Directed differentiation of iPS cells toward various tissue progenitors has been the focus of recent research. Therefore, generation of tissue-specific reporter iPS cell lines provides better understanding of developmental stages in iPS cells. This technical report describes an efficient strategy for generation and validation of knock-in reporter lines in human iPS cells using the Cas9-nickase system. Here, we have generated a knock-in human iPS cell line for the early myogenic lineage specification gene of PAX7. By introduction of site-specific double-stranded breaks (DSB) in the genomic locus of PAX7 using CRISPR/Cas9 nickase pairs, a 2A-GFP reporter with selection markers has been incorporated before the stop codon of the PAX7 gene at the last exon. After positive and negative selection, single cell-derived human iPS clones have been isolated and sequenced for in-frame positioning of the reporter construct. Finally, by using a nuclease-dead Cas9 activator (dCas9-VP160) system, the promoter region of PAX7 has been targeted for transient gene induction to validate the GFP reporter activity. This was confirmed by flow cytometry analysis and immunostaining for PAX7 and GFP. This technical report provides a practical guideline for generation and validation of knock-in reporters using CRISPR/Cas9 system.

  1. Human iPS cell-derived insulin producing cells form vascularized organoids under the kidney capsules of diabetic mice.

    Directory of Open Access Journals (Sweden)

    Sudhanshu P Raikwar

    Full Text Available Type 1 diabetes (T1D is caused by autoimmune disease that leads to the destruction of pancreatic β-cells. Transplantation of cadaveric pancreatic organs or pancreatic islets can restore normal physiology. However, there is a chronic shortage of cadaveric organs, limiting the treatment of the majority of patients on the pancreas transplantation waiting list. Here, we hypothesized that human iPS cells can be directly differentiated into insulin producing cells (IPCs capable of secreting insulin. Using a series of pancreatic growth factors, we successfully generated iPS cells derived IPCs. Furthermore, to investigate the capability of these cells to secrete insulin in vivo, the differentiated cells were transplanted under the kidney capsules of diabetic immunodeficient mice. Serum glucose levels gradually declined to either normal or near normal levels over 150 days, suggesting that the IPCs were secreting insulin. In addition, using MRI, a 3D organoid appeared as a white patch on the transplanted kidneys but not on the control kidneys. These organoids showed neo-vascularization and stained positive for insulin and glucagon. All together, these data show that a pancreatic organ can be created in vivo providing evidence that iPS cells might be a novel option for the treatment of T1D.

  2. Pro-apoptotic effect of rice bran inositol hexaphosphate (IP6) on HT-29 colorectal cancer cells.

    Science.gov (United States)

    Shafie, Nurul Husna; Esa, Norhaizan Mohd; Ithnin, Hairuszah; Saad, Norazalina; Pandurangan, Ashok Kumar

    2013-12-02

    Inositol hexaphosphate (IP6), or phytic acid is a natural dietary ingredient and has been described as a "natural cancer fighter", being an essential component of nutritional diets. The marked anti-cancer effect of IP6 has resulted in our quest for an understanding of its mechanism of action. In particular, our data provided strong evidence for the induction of apoptotic cell death, which may be attributable to the up-regulation of Bax and down-regulation of Bcl-xl in favor of apoptosis. In addition, the up-regulation of caspase-3 and -8 expression and activation of both caspases may also contribute to the apoptotic cell death of human colorectal adenocarcinoma HT-29 cells when exposed to IP6. Collectively, this present study has shown that rice bran IP6 induces apoptosis, by regulating the pro- and anti-apoptotic markers; Bax and Bcl-xl and via the activation of caspase molecules (caspase-3 and -8).

  3. Low-molecular-weight inhibitors of cell differentiation enable efficient growth of mouse iPS cells under feeder-free conditions.

    Science.gov (United States)

    Donai, Kenichiro; Inagaki, Akane; So, Kyoung-Ha; Kuroda, Kengo; Sone, Hideko; Kobayashi, Masayuki; Nishimori, Katsuhiko; Fukuda, Tomokazu

    2015-03-01

    Embryonic stem cells and induced pluripotent stem (iPS) cells are usually maintained on feeder cells derived from mouse embryonic fibroblasts (MEFs). In recent years, the cell culture of iPS cells under serum- and feeder-free conditions is gaining attention in overcoming the biosafety issues for clinical applications. In this study, we report on the use of multiple small-molecular inhibitors (i.e., CHIR99021, PD0325901, and Thiazovivin) to efficiently cultivate mouse iPS cells without feeder cells in a chemically-defined and serum-free condition. In this condition, we showed that mouse iPS cells are expressing the Nanog, Oct3/4, and SSEA-1 pluripotent markers, indicating that the culture condition is optimized to maintain the pluripotent status of iPS cells. Without these small-molecular inhibitors, mouse iPS cells required the adaptation period to start the stable cell proliferation. The application of these inhibitors enabled us the shortcut culture method for the cellular adaptation. This study will be useful to efficiently establish mouse iPS cell lines without MEF-derived feeder cells.

  4. Deletion of inositol hexakisphosphate kinase 1 (IP6K1) reduces cell migration and invasion, conferring protection from aerodigestive tract carcinoma in mice.

    Science.gov (United States)

    Jadav, Rathan S; Kumar, Dharmika; Buwa, Natasha; Ganguli, Shubhra; Thampatty, Sitalakshmi R; Balasubramanian, Nagaraj; Bhandari, Rashna

    2016-08-01

    Inositol hexakisphosphate kinases (IP6Ks), a family of enzymes found in all eukaryotes, are responsible for the synthesis of 5-diphosphoinositol pentakisphosphate (5-IP7) from inositol hexakisphosphate (IP6). Three isoforms of IP6Ks are found in mammals, and gene deletions of each isoform lead to diverse, non-overlapping phenotypes in mice. Previous studies show a facilitatory role for IP6K2 in cell migration and invasion, properties that are essential for the early stages of tumorigenesis. However, IP6K2 also has an essential role in cancer cell apoptosis, and mice lacking this protein are more susceptible to the development of aerodigestive tract carcinoma upon treatment with the oral carcinogen 4-nitroquinoline-1-oxide (4NQO). Not much is known about the functions of the equally abundant and ubiquitously expressed IP6K1 isoform in cell migration, invasion and cancer progression. We conducted a gene expression analysis on mouse embryonic fibroblasts (MEFs) lacking IP6K1, revealing a role for this protein in cell receptor-extracellular matrix interactions that regulate actin cytoskeleton dynamics. Consequently, cells lacking IP6K1 manifest defects in adhesion-dependent signaling, evident by lower FAK and Paxillin activation, leading to reduced cell spreading and migration. Expression of active, but not inactive IP6K1 reverses migration defects in IP6K1 knockout MEFs, suggesting that 5-IP7 synthesis by IP6K1 promotes cell locomotion. Actin cytoskeleton remodeling and cell migration support the ability of cancer cells to achieve their complete oncogenic potential. Cancer cells with lower IP6K1 levels display reduced migration, invasion, and anchorage-independent growth. When fed an oral carcinogen, mice lacking IP6K1 show reduced progression from epithelial dysplasia to invasive carcinoma. Thus, our data reveal that like IP6K2, IP6K1 is also involved in early cytoskeleton remodeling events during cancer progression. However, unlike IP6K2, IP6K1 is essential for 4NQO

  5. Human iPS cell-derived astrocyte transplants preserve respiratory function after spinal cord injury.

    Science.gov (United States)

    Li, Ke; Javed, Elham; Scura, Daniel; Hala, Tamara J; Seetharam, Suneil; Falnikar, Aditi; Richard, Jean-Philippe; Chorath, Ashley; Maragakis, Nicholas J; Wright, Megan C; Lepore, Angelo C

    2015-09-01

    Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSAs) following cervical contusion SCI as a novel strategy for reconstituting GLT1 expression and for protecting diaphragmatic respiratory neural circuitry. Transplant-derived cells showed robust long-term survival post-injection and efficiently differentiated into astrocytes in injured spinal cord of both immunesuppressed mice and rats. However, the majority of transplant-derived astrocytes did not express high levels of GLT1, particularly at early times post-injection. To enhance their ability to modulate extracellular glutamate levels, we engineered hIPSAs with lentivirus to constitutively express GLT1. Overexpression significantly increased GLT1 protein and functional GLT1-mediated glutamate uptake levels in hIPSAs both in vitro and in vivo post-transplantation. Compared to human fibroblast control and unmodified hIPSA transplantation, GLT1-overexpressing hIPSAs reduced (1) lesion size within the injured cervical spinal cord, (2) morphological denervation by respiratory phrenic motor

  6. Reprogramming to iPS cells and their subsequent hematopoietic differentiation is more efficient from MEFs than from preB cells.

    Science.gov (United States)

    Reimer, Andreas; Seiler, Katharina; Tornack, Julia; Tsuneto, Motokazu; Melchers, Fritz

    2012-03-30

    Efficiencies of the generation of induced pluripotent stem (iPS) cells from either mouse embryonic fibroblasts (MEF) or from mouse fetal liver (FL) derived preB cells and their hematogenic potencies were compared. In 10 days approximately 2% of the MEFs transduced with Sox-2, Oct-4 and Klf-4 developed to iPS cells, while only 0.01% of transduced FL-preB cells yielded iPS cells, and only after around 3 weeks. Subsequently, the generated iPS cells were induced to differentiate into hematopoietic cells in vitro. On day 5 of differentiation MEF-iPS yielded numbers and percentages of Flk-1(+) mesodermal-like cells comparable to those developed from embryonic stem (ES) cells. Compared to ES cells further differentiation to hematopoietic and lymphopoietic cells was reduced, possibly because of persistent expression of the reprogramming factors. By contrast, FL-iPS cells developed lower numbers and percentages of Flk-1(+) cells, and no significant further development to hematopoietic or lymphopoietic cells could be induced. These results indicate that the efficiencies of iPS generation and subsequent hematopoietic development depends on the type of differentiated cell from which iPS cells are generated.

  7. [Analysis of Musculoskeletal Systems and Their Diseases. Use of iPS cells in regenerative therapy and drug discovery for cartilage diseases].

    Science.gov (United States)

    Tsumaki, Noriyuki

    2015-08-01

    Induced pluripotent stem (iPS) cells can be generated by transiently expressing defined factors in somatic cells such as dermal fibroblasts and blood cells and culturing them in specific medium. iPS cells are expected to provide new tools for research and development of therapies for various diseases because of two important properties : 1) they can be differentiated into any type of somatic cell (pluripotency) and 2) they can be expanded infinitely (self renew). Research for the transplantation of iPS cell-derived cartilage/chondrocytes and drug discovery by iPS cell-based disease modeling are ongoing.

  8. iPS cells-alternative pluripotent cells to embryo stem cells

    Institute of Scientific and Technical Information of China (English)

    PEI XueTao

    2010-01-01

    @@ Since the first murine and human embryonic stem cell lines were established by Drs.Evans and Kaufman [1] and Thomson et al.[2], respectively, great progress has been make in the field of stem cell research and regenerative medicine that gave promising futures for therapeutic interventions.However, ethical problems and complications from immune rejection have hindered the full development of ES cells into clinical practice for disease treatment.

  9. MOBE-ChIP: a large-scale chromatin immunoprecipitation assay for cell type-specific studies.

    Science.gov (United States)

    Lau, On Sun; Bergmann, Dominique C

    2015-10-01

    Cell type-specific transcriptional regulators play critical roles in the generation and maintenance of multicellularity. As they are often expressed at low levels, in vivo DNA-binding studies of these regulators by standard chromatin immunoprecipitation (ChIP) assays are technically challenging. We describe here an optimized ChIP protocol termed Maximized Objects for Better Enrichment (MOBE)-ChIP, which enhances the sensitivity of ChIP assays for detecting cell type-specific signals. The protocol, which is based on the disproportional increase of target signals over background at higher scales, uses substantially greater volume of starting materials than conventional ChIPs to achieve high signal enrichment. This technique can capture weak binding events that are ambiguous in standard ChIP assays, and is useful both in gene-specific and whole-genome analysis. This protocol has been optimized for Arabidopsis, but should be applicable to other model systems with minor modifications. The full procedure can be completed within 3 days.

  10. Characterization of Mason--Pfizer monkey virus-induced cell fusion. [uv radiation

    Energy Technology Data Exchange (ETDEWEB)

    Chatterjee, S.; Hunter, E.

    1979-06-01

    The characteristics and requirements of multinucleate cell (syncytium) induction by Mason--Pfizer monkey virus (M-PMV) on human and non-human primate cells have been investigated. Multinucleate cell induction by this D-type retrovirus shows single-hit kinetics on human foreskin and rhesus monkey fetal lung cells. The peak of syncytium-forming activity in an isopycnic sucrose gradient coincides with the peak of M-PMV virions as assessed by electron microscopy and analysis of viral polypeptides. Unlike the paramyxoviruses, M-PMV does not induce early cell fusion when added in high concentrations to the target cells. Furthermore, multinucleate cell formation is maximal 48 hr postinfection and the size of the syncytia remains constant after this time. Ultraviolet irradiation of M-PMV reduces its ability to form syncytia and to replicate with single-hit kinetics, suggesting that a functional viral genome is required for syncytium formation. Proviral DNA synthesis and assembly of virions are not necessary for cell fusion since the addition of cytosine arabinoside at concentrations which block virus replication has little effect on multinucleate cell formation. Moreover both multinucleate cells lacking detectable intracellular virus polypeptides, and groups of individual, nonfused but brightly staining cells can be observed in immunofluorescence assays at times when multinucleate cell formation is maximal. Cell fusion is inhibited by the addition of cycloheximide during the first 12 hr of infection, suggesting that de novo protein synthesis is required for multinucleate cell formation. The possibility that the translation of genomic RNA yields a fusion-inducing product is discussed.

  11. Immunopotentiator from Pantoea agglomerans 1 (IP-PA1) Promotes Murine Hair Growth and Human Dermal Papilla Cell Gene Expression.

    Science.gov (United States)

    Wakame, Koji; Okawa, Hiroshi; Komatsu, Ken-Ich; Nakata, Akifumi; Sato, Keisuke; Ingawa, Hiroyuki; Kohchi, Chie; Nishizawa, Takashi; Soma, Gen-Ichiro

    2016-07-01

    The lipopolysaccharide (LPS)-like compound derived from Pantoea agglomerans (immunopotentiator from Pantoea agglomerans 1 (IP-PA1)) has been used not only as dietary supplement or cosmetic for humans, but also by Japanese veterinarians as an anti-tumor, anti-allergy, "keep a fine coat of fur" and hair growth-promoting functional food for dogs and cats. In the present study, we focused on the hair growth-promoting effects of IP-PA1 on a hair-shaved animal model and its mechanism of action. We also investigated its potential on gene expression after stimulating human dermal papilla cells with IP-PA1. The hair on the back of a C3H/HeN mouse was shaved and IP-PA1 was orally administered or applied to the skin. The status of hair growth was observed and recorded for 14 days. Skin was collected and histological tissue examination was performed with respect to hair growth status using hematoxylin and eosin staining. After IP-PA1 administration (2 and 10 μg/ml) to human dermal papilla cell culture system for 24 h, fibroblast growth factor-7 (FGF-7) and vascular endothelial growth factor (VEGF) mRNA expression were measured using real-time polymerase chain reaction (PCR) analysis. IP-PA1, when given orally, showed a tendency to promote hair growth in mice. In addition, skin application also significantly promoted hair growth, while histopathological examinations further demonstrated hair elongation from dermal papilla cells. In the human dermal papilla cell culture system, significant FGF-7 and VEGF mRNA expressions were observed (p<0.05). An underlying mechanism of gene expression by which IP-PA1 promotes hair growth was suggested to be different from that of medicine and traditional hair tonics, such as minoxidil and adenosine. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  12. Neurons Differentiated from Transplanted Stem Cells Respond Functionally to Acoustic Stimuli in the Awake Monkey Brain.

    Science.gov (United States)

    Wei, Jing-Kuan; Wang, Wen-Chao; Zhai, Rong-Wei; Zhang, Yu-Hua; Yang, Shang-Chuan; Rizak, Joshua; Li, Ling; Xu, Li-Qi; Liu, Li; Pan, Ming-Ke; Hu, Ying-Zhou; Ghanemi, Abdelaziz; Wu, Jing; Yang, Li-Chuan; Li, Hao; Lv, Long-Bao; Li, Jia-Li; Yao, Yong-Gang; Xu, Lin; Feng, Xiao-Li; Yin, Yong; Qin, Dong-Dong; Hu, Xin-Tian; Wang, Zheng-Bo

    2016-07-26

    Here, we examine whether neurons differentiated from transplanted stem cells can integrate into the host neural network and function in awake animals, a goal of transplanted stem cell therapy in the brain. We have developed a technique in which a small "hole" is created in the inferior colliculus (IC) of rhesus monkeys, then stem cells are transplanted in situ to allow for investigation of their integration into the auditory neural network. We found that some transplanted cells differentiated into mature neurons and formed synaptic input/output connections with the host neurons. In addition, c-Fos expression increased significantly in the cells after acoustic stimulation, and multichannel recordings indicated IC specific tuning activities in response to auditory stimulation. These results suggest that the transplanted cells have the potential to functionally integrate into the host neural network.

  13. An IP3R3- and NPY-expressing microvillous cell mediates tissue homeostasis and regeneration in the mouse olfactory epithelium.

    Science.gov (United States)

    Jia, Cuihong; Hayoz, Sebastien; Hutch, Chelsea R; Iqbal, Tania R; Pooley, Apryl E; Hegg, Colleen C

    2013-01-01

    Calcium-dependent release of neurotrophic factors plays an important role in the maintenance of neurons, yet the release mechanisms are understudied. The inositol triphosphate (IP3) receptor is a calcium release channel that has a physiological role in cell growth, development, sensory perception, neuronal signaling and secretion. In the olfactory system, the IP3 receptor subtype 3 (IP3R3) is expressed exclusively in a microvillous cell subtype that is the predominant cell expressing neurotrophic factor neuropeptide Y (NPY). We hypothesized that IP3R3-expressing microvillous cells secrete sufficient NPY needed for both the continual maintenance of the neuronal population and for neuroregeneration following injury. We addressed this question by assessing the release of NPY and the regenerative capabilities of wild type, IP3R3(+/-), and IP3R3(-/-) mice. Injury, simulated using extracellular ATP, induced IP3 receptor-mediated NPY release in wild-type mice. ATP-evoked NPY release was impaired in IP3R3(-/-) mice, suggesting that IP3R3 contributes to NPY release following injury. Under normal physiological conditions, both IP3R3(-/-) mice and explants from these mice had fewer progenitor cells that proliferate and differentiate into immature neurons. Although the number of mature neurons and the in vivo rate of proliferation were not altered, the proliferative response to the olfactotoxicant satratoxin G and olfactory bulb ablation injury was compromised in the olfactory epithelium of IP3R3(-/-) mice. The reductions in both NPY release and number of progenitor cells in IP3R3(-/-) mice point to a role of the IP3R3 in tissue homeostasis and neuroregeneration. Collectively, these data suggest that IP3R3 expressing microvillous cells are actively responsive to injury and promote recovery.

  14. An IP3R3- and NPY-expressing microvillous cell mediates tissue homeostasis and regeneration in the mouse olfactory epithelium.

    Directory of Open Access Journals (Sweden)

    Cuihong Jia

    Full Text Available Calcium-dependent release of neurotrophic factors plays an important role in the maintenance of neurons, yet the release mechanisms are understudied. The inositol triphosphate (IP3 receptor is a calcium release channel that has a physiological role in cell growth, development, sensory perception, neuronal signaling and secretion. In the olfactory system, the IP3 receptor subtype 3 (IP3R3 is expressed exclusively in a microvillous cell subtype that is the predominant cell expressing neurotrophic factor neuropeptide Y (NPY. We hypothesized that IP3R3-expressing microvillous cells secrete sufficient NPY needed for both the continual maintenance of the neuronal population and for neuroregeneration following injury. We addressed this question by assessing the release of NPY and the regenerative capabilities of wild type, IP3R3(+/-, and IP3R3(-/- mice. Injury, simulated using extracellular ATP, induced IP3 receptor-mediated NPY release in wild-type mice. ATP-evoked NPY release was impaired in IP3R3(-/- mice, suggesting that IP3R3 contributes to NPY release following injury. Under normal physiological conditions, both IP3R3(-/- mice and explants from these mice had fewer progenitor cells that proliferate and differentiate into immature neurons. Although the number of mature neurons and the in vivo rate of proliferation were not altered, the proliferative response to the olfactotoxicant satratoxin G and olfactory bulb ablation injury was compromised in the olfactory epithelium of IP3R3(-/- mice. The reductions in both NPY release and number of progenitor cells in IP3R3(-/- mice point to a role of the IP3R3 in tissue homeostasis and neuroregeneration. Collectively, these data suggest that IP3R3 expressing microvillous cells are actively responsive to injury and promote recovery.

  15. Amplification of pico-scale DNA mediated by bacterial carrier DNA for small-cell-number transcription factor ChIP-seq

    DEFF Research Database (Denmark)

    Jakobsen, Janus S; Bagger, Frederik O; Hasemann, Marie S

    2015-01-01

    BACKGROUND: Chromatin-Immunoprecipitation coupled with deep sequencing (ChIP-seq) is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. The requirement of nano-scale ChIP DNA for generation of sequencing libraries has impeded ChIP-seq on in vivo tissues of low...... cell numbers. RESULTS: We describe a robust, simple and scalable methodology for ChIP-seq of low-abundant cell populations, verified down to 10,000 cells. By employing non-mammalian genome mapping bacterial carrier DNA during amplification, we reliably amplify down to 50 pg of ChIP DNA from...... transcription factor (CEBPA) and histone mark (H3K4me3) ChIP. We further demonstrate that genomic profiles are highly resilient to changes in carrier DNA to ChIP DNA ratios. CONCLUSIONS: This represents a significant advance compared to existing technologies, which involve either complex steps of pre...

  16. Amplification of pico-scale DNA mediated by bacterial carrier DNA for small-cell-number transcription factor ChIP-seq

    DEFF Research Database (Denmark)

    Jakobsen, Janus S; Bagger, Frederik O; Hasemann, Marie S;

    2015-01-01

    BACKGROUND: Chromatin-Immunoprecipitation coupled with deep sequencing (ChIP-seq) is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. The requirement of nano-scale ChIP DNA for generation of sequencing libraries has impeded ChIP-seq on in vivo tissues of low...... transcription factor (CEBPA) and histone mark (H3K4me3) ChIP. We further demonstrate that genomic profiles are highly resilient to changes in carrier DNA to ChIP DNA ratios. CONCLUSIONS: This represents a significant advance compared to existing technologies, which involve either complex steps of pre...... cell numbers. RESULTS: We describe a robust, simple and scalable methodology for ChIP-seq of low-abundant cell populations, verified down to 10,000 cells. By employing non-mammalian genome mapping bacterial carrier DNA during amplification, we reliably amplify down to 50 pg of ChIP DNA from...

  17. n-Butylidenephthalide (BP) maintains stem cell pluripotency by activating Jak2/Stat3 pathway and increases the efficiency of iPS cells generation.

    Science.gov (United States)

    Liu, Shih-Ping; Harn, Horng-Jyh; Chien, Ying-Jiun; Chang, Cheng-Hsuan; Hsu, Chien-Yu; Fu, Ru-Huei; Huang, Yu-Chuen; Chen, Shih-Yin; Shyu, Woei-Cherng; Lin, Shinn-Zong

    2012-01-01

    In 2006, induced pluripotent stem (iPS) cells were generated from somatic cells by introducing Oct4, Sox2, c-Myc and Klf4. The original process was inefficient; maintaining the pluripotency of embryonic stem (ES) and iPS cell cultures required an expensive reagent-leukemia induced factor (LIF). Our goal is to find a pure compound that not only maintains ES and iPS cell pluripotency, but also increases iPS cell generation efficiency. From 15 candidate compounds we determined that 10 µg/ml n-Butylidenephthalide (BP), an Angelica sinensis extract, triggers the up-regulation of Oct4 and Sox2 gene expression levels in MEF cells. We used ES and iPS cells treated with different concentrations of BP to test its usefulness for maintaining stem cell pluripotency. Results indicate higher expression levels of several stem cell markers in BP-treated ES and iPS cells compared to controls that did not contain LIF, including alkaline phosphatase, SSEA1, and Nanog. Embryoid body formation and differentiation results confirm that BP containing medium culture was capable of maintaining ES cell pluripotency after six time passage. Microarray analysis data identified PPAR, ECM, and Jak-Stat signaling as the top three deregulated pathways. We subsequently determined that phosphorylated Jak2 and phosphorylated Stat3 protein levels increased following BP treatment and suppressed with the Jak2 inhibitor, AG490. The gene expression levels of cytokines associated with the Jak2-Stat3 pathway were also up-regulated. Last, we used pou5f1-GFP MEF cells to test iPS generation efficiency following BP treatment. Our data demonstrate the ability of BP to maintain stem cell pluripotency via the Jak2-Stat3 pathway by inducing cytokine expression levels, at the same time improving iPS generation efficiency.

  18. Selective degeneration of the parvocellular-projecting retinal ganglion cells in a New World monkey, Saimiri sciureus.

    Science.gov (United States)

    Lynch, J J; Eskin, T A; Merigan, W H

    1989-10-16

    Selective degeneration of retinal ganglion cells projecting to parvocellular layers of the dorsal lateral geniculate nucleus (LGN) was observed in squirrel monkeys (Saimiri sciureus) exposed to a range of doses of acrylamide monomer. Similar acrylamide-induced neuronal loss has previously been reported in parvocellular-projecting ganglion cells of macaques, but no such selective degeneration has been found in acrylamide-dosed rats, squirrels, rabbits or cats. The extent of ganglion cell loss observed in the present study suggests that in the squirrel monkey, as in the macaque, a majority of ganglion cells project to parvocellular layers of the LGN. The locus of optic tract degeneration suggests that the squirrel monkey parvocellular pathway passes in dorsolateral optic tract, as does that of the macaque. Patterns of decreases in cytochrome oxidase activity confirm that, in both of these primates, geniculocortical pathways driven by these vulnerable neurons project to cortical layers 4A and 4C beta. These results suggest close parallels in the neuroanatomical projections and toxic vulnerability of the parvocellular-projecting pathway in New and Old World monkeys. They indicate that acrylamide intoxication can be used to selectively damage this pathway in order to study the functional roles of parallel visual pathways in both New and Old World monkeys.

  19. Morphological features of iPS cells generated from Fabry disease skin fibroblasts using Sendai virus vector (SeVdp).

    Science.gov (United States)

    Kawagoe, Shiho; Higuchi, Takashi; Otaka, Manami; Shimada, Yohta; Kobayashi, Hiroshi; Ida, Hiroyuki; Ohashi, Toya; Okano, Hirotaka J; Nakanishi, Mahito; Eto, Yoshikatsu

    2013-08-01

    We generated iPS cells from human dermal fibroblasts (HDFs) of Fabry disease using a Sendai virus (SeVdp) vector; this method has been established by Nakanishi et al. for pathogenic evaluation. We received SeVdp vector from Nakanishi and loaded it simultaneously with four reprogramming factors (Klf4, Oct4, Sox2, and c-Myc) to HDFs of Fabry disease; subsequently, we observed the presence of human iPS-like cells. The Sendai virus nucleocapsid protein was not detected in the fibroblasts by RT-PCR analysis. Additionally, we confirmed an undifferentiated state, alkaline phosphatase staining, and the presence of SSEA-4, TRA-1-60, and TRA-1-81. Moreover, ultrastructural features of these iPS cells included massive membranous cytoplasmic bodies typical of HDFs of Fabry disease. Thus, we successfully generated human iPS cells from HDFs of Fabry disease that retained the genetic conditions of Fabry disease; also, these abnormal iPS cells could not be easily differentiated into mature cell types such as neuronal cells, cardiomyocytes, etc. because of a massive accumulation of membranous cytoplasmic bodies in lysosomes, possibly the persistent damages of intracellular architecture.

  20. A commentary on iPS cells: potential applications in autologous transplantation, study of illnesses and drug screening.

    Science.gov (United States)

    Romano, Gaetano; Morales, Fátima; Marino, Ignazio R; Giordano, Antonio

    2014-02-01

    Undoubtedly, the focus of the field of stem cell research is predominantly aimed at the artificial reprogramming of human somatic cells for the production of induced pluripotent stem (iPS) cells. This relatively new technology may circumvent the ethical issues of using human embryonic stem (hES) cells for the potential applications in cell replacement therapy. Besides such ethical issues, iPS cell technology offers the advantage of obtaining patient-derived tissues and/or cells, which may be utilized for autologous transplantation and tissue regeneration, investigation of a variety of human illnesses and for the screening of new drugs. The field of stem cell research has placed a major emphasis in understanding the genetic and epigenetic codes for pluripotency, in order to control and optimize autologous transplantation techniques and avoid teratoma formation.

  1. Colonization of collagen scaffolds by adipocytes derived from mesenchymal stem cells of the common marmoset monkey

    Energy Technology Data Exchange (ETDEWEB)

    Bernemann, Inga, E-mail: bernemann@imp.uni-hannover.de [Institute for Multiphase Processes, Leibniz Universitaet Hannover, Hannover (Germany); Mueller, Thomas; Blasczyk, Rainer [Institute for Transfusion Medicine, Hannover Medical School, Hannover (Germany); Glasmacher, Birgit; Hofmann, Nicola [Institute for Multiphase Processes, Leibniz Universitaet Hannover, Hannover (Germany)

    2011-07-29

    Highlights: {yields} Marmoset bone marrow-derived MSCs differentiate in suspension into adipogenic, osteogenic and chondrogenic lineages. {yields} Marmoset MSCs integrate in collagen type I scaffolds and differentiate excellently into adipogenic cells. {yields} Common marmoset monkey is a suitable model for soft tissue engineering in human regenerative medicine. -- Abstract: In regenerative medicine, human cell replacement therapy offers great potential, especially by cell types differentiated from immunologically and ethically unproblematic mesenchymal stem cells (MSCs). In terms of an appropriate carrier material, collagen scaffolds with homogeneous pore size of 65 {mu}m were optimal for cell seeding and cultivating. However, before clinical application and transplantation of MSC-derived cells in scaffolds, the safety and efficiency, but also possible interference in differentiation due to the material must be preclinically tested. The common marmoset monkey (Callithrix jacchus) is a preferable non-human primate animal model for this aim due to its genetic and physiological similarities to the human. Marmoset bone marrow-derived MSCs were successfully isolated, cultured and differentiated in suspension into adipogenic, osteogenic and chondrogenic lineages by defined factors. The differentiation capability could be determined by FACS. Specific marker genes for all three cell types could be detected by RT-PCR. Furthermore, MSCs seeded on collagen I scaffolds differentiated in adipogenic lineage showed after 28 days of differentiation high cell viability and homogenous distribution on the material which was validated by calcein AM and EthD staining. As proof of adipogenic cells, the intracellular lipid vesicles in the cells were stained with Oil Red O. The generation of fat vacuoles was visibly extensive distinguishable and furthermore determined on the molecular level by expression of specific marker genes. The results of the study proved both the differential

  2. Aberrant genomic imprinting in rhesus monkey embryonic stem cells.

    Science.gov (United States)

    Fujimoto, Akihisa; Mitalipov, Shoukhrat M; Kuo, Hung-Chih; Wolf, Don P

    2006-03-01

    Genomic imprinting involves modification of a gene or a chromosomal region that results in the differential expression of parental alleles. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. Additionally, abnormal imprinting occurs in mouse embryonic stem cells (ESCs) and in clonally derived animals. Imprinted gene expression patterns in primate ESCs are largely unknown, despite the clinical potential of the latter in the cell-based treatment of human disease. Because of the possible implications of abnormal gene expression to cell or tissue replacement therapies involving ESCs, we examined allele specific expression of four imprinted genes in the rhesus macaque. Genomic and complementary DNA from embryos and ESC lines containing useful single nucleotide polymorphisms were subjected to polymerase chain reaction-based amplification and sequence analysis. In blastocysts, NDN expression was variable indicating abnormal or incomplete imprinting whereas IGF2 and SNRPN were expressed exclusively from the paternal allele and H19 from the maternal allele as expected. In ESCs, both NDN and SNRPN were expressed from the paternal allele while IGF2 and H19 showed loss of imprinting and biallelic expression. In differentiated ESC progeny, these expression patterns were maintained. The implications of aberrant imprinted gene expression to ESC differentiation in vitro and on ESC-derived cell function in vivo after transplantation are unknown.

  3. Immunotherapy against Metastatic Melanoma with Human iPS Cell-Derived Myeloid Cell Lines Producing Type I Interferons.

    Science.gov (United States)

    Miyashita, Azusa; Fukushima, Satoshi; Nakahara, Satoshi; Kubo, Yosuke; Tokuzumi, Aki; Yamashita, Junji; Aoi, Jun; Haruta, Miwa; Senju, Satoru; Nishimura, Yasuharu; Jinnin, Masatoshi; Ihn, Hironobu

    2016-03-01

    In recent years, immunotherapy for advanced melanoma has been gaining increased attention. The efficacy of anti-cytotoxic T-lymphocyte antigen 4 antibodies, anti-programmed cell death 1 antibodies, and the BRAF(V600E) kinase inhibitor has been proven in metastatic melanoma. At the same time, adoptive cell transfer has significant effects against metastatic melanoma; however, it is difficult to apply on a broad scale because of the problems related to cell preparation. To overcome these problems, we developed immune cell therapy using induced pluripotent stem (iPS) cells. The benefit of our method is that a large number of cells can be readily obtained. We focused on macrophages for immune cell therapy because macrophage infiltration is frequently observed in solid cancers. In this study, the efficacy of human iPS cell-derived myeloid cell lines (iPS-ML) genetically modified to express type I IFNs against human melanoma cells was examined. The morphology, phagocytic ability, and surface markers of iPS-ML were similar to those of macrophages. The iPS-ML that express type I IFNs (iPS-ML-IFN) showed significant effects in inhibiting the growth of disseminated human melanoma cells in SCID mice. The infiltration of iPS-ML into the tumor nests was confirmed immunohistologically. The iPS-ML-IFNs increased the expression of CD169, a marker of M1 macrophages that can activate antitumor immunity. The iPS-ML-IFNs could infiltrate into tumor tissue and exert anticancer effects in the local tumor tissue. In conclusion, this method will provide a new therapeutic modality for metastatic melanoma.

  4. Autophagy in retinal ganglion cells in a rhesus monkey chronic hypertensive glaucoma model.

    Directory of Open Access Journals (Sweden)

    Shuifeng Deng

    Full Text Available Primary open angle glaucoma (POAG is a neurodegenerative disease characterized by physiological intraocular hypertension that causes damage to the retinal ganglion cells (RGCs. In the past, RGC damage in POAG was suggested to have been attributed to RGC apoptosis. However, in the present study, we applied a model closer to human POAG through the use of a chronic hypertensive glaucoma model in rhesus monkeys to investigate whether another mode of progressive cell death, autophagy, was activated in the glaucomatous retinas. First, in the glaucomatous retinas, the levels of LC3B-II, LC3B-II/LC3B-I and Beclin 1 increased as demonstrated by Western blot analyses, whereas early or initial autophagic vacuoles (AVi and late or degraded autophagic vacuoles (AVd accumulated in the ganglion cell layer (GCL and in the inner plexiform layer (IPL as determined by transmission electron microscopy (TEM analysis. Second, lysosome activity and autophagosome-lysosomal fusion increased in the RGCs of the glaucomatous retinas, as demonstrated by Western blotting against lysosome associated membrane protein-1 (LAMP1 and double labeling against LC3B and LAMP1. Third, apoptosis was activated in the glaucomatous eyes with increased levels of caspase-3 and cleaved caspase-3 and an increased number of TUNEL-positive RGCs. Our results suggested that autophagy was activated in RGCs in the chronic hypertensive glaucoma model of rhesus monkeys and that autophagy may have potential as a new target for intervention in glaucoma treatment.

  5. Characterization of PrPc-immunoreactive cells in monkey (Macaca fascicularis) gastrointestinal tract.

    Science.gov (United States)

    Marcos, Z; Bodegas, M E; Sesma, M P; Guembe, L

    2005-04-01

    The gastrointestinal tract (GIT) is one of the most likely entry sites for the pathological isoform of prions (PrP(sc)). To understand how PrP(sc) crosses the digestive mucosa, it is crucial to characterize the cells expressing normal prion protein (PrP(c)). By means of double immunofluorescence applied to sections of the monkey GIT, we demonstrated that, in the stomach, PrP(c) immunostaining occurs in subsets of histamine, somatostatin (Som), ghrelin (Ghr), gastrin (G), and serotonin (5HT) cells. In the small and large bowels, PrP(c) cells were found in subpopulations of cells immunolabeled for 5HT, Som, G, and peptide YY (PYY).

  6. Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy

    Directory of Open Access Journals (Sweden)

    Stephanie Friedrichs

    2015-01-01

    Full Text Available Disease-specific induced pluripotent stem (iPS cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.

  7. CAST-ChIP Maps Cell-Type-Specific Chromatin States in the Drosophila Central Nervous System

    Directory of Open Access Journals (Sweden)

    Tamás Schauer

    2013-10-01

    Full Text Available Chromatin organization and gene activity are responsive to developmental and environmental cues. Although many genes are transcribed throughout development and across cell types, much of gene regulation is highly cell-type specific. To readily track chromatin features at the resolution of cell types within complex tissues, we developed and validated chromatin affinity purification from specific cell types by chromatin immunoprecipitation (CAST-ChIP, a broadly applicable biochemical procedure. RNA polymerase II (Pol II CAST-ChIP identifies ∼1,500 neuronal and glia-specific genes in differentiated cells within the adult Drosophila brain. In contrast, the histone H2A.Z is distributed similarly across cell types and throughout development, marking cell-type-invariant Pol II-bound regions. Our study identifies H2A.Z as an active chromatin signature that is refractory to changes across cell fates. Thus, CAST-ChIP powerfully identifies cell-type-specific as well as cell-type-invariant chromatin states, enabling the systematic dissection of chromatin structure and gene regulation within complex tissues such as the brain.

  8. ChIp-seq of bovine cells (MDBK) to study butyrate-induced histone modification with 10 datasets

    Science.gov (United States)

    Next-generation sequencing was combined with chromatin immunoprecipitation (ChIP) technology to analyze histone modification (acetylation) induced by butyrate and to map the epigenomic landscape of normal histone H3, H4 in rumen cells of the cow. Ten variants of histone H3 and H4 modification were m...

  9. Olfactory ensheathing cells of hamsters, rabbits, monkeys, and mice express α-smooth muscle actin.

    Science.gov (United States)

    Rawji, Khalil S; Zhang, Shannon X; Tsai, Ying-Yu; Smithson, Laura J; Kawaja, Michael D

    2013-07-12

    Olfactory ensheathing cells (OECs) are the chief glial population of the mammalian olfactory nervous system, residing in the olfactory mucosa and at the surface of the olfactory bulb. We investigated the neurochemical features of OECs in a variety of mammalian species (including adult hamsters, rabbits, monkeys, and mice, as well as fetal pigs) using three biomarkers: α-smooth muscle actin (αSMA), S100β, and glial fibrillary acidic protein (GFAP). Mucosal and bulbar OECs from all five mammalian species express S100β. Both mucosal and bulbar OECs of monkeys express αSMA, yet only bulbar OECs of hamsters and only mucosal OECs of rabbits express αSMA as well. Mucosal OECs, but not bulbar OECs, also express GFAP in hamsters and monkeys; mice, by comparison, have only a sparse population of OECs expressing GFAP. Though αSMA immunostaining is not detected in OECs of adult mice, GFAP-expressing mucosal OECs isolated from adult mice do coexpress αSMA in vitro. Moreover, mucosal OECs from adult mutant mice lacking αSMA expression display perturbed cellular morphology (i.e., fewer cytoplasmic processes extending among the hundreds of olfactory axons in the olfactory nerve fascicles and nuclei having degenerative features). In sum, these findings highlight the efficacy of αSMA and S100β as biomarkers of OECs from a variety of mammalian species. These observations provide definitive evidence that mammalian OECs express the structural protein αSMA (at various levels of detection), which appears to play a pivotal role in their ensheathment of olfactory axons.

  10. Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells.

    Science.gov (United States)

    Mitalipov, Shoukhrat M; Yeoman, Richard R; Nusser, Kevin D; Wolf, Don P

    2002-05-01

    Production of genetically identical nonhuman primates would reduce the number of animals required for biomedical research and dramatically impact studies pertaining to immune system function, such as development of the human-immunodeficiency-virus vaccine. Our long-term goal is to develop robust somatic cell cloning and/or twinning protocols in the rhesus macaque. The objective of this study was to determine the developmental competence of nuclear transfer (NT) embryos derived from embryonic blastomeres (embryonic cell NT) or fetal fibroblasts (somatic cell NT) as a first step in the production of rhesus monkeys by somatic cell cloning. Development of cleaved embryos up to the 8-cell stage was similar among embryonic and somatic cell NT embryos and comparable to controls created by intracytoplasmic sperm injection (ICSI; mean +/- SEM, 81 +/- 5%, 88 +/- 7%, and 87 +/- 4%, respectively). However, significantly lower rates of development to the blastocyst stage were observed with somatic cell NT embryos (1%) in contrast to embryonic cell NT (34 +/- 15%) or ICSI control embryos (46 +/- 6%). Development of somatic cell NT embryos was not markedly affected by donor cell treatment, timing of activation, or chemical activation protocol. Transfer of embryonic, but not of somatic cell NT embryos, into recipients resulted in term pregnancy. Future efforts will focus on optimizing the production of somatic cell NT embryos that develop in high efficiency to the blastocyst stage in vitro.

  11. Prenatal alcohol exposure affects progenitor cell numbers in olfactory bulbs and dentate gyrus of vervet monkeys

    DEFF Research Database (Denmark)

    Burke, Mark W; Inyatkin, Alexey; Ptito, Maurice

    2016-01-01

    cells in the rostral migratory pathway, while production and migration of postnatal neurons into the dentate gyrus may be more complex. The relatively small size of the olfactory bulb, compared to the hippocampus, potentially makes this structure ideal for a rapid analysis. This study used the St. Kitts...... vervet monkey (Chlorocebus sabeus) to (1) investigate the normal developmental sequence of post-natal proliferation in the olfactory bulb and dentate gyrus and (2) determine the effects of naturalistic prenatal ethanol exposure on proliferation at three different ages (neonate, five months and two years......). Using design-based stereology, we found an age-related decrease of actively proliferating cells in the olfactory bulb and dentate gyrus for both control and FAE groups. Furthermore, at the neonatal time point, the FAE group had fewer actively proliferating cells as compared to the control group...

  12. TATVHL peptide-grafted alginate/poly(γ-glutamic acid) scaffolds with inverted colloidal crystal topology for neuronal differentiation of iPS cells.

    Science.gov (United States)

    Kuo, Yung-Chih; Chung, Chiu-Yen

    2012-12-01

    The neuronal differentiation of induced pluripotent stem (iPS) cells in scaffolding biomaterials is an emerging issue in nervous regeneration and repair. This study presents the production of neuron-lineage cells from iPS cells in inverted colloidal crystal (ICC) scaffolds comprising alginate, poly(γ-glutamic acid) (γ-PGA), and TATVHL peptide. The ability of iPS cells to differentiate toward neurons in the constructs was demonstrated by flow-cytometeric sorting and immunochemical staining. The results revealed that hexagonally arrayed microspheres molded alginate/γ-PGA hydrogel into ICC topology with adequate interconnected pores. An increase in the quantity of surface TATVHL peptide enhanced the atomic ratio of nitrogen and the adhesion efficiency of iPS cells in constructs. However, the effect of TATVHL peptide on the viability of iPS cells was insignificant. The adhesion and viability of iPS cells in ICC constructs was higher than those in freeform ones. TATVHL peptide raised the percentage of β III tubulin-identified cells differentiating from iPS cells, indicating that TATVHL peptide stimulated the neuronal development in alginate/γ-PGA ICC constructs. TATVHL peptide-grafted alginate/γ-PGA ICC scaffolds can be promising for establishing nerve tissue from iPS cells.

  13. Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation.

    Science.gov (United States)

    Jiang, Jing; Lv, Wenjian; Ye, Xiaoying; Wang, Lingbo; Zhang, Man; Yang, Hui; Okuka, Maja; Zhou, Chikai; Zhang, Xuan; Liu, Lin; Li, Jinsong

    2013-01-01

    Induced pluripotent stem (iPS) cells generated using Yamanaka factors have great potential for use in autologous cell therapy. However, genomic abnormalities exist in human iPS cells, and most mouse iPS cells are not fully pluripotent, as evaluated by the tetraploid complementation assay (TCA); this is most likely associated with the DNA damage response (DDR) occurred in early reprogramming induced by Yamanaka factors. In contrast, nuclear transfer can faithfully reprogram somatic cells into embryonic stem (ES) cells that satisfy the TCA. We thus hypothesized that factors involved in oocyte-induced reprogramming may stabilize the somatic genome during reprogramming, and improve the quality of the resultant iPS cells. To test this hypothesis, we screened for factors that could decrease DDR signals during iPS cell induction. We determined that Zscan4, in combination with the Yamanaka factors, not only remarkably reduced the DDR but also markedly promoted the efficiency of iPS cell generation. The inclusion of Zscan4 stabilized the genomic DNA, resulting in p53 downregulation. Furthermore, Zscan4 also enhanced telomere lengthening as early as 3 days post-infection through a telomere recombination-based mechanism. As a result, iPS cells generated with addition of Zscan4 exhibited longer telomeres than classical iPS cells. Strikingly, more than 50% of iPS cell lines (11/19) produced via this "Zscan4 protocol" gave rise to live-borne all-iPS cell mice as determined by TCA, compared to 1/12 for lines produced using the classical Yamanaka factors. Our findings provide the first demonstration that maintaining genomic stability during reprogramming promotes the generation of high quality iPS cells.

  14. Deriving retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells by different sizes of embryoid bodies.

    Science.gov (United States)

    Muñiz, Alberto; Ramesh, Kaini R; Greene, Whitney A; Choi, Jae-Hyek; Wang, Heuy-Ching

    2015-02-04

    Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

  15. Robust Generation of Cardiomyocytes from Human iPS Cells Requires Precise Modulation of BMP and WNT Signaling.

    Science.gov (United States)

    Kadari, Asifiqbal; Mekala, SubbaRao; Wagner, Nicole; Malan, Daniela; Köth, Jessica; Doll, Katharina; Stappert, Laura; Eckert, Daniela; Peitz, Michael; Matthes, Jan; Sasse, Philipp; Herzig, Stefan; Brüstle, Oliver; Ergün, Süleyman; Edenhofer, Frank

    2015-08-01

    Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover, we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling.

  16. Pro-Apoptotic Effect of Rice Bran Inositol Hexaphosphate (IP6 on HT-29 Colorectal Cancer Cells

    Directory of Open Access Journals (Sweden)

    Nurul Husna Shafie

    2013-12-01

    Full Text Available Inositol hexaphosphate (IP6, or phytic acid is a natural dietary ingredient and has been described as a “natural cancer fighter”, being an essential component of nutritional diets. The marked anti-cancer effect of IP6 has resulted in our quest for an understanding of its mechanism of action. In particular, our data provided strong evidence for the induction of apoptotic cell death, which may be attributable to the up-regulation of Bax and down-regulation of Bcl-xl in favor of apoptosis. In addition, the up-regulation of caspase-3 and -8 expression and activation of both caspases may also contribute to the apoptotic cell death of human colorectal adenocarcinoma HT-29 cells when exposed to IP6. Collectively, this present study has shown that rice bran IP6 induces apoptosis, by regulating the pro- and anti-apoptotic markers; Bax and Bcl-xl and via the activation of caspase molecules (caspase-3 and -8.

  17. Ataxia telangiectasia derived iPS cells show preserved x-ray sensitivity and decreased chromosomal instability.

    Science.gov (United States)

    Fukawatase, Yoshihiro; Toyoda, Masashi; Okamura, Kohji; Nakamura, Ken-ichi; Nakabayashi, Kazuhiko; Takada, Shuji; Yamazaki-Inoue, Mayu; Masuda, Akira; Nasu, Michiyo; Hata, Kenichiro; Hanaoka, Kazunori; Higuchi, Akon; Takubo, Kaiyo; Umezawa, Akihiro

    2014-06-27

    Ataxia telangiectasia is a neurodegenerative inherited disease with chromosomal instability and hypersensitivity to ionizing radiation. iPS cells lacking ATM (AT-iPS cells) exhibited hypersensitivity to X-ray irradiation, one of the characteristics of the disease. While parental ataxia telangiectasia cells exhibited significant chromosomal abnormalities, AT-iPS cells did not show any chromosomal instability in vitro for at least 80 passages (560 days). Whole exome analysis also showed a comparable nucleotide substitution rate in AT-iPS cells. Taken together, these data show that ATM is involved in protection from irradiation-induced cell death.

  18. Proto-oncogene PBF/PTTG1IP regulates thyroid cell growth and represses radioiodide treatment.

    Science.gov (United States)

    Read, Martin L; Lewy, Greg D; Fong, Jim C W; Sharma, Neil; Seed, Robert I; Smith, Vicki E; Gentilin, Erica; Warfield, Adrian; Eggo, Margaret C; Knauf, Jeffrey A; Leadbeater, Wendy E; Watkinson, John C; Franklyn, Jayne A; Boelaert, Kristien; McCabe, Christopher J

    2011-10-01

    Pituitary tumor transforming gene (PTTG)-binding factor (PBF or PTTG1IP) is a little characterized proto-oncogene that has been implicated in the etiology of breast and thyroid tumors. In this study, we created a murine transgenic model to target PBF expression to the thyroid gland (PBF-Tg mice) and found that these mice exhibited normal thyroid function, but a striking enlargement of the thyroid gland associated with hyperplastic and macrofollicular lesions. Expression of the sodium iodide symporter (NIS), a gene essential to the radioiodine ablation of thyroid hyperplasia, neoplasia, and metastasis, was also potently inhibited in PBF-Tg mice. Critically, iodide uptake was repressed in primary thyroid cultures from PBF-Tg mice, which could be rescued by PBF depletion. PBF-Tg thyroids exhibited upregulation of Akt and the TSH receptor (TSHR), each known regulators of thyrocyte proliferation, along with upregulation of the downstream proliferative marker cyclin D1. We extended and confirmed findings from the mouse model by examining PBF expression in human multinodular goiters (MNG), a hyperproliferative thyroid disorder, where PBF and TSHR was strongly upregulated relative to normal thyroid tissue. Furthermore, we showed that depleting PBF in human primary thyrocytes was sufficient to increase radioiodine uptake. Together, our findings indicate that overexpression of PBF causes thyroid cell proliferation, macrofollicular lesions, and hyperplasia, as well as repression of the critical therapeutic route for radioiodide uptake.

  19. Proto-oncogene PBF/PTTG1IP regulates thyroid cell growth and represses radioiodide treatment

    Science.gov (United States)

    Read, Martin L.; Lewy, Greg D.; Fong, Jim C.W.; Sharma, Neil; Seed, Robert I.; Smith, Vicki E.; Gentilin, Erica; Warfield, Adrian; Eggo, Margaret C.; Knauf, Jeffrey A.; Leadbeater, Wendy E.; Watkinson, John C.; Franklyn, Jayne A.; Boelaert, Kristien; McCabe, Christopher J.

    2011-01-01

    PTTG Binding Factor (PBF or PTTG1IP) is a little characterised proto-oncogene that has been implicated in the etiology of breast and thyroid tumors. In this study, we created a murine transgenic model to target PBF expression to the thyroid gland (PBF-Tg mice) and found that these mice exhibited normal thyroid function but a striking enlargement of the thyroid gland associated with hyperplastic and macrofollicular lesions. Expression of the sodium iodide symporter (NIS), a gene essential to the radioiodine ablation of thyroid hyperplasia, neoplasia and metastasis, was also potently inhibited in PBF-Tg mice. Critically, iodide uptake was repressed in primary thyroid cultures from PBF-Tg mice, which could be rescued by PBF depletion. PBF-Tg thyroids exhibited upregulation of Akt and the TSH receptor (TSHR), each known regulators of thyrocyte proliferation, along with upregulation of the downstream proliferative marker cyclin D1. We extended and confirmed findings from the mouse model by examining PBF expression in human multinodular goitres (MNG), a hyperproliferative thyroid disorder, where PBF and TSHR was strongly upregulated relative to normal thyroid tissue. Further, we showed that depleting PBF in human primary thyrocytes was sufficient to increase radioiodine uptake. Together, our findings indicate that overexpression of PBF causes thyroid cell proliferation, macrofollicular lesions and hyperplasia, as well as repression of the critical therapeutic route for radioiodide uptake. PMID:21844185

  20. Variants near DMRT1, TERT and ATF7IP are associated with testicular germ cell cancer

    Science.gov (United States)

    Turnbull, Clare; Rapley, Elizabeth A.; Seal, Sheila; Pernet, David; Renwick, Anthony; Hughes, Deborah; Ricketts, Michelle; Linger, Rachel; Nsengimana, Jeremie; Deloukas, Panagiotis; Huddart, Robert A.; Bishop, D Timothy; Easton, Douglas F.; Stratton, Michael R.; Rahman, Nazneen

    2013-01-01

    We conducted a genome-wide association study for testicular germ cell tumor genotyping 298,782 SNPs in 979 cases and 4,947 controls from the UK and replicating associations in a further 664 cases and 3,456 controls. We identified three novel susceptibility loci, two of which include genes that are involved in telomere regulation. We identified two independent signals within the TERT-CLPTM1L locus on chromosome 5 which has been associated with multiple other cancers (rs4635969, OR=1.54 (95%CI 1.33-1.79), P=1.14×10−23 and rs2736100, OR 1.33 (1.18-1.50) P=7.55 ×10−15). We also identified a locus on chromosome 12 (rs2900333, OR=1.27 (95%CI 1.12-1.44), P=6.16×10−10) that contains ATF7IP, a regulator of TERT expression. Finally we identified a locus on chromosome 9 (rs755383, OR=1.37 (95%CI 1.21-1.55), P=1.12×10−23) containing the sex determination gene DMRT1, which has been linked with teratoma susceptibility in mice. PMID:20543847

  1. ChIP-seq Analysis of Human Chronic Myeloid Leukemia Cells.

    Science.gov (United States)

    Anders, Lars; Li, Zhaodong

    2016-01-01

    Many transcription factors, chromatin-associated proteins and regulatory DNA elements are genetically and/or epigenetically altered in cancer, including Chronic Myeloid Leukemia (CML). This leads to deregulation of transcription that is often causally linked to the tumorigenic state. Chromatin-immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) is the key technology to study transcription as it allows in vivo whole-genome mapping of epigenetic modifications and interactions of proteins with DNA or chromatin. However, numerous DNA/chromatin-binding proteins, including EZH2, remain difficult to "ChIP," thus yielding genome-wide binding maps of only suboptimal quality. Here, we describe a ChIP-seq protocol optimized for high-quality protein-genome binding maps that have proven especially useful for studying difficult to 'ChIP' transcription regulatory factors in Chronic Myeloid Leukemia (CML) and related malignancies.

  2. Extracellular Matrix-Dependent Generation of Integration- and Xeno-Free iPS Cells Using a Modified mRNA Transfection Method.

    Science.gov (United States)

    Lee, Kang-In; Lee, Seo-Young; Hwang, Dong-Youn

    2016-01-01

    Human induced pluripotent stem cells (iPS cells) hold great promise in the field of regenerative medicine, especially immune-compatible cell therapy. The most important safety-related issues that must be resolved before the clinical use of iPS cells include the generation of "footprint-free" and "xeno-free" iPS cells. In this study, we sought to examine whether an extracellular matrix- (ECM-) based xeno-free culture system that we recently established could be used together with a microRNA-enhanced mRNA reprogramming method for the generation of clinically safe iPS cells. The notable features of this method are the use of a xeno-free/feeder-free culture system for the generation and expansion of iPS cells rather than the conventional labor-intensive culture systems using human feeder cells or human feeder-conditioned medium and the enhancement of mRNA-mediated reprogramming via the delivery of microRNAs. Strikingly, we observed the early appearance of iPS cell colonies (~11 days), substantial reprogramming efficiency (~0.2-0.3%), and a high percentage of ESC-like colonies among the total colonies (~87.5%), indicating enhanced kinetics and reprogramming efficiency. Therefore, the combined method established in this study provides a valuable platform for the generation and expansion of clinically safe (i.e., integration- and xeno-free) iPS cells, facilitating immune-matched cell therapy in the near future.

  3. Inositol hexaphosphate (IP6) inhibits key events of cancer metastasis: I. In vitro studies of adhesion, migration and invasion of MDA-MB 231 human breast cancer cells.

    Science.gov (United States)

    Tantivejkul, Kwanchanit; Vucenik, Ivana; Shamsuddin, Abulkalam M

    2003-01-01

    The anti-cancer agent inositol hexaphosphate (IP6) is an abundant intrinsic component of both plant and mammalian cells. In addition to inducing differentiation and inhibiting growth of numerous cancer cell lines in vitro, IP6 has been demonstrated to prevent and abrogate both primary tumor and metastasis in vivo. Using MDA-MB 231 human breast cancer cells, we studied the potential of IP6 to inhibit cell adhesion, migration and invasion, the key steps in cancer metastasis, utilizing the extracellular matrix (ECM) proteins, a culture wounding assay, modified Boyden chambers, immunocytochemistry and zymography. IP6 treatment caused a 65% reduction of cell adhesion to fibronection (p = 0.002) and a 37% reduction to collagen (p = 0.005). To determine whether a decrease in cell adhesion leads to a decrease in cell motility, migration assays were performed; IP6 decreased both the number of migrating cells and the distance of cell migration into the denuded area by 72% (p IP6-treated cells as compared to untreated cells, corresponding to a diminished ability of cancer cells to form cellular network as determined by Matrigel outgrowth assay. Likewise, cell invasion also was decreased (by 72% after IP6 treatment, p = 0.001) in a dose-dependent fashion. Additionally, IP6 significantly (p = 0.006) inhibited the secretion of matrix metalloproteinase (MMP)-9 as assessed by zymography. The results of this study show that IP6 inhibits the metastasis of human breast cancer cells in vitro through effects on cancer cell adhesion, migration and invasion.

  4. Production of Retinal Cells from Confluent Human iPS Cells.

    Science.gov (United States)

    Reichman, Sacha; Goureau, Olivier

    2016-01-01

    Human induced pluripotent stem (hiPS) cells could be used as an unlimited source of retinal cells for the treatment of retinal degenerative diseases. Although much progress has been made in the differentiation of pluripotent stem cells towards different retinal lineages, the production of retinal cells from hiPS cells for therapeutic approaches require the development of easy and standardized protocols. In this chapter, we describe a simple and effective protocol for retinal differentiation of hiPS cells bypassing embryoid body formation and the use of exogenous molecules and substrates. In 2 weeks, confluent hiPS cells cultured in pro-neural medium can generate both retinal pigmented epithelial cells and self-forming neural retina-like structures containing retinal progenitor cells. These progenitors can be differentiated into all retinal cell types, including retinal ganglion cells and precursors of photoreceptors, which could find important applications in regenerative medicine. This differentiation system and the resulting hiPS-derived retinal cells will also offer opportunity to study the molecular and cellular mechanisms underlying human retinal development, and the establishment of in vitro models of human retinal degenerative diseases.

  5. Development of feeder-free culture systems for generation of ckit+sca1+ progenitors from mouse iPS cells.

    Science.gov (United States)

    Lin, Jian; Fernandez, Irina; Roy, Krishnendu

    2011-09-01

    Patient-specific therapeutic cells derived from induced pluripotent stem (iPS) cells may bypass the ethical issues associated with embryonic stem (ES) cells and avoid potential immunological reactions associated with allogenic transplantation. It is critical, for the ultimate clinical applicability of iPS cell-derived therapies, to establish feeder-free cultures that ensure efficient differentiation of iPS cells into therapeutic progenitors. It is also necessary to understand if iPS cell-derived progenitors differ from those derived from ES cells. In this study, we compared the efficiency of three different feeder-free cultures for differentiating mouse iPS cells into ckit+sca1+ hematopoietic progenitor cells (HPCs) and compared how differentiation and functionality varies between ES and iPS cells. Our results indicated that both iPS and ES cells can be efficiently differentiated into HPCs in suspension cultures supplemented with secretion factors from mouse bone marrow stromal cells (OP9-DL1 conditioned medium). The functionality of these cells was demonstrated by differentiation into CD11c+ dendritic cells (DCs). Both ES and iPS-derived DCs expressed activation molecules (CD86, CD80) in response to LPS stimulation and stimulated T cell proliferation in a mixed lymphocyte reaction (MLR). Extensive quantitative RT-PCR studies were used to study the differences in gene expression profiles of ckit+sca1+ cells generated from the various culture systems as well as differences between ES-derived and iPS-derived cells. We conclude that a feeder-free system using stromal conditioned medium can efficiently generate HPCs as well as functional DCs from iPS cells and the generated cells have similar gene expression profile as those from ES cells.

  6. Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems

    Science.gov (United States)

    Chen, Silvia S.; Revoltella, Roberto P.; Papini, Sandra; Michelini, Monica; Fitzgerald, Wendy; Zimmerberg, Joshua; Margolis, Leonid

    2003-01-01

    In the course of normal embryogenesis, embryonic stem (ES) cells differentiate along different lineages in the context of complex three-dimensional (3D) tissue structures. In order to study this phenomenon in vitro under controlled conditions, 3D culture systems are necessary. Here, we studied in vitro differentiation of rhesus monkey ES cells in 3D collagen matrixes (collagen gels and porous collagen sponges). Differentiation of ES cells in these 3D systems was different from that in monolayers. ES cells differentiated in collagen matrixes into neural, epithelial, and endothelial lineages. The abilities of ES cells to form various structures in two chemically similar but topologically different matrixes were different. In particular, in collagen gels ES cells formed gland-like circular structures, whereas in collagen sponges ES cells were scattered through the matrix or formed aggregates. Soluble factors produced by feeder cells or added to the culture medium facilitated ES cell differentiation into particular lineages. Coculture with fibroblasts in collagen gel facilitated ES cell differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule, and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ES cell differentiation into cells of an endothelial lineage expressing factor VIII. Exogenous granulocyte-macrophage colony-stimulating factor further enhanced endothelial differentiation. Thus, both soluble factors and the type of extracellular matrix seem to be critical in directing differentiation of ES cells and the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for studying the mechanisms of these phenomena.

  7. Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation

    Institute of Scientific and Technical Information of China (English)

    Jing Jiang; Wenjian Lv; Xiaoying Ye; Lingbo Wang; Man Zhang; Hui Yang; Maja Okuka

    2013-01-01

    Induced pluripotent stem (iPS) cells generated using Yamanaka factors have great potential for use in autologous cell therapy.However,genomic abnormalities exist in human iPS cells,and most mouse iPS cells are not fully pluripotent,as evaluated by the tetraploid complementation assay (TCA); this is most likely associated with the DNA damage response (DDR) occurred in early reprogramming induced by Yamanaka factors.In contrast,nuclear transfer can faithfully reprogram somatic cells into embryonic stem (ES) cells that satisfy the TCA.We thus hypothesized that factors involved in oocyte-induced reprogramming may stabilize the somatic genome during reprogramming,and improve the quality of the resultant iPS cells.To test this hypothesis,we screened for factors that could decrease DDR signals during iPS cell induction.We determined that Zscan4,in combination with the Yamanaka factors,not only remarkably reduced the DDR but also markedly promoted the efficiency of iPS cell generation.The inclusion of Zscan4 stabilized the genomic DNA,resulting in p53 downregulation.Furthermore,Zscan4 also enhanced telomere lengthening as early as 3 days post-infection through a telomere recombination-based mechanism.As a result,iPS cells generated with addition of Zscan4 exhibited longer telomeres than classical iPS cells.Strikingly,more than 50%of iPS cell lines (11/19) produced via this "Zscan4 protocol" gave rise to live-borne all-iPS cell mice as determined by TCA,compared to 1/12 for lines produced using the classical Yamanaka factors.Our findings provide the first demonstration that maintaining genomic stability during reprogramming promotes the generation of high quality iPS cells.

  8. Neuronal differentiation of human iPS cells induced by baicalin via regulation of bHLH gene expression.

    Science.gov (United States)

    Morita, Akihiro; Soga, Kohei; Nakayama, Hironobu; Ishida, Torao; Kawanishi, Shosuke; Sato, Eisuke F

    2015-09-25

    Efficient differentiation is important for regenerative medicine based on pluripotent stem cells, including treatment of neurodegenerative disorders and trauma. Baicalin promotes neuronal differentiation of neural stem/progenitor cells of rats and mice. To evaluate the suitability of baicalin for neuronal differentiation of human iPS cells, we investigated whether it promotes neuronal differentiation in human iPS cells and monitored basic helix-loop-helix (bHLH) gene expression during neuronal differentiation. Baicalin promoted neuronal differentiation and inhibited glial differentiation, suggesting that baicalin can influence the neuronal fate decision in human iPS cells. Notch signaling, which is upstream of bHLH proteins, was not involved in baicalin-induced neuronal differentiation. Baicalin treatment did not down-regulate Hes1 gene expression, but it reduced Hes1 protein levels and up-regulated Ascl1 gene expression. Thus, baicalin promoted neuronal differentiation via modulation of bHLH transcriptional factors. Therefore, baicalin has potential to be used as a small-molecule drug for regenerative treatment of neurodegenerative disorders.

  9. Altered Ca(2+) signaling in cancer cells: proto-oncogenes and tumor suppressors targeting IP3 receptors.

    Science.gov (United States)

    Akl, Haidar; Bultynck, Geert

    2013-04-01

    Proto-oncogenes and tumor suppressors critically control cell-fate decisions like cell survival, adaptation and death. These processes are regulated by Ca(2+) signals arising from the endoplasmic reticulum, which at distinct sites is in close proximity to the mitochondria. These organelles are linked by different mechanisms, including Ca(2+)-transport mechanisms involving the inositol 1,4,5-trisphosphate receptor (IP3R) and the voltage-dependent anion channel (VDAC). The amount of Ca(2+) transfer from the endoplasmic reticulum to mitochondria determines the susceptibility of cells to apoptotic stimuli. Suppressing the transfer of Ca(2+) from the endoplasmic reticulum to the mitochondria increases the apoptotic resistance of cells and may decrease the cellular responsiveness to apoptotic signaling in response to cellular damage or alterations. This can result in the survival, growth and proliferation of cells with oncogenic features. Clearly, proper maintenance of endoplasmic reticulum Ca(2+) homeostasis and dynamics including its links with the mitochondrial network is essential to detect and eliminate altered cells with oncogenic features through the apoptotic pathway. Proto-oncogenes and tumor suppressors exploit the central role of Ca(2+) signaling by targeting the IP3R. There are an increasing number of reports showing that activation of proto-oncogenes or inactivation of tumor suppressors directly affects IP3R function and endoplasmic reticulum Ca(2+) homeostasis, thereby decreasing mitochondrial Ca(2+) uptake and mitochondrial outer membrane permeabilization. In this review, we provide an overview of the current knowledge on the proto-oncogenes and tumor suppressors identified as IP3R-regulatory proteins and how they affect endoplasmic reticulum Ca(2+) homeostasis and dynamics.

  10. Completion of the Entire Hepatitis C Virus Life Cycle in Vero Cells Derived from Monkey Kidney

    Directory of Open Access Journals (Sweden)

    Asako Murayama

    2016-06-01

    Full Text Available A hepatitis C virus (HCV cell culture system incorporating the JFH-1 strain and the human hepatoma cell line HuH-7 enabled the production of infectious HCV particles. Several host factors were identified as essential for HCV replication. Supplementation of these factors in nonhepatic human cell lines enabled HCV replication and particle production. Vero cells established from monkey kidney are commonly used for the production of vaccines against a variety of viruses. In this study, we aimed to establish a novel Vero cell line to reconstruct the HCV life cycle. Unmodified Vero cells did not allow HCV infection or replication. The expression of microRNA 122 (miR-122, an essential factor for HCV replication, is notably low in Vero cells. Therefore, we supplemented Vero cells with miR-122 and found that HCV replication was enhanced. However, Vero cells that expressed miR-122 still did not allow HCV infection. We supplemented HCV receptor molecules and found that scavenger receptor class B type I (SRBI was essential for HCV infection in Vero cells. The supplementation of apolipoprotein E (ApoE, a host factor important for virus production, enabled the production of infectious virus in Vero cells. Finally, we created a Vero cell line that expressed the essential factors miR-122, SRBI, and ApoE; the entire HCV life cycle, including infection, replication, and infectious virus production, was completed in these cells. In conclusion, we demonstrated that miR-122, SRBI, and ApoE were necessary and sufficient for the completion of the entire HCV life cycle in nonhuman, nonhepatic Vero cells.

  11. Distribution of 5-HT1F receptors in monkey vestibular and trigeminal ganglion cells

    Directory of Open Access Journals (Sweden)

    Habiba Usman

    2016-10-01

    Full Text Available Background: Evidence of serotonergic involvement in vestibular pathway contributions to migraine and balance disorders is compelling. Serotonergic 5-HT1B and 5-HT1D receptors are expressed extensively in inner ear ganglia of monkeys and rats. The serotonergic 5-HT1F receptor is also a target of triptans. This study describes its distribution in vestibular and trigeminal ganglia of monkeys. Methods: Using primary polyclonal antibodies raised against oligopeptides specific for the human 5-HT1F receptor, neuronal somatic area and intensity of immunoreactive vestibular and trigeminal ganglia were quantified. Results and Discussion: Virtually all vestibular and considerable trigeminal ganglia showed positive 5-HT1F receptor immunoreactivity. Inferior and superior vestibular ganglia staining appeared confined to distinct cell regions, varying considerably among cells of different sizes: more intense in small, punctate in some medium and regionally polarized in some large cells. Analyses of average somatic vestibular neuronal immunoreactive intensity identified mainly medium sized cells with high standard deviation of intensity corresponding to punctately-stained cells. Less variability occurred in somatic intensity staining and cellular distribution among 5-HT1F receptor immunopositive trigeminal ganglia. Most exhibited similar punctate staining patterns, higher mean somatic immunoreactive intensity and larger neuronal somatic size proportions per size distribution subpopulation compared to vestibular ganglia size distribution populations. Centrally directed vestibular ganglion neuronal processes, cochlear inner hair cells, vestibular hair cells and blood vessels in vestibular maculae and cristae were immunoreactive. The 5-HT1F receptor expression in vestibular ganglia shows complex variable staining intensity patterns associated with cell size of immunopositive neurons, not seen in immunopositive trigeminal ganglia and not previously evident with 5

  12. Early cytoplasmic vacuolization of African green monkey kidney cells by SV40.

    Science.gov (United States)

    Miyamura, T; Kitahara, T

    1975-01-01

    As early as 3--4 hours after infection with SV40 at a high input multiplicity, African green monkey (Cercopithecus aethiops) kidney (AGMK) cells developed cytoplasmic vacuolization. At 10--20 hours after infection, the vacuolization reached its maximal level, then disappeared and SV40 specific cytopathic change followed. This vacuolization developed before the synthesis of the specific T and V antigens. This early cytoplasmic vacuolization (ECV) was prevented by preincubating the virus with specific antiserum, or by heating the virus with MgCl2. The ECV could be induced by UV-irradiated SV40. Addition of metabolic inhibitors had no effect on the induction of the ECV. These results suggest that the capacity to induce the ECV resides in a structural component(s) of SV40 virion and the vacuolization is not associated with the replication of SV40.

  13. Early cytoplasmic vacuolization of African green monkey kidney cells by SV40. [uv radiation

    Energy Technology Data Exchange (ETDEWEB)

    Miyamura, T.; Kitahara, T.

    1975-01-01

    As early as 3 to 4 hours after infection with SV 40 at a high input multiplicity, African green monkey (Cercopithecus aethiops) kidney (AGMK) cells developed cytoplasmic vacuolization. At 10 to 20 hours after infection, the vacuolization reached its maximal level, then disappeared and SV 40 specific cytopathic change followed. This vacuolization developed before the synthesis of the specific T and V antigens. This early cytoplasmic vacuolization (ECV) was prevented by pre-incubating the virus with specific antiserum, or by heating the virus with MgCl/sub 2/. The ECV could be induced by uv-irradiated SV 40. Addition of metabolic inhibitors had no effect on the induction of the ECV. These results suggest that the capacity to induce the ECV resides in a structural component(s) of SV 40 virion and the vacuolization is not associated with the replication of SV 40.

  14. Localization of West Nile Virus in monkey brain: double staining antigens immunohistochemically of neurons, neuroglia cells and West Nile Virus.

    Science.gov (United States)

    He, Xianli; Ren, Junping; Xu, Fangling; Ferguson, Monique R; Li, Guangyu

    2009-11-15

    West Nile virus (WNV) can cause encephalitis or meningitis that affects brain tissue, which can also lead to permanent neurological damage that can be fatal. To our knowledge, no consistent double immunohistochemical staining of neurons, neuroglia cells, and WNV has yet been reported. To establish a method for performing double-label immunohistochemical detection of neurons, neuroglia cells and WNV, examining the pathological characteristics of WNV-infected neurons, neuroglia cells, and investigating distribution of WNV in monkey brain, paraffin-embedded monkey brain tissue were retrospectively studied by immunohistochemical staining of neurons, neuroglia cells and WNV. Antibodies against neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP) and WNV were used to develop the method of double-label immunohistochemical staining, which allowed independent assessment of neuron status and WNV distribution. A range of immunohistochemical WNV infection in monkey brain was observed in both neurons and neuroglia cells in terms of the thickness of lesion staining, and the WNV staining was slightly higher in neuroglia cells than in neurons. All these findings suggest that WNV invasion in the brain plays a crucial role in neurological damage by inducing central nervous system (CNS) cell dysfunction or cell death directly.

  15. Age-related increase of sI(AHP) in prefrontal pyramidal cells of monkeys: relationship to cognition.

    Science.gov (United States)

    Luebke, J I; Amatrudo, J M

    2012-06-01

    Reduced excitability, due to an increase in the slow afterhyperpolarization (and its underlying current sI(AHP)), occurs in CA1 pyramidal cells in aged cognitively-impaired, but not cognitively-unimpaired, rodents. We sought to determine whether similar age-related changes in the sI(AHP) occur in pyramidal cells in the rhesus monkey dorsolateral prefrontal cortex (dlPFC). Whole-cell patch-clamp recordings were obtained from layer 3 and layer 5 pyramidal cells in dlPFC slices prepared from young (9.6 ± 0.7 years old) and aged (22.3 ± 0.7 years old) behaviorally characterized subjects. The amplitude of the sI(AHP) was significantly greater in layer 3 (but not layer 5) cells from aged-impaired compared with both aged-unimpaired and young monkeys, which did not differ. Aged layer 3, but not layer 5, cells exhibited significantly increased action potential firing rates, but there was no relationship between sI(AHP) and firing rate. Thus, in monkey dlPFC layer 3 cells, an increase in sI(AHP) is associated with age-related cognitive decline; however, this increase is not associated with a reduction in excitability.

  16. Testing Brain Overgrowth and Synaptic Models of Autism Using NPC’s and Neurons from Patient-Derived IPS Cells

    Science.gov (United States)

    2014-10-01

    interaction, and limited and repetitive interests and behavior. Recent studies have led to two major hypotheses for autism pathogenesis : early brain...interests and behavior. Recent studies have led to two major hypotheses for autism pathogenesis . First, early brain overgrowth appears to be a critical...Models of Autism Using NPC’s and Neurons from Patient- Derived IPS Cells PRINCIPAL INVESTIGATOR: Fred H. Gage, Ph.D

  17. Analogues of simple and complex cells in rhesus monkey auditory cortex.

    Science.gov (United States)

    Tian, Biao; Kuśmierek, Paweł; Rauschecker, Josef P

    2013-05-01

    Receptive fields (RFs) of neurons in primary visual cortex have traditionally been subdivided into two major classes: "simple" and "complex" cells. Simple cells were originally defined by the existence of segregated subregions within their RF that respond to either the on- or offset of a light bar and by spatial summation within each of these regions, whereas complex cells had ON and OFF regions that were coextensive in space [Hubel DH, et al. (1962) J Physiol 160:106-154]. Although other definitions based on the linearity of response modulation have been proposed later [Movshon JA, et al. (1978) J Physiol 283:53-77; Skottun BC, et al. (1991) Vision Res 31(7-8):1079-1086], the segregation of ON and OFF subregions has remained an important criterion for the distinction between simple and complex cells. Here we report that response profiles of neurons in primary auditory cortex of monkeys show a similar distinction: one group of cells has segregated ON and OFF subregions in frequency space; and another group shows ON and OFF responses within largely overlapping response profiles. This observation is intriguing for two reasons: (i) spectrotemporal dissociation in the auditory domain provides a basic neural mechanism for the segregation of sounds, a fundamental prerequisite for auditory figure-ground discrimination; and (ii) the existence of similar types of RF organization in visual and auditory cortex would support the existence of a common canonical processing algorithm within cortical columns.

  18. Propagation of human hepatitis A virus in African green monkey kidney cell culture: primary isolation and serial passage.

    OpenAIRE

    Daemer, R J; Feinstone, S M; Gust, I D; Purcell, R H

    1981-01-01

    Human hepatitis A virus (HAV) was propagated in primary African Green Monkey (Cercopithecus aethiops) kidney (AGMK) cell cultures. Three strains of HAV were used: MS-1, SD-11, and HM-175. Cells were inoculated with marmoset-passaged material or human clinical specimens and were stained by direct immunofluorescence to establish the identity of the virus. Both clinical samples and marmoset-passaged material produced immunofluorescence. HAV antigen was found scattered throughout the cytoplasm of...

  19. Antibody-directed lentiviral gene transduction for live-cell monitoring and selection of human iPS and hES cells.

    Directory of Open Access Journals (Sweden)

    Dai-tze Wu

    Full Text Available The identification of stem cells within a mixed population of cells is a major hurdle for stem cell biology--in particular, in the identification of induced pluripotent stem (iPS cells during the reprogramming process. Based on the selective expression of stem cell surface markers, a method to specifically infect stem cells through antibody-conjugated lentiviral particles has been developed that can deliver both visual markers for live-cell imaging as well as selectable markers to enrich for iPS cells. Antibodies recognizing SSEA4 and CD24 mediated the selective infection of the iPS cells over the parental human fibroblasts, allowing for rapid expansion of these cells by puromycin selection. Adaptation of the vector allows for the selective marking of human embryonic stem (hES cells for their removal from a population of differentiated cells. This method has the benefit that it not only identifies stem cells, but that specific genes, including positive and negative selection markers, regulatory genes or miRNA can be delivered to the targeted stem cells. The ability to specifically target gene delivery to human pluripotent stem cells has broad applications in tissue engineering and stem cell therapies.

  20. Germ cell dynamics in the testis of the postnatal common marmoset monkey (Callithrix jacchus).

    Science.gov (United States)

    Albert, S; Ehmcke, J; Wistuba, J; Eildermann, K; Behr, R; Schlatt, S; Gromoll, J

    2010-11-01

    The seminiferous epithelium in the nonhuman primate Callithrix jacchus is similarly organized to man. This monkey has therefore been used as a preclinical model for spermatogenesis and testicular stem cell physiology. However, little is known about the developmental dynamics of germ cells in the postnatal primate testis. In this study, we analyzed testes of newborn, 8-week-old, and adult marmosets employing immunohistochemistry using pluripotent stem cell and germ cell markers DDX4 (VASA), POU5F1 (OCT3/4), and TFAP2C (AP-2γ). Stereological and morphometric techniques were applied for quantitative analysis of germ cell populations and testicular histological changes. Quantitative RT-PCR (qRT-PCR) of testicular mRNA was applied using 16 marker genes establishing the corresponding profiles during postnatal testicular development. Testis size increased during the first 8 weeks of life with the main driver being longitudinal outgrowth of seminiferous cords. The number of DDX4-positive cells per testis doubled between birth and 8 weeks of age whereas TFAP2C- and POU5F1-positive cells remained unchanged. This increase in DDX4-expressing cells indicates dynamic growth of the differentiated A-spermatogonial population. The presence of cells expressing POU5F1 and TFAP2C after 8 weeks reveals the persistence of less differentiated germ cells. The mRNA and protein profiles determined by qRT-PCR and western blot in newborn, 8-week-old, and adult marmosets corroborated the immunohistochemical findings. In conclusion, we demonstrated the presence of distinct spermatogonial subpopulations in the primate testis exhibiting different dynamics during early testicular development. Our study demonstrates the suitability of the marmoset testis as a model for human testicular development.

  1. Effect of input multiplicity on the establishment of simian virus 40 persistent infections in rhesus monkey kidney cells.

    Science.gov (United States)

    Norkin, L C

    1977-12-01

    Monolayer cultures of LLC-MK2 rhesus monkey kidney cells become persistently infected with simian virus 40 after infection at input multiplicities of 100, 10, or 1 plaque-forming unit per cell. After 3 weeks, all cells of the cultures infected at a multiplicity of 1 plaque-forming unit per cell produced the simian virus 40 T antigen. In contrast, 8 to 11 weeks elapsed before all the cells in the cultures infected at a multiplicity of 100 plaque-forming units per cell produced T antigen. Defective interfering particles and interferon production were not evident during this time.

  2. Hemopoietic stem cells in rhesus monkeys : surface antigens, radiosensitivity, and responses to GM-CSF

    NARCIS (Netherlands)

    J.J. Wielenga (Jenne)

    1990-01-01

    textabstractRhesus monkeys (Macaca mulatta) were bred at the Primate Center TNO, Rijswijk, The Netherlands!. Both male and female animals were used for the experiments. The monkeys weighed 2.5-4 kg and were 2-4 years old at the time of the experiment. They were all typed for RhLA-A, -B and -DR antig

  3. Mobile IP

    NARCIS (Netherlands)

    Heijenk, Geert; Sallent, S.; Pras, A.

    1999-01-01

    The Internet is growing exponentially, both in the amount of traffic carried, and in the amount of hosts connected. IP technology is becoming more and more important, in company networks (Intranets), and also in the core networks for the next generation mobile networks. Further, wireless access to I

  4. Mobile IP

    NARCIS (Netherlands)

    Heijenk, Gerhard J.; Sallent, S.; Pras, Aiko

    1999-01-01

    The Internet is growing exponentially, both in the amount of traffic carried, and in the amount of hosts connected. IP technology is becoming more and more important, in company networks (Intranets), and also in the core networks for the next generation mobile networks. Further, wireless access to

  5. Association of activating KIR copy number variation of NK cells with containment of SIV replication in rhesus monkeys.

    Directory of Open Access Journals (Sweden)

    Ina Hellmann

    2011-12-01

    Full Text Available While the contribution of CD8⁺ cytotoxic T lymphocytes to early containment of HIV-1 spread is well established, a role for NK cells in controlling HIV-1 replication during primary infection has been uncertain. The highly polymorphic family of KIR molecules expressed on NK cells can inhibit or activate these effector cells and might therefore modulate their activity against HIV-1-infected cells. In the present study, we investigated copy number variation in KIR3DH loci encoding the only activating KIR receptor family in rhesus monkeys and its effect on simian immunodeficiency virus (SIV replication during primary infection in rhesus monkeys. We observed an association between copy numbers of KIR3DH genes and control of SIV replication in Mamu-A*01⁻ rhesus monkeys that express restrictive TRIM5 alleles. These findings provide further evidence for an association between NK cells and the early containment of SIV replication, and underscore the potential importance of activating KIRs in stimulating NK cell responses to control SIV spread.

  6. Myogenic Precursors from iPS Cells for Skeletal Muscle Cell Replacement Therapy

    Directory of Open Access Journals (Sweden)

    Isart Roca

    2015-01-01

    Full Text Available The use of adult myogenic stem cells as a cell therapy for skeletal muscle regeneration has been attempted for decades, with only moderate success. Myogenic progenitors (MP made from induced pluripotent stem cells (iPSCs are promising candidates for stem cell therapy to regenerate skeletal muscle since they allow allogenic transplantation, can be produced in large quantities, and, as compared to adult myoblasts, present more embryonic-like features and more proliferative capacity in vitro, which indicates a potential for more self-renewal and regenerative capacity in vivo. Different approaches have been described to make myogenic progenitors either by gene overexpression or by directed differentiation through culture conditions, and several myopathies have already been modeled using iPSC-MP. However, even though results in animal models have shown improvement from previous work with isolated adult myoblasts, major challenges regarding host response have to be addressed and clinically relevant transplantation protocols are lacking. Despite these challenges we are closer than we think to bringing iPSC-MP towards clinical use for treating human muscle disease and sporting injuries.

  7. Distribution of parvalbumin-immunoreactive cells and fibers in the monkey temporal lobe: the hippocampal formation.

    Science.gov (United States)

    Pitkänen, A; Amaral, D G

    1993-05-01

    The distribution of parvalbumin-immunoreactive cells and fibers in the various fields of the hippocampal formation was studied in the macaque monkey. Parvalbumin-immunoreactive neurons had aspiny or sparsely spiny dendrites that often had a beaded appearance; most resembled classically identified interneurons. Parvalbumin-immunoreactive fibers and terminals were confined to certain laminae in each field and generally had a pericellular distribution. In the dentate gyrus, there was a dense pericellular plexus of immunoreactive terminals in the granule cell layer. Except for a narrow supragranular zone, there was a marked paucity of terminals in the molecular and polymorphic cell layers. Immunoreactive neurons were mainly located immediately subjacent to the granule cell layer and comprised a variety of morphological cell types. The three fields of the hippocampus proper (CA3, CA2, and CA1) demonstrated differences in their parvalbumin staining characteristics. In CA3, there was a prominent pericellular terminal plexus in the pyramidal cell layer that was densest distally (closer to CA2). Immunoreactive cells were located either in the pyramidal cell layer, where many had a pyramidal shape and prominent apical and basal dendrites, or in stratum oriens. CA2 had a staining pattern similar to that in CA3, though both the number of labeled cells and the density of the pericellular terminal plexus were greater in CA2. In CA1, there was a markedly lower number of parvalbumin-labeled cells than in CA3 and CA2 and the cells tended to be located in the deep part of the pyramidal cell layer or in stratum oriens. The pyramidal cell layer of CA1 contained a pericellular terminal plexus that was substantially less dense than in CA3 and CA2. At the border between CA1 and the subiculum there was a marked increase in the number of parvalbumin-immunoreactive neurons. The positive cells were scattered throughout the pyramidal cell layer of the subiculum and comprised a variety of

  8. Monkey adrenal chromaffin cells express α6β4* nicotinic acetylcholine receptors.

    Directory of Open Access Journals (Sweden)

    Alicia Hernández-Vivanco

    Full Text Available Nicotinic acetylcholine receptors (nAChRs that contain α6 and β4 subunits have been demonstrated functionally in human adrenal chromaffin cells, rat dorsal root ganglion neurons, and on noradrenergic terminals in the hippocampus of adolescent mice. In human adrenal chromaffin cells, α6β4* nAChRs (the asterisk denotes the possible presence of additional subunits are the predominant subtype whereas in rodents, the predominant nAChR is the α3β4* subtype. Here we present molecular and pharmacological evidence that chromaffin cells from monkey (Macaca mulatta also express α6β4* receptors. PCR was used to show the presence of transcripts for α6 and β4 subunits and pharmacological characterization was performed using patch-clamp electrophysiology in combination with α-conotoxins that target the α6β4* subtype. Acetylcholine-evoked currents were sensitive to inhibition by BuIA[T5A,P6O] and MII[H9A,L15A]; α-conotoxins that inhibit α6-containing nAChRs. Two additional agonists were used to probe for the expression of α7 and β2-containing nAChRs. Cells with currents evoked by acetylcholine were relatively unresponsive to the α7-selctive agonist choline but responded to the agonist 5-I-A-85380. These studies provide further insights into the properties of natively expressed α6β4* nAChRs.

  9. Lineage-specific diversification of killer cell Ig-like receptors in the owl monkey, a New World primate.

    Science.gov (United States)

    Cadavid, Luis F; Lun, Cheng-Man

    2009-01-01

    Killer cell Ig-like receptors (KIRs) modulate the cytotoxic effects of natural killer cells. In primates, the KIRs are highly diverse as a consequence of variation in gene content, alternative domain composition, and loci polymorphism. We analyzed a bacterial artificial chromosome (BAC) clone draft sequence spanning the owl monkey KIR cluster. The draft sequence had seven ordered yet unconnected contigs containing six full-length and two partial gene models, flanked by the LILRB and FcAR framework genes. Gene models were predicted to encode KIRs with inhibitory, activating, or dual functionality. Four gene models encoded three Ig domain receptors, while three others encoded molecules with four Ig domains. The additional domain resulted from an insertion in tandem of a 2,101 bp fragment containing the last 289 bp of intron 2, exon 3, and intron 3, resulting in molecules with two D0 domains. Re-screening of the owl monkey BAC library and sequencing of partial cDNAs from an owl monkey yielded five additional KIRs, four of which encoded receptors with short cytoplasmic domains with premature stop codons due to either a single nucleotide substitution or deletion or the absence of exon 8. Phylogenetic analysis by domains showed that owl monkey KIRs were monophyletic, clustering independently from other primate KIR lineages. Retroelements found in introns, however, were shared by KIRs from different primate lineages. This suggests that the owl monkey inherited a KIR cluster with a rich history of exon shuffling upon which positive selection for ligand binding operated to diversify the receptors in a lineage-specific fashion.

  10. Use of primary cell cultures to measure the late effects in the skins of rhesus monkeys irradiated with protons

    Science.gov (United States)

    Cox, A. B.; Wood, D. H.; Lett, J. T.

    Previous pilot investigations of the uses of primary cell cultures to study late damage in stem cells of the skin of the New Zealand white (NZW) rabbit and the rhesus monkey /1-3/, have been extended to individual monkeys exposed to 55 MeV protons. Protons of this energy have a larger range in tissue of (~2.6 cm) than the 32 MeV protons (~0.9 cm) to which the animals in our earlier studies had been exposed. Although the primary emphases in the current studies were improvement and simplification in the techniques and logistics of transportation of biopsies to a central analytical facility, comparison of the quantitative measurements obtained thus far for survival of stem cells in the skins from animals irradiated 21 years ago reveals that the effects of both proton energies are similar.

  11. Generation of iPS cells using defined factors linked via the self-cleaving 2A sequences in a single open reading frame

    Science.gov (United States)

    Shao, Lijian; Feng, Wei; Sun, Yan; Bai, Hao; Liu, Jun; Currie, Caroline; Kim, Jaejung; Gama, Rafael; Wang, Zack; Qian, Zhijian; Liaw, Lucy; Wu, Wen-Shu

    2010-01-01

    Generation of induced pluripotent stem (iPS) cells from somatic cells has been achieved successfully by simultaneous viral transduction of defined reprogramming transcription factors (TFs). However, the process requires multiple viral vectors for gene delivery. As a result, generated iPS cells harbor numerous viral integration sites in their genomes. This can increase the probability of gene mutagenesis and genomic instability, and present significant barriers to both research and clinical application studies of iPS cells. In this paper, we present a simple lentivirus reprogramming system in which defined factors are fused in-frame into a single open reading frame (ORF) via self-cleaving 2A sequences. A GFP marker is placed downstream of the transgene to enable tracking of transgene expression. We demonstrate that this polycistronic expression system efficiently generates iPS cells. The generated iPS cells have normal karyotypes and are similar to mouse embryonic stem cells in morphology and gene expression. Moreover, they can differentiate into cell types of the three embryonic germ layers in both in vitro and in vivo assays. Remarkably, most of these iPS cells only harbor a single copy of viral vector. This system provides a valuable tool for generation of iPS cells, and our data suggest that the balance of expression of transduced reprogramming TFs in each cell is essential for the reprogramming process. More importantly, when delivered by non-integrating gene-delivery systems, this re-engineered single ORF will facilitate efficient generation of human iPS cells free of genetic modifications. PMID:19238173

  12. Generation of Ips cells using defined factors linked via the self-cleaving 2A sequences in a single open reading frame

    Institute of Scientific and Technical Information of China (English)

    Lijian Shao; Wei Feng; Yan Sun; Hao Bai; Jun Liu; Caroline Currie; Jaejung Kim; Rafael Gama; Zack Wang; Zhijian Qian; Lucy Liaw; Wen-Shu Wu

    2009-01-01

    Generation of induced pluripotent stem (iPS) cells from somatic cells has been achieved successfully by simultane-ous viral transduction of defined reprogramming transcription factors (TFs). However, the process requires multiple viral vectors for gene delivery. As a result, generated iPS cells harbor numerous viral integration sites in their ge-nomes. This can increase the probability of gene mutagenesis and genomic instability, and present significant barriers to both research and clinical application studies of iPS cells. In this paper, we present a simple lentivirus reprogram-ming system in which defined factors are fused in-frame into a single open reading frame (ORF) via self-cleaving 2A sequences. A GFP marker is placed downstream of the transgene to enable tracking of transgene expression. We demonstrate that this polycistronic expression system efficiently generates iPS cells. The generated iPS cells have nor-mal karyotypes and are similar to mouse embryonic stem cells in morphology and gene expression. Moreover, they can differentiate into cell types of the three embryonic germ layers in both in vitro and in vivo assays. Remarkably, most of these iPS cells only harbor a single copy of viral vector. This system provides a valuable tool for generation of iPS cells, and our data suggest that the balance of expression of transduced reprogramming TFs in each cell is essen-tial for the reprogramming process. More importantly, when delivered by non-integrating gene-delivery systems, this re-engineered single ORF will facilitate efficient generation of human iPS cells free of genetic modifications.

  13. Neurogranin is expressed by principal cells but not interneurons in the rodent and monkey neocortex and hippocampus.

    Science.gov (United States)

    Singec, Ilyas; Knoth, Rolf; Ditter, Margarethe; Volk, Benedikt; Frotscher, Michael

    2004-11-01

    As a substrate of protein kinase C (PKC), neurogranin (NG) is involved in the regulation of calcium signaling and activity-dependent plasticity. Recently, we have shown that, in the rodent cerebellum, NG is exclusively expressed by gamma-aminobutyric acidergic Golgi cells, whereas, in the monkey cerebellum, brush cells were the only neuronal population expressing NG (Singec et al. [2003] J. Comp. Neurol. 459:278-289). In the present study, we analyzed the neocortical and hippocampal expression patterns of NG in adult mouse (C57Bl/6), rat (Wistar), and monkey (Cercopithecus aetiops). By using immunocytochemistry and nonradioactive in situ hybridization, we demonstrate strong NG expression by principal cells in different neocortical layers and in the hippocampus by granule cells of the dentate gyrus and pyramidal neurons of CA1-CA3. In contrast, double-labeling experiments in rodents revealed that neocortical and hippocampal interneurons expressing glutamate decarboxylase 67 (GAD67) were consistently devoid of NG. In addition, by using antibodies against parvalbumin, calbindin, and calretinin, we could demonstrate the absence of NG in interneurons of monkey frontal cortex and hippocampus. Together these findings corroborate the idea of different calcium signaling pathways in excitatory and inhibitory cells that may contribute to different modes of synaptic plasticity in these neurons.

  14. Generation of glucose-responsive functional islets with a three-dimensional structure from mouse fetal pancreatic cells and iPS cells in vitro.

    Directory of Open Access Journals (Sweden)

    Hiroki Saito

    Full Text Available Islets of Langerhans are a pancreatic endocrine compartment consisting of insulin-producing β cells together with several other hormone-producing cells. While some insulin-producing cells or immature pancreatic cells have been generated in vitro from ES and iPS cells, islets with proper functions and a three-dimensional (3D structure have never been successfully produced. To test whether islets can be formed in vitro, we first examined the potential of mouse fetal pancreatic cells. We found that E16.5 pancreatic cells, just before forming islets, were able to develop cell aggregates consisting of β cells surrounded by glucagon-producing α cells, a structure similar to murine adult islets. Moreover, the transplantation of these cells improved blood glucose levels in hyperglycemic mice. These results indicate that functional islets are formed in vitro from fetal pancreatic cells at a specific developmental stage. By adopting these culture conditions to the differentiation of mouse iPS cells, we developed a two-step system to generate islets, i.e. immature pancreatic cells were first produced from iPS cells, and then transferred to culture conditions that allowed the formation of islets from fetal pancreatic cells. The islets exhibited distinct 3D structural features similar to adult pancreatic islets and secreted insulin in response to glucose concentrations. Transplantation of the islets improved blood glucose levels in hyperglycemic mice. In conclusion, the two-step culture system allows the generation of functional islets with a 3D structure from iPS cells.

  15. Cholinergic neuronal differentiation of bone marrow mesenchymal stem cells in rhesus monkeys

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The purpose of the present study was to determine the best cholinergic neuronal differentiation method of rhesus monkey bone marrow mesenchymal stem cells(BMSCs).Four methods were used to induce differentiation,and the groups were assigned accordingly:basal inducing group(culture media,bFGF,and forskolin);SHH inducing group(SHH,inducing group);RA inducing group(RA,basal inducing group);and SHH+RA inducing group(SHH,RA,and basal inducing group).All groups displayed neuronal morphology and increased expression of nestin and neuron-specific enolase.The basal inducing group did not express synapsin,and cells from the SHH inducing group did not exhibit neuronal resting membrane potential.In contrast,results demonstrated that BMSCs from the RA and SHH+RA inducing groups exhibited neuronal resting membrane potential,and cells from the SHH+RA inducing group expressed higher levels of synapsin and acetylcholine.In conclusion,the induction of cholinergic differentiation through SHH+RA was determined to be superior to the other methods.

  16. Global epigenetic changes during somatic cell reprogramming to iPS cells

    Institute of Scientific and Technical Information of China (English)

    Anna Mattout; Alva Biran; Eran Meshorer

    2011-01-01

    Embryonic stem cells (ESCs) exhibit unique chromatin features,including a permissive transcriptional program and an open,decondensed chromatin state.Induced pluripotent stem cells (iPSCs),which are very similar to ESCs,hold great promise for therapy and basic research.However,the mechanisms by which reprogramming occurs and the chromatin organization that underlies the reprogramming process are largely unknown.Here we characterize and compare the epigenetic landscapes of partially and fully reprogrammed iPSCs to mouse embryonic fibroblasts (MEFs) and ESCs,which serves as a standard for pluripotency.Using immunofluorescence and biochemical fractionations,we analyzed the levels and distribution of a battery of histone modifications (H3ac,H4ac,H4KSac,H3Kgac,H3K27ac,H3K4me3,H3K36me2,H3K9me3,H3K27me3,and yH2AX),as well as HP1α and lamin A.We find that fully reprogrammed iPSCs are epigenetically identical to ESCs,and that partially reprogrammed iPSCs are closer to MEFs.Intriguingly,combining both time-course reprogramming experiments and data from the partially reprogrammed iPSCs,we find that heterochromatin reorganization precedes Nanog expression and active histone marking.Together,these data delineate the global epigenetic state of iPSCs in conjunction with their pluripotent state,and demonstrate that heterochromatin precedes euchromatin in reorganization during reprogramming.

  17. Cell-poor septa separate representations of digits in the ventroposterior nucleus of the thalamus in monkeys and prosimian galagos.

    Science.gov (United States)

    Qi, Hui-Xin; Gharbawie, Omar A; Wong, Peiyan; Kaas, Jon H

    2011-03-01

    The architectonic features of the ventroposterior nucleus (VP) were visualized in coronal brain sections from two macaque monkeys, two owl monkeys, two squirrel monkeys, and three galagos that were processed for cytochrome oxidase, Nissl bodies, or the vesicular glutamate transporter 2 (vGluT2). The traditional ventroposterior medial (VPM) and ventroposterior lateral (VPL) subnuclei were easily identified, as well as the forelimb and hindlimb compartments of VPL, as they were separated by poorly staining, cell-poor septa. Septa also separated other cell groups within VPM and VPL, specifically in the medial compartment of VPL representing the hand (hand VPL). In one squirrel monkey and one galago we demonstrated that these five groups of cells represent digits 1-5 in a mediolateral sequence by injecting tracers into the cortical representation of single digits, defined by microelectrode recordings, and relating concentrations of labeled neurons to specific cell groups in hand VPL. The results establish the existence of septa that isolate the representation of the five digits in VPL of primates and demonstrate that the isolated cell groups represent digits 1-5 in a mediolateral sequence. The present results show that the septa are especially prominent in brain sections processed for vGluT2, which is expressed in the synaptic terminals of excitatory neurons in most nuclei of the brainstem and thalamus. As vGluT2 is expressed in the synaptic terminations from dorsal columns and trigeminal brainstem nuclei, the effectiveness of vGluT2 preparations in revealing septa in VP likely reflects a lack of synapses using glutamate in the septa.

  18. Morphology, dendritic field size, somal size, density, and coverage of M and P retinal ganglion cells of dichromatic Cebus monkeys.

    Science.gov (United States)

    Yamada, E S; Silveira, L C; Perry, V H

    1996-01-01

    Male Cebus monkeys are all dichromats, but about two thirds of the females are trichromats. M and P retinal ganglion cells were studied in the male Cebus monkey to investigate the relationship of their morphology to retinal eccentricity. Retinal ganglion cells were retrogradely labeled after optic nerve deposits of biocytin to reveal their entire dendritic tree. Cebus M and P ganglion cell morphology revealed by biocytin retrograde filling is similar to that described for macaque and human M and P ganglion cells obtained by in vitro intracellular injection of HRP and neurobiotin. We measured 264 and 441 M and P ganglion cells, respectively. M ganglion cells have larger dendritic field and cell body size than P ganglion cells at any comparable temporal or nasal eccentricity. Dendritic trees of both M and P ganglion cells are smaller in the nasal than in the temporal region at eccentricities greater than 5 mm and 2 mm for M and P ganglion cells, respectively. The depth of terminal dendrites allows identification of both inner and outer subclasses of M and P ganglion cells. The difference in dendritic tree size between inner and outer cells is small or absent. Comparison between Cebus and Macaca shows that M and P ganglion cells have similar sizes in the central retinal region. The results support the view that M and P pathways are similarly organized in diurnal dichromat and trichromat primates.

  19. Running TCP/IP over ATM Networks.

    Science.gov (United States)

    Witt, Michael

    1995-01-01

    Discusses Internet protocol (IP) and subnets and describes how IP may operate over asynchronous transfer mode (ATM). Topics include TCP (transmission control protocol), ATM cells and adaptation layers, a basic architectural model for IP over ATM, address resolution, mapping IP to a subnet technology, and connection management strategy. (LRW)

  20. The pluripotency factor LIN28 in monkey and human testes: a marker for spermatogonial stem cells?

    Science.gov (United States)

    Aeckerle, N; Eildermann, K; Drummer, C; Ehmcke, J; Schweyer, S; Lerchl, A; Bergmann, M; Kliesch, S; Gromoll, J; Schlatt, S; Behr, R

    2012-10-01

    Mammalian spermatogenesis is maintained by spermatogonial stem cells (SSCs). However, since evidentiary assays and unequivocal markers are still missing in non-human primates (NHPs) and man, the identity of primate SSCs is unknown. In contrast, in mice, germ cell transplantation studies have functionally demonstrated the presence of SSCs. LIN28 is an RNA-binding pluripotent stem cell factor, which is also strongly expressed in undifferentiated mouse spermatogonia. By contrast, two recent reports indicated that LIN28 is completely absent from adult human testes. Here, we analyzed LIN28 expression in marmoset monkey (Callithrix jacchus) and human testes during development and adulthood and compared it with that in mice. In the marmoset, LIN28 was strongly expressed in migratory primordial germ cells and gonocytes. Strikingly, we found a rare LIN28-positive subpopulation of spermatogonia also in adult marmoset testis. This was corroborated by western blotting and quantitative RT-PCR. Importantly, in contrast to previous publications, we found LIN28-positive spermatogonia also in normal adult human and additional adult NHP testes. Some seasonal breeders exhibit a degenerated (involuted) germinal epithelium consisting only of Sertoli cells and SSCs during their non-breeding season. The latter re-initiate spermatogenesis prior to the next breeding-season. Fully involuted testes from a seasonal hamster and NHP (Lemur catta) exhibited numerous LIN28-positive spermatogonia, indicating an SSC identity of the labeled cells. We conclude that LIN28 is differentially expressed in mouse and NHP spermatogonia and might be a marker for a rare SSC population in NHPs and man. Further characterization of the LIN28-positive population is required.

  1. The mitochondrial H(+)-ATP synthase and the lipogenic switch: new core components of metabolic reprogramming in induced pluripotent stem (iPS) cells.

    Science.gov (United States)

    Vazquez-Martin, Alejandro; Corominas-Faja, Bruna; Cufi, Sílvia; Vellon, Luciano; Oliveras-Ferraros, Cristina; Menendez, Octavio J; Joven, Jorge; Lupu, Ruth; Menendez, Javier A

    2013-01-15

    Induced pluripotent stem (iPS) cells share some basic properties, such as self-renewal and pluripotency, with cancer cells, and they also appear to share several metabolic alterations that are commonly observed in human tumors. The cancer cells' glycolytic phenotype, first reported by Otto Warburg, is necessary for the optimal routing of somatic cells to pluripotency. However, how iPS cells establish a Warburg-like metabolic phenotype and whether the metabolic pathways that support the bioenergetics of iPS cells are produced by the same mechanisms that are selected during the tumorigenic process remain largely unexplored. We recently investigated whether the reprogramming-competent metabotype of iPS cells involves changes in the activation/expression status of the H(+)-ATPase, which is a core component of mitochondrial oxidative phosphorylation that is repressed at both the activity and protein levels in human carcinomas, and of the lipogenic switch, which refers to a marked overexpression and hyperactivity of the acetyl-CoA carboxylase (ACACA) and fatty acid synthase (FASN) lipogenic enzymes that has been observed in nearly all examined cancer types. A comparison of a starting population of mouse embryonic fibroblasts and their iPS cell progeny revealed that somatic cell reprogramming involves a significant increase in the expression of ATPase inhibitor factor 1 (IF1), accompanied by extremely low expression levels of the catalytic β-F1-ATPase subunit. The pharmacological inhibition of ACACA and FASN activities markedly decreases reprogramming efficiency, and ACACA and FASN expression are notably upregulated in iPS cells. Importantly, iPS cells exhibited a significant intracellular accumulation of neutral lipid bodies; however, these bodies may be a reflection of intense lysosomal/autophagocytic activity rather than bona fide lipid droplet formation in iPS cells, as they were largely unresponsive to pharmacological modulation of PPARgamma and FASN activities. The

  2. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  3. Generation and Characterization of a MYF5 Reporter Human iPS Cell Line Using CRISPR/Cas9 Mediated Homologous Recombination.

    Science.gov (United States)

    Wu, Jianbo; Hunt, Samuel D; Xue, Haipeng; Liu, Ying; Darabi, Radbod

    2016-01-05

    Human iPS cells hold great promise for disease modeling and treatment of degenerative disorders including muscular dystrophies. Although a few research groups have used them for skeletal muscle differentiation, most were based on gene over-expression or long-term mesenchymal differentiation and retrospective identification of myogenic cells. Therefore, this study was aimed to generate a knock-in reporter human iPS cell line for MYF5, as an early myogenic specification gene, to allow prospective identification and purification of myogenic progenitors from human iPS cells. By using a CRISPR/Cas9 double nickase strategy, a 2A-GFP reporter was inserted before the stop codon of the MYF5 gene using homologous recombination. This approach allowed for highly efficient in-frame targeting of MYF5 in human iPS cells. Furthermore, in order to prove the reporter function, endogenous MYF5 expression was induced using a novel dead Cas9-VP160 transcriptional activator. Induced clones demonstrated appropriate MYF5-GFP co-expression. Finally, to confirm the differentiation potential, reporter human iPS clones were differentiated through embryoid body method and MYF5-GFP(+) myogenic cells were sorted and characterized. These data provides valuable guidelines for generation of knock-in reporter human iPS cell lines for myogenic genes which can be used for disease modeling, drug screening, gene correction and future in vivo applications.

  4. Pharmacodynamic model of interleukin-21 effects on red blood cells in cynomolgus monkeys

    DEFF Research Database (Denmark)

    Overgaard, Rune Viig; Karlsson, M.; Ingwersen, S.H.

    2007-01-01

    Interleukin-21 (IL-21) is a novel cytokine that is currently under clinical investigations as a potential anti-cancer agent. Like many other anti-cancer agents, including other interleukins, IL-21 is seen to produce a broad range of biological effects that may be related to both efficacy and safe...... that the structural model presented here can be used for other types of drug induced loss of red blood cells, whereas the mechanism for sampling related blood loss is relevant for investigations of anaemia in all pharmacological studies with smaller animals.......Interleukin-21 (IL-21) is a novel cytokine that is currently under clinical investigations as a potential anti-cancer agent. Like many other anti-cancer agents, including other interleukins, IL-21 is seen to produce a broad range of biological effects that may be related to both efficacy and safety...... of treatment. The present analysis investigates the observed pharmacodynamics effects on red blood cells following various treatment schedules of human IL-21 administrated to cynomolgus monkeys. These effects are described by a novel non-linear mixed-effects model that enabled separation of drug effects...

  5. Analysis of paired end Pol II ChIP-seq and short capped RNA-seq in MCF-7 cells

    Directory of Open Access Journals (Sweden)

    Adam Scheidegger

    2015-09-01

    Full Text Available While a role of promoter-proximal RNA Polymerase II (Pol II pausing in regulation of eukaryotic gene expression is implied, the mechanisms and dynamics of this process are poorly understood. We performed genome-wide analysis of short capped RNAs (scRNAs and Pol II chromatin immunoprecipitation sequencing (ChIP-seq in human breast cancer MCF-7 cells to better understand Pol II pausing (Samarakkody, A., Abbas, A., Scheidegger, A., Warns, J., Nnoli, O., Jokinen, B., Zarns, K., Kubat, B., Dhasarathy, A. and Nechaev, S. (2015 RNA polymerase II pausing can be retained or acquired during activation of genes involved in the epithelial to mesenchymal transition. Nucleic Acids Res 43, 3938–3949. The data are available at the NCBI Gene Expression Omnibus under accession number GSE67041. For both ChIP and scRNA samples, we used paired end sequencing on the Illumina MiSeq instrument. For ChIP-seq, the use of paired end sequencing allowed us to avoid ambiguities in center-read definition. For scRNA seq, this allowed us to identify both the 5′-end and the 3′-end in the same run that represent, respectively, the transcription start sites and the locations of Pol II pausing. The sharpening of Pol II ChIP-seq metagene profiles when aligned against 5′-ends of scRNAs indicates that these RNAs can be used to define the start sites for the majority of mRNA transcription events.

  6. Global gene expression analysis of peripheral blood mononuclear cells in rhesus monkey infants with CA16 infection-induced HFMD.

    Science.gov (United States)

    Song, Jie; Hu, Yajie; Hu, Yunguang; Wang, Jingjing; Zhang, Xiaolong; Wang, Lichun; Guo, Lei; Wang, Yancui; Ning, Ruotong; Liao, Yun; Zhang, Ying; Zheng, Huiwen; Shi, Haijing; He, Zhanlong; Li, Qihan; Liu, Longding

    2016-03-02

    Coxsackievirus A16 (CA16) is a dominant pathogen that results in hand, foot, and mouth disease and causes outbreaks worldwide, particularly in the Asia-Pacific region. However, the underlying molecular mechanisms remain unclear. Our previous study has demonstrated that the basic CA16 pathogenic process was successfully mimicked in rhesus monkey infant. The present study focused on the global gene expression changes in peripheral blood mononuclear cells of rhesus monkey infants with hand, foot, and mouth disease induced by CA16 infection at different time points. Genome-wide expression analysis was performed with Agilent whole-genome microarrays and established bioinformatics tools. Nine hundred and forty-eight significant differentially expressed genes that were associated with 5 gene ontology categories, including cell communication, cell cycle, immune system process, regulation of transcription and metabolic process were identified. Subsequently, the mapping of genes related to the immune system process by PANTHER pathway analysis revealed the predominance of inflammation mediated by chemokine and cytokine signaling pathways and the interleukin signaling pathway. Ultimately, co-expressed genes and their networks were analyzed. The results revealed the gene expression profile of the immune system in response to CA16 in rhesus monkey infants and suggested that such an immune response was generated as a result of the positive mobilization of the immune system. This initial microarray study will provide insights into the molecular mechanism of CA16 infection and will facilitate the identification of biomarkers for the evaluation of vaccines against this virus.

  7. Phenotype and function of myeloid dendritic cells derived from African green monkey blood monocytes.

    Science.gov (United States)

    Mortara, Lorenzo; Ploquin, Mickaël J-Y; Faye, Abdourahmane; Scott-Algara, Daniel; Vaslin, Bruno; Butor, Cécile; Hosmalin, Anne; Barré-Sinoussi, Françoise; Diop, Ousmane M; Müller-Trutwin, Michaela C

    2006-01-20

    Myeloid dendritic cells probably play an important role in the immune response against HIV and SIV, and in the enhancement of CD4+ T cell infection. Here, we have investigated phenotypic and functional features of myeloid monocyte-derived DC (MDDC) from African green monkeys (AGMs). AGMs are natural hosts of SIV and exhibit no signs of abnormal T cell activation despite high SIV plasma viremia. We identified mAbs that cross-react specifically with homologous molecules expressed on AGM DC. We adapted a protocol to derive AGM MDDC by culture in the presence of GM-CSF and IL-4. The differentiated cells possessed a typical dendritic morphology and the majority were CD11c+ DC-SIGN+. AGM MDDC displayed a high expression of typical maturation markers, such as CD83, CD86 and DC-LAMP, and moderate immunostimulatory capacity, suggesting that the cells were in a semi-mature state. Stimulation resulted in further maturation, as shown by up-regulation of CD80 and decrease of endocytosis ability. However, neither increase of HLA-DR or CD40 expression nor enhanced immunostimulatory capacity was observed. The latter was associated with a low pro-inflammatory cytokine production during mixed lymphocyte reactions and a cytokine balance in favour of IL-10 in contrast to human MDDC. This is the first characterization of AGM MDDC. The tools described here are a crucial step for future studies in vivo or in vitro on the function of myeloid DC using the AGM animal model.

  8. Establishment of LIF-dependent human iPS cells closely related to basic FGF-dependent authentic iPS cells.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Hirai

    Full Text Available Human induced pluripotent stem cells (iPSCs can be divided into a leukemia inhibitory factor (LIF-dependent naïve type and a basic fibroblast growth factor (bFGF-dependent primed type. Although the former are more undifferentiated than the latter, they require signal transduction inhibitors and sustained expression of the transgenes used for iPSC production. We used a transcriptionally enhanced version of OCT4 to establish LIF-dependent human iPSCs without the use of inhibitors and sustained transgene expression. These cells belong to the primed type of pluripotent stem cell, similar to bFGF-dependent iPSCs. Thus, the particular cytokine required for iPSC production does not necessarily define stem cell phenotypes as previously thought. It is likely that the bFGF and LIF signaling pathways converge on unidentified OCT4 target genes. These findings suggest that our LIF-dependent human iPSCs could provide a novel model to investigate the role of cytokine signaling in cellular reprogramming.

  9. Uncertain translation, uncertain benefit and uncertain risk: ethical challenges facing first-in-human trials of induced pluripotent stem (ips) cells.

    Science.gov (United States)

    Fung, Ronald K F; Kerridge, Ian H

    2013-02-01

    The discovery of induced pluripotent stem (iPS) cells in 2006 was heralded as a major breakthrough in stem cell research. Since then, progress in iPS cell technology has paved the way towards clinical application, particularly cell replacement therapy, which has refueled debate on the ethics of stem cell research. However, much of the discourse has focused on questions of moral status and potentiality, overlooking the ethical issues which are introduced by the clinical testing of iPS cell replacement therapy. First-in-human trials, in particular, raise a number of ethical concerns including informed consent, subject recruitment and harm minimisation as well as the inherent uncertainty and risks which are involved in testing medical procedures on humans for the first time. These issues, while a feature of any human research, become more complex in the case of iPS cell therapy, given the seriousness of the potential risks, the unreliability of available animal models, the vulnerability of the target patient group, and the high stakes of such an intensely public area of science. Our paper will present a detailed case study of iPS cell replacement therapy for Parkinson's disease to highlight these broader ethical and epistemological concerns. If we accept that iPS cell technology is fraught with challenges which go far beyond merely refuting the potentiality of the stem cell line, we conclude that iPS cell research should not replace, but proceed alongside embryonic and adult somatic stem cell research to promote cross-fertilisation of knowledge and better clinical outcomes.

  10. Differentiation of rat iPS cells and ES cells into granulosa cell-like cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Juan Zhang; Hui Li; Zhao Wu; XiaoJun Tan; Fengying Liu; Xianghong Huang; Xiaoling Fang

    2013-01-01

    Premature ovarian failure (POF) is an ovarian defect characterized by the premature depletion of ovarian follicles before 40 years of age,representing one major cause of female infertility.Stem cells provide the possibility of a potential treatment for POF.In this study,rat embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) were co-cultured with granulosa cells (GCs) to differentiate to GC-like cells.The level of estradiol (E2) analyzed by radioimmunoassay showed that the E2 concentration of the culture supernatant of co-cultured rat iPSCs and ESCs increased in a time-dependent manner,compared with the GCs group that has an opposite trend.The expression of follicle-stimulating hormone receptor (FSHR) was confirmed by immunostaining.These results indicated that rat iPSCs and ESCs were effectively induced to GC-like cells through indirect cell-to-cell contact.Real-time polymerase chain reaction was performed to analyze the expression level of marker genes in POF,including BMP15,FMR1,FSHR,INHA,AMH,NOBOX,FOXO3,EIF2B,FIGLA,and GDF9.The BMP15,FSHR,INHA,AMH,NOBOX,and GDF9 genes were significantly up-regulated in iPSCs and ESCs cocultured with GCs in comparison with ceils that were not co-cultured.Thus,here we demonstrated an available method to differentiate rat iPSCs and ESCs into GC-like cells in vitro for the possible cell therapy of POF.

  11. Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS).

    Science.gov (United States)

    Breitkopf, Susanne B; Yuan, Min; Pihan, German A; Asara, John M

    2012-10-02

    Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.

  12. Induction of autophagy by valproic acid enhanced lymphoma cell chemosensitivity through HDAC-independent and IP3-mediated PRKAA activation.

    Science.gov (United States)

    Ji, Meng-Meng; Wang, Li; Zhan, Qin; Xue, Wen; Zhao, Yan; Zhao, Xia; Xu, Peng-Peng; Shen, Yang; Liu, Han; Janin, Anne; Cheng, Shu; Zhao, Wei-Li

    2015-01-01

    Autophagy is closely related to tumor cell sensitivity to anticancer drugs. The HDAC (histone deacetylase) inhibitor valproic acid (VPA) interacted synergistically with chemotherapeutic agents to trigger lymphoma cell autophagy, which resulted from activation of AMPK (AMP-activated protein kinase) and inhibition of downstream MTOR (mechanistic target of rapamycin [serine/threonine kinase]) signaling. In an HDAC-independent manner, VPA potentiated the effect of doxorubicin on lymphoma cell autophagy via reduction of cellular inositol 1,4,5 trisphosphate (IP3), blockade of calcium into mitochondria and modulation of PRKAA1/2-MTOR cascade. In murine xenograft models established with subcutaneous injection of lymphoma cells, dual treatment of VPA and doxorubicin initiated IP3-mediated calcium depletion and PRKAA1/2 activation, induced in situ autophagy and efficiently retarded tumor growth. Aberrant genes involving mitochondrial calcium transfer were frequently observed in primary tumors of lymphoma patients. Collectively, these findings suggested an HDAC-independent chemosensitizing activity of VPA and provided an insight into the clinical application of targeting autophagy in the treatment of lymphoma.

  13. Studies of tolerance induction through mixed chimerism in cynomolgus monkeys. Method for detection of chimeric cells and effect of thymic irradiation on induction of tolerance

    Energy Technology Data Exchange (ETDEWEB)

    Hoshino, Tomoaki; Kawai, Tatsuo; Ota, Kazuo [Tokyo Women`s Medical Coll. (Japan)

    1996-12-01

    To establish the method for the detection of chimerism in cynomologus monkeys, we tested cross reactivity of various anti-HLA monoclonal antibodies (mAb) to cynomolgus monkeys. In 29 mAb we tested, only three monoclonal anti-HLA antibodies crossreacted with lymphocytes of monkeys. With these mAb, chimeric cell can be detected up to 1% by flow cytometric analysis (study 1). Utilizing the method we developed in study 1, we applied the regimen that induces mixed chimerism and skin graft tolerance in mice to renal allotransplantation of cynomolgus monkey. Regimen A includes non-lethal dose of total body irradiation (TBI), administration of anti-thymocyte globulin (ATG) for 3 days, donor bone marrow infusion and 45 days course of cyclosporine (CYA) administration. We added 7 Gy of thymic irradiation on day-6 in regimen B and on day-1 in regimen C. Although all monkeys in regimen A and B consistently developed chimerism, they rejected kidney allografts soon after stopping CYA. In contrast, 4 monkeys out of 5 failed to develop chimerism in regimen C, but renal allograft tolerance was induced in one monkey who developed chimerism in regimen C. In conclusion, the induction of chimerism is considered necessary but not sufficient for tolerance induction. (author)

  14. Morphology and connections of intratrigeminal cells and axons in the macaque monkey

    Directory of Open Access Journals (Sweden)

    Susan eWarren

    2013-05-01

    Full Text Available Trigeminal primary afferent fibers have small receptive fields and discrete submodalities, but second order trigeminal neurons often display larger receptive fields with complex, multimodal responses. Moreover, while most large caliber afferents terminate exclusively in the principal trigeminal nucleus, and pars caudalis of the spinal trigeminal nucleus receives almost exclusively small caliber afferents, the characteristics of second order neurons do not always reflect this dichotomy. These surprising characteristics may be due to a network of intratrigeminal connections modifying primary afferent contributions. This study characterizes the distribution and morphology of intratrigeminal cells and axons in a macaque monkeys. Tracer injections centered in the principal nucleus and adjacent pars oralis retrogradely labeled neurons bilaterally in pars interpolaris, but only ipsilaterally, in pars caudalis. Labeled axons terminated contralaterally within pars interpolaris and caudalis. Features of the intratrigeminal cells in ipsilateral pars caudalis suggest that both nociceptive and non-nociceptive neurons project to principalis. A commissural projection to contralateral principalis was also revealed. Injections into pars caudalis labeled cells and terminals in the principal nucleus and pars oralis on both sides, indicating the presence of bilateral reciprocal connections. Labeled terminals and cells were also present bilaterally in pars interpolaris and in contralateral pars caudalis. Interpolaris injections produced labeling patterns similar to those of pars caudalis.Thus, the rostral and caudal poles of the macaque trigeminal complex are richly interconnected by ipsilateral ascending and descending connections providing an anatomical substrate for complex analysis of oro-facial stimuli. Sparser reciprocal crossed intratrigeminal connections may be important for conjugate reflex movements, such as the corneal blink reflex.

  15. Radiation-released histamine in the rhesus monkey as modified by mast cell depletion and antihistamine. Scientific report

    Energy Technology Data Exchange (ETDEWEB)

    Doyle, T.F.; Strike, T.A.

    1975-06-01

    Changes in blood histamine concentrations of rhesus monkeys were measured after a 4000-rad dose of mixed gamma-neutron radiation. All animals were pretreated with amino-guanidine to retard histamine catabolism. Histamine concentrations increased from 26 + or - 13.5 to 235 + or - 16 ng/ml after irradiation. When the animals were pretreated with an antihistamine, chlorpheniramine (3 mg/kg), histamine concentrations changed from 25.7 + or - 13.5 to 462 + or - 226 ng/ml after irradiation. When the monkeys were pretreated with a specific mast cell histamine depleter, compound 48/80 (1mg/kg per day) for four consecutive days and then irradiated (4000 rads), histamine concentrations did not change significantly. When 48/80 was given 20 min after irradiation, histamine concentrations changed from 18 + or - 2 ng/ml to a maximum of 35 + or - 9 ng/ml after 48/80 injection. (Author) (GRA)

  16. Age-related changes in dentate gyrus cell numbers, neurogenesis, and associations with cognitive impairments in the rhesus monkey

    Directory of Open Access Journals (Sweden)

    Laura Benjamin Ngwenya

    2015-07-01

    Full Text Available The generation of new neurons in the adult mammalian brain is well established for the hippocampal dentate gyrus. However, the role of neurogenesis in hippocampal function and cognition, how it changes in aging, and the mechanisms underlying this are yet to be elucidated in the monkey brain. To address this, we investigated adult neurogenesis in the dentate gyrus of 42 rhesus monkeys (39 cognitively tested ranging in age from young adult to the elderly. We report here that there is an age-related decline in proliferation and a delayed development of adult neuronal phenotype. Additionally, we show that many of the new neurons survive throughout the lifetime of the animal and may contribute to a modest increase in total neuron number in the granule cell layer of the dentate gyrus over the adult life span. Lastly, we find that measures of decreased adult neurogenesis are only modestly predictive of age-related cognitive impairment.

  17. Generation of Macaca fascicularis iPS cell line ATCi-MF1 from adult skin fibroblasts using non-integrative Sendai viruses

    Directory of Open Access Journals (Sweden)

    Giulia Coppiello

    2017-05-01

    Full Text Available We generated ATCi-MF1 induced pluripotent stem (iPS cell line from Macaca fascicularis adult skin fibroblasts using non-integrative Sendai viruses carrying OCT3/4, KLF4, SOX2 and c-MYC. Once established, ATCi-MF1 cells present a normal karyotype, are Sendai virus-free and express pluripotency associated markers. Microsatellite markers analysis confirmed the origin of the iPS cells from the parental fibroblasts. Pluripotency was tested with the in vivo teratoma formation assay. ATCi-MF1 cell line may be a useful primate iPS cell model to test different experimental conditions where the use of human cells can imply ethical issues, as microinjection of pluripotent stem cells in pre-implantational embryos.

  18. Mechanism insight into the effect of I/P buffer layer on the performance of NIP-type hydrogenated microcrystalline silicon solar cells

    Science.gov (United States)

    Bai, Lisha; Liu, Bofei; Zhao, Jing; Suo, Song; Hou, Guofu; Zhang, Dekun; Sun, Jian; Wei, Changchun; Zhao, Ying; Zhang, Xiaodan

    2015-05-01

    A simulation and experimental study on the effect of the buffer layer at the I/P interface on the performance of NIP-type hydrogenated microcrystalline silicon (μc-Si:H) single-junction solar cells is presented. Device-quality hydrogenated amorphous silicon (a-Si:H) material as a buffer layer at the I/P interface obviously improves the performance of NIP-type μc-Si:H single-junction solar cells. In addition to the well-known mechanism that an a-Si:H I/P buffer layer can reduce the recombination current density at I/P interfaces, the optically and electrically calibrated simulations and supporting experimental results in this study illustrate that the performance improvement also originates from the mitigation of the electric screening effect due to the reduced defect density at the I/P interfaces, which reinforces the bulk electric field. Integrating an optimized hydrogen profiling strategy and adding a-Si:H I/P buffer layer yielded an initial efficiency of 9.20% for μc-Si:H single-junction solar cells with an active area of 0.27 cm2. This study may provide new ideas of further improving the performance of NIP-type μc-Si:H single-junction solar cells by mitigating the electric screening effect.

  19. Differentiation of mouse iPS cells is dependent on embryoid body size in microwell chip culture.

    Science.gov (United States)

    Miyamoto, Daisuke; Nakazawa, Kohji

    2016-10-01

    A microwell chip possessing microwells of several hundred micrometers is a promising platform for generating embryoid bodies (EBs) of stem cells. Here, we investigated the effects of initial EB size on the growth and differentiation of mouse iPS cells in microwell chip culture. We fabricated a chip that contained 195 microwells in a triangular arrangement at a diameter of 600 μm. To evaluate the effect of EB size, four similar conditions were designed with different seeding cell densities of 100, 500, 1000, and 2000 cells/EB. The cells in each microwell gradually aggregated and then spontaneously formed a single EB within 1 d of culture, and EB size increased with further cell proliferation. EB growth was regulated by the initial EB size, and the growth ability of smaller EBs was higher than that of larger EBs. Furthermore, stem cell differentiation also depended on the initial EB size, and the EBs at more than 500 cells/EB promoted hepatic and cardiac differentiations, but the EBs at 100 cells/EB preferred vascular differentiation. These results indicated that the initial EB size was one of the important factors controlling the proliferation and differentiation of stem cells in the microwell chip culture.

  20. Poly IC triggers a cathepsin D- and IPS-1-dependent pathway to enhance cytokine production and mediate dendritic cell necroptosis.

    Science.gov (United States)

    Zou, Jian; Kawai, Taro; Tsuchida, Tetsuo; Kozaki, Tatsuya; Tanaka, Hiroki; Shin, Kyung-Sue; Kumar, Himanshu; Akira, Shizuo

    2013-04-18

    RIG-I-like receptors (RLRs) sense virus-derived RNA or polyinosinic-polycytidylic acid (poly IC) to exert antiviral immune responses. Here, we examine the mechanisms underlying the adjuvant effects of poly IC. Poly IC was taken up by dendritic cells (DCs), and it induced lysosomal destabilization, which, in turn, activated an RLR-dependent signaling pathway. Upon poly IC stimulation, cathepsin D was released into the cytoplasm from the lysosome to interact with IPS-1, an adaptor molecule for RLRs. This interaction facilitated cathepsin D cleavage of caspase 8 and the activation of the transcription factor NF-κB, resulting in enhanced cytokine production. Further recruitment of the kinase RIP-1 to this complex initiated the necroptosis of a small number of DCs. HMGB1 released by dying cells enhanced IFN-β production in concert with poly IC. Collectively, these findings suggest that cathepsin D-triggered, IPS-1-dependent necroptosis is a mechanism that propagates the adjuvant efficacy of poly IC.

  1. Efficient generation of fully reprogrammed human iPS cells via polycistronic retroviral vector and a new cocktail of chemical compounds.

    Directory of Open Access Journals (Sweden)

    Zhonghui Zhang

    Full Text Available Direct reprogramming of human somatic cells into induced pluripotent stem (iPS cells by defined transcription factors (TFs provides great potential for regenerative medicine and biomedical research. This procedure has many challenges, including low reprogramming efficiency, many partially reprogrammed colonies, somatic coding mutations in the genome, etc. Here, we describe a simple approach for generating fully reprogrammed human iPS cells by using a single polycistronic retroviral vector expressing four human TFs in a single open reading frame (ORF, combined with a cocktail containing three small molecules (Sodium butyrate, SB431542, and PD0325901. Our results demonstrate that human iPS cells generated by this approach express human ES cells markers and exhibit pluripotency demonstrated by their abilities to differentiate into the three germ layers in vitro and in vivo. Notably, this approach not only provides a much faster reprogramming process but also significantly diminishes partially reprogrammed iPS cell colonies, thus facilitating efficient isolation of desired fully reprogrammed iPS cell colonies.

  2. Flexoelectric effect in an in-plane switching (IPS) liquid crystal cell for low-power consumption display devices

    Science.gov (United States)

    Kim, Min Su; Bos, Philip J.; Kim, Dong-Woo; Yang, Deng-Ke; Lee, Joong Hee; Lee, Seung Hee

    2016-10-01

    Technology of displaying static images in portable displays, advertising panels and price tags pursues significant reduction in power consumption and in product cost. Driving at a low-frequency electric field in fringe-field switching (FFS) mode can be one of the efficient ways to save powers of the recent portable devices, but a serious drop of image-quality, so-called image-flickering, has been found in terms of the coupling of elastic deformation to not only quadratic dielectric effect but linear flexoelectric effect. Despite of the urgent requirement of solving the issue, understanding of such a phenomenon is yet vague. Here, we thoroughly analyze and firstly report the flexoelectric effect in in-plane switching (IPS) liquid crystal cell. The effect takes place on the area above electrodes due to splay and bend deformations of nematic liquid crystal along oblique electric fields, so that the obvious spatial shift of the optical transmittance is experimentally observed and is clearly demonstrated based on the relation between direction of flexoelectric polarization and electric field polarity. In addition, we report that the IPS mode has inherent characteristics to solve the image-flickering issue in the low-power consumption display in terms of the physical property of liquid crystal material and the electrode structure.

  3. Prefrontal cell activities related to monkeys' success and failure in adapting to rule changes in a Wisconsin Card Sorting Test analog.

    Science.gov (United States)

    Mansouri, Farshad A; Matsumoto, Kenji; Tanaka, Keiji

    2006-03-08

    The cognitive flexibility to select appropriate rules in a changing environment is essential for survival and is assumed to depend on the integrity of prefrontal cortex (PFC). To explore the contribution of the dorsolateral PFC to flexible rule-based behavior, we recorded the activity of cells in this region of monkeys performing a Wisconsin Card Sorting Test (WCST) analog. The monkey had to match a sample to one of three test items by either color or shape. Liquid reward and a discrete visual signal (error signal) were given as feedback to correct and incorrect target selections, respectively. The relevant rule and its frequent changes were not cued, and the monkeys could find it only by interpreting the feedback. In one-third of cells, cellular activity was modulated by the relevant rule, both throughout the trial and between trials. The magnitude of the modulation correlated with the number of errors that the monkeys committed after each rule change in the course of reestablishing high performance. Activity of other cells differed between correct and error trials independently from the rule-related modulation. This difference appeared during actual responses and before the monkeys faced the problems. Many PFC cells responded to the error-signal presentation, and, in some of them, the magnitude of response depended on the relevant rule. These results suggest that the dorsolateral PFC contributes to WCST performance by maintaining the relevant rule across trials, assessing behavioral outcomes, and monitoring the processes that could lead to success and failure in individual trials.

  4. Discrimination of Stem Cell Status after Subjecting Cynomolgus Monkey Pluripotent Stem Cells to Naïve Conversion

    Science.gov (United States)

    Honda, Arata; Kawano, Yoshihiro; Izu, Haruna; Choijookhuu, Narantsog; Honsho, Kimiko; Nakamura, Tomonori; Yabuta, Yukihiro; Yamamoto, Takuya; Takashima, Yasuhiro; Hirose, Michiko; Sankai, Tadashi; Hishikawa, Yoshitaka; Ogura, Atsuo; Saitou, Mitinori

    2017-01-01

    Experimental animal models have played an indispensable role in the development of human induced pluripotent stem cell (iPSC) research. The derivation of high-quality (so-called “true naïve state”) iPSCs of non-human primates enhances their application and safety for human regenerative medicine. Although several attempts have been made to convert human and non-human primate PSCs into a truly naïve state, it is unclear which evaluation methods can discriminate them as being truly naïve. Here we attempted to derive naïve cynomolgus monkey (Cm) (Macaca fascicularis) embryonic stem cells (ESCs) and iPSCs. Several characteristics of naïve Cm ESCs including colony morphology, appearance of naïve-related mRNAs and proteins, leukaemia inhibitory factor dependency, and mitochondrial respiration were confirmed. Next, we generated Cm iPSCs and converted them to a naïve state. Transcriptomic comparison of PSCs with early Cm embryos elucidated the partial achievement (termed naïve-like) of their conversion. When these were subjected to in vitro neural differentiation, enhanced differentiating capacities were observed after naïve-like conversion, but some lines exhibited heterogeneity. The difficulty of achieving contribution to chimeric mouse embryos was also demonstrated. These results suggest that Cm PSCs could ameliorate their in vitro neural differentiation potential even though they could not display true naïve characteristics. PMID:28349944

  5. A Phase I Study of iPS Cell Generation From Patients With COPD

    Science.gov (United States)

    2016-06-30

    Thoracic Diseases; Respiratory Tract Diseases; Cancer of Lung; Cancer of the Lung; Lung Cancer; Lung Diseases, Obstructive; COPD; Pulmonary Emphysema; Neoplasms, Lung; Neoplasms, Pulmonary; Pulmonary Cancer; Pulmonary Neoplasms; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell

  6. Radiolabeling human peripheral blood stem cells for positron emission tomography (PET imaging in young rhesus monkeys.

    Directory of Open Access Journals (Sweden)

    Alice F Tarantal

    Full Text Available These studies focused on a new radiolabeling technique with copper ((64Cu and zirconium ((89Zr for positron emission tomography (PET imaging using a CD45 antibody. Synthesis of (64Cu-CD45 and (89Zr-CD45 immunoconjugates was performed and the evaluation of the potential toxicity of radiolabeling human peripheral blood stem cells (hPBSC was assessed in vitro (viability, population doubling times, colony forming units. hPBSC viability was maintained as the dose of (64Cu-TETA-CD45 increased from 0 (92% to 160 µCi/mL (76%, p>0.05. Radiolabeling efficiency was not significantly increased with concentrations of (64Cu-TETA-CD45 >20 µCi/mL (p>0.50. Toxicity affecting both growth and colony formation was observed with hPBSC radiolabeled with ≥40 µCi/mL (p0.05, and a trend towards increased radiolabeling efficiency was noted as the dose of (89Zr-Df-CD45 increased, with a greater level of radiolabeling with 160 µCi/mL compared to 0-40 µCi/mL (p<0.05. A greater than 2,000 fold-increase in the level of (89Zr-Df-CD45 labeling efficiency was observed when compared to (64Cu-TETA-CD45. Similar to (64Cu-TETA-CD45, toxicity was noted when hPBSC were radiolabeled with ≥40 µCi/mL (p<0.05 (growth, colony formation. Taken together, 20 µCi/mL resulted in the highest level of radiolabeling efficiency without altering cell function. Young rhesus monkeys that had been transplanted prenatally with 25×10(6 hPBSC expressing firefly luciferase were assessed with bioluminescence imaging (BLI, then 0.3 mCi of (89Zr-Df-CD45, which showed the best radiolabeling efficiency, was injected intravenously for PET imaging. Results suggest that (89Zr-Df-CD45 was able to identify engrafted hPBSC in the same locations identified by BLI, although the background was high.

  7. Increased positive selection pressure within the complementarity determining regions of the T-cell receptor β gene in New World monkeys.

    Science.gov (United States)

    Matsutani, Takaji; Fujii, Yoshiki; Kitaura, Kazutaka; Suzuki, Satsuki; Tsuruta, Yuji; Takasaki, Tomohiko; Ogasawara, Kouetsu; Nishimoto, Norihiro; Kurane, Ichiro; Suzuki, Ryuji

    2011-10-01

    Because of the long-term co-evolution of TCR and MHC molecules, numerous nucleotide substitutions have accumulated within the domains of TCRβ genes. We previously found that nonsynonymous nucleotide substitutions occurred more frequently in complementarity determining region (CDR)β than in CDRα, even though only a limited number of common marmoset (Callithrix jacchus) and human T-cell receptor β variable (TRBV) sequences were compared. This interesting finding raised the question of whether the increased selective pressure within CDRβ was species-specific. In this study, we identified 21 TRBV region sequences from the common marmoset and performed comparative sequence analyses of the T-cell receptor α variable (TRAV) and TRBV regions from human, chimpanzee, rhesus monkey, cotton-top tamarin, Ma's night monkey, and common marmoset. The ratios of the number of nonsynonymous nucleotide substitutions per site (d(N) ) to the d(S) values (d(N) /d(S) ) were less than 1 within the framework regions (FRs) of TRAV and TRBV region sequences, suggesting that purifying selection is largely dominant within the FRs. In contrast, the d(N) values were statistically significantly greater for CDRβ than for CDRα only in New World monkeys. Also, increased d(N) /d(S) ratios (d(N) /d(S) >1) were observed within CDRβ between humans and New World monkeys and, interestingly, between New World monkeys, which share a relatively recent common ancestor. Moreover, phylogenetic analysis by maximum likelihood analysis provided firm evidence to support that positive selection occurred within CDRβ along New World monkey lineages. These results suggest that increased positive selection pressure within CDRβ is common in New World monkeys rather than being species-specific. This study provides an intriguing insight into the co-evolution of TCR and MHC molecules within primates. © 2011 Wiley-Liss, Inc.

  8. Pitfalls in global normalization of ChIP-seq data in CD4(+) T cells treated with butyrate: A possible solution strategy.

    Science.gov (United States)

    Furusawa, Yukihiro; Endo, Takaho A; Obata, Yuuki; Ohara, Osamu; Ohno, Hiroshi; Hase, Koji

    2014-12-01

    Regulatory T cells (Treg) play a central role in the suppression of inflammatory and allergic responses. Colonization of certain gut commensal microbes such as Clostridia class IV and XIVa in the gut can induce development of colonic Treg cells contributing to the maintenance of gut immune homeostasis. Clostridia-derived butyrate promotes the differentiation of naïve T cells into Treg cells through upregulation of Foxp3, the master transcription factor of Treg cells. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that treatment of naïve T cells with butyrate induces Treg-polarizing conditions by enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus. In general, global normalization was utilized for ChIP-seq analysis to compare the data obtained from two or more samples. However, global normalization is not appropriate for the evaluation of ChIP-seq data when treatment can affect the total amount of target protein. Here, we introduce a unique normalization method for ChIP-seq analysis in cells treated with butyrate, a pan-HDAC inhibitor that is likely to affect total acetylation levels of histone H3.

  9. Proteomic and phosphoproteomic comparison of human ES and iPS cells.

    Science.gov (United States)

    Phanstiel, Douglas H; Brumbaugh, Justin; Wenger, Craig D; Tian, Shulan; Probasco, Mitchell D; Bailey, Derek J; Swaney, Danielle L; Tervo, Mark A; Bolin, Jennifer M; Ruotti, Victor; Stewart, Ron; Thomson, James A; Coon, Joshua J

    2011-01-01

    Combining high-mass-accuracy mass spectrometry, isobaric tagging and software for multiplexed, large-scale protein quantification, we report deep proteomic coverage of four human embryonic stem cell and four induced pluripotent stem cell lines in biological triplicate. This 24-sample comparison resulted in a very large set of identified proteins and phosphorylation sites in pluripotent cells. The statistical analysis afforded by our approach revealed subtle but reproducible differences in protein expression and protein phosphorylation between embryonic stem cells and induced pluripotent cells. Merging these results with RNA-seq analysis data, we found functionally related differences across each tier of regulation. We also introduce the Stem Cell-Omics Repository (SCOR), a resource to collate and display quantitative information across multiple planes of measurement, including mRNA, protein and post-translational modifications.

  10. Genetic engineering of human ES and iPS cells using TALE nucleases

    OpenAIRE

    Hockemeyer, Dirk; Wang, Haoyi; Kiani, Samira; Lai, Christine S.; Gao, Qing; Cassady, John P.; Cost, Gregory J.; Zhang, Lei; Santiago, Yolanda; Miller, Jeffrey C; Zeitler, Bryan; Cherone, Jennifer M.; Meng, Xiangdong; Hinkley, Sarah J; Rebar, Edward J.

    2011-01-01

    Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator–like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that T...

  11. Rhesus monkey cumulus cells revert to a mural granulosa cell state after an ovulatory stimulus.

    Science.gov (United States)

    Chaffin, Charles L; Lee, Young S; VandeVoort, Catherine A; Patel, Bela G; Latham, Keith E

    2012-11-01

    Follicular somatic cells (mural granulosa cells and cumulus cells) and the oocyte communicate through paracrine interactions and through direct gap junctions between oocyte and cumulus cells. Considering that mural and cumulus cells arise through a common developmental pathway and that their differentiation is essential to reproductive success, understanding how these cells differ is a key aspect to understanding their critical functions. Changes in global gene expression before and after an ovulatory stimulus were compared between cumulus and mural granulosa cells to test the hypothesis that mural and cumulus cells are highly differentiated at the time of an ovulatory stimulus and further differentiate during the periovulatory interval. The transcriptomes of the two cell types were markedly different (>1500 genes) before an ovulatory hCG bolus but converged after ovulation to become completely overlapping. The predominant transition was for the cumulus cells to become more like mural cells after hCG. This indicates that the differentiated phenotype of the cumulus cell is not stable and irreversibly established but may rather be an ongoing physiological response to the oocyte.

  12. An ES-Like pluripotent state in FGF-dependent murine iPS cells

    NARCIS (Netherlands)

    B. di Stefano (Bruno); C. Buecker (Christa); F. Ungaro (Federica); A. Prigione (Alessandro); H.H. Chen; M. Welling (Maaike); M. Eijpe (Maureen); G. Mostoslavsky (Gustavo); P. Tesar (Paul); J. Adjaye (James); N. Geijsen (Niels); V. Broccoli (Vania)

    2010-01-01

    textabstractRecent data demonstrates that stem cells can exist in two morphologically, molecularly and functionally distinct pluripotent states; a naïve LIF-dependent pluripotent state which is represented by murine embryonic stem cells (mESCs) and an FGFdependent primed pluripotent state represente

  13. Spontaneous mutants of the adenovirus-simian virus 40 hybrid, Ad2/sup +/ND3, that grow efficiently in monkey cells

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, C.W.

    1981-05-01

    An attempt was made to isolate spontaneous mutants of adenovirus type 2 and of the adenovirus-SV40 hybrids, Ad2/sup +/ND3 and Ad2/sup +/ND5, that would grow efficiently on monkey cells. Virus stocks were serially passaged through the semipermissive established monkey line CV-1. After five serial passages in the absence of intentional mutagenesis, only stocks of Ad2/sup +/ND3 yielded significant numbers of variants that plaqued with similar efficiency on human and on monkey cell monolayers. Four independent Ad2/sup +/ND3 variants, designated hr600, hr601, hr602, and hr603, have been isolated and partially characterized. No difference was found between the genomes of these variants and the genome of parental Ad2/sup +/ND3 by restriction enzyme analysis or by the analysis of heteroduplexes between Ad2/sup +/ND3 (or variant) DNA and DNA of the hybrid Ad2/sup +/ND1.

  14. An HDAC2-TET1 switch at distinct chromatin regions significantly promotes the maturation of pre-iPS to iPS cells.

    Science.gov (United States)

    Wei, Tingyi; Chen, Wen; Wang, Xiukun; Zhang, Man; Chen, Jiayu; Zhu, Songcheng; Chen, Long; Yang, Dandan; Wang, Guiying; Jia, Wenwen; Yu, Yangyang; Duan, Tao; Wu, Minjuan; Liu, Houqi; Gao, Shaorong; Kang, Jiuhong

    2015-06-23

    The maturation of induced pluripotent stem cells (iPS) is one of the limiting steps of somatic cell reprogramming, but the underlying mechanism is largely unknown. Here, we reported that knockdown of histone deacetylase 2 (HDAC2) specifically promoted the maturation of iPS cells. Further studies showed that HDAC2 knockdown significantly increased histone acetylation, facilitated TET1 binding and DNA demethylation at the promoters of iPS cell maturation-related genes during the transition of pre-iPS cells to a fully reprogrammed state. We also found that HDAC2 competed with TET1 in the binding of the RbAp46 protein at the promoters of maturation genes and knockdown of TET1 markedly prevented the activation of these genes. Collectively, our data not only demonstrated a novel intrinsic mechanism that the HDAC2-TET1 switch critically regulates iPS cell maturation, but also revealed an underlying mechanism of the interplay between histone acetylation and DNA demethylation in gene regulation.

  15. Effects of ICOS+ T cell depletion via afucosylated monoclonal antibody MEDI-570 on pregnant cynomolgus monkeys and the developing offspring.

    Science.gov (United States)

    Nicholson, Simone M; Carlesso, Gianluca; Cheng, Lily I; Cook, Halie; DaCosta, Karma; Leininger, Joel; McKeever, Kathleen; Scott, Stephen Weasel; Taylor, Devon; Streicher, Katie; Eck, Steve; Reed, Molly; Faggioni, Raffaella; Herbst, Ronald; Dixit, Rakesh; Ryan, Patricia C

    2017-09-13

    MEDI-570 is a fully human afucosylated monoclonal antibody (MAb) against Inducible T-cell costimulator (ICOS), highly expressed on CD4+ T follicular helper (TFH) cells. Effects of MEDI-570 were evaluated in an enhanced pre-postnatal development toxicity (ePPND) study in cynomolgus monkeys. Administration to pregnant monkeys did not cause any abortifacient effects. Changes in hematology and peripheral blood T lymphocyte subsets in maternal animals and infants and the attenuated infant IgG immune response to keyhole limpet hemocyanin (KLH) were attributed to MEDI-570 pharmacology. Adverse findings included aggressive fibromatosis in one dam and two infant losses in the high dose group with anatomic pathology findings suggestive of atypical lymphoid hyperplasia. The margin of safety relative to the no observed adverse effect level (NOAEL) for the highest planned clinical dose in the Phase 1a study was 7. This study suggests that women of child bearing potential employ effective methods of contraception while being treated with MEDI-570. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Profiling of microRNA in human and mouse ES and iPS cells reveals overlapping but distinct microRNA expression patterns.

    Science.gov (United States)

    Razak, Siti Razila Abdul; Ueno, Kazuko; Takayama, Naoya; Nariai, Naoki; Nagasaki, Masao; Saito, Rika; Koso, Hideto; Lai, Chen-Yi; Murakami, Miyako; Tsuji, Koichiro; Michiue, Tatsuo; Nakauchi, Hiromitsu; Otsu, Makoto; Watanabe, Sumiko

    2013-01-01

    Using quantitative PCR-based miRNA arrays, we comprehensively analyzed the expression profiles of miRNAs in human and mouse embryonic stem (ES), induced pluripotent stem (iPS), and somatic cells. Immature pluripotent cells were purified using SSEA-1 or SSEA-4 and were used for miRNA profiling. Hierarchical clustering and consensus clustering by nonnegative matrix factorization showed two major clusters, human ES/iPS cells and other cell groups, as previously reported. Principal components analysis (PCA) to identify miRNAs that segregate in these two groups identified miR-187, 299-3p, 499-5p, 628-5p, and 888 as new miRNAs that specifically characterize human ES/iPS cells. Detailed direct comparisons of miRNA expression levels in human ES and iPS cells showed that several miRNAs included in the chromosome 19 miRNA cluster were more strongly expressed in iPS cells than in ES cells. Similar analysis was conducted with mouse ES/iPS cells and somatic cells, and several miRNAs that had not been reported to be expressed in mouse ES/iPS cells were suggested to be ES/iPS cell-specific miRNAs by PCA. Comparison of the average expression levels of miRNAs in ES/iPS cells in humans and mice showed quite similar expression patterns of human/mouse miRNAs. However, several mouse- or human-specific miRNAs are ranked as high expressers. Time course tracing of miRNA levels during embryoid body formation revealed drastic and different patterns of changes in their levels. In summary, our miRNA expression profiling encompassing human and mouse ES and iPS cells gave various perspectives in understanding the miRNA core regulatory networks regulating pluripotent cells characteristics.

  17. Müller cells express the cannabinoid CB2 receptor in the vervet monkey retina

    DEFF Research Database (Denmark)

    Bouskila, Joseph; Javadi, Pasha; Casanova, Christian

    2013-01-01

    The presence of the cannabinoid receptor type 1 (CB1R) has been largely documented in the rodent and primate retinae in recent years. There is, however, some controversy concerning the presence of the CB2 receptor (CB2R) within the central nervous system. Only recently, CB2R has been found...... in the rodent retina, but its presence in the primate retina has not yet been demonstrated. The aim of this study was twofold: 1) to characterize the distribution patterns of CB2R in the monkey retina and compare this distribution with that previously reported for CB1R and 2) to resolve the controversy...... on the presence of CB2R in the neural component of the retina. We therefore thoroughly examined the cellular localization of CB2R in the vervet monkey (Chlorocebus sabeus) retina, using confocal microscopy. Our results demonstrate that CB2R, like CB1R, is present throughout the retinal layers, but with striking...

  18. Gate leakage current reduction in IP3 SRAM cells at 45 nm CMOS technology for multimedia applications

    Institute of Scientific and Technical Information of China (English)

    R.K.Singh; Neeraj Kr.Shukla; Manisha Pattanaik

    2012-01-01

    We have presented an analysis of the gate leakage current of the IP3 static random access memory (SRAM) cell structure when the cell is in idle mode (performs no data read/write operations) and active mode (performs data read/write operations),along with the requirements for the overall standby leakage power,active write and read powers.A comparison has been drawn with existing SRAM cell structures,the conventional 6T,PP,P4 and P3 cells.At the supply voltage,VDD =0.8 V,a reduction of 98%,99%,92% and 94% is observed in the gate leakage current in comparison with the 6T,PP P4 and P3 SRAM cells,respectively,while at VDD =0.7 V,it is 97%,98%,87% and 84%.A significant reduction is also observed in the overall standby leakage power by 56%,the active write power by 44% and the active read power by 99%,compared with the conventional 6T SRAM cell at VDD =0.8 V,with no loss in cell stability and performance with a small area penalty.The simulation environment used for this work is 45 nm deep sub-micron complementary metal oxide semiconductor (CMOS) technology,tox =2.4 nm,Vthn =0.22 V,Vthp =0.224 V,VDD =0.7 V and 0.8 V,at T =300 K.

  19. Isolation of single-base genome-edited human iPS cells without antibiotic selection.

    Science.gov (United States)

    Miyaoka, Yuichiro; Chan, Amanda H; Judge, Luke M; Yoo, Jennie; Huang, Miller; Nguyen, Trieu D; Lizarraga, Paweena P; So, Po-Lin; Conklin, Bruce R

    2014-03-01

    Precise editing of human genomes in pluripotent stem cells by homology-driven repair of targeted nuclease-induced cleavage has been hindered by the difficulty of isolating rare clones. We developed an efficient method to capture rare mutational events, enabling isolation of mutant lines with single-base substitutions without antibiotic selection. This method facilitates efficient induction or reversion of mutations associated with human disease in isogenic human induced pluripotent stem cells.

  20. ChIP-on-chip analysis identifies IL-22 as direct target gene of ectopically expressed FOXP3 transcription factor in human T cells

    Directory of Open Access Journals (Sweden)

    Jeron Andreas

    2012-12-01

    Full Text Available Abstract Background The transcription factor (TF forkhead box P3 (FOXP3 is constitutively expressed at high levels in naturally occurring CD4+CD25+ regulatory T cells (nTregs. It is not only the most accepted marker for that cell population but is also considered lineage determinative. Chromatin immunoprecipitation (ChIP of TFs in combination with genomic tiling microarray analysis (ChIP-on-chip has been shown to be an appropriate tool for identifying FOXP3 transcription factor binding sites (TFBSs on a genome-wide scale. In combination with microarray expression analysis, the ChIP-on-chip technique allows identification of direct FOXP3 target genes. Results ChIP-on-chip analysis of the human FOXP3 expressed in resting and PMA/ionomycin–stimulated Jurkat T cells revealed several thousand putative FOXP3 binding sites and demonstrated the importance of intronic regions for FOXP3 binding. The analysis of expression data showed that the stimulation-dependent down-regulation of IL-22 was correlated with direct FOXP3 binding in the IL-22 promoter region. This association was confirmed by real-time PCR analysis of ChIP-DNA. The corresponding ChIP-region also contained a matching FOXP3 consensus sequence. Conclusions Knowledge of the general distribution patterns of FOXP3 TFBSs in the human genome under resting and activated conditions will contribute to a better understanding of this TF and its influence on direct target genes, as well as its importance for the phenotype and function of Tregs. Moreover, FOXP3-dependent repression of Th17-related IL-22 may be relevant to an understanding of the phenomenon of Treg/Th17 cell plasticity.

  1. ChIP-on-chip analysis identifies IL-22 as direct target gene of ectopically expressed FOXP3 transcription factor in human T cells.

    Science.gov (United States)

    Jeron, Andreas; Hansen, Wiebke; Ewert, Franziska; Buer, Jan; Geffers, Robert; Bruder, Dunja

    2012-12-17

    The transcription factor (TF) forkhead box P3 (FOXP3) is constitutively expressed at high levels in naturally occurring CD4+CD25+ regulatory T cells (nTregs). It is not only the most accepted marker for that cell population but is also considered lineage determinative. Chromatin immunoprecipitation (ChIP) of TFs in combination with genomic tiling microarray analysis (ChIP-on-chip) has been shown to be an appropriate tool for identifying FOXP3 transcription factor binding sites (TFBSs) on a genome-wide scale. In combination with microarray expression analysis, the ChIP-on-chip technique allows identification of direct FOXP3 target genes. ChIP-on-chip analysis of the human FOXP3 expressed in resting and PMA/ionomycin-stimulated Jurkat T cells revealed several thousand putative FOXP3 binding sites and demonstrated the importance of intronic regions for FOXP3 binding. The analysis of expression data showed that the stimulation-dependent down-regulation of IL-22 was correlated with direct FOXP3 binding in the IL-22 promoter region. This association was confirmed by real-time PCR analysis of ChIP-DNA. The corresponding ChIP-region also contained a matching FOXP3 consensus sequence. Knowledge of the general distribution patterns of FOXP3 TFBSs in the human genome under resting and activated conditions will contribute to a better understanding of this TF and its influence on direct target genes, as well as its importance for the phenotype and function of Tregs. Moreover, FOXP3-dependent repression of Th17-related IL-22 may be relevant to an understanding of the phenomenon of Treg/Th17 cell plasticity.

  2. Modelling of pressurised hybrid systems based on integrated planar solid oxide fuel cell (IP-SOFC) technology

    Energy Technology Data Exchange (ETDEWEB)

    Magistri, L.; Traverso, A.; Massardo, A.F. [TPG-DIMSET, University of Genoa, Via Montallegro 1, 16145 Genova (Italy); Cerutti, F.; Costamagna, P. [TPG-DICHEP, University of Genoa, Via Opera Pia 15, 16145 Genova (Italy); Bozzolo, M. [Rolls-Royce Fuel Cell Systems Ltd, PO Box 31, Derby DE24 8BJ (United Kingdom)

    2005-02-01

    This work describes different models, developed by the Thermochemical Power Group at the University of Genoa (Italy), for the simulation of solid oxide fuel cell and gas turbine hybrid systems. The paper focuses on both ''cores'' of the system: the fuel cell stack on the one hand and the turbomachinery and the auxiliaries on the other hand. Therefore, in the first part of the paper the models developed for the analysis of the Rolls-Royce Integrated Planar SOFC cells are presented; the results are compared to experimental data, and carefully analysed and discussed. In the second part of the paper, design and off design models of IP-SOFC pressurised hybrid systems in the range 250 kW-20 MW are presented; the hybrid performance results are presented and discussed, also taking ambient condition effects and a possible control strategy into account. Finally, using an in-house general purpose transient system analysis code (TRANSEO code), where chemical composition, heat transfer, and fluid dynamic influences vs. time are considered in detail, a preliminary time dependent investigation of a pressurised hybrid system behaviour is presented. (Abstract Copyright [2005], Wiley Periodicals, Inc.)

  3. Identifying Cell Type-Specific Transcription Factors by Integrating ChIP-seq and eQTL Data-Application to Monocyte Gene Regulation.

    Science.gov (United States)

    Choudhury, Mudra; Ramsey, Stephen A

    2016-01-01

    We describe a novel computational approach to identify transcription factors (TFs) that are candidate regulators in a human cell type of interest. Our approach involves integrating cell type-specific expression quantitative trait locus (eQTL) data and TF data from chromatin immunoprecipitation-to-tag-sequencing (ChIP-seq) experiments in cell lines. To test the method, we used eQTL data from human monocytes in order to screen for TFs. Using a list of known monocyte-regulating TFs, we tested the hypothesis that the binding sites of cell type-specific TF regulators would be concentrated in the vicinity of monocyte eQTLs. For each of 397 ChIP-seq data sets, we obtained an enrichment ratio for the number of ChIP-seq peaks that are located within monocyte eQTLs. We ranked ChIP-seq data sets according to their statistical significances for eQTL overlap, and from this ranking, we observed that monocyte-regulating TFs are more highly ranked than would be expected by chance. We identified 27 TFs that had significant monocyte enrichment scores and mapped them into a protein interaction network. Our analysis uncovered two novel candidate monocyte-regulating TFs, BCLAF1 and SIN3A. Our approach is an efficient method to identify candidate TFs that can be used for any cell/tissue type for which eQTL data are available.

  4. Monkey Business

    Science.gov (United States)

    Blackwood, Christine Horvatis

    2012-01-01

    A ballerina, a gladiator, a camper, a baseball player, a surfer, and a shopper; these are just a few of the amazing monkeys that the author's seventh graders created from papier-mache. This project provided an opportunity for students to express themselves through the creation of sculptural characters based on their own interests, hobbies, and…

  5. Monkey Business

    Science.gov (United States)

    Blackwood, Christine Horvatis

    2012-01-01

    A ballerina, a gladiator, a camper, a baseball player, a surfer, and a shopper; these are just a few of the amazing monkeys that the author's seventh graders created from papier-mache. This project provided an opportunity for students to express themselves through the creation of sculptural characters based on their own interests, hobbies, and…

  6. Detection of thalidomide embryotoxicity by in vitro embryotoxicity testing based on human iPS cells.

    Science.gov (United States)

    Aikawa, Nobuo; Kunisato, Atsushi; Nagao, Kenji; Kusaka, Hideaki; Takaba, Katsumi; Ohgami, Kinya

    2014-01-01

    The mouse embryonic stem cell test (mEST) is used to assess the embryotoxicity of drug candidates by evaluating the effects on the cardiac differentiation of stem cells. However, thalidomide embryotoxicity has not yet been reported using the mEST. To detect the effects of thalidomide, we used human induced pluripotent stem cells (hiPSCs) instead of mouse embryonic stem cells, and assessed three endpoints: the inhibition of cardiac differentiation, the cytotoxicity to hiPSCs, and the cytotoxicity to human dermal fibroblasts, according to the mEST. From these data (IC50 values), the embryotoxicity was classified into one of three different classes based on the mEST and our criteria. Valproate was used as a positive control and ascorbic acid was used as a negative control, and their effects were assessed. Similar to valproate, thalidomide was classified as a Class 2 agent, with weak embryotoxicity, by the mEST criteria, and was classified as Category 3 embryotoxic based on our criteria. Ascorbic acid was classified as a Class 1 / Category 1, non-embryotoxic agent, based on both criteria. Thalidomide embryotoxicity was detected in the embryonic stem cell test based on hiPSCs. This test system is thus considered to have a much greater predictive ability than the mEST.

  7. iPS Cells for Modelling and Treatment of Retinal Diseases

    Directory of Open Access Journals (Sweden)

    Fred K. Chen

    2014-12-01

    Full Text Available For many decades, we have relied on immortalised retinal cell lines, histology of enucleated human eyes, animal models, clinical observation, genetic studies and human clinical trials to learn more about the pathogenesis of retinal diseases and explore treatment options. The recent availability of patient-specific induced pluripotent stem cells (iPSC for deriving retinal lineages has added a powerful alternative tool for discovering new disease-causing mutations, studying genotype-phenotype relationships, performing therapeutics-toxicity screening and developing personalised cell therapy. This review article provides a clinical perspective on the current and potential benefits of iPSC for managing the most common blinding diseases of the eye: inherited retinal diseases and age-related macular degeneration.

  8. Generation of Col2a1-EGFP iPS cells for monitoring chondrogenic differentiation.

    Directory of Open Access Journals (Sweden)

    Taku Saito

    Full Text Available Induced pluripotent stem cells (iPSC are a promising cell source for cartilage regenerative medicine; however, the methods for chondrocyte induction from iPSC are currently developing and not yet sufficient for clinical application. Here, we report the establishment of a fluorescent indicator system for monitoring chondrogenic differentiation from iPSC to simplify screening for effective factors that induce chondrocytes from iPSC. We generated iPSC from embryonic fibroblasts of Col2a1-EGFP transgenic mice by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. Among the 30 clones of Col2a1-EGFP iPSC we established, two clones showed high expression levels of embryonic stem cell (ESC marker genes, similar to control ESC. A teratoma formation assay showed that the two clones were pluripotent and differentiated into cell types from all three germ layers. The fluorescent signal was observed during chondrogenic differentiation of the two clones concomitant with the increase in chondrocyte marker expression. In conclusion, Col2a1-EGFP iPSC are useful for monitoring chondrogenic differentiation and will contribute to research in cartilage regenerative medicine.

  9. Cloning Mice and Men: Prohibiting the Use of iPS Cells for Human Reproductive Cloning

    OpenAIRE

    Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

    2010-01-01

    The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation.

  10. Cloning mice and men: prohibiting the use of iPS cells for human reproductive cloning.

    Science.gov (United States)

    Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

    2010-01-08

    The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation.

  11. Juxtaglomerular cell CaSR stimulation decreases renin release via activation of the PLC/IP(3) pathway and the ryanodine receptor.

    Science.gov (United States)

    Ortiz-Capisano, M Cecilia; Reddy, Mahendranath; Mendez, Mariela; Garvin, Jeffrey L; Beierwaltes, William H

    2013-02-01

    The calcium-sensing receptor (CaSR) is a G-coupled protein expressed in renal juxtaglomerular (JG) cells. Its activation stimulates calcium-mediated decreases in cAMP content and inhibits renin release. The postreceptor pathway for the CaSR in JG cells is unknown. In parathyroids, CaSR acts through G(q) and/or G(i). Activation of G(q) stimulates phospholipase C (PLC), and inositol 1,4,5-trisphosphate (IP(3)), releasing calcium from intracellular stores. G(i) stimulation inhibits cAMP formation. In afferent arterioles, the ryanodine receptor (RyR) enhances release of stored calcium. We hypothesized JG cell CaSR activation inhibits renin via the PLC/IP(3) and also RyR activation, increasing intracellular calcium, suppressing cAMP formation, and inhibiting renin release. Renin release from primary cultures of isolated mouse JG cells (n = 10) was measured. The CaSR agonist cinacalcet decreased renin release 56 ± 7% of control (P PLC inhibitor U73122 reversed cinacalcet inhibition of renin (104 ± 11% of control). The IP(3) inhibitor 2-APB also reversed inhibition of renin from 56 ± 6 to 104 ± 11% of control (P PLC/IP(3) pathway, activating RyR, increasing intracellular calcium, and resulting in calcium-mediated renin inhibition.

  12. Boron Concentration Measurements at the I/P Interface in Nip a-Si Solar Cells

    DEFF Research Database (Denmark)

    Aken, Van; Duchamp, Martial; Boothroyd, Chris

    2011-01-01

    The p-type Si layer in n-i-p a-Si and μc-Si solar cells on foil has several important requirements with respect to conductivity and optical transmission. We control the optical band gap and activation energy of p-a-SiC by varying the B2H6 and CH4 flows in the process chamber. Modelling shows...... that the optimum efficiency in n-i-p solar cells is obtained when the p-a-SiC band gap is just above the band gap of the absorber layer. We have assessed the potential of core-loss electron energy-loss spectroscopy (EELS) for detecting B and C and of low-loss EELS, in a spatially resolved manner, as probe of local...

  13. Boron Concentration Measurements at the I/P Interface in Nip a-Si Solar Cells

    DEFF Research Database (Denmark)

    Aken, Van; Duchamp, Martial; Boothroyd, Chris

    2011-01-01

    The p-type Si layer in n-i-p a-Si and μc-Si solar cells on foil has several important requirements with respect to conductivity and optical transmission. We control the optical band gap and activation energy of p-a-SiC by varying the B2H6 and CH4 flows in the process chamber. Modelling shows...... that the optimum efficiency in n-i-p solar cells is obtained when the p-a-SiC band gap is just above the band gap of the absorber layer. We have assessed the potential of core-loss electron energy-loss spectroscopy (EELS) for detecting B and C and of low-loss EELS, in a spatially resolved manner, as probe of local...

  14. DNA repair and replication links to pluripotency and differentiation capacity of pig iPS cells

    Science.gov (United States)

    Song, Lipu; Fan, Anran; Zhang, Sheng; Wang, Jianyu; Fan, Nana; Liu, Na; Ye, Xiaoying; Fu, Haifeng; Zhou, Zhongcheng; Wang, Yong; Wei, Hong; Liu, Zhonghua; Li, Ziyi; Lai, Liangxue; Wang, Xumin; Liu, Lin

    2017-01-01

    Pigs are proposed to be suitable large animal models for test of the efficacy and safety of induced pluripotent stem cells (iPSCs) for stem cell therapy, but authentic pig ES/iPS cell lines with germline competence are rarely produced. The pathways or signaling underlying the defective competent pig iPSCs remain poorly understood. By improving induction conditions using various small chemicals, we generated pig iPSCs that exhibited high pluripotency and differentiation capacity that can contribute to chimeras. However, their potency was reduced with increasing passages by teratoma formation test, and correlated with declined expression levels of Rex1, an important marker for naïve state. By RNA-sequencing analysis, genes related to WNT signaling were upregulated and MAPK signaling and TGFβ pathways downregulated in pig iPSCs compared to fibroblasts, but they were abnormally expressed during passages. Notably, pathways involving in DNA repair and replication were upregulated at early passage, but downregulated in iPSCs during prolonged passage in cluster with fibroblasts. Our data suggests that reduced DNA repair and replication capacity links to the instability of pig iPSCs. Targeting these pathways may facilitate generation of truly pluripotent pig iPSCs, with implication in translational studies. PMID:28253351

  15. Mapping mammalian cell-type-specific transcriptional regulatory networks using KD-CAGE and ChIP-seq data in the TC-YIK cell line.

    Directory of Open Access Journals (Sweden)

    Marina eLizio

    2015-11-01

    Full Text Available Mammals are composed of hundreds of different cell types with specialized functions. Each of these cellular phenotypes are controlled by different combinations of transcription factors. Using a human non islet cell insulinoma cell line (TC-YIK which expresses insulin and the majority of known pancreatic beta cell specific genes as an example, we describe a general approach to identify key cell-type-specific transcription factors (TFs and their direct and indirect targets. By ranking all human TFs by their level of enriched expression in TC-YIK relative to a broad collection of samples (FANTOM5, we confirmed known key regulators of pancreatic function and development. Systematic siRNA mediated perturbation of these TFs followed by qRT-PCR revealed their interconnections with NEUROD1 at the top of the regulation hierarchy and its depletion drastically reducing insulin levels. For 15 of the TF knock-downs (KD, we then used Cap Analysis of Gene Expression (CAGE to identify thousands of their targets genome-wide (KD-CAGE. The data confirm NEUROD1 as a key positive regulator in the transcriptional regulatory network (TRN, and ISL1 and PROX1 as antagonists. As a complimentary approach we used ChIP-seq on four of these factors to identify NEUROD1, LMX1A, PAX6 and RFX6 binding sites in the human genome. Examining the overlap between genes perturbed in the KD-CAGE experiments and genes with a ChIP-seq peak within 1kb of their promoter, we identified direct transcriptional targets of these TFs. Integration of KD-CAGE and ChIP-seq data shows that both NEUROD1 and LMX1A work as the main transcriptional activators. In the core TRN (i.e. TF-TF only, NEUROD1 directly transcriptionally activates the pancreatic TFs HSF4, INSM1, MLXIPL, MYT1, NKX6-3, ONECUT2, PAX4, PROX1, RFX6, ST18, DACH1 and SHOX2, while LMX1A directly transcriptionally activates DACH1, SHOX2, PAX6 and PDX1. Analysis of these complementary datasets suggests the need for caution in interpreting

  16. Mapping Mammalian Cell-type-specific Transcriptional Regulatory Networks Using KD-CAGE and ChIP-seq Data in the TC-YIK Cell Line.

    Science.gov (United States)

    Lizio, Marina; Ishizu, Yuri; Itoh, Masayoshi; Lassmann, Timo; Hasegawa, Akira; Kubosaki, Atsutaka; Severin, Jessica; Kawaji, Hideya; Nakamura, Yukio; Suzuki, Harukazu; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R R

    2015-01-01

    Mammals are composed of hundreds of different cell types with specialized functions. Each of these cellular phenotypes are controlled by different combinations of transcription factors. Using a human non islet cell insulinoma cell line (TC-YIK) which expresses insulin and the majority of known pancreatic beta cell specific genes as an example, we describe a general approach to identify key cell-type-specific transcription factors (TFs) and their direct and indirect targets. By ranking all human TFs by their level of enriched expression in TC-YIK relative to a broad collection of samples (FANTOM5), we confirmed known key regulators of pancreatic function and development. Systematic siRNA mediated perturbation of these TFs followed by qRT-PCR revealed their interconnections with NEUROD1 at the top of the regulation hierarchy and its depletion drastically reducing insulin levels. For 15 of the TF knock-downs (KD), we then used Cap Analysis of Gene Expression (CAGE) to identify thousands of their targets genome-wide (KD-CAGE). The data confirm NEUROD1 as a key positive regulator in the transcriptional regulatory network (TRN), and ISL1, and PROX1 as antagonists. As a complimentary approach we used ChIP-seq on four of these factors to identify NEUROD1, LMX1A, PAX6, and RFX6 binding sites in the human genome. Examining the overlap between genes perturbed in the KD-CAGE experiments and genes with a ChIP-seq peak within 50 kb of their promoter, we identified direct transcriptional targets of these TFs. Integration of KD-CAGE and ChIP-seq data shows that both NEUROD1 and LMX1A work as the main transcriptional activators. In the core TRN (i.e., TF-TF only), NEUROD1 directly transcriptionally activates the pancreatic TFs HSF4, INSM1, MLXIPL, MYT1, NKX6-3, ONECUT2, PAX4, PROX1, RFX6, ST18, DACH1, and SHOX2, while LMX1A directly transcriptionally activates DACH1, SHOX2, PAX6, and PDX1. Analysis of these complementary datasets suggests the need for caution in interpreting ChIP

  17. Inositol hexaphosphate (IP6) blocks proliferation of human breast cancer cells through a PKCdelta-dependent increase in p27Kip1 and decrease in retinoblastoma protein (pRb) phosphorylation.

    Science.gov (United States)

    Vucenik, Ivana; Ramakrishna, Gayatri; Tantivejkul, Kwanchanit; Anderson, Lucy M; Ramljak, Danica

    2005-05-01

    Inositol hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate with demonstrated anti-proliferative and anti-cancer activity in mammary cells. We hypothesized that IP6 modulates cell cycle proteins by action on cytoplasmic signaling molecules. The effects of both pharmacological (2 mM) and physiological (100 microM) doses of IP6 on major PKC isoforms (PKCalpha, delta, epsilon, beta and zeta), PI3-K/Akt and ras/Erk1/2 were evaluated. Treatment of MCF-7 human breast cancer cells with 2 mM IP6 for 24 h caused a 3.1-fold increase in the expression of anti-proliferative PKCdelta. Similar results were observed with 100 microM IP6 at only 30-60 min post-treatment. IP6 also caused an increase in PKCdelta activity, shown by its translocation from cytosol to membrane. No changes in expression of PKC alpha, delta, epsilon, beta and zeta were detected. Additionally, IP6 caused a decrease of Erk1/2 and Akt activity. Among cell cycle control proteins, IP6 resulted in increased p27Kip1 protein levels and marked reduction of pRb phosphorylation. Specificity of the IP6 effects on p27Kip1 and pRb in MCF-7 cells (hormone-dependent) were additionally confirmed in highly invasive hormone-independent MDA-MB 231 breast cancer cells. Use of specific pharmaclogical inhibitors of PKC delta, MEK/Erk, and PI3K/Akt pathways indicated that the IP6-mediated effects on PKC delta were responsible for up-regulation of p27Kip, and pRb hypo-phosphorylation. In addition, IP6-induced apoptosis detected in MCF-7 cells appeared also to be PKC delta-dependent. Our data suggest potential usefulness of IP6 as a novel therapeutic modulator of PKC delta and p27Kip1, an important prognostic factor in human breast cancers.

  18. Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1.

    Science.gov (United States)

    Solanes, Paola; Heuzé, Mélina L; Maurin, Mathieu; Bretou, Marine; Lautenschlaeger, Franziska; Maiuri, Paolo; Terriac, Emmanuel; Thoulouze, Maria-Isabel; Launay, Pierre; Piel, Matthieu; Vargas, Pablo; Lennon-Duménil, Ana-Maria

    2015-03-12

    Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP₃ receptors (IP₃Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP₃R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP₃R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP₃R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment.

  19. Boron concentration measurements at the I/P interface in nip a-Si solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Van Aken, B.B.; Soppe, W.J. [ECN Solar Energy, P.O. Box 1, NL-1755 ZG Petten (Netherlands); Duchamp, M.; Boothroyd, C.B. [Center for Electron Nanoscopy, Technical University of Denmark, DK-2800 Kongens Lyngby (Denmark); Dunin-Borkowski, R.E. [Institute for Microstructure Research, Forschungszentrum Juelich, D-52425 Juelich (Germany); Barnes, J.P.; Veillerot, M. [CEA-Leti, MINATEC Campus, 17 rue des Martyrs, FR-38054 Grenoble Cedex 9 (France)

    2011-09-15

    The p-type Si layer in n-i-p a-Si and {mu}c-Si solar cells on foil has several important requirements with respect to conductivity and optical transmission. We control the optical band gap and activation energy of p-a-SiC by varying the B2H6 and CH4 flows in the process chamber. Modelling shows that the optimum efficiency in n-i-p solar cells is obtained when the p-a-SiC band gap is just above the band gap of the absorber layer. We have assessed the potential of core-loss electron energy-loss spectroscopy (EELS) for detecting B and C and of low-loss EELS, in a spatially resolved manner, as probe of local variations in bulk plasmon energy. EELS in the transmission electron microscope (TEM) combines the necessary spatial resolution to investigate the boundary between p-a-SiC and i-a-Si with sufficient sensitivity to the boron content.

  20. bFGF and Activin A function to promote survival and proliferation of single iPS cells in conditioned half-exchange mTeSR1 medium.

    Science.gov (United States)

    Guo, Xiaoling; Lian, Ruiling; Guo, Yonglong; Liu, Qing; Ji, Qingshan; Chen, Jiansu

    2015-07-01

    Human induced pluripotent stem (iPS) cells can be well maintained by clonal growth. The pluripotent growth of single iPS cells is limited by low survival. To facilitate robust single iPS cells cultured in vitro, half-exchange mTeSR1 medium (HM), whole-exchange medium (WM) and iPS cell-derived conditioned medium (iPS-CM) culture were used. The effects of bFGF and Activin A on the growth of single iPS cells were explored. The dissociation and propagation of single iPS cells also included Accutase enzymatic isolation, Rho-associated protein kinase (ROCK) inhibitor Y27632 protection and high-density single-cell seeding (1 × 10(6) cells/well). CCK-8 assays demonstrated that the viability of clonal iPS cells in mTeSR1 medium and single iPS cells in HM, iPS-CM or WM supplemented with 100 ng/ml bFGF and 10 ng/ml Activin A was significantly higher than that in WM. Annexin v and propidium iodide (PI) assay, Calcein AM and EthD-III double staining also confirmed the similar results. ELISA assays showed that the levels of bFGF and Activin A of single iPS cells in HM and iPS-CM were higher than single iPS cells in WM. Meanwhile, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), quantitative Polymerase Chain Reaction (qPCR), Western Blotting (WB), Immunofluorescence (IF) and karyotype analysis revealed that HM culture was able to maintain undifferentiated markers of Nanog, Klf4, Sox2, Oct4, and did not affect the karyotype of iPS cells. Undifferentiated single iPS cells in HM displayed homogenized growth. These findings demonstrate that bFGF and Activin A are important for the survival and growth of single iPS cells. HM culture system combined Accutase, Y27632 and high-density single-cell seeding can facilitate short-term growth of single iPS cells in vitro.

  1. Rhinovirus-16 induced release of IP-10 and IL-8 is augmented by Th2 cytokines in a pediatric bronchial epithelial cell model.

    Directory of Open Access Journals (Sweden)

    Julie A Cakebread

    Full Text Available BACKGROUND: In response to viral infection, bronchial epithelial cells increase inflammatory cytokine release to activate the immune response and curtail viral replication. In atopic asthma, enhanced expression of Th2 cytokines is observed and we postulated that Th2 cytokines may augment the effects of rhinovirus-induced inflammation. METHODS: Primary bronchial epithelial cell cultures from pediatric subjects were treated with Th2 cytokines for 24 h before infection with RV16. Release of IL-8, IP-10 and GM-CSF was measured by ELISA. Infection was quantified using RTqPCR and TCID50. Phosphatidyl inositol 3-kinase (PI3K and P38 mitogen activated protein kinase (MAPK inhibitors and dexamethasone were used to investigate differences in signaling pathways. RESULTS: The presence of Th2 cytokines did not affect RV replication or viral titre, yet there was a synergistic increase in IP-10 release from virally infected cells in the presence of Th2 cytokines. Release of IL-8 and GM-CSF was also augmented. IP-10 release was blocked by a PI3K inhibitor and IL-8 by dexamethasone. CONCLUSION: Th2 cytokines increase release of inflammatory cytokines in the presence of rhinovirus infection. This increase is independent of effects of virus replication. Inhibition of the PI3K pathway inhibits IP-10 expression.

  2. Rabies virus infection induces type I interferon production in an IPS-1 dependent manner while dendritic cell activation relies on IFNAR signaling.

    Science.gov (United States)

    Faul, Elizabeth J; Wanjalla, Celestine N; Suthar, Mehul S; Gale, Michael; Wirblich, Christoph; Schnell, Matthias J

    2010-07-22

    As with many viruses, rabies virus (RABV) infection induces type I interferon (IFN) production within the infected host cells. However, RABV has evolved mechanisms by which to inhibit IFN production in order to sustain infection. Here we show that RABV infection of dendritic cells (DC) induces potent type I IFN production and DC activation. Although DCs are infected by RABV, the viral replication is highly suppressed in DCs, rendering the infection non-productive. We exploited this finding in bone marrow derived DCs (BMDC) in order to differentiate which pattern recognition receptor(s) (PRR) is responsible for inducing type I IFN following infection with RABV. Our results indicate that BMDC activation and type I IFN production following a RABV infection is independent of TLR signaling. However, IPS-1 is essential for both BMDC activation and IFN production. Interestingly, we see that the BMDC activation is primarily due to signaling through the IFNAR and only marginally induced by the initial infection. To further identify the receptor recognizing RABV infection, we next analyzed BMDC from Mda-5-/- and RIG-I-/- mice. In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection. However, only RIG-I-/- cells exhibit a delay in type I IFN production. In order to determine the role that IPS-1 plays in vivo, we infected mice with pathogenic RABV. We see that IPS-1-/- mice are more susceptible to infection than IPS-1+/+ mice and have a significantly increased incident of limb paralysis.

  3. Integrated transcriptome analysis of human iPS cells derived from a fragile X syndrome patient during neuronal differentiation.

    Science.gov (United States)

    Lu, Ping; Chen, Xiaolong; Feng, Yun; Zeng, Qiao; Jiang, Cizhong; Zhu, Xianmin; Fan, Guoping; Xue, Zhigang

    2016-11-01

    Fragile X syndrome (FXS) patients carry the expansion of over 200 CGG repeats at the promoter of fragile X mental retardation 1 (FMR1), leading to decreased or absent expression of its encoded fragile X mental retardation protein (FMRP). However, the global transcriptional alteration by FMRP deficiency has not been well characterized at single nucleotide resolution, i.e., RNA-seq. Here, we performed in-vitro neuronal differentiation of human induced pluripotent stem (iPS) cells that were derived from fibroblasts of a FXS patient (FXS-iPSC). We then performed RNA-seq and examined the transcriptional misregulation at each intermediate stage during in-vitro differentiation of FXS-iPSC into neurons. After thoroughly analyzing the transcriptomic data and integrating them with those from other platforms, we found up-regulation of many genes encoding TFs for neuronal differentiation (WNT1, BMP4, POU3F4, TFAP2C, and PAX3), down-regulation of potassium channels (KCNA1, KCNC3, KCNG2, KCNIP4, KCNJ3, KCNK9, and KCNT1) and altered temporal regulation of SHANK1 and NNAT in FXS-iPSC derived neurons, indicating impaired neuronal differentiation and function in FXS patients. In conclusion, we demonstrated that the FMRP deficiency in FXS patients has significant impact on the gene expression patterns during development, which will help to discover potential targeting candidates for the cure of FXS symptoms.

  4. Planckian Information (Ip): A New Measure of Order in Atoms, Enzymes, Cells, Brains, Human Societies, and the Cosmos

    Science.gov (United States)

    Ji, Sungchul

    A new mathematical formula referred to as the Planckian distribution equation (PDE) has been found to fit long-tailed histograms generated in various fields of studies, ranging from atomic physics to single-molecule enzymology, cell biology, brain neurobiology, glottometrics, econophysics, and to cosmology. PDE can be derived from a Gaussian-like equation (GLE) by non-linearly transforming its variable, x, while keeping the y coordinate constant. Assuming that GLE represents a random distribution (due to its symmetry), it is possible to define a binary logarithm of the ratio between the areas under the curves of PDE and GLE as a measure of the non-randomness (or order) underlying the biophysicochemical processes generating long-tailed histograms that fit PDE. This new function has been named the Planckian information, IP, which (i) may be a new measure of order that can be applied widely to both natural and human sciences and (ii) can serve as the opposite of the Boltzmann-Gibbs entropy, S, which is a measure of disorder. The possible rationales for the universality of PDE may include (i) the universality of the wave-particle duality embedded in PDE, (ii) the selection of subsets of random processes (thereby breaking the symmetry of GLE) as the basic mechanism of generating order, organization, and function, and (iii) the quantity-quality complementarity as the connection between PDE and Peircean semiotics.

  5. Promoted differentiation of cynomolgus monkey ES cells into hepatocyte-like cells by co-culture with mouse fetal liver-derived cells

    Institute of Scientific and Technical Information of China (English)

    Ko Saito; Masahide Yoshikawa; Yukiteru Ouji; Kei Moriya; Mariko Nishiofuku; Shigehiko Ueda; Noriko Hayashi; Shigeaki Ishizaka; Hiroshi Fukui

    2006-01-01

    AIM:To explore whether a co-culture of cynomolgus monkey embryonic stem (cES) cells with embryonic liver cells could promote their differentiation into hepatocytes.METHODS:Mouse fetal liver-derived cells (MFLCs) were prepared as adherent cells from mouse embryos on embryonic d (ED) 14, after which undifferentiated cES cells were co-cultured with MFLCs. The induction of cES cells along a hepatic lineage was examined in MFLCassisted differentiation, spontaneous differentiation,and growth factors (GF) and chemicals-induced differentiations (GF-induced differentiation) using retinoic acid, leukemia inhibitory factor (LIF), FGF2, FGF4,hepatocyte growth factor (HGF), oncostatin M (OSM),and dexamethasone.RESULTS:The mRNA expression of α-fetoprotein,albumin (ALB), α-1-antitrypsin, and hepatocyte nuclear factor 4α was observed earlier in the differentiating cES cells co-cultured with MFLCs, as compared to cES cells undergoing spontaneous differentiation and those subjected to GF-induced differentiation. The expression of cytochrome P450 7a1, a possible marker for embryonic endoderm-derived mature hepatocytes,was only observed in cES cells that had differentiated in a co-culture with MFLCs. Further, the disappearance of Oct3/4, a representative marker of an undifferentiated state, was noted in cells co-cultured with MFLCs, but not in those undergoing spontaneous or GF-induced differentiation. Tmmunocytochemical analysis revealed an increased ratio of ALS-immunopositive cells among cES cells co-cultured with MFLCs, while glycogen storage and urea synthesis were also demonstrated.CONCLUSION:MFLCs showed an ability to induce cES cells to differentiate toward hepatocytes. The co-culture system with MFLCs is a useful method for induction of hepatocyte-like cells from undifferentiated cES cells.

  6. Effect of oxygen on cardiac differentiation in mouse iPS cells: role of hypoxia inducible factor-1 and Wnt/beta-catenin signaling.

    Directory of Open Access Journals (Sweden)

    Tanya L Medley

    Full Text Available BACKGROUND: Disturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes. OBJECTIVE: We investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS cells, assessed the involvement of HIF-1alpha (hypoxia-inducible factor-1alpha and the canonical Wnt pathway in this process. METHODS: Embryoid bodies (EBs derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture. RESULTS: At 14 days of differentiation, 59 ± 2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway. CONCLUSION: Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis.

  7. Inhibition of stearoyl-coA desaturase selectively eliminates tumorigenic Nanog-positive cells: improving the safety of iPS cell transplantation to myocardium.

    Science.gov (United States)

    Zhang, Lan; Pan, Yaohua; Qin, Gangjian; Chen, Lijuan; Chatterjee, Tapan K; Weintraub, Neal L; Tang, Yaoliang

    2014-01-01

    Induced pluripotent stem cells (iPS) can differentiate into cardiomyocytes (CM) and represent a promising form of cellular therapy for heart regeneration. However, residual undifferentiated iPS derivates (iPSD), which are not fully eliminated by cell differentiation or purification protocols, may form tumors after transplantation, thus compromising therapeutic application. Inhibition of stearoyl-coA desaturase (SCD) has recently been reported to eliminate undifferentiated human embryonic stem cells, which share many features with iPSD. Here, we tested the effects of PluriSin#1, a small-molecule inhibitor of SCD, on iPS-derived CM. We found that plurisin#1 treatment significantly decreased the mRNA and protein level of Nanog, a marker for both cell pluripotency and tumor progression; importantly, we provide evidence that PluriSin#1 treatment at 20 µM for 1 day significantly induces the apoptosis of Nanog-positive iPSD. In addition, PluriSin#1 treatment at 20 µM for 4 days diminished Nanog-positive stem cells in cultured iPSD while not increasing apoptosis of iPS-derived CM. To investigate whether PluriSin#1 treatment prevents tumorigenicity of iPSD after cell transplantation, we intramyocardially injected PluriSin#1- or DMSO-treated iPSD in a mouse model of myocardial infarction (MI). DMSO-treated iPSD readily formed Nanog-expressing tumors 2 weeks after injection, which was prevented by treatment with PluriSin#1. Moreover, treatment with PluriSin#1 did not change the expression of cTnI, α-MHC, or MLC-2v, markers of cardiac differentiation (P>0.05, n = 4). Importantly, pluriSin#1-treated iPS-derived CM exhibited the ability to engraft and survive in the infarcted myocardium. We conclude that inhibition of SCD holds the potential to enhance the safety of therapeutic application of iPS cells for heart regeneration.

  8. Generation and characterization of the human iPSC line PBMC1-iPS4F1 from adult peripheral blood mononuclear cells

    Directory of Open Access Journals (Sweden)

    Rosa Montes

    2015-11-01

    Full Text Available Here we describe the generation and characterization of the human induced pluripotent stem cell (iPSC line PBMC1-iPS4F1 from peripheral blood mononuclear cells from a healthy female with Spanish background. We used heat sensitive, non-integrative Sendai viruses containing the reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc, whose expression was silenced in the established iPSC line. Characterization of the PBMC1-iPS4F1 cell line included analysis of typical pluripotency-associated factors at mRNA and protein level, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation studies.

  9. Variation of T cell subset during acute rejection after liver transplantation in rhesus monkeys

    Institute of Scientific and Technical Information of China (English)

    Ran Jiang-hua; Liu Jing; Zhang Xi-bing; Zhang Sheng-ning; Wu Shu-yuan; Li Lai-bang; Li Wang; Li Li

    2014-01-01

    Abstract BACKGROUND: Looking for the early diagnosis of acute rejection indicators after liver transplantation can assess the risk after liver transplantation quickly and effectively, and T lymphocytes play the significant role in acute rejection. OBJECTIVE:To observe the relationship between acute rejection and variation of expression of T cel subset in blood after liver transplantation in rhesus monkey. METHODS: The sixteen liver transplant models in rhesus monkey which were constructed successfuly by the method of “double-cuff and one support tube” were divided into two groups randomly: experiment group (no treated by immunosuppressant in perioperative period) and control group (treated by immunosuppressant in perioperative period). Then the blood specimen and liver tissue respectively were colected at 6, 12, 24 and 72 hours after operation. The levels of alanine transferase, aspartate aminotransferase, and total bilirubin were detected with the fuly automatic biochemical analyser. The levels of CD4+/CD8+were tested by flow cytometry. The liver tissue in rhesus monkey after liver transplantation was detected by hematoxylin-eosin staining. The degree of acute rejection was evaluated by Banff Score System. RESULTS AND CONCLUSION: Acute rejection appeared in the experiment group at 12, 24, and 72 hours after liver transplantation. Levels of alanine transferase, aspartate aminotransferase, and total bilirubin were significantly higher in the experimental group than in the control group at 24 and 72 hours after transplantation (P < 0.05). The expression of CD4+/CD8+of the experiment group and control group began to rise at 6 hours after surgery, but the experiment group increased the most obvious. CD4+/CD8+ expression was significantly greater in the experimental group than in the control group at 24 and 72 hours after transplantation (P < 0.05). Morphological pathology was severer, and Banff score was higher in the experiment group than in the control group at

  10. Promotion on Nucleation and Aggregation of Calcium Oxalate Crystals by Injured African Green Monkey Renal Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    张燊; 彭花; 姚秀琼; 苏泽轩; 欧阳健明

    2012-01-01

    The purpose of this work was to detect the properties of African green monkey renal epithelial cells (Vero) after oxidative injury and to study the mediation of the injured Vero on aggregation and formation of calcium oxalate crystals. This injury model was induced by 0.15 mmol/L H2O2 according to the pretest evaluation. The results suggested that H2O2 could injure Vero significantly and decrease cell viability in a time-dependent manner for exposure time of 0.5--2 h. After cell injury, the indexes connected with oxidative injury changed. The malondialdehyde (MDA) content and osteopontin (OPN) expression increased, while superoxide dismutase (SOD) level decreased. It resulted in the increase of both the amount of CaOxa crystals and the degree of crystal aggregation on the injured cells. This work indicated that injured cells promoted the formation of calcium oxalate monohydrate (COM) crystals, thus increased the risk of formation of urinary stone.

  11. IPS-1 is essential for type III IFN production by hepatocytes and dendritic cells in response to hepatitis C virus infection.

    Science.gov (United States)

    Okamoto, Masaaki; Oshiumi, Hiroyuki; Azuma, Masahiro; Kato, Nobuyuki; Matsumoto, Misako; Seya, Tsukasa

    2014-03-15

    Hepatitis C virus (HCV) is a major cause of liver disease. The innate immune system is essential for controlling HCV replication, and HCV is recognized by RIG-I and TLR3, which evoke innate immune responses through IPS-1 and TICAM-1 adaptor molecules, respectively. IL-28B is a type III IFN, and genetic polymorphisms upstream of its gene are strongly associated with the efficacy of polyethylene glycol-IFN and ribavirin therapy. As seen with type I IFNs, type III IFNs induce antiviral responses to HCV. Recent studies established the essential role of TLR3-TICAM-1 pathway in type III IFN production in response to HCV infection. Contrary to previous studies, we revealed an essential role of IPS-1 in type III IFN production in response to HCV. First, using IPS-1 knockout mice, we revealed that IPS-1 was essential for type III IFN production by mouse hepatocytes and CD8(+) dendritic cells (DCs) in response to cytoplasmic HCV RNA. Second, we demonstrated that type III IFN induced RIG-I but not TLR3 expression in CD8(+) DCs and augmented type III IFN production in response to cytoplasmic HCV RNA. Moreover, we showed that type III IFN induced cytoplasmic antiviral protein expression in DCs and hepatocytes but failed to promote DC-mediated NK cell activation or cross-priming. Our study indicated that IPS-1-dependent pathway plays a crucial role in type III IFN production by CD8(+) DCs and hepatocytes in response to HCV, leading to cytoplasmic antiviral protein expressions.

  12. Dengue type four viruses with E-Glu345Lys adaptive mutation from MRC-5 cells induce low viremia but elicit potent neutralizing antibodies in rhesus monkeys.

    Science.gov (United States)

    Lin, Hsiao-Han; Lee, Hsiang-Chi; Li, Xiao-Feng; Tsai, Meng-Ju; Hsiao, Hung-Ju; Peng, Jia-Guan; Sue, Shih-Che; Qin, Cheng-Feng; Wu, Suh-Chin

    2014-01-01

    Knowledge of virulence and immunogenicity is important for development of live-attenuated dengue vaccines. We previously reported that an infectious clone-derived dengue type 4 virus (DENV-4) passaged in MRC-5 cells acquired a Glu345Lys (E-E345K) substitution in the E protein domain III (E-DIII). The same cloned DENV-4 was found to yield a single E-Glu327Gly (E-E327G) mutation after passage in FRhL cells and cause the loss of immunogenicity in rhesus monkeys. Here, we used site-directed mutagenesis to generate the E-E345K and E-E327G mutants from DENV-4 and DENV-4Δ30 infectious clones and propagated in Vero or MRC-5 cells. The E-E345K mutations were consistently presented in viruses recovered from MRC-5 cells, but not Vero cells. Recombinant E-DIII proteins of E345K and E327G increased heparin binding correlated with the reduced infectivity by heparin treatment in cell cultures. Different from the E-E327G mutant viruses to lose the immunogencity in rhesus monkeys, the E-E345K mutant viruses were able to induce neutralizing antibodies in rhesus monkeys with an almost a 10-fold lower level of viremia as compared to the wild type virus. Monkeys immunized with the E-E345K mutant virus were completely protected with no detectable viremia after live virus challenges with the wild type DENV-4. These results suggest that the E-E345K mutant virus propagated in MRC-5 cells may have potential for the use in live-attenuated DENV vaccine development.

  13. The PTTG1-binding factor (PBF/PTTG1IP) regulates p53 activity in thyroid cells.

    Science.gov (United States)

    Read, Martin L; Seed, Robert I; Fong, Jim C W; Modasia, Bhavika; Ryan, Gavin A; Watkins, Rachel J; Gagliano, Teresa; Smith, Vicki E; Stratford, Anna L; Kwan, Perkin K; Sharma, Neil; Dixon, Olivia M; Watkinson, John C; Boelaert, Kristien; Franklyn, Jayne A; Turnell, Andrew S; McCabe, Christopher J

    2014-04-01

    The PTTG1-binding factor (PBF/PTTG1IP) has an emerging repertoire of roles, especially in thyroid biology, and functions as a protooncogene. High PBF expression is independently associated with poor prognosis and lower disease-specific survival in human thyroid cancer. However, the precise role of PBF in thyroid tumorigenesis is unclear. Here, we present extensive evidence demonstrating that PBF is a novel regulator of p53, a tumor suppressor protein with a key role in maintaining genetic stability, which is infrequently mutated in differentiated thyroid cancer. By coimmunoprecipitation and proximity-ligation assays, we show that PBF binds specifically to p53 in thyroid cells and significantly represses transactivation of responsive promoters. Further, we identify that PBF decreases p53 stability by enhancing ubiquitination, which appears dependent on the E3 ligase activity of Mdm2. Impaired p53 function was evident in a transgenic mouse model with thyroid-specific PBF overexpression (transgenic PBF mice), which had significantly increased genetic instability as indicated by fluorescent inter simple sequence repeat-PCR analysis. Consistent with this, approximately 40% of all DNA repair genes examined were repressed in transgenic PBF primary cultures, including genes with critical roles in maintaining genomic integrity such as Mgmt, Rad51, and Xrcc3. Our data also revealed that PBF induction resulted in up-regulation of the E2 enzyme Rad6 in murine thyrocytes and was associated with Rad6 expression in human thyroid tumors. Overall, this work provides novel insights into the role of the protooncogene PBF as a negative regulator of p53 function in thyroid tumorigenesis, in which PBF is generally overexpressed and p53 mutations are rare compared with other tumor types.

  14. iPS cell modeling of Best disease: insights into the pathophysiology of an inherited macular degeneration.

    Science.gov (United States)

    Singh, Ruchira; Shen, Wei; Kuai, David; Martin, Jessica M; Guo, Xiangrong; Smith, Molly A; Perez, Enio T; Phillips, M Joseph; Simonett, Joseph M; Wallace, Kyle A; Verhoeven, Amelia D; Capowski, Elizabeth E; Zhang, Xiaoqing; Yin, Yingnan; Halbach, Patrick J; Fishman, Gerald A; Wright, Lynda S; Pattnaik, Bikash R; Gamm, David M

    2013-02-01

    Best disease (BD) is an inherited degenerative disease of the human macula that results in progressive and irreversible central vision loss. It is caused by mutations in the retinal pigment epithelium (RPE) gene BESTROPHIN1 (BEST1), which, through mechanism(s) that remain unclear, lead to the accumulation of subretinal fluid and autofluorescent waste products from shed photoreceptor outer segments (POSs). We employed human iPS cell (hiPSC) technology to generate RPE from BD patients and unaffected siblings in order to examine the cellular and molecular processes underlying this disease. Consistent with the clinical phenotype of BD, RPE from mutant hiPSCs displayed disrupted fluid flux and increased accrual of autofluorescent material after long-term POS feeding when compared with hiPSC-RPE from unaffected siblings. On a molecular level, RHODOPSIN degradation after POS feeding was delayed in BD hiPSC-RPE relative to unaffected sibling hiPSC-RPE, directly implicating impaired POS handling in the pathophysiology of the disease. In addition, stimulated calcium responses differed between BD and normal sibling hiPSC-RPE, as did oxidative stress levels after chronic POS feeding. Subcellular localization, fractionation and co-immunoprecipitation experiments in hiPSC-RPE and human prenatal RPE further linked BEST1 to the regulation and release of endoplasmic reticulum calcium stores. Since calcium signaling and oxidative stress are critical regulators of fluid flow and protein degradation, these findings likely contribute to the clinical picture of BD. In a larger context, this report demonstrates the potential to use patient-specific hiPSCs to model and study maculopathies, an important class of blinding disorders in humans.

  15. Mobile IP: Security & application

    NARCIS (Netherlands)

    Tuquerres, Gloria; Salvador, Marcos Rogério; Sprenkels, Ron

    1999-01-01

    As required in the TGS Mobile IP Advanced Module, this paper presents a survey of common security threats which mobile IP networks are exposed to as well as some proposed solutions to deal with such threats.

  16. Propagation of human hepatitis A virus in African green monkey kidney cell culture: primary isolation and serial passage.

    Science.gov (United States)

    Daemer, R J; Feinstone, S M; Gust, I D; Purcell, R H

    1981-04-01

    Human hepatitis A virus (HAV) was propagated in primary African Green Monkey (Cercopithecus aethiops) kidney (AGMK) cell cultures. Three strains of HAV were used: MS-1, SD-11, and HM-175. Cells were inoculated with marmoset-passaged material or human clinical specimens and were stained by direct immunofluorescence to establish the identity of the virus. Both clinical samples and marmoset-passaged material produced immunofluorescence. HAV antigen was found scattered throughout the cytoplasm of inoculated cultures. The HM-175 strain produced the most intense immunofluorescence. This strain of HAV had been serially passaged in cell culture seven times. Blocking experiments with paired human sera from naturally acquired HAV infections and hyperimmune chimpanzee serum from an experimentally infected animal established that the immunofluorescence was specific. The viral antigen was found to be exclusively intracellular. The interval to maximum HAV antigen expression was decreased by serial passage. The HAV strain described herein, which was recovered directly from the stool specimen of a patient with HAV in primary AGMK cell culture, may prove useful as a source of antigen for serological tests and as a candidate vaccine strain.

  17. Anti-Aβ drug screening platform using human iPS cell-derived neurons for the treatment of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Naoki Yahata

    Full Text Available BACKGROUND: Alzheimer's disease (AD is a neurodegenerative disorder that causes progressive memory and cognitive decline during middle to late adult life. The AD brain is characterized by deposition of amyloid β peptide (Aβ, which is produced from amyloid precursor protein by β- and γ-secretase (presenilin complex-mediated sequential cleavage. Induced pluripotent stem (iPS cells potentially provide an opportunity to generate a human cell-based model of AD that would be crucial for drug discovery as well as for investigating mechanisms of the disease. METHODOLOGY/PRINCIPAL FINDINGS: We differentiated human iPS (hiPS cells into neuronal cells expressing the forebrain marker, Foxg1, and the neocortical markers, Cux1, Satb2, Ctip2, and Tbr1. The iPS cell-derived neuronal cells also expressed amyloid precursor protein, β-secretase, and γ-secretase components, and were capable of secreting Aβ into the conditioned media. Aβ production was inhibited by β-secretase inhibitor, γ-secretase inhibitor (GSI, and an NSAID; however, there were different susceptibilities to all three drugs between early and late differentiation stages. At the early differentiation stage, GSI treatment caused a fast increase at lower dose (Aβ surge and drastic decline of Aβ production. CONCLUSIONS/SIGNIFICANCE: These results indicate that the hiPS cell-derived neuronal cells express functional β- and γ-secretases involved in Aβ production; however, anti-Aβ drug screening using these hiPS cell-derived neuronal cells requires sufficient neuronal differentiation.

  18. Optimization of n/i and i/p buffer layers in n-i-p hydrogenated microcrystalline silicon solar cells

    Institute of Scientific and Technical Information of China (English)

    Yuan Yujie; Hou Guofu; Zhang Jianjun; Xue Junming; Cao Liran; Zhao Ying; Geng Xinhua

    2009-01-01

    Hydrogenated microcrystalline silicon (μc-Si:H) intrinsic films and solar cells with n-i-p configuration were prepared by plasma enhanced chemical vapor deposition (PECVD). The influence of n/i and i/p buffer layerson the μc-Si:H cell performance was studied in detail. The experimental results demonstrated that the efficiency is much improved when there is a higher crystallinity at n/i interface and an optimized a-Si:H buffer layer at i/p interface. By combining the above methods, the performance ofμc-Si:H single-junction and a-Si:H/μc-Si:H tandemsolar ceils has been significantly improved.

  19. Morphology of horizontal cells in the retina of the capuchin monkey, Cebus apella: how many horizontal cell classes are found in dichromatic primates?

    Science.gov (United States)

    dos Reis, José Wesley L; de Carvalho, Walther Augusto; Saito, Cézar A; Silveira, Luiz Carlos L

    2002-02-04

    The morphology of horizontal cells was studied in the retina of dichromatic capuchin monkeys, Cebus apella. The cells were labeled with the carbocyanine dye, 1,1',dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), and the labeling was then photoconverted to a stable product by using a diaminobenzidine reaction. The sizes of cell body, dendritic field, and axon terminal, as well as the number of dendritic clusters and cone convergence, were measured at increasing distance from the fovea. Three distinct morphological classes of horizontal cells were identified. Their dendritic and axonal morphology resembles those of H1, H2, and H3 cells described in trichromatic primates. The size of the cell bodies, dendritic fields, and axon terminals of all cell classes increases towards retinal periphery. H3 cells have larger dendritic fields and more dendritic clusters than H1 cells. All labeled horizontal cells located in selected patches of retina were further analyzed to quantify the differences between H1 and H3 cells. H1 cells have smaller dendritic field area, smaller total length of primary dendrites, more dendritic branching points, and larger fractal dimension than H3 cells. We have distinguished H1 and H3 cells based solely in morphological criteria. Their physiology should be further analyzed with detail, but their presence in both dichromatic and trichromatic primates suggests that neither of them have a specialized role in the red-green color opponent channel of color vision.

  20. Ensuring Software IP Cleanliness

    Directory of Open Access Journals (Sweden)

    Mahshad Koohgoli

    2007-12-01

    Full Text Available At many points in the life of a software enterprise, determination of intellectual property (IP cleanliness becomes critical. The value of an enterprise that develops and sells software may depend on how clean the software is from the IP perspective. This article examines various methods of ensuring software IP cleanliness and discusses some of the benefits and shortcomings of current solutions.

  1. Evaluation of IP Portfolios

    DEFF Research Database (Denmark)

    Søberg, Peder Veng

    2009-01-01

    As a result of an inquiry concerning how to evaluate IP (intellectual property) portfolios in order to enable the best possible use of IP resources within organizations, an IP evaluation approach primarily applicable for patents and utility models is developed. The developed approach is useful...

  2. Highly efficient differentiation and enrichment of spinal motor neurons derived from human and monkey embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Tamaki Wada

    Full Text Available BACKGROUND: There are no cures or efficacious treatments for severe motor neuron diseases. It is extremely difficult to obtain naïve spinal motor neurons (sMNs from human tissues for research due to both technical and ethical reasons. Human embryonic stem cells (hESCs are alternative sources. Several methods for MN differentiation have been reported. However, efficient production of naïve sMNs and culture cost were not taken into consideration in most of the methods. METHODS/PRINCIPAL FINDINGS: We aimed to establish protocols for efficient production and enrichment of sMNs derived from pluripotent stem cells. Nestin+ neural stem cell (NSC clusters were induced by Noggin or a small molecule inhibitor of BMP signaling. After dissociation of NSC clusters, neurospheres were formed in a floating culture containing FGF2. The number of NSCs in neurospheres could be expanded more than 30-fold via several passages. More than 33% of HB9+ sMN progenitor cells were observed after differentiation of dissociated neurospheres by all-trans retinoic acid (ATRA and a Shh agonist for another week on monolayer culture. HB9+ sMN progenitor cells were enriched by gradient centrifugation up to 80% purity. These HB9+ cells differentiated into electrophysiologically functional cells and formed synapses with myotubes during a few weeks after ATRA/SAG treatment. CONCLUSIONS AND SIGNIFICANCE: The series of procedures we established here, namely neural induction, NSC expansion, sMN differentiation and sMN purification, can provide large quantities of naïve sMNs derived from human and monkey pluripotent stem cells. Using small molecule reagents, reduction of culture cost could be achieved.

  3. Highly Efficient Differentiation and Enrichment of Spinal Motor Neurons Derived from Human and Monkey Embryonic Stem Cells

    Science.gov (United States)

    Wada, Tamaki; Honda, Makoto; Minami, Itsunari; Tooi, Norie; Amagai, Yuji; Nakatsuji, Norio; Aiba, Kazuhiro

    2009-01-01

    Background There are no cures or efficacious treatments for severe motor neuron diseases. It is extremely difficult to obtain naïve spinal motor neurons (sMNs) from human tissues for research due to both technical and ethical reasons. Human embryonic stem cells (hESCs) are alternative sources. Several methods for MN differentiation have been reported. However, efficient production of naïve sMNs and culture cost were not taken into consideration in most of the methods. Methods/Principal Findings We aimed to establish protocols for efficient production and enrichment of sMNs derived from pluripotent stem cells. Nestin+ neural stem cell (NSC) clusters were induced by Noggin or a small molecule inhibitor of BMP signaling. After dissociation of NSC clusters, neurospheres were formed in a floating culture containing FGF2. The number of NSCs in neurospheres could be expanded more than 30-fold via several passages. More than 33% of HB9+ sMN progenitor cells were observed after differentiation of dissociated neurospheres by all-trans retinoic acid (ATRA) and a Shh agonist for another week on monolayer culture. HB9+ sMN progenitor cells were enriched by gradient centrifugation up to 80% purity. These HB9+ cells differentiated into electrophysiologically functional cells and formed synapses with myotubes during a few weeks after ATRA/SAG treatment. Conclusions and Significance The series of procedures we established here, namely neural induction, NSC expansion, sMN differentiation and sMN purification, can provide large quantities of naïve sMNs derived from human and monkey pluripotent stem cells. Using small molecule reagents, reduction of culture cost could be achieved. PMID:19701462

  4. Whole-exome sequencing of fibroblast and its iPS cell lines derived from a patient diagnosed with xeroderma pigmentosum

    Directory of Open Access Journals (Sweden)

    Kohji Okamura

    2015-12-01

    Full Text Available Cells from a patient with a DNA repair-deficiency disorder are anticipated to bear a large number of somatic mutations. Because such mutations occur independently in each cell, there is a high degree of mosaicism in patients' tissues. While major mutations that have been expanded in many cognate cells are readily detected by sequencing, minor ones are overlaid with a large depth of non-mutated alleles and are not detected. However, cell cloning enables us to observe such cryptic mutations as well as major mutations. In the present study, we focused on a fibroblastic cell line that is derived from a patient diagnosed with xeroderma pigmentosum (XP, which is an autosomal recessive disorder caused by a deficiency in nucleotide excision repair. By making a list of somatic mutations, we can expect to see a characteristic pattern of mutations caused by the hereditary disorder. We cloned a cell by generating an iPS cell line and performed a whole-exome sequencing analysis of the progenitor and its iPS cell lines. Unexpectedly, we failed to find causal mutations in the XP-related genes, but we identified many other mutations including homozygous deletion of GSTM1 and GSTT1. In addition, we found that the long arm of chromosome 9 formed uniparental disomy in the iPS cell line, which was also confirmed by a structural mutation analysis using a SNP array. Type and number of somatic mutations were different from those observed in XP patients. Taken together, we conclude that the patient might be affected by a different type of the disorder and that some of the mutations that we identified here may be responsible for exhibiting the phenotype. Sequencing and SNP-array data have been submitted to SRA and GEO under accession numbers SRP059858 and GSE55520, respectively.

  5. Beyond iPS!

    Directory of Open Access Journals (Sweden)

    Editorial

    2012-01-01

    Full Text Available It’s undoubtedly a jubilant moment for scientists and clinicians working in the stem cell arena as Prof. Gurdon and Prof. Shinya Yamanaka have been chosen for the Nobel Prize in Physiology & Medicine this year. The mystery of cell biology is something unfathomable and probably the work of this duo as well as the other scientists, who have put their hands on in- vitro de-differentiation have opened our eyes to a new window or a new paradigm in cell biology. The iPS invention has brought a lot of hope in terms of potential direct benefits to treat several diseases, which have no definite options at the moment. But, we envisage that several spin-offs could come out of this invention and one very significant spin-off finding recently witnessed is the finding by Prof. Masaharu Seno and his team of researchers at the Okayama University, Japan (Chen L, et al. 2012, PLoS ONE 7(4:e33544.doi:10.1371/journal.pone.0033544. According to Prof. Seno, mouse iPS cells (miPS when cultured in the conditioned medium derived from cancer cell lines, differentiate into cancer stem cells (CSCs. While differentiating into CSCs, they do retain the potential to develop endothelial progenitor cells. Several questions arise here: 1.Are these miPS derived CSCs really pluripotent, even if the terminal differentiation destined to specific phenotypes? 2.Shouldn’t the Cancer Stem Cells be termed as cancer progenitor cells, as till date they are considered to be producing only cancer cells but not pluripotent to yield other types of normal tissues? The spin-offs could be infinite as the process of differentiation and de-differentiation happening due to trillions of signals and pathways, most still remaining not-so-well understood. A special mention should be made to Prof. Shinya Yamanaka as he has several sterling qualities to be a role-model for budding scientists. Apart from his passion for science, which made him shift his career from orthopedics to a cell biologist, his

  6. Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.

    Science.gov (United States)

    Haraguchi, Yuji; Matsuura, Katsuhisa; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

    2015-12-01

    In this study, a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT-PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A, BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes, including HCN4, MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering.

  7. [Endoplasmic-mitochondrial Ca(2+)-functional unit: dependence of respiration of secretory cells on activity of ryanodine- and IP3 - sensitive Ca(2+)-channels].

    Science.gov (United States)

    Velykopols'ka, O Iu; Man'ko, B O; Man'ko, V V

    2012-01-01

    Using Clark oxygen electrode, dependence of mitochondrial functions on Ca(2+)-release channels activity of Chironomus plumosus L. larvae salivary glands suspension was investigated. Cells were ATP-permeabilized in order to enable penetration of exogenous oxidative substrates. Activation of plasmalemmal P2X-receptors (as well as P2Y-receptors) per se does not modify the endogenous respiration of salivary gland suspension. That is, Ca(2+)-influx from extracellular medium does not influence functional activity of mitochondria, although they are located along the basal part of the plasma membrane. Activation of RyRs intensifies endogenous respiration and pyruvate-malate-stimulated respiration, but not succinate-stimulated respiration. Neither activation of IP3Rs (via P2Y-receptors activation), nor their inhibition alters endogenous respiration. Nevertheless, IP3Rs inhibition by 2-APB intensifies succinate-stimulated respiration. All abovementioned facts testify that Ca2+, released from stores via channels, alters functional activity of mitochondria, and undoubtedly confirm the existence of endoplasmic-mitochondrial Ca(2+)-functional unit in Ch. plumosus larvae salivary glands secretory cells. In steady state of endoplasmic-mitochondrial Ca(2+)-functional unit the spontaneous activity of IP3Rs is observed; released through IP3Rs, Ca2+ is accumulated in mitochondria via uniporter and modulates oxidative processes. Activation of RyRs induces the transition of endoplasmic-mitochondrial Ca(2+)-functional unit to the active state, which is required to intensify cell respiration and oxidative phosphorylation. As expected, the transition of endoplasmic-mitochondrial Ca(2+)-functional unit to inactivated state (i. e. inhibition of Ca(2+)-release channels at excessive [Ca2+]i) limits the duration of signal transduction, has protective nature and prevents apoptosis.

  8. Rabies virus infection induces type I interferon production in an IPS-1 dependent manner while dendritic cell activation relies on IFNAR signaling.

    Directory of Open Access Journals (Sweden)

    Elizabeth J Faul

    Full Text Available As with many viruses, rabies virus (RABV infection induces type I interferon (IFN production within the infected host cells. However, RABV has evolved mechanisms by which to inhibit IFN production in order to sustain infection. Here we show that RABV infection of dendritic cells (DC induces potent type I IFN production and DC activation. Although DCs are infected by RABV, the viral replication is highly suppressed in DCs, rendering the infection non-productive. We exploited this finding in bone marrow derived DCs (BMDC in order to differentiate which pattern recognition receptor(s (PRR is responsible for inducing type I IFN following infection with RABV. Our results indicate that BMDC activation and type I IFN production following a RABV infection is independent of TLR signaling. However, IPS-1 is essential for both BMDC activation and IFN production. Interestingly, we see that the BMDC activation is primarily due to signaling through the IFNAR and only marginally induced by the initial infection. To further identify the receptor recognizing RABV infection, we next analyzed BMDC from Mda-5-/- and RIG-I-/- mice. In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection. However, only RIG-I-/- cells exhibit a delay in type I IFN production. In order to determine the role that IPS-1 plays in vivo, we infected mice with pathogenic RABV. We see that IPS-1-/- mice are more susceptible to infection than IPS-1+/+ mice and have a significantly increased incident of limb paralysis.

  9. Effect of dietary fat saturation and cholesterol on low density lipoprotein degradation by mononuclear cells of Cebus monkeys.

    Science.gov (United States)

    Kuo, P C; Rudd, M A; Nicolosi, R; Loscalzo, J

    1989-01-01

    The mechanism by which dietary unsaturated fatty acids lower low density lipoprotein (LDL) cholesterol is unknown. Unsaturated fatty acids incorporated into the cell membrane can increase membrane fluidity and, as a result, dramatically alter membrane-dependent cell functions. Therefore, we examined the effect of long-term dietary consumption of corn oil and coconut oil with and without cholesterol in amounts equivalent to those of a typical Western diet on the degradation of human LDL by peripheral blood mononuclear cells in Cebus albifrons monkeys. Cellular LDL degradation was dramatically enhanced in the mononuclear cells isolated from animals fed corn oil in comparison with those from animals fed coconut oil. The addition of cholesterol to the diets resulted in a slight attenuation of LDL degradation in the corn oil group while no effect was noted in the coconut oil group. Crossover LDL binding and degradation experiments with LDL isolated from animals fed corn oil diets and coconut oil diets demonstrated increased binding and degradation of LDL in mononuclear cells from animals fed corn oil diets. Enhanced mononuclear cell LDL degradation was accompanied by increased cellular cis-unsaturated fatty acyl content, increased membrane fluidity, and decreased plasma cholesterol. Increased cellular cis-unsaturated fatty acyl content with its concomitant increase in membrane fluidity mirrored the dietary lipid profile of the host animal. A linear relationship was observed between cellular LDL degradation and both cellular cis-unsaturated fatty acyl content and membrane fluidity. These observations parallel results noted in whole-animal LDL catabolic studies with these same animals described elsewhere. These data suggest a novel mechanism by which dietary unsaturated fatty acids exert their LDL-lowering effect.

  10. MULTICELLULAR-MEDIATED EXPRESSION OF P-GP AND MRP AND RELATIONSHIP WITH CELL CYCLE PROFILES IN HUMAN OVARIAN CANCER SK-OV-3ip1 MULTICELLULAR AGGREGATES

    Institute of Scientific and Technical Information of China (English)

    陈建利; 丰有吉; 张琴

    2001-01-01

    To investigate the expression of P-glycoprotein (P-gp) and multidrug resistance- associated protein (MRP) and the relationship with cell cycle profiles in ovarian cancer SK-OV-3ip1 multicellular aggregates. Methods: Liquid overlay system was employed to obtain multicellular aggregates. Expression of P-gp and MRP was detected with flow cytometry (FCM). Outer, intermediate and inner cells from multicellular aggregates were collected by layer-trypsinized method. Cell cycle profiles were also analyzed by FCM. Results: Compared with control cells, no expression of P-gp and MRP was detected in monolyer cells (P=0.128 and P=0.604), but expression of P-gp and MRP in aggregate cells was significantly elevated (P<0.01). P-gp expression in every layer cells was also obviously increased (P<0.01). Furthermore, P-gp expression in every layer cells was also obviously increased (P=0.071). Tendency to increased G0-G1 phase and reduced S phase cells existed from outer through intermediate to inner layers in multicellular aggregates but with no statistical difference. Cell percentages in G2-M phase also had no difference. However, compared with monolayer cells, cells in G0-G1 phase increased and cells in S and G2-M phases lowered significantly in every layer and in the whole multicellular aggregates. Expression elevation of P-gp and MRP was consistent with increased G0-G1 percentage in aggregate cells. Conclusion: Expression of P-gp and MRP increases in cells of SK-OV-3ip1 multicellular aggregates and is consistent with increased G0-G1 percentage, which implies the possible relationship between them and the possible role in multicellular-mediated drug resistance.

  11. CtIP-Specific Roles during Cell Reprogramming Have Long-Term Consequences in the Survival and Fitness of Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Daniel Gómez-Cabello

    2017-02-01

    Full Text Available Acquired genomic instability is one of the major concerns for the clinical use of induced pluripotent stem cells (iPSCs. All reprogramming methods are accompanied by the induction of DNA damage, of which double-strand breaks are the most cytotoxic and mutagenic. Consequently, DNA repair genes seem to be relevant for accurate reprogramming to minimize the impact of such DNA damage. Here, we reveal that reprogramming is associated with high levels of DNA end resection, a critical step in homologous recombination. Moreover, the resection factor CtIP is essential for cell reprogramming and establishment of iPSCs, probably to repair reprogramming-induced DNA damage. Our data reveal a new role for DNA end resection in maintaining genomic stability during cell reprogramming, allowing DNA repair fidelity to be retained in both human and mouse iPSCs. Moreover, we demonstrate that reprogramming in a resection-defective environment has long-term consequences on stem cell self-renewal and differentiation.

  12. Vector-free and transgene-free human iPS cells differentiate into functional neurons and enhance functional recovery after ischemic stroke in mice.

    Directory of Open Access Journals (Sweden)

    Osama Mohamad

    Full Text Available Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited, stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently, the creation of induced pluripotent stem (iPS cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition, the creation of vector-free and transgene-free human iPS (hiPS cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However, the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation, we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice, hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling, increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice.

  13. Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells.

    Science.gov (United States)

    Adams, Rebecca L; Mason, Aaron C; Glass, Laura; Aditi; Wente, Susan R

    2017-09-04

    The mRNA lifecycle is driven through spatiotemporal changes in the protein composition of mRNA particles (mRNPs) that are triggered by RNA-dependent DEAD-box protein (Dbp) ATPases. As mRNPs exit the nuclear pore complex (NPC) in Saccharomyces cerevisiae, this remodeling occurs through activation of Dbp5 by inositol hexakisphosphate (IP6 )-bound Gle1. At the NPC, Gle1 also binds Nup42, but Nup42's molecular function is unclear. Here we employ the power of structure-function analysis in S. cerevisiae and human (h) cells, and find that the high-affinity Nup42-Gle1 interaction is integral to Dbp5 (hDDX19B) activation and efficient mRNA export. The Nup42 carboxy-terminal domain (CTD) binds Gle1/hGle1B at an interface distinct from the Gle1-Dbp5/hDDX19B interaction site. A nup42-CTD/gle1-CTD/Dbp5 trimeric complex forms in the presence of IP6 . Deletion of NUP42 abrogates Gle1-Dbp5 interaction, and disruption of the Nup42 or IP6 binding interfaces on Gle1/hGle1B leads to defective mRNA export in S. cerevisiae and human cells. In vitro, Nup42-CTD and IP6 stimulate Gle1/hGle1B activation of Dbp5 and DDX19B recombinant proteins in similar, non-additive manners, demonstrating complete functional conservation between humans and S. cerevisiae. Together, a highly conserved mechanism governs spatial coordination of mRNP remodeling during export. This has implications for understanding human disease mutations that perturb the Nup42-hGle1B interaction. This article is protected by copyright. All rights reserved.

  14. Activation of the MDA-5-IPS-1 Viral Sensing Pathway Induces Cancer Cell Death and Type I IFN-Dependent Antitumor Immunity.

    Science.gov (United States)

    Yu, Xiaofei; Wang, Hongxia; Li, Xia; Guo, Chunqing; Yuan, Fang; Fisher, Paul B; Wang, Xiang-Yang

    2016-04-15

    Melanoma differentiation-associated gene 5 (MDA-5, IFIH1), a cytosolic innate pattern recognition receptor, functions as a first line of defense against viral infection by sensing double-stranded RNA. Ectopic expression of MDA-5 has been shown to induce cancer cell death, but the mechanism of action by which MDA-5 exerts these cytotoxic effects is unclear. Here, we demonstrate that ectopic expression of MDA-5 via replication-incompetent adenovirus (Ad.Mda-5) initiates multiple signaling cascades, culminating in cytotoxicity and type I IFN production in mouse and human prostate cancer cells. This intrinsic dual activity of MDA-5 required the adaptor protein IFNβ promoter stimulator 1 (IPS-1, MAVS) and could be functionally uncoupled. MDA-5 lacking N-terminal caspase recruitment domains (CARD) engaged an intracellular death program in cancer cells but was unable to efficiently stimulate the expression of IFNβ. In contrast to cancer cells susceptible to MDA-5-mediated cytotoxicity, normal cells were highly resistant and instead developed a robust type I IFN response. Strikingly, intratumoral delivery of Ad.Mda-5 led to regression of preestablished prostate cancers and development of long-lasting antitumor immune memory, which was primarily attributed to the activation of tumor-reactive cytotoxic T lymphocytes and/or natural killer cells. Using the CARD-truncated MDA-5 mutant, silencing of IPS-1, and antibody blockade of the IFNα/β receptor, we further demonstrate that type I IFN signaling was crucial for in situ MDA-5-induced protective antitumor immunity. Therefore, deliberately targeting the evolutionarily conserved MDA-5-IPS-1 antiviral pathway in tumors can provoke parallel tumoricidal and immunostimulatory effects that bridge innate and adaptive immune responses for the therapeutic treatment of cancer. Cancer Res; 76(8); 2166-76. ©2016 AACR. ©2016 American Association for Cancer Research.

  15. Generation of GFAP::GFP astrocyte reporter lines from human adult fibroblast-derived iPS cells using zinc-finger nuclease technology.

    Science.gov (United States)

    Zhang, Ping-Wu; Haidet-Phillips, Amanda M; Pham, Jacqueline T; Lee, Youngjin; Huo, Yuqing; Tienari, Pentti J; Maragakis, Nicholas J; Sattler, Rita; Rothstein, Jeffrey D

    2016-01-01

    Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100β, NFIA and ALDH1L1. In addition, mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy.

  16. Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells

    Directory of Open Access Journals (Sweden)

    Han Yong-Mahn

    2009-07-01

    Full Text Available Abstract Background Interspecies somatic cell nuclear transfer (iSCNT has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. Results No apparent difference in cleavage rate was found among IVF, monkey-bovine (MB-iSCNT, and bovine-bovine (BB-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM, we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively. Conclusion The down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

  17. Model for long QT syndrome type 2 using human iPS cells demonstrates arrhythmogenic characteristics in cell culture

    Directory of Open Access Journals (Sweden)

    Anna L. Lahti

    2012-03-01

    Long QT syndrome (LQTS is caused by functional alterations in cardiac ion channels and is associated with prolonged cardiac repolarization time and increased risk of ventricular arrhythmias. Inherited type 2 LQTS (LQT2 and drug-induced LQTS both result from altered function of the hERG channel. We investigated whether the electrophysiological characteristics of LQT2 can be recapitulated in vitro using induced pluripotent stem cell (iPSC technology. Spontaneously beating cardiomyocytes were differentiated from two iPSC lines derived from an individual with LQT2 carrying the R176W mutation in the KCNH2 (HERG gene. The individual had been asymptomatic except for occasional palpitations, but his sister and father had died suddenly at an early age. Electrophysiological properties of LQT2-specific cardiomyocytes were studied using microelectrode array and patch-clamp, and were compared with those of cardiomyocytes derived from control cells. The action potential duration of LQT2-specific cardiomyocytes was significantly longer than that of control cardiomyocytes, and the rapid delayed potassium channel (IKr density of the LQT2 cardiomyocytes was significantly reduced. Additionally, LQT2-derived cardiac cells were more sensitive than controls to potentially arrhythmogenic drugs, including sotalol, and demonstrated arrhythmogenic electrical activity. Consistent with clinical observations, the LQT2 cardiomyocytes demonstrated a more pronounced inverse correlation between the beating rate and repolarization time compared with control cells. Prolonged action potential is present in LQT2-specific cardiomyocytes derived from a mutation carrier and arrhythmias can be triggered by a commonly used drug. Thus, the iPSC-derived, disease-specific cardiomyocytes could serve as an important platform to study pathophysiological mechanisms and drug sensitivity in LQT2.

  18. Hepatic maturation of human iPS cell-derived hepatocyte-like cells by ATF5, c/EBPα, and PROX1 transduction.

    Science.gov (United States)

    Nakamori, Daiki; Takayama, Kazuo; Nagamoto, Yasuhito; Mitani, Seiji; Sakurai, Fuminori; Tachibana, Masashi; Mizuguchi, Hiroyuki

    2016-01-15

    Hepatocyte-like cells differentiated from human iPS cells (human iPS-HLCs) are expected to be utilized in drug development and research. However, recent hepatic characterization of human iPS-HLCs showed that these cells resemble fetal hepatocytes rather than adult hepatocytes. Therefore, in this study, we aimed to develop a method to enhance the hepatic function of human iPS-HLCs. Because the gene expression levels of the hepatic transcription factors (activating transcription factor 5 (ATF5), CCAAT/enhancer-binding protein alpha (c/EBPα), and prospero homeobox protein 1 (PROX1)) in adult liver were significantly higher than those in human iPS-HLCs and fetal liver, we expected that the hepatic functions of human iPS-HLCs could be enhanced by adenovirus (Ad) vector-mediated ATF5, c/EBPα, and PROX1 transduction. The gene expression levels of cytochrome P450 (CYP) 2C9, 2E1, alpha-1 antitrypsin, transthyretin, Na+/taurocholate cotransporting polypeptide, and uridine diphosphate glucuronosyl transferase 1A1 and protein expression levels of CYP2C9 and CYP2E1 were upregulated by ATF5, c/EBPα, and PROX1 transduction. These results suggest that the hepatic functions of the human iPS-HLCs could be enhanced by ATF5, c/EBPα, and PROX1 transduction. Our findings would be useful for the hepatic maturation of human iPS-HLCs.

  19. Apoptotic effect of IP6 was not enhanced by co-treatment with myo-inositol in prostate carcinoma PC3 cells

    OpenAIRE

    Kim, Hyun-Jung; Jang, Yu-mi; Kim, Harriet; Kwon, Young Hye

    2007-01-01

    Inositol hexaphosphate (IP6) is a major constituent of most cereals, legumes, nuts, oil seeds and soybean. Previous studies reported the anticancer effect of IP6 and suggested that co-treatment of IP6 with inositol may enhance anticancer effect of IP6. Although the anticancer effect of IP6 has been intensively studied, the combinational effect of IP6 and inositol and involved mechanisms are not well understood so far. In the present study, we investigated the effect of IP6 and myo-inositol (M...

  20. Effects of Overexpression of IP-10 Gene on MA-10 Mouse Leydig Tumor Cell Steroidogenesis and Cell Proliferation%IP-10基因过量表达对MA-10小鼠Leydig肿瘤细胞类固醇合成及细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    陈粤; 苏文金; LIN T; NAGPAL ML

    2005-01-01

    背景与目的:研究在体外培养的MA-10小鼠Leydig肿瘤细胞中,IP-10基因的过表达对细胞类固醇合成以及细胞增殖的影响作用.材料与方法:采用细胞转染实验,将含有IP-10基因cDNA的重组质粒转入MA-10小鼠Leydig肿瘤细胞中,用Western blotting法检测细胞IP-10基因的过表达,用放免分析(Radioimmuno assay,RIA)方法检测IP-10基因过表达对MA-10细胞孕酮合成的影响,用3H-Thymidine掺入DNA合成实验研究IP-10基因过表达对MA-10细胞增殖的作用.结果:实验数据表明,成功地在MA-10细胞中过量表达了IP-10蛋白;MA-10细胞内转染的IP-10基因的过量表达可显著抑制8-bromo-cAMP(0.2 mmol/L)诱导的孕酮生成,加入1.0 μgIP-10重组质粒转染细胞时,可以使8-bromo-cAMP诱导的孕酮的合成水平由对照组的(38.5±1.7)ng/mL显著降低到(23.2±1.5)ng/ml(1.5×105cells/ml),且抑制作用随所用IP-10重组质粒的浓度增加而增加,它的抑制作用可能是通过减少了类固醇合成急性调节蛋白StAR基因的表达而达到的.3H-Thymidine掺入实验结果还显示,IP-10基因在转染的MA-10细胞中过量表达能够显著抑制MA-10细胞的增殖.结论:过量表达的IP-10基因对MA-10小鼠Leydig肿瘤细胞的类固醇合成及生长具有显著的抑制作用,此结果提示我们可以考虑使用转入IP-10基因的基因疗法治疗前列腺癌等肿瘤疾病,而其抑制细胞增殖的机制还需进一步探讨.

  1. No monkey business: why studying NK cells in nonhuman primates pays off

    Directory of Open Access Journals (Sweden)

    Henoch Sangjoon Hong

    2013-02-01

    Full Text Available Human NK cells play a key role in mediating host immune responses against various infectious diseases. For practical reasons, the majority of the data on human NK cells has been generated using peripheral blood lymphocytes. In contrast, our knowledge of NK cells in human tissues is limited, and not much is known about developmental pathways of human NK cell subpopulations in vivo. Although research in mice has elucidated a number of fundamental features of NK cell biology, mouse and human NK cells significantly differ in their subpopulations, functions and receptor repertoires. Thus, there is a need for a model that is more closely related to humans and yet allows experimental manipulations. Nonhuman primate models offer numerous opportunities for the study of NK cells, including the study of the role of NK cells after solid organ and stem cell transplantation, as well as in acute viral infection. Macaque NK cells can be depleted in vivo or adoptively transferred in an autologous system. All of these studies are either difficult or unethical to carry out in humans. Here we highlight recent advances in rhesus NK cell research and their parallels in humans. Using high-throughput transcriptional profiling, we demonstrate that the human CD56bright and CD56dim NK cell subsets have phenotypically and functionally analogous counterparts in rhesus macaques. Thus, the use of nonhuman primate models offers the potential to substantially advance human NK cell research.

  2. ES cells derived from cloned embryos in monkey - a jump toward human therapeutic cloning

    Institute of Scientific and Technical Information of China (English)

    Xiangzhong Yang; Sadie L Smith

    2007-01-01

    @@ Therapeutic cloning refers to the derivation of embryonic stem cells (ntESC) from embryos derived from somatic cell nuclear transfer (SCNT) also known as cloning. Cloning involves transplanting a differentiated cell into an oocyte that has had its nucleus (DNA) removed.

  3. No Monkeying Around : Clonal Tracking of Stem Cells and Progenitors in the Macaque

    NARCIS (Netherlands)

    Dykstra, Brad; Bystrykh, Leonid V.

    2014-01-01

    Clonal tracking of hematopoietic stem and progenitor cells (HSPCs) has proven valuable for studying their behavior in murine recipients. Now in Cell Stem Cell, Kim et al. (2014) and Wu et al. (2014) extend these analyses to nonhuman primates, providing insights into dynamics of HSPC expansion and li

  4. Rhesus monkeys kidney cells persistently infected with Simian Virus 40: production of defective interfering virus and acquisition of the transformed phenotype.

    Science.gov (United States)

    Norkin, L C

    1976-09-01

    Monolayer cultures of LLC-MK2 rhesus monkey kidney cells became persistently infected with simian virus 40 (SV40) when infected at a multiplicity of infection of 100 plaque-forming units/cell. A stable carrier state developed characterized by extensive viral proliferation without obvious cytopathic effect other than the slow growth of these cultures. By 11 weeks all cells produced the SV40 T antigen. In contrast, less than 5% of the cells produced V antigen. Virus-free clonal isolates were obtained by cloning in SV40 antiserum. Continuous cultivation in antiserum resulted in a temporary cure of unclone cultures. When virus did eventually reappear in the "cured" cultures the titers remained low. The virus produced by the carrier culture was defective at both 31 and 37% c, and it interfered with the growth of standard s40 during mixed infection of CV-1 green monkey kidney cells. All of the interfering activity in carrier culture homogenates could be sedimented by centrifugation at 109,000 x g for 3 h. These cultures were completely susceptible to vesicular stomatitis virus. Extensive viral deoxyribonucleic acid synthesis occurred in CV-1 cells infected with carrier culture virus. Carrier culture homogenates are only slightly less cytopathic to CV-1 cells than standard SV40. The carrier culture express several properties of SV40 transformation.

  5. Multimodal convergence within the intraparietal sulcus of the macaque monkey.

    Science.gov (United States)

    Guipponi, Olivier; Wardak, Claire; Ibarrola, Danielle; Comte, Jean-Christophe; Sappey-Marinier, Dominique; Pinède, Serge; Ben Hamed, Suliann

    2013-02-27

    The parietal cortex is highly multimodal and plays a key role in the processing of objects and actions in space, both in human and nonhuman primates. Despite the accumulated knowledge in both species, we lack the following: (1) a general description of the multisensory convergence in this cortical region to situate sparser lesion and electrophysiological recording studies; and (2) a way to compare and extrapolate monkey data to human results. Here, we use functional magnetic resonance imaging (fMRI) in the monkey to provide a bridge between human and monkey studies. We focus on the intraparietal sulcus (IPS) and specifically probe its involvement in the processing of visual, tactile, and auditory moving stimuli around and toward the face. We describe three major findings: (1) the visual and tactile modalities are strongly represented and activate mostly nonoverlapping sectors within the IPS. The visual domain occupies its posterior two-thirds and the tactile modality its anterior one-third. The auditory modality is much less represented, mostly on the medial IPS bank. (2) Processing of the movement component of sensory stimuli is specific to the fundus of the IPS and coincides with the anatomical definition of monkey ventral intraparietal area (VIP). (3) A cortical sector within VIP processes movement around and toward the face independently of the sensory modality. This amodal representation of movement may be a key component in the construction of peripersonal space. Overall, our observations highlight strong homologies between macaque and human VIP organization.

  6. Differentiation of monkey embryonic stem cells to hepatocytes by feeder-free dispersion culture and expression analyses of cytochrome p450 enzymes responsible for drug metabolism.

    Science.gov (United States)

    Maruyama, Junya; Matsunaga, Tamihide; Yamaori, Satoshi; Sakamoto, Sakae; Kamada, Noboru; Nakamura, Katsunori; Kikuchi, Shinji; Ohmori, Shigeru

    2013-01-01

    We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.

  7. Transmission of colour and acuity signals by parvocellular cells in marmoset monkeys.

    Science.gov (United States)

    Martin, Paul R; Blessing, Esther M; Buzás, Péter; Szmajda, Brett A; Forte, Jason D

    2011-06-01

    The red-green axis of colour vision evolved recently in primate evolutionary history. Signals serving red-green colour vision travel together with signals serving spatial vision, in the parvocellular (PC) division of the subcortical visual pathway. However, the question of whether receptive fields of PC pathway cells are specialized to transmit red-green colour signals remains unresolved. We addressed this question in single-cell recordings from the lateral geniculate nucleus of anaesthetized marmosets. Marmosets show a high proportion of dichromatic (red-green colour-blind) individuals, allowing spatial and colour tuning properties of PC cells to be directly compared in dichromatic and trichromatic visual systems. We measured spatial frequency tuning for sine gratings that provided selective stimulation of individual photoreceptor types. We found that in trichromatic marmosets, the foveal visual field representation is dominated by red-green colour-selective PC cells. Colour selectivity of PC cells is reduced at greater eccentricities, but cone inputs to centre and surround are biased to create more selectivity than predicted by a purely 'random wiring' model. Thus, one-to-one connections in the fovea are sufficient, but not necessary, to create colour-selective responses. The distribution of spatial tuning properties for achromatic stimuli shows almost complete overlap between PC cells recorded in dichromatic and trichromatic marmosets. These data indicate that transmission of red-green colour signals has been enabled by centre-surround receptive fields of PC cells, and has not altered the capacity of PC cells to serve high-acuity vision at high stimulus contrast.

  8. Refinement of the androgen response element based on ChIP-Seq in androgen-insensitive and androgen-responsive prostate cancer cell lines.

    Science.gov (United States)

    Wilson, Stephen; Qi, Jianfei; Filipp, Fabian V

    2016-09-14

    Sequence motifs are short, recurring patterns in DNA that can mediate sequence-specific binding for proteins such as transcription factors or DNA modifying enzymes. The androgen response element (ARE) is a palindromic, dihexameric motif present in promoters or enhancers of genes targeted by the androgen receptor (AR). Using chromatin immunoprecipitation sequencing (ChIP-Seq) we refined AR-binding and AREs at a genome-scale in androgen-insensitive and androgen-responsive prostate cancer cell lines. Model-based searches identified more than 120,000 ChIP-Seq motifs allowing for expansion and refinement of the ARE. We classified AREs according to their degeneracy and their transcriptional involvement. Additionally, we quantified ARE utilization in response to somatic copy number amplifications, AR splice-variants, and steroid treatment. Although imperfect AREs make up 99.9% of the motifs, the degree of degeneracy correlates negatively with validated transcriptional outcome. Weaker AREs, particularly ARE half sites, benefit from neighboring motifs or cooperating transcription factors in regulating gene expression. Taken together, ARE full sites generate a reliable transcriptional outcome in AR positive cells, despite their low genome-wide abundance. In contrast, the transcriptional influence of ARE half sites can be modulated by cooperating factors.

  9. A one-day, dispense-only IP-One HTRF assay for high-throughput screening of Galphaq protein-coupled receptors: towards cells as reagents.

    Science.gov (United States)

    Bergsdorf, Christian; Kropp-Goerkis, Carmen; Kaehler, Irene; Ketscher, Lars; Boemer, Ulf; Parczyk, Karsten; Bader, Benjamin

    2008-02-01

    Abstract: Compared to biochemical high-throughput screening (HTS) assays, cell-based functional assays are generally thought to be more time consuming and complex because of additional efforts for running continuous cell cultures as well as the numerous assay steps when transferring media and compounds. A common strategy to compensate the anticipated reduction in overall throughput is to implement highly automated cell culture and screening systems. However, such systems require substantial investments in sophisticated hardware and highly specialized personnel. In trying to set up alternatives to increasing throughput in functional cell-based screening, we combined several approaches. By using (1) cryopreserved cell aliquots instead of continuous cell culture, (2) cells in suspension instead of adherent cells, and (3) "ready-to-screen" assay plates with nanoliter aliquots of test compounds, an assay procedure was developed that very much resembles a standard biochemical, enzymatic assay comprising only a few dispense steps. Chinese hamster ovary cells stably overexpressing a Galphaq-coupled receptor were used as a model system to measure receptor activation by detection of intracellular D-myo-inositol 1-phosphate with the help of homogeneous time-resolved fluorescence (HTRF, CISbio International, Bagnols-sur-Cèze, France). Initially established in 384-well adherent cell format, the assay was successfully transferred to 1,536-well format. The assay quality was sufficient to run HTS campaigns in both formats with good Z'-factors and excellent reproducibility of antagonists. Subsequently, the assay procedure was optimized for usage of suspension cells. The influences of cell culture media, plate type, cell number, and incubation time were assessed. Finally, the suspension cell assay was applied to pharmacological characterization of a small molecule antagonist by Schild plot analysis. Our data demonstrate not only the application of the IP-One HTRF assay (CISbio

  10. Voice over IP Security

    CERN Document Server

    Keromytis, Angelos D

    2011-01-01

    Voice over IP (VoIP) and Internet Multimedia Subsystem technologies (IMS) are rapidly being adopted by consumers, enterprises, governments and militaries. These technologies offer higher flexibility and more features than traditional telephony (PSTN) infrastructures, as well as the potential for lower cost through equipment consolidation and, for the consumer market, new business models. However, VoIP systems also represent a higher complexity in terms of architecture, protocols and implementation, with a corresponding increase in the potential for misuse. In this book, the authors examine the

  11. IP Routing Protocols

    Science.gov (United States)

    Nolasco Pinto, Armando

    2002-03-01

    Uyless Black is a widely known expert in computer networks and data communications. He is author of more than ten books in the communication technologies field, which puts him in a good position to address this topic. In IP Routing Protocols he starts by providing the background and concepts required for understanding TCP/IP technology. This is done clearly and assumes little prior knowledge of the area. As might be expected, he emphasizes the IP route discovery problem. Later he details several routing protocols.

  12. Time course and role of LH and FSH in the expansion of the Leydig cell population at the time of puberty in the rhesus monkey (Macaca mulatta)

    Science.gov (United States)

    Verhagen, I.; Ramaswamy, S.; Teerds, K.J.; Keijer, J.; Plant, T. M.

    2014-01-01

    In higher primates development of the adult population of Leydig cells has received little attention. Here, the emergence of 3β-hydroxysteroid dehydrogenase (HSD3B) positive cells in the testis of the rhesus monkey was examined during spontaneous puberty, and correlated with S-phase labeling in the interstitium at this critical stage of development. In addition, the relative role of LH and FSH in initiating the pubertal expansion of Leydig cells was studied by precociously stimulating the juvenile testis in vivo with pulsatile 11-day infusions of recombinant LH and FSH, either alone or in combination. At the time of castration, testes were immersion fixed in Bouin’s, embedded in paraffin and sectioned at 5 μm. Leydig cells/testis were enumerated using HSD3B as a Leydig cell marker. Leydig cell number per testis increased progressively during puberty to reach values in the adult approximately 10 fold greater than in early pubertal animals. The rise in cell number was associated with an increase in nuclear diameter. That the pubertal expansion of Leydig cell number was driven primarily by the increase in LH secretion at this stage of development was suggested by the finding that precocious stimulation of mid juvenile monkeys with LH, either alone or in combination with that of FSH, resulted in a 20 to 30 fold increase in the number of HSD3B positive cells. Interestingly, precocious FSH stimulation, alone, also resulted in appearance of Leydig cells as indicated by the occasional HSD3B positive cell in the interstitium. The nuclear diameter of these Leydig cells, however was less than that of those generated in response to LH. PMID:25269763

  13. Stimulation of inositol 1,4,5-trisphosphate (IP3 receptor subtypes by analogues of IP3.

    Directory of Open Access Journals (Sweden)

    Huma Saleem

    Full Text Available Most animal cells express mixtures of the three subtypes of inositol 1,4,5-trisphosphate receptor (IP(3R encoded by vertebrate genomes. Activation of each subtype by different agonists has not hitherto been examined in cells expressing defined homogenous populations of IP(3R. Here we measure Ca(2+ release evoked by synthetic analogues of IP(3 using a Ca(2+ indicator within the lumen of the endoplasmic reticulum of permeabilized DT40 cells stably expressing single subtypes of mammalian IP(3R. Phosphorylation of (1,4,5IP(3 to (1,3,4,5IP(4 reduced potency by ~100-fold. Relative to (1,4,5IP(3, the potencies of IP(3 analogues modified at the 1-position (malachite green (1,4,5IP(3, 2-position (2-deoxy(1,4,5IP(3 or 3-position (3-deoxy(1,4,5IP(3, (1,3,4,5IP(4 were similar for each IP(3R subtype. The potency of an analogue, (1,4,6IP(3, in which the orientations of the 2- and 3-hydroxyl groups were inverted, was also reduced similarly for all three IP(3R subtypes. Most analogues of IP(3 interact similarly with the three IP(3R subtypes, but the decrease in potency accompanying removal of the 1-phosphate from (1,4,5IP(3 was least for IP(3R3. Addition of a large chromophore (malachite green to the 1-phosphate of (1,4,5IP(3 only modestly reduced potency suggesting that similar analogues could be used to measure (1,4,5IP(3 binding optically. These data provide the first structure-activity analyses of key IP(3 analogues using homogenous populations of each mammalian IP(3R subtype. They demonstrate broadly similar structure-activity relationships for all mammalian IP(3R subtypes and establish the potential utility of (1,4,5IP(3 analogues with chromophores attached to the 1-position.

  14. Antiplatelet activity of inositol hexaphosphate (IP6).

    Science.gov (United States)

    Vucenik, I; Podczasy, J J; Shamsuddin, A M

    1999-01-01

    Platelet adhesion to endothelial cells, their aggregation and subsequent release of platelet-derived mediators are key steps in the pathogenesis of thrombosis and atherosclerosis. Using impedance technology the effect of inositol hexaphosphate (IP6) on platelet aggregation and adenosine triphosphate (ATP) release were simultaneously measured in whole blood obtained from healthy volunteers (n = 10). The platelets were activated with adenosine diphosphate (ADP) (10 microM), collagen (2 micrograms/mL), or thrombin (1 U/mL) in the presence or absence of IP6. IP6 significantly inhibited platelet aggregation induced with all agonists in a dose-response manner (p IP6 strongly and significantly reduced agonist-induced ATP release (p = 0.00247 for ADP; p = 0.0074 for collagen; p = 0.0069 for thrombin). These data demonstrate that IP6 effectively inhibits human platelet aggregation in vitro, suggesting its potential in reducing the risk for cardiovascular disease.

  15. IP Rights and Technological Platforms

    OpenAIRE

    Robert P. Merges

    2008-01-01

    This paper is about intellectual property rights (IPRs) and platform technologies. After a brief introduction explaining some basics of networks, standards and platforms, I turn to three policy issues. The first is the role of IP in what might be termed platform policies, the decisions by courts and regulators concerning whether and how to promote multi-party access to important digital platforms such as media player hardware, cell phones, PCs, and the like. I argue that for the most part the...

  16. Analysis list: Atf7ip [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Atf7ip Pluripotent stem cell + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Atf7ip....1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Atf7ip.5.tsv http://dbarchive.biosc...iencedbc.jp/kyushu-u/mm9/target/Atf7ip.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Atf7ip.Plurip...otent_stem_cell.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Pluripotent_stem_cell.gml ...

  17. Radiation-Released Histamine in the Rhesus Monkey as Modified by Mast Cell Depletion and Antihistamine

    Science.gov (United States)

    1975-06-01

    mast cell histamine depleter, compound 48/80 (1mg/kg per day) for four consecutive days and then irradiated (4000 rads), histamine concentrations did not change significantly. When 48/80 was given 20 min after irradiation, histamine concentrations changed from 18 + or - 2 ng/ml to a maximum of 35 + or - 9 ng/ml after 48/80 injection.

  18. Voice over IP

    OpenAIRE

    Mantula, Juha

    2006-01-01

    Tämä opinnäytetyö käsittelee Voice over Internet Protocol -tekniikkaa ja sen tuomia mahdollisuuksia yrityselämässä. Teoriaosa käsittelee VoIP:n kannalta tärkeitä pro-tokollia ja standardeja, VoIP:n ominaisuuksia sekä esittelee erilaisia puheohjelmia, jotka käyttävät VoIP-tekniikkaa hyväkseen. Empiirinen osuus tutkii Viestintä Ky Pitkärannan Skype-ohjelman käyttöä. Työn tarkoituksena on selvittää VoIP:n hyviä ja huonoja puolia ja sitä kuinka tek-niikkaa voidaan käyttää hyväksi päivittäisessä ...

  19. Wireless IP Telephony

    CERN Document Server

    Gerami, Mohsen

    2010-01-01

    The convergence of traditional telecommunications and the Internet is creating new network-based service delivery opportunities for telecommunications companies carriers, service providers, and network equipment providers. Voice over Wireless IP is one of the most exciting new developments emerging within the telephony market. It is set to revolutionize the delivery of mobile voice Services and provide exciting new opportunities for operators and service providers alike. This survey discusses principal of Wireless IP Telephony.

  20. Potentiation of nerve growth factor-induced neurite outgrowth in PC12 cells by ifenprodil: the role of sigma-1 and IP3 receptors.

    Directory of Open Access Journals (Sweden)

    Tamaki Ishima

    Full Text Available In addition to both the α1 adrenergic receptor and N-methyl-D-aspartate (NMDA receptor antagonists, ifenprodil binds to the sigma receptor subtypes 1 and 2. In this study, we examined the effects of ifenprodil on nerve growth factor (NGF-induced neurite outgrowth in PC12 cells. Ifenprodil significantly potentiated NGF-induced neurite outgrowth, in a concentration-dependent manner. In contrast, the α1 adrenergic receptor antagonist, prazosin and the NMDA receptor NR2B antagonist, Ro 25-6981 did not alter NGF-induced neurite outgrowth. Potentiation of NGF-induced neurite outgrowth mediated by ifenprodil was significantly antagonized by co-administration of the selective sigma-1 receptor antagonist, NE-100, but not the sigma-2 receptor antagonist, SM-21. Similarly, ifenprodil enhanced NGF-induced neurite outgrowth was again significantly reduced by the inositol 1,4,5-triphosphate (IP(3 receptor antagonists, xestospongin C and 2-aminoethoxydiphenyl borate (2-APB treatment. Furthermore, BAPTA-AM, a chelator of intracellular Ca(2+, blocked the effects of ifenprodil on NGF-induced neurite outgrowth, indicating the role of intracellular Ca(2+ in the neurite outgrowth. These findings suggest that activation at sigma-1 receptors and subsequent interaction with IP(3 receptors may mediate the pharmacological effects of ifenprodil on neurite outgrowth.

  1. Isolation of saffold virus type 2 in green monkey kidney cells.

    Science.gov (United States)

    Blomqvist, Soile; Lappalainen, Maija; Paananen, Anja; Ylipaasto, Petri; Roivainen, Merja

    2012-09-01

    Saffold viruses (SAFV) have been discovered recently and they are classified into Theilovirus species in genus Cardiovirus in the Picornaviridae family. SAFV, especially those belonging to the genotype 2, have been difficult to propagate in laboratory cell lines. This study describes the successful isolation of an efficiently growing SAFV-2 strain directly from a stool specimen by standard virological methods. The availability of SAFV isolates that can be propagated to high titers is crucial to the future studies on pathogenesis and epidemiology of these novel human viruses. Copyright © 2012 Wiley Periodicals, Inc.

  2. Combined expression of CTGF and tissue inhibitor of metalloprotease-1 promotes synthesis of proteoglycan and collagen type Ⅱ in rhesus monkey lumbar intervertebral disc cells in vitro

    Institute of Scientific and Technical Information of China (English)

    LIU Yong; KONG Jie; CHEN Bo-hua; HU You-gu

    2010-01-01

    Background Low back pain has emerged as a widespread disease often caused by intervertebral disc degeneration.This study aimed to establish an in vitro cell culture model of rhesus monkey lumbar intervertebral discs and to investigate the effect of combined connective tissue growth factor (CTGF) and tissue inhibitor of metalloprotease-1(TIMP-1) expression mediated by adeno-associated virus (AAV) on collagen type Ⅱ and proteoglycan levels.The purpose of these investigations was to explore potential methods for relieving the degeneration of lumbar intervertebral disc cells.Methods Rhesus monkey lumbar intervertebral disc nucleus pulposus cells (NPCs) were isolated by enzyme digestion,cultured, and transduced with rAAV2-CTGF-IRES-TIMP-1, rAAV2-CTGF, or rAAV2-TIMP-1 at a multiplicity of infection (MOl) of 106.The expression of collagen type Ⅱ and proteoglycan was measured using RT-PCR and Western blotting.The synthetic rate of proteoglycan was measured using 35S incorporation.Results Rhesus monkey lumbar intervertebral disc NPCs were transduced with rAAV2-CTGF-IRES-TIMP-1,rAAV2-CTGF, and rAAV2-TIMP-1 and the transduced genes were expressed and detected.Compared to the control,CTGF promoted the synthesis of collagen type Ⅱ and proteoglycan.TIMP-1 showed an enhancing effect on the expression of proteoglycan but no effect on collagen type Ⅱ.Expression of both genes in rhesus monkey lumbar intervertebral disc NPCs significantly enhances the synthesis of proteoglycan and collagen type Ⅱ.Conclusions Single gene transduction of CTGF or TIMP-1 can enhanced synthesis of proteoglycan.CTGF expression can also enhance collagen type Ⅱ protein synthesis.Combined transduction of both CTGF and TIMP1 can significantly promote the expression of proteoglycan and collagen type Ⅱ to levels greater than transduction of a single gene alone.Our study provides a good basis for multi-gene therapy to treat lumbar intervertebral disc degeneration.

  3. Nbs1 ChIP-Seq Identifies Off-Target DNA Double-Strand Breaks Induced by AID in Activated Splenic B Cells.

    Directory of Open Access Journals (Sweden)

    Lyne Khair

    2015-08-01

    Full Text Available Activation-induced cytidine deaminase (AID is required for initiation of Ig class switch recombination (CSR and somatic hypermutation (SHM of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq. We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID.

  4. Advantages of COS-1 monkey kidney epithelial cells as packaging host for small-volume production of high-quality recombinant lentiviruses.

    Science.gov (United States)

    Smith, Shannon L; Shioda, Toshi

    2009-04-01

    The HEK293T human embryonic kidney cells have been used widely as a packaging host for transfection-based production of recombinant lentiviruses. The present study describes advantages of using COS-1 African green monkey kidney cells versus HEK293T cells as a packaging host for small-volume production of high-quality recombinant lentiviruses. The particle performance index, defined as the ratio of infection-competent viral particles to the total number of particles, was three- to four-fold greater in transfection supernatants generated using COS-1 cells than that generated using HEK293T cells. Adhesion of HEK293T cells to the cell culture-treated plastic surface was weak, causing significant HEK293T cell contamination in the transfection supernatants produced by laboratory automation using the 96-well cell culture plates. In contrast, COS-1 cells adhered strongly to the plastic surface, and cell contamination was not detected in the transfection supernatants. These results suggest that COS-1 cells may be a useful alternative packaging host for use for automated generation of large numbers of high-quality lentivirus reagents, particularly because they eliminate the need for additional purification steps to remove viral particles from cell culture supernatant.

  5. Comparative study of the immunogenicity in mice and monkeys of an inactivated CA16 vaccine made from a human diploid cell line.

    Science.gov (United States)

    Yang, Erxia; Cheng, Chen; Zhang, Ying; Wang, Jingjing; Che, Yanchun; Pu, Jing; Dong, Chenghong; Liu, Longding; He, Zhanlong; Lu, Shuaiyao; Zhao, Yuan; Jiang, Li; Liao, Yun; Shao, Congwen; Li, Qihan

    2014-01-01

    The coxsackie A16 virus (CA16), along with enterovirus 71 (EV71), is a primary pathogen that causes hand, foot, and mouth disease (HFMD). To control HFMD, CA16, and EV71 vaccines are needed. In this study, an experimental inactivated CA16 vaccine was prepared using human diploid cells, and the vaccine's immunogenicity was analyzed in mice and rhesus monkeys. The results showed that the neutralizing antibody was developed in a dose-dependent manner, and was sustained for 70 days with an average GMT (geometric mean titer) level of 80 to 90 in immunized mouse and for 56 days with GMT of higher than 300 in monkeys. The neutralizing antibody had a cross-neutralizing activity against different viral strains (genotype A and B), and the specific IFN-γ-secreting cell response was activated by these virus strains in an ELISPOT assay. This study provides evidence for the potential use of inactivated CA16 as a candidate for use in vaccines.

  6. MECP2e1 isoform mutation affects the form and function of neurons derived from Rett syndrome patient iPS cells.

    Science.gov (United States)

    Djuric, Ugljesa; Cheung, Aaron Y L; Zhang, Wenbo; Mok, Rebecca S; Lai, Wesley; Piekna, Alina; Hendry, Jason A; Ross, P Joel; Pasceri, Peter; Kim, Dae-Sung; Salter, Michael W; Ellis, James

    2015-04-01

    MECP2 mutations cause the X-linked neurodevelopmental disorder Rett Syndrome (RTT) by consistently altering the protein encoded by the MECP2e1 alternative transcript. While mutations that simultaneously affect both MECP2e1 and MECP2e2 isoforms have been widely studied, the consequence of MECP2e1 deficiency on human neurons remains unknown. Here we report the first isoform-specific patient induced pluripotent stem cell (iPSC) model of RTT. RTTe1 patient iPS cell-derived neurons retain an inactive X-chromosome and express only the mutant allele. Single-cell mRNA analysis demonstrated they have a molecular signature of cortical neurons. Mutant neurons exhibited a decrease in soma size, reduced dendritic complexity and decreased cell capacitance, consistent with impaired neuronal maturation. The soma size phenotype was rescued cell-autonomously by MECP2e1 transduction in a level-dependent manner but not by MECP2e2 gene transfer. Importantly, MECP2e1 mutant neurons showed a dysfunction in action potential generation, voltage-gated Na(+) currents, and miniature excitatory synaptic current frequency and amplitude. We conclude that MECP2e1 mutation affects soma size, information encoding properties and synaptic connectivity in human neurons that are defective in RTT.

  7. Human neural stem cells survive long term in the midbrain of dopamine-depleted monkeys after GDNF overexpression and project neurites toward an appropriate target.

    Science.gov (United States)

    Wakeman, Dustin R; Redmond, D Eugene; Dodiya, Hemraj B; Sladek, John R; Leranth, Csaba; Teng, Yang D; Samulski, R Jude; Snyder, Evan Y

    2014-06-01

    Transplanted multipotent human fetal neural stem cells (hfNSCs) significantly improved the function of parkinsonian monkeys in a prior study primarily by neuroprotection, with only 3%-5% of cells expressing a dopamine (DA) phenotype. In this paper, we sought to determine whether further manipulation of the neural microenvironment by overexpression of a developmentally critical molecule, glial cell-derived neurotrophic factor (GDNF), in the host striatum could enhance DA differentiation of hfNSCs injected into the substantia nigra and elicit growth of their axons to the GDNF-expressing target. hfNSCs were transplanted into the midbrain of 10 green monkeys exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine. GDNF was delivered concomitantly to the striatum via an adeno-associated virus serotype 5 vector, and the fate of grafted cells was assessed after 11 months. Donor cells remained predominantly within the midbrain at the injection site and sprouted numerous neurofilament-immunoreactive fibers that appeared to course rostrally toward the striatum in parallel with tyrosine hydroxylase-immunoreactive fibers from the host substantia nigra but did not mature into DA neurons. This work suggests that hfNSCs can generate neurons that project long fibers in the adult primate brain. However, in the absence of region-specific signals and despite GDNF overexpression, hfNSCs did not differentiate into mature DA neurons in large numbers. It is encouraging, however, that the adult primate brain appeared to retain axonal guidance cues. We believe that transplantation of stem cells, specifically instructed ex vivo to yield DA neurons, could lead to reconstruction of some portion of the nigrostriatal pathway and prove beneficial for the parkinsonian condition. ©AlphaMed Press.

  8. IP over DWDM

    Science.gov (United States)

    Allen, Mark E.

    2003-04-01

    The industry has been full of many lively debates about exactly how the functions of layer 1 and 2 (physical layer and data-link layer) will be orchestrated to support the growing amount of IP traffic. In effect, this book must simultaneously address layers 1 through 3. Two main strengths of the book are in describing current state-of-the-art research in very-high-speed IP networks and in detailing the standards activities related to net-work management, software, and signaling.

  9. From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium.

    Science.gov (United States)

    Reichman, Sacha; Terray, Angélique; Slembrouck, Amélie; Nanteau, Céline; Orieux, Gaël; Habeler, Walter; Nandrot, Emeline F; Sahel, José-Alain; Monville, Christelle; Goureau, Olivier

    2014-06-10

    Progress in retinal-cell therapy derived from human pluripotent stem cells currently faces technical challenges that require the development of easy and standardized protocols. Here, we developed a simple retinal differentiation method, based on confluent human induced pluripotent stem cells (hiPSC), bypassing embryoid body formation and the use of exogenous molecules, coating, or Matrigel. In 2 wk, we generated both retinal pigmented epithelial cells and self-forming neural retina (NR)-like structures containing retinal progenitor cells (RPCs). We report sequential differentiation from RPCs to the seven neuroretinal cell types in maturated NR-like structures as floating cultures, thereby revealing the multipotency of RPCs generated from integration-free hiPSCs. Furthermore, Notch pathway inhibition boosted the generation of photoreceptor precursor cells, crucial in establishing cell therapy strategies. This innovative process proposed here provides a readily efficient and scalable approach to produce retinal cells for regenerative medicine and for drug-screening purposes, as well as an in vitro model of human retinal development and disease.

  10. Generation of chimeric rhesus monkeys.

    Science.gov (United States)

    Tachibana, Masahito; Sparman, Michelle; Ramsey, Cathy; Ma, Hong; Lee, Hyo-Sang; Penedo, Maria Cecilia T; Mitalipov, Shoukhrat

    2012-01-20

    Totipotent cells in early embryos are progenitors of all stem cells and are capable of developing into a whole organism, including extraembryonic tissues such as placenta. Pluripotent cells in the inner cell mass (ICM) are the descendants of totipotent cells and can differentiate into any cell type of a body except extraembryonic tissues. The ability to contribute to chimeric animals upon reintroduction into host embryos is the key feature of murine totipotent and pluripotent cells. Here, we demonstrate that rhesus monkey embryonic stem cells (ESCs) and isolated ICMs fail to incorporate into host embryos and develop into chimeras. However, chimeric offspring were produced following aggregation of totipotent cells of the four-cell embryos. These results provide insights into the species-specific nature of primate embryos and suggest that a chimera assay using pluripotent cells may not be feasible.

  11. [Application of induced pluripotent stem (iPS) cells for nerve injury in the central nervous system].

    Science.gov (United States)

    Kinoshita, Hiroyuki

    2013-03-01

    Induced pluripotent stem cells (iPSCs), with both pluripotency and replication competence similar to embryonic stem cells (ESCs), have been developed from mouse fibroblasts in 2006 by Yamanaka et al. iPSCs are unique in employing somatic cells for their production, and can avoid ethical issues existing in ESCs. It is clear that progress in technology to produce iPSCs is one of the most crucial achievements of medicine in this century. Technology with the new pluripotent cells will offer many advantages in the field of regeneration medicine supplying new tissues to the injured organ and/or development of methodology to uncover many genetic diseases. On the other hand, we have to await adequate progress in issues regarding iPSCs, including enhanced efficiency to obtain iPSCs, the technology to produce organs from the cells, avoidance of tumorigenesis and decrease in immunity in response to iPSCs.

  12. Representation of the visual field in the primary visual area of the marmoset monkey: magnification factors, point-image size, and proportionality to retinal ganglion cell density.

    Science.gov (United States)

    Chaplin, Tristan A; Yu, Hsin-Hao; Rosa, Marcello G P

    2013-04-01

    The primary visual area (V1) forms a systematic map of the visual field, in which adjacent cell clusters represent adjacent points of visual space. A precise quantification of this map is key to understanding the anatomical relationships between neurons located in different stations of the visual pathway, as well as the neural bases of visual performance in different regions of the visual field. We used computational methods to quantify the visual topography of V1 in the marmoset (Callithrix jacchus), a small diurnal monkey. The receptive fields of neurons throughout V1 were mapped in two anesthetized animals using electrophysiological recordings. Following histological reconstruction, precise 3D reconstructions of the V1 surface and recording sites were generated. We found that the areal magnification factor (M(A) ) decreases with eccentricity following a function that has the same slope as that observed in larger diurnal primates, including macaque, squirrel, and capuchin monkeys, and humans. However, there was no systematic relationship between M(A) and polar angle. Despite individual variation in the shape of V1, the relationship between M(A) and eccentricity was preserved across cases. Comparison between V1 and the retinal ganglion cell density demonstrated preferential magnification of central space in the cortex. The size of the cortical compartment activated by a punctiform stimulus decreased from the foveal representation towards the peripheral representation. Nonetheless, the relationship between the receptive field sizes of V1 cells and the density of ganglion cells suggested that each V1 cell receives information from a similar number of retinal neurons, throughout the visual field.

  13. Induced adult stem (iAS) cells and induced transit amplifying progenitor (iTAP) cells-a possible alternative to induced pluripotent stem (iPS) cells?

    Science.gov (United States)

    Heng, Boon Chin; Richards, Mark; Ge, Zigang; Shu, Yimin

    2010-02-01

    The successful derivation of iPSC lines effectively demonstrates that it is possible to reset the 'developmental clock' of somatic cells all the way back to the initial embryonic state. Hence, it is plausible that this clock may instead be turned back half-way to a less immature developmental stage that is more directly applicable to clinical therapeutic applications or for in vitro pharmacology/toxicology screening assays. Such a suitable developmental state is postulated to be either the putative transit amplifying progenitor stage or adult stem cell stage. It is hypothetically possible to reprogram mature and terminally differentiated somatic cells back to the adult stem cell or transit amplifying progenitor stage, in a manner similar to the derivation of iPSC. It is proposed that the terminology 'Induced Adult Stem Cells' (iASC) or 'Induced Transit Amplifying Progenitor Cells' (iTAPC) be used to described such reprogrammed somatic cells. Of particular interest, is the possibility of resetting the developmental clock of mature differentiated somatic cells of the mesenchymal lineage, explanted from adipose tissue, bone marrow and cartilage. The putative adult stem cell sub-population from which these cells are derived, commonly referred to as 'mesenchymal stem cells', are highly versatile and hold much therapeutic promise in regenerative medicine, as attested to by numerous human clinical trials and animal studies. Perhaps it may be appropriate to term such reprogrammed cells as 'Induced Mesenchymal Stem Cells' (iMSC) or as 'Induced Mesenchumal Progenitor Cells' (iMPC). Given that cells from the same organ/tissue will share some commonalities in gene expression, we hypothesize that the generation of iASC or iTAPC would be more efficient as compared to iPSC generation, since a common epigenetic program must exist between the reprogrammed cells, adult stem cell or progenitor cell types and terminally differentiated cell types from the same organ/tissue.

  14. Kenya's Monkeys

    Institute of Scientific and Technical Information of China (English)

    唐天麒

    2004-01-01

    It's difficult to get close to patas monkeys(花脸猴). Clever and nervous, they run away at the sight of humans. The long-legged monkeys, clocked ( 记录 [ 速度 ] ) at 34 miles an hour, easily escaped from the zoologist Lynne Isbell when she arrived in Kenya in 1992.

  15. Mobile IP and protocol authentication extension

    CERN Document Server

    Nguyen, Phuc V

    2011-01-01

    Mobile IP is an open standard, defined by the Internet Engineering Task Force (IETF) RFC 3220. By using Mobile IP, you can keep the same IP address, stay connected, and maintain ongoing applications while roaming between IP networks. Mobile IP is scalable for the Internet because it is based on IP - any media that can support IP can support Mobile IP.

  16. Digital signage sobre IP

    OpenAIRE

    Casademont Filella, Albert

    2012-01-01

    Projecte que versa sobre el disseny i la implementació d'un sistema de Digital Signage (Cartelleria Digital) sobre IP, creant un gestor que permeti controlar de forma remota tot un conjunt de petits dispositius connectats a pantalles que emeten continguts multimèdia com vídeos, imatges, feeds rss...

  17. RelationshipbetweentheexpressionofIP-10and IP-10mRNAinperipheralbloodandHBVDNA levelinpatientswithcirrhosis

    Institute of Scientific and Technical Information of China (English)

    Jian Wang; Ping-Ping Wang; Gui-Ju Xiang; Xiao-Bin Hu

    2010-01-01

    BACKGROUND:Post-hepatitic cirrhosis is regarded as common and severe form of liver damage. Interferonγ-inducible protein 10 (IP-10), a member of the non-ELR (glutamic-leucine-arginine) motif CXC chemokine family, has recently been shown to recruit and activate speciifc subsets of leukocytes to sites of inlfammation or an immune response during the development of hepatic cirrhosis. However, the effects of IP-10 and IP-10 mRNA on inlfammatory inifltration at local sites and in the peripheral blood of patients with post-hepatitic cirrhosis as well as their relationship with viral load are still poorly deifned. This study aimed to detect the relationship between the expression of IP-10 in serum, IP-10 mRNA in peripheral blood mononuclear cells (PBMCs), and the levels of HBV DNA in the serum of patients, and to explore their role in the pathogenesis of cirrhosis. METHODS:Typical patients with cirrhosis after HBV infection were selected, and their serum IP-10 concentrations were evaluated with ELISA, the content of IP-10 mRNA in PBMCs was measured by real-time PCR, and the load of HBV DNA in serum and PBMCs was assessed by semi-quantitative analysis of gel imaging. RESULTS:The levels of IP-10 in serum and IP-10 mRNA in PBMCs of patients with cirrhosis were 299.9±77.2 pg/ml and 0.7500±0.1495, respectively. They were higher than those of controls (P CONCLUSIONS:The levels of IP-10 and IP-10 mRNA in the peripheral blood of patients with cirrhosis increase are closely correlated with the load of HBV DNA in serum, and play a key role in the progression of post-hepatitic cirrhosis.

  18. [Raman spectra of monkey cerebral cortex tissue].

    Science.gov (United States)

    Zhu, Ji-chun; Guo, Jian-yu; Cai, Wei-ying; Wang, Zu-geng; Sun, Zhen-rong

    2010-01-01

    Monkey cerebral cortex, an important part in the brain to control action and thought activities, is mainly composed of grey matter and nerve cell. In the present paper, the in situ Raman spectra of the cerebral cortex of the birth, teenage and aged monkeys were achieved for the first time. The results show that the Raman spectra for the different age monkey cerebral cortex exhibit most obvious changes in the regions of 1000-1400 and 2800-3000 cm(-1). With monkey growing up, the relative intensities of the Raman bands at 1313 and 2885 cm(-1) mainly assigned to CH2 chain vibrational mode of lipid become stronger and stronger whereas the relative intensities of the Raman bands at 1338 and 2932 cm(-1) mainly assigned to CH3 chain vibrational mode of protein become weaker and weaker. In addition, the two new Raman bands at 1296 and 2850 cm(-1) are only observed in the aged monkey cerebral cortex, therefore, the two bands can be considered as a character or "marker" to differentiate the caducity degree with monkey growth In order to further explore the changes, the relative intensity ratios of the Raman band at 1313 cm(-1) to that at 1338 cm(-1) and the Raman band at 2885 cm(-1) to that at 2 932 cm(-1), I1313/I1338 and I2885/I2932, which are the lipid-to-protein ratios, are introduced to denote the degree of the lipid content. The results show that the relative intensity ratios increase significantly with monkey growth, namely, the lipid content in the cerebral cortex increases greatly with monkey growth. So, the authors can deduce that the overmuch lipid is an important cause to induce the caducity. Therefore, the results will be a powerful assistance and valuable parameter to study the order of life growth and diagnose diseases.

  19. Microarray and ChIP-seq data analysis revealed changes in p53-mediated transcriptional regulation in Nutlin-3-treated U2OS cells.

    Science.gov (United States)

    Zhao, Song; Niu, Feng; Xu, Chang-Yan; Ye, Long; Bi, Gui-Bin; Chen, Lin; Gong, Ping; Tian, Gang; Nie, Tian-Hong

    2015-09-01

    Integrative analysis of chromatin immunoprecipitation-sequencing (ChIP-seq) data and microarray data was performed to illustrate the effect of Nutlin‑3 on promoter selectivity and transcriptional regulation by the tumor suppressor p53 in U2OS human osteosarcoma cells. Raw data (accession number, GSE46642) were downloaded from Gene Expression Omnibus. Differential analyses were performed using package limma of R software. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed for the differentially expressed genes (DEGs) using the Database for Annotation, Visualization and Integration Discovery. Integrative analysis of ChIP‑seq data and microarray data were confirmed with ChIP‑Array. A total of 565 DEGs were identified, including 373 upregulated genes and 192 downregulated genes. Genes involved in the p53 signaling pathway, cell cycle, DNA replication, cytokine‑cytokine receptor interaction and melanoma were markedly over‑represented in the DEGs. A total of 39 DEGs were directly regulated by p53 and two were the transcription factors (TFs), E2F2 and HOXA1. E2F2 regulated 25 DEGs, while HOXA1 regulated one DEG. The cell cycle, p53 signaling pathway, melanoma and pathways involved in cancer were enriched in the direct and indirect target genes. Changes in the p53‑binding pattern induced by Nutlin‑3 were described in the present study, which may advance the understanding of the regulatory network of p53 in osteosarcoma and aid in the development of novel therapies.

  20. New type of Sendai virus vector provides transgene-free iPS cells derived from chimpanzee blood.

    Directory of Open Access Journals (Sweden)

    Yasumitsu Fujie

    Full Text Available Induced pluripotent stem cells (iPSCs are potentially valuable cell sources for disease models and future therapeutic applications; however, inefficient generation and the presence of integrated transgenes remain as problems limiting their current use. Here, we developed a new Sendai virus vector, TS12KOS, which has improved efficiency, does not integrate into the cellular DNA, and can be easily eliminated. TS12KOS carries KLF4, OCT3/4, and SOX2 in a single vector and can easily generate iPSCs from human blood cells. Using TS12KOS, we established iPSC lines from chimpanzee blood, and used DNA array analysis to show that the global gene-expression pattern of chimpanzee iPSCs is similar to those of human embryonic stem cell and iPSC lines. These results demonstrated that our new vector is useful for generating iPSCs from the blood cells of both human and chimpanzee. In addition, the chimpanzee iPSCs are expected to facilitate unique studies into human physiology and disease.

  1. No Immunogenicity of IPS Cells in Syngeneic Host Studied by In Vivo Injection and 3D Scaffold Experiments

    Directory of Open Access Journals (Sweden)

    Suganya Thanasegaran

    2013-01-01

    Full Text Available Induced Pluripotent Stem Cells (IPSCs open the great possibility to employ patient’s own tissue to the previously incurable diseases. However these cells can be used in cell therapy only if they are not rejected when transplanted back into the syngeneic host. We found that the injection of iPSCs derived from different ages of mice into syngeneic C57BL/6 mice produced teratoma and was not rejected. Then we cultured iPSCs and myeloid differentiated iPSCs in three-dimensional porous scaffold and transplanted to C57BL/6 mice and BALB/C mice. After transplantation, we could observe the cell density inside the scaffold increased rapidly in syngeneic mice compared to the allogeneic mice indicating the favorable conditions supporting the growth of iPSCs in vivo. Unlike the allogeneic counterpart, we could not observe few infiltrating T cells inside the scaffold of syngeneic mice. These results contribute to the optimistic view of iPSCs for regenerative medicine in near future.

  2. iPS cell technologies and their prospect for bone regeneration and disease modeling: A mini review

    Directory of Open Access Journals (Sweden)

    Maria Csobonyeiova

    2017-07-01

    Full Text Available Bone disorders are a group of varied acute and chronic traumatic, degenerative, malignant or congenital conditions affecting the musculoskeletal system. They are prevalent in society and, with an ageing population, the incidence and impact on the population’s health is growing. Severe persisting pain and limited mobility are the major symptoms of the disorder that impair the quality of life in affected patients. Current therapies only partially treat the disorders, offering management of symptoms, or temporary replacement with inert materials. However, during the last few years, the options for the treatment of bone disorders have greatly expanded, thanks to the advent of regenerative medicine. Skeletal cell-based regeneration medicine offers promising reparative therapies for patients. Mesenchymal stem (stromal cells from different tissues have been gradually translated into clinical practice; however, there are a number of limitations. The introduction of reprogramming methods and the subsequent production of induced pluripotent stem cells provides a possibility to create human-specific models of bone disorders. Furthermore, human-induced pluripotent stem cell-based autologous transplantation is considered to be future breakthrough in the field of regenerative medicine. The main goal of the present paper is to review recent applications of induced pluripotent stem cells in bone disease modeling and to discuss possible future therapy options. The present article contributes to the dissemination of scientific and pre-clinical results between physicians, mainly orthopedist and thus supports the translation to clinical practice.

  3. Cryopreservation of human induced pluripotent stem cells (iPS cells) through vitrification in mini straw%麦管玻璃化法冷冻保存人诱导多能干细胞

    Institute of Scientific and Technical Information of China (English)

    蒋春艳; 董娟; 廖婷婷; 宁松; 范国平; 曾桥; 薛志刚; 刘嘉茵

    2011-01-01

    Objective: To explore the method of cryopreservating human induced pluripotent. Methods:Colonies of induced pluripotent stem cells (iPS cells) were dissected into pieces (containing approximately 100~200 cells) using mechanical methods, then sequentially treated with 10%,20% vitrification solution, and sealed in mini straws with the second solution. Mini straws were then dropped into liquid nitrogen imInediately for vitrification and cryopreservation. After vitrificated preservation, iPS cells were thawed in 37℃ water bath, and were immediately balanced in 0.2 mol/L sucrose solution and then in 0.1 mol/L sucrose solution, and at last plated on feeder layer cells. Alkaline phosphatase activity, expressions of OCT4, SOX2 in iPS cells and cell differentiation were evaluated. Results:Post vitrification and thawing, iPS cells maintain properties of pluripotent stem cells, including normal morphology, alkaline phosphatase staining,OCT4 and SOX2 expression. Calculation of colony recovery rates indicates that approximate (77.40±13.12)% of iPS cell colonies survived the freezing and thaw procedures. Conclusion:Vitrification with ministraws is a very useful and effective cryopresorvation method for iPS cells.%目的:麦管玻璃化法冷冻保存人诱导多能干细胞(induced pluripotent cells,iPS细胞).方法:将机械法切割的iPS细胞团块,依次在10%玻璃化冷冻液和20%玻璃化冷冻液中处理后保存于20%的冷冻液中,封装于0.25 ml麦管并快速置于液氮中保存.解冻时将iPS团块依次置入0.2 mol/L蔗糖溶液和0.1 mol/L蔗糖溶液后,接种至饲养层上培养.检测复苏效率,并对解冻后长期培养的iPS细胞进行特性鉴定,包括:碱性磷酸酶染色、OCT4等免疫荧光染色、RT-PCR法检测内源性oct4和sox2的表达、拟胚体(embryoid,EB)分化、神经细胞分化和畸胎瘤分化能力检测等.结果:麦管玻璃化法冷冻保存的iPS细胞解冻后复苏率可达(77.40±13.12)%,解冻后iPS

  4. Chemoprophylaxis with sporozoite immunization in P. knowlesi rhesus monkeys confers protection and elicits sporozoite-specific memory T cells in the liver

    Science.gov (United States)

    Spring, Michele D.; Yongvanitchit, Kosol; Kum-Arb, Utaiwan; Limsalakpetch, Amporn; Im-Erbsin, Rawiwan; Ubalee, Ratawan; Vanachayangkul, Pattaraporn; Remarque, Edmond J.; Angov, Evelina; Smith, Philip L.; Saunders, David L.

    2017-01-01

    Whole malaria sporozoite vaccine regimens are promising new strategies, and some candidates have demonstrated high rates of durable clinical protection associated with memory T cell responses. Little is known about the anatomical distribution of memory T cells following whole sporozoite vaccines, and immunization of nonhuman primates can be used as a relevant model for humans. We conducted a chemoprophylaxis with sporozoite (CPS) immunization in P. knowlesi rhesus monkeys and challenged via mosquito bites. Half of CPS immunized animals developed complete protection, with a marked delay in parasitemia demonstrated in the other half. Antibody responses to whole sporozoites, CSP, and AMA1, but not CelTOS were detected. Peripheral blood T cell responses to whole sporozoites, but not CSP and AMA1 peptides were observed. Unlike peripheral blood, there was a high frequency of sporozoite-specific memory T cells observed in the liver and bone marrow. Interestingly, sporozoite-specific CD4+ and CD8+ memory T cells in the liver highly expressed chemokine receptors CCR5 and CXCR6, both of which are known for liver sinusoid homing. The majority of liver sporozoite-specific memory T cells expressed CD69, a phenotypic marker of tissue-resident memory (TRM) cells, which are well positioned to rapidly control liver-stage infection. Vaccine strategies that aim to elicit large number of liver TRM cells may efficiently increase the efficacy and durability of response against pre-erythrocytic parasites. PMID:28182750

  5. Experimental protein malnutrition as a causative factor in the histological and histochemical disruption of the ependymal cells of the third ventricla and cervical central canal of squirrel monkeys.

    Science.gov (United States)

    Sharma, S P; Manocha, S L

    1977-01-01

    The histologic disturbances of the cuboidal and columnar ependymal cells of the neonates under the extrinsic influence of maternal protein deprivation during most of the gestation period have been demonstrated in the central canal of the cervical spinal cord and III ventricle of the squirrel monkey brain. The control animals whose mothers were maintained on high protein diets showed an unbroken ependymal layer with an intact glial fiber layer and the subependymal cell plate. Taking birth-weight as an indication of the degree of malnutrition, maximum disruption of the ependymal layer was observed in those animals born around 80 g birth-weight. In the latter, most of the ependymal cells lost their characteristic arrangement and columnar shapes. The ependymal cells of the manourished animals show a marked reduction of oxidative enzyme content and relatively active glycogen metabolism as compared to the healthy controls. The significance of these changes has been discussed in the light of parallels in the embryological development and, to some extent, functions between the orinary neurons, ependymal cells of the choroid plexus, other ependymal cells and the glial cells. In this context, the changes observed in the present studies under the effect of protein malnutrition must be taken as part of overall changes experienced by various kinds of cells comprising the nervous system.

  6. Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential

    Directory of Open Access Journals (Sweden)

    Kristi A. Hohenstein Elliott

    2012-01-01

    Full Text Available Proper maintenance of stem cells is essential for successful utilization of ESCs/iPSCs as tools in developmental and drug discovery studies and in regenerative medicine. Standardization is critical for all future applications of stem cells and necessary to fully understand their potential. This study reports a novel approach for the efficient, consistent expansion of human ESCs and iPSCs using laser sectioning, instead of mechanical devices or enzymes, to divide cultures into defined size clumps for propagation. Laser-mediated propagation maintained the pluripotency, quality, and genetic stability of ESCs/iPSCs and led to enhanced differentiation potential. This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines. Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.

  7. Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

    Science.gov (United States)

    Tanaka, Akihito; Woltjen, Knut; Miyake, Katsuya; Hotta, Akitsu; Ikeya, Makoto; Yamamoto, Takuya; Nishino, Tokiko; Shoji, Emi; Sehara-Fujisawa, Atsuko; Manabe, Yasuko; Fujii, Nobuharu; Hanaoka, Kazunori; Era, Takumi; Yamashita, Satoshi; Isobe, Ken-ichi; Kimura, En; Sakurai, Hidetoshi

    2013-01-01

    The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs. PMID:23626698

  8. Procedures for the isolation and culture of Sertoli cells from the testes of infant, juvenile, and adult rhesus monkeys (Macaca mulatta).

    Science.gov (United States)

    Majumdar, S S; Winters, S J; Plant, T M

    1998-03-01

    The purpose of the present study was to establish culture conditions for the in vitro study of the rhesus monkey Sertoli cell (Sc) at three major stages of development, namely infancy, adulthood, and the intervening prepubertal period. Conditions for the culture of Sc from juveniles were first established using collagenase and pancreatin digestion of seminiferous tubules. The addition of 1% fetal bovine serum for the first 24 h of culture was necessary for attachment of Sc clusters. Confluency of Sc from juveniles was reached as early as 4 days of culture. Histochemical and ultrastructural observations confirmed that the cultures were enriched with Sc and that contamination by peritubular cells was minimal (2%). Although application of similar culture conditions was successful in establishing cultures of Sc from infants, significant modification of the procedure was required before Sc from adults could be cultured. Specifically, adult testicular tissue required two sequential collagenase digestions at elevated temperature. The yield of adult Sc, however, remained low. Cultures of juvenile Sc produced substantial quantities of 31-kDa inhibin, which was bioactive as reflected by its ability to suppress FSH secretion from rat pituitary cells in vitro. Although aromatase activity in juvenile Sc cultures was stimulated by FSH, inhibin synthesis, as reflected by immunoactive inhibin production and steady-state levels of alpha inhibin mRNA, was not increased by FSH. The establishment of conditions for the culture of infant, juvenile, and adult Sc from the rhesus monkey will provide a model for study of the postnatal ontogeny of Sc function in higher primates.

  9. 诱导多能干细胞体外定向分化为雄性生殖细胞研究进展%Advances in Differentiation of iPS Cells into Male Germ Cells

    Institute of Scientific and Technical Information of China (English)

    杜军慧; 曹文广

    2014-01-01

    Induced pluripotent stem cells (iPS cells) refers to reprograming animals or human somatic cells by gene transfer technology, i.e. using so-called packed viral vectors or particles to infect the cells to be induced. Different somatic cells can be induced into iPS cells through different vectors or different transcription factors combination, such as fibroblasts, hepatocytes, keratinocytes and cord blood, etc. As a new member, iPS cells were similar to embryonic stem cells, i.e. clonal morphology, gene expression pattern, surface marker, embryoid bodies formation, differentiative capacity, teratoma and chimeras (in mice) and so on. Just like ESCs, studies have shown that iPS cells can be induced into different cellsin vitro under the conditions of specific induction, including myocardial cells, blood cells and germ cells. It shortens the distance between stem cells and clinical disease, Therefore iPS cells have become the most promising seed cells in cell therapy and tissue organ regeneration, and they have a potential value in the cells’ alternative treatment, pathogenesis research and new drug screening simultaneously. Male sterility not only affects human normal life, but is also very adverse to the development of animal husbandry. Great progress has been made inthat iPS cells from humans and mice can be differentiated into primordial germ cells (PGCs), spermatozoa and their precursors. These findings can help not only avoid the difficulties in obtaining ESCs, immune rejection and ethical problems, which may be aroused by using embryonic stem cells, but also provide a good research platform for revealing the developmental mechanism of male germ cells. Patients can use their iPS cells derived male gametes to reproduction. Therefore, differentiation of iPS cells into male germ cells brings new hope for the treatment of male sterility in the future. In addition, differentiation of iPS cellsin vitro has broad application potentials for modern animal husbandry. It

  10. miR-140-5p attenuates chemotherapeutic drug-induced cell death by regulating autophagy through inositol 1,4,5-trisphosphate kinase 2 (IP3k2) in human osteosarcoma cells

    Science.gov (United States)

    Wei, Renxiong; Cao, Gang; Deng, Zhouming; Su, Jiajia; Cai, Lin

    2016-01-01

    Acquisition of drug-resistant phenotypes is often associated with chemotherapy in osteosarcoma. A number of studies have demonstrated a critical role for autophagy in osteosarcoma development, therapy and drug resistance. However, the molecular mechanisms underlying the autophagy-mediated chemotherapy resistance of osteosarcoma cells remain largely unknown. In the present study, we determined the autophagy and microRNA-140 (miR-140-5p, miRBase ID: MIMAT0000431) expression induced by chemotherapeutic drugs in osteosarcoma cells. Then we determined the promotory role of miR-140-5p to the chemotherapy-induced autophagy. Our results demonstrated that miR-140-5p expression was highly induced during chemotherapy of osteosarcoma cells, and this was accompanied by up-regulated autophagy. The increased miR-140-5p expression levels up-regulated anticancer drug-induced autophagy in osteosarcoma cells and ameliorated the anticancer drug-induced cell proliferation and viability decrease. Importantly, miR-140-5p regulates this context-specific autophagy through its target, inositol 1,4,5-trisphosphate kinase 2 (IP3k2). Therefore, the results of the present study demonstrated that miR-140-5p mediated drug-resistance in osteosarcoma cells by inducing autophagy. The present study provides evidence of miRNA regulation of autophagy through modulation of IP3 signalling. The present study recognized a novel mechanism of chemoresistance in osteosarcoma cancers. PMID:27582507

  11. Inherited heart disease - what can we expect from the second decade of human iPS cell research?

    Science.gov (United States)

    Bellin, Milena; Mummery, Christine L

    2016-08-01

    Induced pluripotent stem cells (iPSCs) were first generated 10 years ago. Their ability to differentiate into any somatic cell type of the body including cardiomyocytes has already made them a valuable resource for modelling cardiac disease and drug screening. Initially human iPSCs were used mostly to model known disease phenotypes; more recently, and despite a number of recognised shortcomings, they have proven valuable in providing fundamental insights into the mechanisms of inherited heart disease with unknown genetic cause using surprisingly small cohorts. In this review, we summarise the progress made with human iPSCs as cardiac disease models with special focus on the latest mechanistic insights and related challenges. Furthermore, we suggest emerging solutions that will likely move the field forward.

  12. Bioengineering a 3D integumentary organ system from iPS cells using an in vivo transplantation model.

    Science.gov (United States)

    Takagi, Ryoji; Ishimaru, Junko; Sugawara, Ayaka; Toyoshima, Koh-Ei; Ishida, Kentaro; Ogawa, Miho; Sakakibara, Kei; Asakawa, Kyosuke; Kashiwakura, Akitoshi; Oshima, Masamitsu; Minamide, Ryohei; Sato, Akio; Yoshitake, Toshihiro; Takeda, Akira; Egusa, Hiroshi; Tsuji, Takashi

    2016-04-01

    The integumentary organ system is a complex system that plays important roles in waterproofing, cushioning, protecting deeper tissues, excreting waste, and thermoregulation. We developed a novel in vivo transplantation model designated as a clustering-dependent embryoid body transplantation method and generated a bioengineered three-dimensional (3D) integumentary organ system, including appendage organs such as hair follicles and sebaceous glands, from induced pluripotent stem cells. This bioengineered 3D integumentary organ system was fully functional following transplantation into nude mice and could be properly connected to surrounding host tissues, such as the epidermis, arrector pili muscles, and nerve fibers, without tumorigenesis. The bioengineered hair follicles in the 3D integumentary organ system also showed proper hair eruption and hair cycles, including the rearrangement of follicular stem cells and their niches. Potential applications of the 3D integumentary organ system include an in vitro assay system, an animal model alternative, and a bioengineered organ replacement therapy.

  13. Genome-wide ChIP-seq analysis of human TOP2B occupancy in MCF7 breast cancer epithelial cells

    Directory of Open Access Journals (Sweden)

    Catriona M. Manville

    2015-11-01

    Full Text Available We report the whole genome ChIP seq for human TOP2B from MCF7 cells. Using three different peak calling methods, regions of binding were identified in the presence or absence of the nuclear hormone estradiol, as TOP2B has been reported to play a role in ligand-induced transcription. TOP2B peaks were found across the whole genome, 50% of the peaks fell either within a gene or within 5 kb of a transcription start site. TOP2B peaks coincident with gene promoters were less frequently associated with epigenetic features marking active promoters in estradiol treated than in untreated cells. Significantly enriched transcription factor motifs within the DNA sequences underlying the peaks were identified. These included SP1, KLF4, TFAP2A, MYF, REST, CTCF, ESR1 and ESR2. Gene ontology analysis of genes associated with TOP2B peaks found neuronal development terms including axonogenesis and axon guidance were significantly enriched. In the absence of functional TOP2B there are errors in axon guidance in the zebrafish eye. Specific heparin sulphate structures are involved in retinal axon targeting. The glycosaminoglycan biosynthesis–heparin sulphate/heparin pathway is significantly enriched in the TOP2B gene ontology analysis, suggesting changes in this pathway in the absence of TOP2B may cause the axon guidance faults.

  14. Sequence and diversity of T-cell receptor alpha V, J, and C genes of the owl monkey Aotus nancymaae.

    Science.gov (United States)

    Favre, N; Daubenberger, C; Marfurt, J; Moreno, A; Patarroyo, M; Pluschke, G

    1998-09-01

    We cloned and sequenced TcR alpha chain cDNA of three healthy Aotus nancymaae monkeys. Fifteen different TRAJ segments and 9 different TRAV genes were identified in the 29 rearrangements analyzed. As expected from the greater phylogenetic distance, A. nancymaae TRA gene sequences diverged more from the human sequences than those of the chimpanzee or the rhesus macaque. However, no Aotus TRAJ segment or TRAV gene was found which lacked a human counterpart. These counterparts were AJ02, AJ05, AJ09, AJ15, AJ22, AJ23, AJ28, AJ30, AJ32, AJ34, AJ37, AJ40, AJ42, AJ45, AJ52 and AV2S1, AV2S3, AV3S1, AV8S1, AV12S1, AV15S1, ADV21S1/DV5, AV22S1S and AV23S1, respectively. In most cases the identity of amino acid sequences between corresponding Aotus and human genes was greater than 80%. This marked conservation of TRA gene sequences indicates a close structural relationship of Aotus and human TcR and demonstrates that the TcR repertoire in primates is remarkably stable. The results support the concept of using Aotus monkeys, which are susceptible to infection with the human malaria parasite Plasmodium falciparum, as an animal model for the evaluation of molecularly defined malaria vaccine candidates.

  15. IP and ATM Integration

    OpenAIRE

    Mannie, E.; Corridoni, J.; Cremonese, P.; Giordano, Silvia

    1999-01-01

    The performance limits of the current Internet make the integration of IP with ATM a hotly debated issue in the networking arena, leading to various competing approaches and products. Legitimate technical and market issues are, however, often intertwined with biased views and hype, with vendors competing in the standards arena as well as on the markets. Together with the speed of technical evolution, this causes confusion for purchasers of networking equipment who, usually preferring a single...

  16. CtIP-BRCA1 modulates the choice of DNA double-strand break repair pathway throughout the cell cycle

    OpenAIRE

    Yun, Maximina H.; Hiom, Kevin

    2009-01-01

    The repair of DNA double-strand breaks (DSB) is tightly regulated during the cell cycle. In G1 phase, the absence of a sister chromatid means that repair of DSB occurs through non-homologous end-joining (NHEJ) or microhomology-mediated end-joining (MMEJ)1. These pathways often involve loss of DNA sequences at the break site and are therefore error-prone. In late S and G2 phases, even though DNA end-joining pathways remain functional2, there is an increase in repair of DSB by homologous recomb...

  17. Human induced hepatic lineage-oriented stem cells: autonomous specification of human iPS cells toward hepatocyte-like cells without any exogenous differentiation factors.

    Directory of Open Access Journals (Sweden)

    Tetsuya Ishikawa

    Full Text Available Preparing targeted cells for medical applications from human induced pluripotent stem cells (hiPSCs using growth factors, compounds, or gene transfer has been challenging. Here, we report that human induced hepatic lineage-oriented stem cells (hiHSCs were generated and expanded as a new type of hiPSC under non-typical coculture with feeder cells in a chemically defined hiPSC medium at a very high density. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs and hepatocytes. Those cells were highly expandable, markedly enhancing gene expression of serum hepatic proteins and cytochrome P450 enzymes with the omission of FGF-2 from an undefined hiPSC medium. The hepatic specification of hiHSCs was not attributable to the genetic and epigenetic backgrounds of the starting cells, as they were established from distinct donors and different types of cells. Approximately 90% of hiHSCs autonomously differentiated to hepatocyte-like cells, even in a defined minimum medium without any of the exogenous growth factors necessary for hepatic specification. After 12 days of this culture, the differentiated cells significantly enhanced gene expression of serum hepatic proteins (ALB, SERPINA1, TTR, TF, FABP1, FGG, AGT, RBP4, and AHSG, conjugating enzymes (UGT2B4, UGT2B7, UGT2B10, GSTA2, and GSTA5, transporters (SULT2A1, SLC13A5, and SLCO2B1, and urea cycle-related enzymes (ARG1 and CPS1. In addition, the hepatocyte-like cells performed key functions of urea synthesis, albumin secretion, glycogen storage, indocyanine green uptake, and low-density lipoprotein uptake. The autonomous hepatic specification of hiHSCs was due to their culture conditions (coculture with feeder cells in a defined hiPSC medium at a very high density in self-renewal rather than in differentiation. These results suggest the feasibility of preparing large quantities of hepatocytes as a convenient and inexpensive hiPSC differentiation. Our study also suggests the

  18. Integrative ChIP-seq/microarray analysis identifies a CTNNB1 target signature enriched in intestinal stem cells and colon cancer.

    Directory of Open Access Journals (Sweden)

    Kazuhide Watanabe

    Full Text Available BACKGROUND: Deregulation of canonical Wnt/CTNNB1 (beta-catenin pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells. RESULTS: We observed 3629 CTNNB1 binding peaks across the genome and a significant correlation between CTNNB1 binding and knockdown-induced gene expression change. Our integrative analysis led to the discovery of a direct Wnt target signature composed of 162 genes. Gene ontology analysis of this signature revealed a significant enrichment of Wnt pathway genes, suggesting multiple feedback regulations of the pathway. We provide evidence that this gene signature partially overlaps with the Lgr5+ intestinal stem cell signature, and is significantly enriched in normal intestinal stem cells as well as in clinical colorectal cancer samples. Interestingly, while the expression of the CTNNB1 target gene set does not correlate with survival, elevated expression of negative feedback regulators within the signature predicts better prognosis. CONCLUSION: Our data provide a genome-wide view of chromatin occupancy and gene regulation of Wnt/CTNNB1 signaling in colon cancer cells.

  19. Highly sensitive in vitro methods for detection of residual undifferentiated cells in retinal pigment epithelial cells derived from human iPS cells.

    Directory of Open Access Journals (Sweden)

    Takuya Kuroda

    Full Text Available Human induced pluripotent stem cells (hiPSCs possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells (hESCs. These characteristics make hiPSCs a promising choice for future regenerative medicine research. There are significant obstacles, however, preventing the clinical use of hiPSCs. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. To locate residual undifferentiated cells, in vivo teratoma formation assays have been performed with immunodeficient animals, which is both costly and time-consuming. Here, we examined three in vitro assay methods to detect undifferentiated cells (designated an in vitro tumorigenicity assay: soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR. Although the soft agar colony formation assay was unable to detect hiPSCs even in the presence of a ROCK inhibitor that permits survival of dissociated hiPSCs/hESCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of Lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×10⁴ RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived products for future regenerative medicine research.

  20. Spinogenesis and pruning in the anterior ventral inferotemporal cortex of the macaque monkey: an intracellular injection study of layer III pyramidal cells

    Directory of Open Access Journals (Sweden)

    Guy N. Elston

    2011-07-01

    Full Text Available Cortical pyramidal cells grow and mature at different rates in visual, auditory and prefrontal cortex of the macaque monkey. In particular, differences across the areas have been reported in both the timing and magnitude of growth, branching, spinogenesis and pruning in the basal dendritic trees of cells in layer III. Presently available data suggest that these different growth profiles reflect the type of functions performed by these cells in the adult brain. However, to date, studies have focussed on only a relatively few cortical areas. In the present investigation we quantified the growth of the dendritic trees of layer III pyramidal cells in the anterior ventral portion of cytoarchitectonic area TE (TEav to better comprehend developmental trends in the cerebral cortex. We quantified the growth and branching of the dendrities, and spinogenesis and pruning of spines, from post-natal day 2 (PND2 to four and a half years of age. We found that the dendritic trees increase in size from PND2 to 7 months of age and thereafter become smaller. The dendritic trees became increasingly more branched from PND2 into adulthood. There was a 2-fold increase in the number of spines in the basal dendritic trees of pyramidal cells from PND2 to 3½ months of age and then a 10% net decrease in spine number into adulthood. Thus, the growth profile of layer III pyramidal cells in the anterior ventral portion of the inferotemporal cortex differs to that in other cortical areas associated with visual processing.

  1. Postnatal Dendritic Growth and Spinogenesis of Layer-V Pyramidal Cells Differ between Visual, Inferotemporal, and Prefrontal Cortex of the Macaque Monkey

    Science.gov (United States)

    Oga, Tomofumi; Elston, Guy N.; Fujita, Ichiro

    2017-01-01

    Pyramidal cells in the primate cerebral cortex, particularly those in layer III, exhibit regional variation in both the time course and magnitude of postnatal growth and pruning of dendrites and spines. Less is known about the development of pyramidal cell dendrites and spines in other cortical layers. Here we studied dendritic morphology of layer-V pyramidal cells in primary visual cortex (V1, sensory), cytoarchitectonic area TE in the inferotemporal cortex (sensory association), and granular prefrontal cortex (Walker's area 12, executive) of macaque monkeys at the ages of 2 days, 3 weeks, 3.5 months, and 4.5 years. We found that changes in the basal dendritic field area of pyramidal cells were different across the three areas. In V1, field size became smaller over time (largest at 2 days, half that size at 4.5 years), in TE it did not change, and in area 12 it became larger over time (smallest at 2 days, 1.5 times greater at 4.5 years). In V1 and TE, the total number of branch points in the basal dendritic trees was similar between 2 days and 4.5 years, while in area 12 the number was greater in the adult monkeys than in the younger ones. Spine density peaked at 3 weeks and declined in all areas by adulthood, with V1 exhibiting a faster decline than area TE or area 12. Estimates of the total number of spines in the dendritic trees revealed that following the onset of visual experience, pyramidal cells in V1 lose more spines than they grow, whereas those in TE and area 12 grow more spines than they lose during the same period. These data provide further evidence that the process of synaptic refinement in cortical pyramidal cells differs not only according to time, but also location within the cortex. Furthermore, given the previous finding that layer-III pyramidal cells in all these areas exhibit the highest density and total number of spines at 3.5 months, the current results indicate that pyramidal cells in layers III and V develop spines at different rates.

  2. Adrenergic Stress Protection of Human iPS Cell-Derived Cardiomyocytes by Fast Kv7.1 Recycling

    Directory of Open Access Journals (Sweden)

    Ilaria Piccini

    2017-09-01

    Full Text Available The fight-or-flight response (FFR, a physiological acute stress reaction, involves positive chronotropic and inotropic effects on heart muscle cells mediated through β-adrenoceptor activation. Increased systolic calcium is required to enable stronger heart contractions whereas elevated potassium currents are to limit the duration of the action potentials and prevent arrhythmia. The latter effect is accomplished by an increased functional activity of the Kv7.1 channel encoded by KCNQ1. Current knowledge, however, does not sufficiently explain the full extent of rapid Kv7.1 activation and may hence be incomplete. Using inducible genetic KCNQ1 complementation in KCNQ1-deficient human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs, we here reinvestigate the functional role of Kv7.1 in adapting human CMs to adrenergic stress. Under baseline conditions, Kv7.1 was barely detectable at the plasma membrane of hiPSC-CMs, yet it fully protected these from adrenergic stress-induced beat-to-beat variability of repolarization and torsade des pointes-like arrhythmia. Furthermore, isoprenaline treatment increased field potential durations specifically in KCNQ1-deficient CMs to cause these adverse macroscopic effects. Mechanistically, we find that the protective action by Kv7.1 resides in a rapid translocation of channel proteins from intracellular stores to the plasma membrane, induced by adrenergic signaling. Gene silencing experiments targeting RAB GTPases, mediators of intracellular vesicle trafficking, showed that fast Kv7.1 recycling under acute stress conditions is RAB4A-dependent.Our data reveal a key mechanism underlying the rapid adaptation of human cardiomyocytes to adrenergic stress. These findings moreover aid to the understanding of disease pathology in long QT syndrome and bear important implications for safety pharmacological screening.

  3. Inositol hexaphosphate (IP6): a novel treatment for pancreatic cancer.

    Science.gov (United States)

    Somasundar, Ponnandai; Riggs, Dale R; Jackson, Barbara J; Cunningham, Cynthia; Vona-Davis, Linda; McFadden, David W

    2005-06-15

    Inositol hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate found in food sources high in fiber content. IP6 has been reported to have significant inhibitory effects against a variety of primary tumors including breast and colon. The effects of IP6 have not been evaluated in pancreatic cancer. We hypothesized that IP6 would significantly inhibit cell growth and increase the apoptotic rate of pancreatic cancer in vitro. Two pancreatic cancer cell lines (MIAPACA and PANC1) were cultured using standard techniques and treated with IP6 at doses of 0.5, 1.0, and 5.0 mm. Cell viability was measured by MTT at 24 and 72 h. Apoptosis was evaluated by Annexin V-FITC and results calculated using FACS analysis. Statistical analysis was performed by ANOVA. Significant reductions (P IP6 concentrations tested in both cell lines and at both time points. Reductions in cell proliferation ranged from 37.1 to 91.5%. IP6 increased early and late apoptotic activity (P IP6 significantly decreased cellular growth and increased apoptosis. Our findings suggest that IP6 has the potential to become an effective adjunct for pancreatic cancer treatment. Further in vivo and human studies are needed to evaluate safety and clinical utility of this agent in patients with pancreatic cancer.

  4. CPM Is a Useful Cell Surface Marker to Isolate Expandable Bi-Potential Liver Progenitor Cells Derived from Human iPS Cells.

    Science.gov (United States)

    Kido, Taketomo; Koui, Yuta; Suzuki, Kaori; Kobayashi, Ayaka; Miura, Yasushi; Chern, Edward Y; Tanaka, Minoru; Miyajima, Atsushi

    2015-10-13

    To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM(+) cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM(+) cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM(+) cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes.

  5. KAJIAN INDUCED PLURIPOTENT STEMCELL (iPS (HARAPAN DAN TANTANGAN

    Directory of Open Access Journals (Sweden)

    Masagus Zainuri

    2014-05-01

    Full Text Available AbstractInduced Pluripotent Stemcell (iPS are adult cells which the genetic information in the nucleus of those cells being reprogrammed (reprogram by inserting exogenous pluripotential genes. The exogenous gene transduction is using vectors, such as lentivirus, retrovirus, or adenovirus, which suppressed the gene expression of the original cells, so they will express the transduced exogenous gene. Viral vectors are then used to reprogramming and producing iPS clones that are pluripotent. iPS derived from adult cells of patient with certain diseases will be used as a tool to study the mechanisms of those specific diseases and the effects of selected drugs against the diseases. Several previous studies have shown that iPS clones developed from specific genetic disease have its original genotype and retain the character of the response to the drug that similar as the original adult cells. Opportunities for the utilization of autologous iPS cell therapy in the future is wide open as expected iPS transplant will not be rejected when transplanted back to the patient. Behind all its potential, iPS production is still facing some problems to be applicable clinically. The use of viruses as vectors may cause problems due to virus gene sequences may be integrated into the genome of the DNA donor cell, thereby causing mutations of the iPS clones. Several subsequent studies have succeeded in replacing the use of viruses as vectors, but the level of efficiency obtained is still very low. Another problem that arises is that epigenetic changes may occur in iPS cultures. Many advanced research related to iPS may be developed in Indonesia and is necessary to improve the production efficiency of iPS and solve iPS clones epigenetic changes problems in the future.Keywords: iPS, pluripotency, transduction, transfection.AbstrakInduced Pluripotent Stemcell (iPS adalah sel somatic dewasa yang informasi genetika dalam inti selnyadiprogram ulang (reprogram dengan cara

  6. Hepatitis E genotype 4 virus from feces of monkeys infected experimentally can be cultured in PLC/PRF/5 cells and upregulate host interferon-inducible genes.

    Science.gov (United States)

    Zhang, Feng; Qi, Ying; Harrison, Tim J; Luo, Baobin; Zhou, Yan; Li, Xiuhua; Song, Aijing; Huang, Weijin; Wang, Youchun

    2014-10-01

    The understanding of the interaction between hepatitis E virus (HEV) and its host cells has been impeded greatly by the absence of a cell culture system. In this study, an efficient cultivation method was developed in PLC/PRF/5 cells for HEV genotype 4 from the feces of monkeys infected experimentally. Compared to minimal essential medium (MEM), mixed Dulbecco's Modified Eagle's Medium (DMEM)/M199 improved the infection efficiency of HEV in PLC/PRF/5 cells. The incubation time and temperature were set at 6 hr and 40°C, respectively. Compared to a 100% ELISA positive ratio (EPR) of 1 × 10(6)  copies/ml HEV inoculated flasks, the ELISA positive ratio was 100%, 75%, 37.5%, and 100% for flasks inoculated with HEV incubated for 30 min under the conditions of pH 3.0, pH 11.0, 56°C and delipidation treatment, respectively. Gene expression profiles of HEV inoculated and control PLC/PRF/5 cells were assayed using a microarray. Four interferon-inducible genes, IFI27, IFI6, Mx1, and CMPK2, were up-regulated during HEV-infection. Furthermore, the replication of HEV was inhibited at 3-14 days after treatment with 500 IU/ml IFN-α2b.

  7. Mosaic vaccines elicit CD8+ T cell responses in monkeys that confer immune coverage of diverse HIV strains

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, Will [Los Alamos National Laboratory; Korber, Bette [Los Alamos National Laboratory

    2009-01-01

    Creation of a successful HIV vaccine will require the development of a strategy to generate cellular immunity with sufficient cross-clade breadth to deal with the extreme genetic diversity of the virus. Polyvalent mosaic immunogens derived from in silica recombination of natural strains of HIV are designed to induce cellular immune responses that maximally cover the sequence diversity of circulating virus isolates. Immunization of rhesus monkeys with plasmid DNA and recombinant vaccinia virus vaccine constructs expressing either consensus immunogens or polyvalent mosaic immunogens elicited a CD4+ T lymphocyte-biased response with comparably broad epitope-specific total T lymphocyte specificities. However, immunization with the mosaic immunogens induced HIV-specific CD8+ T lymphocyte responses with markedly greater depth and breadth. Therefore, the use of polyvalent mosaic immunogens is a promising strategy for a global vaccine for HIV.

  8. Inositol hexaphosphate (IP6) inhibits cellular proliferation in melanoma.

    Science.gov (United States)

    Rizvi, Irfan; Riggs, Dale R; Jackson, Barbara J; Ng, Alex; Cunningham, Cynthia; McFadden, David W

    2006-06-01

    Inositol Hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate found in food sources high in fiber content. We have previously reported IP6 to have significant inhibitory effects against pancreatic cancer in vitro. We hypothesized that the IP6 would significantly inhibit cell growth of cutaneous melanoma in vitro. The melanoma line HTB68 was cultured using standard techniques and treated with IP6 at doses ranging from 0.2 to 1.0 mM/well. Cell viability was measured by MTT at 72 h. VEGF production was measured in the cell supernatants by ELISA. Apoptosis was evaluated by Annexin V-FITC and results calculated using FACS analysis. Statistical analysis was performed by ANOVA. Significant reductions (P IP6. Overall, IP6 exhibited a mean inhibition of cell growth of 52.1 +/- 11.5% (range, 1.6-83.0%) at 72 h of incubation. VEGF production was significantly reduced (P IP6 (7.5 pg/ml) compared to control (40.9 pg/ml). IP6 significantly increased (P = 0.029) late apoptosis from 5.3 to 7.0% gated events. No changes in necrosis or early apoptosis were observed. Adjuvant treatment of melanoma continues to challenge clinicians and patients. Our findings that IP6 significantly decreased cellular growth, VEGF production and increased late apoptosis in melanoma suggest its potential therapeutic value. Further in vivo studies are planned to evaluate safety and clinical utility of this agent.

  9. Embryotoxicity of arsenite and arsenate. Distribution in pregnant mice and monkeys and effects on embryonic cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Lindgren, A.; Danielsson, R.G.; Dencker, L. (Department of Toxicology, Biomedical Center, Uppsala University, Sweden); Vahter, M. (National Institute of Environmental Medicine, Stockholm, Sweden)

    1984-01-01

    The distribution of /sup 74/As-labelled and arsenite in pregnant mice and a monkey has been studied by autoradiography and gamma counting of isolated tissues, and their in vitro toxicity to a chondrogenic system has been investigated. With both arsenic forms, given as single intravenous injections to the mother, the /sup 74/As-arsenic appeared to pass the mouse placenta relatively freely and approximately to the same extent. The retention time in material tissues including the placenta was, however, around three times longer with arsenite than with arsenate. In early gestation, high activity was registered in the embryonic neuroepithelium, which correlates well with reported CNS malformations in rodents. In late gestation, the distribution pattern was more like that in the adults. Accumulation in skin and squamous epithelia of the upper gastrointestinal tract (oral cavity, oesophagus and oesophageal region of stomach) dominated the distribution picture, especially at a long survival interval. Arsenate, but not arsenite, showed affinity for the calcified areas of the skeleton. A marmoset monkey in late gestation receiving arsenite showed a somewhat lower rate of placental transfer than the mice. Skin and liver had the highest concentrations (at 8 hrs), both in mother and foetuses. This species is known not to methylate arsenic, resulting in stronger binding and longer retention times of arsenic as compared with other species. The stronger binding in maternal tissues may possibly explain the lower rate of placental transfer. Arsenite was shown to inhibit cartilage formation in a chick limb bud mesenchymal spot culture system (ED50 approximately 5-10..mu..M) while arsenate seemed to be without effect at concentrations up to 200 ..mu..M (highest tested). Arsenate, however, showed a potential of the arsenite toxicity.

  10. Embryotoxicity of arsenite and arsenate. Distribution in pregnant mice and monkeys and effects on embryonic cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Lindgren, A.; Danielsson, R.G.; Dencker, L. (Department of Toxicology, Biomedical Center, Uppsala University, Sweden); Vahter, M. (National Institute of Environmental Medicine, Stockholm, Sweden)

    1984-01-01

    The distribution of /sup 74/As-labelled arsenate and arsenite in pregnant mice and a monkey has been studied by autoradiography and gamma counting of isolated tissues, and their in vitro toxicity to a chondrogenic system has been investigated. With both arsenic forms, given as single intravenous injections to the mother, the /sup 74/As-arsenic appeared to pass the mouse placenta relatively freely and approximately to the same extent. The retention time in material tissues including the placenta was, however, around three times longer with arsenite than with arsenate. In early gestation, high activity was registered in the embryonic neuroepithelium, which correlates well with reported CNS malformations in rodents. In late gestation, the distribution pattern was more like that in the adults. Accumulation in skin and squamous epithelia of the upper gastrointestinal tract (oral cavity, oesophagus and oesophageal region of stomach) dominated the distribution pucture, especially at a long survival interval. Arsenate, but not arsenite, showed affinity for the calcified areas of the skeleton. A marmoset monkey in late gestation receiving arsenite showed a somewhat lower rate of placental transfer than the mice. Skin and liver had the highest concentrations (at 8 hrs), both in mother and foetuses. This species is known not to methylate arsenic, resulting in stronger binding and longer retention times of arsenic as compared with other species. The stronger binding in maternal tissues may possibly explain the lower rate of placental transfer. Arsenite was shown to inhibit cartilage formation in a chick limb bud mesenchymal spot culture system (ED50 approximately 5-10..mu..M) while arsenate seemed to be without effect at concentrations up to 200 ..mu..M (highest tested). Arsenate, however, showed a potential of the arsenite toxicity.

  11. IP Phone 综述

    Institute of Scientific and Technical Information of China (English)

    夏华; 陈常嘉

    2000-01-01

    IP Phone代表下一代电话技术,是目前的热点,它通过分组交换网络来传输话音,是开放式分组业务的一种。本文从IP Phone的出现谈起,简要介绍了IP Phone的优势,工作原理和相关标准,着重分析了IP Phone面临的挑战与机遇,并展望了IP Phone的发展前景。

  12. Protection against cancer by dietary IP6 and inositol.

    Science.gov (United States)

    Vucenik, Ivana; Shamsuddin, AbulKalam M

    2006-01-01

    Inositol hexaphosphate (IP(6)) is a naturally occurring polyphosphorylated carbohydrate, abundantly present in many plant sources and in certain high-fiber diets, such as cereals and legumes. In addition to being found in plants, IP(6) is contained in almost all mammalian cells, although in much smaller amounts, where it is important in regulating vital cellular functions such as signal transduction, cell proliferation, and differentiation. For a long time IP(6) has been recognized as a natural antioxidant. Recently IP(6) has received much attention for its role in cancer prevention and control of experimental tumor growth, progression, and metastasis. In addition, IP(6) possesses other significant benefits for human health, such as the ability to enhance immune system, prevent pathological calcification and kidney stone formation, lower elevated serum cholesterol, and reduce pathological platelet activity. In this review we show the efficacy and discuss some of the molecular mechanisms that govern the action of this dietary agent. Exogenously administered IP(6) is rapidly taken up into cells and dephosphorylated to lower inositol phosphates, which further affect signal transduction pathways resulting in cell cycle arrest. A striking anticancer action of IP(6) was demonstrated in different experimental models. In addition to reducing cell proliferation, IP(6) also induces differentiation of malignant cells. Enhanced immunity and antioxidant properties also contribute to tumor cell destruction. Preliminary studies in humans show that IP(6) and inositol, the precursor molecule of IP(6), appear to enhance the anticancer effect of conventional chemotherapy, control cancer metastases, and improve quality of life. Because it is abundantly present in regular diet, efficiently absorbed from the gastrointestinal tract, and safe, IP(6) + inositol holds great promise in our strategies for cancer prevention and therapy. There is clearly enough evidence to justify the

  13. TCP/IP foundations

    CERN Document Server

    Blank, Andrew G

    2006-01-01

    The world of IT is always evolving, but in every area there are stable, core concepts that anyone just setting out needed to know last year, needs to know this year, and will still need to know next year. The purpose of the Foundations series is to identify these concepts and present them in a way that gives you the strongest possible starting point, no matter what your endeavor. TCP/IP Foundations provides essential knowledge about the two protocols that form the basis for the Internet, as well as many other networks. What you learn here will benefit you in the short term, as you acquire and

  14. Functional characterizations of IPS-1 in CIK cells: Potential roles in regulating IFN-I response dependent on IRF7 but not IRF3.

    Science.gov (United States)

    Feng, Xiaoli; Zhang, Yixuan; Yang, Chunrong; Liao, Lanjie; Wang, Yaping; Su, Jianguo

    2015-11-01

    IPS-1, as the sole adaptor of RIG-I and MDA5, plays a central role in innate antiviral immunity. In this study, we investigated potential roles of IPS-1 in innate immunity and the domain-requirement of IPS-1 for its signaling in grass carp (Ctenopharyngodon idella). Overexpression experiment showed that CiIPS-1 mediated IFN-I signal possibly dependent on CiIRF7 but not CiIRF3. Post GCRV challenge, CiIPS-1 could enhance antiviral immune responses. CARD and TM domains were crucial for antiviral function of CiIPS-1, and TRAF motif played an assistant role. PRO domain seemed as a negative regulator but was pivotal for the initiation of CiIFN-I and CiMx1. Post viral/bacterial PAMPs stimulation, CiIPS-1-mediated signaling was tightly controlled. CARD domain of CiIPS-1 could significantly elicit poly I:C/LPS/PGN-mediated signaling. PRO domain negatively regulated CiIRF7 and CiIFN-I but was indispensable for inductions of CiMx1 and CiIL-1β. TRAF motif and TM domain regulated the signaling presumably in a cooperative fashion. Post poly I:C stimulation, TRAF motif negatively regulated CiIRF7, CiIFN-I and CiIL-1β at a relative early time while TM domain functioned at a relative late time. TRAF motif was indispensable for the production of CiMx1, while TM domain slightly negatively regulated the expression. Post LPS and PGN stimulation, TRAF motif excited an assistant and persistent negative role on CiIFN-I, CiIRF7 and CiIL-1β induction, but was crucial for induction of CiMx1. TM domain slightly negatively regulated LPS- and PGN-triggered signaling. Taken together, CiIPS-1 not only exerted important functions in antiviral immune response but also participated in viral/bacterial PAMPs-triggered immune response which was tightly controlled to prevent harmful effects resulting from excessive activation. This study provided novel insights into the pivotal role of IPS-1 in innate immunity.

  15. Glutathione deficiency induced by cystine and/or methionine deprivation does not affect thyroid hormone deiodination in cultured rat hepatocytes and monkey hepatocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Sato, K.; Robbins, J.

    1981-09-01

    To elucidate the recently advanced hypothesis that glutathione (L-gamma-glutamyl-L-cysteinyl glycine (GSH)) regulates deiodinating enzyme activities, accounting for the decreased conversion of T4 to T3 in the liver of fetal and starved animals, we investigated thyroid hormone metabolism in GSH-depleted neoplastic and normal hepatocytes. In monkey hepatocarcinoma cells, intracellular total GSH decreased below 10% of the control value (approximately 25 micrograms/mg protein) when cells were grown for 44 h in medium deficient in cystine and methionine or in cystine alone. The latter finding indicated that transsulfuration from methionine to cysteine was defective in these neoplastic cells. In primary cultured adult rat hepatocytes, on the other hand, the transsulfuration pathway was intact, and total GSH decreased below 10% of control (approximately 20 micrograms/mg protein) only in cells grown in cystine- and methionine-deficient medium. In both cell types, the oxidized GSH fraction remained constant (2-5% of total). Incubation with 125I-labeled T4 and T3, followed by chromatography, was used to evaluate 5-deiodination in hepatocarcinoma cells and both 5- and 5'-deiodination in normal hepatocytes. Deiodination was not decreased by GSH deficiency in either case, but was actually increased in hepatocarcinoma cells. This resulted from an increase in the Vmax of 5-deiodinase related to growth arrest. Diamide at 2 mM reversibly inhibited both 5'- and 5'-deiodination in rat hepatocytes, accompanied by decreased total GSH as well as increased GSH disulfide (27% of total). The data suggest that GSH is so abundant in the liver that hepatocytes can tolerate a greater than 90% decrease in intracellular concentration without any change in thyroid hormone deiodination and indicate that altered thyroid hormone metabolism in the fetus and in starvation cannot be accounted for by a decreased hepatic GSH concentration.

  16. 脑缺血发生后 IP-10趋化 NK 细胞通过血脑屏障%Chemotactic effect of IP-10 to natural killer cells getting through blood brain barrier after cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    张瑶; 冯涛; 祝鸿雁; 王广友; 王丹丹; 李呼伦

    2014-01-01

    Objective To research the chemotactic effect of IP-10 on natural killer cells after cerebral ischemia .Methods Experiments in vivo: NK infiltration and IP-10 and CXCR3 ex-pression in pMCAO mouse brain were detected by immunofluorescence;NK infiltration in pM-CAO brains was detected by flowcytometry;IP-10 total expression level in brain were investiga-ted by RT-PCR.Experiments in vitro:Established blood brain barrier ( B.B.B.) in vitro;IP-10 expression of the supernatant from brain microvascular endothclial cell ( BMVEC ) and neu-ral cells after oxygen-gluoose depriuation ( OGD) were detected by ELISA; B.B.B.in vitro were separated into NK , NK+IP-10 blockade two groups , after OGD 6 h, NK cells migration counts were detected by flowcytometry;NK cells were cultured together with IP -10 ( 10 ng/mL), IP-10 (50 ng/mL), IP-10(100 ng/mL), then CXCR3 geomean.MFI value were de-tected by flowcytometry .Results Experiments in vivo:NK cells were found in pMCAO brain section and expressing CXCR 3 and IP-10; NK cells infiltration were higher in ischemia-hemi-sphere , and got a peak at 12 h ( P<0.05 );NK cells had anobviously increase by 12 h at in-filtration frequency (P<0.01 );IP-10 had a highest level at 12 h ( P<0.001 ) .Experiments in vitro:Neural cell had higher expression of IP-10 after OGD than BMVEC ( P <0.001 );Compared with group NK , group NK+IP-10 blockade had lower NK cells migration counts ( P<0.05 ) and permeability ( P<0.001 ) .Conclusion ①NK cells plays an important role dur-ing cerebral ischemia;②IP-10 can absorb NK cells across B .B.B.when ischemia happens by combined with its receptor CXCR 3 and has dose dependent .%目的:探讨脑缺血发生后 IP-10对NK细胞的趋化作用。方法体内实验:免疫荧光染色观察永久性大脑中动脉栓塞小鼠模型( pMCAO)脑组织中NK细胞浸润情况以及IP-10、CXCR3表达情况;流式细胞术检测pMCAO小鼠模型脑中NK细胞浸润数目;RT-PCR

  17. ChIP-seq analysis of histone H3K9 trimethylation in peripheral blood mononuclear cells of membranous nephropathy patients

    Energy Technology Data Exchange (ETDEWEB)

    Sui, W.G. [Guangxi Key Laboratory of Metabolic Diseases Research, Nephrology Department, 181st Hospital, Guilin, Guangxi (China); He, H.Y. [The Life Science College, Guangxi Normal University, Guilin, Guangxi (China); Yan, Q.; Chen, J.J. [Guangxi Key Laboratory of Metabolic Diseases Research, Nephrology Department, 181st Hospital, Guilin, Guangxi (China); Zhang, R.H. [The Life Science College, Guangxi Normal University, Guilin, Guangxi (China); Dai, Y. [Clinical Medical Research Center, The Second Clinical Medical College, Shenzhen People’s Hospital, Jinan University, Shenzhen, Guangdong (China)

    2013-12-12

    Membranous nephropathy (MN), characterized by the presence of diffuse thickening of the glomerular basement membrane and subepithelial in situ immune complex disposition, is the most common cause of idiopathic nephrotic syndrome in adults, with an incidence of 5-10 per million per year. A number of studies have confirmed the relevance of several experimental insights to the pathogenesis of human MN, but the specific biomarkers of MN have not been fully elucidated. As a result, our knowledge of the alterations in histone methylation in MN is unclear. We used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to analyze the variations in a methylated histone (H3K9me3) in peripheral blood mononuclear cells from 10 MN patients and 10 healthy subjects. There were 108 genes with significantly different expression in the MN patients compared with the normal controls. In MN patients, significantly increased activity was seen in 75 H3K9me3 genes, and decreased activity was seen in 33, compared with healthy subjects. Five positive genes, DiGeorge syndrome critical region gene 6 (DGCR6), sorting nexin 16 (SNX16), contactin 4 (CNTN4), baculoviral IAP repeat containing 3 (BIRC3), and baculoviral IAP repeat containing 2 (BIRC2), were selected and quantified. There were alterations of H3K9me3 in MN patients. These may be candidates to help explain pathogenesis in MN patients. Such novel findings show that H3K9me3 may be a potential biomarker or promising target for epigenetic-based MN therapies.

  18. Inhibition of tumor-associated antigens secreted from cancer cell lines by Taimatsu fermented rice germ solution containing inositol hexaphosphate (IP6)

    OpenAIRE

    池脇, 信直; 石附, 亨; 中村, 雅彦; 高木, 隆太郎; 金井, 美樹; 樋口, 元剛; 近藤, 照義; イケワキ, ノブナオ; イシズキ, トオル; ナカムラ, マサヒコ; タカキ, リュウタロウ; カナイ, ミキ; ヒグチ, ゲンゴウ; コンドウ, テルヨシ; Nobunao, IKEWAKI

    2011-01-01

    本研究では、我々が開発したIP6含有たいまつ米胚芽発酵液(T-IP6)の癌細胞株(MKN-45およびHepG2)から分泌される腫瘍関連抗原(CEAまたはPIVKA-II)に対する影響を化学発光および電気化学発光酵素免疫測定法で解析した。その結果、T-IP6で培養したMKN-45およびHepG2細胞から分泌されるCEAまたはPIVKA-IIは、細胞の形態変化を伴いながら有意に抑制された(P

  19. Foundations of hardware IP protection

    CERN Document Server

    Torres, Lionel

    2017-01-01

    This book provides a comprehensive and up-to-date guide to the design of security-hardened, hardware intellectual property (IP). Readers will learn how IP can be threatened, as well as protected, by using means such as hardware obfuscation/camouflaging, watermarking, fingerprinting (PUF), functional locking, remote activation, hidden transmission of data, hardware Trojan detection, protection against hardware Trojan, use of secure element, ultra-lightweight cryptography, and digital rights management. This book serves as a single-source reference to design space exploration of hardware security and IP protection. · Provides readers with a comprehensive overview of hardware intellectual property (IP) security, describing threat models and presenting means of protection, from integrated circuit layout to digital rights management of IP; · Enables readers to transpose techniques fundamental to digital rights management (DRM) to the realm of hardware IP security; · Introduce designers to the concept of salutar...

  20. The Genial Monkeys of Emei

    Institute of Scientific and Technical Information of China (English)

    CAOHONG

    2004-01-01

    MANY of China's beautiful mountainous areas are home to monkeys,the most famous monkey resort being Emei Mountain. Perhaps affected by the mountain's Buddhist atmosphere, Emei's monkeys are gentle and often approach tourists for food and play. Cute and impish, these delightful creatures are the main attraction for many visitors.

  1. A Mason-Pfizer Monkey virus Gag-GFP fusion vector allows visualization of capsid transport in live cells and demonstrates a role for microtubules.

    Directory of Open Access Journals (Sweden)

    Jasmine Clark

    Full Text Available Immature capsids of the Betaretrovirus, Mason-Pfizer Monkey virus (M-PMV, are assembled in the pericentriolar region of the cell, and are then transported to the plasma membrane for budding. Although several studies, utilizing mutagenesis, biochemistry, and immunofluorescence, have defined the role of some viral and host cells factors involved in these processes, they have the disadvantage of population analysis, rather than analyzing individual capsid movement in real time. In this study, we created an M-PMV vector in which the enhanced green fluorescent protein, eGFP, was fused to the carboxyl-terminus of the M-PMV Gag polyprotein, to create a Gag-GFP fusion that could be visualized in live cells. In order to express this fusion protein in the context of an M-PMV proviral backbone, it was necessary to codon-optimize gag, optimize the Kozak sequence preceding the initiating methionine, and mutate an internal methionine codon to one for alanine (M100A to prevent internal initiation of translation. Co-expression of this pSARM-Gag-GFP-M100A vector with a WT M-PMV provirus resulted in efficient assembly and release of capsids. Results from fixed-cell immunofluorescence and pulse-chase analyses of wild type and mutant Gag-GFP constructs demonstrated comparable intracellular localization and release of capsids to untagged counterparts. Real-time, live-cell visualization and analysis of the GFP-tagged capsids provided strong evidence for a role for microtubules in the intracellular transport of M-PMV capsids. Thus, this M-PMV Gag-GFP vector is a useful tool for identifying novel virus-cell interactions involved in intracellular M-PMV capsid transport in a dynamic, real-time system.

  2. Development of Eimeria bovis in vitro: suitability of several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal, Madin-Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells.

    Science.gov (United States)

    Hermosilla, C; Barbisch, B; Heise, A; Kowalik, S; Zahner, H

    2002-04-01

    Several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal cells (BFGC), Madin-Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells were exposed in vitro to sporozoites of Eimeria bovis. Parasites invaded all cells used and changed their shape to more stumpy forms within 12 h. Sporozoites left their host cells and invaded new ones frequently within the first 12 h post-infection. Further development took place only in bovine cells, although parasites survived in the other cells for at least 3 weeks. Within the non-bovine cells, conspicuously enlarged parasitophorous vacuoles developed in VERO cells and reached a diameter of 15-20 microm. The best development to first generation schizonts with regard both to time required to mature, to schizont size and to merozoite yields was observed in BFGC, followed by bovine umbilical vein and bovine spleen lymphatic endothelial cells. MDBK cells were less suitable. The life cycle was completed (development of oocysts) only occasionally in BFGC. Results are considered under several aspects. Thus, infected VERO cells may represent a suitable tool for studying the parasitophorous vacuole, while infected endothelial cells represent a system quite narrow to the in vivo situation and should allow basic studies on parasite/host cell interactions and BFGC can be used for the mass production of E. bovis first generation merozoites.

  3. Quantitative analysis of ChIP-seq data uncovers dynamic and sustained H3K4me3 and H3K27me3 modulation in cancer cells under hypoxia.

    Science.gov (United States)

    Adriaens, Michiel E; Prickaerts, Peggy; Chan-Seng-Yue, Michelle; van den Beucken, Twan; Dahlmans, Vivian E H; Eijssen, Lars M; Beck, Timothy; Wouters, Bradly G; Voncken, Jan Willem; Evelo, Chris T A

    2016-01-01

    A comprehensive assessment of the epigenetic dynamics in cancer cells is the key to understanding the molecular mechanisms underlying cancer and to improving cancer diagnostics, prognostics and treatment. By combining genome-wide ChIP-seq epigenomics and microarray transcriptomics, we studied the effects of oxygen deprivation and subsequent reoxygenation on histone 3 trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3) in a breast cancer cell line, serving as a model for abnormal oxygenation in solid tumors. A priori, epigenetic markings and gene expression levels not only are expected to vary greatly between hypoxic and normoxic conditions, but also display a large degree of heterogeneity across the cell population. Where traditionally ChIP-seq data are often treated as dichotomous data, the model and experiment here necessitate a quantitative, data-driven analysis of both datasets. We first identified genomic regions with sustained epigenetic markings, which provided a sample-specific reference enabling quantitative ChIP-seq data analysis. Sustained H3K27me3 marking was located around centromeres and intergenic regions, while sustained H3K4me3 marking is associated with genes involved in RNA binding, translation and protein transport and localization. Dynamic marking with both H3K4me3 and H3K27me3 (hypoxia-induced bivalency) was found in CpG-rich regions at loci encoding factors that control developmental processes, congruent with observations in embryonic stem cells. In silico-identified epigenetically sustained and dynamic genomic regions were confirmed through ChIP-PCR in vitro, and obtained results are corroborated by published data and current insights regarding epigenetic regulation.

  4. Introduction à TCP/IP

    OpenAIRE

    Archimbaud, Jean-Luc

    1993-01-01

    • Ce qu'il faut savoir sur les réseaux • Ce qu'il faut savoir sur Ethernet • Généralités sur TCP/IPIP et consort • TCP et consort • Le routage • Interconnexion de réseaux Ethernet et IP • Nommage • Exemples d'applications • Enchaînement des opérations • Daemons et commandes Unix • Glossaire; Engineering school; Cours d'introduction à TCP/IP

  5. Anti-angiogenic activity of inositol hexaphosphate (IP6).

    Science.gov (United States)

    Vucenik, Ivana; Passaniti, Antonino; Vitolo, Michele I; Tantivejkul, Kwanchanit; Eggleton, Paul; Shamsuddin, Abulkalam M

    2004-11-01

    A significant anticancer activity of the naturally occurring carbohydrate inositol hexaphosphate (IP(6)) has been reported against numerous cancer models. Since tumors require angiogenesis for growth and metastasis, we hypothesize that IP(6) reduces tumor growth by inhibiting angiogenesis. Because angiogenesis depends on the interaction between endothelial and tumor cells, we investigated the effect of IP(6) on both. IP(6) inhibited the proliferation and induced the differentiation of endothelial cells in vitro; the growth of bovine aortic endothelial cells (BAECs) evaluated by MTT proliferation assay was inhibited in a dose-dependent manner (IC(50) = 0.74 mM). The combination of IP(6) and vasostatin, a calreticulin fragment with anti-angiogenic activity, was synergistically superior in growth inhibition than either compound. IP(6) inhibited human umbilical vein endothelial cell (HUVEC) tube formation (in vitro capillary differentiation) on a reconstituted extracellular matrix, Matrigel, and disrupted pre-formed tubes. IP(6) significantly reduced basic fibroblast growth factor (bFGF)-induced vessel formation (P < 0.01) in vivo in Matrigel plug assay. Exposure of HepG2, a human hepatoma cell line, to IP(6) for 8 h, resulted in a dose-dependent decrease in the mRNA levels of vascular endothelial growth factor (VEGF), as assessed by RT-PCR. IP(6) treatment of HepG2 cells for 24 h also significantly reduced the VEGF protein levels in conditioned medium, in a concentration-dependent manner (P = 0.012). Thus, IP(6) has an inhibitory effect on induced angiogenesis.

  6. 人诱导性多潜能干细胞(iPS细胞)系的建立%Generation of A Human Induced Pluripotent Stem (iPS) Cells Line

    Institute of Scientific and Technical Information of China (English)

    姚志芳; 史俊文; 贾俊双; 申红芬; 林晓琳; 肖高芳; 张晟; 肖东; 姚开泰

    2011-01-01

    To master the technology of reprogramming human somatic cells to iPS cells and set up a technical platform to reprogram human cancer cells into iPS cells. Under human embryonic stem cell (hES cell) culture conditions, human skin fibroblasts (CCD-1079SK) cells infected by lentivirus mixture harboring Oct4, Sox2, c-Myc and Klf4 genes were induced into hES cell-like colonies, following characterization were done by comparatively analyzing hES cells markers: colony morphology, alkaline phosphatase (AP) activity, the expression profile of ES cell-marker genes, karyotype and embryoid body (EB)-mediated in vitro differentiation of CCD-1079SK cell-derived hES cell-like colonies. The results showed that human iPS cell line (iPSC-1) was generated from CCD-1079SK cells by introducing four genes, Oct4, Sox2, c-Myc and Klf4, under hES cell culture conditions. iPSC was similar to hES cells in morphology, proliferation, hES cell-specific surfaceanti-gens and gene expression. Additionally, iPSC could be cultured in suspension to form embryoid bodies (Ebs) and differentiate into cell types of the three germ layers in vitro, in addition, human iPS cell line is successfully established, which will lay a solid foundation for performing cancer cell reprogramming by using iPS cell technology.%掌握建立人iPS细胞系(induced pluripotent stem cells,iPSCs)的技术,以便为人肿瘤细胞重编程为iPS细胞建立技术平台.在人胚胎干细胞的培养条件下,通过携带Oct4、Sox2、c-Myc、Klf44个混合因子的慢病毒感染人皮肤成纤维细胞(CCD-1079SK细胞),从而诱导成干细胞样的克隆.根据人胚胎干细胞的特性进行如下鉴定:克隆形态、碱性磷酸酶活性、核型和CCD-1079SK细胞来源的克隆拟胚体(embryoid bodies,EBs)形成及分化等.结果显示,在人胚胎干细胞的培养环境中,导入Oct4、Sox2、c-Myc、Klf44个因子的CCD-1079SK细胞产生了一株iPSC克隆,这株iPSC克隆在细胞形态、增殖能力、胚胎

  7. Improving T-cell assays for the diagnosis of latent TB infection: potential of a diagnostic test based on IP-10

    DEFF Research Database (Denmark)

    Ruhwald, Morten; Petersen, Janne; Kofoed, Kristian

    2008-01-01

    BACKGROUND: There is a need for simple tools such as the M.tuberculosis specific IFN-gamma release assays (IGRA) to improve diagnosis of M.tuberculosis-infection in children. The aim of the study was to evaluate the performance of an IP-10 and IL-2 based tests for the diagnosis of M.tuberculosis-...

  8. Development of iPS (induced pluripotent stem cells) using natural product from extract of fish oocyte to provide stem cell for regenerative therapy

    Science.gov (United States)

    Meilany, Sofy; Firdausiyah, Qonitha S.; Naroeni, Aroem

    2017-02-01

    In this study, we developed a method to induce pluripotency of adult cells (fibroblast) into stem cells using a natural product, extract of fish oocyte, by comparing the extract concentration, 1 mg/ml and 2 mg/ml. The analyses were done by measuring the Nanog gene expression in cells using qPCR and detecting fibroblast marker anti H2-KK. The results revealed existence of a colony of stem cells in the cell that was induced with 2mg/ml concentration of oocytes. Nanoggene expression was analyzed by qPCR and the results showed expression of Nanog gene compared to the control. Analysis of result of fibroblast using Tali Cytometer and anti H2KK antibody showed loss of expression of Anti H2KK meaning there was transformation from fibroblast type cell to pluripotent cell type.

  9. Identification of a major IP5 kinase in Cryptococcus neoformans confirms that PP-IP5/IP7, not IP6, is essential for virulence.

    Science.gov (United States)

    Li, Cecilia; Lev, Sophie; Saiardi, Adolfo; Desmarini, Desmarini; Sorrell, Tania C; Djordjevic, Julianne T

    2016-04-01

    Fungal inositol polyphosphate (IP) kinases catalyse phosphorylation of IP3 to inositol pyrophosphate, PP-IP5/IP7, which is essential for virulence of Cryptococcus neoformans. Cryptococcal Kcs1 converts IP6 to PP-IP5/IP7, but the kinase converting IP5 to IP6 is unknown. Deletion of a putative IP5 kinase-encoding gene (IPK1) alone (ipk1Δ), and in combination with KCS1 (ipk1Δkcs1Δ), profoundly reduced virulence in mice. However, deletion of KCS1 and IPK1 had a greater impact on virulence attenuation than that of IPK1 alone. ipk1Δkcs1Δ and kcs1Δ lung burdens were also lower than those of ipk1Δ. Unlike ipk1Δ, ipk1Δkcs1Δ and kcs1Δ failed to disseminate to the brain. IP profiling confirmed Ipk1 as the major IP5 kinase in C. neoformans: ipk1Δ produced no IP6 or PP-IP5/IP7 and, in contrast to ipk1Δkcs1Δ, accumulated IP5 and its pyrophosphorylated PP-IP4 derivative. Kcs1 is therefore a dual specificity (IP5 and IP6) kinase producing PP-IP4 and PP-IP5/IP7. All mutants were similarly attenuated in virulence phenotypes including laccase, urease and growth under oxidative/nitrosative stress. Alternative carbon source utilisation was also reduced significantly in all mutants except ipk1Δ, suggesting that PP-IP4 partially compensates for absent PP-IP5/IP7 in ipk1Δ grown under this condition. In conclusion, PP-IP5/IP7, not IP6, is essential for fungal virulence.

  10. THE CLEVER MONKEYS

    Institute of Scientific and Technical Information of China (English)

    付惠娟

    2004-01-01

    A man was walking through a forest. He had a few caps in his hands. In the forest there were a lot of monkeys. The day was hot, so he decided to have a rest under a tree. I-le put one cap on his head and lay down to sleep.

  11. In silico pooling of ChIP-seq control experiments.

    Science.gov (United States)

    Sun, Guannan; Srinivasan, Rajini; Lopez-Anido, Camila; Hung, Holly A; Svaren, John; Keleş, Sündüz

    2014-01-01

    As next generation sequencing technologies are becoming more economical, large-scale ChIP-seq studies are enabling the investigation of the roles of transcription factor binding and epigenome on phenotypic variation. Studying such variation requires individual level ChIP-seq experiments. Standard designs for ChIP-seq experiments employ a paired control per ChIP-seq sample. Genomic coverage for control experiments is often sacrificed to increase the resources for ChIP samples. However, the quality of ChIP-enriched regions identifiable from a ChIP-seq experiment depends on the quality and the coverage of the control experiments. Insufficient coverage leads to loss of power in detecting enrichment. We investigate the effect of in silico pooling of control samples within multiple biological replicates, multiple treatment conditions, and multiple cell lines and tissues across multiple datasets with varying levels of genomic coverage. Our computational studies suggest guidelines for performing in silico pooling of control experiments. Using vast amounts of ENCODE data, we show that pairwise correlations between control samples originating from multiple biological replicates, treatments, and cell lines/tissues can be grouped into two classes representing whether or not in silico pooling leads to power gain in detecting enrichment between the ChIP and the control samples. Our findings have important implications for multiplexing samples.

  12. Wide-field diffuse amacrine cells in the monkey retina contain immunoreactive Cocaine- and Amphetamine-Regulated Transcript (CART).

    Science.gov (United States)

    Long, Ye; Bordt, Andrea S; Liu, Weiley S; Davis, Elizabeth P; Lee, Stephen J; Tseng, Luke; Chuang, Alice Z; Whitaker, Christopher M; Massey, Stephen C; Sherman, Michael B; Marshak, David W

    2016-10-01

    The goals of this study were to localize the neuropeptide Cocaine- and Amphetamine-Regulated Transcript (CART) in primate retinas and to describe the morphology, neurotransmitter content and synaptic connections of the neurons that contain it. Using in situ hybridization, light and electron microscopic immunolabeling, CART was localized to GABAergic amacrine cells in baboon retinas. The CART-positive cells had thin, varicose dendrites that gradually descended through the inner plexiform layer and ramified extensively in the innermost stratum. They resembled two types of wide-field diffuse amacrine cells that had been described previously in macaque retinas using the Golgi method and also A17, serotonin-accumulating and waterfall cells of other mammals. The CART-positive cells received synapses from rod bipolar cell axons and made synapses onto the axons in a reciprocal configuration. The CART-positive cells also received synapses from other amacrine cells. Some of these were located on their primary dendrites, and the presynaptic cells there included dopaminergic amacrine cells. Although some CART-positive somas were localized in the ganglion cell layer, they did not contain the ganglion cell marker RNA binding protein with multiple splicing (RBPMS). Based on these results and electrophysiological studies in other mammals, the CART-positive amacrine cells would be expected to play a major role in the primary rod pathway of primates, providing feedback inhibition to rod bipolar cells.

  13. Mitochondrial-associated cell death mechanisms are reset to an embryonic-like state in aged donor-derived iPS cells harboring chromosomal aberrations.

    Science.gov (United States)

    Prigione, Alessandro; Hossini, Amir M; Lichtner, Björn; Serin, Akdes; Fauler, Beatrix; Megges, Matthias; Lurz, Rudi; Lehrach, Hans; Makrantonaki, Eugenia; Zouboulis, Christos C; Adjaye, James

    2011-01-01

    Somatic cells reprogrammed into induced pluripotent stem cells (iPSCs) acquire features of human embryonic stem cells (hESCs) and thus represent a promising source for cellular therapy of debilitating diseases, such as age-related disorders. However, reprogrammed cell lines have been found to harbor various genomic alterations. In addition, we recently discovered that the mitochondrial DNA of human fibroblasts also undergoes random mutational events upon reprogramming. Aged somatic cells might possess high susceptibility to nuclear and mitochondrial genome instability. Hence, concerns over the oncogenic potential of reprogrammed cells due to the lack of genomic integrity may hinder the applicability of iPSC-based therapies for age-associated conditions. Here, we investigated whether aged reprogrammed cells harboring chromosomal abnormalities show resistance to apoptotic cell death or mitochondrial-associated oxidative stress, both hallmarks of cancer transformation. Four iPSC lines were generated from dermal fibroblasts derived from an 84-year-old woman, representing the oldest human donor so far reprogrammed to pluripotency. Despite the presence of karyotype aberrations, all aged-iPSCs were able to differentiate into neurons, re-establish telomerase activity, and reconfigure mitochondrial ultra-structure and functionality to a hESC-like state. Importantly, aged-iPSCs exhibited high sensitivity to drug-induced apoptosis and low levels of oxidative stress and DNA damage, in a similar fashion as iPSCs derived from young donors and hESCs. Thus, the occurrence of chromosomal abnormalities within aged reprogrammed cells might not be sufficient to over-ride the cellular surveillance machinery and induce malignant transformation through the alteration of mitochondrial-associated cell death. Taken together, we unveiled that cellular reprogramming is capable of reversing aging-related features in somatic cells from a very old subject, despite the presence of genomic

  14. Mitochondrial-associated cell death mechanisms are reset to an embryonic-like state in aged donor-derived iPS cells harboring chromosomal aberrations.

    Directory of Open Access Journals (Sweden)

    Alessandro Prigione

    Full Text Available Somatic cells reprogrammed into induced pluripotent stem cells (iPSCs acquire features of human embryonic stem cells (hESCs and thus represent a promising source for cellular therapy of debilitating diseases, such as age-related disorders. However, reprogrammed cell lines have been found to harbor various genomic alterations. In addition, we recently discovered that the mitochondrial DNA of human fibroblasts also undergoes random mutational events upon reprogramming. Aged somatic cells might possess high susceptibility to nuclear and mitochondrial genome instability. Hence, concerns over the oncogenic potential of reprogrammed cells due to the lack of genomic integrity may hinder the applicability of iPSC-based therapies for age-associated conditions. Here, we investigated whether aged reprogrammed cells harboring chromosomal abnormalities show resistance to apoptotic cell death or mitochondrial-associated oxidative stress, both hallmarks of cancer transformation. Four iPSC lines were generated from dermal fibroblasts derived from an 84-year-old woman, representing the oldest human donor so far reprogrammed to pluripotency. Despite the presence of karyotype aberrations, all aged-iPSCs were able to differentiate into neurons, re-establish telomerase activity, and reconfigure mitochondrial ultra-structure and functionality to a hESC-like state. Importantly, aged-iPSCs exhibited high sensitivity to drug-induced apoptosis and low levels of oxidative stress and DNA damage, in a similar fashion as iPSCs derived from young donors and hESCs. Thus, the occurrence of chromosomal abnormalities within aged reprogrammed cells might not be sufficient to over-ride the cellular surveillance machinery and induce malignant transformation through the alteration of mitochondrial-associated cell death. Taken together, we unveiled that cellular reprogramming is capable of reversing aging-related features in somatic cells from a very old subject, despite the presence

  15. Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype.

    Science.gov (United States)

    Katt, Moriah E; Xu, Zinnia S; Gerecht, Sharon; Searson, Peter C

    2016-01-01

    The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2-4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.

  16. Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype

    OpenAIRE

    Katt, Moriah E.; Xu, Zinnia S.; Gerecht, Sharon; Searson, Peter C.

    2016-01-01

    The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial...

  17. Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency

    Science.gov (United States)

    Lee, Kiho; Kwon, Deug-Nam; Ezashi, Toshihiko; Choi, Yun-Jung; Park, Chankyu; Ericsson, Aaron C.; Brown, Alana N.; Samuel, Melissa S.; Park, Kwang-Wook; Walters, Eric M.; Kim, Dae Young; Kim, Jae-Hwan; Franklin, Craig L.; Murphy, Clifton N.; Roberts, R. Michael; Prather, Randall S.; Kim, Jin-Hoi

    2014-01-01

    Pigs with severe combined immunodeficiency (SCID) may provide useful models for regenerative medicine, xenotransplantation, and tumor development and will aid in developing therapies for human SCID patients. Using a reporter-guided transcription activator-like effector nuclease (TALEN) system, we generated targeted modifications of recombination activating gene (RAG) 2 in somatic cells at high efficiency, including some that affected both alleles. Somatic-cell nuclear transfer performed with the mutated cells produced pigs with RAG2 mutations without integrated exogenous DNA. Biallelically modified pigs either lacked a thymus or had one that was underdeveloped. Their splenic white pulp lacked B and T cells. Under a conventional housing environment, the biallelic RAG2 mutants manifested a “failure to thrive” phenotype, with signs of inflammation and apoptosis in the spleen compared with age-matched wild-type animals by the time they were 4 wk of age. Pigs raised in a clean environment were healthier and, following injection of human induced pluripotent stem cells (iPSCs), quickly developed mature teratomas representing all three germ layers. The pigs also tolerated grafts of allogeneic porcine trophoblast stem cells. These SCID pigs should have a variety of uses in transplantation biology. PMID:24799706

  18. Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency.

    Science.gov (United States)

    Lee, Kiho; Kwon, Deug-Nam; Ezashi, Toshihiko; Choi, Yun-Jung; Park, Chankyu; Ericsson, Aaron C; Brown, Alana N; Samuel, Melissa S; Park, Kwang-Wook; Walters, Eric M; Kim, Dae Young; Kim, Jae-Hwan; Franklin, Craig L; Murphy, Clifton N; Roberts, R Michael; Prather, Randall S; Kim, Jin-Hoi

    2014-05-20

    Pigs with severe combined immunodeficiency (SCID) may provide useful models for regenerative medicine, xenotransplantation, and tumor development and will aid in developing therapies for human SCID patients. Using a reporter-guided transcription activator-like effector nuclease (TALEN) system, we generated targeted modifications of recombination activating gene (RAG) 2 in somatic cells at high efficiency, including some that affected both alleles. Somatic-cell nuclear transfer performed with the mutated cells produced pigs with RAG2 mutations without integrated exogenous DNA. Biallelically modified pigs either lacked a thymus or had one that was underdeveloped. Their splenic white pulp lacked B and T cells. Under a conventional housing environment, the biallelic RAG2 mutants manifested a "failure to thrive" phenotype, with signs of inflammation and apoptosis in the spleen compared with age-matched wild-type animals by the time they were 4 wk of age. Pigs raised in a clean environment were healthier and, following injection of human induced pluripotent stem cells (iPSCs), quickly developed mature teratomas representing all three germ layers. The pigs also tolerated grafts of allogeneic porcine trophoblast stem cells. These SCID pigs should have a variety of uses in transplantation biology.

  19. Gevaar VoIP voor telecomsector overdreven

    NARCIS (Netherlands)

    Deventer, M.O. van; Wegberg, M. van

    2004-01-01

    Het is een hype Voice-over-IP (VoIP) voor te stellen als een ontwrichtende technologie die de telecomindustrie ingrijpend zal veranderen. Maar hoe ontwrichtend is VoIP eigenlijk? Oskar van Deventer en Marc van Wegberg analyseren drie vormen van VoIP en laten zien dat alleen ‘VoIP-chat’ potentieel

  20. Dyscoria Associated with Herpesvirus Infection in Owl Monkeys (Aotus nancymae)

    OpenAIRE

    Gozalo, Alfonso S; Montoya, Enrique J; Weller, Richard E

    2008-01-01

    Dyscoria was noted in a female owl monkey and 2 of her offspring. The third offspring was found dead with necrohemorrhagic encephalitis. Two male monkeys paired with the female died, 1 of which showed oral ulcers at necropsy. Histologic examination of the oral ulcers revealed syncytia and eosinophilic intranuclear inclusion bodies in epithelial cells. Ocular examination revealed posterior synechia associated with the dyscoria in all 3 animals. Serum samples from the female and her offspring w...

  1. Dynamic IP Reputation from DNS

    Science.gov (United States)

    2016-06-21

    management systems for Internet principals and services – E.g., IP addresses, DNS domains/servers, BGP/AS, etc. – Avoid connections to/from malicious...Operational; data sources for dynamic trust management – Dynamic IP reputation using DNS data 11/4/2009 1 Overview and Motivation • Dynamic Domain...it hosts a few number of bad domains • Ultimate goal: – An on-line dynamic trust/reputation service for IP/Domain 11/4/2009 19 Credits and

  2. 食蟹猴胚胎干细胞向间充质前体细胞诱导分化及鉴定%Induction and Identification of Mesenchymal Precursor Cells from Cynomolgus Monkey Embryonic Stem Cells

    Institute of Scientific and Technical Information of China (English)

    任振华; 王佳茵; 朱宛宛; 邹春林; 张愚

    2011-01-01

    间充质干细胞(mesenchymal stem cells,MSCs)可以诱导分化成脂肪、软骨、骨骼和骨骼肌细胞,并可作为骨骼、软骨或肌肉移植中的再生干细胞,广泛应用于细胞治疗和组织工程.胚胎干细胞(embryonic stem cells,ESCs)具有体外培养无限增殖和多向分化的特性,能被诱导分化为机体几乎所有的细胞类型.该研究通过无血清条件下诱导食蟹猴ESCs形成类胚体(embryoid bodies,EBs),然后在血清条件下贴壁分化EBs成间充质前体细胞(mesenchymal precursor cells,MPCs),再经过长期体外培养,纯化和扩增MPCSo结果显示,纯化后的MPCs具有MSCs生物学特征,并能在体外诱导分化成脂肪细胞和骨细胞.将这些细胞皮下注射给SCID小鼠,并未发现形成肿瘤,提示食蟹猴ESCs来源的MPCs具有一定的安全性.%Mesenchymal stem cells (MSCs) have received considerable attention in recent years for various therapeutic approaches. The reason is that MSCs have the ability to expand in vitro and differentiate into osteo-genic, adipogenic and chondrogenic lineages, which could then be used for cell therapy and tissue engineering. Embryonic stem cells (ESCs) are pluripotent cells with unlimited proliferation and differentiating into any cell type of the body. In this study, cynomolgus monkey ESCs were cultured in the absence of serum to form embryoid bodies (Ebs), and then Ebs were transferred for adherent culture in the presence of serum. Mesenchymal precursor cells (MPCs) migrated from Ebs were selectively isolated and expanded. The results showed that MPCs derived from ESCs had the biological characteristics of mesenchymal stem cells (MSCs), and could be differentiated into osteo-genic and adipogenic cells. NOD/SCID mice injected with MPCs did not show signs of tumor development. The results suggested that MPCs derived from cynomolgus monkey ESCs had certain the safety of its clinical application.

  3. Light-induced translocation of Pyronine G from mitochondria to nucleoli in monkey kidney CV-1 cells

    Science.gov (United States)

    Geze, Marc; Dellinger, M.; Bazin, M.; Santus, Rene C.

    1996-12-01

    Pyronine G (3,6-bis-N,N-dimethylaminoxanthylium chloride; PG) is a cationic dye that concentrates in mitochondria of living cells due to the high membrane potential of these organelles, similarly to rhodamine 123 and many other cationic dyes. Pyronine G also shows a preferential affinity for RNA. Upon light irradiation PG has been shown to induce cell death, but the photosensitizing properties of this molecule and the mechanism of cell death are not well understood. Microfluorometry and most particularly microspectrofluorometry are now powerful non-invasive techniques for quantitative studies of single living cells in real time which allow, for example, knowing how living cells are affected by photosensitization. To demonstrate the usefulness of image acquisition with high resolution and high sensitive camera, we present data on photosensitizer relocalization during illumination leading to functional and structural damage in the cells.

  4. Modeling of drug-mediated CYP3A4 induction by using human iPS cell-derived enterocyte-like cells

    Energy Technology Data Exchange (ETDEWEB)

    Negoro, Ryosuke [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Takayama, Kazuo [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); The Keihanshin Consortium for Fostering the Next Generation of Global Leaders in Research (K-CONNEX), Kyoto University, Kyoto 606-8302 (Japan); Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085 (Japan); Nagamoto, Yasuhito [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085 (Japan); Sakurai, Fuminori [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Laboratory of Regulatory Sciences for Oligonucleotide Therapeutics, Clinical Drug Development Project, Graduate School of Pharmaceutical Sciences, Osaka University Osaka 565-0871 (Japan); Tachibana, Masashi [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Mizuguchi, Hiroyuki, E-mail: mizuguch@phs.osaka-u.ac.jp [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085 (Japan); Global Center for Medical Engineering and Informatics, Osaka University, Osaka 565-0871 (Japan)

    2016-04-15

    Many drugs have potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in small intestinal enterocytes. Therefore, a model that can accurately evaluate drug-mediated CYP3A4 induction is urgently needed. In this study, we overlaid Matrigel on the human induced pluripotent stem cells-derived enterocyte-like cells (hiPS-ELCs) to generate the mature hiPS-ELCs that could be applied to drug-mediated CYP3A4 induction test. By overlaying Matrigel in the maturation process of enterocyte-like cells, the gene expression levels of intestinal markers (VILLIN, sucrase-isomaltase, intestine-specific homeobox, caudal type homeobox 2, and intestinal fatty acid-binding protein) were enhanced suggesting that the enterocyte-like cells were maturated by Matrigel overlay. The percentage of VILLIN-positive cells in the hiPS-ELCs found to be approximately 55.6%. To examine the CYP3A4 induction potential, the hiPS-ELCs were treated with various drugs. Treatment with dexamethasone, phenobarbital, rifampicin, or 1α,25-dihydroxyvitamin D3 resulted in 5.8-fold, 13.4-fold, 9.8-fold, or 95.0-fold induction of CYP3A4 expression relative to that in the untreated controls, respectively. These results suggest that our hiPS-ELCs would be a useful model for CYP3A4 induction test. - Highlights: • The hiPS-ELCs were matured by Matrigel overlay. • The hiPS-ELCs expressed intestinal nuclear receptors, such as PXR, GR and VDR. • The hiPS-ELC is a useful model for the drug-mediated CYP3A4 induction test.

  5. Cellular Functions and Gene and Protein Expression Profiles in Endothelial Cells Derived from Moyamoya Disease-Specific iPS Cells

    Science.gov (United States)

    Hamauchi, Shuji; Shichinohe, Hideo; Uchino, Haruto; Yamaguchi, Shigeru; Nakayama, Naoki; Kazumata, Ken; Osanai, Toshiya; Abumiya, Takeo; Houkin, Kiyohiro; Era, Takumi

    2016-01-01

    Background and purpose Moyamoya disease (MMD) is a slow, progressive steno-occlusive disease, arising in the terminal portions of the cerebral internal carotid artery. However, the functions and characteristics of the endothelial cells (ECs) in MMD are unknown. We analyzed these features using induced pluripotent stem cell (iPSC)-derived ECs. Methods iPSC lines were established from the peripheral blood of three patients with MMD carrying the variant RNF213 R4810K, and three healthy persons used as controls. After the endothelial differentiation of iPSCs, CD31+CD144+ cells were purified as ECs using a cell sorter. We analyzed their proliferation, angiogenesis, and responses to some angiogenic factors, namely VEGF, bFGF, TGF-β, and BMP4. The ECs were also analyzed using DNA microarray and proteomics to perform comprehensive gene and protein expression analysis. Results Angiogenesis was significantly impaired in MMD regardless of the presence of any angiogenic factor. On the contrary, endothelial proliferation was not significant between control- and MMD-derived cells. Regarding DNA microarray, pathway analysis illustrated that extracellular matrix (ECM) receptor-related genes, including integrin β3, were significantly downregulated in MMD. Proteomic analysis revealed that cytoskeleton-related proteins were downregulated and splicing regulation-related proteins were upregulated in MMD. Conclusions Downregulation of ECM receptor-related genes may be associated with impaired angiogenic activity in ECs derived from iPSCs from patients with MMD. Upregulation of splicing regulation-related proteins implied differences in splicing patterns between control and MMD ECs. PMID:27662211

  6. 恒河猴和树鼩角膜内皮细胞的比较分析%Comparison of the corneal endothelial cells of rhesus monkeys and tree shrews

    Institute of Scientific and Technical Information of China (English)

    吴敏; 李娜; 孙晓梅; 胡竹林

    2016-01-01

    的合适实验动物。%Objective To analyze and compare the characteristics and differences of corneal endothelial cells of rhesus monkey and tree shrew eyes.Methods Corneal endothelial cells of 6 healthy rhesus monkeys (12 eyes) and 20 healthy tree shrews (40 eyes) were measured using a non-contact SP3000P specular microscope.Eight parameters were de-termined and compared with relevant parameters of human eyes reported in the literature, including minimum cell area (Smin), maximum cell area (Smax), average cell area (Savg), standard deviation of cell area (SD), coefficient of variabili-ty ( CV) , cell density ( CD) , hexagonality percentage ( HG%) and central corneal thickness ( CCT) .Results The ima-ging and measurement of all parameters could be completed in a short time both in rhesus monkeys and tree shrews.The time spent in the two kinds of animals was not significantly different.The CCT was ( 449.2 ±12.8 ) μm and ( 262.4 ± 24.6) μm, Smin was (120.4 ±26.3) S/μm2 and (153.2 ±42.9) S/μm2 , Smax was (705.0 ±130.8) S/μm2 and (468.7 ±109.3) S/μm2 , Savg was (351.1 ±26.1) and (295.4 ±18.9) S/μm2 , SSD was (113.1 ±27.4) and (75.9 ±27.3) S/μm2, CV was (31.9 ±6.0) and (25.3 ±8.3), CD was (2874.2 ±203.8) p/cell· mm-2 and (3399.3 ±224.7) p/cell· mm-2 , and the HG% was (58.6 ±9.1) and (94.0 ±9.7) in the rhesus monkeys andt tree shrews, respectively. The differences of all the above parameters between rhesus monkeys and tree shrews were statistically significant ( P<0.05 for all) .The cornea of tree shrews was significantly thinner than that of rhesus monkeys.The area and coefficient of varia-bility of tree shrews were smaller to those of rhesus monkeys, while the cell density and hexagonality percentage were higher than those of rhesus monkeys.Compared with human eyes, the CCT, CV and HG%in rhesus monkeys were highly simi-lar, while the CD was lower than that of human eyes.The CCT in tree shrew was only 60%of the rhesus monkey eyes and 50%of human eyes, while the CD and Savg

  7. Qos and Voice Over IP

    Directory of Open Access Journals (Sweden)

    Adrian GHENCEA

    Full Text Available As Voice over Internet Protocol (VoIP technology matures, companies are increasingly adopting it to cut costs, improve efficiency and enhance customer service. Using the Internet as an existing network for integrating data and telecom systems through intelligent VoIP, a range of benefits results: lower long distance costs, cost cuts in cabling processes and more flexible telephony management. However, as voice over IP services grow in popularity, major threats arise: this rapid growth leads to traffic congestion, security is jeopardizedand the poor quality of calls affects communication. The objective of this article is to present all the elements that can affect voicequality in a VoIP network and to provide methods for solving them. A detailed analysis to minimize the impact of implementation of QoS will be made, and at the end solutions to management strategies will be proposed.

  8. Hardware IP security and trust

    CERN Document Server

    Bhunia, Swarup; Tehranipoor, Mark

    2017-01-01

    This book provides an overview of current Intellectual Property (IP) based System-on-Chip (SoC) design methodology and highlights how security of IP can be compromised at various stages in the overall SoC design-fabrication-deployment cycle. Readers will gain a comprehensive understanding of the security vulnerabilities of different types of IPs. This book would enable readers to overcome these vulnerabilities through an efficient combination of proactive countermeasures and design-for-security solutions, as well as a wide variety of IP security and trust assessment and validation techniques. This book serves as a single-source of reference for system designers and practitioners for designing secure, reliable and trustworthy SoCs.

  9. The Elephant and the Monkey

    Institute of Scientific and Technical Information of China (English)

    朱妤

    2009-01-01

    @@ Once an Elephant met a Monkey."Look how big and strong I am!"he said."I can break a tree.Can you break a tree?" "Look how quickly I can run and climb!"said the Monkey."Can you climb a tree?" The elephant was proud because he was so strong,and the Monkey Was proud because she was so quick.

  10. Selective Blockade of CD28-Mediated T Cell Costimulation Protects Rhesus Monkeys against Acute Fatal Experimental Autoimmune Encephalomyelitis

    NARCIS (Netherlands)

    Haanstra, Krista G.; Dijkman, Karin; Bashir, Noun; Bauer, Jan; Mary, Caroline; Poirier, Nicolas; Baker, Paul; Scobie, Linda; 't Hart, Bert A.; Vanhove, Bernard

    2015-01-01

    Costimulatory and coinhibitory receptor-ligand pairs on T cells and APC control the immune response. We have investigated whether selective blockade of CD28-CD80/86 costimulatory interactions, which preserves the coinhibitory CTLA4-CD80/86 interactions and the function of regulatory T (Treg) cells,

  11. VoIP Forensic Analyzer

    Directory of Open Access Journals (Sweden)

    M Mohemmed Sha

    2016-01-01

    Full Text Available People have been utilizing Voice over Internet Protocol (VoIP in most of the conventional communication facilities which has been of assistance in the enormous attenuation of operating costs, as well as the promotion of next- generation communication services-based IP. As an intimidating upshot, cyber criminals have correspondingly started interjecting the environment and creating new challenges for the law enforcement system in any Country. This paper presents an idea of a framework for the forensic analysis of the VoIP traffic over the network. This forensic activity includes spotting and scrutinizing the network patterns of VoIP-SIP stream, which is used to initiate a session for the communication, and regenerate the content from VoIP-RTP stream, which is employed to convey the data. Proposed network forensic investigation framework also accentuates on developing an efficient packet restructuring algorithm for tracing the depraved users involved in a conversation. Network forensics is the basis of proposed work, and performs packet level surveillance of VoIP followed by reconstruction of original malicious content or network session between users for their prosecution in the court.

  12. Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function.

    Science.gov (United States)

    Scherer, Paul C; Ding, Yan; Liu, Zhiqing; Xu, Jing; Mao, Haibin; Barrow, James C; Wei, Ning; Zheng, Ning; Snyder, Solomon H; Rao, Feng

    2016-03-29

    The family of cullin-RING E3 Ligases (CRLs) and the constitutive photomorphogenesis 9 (COP9) signalosome (CSN) form dynamic complexes that mediate ubiquitylation of 20% of the proteome, yet regulation of their assembly/disassembly remains poorly understood. Inositol polyphosphates are highly conserved signaling molecules implicated in diverse cellular processes. We now report that inositol hexakisphosphate (IP6) is a major physiologic determinant of the CRL-CSN interface, which includes a hitherto unidentified electrostatic interaction between the N-terminal acidic tail of CSN subunit 2 (CSN2) and a conserved basic canyon on cullins. IP6, with an EC50 of 20 nM, acts as an intermolecular "glue," increasing cullin-CSN2 binding affinity by 30-fold, thereby promoting assembly of the inactive CRL-CSN complexes. The IP6 synthase, Ins(1,3,4,5,6)P5 2-kinase (IPPK/IP5K) binds to cullins. Depleting IP5K increases the percentage of neddylated, active Cul1 and Cul4A, and decreases levels of the Cul1/4A substrates p27 and p21. Besides dysregulating CRL-mediated cell proliferation and UV-induced apoptosis, IP5K depletion potentiates by 28-fold the cytotoxic effect of the neddylation inhibitor MLN4924. Thus, IP5K and IP6 are evolutionarily conserved components of the CRL-CSN system and are potential targets for cancer therapy in conjunction with MLN4924.

  13. The relationship between the production and the anti-gonadotrophic action of prostaglandin F 2 alpha in luteal cells from the marmoset monkey (Callithrix jacchus) in the early and mid-luteal phase.

    Science.gov (United States)

    Webley, G E; Michael, A E; Abayasekara, D R E

    2010-04-01

    To address the potential luteolytic role for prostaglandin F(2 alpha) (PGF(2 alpha)) in the corpus luteum of the common marmoset monkey (Callithrix jacchus), the ability of marmoset luteal cells, maintained in monolayer culture, to produce PGF(2 alpha) was determined in vitro in the presence and absence of human chorionic gonadotrophin (hCG) and other established pharmacological modulators of PGF(2 alpha) synthesis. We also assessed the effects of the PGF(2 alpha) analogue, cloprostenol, on progesterone output from luteal cells isolated in the early luteal phase versus the mid-luteal phase (days 3 and 14 post ovulation, respectively). Cloprostenol had no effect on progesterone output from luteal cells isolated on day 3 of the luteal phase, whereas it significantly inhibited both basal and hCG-stimulated progesterone synthesis by day 14 luteal cells during the culture period 48-72 h (Pmarmoset monkey appears to involve changes in both the luteal cell response to and production of PGF(2 alpha).

  14. 建立骨质疏松症 iPS 细胞的重要性及其应用前景%The importance of the establishment of induced pluripotent stem cells and its therapeutic potential in osteoporosis

    Institute of Scientific and Technical Information of China (English)

    石梦琪; 刘文佳; 张立强

    2015-01-01

    Osteoporosis ( OP) is a complex disease characterized by descending of bone strength and increasing of bone fracture risk, which is associated with genetic and environmental factors.Currently the incidence of osteoporosis has risen year by year, which severely influences the health of the elderly.However, the pathogenic mechanism of OP is not clear, and there still remain many problems in drug therapies, such as long treatment cycle, high cost, and many adverse drug effects.Recently the emergence of induced pluripotent stem ( iPS) cells creates a novel platform for OP.It is reported that iPS cells have been successfully used in Parkinson’ s disease, Alzheimer’ s disease, Schizophrenia, spinal cord injury and so on.Modeling the osteoporosis with iPS cells will help to make some vital breakthroughs in the research of pathogenic mechanism and drug screening.This review describes the importance of modeling the osteoporosis with iPS cells and predicts its potentials in the basic experimental research and clinical treatment.%骨质疏松症( osteoporosis, OP)是一种以骨强度下降、骨折危险性增加为特征,受遗传和环境因素共同作用的复杂疾病。目前OP的发病率逐年升高,严重影响了老年人的健康。然而由于发病机制尚不明确,药物治疗还存在周期长、费用高、不良反应多等问题。近年来诱导多功能干细胞( induced pluripotent stem cells, iPS细胞)技术的诞生为OP的防治开创了新平台。研究报道iPS细胞模型已成功用于帕金森病、阿尔茨海默症、精神分裂症、脊髓损伤等众多疾病,建立OP的iPS细胞模型将有助于在其致病机制研究和药物筛选中取得新突破。这篇综述主要介绍建立OP的iPS细胞模型的重要性,并预测它在OP的基础研究与临床治疗上的应用前景。

  15. A native IP satellite communications system

    Science.gov (United States)

    Koudelka, O.; Schmidt, M.; Ebert, J.; Schlemmer, H.; Kastner-Puschl, S.; Riedler, W.

    2004-08-01

    ≪ In the framework of ESA's ARTES-5 program the Institute of Applied Systems Technology (Joanneum Research) in cooperation with the Department of Communications and Wave Propagation has developed a novel meshed satellite communications system which is optimised for Internet traffic and applications (L*IP—Local Network Interconnection via Satellite Systems Using the IP Protocol Suite). Both symmetrical and asymmetrical connections are supported. Bandwidth on demand and guaranteed quality of service are key features of the system. A novel multi-frequency TDMA access scheme utilises efficient methods of IP encapsulation. In contrast to other solutions it avoids legacy transport network techniques. While the DVB-RCS standard is based on ATM or MPEG transport cells, the solution of the L*IP system uses variable-length cells which reduces the overhead significantly. A flexible and programmable platform based on Linux machines was chosen to allow the easy implementation and adaptation to different standards. This offers the possibility to apply the system not only to satellite communications, but provides seamless integration with terrestrial fixed broadcast wireless access systems. The platform is also an ideal test-bed for a variety of interactive broadband communications systems. The paper describes the system architecture and the key features of the system.

  16. Study of CCNB1IP1 and c-Cbl gene mutation in peripheral blood of patients with non-small cell lung cancer%非小细胞肺癌患者外周血CCNB1IP1及c-Cbl基因突变研究

    Institute of Scientific and Technical Information of China (English)

    冯士生; 梁建琴; 王金河; 史晓朋

    2013-01-01

    目的 检测NSCLC患者外周血c-Cbl和CCNB1IP1基因突变情况.方法收集20例健康人、20例肺结核和40例NSCLC患者外周血标本,采用RT-PCR和基因测序方法,检测c-Cbl和CCNB1IP1基因突变.结果 所有标本中均未检测出CCNB1IP1基因突变;而仅在NSCLC患者外周血检测到4种c-Cbl基因突变,突变率为20%;突变率与患者的性别、年龄及病理学分型均无相关性(P>0.05);而不同临床分期间突变率不同,差异有统计学意义(P<0.05).结论 NSCLC患者外周血中检测到c-Cbl基因突变,且不同临床分期间突变率不同.%Objective To observe the changes of GGNB11P1 and c-Gbl gene mutations in peripheral blood of non-small cell lung cancer ( NSCLC ) patients. Methods The peripheral blood was collected from 40 patients with NSCLC, 20 TB patients, and 20 healthy people. The gene mutations of GGNB11P1 and c-Gbl were tested by RT-PGR and gene sequencing method. Results The gene mutations of GGNB11P1 were not detected in peripheral blood of all selected cases. 4 kinds of gene mutations of c-Gbl were detected in peripheral blood of NSGLG patients, with a mutation rate of 20% . The mutation rate of c-Gbl had no obvious relationship with gender, age and pathology types ( P >0. 05 ), but it was closely related to different clinical stages with statistical significance ( P < 0. 05 ). Conclusion The gene mutations of c-Gbl are detected in peripheral blood of NSGLG patients, and its mutation rate is closely related to different clinical stages with statistical significance.

  17. Modification of a loop sequence between α-helices 6 and 7 of virus capsid (CA protein in a human immunodeficiency virus type 1 (HIV-1 derivative that has simian immunodeficiency virus (SIVmac239 vif and CA α-helices 4 and 5 loop improves replication in cynomolgus monkey cells

    Directory of Open Access Journals (Sweden)

    Adachi Akio

    2009-08-01

    Full Text Available Abstract Background Human immunodeficiency virus type 1 (HIV-1 productively infects only humans and chimpanzees but not cynomolgus or rhesus monkeys while simian immunodeficiency virus isolated from macaque (SIVmac readily establishes infection in those monkeys. Several HIV-1 and SIVmac chimeric viruses have been constructed in order to develop an animal model for HIV-1 infection. Construction of an HIV-1 derivative which contains sequences of a SIVmac239 loop between α-helices 4 and 5 (L4/5 of capsid protein (CA and the entire SIVmac239 vif gene was previously reported. Although this chimeric virus could grow in cynomolgus monkey cells, it did so much more slowly than did SIVmac. It was also reported that intrinsic TRIM5α restricts the post-entry step of HIV-1 replication in rhesus and cynomolgus monkey cells, and we previously demonstrated that a single amino acid in a loop between α-helices 6 and 7 (L6/7 of HIV type 2 (HIV-2 CA determines the susceptibility of HIV-2 to cynomolgus monkey TRIM5α. Results In the study presented here, we replaced L6/7 of HIV-1 CA in addition to L4/5 and vif with the corresponding segments of SIVmac. The resultant HIV-1 derivatives showed enhanced replication capability in established T cell lines as well as in CD8+ cell-depleted primary peripheral blood mononuclear cells from cynomolgus monkey. Compared with the wild type HIV-1 particles, the viral particles produced from a chimeric HIV-1 genome with those two SIVmac loops were less able to saturate the intrinsic restriction in rhesus monkey cells. Conclusion We have succeeded in making the replication of simian-tropic HIV-1 in cynomolgus monkey cells more efficient by introducing into HIV-1 the L6/7 CA loop from SIVmac. It would be of interest to determine whether HIV-1 derivatives with SIVmac CA L4/5 and L6/7 can establish infection of cynomolgus monkeys in vivo.

  18. Induction of corneal epithelial progenitors from bone-marrow mesenchymal stem cells of rhesus monkeys in vitro

    Institute of Scientific and Technical Information of China (English)

    YUAN Jing; YU JianXiong; HUANG Bing; LIU BingQian; LIU JingBo; JIANG RuZhang; GE Jian

    2007-01-01

    Bioengineered corneas are substitutes for human donor tissue that are designed to treat severe disease affecting ocular surfaces.However, a shortage of candidate seed cells for bioengineering corneas is still a problem.Bone-marrow mesenchymal stem cells (MSCs) are capable of multilineage differentiation.Therefore, we determined whether MSCs differentiate into corneal epithelial cells (ECs).We applied three exoteric-microenvironmental systems to induce MSCs to become ECs.Induced MSC were identified by means of morphologic examination, immunocytochemical analysis, and flow cytometry.MSCs grown in one microenvironment had characteristics similar to those of corneal epithelial progenitors.Induced MSCs expressed markers for EC, including integrin β1, Cx43, Pax6, and P63.MSCs were successfully induced to become corneal epithelial progenitors.Therefore, the use of MSCs may hold substantial promise for reconstructing the ocular surface after corneal injury.

  19. Protection by beverages, fruits, vegetables, herbs, and flavonoids against genotoxicity of 2-acetylaminofluorene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in metabolically competent V79 cells.

    Science.gov (United States)

    Edenharder, R; Sager, J W; Glatt, H; Muckel, E; Platt, K L

    2002-11-26

    Chinese hamster lung fibroblasts, genetically engineered for the expression of rat cytochrome P450 dependent monooxygenase 1A2 and rat sulfotransferase 1C1 (V79-rCYP1A2-rSULT1C1 cells), were utilized to check for possible protective effects of beverages of plant origin, fruits, vegetables, and spices against genotoxicity induced by 2-acetylaminofluorene (AAF) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Antigenotoxic activities of juices from spinach and red beets against AAF could be monitored with similar effectivity by the HPRT-mutagenicity test (IC(50)=0.64%; 2.57%) and alkaline single cell gel electrophoresis (comet assay; IC(50)=0.12%; 0.89%) which detects DNA strand breaks and abasic sites. Applying the comet assay, genotoxicity of PhIP could, however, be demonstrated only in the presence of hydroxyurea and 1-[beta-D-arabinofuranosyl]cytosine, known inhibitors of DNA repair synthesis. As expected, AAF and PhIP were unable to induce any genotoxic effects in the parent V79 cells. Genotoxic activity of PhIP was strongly reduced in a dose-related manner by green tea and red wine, by blueberries, blackberries, red grapes, kiwi, watermelon, parsley, and spinach, while two brands of beer, coffee, black tea, rooibos tea, morellos, black-currants, plums, red beets, broccoli (raw and cooked), and chives were somewhat less active. One brand of beer was only moderately active while white wine, bananas, white grapes, and strawberries were inactive. Similarly, genotoxicity of AAF was strongly reduced by green, black, and rooibos tea, red wine, morellos, black-currants, kiwi, watermelon, and spinach while plums, red beets, and broccoli (raw) were less potent. Broccoli cooked exerted only moderate and white wine weak antigenotoxic activity. With respect to the possible mechanism(s) of inhibition of genotoxicity, benzo[a]pyrene-7,8-dihydrodiol (BaP-7,8-OH) and N-OH-PhIP were applied as substrates for the CYP1A family and for rSULT 1C1, respectively. Morellos

  20. Attention Induced Gain Stabilization in Broad and Narrow-Spiking Cells in the Frontal Eye-Field of Macaque Monkeys.

    Science.gov (United States)

    Thiele, Alexander; Brandt, Christian; Dasilva, Miguel; Gotthardt, Sascha; Chicharro, Daniel; Panzeri, Stefano; Distler, Claudia

    2016-07-20

    Top-down attention increases coding abilities by altering firing rates and rate variability. In the frontal eye field (FEF), a key area enabling top-down attention, attention induced firing rate changes are profound, but its effect on different cell types is unknown. Moreover, FEF is the only cortical area investigated in which attention does not affect rate variability, as assessed by the Fano factor, suggesting that task engagement affects cortical state nonuniformly. We show that putative interneurons in FEF of Macaca mulatta show stronger attentional rate modulation than putative pyramidal cells. Partitioning rate variability reveals that both cell types reduce rate variability with attention, but more strongly so in narrow-spiking cells. The effects are captured by a model in which attention stabilizes neuronal excitability, thereby reducing the expansive nonlinearity that links firing rate and variance. These results show that the effect of attention on different cell classes and different coding properties are consistent across the cortical hierarchy, acting through increased and stabilized neuronal excitability. Cortical processing is critically modulated by attention. A key feature of this influence is a modulation of "cortical state," resulting in increased neuronal excitability and resilience of the network against perturbations, lower rate variability, and an increased signal-to-noise ratio. In the frontal eye field (FEF), an area assumed to control spatial attention in human and nonhuman primates, firing rate changes with attention occur, but rate variability, quantified by the Fano factor, appears to be unaffected by attention. Using recently developed analysis tools and models to quantify attention effects on narrow- and broad-spiking cell activity, we show that attention alters cortical state strongly in the FEF, demonstrating that its effect on the neuronal network is consistent across the cortical hierarchy. Copyright © 2016 Thiele et al.

  1. Induction of Encephalitis in Rhesus Monkeys Infused with Lymphocryptovirus-Infected B-Cells Presenting MOG(34-56) Peptide

    NARCIS (Netherlands)

    Haanstra, Krista G.; Wubben, Jacqueline A. M.; Jonker, Margreet; 't Hart, Bert A.

    2013-01-01

    The overlapping epidemiology of multiple sclerosis (MS) and Epstein-Barr virus (EBV), the increased risk to develop MS after infectious mononucleosis (IM) and the localization of EBV-infected B-cells within the MS brain suggest a causal link between EBV and MS. However, the underlying mechanism is u

  2. Experimental depletion of CD8+ cells in acutely SIVagm-Infected African Green Monkeys results in increased viral replication

    Directory of Open Access Journals (Sweden)

    Apetrei Cristian

    2010-05-01

    Full Text Available Abstract Background In vivo CD8+ cell depletions in pathogenic SIV infections identified a key role for cellular immunity in controlling viral load (VL and disease progression. However, similar studies gave discordant results in chronically-infected SMs, leading some authors to propose that in natural hosts, SIV replication is independent of cellular immunity. To assess the role of cellular immune responses in the control of SIV replication in natural hosts, we investigated the impact of CD8+ cell depletion during acute SIV infection in AGMs. Results Nine AGMs were infected with SIVagm.sab and were followed up to day 225 p.i. Four were intravenously infused with the cM-T807 antibody on days 0 (50 mg/kg, 6, and 13 (10 mg/kg, respectively post infection (p.i.. CD8+ cells were depleted for up to 28 days p.i. in peripheral blood and LNs in all treated AGMs. Partial CD8+ T cell depletion occurred in the intestine. SIVagm VLs peaked at similar levels in both groups (107-108 RNA copies/ml. However, while VLs were controlled in undepleted AGMs, reaching set-point levels (104-105 RNA copies/ml by day 28 p.i., high VLs (>106 RNA copies/ml were maintained by day 21 p.i. in CD8-depleted AGMs. By day 42 p.i., VLs were comparable between the two groups. The levels of immune activation and proliferation remained elevated up to day 72 p.i. in CD8-depleted AGMs and returned to preinfection levels in controls by day 28 p.i. None of the CD8-depleted animals progressed to AIDS. Conclusion CD8+ cells are responsible for a partial control of postacute viral replication in SIVagm.sab-infected AGMs. In contrast to macaques, the SIVagm-infected AGMs are able to control viral replication after recovery of the CD8+ T cells and avoid disease progression.

  3. Non-Linear Relationships between Aflatoxin B1 Levels and the Biological Response of Monkey Kidney Vero Cells

    Directory of Open Access Journals (Sweden)

    Mendel Friedman

    2013-08-01

    Full Text Available Aflatoxin-producing fungi contaminate food and feed during pre-harvest, storage and processing periods. Once consumed, aflatoxins (AFs accumulate in tissues, causing illnesses in animals and humans. Most human exposure to AF seems to be a result of consumption of contaminated plant