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Sample records for human feeder cells

  1. Human induced pluripotent stem cells on autologous feeders.

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    Kazutoshi Takahashi

    Full Text Available BACKGROUND: For therapeutic usage of induced Pluripotent Stem (iPS cells, to accomplish xeno-free culture is critical. Previous reports have shown that human embryonic stem (ES cells can be maintained in feeder-free condition. However, absence of feeder cells can be a hostile environment for pluripotent cells and often results in karyotype abnormalities. Instead of animal feeders, human fibroblasts can be used as feeder cells of human ES cells. However, one still has to be concerned about the existence of unidentified pathogens, such as viruses and prions in these non-autologous feeders. METHODOLOGY/PRINCIPAL FINDINGS: This report demonstrates that human induced Pluripotent Stem (iPS cells can be established and maintained on isogenic parental feeder cells. We tested four independent human skin fibroblasts for the potential to maintain self-renewal of iPS cells. All the fibroblasts tested, as well as their conditioned medium, were capable of maintaining the undifferentiated state and normal karyotypes of iPS cells. Furthermore, human iPS cells can be generated on isogenic parental fibroblasts as feeders. These iPS cells carried on proliferation over 19 passages with undifferentiated morphologies. They expressed undifferentiated pluripotent cell markers, and could differentiate into all three germ layers via embryoid body and teratoma formation. CONCLUSIONS/SIGNIFICANCE: These results suggest that autologous fibroblasts can be not only a source for iPS cells but also be feeder layers. Our results provide a possibility to solve the dilemma by using isogenic fibroblasts as feeder layers of iPS cells. This is an important step toward the establishment of clinical grade iPS cells.

  2. Effects of Feeder Cells on Dopaminergic Differentiation of Human Embryonic Stem Cells

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    Zhenqiang Zhao

    2016-12-01

    Full Text Available Mouse embryonic fibroblasts (MEFs and human foreskin fibroblasts (HFFs are used for the culture of human embryonic stem cells (hESCs. MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation, two hESCs lines were cultured on mixed feeder cells (MFCs, MEFs: HFFs =1:1 and HFFs feeder respectively, and then were differentiated into DA neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry, quantitative fluorescent real-time PCR (qRT-PCR, transmission and scanning electron microscopy, and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However, compared to hESCs line on MFCs feeder, hESCs line on HFFs feeder had a higher proportion of TH positive cells and expressed higher levels of FOXA2, PITX3, NURR1 and TH genes. In addition, the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion, HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons, but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore, feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines, but also electrophysiological properties of hESCs-derived DA neurons.

  3. Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells

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    Anisimov, Sergey V.; Christophersen, Nicolaj S.; Correia, Ana S.

    2011-01-01

    The majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both...... the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early...... foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates....

  4. Utilization of human amniotic mesenchymal cells as feeder layers to sustain propagation of human embryonic stem cells in the undifferentiated state.

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    Zhang, Kehua; Cai, Zhe; Li, Yang; Shu, Jun; Pan, Lin; Wan, Fang; Li, Hong; Huang, Xiaojie; He, Chun; Liu, Yanqiu; Cui, Xiaohui; Xu, Yang; Gao, Yan; Wu, Liqun; Cao, Shanxia; Li, Lingsong

    2011-08-01

    Human embryonic stem (ES) cells are usually maintained in the undifferentiated state by culturing on feeder cells layers of mouse embryonic fibroblasts (MEFs). However, MEFs are not suitable to support human ES cells used for clinical purpose because of risk of zoonosis from animal cells. Therefore, human tissue-based feeder layers need to be developed for human ES cells for clinical purpose. Hereof we report that human amniotic mesenchymal cells (hAMCs) could act as feeder cells for human ES cells, because they are easily obtained and relatively exempt from ethical problem. Like MEFs, hAMCs could act as feeder cells for human ES cells to grow well on. The self-renewal rate of human ES cells cultured on hAMCs feeders was higher than that on MEFs and human amniotic epithelial cells determined by measurement of colonial diameters and growth curve as well as cell cycle analysis. Both immunofluorescence staining and immunoblotting showed that human ES cells cultured on hAMCs expressed stem cell markers such as Oct-3/4, Sox2, and NANOG. Verified by embryoid body formation in vitro and teratoma formation in vivo, we found out that after 20 passages of culture, human ES cells grown on hAMCs feeders could still retain the potency of differentiating into three germ layers. Taken together, our data suggested hAMCs may be safe feeder cells to sustain the propagation of human ES cells in undifferentiated state for future therapeutic use.

  5. Tumorigenic risk of human induced pluripotent stem cell explants cultured on mouse SNL76/7 feeder cells

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    Kamada, Mizuna; Mitsui, Youji, E-mail: y-mitsui8310@hb.tp1.jp; Kumazaki, Tsutomu; Kawahara, Yuta; Matsuo, Taira; Takahashi, Tomoko, E-mail: t-takahashi@kph.bunri-u.ac.jp

    2014-10-24

    Highlights: • hiPS cell explants formed malignant tumors when SNL76/7 feeder cells were used. • Multi type tumors developed by interaction of SNL76/7 feeder cells with hiPS cells. • Tumorigenic risk occurs by co-culture of hiPS cells with SNL76/7 feeder cells. - Abstract: The potential for tumor formation from transplanted human induced pluripotent stem cell (hiPSC) derivatives represents a high risk in their application to regenerative medicine. We examined the genetic origin and characteristics of tumors, that were formed when 13 hiPSC lines, established by ourselves, and 201B7 hiPSC from Kyoto University were transplanted into severe combined immune-deficient (SCID) mice. Though teratomas formed in 58% of mice, five angiosarcomas, one malignant solitary fibrous tumor and one undifferentiated pleomorphic sarcoma formed in the remaining mice. Three malignant cell lines were established from the tumors, which were derived from mitomycin C (MMC)-treated SNL76/7 (MMC-SNL) feeder cells, as tumor development from fusion cells between MMC-SNL and hiPSCs was negative by genetic analysis. While parent SNL76/7 cells produced malignant tumors, neither MMC-SNL nor MMC-treated mouse embryo fibroblast (MEF) produced malignant tumors. When MMC-SNL feeder cells were co-cultured with hiPSCs, growing cell lines were generated, that expressed genes similar to the parent SNL76/7 cells. Thus, hiPSCs grown on MMC-SNL feeder cells have a high risk of generating feeder-derived malignant tumors. The possible mechanism(s) of growth restoration and the formation of multiple tumor types are discussed with respect of the interactions between MMC-SNL and hiPSC.

  6. Establishment of feeder-free culture system for human induced pluripotent stem cell on DAS nanocrystalline graphene

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    Lee, Hyunah; Nam, Donggyu; Choi, Jae-Kyung; Araúzo-Bravo, Marcos J.; Kwon, Soon-Yong; Zaehres, Holm; Lee, Taehee; Park, Chan Young; Kang, Hyun-Wook; Schöler, Hans R.; Kim, Jeong Beom

    2016-02-01

    The maintenance of undifferentiated human pluripotent stem cells (hPSC) under xeno-free condition requires the use of human feeder cells or extracellular matrix (ECM) coating. However, human-derived sources may cause human pathogen contamination by viral or non-viral agents to the patients. Here we demonstrate feeder-free and xeno-free culture system for hPSC expansion using diffusion assisted synthesis-grown nanocrystalline graphene (DAS-NG), a synthetic non-biological nanomaterial which completely rule out the concern of human pathogen contamination. DAS-NG exhibited advanced biocompatibilities including surface nanoroughness, oxygen containing functional groups and hydrophilicity. hPSC cultured on DAS-NG could maintain pluripotency in vitro and in vivo, and especially cell adhesion-related gene expression profile was comparable to those of cultured on feeders, while hPSC cultured without DAS-NG differentiated spontaneously with high expression of somatic cell-enriched adhesion genes. This feeder-free and xeno-free culture method using DAS-NG will facilitate the generation of clinical-grade hPSC.

  7. A feeder-free, human plasma-derived hydrogel for maintenance of a human embryonic stem cell phenotype in vitro

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    Lewis Fiona C

    2012-08-01

    Full Text Available Abstract Background Human embryonic stem cells (hESCs represent a tremendous resource for cell therapies and the study of human development; however to maintain their undifferentiated state in vitro they routinely require the use of mouse embryonic fibroblast (MEF feeder-layers and exogenous protein media supplementation. Results These well established requirements can be overcome and in this study, it will be demonstrated that phenotypic stability of hESCs can be maintained using a novel, human plasma protein-based hydrogel as an extracellular culture matrix without the use of feeder cell co-culture. hESCs were resuspended in human platelet poor plasma (PPP, which was gelled by the addition of calcium containing DMEM-based hESC culture medium. Phenotypic and genomic expression of the pluripotency markers OCT4, NANOG and SOX2 were measured using immunohistochemistry and qRT-PCR respectively. Typical hESC morphology was demonstrated throughout in vitro culture and both viability and phenotypic stability were maintained throughout extended culture, up to 25 passages. Conclusions PPP-derived hydrogel has demonstrated to be an efficacious alternative to MEF co-culture with its hydrophilicity allowing for this substrate to be delivered via minimally invasive procedures in a liquid phase with polymerization ensuing in situ. Together this provides a novel technique for the study of this unique group of stem cells in either 2D or 3D both in vitro and in vivo.

  8. Irradiated murine fibroblasts as feeder layer used in human cell culture

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    Almeida, Tiago L.; Klingbeil, Fatima G.; Yoshito, Daniele; Caproni, Priscila; Mathor, Monica B.; Herson, Marisa R.

    2007-01-01

    In 1975, Rheinwald and Green published an in vitro model for keratinocyte cell cultures in which the use of murine fibroblasts, as a feeder layer was introduced. These cells are modified fibroblasts, which presence render keratinocyte cells to remain proliferative for longer periods of time. This optimization of culture outputs has allowed for several clinical applications of confluent keratinocyte cultures as skin substitutes or wound dressings in situations such as post burn extensive skin loss, loss of oral mucosa, and other skin disorders. Nevertheless, proliferation of fibroblast in co-culture with keratinocytes must be controlled by anti-proliferative measures such as irradiation; at the same time, keratinocytes require specific nutrients in the culture medium, which may interfere with the fibroblast feeder layer viability. Therefore, the thorough understanding of the impact of different issues such as culture media composition, irradiation dose and pre-plating storage conditions of irradiated fibroblast to be used as feeder layer in these co-culture systems is important. In this work, changes as far as viability and proliferative rates of irradiated fibroblasts in culture were evaluated in relation to the type of culture medium used, dose of gamma radiation exposure, storage and timing of cell plating post irradiation. Results indicate that the type of culture medium used and time-lag between irradiation, refrigeration and plating of irradiated cells do not have significant impact in culture outcomes. However, the dose of gamma radiation administered to the cells may influence the final quality of these cells if to be used as a feeder layer. (author)

  9. A feeder-free culture using autogeneic conditioned medium for undifferentiated growth of human embryonic stem cells: Comparative expression profiles of mRNAs, microRNAs and proteins among different feeders and conditioned media

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    Chou Chi-Hsien

    2010-10-01

    Full Text Available Abstract Background Human embryonic stem (hES cell lines were derived from the inner cell mass of human blastocysts, and were cultured on mouse embryonic fibroblast (MEF feeder to maintain undifferentiated growth, extensive renewal capacity, and pluripotency. The hES-T3 cell line with normal female karyotype was previously used to differentiate into autogeneic fibroblast-like cells (T3HDF as feeder to support the undifferentiated growth of hES-T3 cells (T3/HDF for 14 passages. Results A feeder-free culture on Matrigel in hES medium conditioned by the autogeneic feeder cells (T3HDF was established to maintain the undifferentiated growth of hES-T3 cells (T3/CMHDF for 8 passages in this investigation. The gene expression profiles of mRNAs, microRNAs and proteins between the undifferentiated T3/HDF and T3/CMHDF cells were shown to be very similar, and their expression profiles were also found to be similar to those of T3/MEF and T3/CMMEF cells grown on MEF feeder and feeder-free Matrigel in MEF-conditioned medium, respectively. The undifferentiated state of T3/HDF and T3/CMHDF as well as T3/MEF andT3/CMMEF cells was evidenced by the very high expression levels of "stemness" genes and low expression levels of differentiation markers of ectoderm, mesoderm and endoderm in addition to the strong staining of OCT4 and NANOG. Conclusion The T3HDF feeder and T3HDF-conditioned medium were able to support the undifferentiated growth of hES cells, and they would be useful for drug development and toxicity testing in addition to the reduced risks of xenogeneic pathogens when used for medical applications such as cell therapies.

  10. Derivation and characterization of the NIH registry human stem cell line NYSCF100 line under defined feeder-free conditions

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    Ana Sevilla

    2018-05-01

    Full Text Available The human embryonic stem cell line NYSCFe001-A was derived from a day 6 blastocyst in feeder-free and antibiotic free conditions. The blastocyst was voluntarily donated for research as surplus after in vitro fertilization treatment following informed consent. The NYSCFe001-A line, registered as NYSCF100 on the NIH registry, presents normal karyotype, is mycoplasma free, expresses all the pluripotency markers and has the potential to differentiate into all three germ layers in vitro.

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

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    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

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

    International Nuclear Information System (INIS)

    Liu, Te; Cheng, Weiwei; Huang, Yongyi; Huang, Qin; Jiang, Lizhen; Guo, Lihe

    2012-01-01

    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: ► microRNA-145 inhibits Sox2 expression in human iPS cells. ► microRNA-145 suppresses the self-renewal and pluripotency of human iPS cells. ► HuAECs regulate expression of microRNA-145 and Sox2 in human iPS cells. ► HuAECs feeder layers maintain human iPS cells pluripotency. ► HuAECs negatively regulates the synthesis of

  13. Immunoflourescence and mRNA analysis of human embryonic stem cells (hESCs) grown under feeder-free conditions

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    Awan, Aashir; Oliveri, Roberto S; Jensen, Pernille L

    2010-01-01

    onto 16-well glass chambers, and continuing with the general IF and qPCR steps will be provided. The techniques will be illustrated with new results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, and PDGF signaling pathways to primary cilia in stem cell......This chapter describes the procedures in order to do immunofluorescence (IF) microscopy and quantitative PCR (qPCR) analysis of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol outlining the steps from initially growing the cells, passaging...

  14. Neural stem cells achieve and maintain pluripotency without feeder cells.

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    Hyun Woo Choi

    Full Text Available BACKGROUND: Differentiated cells can be reprogrammed into pluripotency by transduction of four defined transcription factors. Induced pluripotent stem cells (iPS cells are expected to be useful for regenerative medicine as well as basic research. Recently, the report showed that mouse embryonic fibroblasts (MEF cells are not essential for reprogramming. However, in using fibroblasts as donor cells for reprogramming, individual fibroblasts that had failed to reprogram could function as feeder cells. METHODOLOGY/PRINCIPAL FINDING: Here, we show that adult mouse neural stem cells (NSCs, which are not functional feeder cells, can be reprogrammed into iPS cells using defined four factors (Oct4, Sox2, Klf4, and c-Myc under feeder-free conditions. The iPS cells, generated from NSCs expressing the Oct4-GFP reporter gene, could proliferate for more than two months (passage 20. Generated and maintained without feeder cells, these iPS cells expressed pluripotency markers (Oct4 and Nanog, the promoter regions of Oct4 and Nanog were hypomethylated, could differentiated into to all three germ layers in vitro, and formed a germline chimera. These data indicate that NSCs can achieve and maintain pluripotency under feeder-free conditions. CONCLUSION/SIGNIFICANCE: This study suggested that factors secreted by feeder cells are not essential in the initial/early stages of reprogramming and for pluripotency maintenance. This technology might be useful for a human system, as a feeder-free reprogramming system may help generate iPS cells of a clinical grade for tissue or organ regeneration.

  15. Robust and highly-efficient differentiation of functional monocytic cells from human pluripotent stem cells under serum- and feeder cell-free conditions.

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    Masakatsu D Yanagimachi

    Full Text Available Monocytic lineage cells (monocytes, macrophages and dendritic cells play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3 × 10(6 ± 0.3 × 10(6 floating monocytes from approximately 30 clusters of ESCs/iPSCs 5-6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.

  16. Cultivation and irradiation of human fibroblasts in a medium enriched with platelet lysate for obtaining feeder layer in epidermal cell culture

    International Nuclear Information System (INIS)

    Yoshito, Daniele

    2011-01-01

    For over 30 years, the use of culture medium, enriched with bovine serum, and murines fibroblasts, with the rate of proliferation controlled by irradiation or by share anticarcinogenic drugs, has been playing successfully its role in assisting in the development of keratinocytes in culture, for clinical purposes. However, currently there is a growing concern about the possibility of transmitting prions and animals viruses to transplanted patients. Taking into account this concern, the present work aims to cultivate human fibroblasts in a medium enriched with human platelets lysate and determine the irradiation dose of these cells, for obtaining feeder layer in epidermal cell culture. For carrying out the proposed objective, platelets lysis has standardized, this lysate was used for human fibroblasts cultivation and the irradiation dose enough to inhibit its duplication was evaluated. Human keratinocytes were cultivated in these feeder layers, in culture medium enriched with the lysate. With these results we conclude that the 10% platelets lysate promoted a better adhesion and proliferation of human fibroblasts and in all dose levels tested (60 to 300 Gy), these had their mitotic activity inactivated by ionizing irradiation, being that the feeder layers obtained with doses from 70 to 150 Gy were those that provided the best development of keratinocytes in medium containing 2.5% of human platelet lysate. Therefore, it was possible to standardize both the cultivation of human fibroblasts as its inactivation for use as feeder layer in culture of keratinocytes, so as to eliminate xenobiotics components. (author)

  17. Generation of megakaryocytic progenitors from human embryonic stem cells in a feeder- and serum-free medium.

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    Marjorie Pick

    Full Text Available BACKGROUND: The production of human platelets from embryonic stem cells in a defined culture system is a prerequisite for the generation of platelets for therapeutic use. As an important step towards this goal, we report the differentiation of human embryonic stem cells (hESCs towards the megakaryocyte (Mk lineage using a 'spin embryoid body' method in serum-free differentiation medium. METHODOLOGY AND PRINCIPAL FINDINGS: Immunophenotypic analyses of differentiating hESC identified a subpopulation of cells expressing high levels of CD41a that expressed other markers associated with the Mk lineage, including CD110, CD42b and CD61. Differentiated cells were sorted on the basis of their expression of CD41a, CD34 and CD45 and assessed for Mk colony formation, expression of myeloid and Mk genes and ability to endoreplicate DNA. In a collagen-based colony assay, the CD41a⁺ cells sorted from these differentiation cultures produced 100-800 Mk progenitors at day 13 and 25-160 Mk progenitors at day 20 of differentiation per 100,000 cells assayed. Differentiated Mk cells produced platelet-like particles which expressed CD42b and were activated by ADP, similar to platelets generated from precursors in cord blood. These studies were complemented by real time PCR analyses showing that subsets of cells enriched for CD41a⁺ Mk precursors expressed high levels of Mk associated genes such as PF4 and MPL. Conversely, high levels of myeloid and erythroid related transcripts, such as GATA1, TAL1/SCL and PU.1, were detected in sorted fractions containing CD34⁺ and CD45⁺ cells. CONCLUSIONS: We describe a serum- and feeder-free culture system that enabled the generation of Mk progenitors from human embryonic stem cells. These cells formed colonies that included differentiated Mks that fragmented to form platelet-like particles. This protocol represents an important step towards the generation of human platelets for therapeutic use.

  18. A feeder- and xeno-free human induced pluripotent stem cell line obtained from primary human dermal fibroblasts with epigenetic repression of reprogramming factors expression: GPCCi001-A

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    Michał Stefan Lach

    2017-04-01

    Full Text Available The primary human dermal fibroblasts (PHDFs from breast cancer patient were obtained to generate the human induced pluripotent stem cell line GPCCi001-A via lentiviral transfection. Thus, a modified EF1a-hSTEMCCA-loxP with tetO operator which regulates transgene expression was used. This method takes advantage of epigenetic regulation of transcription and allows for stable silencing of the reprogramming factors in obtained hiPS cells. To increase the potential utility of hiPSCs for clinical applications, they were adapted to feeder- and xeno-free conditions. The pluripotency of GPCCi001-A cell line and ability to differentiate into three germ layers was confirmed.

  19. Human amniotic epithelial cell feeder layers maintain mouse embryonic stem cell pluripotency via epigenetic regulation of the c-Myc promoter.

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    Liu, Te; Cheng, Weiwei; Liu, Tianjin; Guo, Lihe; Huang, Qin; Jiang, Lizhen; Du, Xiling; Xu, Fuhui; Liu, Zhixue; Lai, Dongmei

    2010-02-01

    Mouse embryonic stem cells (ESCs) are typically cultured on a feeder layer of mouse embryonic fibroblasts (MEFs), with leukemia inhibitory factor (LIF) added to maintain them in an undifferentiated state. We have previously shown that human amniotic epithelial cells (hAECs) can be used as feeder cells to maintain mouse ESC pluripotency, but the mechanism for this is unknown. In the present study, we found that CpG islands 5' of the c-Myc gene remain hypomethylated in mouse ESCs cultured on hAECs. In addition, levels of acetylation of histone H3 and trimethylation of histone H3K4 in the c-Myc gene promoter were higher in ES cells cultured on hAECs than those in ES cells cultured on MEFs. These data suggested that hAECs can alter mouse ESC gene expression via epigenetic modification of c-Myc, providing a possible mechanism for the hAEC-induced maintenance of ESCs in an undifferentiated state.

  20. Mannosides as crucial part of bioactive supports for cultivation of human epidermal keratinocytes without feeder cells

    Czech Academy of Sciences Publication Activity Database

    Labský, Jiří; Dvořánková, B.; Smetana, Karel; Holíková, Z.; Brož, L.; Gabius, H.J.

    2003-01-01

    Roč. 24, č. 5 (2003), s. 863-872 ISSN 0142-9612 R&D Projects: GA ČR GA203/00/1310; GA MŠk LN00A065; GA MZd ND6340 Institutional research plan: CEZ:AV0Z4050913 Keywords : cell therapy * keratinocyte * mannose Subject RIV: CD - Macromolecular Chemistry Impact factor: 2.903, year: 2003

  1. Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes.

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    Tan, Xiaobing; Dai, Qingli; Guo, Tao; Xu, Jingshu; Dai, Qingyuan

    2018-01-22

    Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy. Copyright © 2017. Published by Elsevier Inc.

  2. Induced pluripotent stem cells generated from human adipose-derived stem cells using a non-viral polycistronic plasmid in feeder-free conditions.

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    Xinjian Qu

    Full Text Available Induced pluripotent stem cells (iPSCs can be generated from somatic cells by ectopic expression of defined transcription factors (TFs. However, the optimal cell type and the easy reprogramming approaches that minimize genetic aberrations of parent cells must be considered before generating the iPSCs. This paper reports a method to generate iPSCs from adult human adipose-derived stem cells (hADSCs without the use of a feeder layer, by ectopic expression of the defined transcription factors OCT4, SOX2, KLF4 and C-MYC using a polycistronic plasmid. The results, based on the expression of pluripotent marker, demonstrated that the iPSCs have the characteristics similar to those of embryonic stem cells (ESCs. The iPSCs differentiated into three embryonic germ layers both in vitro by embryoid body generation and in vivo by teratoma formation after being injected into immunodeficient mice. More importantly, the plasmid DNA does not integrate into the genome of human iPSCs as revealed by Southern blotting experiments. Karyotypic analysis also demonstrated that the reprogramming of hADSCs by the defined factors did not induce chromosomal abnormalities. Therefore, this technology provides a platform for studying the biology of iPSCs without viral vectors, and can hopefully overcome immune rejection and ethical concerns, which are the two important barriers of ESC applications.

  3. Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea.

    Directory of Open Access Journals (Sweden)

    Djida Ghoubay-Benallaoua

    Full Text Available Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC and epithelial limbal (LSC stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8, E8 supplemented with EGF (E8+ or Green's medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green's media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes.

  4. Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System.

    Science.gov (United States)

    Nandal, Anjali; Mallon, Barbara; Telugu, Bhanu P

    2017-11-08

    Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide "NGG" protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

  5. An evaluation of the choice of feeder cell growth arrest for the production of cultured epidermis.

    Science.gov (United States)

    Chugh, Rishi Man; Chaturvedi, Madhusudan; Yerneni, Lakshmana Kumar

    2015-12-01

    Growth arrested 3T3 cells have been used as feeder cells in human epidermal keratinocyte cultures to produce cultured epidermal autografts for the treatment of burns. The feeder cells were ideally growth-arrested by gamma-irradiation. Alternatively, growth arrest by mitomycin C treatment is a cost effective option. We compared the functional efficacy of these two approaches in keratinocyte cultures by colony forming efficiency, the net growth area of colonies, BrdU labeling and histological features of cultured epidermal sheets. The growth area estimation involved a semi-automated digital technique using the Adobe Photoshop and comprised of isolation and enumeration of red pixels in Rhodamine B-stained keratinocyte colonies. A further refinement of the technique led to the identification of critical steps to increasing the degree of accuracy and enabling its application as an extension of colony formation assay. The results on feeder cell functionality revealed that the gamma irradiated feeders influenced significantly higher colony forming efficiency and larger growth area than the mitomycin C treated feeders. The BrdU labeling study indicated significant stimulation of the overall keratinocyte proliferation by the gamma irradiated feeders. The cultured epidermal sheets produced by gamma feeders were relatively thicker than those produced by mitomycin C feeders. We discussed the clinical utility of mitomycin C feeders from the viewpoint of cost-effective burn care in developing countries. Copyright © 2015 Elsevier Ltd and ISBI. All rights reserved.

  6. The influence of interleukin-2, feeder cells, and timing of irradiation on the radiosensitivity of human T lymphocytes assessed by the colony-forming assay

    International Nuclear Information System (INIS)

    Gerber, M.; Guichard, M.; Pioch, Y.; Dubois, J.B.

    1989-01-01

    The radiosensitivity of human lymphocytes was investigated by the method of colony formation in the absence of interleukin-2 (IL2) and feeder cells, both of which enhance growth of T-cell colonies. The shape of the survival curve and the radiosensitivity was shown to depend upon the ability of lymphocytes to produce IL2: the survival curve for lymphocytes that were the most competent producers of IL2 is the closest to linearity; the lymphocytes that were poor producers show biphasic survival curves. The radiosensitivity of the lymphocytes from the first group is less than that of the latter, when the comparison is based on the first part of the biphasic survival curve. This is more easily seen when cultures are irradiated 24 h after stimulation by phytohemagglutinin (the time of the peak IL2 production) than when cultures are irradiated 2 h before stimulation. This study demonstrates that growth conditions influence the response of lymphocytes to irradiation and that optimal growth conditions result in a linear survival curve

  7. "The good into the pot, the bad into the crop!"--a new technology to free stem cells from feeder cells.

    Directory of Open Access Journals (Sweden)

    Annette Schneider

    Full Text Available A variety of embryonic and adult stem cell lines require an initial co-culturing with feeder cells for non-differentiated growth, self renewal and maintenance of pluripotency. However for many downstream ES cell applications the feeder cells have to be considered contaminations that might interfere not just with the analysis of experimental data but also with clinical application and tissue engineering approaches. Here we introduce a novel technique that allows for the selection of pure feeder-freed stem cells, following stem cell proliferation on feeder cell layers. Complete and reproducible separation of feeder and embryonic stem cells was accomplished by adaptation of an automated cell selection system that resulted in the aspiration of distinct cell colonies or fraction of colonies according to predefined physical parameters. Analyzing neuronal differentiation we demonstrated feeder-freed stem cells to exhibit differentiation potentials comparable to embryonic stem cells differentiated under standard conditions. However, embryoid body growth as well as differentiation of stem cells into cardiomyocytes was significantly enhanced in feeder-freed cells, indicating a feeder cell dependent modulation of lineage differentiation during early embryoid body development. These findings underline the necessity to separate stem and feeder cells before the initiation of in vitro differentiation. The complete separation of stem and feeder cells by this new technology results in pure stem cell populations for translational approaches. Furthermore, a more detailed analysis of the effect of feeder cells on stem cell differentiation is now possible, that might facilitate the identification and development of new optimized human or genetically modified feeder cell lines.

  8. Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

    Science.gov (United States)

    Yamashita, Tomoko; Miyamoto, Yuki; Bando, Yoshio; Ono, Takashi; Kobayashi, Sakurako; Doi, Ayano; Araki, Toshihiro; Kato, Yosuke; Shirakawa, Takayuki; Suzuki, Yutaka; Yamauchi, Junji; Yoshida, Shigetaka; Sato, Naoya

    2017-01-01

    Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a

  9. Feeder cells support the culture of induced pluripotent stem cells even after chemical fixation.

    Directory of Open Access Journals (Sweden)

    Xiao-Shan Yue

    Full Text Available Chemically fixed mouse embryonic fibroblasts (MEFs, instead of live feeder cells, were applied to the maintenance of mouse induced pluripotent stem (miPS cells. Formaldehyde and glutaraldehyde were used for chemical fixation. The chemically fixed MEF feeders maintained the pluripotency of miPS cells, as well as their undifferentiated state. Furthermore, the chemically fixed MEF feeders were reused several times without affecting their functions. These results indicate that chemical fixation can be applied to modify biological feeders chemically, without losing their original functions. Chemically fixed MEF feeders will be applicable to other stem cell cultures as a reusable extracellular matrix candidate that can be preserved on a long-term basis.

  10. Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells.

    Science.gov (United States)

    Klöß, Stephan; Oberschmidt, Olaf; Morgan, Michael; Dahlke, Julia; Arseniev, Lubomir; Huppert, Volker; Granzin, Markus; Gardlowski, Tanja; Matthies, Nadine; Soltenborn, Stephanie; Schambach, Axel; Koehl, Ulrike

    2017-10-01

    The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56 + CD3 - ) was carried out with the CliniMACS Prodigy ® in a single process, starting with approximately 1.2 × 10 9 leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 10 6 effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO ™ 10, CellGro ® , TexMACS ™ , and NK MACS ® ). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56 + CD3 - target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including CD3

  11. Cultivation and irradiation of human fibroblasts in a medium enriched with platelet lysate for obtaining feeder layer in epidermal cell culture; Cultivo e irradiacao de fibroblastos humanos em meio enriquecido com lisado de plaquetas para obtencao de camada de sustentacao em culturas de celulas da epiderme

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele

    2011-07-01

    For over 30 years, the use of culture medium, enriched with bovine serum, and murines fibroblasts, with the rate of proliferation controlled by irradiation or by share anticarcinogenic drugs, has been playing successfully its role in assisting in the development of keratinocytes in culture, for clinical purposes. However, currently there is a growing concern about the possibility of transmitting prions and animals viruses to transplanted patients. Taking into account this concern, the present work aims to cultivate human fibroblasts in a medium enriched with human platelets lysate and determine the irradiation dose of these cells, for obtaining feeder layer in epidermal cell culture. For carrying out the proposed objective, platelets lysis has standardized, this lysate was used for human fibroblasts cultivation and the irradiation dose enough to inhibit its duplication was evaluated. Human keratinocytes were cultivated in these feeder layers, in culture medium enriched with the lysate. With these results we conclude that the 10% platelets lysate promoted a better adhesion and proliferation of human fibroblasts and in all dose levels tested (60 to 300 Gy), these had their mitotic activity inactivated by ionizing irradiation, being that the feeder layers obtained with doses from 70 to 150 Gy were those that provided the best development of keratinocytes in medium containing 2.5% of human platelet lysate. Therefore, it was possible to standardize both the cultivation of human fibroblasts as its inactivation for use as feeder layer in culture of keratinocytes, so as to eliminate xenobiotics components. (author)

  12. Developmental Competence of Buffalo (Bubalus bubalis) Pluripotent Embryonic Stem Cells Over Different Homologous Feeder Layers and the Comparative Evaluation with Various Extracellular Matrices.

    Science.gov (United States)

    Sharma, Manjinder; Dubey, Pawan K; Kumar, Rajesh; Nath, Amar; Kumar, G Sai; Sharma, G Taru

    2013-05-01

    Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA- 4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.

  13. Feeder Cell Type Affects the Growth of In Vitro Cultured Bovine Trophoblast Cells

    Directory of Open Access Journals (Sweden)

    Islam M. Saadeldin

    2017-01-01

    Full Text Available Trophectoderm cells are the foremost embryonic cells to differentiate with prospective stem-cell properties. In the current study, we aimed at improving the current approach for trophoblast culture by using granulosa cells as feeders. Porcine granulosa cells (PGCs compared to the conventional mouse embryonic fibroblasts (MEFs were used to grow trophectoderm cells from hatched bovine blastocysts. Isolated trophectoderm cells were monitored and displayed characteristic epithelial/cuboidal morphology. The isolated trophectoderm cells expressed mRNA of homeobox protein (CDX2, cytokeratin-8 (KRT8, and interferon tau (IFNT. The expression level was higher on PGCs compared to MEFs throughout the study. In addition, primary trophectoderm cell colonies grew faster on PGCs, with a doubling time of approximately 48 hrs, compared to MEFs. PGCs feeders produced a fair amount of 17β-estradiol and progesterone. We speculated that the supplementation of sex steroids and still-unknown factors during the trophoblasts coculture on PGCs have helped to have better trophectoderm cell’s growth than on MEFs. This is the first time to use PGCs as feeders to culture trophectoderm cells and it proved superior to MEFs. We propose PGCs as alternative feeders for long-term culture of bovine trophectoderm cells. This model will potentially benefit studies on the early trophoblast and embryonic development in bovines.

  14. Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method.

    Science.gov (United States)

    Nakajima, Ryota; Takeda, Shizu

    2014-01-01

    The use of murine 3T3 feeder cells needs to be avoided when fabricating corneal epithelial cell sheets for use in treating ocular surface diseases. However, the expression level of the epithelial stem/progenitor cell marker, p63, is down-regulated in feeder-free culture systems. In this study, in order to fabricate corneal epithelial cell sheets that maintain colony-forming cells without using any feeder cells, we investigated the use of an oxygen-controlled method that was developed previously to fabricate cell sheets efficiently. Rabbit limbal epithelial cells were cultured under hypoxia (1-10% O2) and under normoxia during stratification after reaching confluence. Multilayered corneal epithelial cell sheets were fabricated using an oxygen-controlled method, and immunofluorescence analysis showed that cytokeratin 3 and p63 was expressed in appropriate localization in the cell sheets. The colony-forming efficiency of the cell sheets fabricated by the oxygen-controlled method without feeder cells was significantly higher than that of cell sheets fabricated under 20% O2 without feeder cells. These results indicate that the oxygen-controlled method has the potential to achieve a feeder-free culture system for fabricating corneal epithelial cell sheets for corneal regeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro.

    Science.gov (United States)

    Cong, Shan; Cao, Guifang; Liu, Dongjun

    2014-12-01

    To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1-5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

  16. Advanced feeder-free generation of induced pluripotent stem cells directly from blood cells.

    Science.gov (United States)

    Trokovic, Ras; Weltner, Jere; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Salomaa, Veikko; Jalanko, Anu; Otonkoski, Timo; Kyttälä, Aija

    2014-12-01

    Generation of validated human induced pluripotent stem cells (iPSCs) for biobanking is essential for exploring the full potential of iPSCs in disease modeling and drug discovery. Peripheral blood mononuclear cells (PBMCs) are attractive targets for reprogramming, because blood is collected by a routine clinical procedure and is a commonly stored material in biobanks. Generation of iPSCs from blood cells has previously been reported using integrative retroviruses, episomal Sendai viruses, and DNA plasmids. However, most of the published protocols require expansion and/or activation of a specific cell population from PBMCs. We have recently collected a PBMC cohort from the Finnish population containing more than 2,000 subjects. Here we report efficient generation of iPSCs directly from PBMCs in feeder-free conditions in approximately 2 weeks. The produced iPSC clones are pluripotent and transgene-free. Together, these properties make this novel method a powerful tool for large-scale reprogramming of PBMCs and for iPSC biobanking. ©AlphaMed Press.

  17. Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro

    OpenAIRE

    Cong, Shan; Cao, Guifang; Liu, Dongjun

    2014-01-01

    To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic ...

  18. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    International Nuclear Information System (INIS)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B.; Altran, Silvana C.; Isaac, Cesar

    2011-01-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  19. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Altran, Silvana C.; Isaac, Cesar [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Medicina. Lab. de Microcirurgia Plastica; Esteves-Pedro, Natalia M. [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Ciencias Farmaceuticas. Lab. de Controle Biologico; Herson, Marisa R. [DonorTissue Bank of Victoria (Australia)

    2011-07-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  20. Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31

    Science.gov (United States)

    The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the subclonal cell line, PICM-31A1. P...

  1. A single-cell and feeder-free culture system for monkey embryonic stem cells.

    Science.gov (United States)

    Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

    2014-01-01

    Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

  2. Evaluation of the effect of radiation levels and dose rates in irradiation of murine fibroblasts used as a feeder layer in the culture of human keratinocytes

    International Nuclear Information System (INIS)

    Yoshito, Daniele; Almeida, Tiago L.; Santin, Stefany Plumeri; Somessari, Elizabeth S.R.; Silveira, Carlos G. da; Mathor, Monica B.; Altran, Silvana C.; Isaac, Cesar

    2009-01-01

    In 1975, Rheinwald and Green published an effective methodology for obtaining and cultivating human keratinocytes. This methodology consisted of seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate of which is then controlled through the action of ionizing radiation. The presence of the feeder layer encourages the development of keratinocyte colonies and their propagation in similar cultures, becoming possible several clinical applications as skin substitutes or wound dressings in situations such as post burn extensive skin loss and other skin disorders. However, good development of these keratinocytes depends on a high quality feeder layer among other factors. In the present work, we evaluated the relationship between radiation levels and dose rates applied to fibroblasts used in construction of feeder layers and the radiation effect on keratinocytes colonies forming efficiency. Results indicate 3T3 lineage murine fibroblasts irradiated with doses varying between 60 and 100 Gy can be used as a feeder layer immediately after irradiation or storage of the irradiated cells in suspension at 4 g C for 24 hours with similar results. The exception is when the irradiation dose rate is 2.75 Gyh -1 ; in this case, results suggested that the fibroblasts should be used immediately after irradiation. (author)

  3. A defined, feeder-free, serum-free system to generate in vitro hematopoietic progenitors and differentiated blood cells from hESCs and hiPSCs.

    Directory of Open Access Journals (Sweden)

    Giorgia Salvagiotto

    2011-03-01

    Full Text Available Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimensional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells.

  4. Using Human Gestures and Generic Skills to Instruct a Mobile Robot Arm in a Feeder Filling Scenario

    DEFF Research Database (Denmark)

    Pedersen, Mikkel Rath; Høilund, Carsten; Krüger, Volker

    2012-01-01

    Mobile robots that have the ability to cooperate with humans are able to provide new possibilities to manufac- turing industries. In this paper, we discuss our mobile robot arm that a) can provide assistance at different locations in a factory and b) that can be programmed using complex human...... actions such as pointing in Take this object. In this paper, we discuss the use of the mobile robot for a feeding scenario where a human operator specifies the parts and the feeders through pointing gestures. The system is partially built using generic robotic skills. Through extensive experiments, we...

  5. Growth factor expression pattern of homologous feeder layer for culturing buffalo embryonic stem cell-like cells.

    Science.gov (United States)

    Sharma, Ruchi; George, Aman; Kamble, Nitin M; Chauhan, Manmohan S; Singla, Suresh; Manik, Radhey S; Palta, Prabhat

    2012-01-01

    The present study examined the expression profile of buffalo fetal fibroblasts (BFF) used as a feeder layer for embryonic stem (ES) cell-like cells. The expression of important growth factors was detected in cells at different passages. Mitomycin-C inactivation increased relative expression levels of ACTIVIN-A, TGF-β1, BMP-4 and GREMLIN but not of fibroblast growth factor-2 (FGF-2). The expression level of ACTIVIN-A, transforming growth factor-β1 (TGF-β1), bone morphogenetic protein-4 (BMP-4) and FGF-2 was similar in buffalo fetal fibroblast (BFF) cultured in stem cell medium (SCM), SCM+1000IU mL(-1) leukemia inhibitory factor (LIF), SCM+5 ngmL(-1) FGF-2 or SCM+LIF+FGF-2 for 24 h whereas GREMLIN expression was higher in FGF-2-supplemented groups. In spent medium, the concentration of ACTIVIN-A was higher in FGF-2-supplemented groups whereas that of TGF-β1 was similar in SCM and LIF+FGF-2, which was higher than when either LIF or FGF-2 was used alone. Following culture of ES cell-like cells on a feeder layer for 24 h, the TGF-β1 concentration was higher with LIF+FGF-2 than with LIF or FGF-2 alone which, in turn, was higher than that in SCM. In the LIF+FGF-2 group, the concentration of TGF-β1 was lower and that of ACTIVIN-A was higher in spent medium at 24 h than at 48 h of culture. These results suggest that BFF produce signalling molecules that may help in self-renewal of buffalo ES cell-like cells.

  6. Establishment of rat embryonic stem-like cells from the morula using a combination of feeder layers.

    Science.gov (United States)

    Sano, Chiaki; Matsumoto, Asako; Sato, Eimei; Fukui, Emiko; Yoshizawa, Midori; Matsumoto, Hiromichi

    2009-08-01

    Embryonic stem (ES) cells are characterized by pluripotency, in particular the ability to form a germline on injection into blastocysts. Despite numerous attempts, ES cell lines derived from rat embryos have not yet been established. The reason for this is unclear, although certain intrinsic biological differences among species and/or strains have been reported. Herein, using Wistar-Imamichi rats, specific characteristics of preimplantation embryos are described. At the blastocyst stage, Oct4 (also called Pou5f1) was expressed in both the inner cell mass (ICM) and the trophectoderm (TE), whereas expression of Cdx2 was localized to the TE. In contrast, at an earlier stage, expression of Oct4 was detected in all the nuclei in the morula. These stages were examined using a combination of feeder layers (rat embryonic fibroblast [REF] for primary outgrowth and SIM mouse embryo-derived thioguanine- and ouabain-resistant [STO] cells for passaging) to establish rat ES-like cell lines. The rat ES-like cell lines obtained from the morula maintained expression of Oct4 over long-term culture, whereas cell lines derived from blastocysts lost pluripotency during early passage. The morula-derived ES-like cell lines showed Oct4 expression in a long-term culture, even after cryogenic preservation, thawing and EGFP transfection. These results indicate that rat ES-like cell lines with long-term Oct4 expression can be established from the morula of Wistar-Imamichi rats using a combination of feeder layers.

  7. Operation of a cell feeder for coal controlled by a frequency converter. [Czechoslovakia

    Energy Technology Data Exchange (ETDEWEB)

    Stonawski, J; Sikora, G

    1984-02-01

    Steam boiler of the Trinec coal-fired power plant with a capacity of 125 t/h, 545 C, 9.5 MPa was equipped with a screw coal feeder with the KT26A-6 Winter-Eichberg motor with a transformer. The screw fee

  8. CATTLE FEEDER BEHAVIOR AND FEEDER CATTLE PLACEMENTS

    OpenAIRE

    Kastens, Terry L.; Schroeder, Ted C.

    1994-01-01

    Cattle feeders appear irrational when they place cattle on feed when projected profit is negative. Long futures positions appear to offer superior returns to cattle feeding investment. Cattle feeder behavior suggests that they believe a downward bias in live cattle futures persists and that cattle feeders use different expectations than the live cattle futures market price when making placement decisions. This study examines feeder cattle placement determinants, comparing performance of expec...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-10-28

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

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

    International Nuclear Information System (INIS)

    Varga, Nóra; Veréb, Zoltán; Rajnavölgyi, Éva; Német, Katalin; Uher, Ferenc; Sarkadi, Balázs; Apáti, Ágota

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jiafei Xi

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

  12. Stepwise Differentiation of Pluripotent Stem Cells into Osteoblasts Using Four Small Molecules under Serum-free and Feeder-free Conditions

    Directory of Open Access Journals (Sweden)

    Kosuke Kanke

    2014-06-01

    Full Text Available Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenylpyrido[4′,3′:4,5]thieno[2,3-b]pyridine-2-carboxamide [TH] under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2, a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.

  13. 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.

  14. Variations in Humanized and Defined Culture Conditions Supporting Derivation of New Human Embryonic Stem Cell Lines

    DEFF Research Database (Denmark)

    Fletcher, Judy M; Ferrier, Patricia M; Gardner, John O

    2006-01-01

    matrix substrate of purified human laminin (Ln) with transitional reliance on mitotically inactivated human fibroblast (HDF) feeder cells. With this integrated system hESC lines were isolated using either HDF conditioned medium supplemented with a bovine-sourced serum replacement (bSRM), or a defined...

  15. Design of apron feeders

    Energy Technology Data Exchange (ETDEWEB)

    Ramos, C.M.

    1987-12-01

    This paper discusses practical aspects of apron feeder design, and includes information on how to make the first selection. Various aspects of design such as feeder speed, material depth, chain-pitch, roller loading, wheels/sprockets and chain roller friction are elaborated in detail. 8 refs., 14 figs., 12 tabs.

  16. Human embryonic stem cells and microenvironment

    Directory of Open Access Journals (Sweden)

    Banu İskender

    2014-09-01

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

  17. Feederism in a woman.

    Science.gov (United States)

    Terry, Lesley L; Vasey, Paul L

    2011-06-01

    Feederism is a fat fetish subculture in which individuals eroticize weight gain and feeding. Feeders are individuals who claim to become sexually aroused by feeding their partners and encouraging them to gain weight. Conversely, Feedees are individuals who claim to become sexually aroused by eating, being fed, and the idea or act of gaining weight. Very little is known about this population. This report describes a woman who self-identified as a Feedee. It is unclear, at present, whether female Feederism represents a unique paraphilia or a thematic variation of morphophilia or sexual masochism.

  18. Identification of pectenotoxins in plankton, filter feeders, and isolated cells of a Dinophysis acuminata with an atypical toxin profile, from Chile.

    Science.gov (United States)

    Blanco, Juan; Alvarez, Gonzalo; Uribe, Eduardo

    2007-04-01

    A bloom of Dinophysis acuminata produced, in autumn of 2005, a closure of the scallop harvesting in Bahía Inglesa, in the Chilean III region. Isolated cells of this Dinophysis species were shown to contain 180 pg cell(-1) of pectenotoxin 2 but neither okadaic acid nor any of its analogs or derivatives (at least at a detectable level). Examination of plankton and filter-feeder samples covering an area of ca. 350 km, from the location where the toxicity was recorded to Bahía Tongoy, showed that the unique toxin profile found in the first bloom was widespread over that part of Chile and persisted for months. The analysis were carried out by HPLC-ESI-MS using positive ionization mode, with a detection limit below 2 ng of OA mL(-1) of methanolic extract. This is the first report of the presence of pectenotoxins in the plankton of the Pacific coast of America and in the studied filter feeders. This is also the first report of a Dinophysis species containing pectenotoxins and not any toxin of the okadaic acid group.

  19. Dry piston coal feeder

    Science.gov (United States)

    Hathaway, Thomas J.; Bell, Jr., Harold S.

    1979-01-01

    This invention provides a solids feeder for feeding dry coal to a pressurized gasifier at elevated temperatures substantially without losing gas from the gasifier by providing a lock having a double-acting piston that feeds the coals into the gasifier, traps the gas from escaping, and expels the trapped gas back into the gasifier.

  20. Feeder replacement tooling and processes

    International Nuclear Information System (INIS)

    Mallozzi, R.; Goslin, R.; Pink, D.; Askari, A.

    2008-01-01

    Primary heat transport system feeder integrity has become a concern at some CANDU nuclear plants as a result of thinning caused by flow accelerated corrosion (FAC). Feeder inspections are indicating that life-limiting wall thinning can occur in the region between the Grayloc hub weld and second elbow of some outlet feeders. In some cases it has become necessary to replace thinned sections of affected feeders to restore feeder integrity to planned end of life. Atomic Energy of Canada Limited (AECL) and Babcock and Wilcox Canada Ltd. (B and W) have developed a new capability for replacement of single feeders at any location on the reactor face without impacting or interrupting operation of neighbouring feeders. This new capability consists of deploying trained crews with specialized tools and procedures for feeder replacements during planned outages. As may be expected, performing single feeder replacement in the congested working environment of an operational CANDU reactor face involves overcoming many challenges with respect to access to feeders, available clearances for tooling, and tooling operation and performance. This paper describes some of the challenges encountered during single feeder replacements and actions being taken by AECL and B and W to promote continuous improvement of feeder replacement tooling and processes and ensure well-executed outages. (author)

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

    OpenAIRE

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

    2012-01-01

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

  2. A review of human cell radiosensitivity in vitro

    International Nuclear Information System (INIS)

    Deschavanne, Patrick J.; Fertil, Bernard

    1996-01-01

    The survival curves of 694 human cell lines irradiated in exponentially growing phase in vitro were collected from the literature. Among them, 271 were derived from tumors, 423 were nontransformed fibroblasts and other normal cell strains from healthy people or people with some genetic disorders. Seventy-six different cell types are identified, and a specific radiosensitivity could be associated with each, using D-bar and surviving fraction at 2 Gy. Technical factors such as culture medium, feeder cells, and scoring method were found to affect intrinsic radiosensitivity. In particular, the cell type is not a discriminating factor when cells are studied in agar. Results obtained with cells irradiated in agar must be used cautiously, depending on how the cells were prepared for the experiments. The use of feeder cells narrows the range of radiosensitivity of human cells. For cells irradiated as monolayer, it was possible to build a scale of radiosensitivity according to cell type, ranging, in terms of D-bar from 0.6 Gy for the most sensitive cell lines to more than 4 Gy for the most resistant. Considering that, in most cases, we could estimate the variation of radiosensitivity within each cell type, our classification among cell types can be used by researchers to place their results in the context of the literature

  3. Cloning of a novel cell type from human fetal liver expressing cytoplasmic CD3 delta and epsilon but not membrane CD3

    NARCIS (Netherlands)

    Hori, T.; de Waal Malefyt, R.; Duncan, B. W.; Harrison, M. R.; Roncarolo, M. G.; Spits, H.

    1991-01-01

    Seventeen-week human fetal liver cells cultured with a feeder cell mixture of irradiated PBL, irradiated JY cells (an EBV-transformed B cell line) and PHA contained a subpopulation of CD3- cells in addition to a major population of T cells with the mature phenotype. After 12 days in culture, CD3-

  4. Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance*

    Science.gov (United States)

    Soteriou, Despina; Iskender, Banu; Byron, Adam; Humphries, Jonathan D.; Borg-Bartolo, Simon; Haddock, Marie-Claire; Baxter, Melissa A.; Knight, David; Humphries, Martin J.; Kimber, Susan J.

    2013-01-01

    Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal. PMID:23658023

  5. Colour preferences of UK garden birds at supplementary seed feeders.

    Directory of Open Access Journals (Sweden)

    Luke Rothery

    Full Text Available Supplementary feeding of garden birds generally has benefits for both bird populations and human wellbeing. Birds have excellent colour vision, and show preferences for food items of particular colours, but research into colour preferences associated with artificial feeders is limited to hummingbirds. Here, we investigated the colour preferences of common UK garden birds foraging at seed-dispensing artificial feeders containing identical food. We presented birds simultaneously with an array of eight differently coloured feeders, and recorded the number of visits made to each colour over 370 30-minute observation periods in the winter of 2014/15. In addition, we surveyed visitors to a garden centre and science festival to determine the colour preferences of likely purchasers of seed feeders. Our results suggest that silver and green feeders were visited by higher numbers of individuals of several common garden bird species, while red and yellow feeders received fewer visits. In contrast, people preferred red, yellow, blue and green feeders. We suggest that green feeders may be simultaneously marketable and attractive to foraging birds.

  6. Generation of functional cardiomyocytes from rat embryonic and induced pluripotent stem cells using feeder-free expansion and differentiation in suspension culture.

    Science.gov (United States)

    Dahlmann, Julia; Awad, George; Dolny, Carsten; Weinert, Sönke; Richter, Karin; Fischer, Klaus-Dieter; Munsch, Thomas; Leßmann, Volkmar; Volleth, Marianne; Zenker, Martin; Chen, Yaoyao; Merkl, Claudia; Schnieke, Angelika; Baraki, Hassina; Kutschka, Ingo; Kensah, George

    2018-01-01

    The possibility to generate cardiomyocytes from pluripotent stem cells in vitro has enormous significance for basic research, disease modeling, drug development and heart repair. The concept of heart muscle reconstruction has been studied and optimized in the rat model using rat primary cardiovascular cells or xenogeneic pluripotent stem cell derived-cardiomyocytes for years. However, the lack of rat pluripotent stem cells (rPSCs) and their cardiovascular derivatives prevented the establishment of an authentic clinically relevant syngeneic or allogeneic rat heart regeneration model. In this study, we comparatively explored the potential of recently available rat embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs) as a source for cardiomyocytes (CMs). We developed feeder cell-free culture conditions facilitating the expansion of undifferentiated rPSCs and initiated cardiac differentiation by embryoid body (EB)-formation in agarose microwell arrays, which substituted the robust but labor-intensive hanging drop (HD) method. Ascorbic acid was identified as an efficient enhancer of cardiac differentiation in both rPSC types by significantly increasing the number of beating EBs (3.6 ± 1.6-fold for rESCs and 17.6 ± 3.2-fold for riPSCs). These optimizations resulted in a differentiation efficiency of up to 20% cTnTpos rPSC-derived CMs. CMs showed spontaneous contractions, expressed cardiac markers and had typical morphological features. Electrophysiology of riPSC-CMs revealed different cardiac subtypes and physiological responses to cardio-active drugs. In conclusion, we describe rPSCs as a robust source of CMs, which is a prerequisite for detailed preclinical studies of myocardial reconstruction in a physiologically and immunologically relevant small animal model.

  7. Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

    Science.gov (United States)

    Hertsenberg, Andrew J; Funderburgh, James L

    2016-01-01

    Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.

  8. Development of dry coal feeders

    Science.gov (United States)

    Bonin, J. H.; Cantey, D. E.; Daniel, A. D., Jr.; Meyer, J. W.

    1977-01-01

    Design and fabrication of equipment of feed coal into pressurized environments were investigated. Concepts were selected based on feeder system performance and economic projections. These systems include: two approaches using rotating components, a gas or steam driven ejector, and a modified standpipe feeder concept. Results of development testing of critical components, design procedures, and performance prediction techniques are reviewed.

  9. In Vitro Modeling of Human Germ Cell Development Using Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Yuncheng Zhao

    2018-02-01

    Full Text Available Summary: Due to differences across species, the mechanisms of cell fate decisions determined in mice cannot be readily extrapolated to humans. In this study, we developed a feeder- and xeno-free culture protocol that efficiently induced human pluripotent stem cells (iPSCs into PLZF+/GPR125+/CD90+ spermatogonium-like cells (SLCs. These SLCs were enriched with key genes in germ cell development such as MVH, DAZL, GFRα1, NANOS3, and DMRT1. In addition, a small fraction of SLCs went through meiosis in vitro to develop into haploid cells. We further demonstrated that this chemically defined induction protocol faithfully recapitulated the features of compromised germ cell development of PSCs with NANOS3 deficiency or iPSC lines established from patients with non-obstructive azoospermia. Taken together, we established a powerful experimental platform to investigate human germ cell development and pathology related to male infertility. : In this article, Wang and colleagues established a feeder- and xeno-free system to robustly induce human pluripotent stem cells (PSCs into spermatogonia-like cells. This chemically defined induction protocol faithfully recapitulated the features of compromised germ cell development of PSCs with NANOS3 deficiency or iPSC lines established from patients with non-obstructive azoospermia. Keywords: pluripotent stem cells, spermatogonia, infertility, non-obstructive azoospermia

  10. Primary chondrocytes enhance cartilage tissue formation upon co-culture with expanded chondrocytes, dermal fibroblasts, 3T3 feeder cells and embryonic stem cells

    NARCIS (Netherlands)

    Hendriks, J.A.A.; Miclea, Razvan L.; Schotel, Roka; de Bruijn, Ewart; Moroni, Lorenzo; Karperien, Hermanus Bernardus Johannes; Riesle, J.U.; van Blitterswijk, Clemens

    2010-01-01

    Co-culture models have been increasingly used in tissue engineering applications to understand cell–cell interactions and consequently improve regenerative medicine strategies. Aiming at further elucidating cartilage tissue formation, we co-cultured bovine primary chondrocytes (BPCs) with human

  11. Generating a non-integrating human induced pluripotent stem cell bank from urine-derived cells.

    Directory of Open Access Journals (Sweden)

    Yanting Xue

    Full Text Available Induced pluripotent stem cell (iPS cell holds great potential for applications in regenerative medicine, drug discovery, and disease modeling. We describe here a practical method to generate human iPS cells from urine-derived cells (UCs under feeder-free, virus-free, serum-free condition and without oncogene c-MYC. We showed that this approach could be applied in a large population with different genetic backgrounds. UCs are easily accessible and exhibit high reprogramming efficiency, offering advantages over other cell types used for the purpose of iPS generation. Using the approach described in this study, we have generated 93 iPS cell lines from 20 donors with diverse genetic backgrounds. The non-viral iPS cell bank with these cell lines provides a valuable resource for iPS cells research, facilitating future applications of human iPS cells.

  12. Multi-objective stochastic distribution feeder reconfiguration problem considering hydrogen and thermal energy production by fuel cell power plants

    International Nuclear Information System (INIS)

    Niknam, Taher; Kavousi Fard, Abdollah; Baziar, Aliasghar

    2012-01-01

    This paper assesses the operation and management of electrical energy, hydrogen production and thermal load supplement by the Fuel Cell Power Plants (FCPP) in the distribution systems with regard to the uncertainties which exist in the load demand as well as the price of buying natural gas for FCPPs, fuel cost for residential loads, tariff for purchasing electricity, tariff for selling electricity, hydrogen selling price, operation and maintenance cost and the price of purchasing power from the grid. Therefore, a new modified multi-objective optimization algorithm called Teacher-Learning Algorithm (TLA) is proposed to integrate the optimal operation management of Proton Exchange Membrane FCPPs (PEM-FCPPs) and the optimal configuration of the system through an economic model of the PEM-FCPP. In order to improve the total ability of TLA for global search and exploration, a new modification process is suggested such that the algorithm will search the total search space globally. Also, regarding the uncertainties of the new complicated power systems, in this paper for the first time, the DFR problem is investigated in a stochastic environment by the use of probabilistic load flow technique based on Point Estimate Method (PEM). In order to see the feasibility and the superiority of the proposed method, a standard test system is investigated as the case study. The simulation results are investigated in four different scenarios to show the effect of hydrogen production and thermal recovery more evidently. -- Highlights: ► Present an economical and thermal modeling of PEM-FCPPs. ► Present an approach for optimal operation of PEM-FCPPs in a stochastic environment. ► Consider benefits of thermal recovery and Hydrogen production for PEM-FCPPs. ► Present several scenarios for management of PEM-FCPPs.

  13. The Human Cell Atlas.

    Science.gov (United States)

    Regev, Aviv; Teichmann, Sarah A; Lander, Eric S; Amit, Ido; Benoist, Christophe; Birney, Ewan; Bodenmiller, Bernd; Campbell, Peter; Carninci, Piero; Clatworthy, Menna; Clevers, Hans; Deplancke, Bart; Dunham, Ian; Eberwine, James; Eils, Roland; Enard, Wolfgang; Farmer, Andrew; Fugger, Lars; Göttgens, Berthold; Hacohen, Nir; Haniffa, Muzlifah; Hemberg, Martin; Kim, Seung; Klenerman, Paul; Kriegstein, Arnold; Lein, Ed; Linnarsson, Sten; Lundberg, Emma; Lundeberg, Joakim; Majumder, Partha; Marioni, John C; Merad, Miriam; Mhlanga, Musa; Nawijn, Martijn; Netea, Mihai; Nolan, Garry; Pe'er, Dana; Phillipakis, Anthony; Ponting, Chris P; Quake, Stephen; Reik, Wolf; Rozenblatt-Rosen, Orit; Sanes, Joshua; Satija, Rahul; Schumacher, Ton N; Shalek, Alex; Shapiro, Ehud; Sharma, Padmanee; Shin, Jay W; Stegle, Oliver; Stratton, Michael; Stubbington, Michael J T; Theis, Fabian J; Uhlen, Matthias; van Oudenaarden, Alexander; Wagner, Allon; Watt, Fiona; Weissman, Jonathan; Wold, Barbara; Xavier, Ramnik; Yosef, Nir

    2017-12-05

    The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

  14. Generation of Megakaryocytes and Platelets from Human Pluripotent Stem Cells.

    Science.gov (United States)

    Pick, Marjorie

    2016-01-01

    Human pluripotent stem cells (hPSC) have the potential to produce any tissue type in the body and thus represent a source of cells for regenerative medicine. Here we have shown that human platelets can be produced from embryonic or induced pluripotent stem cells in a defined culture system. We describe a serum- and feeder-free culture system that enabled the generation of megakaryocyte (Mk) progenitors and functional platelets from hPSCs. After 13 days the differentiated population included precursor cells that formed colonies containing differentiated Mks, and after 20 days these Mks were able to fragment into platelet-like particles that were functional. This protocol represents an important step towards the generation of human platelets for therapeutic use.

  15. Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.

    Science.gov (United States)

    Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

    2014-01-01

    Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

  16. The human cell atlas

    DEFF Research Database (Denmark)

    Regev, Aviv; Teichmann, Sarah A.; Lander, Eric S.

    2017-01-01

    The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international...... collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells...... in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early...

  17. The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers.

    Directory of Open Access Journals (Sweden)

    Nil Emre

    Full Text Available BACKGROUND: Due to the inherent sensitivity of human embryonic stem cells (hESCs to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions. METHODOLOGY/PRINCIPAL FINDINGS: HESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions. CONCLUSIONS/SIGNIFICANCE: The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types

  18. Derivation of Huntington Disease affected Genea046 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea046 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying HTT gene CAG expansion of 45 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 85% of cells expressed Nanog, 92% Oct4, 75% Tra1–60 and 99% SSEA4 and demonstrated Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and visible contamination.

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

    DEFF Research Database (Denmark)

    Mahmood, Amer; Harkness, Linda; Abdallah, Basem

    2012-01-01

    EBs using BMP2 (bone morphogenic protein 2) combined with standard osteoblast induction medium led to weak osteoblastic induction. Conversely, subcutaneous in vivo implantation of day 20 hEBs in immune deficient mice, mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) as an osteoconductive scaffold......Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESC) is a pre-requisite for their use in clinical applications. However, there is no standard protocol for differentiating hESC into osteoblastic cells. The aim of this study was to identify the emergence of a human...... stromal (mesenchymal, skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESC in a feeder-free environment using serum replacement and as suspension aggregates (embryoid...

  20. Derivation of transgene-free human induced pluripotent stem cells from human peripheral T cells in defined culture conditions.

    Directory of Open Access Journals (Sweden)

    Yoshikazu Kishino

    Full Text Available Recently, induced pluripotent stem cells (iPSCs were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.

  1. Derivation and characterization of the NYSCFe003-A human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Ana Sevilla

    2017-12-01

    Full Text Available The human embryonic stem cell line NYSCFe003-A was derived from a day 5 to day 6 blastocyst in feeder-free and antibiotic free conditions. The blastocyst was voluntarily donated for research as surplus after in vitro fertilization treatment following informed consent. The NYSCFe003-A line expresses all the pluripotency markers and has the potential to differentiate into all three germ layers in vitro. The line presents normal karyotype and is mycoplasma free.

  2. Efficient and Cost-Effective Generation of Mature Neurons From Human Induced Pluripotent Stem Cells

    OpenAIRE

    Badja , Cherif; Maleeva , Galyna; El-Yazidi , Claire; Barruet , Emilie; Lasserre , Manon; Tropel , Philippe; Binetruy , Bernard; Bregestovski , Piotr; Magdinier , Frédérique

    2014-01-01

    The authors describe a feeder-free method of generating induced pluripotent stem cells by relying on the use of a chemically defined medium that overcomes the need for embryoid body formation and neuronal rosette isolation for neuronal precursors and terminally differentiated neuron production. This specific and efficient single-step strategy allows the production of mature neurons in 20–40 days with multiple applications, especially for modeling human pathologies.

  3. Leaky feeder: the communication backbone

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1999-01-01

    The need to communicate with all areas of the underground operation in monitoring the movement of men, materials and vehicles, as well as the optimum performance of conveyor systems, has been effectively met with the installation of a Flexcom leaky feeder cable network at a coliery in Mpumalanga. Installed by the South African subsidiary of Mine Radio Systems (MRS), based in Canada, Flexcom is an RF communications highway for underground mines. The system can provide up to 32 voice/data control channels and up to 16 video channels, all operating simultaneously. The system uses a series of bi-directional amplifiers (or signal boosters) spaced at 350 m intervals along the leaky feeder cable, with branching units and termination units added as required. Communication is possible within 50 m of the leaky feeder cable. MRS has 11 conveyors monitored via the SCADA program at the mine and the system produces reports as required which are accessible via cellphone from anywhere in the world. The wireless monitoring of miners and equipment contributes to mine safety. 3 figs.

  4. NCR1 Expression Identifies Canine Natural Killer Cell Subsets with Phenotypic Similarity to Human Natural Killer Cells

    Directory of Open Access Journals (Sweden)

    Jennifer Ann Foltz

    2016-11-01

    Full Text Available Canines spontaneously develop many cancers similar to humans - including osteosarcoma, leukemia, and lymphoma - offering the opportunity to study immune therapies in a genetically heterogeneous and immunocompetent environment. However, a lack of antibodies recognizing canine NK cell markers has resulted in suboptimal characterization and unknown purity of NK cell products, hindering the development of canine models of NK cell adoptive immunotherapy. To this end, we generated a novel antibody to canine NCR1 (NKp46, the putative species-wide marker of NK cells, enabling purification of NK cells for further characterization. We demonstrate that CD3-/NKp46+ cells in healthy and osteosarcoma-bearing canines have phenotypic similarity to human CD3-/NKp46+ NK cells, expressing mRNA for CD16 and the natural cytotoxicity receptors NKp30, NKp44, and NKp80. Functionally, we demonstrate with the calcein release assay that canine CD3-/NKp46+ cells kill canine tumor cell lines without prior sensitization and secrete IFN-γ, TNF-α, IL-8, IL-10, and GM-CSF as measured by Luminex. Like human NK cells, CD3-/NKp46+ cells expand rapidly on feeder cells expressing 4-1BBL and membrane-bound IL-21 (median= 20,283-fold in 21 days. Further, we identify a minor Null population (CD3-/CD21-/CD14-/NKp46- with reduced cytotoxicity against osteosarcoma cells, but similar cytokine secretion as CD3-/NKp46+ cells. Null cells in canines and humans have reduced expression of NKG2D, NKp44, and CD16 compared to NKp46+ NK cells, and can be induced to express NKp46 with further expansion on feeder cells. In conclusion, we have identified and characterized canine NK cells, including an NKp46- subset of canine and human NK cells, using a novel anti-canine NKp46 antibody, and report robust ex vivo expansion of canine NK cells sufficient for adoptive immunotherapy.

  5. Keratinocytes propagated in serum-free, feeder-free culture conditions fail to form stratified epidermis in a reconstituted skin model.

    Directory of Open Access Journals (Sweden)

    Rebecca Lamb

    Full Text Available Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification.

  6. Human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Abdallah, Basem; Kassem, Moustapha

    2008-01-01

    Mesenchymal stem cells (MSC) are a group of clonogenic cells present among the bone marrow stroma and capable of multilineage differentiation into mesoderm-type cells such as osteoblasts, adipocytes and chondrocytes. Due to their ease of isolation and their differentiation potential, MSC are being...... introduced into clinical medicine in variety of applications and through different ways of administration. Here, we discuss approaches for isolation, characterization and directing differentiation of human mesenchymal stem cells (hMSC). An update of the current clinical use of the cells is also provided....

  7. Elimination of remaining undifferentiated induced pluripotent stem cells in the process of human cardiac cell sheet fabrication using a methionine-free culture condition.

    Science.gov (United States)

    Matsuura, Katsuhisa; Kodama, Fumiko; Sugiyama, Kasumi; Shimizu, Tatsuya; Hagiwara, Nobuhisa; Okano, Teruo

    2015-03-01

    Cardiac tissue engineering is a promising method for regenerative medicine. Although we have developed human cardiac cell sheets by integration of cell sheet-based tissue engineering and scalable bioreactor culture, the risk of contamination by induced pluripotent stem (iPS) cells in cardiac cell sheets remains unresolved. In the present study, we established a novel culture method to fabricate human cardiac cell sheets with a decreased risk of iPS cell contamination while maintaining viabilities of iPS cell-derived cells, including cardiomyocytes and fibroblasts, using a methionine-free culture condition. When cultured in the methionine-free condition, human iPS cells did not survive without feeder cells and could not proliferate or form colonies on feeder cells or in coculture with cells for cardiac cell sheet fabrication. When iPS cell-derived cells after the cardiac differentiation were transiently cultured in the methionine-free condition, gene expression of OCT3/4 and NANOG was downregulated significantly compared with that in the standard culture condition. Furthermore, in fabricated cardiac cell sheets, spontaneous and synchronous beating was observed in the whole area while maintaining or upregulating the expression of various cardiac and extracellular matrix genes. These findings suggest that human iPS cells are methionine dependent and a methionine-free culture condition for cardiac cell sheet fabrication might reduce the risk of iPS cell contamination.

  8. Generation of Human Induced Pluripotent Stem Cells Using RNA-Based Sendai Virus System and Pluripotency Validation of the Resulting Cell Population.

    Science.gov (United States)

    Chichagova, Valeria; Sanchez-Vera, Irene; Armstrong, Lyle; Steel, David; Lako, Majlinda

    2016-01-01

    Human induced pluripotent stem cells (hiPSCs) provide a platform for studying human disease in vitro, increase our understanding of human embryonic development, and provide clinically relevant cell types for transplantation, drug testing, and toxicology studies. Since their discovery, numerous advances have been made in order to eliminate issues such as vector integration into the host genome, low reprogramming efficiency, incomplete reprogramming and acquisition of genomic instabilities. One of the ways to achieve integration-free reprogramming is by using RNA-based Sendai virus. Here we describe a method to generate hiPSCs with Sendai virus in both feeder-free and feeder-dependent culture systems. Additionally, we illustrate methods by which to validate pluripotency of the resulting stem cell population.

  9. Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.

    Science.gov (United States)

    Saha, Krishanu; Mei, Ying; Reisterer, Colin M; Pyzocha, Neena Kenton; Yang, Jing; Muffat, Julien; Davies, Martyn C; Alexander, Morgan R; Langer, Robert; Anderson, Daniel G; Jaenisch, Rudolf

    2011-11-15

    The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications.

  10. Human innate lymphoid cells.

    Science.gov (United States)

    Mjösberg, Jenny; Spits, Hergen

    2016-11-01

    Innate lymphoid cells (ILCs) are increasingly acknowledged as important mediators of immune homeostasis and pathology. ILCs act as early orchestrators of immunity, responding to epithelium-derived signals by expressing an array of cytokines and cell-surface receptors, which shape subsequent immune responses. As such, ILCs make up interesting therapeutic targets for several diseases. In patients with allergy and asthma, group 2 innate lymphoid cells produce high amounts of IL-5 and IL-13, thereby contributing to type 2-mediated inflammation. Group 3 innate lymphoid cells are implicated in intestinal homeostasis and psoriasis pathology through abundant IL-22 production, whereas group 1 innate lymphoid cells are accumulated in chronic inflammation of the gut (inflammatory bowel disease) and lung (chronic obstructive pulmonary disease), where they contribute to IFN-γ-mediated inflammation. Although the ontogeny of mouse ILCs is slowly unraveling, the development of human ILCs is far from understood. In addition, the growing complexity of the human ILC family in terms of previously unrecognized functional heterogeneity and plasticity has generated confusion within the field. Here we provide an updated view on the function and plasticity of human ILCs in tissue homeostasis and disease. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  11. in meat production III. Feeder - breeder dimorphism

    African Journals Online (AJOL)

    Feeder- breeder dimorphism is advantageous when large offspring for slaughter is obtained from small breeding animals. The effect of feeder- breeder dimorphism on herd efficiency is evaluated for terminal crossbreeding and growth modification by biotechnological or dietary means. Selection criteria for breeds or lines in ...

  12. OP9 Feeder Cells Are Superior to M2-10B4 Cells for the Generation of Mature and Functional Natural Killer Cells from Umbilical Cord Hematopoietic Progenitors

    Directory of Open Access Journals (Sweden)

    Lara Herrera

    2017-06-01

    Full Text Available Adoptive natural killer (NK cell therapy relies on the acquisition of large numbers of mature and functional NK cells. An option for future immunotherapy treatments is to use large amounts of NK cells derived and differentiated from umbilical cord blood (UCB CD34+ hematopoietic stem cells (HSCs, mainly because UCB is one of the most accessible HSC sources. In our study, we compared the potential of two stromal cell lines, OP9 and M2-10B4, for in vitro generation of mature and functional CD56+ NK cells from UCB CD34+ HSC. We generated higher number of CD56+ NK cells in the presence of the OP9 cell line than when they were generated in the presence of M2-10B4 cells. Furthermore, higher frequency of CD56+ NK cells was achieved earlier when cultures were performed with the OP9 cells than with the M2-10B4 cells. Additionally, we studied in detail the maturation stages of CD56+ NK cells during the in vitro differentiation process. Our data show that by using both stromal cell lines, CD34+ HSC in vitro differentiated into the terminal stages 4–5 of maturation resembled the in vivo differentiation pattern of human NK cells. Higher frequencies of more mature NK cells were reached earlier by using OP9 cell line than M2-10B4 cells. Alternatively, we observed that our in vitro NK cells expressed similar levels of granzyme B and perforin, and there were no significant differences between cultures performed in the presence of OP9 cell line or M2-10B4 cell line. Likewise, degranulation and cytotoxic activity against K562 target cells were very similar in both culture conditions. The results presented here provide an optimal strategy to generate high numbers of mature and functional NK cells in vitro, and point toward the use of the OP9 stromal cell line to accelerate the culture procedure to obtain them. Furthermore, this method could establish the basis for the generation of mature NK cells ready for cancer immunotherapy.

  13. Human leukaemic cells

    International Nuclear Information System (INIS)

    Andronikashvili, E.L.; Mosulishvili, L.M.; Belokobil'skiy, A.I.; Kharabadze, N.E.; Shonia, N.I.; Desai, L.S.; Foley, G.E.

    1976-01-01

    The results of the determination of trace elements in nucleic acids and histones in human leukaemic cells by activation analysis are reported. The Cr 2+ , Fe 2+ , Zn 2+ , Co 2+ and Sb 2+ content of DNA and RNA of leukaemic cells compared to that of lymphocytes from a patient with infectious mononucleosis or a normal donor are shown tabulated. Similar comparisons are shown for the same trace metal content of histones isolated from the same type of cells. It is felt that the results afford further interesting speculation that trace metals may be involved in the interactions between histones and DNA (especially at the binding sites of histones to DNA), which affect transcription characteristics. (U.K.)

  14. Human spermatogonial stem cells display limited proliferation in vitro under mouse spermatogonial stem cell culture conditions.

    Science.gov (United States)

    Medrano, Jose V; Rombaut, Charlotte; Simon, Carlos; Pellicer, Antonio; Goossens, Ellen

    2016-11-01

    To study the ability of human spermatogonial stem cells (hSSCs) to proliferate in vitro under mouse spermatogonial stem cell (mSSC) culture conditions. Experimental basic science study. Reproductive biology laboratory. Cryopreserved testicular tissue with normal spermatogenesis obtained from three donors subjected to orchiectomy due to a prostate cancer treatment. Testicular cells used to create in vitro cell cultures corresponding to the following groups: [1] unsorted human testicular cells, [2] differentially plated human testicular cells, and [3] cells enriched with major histocompatibility complex class 1 (HLA - )/epithelial cell surface antigen (EPCAM + ) in coculture with inactivated testicular feeders from the same patient. Analyses and characterization including immunocytochemistry and quantitative reverse-transcription polymerase chain reaction for somatic and germ cell markers, testosterone and inhibin B quantification, and TUNEL assay. Putative hSSCs appeared in singlets, doublets, or small groups of up to four cells in vitro only when testicular cells were cultured in StemPro-34 medium supplemented with glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Fluorescence-activated cell sorting with HLA - /EPCAM + resulted in an enrichment of 27% VASA + /UTF1 + hSSCs, compared to 13% in unsorted controls. Coculture of sorted cells with inactivated testicular feeders gave rise to an average density of 112 hSSCs/cm 2 after 2 weeks in vitro compared with unsorted cells (61 hSSCs/cm 2 ) and differentially plated cells (49 hSSCS/cm 2 ). However, putative hSSCs rarely stained positive for the proliferation marker Ki67, and their presence was reduced to the point of almost disappearing after 4 weeks in vitro. We found that hSSCs show limited proliferation in vitro under mSSC culture conditions. Coculture of HLA - /EPCAM + sorted cells with testicular

  15. Directed Differentiation of Human-Induced Pluripotent Stem Cells to Mesenchymal Stem Cells.

    Science.gov (United States)

    Lian, Qizhou; Zhang, Yuelin; Liang, Xiaoting; Gao, Fei; Tse, Hung-Fat

    2016-01-01

    Multipotent stromal cells, also known as mesenchymal stem cells (MSCs), possess great potential to generate a wide range of cell types including endothelial cells, smooth muscle cells, bone, cartilage, and lipid cells. This protocol describes in detail how to perform highly efficient, lineage-specific differentiation of human-induced pluripotent stem cells (iPSCs) with an MSCs fate. The approach uses a clinically compliant protocol with chemically defined media, feeder-free conditions, and a CD105 positive and CD24 negative selection to achieve a single cell-based MSCs derivation from differentiating human pluripotent cells in approximately 20 days. Cells generated with this protocol express typical MSCs surface markers and undergo adipogenesis, osteogenesis, and chondrogenesis similar to adult bone marrow-derived MSCs (BM-MSCs). Nonetheless, compared with adult BM-MSCs, iPSC-MSCs display a higher proliferative capacity, up to 120 passages, without obvious loss of self-renewal potential and constitutively express MSCs surface antigens. MSCs generated with this protocol have numerous applications, including expansion to large scale cell numbers for tissue engineering and the development of cellular therapeutics. This approach has been used to rescue limb ischemia, allergic disorders, and cigarette smoke-induced lung damage and to model mesenchymal and vascular disorders of Hutchinson-Gilford progeria syndrome (HGPS).

  16. An RFID Based Smart Feeder for Hummingbirds.

    Science.gov (United States)

    Ibarra, Vicente; Araya-Salas, Marcelo; Tang, Yu-ping; Park, Charlie; Hyde, Anthony; Wright, Timothy F; Tang, Wei

    2015-12-16

    We present an interdisciplinary effort to record feeding behaviors and control the diet of a hummingbird species (Phaethornis longirostris, the long-billed hermit or LBH) by developing a Radio Frequency Identification (RFID) based smart feeder. The system contains an RFID reader, a microcontroller, and a servo-controlled hummingbird feeder opener; the system is presented as a tool for studying the cognitive ability of the LBH species. When equipped with glass capsule RFID tags (which are mounted on the hummingbird), the smart feeder can provide specific diets for predetermined sets of hummingbirds at the discretion of biologists. This is done by reading the unique RFID tag on the hummingbirds and comparing the ID number with the pre-programmed ID numbers stored in the smart feeder. The smart feeder records the time and ID of each hummingbird visit. The system data is stored in a readily available SD card and is powered by two 9 V batteries. The detection range of the system is approximately 9-11 cm. Using this system, biologists can assign the wild hummingbirds to different experimental groups and monitor their diets to determine if they develop a preference to any of the available nectars. During field testing, the smart feeder system has demonstrated consistent detection (when compared to detections observed by video-recordings) of RFID tags on hummingbirds and provides pre-designed nectars varying water and sugar concentrations to target individuals. The smart feeder can be applied to other biological and environmental studies in the future.

  17. In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells.

    Science.gov (United States)

    Szaraz, Peter; Gratch, Yarden S; Iqbal, Farwah; Librach, Clifford L

    2017-08-09

    Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells. Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein α, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. Our results demonstrate that this

  18. Human leukaemic cells

    International Nuclear Information System (INIS)

    Andronikashvili, E.L.; Mosulishvili, L.M.; Belokobil'skiy, A.I.; Kharabadze, N.E.; Shonia, N.I.; Desai, L.S.; Foley, G.E.

    1976-01-01

    Trace metals were measured by neutron-activation analyses in purified nucleic acids and histone(s) of lymphocytes from patients with acute lymphocytic leukaemia or infectious mononucleosis, and from normal donors. DNA isolated from lymphocytes of a patient with infectious mononucleosis and a normal donor showed a high content of Cr 2+ , Sb 2+ , Fe 2+ , Zn 2+ , whereas DNA of lymphoblasts from a patient with acute lymphocytic leukaemia had a lower content of these trace metals, but the Co 2+ content was 20-fold higher than in DNA of normal donor lymphocytic cells. Total histones from leukaemic cells had higher contents of most of the trace metals except for Zn 2+ , which was present in lesser concentration than in histones from normal donor lymphocytic cells. Lysine-rich (F1) histones showed lower contents of Cr 2+ , Sb 2+ and Co 2+ , whereas arginine-rich (F3) histones had significantly higher contents of these trace metals. These observations may be of interest in that F3 histones more effectively inhibit RNA synthesis in human lymphocytic cells than do other species of histones. (author)

  19. Protein biosynthesis in cultured human hair follicle cells.

    Science.gov (United States)

    Weterings, P J; Vermorken, A J; Bloemendal, H

    1980-10-31

    A new technique has been used for culturing human keratinocytes. The cells grow on the basement membrane-like capsules of bovine lenses. Lens cells were removed from the capsules by rigid trypsinization. In order to exclude any contamination with remaining living cells the isolated capsules were irradiated with X-rays at a dose of 10,000 rad. In this way human epithelial cells can be brought in culture from individual hair follicles. Since feeder cells are not used in this culture technique, the biosynthesis of keratinocyte proteins can be studied in these cultures. The newly synthesized proteins can be separated into a water-soluble, a urea-soluble, and a urea-insoluble fraction. Product analysis has been performed on the first two fractions revealing protein patterns identical to those of intact hair follicles. Product analysis of the urea-soluble fractions of microdissected hair follicles shows that the protein pattern of the cultured keratinocytes resembles the protein pattern of the hair follicle sheath. Studies on the metabolism of benzo(a)pyrene revealed that the enzyme aryl hydrocarbon hydroxylase (AHH) is present in cultured hair follicle cells. A possible use of our culture system for eventual detection of inherited predisposition for smoking-dependent lung cancer is discussed.

  20. Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.

    Directory of Open Access Journals (Sweden)

    Kathryn L McCabe

    Full Text Available To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs for transplantation in patients with corneal endothelial dystrophies.Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1 and Na+/K+ATPaseα1 (ATPA1 on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2. Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.

  1. A study of inter linkage effects on Candu feeder piping

    International Nuclear Information System (INIS)

    Li, M.; Aggarwal, M.L.; Meysner, A.

    2005-01-01

    A CANDU (Canadian Deuterium Uranium) reactor core consists of a large number of fuel channels where heat is generated. Two feeder pipes are connected to each fuel channel to transport D 2 O coolant into and out of the reactor core. The feeder piping is designed to the requirements of Class 1 piping of Section III NB of the ASME Boiler and Pressure Vessel and CSA Codes. Feeder piping stress analysis is being performed to demonstrate the code compliance check and the fitness for service of feeders. In the past, stress analyses were conducted for each individual feeder without including interaction effects among connected feeders. Interaction effects occur as a result of linkages that exist between feeders to prevent fretting and impacting damage during normal, abnormal and accident conditions. In this paper, a 'combined' approach is adopted to include all feeders connected by inter linkages into one feeder piping model. MSC/NASTRAN finite element software was used in the stress simulation, which contains up to 127 feeder pipes. The ASME Class 1 piping analysis was conducted to investigate the effects of the linkages between feeders. Both seismic time history and broadened response spectra methods were used in the seismic stress calculation. The results show that the effect of linkages is significant in dynamic stresses for all feeder configurations, as well as in static stresses for certain feeder configurations. The single feeder analysis could either underestimate or overestimate feeder stresses depending on the pipe geometry and bend wall thickness. (authors)

  2. An RFID Based Smart Feeder for Hummingbirds

    Directory of Open Access Journals (Sweden)

    Vicente Ibarra

    2015-12-01

    Full Text Available We present an interdisciplinary effort to record feeding behaviors and control the diet of a hummingbird species (Phaethornis longirostris, the long-billed hermit or LBH by developing a Radio Frequency Identification (RFID based smart feeder. The system contains an RFID reader, a microcontroller, and a servo-controlled hummingbird feeder opener; the system is presented as a tool for studying the cognitive ability of the LBH species. When equipped with glass capsule RFID tags (which are mounted on the hummingbird, the smart feeder can provide specific diets for predetermined sets of hummingbirds at the discretion of biologists. This is done by reading the unique RFID tag on the hummingbirds and comparing the ID number with the pre-programmed ID numbers stored in the smart feeder. The smart feeder records the time and ID of each hummingbird visit. The system data is stored in a readily available SD card and is powered by two 9 V batteries. The detection range of the system is approximately 9–11 cm. Using this system, biologists can assign the wild hummingbirds to different experimental groups and monitor their diets to determine if they develop a preference to any of the available nectars. During field testing, the smart feeder system has demonstrated consistent detection (when compared to detections observed by video-recordings of RFID tags on hummingbirds and provides pre-designed nectars varying water and sugar concentrations to target individuals. The smart feeder can be applied to other biological and environmental studies in the future.

  3. Efficient generation of functional pancreatic β-cells from human induced pluripotent stem cells.

    Science.gov (United States)

    Yabe, Shigeharu G; Fukuda, Satsuki; Takeda, Fujie; Nashiro, Kiyoko; Shimoda, Masayuki; Okochi, Hitoshi

    2017-02-01

    Insulin-secreting cells have been generated from human embryonic or induced pluripotent stem cells (iPSCs) by mimicking developmental processes. However, these cells do not always secrete glucose-responsive insulin, one of the most important characteristics of pancreatic β-cells. We focused on the importance of endodermal differentiation from human iPSCs in order to obtain functional pancreatic β-cells. A six-stage protocol was established for the differentiation of human iPSCs to pancreatic β-cells using defined culture media without feeders or serum. The effects of CHIR99021, a selective glycogen synthase kinase-3β inhibitor, were examined in the presence of fibroblast growth factor 2, activin, and bone morphogenetic protein 4 (FAB) during definitive endodermal induction by immunostaining for SRY (sex determining region Y)-box 17 (SOX17) and Forkhead box protein A2 (FOXA2). Insulin secretion was compared between the last stage of monolayer culture and spheroid culture conditions. Cultured cells were transplanted under kidney capsules of streptozotocin-diabetic non-obese diabetic-severe combined immunodeficiency mice, and blood glucose levels were measured once a week. Immunohistochemical analyses were performed 4 and 12 weeks after transplantation. Addition of CHIR99021 (3 μmol/L) in the presence of FAB for 2 days improved endodermal cell viability, maintaining the high SOX17-positive rate. Spheroid formation after the endocrine progenitor stage showed more efficient insulin secretion than did monolayer culture. After cell transplantation, diabetic mice had lower blood glucose levels, and islet-like structures were detected in vivo. Functional pancreatic β-cells were generated from human iPSCs. Induction of definitive endoderm and spheroid formation may be key steps for producing these cells. © 2016 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

  4. Rat embryonic fibroblasts improve reprogramming of human keratinocytes into induced pluripotent stem cells.

    Science.gov (United States)

    Linta, Leonhard; Stockmann, Marianne; Kleinhans, Karin N; Böckers, Anja; Storch, Alexander; Zaehres, Holm; Lin, Qiong; Barbi, Gotthold; Böckers, Tobias M; Kleger, Alexander; Liebau, Stefan

    2012-04-10

    Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is, however, limited and thereby further optimization in terms of time, efficiency, and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1, Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells. © Mary Ann Liebert, Inc.

  5. Genome engineering in human cells.

    Science.gov (United States)

    Song, Minjung; Kim, Young-Hoon; Kim, Jin-Soo; Kim, Hyongbum

    2014-01-01

    Genome editing in human cells is of great value in research, medicine, and biotechnology. Programmable nucleases including zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases recognize a specific target sequence and make a double-strand break at that site, which can result in gene disruption, gene insertion, gene correction, or chromosomal rearrangements. The target sequence complexities of these programmable nucleases are higher than 3.2 mega base pairs, the size of the haploid human genome. Here, we briefly introduce the structure of the human genome and the characteristics of each programmable nuclease, and review their applications in human cells including pluripotent stem cells. In addition, we discuss various delivery methods for nucleases, programmable nickases, and enrichment of gene-edited human cells, all of which facilitate efficient and precise genome editing in human cells.

  6. YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers

    DEFF Research Database (Denmark)

    Brøchner, Christian B; Johansen, Julia S; Larsen, Lars A

    2012-01-01

    oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted......The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high...... YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3ß, PDX1, CD34, p63, nestin, PAX6) markers. Double...

  7. Economic feeder for recharging and ``topping off''

    Science.gov (United States)

    Fickett, Bryan; Mihalik, G.

    2000-04-01

    Increasing the size of the melt charge significantly increases yield and reduces costs. Siemens Solar Industries is optimizing a method to charge additional material after meltdown (top-off) using an external feeder system. A prototype feeder system was fabricated consisting of a hopper and feed delivery system. The low-cost feeder is designed for simple operation and maintenance. The system is capable of introducing up to 60 kg of granular silicon while under vacuum. An isolation valve permits refilling of the hopper while maintaining vacuum in the growth furnace. Using the feeder system in conjunction with Siemens Solar Industries' energy efficient hot zone dramatically reduces power and argon consumption. Throughput is also improved as faster pull speeds can be attained. The increased pull speeds have an even greater impact when the charge size is increased. Further cost reduction can be achieved by refilling the crucible after crystal growth and pulling a second ingot run. Siemens Solar Industries is presently testing the feeder in production.

  8. Derivation and characterisation of the human embryonic stem cell lines, NOTT1 and NOTT2.

    Science.gov (United States)

    Priddle, Helen; Allegrucci, Cinzia; Burridge, Paul; Munoz, Maria; Smith, Nigel M; Devlin, Lyndsey; Sjoblom, Cecilia; Chamberlain, Sarah; Watson, Sue; Young, Lorraine E; Denning, Chris

    2010-04-01

    The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.

  9. Derivation of NEM2 affected human embryonic stem cell line Genea079

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea079 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying compound heterozygous mutations in the NEB gene, exon 55 deletion & c.15110dupA, indicative of Nemaline Myopathy Type 2 (NEM2. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male Allele pattern. The hESC line had pluripotent cell morphology, 86% of cells expressed Nanog, 95% Oct4, 54% Tra1-60 and 98% SSEA4 and gave a PluriTest Pluripotency score of 30.25, Novelty of 1.21. The cell line was negative for Mycoplasma and visible contamination.

  10. Advances in technologies for feeder pipe inspections

    Energy Technology Data Exchange (ETDEWEB)

    Ten Grotenhuis, R.; Verma, Y.; Hitchcox, T.; Sakuta, A. [Ontario Power Generation, Inspection and Maintenance Div., Toronto, Ontario (Canada)

    2014-07-15

    The successful development of the Matrix Inspection Technique (MIT) for feeder weld area applications has been followed up with a project to leverage the technology to address other aspects of feeder inspection. The goal of the project is to adapt the technology to provide full circumferential inspection of feeder pipes for FAC thinning and, potentially, for axial cracking. The project necessitated evolving a new generation of high speed, high element count data acquisition instruments. It also required the development of custom inspection arrays, innovative approaches to sealing the water column, use of inertial motion sensors to synthesize encoder inputs, real-time visual feedback for the operator, and enhanced automated analysis software capable of plotting the inspected configuration in 3D. The individual components of the system are currently being integrated into a whole. The results obtained to date demonstrate the approach to be fundamentally sound. (author)

  11. Vertical-Screw-Auger Conveyer Feeder

    Science.gov (United States)

    Walton, Otis (Inventor); Vollmer, Hubert J. (Inventor)

    2016-01-01

    A conical feeder is attached to a vertically conveying screw auger. The feeder is equipped with scoops and rotated from the surface to force-feed regolith the auger. Additional scoops are possible by adding a cylindrical section above the conical funnel section. Such then allows the unit to collect material from swaths larger in diameter than the enclosing casing pipe of the screw auger. A third element includes a flexible screw auger. All three can be used in combination in microgravity and zero atmosphere environments to drill and recover a wide area of subsurface regolith and entrained volatiles through a single access point on the surface.

  12. Small-Molecule Induction Promotes Corneal Epithelial Cell Differentiation from Human Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Alexandra Mikhailova

    2014-02-01

    Full Text Available Human induced pluripotent stem cells (hiPSCs offer unique opportunities for developing novel cell-based therapies and disease modeling. In this study, we developed a directed differentiation method for hiPSCs toward corneal epithelial progenitor cells capable of terminal differentiation toward mature corneal epithelial-like cells. In order to improve the efficiency and reproducibility of our method, we replicated signaling cues active during ocular surface ectoderm development with the help of two small-molecule inhibitors in combination with basic fibroblast growth factor (bFGF in serum-free and feeder-free conditions. First, small-molecule induction downregulated the expression of pluripotency markers while upregulating several transcription factors essential for normal eye development. Second, protein expression of the corneal epithelial progenitor marker p63 was greatly enhanced, with up to 95% of cells being p63 positive after 5 weeks of differentiation. Third, corneal epithelial-like cells were obtained upon further maturation.

  13. Human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Zaher, Walid; Al-Nbaheen, May

    2012-01-01

    Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self......-renewal and multi-lineage differentiation into mesoderm-type of cells, e.g., to osteoblasts, adipocytes, chondrocytes and possibly other cell types including hepatocytes and astrocytes. Due to their ease of culture and multipotentiality, hMSC are increasingly employed as a source for cells suitable for a number...

  14. Counseling in the Elementary Feeder Schools.

    Science.gov (United States)

    Dunham, Virginia

    This brief paper presents the concept of transition counseling between a junior high school and its feeder school(s), designed to make the change from elementary into junior high less traumatic. Aside from routine sixth grade counseling, the counselors expanded their base of counseling to include all types of problems as well as all grade levels.…

  15. Human innate lymphoid cells

    NARCIS (Netherlands)

    Hazenberg, Mette D.; Spits, Hergen

    2014-01-01

    Innate lymphoid cells (ILCs) are lymphoid cells that do not express rearranged receptors and have important effector and regulatory functions in innate immunity and tissue remodeling. ILCs are categorized into 3 groups based on their distinct patterns of cytokine production and the requirement of

  16. Human innate lymphoid cells

    NARCIS (Netherlands)

    Mjösberg, Jenny; Spits, Hergen

    2016-01-01

    Innate lymphoid cells (ILCs) are increasingly acknowledged as important mediators of immune homeostasis and pathology. ILCs act as early orchestrators of immunity, responding to epithelium-derived signals by expressing an array of cytokines and cell-surface receptors, which shape subsequent immune

  17. The development and manufacture of size for size feeder pipe for feeder replacement

    International Nuclear Information System (INIS)

    Legate, G.; Schreiter, D.; Townley, N.

    2008-01-01

    The recently recognised problem of feeder pipe thinning created a unique sourcing problem. Operators require relatively small quantities of nuclear class 1 seamless feeder pipe for such replacement which prior to the introduction of this product in 2006 was not available. It was desired that the pipe be produced at the exact size of the pipe currently in use at the specific reactor site (feeder pipe size varies from site to site). Secondly the pipe had to be made in conformance to the original code year of issue and to conform to the intent of the original material specifications. Finally a supply strategy had to be implemented allowing for timely manufacture of replacement piping. This presentation will report upon how replacement size for size feeder tube was developed and is currently manufactured at Nu-Tech Precision Metals. The paper will also detail the current supply strategy to ensure timely manufacture of the product.

  18. Expectations of Cattle Feeding Investors in Feeder Cattle Placements

    OpenAIRE

    Kastens, Terry L.; Schroeder, Ted C.

    1993-01-01

    Cattle feeders appear irrational when they place cattle on feed when projected profits are negative. Long futures positions appear to offer superior returns to cattle feeding investment. Cattle feeder behavior suggests that they believe a downward bias in live cattle futures persists and that cattle feeders use different information than the live cattle futures market price when making placement decisions. This paper examines feeder cattle placement determinants and compares performance of ex...

  19. Differentiation of human pluripotent stem cells into highly functional classical brown adipocytes.

    Science.gov (United States)

    Nishio, Miwako; Saeki, Kumiko

    2014-01-01

    We describe a detailed method for directed differentiation of human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), into functional classical brown adipocytes (BAs) under serum-free and feeder-free conditions. It is a two-tiered culture system, based on very simple techniques, a floating culture and a subsequent adherent culture. It does not require gene transfer. The entire process can be carried out in about 10 days. The key point is the usage of our special hematopoietic cytokine cocktail. Almost all the differentiated cells express uncoupling protein 1, a BA-selective marker, as determined by immunostaining. The differentiated cells show characteristics of classical BA as assessed by morphology and gene/protein expression. Moreover, the expression of myoblast marker genes is transiently induced during the floating culture step. hESC/hiPSC-derived BAs show significantly higher oxygen consumption rates (OCRs) than white adipocytes generated from human mesenchymal stem cell. They also show responsiveness to adrenergic stimuli, with about twofold upregulation in OCR by β-adrenergic receptor (β-AR) agonist treatments. hESC/hiPSC-derived BAs exert in vivo calorigenic activities in response to β-AR agonist treatments as assessed by thermography. Finally, lipid and glucose metabolisms are significantly improved in hESC/hiPSC-derived BA-transplanted mice. Our system provides a highly feasible way to produce functional classical BA bearing metabolism-improving capacities from hESC/hiPSC under a feeder-free and serum-free condition without gene transfer. © 2014 Elsevier Inc. All rights reserved.

  20. Expression and secretory profile of buffalo fetal fibroblasts and Wharton's jelly feeder layers.

    Science.gov (United States)

    Parmar, Mehtab S; Mishra, Smruti Ranjan; Somal, Anjali; Pandey, Sriti; Kumar, G Sai; Sarkar, Mihir; Chandra, Vikash; Sharma, G Taru

    2017-05-01

    The present study examined the comparative expression and secretory profile of vital signaling molecules in buffalo fetal fibroblasts (BFF) and Wharton's jelly (BWJ) feeder layers at different passages. Both feeder layers were expanded up to 8th passage. Signaling molecules viz. bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF) and transforming growth factor beta 1 (TGFB1) and pluripotency-associated transcriptional factors (POU5F1, SOX2, NANOG, KLF4, MYC and FOXD3) were immunolocalized in the both feeder types. A clear variation in the expression pattern of key signaling molecules with passaging was registered in both feeders compared to primary culture (0 passage). The conditioned media (CM) was collected from different passages (2, 4, 6, 8) of both the feeder layers and was quantified using enzyme-linked immunosorbent assay (ELISA). Concomitant to expression profile, protein quantification also revealed differences in the concentration of signaling molecules at different time points. Conjointly, expression and secretory profile revealed that 2nd passage of BFF and 6th passage of BWJ exhibit optimal levels of key signaling molecules thus may be selected as best passages for embryonic stem cells (ESCs) propagation. Further, the effect of mitomycin-C (MMC) treatment on the expression profile of signaling molecules in the selected passages of BFF and BWJ revealed that MMC modulates the expression profile of these molecules. In conclusion, the results indicate that feeder layers vary in expression and secretory pattern of vital signaling molecules with passaging. Based on these findings, the appropriate feeder passages may be selected for the quality propagation of buffalo ESCs. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. A bend thickness sensitivity study of Candu feeder piping

    International Nuclear Information System (INIS)

    Li, M.; Aggarwal, M.L.; Meysner, A.; Micelotta, C.

    2005-01-01

    In CANDU reactors, feeder bends close to the connection at the fuel channel may be subjected to the highest Flow Accelerated Corrosion (FAC) and stresses. Feeder pipe stress analysis is crucial in the life extension of aging CANDU plants. Typical feeder pipes are interconnected by upper link plates and spacers. It is well known that the stresses at the bends are sensitive to the local bend thicknesses. It is also known from the authors' study (Li and et al, 2005) that feeder inter linkage effect is significant and cannot be ignored. The field measurement of feeder bend thickness is difficult and may be subjected to uncertainty in accuracy. Hence, it is desirable to know how the stress on a subject feeder could be affected by the bend thickness variation of the neighboring feeders. This effect cannot be evaluated by the traditional 'single' feeder model approach. In this paper, the 'row' and 'combined' models developed in the previous study (Li and et al, 2005), which include the feeder interactions, are used to investigate the sensitivity of bend thickness. A series of random thickness bounded by maximum and minimum measured values were applied to feeders in the model. The results show that an individual feeder is not sensitive to the bend thickness variation of the remaining feeders in the model, but depends primarily on its own bend thickness. The highest stress at a feeder always occurs when the feeder has the smallest possible bend thickness. A minimum acceptable bend thickness for individual feeders can be computed by an iterative computing process. The dependency of field thickness measurement and the amount of required analysis work can be greatly reduced. (authors)

  2. Differential effects of the extracellular microenvironment on human embryonic stem cell differentiation into keratinocytes and their subsequent replicative life span.

    Science.gov (United States)

    Movahednia, Mohammad Mehdi; Kidwai, Fahad Karim; Zou, Yu; Tong, Huei Jinn; Liu, Xiaochen; Islam, Intekhab; Toh, Wei Seong; Raghunath, Michael; Cao, Tong

    2015-04-01

    Culture microenvironment plays a critical role in the propagation and differentiation of human embryonic stem cells (hESCs) and their differentiated progenies. Although high efficiency of hESC differentiation to keratinocytes (hESC-Kert) has been achieved, little is known regarding the effects of early culture microenvironment and pertinent extracellular matrix (ECM) interactions during epidermal commitment on subsequent proliferative capacity of hESC-Kert. The aim of this study is to evaluate the effects of the different ECM microenvironments during hESC differentiation on subsequent replicative life span of hESC-Kert. In doing so, H1-hESCs were differentiated to keratinocytes (H1-Kert) in two differentiation systems. The first system employed autologous fibroblast feeder support, in which keratinocytes (H1-Kert(ACC)) were derived by coculture of hESCs with hESC-derived fibroblasts (H1-ebFs). The second system employed a novel decellularized matrix from H1-ebFs to create a dermoepidermal junction-like (DEJ) matrix. H1-Kert(AFF) were derived by differentiation of hESCs on the feeder-free system employing the DEJ matrix. Our study indicated that the feeder-free system with the use of DEJ matrix was more efficient in differentiation of hESCs toward epidermal progenitors. However, the feeder-free system was not sufficient to support the subsequent replicative capacity of differentiated keratinocytes. Of note, H1-Kert(AFF) showed limited replicative capacity with reduced telomere length and early cellular senescence. We further showed that the lack of cell-cell interactions during epidermal commitment led to heightened production of TGF-β1 by hESC-Kert during extended culture, which in turn was responsible for resulting in the limited replicative life span with cellular senescence of hESC-Kert derived under the feeder-free culture system. This study highlights for the first time the importance of the culture microenvironment and cell-ECM interactions during

  3. Stimulated human fibroblast cell survival

    International Nuclear Information System (INIS)

    Smith, B.P.; Gale, K.L.; Einspenner, M.; Greenstock, C.L.; Gentner, N.E.

    1992-01-01

    Techniques for cloning cultured mammalian cells have supported the most universally-accepted method for measuring the induction of lethality by geno-toxicants such as ionizing radiation: the 'survival of colony-forming ability (CFA)' assay. Since most cultured human cell lines exhibit plating efficiency (i.e. the percentage of cells that are capable of reproductively surviving and dividing to form visible colonies) well below 100%, such assays are in essence 'survival of plating efficiency' assays, since they are referred to the plating (or cloning) efficiency of control (i.e. unirradiated) cells. (author). 8 refs., 2 figs

  4. Pressurized feeder for biofuels; Paineistettu syoetin biopolttoaineille

    Energy Technology Data Exchange (ETDEWEB)

    Lundqvist, R; Makkonen, P [Foster Wheeler Energia Oy, Karhula (Finland). Karhula R and D Center

    1996-12-31

    A new type of pressurized piston feeder has been tested for further development of the IGCC concept. The project is funded by the Finnish Ministry of Trade and Industry. The objective has been to develop a substitute for lock-hopper systems, which are commercial, but far too expensive from an operational point of view, due to high inert gas consumption for pressurization. The new feeder system, which is of dual piston type, uses little inert gas, because the pressurization sequence is performed partially by the piston movement. The project comprised further development of the concept, mechanical testing under atmospheric and pressurized conditions and testing with wood chips, peat and coal at elevated pressures. The main concern has been the wear of seals and gaskets during long term operation and for this reason over 600 hours of testing has been carried out. The tests were carried out between May 1994 and January 1995 with wood chips, peat and coal mainly at 17 bar(a) pressure. During the operation, the system was fine-tuned, and generally, the testing was successful. At the early stage of the tests, the ring seal design was modified and several alternative seal constructions were tested. During the tests, the seal was developed further and improved. The present ring seal design has been proven reliable and it meets the commercial requirements. After the tests, the feeder system was inspected thoroughly and found to be in good condition. A commercially available concept of the feeder has been developed on the basis of the knowledge obtained during the test runs

  5. Pressurized feeder for biofuels; Paineistettu syoetin biopolttoaineille

    Energy Technology Data Exchange (ETDEWEB)

    Lundqvist, R.; Makkonen, P. [Foster Wheeler Energia Oy, Karhula (Finland). Karhula R and D Center

    1995-12-31

    A new type of pressurized piston feeder has been tested for further development of the IGCC concept. The project is funded by the Finnish Ministry of Trade and Industry. The objective has been to develop a substitute for lock-hopper systems, which are commercial, but far too expensive from an operational point of view, due to high inert gas consumption for pressurization. The new feeder system, which is of dual piston type, uses little inert gas, because the pressurization sequence is performed partially by the piston movement. The project comprised further development of the concept, mechanical testing under atmospheric and pressurized conditions and testing with wood chips, peat and coal at elevated pressures. The main concern has been the wear of seals and gaskets during long term operation and for this reason over 600 hours of testing has been carried out. The tests were carried out between May 1994 and January 1995 with wood chips, peat and coal mainly at 17 bar(a) pressure. During the operation, the system was fine-tuned, and generally, the testing was successful. At the early stage of the tests, the ring seal design was modified and several alternative seal constructions were tested. During the tests, the seal was developed further and improved. The present ring seal design has been proven reliable and it meets the commercial requirements. After the tests, the feeder system was inspected thoroughly and found to be in good condition. A commercially available concept of the feeder has been developed on the basis of the knowledge obtained during the test runs

  6. Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

    International Nuclear Information System (INIS)

    Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

    2011-01-01

    Highlights: → Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. → The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. → Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

  7. Improvement of automatic fish feeder machine design

    Science.gov (United States)

    Chui Wei, How; Salleh, S. M.; Ezree, Abdullah Mohd; Zaman, I.; Hatta, M. H.; Zain, B. A. Md; Mahzan, S.; Rahman, M. N. A.; Mahmud, W. A. W.

    2017-10-01

    Nation Plan of action for management of fishing is target to achieve an efficient, equitable and transparent management of fishing capacity in marine capture fisheries by 2018. However, several factors influence the fishery production and efficiency of marine system such as automatic fish feeder machine could be taken in consideration. Two latest fish feeder machines have been chosen as the reference for this study. Based on the observation, it has found that the both machine was made with heavy structure, low water and temperature resistance materials. This research’s objective is to develop the automatic feeder machine to increase the efficiency of fish feeding. The experiment has conducted to testing the new design of machine. The new machine with maximum storage of 5 kg and functioning with two DC motors. This machine able to distribute 500 grams of pellets within 90 seconds and longest distance of 4.7 meter. The higher speed could reduce time needed and increase the distance as well. The minimum speed range for both motor is 110 and 120 with same full speed range of 255.

  8. GROα regulates human embryonic stem cell self-renewal or adoption of a neuronal fate

    Science.gov (United States)

    Krtolica, Ana; Larocque, Nick; Genbacev, Olga; Ilic, Dusko; Coppe, Jean-Philippe; Patil, Christopher K.; Zdravkovic, Tamara; McMaster, Michael; Campisi, Judith; Fisher, Susan J.

    2012-01-01

    Previously we reported that feeders formed from human placental fibroblasts (hPFs) support derivation and long-term self-renewal of human embryonic stem cells (hESCs) under serum-free conditions. Here, we show, using antibody array and ELISA platforms, that hPFs secrete ~6-fold higher amounts of the CXC-type chemokine, GROα, than IMR 90, a human lung fibroblast line, which does not support hESC growth. Furthermore, immunocytochemistry and immunoblot approaches revealed that hESCs express CXCR, a GROα receptor. We used this information to develop defined culture medium for feeder-free propagation of hESCs in an undifferentiated state. Cells passaged as small aggregates and maintained in the GROα-containing medium had a normal karyotype, expressed pluripotency markers, and exhibited apical–basal polarity, i.e., had the defining features of pluripotent hESCs. They also differentiated into the three primary (embryonic) germ layers and formed teratomas in immunocompromised mice. hESCs cultured as single cells in the GROα-containing medium also had a normal karyotype, but they downregulated markers of pluripotency, lost apical–basal polarity, and expressed markers that are indicative of the early stages of neuronal differentiation—βIII tubulin, vimentin, radial glial protein, and nestin. These data support our hypothesis that establishing and maintaining cell polarity is essential for the long-term propagation of hESCs in an undifferentiated state and that disruption of cell–cell contacts can trigger adoption of a neuronal fate. PMID:21396766

  9. Human embryonic stem cells handbook

    Directory of Open Access Journals (Sweden)

    Carlo Alberto Redi

    2013-03-01

    Full Text Available After the Nobel prize in physiology or medicine was awarded jointly to Sir John Gurdon and Shinya Yamanaka for the discovery that mature cells can be reprogrammed to become pluripotent it became imperative to write down the review for a book entirely devoted to human embryonic stem cells (hES, those cells that are a urgent need for researchers, those cells that rekindle the ethical debates and finally, last but not least, those cells whose study paved the way to obtain induced pluripotent stem cells by the OSKC’s Yamanaka method (the OSKC acronim refers, for those not familiar with the topic, to the four stemness genes used to transfect somatic fibroblasts: Oct4, Sox2, Klf4 and c-Myc....

  10. Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture

    Science.gov (United States)

    Pijuan-Galitó, Sara; Tamm, Christoffer; Schuster, Jens; Sobol, Maria; Forsberg, Lars; Merry, Catherine L. R.; Annerén, Cecilia

    2016-01-01

    Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications. PMID:27405751

  11. Feeder density enhances house finch disease transmission in experimental epidemics.

    Science.gov (United States)

    Moyers, Sahnzi C; Adelman, James S; Farine, Damien R; Thomason, Courtney A; Hawley, Dana M

    2018-05-05

    Anthropogenic food provisioning of wildlife can alter the frequency of contacts among hosts and between hosts and environmental sources of pathogens. Despite the popularity of garden bird feeding, few studies have addressed how feeders influence host contact rates and disease dynamics. We experimentally manipulated feeder density in replicate aviaries containing captive, pathogen-naive, groups of house finches ( Haemorhous mexicanus ) and continuously tracked behaviours at feeders using radio-frequency identification devices. We then inoculated one bird per group with Mycoplasma gallisepticum (Mg), a common bacterial pathogen for which feeders are fomites of transmission, and assessed effects of feeder density on house finch behaviour and pathogen transmission. We found that pathogen transmission was significantly higher in groups with the highest density of bird feeders, despite a significantly lower rate of intraspecific aggressive interactions relative to the low feeder density groups. Conversely, among naive group members that never showed signs of disease, we saw significantly higher concentrations of Mg-specific antibodies in low feeder density groups, suggesting that birds in low feeder density treatments had exposure to subclinical doses of Mg. We discuss ways in which the density of garden bird feeders could play an important role in mediating the intensity of Mg epidemics.This article is part of the theme issue 'Anthropogenic resource subsidies and host-parasite dynamics in wildlife'. © 2018 The Author(s).

  12. Human T cell colony formation in microculture: analysis of growth requirements and functional activities.

    Science.gov (United States)

    Gelfand, E W; Lee, J W; Dosch, H M; Price, G B

    1981-03-01

    A microculture method in methylcellulose has been developed for the study of human T cell colony formation. The technique is simple, reliable, does not require preincubation with lectin and requires small numbers of cells. Colony formation was dependent on the presence of phytohemagglutin-conditioned medium, a T colony precursor cell (TCPC), and a "helper" or accessory T cell. Plating efficiency was increased 10-fold in the presence of irradiated feeder cells. Progenitors of the T colony cells were identified in peripheral blood, tonsil, and spleen but not in thymus or thoracic duct. They were isolated in the E-rosetting, theophylline-resistant, Fc-IgG-negative cell populations. In peripheral blood the frequency of TCPC and accessory cells, the T colony forming unit, was estimated to be 8 X 10(-3). Colony cells proliferated in response to lectins and allogeneic cells. Forty to 80% of the cells were Ia-positive and stimulated both autologous and allogeneic mixed lymphocyte responses. They were incapable of mediating antibody-dependent cytotoxicity. In contrast, they were effective in assays of spontaneous cytotoxicity but only against certain target cells. This method for the analysis of T colony formation should prove valuable in the functional analysis of T cell subsets in immunodeficiency states or the transplant recipient.

  13. A reliable and economical method for gaining mouse embryonic fibroblasts capable of preparing feeder layers.

    Science.gov (United States)

    Jiang, Guangming; Wan, Xiaoju; Wang, Ming; Zhou, Jianhua; Pan, Jian; Wang, Baolong

    2016-08-01

    Mouse embryonic fibroblasts (MEFs) are widely used to prepare feeder layers for culturing embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in vitro. Transportation lesions and exorbitant prices make the commercially obtained MEFs unsuitable for long term research. The aim of present study is to establish a method, which enables researchers to gain MEFs from mice and establish feeder layers by themselves in ordinary laboratories. MEFs were isolated from ICR mouse embryos at 12.5-17.5 day post-coitum (DPC) and cultured in vitro. At P2-P7, the cells were inactivated with mitomycin C or by X-ray irradiation. Then they were used to prepare feeder layers. The key factors of the whole protocol were analyzed to determine the optimal conditions for the method. The results revealed MEFs isolated at 12.5-13.5 DPC, and cultured to P3 were the best choice for feeder preparation, those P2 and P4-P5 MEFs were also suitable for the purpose. The P3-P5 MEFs treated with 10 μg/ml of mitomycin C for 3 h, or irradiated with X-ray at 1.5 Gy/min for 25 Gy were the most suitable feeder cells. Treating MEFs with 10 μg/ml of mitomycin C for 2.5 h, 15 μg/ml for 2.0 h, or irradiating the cells with 20 Gy of X-ray at 2.0 Gy/min could all serve as alternative methods for P3-P4 cells. Our study provides a reliable and economical way to obtain large amount of qualified MEFs for long term research of ESCs or iPSCs.

  14. Impact of flow accelerated corrosion (FAC) on feeder refurbishment planning

    International Nuclear Information System (INIS)

    Jyrkama, M.; Pandey, M.

    2010-01-01

    Feeder wall thinning due to flow accelerated corrosion (FAC) may result in a large number of feeder replacements in the future. In this study, the process of FAC is modelled using a probabilistic approach and used to predict the expected number of degraded feeders and their replacements in the future. Because of the high cost associated with feeder replacements, it may be optimal to replace the entire feeder population during a single refurbishment outage when the unit cost of replacement is likely to be less. The results of this study demonstrate, however, that the unit cost of feeder replacement must be sufficiently lower than the standard replacement cost and the refurbishment performed at an optimal time to realize the economic benefits associated with the refurbishment. (author)

  15. System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer T.G.; Prokhorova, Tatyana; Akimov, Vyacheslav

    2011-01-01

    by feeder cells. We profiled 6521 proteins and 23,522 phosphorylation sites, of which almost 50% displayed dynamic changes in phosphorylation status during 24 hours of differentiation. These data are a resource for studies of the events associated with the maintenance of hESC pluripotency and those...... of the matching sequence motif. In addition to identifying previously unknown phosphorylation sites on factors associated with differentiation, such as kinases and transcription factors, we observed dynamic phosphorylation of DNA methyltransferases (DNMTs). We found a specific interaction of DNMTs during early......To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned...

  16. A HYBRID APPROACH FOR RURAL FEEDER DESIGN

    Directory of Open Access Journals (Sweden)

    DAMANJEET KAUR

    2012-08-01

    Full Text Available In this paper, a population based approach for conductor size selection in rural radial distribution system is presented. The proposed hybrid approach implies a particle swarm optimization (PSO approach in combination with mutant property of differential evolution (DE for conductor size selection in radial distribution system. The conductor size for each feeder segment is selected such that the total cost of capital investment and capitalized cost of energy losses is minimized while constraints of voltage at each node and current carrying capacity of conductor is within the limits. The applicability and effectiveness of the proposed method is demonstrated with the help of 32-node test system.

  17. Immunofluorescence Microscopy and mRNA Analysis of Human Embryonic Stem Cells (hESCs) Including Primary Cilia Associated Signaling Pathways

    DEFF Research Database (Denmark)

    Vestergaard, Maj Linea; Awan, Aashir; Warzecha, Caroline Becker

    2016-01-01

    onto 16-well glass chambers, and continuing with the general IFM and qPCR anlysis. The techniques are illustrated with results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, PDGF, and TGFβ signaling pathways to primary cilia in stem cell maintenance......This chapter describes the procedures for immunofluorescence microscopy (IFM) and quantitative PCR (qPCR) analyses of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol is provided outlining the steps from initially growing the cells, passaging...

  18. Functional vascular smooth muscle cells derived from human induced pluripotent stem cells via mesenchymal stem cell intermediates

    Science.gov (United States)

    Bajpai, Vivek K.; Mistriotis, Panagiotis; Loh, Yuin-Han; Daley, George Q.; Andreadis, Stelios T.

    2012-01-01

    Aims Smooth muscle cells (SMC) play an important role in vascular homeostasis and disease. Although adult mesenchymal stem cells (MSC) have been used as a source of contractile SMC, they suffer from limited proliferation potential and culture senescence, particularly when originating from older donors. By comparison, human induced pluripotent stem cells (hiPSC) can provide an unlimited source of functional SMC for autologous cell-based therapies and for creating models of vascular disease. Our goal was to develop an efficient strategy to derive functional, contractile SMC from hiPSC. Methods and results We developed a robust, stage-wise, feeder-free strategy for hiPSC differentiation into functional SMC through an intermediate stage of multipotent MSC, which could be coaxed to differentiate into fat, bone, cartilage, and muscle. At this stage, the cells were highly proliferative and displayed higher clonogenic potential and reduced senescence when compared with parental hair follicle mesenchymal stem cells. In addition, when exposed to differentiation medium, the myogenic proteins such as α-smooth muscle actin, calponin, and myosin heavy chain were significantly upregulated and displayed robust fibrillar organization, suggesting the development of a contractile phenotype. Indeed, tissue constructs prepared from these cells exhibited high levels of contractility in response to receptor- and non-receptor-mediated agonists. Conclusion We developed an efficient stage-wise strategy that enabled hiPSC differentiation into contractile SMC through an intermediate population of clonogenic and multipotent MSC. The high yield of MSC and SMC derivation suggests that our strategy may facilitate an acquisition of the large numbers of cells required for regenerative medicine or for studying vascular disease pathophysiology. PMID:22941255

  19. Risk-informed prediction of feeder end of life

    International Nuclear Information System (INIS)

    Jyrkama, M.; Pandey, M.

    2011-01-01

    The operating life of feeder piping is negatively impacted by flow accelerated corrosion (FAC). In this study, an assessment of a large set of inspection data reveals that FAC in feeders is a relatively stationary process, with variability only at the local scale. Given the added uncertainty from inspection coverage, a new method for estimating the thinning rate and feeder EOL is developed using a probabilistic approach. The results of the study illustrate the benefits of the methodology in supporting risk-informed decision making at the station by quantifying the present and incremental risk in the feeder system over time. (author)

  20. Determination of representative CANDU feeder dimensions for engineering simulator

    International Nuclear Information System (INIS)

    Cho, S.; Muzumdar, A.

    1996-01-01

    This paper describes a logic for selection of representative channel groups and a methodology for determination of representative CANDU feeder dimensions and the pressure drops between inlet/outlet header and fuel channel in the primary loop. A code, MEDOC, was developed based on this logic and methodology and helps perform a calculation of representative feeder dimensions for a selected channel group on the basis of feeder geometry data (fluid volume, mass flow rate, loss factor) and given property data (pressure, quality, density) at inlet/outlet header. The representative feeder dimensions calculated based on this methodology will be useful for the engineering simulator for the CANDU type reactor. (author)

  1. Risk-informed prediction of feeder end of life

    Energy Technology Data Exchange (ETDEWEB)

    Jyrkama, M.; Pandey, M. [Univ. of Waterloo, Ontario (Canada)

    2011-07-01

    The operating life of feeder piping is negatively impacted by flow accelerated corrosion (FAC). In this study, an assessment of a large set of inspection data reveals that FAC in feeders is a relatively stationary process, with variability only at the local scale. Given the added uncertainty from inspection coverage, a new method for estimating the thinning rate and feeder EOL is developed using a probabilistic approach. The results of the study illustrate the benefits of the methodology in supporting risk-informed decision making at the station by quantifying the present and incremental risk in the feeder system over time. (author)

  2. Tuft (caveolated) cells in two human colon carcinoma cell lines.

    OpenAIRE

    Barkla, D. H.; Whitehead, R. H.; Foster, H.; Tutton, P. J.

    1988-01-01

    The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting f...

  3. Chemosensitivity and radiosensitivity testing of freshly explanted human tumour cells in vitro

    International Nuclear Information System (INIS)

    Wells, J.

    1977-10-01

    In this thesis, in vitro testing for the chemosensitivity and radiosensitivity of freshly explanted human tumour cells is described. The cells were incubated with anti-tumour drugs and either a 6-day growth test performed or a clonal growth test as a measure of survival of cell reproductive capacity. It was shown that if one aims to develop a suitable in vitro method for predicting the subsequent response of human tumour cells in situ to cytotoxic chemotherapy, the test procedure must be initiated before the explanted cells have undergone significant growth in vitro. The survival of the reproductive capacity of tumour cell explants following X-radiation was also studied. Using a 'feeder' layer technique, values for the survival curve parameter Dsub(q) were in the range 400-610 rad and the values for D 0 were in the range 120-160 rad. The shape of the X-ray survival curves did not change when cells were retested after repeated subculturing in vitro. Therefore, unlike chemosensitivity measured by the same biological end-point, radiosensitivity apparently does not change once cells have reached their maximum growth potential. (UK)

  4. Rapid fabricating technique for multi-layered human hepatic cell sheets by forceful contraction of the fibroblast monolayer.

    Directory of Open Access Journals (Sweden)

    Yusuke Sakai

    Full Text Available Cell sheet engineering is attracting attention from investigators in various fields, from basic research scientists to clinicians focused on regenerative medicine. However, hepatocytes have a limited proliferation potential in vitro, and it generally takes a several days to form a sheet morphology and multi-layered sheets. We herein report our rapid and efficient technique for generating multi-layered human hepatic cell (HepaRG® cell sheets using pre-cultured fibroblast monolayers derived from human skin (TIG-118 cells as a feeder layer on a temperature-responsive culture dish. Multi-layered TIG-118/HepaRG cell sheets with a thick morphology were harvested on day 4 of culturing HepaRG cells by forceful contraction of the TIG-118 cells, and the resulting sheet could be easily handled. In addition, the human albumin and alpha 1-antitrypsin synthesis activities of TIG-118/HepaRG cells were approximately 1.2 and 1.3 times higher than those of HepaRG cells, respectively. Therefore, this technique is considered to be a promising modality for rapidly fabricating multi-layered human hepatocyte sheets from cells with limited proliferation potential, and the engineered cell sheet could be used for cell transplantation with highly specific functions.

  5. Generation of clinical-grade human induced pluripotent stem cells in Xeno-free conditions.

    Science.gov (United States)

    Wang, Juan; Hao, Jie; Bai, Donghui; Gu, Qi; Han, Weifang; Wang, Lei; Tan, Yuanqing; Li, Xia; Xue, Ke; Han, Pencheng; Liu, Zhengxin; Jia, Yundan; Wu, Jun; Liu, Lei; Wang, Liu; Li, Wei; Liu, Zhonghua; Zhou, Qi

    2015-11-12

    Human induced pluripotent stem cells (hiPSCs) are considered as one of the most promising seed cell sources in regenerative medicine. Now hiPSC-based clinical trials are underway. To ensure clinical safety, cells used in clinical trials or therapies should be generated under GMP conditions, and with Xeno-free culture media to avoid possible side effects like immune rejection that induced by the Xeno reagents. However, up to now there are no reports for hiPSC lines developed completely under GMP conditions using Xeno-free reagents. Clinical-grade human foreskin fibroblast (HFF) cells used as feeder cells and parental cells of the clinical-grade hiPSCs were isolated from human foreskin tissues and cultured in Xeno-free media. Clinical-grade hiPSCs were derived by integration-free Sendai virus-based reprogramming kit in Xeno-free pluriton™ reprogramming medium or X medium. Neural cells and cardiomyocytes differentiation were conducted following a series of spatial and temporal specific signals induction according to the corresponding lineage development signals. Biological safety evaluation of the clinical-grade HFF cells and hiPSCs were conducted following the guidance of the "Pharmacopoeia of the People's Republic of China, Edition 2010, Volume III". We have successfully derived several integration-free clinical-grade hiPSC lines under GMP-controlled conditions and with Xeno-free reagents culture media in line with the current guidance of international and national evaluation criteria. As for the source of hiPSCs and feeder cells, biological safety evaluation of the HFF cells have been strictly reviewed by the National Institutes for Food and Drug Control (NIFDC). The hiPSC lines are pluripotent and have passed the safety evaluation. Moreover, one of the randomly selected hiPSC lines was capable of differentiating into functional neural cells and cardiomyocytes in Xeno-free culture media. The clinical-grade hiPSC lines therefore could be valuable sources for

  6. Human glomerular epithelial cell proteoglycans

    International Nuclear Information System (INIS)

    Thomas, G.J.; Jenner, L.; Mason, R.M.; Davies, M.

    1990-01-01

    Proteoglycans synthesized by cultures of human glomerular epithelial cells have been isolated and characterized. Three types of heparan sulfate were detected. Heparan sulfate proteoglycan I (HSPG-I; Kav 6B 0.04) was found in the cell layer and medium and accounted for 12% of the total proteoglycans synthesized. HSPG-II (Kav 6B 0.25) accounted for 18% of the proteoglycans and was located in the medium and cell layer. A third population (9% of the proteoglycan population), heparan sulfate glycosaminoglycan (HS-GAG; Kav 6B 0.4-0.8), had properties consistent with single glycosaminoglycan chains or their fragments and was found only in the cell layer. HSPG-I and HSPG-II from the cell layer had hydrophobic properties; they were released from the cell layer by mild trypsin treatment. HS-GAG lacked these properties, consisted of low-molecular-mass heparan sulfate oligosaccharides, and were intracellular. HSPG-I and -II released to the medium lacked hydrophobic properties. The cells also produced three distinct types of chondroitin sulfates. The major species, chondroitin sulfate proteoglycan I (CSPG-I) eluted in the excluded volume of a Sepharose CL-6B column, accounted for 30% of the proteoglycans detected, and was found in both the cell layer and medium. Cell layer CSPG-I bound to octyl-Sepharose. It was released from the cell layer by mild trypsin treatment. CSPG-II (Kav 6B 0.1-0.23) accounted for 10% of the total 35S-labeled macromolecules and was found predominantly in the culture medium. A small amount of CS-GAG (Kav 6B 0.25-0.6) is present in the cell extract and like HS-GAG is intracellular. Pulse-chase experiments indicated that HSPG-I and -II and CSPG-I and -II are lost from the cell layer either by direct release into the medium or by internalization where they are metabolized to single glycosaminoglycan chains and subsequently to inorganic sulfate

  7. Discarded human fetal tissue and cell cultures for transplantation research

    International Nuclear Information System (INIS)

    Hay, R.J.; Phillips, T.; Thompson, A.; Vilner, L.; Cleland, M.; Tchaw-ren Chen; Zabrenetzky, V.

    1999-01-01

    A feasibility study has been performed to explore the utility of various tissues from discarded human abortuses for transplantation and related research. Specifically, aborted fetuses plus parental blood samples and all relevant clinical data were obtained through a local hospital complex. Whenever possible, pancreas, skin and skeletal muscle, heart, liver, kidney, cartilage and lung tissues were removed, dissociated and subfractionated for cryopreservation, characterization and cultivation trials in vitro. Existing protocols for these manipulations were compared and improved upon as required. Clonal culture, cell aggregate maintenance techniques and use of feeder cell populations have been utilized where appropriate to develop quantitative comparative data. Histological and biochemical assays were applied both to evaluate separation/cultivation methods and to identify optimal culture conditions for maintaining functional cells. Immunochemical and molecular biological procedures were applied to study expression of Major Histocompatibility Vomplex (MHC) class 1 and 11 molecules on cell lines derived. Tissue and cell culture populations were examined for infections with bacteria, ftingi, mycoplasma, HIV, CMV, hepatitis B and other viruses. Only 1% of the abortuses tested were virally infected. Cytogenetic analyses confin-ned the normal diploid status in the vast majority (>98%) of lines tested. A total of over 250 abortuses have been obtained and processed. Only 25 were found to be contaminated with bacteria or fungi and unsuitable for further cultivation trials. A total of over 200 cell populations were isolated, characterized and cryopreserved for further study. Included were kidney, lung, liver and epidermal epithelia: cartilage-derived cells from the spine and epiphyses plus myogenic myoblasts. Selected lines have been immortalized using HPV I 6E6/E7 sequences. Epithelia from the liver and pancreas and cardiac myocytes were the most problematic in that initial

  8. Derivation of Stromal (Skeletal and Mesenchymal) Stem-Like Cells from Human Embryonic Stem Cells

    Science.gov (United States)

    Harkness, Linda; Abdallah, Basem M.; Elsafadi, Mona; Al-Nbaheen, May S.; Aldahmash, Abdullah; Kassem, Moustapha

    2012-01-01

    Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESCs) is a prerequisite for their use in clinical applications. However, there is no standard protocol for differentiating hESCs into osteoblastic cells. The aim of this study was to identify the emergence of a human stromal (mesenchymal and skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESCs in a feeder-free environment using serum replacement and as suspension aggregates (embryoid bodies; hEBs). Over a 20 day developmental period, the hEBs demonstrated increasing enrichment for cells expressing hMSC markers: CD29, CD44, CD63, CD56, CD71, CD73, CD105, CD106, and CD166 as revealed by immunohistochemical staining and flow cytometry (fluorescence-activated cell sorting) analysis. Ex vivo differentiation of hEBs using bone morphogenic protein 2 (BMP2) combined with standard osteoblast induction medium led to weak osteoblastic induction. Conversely, subcutaneous in vivo implantation of day 20 hEBs in immune deficient mice, mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) as an osteoconductive scaffold, revealed bone and cartilage, and fibrous tissue elements after 8 weeks. These tissues were of human origin and there was no evidence of differentiation to nonmesodermal tissues. hEBs implanted in the absence of HA/TCP formed vacuolated tissue containing glandular, fibrous and muscle-like tissue elements. Conversely, implantation of undifferentiated hESCs resulted in the formation of a teratoma containing a mixture of endodermal, mesodermal, and ectodermal tissues. Our study demonstrates that hMSC-like cells can be obtained from hESCs and they can be induced to form skeletal tissues in vivo when combined with HA/TCP. These findings are relevant for tissue engineering and suggest that differentiated hEBs can provide an unlimited source for

  9. The role of NF-κB signaling in the maintenance of pluripotency of human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Osamu Takase

    Full Text Available NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.

  10. The Use of Filter-feeders to Manage Disease in a Changing World.

    Science.gov (United States)

    Burge, Colleen A; Closek, Collin J; Friedman, Carolyn S; Groner, Maya L; Jenkins, Cody M; Shore-Maggio, Amanda; Welsh, Jennifer E

    2016-10-01

    Rapid environmental change is linked to increases in aquatic disease heightening the need to develop strategies to manage disease. Filter-feeding species are effective biofilters and can naturally mitigate disease risk to humans and wildlife. We review the role of filter-feeders, with an emphasis on bivalves, in altering disease outcomes via augmentation and reduction. Filtration can reduce transmission by removing pathogens from the water column via degradation and release of pathogens in pseudofeces. In other cases, filtration can increase pathogen transmission and disease risk. The effect of filtration on pathogen transmission depends on the selectivity of the filter-feeder, the degree of infectivity by the pathogen, the mechanism(s) of pathogen transmission and the ability of the pathogen to resist degradation. For example, some bacteria and viruses can resist degradation and accumulate within a filter-feeder leading to disease transmission to humans and other wildlife upon ingestion. Since bivalves can concentrate microorganisms, they are also useful as sentinels for the presence of pathogenic microorganisms. While somewhat less studied, other invertebrates, including ascidians and sponges may also provide ecosystem services by altering pathogen transmission. In all scenarios, climate change may affect the potential for filter-feeders to mitigate disease risk. We conclude that an assessment including empirical data and modeling of system-wide impacts should be conducted before selection of filter-feeders to mitigate disease. Such studies should consider physiology of the host and microbe and risk factors for negative impacts including augmentation of other pathogens. © The Author 2016. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

  11. 26 CFR 1.502-1 - Feeder organizations.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 7 2010-04-01 2010-04-01 true Feeder organizations. 1.502-1 Section 1.502-1...) INCOME TAXES (CONTINUED) Exempt Organizations § 1.502-1 Feeder organizations. (a) In the case of an organization operated for the primary purpose of carrying on a trade or business for profit, exemption is not...

  12. DNA repair in human cells

    International Nuclear Information System (INIS)

    Regan, J.D.; Carrier, W.L.; Kusano, I.; Furuno-Fukushi, I.; Dunn, W.C. Jr.; Francis, A.A.; Lee, W.H.

    1982-01-01

    Our primary objective is to elucidate the molecular events in human cells when cellular macromolecules such as DNA are damaged by radiation or chemical agents. We study and characterize (i) the sequence of DNA repair events, (ii) the various modalities of repair, (iii) the genetic inhibition of repair due to mutation, (iv) the physiological inhibition of repair due to mutation, (v) the physiological inhibition of repair due to biochemical inhibitors, and (vi) the genetic basis of repair. Our ultimate goals are to (i) isolate and analyze the repair component of the mutagenic and/or carcinogenic event in human cells, and (ii) elucidate the magnitude and significance of this repair component as it impinges on the practical problems of human irradiation or exposure to actual or potential chemical mutagens and carcinogens. The significance of these studies lies in (i) the ubiquitousness of repair (most organisms, including man, have several complex repair systems), (ii) the belief that mutagenic and carcinogenic events may arise only from residual (nonrepaired) lesions or that error-prone repair systems may be the major induction mechanisms of the mutagenic or carcinogenic event, and (iii) the clear association of repair defects and highly carcinogenic disease states in man [xeroderma pigmentosum (XP)

  13. [The characters and specific features of new human embryonic stem cells lines].

    Science.gov (United States)

    Krylova, T A; Kol'tsova, A M; Zenin, V V; Gordeeva, O F; Musorina, A S; Goriachaia, T S; Shlykova, S A; Kamenetskaia, Iu K; Pinaev, G P; Polianskaia, G G

    2009-01-01

    Four continuous human embryonic stem cell lines (SC1, SC2, SC3 and SC4), derived from the blastocysts has been described. The cell lines were cultivated on mitotically inactivated human feeder cells. The cell lines SC1 and SC2 have passed through 150 population doublings and the cell lines SC3 and SC4 -- near 120 populations doublings, which exceeds Hayflick limit sufficiently. These cell lines maintain high activity of alkaline phosphatase, expression of transcription factor OCT-4 and cell surface antigens (SSEA-4, TRA-1-60 and TRA-1-81), confirming their ESC status and human specificity. Immunofluorescent detection of antigens, characteristic of ectoderm, endoderm and mesoderm confirms the ability of these cells to retain their pluripotency under in vitro condition. PCR analysis revealed expression of six genes specific for pluripotent cells (OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO and LEFTYA). Correlation between the level of proliferative activity and the character of DNA-bound fluorescent staining was found. Fluorescent dyes, Hoechst 33342 and PI, produced diffuse staining of the nuclei in slowly proliferating cells of the SC1 and SC2 lines. In contrast, in actively proliferating cells of the SC3 and SC4 lines, the clear staining of the nuclei was observed. Upon changing the cultivation condition, proliferative activity of SC3 and SC4 lines decreased and became similar to that of SC1 and SC2 lines. The character of the fluorescent staining of all these lines was also shown to be similar. These results show that quality of the fluorescent staining with Hoechst 33342 and PI reflects the level of proliferation. Possible causes and mechanisms of this feature of human ESC are discussed.

  14. Development of ceramic-free antenna feeder

    International Nuclear Information System (INIS)

    Moriyama, S.; Kimura, H.; Fujii, T.; Saigusa, M.; Arai, H.

    1994-01-01

    We have proposed a ceramics-free antenna feeder line employing a ridged waveguide as a local support for IC antenna of next-generation tokamaks. One fourth mock-up model of the all metal waveguide designed for the ITER ICRF system is fabricated and electrical characteristics of the model including the coaxial line - waveguide converter are measured. Power reflection coefficient of the model including the coax-waveguide converter to the input coaxial line is estimated to be less than 15% below the cut-off frequency of 107 MHz and less than 3% above the cut-off frequency. It is found that this ceramics-free antenna support employing a ridged waveguide is quite available for IC antenna of next-generation tokamaks. (author)

  15. Generation of a human induced pluripotent stem cell line (MUSIi001-A from caesarean section scar fibroblasts using Sendai viral vectors

    Directory of Open Access Journals (Sweden)

    Methichit Wattanapanitch

    2018-03-01

    Full Text Available We generated a human induced pluripotent stem cell (iPSC line from caesarean section scar fibroblasts of a 33-year-old healthy woman using transgene-free Sendai viral vectors under feeder-free condition. The established iPSC line, designated as MUSIi001-A, exhibited a normal karyotype, expressed pluripotent markers, differentiated into cells of three embryonic germ layers. Further analyses showed that the Sendai viral genome was absent at passage 25. The MUSIi001-A line can serve as a control for studying developmental biology and phenotypic comparison with disease-specific iPSCs.

  16. The human airway epithelial basal cell transcriptome.

    Directory of Open Access Journals (Sweden)

    Neil R Hackett

    2011-05-01

    Full Text Available The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population.Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the "human airway basal cell signature" as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels.The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem/progenitor cells of the human airway epithelium.

  17. Human regulatory B cells control the TFH cell response.

    Science.gov (United States)

    Achour, Achouak; Simon, Quentin; Mohr, Audrey; Séité, Jean-François; Youinou, Pierre; Bendaoud, Boutahar; Ghedira, Ibtissem; Pers, Jacques-Olivier; Jamin, Christophe

    2017-07-01

    Follicular helper T (T FH ) cells support terminal B-cell differentiation. Human regulatory B (Breg) cells modulate cellular responses, but their control of T FH cell-dependent humoral immune responses is unknown. We sought to assess the role of Breg cells on T FH cell development and function. Human T cells were polyclonally stimulated in the presence of IL-12 and IL-21 to generate T FH cells. They were cocultured with B cells to induce their terminal differentiation. Breg cells were included in these cultures, and their effects were evaluated by using flow cytometry and ELISA. B-cell lymphoma 6, IL-21, inducible costimulator, CXCR5, and programmed cell death protein 1 (PD-1) expressions increased on stimulated human T cells, characterizing T FH cell maturation. In cocultures they differentiated B cells into CD138 + plasma and IgD - CD27 + memory cells and triggered immunoglobulin secretions. Breg cells obtained by Toll-like receptor 9 and CD40 activation of B cells prevented T FH cell development. Added to T FH cell and B-cell cocultures, they inhibited B-cell differentiation, impeded immunoglobulin secretions, and expanded Foxp3 + CXCR5 + PD-1 + follicular regulatory T cells. Breg cells modulated IL-21 receptor expressions on T FH cells and B cells, and their suppressive activities involved CD40, CD80, CD86, and intercellular adhesion molecule interactions and required production of IL-10 and TGF-β. Human Breg cells control T FH cell maturation, expand follicular regulatory T cells, and inhibit the T FH cell-mediated antibody secretion. These novel observations demonstrate a role for the Breg cell in germinal center reactions and suggest that deficient activities might impair the T FH cell-dependent control of humoral immunity and might lead to the development of aberrant autoimmune responses. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  18. Endocannabinoids and Human Sperm Cells

    Directory of Open Access Journals (Sweden)

    Giovanna Zolese

    2010-10-01

    Full Text Available N-acylethanolamides (NAEs are naturally occurring signaling lipids consisting of amides and esters of long-chain polyunsaturated fatty acids. Usually they are present in a very small amounts in many mammalian tissues and cells, including human reproductive tracts and fluids. Recently, the presence of N-arachidonoylethanolamide (anandamide, AEA, the most characterised member of endocannabinoids, and its congeners palmitoylethanolamide (PEA and oleylethanolamide (OEA in seminal plasma, oviductal fluid, and follicular fluids was demonstrated. AEA has been shown to bind not only type-1 (CB1 and type-2 (CB2 cannabinoid receptors, but also type-1 vanilloid receptor (TRPV1, while PEA and OEA are inactive with respect to classical cannabinoid CB1 and CB2 but activate TRPV1 or peroxisome proliferator activate receptors (PPARs. This review concerns the most recent experimental data on PEA and OEA, endocannabinoid-like molecules which appear to exert their action exclusively on sperm cells with altered features, such as membrane characteristics and kinematic parameters. Their beneficial effects on these cells could suggest a possible pharmacological use of PEA and OEA on patients affected by some forms of idiopathic infertility.

  19. Generation of human-induced pluripotent stem cells from burn patient-derived skin fibroblasts using a non-integrative method.

    Science.gov (United States)

    Fu, Shangfeng; Ding, Jianwu; Liu, Dewu; Huang, Heping; Li, Min; Liu, Yang; Tu, Longxiang; Liu, Deming

    2018-01-01

    Patient specific induced pluripotent stem cells (iPSCs) have been recognized as a possible source of cells for skin tissue engineering. They have the potential to greatly benefit patients with large areas of burned skin or skin defects. However, the integration virus-based reprogramming method is associated with a high risk of genetic mutation and mouse embryonic fibroblast feeder-cells may be a pollutant. In the present study, human skin fibroblasts (HSFs) were successfully harvested from patients with burns and patient-specific iPSCs were generated using a non-integration method with a feeder-free approach. The octamer-binding transcription factor 4 (OCT4), sex-determining region Y box 2 (SOX2) and NANOG transcription factors were delivered using Sendai virus vectors. iPSCs exhibited representative human embryonic stem cell-like morphology and proliferation characteristics. They also expressed pluripotent markers, including OCT4, NANOG, SOX2, TRA181, stage-specific embryonic antigen 4 and TRA-160, and exhibited a normal karyotype. Teratoma and embryoid body formation revealed that iPSCs were able to differentiate into cells of all three germ layers in vitro and in vivo. The results of the present study demonstrate that HSFs derived from patients with burns, may be reprogrammed into stem cells with pluripotency, which provides a basis for cell‑based skin tissue engineering in the future.

  20. A Dynamic and Heuristic Phase Balancing Method for LV Feeders

    Directory of Open Access Journals (Sweden)

    Samad Taghipour Boroujeni

    2016-01-01

    Full Text Available Due to the single-phase loads and their stochastic behavior, the current in the distribution feeders is not balanced. In addition, the single-phase loads are located in different positions along the LV feeders. So the amount of the unbalanced load and its location affect the feeder losses. An unbalanced load causes the feeder losses and the voltage drop. Because of time-varying behavior of the single-phase loads, phase balancing is a dynamic and combinatorial problem. In this research, a heuristic and dynamic solution for the phase balancing of the LV feeders is proposed. In this method, it is supposed that the loads’ tie could be connected to all phases through a three-phase switch. The aim of the proposed method is to make the feeder conditions as balanced as possible. The amount and the location of single-phase loads are considered in the proposed phase balancing method. Since the proposed method needs no communication interface or no remote controller, it is inexpensive, simple, practical, and robust. Applying this method provides a distributed and dynamic phase balancing control. In addition, the feasibility of reducing the used switches is investigated. The ability of the proposed method in the phase balancing of the LV feeders is approved by carrying out some simulations.

  1. Effects of mechanical stimulation on the reprogramming of somatic cells into human-induced pluripotent stem cells.

    Science.gov (United States)

    Kim, Young Mi; Kang, Yun Gyeong; Park, So Hee; Han, Myung-Kwan; Kim, Jae Ho; Shin, Ji Won; Shin, Jung-Woog

    2017-06-08

    Mechanical stimuli play important roles in the proliferation and differentiation of adult stem cells. However, few studies on their effects on induced pluripotent stem cells (iPSCs) have been published. Human dermal fibroblasts were seeded onto flexible membrane-bottom plates, and infected with retrovirus expressing the four reprogramming factors OCT4, SOX2, KLF, and c-MYC (OSKM). The cells were subjected to equiaxial stretching (3% or 8% for 2, 4, or 7 days) and seeded on feeder cells (STO). The reprogramming into iPSCs was evaluated by the expression of pluripotent markers, in vitro differentiation into three germ layers, and teratoma formation. Equiaxial stretching enhanced reprogramming efficiency without affecting the viral transduction rate. iPSCs induced by transduction of four reprogramming factors and application of equiaxial stretching had characteristics typical of iPSCs in terms of pluripotency and differentiation potentials. This is the first study to show that mechanical stimuli can increase reprogramming efficiency. However, it did not enhance the infection rate, indicating that mechanical stimuli, defined as stretching in this study, have positive effects on reprogramming rather than on infection. Additional studies should evaluate the mechanism underlying the modulation of reprogramming of somatic cells into iPSCs.

  2. Stem cells in the human breast

    DEFF Research Database (Denmark)

    Petersen, Ole William; Polyak, Kornelia

    2010-01-01

    The origins of the epithelial cells participating in the development, tissue homeostasis, and cancer of the human breast are poorly understood. However, emerging evidence suggests a role for adult tissue-specific stem cells in these processes. In a hierarchical manner, these generate the two main...... mammary cell lineages, producing an increasing number of cells with distinct properties. Understanding the biological characteristics of human breast stem cells and their progeny is crucial in attempts to compare the features of normal stem cells and cancer precursor cells and distinguish these from...... nonprecursor cells and cells from the bulk of a tumor. A historical overview of research on human breast stem cells in primary tissue and in culture reveals the progress that has been made in this area, whereas a focus on the cell-of-origin and reprogramming that occurs during neoplastic conversion provides...

  3. Satellite cells in human skeletal muscle plasticity.

    Science.gov (United States)

    Snijders, Tim; Nederveen, Joshua P; McKay, Bryon R; Joanisse, Sophie; Verdijk, Lex B; van Loon, Luc J C; Parise, Gianni

    2015-01-01

    Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models.

  4. A Decentralized Storage Strategy for Residential Feeders with Photovoltaics

    DEFF Research Database (Denmark)

    Marra, Francesco; Yang, Guangya; Træholt, Chresten

    2014-01-01

    . The power sizing of the ESSs is performed with linear programming (LP) method, based on voltage sensitivity analysis. A Belgian residential LV feeder with private PV systems is used as a case study to demonstrate the effectiveness of the proposed strategy. Quantification of the required energy levels...... domestic energy storage systems (ESS). The traditional way of operating a domestic ESS to increase the local consumption rate does not take into account the need of voltage support in a feeder; the proposed storage concept improves the traditional one, by mitigating voltage rise due to PV in the feeder...

  5. Automation Selection and Sequencing of Traps for Vibratory Feeders

    DEFF Research Database (Denmark)

    Mathiesen, Simon; Ellekilde, Lars-Peter

    2017-01-01

    Vibratory parts feeders with mechanical orienting devices are used extensively in the assembly automation industry. Even so, the design process is based on trial-and-error approaches and is largely manual. In this paper, a methodology is presented for automatic design of this type of feeder....... The approach uses dynamic simulation for generating the necessary data for configuring a feeder with a sequence of mechanical orienting devices called traps, with the goal of reorienting all parts from a random to fixed orientation. Then, a fast algorithm for facilitating this configuration task automatically...

  6. YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers.

    Science.gov (United States)

    Brøchner, Christian B; Johansen, Julia S; Larsen, Lars A; Bak, Mads; Mikkelsen, Hanne B; Byskov, Anne Grete; Andersen, Claus Yding; Møllgård, Kjeld

    2012-03-01

    The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3β, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3β-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.

  7. c-Myc-Dependent Cell Competition in Human Cancer Cells.

    Science.gov (United States)

    Patel, Manish S; Shah, Heta S; Shrivastava, Neeta

    2017-07-01

    Cell Competition is an interaction between cells for existence in heterogeneous cell populations of multicellular organisms. This phenomenon is involved in initiation and progression of cancer where heterogeneous cell populations compete directly or indirectly for the survival of the fittest based on differential gene expression. In Drosophila, cells having lower dMyc expression are eliminated by cell competition through apoptosis when present in the milieu of cells having higher dMyc expression. Thus, we designed a study to develop c-Myc (human homolog) dependent in vitro cell competition model of human cancer cells. Cells with higher c-Myc were transfected with c-myc shRNA to prepare cells with lower c-Myc and then co-cultured with the same type of cells having a higher c-Myc in equal ratio. Cells with lower c-Myc showed a significant decrease in numbers when compared with higher c-Myc cells, suggesting "loser" and "winner" status of cells, respectively. During microscopy, engulfment of loser cells by winner cells was observed with higher expression of JNK in loser cells. Furthermore, elimination of loser cells was prevented significantly, when co-cultured cells were treated with the JNK (apoptosis) inhibitor. Above results indicate elimination of loser cells in the presence of winner cells by c-Myc-dependent mechanisms of cell competition in human cancer cells. This could be an important mechanism in human tumors where normal cells are eliminated by c-Myc-overexpressed tumor cells. J. Cell. Biochem. 118: 1782-1791, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. An in vitro expansion system for generation of human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential.

    Directory of Open Access Journals (Sweden)

    Ayaka Yanagida

    Full Text Available Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13(highCD133(+ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632, individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein and a cholangiocytic marker gene (cytokeratin 7, and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that

  9. Signaling hierarchy regulating human endothelial cell development.

    Science.gov (United States)

    Kelly, Melissa A; Hirschi, Karen K

    2009-05-01

    Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies. Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via coculture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that coexpress multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development. Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.

  10. Symmetry breaking in human neuroblastoma cells

    Science.gov (United States)

    Izumi, Hideki; Kaneko, Yasuhiko

    2014-01-01

    Asymmetric cell division (ACD) is a characteristic of cancer stem cells, which exhibit high malignant potential. However, the cellular mechanisms that regulate symmetric (self-renewal) and asymmetric cell divisions are mostly unknown. Using human neuroblastoma cells, we found that the oncosuppressor protein tripartite motif containing 32 (TRIM32) positively regulates ACD. PMID:27308367

  11. Damping considerations in CANDU feeder pipe design and analysis

    International Nuclear Information System (INIS)

    Usmani, S.A.; Saleem, M.A.; So, G.

    1990-01-01

    Recent developments in pipe damping indicate a trend towards more realistic and less conservative values, which result in less rigid and safer pipe designs. The CANDU-PHW (Canada deuterium uranium, pressurized heavy water) reactor feeder pipe designs have applied similar approaches which permit seismic qualifications without overly restraining these compact arrays of pipes to cater for the large creep and thermal anchor movement. This paper reviews the feeder design aspects, especially pertaining to the design provisions, experimental verification and analytical modelling for seismic qualification in the light of recent pipe dynamic developments. Using illustrative examples, comparison of seismic analysis results is provided for the ASME Code Case N-411 dampings, and those traditionally used in the feeder seismic qualification. The results confirm acceptability of the traditional approach which permit simplified analysis to demonstrate seismic qualificationqualification of CANDU feeder pipes

  12. Faecal nitrogen of browser and mixed feeder game species during ...

    African Journals Online (AJOL)

    Faecal nitrogen of browser and mixed feeder game species during different seasons. ... A practical measure of assessing periods of potential nutritional stress of game species is needed in the management of ... AJOL African Journals Online.

  13. Structural integrity investigations of feeder pipe ice plugging procedures

    International Nuclear Information System (INIS)

    Flaman, M.T.; Shah, N.N.

    1985-03-01

    A procedure involving the use of a liquid nitrogen cooled heat exchanger to form internal ice plugs in feeder pipes is routinely used in nuclear generating stations. The use of this procedure has caused concerns with regard to the safety of station maintenance personnel, and in regard to the integrity of the feeder pipes. This report describes the results of laboratory stress and pressure measurements which were performed on a feeder pipe section during ice plugging operations to investigate these concerns. From the results of this study, and from the results of previous studies of material behaviour at low temperatures, it has been determined that the ice plugging procedure can be performed on feeder pipes in a safe and effective manner

  14. Trophoblast lineage cells derived from human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying, E-mail: ying.chen@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Wang, Kai; Chandramouli, Gadisetti V.R. [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Knott, Jason G. [Developmental Epigenetics Laboratory, Department of Animal Science, Michigan State University (United States); Leach, Richard, E-mail: Richard.leach@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Department of Obstetrics, Gynecology and Women’s Health, Spectrum Health Medical Group (United States)

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  15. Trophoblast lineage cells derived from human induced pluripotent stem cells

    International Nuclear Information System (INIS)

    Chen, Ying; Wang, Kai; Chandramouli, Gadisetti V.R.; Knott, Jason G.; Leach, Richard

    2013-01-01

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro

  16. SAFIRE - a robotic inspection system for CANDU feeders

    International Nuclear Information System (INIS)

    Buckingham, R.

    2011-01-01

    The condition of primary circuit feeder pipes in CANDU reactors is relevant to the commercial viability and plant life. One known wear mechanism is external fretting between feeder pipes and adjacent services or support structures, particularly within the Upper Feeder Cabinet (UFC). Fretting leads to wall thinning which must not exceed certain agreed limits. Chafe shields have been added to protect the feeder pipes. Regular inspections are required of the chafe shields, feeder pipes and other structures that may cause feeder damage. Historically, the dose received by inspectors conducting this work has been significant. For this reason Ontario Power Generation has invested in a remotely operated robot system to conduct visual inspections within the UFC. This system, called SAFIRE for 'Snake-Arm Feeder Inspection Robot Equipment' has been deployed at Pickering during 2010 and 2011 and has been used to inspect areas that are extremely difficult to inspect with existing manual techniques. The 2011 scope of work included inspection of a total of 660 feeder pipes in three UFC quadrants, in two reactors. The full scope was completed over a one-month period in Autumn 2011 in which SAFIRE was used during 23, twelve hour shifts. This included two periods each of 72 hours of continuous operation using multiple teams of operators. SAFIRE is remote controlled delivery system for multiple cameras to record still images and video. The main system elements include a snake-arm robot mounted on a mobile vehicle. It can be controlled from up to 500m away using a fibre/copper connection. The snake-arm is 2.2m long, 25mm wide and has 18 degrees of freedom. It is designed to snake between the rows of feeder pipes to inspect feeder/hanger interfaces, both above and below the feeder cabinet catwalks. Future upgrades offer the potential to add additional tools to increase functionality. This paper describes the SAFIRE development process from inception to operational experience gained

  17. SAFIRE - a robotic inspection system for CANDU feeders

    Energy Technology Data Exchange (ETDEWEB)

    Buckingham, R. [OC Robotics, Bristol (United Kingdom)

    2011-07-01

    The condition of primary circuit feeder pipes in CANDU reactors is relevant to the commercial viability and plant life. One known wear mechanism is external fretting between feeder pipes and adjacent services or support structures, particularly within the Upper Feeder Cabinet (UFC). Fretting leads to wall thinning which must not exceed certain agreed limits. Chafe shields have been added to protect the feeder pipes. Regular inspections are required of the chafe shields, feeder pipes and other structures that may cause feeder damage. Historically, the dose received by inspectors conducting this work has been significant. For this reason Ontario Power Generation has invested in a remotely operated robot system to conduct visual inspections within the UFC. This system, called SAFIRE for 'Snake-Arm Feeder Inspection Robot Equipment' has been deployed at Pickering during 2010 and 2011 and has been used to inspect areas that are extremely difficult to inspect with existing manual techniques. The 2011 scope of work included inspection of a total of 660 feeder pipes in three UFC quadrants, in two reactors. The full scope was completed over a one-month period in Autumn 2011 in which SAFIRE was used during 23, twelve hour shifts. This included two periods each of 72 hours of continuous operation using multiple teams of operators. SAFIRE is remote controlled delivery system for multiple cameras to record still images and video. The main system elements include a snake-arm robot mounted on a mobile vehicle. It can be controlled from up to 500m away using a fibre/copper connection. The snake-arm is 2.2m long, 25mm wide and has 18 degrees of freedom. It is designed to snake between the rows of feeder pipes to inspect feeder/hanger interfaces, both above and below the feeder cabinet catwalks. Future upgrades offer the potential to add additional tools to increase functionality. This paper describes the SAFIRE development process from inception to operational experience

  18. Diffusion inside living human cells

    DEFF Research Database (Denmark)

    Leijnse, N.; Jeon, J. -H.; Loft, Steffen

    2012-01-01

    of the cell or within the nucleus. Also, granules in cells which are stressed by intense laser illumination or which have attached to a surface for a long period of time move in a more restricted fashion than those within healthy cells. For granules diffusing in healthy cells, in regions away from the cell...... cells. For these cells the exact diffusional pattern of a particular granule depends on the physiological state of the cell and on the localization of the granule within the cytoplasm. Granules located close to the actin rich periphery of the cell move less than those located towards to the center...

  19. Urban Bird Feeders Dominated by a Few Species and Individuals

    Directory of Open Access Journals (Sweden)

    Josie A. Galbraith

    2017-08-01

    Full Text Available The practice of garden bird feeding is a global phenomenon, involving millions of people and vast quantities of food annually. Many people engage in the practice of feeding assuming that birds gain some benefit from the food they provide, yet recent studies have revealed the potential for detrimental impacts as well. However, there is still a paucity of information on the impacts of feeding, including the ubiquity of these impacts among and within feeder-visiting species. Consistency in feeder use among birds is likely an important determinant of this. Individual birds and species that make frequent use of feeders are more likely to experience both the benefits and detrimental impacts of supplementary food. We investigated patterns of feeder use by garden birds visiting experimental feeding stations in Auckland, New Zealand, with the specific aim of determining whether use of supplementary food was consistent or variable among individuals and species. We used camera traps as well as Radio Frequency Identification (RFID technology to examine intra- and interspecific feeder visitation patterns and to discern species associations. Eleven bird species were detected using feeding stations, however, two introduced species (house sparrow Passer domesticus and spotted dove Streptopelia chinensis dominated visitation events. These species were present at feeders most frequently, with the largest conspecific group sizes. Significant associations were detected among a number of species, suggesting interspecific interactions are important in determining feeder use. We also found within-species differences in feeder use for all focal species, with individual variation greatest in house sparrows. Furthermore, season had an important influence on most visitation parameters. The observed individual and species-specific differences in supplementary food resource use imply that the impacts of garden bird feeding are not universal. Crucially, particularly given

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

    Science.gov (United States)

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

    2017-03-01

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

  1. Comparison of the Ex Vivo Expansion of UCB-Derived CD34+ in 3D DBM/MBA Scaffolds with USSC as a Feeder Layer.

    Science.gov (United States)

    Sadat Hashemi, Zahra; Forouzandeh Moghadam, Mahdi; Soleimani, Masoud

    2013-10-01

    Ex vivo expansion of hematopoitic stem cells is an alternative way to increase umbilical cord blood (UCB)-CD34+ cells for bone marrow transplantation. For this purpose demineralized bone matrix (DBM) and mineralized bone allograft (MBA) as two scaffolds based on bone matrix and stem cell niche, were simultaneously used to enhance the effect of human mesenchymal progenitor cells (MPCs) - unrestricted somatic stem cells (USSCs) - as a feeder layer. USSCs were isolated and characterized by morphological and immunological analysis then seeded on both scaffolds as a feeder layer. UCB-CD34(+) were isolated by MACS method and were co-culture expanded by USSC in 3D and 2D environments. After 3 weeks expansion, cells were counted and were assessed by karyotype, flow cytometry, clonogenic activity, and long-term culture-initiating cells (LTC-IC). Co-culture expansion in DBM and MBA was 29.22-fold and 27.77-fold, no significant differences in colony and LTC-IC were obtained. Maximum number of colonies belonged to the day 14 with the 73% CFU-GM (Colony Forming Unit- Granulocyte/Macrophage) in contrast to the day 0 which was BFU-E/CFU-E (Burst/Colony Forming Unit-Erythroid). Flow cytometry indicated that the percentage of CD34+ marker was decreased in USSC co-culture and the highest percentage was observed in simple 2D culture. Because of acid extraction in the DBM production process, mineral materials were removed and the protein background that was more flexible was presented. Therefore these results suggest that USSC-DBM can be a suitable ex vivo mimicry niche by intensifying of surface/volume ratio and supporting the stem cell differentiation and expansion.

  2. Molecular regulation of human hematopoietic stem cells

    NARCIS (Netherlands)

    van Galen, P.L.J.

    2014-01-01

    Peter van Galen focuses on understanding the determinants that maintain the stem cell state. Using human hematopoietic stem cells (HSCs) as a model, processes that govern self-renewal and tissue regeneration were investigated. Specifically, a role for microRNAs in balancing the human HSC

  3. Scalable Generation of Universal Platelets from Human Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Qiang Feng

    2014-11-01

    Full Text Available Human induced pluripotent stem cells (iPSCs provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid “surge” capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the β2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness.

  4. Assessment of in vitro drug resistance of human breast cancer cells subcultured from biopsy specimens

    Czech Academy of Sciences Publication Activity Database

    Krásná, Luboslava; Netíková, I.; Chaloupková, Alena; Taišlová, Eva; Zimovjanová, M.; Veselý, Pavel; Daneš, J.; Petruželka, L.; Matoušková, Eva

    2003-01-01

    Roč. 23, 3B (2003), s. 2593-2600 ISSN 0250-7005 R&D Projects: GA MZd NC6734 Institutional research plan: CEZ:AV0Z5052915 Keywords : Breast cancer * 3T3 feeder-layer culture technique * MTT test Subject RIV: EA - Cell Biology Impact factor: 1.347, year: 2003

  5. Clonal proliferation of cultured nonmalignant and malignant human breast epithelia

    International Nuclear Information System (INIS)

    Smith, H.S.; Lan, S.; Ceriani, R.; Hackett, A.J.; Stampfer, M.R.

    1981-01-01

    We have developed a method for clonal growth of human mammary epithelial cells of both nonmalignant and malignant origin. Plating efficiencies of 1 to 50% were obtained by seeding second-passage mammary epithelial cells on fibroblast feeder layers in an enriched medium composed of various hormones and growth factors, as well as conditioned media from three specific human cell lines. Single mammary epithelial cells seeded sparsely onto the fibroblasts underwent at least eight population doublings to form large, readily visible colonies. Optimal colony formation required both feeder cells and the enriched medium. Epithelial colonies containing at least 16 cells were visible 5 days postseeding, and these colonies continued to grow progressively. Plating efficiency and colony size were similar on ultraviolet-irradiated or nonirradiated fibroblasts. The number of colonies formed was proportional to the number of epithelial cells plated. The colonies were identified as epithelial by the presence of human mammary epithelial antigens

  6. Directed differentiation of basal forebrain cholinergic neurons from human pluripotent stem cells.

    Science.gov (United States)

    Hu, Yao; Qu, Zhuang-Yin; Cao, Shi-Ying; Li, Qi; Ma, Lixiang; Krencik, Robert; Xu, Min; Liu, Yan

    2016-06-15

    Basal forebrain cholinergic neurons (BFCNs) play critical roles in learning, memory and cognition. Dysfunction or degeneration of BFCNs may connect to neuropathology, such as Alzheimer's disease, Down's syndrome and dementia. Generation of functional BFCNs may contribute to the studies of cell-based therapy and pathogenesis that is related to learning and memory deficits. Here we describe a detail method for robust generation of BFCNs from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). In this method, BFCN progenitors are patterned from hESC or hiPSC-derived primitive neuroepithelial cells, with the treatment of sonic hedgehog (SHH) or combination with its agonist Purmorphamine, and by co-culturing with human astrocytes. At day 20, ∼90% hPSC-derived progenitors expressed NKX2.1, which is a transcriptional marker for MGE. Moreover, around 40% of NKX2.1+ cells co-expressed OLIG2 and ∼15% of NKX2.1+ cells co-expressed ISLET1, which are ventral markers. At day 35, ∼40% neurons robustly express ChAT, most of which are co-labeled with NKX2.1, ISLET1 and FOXG1, indicating the basal forebrain-like identity. At day 45, these neurons express mature neuronal markers MAP2, Synapsin, and VAChT. In this method, undefined conditions including genetic modification or cell-sorting are avoided. As a choice, feeder free conditions are used to avoid ingredients of animal origin. Moreover, Purmorphamine can be substituted for SHH to induce ventral progenitors effectively and economically. We provide an efficient method to generate BFCNs from multiple hPSC lines, which offers the potential application for disease modeling and pharmacological studies. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Generation of Hepatocyte-like Cells from Human Induced Pluripotent Stem (iPS) Cells By Co-culturing Embryoid Body Cells with Liver Non-parenchymal Cell Line TWNT-1

    International Nuclear Information System (INIS)

    Javed, M. S.; Yaqoob, N.; Iwamuro, M.; Kobayashi, N.; Fujiwara, T.

    2014-01-01

    Objective: To generate a homogeneous population of patient-specific hepatocyte-like cells (HLCs) from human iPS cells those show the morphologic and phenotypic properties of primary human hepatocytes. Study Design: An experimental study. Place and Duration of Study: Department of Surgery, Okayama University, Graduate School of Medicine, Japan, from April to December 2011. Methodology: Human iPS cells were generated and maintained on ES qualified matrigel coated plates supplemented with mTeSR medium or alternatively on mitotically inactivated MEF feeder layer in DMEM/F12 medium containing 20% KOSR, 4ng/ml bFGF-2, 1 x 10-4 M 2-mercaptoethanol, 1 mmol/L NEAA, 2mM L-glutamine and 1% penicillin-streptomycin. iPS cells were differentiated to HLCs by sequential culture using a four step differentiation protocol: (I) Generation of embryoid bodies (EBs) in suspension culture; (II) Induction of definitive endoderm (DE) from 2 days old EBs by growth in human activin-A (100 ng/ml) and basic fibroblasts growth factor (bFGF2) (100 ng/ml) on matrigel coated plates; (III) Induction of hepatic progenitors by co-culture with non-parenchymal human hepatic stellate cell line (TWNT-1); and (IV) Maturation by culture in dexamethasone. Characterization was performed by RT-PCR and functional assays. Results: The generated HLCs showed microscopically morphological phenotype of human hepatocytes, expressed liver specific genes (ASGPR, Albumin, AFP, Sox17, Fox A2), secreted human liver-specific proteins such as albumin, synthesized urea and metabolized ammonia. Conclusion: Functional HLCs were generated from human iPS cells, which could be used for autologus hepatocyte transplantation for liver failure and as in vitro model for determining the metabolic and toxicological properties of drug compounds. (author)

  8. Human monoclonal antibodies reactive with human myelomonocytic leukemia cells.

    Science.gov (United States)

    Posner, M R; Santos, D J; Elboim, H S; Tumber, M B; Frackelton, A R

    1989-04-01

    Peripheral blood mononuclear cells from a patient with chronic myelogenous leukemia (CML), in remission, were depleted of CD8-positive T-cells and cultured with Epstein-Barr virus. Four of 20 cultures (20%) secreted human IgG antibodies selectively reactive with the cell surfaces of certain human leukemia cell lines. Three polyclonal, Epstein-Barr virus-transformed, B-cell lines were expanded and fused with the human-mouse myeloma analogue HMMA2.11TG/O. Antibody from secreting clones HL 1.2 (IgG1), HL 2.1 (IgG3), and HL 3.1 (IgG1) have been characterized. All three react with HL-60 (promyelocytic), RWLeu4 (CML promyelocytic), and U937 (monocytic), but not with KG-1 (myeloblastic) or K562 (CML erythroid). There is no reactivity with T-cell lines, Burkitt's cell lines, pre-B-leukemia cell lines, or an undifferentiated CML cell line, BV173. Leukemic cells from two of seven patients with acute myelogenous leukemia and one of five with acute lymphocytic leukemia react with all three antibodies. Normal lymphocytes, monocytes, polymorphonuclear cells, red blood cells, bone marrow cells, and platelets do not react. Samples from patients with other diverse hematopoietic malignancies showed no reactivity. Immunoprecipitations suggest that the reactive antigen(s) is a lactoperoxidase iodinatable series of cell surface proteins with molecular weights of 42,000-54,000 and a noniodinatable protein with a molecular weight of 82,000. Based on these data these human monoclonal antibodies appear to react with myelomonocytic leukemic cells and may detect a leukemia-specific antigen or a highly restricted differentiation antigen.

  9. Human hematopoietic cell culture, transduction, and analyses

    DEFF Research Database (Denmark)

    Bonde, Jesper; Wirthlin, Louisa; Kohn, Donald B

    2008-01-01

    This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34(+) cells, transduction of these cells with a retroviral vector on fibronectin-coated plates, assaying the efficiency of transduction, and establishing long-te...

  10. The lifetime of hypoxic human tumor cells

    International Nuclear Information System (INIS)

    Durand, Ralph E.; Sham, Edward

    1998-01-01

    Purpose: For hypoxic and anoxic cells in solid tumors to be a therapeutic problem, they must live long enough to be therapeutically relevant, or else be rapidly recruited into the proliferating compartment during therapy. We have, therefore, estimated lifetime and recruitment rate of hypoxic human tumor cells in multicell spheroids in vitro, or in xenografted tumors in SCID mice. Materials and Methods: Cell turnover was followed by flow cytometry techniques, using antibodies directed at incorporated halogenated pyrimidines. The disappearance of labeled cells was quantified, and verified to be cell loss rather than label dilution. Repopulation was studied in SiHa tumor xenografts during twice-daily 2.5-Gy radiation exposures. Results: The longevity of hypoxic human tumor cells in spheroids or xenografts exceeded that of rodent cell lines, and cell turnover was slower in xenografts than under static growth as spheroids. Human tumor cells remained viable in the hypoxic regions of xenografts for 4-10 days, compared to 3-5 days in spheroids, and 1-3 days for most rodent cells in spheroids. Repopulation was observed within the first few radiation treatments for the SiHa xenografts and, with accumulated doses of more than 10 Gy, virtually all recovered cells had progressed through at least one S-phase. Conclusion: Our results suggest an important difference in the ability of human vs. rodent tumor cells to withstand hypoxia, and raise questions concerning the increased longevity seen in vivo relative to the steady-state spheroid system

  11. Feeder use predicts both acquisition and transmission of a contagious pathogen in a North American songbird.

    Science.gov (United States)

    Adelman, James S; Moyers, Sahnzi C; Farine, Damien R; Hawley, Dana M

    2015-09-22

    Individual heterogeneity can influence the dynamics of infectious diseases in wildlife and humans alike. Thus, recent work has sought to identify behavioural characteristics that contribute disproportionately to individual variation in pathogen acquisition (super-receiving) or transmission (super-spreading). However, it remains unknown whether the same behaviours enhance both acquisition and transmission, a scenario likely to result in explosive epidemics. Here, we examined this possibility in an ecologically relevant host-pathogen system: house finches and their bacterial pathogen, Mycoplasma gallisepticum, which causes severe conjunctivitis. We examined behaviours likely to influence disease acquisition (feeder use, aggression, social network affiliations) in an observational field study, finding that the time an individual spends on bird feeders best predicted the risk of conjunctivitis. To test whether this behaviour also influences the likelihood of transmitting M. gallisepticum, we experimentally inoculated individuals based on feeding behaviour and tracked epidemics within captive flocks. As predicted, transmission was fastest when birds that spent the most time on feeders initiated the epidemic. Our results suggest that the same behaviour underlies both pathogen acquisition and transmission in this system and potentially others. Identifying individuals that exhibit such behaviours is critical for disease management. © 2015 The Author(s).

  12. Urbanization affects neophilia and risk-taking at bird-feeders

    Science.gov (United States)

    Tryjanowski, Piotr; Møller, Anders Pape; Morelli, Federico; Biaduń, Waldemar; Brauze, Tomasz; Ciach, Michał; Czechowski, Paweł; Czyż, Stanisław; Dulisz, Beata; Goławski, Artur; Hetmański, Tomasz; Indykiewicz, Piotr; Mitrus, Cezary; Myczko, Łukasz; Nowakowski, Jacek J.; Polakowski, Michał; Takacs, Viktoria; Wysocki, Dariusz; Zduniak, Piotr

    2016-06-01

    Urban environments cover vast areas with a high density of humans and their dogs and cats causing problems for exploitation of new resources by wild animals. Such resources facilitate colonization by individuals with a high level of neophilia predicting that urban animals should show more neophilia than rural conspecifics. We provided bird-feeders across urban environments in 14 Polish cities and matched nearby rural habitats, testing whether the presence of a novel item (a brightly coloured green object made out of gum with a tuft of hair) differentially delayed arrival at feeders in rural compared to urban habitats. The presence of a novel object reduced the number of great tits Parus major, but also the total number of all species of birds although differentially so in urban compared to rural areas. That was the case independent of the potentially confounding effects of temperature, population density of birds, and the abundance of cats, dogs and pedestrians. The number of great tits and the total number of birds attending feeders increased in urban compared to rural areas independent of local population density of birds. This implies that urban birds have high levels of neophilia allowing them to readily exploit unpredictable resources in urban environments.

  13. Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells.

    Science.gov (United States)

    Burton, Peter; Adams, David R; Abraham, Achamma; Allcock, Robert W; Jiang, Zhong; McCahill, Angela; Gilmour, Jane; McAbney, John; Kaupisch, Alexandra; Kane, Nicole M; Baillie, George S; Baker, Andrew H; Milligan, Graeme; Houslay, Miles D; Mountford, Joanne C

    2010-12-15

    hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure-activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.

  14. Human Pluripotent Stem Cell Differentiation into Functional Epicardial Progenitor Cells

    NARCIS (Netherlands)

    Guadix, Juan Antonio; Orlova, Valeria V.; Giacomelli, Elisa; Bellin, Milena; Ribeiro, Marcelo C.; Mummery, Christine L.; Pérez-Pomares, José M.; Passier, Robert

    2017-01-01

    Human pluripotent stem cells (hPSCs) are widely used to study cardiovascular cell differentiation and function. Here, we induced differentiation of hPSCs (both embryonic and induced) to proepicardial/epicardial progenitor cells that cover the heart during development. Addition of retinoic acid (RA)

  15. Assessment of CANDU feeders subject to flow accelerated corrosion

    Energy Technology Data Exchange (ETDEWEB)

    Iyer, S. [Atomic Energy of Canada Ltd., Mississauga, Ontario (Canada); Slade, J.P. [New Brunswick Power, Point Lepreau Generating Station, Lepreau, New Brunswick (Canada)

    2003-07-01

    'Full Text:' Inspections of CANDU feeders have indicated greater than expected wall thinning of outlet feeders. This wall thinning is attributed to Flow-Accelerated Corrosion (FAC). The rate of wall loss due to FAC is highest in the close radius bends near the end fitting. The minimum allowed thickness for a feeder pipes is based on design pressure during the design stage. Extended operation of the thinned feeders beyond their design basis, i.e., operation of feeders with thickness below design pressure based minimum thickness has economic benefits for the utilities. In such cases, it is important to establish the remaining life and evaluate the adequacy of the components for safe operation. ASME Code Case N-597 provides the guidelines for acceptance for continued service of Classes 2 and 3 piping components experiencing wall thinning during operation. However, for Class 1 systems, the Code Case recommends that the owner develop the methodology and criteria for the assessment of wall thinning. Therefore, under the CANDU Owner's Group's (COG) Feeder Integrity Joint Program (FIJP), the 'Fitness for Service Guidelines (FFSG) for Feeders Affected by Wall Thinning in Operating CANDU Reactors' was developed and subsequently conditionally approved by CNSC. This paper illustrates the underlying concepts in the FFSG methodology and its benefits to utilities. Specific examples of the application and benefits of the FFSG at Point Lepreau G.S. are described in this paper. The assessment of feeders is based on the requirements of the construction Code (Section III of the ASME Boiler and Pressure Vessel Code): The following points briefly describe the assessment methodology. Satisfying the requirements of NB-3650 for design and service loadings are sufficient for continued service and extended life if the predicted minimum wall thickness of the component is greater than or equal to 90% of the pressure based thickness calculated as per NB-3641. The B

  16. Radiation effects on cultured human lymphoid cells

    International Nuclear Information System (INIS)

    Johansson, L.; Nilsson, K.; Carlsson, J.; Larsson, B.; Jakobsson, P.

    1981-01-01

    The cloning efficiency of human normal and malignant lymphoid cells is usually low. Radiation effects in vitro on such cells can therefore not be analysed with conventional cloning. However, this problem can be circumscribed by using the growth extrapolation method. A panel of human leukemia-lymphoma cell-lines representing Epstein-Barr virus carrying lymphoblastoid cells of presumed non-neoplastic derivation and neoplastic T- and B-lymphocytes was used to test the efficiency of this method. The sensitivity to radiation could be determined for all these cell types. The growth extrapolation method gave generally the same result as conventional cloning demonstrated by comparison with one exceptional cell-line with capacity for cloning in agar. The sensitivity varied largely between the different cell types. A common feature was that none of the cell lines had a good capacity to accumulate sublethal radiation injury. (Auth.)

  17. Energy Generation in the Human Body by the Human Cells ...

    African Journals Online (AJOL)

    We adapted the thermodynamics equation for energy generation in a diesel engine in modeling energy generation in human body by the human cells by doing a thorough study on both systems and saw that the process of energy generation is the same in them. We equally saw that the stages involved in energy generation ...

  18. Object categorization by wild ranging birds-Winter feeder experiments.

    Science.gov (United States)

    Nováková, Nela; Veselý, Petr; Fuchs, Roman

    2017-10-01

    The object categorization is only scarcely studied using untrained wild ranging animals and relevant stimuli. We tested the importance of the spatial position of features salient for categorization of a predator using wild ranging birds (titmice) visiting a winter feeder. As a relevant stimulus we used a dummy of a raptor, the European sparrowhawk (Accipiter nisus), placed at the feeding location. This dummy was designed to be dismantled into three parts and rearranged with the head in the correct position, in the middle or at the bottom of the dummy. When the birds had the option of visiting an alternative feeder with a dummy pigeon, they preferred this option to visiting the feeder with the dummy sparrowhawk with the head in any of the three positions. When the birds had the option of visiting an alternative feeder with an un-rearranged dummy sparrowhawk, they visited both feeders equally often, and very scarcely. This suggests that the titmice considered all of the sparrowhawk modifications as being dangerous, and equally dangerous as the un-rearranged sparrowhawk. The position of the head was not the most important cue for categorization. The presence of the key features was probably sufficient for categorization, and their mutual spatial position was of lower importance. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Seismic analysis of ITER fourth PF (Poloidal Field Coil) feeder

    International Nuclear Information System (INIS)

    Liu, Sumei; Chen, Wei; Song, Yuntao; Ni, Xiaojun; Wang, Zhongwei; Chen, Yonghua; Gong, Chenyu

    2014-01-01

    The ITER feeder systems connect the ITER magnet systems located inside the main cryostat to the cryo-plant, power-supply and control system interfaces outside the cryostat. The main purpose of the feeders is to convey the cryogenic supply and electrical power to the coils as well as house the instrumentation wiring. The PF busbar which carries 52 kA current will suffer from high Lorentz force due to the background magnetic field inspired by the coils and the self-field between every pair of busbars. Except their mechanical strength and thermal insulation performance must be achieved, the dynamic mechanism on PF structure should be assessed. This paper presents the simulation and seismic analysis on ITER 4th PF feeder including the Coil Terminal Box and S-bend Box (CTB and SBB), the Cryostat Feed-through (CFT), the In-Cryostat-Feeder (ICF), especially for the ground supports and main outer-tube firstly. This analysis aims to study seismic resistance on system design under local seismograms with floor response spectrum, the structural response vibration mode and response duration results of displacement, membrane stress, and bending stress on structure under different directions actuating signals were obtained by using the single-seismic spectrum analysis and Dead Weight analysis respectively. Based on the simulative and analytical results, the system seismic resistance and the integrity of the support structure in the 4th PF feeder have been studied and the detail design confirmed

  20. Tuft (caveolated) cells in two human colon carcinoma cell lines.

    Science.gov (United States)

    Barkla, D H; Whitehead, R H; Foster, H; Tutton, P J

    1988-09-01

    The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting from the apical surface. The microvilli are attached by a core of long microfilaments passing deep into the apical cytoplasm. Between the microvilli are parallel arrays of vesicles (caveoli) containing flocculent material. Two different but not mutually exclusive explanations for the presence of tuft cells are proposed. The first explanation is that tuft cells came from the resected tumour and have survived by mitotic division during subsequent passages. The second explanation suggests that tuft cells are the progeny of undifferentiated tumour cells. Descriptions of tuft cells in colon carcinomas are uncommon and possible reasons for this are presented. The morphology of tuft cells is consistent with that of a highly differentiated cell specialised for absorption, and these new models provide an opportunity to further investigate the structure and function of tuft cells.

  1. A Simple and Robust Method for Culturing Human-Induced Pluripotent Stem Cells in an Undifferentiated State Using Botulinum Hemagglutinin.

    Science.gov (United States)

    Kim, Mee-Hae; Matsubara, Yoshifumi; Fujinaga, Yukako; Kino-Oka, Masahiro

    2018-02-01

    Clinical and industrial applications of human-induced pluripotent stem cells (hiPSCs) is hindered by the lack of robust culture strategies capable of sustaining a culture in an undifferentiated state. Here, a simple and robust hiPSC-culture-propagation strategy incorporating botulinum hemagglutinin (HA)-mediated selective removal of cells deviating from an undifferentiated state is developed. After HA treatment, cell-cell adhesion is disrupted, and deviated cells detached from the central region of the colony to subsequently form tight monolayer colonies following prolonged incubation. The authors find that the temporal and dose-dependent activity of HA regulated deviated-cell removal and recoverability after disruption of cell-cell adhesion in hiPSC colonies. The effects of HA are confirmed under all culture conditions examined, regardless of hiPSC line and feeder-dependent or -free culture conditions. After routine application of our HA-treatment paradigm for serial passages, hiPSCs maintains expression of pluripotent markers and readily forms embryoid bodies expressing markers for all three germ-cell layers. This method enables highly efficient culturing of hiPSCs and use of entire undifferentiated portions without having to pick deviated cells manually. This simple and readily reproducible culture strategy is a potentially useful tool for improving the robust and scalable maintenance of undifferentiated hiPSC cultures. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Urban and rural habitats differ in number and type of bird feeders and in bird species consuming supplementary food.

    Science.gov (United States)

    Tryjanowski, Piotr; Skórka, Piotr; Sparks, Tim H; Biaduń, Waldemar; Brauze, Tomasz; Hetmański, Tomasz; Martyka, Rafał; Indykiewicz, Piotr; Myczko, Łukasz; Kunysz, Przemysław; Kawa, Piotr; Czyż, Stanisław; Czechowski, Paweł; Polakowski, Michał; Zduniak, Piotr; Jerzak, Leszek; Janiszewski, Tomasz; Goławski, Artur; Duduś, Leszek; Nowakowski, Jacek J; Wuczyński, Andrzej; Wysocki, Dariusz

    2015-10-01

    Bird feeding is one of the most widespread direct interactions between man and nature, and this has important social and environmental consequences. However, this activity can differ between rural and urban habitats, due to inter alia habitat structure, human behaviour and the composition of wintering bird communities. We counted birds in 156 squares (0.25 km(2) each) in December 2012 and again in January 2013 in locations in and around 26 towns and cities across Poland (in each urban area, we surveyed 3 squares and also 3 squares in nearby rural areas). At each count, we noted the number of bird feeders, the number of bird feeders with food, the type of feeders, additional food supplies potentially available for birds (bread offered by people, bins) and finally the birds themselves. In winter, urban and rural areas differ in the availability of food offered intentionally and unintentionally to birds by humans. Both types of food availability are higher in urban areas. Our findings suggest that different types of bird feeder support only those species specialized for that particular food type and this relationship is similar in urban and rural areas.

  3. Sensing radiosensitivity of human epidermal stem cells

    International Nuclear Information System (INIS)

    Rachidi, Walid; Harfourche, Ghida; Lemaitre, Gilles; Amiot, Franck; Vaigot, Pierre; Martin, Michele T.

    2007-01-01

    Purpose: Radiosensitivity of stem cells is a matter of debate. For mouse somatic stem cells, both radiosensitive and radioresistant stem cells have been described. By contrast, the response of human stem cells to radiation has been poorly studied. As epidermis is a radiosensitive tissue, we evaluated in the present work the radiosensitivity of cell populations enriched for epithelial stem cells of human epidermis. Methods and materials: The total keratinocyte population was enzymatically isolated from normal human skin. We used flow cytometry and antibodies against cell surface markers to isolate basal cell populations from human foreskin. Cell survival was measured after a dose of 2 Gy with the XTT assay at 72 h after exposure and with a clonogenic assay at 2 weeks. Transcriptome analysis using oligonucleotide microarrays was performed to assess the genomic cell responses to radiation. Results: Cell sorting based on two membrane proteins, α6 integrin and the transferrin receptor CD71, allowed isolation of keratinocyte populations enriched for the two types of cells found in the basal layer of epidermis: stem cells and progenitors. Both the XTT assay and the clonogenic assay showed that the stem cells were radioresistant whereas the progenitors were radiosensitive. We made the hypothesis that upstream DNA damage signalling might be different in the stem cells and used microarray technology to test this hypothesis. The stem cells exhibited a much more reduced gene response to a dose of 2 Gy than the progenitors, as we found that 6% of the spotted genes were regulated in the stem cells and 20% in the progenitors. Using Ingenuity Pathway Analysis software, we found that radiation exposure induced very specific pathways in the stem cells. The most striking responses were the repression of a network of genes involved in apoptosis and the induction of a network of cytokines and growth factors. Conclusion: These results show for the first time that keratinocyte

  4. Human Pluripotent Stem Cell Differentiation into Functional Epicardial Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Juan Antonio Guadix

    2017-12-01

    Full Text Available Summary: Human pluripotent stem cells (hPSCs are widely used to study cardiovascular cell differentiation and function. Here, we induced differentiation of hPSCs (both embryonic and induced to proepicardial/epicardial progenitor cells that cover the heart during development. Addition of retinoic acid (RA and bone morphogenetic protein 4 (BMP4 promoted expression of the mesodermal marker PDGFRα, upregulated characteristic (proepicardial progenitor cell genes, and downregulated transcription of myocardial genes. We confirmed the (proepicardial-like properties of these cells using in vitro co-culture assays and in ovo grafting of hPSC-epicardial cells into chick embryos. Our data show that RA + BMP4-treated hPSCs differentiate into (proepicardial-like cells displaying functional properties (adhesion and spreading over the myocardium of their in vivo counterpart. The results extend evidence that hPSCs are an excellent model to study (proepicardial differentiation into cardiovascular cells in human development and evaluate their potential for cardiac regeneration. : The authors have shown that hPSCs can be instructed in vitro to differentiate into a specific cardiac embryonic progenitor cell population called the proepicardium. Proepicardial cells are required for normal formation of the heart during development and might contribute to the development of cell-based therapies for heart repair. Keywords: human pluripotent stem cells, proepicardium, progenitor cells, cardiovascular, differentiation

  5. Toxicity of diuron in human cancer cells.

    Science.gov (United States)

    Huovinen, Marjo; Loikkanen, Jarkko; Naarala, Jonne; Vähäkangas, Kirsi

    2015-10-01

    Diuron is a substituted phenylurea used as a herbicide to control broadleaf and grass weeds and as a biocidal antifouling agent. Diuron is carcinogenic in rat urinary bladder and toxic to the reproductive system of oysters, sea urchins and lizards. The few studies carried out in human cells do not include the genotoxicity of diuron. We have investigated the toxicity of diuron in human breast adenocarcinoma (MCF-7) and human placental choriocarcinoma (BeWo) cells. The production of reactive oxygen species (ROS) was statistically significantly increased in both cell lines but only at the highest 200 μM concentration. Diuron clearly reduced the viability of BeWo, but not MCF-7 cells. The relative cell number was decreased in both cell lines indicative of inhibition of cell proliferation. In the Comet assay, diuron increased DNA fragmentation in MCF-7 but not in BeWo cells. The expressions of p53 protein, a marker for cell stress, and p21 protein, a transcriptional target of p53, were increased, but only in MCF-7 cells. In conclusion, our results suggest that diuron is cytotoxic and potentially genotoxic in a tissue-specific manner and that ROS play a role in its toxicity. Thus, exposure to diuron may exert harmful effects on fetal development and damage human health. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Regulatory T Cells in Human Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Dong-Jun Peng

    2012-01-01

    Full Text Available Multiple layers of suppressive components including regulatory T (TReg cells, suppressive antigen-presenting cells, and inhibitory cytokines form suppressive networks in the ovarian cancer microenvironment. It has been demonstrated that as a major suppressive element, TReg cells infiltrate tumor, interact with several types of immune cells, and mediate immune suppression through different molecular and cellular mechanisms. In this paper, we focus on human ovarian cancer and will discuss the nature of TReg cells including their subsets, trafficking, expansion, and function. We will briefly review the development of manipulation of TReg cells in preclinical and clinical settings.

  7. Genome Editing in Human Pluripotent Stem Cells.

    Science.gov (United States)

    Carlson-Stevermer, Jared; Saha, Krishanu

    2017-01-01

    Genome editing in human pluripotent stem cells (hPSCs) enables the generation of reporter lines and knockout cell lines. Zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 technology have recently increased the efficiency of proper gene editing by creating double strand breaks (DSB) at defined sequences in the human genome. These systems typically use plasmids to transiently transcribe nucleases within the cell. Here, we describe the process for preparing hPSCs for transient expression of nucleases via electroporation and subsequent analysis to create genetically modified stem cell lines.

  8. Modeling human infertility with pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Di Chen

    2017-05-01

    Full Text Available Human fertility is dependent upon the correct establishment and differentiation of the germline. This is because no other cell type in the body is capable of passing a genome and epigenome from parent to child. Terminally differentiated germline cells in the adult testis and ovary are called gametes. However, the initial specification of germline cells occurs in the embryo around the time of gastrulation. Most of our knowledge regarding the cell and molecular events that govern human germline specification involves extrapolating scientific principles from model organisms, most notably the mouse. However, recent work using next generation sequencing, gene editing and differentiation of germline cells from pluripotent stem cells has revealed that the core molecular mechanisms that regulate human germline development are different from rodents. Here, we will discuss the major molecular pathways required for human germline differentiation and how pluripotent stem cells have revolutionized our ability to study the earliest steps in human embryonic lineage specification in order to understand human fertility.

  9. Changes in thunderstorm characteristics due to feeder cloud merging

    Science.gov (United States)

    Sinkevich, Andrei A.; Krauss, Terrence W.

    2014-06-01

    Cumulus cloud merging is a complex dynamical and microphysical process in which two convective cells merge into a single cell. Previous radar observations and numerical simulations have shown a substantial increase in the maximum area, maximum echo top and maximum reflectivity as a result of the merging process. Although the qualitative aspects of merging have been well documented, the quantitative effects on storm properties remain less defined. Therefore, a statistical assessment of changes in storm characteristics due to merging is of importance. Further investigation into the effects of cloud merging on precipitation flux (Pflux) in a statistical manner provided the motivation for this study in the Asir region of Saudi Arabia. It was confirmed that merging has a strong effect on storm development in this region. The data analysis shows that an increase in the median of the distribution of maximum reflectivity was observed just after merging and was equal to 3.9 dBZ. A detailed analysis of the individual merge cases compared the merged storm Pflux and mass to the sum of the individual Feeder and Storm portions just before merging for each case. The merged storm Pflux increased an average of 106% over the 20-min period after merging, and the mass increased on average 143%. The merged storm clearly became larger and more severe than the sum of the two parts prior to merging. One consequence of this study is that any attempts to evaluate the precipitation enhancement effects of cloud seeding must also include the issue of cloud mergers because merging can have a significant effect on the results.

  10. A Markov chain model for CANDU feeder pipe degradation

    International Nuclear Information System (INIS)

    Datla, S.; Dinnie, K.; Usmani, A.; Yuan, X.-X.

    2008-01-01

    There is need for risk based approach to manage feeder pipe degradation to ensure safe operation by minimizing the nuclear safety risk. The current lack of understanding of some fundamental degradation mechanisms will result in uncertainty in predicting the rupture frequency. There are still concerns caused by uncertainties in the inspection techniques and engineering evaluations which should be addressed in the current procedures. A probabilistic approach is therefore useful in quantifying the risk and also it provides a tool for risk based decision making. This paper discusses the application of Markov chain model for feeder pipes in order to predict and manage the risks associated with the existing and future aging-related feeder degradation mechanisms. The major challenge in the approach is the lack of service data in characterizing the transition probabilities of the Markov model. The paper also discusses various approaches in estimating plant specific degradation rates. (author)

  11. Plasma membrane proteomics of human embryonic stem cells and human embryonal carcinoma cells.

    NARCIS (Netherlands)

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

    2008-01-01

    Human embryonic stem cells (hESCs) are of immense interest in regenerative medicine as they can self-renew indefinitely and can give rise to any adult cell type. Human embryonal carcinoma cells (hECCs) are the malignant counterparts of hESCs found in testis tumors. hESCs that have acquired

  12. Guidelines for human embryonic stem cell research

    National Research Council Canada - National Science Library

    Committee on Guidelines for Human Embryonic Stem Cell Research, National Research Council

    2005-01-01

    Since 1998, the volume of research being conducted using human embryonic stem (hES) cells has expanded primarily using private funds because of restrictions on the use of federal funds for such research...

  13. Satellite cells in human skeletal muscle plasticity

    Directory of Open Access Journals (Sweden)

    Tim eSnijders

    2015-10-01

    Full Text Available Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodelling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodelling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodelling in the wider context of satellite cell biology whose literature is largely based on animal and cell models.

  14. Configuration System for Simulation Based Design of Vibratory Bowl Feeders

    DEFF Research Database (Denmark)

    Hansson, Michael Natapon; Mathiesen, Simon; Ellekilde, Lars-Peter

    2017-01-01

    Vibratory bowl feeders are still among the most commonly used production equipment for automated part feeding, where parts are correctly oriented for further manipulation by being conveyed through a set of orienting devices. Designing vibratory bowl feeders involves selecting and sequencing...... a number of these devices that either reorients or rejects the part until a desirable orientation is achieved. To aid the designer in this task, this work presents a configuration system where knowledge of the behaviour for each device is acquired through dynamic simulation, and used to solve...

  15. Human stem cells for craniomaxillofacial reconstruction.

    Science.gov (United States)

    Jalali, Morteza; Kirkpatrick, William Niall Alexander; Cameron, Malcolm Gregor; Pauklin, Siim; Vallier, Ludovic

    2014-07-01

    Human stem cell research represents an exceptional opportunity for regenerative medicine and the surgical reconstruction of the craniomaxillofacial complex. The correct architecture and function of the vastly diverse tissues of this important anatomical region are critical for life supportive processes, the delivery of senses, social interaction, and aesthetics. Craniomaxillofacial tissue loss is commonly associated with inflammatory responses of the surrounding tissue, significant scarring, disfigurement, and psychological sequelae as an inevitable consequence. The in vitro production of fully functional cells for skin, muscle, cartilage, bone, and neurovascular tissue formation from human stem cells, may one day provide novel materials for the reconstructive surgeon operating on patients with both hard and soft tissue deficit due to cancer, congenital disease, or trauma. However, the clinical translation of human stem cell technology, including the application of human pluripotent stem cells (hPSCs) in novel regenerative therapies, faces several hurdles that must be solved to permit safe and effective use in patients. The basic biology of hPSCs remains to be fully elucidated and concerns of tumorigenicity need to be addressed, prior to the development of cell transplantation treatments. Furthermore, functional comparison of in vitro generated tissue to their in vivo counterparts will be necessary for confirmation of maturity and suitability for application in reconstructive surgery. Here, we provide an overview of human stem cells in disease modeling, drug screening, and therapeutics, while also discussing the application of regenerative medicine for craniomaxillofacial tissue deficit and surgical reconstruction.

  16. Human corneal epithelial subpopulations

    DEFF Research Database (Denmark)

    Søndergaard, Chris Bath

    2013-01-01

    Corneal epithelium is being regenerated throughout life by limbal epithelial stem cells (LESCs) believed to be located in histologically defined stem cell niches in corneal limbus. Defective or dysfunctional LESCs result in limbal stem cell deficiency (LSCD) causing pain and decreased visual acuity...... subpopulations in human corneal epithelium using a combination of laser capture microdissection and RNA sequencing for global transcriptomic profiling. We compared dissociation cultures, using either expansion on γ-irradiated NIH/3T3 feeder cells in serum-rich medium or expansion directly on plastic in serum...

  17. Human neutrophils facilitate tumor cell transendothelial migration.

    LENUS (Irish Health Repository)

    Wu, Q D

    2012-02-03

    Tumor cell extravasation plays a key role in tumor metastasis. However, the precise mechanisms by which tumor cells migrate through normal vascular endothelium remain unclear. In this study, using an in vitro transendothelial migration model, we show that human polymorphonuclear neutrophils (PMN) assist the human breast tumor cell line MDA-MB-231 to cross the endothelial barrier. We found that tumor-conditioned medium (TCM) downregulated PMN cytocidal function, delayed PMN apoptosis, and concomitantly upregulated PMN adhesion molecule expression. These PMN treated with TCM attached to tumor cells and facilitated tumor cell migration through different endothelial monolayers. In contrast, MDA-MB-231 cells alone did not transmigrate. FACScan analysis revealed that these tumor cells expressed high levels of intercellular adhesion molecule-1 (ICAM-1) but did not express CD11a, CD11b, or CD18. Blockage of CD11b and CD18 on PMN and of ICAM-1 on MDA-MB-231 cells significantly attenuated TCM-treated, PMN-mediated tumor cell migration. These tumor cells still possessed the ability to proliferate after PMN-assisted transmigration. These results indicate that TCM-treated PMN may serve as a carrier to assist tumor cell transendothelial migration and suggest that tumor cells can exploit PMN and alter their function to facilitate their extravasation.

  18. HLA engineering of human pluripotent stem cells.

    Science.gov (United States)

    Riolobos, Laura; Hirata, Roli K; Turtle, Cameron J; Wang, Pei-Rong; Gornalusse, German G; Zavajlevski, Maja; Riddell, Stanley R; Russell, David W

    2013-06-01

    The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their human leukocyte antigen (HLA) genes. This has led to the proposed use of histocompatible, patient-specific stem cells; however, the preparation of many different stem cell lines for clinical use is a daunting task. Here, we develop two distinct genetic engineering approaches that address this problem. First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line. A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population. Second, we derive HLA class I-negative cells by targeted disruption of both alleles of the Beta-2 Microglobulin (B2M) gene in ESCs. Mixed leukocyte reactions and peptide-specific HLA-restricted CD8(+) T cell responses were reduced in class I-negative cells that had undergone differentiation in embryoid bodies. These B2M(-/-) ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes. Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines.

  19. HLA Engineering of Human Pluripotent Stem Cells

    Science.gov (United States)

    Riolobos, Laura; Hirata, Roli K; Turtle, Cameron J; Wang, Pei-Rong; Gornalusse, German G; Zavajlevski, Maja; Riddell, Stanley R; Russell, David W

    2013-01-01

    The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their human leukocyte antigen (HLA) genes. This has led to the proposed use of histocompatible, patient-specific stem cells; however, the preparation of many different stem cell lines for clinical use is a daunting task. Here, we develop two distinct genetic engineering approaches that address this problem. First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line. A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population. Second, we derive HLA class I–negative cells by targeted disruption of both alleles of the Beta-2 Microglobulin (B2M) gene in ESCs. Mixed leukocyte reactions and peptide-specific HLA-restricted CD8+ T cell responses were reduced in class I–negative cells that had undergone differentiation in embryoid bodies. These B2M−/− ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes. Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines. PMID:23629003

  20. DNA fork displacement rates in human cells

    International Nuclear Information System (INIS)

    Kapp, L.N.; Painter, R.B.

    1981-01-01

    DNA fork displacement rates were measured in 20 human cell lines by a bromodeoxyuridine-313 nm photolysis technique. Cell lines included representatives of normal diploid, Fanconi's anemia, ataxia telangiectasia, xeroderma pigmentosum, trisomy-21 and several transformed lines. The average value for all the cell lines was 0.53 +- 0.08 μm/min. The average value for individual cell lines, however, displayed a 30% variation. Less than 10% of variation in the fork displacement rate appears to be due to the experimental technique; the remainder is probably due to true variation among the cell types and to culture conditions. (Auth.)

  1. DNA fork displacement rates in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Kapp, L.N.; Painter, R.B. (California Univ., San Francisco (USA). Lab. of Radiobiology)

    1981-11-27

    DNA fork displacement rates were measured in 20 human cell lines by a bromodeoxyuridine-313 nm photolysis technique. Cell lines included representatives of normal diploid, Fanconi's anemia, ataxia telangiectasia, xeroderma pigmentosum, trisomy-21 and several transformed lines. The average value for all the cell lines was 0.53 +- 0.08 ..mu..m/min. The average value for individual cell lines, however, displayed a 30% variation. Less than 10% of variation in the fork displacement rate appears to be due to the experimental technique; the remainder is probably due to true variation among the cell types and to culture conditions.

  2. Signaling hierarchy regulating human endothelial cell development

    Science.gov (United States)

    Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these stud...

  3. The biology of human innate lymphoid cells

    NARCIS (Netherlands)

    Bernink, J.H.J.

    2016-01-01

    In this thesis I performed studies to investigate the contribution of human innate lymphoid cells (ILCs) in maintaining the mucosal homeostasis, initiating and/or propagating inflammatory responses, but also - when not properly regulated - how these cells contribute to immunopathology. First I

  4. Haematopoietic stem and progenitor cells from human pluripotent stem cells

    Science.gov (United States)

    Sugimura, Ryohichi; Jha, Deepak Kumar; Han, Areum; Soria-Valles, Clara; da Rocha, Edroaldo Lummertz; Lu, Yi-Fen; Goettel, Jeremy A.; Serrao, Erik; Rowe, R. Grant; Malleshaiah, Mohan; Wong, Irene; Sousa, Patricia; Zhu, Ted N.; Ditadi, Andrea; Keller, Gordon; Engelman, Alan N.; Snapper, Scott B.; Doulatov, Sergei; Daley, George Q.

    2018-01-01

    A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. PMID:28514439

  5. Intrinsic radiation resistance in human chondrosarcoma cells

    International Nuclear Information System (INIS)

    Moussavi-Harami, Farid; Mollano, Anthony; Martin, James A.; Ayoob, Andrew; Domann, Frederick E.; Gitelis, Steven; Buckwalter, Joseph A.

    2006-01-01

    Human chondrosarcomas rarely respond to radiation treatment, limiting the options for eradication of these tumors. The basis of radiation resistance in chondrosarcomas remains obscure. In normal cells radiation induces DNA damage that leads to growth arrest or death. However, cells that lack cell cycle control mechanisms needed for these responses show intrinsic radiation resistance. In previous work, we identified immortalized human chondrosarcoma cell lines that lacked p16 ink4a , one of the major tumor suppressor proteins that regulate the cell cycle. We hypothesized that the absence of p16 ink4a contributes to the intrinsic radiation resistance of chondrosarcomas and that restoring p16 ink4a expression would increase their radiation sensitivity. To test this we determined the effects of ectopic p16 ink4a expression on chondrosarcoma cell resistance to low-dose γ-irradiation (1-5 Gy). p16 ink4a expression significantly increased radiation sensitivity in clonogenic assays. Apoptosis did not increase significantly with radiation and was unaffected by p16 ink4a transduction of chondrosarcoma cells, indicating that mitotic catastrophe, rather than programmed cell death, was the predominant radiation effect. These results support the hypothesis that p16 ink4a plays a role in the radiation resistance of chondrosarcoma cell lines and suggests that restoring p16 expression will improve the radiation sensitivity of human chondrosarcomas

  6. DNA repair in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Lechner, J.F.; Grafstrom, R.C.; Harris, C.C.

    1982-01-01

    The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz[a]anthracene; benzo[a]pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents

  7. Interaction of Staphylococci with Human B cells.

    Directory of Open Access Journals (Sweden)

    Tyler K Nygaard

    Full Text Available Staphylococcus aureus is a leading cause of human infections worldwide. The pathogen produces numerous molecules that can interfere with recognition and binding by host innate immune cells, an initial step required for the ingestion and subsequent destruction of microbes by phagocytes. To better understand the interaction of this pathogen with human immune cells, we compared the association of S. aureus and S. epidermidis with leukocytes in human blood. We found that a significantly greater proportion of B cells associated with S. epidermidis relative to S. aureus. Complement components and complement receptors were important for the binding of B cells with S. epidermidis. Experiments using staphylococci inactivated by ultraviolet radiation and S. aureus isogenic deletion mutants indicated that S. aureus secretes molecules regulated by the SaeR/S two-component system that interfere with the ability of human B cells to bind this bacterium. We hypothesize that the relative inability of B cells to bind S. aureus contributes to the microbe's success as a human pathogen.

  8. Does proximity to urban centres affect the dietary regime of marine benthic filter feeders?

    Science.gov (United States)

    Puccinelli, Eleonora; Noyon, Margaux; McQuaid, Christopher D.

    2016-02-01

    Threats to marine ecosystems include habitat destruction and degradation of water quality, resulting from land- and ocean-based human activities. Anthropogenic input causing modification of water quality, can affect primary productivity and thus food availability and quality for higher trophic levels. This is especially important for sedentary benthic intertidal communities, which rely on local food availability. We investigated the effect of urbanization on the dietary regime of four species of intertidal filter feeders (three barnacles and one mussel) at sites close to high-density cities and at sites far from heavily urbanized areas using fatty acid and stable isotope techniques. δ15N was significantly higher at urbanized sites compared to their corresponding control sites for all species with few exceptions, while no effect on δ13C was recorded. Barnacle fatty acid profiles were not affected by cities, while mussels from sites close to cities had fatty acid signatures with a higher proportion of polyunsaturated fatty acids (PUFA). We suggest that the enrichment in δ15N at urbanised sites reflects the influence of anthropogenically derived nitrogen directly linked to wastewater input from domestic and industrial sewage. Linked to this, the high proportion of PUFA in mussels at urbanized sites may reflect the influence of increased nitrogen concentrations on primary production and enhanced growth of large phytoplankton cells. The results indicate that anthropogenic effects can strongly influence the diets of benthic organisms, but these effects differ among taxa. Changes in the diet of such habitat forming species can affect their fitness and survival with potential effects on the populations associated with them.

  9. Microplastic in a macro filter feeder: humpback whale Megaptera novaeangliae

    NARCIS (Netherlands)

    Besseling, E.; Foekema, E.M.; Franeker, van J.A.; Leopold, M.F.; Bravo Rebolledo, E.; Kuehn, S.; Mielke, L.; Heberle-Bors, E.; Ijzer, J.; Kamminga, P.; Koelmans, A.A.

    2015-01-01

    Marine filter feeders are exposed to microplastic because of their selection of small particles as food source. Baleen whales feed by filtering small particles from large water volumes. Macroplastic was found in baleen whales before. This study is the first to show the presence of microplastic in

  10. Optimisation of Trap Design for Vibratory Bowl Feeders

    DEFF Research Database (Denmark)

    Mathiesen, Simon; Sørensen, Lars Carøe; Kraft, Dirk

    2018-01-01

    Vibratory bowl feeders (VBFs) are a widely used option for industrial part feeding, but their design is still largely manual. A subtask of VBF design is determining an optimal parameter set for the passive devices, called traps, which the VBF uses to ensure correct part orientation. This paper...

  11. Microplastic in a macro filter feeder: Humpback whale Megaptera novaeangliae.

    NARCIS (Netherlands)

    E., Besseling,; E.M., Foekema,; J.A. van, Franeker; Leopold, Mardik F; Kuhn, S.; Bravo Rebolledo, E.L.; Hese, E.; Mielke, L.; IJzer, J.|info:eu-repo/dai/nl/304839663; Kamminga, P.; Koelmans, A.A.

    2015-01-01

    Marine filter feeders are exposed to microplastic because of their selection of small particles as food source. Baleen whales feed by filtering small particles from large water volumes. Macroplastic was found in baleen whales before. This study is the first to show the presence of microplastic in

  12. A hybrid evolutionary algorithm for distribution feeder reconfiguration

    Indian Academy of Sciences (India)

    the reconfiguration of distribution networks has been proposed by .... An effective strategy to increase the loading margin of heavily loaded feeders is to ... social animals such as a flock of birds, a school of fish or a group of people that pursue.

  13. Isolation of Human Innate Lymphoid Cells.

    Science.gov (United States)

    Krabbendam, Lisette; Nagasawa, Maho; Spits, Hergen; Bal, Suzanne M

    2018-06-29

    Innate lymphoid cells (ILCs) are innate immune cells of lymphoid origin that have important effector and regulatory functions in the first line of defense against pathogens, but also regulate tissue homeostasis, remodeling, and repair. Their function mirrors T helper cells and cytotoxic CD8 + T lymphocytes, but they lack expression of rearranged antigen-specific receptors. Distinct ILC subsets are classified in group 1 ILCs (ILC1s), group 2 ILCs (ILC2s), and group 3 ILCs (ILC3s and lymphoid tissue-inducer cells), based on the expression of transcription factors and the cytokines they produce. As the frequency of ILCs is low, their isolation requires extensive depletion of other cell types. The lack of unique cell surface antigens further complicates the identification of these cells. Here, methods for ILC isolation and characterization from human peripheral blood and different tissues are described. © 2018 by John Wiley & Sons, Inc. © 2018 John Wiley & Sons, Inc.

  14. Higher-Density Culture in Human Embryonic Stem Cells Results in DNA Damage and Genome Instability

    Directory of Open Access Journals (Sweden)

    Kurt Jacobs

    2016-03-01

    Full Text Available Human embryonic stem cells (hESC show great promise for clinical and research applications, but their well-known proneness to genomic instability hampers the development to their full potential. Here, we demonstrate that medium acidification linked to culture density is the main cause of DNA damage and genomic alterations in hESC grown on feeder layers, and this even in the short time span of a single passage. In line with this, we show that increasing the frequency of the medium refreshments minimizes the levels of DNA damage and genetic instability. Also, we show that cells cultured on laminin-521 do not present this increase in DNA damage when grown at high density, although the (long-term impact on their genomic stability remains to be elucidated. Our results explain the high levels of genome instability observed over the years by many laboratories worldwide, and show that the development of optimal culture conditions is key to solving this problem.

  15. Human CD56bright NK Cells

    DEFF Research Database (Denmark)

    Michel, Tatiana; Poli, Aurélie; Cuapio, Angelica

    2016-01-01

    Human NK cells can be subdivided into various subsets based on the relative expression of CD16 and CD56. In particular, CD56(bright)CD16(-/dim) NK cells are the focus of interest. They are considered efficient cytokine producers endowed with immunoregulatory properties, but they can also become c...... NK cell subsets is not fully defined, nor is their precise hematopoietic origin. In this article, we summarize recent studies about CD56(bright) NK cells in health and disease and briefly discuss the current controversies surrounding them....

  16. Evaluating human cancer cell metastasis in zebrafish

    International Nuclear Information System (INIS)

    Teng, Yong; Xie, Xiayang; Walker, Steven; White, David T; Mumm, Jeff S; Cowell, John K

    2013-01-01

    In vivo metastasis assays have traditionally been performed in mice, but the process is inefficient and costly. However, since zebrafish do not develop an adaptive immune system until 14 days post-fertilization, human cancer cells can survive and metastasize when transplanted into zebrafish larvae. Despite isolated reports, there has been no systematic evaluation of the robustness of this system to date. Individual cell lines were stained with CM-Dil and injected into the perivitelline space of 2-day old zebrafish larvae. After 2-4 days fish were imaged using confocal microscopy and the number of metastatic cells was determined using Fiji software. To determine whether zebrafish can faithfully report metastatic potential in human cancer cells, we injected a series of cells with different metastatic potential into the perivitelline space of 2 day old embryos. Using cells from breast, prostate, colon and pancreas we demonstrated that the degree of cell metastasis in fish is proportional to their invasion potential in vitro. Highly metastatic cells such as MDA231, DU145, SW620 and ASPC-1 are seen in the vasculature and throughout the body of the fish after only 24–48 hours. Importantly, cells that are not invasive in vitro such as T47D, LNCaP and HT29 do not metastasize in fish. Inactivation of JAK1/2 in fibrosarcoma cells leads to loss of invasion in vitro and metastasis in vivo, and in zebrafish these cells show limited spread throughout the zebrafish body compared with the highly metastatic parental cells. Further, knockdown of WASF3 in DU145 cells which leads to loss of invasion in vitro and metastasis in vivo also results in suppression of metastasis in zebrafish. In a cancer progression model involving normal MCF10A breast epithelial cells, the degree of invasion/metastasis in vitro and in mice is mirrored in zebrafish. Using a modified version of Fiji software, it is possible to quantify individual metastatic cells in the transparent larvae to correlate with

  17. Novel Methods to Determine Feeder Locational PV Hosting Capacity and PV Impact Signatures

    Energy Technology Data Exchange (ETDEWEB)

    Reno, Matthew J. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Coogan, Kyle [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Seuss, John [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Broderick, Robert Joseph [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2017-05-01

    Often PV hosting capacity analysis is performed for a limited number of distribution feeders. For medium - voltage distribution feeders, previous results generally analyze less than 20 feeders, and then the results are extrapolated out to similar types of feeders. Previous hosting capacity research has often focused on determining a single value for the hosting capacity for the entire feeder, whereas this research expands previous hosting capacity work to investigate all the regions of the feeder that may allow many different hosting capacity values wit h an idea called locational hosting capacity (LHC)to determine the largest PV size that can be interconnected at different locations (buses) on the study feeders. This report discusses novel methods for analyzing PV interconnections with advanced simulati on methods. The focus is feeder and location - specific impacts of PV that determine the locational PV hosting capacity. Feeder PV impact signature are used to more precisely determine the local maximum hosting capacity of individual areas of the feeder. T he feeder signature provides improved interconnection screening with certain zones that show the risk of impact to the distribution feeder from PV interconnections.

  18. Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins

    OpenAIRE

    Hayato Fukusumi; Tomoko Shofuda; Yohei Bamba; Atsuyo Yamamoto; Daisuke Kanematsu; Yukako Handa; Keisuke Okita; Masaya Nakamura; Shinya Yamanaka; Hideyuki Okano; Yonehiro Kanemura

    2016-01-01

    Human neural progenitor cells (hNPCs) have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC) clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB) formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi). Our results showed that expandable hNPCs could be generated from hiPS...

  19. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  20. 3 CFR - Guidelines for Human Stem Cell Research

    Science.gov (United States)

    2010-01-01

    ... 3 The President 1 2010-01-01 2010-01-01 false Guidelines for Human Stem Cell Research Presidential Documents Other Presidential Documents Memorandum of July 30, 2009 Guidelines for Human Stem Cell Research..., scientifically worthy human stem cell research, including human embryonic stem cell research, to the extent...

  1. 21 CFR 864.2280 - Cultured animal and human cells.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cultured animal and human cells. 864.2280 Section... Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell lines from the tissue of humans or other animals which are used in various diagnostic...

  2. Round-bale feeder design affects hay waste and economics during horse feeding.

    Science.gov (United States)

    Martinson, K; Wilson, J; Cleary, K; Lazarus, W; Thomas, W; Hathaway, M

    2012-03-01

    Many horse owners find round bales convenient, less labor intensive, and more affordable than other hay types, but report an inability to control horse BW gain and excessive hay waste. The objectives were to compare hay waste, hay intake, and payback of 9 round-bale feeders and a no-feeder control when used during horse feeding. Nine round-bale feeders were tested: Cinch Net, Cone, Covered Cradle, Hayhut, Hay Sleigh, Ring, Tombstone, Tombstone Saver, and Waste Less. Each feeder design was placed on the ground in a dirt paddock. Five groups of 5 horses were fed in rotation for a 4-d period with each feeder. Every fourth day, groups were rotated among paddocks and a new round bale was placed in each feeder. In the 5 paddocks used, 5 feeders were installed for d 1 through 20, and the remaining 4 feeders and no-feeder control were installed for d 21 through 40. Groups of horses were sequentially assigned to feeders using two 5 × 5 Latin squares, the first for d 1 through 20, the second for d 21 through 40. Horse groups of similar age, BW, breed, and sex were formed from 25 Quarter Horse and Thoroughbred geldings and open mares (means: 11 yr; 541 kg of BW). Hay on the ground surrounding the feeder was collected daily, dried, and weighed. The total amount of hay removed around each feeder for a 4-d period was considered waste. Dry matter intake was estimated as the difference between hay disappearance and waste. Number of months for the reduction in waste to repay feeder cost (payback) were calculated using hay valued at $110/t, and improved feeder efficiency over the control. Feeder design did not affect hay intake (P > 0.05); all feeders resulted in an estimated hay intake of 2.0 to 2.4% BW; the no-feeder control resulted in a reduced intake of 1.3% BW (P = 0.001). Mean percentage of hay waste differed among feeders (P feeder control, 57%. Feeder design also affected payback (P feeder design affected hay waste and payback, but not estimated hay intake or BW change

  3. Cell pattern in adult human corneal endothelium.

    Directory of Open Access Journals (Sweden)

    Carlos H Wörner

    Full Text Available A review of the current data on the cell density of normal adult human endothelial cells was carried out in order to establish some common parameters appearing in the different considered populations. From the analysis of cell growth patterns, it is inferred that the cell aging rate is similar for each of the different considered populations. Also, the morphology, the cell distribution and the tendency to hexagonallity are studied. The results are consistent with the hypothesis that this phenomenon is analogous with cell behavior in other structures such as dry foams and grains in polycrystalline materials. Therefore, its driving force may be controlled by the surface tension and the mobility of the boundaries.

  4. Merkel cell distribution in the human eyelid

    Directory of Open Access Journals (Sweden)

    C.A. May

    2013-10-01

    Full Text Available Although Merkel cell carcinoma of the eye lid is reported frequently in the literature, only limited information exists about the distribution of Merkel cells in this tissue. Therefore, serial sections of 18 human cadaver eye lids (donors ages ranging between 63 and 97 years were stained for cytokeratin 20 in various planes. The overall appearance of Merkel cells in these samples was low and mainly located in the outer root layer of the cilia hair follicles. Merkel cells were more frequent in the middle, and almost not detectable at the nasal and temporal edges. The localization is in accordance with that of Merkel cell carcinoma, but concerning the scarce appearance within this adulthood group, a specific physiological role of these cells in the eye lid is difficult to establish.

  5. Haemopoietic progenitor cells in human peripheral blood

    International Nuclear Information System (INIS)

    Zwaan, F.E.

    1980-01-01

    The purpose of the investigation reported is to purify haemopoietic progenitor cells from human peripheral blood using density gradient centrifugation in order to isolate a progenitor cell fraction without immunocompetent cells. The purification technique of peripheral blood flow colony forming unit culture (CFU-c) by means of density gradient centrifugation and a combined depletion of various rosettes is described. The results of several 'in vitro' characteristics of purified CFU-c suspensions and of the plasma clot diffusion chamber culture technique are presented. Irradiation studies revealed that for both human bone marrow and peripheral blood the CFU-c were less radioresistant than clusters. Elimination of monocytes (and granulocytes) from the test suspensions induced an alteration in radiosensitivity pararmeters. The results obtained with the different techniques are described by analysing peripheral progenitor cell activity in myeloproliferative disorders. (Auth.)

  6. T-cell response in human leishmaniasis

    DEFF Research Database (Denmark)

    Kharazmi, A; Kemp, K; Ismail, A

    1999-01-01

    In the present communication we provide evidence for the existence of a Th1/Th2 dichotomy in the T-cell response to Leishmania antigens in human leishmaniasis. Our data suggest that the pattern of IL-4 and IFN-gamma response is polarised in these patients. Lymphocytes from individuals recovered...... from cutaneous leishmaniasis (CL) responded by IFN-gamma production following stimulation with Leishmania antigens whereas cells from patients recovered from visceral leishmaniasis (VL) showed a mixed pattern of IFN-gamma and IL-4 responses. The cells producing these cytokines were predominantly CD4......+. Furthermore, IL-10 plays an important role in the development of post kala azar dermal leishmaniasis (PKDL) from VL. The balance between the parasitic-specific T-cell response plays an important regulatory role in determining the outcome of Leishmania infections in humans....

  7. Lobaplatin arrests cell cycle progression in human hepatocellular carcinoma cells

    Directory of Open Access Journals (Sweden)

    Chen Chang-Jie

    2010-10-01

    Full Text Available Abstract Background Hepatocellular carcinoma (HCC still is a big burden for China. In recent years, the third-generation platinum compounds have been proposed as potential active agents for HCC. However, more experimental and clinical data are warranted to support the proposal. In the present study, the effect of lobaplatin was assessed in five HCC cell lines and the underlying molecular mechanisms in terms of cell cycle kinetics were explored. Methods Cytotoxicity of lobaplatin to human HCC cell lines was examined using MTT cell proliferation assay. Cell cycle distribution was determined by flow cytometry. Expression of cell cycle-regulated genes was examined at both the mRNA (RT-PCR and protein (Western blot levels. The phosphorylation status of cyclin-dependent kinases (CDKs and retinoblastoma (Rb protein was also examined using Western blot analysis. Results Lobaplatin inhibited proliferation of human HCC cells in a dose-dependent manner. For the most sensitive SMMC-7721 cells, lobaplatin arrested cell cycle progression in G1 and G2/M phases time-dependently which might be associated with the down-regulation of cyclin B, CDK1, CDC25C, phosphorylated CDK1 (pCDK1, pCDK4, Rb, E2F, and pRb, and the up-regulation of p53, p21, and p27. Conclusion Cytotoxicity of lobaplatin in human HCC cells might be due to its ability to arrest cell cycle progression which would contribute to the potential use of lobaplatin for the management of HCC.

  8. Defined Conditions for the Isolation and Expansion of Basal Prostate Progenitor Cells of Mouse and Human Origin

    Directory of Open Access Journals (Sweden)

    Thomas Höfner

    2015-03-01

    Full Text Available Methods to isolate and culture primary prostate epithelial stem/progenitor cells (PESCs have proven difficult and ineffective. Here, we present a method to grow and expand both murine and human basal PESCs long term in serum- and feeder-free conditions. The method enriches for adherent mouse basal PESCs with a Lin−SCA-1+CD49f+TROP2high phenotype. Progesterone and sodium selenite are additionally required for the growth of human Lin−CD49f+TROP2high PESCs. The gene-expression profiles of expanded basal PESCs show similarities to ESCs, and NF-kB function is critical for epithelial differentiation of sphere-cultured PESCs. When transplanted in combination with urogenital sinus mesenchyme, expanded mouse and human PESCs generate ectopic prostatic tubules, demonstrating their stem cell activity in vivo. This novel method will facilitate the molecular, genomic, and functional characterization of normal and pathologic prostate glands of mouse and human origin.

  9. In vitro study of cell differentiation by two type mouse embryo stem cells on mono- and multilayer nanocarbon tubes

    Science.gov (United States)

    Imai, Koichi; Akasaka, Tsukasa; Watari, Fumio; Tanoue, Akito; Nakamura, Kazuaki; Suese, Kazuhiko; Takashima, Hiromasa; Nishikawa, Tetsunari; Tanaka, Akio; Takeda, Shoji

    2012-09-01

    The effects of nanomaterials on human reproduction and development remain unknown. The risks of nanomaterials for future generations should be elucidated. Thus, it is important to establish an experimental method to accurately examine embryotoxicity. We previously investigated the myocardial cell differentiation of ES-D3 cells using monolayer (SWCNTs) and multilayer (MWCNTs) nanocarbon tubes. As a result, in spite of having the same carbon composition, the effects on the cell differentiation levels differed between the tubes. We investigated their cell differentiation and cytotoxic effects on EL M3 and ES-R1-EGFP B2/EGFP cells, which require feeder cells. As a result, myocardial pulse rates differed between the presence of SWCNTs and MWCNTs even when feeder cells existed between the samples and cells. The different surface structures of SWCNTs and MWCNTs may have influenced ES cell differentiation.

  10. Shape Memory of Human Red Blood Cells

    OpenAIRE

    Fischer, Thomas M.

    2004-01-01

    The human red cell can be deformed by external forces but returns to the biconcave resting shape after removal of the forces. If after such shape excursions the rim is always formed by the same part of the membrane, the cell is said to have a memory of its biconcave shape. If the rim can form anywhere on the membrane, the cell would have no shape memory. The shape memory was probed by an experiment called go-and-stop. Locations on the membrane were marked by spontaneously adhering latex spher...

  11. Feeding Against Gravity with Spot Feeders in High Silicon Ductile Iron

    DEFF Research Database (Denmark)

    Vedel-Smith, Nikolaj Kjelgaard; Tiedje, Niels Skat

    2014-01-01

    and classified using X-ray imaging and ultrasound analysis. The effect of the different feeder configurations were classified in reference to defect location, sleeve material, and feeder type, modulus, and location. The analysis showed that exothermal feeder sleeves with the right configurations can feed up......-hill against gravity. This effect may contribute to the thermal expansion created by the exothermal reaction. It was also found that the optimum feeder size does not scale linearly with the casting modulus but that larger casting modulus requires relatively smaller modulus feeders. The thermal gradient created...

  12. Human embryonic stem cell lines model experimental human cytomegalovirus latency.

    Science.gov (United States)

    Penkert, Rhiannon R; Kalejta, Robert F

    2013-05-28

    Herpesviruses are highly successful pathogens that persist for the lifetime of their hosts primarily because of their ability to establish and maintain latent infections from which the virus is capable of productively reactivating. Human cytomegalovirus (HCMV), a betaherpesvirus, establishes latency in CD34(+) hematopoietic progenitor cells during natural infections in the body. Experimental infection of CD34(+) cells ex vivo has demonstrated that expression of the viral gene products that drive productive infection is silenced by an intrinsic immune defense mediated by Daxx and histone deacetylases through heterochromatinization of the viral genome during the establishment of latency. Additional mechanistic details about the establishment, let alone maintenance and reactivation, of HCMV latency remain scarce. This is partly due to the technical challenges of CD34(+) cell culture, most notably, the difficulty in preventing spontaneous differentiation that drives reactivation and renders them permissive for productive infection. Here we demonstrate that HCMV can establish, maintain, and reactivate in vitro from experimental latency in cultures of human embryonic stem cells (ESCs), for which spurious differentiation can be prevented or controlled. Furthermore, we show that known molecular aspects of HCMV latency are faithfully recapitulated in these cells. In total, we present ESCs as a novel, tractable model for studies of HCMV latency.

  13. Human embryonic stem cells: preclinical perspectives

    Directory of Open Access Journals (Sweden)

    Sarda Kanchan

    2008-01-01

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

  14. Neocortical glial cell numbers in human brains

    DEFF Research Database (Denmark)

    Pelvig, D.P.; Pakkenberg, H.; Stark, A.K.

    2008-01-01

    Stereological cell counting was applied to post-mortem neocortices of human brains from 31 normal individuals, age 18-93 years, 18 females (average age 65 years, range 18-93) and 13 males (average age 57 years, range 19-87). The cells were differentiated in astrocytes, oligodendrocytes, microglia...... while the total astrocyte number is constant through life; finally males have a 28% higher number of neocortical glial cells and a 19% higher neocortical neuron number than females. The overall total number of neocortical neurons and glial cells was 49.3 billion in females and 65.2 billion in males...... and neurons and counting were done in each of the four lobes. The study showed that the different subpopulations of glial cells behave differently as a function of age; the number of oligodendrocytes showed a significant 27% decrease over adult life and a strong correlation to the total number of neurons...

  15. Melanopsin expressing human retinal ganglion cells

    DEFF Research Database (Denmark)

    Hannibal, Jens; Christiansen, Anders Tolstrup; Heegaard, Steffen

    2017-01-01

    microscopy and 3D reconstruction of melanopsin immunoreactive (-ir) RGCs, we applied the criteria used in mouse on human melanopsin-ir RGCs. We identified M1, displaced M1, M2, and M4 cells. We found two other subtypes of melanopsin-ir RGCs, which were named "gigantic M1 (GM1)" and "gigantic displaced M1...

  16. Clinical potentials of human pluripotent stem cells.

    Science.gov (United States)

    Mora, Cristina; Serzanti, Marialaura; Consiglio, Antonella; Memo, Maurizio; Dell'Era, Patrizia

    2017-08-01

    Aging, injuries, and diseases can be considered as the result of malfunctioning or damaged cells. Regenerative medicine aims to restore tissue homeostasis by repairing or replacing cells, tissues, or damaged organs, by linking and combining different disciplines including engineering, technology, biology, and medicine. To pursue these goals, the discipline is taking advantage of pluripotent stem cells (PSCs), a peculiar type of cell possessing the ability to differentiate into every cell type of the body. Human PSCs can be isolated from the blastocysts and maintained in culture indefinitely, giving rise to the so-called embryonic stem cells (ESCs). However, since 2006, it is possible to restore in an adult cell a pluripotent ESC-like condition by forcing the expression of four transcription factors with the rejuvenating reprogramming technology invented by Yamanaka. Then the two types of PSC can be differentiated, using standardized protocols, towards the cell type necessary for the regeneration. Although the use of these derivatives for therapeutic transplantation is still in the preliminary phase of safety and efficacy studies, a lot of efforts are presently taking place to discover the biological mechanisms underlying genetic pathologies, by differentiating induced PSCs derived from patients, and new therapies by challenging PSC-derived cells in drug screening.

  17. The negative impact of Wnt signaling on megakaryocyte and primitive erythroid progenitors derived from human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Prasuna Paluru

    2014-03-01

    Full Text Available The Wnt gene family consists of structurally related genes encoding secreted signaling molecules that have been implicated in many developmental processes, including regulation of cell fate and patterning during embryogenesis. Previously, we found that Wnt signaling is required for primitive or yolk sac-derived-erythropoiesis using the murine embryonic stem cell (ESC system. Here, we examine the effect of Wnt signaling on the formation of early hematopoietic progenitors derived from human ESCs. The first hematopoietic progenitor cells in the human ESC system express the pan-hematopoietic marker CD41 and the erythrocyte marker, glycophorin A or CD235. We have developed a novel serum-free, feeder-free, adherent differentiation system that can efficiently generate large numbers of CD41 + CD235+ cells. We demonstrate that this cell population contains progenitors not just for primitive erythroid and megakaryocyte cells but for the myeloid lineage as well and term this population the primitive common myeloid progenitor (CMP. Treatment of mesoderm-specified cells with Wnt3a led to a loss of hematopoietic colony-forming ability while the inhibition of canonical Wnt signaling with DKK1 led to an increase in the number of primitive CMPs. Canonical Wnt signaling also inhibits the expansion and/or survival of primitive erythrocytes and megakaryocytes, but not myeloid cells, derived from this progenitor population. These findings are in contrast to the role of Wnt signaling during mouse ESC differentiation and demonstrate the importance of the human ESC system in studying species-specific differences in development.

  18. Pigment Production Analysis in Human Melanoma Cells.

    Science.gov (United States)

    Hopkin, Amelia Soto; Paterson, Elyse K; Ruiz, Rolando; Ganesan, Anand K

    2016-05-25

    The human epidermal melanocyte is a highly specialized pigmented cell that serves to protect the epidermis from ultraviolet (UV) damage through the production of melanin, or melanogenesis. Misregulation in melanogenesis leading to either hyper- or hypo-pigmentation is found in human diseases such as malasma and vitiligo. Current therapies for these diseases are largely unsuccessful and the need for new therapies is necessary. In order to identify genes and or compounds that can alter melanogenesis, methods are required that can detect changes in pigment production as well as expression of key melanogenesis transcription factors and enzymes. Here we describe methods to detect changes in melanogenesis in a human melanoma cell line, MNT-1, by (1) analyzing pigment production by measuring the absorbance of melanin present by spectrophotometry, (2) analyzing transcript expression of potent regulators of melanogenesis by qunatitative reverse-transcription (RT)PCR and (3) analyzing protein expression of potent regulators of melanogenesis by Western blot (WB).

  19. Feederism: an exaggeration of a normative mate selection preference?

    Science.gov (United States)

    Terry, Lesley L; Suschinsky, Kelly D; Lalumière, Martin L; Vasey, Paul L

    2012-02-01

    Quinsey and Lalumière (1995) suggested that some, if not most, paraphilias are exaggerated manifestations of more normative and functional mate selection preferences. The present study tested whether Feederism, a fat fetish focused on erotic eating, feeding, and gaining weight, is an exaggeration of a sexual arousal pattern commonly seen in the general population. Thirty participants (15 men and 15 women) recruited from the general population were assessed using penile plethysmography and vaginal photoplethysmography, respectively. None of the participants were self-identified Feeders or Feedees. Participants were shown sexual, neutral, and feeding still images while listening to audio recordings of sexual, neutral, and feeding stories. Participants did not genitally respond to feeding stimuli. However, both men and women subjectively rated feeding stimuli as more sexually arousing than neutral stimuli. We discuss the discordance between physiological and self-reported sexual arousal in the context of sex differences in sexual concordance and implications for future research.

  20. Digital optical feeder links system for broadband geostationary satellite

    Science.gov (United States)

    Poulenard, Sylvain; Mège, Alexandre; Fuchs, Christian; Perlot, Nicolas; Riedi, Jerome; Perdigues, Josep

    2017-02-01

    An optical link based on a multiplex of wavelengths at 1.55μm is foreseen to be a valuable solution for the feeder link of the next generation of high-throughput geostationary satellite. The main satellite operator specifications for such link are an availability of 99.9% over the year, a capacity around 500Gbit/s and to be bent-pipe. Optical ground station networks connected to Terabit/s terrestrial fibers are proposed. The availability of the optical feeder link is simulated over 5 years based on a state-of-the-art cloud mask data bank and an atmospheric turbulence strength model. Yearly and seasonal optical feeder link availabilities are derived and discussed. On-ground and on-board terminals are designed to be compliant with 10Gbit/s per optical channel data rate taking into account adaptive optic systems to mitigate the impact of atmospheric turbulences on single-mode optical fiber receivers. The forward and return transmission chains, concept and implementation, are described. These are based on a digital transparent on-off keying optical link with digitalization of the DVB-S2 and DVB-RCS signals prior to the transmission, and a forward error correcting code. In addition, the satellite architecture is described taking into account optical and radiofrequency payloads as well as their interfaces.

  1. Human T Cell Memory: A Dynamic View

    Directory of Open Access Journals (Sweden)

    Derek C. Macallan

    2017-02-01

    Full Text Available Long-term T cell-mediated protection depends upon the formation of a pool of memory cells to protect against future pathogen challenge. In this review we argue that looking at T cell memory from a dynamic viewpoint can help in understanding how memory populations are maintained following pathogen exposure or vaccination. For example, a dynamic view resolves the apparent paradox between the relatively short lifespans of individual memory cells and very long-lived immunological memory by focussing on the persistence of clonal populations, rather than individual cells. Clonal survival is achieved by balancing proliferation, death and differentiation rates within and between identifiable phenotypic pools; such pools correspond broadly to sequential stages in the linear differentiation pathway. Each pool has its own characteristic kinetics, but only when considered as a population; single cells exhibit considerable heterogeneity. In humans, we tend to concentrate on circulating cells, but memory T cells in non-lymphoid tissues and bone marrow are increasingly recognised as critical for immune defence; their kinetics, however, remain largely unexplored. Considering vaccination from this viewpoint shifts the focus from the size of the primary response to the survival of the clone and enables identification of critical system pinch-points and opportunities to improve vaccine efficacy.

  2. Human neural progenitor cells decrease photoreceptor degeneration, normalize opsin distribution and support synapse structure in cultured porcine retina.

    Science.gov (United States)

    Mollick, Tanzina; Mohlin, Camilla; Johansson, Kjell

    2016-09-01

    Retinal neurodegenerative disorders like retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy and retinal detachment decrease retinal functionality leading to visual impairment. The pathological events are characterized by photoreceptor degeneration, synaptic disassembly, remodeling of postsynaptic neurons and activation of glial cells. Despite intense research, no effective treatment has been found for these disorders. The current study explores the potential of human neural progenitor cell (hNPC) derived factors to slow the degenerative processes in adult porcine retinal explants. Retinas were cultured for 3 days with or without hNPCs as a feeder layer and investigated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), immunohistochemical, western blot and quantitative real time-polymerase chain reaction (qRT-PCR) techniques. TUNEL showed that hNPCs had the capacity to limit photoreceptor cell death. Among cone photoreceptors, hNPC coculture resulted in better maintenance of cone outer segments and reduced opsin mislocalization. Additionally, maintained synaptic structural integrity and preservation of second order calbindin positive horizontal cells was also observed. However, Müller cell gliosis only seemed to be alleviated in terms of reduced Müller cell density. Our observations indicate that at 3 days of coculture, hNPC derived factors had the capacity to protect photoreceptors, maintain synaptic integrity and support horizontal cell survival. Human neural progenitor cell applied treatment modalities may be an effective strategy to help maintain retinal functionality in neurodegenerative pathologies. Whether hNPCs can independently hinder Müller cell gliosis by utilizing higher concentrations or by combination with other pharmacological agents still needs to be determined. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Development and function of human innate immune cells in a humanized mouse model.

    Science.gov (United States)

    Rongvaux, Anthony; Willinger, Tim; Martinek, Jan; Strowig, Till; Gearty, Sofia V; Teichmann, Lino L; Saito, Yasuyuki; Marches, Florentina; Halene, Stephanie; Palucka, A Karolina; Manz, Markus G; Flavell, Richard A

    2014-04-01

    Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models cannot support development of human innate immune cells, including myeloid cells and natural killer (NK) cells. Here we describe two mouse strains called MITRG and MISTRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked into their respective mouse loci. The human cytokines support the development and function of monocytes, macrophages and NK cells derived from human fetal liver or adult CD34(+) progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MITRG and MISTRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

  4. Isolation of Human Skin Dendritic Cell Subsets.

    Science.gov (United States)

    Gunawan, Merry; Jardine, Laura; Haniffa, Muzlifah

    2016-01-01

    Dendritic cells (DCs) are specialized leukocytes with antigen-processing and antigen-presenting functions. DCs can be divided into distinct subsets by anatomical location, phenotype and function. In human, the two most accessible tissues to study leukocytes are peripheral blood and skin. DCs are rare in human peripheral blood (skin covering an average total surface area of 1.8 m(2) has approximately tenfold more DCs than the average 5 L of total blood volume (Wang et al., J Invest Dermatol 134:965-974, 2014). DCs migrate spontaneously from skin explants cultured ex vivo, which provide an easy method of cell isolation (Larsen et al., J Exp Med 172:1483-1493, 1990; Lenz et al., J Clin Invest 92:2587-2596, 1993; Nestle et al., J Immunol 151:6535-6545, 1993). These factors led to the extensive use of skin DCs as the "prototype" migratory DCs in human studies. In this chapter, we detail the protocols to isolate DCs and resident macrophages from human skin. We also provide a multiparameter flow cytometry gating strategy to identify human skin DCs and to distinguish them from macrophages.

  5. Neocortical glial cell numbers in human brains.

    Science.gov (United States)

    Pelvig, D P; Pakkenberg, H; Stark, A K; Pakkenberg, B

    2008-11-01

    Stereological cell counting was applied to post-mortem neocortices of human brains from 31 normal individuals, age 18-93 years, 18 females (average age 65 years, range 18-93) and 13 males (average age 57 years, range 19-87). The cells were differentiated in astrocytes, oligodendrocytes, microglia and neurons and counting were done in each of the four lobes. The study showed that the different subpopulations of glial cells behave differently as a function of age; the number of oligodendrocytes showed a significant 27% decrease over adult life and a strong correlation to the total number of neurons while the total astrocyte number is constant through life; finally males have a 28% higher number of neocortical glial cells and a 19% higher neocortical neuron number than females. The overall total number of neocortical neurons and glial cells was 49.3 billion in females and 65.2 billion in males, a difference of 24% with a high biological variance. These numbers can serve as reference values in quantitative studies of the human neocortex.

  6. The Human Cell Surfaceome of Breast Tumors

    Science.gov (United States)

    da Cunha, Júlia Pinheiro Chagas; Galante, Pedro Alexandre Favoretto; de Souza, Jorge Estefano Santana; Pieprzyk, Martin; Carraro, Dirce Maria; Old, Lloyd J.; Camargo, Anamaria Aranha; de Souza, Sandro José

    2013-01-01

    Introduction. Cell surface proteins are ideal targets for cancer therapy and diagnosis. We have identified a set of more than 3700 genes that code for transmembrane proteins believed to be at human cell surface. Methods. We used a high-throuput qPCR system for the analysis of 573 cell surface protein-coding genes in 12 primary breast tumors, 8 breast cell lines, and 21 normal human tissues including breast. To better understand the role of these genes in breast tumors, we used a series of bioinformatics strategies to integrates different type, of the datasets, such as KEGG, protein-protein interaction databases, ONCOMINE, and data from, literature. Results. We found that at least 77 genes are overexpressed in breast primary tumors while at least 2 of them have also a restricted expression pattern in normal tissues. We found common signaling pathways that may be regulated in breast tumors through the overexpression of these cell surface protein-coding genes. Furthermore, a comparison was made between the genes found in this report and other genes associated with features clinically relevant for breast tumorigenesis. Conclusions. The expression profiling generated in this study, together with an integrative bioinformatics analysis, allowed us to identify putative targets for breast tumors. PMID:24195083

  7. Shape memory of human red blood cells.

    Science.gov (United States)

    Fischer, Thomas M

    2004-05-01

    The human red cell can be deformed by external forces but returns to the biconcave resting shape after removal of the forces. If after such shape excursions the rim is always formed by the same part of the membrane, the cell is said to have a memory of its biconcave shape. If the rim can form anywhere on the membrane, the cell would have no shape memory. The shape memory was probed by an experiment called go-and-stop. Locations on the membrane were marked by spontaneously adhering latex spheres. Shape excursions were induced by shear flow. In virtually all red cells, a shape memory was found. After stop of flow and during the return of the latex spheres to the original location, the red cell shape was biconcave. The return occurred by a tank-tread motion of the membrane. The memory could not be eliminated by deforming the red cells in shear flow up to 4 h at room temperature as well as at 37 degrees C. It is suggested that 1). the characteristic time of stress relaxation is >80 min and 2). red cells in vivo also have a shape memory.

  8. Human somatic cell nuclear transfer and cloning.

    Science.gov (United States)

    2012-10-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer (cloning)," last published in Fertil Steril 2000;74:873-6. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  9. Human induced pluripotent stem cell-derived models to investigate human cytomegalovirus infection in neural cells.

    Directory of Open Access Journals (Sweden)

    Leonardo D'Aiuto

    Full Text Available Human cytomegalovirus (HCMV infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs, neural progenitor cells (NPCs and neurons suggests that (i iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii Neural stem cells have impaired differentiation when infected by HCMV; (iii NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv most iPS-derived neurons are not permissive to HCMV infection; and (v infected neurons have impaired calcium influx in response to glutamate.

  10. Dopamine receptor repertoire of human granulosa cells

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    Kunz Lars

    2007-10-01

    Full Text Available Abstract Background High levels of dopamine (DA were described in human ovary and recently evidence for DA receptors in granulosa and luteal cells has been provided, as well. However, neither the full repertoire of ovarian receptors for DA, nor their specific role, is established. Human granulosa cells (GCs derived from women undergoing in vitro fertilization (IVF are an adequate model for endocrine cells of the follicle and the corpus luteum and were therefore employed in an attempt to decipher their DA receptor repertoire and functionality. Methods Cells were obtained from patients undergoing IVF and examined using cDNA-array, RT-PCR, Western blotting and immunocytochemistry. In addition, calcium measurements (with FLUO-4 were employed. Expression of two DA receptors was also examined by in-situ hybridization in rat ovary. Effects of DA on cell viability and cell volume were studied by using an ATP assay and an electronic cell counter system. Results We found members of the two DA receptor families (D1- and D2 -like associated with different signaling pathways in human GCs, namely D1 (as expected and D5 (both are Gs coupled and linked to cAMP increase and D2, D4 (Gi/Gq coupled and linked to IP3/DAG. D3 was not found. The presence of the trophic hormone hCG (10 IU/ml in the culture medium for several days did not alter mRNA (semiquantitative RT-PCR or protein levels (immunocytochemistry/Western blotting of D1,2,4,5 DA receptors. Expression of prototype receptors for the two families, D1 and D2, was furthermore shown in rat granulosa and luteal cells by in situ hybridization. Among the DA receptors found in human GCs, D2 expression was marked both at mRNA and protein levels and it was therefore further studied. Results of additional RT-PCR and Western blots showed two splice variants (D2L, D2S. Irrespective of these variants, D2 proved to be functional, as DA raised intracellular calcium levels. This calcium mobilizing effect of DA was observed

  11. Some Ethical Concerns About Human Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Zheng, Yue Liang

    2016-10-01

    Human induced pluripotent stem cells can be obtained from somatic cells, and their derivation does not require destruction of embryos, thus avoiding ethical problems arising from the destruction of human embryos. This type of stem cell may provide an important tool for stem cell therapy, but it also results in some ethical concerns. It is likely that abnormal reprogramming occurs in the induction of human induced pluripotent stem cells, and that the stem cells generate tumors in the process of stem cell therapy. Human induced pluripotent stem cells should not be used to clone human beings, to produce human germ cells, nor to make human embryos. Informed consent should be obtained from patients in stem cell therapy.

  12. Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

    Science.gov (United States)

    Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

    2013-03-01

    Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

  13. Cell Culture Assay for Human Noroviruses [response

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    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  14. Nicotinamide extends replicative lifespan of human cells.

    Science.gov (United States)

    Kang, Hyun Tae; Lee, Hyung Il; Hwang, Eun Seong

    2006-10-01

    We found that an ongoing application of nicotinamide to normal human fibroblasts not only attenuated expression of the aging phenotype but also increased their replicative lifespan, causing a greater than 1.6-fold increase in the number of population doublings. Although nicotinamide by itself does not act as an antioxidant, the cells cultured in the presence of nicotinamide exhibited reduced levels of reactive oxygen species (ROS) and oxidative damage products associated with cellular senescence, and a decelerated telomere shortening rate without a detectable increase in telomerase activity. Furthermore, in the treated cells growing beyond the original Hayflick limit, the levels of p53, p21WAF1, and phospho-Rb proteins were similar to those in actively proliferating cells. The nicotinamide treatment caused a decrease in ATP levels, which was stably maintained until the delayed senescence point. Nicotinamide-treated cells also maintained high mitochondrial membrane potential but a lower respiration rate and superoxide anion level. Taken together, in contrast to its demonstrated pro-aging effect in yeast, nicotinamide extends the lifespan of human fibroblasts, possibly through reduction in mitochondrial activity and ROS production.

  15. Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins

    Directory of Open Access Journals (Sweden)

    Hayato Fukusumi

    2016-01-01

    Full Text Available Human neural progenitor cells (hNPCs have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi. Our results showed that expandable hNPCs could be generated from hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes.

  16. Immortalization of human myogenic progenitor cell clone retaining multipotentiality

    International Nuclear Information System (INIS)

    Hashimoto, Naohiro; Kiyono, Tohru; Wada, Michiko R.; Shimizu, Shirabe; Yasumoto, Shigeru; Inagawa, Masayo

    2006-01-01

    Human myogenic cells have limited ability to proliferate in culture. Although forced expression of telomerase can immortalize some cell types, telomerase alone delays senescence of human primary cultured myogenic cells, but fails to immortalize them. In contrast, constitutive expression of both telomerase and the E7 gene from human papillomavirus type 16 immortalizes primary human myogenic cells. We have established an immortalized primary human myogenic cell line preserving multipotentiality by ectopic expression of telomerase and E7. The immortalized human myogenic cells exhibit the phenotypic characteristics of their primary parent, including an ability to undergo myogenic, osteogenic, and adipogenic terminal differentiation under appropriate culture conditions. The immortalized cells will be useful for both basic and applied studies aimed at human muscle disorders. Furthermore, immortalization by transduction of telomerase and E7 represents a useful method by which to expand human myogenic cells in vitro without compromising their ability to differentiate

  17. Scalable cultivation of human pluripotent stem cells on chemically-defined surfaces

    Science.gov (United States)

    Hsiung, Michael Chi-Wei

    Human stem cells (SCs) are classified as self-renewing cells possessing great ability in therapeutic applications due of their ability to differentiate along any major cell lineage in the human body. Despite their restorative potential, widespread use of SCs is hampered by strenuous control issues. Along with the need for strict xeno-free environments to sustain growth in culture, current methods for growing human pluripotent stem cells (hPSCs) rely on platforms which impede large-scale cultivation and therapeutic delivery. Hence, any progress towards development of large-scale culture systems is severely hindered. In a concentrated effort to develop a scheme that can serve as a model precursor for large scale SC propagation in clinical use, we have explored methods for cultivating hPSCs on completely defined surfaces. We discuss novel approaches with the potential to go beyond the limitations presented by current methods. In particular, we studied the cultivation of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) on surface which underwent synthetic or chemical modification. Current methods for hPSCs rely on animal-based extracellular matrices (ECMs) such as mouse embryonic fibroblasts (MEFs) or feeders and murine sacoma cell-derived substrates to facilitate their growth. While these layers or coatings can be used to maximize the output of hPSC production, they cannot be considered for clinical use because they risk introducing foreign pathogens into culture. We have identified and developed conditions for a completely defined xeno-free substrate used for culturing hPSCs. By utilizing coupling chemistry, we can functionalize ester groups on a given surface and conjugate synthetic peptides containing the arginine-glycine-aspartic acid (RGD) motif, known for their role in cell adhesion. This method offers advantages over traditional hPSC culture by keeping the modified substrata free of xenogenic response and can be scaled up in

  18. Human prostatic cancer cells, PC3, elaborate mitogenic activity which selectively stimulates human bone cells

    International Nuclear Information System (INIS)

    Perkel, V.S.; Mohan, S.; Herring, S.J.; Baylink, D.J.; Linkhart, T.A.

    1990-01-01

    Prostatic cancer typically produces osteoblastic metastases which are not attended by marrow fibrosis. In the present study we sought to test the hypothesis that prostatic cancer cells produce factor(s) which act selectively on human osteoblasts. Such a paracrine mechanism would explain the observed increase in osteoblasts, unaccompanied by an increase in marrow fibroblasts. To test this hypothesis we investigated the mitogenic activity released by the human prostatic tumor cell line, PC3. PC3 cells have been reported previously to produce mitogenic activity for cells that was relatively specific for rat osteoblasts compared to rat fibroblasts. However, the effects of this activity on human cells has not been examined previously. PC3-conditioned medium (CM) (5-50 micrograms CM protein/ml) stimulated human osteoblast proliferation by 200-950% yet did not stimulate human fibroblast proliferation ([3H]thymidine incorporation). PC3 CM also increased cell numbers in human osteoblast but not fibroblast cell cultures. To determine whether the osteoblast-specific mitogenic activity could be attributed to known bone growth factors, specific assays for these growth factors were performed. PC3 CM contained 10 pg insulin-like growth factor (IGF) I, less than 2 pg IGF II, 54 pg basic fibroblast growth factor, and 16 pg transforming growth factor beta/microgram CM protein. None of these growth factors alone or in combination could account for the observed osteoblast-specific PC3 cell-derived mitogenic activity. Furthermore, when 5 micrograms/ml PC3 CM was tested in combination with maximally effective concentrations of either basic fibroblast growth factor, IGF I, IGF II, or transforming growth factor beta, it produced an additive effect suggesting that PC3 CM stimulates osteoblast proliferation by a mechanism independent of these bone mitogens

  19. Efficient generation of megakaryocytes from human induced pluripotent stem cells using food and drug administration-approved pharmacological reagents.

    Science.gov (United States)

    Liu, Yanfeng; Wang, Ying; Gao, Yongxing; Forbes, Jessica A; Qayyum, Rehan; Becker, Lewis; Cheng, Linzhao; Wang, Zack Z

    2015-04-01

    Megakaryocytes (MKs) are rare hematopoietic cells in the adult bone marrow and produce platelets that are critical to vascular hemostasis and wound healing. Ex vivo generation of MKs from human induced pluripotent stem cells (hiPSCs) provides a renewable cell source of platelets for treating thrombocytopenic patients and allows a better understanding of MK/platelet biology. The key requirements in this approach include developing a robust and consistent method to produce functional progeny cells, such as MKs from hiPSCs, and minimizing the risk and variation from the animal-derived products in cell cultures. In this study, we developed an efficient system to generate MKs from hiPSCs under a feeder-free and xeno-free condition, in which all animal-derived products were eliminated. Several crucial reagents were evaluated and replaced with Food and Drug Administration-approved pharmacological reagents, including romiplostim (Nplate, a thrombopoietin analog), oprelvekin (recombinant interleukin-11), and Plasbumin (human albumin). We used this method to induce MK generation from hiPSCs derived from 23 individuals in two steps: generation of CD34(+)CD45(+) hematopoietic progenitor cells (HPCs) for 14 days; and generation and expansion of CD41(+)CD42a(+) MKs from HPCs for an additional 5 days. After 19 days, we observed abundant CD41(+)CD42a(+) MKs that also expressed the MK markers CD42b and CD61 and displayed polyploidy (≥16% of derived cells with DNA contents >4N). Transcriptome analysis by RNA sequencing revealed that megakaryocytic-related genes were highly expressed. Additional maturation and investigation of hiPSC-derived MKs should provide insights into MK biology and lead to the generation of large numbers of platelets ex vivo. ©AlphaMed Press.

  20. Differentiation of blood T cells: Reprogramming human induced pluripotent stem cells into neuronal cells

    Directory of Open Access Journals (Sweden)

    Ping-Hsing Tsai

    2015-06-01

    Conclusion: We have developed a safer method to generate integration-free and nonviral human iPSCs from adult somatic cells. This induction method will be useful for the derivation of human integration-free iPSCs and will also be applicable to the generation of iPSCs-derived neuronal cells for drug screening or therapeutics in the near future.

  1. Lake Bacterial Assemblage Composition Is Sensitive to Biological Disturbance Caused by an Invasive Filter Feeder.

    Science.gov (United States)

    Denef, Vincent J; Carrick, Hunter J; Cavaletto, Joann; Chiang, Edna; Johengen, Thomas H; Vanderploeg, Henry A

    2017-01-01

    One approach to improve forecasts of how global change will affect ecosystem processes is to better understand how anthropogenic disturbances alter bacterial assemblages that drive biogeochemical cycles. Species invasions are important contributors to global change, but their impacts on bacterial community ecology are rarely investigated. Here, we studied direct impacts of invasive dreissenid mussels (IDMs), one of many invasive filter feeders, on freshwater lake bacterioplankton. We demonstrated that direct effects of IDMs reduced bacterial abundance and altered assemblage composition by preferentially removing larger and particle-associated bacteria. While this increased the relative abundances of many free-living bacterial taxa, some were susceptible to filter feeding, in line with efficient removal of phytoplankton cells of <2 μm. This selective removal of particle-associated and larger bacteria by IDMs altered inferred bacterial functional group representation, defined by carbon and energy source utilization. Specifically, we inferred an increased relative abundance of chemoorganoheterotrophs predicted to be capable of rhodopsin-dependent energy generation. In contrast to the few previous studies that have focused on the longer-term combined direct and indirect effects of IDMs on bacterioplankton, our study showed that IDMs act directly as a biological disturbance to which freshwater bacterial assemblages are sensitive. The negative impacts on particle-associated bacteria, which have been shown to be more active than free-living bacteria, and the inferred shifts in functional group representation raise the possibility that IDMs may directly alter bacterially mediated ecosystem functions. IMPORTANCE Freshwater bacteria play fundamental roles in global elemental cycling and are an intrinsic part of local food webs. Human activities are altering freshwater environments, and much has been learned regarding the sensitivity of bacterial assemblages to a variety of

  2. Critical evaluation of gamma-irradiated serum used as feeder in the culture and demonstration of putative nanobacteria and calcifying nanoparticles.

    Directory of Open Access Journals (Sweden)

    Jan Martel

    Full Text Available The culture and demonstration of putative nanobacteria (NB and calcifying nanoparticles (CNP from human and animal tissues has relied primarily on the use of a culture supplement consisting of FBS that had been gamma-irradiated at a dose of 30 kGy (gamma-FBS. The use of gamma-FBS is based on the assumption that this sterilized fluid has been rid entirely of any residual NB/CNP, while it continues to promote the slow growth in culture of NB/CNP from human/animal tissues. We show here that gamma-irradiation (5-50 kGy produces extensive dose-dependent serum protein breakdown as demonstrated through UV and visible light spectrophotometry, fluorometry, Fourier-transformed infrared spectroscopy, and gel electrophoresis. Yet, both gamma-FBS and gamma-irradiated human serum (gamma-HS produce NB/CNP in cell culture conditions that are morphologically and chemically indistinguishable from their normal serum counterparts. Contrary to earlier claims, gamma-FBS does not enhance the formation of NB/CNP from several human body fluids (saliva, urine, ascites, and synovial fluid tested. In the presence of additional precipitating ions, both gamma-irradiated serum (FBS and HS and gamma-irradiated proteins (albumin and fetuin-A retain the inherent dual NB inhibitory and seeding capabilities seen also with their untreated counterparts. By gel electrophoresis, the particles formed from both gamma-FBS and gamma-HS are seen to have assimilated into their scaffold the same smeared protein profiles found in the gamma-irradiated sera. However, their protein compositions as identified by proteomics are virtually identical to those seen with particles formed from untreated serum. Moreover, particles derived from human fluids and cultured in the presence of gamma-FBS contain proteins derived from both gamma-FBS and the human fluid under investigation-a confusing and unprecedented scenario indicating that these particles harbor proteins from both the host tissue and the FBS

  3. Markers of T Cell Senescence in Humans

    Directory of Open Access Journals (Sweden)

    Weili Xu

    2017-08-01

    Full Text Available Many countries are facing the aging of their population, and many more will face a similar obstacle in the near future, which could be a burden to many healthcare systems. Increased susceptibility to infections, cardiovascular and neurodegenerative disease, cancer as well as reduced efficacy of vaccination are important matters for researchers in the field of aging. As older adults show higher prevalence for a variety of diseases, this also implies higher risk of complications, including nosocomial infections, slower recovery and sequels that may reduce the autonomy and overall quality of life of older adults. The age-related effects on the immune system termed as “immunosenescence” can be exemplified by the reported hypo-responsiveness to influenza vaccination of the elderly. T cells, which belong to the adaptive arm of the immune system, have been extensively studied and the knowledge gathered enables a better understanding of how the immune system may be affected after acute/chronic infections and how this matters in the long run. In this review, we will focus on T cells and discuss the surface and molecular markers that are associated with T cell senescence. We will also look at the implications that senescent T cells could have on human health and diseases. Finally, we will discuss the benefits of having these markers for investigators and the future work that is needed to advance the field of T cell senescence markers.

  4. Some software issues in mapping of power distribution feeders

    International Nuclear Information System (INIS)

    Mufti, I.A.

    1994-01-01

    This paper is about the in-house developed software for distribution feeders mapping project. It first gives birds eye view of the project, highlight its technical complexity in management and logistics, introduced by sheer size of the project. It gives an overview of the software developed and the moves on to describe circuit tracing, circuit model, leaves isolation (for tree structured network) and backtracking in more detail among many different parts of software, description of all which is not possible because of space limitations. (author)

  5. Generation and periodontal differentiation of human gingival fibroblasts-derived integration-free induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Xiaohui [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China); Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Li, Yang [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Li, Jingwen [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China); Li, Peng [Faculty of Dentistry, The University of Hong Kong, 34 Hospital Road, Hong Kong SAR (China); Liu, Yinan [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Wen, Jinhua, E-mail: jhwen@bjmu.edu.cn [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Luan, Qingxian, E-mail: kqluanqx@126.com [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China)

    2016-05-06

    Induced pluripotent stem cells (iPSCs) have been recognized as a promising cell source for periodontal tissue regeneration. However, the conventional virus-based reprogramming approach is associated with a high risk of genetic mutation and limits their therapeutic utility. Here, we successfully generated iPSCs from readily accessible human gingival fibroblasts (hGFs) through an integration-free and feeder-free approach via delivery of reprogramming factors of Oct4, Sox2, Klf4, L-myc, Lin28 and TP53 shRNA with episomal plasmid vectors. The iPSCs presented similar morphology and proliferation characteristics as embryonic stem cells (ESCs), and expressed pluripotent markers including Oct4, Tra181, Nanog and SSEA-4. Additionally, these cells maintained a normal karyotype and showed decreased CpG methylation ratio in the promoter regions of Oct4 and Nanog. In vivo teratoma formation assay revealed the development of tissues representative of three germ layers, confirming the acquisition of pluripotency. Furthermore, treatment of the iPSCs in vitro with enamel matrix derivative (EMD) or growth/differentiation factor-5 (GDF-5) significantly up-regulated the expression of periodontal tissue markers associated with bone, periodontal ligament and cementum respectively. Taken together, our data demonstrate that hGFs are a valuable cell source for generating integration-free iPSCs, which could be sequentially induced toward periodontal cells under the treatment of EMD and GDF-5. - Highlights: • Integration-free iPSCs are successfully generated from hGFs via an episomal approach. • EMD promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • GDF-5 promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • hGFs-derived iPSCs could be a promising cell source for periodontal regeneration.

  6. Generation and periodontal differentiation of human gingival fibroblasts-derived integration-free induced pluripotent stem cells

    International Nuclear Information System (INIS)

    Yin, Xiaohui; Li, Yang; Li, Jingwen; Li, Peng; Liu, Yinan; Wen, Jinhua; Luan, Qingxian

    2016-01-01

    Induced pluripotent stem cells (iPSCs) have been recognized as a promising cell source for periodontal tissue regeneration. However, the conventional virus-based reprogramming approach is associated with a high risk of genetic mutation and limits their therapeutic utility. Here, we successfully generated iPSCs from readily accessible human gingival fibroblasts (hGFs) through an integration-free and feeder-free approach via delivery of reprogramming factors of Oct4, Sox2, Klf4, L-myc, Lin28 and TP53 shRNA with episomal plasmid vectors. The iPSCs presented similar morphology and proliferation characteristics as embryonic stem cells (ESCs), and expressed pluripotent markers including Oct4, Tra181, Nanog and SSEA-4. Additionally, these cells maintained a normal karyotype and showed decreased CpG methylation ratio in the promoter regions of Oct4 and Nanog. In vivo teratoma formation assay revealed the development of tissues representative of three germ layers, confirming the acquisition of pluripotency. Furthermore, treatment of the iPSCs in vitro with enamel matrix derivative (EMD) or growth/differentiation factor-5 (GDF-5) significantly up-regulated the expression of periodontal tissue markers associated with bone, periodontal ligament and cementum respectively. Taken together, our data demonstrate that hGFs are a valuable cell source for generating integration-free iPSCs, which could be sequentially induced toward periodontal cells under the treatment of EMD and GDF-5. - Highlights: • Integration-free iPSCs are successfully generated from hGFs via an episomal approach. • EMD promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • GDF-5 promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • hGFs-derived iPSCs could be a promising cell source for periodontal regeneration.

  7. Efficient, long term production of monocyte-derived macrophages from human pluripotent stem cells under partly-defined and fully-defined conditions.

    Directory of Open Access Journals (Sweden)

    Bonnie van Wilgenburg

    Full Text Available Human macrophages are specialised hosts for HIV-1, dengue virus, Leishmania and Mycobacterium tuberculosis. Yet macrophage research is hampered by lack of appropriate cell models for modelling infection by these human pathogens, because available myeloid cell lines are, by definition, not terminally differentiated like tissue macrophages. We describe here a method for deriving monocytes and macrophages from human Pluripotent Stem Cells which improves on previously published protocols in that it uses entirely defined, feeder- and serum-free culture conditions and produces very consistent, pure, high yields across both human Embryonic Stem Cell (hESC and multiple human induced Pluripotent Stem Cell (hiPSC lines over time periods of up to one year. Cumulatively, up to ∼3×10(7 monocytes can be harvested per 6-well plate. The monocytes produced are most closely similar to the major blood monocyte (CD14(+, CD16(low, CD163(+. Differentiation with M-CSF produces macrophages that are highly phagocytic, HIV-1-infectable, and upon activation produce a pro-inflammatory cytokine profile similar to blood monocyte-derived macrophages. Macrophages are notoriously hard to genetically manipulate, as they recognise foreign nucleic acids; the lentivector system described here overcomes this, as pluripotent stem cells can be relatively simply genetically manipulated for efficient transgene expression in the differentiated cells, surmounting issues of transgene silencing. Overall, the method we describe here is an efficient, effective, scalable system for the reproducible production and genetic modification of human macrophages, facilitating the interrogation of human macrophage biology.

  8. Inhibition of mitogen-activated protein kinase kinase, DNA methyltransferase, and transforming growth factor-β promotes differentiation of human induced pluripotent stem cells into enterocytes.

    Science.gov (United States)

    Kodama, Nao; Iwao, Takahiro; Kabeya, Tomoki; Horikawa, Takashi; Niwa, Takuro; Kondo, Yuki; Nakamura, Katsunori; Matsunaga, Tamihide

    2016-06-01

    We previously reported that small-molecule compounds were effective in generating pharmacokinetically functional enterocytes from human induced pluripotent stem (iPS) cells. In this study, to determine whether the compounds promote the differentiation of human iPS cells into enterocytes, we investigated the effects of a combination of mitogen-activated protein kinase kinase (MEK), DNA methyltransferase (DNMT), and transforming growth factor (TGF)-β inhibitors on intestinal differentiation. Human iPS cells cultured on feeder cells were differentiated into endodermal cells by activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, the cells were differentiated into enterocyte cells by epidermal growth factor and small-molecule compounds. After differentiation, mRNA expression levels and drug-metabolizing enzyme activities were measured. The mRNA expression levels of the enterocyte marker sucrase-isomaltase and the major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 were increased by a combination of MEK, DNMT, and TGF-β inhibitors. The mRNA expression of CYP3A4 was markedly induced by 1α,25-dihydroxyvitamin D3. Metabolic activities of CYP1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP3A4/5, UDP-glucuronosyltransferase, and sulfotransferase were also observed in the differentiated cells. In conclusion, MEK, DNMT, and TGF-β inhibitors can be used to promote the differentiation of human iPS cells into pharmacokinetically functional enterocytes. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  9. Stress analysis of feeder bends using neutrons: new results and cumulative impacts

    Energy Technology Data Exchange (ETDEWEB)

    Banks, D.; Donaberger, R. [Canadian Neutron Beam Centre, Chalk River, ON (Canada); Leitch, B. [Atomic Energy of Canada Limited, Chalk River, ON (Canada); Rogge, R.B. [Canadian Neutron Beam Centre, Chalk River, ON (Canada)

    2014-07-01

    Neutron diffraction has played a vital role in stress analysis of bends in carbon steel pipes, known as feeder pipes, in CANDU reactors. Due to incidents of cracking of feeders, extensive R&D programs to manage feeder cracking have been implemented over about ten years. We review the cumulative impacts of this research from the view point of the stress analysis using neutrons, and present new results by examining a feeder bend with a partial crack both experimentally using neutron diffraction and theoretically using a finite element model. (author)

  10. limit loads for wall-thinning feeder pipes under combined bending and internal pressure

    International Nuclear Information System (INIS)

    Je, Jin Ho; Lee, Kuk Hee; Chung, Ha Joo; Kim, Ju Hee; Han, Jae Jun; Kim, Yun Jae

    2009-01-01

    Flow Accelerated Corrosion (FAC) during inservice conditions produces local wall-thinning in the feeder pipes of CANDU. The Wall-thinning in the feeder pipes is main degradation mechanisms affecting the integrity of piping systems. This paper discusses the integrity assessment of wall-thinned feeder pipes using limit load analysis. Based on finite element limit analyses, this paper compare limit loads for wall-thinning feeder pipes under combined bending and internal pressure with proposed limit loads. The limit loads are determined from limit analyses based on rectangular wall-thinning and elastic-perfectly-plastic materials using the large geometry change.

  11. S08: investigation and repair of a cracked feeder at Point Lepreau GS

    International Nuclear Information System (INIS)

    A Celovsky, A.; Wright, M.D.; Gendron, T.S.

    1997-01-01

    Early in 1997 investigation of a low level leak in the Point Lepreau GS (PLGS) PHTS revealed that an outlet feeder, S08, was leaking. Ultrasonic inspection, and subsequent failure analysis, revealed that the leak was a consequence of a crack. Given the unusual nature of this event, and current concerns over feeder thinning, a detailed and careful removal and examination procedure was developed. The S08 outlet feeder was removed and shipped to Chalk River Laboratories for examination. The examination confirmed that the failure was a through-wall crack, most likely the consequence of stress corrosion cracking. A critical point of the analysis was to determine how the crack initiated, and subsequently propagated. High residual stresses and possible abnormal loading in conjunction with chemistry environments resulted in the Stress Corrosion Cracking (SCC) of the S08 outlet feeder bend. It is recognized that some of the causative factors implicated in the S08 failure apply to other outlet feeders. In particular, residual stresses in the non-stress-relieved, short-radius cold bent pipes will remain relatively high over the future life of the feeders. However, the risk of CANDU feeder failure by SCC is judged to be extremely low based on the evidence of the inspections carried out to date and the good performance record of feeder pipe in the CANDU industry. The channel was restored to its locked configuration, and the failed section of feeder replaced. (author)

  12. The Cultivation of Human Granulosa Cells

    Directory of Open Access Journals (Sweden)

    Lenka Brůčková

    2008-01-01

    Full Text Available The major functions of granulosa cells (GCs include the production of steroids, as well as a myriad of growth factors to interact with the oocyte during its development within the ovarian follicle. Also FSH stimulates GCs to convert androgens (coming from the thecal cells to estradiol by aromatase. However, after ovulation the GCs produce progesterone that may maintain a potential pregnancy. Experiments with human GCs are mainly focused on the purification of GCs from ovarian follicular fluid followed by FACS analysis or short-term cultivation. The aim of our study was to cultivate GCs for a long period, to characterize their morphology and phenotype. Moreover, we have cultivated GCs under gonadotropin stimulation in order to simulate different pathological mechanisms during folliculogenesis (e.g. ovarian hyperstimulation syndrome. GCs were harvested from women undergoing in vitro fertilization. Complex oocyte-cumulus oophorus was dissociated by hyaluronidase. The best condition for transport of GCs was optimized as short transport in follicular fluid at 37 °C. GCs expansion medium consisted of DMEM/F12, 2 % FCS, ascorbic acid, dexamethasone, L-glutamine, gentamycine, penicillin, streptomycin and growth factors (EGF, bFGF. GCs transported in follicular fluid and cultivated in 2 % FCS containing DMEM/F12 medium supplemented with follicular fluid presented increased adhesion, proliferation, viability and decreased doubling time. Cell viability was 92 % and mean cell doubling time was 52 hrs. We have optimized transport and cultivation protocols for long-term cultivation of GCs.

  13. Experience of superconducting current feeders system of SST-1

    International Nuclear Information System (INIS)

    Gupta, N.C.; Garg, A.; Sonara, D.

    2014-01-01

    The superconducting current feeder system for SST-1 which has been installed and commissioned recently along with SST-1, felicitates to energize the SST-1. The CFS consists of ten pairs of 10,000 Ampere (A) rating helium vapor cooled conventional current leads, interconnecting Cu-SC joints, three numbers of cryo-compatible SC feeders ducts, current leads assembly chamber, hydraulic network and three numbers of joint boxes operated at different current rating to charge Toroidal Field and Poloidal Field coils separately. During the last three campaigns, it was possible to achieve a controlled cool down up to 4 K and showed its rated operational performance. Actively cooled liquid nitrogen shield showed temperature profile in the temperature range of 80-85K and the whole system was evacuated up to 6x10 -6 mbar. The measured LHe consumption rates from TF VCCL were 0.3 g/s and 0.35 g/s at zero current and 1 kA respectively. (author)

  14. Single Cell Dissection of Human Pancreatic Islet Dysfunction in Diabetes

    Science.gov (United States)

    2017-06-01

    of memory T cells , innate cells and the differentiation potential of naive T cells during ME/CFS; and 3) To determine the T cell and innate cell ...apoptosis and the innate immune response in human pancreatic β- cells . Diabetes 64: 3808–3817. Marselli L, Thorne J, Dahiya S, Sgroi DC, Sharma A, Bonner-Weir...interactive nature of CellView aids in cell doublet identification. In the PBMC data, ‘Subcluster-analysis’ reveals a mixture of lymphoid and myeloid

  15. Cytokine-free directed differentiation of human pluripotent stem cells efficiently produces hemogenic endothelium with lymphoid potential.

    Science.gov (United States)

    Galat, Yekaterina; Dambaeva, Svetlana; Elcheva, Irina; Khanolkar, Aaruni; Beaman, Kenneth; Iannaccone, Philip M; Galat, Vasiliy

    2017-03-17

    The robust generation of human hematopoietic progenitor cells from induced or embryonic pluripotent stem cells would be beneficial for multiple areas of research, including mechanistic studies of hematopoiesis, the development of cellular therapies for autoimmune diseases, induced transplant tolerance, anticancer immunotherapies, disease modeling, and drug/toxicity screening. Over the past years, significant progress has been made in identifying effective protocols for hematopoietic differentiation from pluripotent stem cells and understanding stages of mesodermal, endothelial, and hematopoietic specification. Thus, it has been shown that variations in cytokine and inhibitory molecule treatments in the first few days of hematopoietic differentiation define primitive versus definitive potential of produced hematopoietic progenitor cells. The majority of current feeder-free, defined systems for hematopoietic induction from pluripotent stem cells include prolonged incubations with various cytokines that make the differentiation process complex and time consuming. We established that the application of Wnt agonist CHIR99021 efficiently promotes differentiation of human pluripotent stem cells in the absence of any hematopoietic cytokines to the stage of hemogenic endothelium capable of definitive hematopoiesis. The hemogenic endothelium differentiation was accomplished in an adherent, serum-free culture system by applying CHIR99021. Hemogenic endothelium progenitor cells were isolated on day 5 of differentiation and evaluated for their endothelial, myeloid, and lymphoid potential. Monolayer induction based on GSK3 inhibition, described here, yielded a large number of CD31 + CD34 + hemogenic endothelium cells. When isolated and propagated in adherent conditions, these progenitors gave rise to mature endothelium. When further cocultured with OP9 mouse stromal cells, these progenitors gave rise to various cells of myeloid lineages as well as natural killer lymphoid, T

  16. 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.

  17. Proteomic Analysis of Human Blastocoel Fluid and Blastocyst Cells

    DEFF Research Database (Denmark)

    Linnert Jensen, Pernille; Beck, Hans Christian; Petersen, Jørgen

    The human blastocyst consists of 100-200 cells that are organized in an outer layer of differentiated trophectoderm (TE) cells lining the blastocyst cavity into which the undifferentiated inner cell mass (ICM) protrudes. The cavity of the blastocyst is filled with blastocoel fluid to which all...... the cells of the blastocyst are exposed. The ICM is the starting point for the development of undifferentiated human embryonic stem cells (hESCs), which posses the potential to develop into any cell type present in the adult human body [1,2]. This ability makes hESCs a potential source of cells...

  18. Studies in human skin epithelial cell carcinogenesis

    International Nuclear Information System (INIS)

    Lehman, T.A.

    1987-01-01

    Metabolism and DNA adduct formation of benzo[a]pyrene (BP) by human epidermal keratinocytes pretreated with inhibitors or inducer of cytochrame P450 was studied. To study DNA adduct analysis, cultures were pretreated as described above, and then treated with non-radiolabeled BP. DNA was prepared from these cultures, digested to the nucleotide level, and 32 P-postlabeled for adduct analysis. Cultures pretreated with BHA, 7,8-BF or disulfiralm formed significantly fewer BPDE I-dB adducts than non-pretreated cultures, while cultures pretreated with MeBHA formed more BPDE-I-dG adducts. MeBHA increased BP activation and adduct formation inhuman keratinocyte in cultures by inducing a specific isoenzyme of cytochrome P450 which preferentially increases the oxidative metabolism of BP to 7,8 diol BP and 7,8 diol BP to BPDE I. To approximate an in vivo human system, metabolism of BPDE I by human skin xenografts treated with cell cycles modulators was studied. When treated with BPDE I, specific carcinogen-DNA adducts were formed. Separation and identification of these adducts by the 32 P-postlabeling technique indicated that the 7R- and 7S-BPDE I-dG adducts were the major adducts

  19. Technical Challenges in the Derivation of Human Pluripotent Cells

    Directory of Open Access Journals (Sweden)

    Parinya Noisa

    2011-01-01

    Full Text Available It has long been discovered that human pluripotent cells could be isolated from the blastocyst state of embryos and called human embryonic stem cells (ESCs. These cells can be adapted and propagated indefinitely in culture in an undifferentiated manner as well as differentiated into cell representing the three major germ layers: endoderm, mesoderm, and ectoderm. However, the derivation of human pluripotent cells from donated embryos is limited and restricted by ethical concerns. Therefore, various approaches have been explored and proved their success. Human pluripotent cells can also be derived experimentally by the nuclear reprogramming of somatic cells. These techniques include somatic cell nuclear transfer (SCNT, cell fusion and overexpression of pluripotent genes. In this paper, we discuss the technical challenges of these approaches for nuclear reprogramming, involving their advantages and limitations. We will also highlight the possible applications of these techniques in the study of stem cell biology.

  20. Stereological quantification of mast cells in human synovium

    DEFF Research Database (Denmark)

    Damsgaard, T E; Sørensen, Flemming Brandt; Herlin, T

    1999-01-01

    Mast cells participate in both the acute allergic reaction as well as in chronic inflammatory diseases. Earlier studies have revealed divergent results regarding the quantification of mast cells in the human synovium. The aim of the present study was therefore to quantify these cells in the human...... synovium, using stereological techniques. Different methods of staining and quantification have previously been used for mast cell quantification in human synovium. Stereological techniques provide precise and unbiased information on the number of cell profiles in two-dimensional tissue sections of......, in this case, human synovium. In 10 patients suffering from osteoarthritis a median of 3.6 mast cells/mm2 synovial membrane was found. The total number of cells (synoviocytes, fibroblasts, lymphocytes, leukocytes) present was 395.9 cells/mm2 (median). The mast cells constituted 0.8% of all the cell profiles...

  1. DYNAMICS OF VIBRATION FEEDERS WITH A NONLINEAR ELASTIC CHARACTERISTIC

    Directory of Open Access Journals (Sweden)

    V. I. Dyrda

    2017-04-01

    Full Text Available Purpose. Subject to the smooth and efficient operation of each production line, is the use of vehicles transporting high specification. It worked well in practice for transporting construction machines, which are used during the vibration. The use of vibration machines requires optimization of their operation modes. In the form of elastic link in them are increasingly using rubber-metallic elements, which are characterized by nonlinear damping properties. So it is necessary to search for new, more modern, methods of calculation of dynamic characteristics of the vibration machines on the properties of rubber as a cushioning material. Methodology. The dynamics of vibration machine that is as elastic rubber block units and buffer shock absorbers limiting the amplitude of the vibrations of the working body. The method of determining amplitude-frequency characteristics of the vibrating feeder is based on the principle of Voltaire, who in the calculations of the damping properties of the dampers will allow for elastic-hereditary properties of rubber. When adjusting the basic dynamic stiffness of the elastic ties and vibratory buffers, using the principle of heredity rubber properties, determine the dependence of the amplitude of the working body of the machine vibrations. This method is called integro-operator using the fractional-exponential kernels of relaxation. Findings. Using the derived formula for determining the amplitude of the resonance curve is constructed one-mass nonlinear system. It is established that the use of the proposed method of calculation will provide a sufficiently complete description of the damping parameters of rubber-metallic elements and at the same time be an effective means of calculating the amplitude-frequency characteristics of nonlinear vibration systems. Originality. The authors improved method of determining damping characteristics of rubber-metallic elements and the amplitude-frequency characteristics of nonlinear

  2. A Chemical Probe that Labels Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Nao Hirata

    2014-03-01

    Full Text Available A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1] that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1 and ABCG2 (BCRP, both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.

  3. Alternative Sources of Adult Stem Cells: Human Amniotic Membrane

    Science.gov (United States)

    Wolbank, Susanne; van Griensven, Martijn; Grillari-Voglauer, Regina; Peterbauer-Scherb, Anja

    Human amniotic membrane is a highly promising cell source for tissue engineering. The cells thereof, human amniotic epithelial cells (hAEC) and human amniotic mesenchymal stromal cells (hAMSC), may be immunoprivileged, they represent an early developmental status, and their application is ethically uncontroversial. Cell banking strategies may use freshly isolated cells or involve in vitro expansion to increase cell numbers. Therefore, we have thoroughly characterized the effect of in vitro cultivation on both phenotype and differentiation potential of hAEC. Moreover, we present different strategies to improve expansion including replacement of animal-derived supplements by human platelet products or the introduction of the catalytic subunit of human telomerase to extend the in vitro lifespan of amniotic cells. Characterization of the resulting cultures includes phenotype, growth characteristics, and differentiation potential, as well as immunogenic and immunomodulatory properties.

  4. In Vitro Cardiomyogenic Potential of Human Amniotic Fluid Stem Cells

    OpenAIRE

    Guan, Xuan; Delo, Dawn M.; Atala, Anthony; Soker, Shay

    2011-01-01

    Stem cell therapy for damaged cardiac tissue is currently limited by a number of factors, including the inability to obtain sufficient cell numbers, the potential tumorigenicity of certain types of stem cells, and the possible link between stem cell therapy and the development of malignant arrhythmias. In this study, we investigated whether human amniotic fluid-derived stem (hAFS) cells could be a potential source of cells for cardiac cell therapy by testing the in vitro differentiation capab...

  5. Trichloroethylene toxicity in a human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Thevenin, E.; McMillian, J. [Medical Univ. of Charleston South Carolina, SC (United States)

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  6. Identification of human tissue cross-presenting dendritic cells

    OpenAIRE

    Haniffa, Muzlifah; Collin, Matthew; Ginhoux, Florent

    2013-01-01

    Dendritic cells (DCs) are a heterogeneous group of functionally specialized antigen-presenting cells. We recently characterized the human tissue cross-presenting DCs and aligned the human and mouse DC subsets. Our findings will facilitate the translation of murine DC studies to the human setting and aid the design of DC-based vaccine strategies for infection and cancer immunotherapy.

  7. Response of deposit-feeders to exclusion of epibenthic predators in a Mediterranean intertidal flat

    NARCIS (Netherlands)

    Como, S.; Rossi, F.; Lardicci, C.

    2004-01-01

    Deposit-feeders are common components of macrofaunal assemblages in intertidal soft sediments. Predation has been considered to have a central role in affecting their distribution and population dynamics. This study investigates the effect of epibenthic predators on deposit-feeders, inhabiting the

  8. harmonic load modeling: a case study of 33 kv abuja steel mill feeder

    African Journals Online (AJOL)

    HOD

    techniques are adopted. This paper studied the harmonic orders of the 33 kV Abuja Steel Feeder modeled as a ... (ETAP) software package was deployed to perform Discrete Fast Transform (DFT) while the input ... and documented in research and development articles ... network with 33kV Abuja Steel feeder as case study.

  9. Final report on the evolution of supporting conditions for the feeders of 500 MWe PHWR

    International Nuclear Information System (INIS)

    Mishra, Rajesh; Soni, R.S.; Kushawaha, H.S.; Mahajan, S.C.; Kakodkar, A.; Hariprasad, K.

    1994-01-01

    This report deals with the evolution of generic supporting conditions for the feeders of 500 MWe PHWR based on the analysis and qualification of a few representative feeders. There are 196 different feeder pipe configurations for a total of 748 feeders. The present analysis was aimed at evolving a generalised supporting criteria based on the analysis of some representative feeders. The analysis was carried out for various loadings viz. pressure, temperature, dead weight, operating basis earthquake (OBE), safe shutdown earthquake (SSE) and creep loadings. The analysis for OBE and SSE loadings were carried out using response spectrum method. The effect of spacers between various feeders was modelled using higher damping values than those prescribed in ASME code. Based on the above analyses, generic supporting arrangements for the feeders of various groups have been finalized. This report gives details about the mathematical modelling, the analysis approach, the optimised supporting criteria, finalization of grouping and fixing of boundaries between various groups of feeders. (author). 34 refs., 51 figs., 69 tabs

  10. Development of I and C system for the coal feeder of coal firing plant

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Teak Soo; Park, Chan Ho [Korea Electric Power Corp. (KEPCO), Taejon (Korea, Republic of). Research Center

    1996-12-31

    KECC(Kepco Coal Feeder Control System) receives coal weight, conveyor speed and boiler demand signals. It controls coal flow by generating speed signal of feeder which conveys coal in hopper to pulverizer, displaying measured coal quantity and providing local auto and manual manipulator (author). 33 figs.

  11. Evaluation of options for life cycle management of feeder cracking at the Point Lepreau Generating Station

    International Nuclear Information System (INIS)

    Gendron, T.S.; Slade, J.P.

    2003-01-01

    The CANDU industry has a predictive capability for most Heat Transport System (HTS) degradation issues that allows utilities to apply cost-effective maintenance programs. The standard approach for maintenance programs is focussed inspection and planned replacement. Some examples of degradation issues with deterministic failure rates are feeder thinning, and pressure tube elongation and deuterium ingress. However, the cracking observed in Point Lepreau Generating Station (PLGS) outlet feeder first bends is one notable exception to this behaviour. A predictive capability for feeder cracking does not currently exist for several reasons. First, the mechanism of feeder cracking, stress corrosion cracking (SCC), has to some degree a random nature. Second, although a probable environment causing cracking has been identified, the precise stress and environmental conditions for feeder crack initiation and propagation have not been defined. Finally, the very low incidence of feeder cracking observed to-date (four, all at PLGS) precludes a probabilistic or statistical prediction of failure rate. Generally, utilities select a Life Cycle Management Plan that ensures safe operation and has the lowest Net Present Value cost. In preparing a Feeder Life Cycle Management Plan, New Brunswick Power (NBP) has recognized that the Net Present Value cost is very sensitive to failure rate. Since the failure rate for feeder cracking is not well defined, the following three scenarios were considered to bound the probability of future failures at PLGS. (author)

  12. Enhanced casein kinase II activity in human tumour cell cultures

    DEFF Research Database (Denmark)

    Prowald, K; Fischer, H; Issinger, O G

    1984-01-01

    Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells...

  13. MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

    NARCIS (Netherlands)

    KOLODZIEJ, H; HABERLAND, C; WOERDENBAG, HJ; KONINGS, AWT

    In the present study the cytotoxicity of 16 proanthocyanidins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values ranging from 18 to >200 mu m following continuous

  14. Isolation and in vitro expansion of human colonic stem cells

    NARCIS (Netherlands)

    Jung, P.; Sato, T.; Merlos-Suarez, A.; Barriga, F.M.; Iglesias, M.; Rossell, D.; Auer, H.; Gallardo, M.; Blasco, M.A.; Sancho, E.; Clevers, H.; Batlle, E.

    2011-01-01

    Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic

  15. The development of human mast cells. An historical reappraisal

    International Nuclear Information System (INIS)

    Ribatti, Domenico

    2016-01-01

    The understanding of mast cell (MC) differentiation is derived mainly from in vitro studies of different stages of stem and progenitor cells. The hematopoietic lineage development of human MCs is unique compared to other myeloid-derived cells. Human MCs originate from CD34"+/CD117"+/CD13"+multipotent hematopoietic progenitors, which undergo transendothelial recruitment into peripheral tissues, where they complete differentiation. Stem cell factor (SCF) is a major chemotactic factor for MCs and their progenitors. SCF also elicits cell-cell and cell-substratum adhesion, facilitates the proliferation, and sustains the survival, differentiation, and maturation, of MCs. Because MC maturation is influenced by local microenvironmental factors, different MC phenotypes can develop in different tissues and organs. - Highlights: • Human mast cells originate from CD34/CD117/CD13 positive multipotent hematopoietic progenitors. • Stem cell factor is a major chemotactic factor for mast cells and their progenitors. • Different mast cell phenotypes can develop in different tissues and organs.

  16. Improved Survival and Initiation of Differentiation of Human Induced Pluripotent Stem Cells to Hepatocyte-Like Cells upon Culture in William's E Medium followed by Hepatocyte Differentiation Inducer Treatment.

    Directory of Open Access Journals (Sweden)

    Minoru Tomizawa

    Full Text Available Hepatocyte differentiation inducer (HDI lacks both glucose and arginine, but is supplemented with galactose and ornithine, and is added together with other reagents such as apoptosis inhibitor and oncostatin M. Although human induced pluripotent stem (iPS cells initiate hepatocyte differentiation, most die within 7 days. In this study, we investigated both HDI and conventional media for their potential to improve cell survival.201B7 iPS cells were cultured in conventional media. This consisted of three cycles of 5-day culture in William's E (WE medium, followed by a 2-day culture in HDI.Expression levels of α-feto protein (AFP were higher in cells cultured in WE and in Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham (DF12. 201B7 cells expressed the highest AFP and albumin (ALB when cultured in HDI for 2 days following 7-day culture in WE. After three cycles of 5-day culture in WE followed by 2 days in HDI, 201B7 cells expressed AFP and ALB 54 ± 2.3 (average ± standard deviation and 73 ± 15.1 times higher, respectively, than those cultured in ReproFF (feeder-free condition.201B7 cells survived culture in WE for 7 days followed HDI for 2 days. After three cycles of culture under these conditions, hepatocyte differentiation was enhanced, as evidenced by increased AFP and ALB expression.

  17. Preliminary analysis of accident in SST-1 current feeder system

    International Nuclear Information System (INIS)

    Roy, Swati; Kanabar, Deven; Garg, Atul; Singh, Amit; Tanna, Vipul; Prasad, Upendra; Srinivasan, R.

    2017-01-01

    Steady-state Tokamak-1 (SST-1) has 16 superconducting Toroidal field (TF) and 9 superconducting poloidal field (PF) coils rated for 10kA DC. All the TF are connected in series and are operated in DC condition whereas PF coils are individually operated in pulse mode during SST-1 campaigns. SST-1 current feeder system (CFS) houses 9 pairs of PF current leads and 1 pair of TF current leads. During past SST-1 campaign, there were arcing incidents within SST-1 CFS chamber which caused significant damage to PF superconducting current leads as well as its Helium cooling lines of the current leads. This paper brings out the preliminary analysis of the mentioned arcing incident, possible reasons and its investigation thereby laying out the sequence of events. From this analysis and observations, various measures to avoid such arcing incidents have also been proposed. (author)

  18. Microplastic in a macro filter feeder: Humpback whale Megaptera novaeangliae.

    Science.gov (United States)

    Besseling, E; Foekema, E M; Van Franeker, J A; Leopold, M F; Kühn, S; Bravo Rebolledo, E L; Heße, E; Mielke, L; IJzer, J; Kamminga, P; Koelmans, A A

    2015-06-15

    Marine filter feeders are exposed to microplastic because of their selection of small particles as food source. Baleen whales feed by filtering small particles from large water volumes. Macroplastic was found in baleen whales before. This study is the first to show the presence of microplastic in intestines of a baleen whale (Megaptera novaeangliae). Contents of its gastrointestinal tract were sieved, dissolved in 10% potassium hydroxide and washed. From the remaining dried material, potential synthetic polymer particles were selected based on density and appearance, and analysed by Fourier transform infrared (FTIR) spectroscopy. Several polymer types (polyethylene, polypropylene, polyvinylchloride, polyethylene terephthalate, nylon) were found, in varying particle shapes: sheets, fragments and threads with a size of 1mm to 17cm. This diversity in polymer types and particle shapes, can be interpreted as a representation of the varying characteristics of marine plastic and the unselective way of ingestion by M. novaeangliae. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.

    Science.gov (United States)

    Slamecka, Jaroslav; Salimova, Lilia; McClellan, Steven; van Kelle, Mathieu; Kehl, Debora; Laurini, Javier; Cinelli, Paolo; Owen, Laurie; Hoerstrup, Simon P; Weber, Benedikt

    2016-01-01

    Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

  20. The commitment of human cells to senescence.

    Science.gov (United States)

    Holliday, Robin

    2014-01-01

    Fifty years ago, it was demonstrated by Leonard Hayflick that human diploid fibroblasts grown in culture have a finite lifespan. Since that time, innumerable experiments have been published to discover the mechanism(s) that are responsible for this 'Hayflick limit' to continuous growth. Much new information has been gained, but there are certain features of this experimental system which have not been fully understood. One is the fact that different populations of the foetal lung strains WI-38 and MRC-5 have a range in division potential of at least a millionfold. The commitment theory of cellular aging, published more than 30 years ago, is able to explain this, but it has been consistently ignored. The theory predicts that bottlenecks, which are transient reductions in population size, can significantly reduce lifespan, or increase variability of lifespans. Computer simulations specify the effects of bottlenecks on longevity, and these were confirmed in two series of experiments. Commitment to senescence may be the loss of telomerase, which leads to the erosion of telomeres and the inability to grow indefinitely. Many experiments have been done with skin fibroblasts from human donors of different age, and it was originally thought that in vitro lifespan was inversely correlated with donor age. In these experiments, a single skin biopsy produces a population of cells that are grown to senescence. However, there is no reason to believe that skin fibroblasts are less variable in their in vitro lifespan than foetal lung strains, in which case the data points with skin cells are so variable that they may completely obscure any inverse correlation between culture lifespans and donor age.

  1. Development of an extruder-feeder biomass direct liquefaction process

    Energy Technology Data Exchange (ETDEWEB)

    White, D.H.; Wolf, D. (Arizona Univ., Tucson, AZ (United States). Dept. of Chemical Engineering)

    1991-10-01

    As an abundant, renewable, domestic energy resource, biomass could help the United States reduce its dependence on imported oil. Biomass is the only renewable energy technology capable of addressing the national need for liquid transportation fuels. Thus, there is an incentive to develop economic conversion processes for converting biomass, including wood, into liquid fuels. Through research sponsored by the US DOE's Biomass Thermochemical Conversion Program, the University of Arizona has developed a unique biomass direct liquefaction system. The system features a modified single-screw extruder capable of pumping solid slurries containing as high as 60 wt% wood flour in wood oil derived vacuum bottoms at pressures up to 3000 psi. The extruder-feeder has been integrated with a unique reactor by the University to form a system which offers potential for improving high pressure biomass direct liquefaction technology. The extruder-feeder acts simultaneously as both a feed preheater and a pumping device for injecting wood slurries into a high pressure reactor in the biomass liquefaction process. An experimental facility was constructed and following shakedown operations, wood crude oil was produced by mid-1985. By July 1988, a total of 57 experimental continuous biomass liquefaction runs were made using White Birch wood feedstock. Good operability was achieved at slurry feed rates up to 30 lb/hr, reactor pressures from 800 to 3000 psi and temperatures from 350{degree}C to 430{degree}C under conditions covering a range of carbon monoxide feed rates and sodium carbonate catalyst addition. Crude wood oils containing as little as 6--10 wt% residual oxygen were produced. 38 refs., 82 figs., 26 tabs.

  2. Nitrogen fixed by cyanobacteria is utilized by deposit-feeders.

    Science.gov (United States)

    Karlson, Agnes M L; Gorokhova, Elena; Elmgren, Ragnar

    2014-01-01

    Benthic communities below the photic zone depend for food on allochthonous organic matter derived from seasonal phytoplankton blooms. In the Baltic Sea, the spring diatom bloom is considered the most important input of organic matter, whereas the contribution of the summer bloom dominated by diazotrophic cyanobacteria is less understood. The possible increase in cyanobacteria blooms as a consequence of eutrophication and climate change calls for evaluation of cyanobacteria effects on benthic community functioning and productivity. Here, we examine utilization of cyanobacterial nitrogen by deposit-feeding benthic macrofauna following a cyanobacteria bloom at three stations during two consecutive years and link these changes to isotopic niche and variations in body condition (assayed as C:N ratio) of the animals. Since nitrogen-fixing cyanobacteria have δ(15)N close to -2‰, we expected the δ(15)N in the deposit-feeders to decrease after the bloom if their assimilation of cyanobacteria-derived nitrogen was substantial. We also expected the settled cyanobacteria with their associated microheterotrophic community and relatively high nitrogen content to increase the isotopic niche area, trophic diversity and dietary divergence between individuals (estimated as the nearest neighbour distance) in the benthic fauna after the bloom. The three surface-feeding species (Monoporeia affinis, Macoma balthica and Marenzelleria arctia) showed significantly lower δ(15)N values after the bloom, while the sub-surface feeder Pontoporeia femorata did not. The effect of the bloom on isotopic niche varied greatly between stations; populations which increased niche area after the bloom had better body condition than populations with reduced niche, regardless of species. Thus, cyanobacterial nitrogen is efficiently integrated into the benthic food webs in the Baltic, with likely consequences for their functioning, secondary production, transfer efficiency, trophic interactions, and

  3. Nitrogen fixed by cyanobacteria is utilized by deposit-feeders.

    Directory of Open Access Journals (Sweden)

    Agnes M L Karlson

    Full Text Available Benthic communities below the photic zone depend for food on allochthonous organic matter derived from seasonal phytoplankton blooms. In the Baltic Sea, the spring diatom bloom is considered the most important input of organic matter, whereas the contribution of the summer bloom dominated by diazotrophic cyanobacteria is less understood. The possible increase in cyanobacteria blooms as a consequence of eutrophication and climate change calls for evaluation of cyanobacteria effects on benthic community functioning and productivity. Here, we examine utilization of cyanobacterial nitrogen by deposit-feeding benthic macrofauna following a cyanobacteria bloom at three stations during two consecutive years and link these changes to isotopic niche and variations in body condition (assayed as C:N ratio of the animals. Since nitrogen-fixing cyanobacteria have δ(15N close to -2‰, we expected the δ(15N in the deposit-feeders to decrease after the bloom if their assimilation of cyanobacteria-derived nitrogen was substantial. We also expected the settled cyanobacteria with their associated microheterotrophic community and relatively high nitrogen content to increase the isotopic niche area, trophic diversity and dietary divergence between individuals (estimated as the nearest neighbour distance in the benthic fauna after the bloom. The three surface-feeding species (Monoporeia affinis, Macoma balthica and Marenzelleria arctia showed significantly lower δ(15N values after the bloom, while the sub-surface feeder Pontoporeia femorata did not. The effect of the bloom on isotopic niche varied greatly between stations; populations which increased niche area after the bloom had better body condition than populations with reduced niche, regardless of species. Thus, cyanobacterial nitrogen is efficiently integrated into the benthic food webs in the Baltic, with likely consequences for their functioning, secondary production, transfer efficiency, trophic

  4. A Feeder-Bus Dispatch Planning Model for Emergency Evacuation in Urban Rail Transit Corridors

    Science.gov (United States)

    Wang, Yun; Yan, Xuedong; Zhou, Yu; Zhang, Wenyi

    2016-01-01

    The mobility of modern metropolises strongly relies on urban rail transit (URT) systems, and such a heavy dependence causes that even minor service interruptions would make the URT systems unsustainable. This study aims at optimally dispatching the ground feeder-bus to coordinate with the urban rails’ operation for eliminating the effect of unexpected service interruptions in URT corridors. A feeder-bus dispatch planning model was proposed for the collaborative optimization of URT and feeder-bus cooperation under emergency situations and minimizing the total evacuation cost of the feeder-buses. To solve the model, a concept of dummy feeder-bus system is proposed to transform the non-linear model into traditional linear programming (ILP) model, i.e., traditional transportation problem. The case study of Line #2 of Nanjing URT in China was adopted to illustrate the model application and sensitivity analyses of the key variables. The modeling results show that as the evacuation time window increases, the total evacuation cost as well as the number of dispatched feeder-buses decrease, and the dispatched feeder-buses need operate for more times along the feeder-bus line. The number of dispatched feeder-buses does not show an obvious change with the increase of parking spot capacity and time window, indicating that simply increasing the parking spot capacity would cause huge waste for the emergent bus utilization. When the unbalanced evacuation demand exists between stations, the more feeder-buses are needed. The method of this study will contribute to improving transportation emergency management and resource allocation for URT systems. PMID:27676179

  5. Human Stem Cell Derived Cardiomyocytes: An Alternative ...

    Science.gov (United States)

    Chemical spills and associated deaths in the US has increased 2.6-fold and 16-fold from 1983 to 2012, respectfully. In addition, the number of chemicals to which humans are exposed to in the environment has increased almost 10-fold from 2001 to 2013 within the US. Internationally, a WHO report on the global composite impact of chemicals on health reported that 16% of the total burden of cardiovascular disease was attributed to environmental chemical exposure with 2.5 million deaths per year. Clearly, the cardiovascular system, at all its various developmental and life stages, represents a critical target organ system that can be adversely affected by existing and emerging chemicals (e.g., engineered nanomaterials) in a variety of environmental media. The ability to assess chemical cardiac risk and safety is critically needed but extremely challenging due to the number and categories of chemicals in commerce, as indicated. This presentation\\session will evaluate the use of adult human stem cell derived cardiomyocytes, and existing platforms, as an alternative model to evaluate environmental chemical cardiac toxicity as well as provide key information for the development of predictive adverse outcomes pathways associated with environmental chemical exposures. (This abstract does not represent EPA policy) Rapid and translatable chemical safety screening models for cardiotoxicity current status for informing regulatory decisions, a workshop sponsored by the Society

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

    Directory of Open Access Journals (Sweden)

    Xinxin Han

    2017-01-01

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

  7. New frontiers in human cell biology and medicine: can pluripotent stem cells deliver?

    Science.gov (United States)

    Goldstein, Lawrence S B

    2012-11-12

    Human pluripotent stem cells provide enormous opportunities to treat disease using cell therapy. But human stem cells can also drive biomedical and cell biological discoveries in a human model system, which can be directly linked to understanding disease or developing new therapies. Finally, rigorous scientific studies of these cells can and should inform the many science and medical policy issues that confront the translation of these technologies to medicine. In this paper, I discuss these issues using amyotrophic lateral sclerosis as an example.

  8. Human-induced pluripotent stem cell-derived macrophages and their immunological function in response to tuberculosis infection.

    Science.gov (United States)

    Hong, Danping; Ding, Jiongyan; Li, Ouyang; He, Quan; Ke, Minxia; Zhu, Mengyi; Liu, Lili; Ou, Wen-Bin; He, Yulong; Wu, Yuehong

    2018-02-26

    Induced pluripotent stem cells (iPS) represent an innovative source for the standardized in vitro generation of macrophages (Mφ). Mφ show great promise in disease pathogenesis, particularly tuberculosis. However, there is no information about human iPS-derived (hiPS) macrophages (hiPS-Mφ) in response to tuberculosis infection. In the present study, macrophages derived from hiPS were established via embryoid body (EB) formation by using feeder-free culture conditions, and the human monocyte cell line THP-1 (THP-1-Mφ) was used as control. iPS-Mφ were characterized by using morphology, Giemsa staining, nonspecific esterase staining (α-NAE), phagocytosis, and surface phenotype. Additionally, after treatment with Bacillus Calmette-Guérin (BCG) for 24 h, cell apoptosis was detected by using an Annexin V-FITC Apoptosis Detection assay. The production of nitric oxide (NO), expression of tumor necrosis factor alpha (TNF-α), activity of apoptosis-related protein cysteine-3 (Caspase-3) and expression of B-cell lymphoma-2 (Bcl-2) were analyzed. With respect to morphology, surface phenotype, and function, the iPS-Mφ closely resembled their counterparts generated in vitro from a human monocyte cell line. iPS-Mφ exhibited the typically morphological characteristics of macrophages, such as round, oval, fusiform and irregular characteristics. The cells were Giemsa-stained-positive, α-NAE-positive, and possessed phagocytic ability. iPS-Mφ express high levels of CD14, CD11b, CD40, CD68, and major histocompatibility complex II (MHC-II). Moreover, with regard to the apoptotic rate, the production of NO, expression of TNF-α, and activity of Caspase-3 and Bcl-2, iPS-Mφ closely resemble that of their counterparts generated in vitro from human monocyte cell line in response to BCG infection. The rate of apoptosis of BCG-treated iPS-Mφ was 37.77 ± 7.94% compared to that of the untreated group at 4.97 ± 1.60% (P immunological function in response to Bacillus Calmette

  9. Effects of Human Umbilical Cord Mesenchymal Stem Cells on Human Trophoblast Cell Functions In Vitro

    Directory of Open Access Journals (Sweden)

    Yajing Huang

    2016-01-01

    Full Text Available Trophoblast cell dysfunction is involved in many disorders during pregnancy such as preeclampsia and intrauterine growth restriction. Few treatments exist, however, that target improving trophoblast cell function. Human umbilical cord mesenchymal stem cells (hUCMSCs are capable of self-renewing, can undergo multilineage differentiation, and have homing abilities; in addition, they have immunomodulatory effects and paracrine properties and thus are a prospective source for cell therapy. To identify whether hUCMSCs can regulate trophoblast cell functions, we treated trophoblast cells with hUCMSC supernatant or cocultured them with hUCMSCs. Both treatments remarkably enhanced the migration and invasion abilities of trophoblast cells and upregulated their proliferation ability. At a certain concentration, hUCMSCs also modulated hCG, PIGF, and sEndoglin levels in the trophoblast culture medium. Thus, hUCMSCs have a positive effect on trophoblast cellular functions, which may provide a new avenue for treatment of placenta-related diseases during pregnancy.

  10. CD56 marks human dendritic cell subsets with cytotoxic potential

    NARCIS (Netherlands)

    Roothans, D.; Smits, E.; Lion, E.; Tel, J.; Anguille, S.

    2013-01-01

    Human plasmacytoid and myeloid dendritic cells (DCs), when appropriately stimulated, can express the archetypal natural killer (NK)-cell surface marker CD56. In addition to classical DC functions, CD56(+) DCs are endowed with an unconventional cytotoxic capacity.

  11. Bacteria, fungi, and viruses outnumber human cells 10:1

    Indian Academy of Sciences (India)

    Karen Nelson

    Fig 2. The distribution of the number of human cells by cell type. Sender R ... Type 2 diabetes ... Development of new predictive biomarkers so that preventive ... Microbiome, irrespective of lifestyle and age, which is distinct from races and.

  12. CD133-enriched Xeno-Free human embryonic-derived neural stem cells expand rapidly in culture and do not form teratomas in immunodeficient mice

    Directory of Open Access Journals (Sweden)

    Daniel L. Haus

    2014-09-01

    Full Text Available Common methods for the generation of human embryonic-derived neural stem cells (hNSCs result in cells with potentially compromised safety profiles due to maintenance of cells in conditions containing non-human proteins (e.g. in bovine serum or on mouse fibroblast feeders. Additionally, sufficient expansion of resulting hNSCs for scaling out or up in a clinically relevant time frame has proven to be difficult. Here, we report a strategy that produces hNSCs in completely “Xeno-Free” culture conditions. Furthermore, we have enriched the hNSCs for the cell surface marker CD133 via magnetic sorting, which has led to an increase in the expansion rate and neuronal fate specification of the hNSCs in vitro. Critically, we have also confirmed neural lineage specificity upon sorted hNSC transplantation into the immunodeficient NOD-scid mouse brain. The future use or adaptation of these protocols has the potential to better facilitate the advancement of pre-clinical strategies from the bench to the bedside.

  13. Microspectroscopy of spectral biomarkers associated with human corneal stem cells

    OpenAIRE

    Nakamura, Takahiro; Kelly, Jemma G.; Trevisan, J?lio; Cooper, Leanne J.; Bentley, Adam J.; Carmichael, Paul L.; Scott, Andrew D.; Cotte, Marine; Susini, Jean; Martin-Hirsch, Pierre L.; Kinoshita, Shigeru; Fullwood, Nigel J.; Martin, Francis L.

    2010-01-01

    Purpose Synchrotron-based radiation (SRS) Fourier-transform infrared (FTIR) microspectroscopy potentially provides novel biomarkers of the cell differentiation process. Because such imaging gives a ?biochemical-cell fingerprint? through a cell-sized aperture, we set out to determine whether distinguishing chemical entities associated with putative stem cells (SCs), transit-amplifying (TA) cells, or terminally-differentiated (TD) cells could be identified in human corneal epithelium. Methods D...

  14. Measurements of local mass transfer coefficient of Flow Accelerated Corrosion at feeder outlet pipes in CANDU using Plaster of Paris method

    International Nuclear Information System (INIS)

    Hyuk Kwon; Dong Un Seo; Goon-Cherl Park

    2005-01-01

    Full text of publication follows: Flow Accelerated Corrosion (FAC) is a corrosion mechanism that results in wall thinning in piping systems and components. FAC on carbon steels in pure water has occurred in a number of industry and power plant. A pipe wall thinning due to the FAC at nuclear power plant is just reported in confined to carbon steel pipe on the secondary system which does not equip a radioactive component. Recently, at Canadian Deuterium Uranium type reactor, it is reported that the feeder lines suffer the wall thinning on the primary system to equip 380 feeder pipes. Differently from the fast FAC rate of the secondary system on PWR, FAC rate of feeder pipes on CANDU is not more than the 0.2 mm/year. Although the FAC rate of feeder pipe is relatively slow, the narrow thickness margin of 2.6089 mm is endangered sufficiently by only the slow FAC rate. The FAC is governed by the mass transfer coefficient which is determined by the flow field. To well predict the FAC rate, mass transfer coefficient should be well measured or calculated. New measurement method of mass transfer coefficient is developed to obtain the coefficient for the complex shape like feeder pipe. The method evaluated the naphthalene sublimation method to apply it under the water flow. The naphthalene sublimation method can be used to study mass and heat transfer with confidence for a variety of applications, but with certain restrictions. At present, Plaster of paris method can modified the sublimation method to overcome the restrictions. This method is particularly useful in complex flows on geometries and for flows with large gradients in wall transport rate. The test specimen can be easily prepared by several methods, including dipping, machining, spraying, and casting. The local transfer coefficients can be determined with high accuracy and in detail by automated measurement systems that eliminate most human errors during the measurement process. To evaluate the method, the

  15. Intestinal Stem Cell Dynamics: A Story of Mice and Humans.

    Science.gov (United States)

    Hodder, Michael C; Flanagan, Dustin J; Sansom, Owen J

    2018-06-01

    Stem cell dynamics define the probability of accumulating mutations within the intestinal epithelium. In this issue of Cell Stem Cell, Nicholson et al. (2018) report that human intestinal stem cell dynamics differ significantly from those of mice and establish that oncogenic mutations are more likely to expand; therefore, "normal" epithelium may carry multiple mutations. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Comparative mutagenesis of human cells in vivo and in vitro

    International Nuclear Information System (INIS)

    Thilly, W.G.

    1992-05-01

    This report discusses measuring methods of point mutations; high density cell cultures for low dose studies; measurement and sequence determination of mutations in DNA; the mutational spectra of styrene oxide and ethlyene oxide in TK-6 cells; mutational spectrum of Cr in human lymphoblast cells; mutational spectra of radon in TK-6 cells; and the mutational spectra of smokeless tobacco

  17. Characterizing the radioresponse of pluripotent and multipotent human stem cells.

    Directory of Open Access Journals (Sweden)

    Mary L Lan

    Full Text Available The potential capability of stem cells to restore functionality to diseased or aged tissues has prompted a surge of research, but much work remains to elucidate the response of these cells to genotoxic agents. To more fully understand the impact of irradiation on different stem cell types, the present study has analyzed the radioresponse of human pluripotent and multipotent stem cells. Human embryonic stem (ES cells, human induced pluripotent (iPS cells, and iPS-derived human neural stem cells (iPS-hNSCs cells were irradiated and analyzed for cell survival parameters, differentiation, DNA damage and repair and oxidative stress at various times after exposure. While irradiation led to dose-dependent reductions in survival, the fraction of surviving cells exhibited dose-dependent increases in metabolic activity. Irradiation did not preclude germ layer commitment of ES cells, but did promote neuronal differentiation. ES cells subjected to irradiation exhibited early apoptosis and inhibition of cell cycle progression, but otherwise showed normal repair of DNA double-strand breaks. Cells surviving irradiation also showed acute and persistent increases in reactive oxygen and nitrogen species that were significant at nearly all post-irradiation times analyzed. We suggest that stem cells alter their redox homeostasis to adapt to adverse conditions and that radiation-induced oxidative stress plays a role in regulating the function and fate of stem cells within tissues compromised by radiation injury.

  18. Sequential cancer mutations in cultured human intestinal stem cells

    NARCIS (Netherlands)

    Drost, Jarno; van Jaarsveld, Richard H.; Ponsioen, Bas; Zimberlin, Cheryl; van Boxtel, Ruben; Buijs, Arjan; Sachs, Norman; Overmeer, René M.; Offerhaus, G. Johan; Begthel, Harry; Korving, Jeroen; van de Wetering, Marc; Schwank, Gerald; Logtenberg, Meike; Cuppen, Edwin; Snippert, Hugo J.; Medema, Jan Paul; Kops, Geert J. P. L.; Clevers, Hans

    2015-01-01

    Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain

  19. Alloimmune Responses of Humanized Mice to Human Pluripotent Stem Cell Therapeutics

    Directory of Open Access Journals (Sweden)

    Nigel G. Kooreman

    2017-08-01

    Full Text Available There is growing interest in using embryonic stem cell (ESC and induced pluripotent stem cell (iPSC derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an “allogenized” mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts.

  20. Nanoscale Mechanical Stimulation of Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    H Nikukar

    2014-05-01

    We observed significant responses after 1 and 2-week stimulations in cell number, cell shapes and phenotypical markers. Microarray was performed for all groups. Cell count showed normal cell growth with stimulation. However, cell surface area, cell perimeter, and arboration after 1-week stimulation showed significant increases. Immunofluorescent studies have showed significant increase in osteocalcin production after stimulation. Conclusions: Nanoscale mechanical vibration showed significant changes in human mesenchymal stem cell behaviours. Cell morphology changed to become more polygonal and increased expression of the osteoblast markers were noted. These findings with gene regulation changes suggesting nanoscale mechanostimulation has stimulated osteoblastogenesis.  Keywords:  Mesenchymal, Nanoscale, Stem Cells.

  1. Canthin-6-one induces cell death, cell cycle arrest and differentiation in human myeloid leukemia cells.

    Science.gov (United States)

    Vieira Torquato, Heron F; Ribeiro-Filho, Antonio C; Buri, Marcus V; Araújo Júnior, Roberto T; Pimenta, Renata; de Oliveira, José Salvador R; Filho, Valdir C; Macho, Antonio; Paredes-Gamero, Edgar J; de Oliveira Martins, Domingos T

    2017-04-01

    Canthin-6-one is a natural product isolated from various plant genera and from fungi with potential antitumor activity. In the present study, we evaluate the antitumor effects of canthin-6-one in human myeloid leukemia lineages. Kasumi-1 lineage was used as a model for acute myeloid leukemia. Cells were treated with canthin-6-one and cell death, cell cycle and differentiation were evaluated in both total cells (Lin + ) and leukemia stem cell population (CD34 + CD38 - Lin -/low ). Among the human lineages tested, Kasumi-1 was the most sensitive to canthin-6-one. Canthin-6-one induced cell death with apoptotic (caspase activation, decrease of mitochondrial potential) and necrotic (lysosomal permeabilization, double labeling of annexin V/propidium iodide) characteristics. Moreover, canthin-6-one induced cell cycle arrest at G 0 /G 1 (7μM) and G 2 (45μM) evidenced by DNA content, BrdU incorporation and cyclin B1/histone 3 quantification. Canthin-6-one also promoted differentiation of Kasumi-1, evidenced by an increase in the expression of myeloid markers (CD11b and CD15) and the transcription factor PU.1. Furthermore, a reduction of the leukemic stem cell population and clonogenic capability of stem cells were observed. These results show that canthin-6-one can affect Kasumi-1 cells by promoting cell death, cell cycle arrest and cell differentiation depending on concentration used. Canthin-6-one presents an interesting cytotoxic activity against leukemic cells and represents a promising scaffold for the development of molecules for anti-leukemic applications, especially by its anti-leukemic stem cell activity. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2017-04-01

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

  3. A DNA Vaccine Protects Human Immune Cells against Zika Virus Infection in Humanized Mice

    Directory of Open Access Journals (Sweden)

    Guohua Yi

    2017-11-01

    Full Text Available A DNA vaccine encoding prM and E protein has been shown to induce protection against Zika virus (ZIKV infection in mice and monkeys. However, its effectiveness in humans remains undefined. Moreover, identification of which immune cell types are specifically infected in humans is unclear. We show that human myeloid cells and B cells are primary targets of ZIKV in humanized mice. We also show that a DNA vaccine encoding full length prM and E protein protects humanized mice from ZIKV infection. Following administration of the DNA vaccine, humanized DRAG mice developed antibodies targeting ZIKV as measured by ELISA and neutralization assays. Moreover, following ZIKV challenge, vaccinated animals presented virtually no detectable virus in human cells and in serum, whereas unvaccinated animals displayed robust infection, as measured by qRT-PCR. Our results utilizing humanized mice show potential efficacy for a targeted DNA vaccine against ZIKV in humans.

  4. Molecular aging and rejuvenation of human muscle stem cells

    DEFF Research Database (Denmark)

    Carlson, Morgan E; Suetta, Charlotte; Conboy, Michael J

    2009-01-01

    . Our findings establish key evolutionarily conserved mechanisms of human stem cell aging. We find that satellite cells are maintained in aged human skeletal muscle, but fail to activate in response to muscle attrition, due to diminished activation of Notch compounded by elevated transforming growth...... factor beta (TGF-beta)/phospho Smad3 (pSmad3). Furthermore, this work reveals that mitogen-activated protein kinase (MAPK)/phosphate extracellular signal-regulated kinase (pERK) signalling declines in human muscle with age, and is important for activating Notch in human muscle stem cells. This molecular......Very little remains known about the regulation of human organ stem cells (in general, and during the aging process), and most previous data were collected in short-lived rodents. We examined whether stem cell aging in rodents could be extrapolated to genetically and environmentally variable humans...

  5. In vitro proliferation of adult human beta-cells.

    Directory of Open Access Journals (Sweden)

    Sabine Rutti

    Full Text Available A decrease in functional beta-cell mass is a key feature of type 2 diabetes. Glucagon-like peptide 1 (GLP-1 analogues induce proliferation of rodent beta-cells. However, the proliferative capacity of human beta-cells and its modulation by GLP-1 analogues remain to be fully investigated. We therefore sought to quantify adult human beta-cell proliferation in vitro and whether this is affected by the GLP-1 analogue liraglutide.Human islets from 7 adult cadaveric organ donors were dispersed into single cells. Beta-cells were purified by FACS. Non-sorted cells and the beta-cell enriched ("beta-cells" population were plated on extracellular matrix from rat (804G and human bladder carcinoma cells (HTB9 or bovine corneal endothelial ECM (BCEC. Cells were maintained in culture+/-liraglutide for 4 days in the presence of BrdU.Rare human beta-cell proliferation could be observed either in the purified beta-cell population (0.051±0.020%; 22 beta-cells proliferating out of 84'283 beta-cells counted or in the non-sorted cell population (0.055±0.011%; 104 proliferating beta-cells out of 232'826 beta-cells counted, independently of the matrix or the culture conditions. Liraglutide increased human beta-cell proliferation on BCEC in the non-sorted cell population (0.082±0.034% proliferating beta-cells vs. 0.017±0.008% in control, p<0.05.These results indicate that adult human beta-cell proliferation can occur in vitro but remains an extremely rare event with these donors and particular culture conditions. Liraglutide increases beta-cell proliferation only in the non-sorted cell population and only on BCEC. However, it cannot be excluded that human beta-cells may proliferate to a greater extent in situ in response to natural stimuli.

  6. Hybrid clone cells derived from human breast epithelial cells and human breast cancer cells exhibit properties of cancer stem/initiating cells.

    Science.gov (United States)

    Gauck, Daria; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S; Dittmar, Thomas

    2017-08-02

    The biological phenomenon of cell fusion has been associated with cancer progression since it was determined that normal cell × tumor cell fusion-derived hybrid cells could exhibit novel properties, such as enhanced metastatogenic capacity or increased drug resistance, and even as a mechanism that could give rise to cancer stem/initiating cells (CS/ICs). CS/ICs have been proposed as cancer cells that exhibit stem cell properties, including the ability to (re)initiate tumor growth. Five M13HS hybrid clone cells, which originated from spontaneous cell fusion events between M13SV1-EGFP-Neo human breast epithelial cells and HS578T-Hyg human breast cancer cells, and their parental cells were analyzed for expression of stemness and EMT-related marker proteins by Western blot analysis and confocal laser scanning microscopy. The frequency of ALDH1-positive cells was determined by flow cytometry using AldeRed fluorescent dye. Concurrently, the cells' colony forming capabilities as well as the cells' abilities to form mammospheres were investigated. The migratory activity of the cells was analyzed using a 3D collagen matrix migration assay. M13HS hybrid clone cells co-expressed SOX9, SLUG, CK8 and CK14, which were differently expressed in parental cells. A variation in the ALDH1-positive putative stem cell population was observed among the five hybrids ranging from 1.44% (M13HS-7) to 13.68% (M13HS-2). In comparison to the parental cells, all five hybrid clone cells possessed increased but also unique colony formation and mammosphere formation capabilities. M13HS-4 hybrid clone cells exhibited the highest colony formation capacity and second highest mammosphere formation capacity of all hybrids, whereby the mean diameter of the mammospheres was comparable to the parental cells. In contrast, the largest mammospheres originated from the M13HS-2 hybrid clone cells, whereas these cells' mammosphere formation capacity was comparable to the parental breast cancer cells. All M13HS

  7. Impacting effects of seismic loading in feeder pipes of PHWR power plants

    International Nuclear Information System (INIS)

    Kumar, R.

    1996-01-01

    The core of a pressurized heavy water reactor (PHWR) consists of a large number of fuel channels. These fuel channels are connected to the feeder pipes through which the heavy water flows and transports heat from the reactor core to the steam generators. The feeder pipes are several hundreds in number. They run close to each other with small gaps and have several bends. Thus they represent a complex piping system. Under seismic loading, the adjacent feeder pipes may impact each other. In this paper a simplified procedure has been established to assess such impacting effects. The results of the proposed analysis include bending moment and impact force, which provide the stresses due to impacting effects. These results are plotted in nondimensional form so that they could be utilized for any set of feeder pipes. The procedure used for studying the impacting effects includes seismic analysis of individual feeder pipes without impacting effects, selection of pipes for impact analysis, and estimating their maximum impact velocity. Based on the static and dynamic characteristics of the selected feeder pipes, the maximum bending moment, impact force, and stresses are obtained. The results of this study are useful for quick evaluation of the impacting effects in feeder pipes

  8. Nucleosome Organization in Human Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Puya G Yazdi

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

  9. Human interleukin for DA cells or leukemia inhibitory factor is released by Vero cells in human embryo coculture.

    Science.gov (United States)

    Papaxanthos-Roche, A; Taupin, J L; Mayer, G; Daniel, J Y; Moreau, J F

    1994-09-01

    In the light of the newly discovered implications of human interleukin for DA cells and leukemia inhibitory factor in embryology, we searched for the presence of this soluble cytokine in the supernatant of Vero cell coculture systems. Using a bioassay as well as a specific ELISA, we demonstrated that Vero cells are able to release large quantities of human interleukin for DA cells and leukemia inhibitory factor in the embryo-growing medium of such cocultures.

  10. A novel method for decomposing electricity feeder load into elementary profiles from customer information

    International Nuclear Information System (INIS)

    Gerossier, Alexis; Barbier, Thibaut; Girard, Robin

    2017-01-01

    Highlights: •Use of aggregated electricity load profiles and customer description at feeder level. •Statistical recovery of elementary load profiles with customer categorization. •Generation of load demand profiles for unknown feeders and new local areas. •Relevancy of the different categorizations. -- Abstract: To plan a distribution grid involves making a long-term forecast of sub-hourly demand, which requires modeling the demand and its dynamics with aggregated measurement data. Distribution system operators (DSOs) have been recording electricity sub-hourly demand delivered by their medium-voltage feeders (around 1000—10,000 customers) for several years. Demand profiles differ widely among the various considered feeders. This is partly due to the varying mix of customer categories from one feeder to another. To overcome this issue, elementary demand profiles are often associated with customer categories and then combined according to a mix description. This paper presents a novel method to estimate elementary profiles that only requires several feeder demand curves and a description of customers. The method relies on a statistical blind source model and a new estimation procedure based on the augmented Lagrangian method. The use of feeders to estimate elementary profiles means that measurements are fully representative and continuously updated. We illustrate the proposed method through a case study comprising around 1000 feeder demand curves operated by the main French DSO Enedis. We propose an application o that uses the obtained profiles to evaluate the contribution of any set of new customers to a feeder peak load. We show that profiles enable a simulation of new unmeasured areas with errors of around 20%. We also show how our method can be used to evaluate the relevancy of different customer categorizations.

  11. Derivation of xeno-free and GMP-grade human embryonic stem cells--platforms for future clinical applications.

    Directory of Open Access Journals (Sweden)

    Shelly E Tannenbaum

    Full Text Available Clinically compliant human embryonic stem cells (hESCs should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs.

  12. Current applications of human pluripotent stem cells: possibilities and challenges.

    Science.gov (United States)

    Ho, Pai-Jiun; Yen, Men-Luh; Yet, Shaw-Fang; Yen, B Linju

    2012-01-01

    Stem cells are self-renewable cells with the differentiation capacity to develop into somatic cells with biological functions. This ability to sustain a renewable source of multi- and/or pluripotential differentiation has brought new hope to the field of regenerative medicine in terms of cell therapy and tissue engineering. Moreover, stem cells are invaluable tools as in vitro models for studying diverse fields, from basic scientific questions such as developmental processes and lineage commitment, to practical application including drug screening and testing. The stem cells with widest differentiation potential are pluripotent stem cells (PSCs), which are rare cells with the ability to generate somatic cells from all three germ layers. PSCs are considered the most optimal choice for therapeutic potential of stem cells, bringing new impetus to the field of regenerative medicine. In this article, we discuss the therapeutic potential of human PSCs (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), reviewing the current preclinical and clinical data using these stem cells. We describe the classification of different sources of hPSCs, ongoing research, and currently encountered clinical obstacles of these novel and versatile human stem cells.

  13. Evaluating the potential of poly(beta-amino ester nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Bhise NS

    2013-12-01

    Full Text Available Nupura S Bhise,1,* Karl J Wahlin,2,* Donald J Zack,2–4 Jordan J Green1,21Department of Biomedical Engineering, Translational Tissue Engineering Center, and Institute for Nanobiotechnology, 2Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, MD, 3Solomon H Snyder Department of Neuroscience, Department of Molecular Biology and Genetics, and Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; 4Institut de la Vision, Paris, France*These authors contributed equally to this workBackground: Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs from human fibroblasts.Methods: A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling.Results: 1-(3-aminopropyl-4-methylpiperazine end-terminated poly(1,4-butanediol diacrylate-co-4-amino-1-butanol polymer (B4S4E7 self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available

  14. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

    Directory of Open Access Journals (Sweden)

    Chen Lei

    2011-06-01

    Full Text Available Abstract Background Cancer stem cells (CSCs are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44. Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

  15. Human dental pulp stem cells: Applications in future regenerative medicine

    Science.gov (United States)

    Potdar, Pravin D; Jethmalani, Yogita D

    2015-01-01

    Stem cells are pluripotent cells, having a property of differentiating into various types of cells of human body. Several studies have developed mesenchymal stem cells (MSCs) from various human tissues, peripheral blood and body fluids. These cells are then characterized by cellular and molecular markers to understand their specific phenotypes. Dental pulp stem cells (DPSCs) are having a MSCs phenotype and they are differentiated into neuron, cardiomyocytes, chondrocytes, osteoblasts, liver cells and β cells of islet of pancreas. Thus, DPSCs have shown great potentiality to use in regenerative medicine for treatment of various human diseases including dental related problems. These cells can also be developed into induced pluripotent stem cells by incorporation of pluripotency markers and use for regenerative therapies of various diseases. The DPSCs are derived from various dental tissues such as human exfoliated deciduous teeth, apical papilla, periodontal ligament and dental follicle tissue. This review will overview the information about isolation, cellular and molecular characterization and differentiation of DPSCs into various types of human cells and thus these cells have important applications in regenerative therapies for various diseases. This review will be most useful for postgraduate dental students as well as scientists working in the field of oral pathology and oral medicine. PMID:26131314

  16. Comprehensive evaluation of leukocyte lineage derived from human hematopoietic cells in humanized mice.

    Science.gov (United States)

    Takahashi, Masayuki; Tsujimura, Noriyuki; Otsuka, Kensuke; Yoshino, Tomoko; Mori, Tetsushi; Matsunaga, Tadashi; Nakasono, Satoshi

    2012-04-01

    Recently, humanized animals whereby a part of the animal is biologically engineered using human genes or cells have been utilized to overcome interspecific differences. Herein, we analyzed the detail of the differentiation states of various human leukocyte subpopulations in humanized mouse and evaluated comprehensively the similarity of the leukocyte lineage between humanized mice and humans. Humanized mice were established by transplanting human CD34(+) cord blood cells into irradiated severely immunodeficient NOD/Shi-scid/IL2Rγ(null) (NOG) mice, and the phenotypes of human cells contained in bone marrow, thymus, spleen and peripheral blood from the mice were analyzed at monthly intervals until 4 months after cell transplantation. The analysis revealed that transplanted human hematopoietic stem cells via the caudal vein homed and engrafted themselves successfully at the mouse bone marrow. Subsequently, the differentiated leukocytes migrated to the various tissues. Almost all of the leukocytes within the thymus were human cells. Furthermore, analysis of the differentiation states of human leukocytes in various tissues and organs indicated that it is highly likely that the human-like leukocyte lineage can be developed in mice. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  17. Improvement of Local Voltage in Feeders with Photovoltaic using Electric Vehicles

    DEFF Research Database (Denmark)

    Marra, Francesco; Yang, Guangya; Fawzy, Y. T.

    2013-01-01

    In low-voltage (LV) feeders with high penetration of photovoltaic (PV), a major issue to be solved is voltage rise due to the active power injection. If no measures are taken, this may lead to generation’s interruptions and to the malfunctioning of domestic appliances due to non-standard voltage...... profiles. This paper proposes a storage strategy to alleviate voltage rise in feeders with PV, using coordinated electric vehicle (EV) load as the storage solution. The voltage support strategy is easy to implement practically and it is demonstrated on a test feeder emulating a household with roof...

  18. Wharton's Jelly Derived Mesenchymal Stem Cells: Comparing Human and Horse.

    Science.gov (United States)

    Merlo, Barbara; Teti, Gabriella; Mazzotti, Eleonora; Ingrà, Laura; Salvatore, Viviana; Buzzi, Marina; Cerqueni, Giorgia; Dicarlo, Manuela; Lanci, Aliai; Castagnetti, Carolina; Iacono, Eleonora

    2018-08-01

    Wharton's jelly (WJ) is an important source of mesenchymal stem cells (MSCs) both in human and other animals. The aim of this study was to compare human and equine WJMSCs. Human and equine WJMSCs were isolated and cultured using the same protocols and culture media. Cells were characterized by analysing morphology, growth rate, migration and adhesion capability, immunophenotype, differentiation potential and ultrastructure. Results showed that human and equine WJMSCs have similar ultrastructural details connected with intense synthetic and metabolic activity, but differ in growth, migration, adhesion capability and differentiation potential. In fact, at the scratch assay and transwell migration assay, the migration ability of human WJMSCs was higher (P cells, while the volume of spheroids obtained after 48 h of culture in hanging drop was larger than the volume of equine ones (P cell adhesion ability. This can also revealed in the lower doubling time of equine cells (3.5 ± 2.4 days) as compared to human (6.5 ± 4.3 days) (P cell doubling after 44 days of culture observed for the equine (20.3 ± 1.7) as compared to human cells (8.7 ± 2.4) (P cells showed an higher chondrogenic and osteogenic differentiation ability (P staminal phenotype in human and equine WJMSCs, they showed different properties reflecting the different sources of MSCs.

  19. induced acute cytotoxicity in human cervical epithelial carcinoma cells

    African Journals Online (AJOL)

    Molecular basis of arsenite (As +3 )-induced acute cytotoxicity in human cervical epithelial carcinoma cells. ... Libyan Journal of Medicine ... Methods: After performing cytotoxic assays on a human epithelial carcinoma cell line, expression analysis was done by quantitative polymerase chain reaction, western blotting, and ...

  20. Novel human multiple myeloma cell line UHKT-893

    Czech Academy of Sciences Publication Activity Database

    Uherková, L.; Vančurová, I.; Vyhlídalová, I.; Pleschnerová, M.; Špička, I.; Mihalová, R.; Březinová, J.; Hodný, Zdeněk; Čermáková, K.; Polanská, V.; Marinov, I.; Jedelský, P.L.; Kuželová, K.; Stöckbauer, P.

    2013-01-01

    Roč. 37, č. 3 (2013), s. 320-326 ISSN 0145-2126 Institutional support: RVO:68378050 Keywords : human myeloma cell line * human multiple myeloma * plasma cell * IL-6 dependence * immunoglobulin * free light chain Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.692, year: 2013

  1. The association between human papillomavirus and oropharyngeal squamous cell Carcinoma

    DEFF Research Database (Denmark)

    Walvik, Lena; Svensson, Amanda Björk; Friborg, Jeppe

    2016-01-01

    carcinoma using the Bradford Hill criteria. The strength of the association is supported by, detection of human papillomavirus infection and antibodies prior to oropharyngeal squamous cell carcinoma. This is furthermore reinforced by the absence of human papillomavirus DNA in healthy tonsils...... incidence in human papillomavirus positive oropharyngeal squamous cell carcinoma is associated with sexual behaviour. These associations have been repeatedly observed and are in accordance with our current knowledge. The time relation between cause and effect remains the main challenge, due to the lack...... of well-defined premalignant lesions. However, a causal relationship between human papillomavirus infection and oropharyngeal squamous cell carcinoma seems evident....

  2. Freeze-Dried Human Red Blood Cells

    Science.gov (United States)

    1992-04-15

    period in the liquid state. 2. The levels of glycolytic intermediates (ATP, adenosine 5’triphosphate; 2,3-DPG 2, 3- diphosphoglycerate ) in rehydrated...8217 diphosphate, ADP; adenosine 5 monophosphate, AMP; 2,3- diphosphoglycerate . 2.3-DPG and lactate: (2) measurement of cell indices (mean cell volume (MCV), mean...monophosphate: 2,3-DPG. 2.3- diphosphoglycerate : MCV. Mean Cell Volume: MCH, Mean Cell Hemoglobin: MCHC, Mean Cell Hemoglobin Concentrations. ** Lactate levels

  3. CD1 and mycobacterial lipids activate human T cells.

    Science.gov (United States)

    Van Rhijn, Ildiko; Moody, D Branch

    2015-03-01

    For decades, proteins were thought to be the sole or at least the dominant source of antigens for T cells. Studies in the 1990s demonstrated that CD1 proteins and mycobacterial lipids form specific targets of human αβ T cells. The molecular basis by which T-cell receptors (TCRs) recognize CD1-lipid complexes is now well understood. Many types of mycobacterial lipids function as antigens in the CD1 system, and new studies done with CD1 tetramers identify T-cell populations in the blood of tuberculosis patients. In human populations, a fundamental difference between the CD1 and major histocompatibility complex systems is that all humans express nearly identical CD1 proteins. Correspondingly, human CD1 responsive T cells show evidence of conserved TCRs. In addition to natural killer T cells and mucosal-associated invariant T (MAIT cells), conserved TCRs define other subsets of human T cells, including germline-encoded mycolyl-reactive (GEM) T cells. The simple immunogenetics of the CD1 system and new investigative tools to measure T-cell responses in humans now creates a situation in which known lipid antigens can be developed as immunodiagnostic and immunotherapeutic reagents for tuberculosis disease. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. On the development of extragonadal and gonadal human germ cells

    Directory of Open Access Journals (Sweden)

    A. Marijne Heeren

    2016-02-01

    Full Text Available Human germ cells originate in an extragonadal location and have to migrate to colonize the gonadal primordia at around seven weeks of gestation (W7, or five weeks post conception. Many germ cells are lost along the way and should enter apoptosis, but some escape and can give rise to extragonadal germ cell tumors. Due to the common somatic origin of gonads and adrenal cortex, we investigated whether ectopic germ cells were present in the human adrenals. Germ cells expressing DDX4 and/or POU5F1 were present in male and female human adrenals in the first and second trimester. However, in contrast to what has been described in mice, where ‘adrenal’ and ‘ovarian’ germ cells seem to enter meiosis in synchrony, we were unable to observe meiotic entry in human ‘adrenal’ germ cells until W22. By contrast, ‘ovarian’ germ cells at W22 showed a pronounced asynchronous meiotic entry. Interestingly, we observed that immature POU5F1+ germ cells in both first and second trimester ovaries still expressed the neural crest marker TUBB3, reminiscent of their migratory phase. Our findings highlight species-specific differences in early gametogenesis between mice and humans. We report the presence of a population of ectopic germ cells in the human adrenals during development.

  5. Defective repair of UV-damaged DNA in human tumor and SV40-transformed human cells but not in adenovirus-transformed human cells

    International Nuclear Information System (INIS)

    Rainbow, A.J.

    1989-01-01

    The DNA repair capacities of five human tumor cell lines, one SV40-transformed human cell line and one adenovirus-transformed human cell line were compared with that of normal human fibroblasts using a sensitive host cell reactivation (HCR) technique. Unirradiated and UV-irradiated suspensions of adenovirus type 2 (Ad 2) were assayed for their ability to form viral structural antigens (Vag) in the various cell types using immunofluorescent staining. The survival of Vag formation for UV-irradiated Ad 2 was significantly reduced in all the human tumor cell lines and the SV40-transformed human line compared to the normal human fibroblasts, but was apparently normal in the adenovirus-transformed human cells. D 0 values for the UV survival of Ad 2 Vag synthesis in the tumor and virally transformed lines expressed as a percentage of that obtained on normal fibroblast strains were used as a measure of DNA repair capacity. Percent HCR values ranged from 26 to 53% in the tumor cells. These results indicate a deficiency in the repair of UV-induced DNA damage associated with human tumorigenesis and the transformation of human cells by SV40 but not the transformation of human cells by adenovirus. (author)

  6. Generation of hepatocyte- and endocrine pancreatic-like cells from human induced endodermal progenitor cells

    NARCIS (Netherlands)

    Sambathkumar, Rangarajan; Akkerman, Renate; Dastidar, Sumitava; Roelandt, Philip; Kumar, Manoj; Bajaj, Manmohan; Mestre Rosa, Ana Rita; Helsen, Nicky; Vanslembrouck, Veerle; Kalo, Eric; Khurana, Satish; Laureys, Jos; Gysemans, Conny; Faas, Marijke M; de Vos, Paul; Verfaillie, Catherine M

    2018-01-01

    Multipotent Adult Progenitor Cells (MAPCs) are one potential stem cell source to generate functional hepatocytes or β-cells. However, human MAPCs have less plasticity than pluripotent stem cells (PSCs), as their ability to generate endodermal cells is not robust. Here we studied the role of 14

  7. Proteomic analysis of human blastocoel fluid and blastocyst cells

    DEFF Research Database (Denmark)

    Jensen, Pernille; Beck, Hans Christian; Petersen, Jørgen

    2013-01-01

    Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the blastocyst and can differentiate into any cell type in the human body. These cells hold a great potential for regenerative medicine, but to obtain enough cells needed for medical treatment, culture is required......, the blastocoel fluid, which is in contact with all the cells in the blastocyst, including hESCs. Fifty-three surplus human blastocysts were donated after informed consent, and blastocoel fluid was isolated by micromanipulation. Using highly sensitive nano-high-pressure liquid chromatography-tandem mass...... from the ICM of the human blastocyst are exposed to via the blastocoel fluid. These results can be an inspiration for the development of improved culture conditions for hESCs....

  8. Abnormal number cell division of human thyroid anaplastic carcinoma cell line, SW 1736

    Directory of Open Access Journals (Sweden)

    Keiichi Ikeda

    2015-12-01

    Full Text Available Cell division, during which a mother cell usually divides into two daughter cells during one cell cycle, is the most important physiological event of cell biology. We observed one-to-four cell division during imaging of live SW1736 human thyroid anaplastic carcinoma cells transfected with a plasmid expressing the hybrid protein of green fluorescent protein and histone 2B (plasmid eGFP-H2B. Analysis of the images revealed a mother cell divided into four daughter cells. And one of the abnormally divided daughter cells subsequently formed a dinucleate cell.

  9. The use of human cells in biomedical research and testing.

    Science.gov (United States)

    Combes, Robert D

    2004-06-01

    The ability to use human cells in biomedical research and testing has the obvious advantage over the use of laboratory animals that the need for species extrapolation is obviated, due to the presence of more-relevant morphological, physiological and biochemical properties, including receptors. Moreover, human cells exhibit the same advantages as animal cells in culture in that different cell types can be used, from different tissues, with a wide range of techniques, to investigate a wide variety of biological phenomena in tissue culture. Human cells can also be grown as organotypic cultures to facilitate the extrapolation from cells to whole organisms. Human cell lines have been available for many years on an ad hoc basis from individual researchers, and also from recognised sources, such as the European Collection of Animal Cell Cultures (ECACC) and, in the USA, the Human Cell Culture Centre (HCCC). Such cells have usually been derived from tumours and this has restricted the variety of types of cells available. This problem has been addressed by using primary human cells that can be obtained from a variety of sources, such as cadavers, diseased tissue, skin strips, peripheral blood, buccal cavity smears, hair follicles and surgical waste from biopsy material that is unsuitable for transplantation purposes. However, primary human cells need to be obtained, processed, distributed and handled in a safe and ethical manner. They also have to be made available at the correct time to researchers very shortly after they become available. It is only comparatively recently that the safe and controlled acquisition of surgical waste and non-transplantable human tissues has become feasible with the establishment of several human tissue banks. Recently, the formation of a UK and European centralised network for human tissue supply has been initiated. The problems of short longevity and loss of specialisation in culture are being approached by: a) cell immortalisation to

  10. The response of human and rodent cells to hyperthermia

    International Nuclear Information System (INIS)

    Roizin-Towle, L.; Pirro, J.P.

    1991-01-01

    Inherent cellular radiosensitivity in vitro has been shown to be a good predictor of human tumor response in vivo. In contrast, the importance of the intrinsic thermosensitivity of normal and neoplastic human cells as a factor in the responsiveness of human tumors to adjuvant hyperthermia has never been analyzed systematically. A comparison of thermal sensitivity and thermo-radiosensitization in four rodent and eight human-derived cell lines was made in vitro. Arrhenius plots indicated that the rodent cells were more sensitive to heat killing than the human, and the break-point was 0.5 degrees C higher for the human than rodent cells. The relationship between thermal sensitivity and the interaction of heat with X rays at low doses was documented by thermal enhancement ratios (TER's). Cells received either a 1 hr exposure to 43 degrees C or a 20 minute treatment at 45 degrees C before exposure to 300 kVp X rays. Thermal enhancement ratios ranged from 1.0 to 2.7 for human cells heated at 43 degrees C and from 2.1 to 5.3 for heat exposures at 45 degrees C. Thermal enhancement ratios for rodent cells were generally 2 to 3 times higher than for human cells, because of the fact that the greater thermosensitivity of rodent cells results in a greater enhancement of radiation damage. Intrinsic thermosensitivity of human cells has relevance to the concept of thermal dose; intrinsic thermo-radiosensitization of a range of different tumor cells is useful in documenting the interactive effects of radiation combined with heat

  11. Minimal incorporation of Deepwater Horizon oil by estuarine filter feeders

    International Nuclear Information System (INIS)

    Fry, Brian; Anderson, Laurie C.

    2014-01-01

    Highlights: • Oil from the Deepwater Horizon oil spill entered Louisiana bays in mid-2010. • Oil was used minimally (<1%) in diets of mussels and barnacles. • Also, oil did not enhance planktonic respiration rates. • Use of oil carbon was relatively small in these productive estuarine food webs. - Abstract: Natural abundance carbon isotope analyses are sensitive tracers for fates and use of oil in aquatic environments. Use of oil carbon in estuarine food webs should lead to isotope values approaching those of oil itself, −27‰ for stable carbon isotopes reflecting oil origins and −1000‰ for carbon-14 reflecting oil age. To test for transfer of oil from the 2010 Deepwater Horizon spill into estuarine food webs, filter-feeding barnacles (Balanus sp.) and marsh mussels (Geukensia demissa) were collected from Louisiana estuaries near the site of the oil spill. Carbon-14 analyses of these animals from open waters and oiled marshes showed that oil use was <1% and near detection limits estimated at 0.3% oil incorporation. Respiration studies showed no evidence for enhanced microbial activity in bay waters. Results are consistent with low dietary impacts of oil for filter feeders and little overall impact on respiration in the productive Louisiana estuarine systems

  12. Automatic feeder for small fish held in tanks

    Science.gov (United States)

    Joeris, Leonard S.

    1965-01-01

    The Northville (Michigan) Biological Station has been a center for study of the developmental morphology of coregonid fishes. This work requires the production of numerous individual series of lake herring, lake whitefish, and several species of chubs from parent fish of positively known identity. The offspring of individual pairs or groups of fish must be held in individual tanks from the time they hatch until they reach maturity. One of the important problems in this project has been the poor growth of most fish. Though some have grown well, their growth has been less than that of the same species in nature, and a few fish from each hatch have grown very slowly. Irregularity of feeding may contribute to the slow growth of laboratory fish. The hatchery caretaker feeds them several times during his 8-hour workday, but they must go without food during the remaining 16 hours. The high metabolic rate of small fish, however, appears to make them strongly inclined toward almost continual feeding. Belief that greater, more regular food consumption would result from a mechanical feeder providing a continuous supply of food over a longer period of the day led to development of the equipment described in this paper.

  13. Generation of Functional Thymic Epithelium from Human Embryonic Stem Cells that Supports Host T Cell Development

    OpenAIRE

    Parent, Audrey V.; Russ, Holger A.; Khan, Imran S.; LaFlam, Taylor N.; Metzger, Todd C.; Anderson, Mark S.; Hebrok, Matthias

    2013-01-01

    Inducing immune tolerance to prevent rejection is a key step toward successful engraftment of stem-cell-derived tissue in a clinical setting. Using human pluripotent stem cells to generate thymic epithelial cells (TECs) capable of supporting T cell development represents a promising approach to reach this goal; however, progress toward generating functional TECs has been limited. Here, we describe a robust in vitro method to direct differentiation of human embryonic stem cells (hESCs) into th...

  14. Selective interactions between epithelial tumour cells and bone marrow mesenchymal stem cells

    OpenAIRE

    Hombauer, H; Minguell, J J

    2000-01-01

    This work is a comparative study on the features displayed by an epithelial metastatic breast cancer cell line (MCF-7) when set in co-culture with human bone marrow mesenchymal stem cells (MSC) or a feeder layer of 3T3 fibroblasts. MSC, a subset of non-haematopoietic cells in the marrow stroma, display a potential for self-renewal, proliferation and differentiation into precursors for bone, cartilage, connective and muscular tissue. Adhesion of MCF-7 cells to monolayers of MSC or 3T3 was high...

  15. Stepwise development of MAIT cells in mouse and human.

    Directory of Open Access Journals (Sweden)

    Emmanuel Martin

    2009-03-01

    Full Text Available Mucosal-associated invariant T (MAIT cells display two evolutionarily conserved features: an invariant T cell receptor (TCRalpha (iTCRalpha chain and restriction by the nonpolymorphic class Ib major histocompatibility complex (MHC molecule, MHC-related molecule 1 (MR1. MR1 expression on thymus epithelial cells is not necessary for MAIT cell development but their accumulation in the gut requires MR1 expressing B cells and commensal flora. MAIT cell development is poorly known, as these cells have not been found in the thymus so far. Herein, complementary human and mouse experiments using an anti-humanValpha7.2 antibody and MAIT cell-specific iTCRalpha and TCRbeta transgenic mice in different genetic backgrounds show that MAIT cell development is a stepwise process, with an intra-thymic selection followed by peripheral expansion. Mouse MAIT cells are selected in an MR1-dependent manner both in fetal thymic organ culture and in double iTCRalpha and TCRbeta transgenic RAG knockout mice. In the latter mice, MAIT cells do not expand in the periphery unless B cells are added back by adoptive transfer, showing that B cells are not required for the initial thymic selection step but for the peripheral accumulation. In humans, contrary to natural killer T (NKT cells, MAIT cells display a naïve phenotype in the thymus as well as in cord blood where they are in low numbers. After birth, MAIT cells acquire a memory phenotype and expand dramatically, up to 1%-4% of blood T cells. Finally, in contrast with NKT cells, human MAIT cell development is independent of the molecular adaptor SAP. Interestingly, mouse MAIT cells display a naïve phenotype and do not express the ZBTB16 transcription factor, which, in contrast, is expressed by NKT cells and the memory human MAIT cells found in the periphery after birth. In conclusion, MAIT cells are selected by MR1 in the thymus on a non-B non-T hematopoietic cell, and acquire a memory phenotype and expand in the

  16. Human induced pluripotent stem cells: A disruptive innovation.

    Science.gov (United States)

    De Vos, J; Bouckenheimer, J; Sansac, C; Lemaître, J-M; Assou, S

    2016-01-01

    This year (2016) will mark the 10th anniversary of the discovery of induced pluripotent stem cells (iPSCs). The finding that the transient expression of four transcription factors can radically remodel the epigenome, transcriptome and metabolome of differentiated cells and reprogram them into pluripotent stem cells has been a major and groundbreaking technological innovation. In this review, we discuss the major applications of this technology that we have grouped in nine categories: a model to study cell fate control; a model to study pluripotency; a model to study human development; a model to study human tissue and organ physiology; a model to study genetic diseases in a dish; a tool for cell rejuvenation; a source of cells for drug screening; a source of cells for regenerative medicine; a tool for the production of human organs in animals. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  17. Chromosome aberration induction in human diploid fibroblast and epithelial cells

    International Nuclear Information System (INIS)

    Scott, D.

    1986-01-01

    The relative sensitivity of cultured human fibroblasts and epithelial cells to radiation-induced chromosomal aberrations was investigated. Lung fibroblast and kidney epithelial cells from the same fetus were compared, as were skin fibroblasts and epithelial keratinocytes from the same foreskin sample. After exposure of proliferating fetal cells to 1.5 Gy X-rays there was a very similar aberration yield in the fibroblasts and epithelial cells. Observations of either little or no difference in chromosomal sensitivity between human fibroblasts and epithelial cells give added confidence that quantitative cytogenetic data obtained from cultured fibroblasts are relevant to the question of sensitivity of epithelial cells which are the predominant cell type in human cancers. (author)

  18. Genome editing: a robust technology for human stem cells.

    Science.gov (United States)

    Chandrasekaran, Arun Pandian; Song, Minjung; Ramakrishna, Suresh

    2017-09-01

    Human pluripotent stem cells comprise induced pluripotent and embryonic stem cells, which have tremendous potential for biological and therapeutic applications. The development of efficient technologies for the targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. Genome editing of stem cells is possible with the help of synthetic nucleases that facilitate site-specific modification of a gene of interest. Recent advances in genome editing techniques have improved the efficiency and speed of the development of stem cells for human disease models. Zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system are powerful tools for editing DNA at specific loci. Here, we discuss recent technological advances in genome editing with site-specific nucleases in human stem cells.

  19. Applied Developmental Biology: Making Human Pancreatic Beta Cells for Diabetics.

    Science.gov (United States)

    Melton, Douglas A

    2016-01-01

    Understanding the genes and signaling pathways that determine the differentiation and fate of a cell is a central goal of developmental biology. Using that information to gain mastery over the fates of cells presents new approaches to cell transplantation and drug discovery for human diseases including diabetes. © 2016 Elsevier Inc. All rights reserved.

  20. Human papilloma virus prevalence in laryngeal squamous cell carcinoma.

    Science.gov (United States)

    Gungor, A; Cincik, H; Baloglu, H; Cekin, E; Dogru, S; Dursun, E

    2007-08-01

    To determine the prevalence and type of human papilloma virus deoxyribonucleic acid (DNA) in cases of laryngeal squamous cell carcinoma. We analysed the prevalence of human papilloma virus infection in archived paraffin block specimens taken from 99 cases of laryngeal squamous cell carcinoma between 1990 and 2005, using polymerase chain reaction techniques. Biopsy specimens from five proven verrucous skin lesions were used as positive controls, and peripheral blood samples from five healthy volunteers were used as negative controls. Four test samples were found to have inadequate deoxyribonucleic acid purity and were therefore excluded from the study. Human papilloma virus deoxyribonucleic acid was detected in seven of 95 cases of laryngeal squamous cell carcinoma (7.36 per cent). Human papilloma virus genotyping revealed double human papilloma virus infection in three cases and single human papilloma virus infection in the remaining four cases. The human papilloma virus genotypes detected were 6, 11 and 16 (the latter detected in only one case). In our series, a very low human papilloma virus prevalence was found among laryngeal squamous cell carcinoma cases. The human papilloma virus genotypes detected were mostly 6 and/or 11, and 16 in only one case. To the best of our knowledge, this is the first report of human papilloma virus prevalence in laryngeal squamous cell carcinoma, based on polymerase chain reaction genotyping in a Turkish population.

  1. Decision Support for Optimal Repositioning of Containers in a Feeder System

    Directory of Open Access Journals (Sweden)

    Danijela Tuljak-Suban

    2008-03-01

    Full Text Available The transport of empty containers represents a seriousproblem in the fast growing sphere of maritime container transport.The most widespread type of container transport organizationin maritime transport is the hub and spoke mode, whichenables the transport of a great number of containers via largevessels between hub ports, from where feeder ships transport tosmaller ports that thus gravitate to the central hub port. The articlecontains a detailed analysis of the northern Adriatic portsand the feeder connections with the hub ports of the Mediterranean.A two-level VRPPD (Vehicle Routing Problem withPickup and Delivery problem is modelled on a graph, wherethe transport of full containers is privileged over the transport ofempty containers. This enables the simulation of the feeder systemin the n01them Adriatic, meaning that it shows the ship'soperator the movement programme with minimal transportcosts for the superfluous empty containers in the complex of theregular transports of full containers in the feeder system.

  2. Hexavalent chromium induces chromosome instability in human urothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Wise, Sandra S. [Wise Laboratory of Environmental and Genetic Toxicology, Maine Center for Toxicology and Environmental Health, Department of Applied Medical Science, University of Southern Maine, Science Building, 96 Falmouth Street, Portland, ME 04103 (United States); Holmes, Amie L. [Wise Laboratory of Environmental and Genetic Toxicology, Maine Center for Toxicology and Environmental Health, Department of Applied Medical Science, University of Southern Maine, Science Building, 96 Falmouth Street, Portland, ME 04103 (United States); Department of Radiation Oncology, Dana Farber Cancer Institute, 450 Brookline Ave., Boston, MA 02215 (United States); Liou, Louis [Department of Pathology, Boston University School of Medicine, 670 Albany St., Boston, MA 02118 (United States); Adam, Rosalyn M. [Department of Surgery, Harvard Medical School, Boston, MA 02115 (United States); Wise, John Pierce Sr., E-mail: john.wise@louisville.edu [Wise Laboratory of Environmental and Genetic Toxicology, Maine Center for Toxicology and Environmental Health, Department of Applied Medical Science, University of Southern Maine, Science Building, 96 Falmouth Street, Portland, ME 04103 (United States)

    2016-04-01

    Numerous metals are well-known human bladder carcinogens. Despite the significant occupational and public health concern of metals and bladder cancer, the carcinogenic mechanisms remain largely unknown. Chromium, in particular, is a metal of concern as incidences of bladder cancer have been found elevated in chromate workers, and there is an increasing concern for patients with metal hip implants. However, the impact of hexavalent chromium (Cr(VI)) on bladder cells has not been studied. We compared chromate toxicity in two bladder cell lines; primary human urothelial cells and hTERT-immortalized human urothelial cells. Cr(VI) induced a concentration- and time-dependent increase in chromosome damage in both cell lines, with the hTERT-immortalized cells exhibiting more chromosome damage than the primary cells. Chronic exposure to Cr(VI) also induced a concentration-dependent increase in aneuploid metaphases in both cell lines which was not observed after a 24 h exposure. Aneuploidy induction was higher in the hTERT-immortalized cells. When we correct for uptake, Cr(VI) induces a similar amount of chromosome damage and aneuploidy suggesting that the differences in Cr(VI) sensitivity between the two cells lines were due to differences in uptake. The increase in chromosome instability after chronic chromate treatment suggests this may be a mechanism for chromate-induced bladder cancer, specifically, and may be a mechanism for metal-induced bladder cancer, in general. - Highlights: • Hexavalent chromium is genotoxic to human urothelial cells. • Hexavalent chromium induces aneuploidy in human urothelial cells. • hTERT-immortalized human urothelial cells model the effects seen in primary urothelial cells. • Hexavalent chromium has a strong likelihood of being carcinogenic for bladder tissue.

  3. Male germline stem cells in non-human primates

    Directory of Open Access Journals (Sweden)

    S. Sharma

    2017-09-01

    Full Text Available Over the past few decades, several studies have attempted to decipher the biology of mammalian germline stem cells (GSCs. These studies provide evidence that regulatory mechanisms for germ cell specification and migration are evolutionarily conserved across species. The characteristics and functions of primate GSCs are highly distinct from rodent species; therefore the findings from rodent models cannot be extrapolated to primates. Due to limited availability of human embryonic and testicular samples for research purposes, two non-human primate models (marmoset and macaque monkeys are extensively employed to understand human germline development and differentiation. This review provides a broader introduction to the in vivo and in vitro germline stem cell terminology from primordial to differentiating germ cells. Primordial germ cells (PGCs are the most immature germ cells colonizing the gonad prior to sex differentiation into testes or ovaries. PGC specification and migratory patterns among different primate species are compared in the review. It also reports the distinctions and similarities in expression patterns of pluripotency markers (OCT4A, NANOG, SALL4 and LIN28 during embryonic developmental stages, among marmosets, macaques and humans. This review presents a comparative summary with immunohistochemical and molecular evidence of germ cell marker expression patterns during postnatal developmental stages, among humans and non-human primates. Furthermore, it reports findings from the recent literature investigating the plasticity behavior of germ cells and stem cells in other organs of humans and monkeys. The use of non-human primate models would enable bridging the knowledge gap in primate GSC research and understanding the mechanisms involved in germline development. Reported similarities in regulatory mechanisms and germ cell expression profile in primates demonstrate the preclinical significance of monkey models for development of

  4. Cytotoxic human CD4(+) T cells

    NARCIS (Netherlands)

    van de Berg, Pablo J.; van Leeuwen, Ester M.; ten Berge, Ineke J.; van Lier, Rene

    2008-01-01

    The induction of adaptive immune responses critically depends on helper signals provided by CD4(+) T cells. These signals not only license antigen presenting cells (APC) to activate naïve CD8(+) T cells leading to the formation of vast numbers of cytotoxic T lymphocytes but also support the

  5. Human Red Cells With Paroxysmal Nocturnal Haemoglobinuria ...

    African Journals Online (AJOL)

    The purified cells were used as hosts for the culture of P.falciparum in vitro. Results show that GPI-linked molecules on the red cell surface are not required for the efficient entry of the parasites, and that the PNH red cells are competent to sustain the growth of P.falciparum. Nigerian Quarterly Journal of Hospital Medicine Vol ...

  6. Human papillomavirus 16 E5 induces bi-nucleated cell formation by cell-cell fusion

    International Nuclear Information System (INIS)

    Hu Lulin; Plafker, Kendra; Vorozhko, Valeriya; Zuna, Rosemary E.; Hanigan, Marie H.; Gorbsky, Gary J.; Plafker, Scott M.; Angeletti, Peter C.; Ceresa, Brian P.

    2009-01-01

    Human papillomaviruses (HPV) 16 is a DNA virus encoding three oncogenes - E5, E6, and E7. The E6 and E7 proteins have well-established roles as inhibitors of tumor suppression, but the contribution of E5 to malignant transformation is controversial. Using spontaneously immortalized human keratinocytes (HaCaT cells), we demonstrate that expression of HPV16 E5 is necessary and sufficient for the formation of bi-nucleated cells, a common characteristic of precancerous cervical lesions. Expression of E5 from non-carcinogenic HPV6b does not produce bi-nucleate cells. Video microscopy and biochemical analyses reveal that bi-nucleates arise through cell-cell fusion. Although most E5-induced bi-nucleates fail to propagate, co-expression of HPV16 E6/E7 enhances the proliferation of these cells. Expression of HPV16 E6/E7 also increases bi-nucleated cell colony formation. These findings identify a new role for HPV16 E5 and support a model in which complementary roles of the HPV16 oncogenes lead to the induction of carcinogenesis

  7. Demonstration of Market-Based Real-Time Electricity Pricing on a Congested Feeder

    DEFF Research Database (Denmark)

    Larsen, Emil Mahler; Pinson, Pierre; le Ray, Guillaume

    2015-01-01

    Congestion management can delay grid reinforcements needed due to the growth of distributed technologies like photovoltaics and electric vehicles. This paper presents a method of congestion management for low voltage feeders using indirect control from the smart grid demonstration EcoGrid EU, where...... prices to 1900 houses, with a virtual feeder of 28 houses receiving congestion pricing. Simulations are used to calculate the cost from using this congestion management method, while demonstration results indicate that congestion can be managed successfully....

  8. Propagation of human spermatogonial stem cells in vitro.

    Science.gov (United States)

    Sadri-Ardekani, Hooman; Mizrak, Sefika C; van Daalen, Saskia K M; Korver, Cindy M; Roepers-Gajadien, Hermien L; Koruji, Morteza; Hovingh, Suzanne; de Reijke, Theo M; de la Rosette, Jean J M C H; van der Veen, Fulco; de Rooij, Dirk G; Repping, Sjoerd; van Pelt, Ans M M

    2009-11-18

    Young boys treated with high-dose chemotherapy are often confronted with infertility once they reach adulthood. Cryopreserving testicular tissue before chemotherapy and autotransplantation of spermatogonial stem cells at a later stage could theoretically allow for restoration of fertility. To establish in vitro propagation of human spermatogonial stem cells from small testicular biopsies to obtain an adequate number of cells for successful transplantation. Study performed from April 2007 to July 2009 using testis material donated by 6 adult men who underwent orchidectomy as part of prostate cancer treatment. Testicular cells were isolated and cultured in supplemented StemPro medium; germline stem cell clusters that arose were subcultured on human placental laminin-coated dishes in the same medium. Presence of spermatogonia was determined by reverse transcriptase polymerase chain reaction and immunofluorescence for spermatogonial markers. To test for the presence of functional spermatogonial stem cells in culture, xenotransplantation to testes of immunodeficient mice was performed, and migrated human spermatogonial stem cells after transplantation were detected by COT-1 fluorescence in situ hybridization. The number of colonized spermatogonial stem cells transplanted at early and later points during culture were counted to determine propagation. Propagation of spermatogonial stem cells over time. Testicular cells could be cultured and propagated up to 15 weeks. Germline stem cell clusters arose in the testicular cell cultures from all 6 men and could be subcultured and propagated up to 28 weeks. Expression of spermatogonial markers on both the RNA and protein level was maintained throughout the entire culture period. In 4 of 6 men, xenotransplantation to mice demonstrated the presence of functional spermatogonial stem cells, even after prolonged in vitro culture. Spermatogonial stem cell numbers increased 53-fold within 19 days in the testicular cell culture and

  9. Identification of Human Cutaneous Basal Cell Carcinoma Cancer Stem Cells.

    Science.gov (United States)

    Morgan, Huw; Olivero, Carlotta; Patel, Girish K

    2018-04-20

    The cancer stem cell model states that a subset of tumor cells, called "cancer stem cells," can initiate and propagate tumor growth through self-renewal, high proliferative capacity, and their ability to recreate tumor heterogeneity. In basal cell carcinoma (BCC), we have shown that tumor cells that express the cell surface protein CD200 fulfill the cancer stem cell hypothesis. CD200+ CD45- BCC cells represent 0.05-3.96% of all BCC cells and reside in small clusters at the tumor periphery. Using a novel, reproducible in vivo xenograft growth assay, we determined that tumor-initiating cell (TIC) frequencies are approximately 1 per 1.5 million unsorted BCC cells. The CD200+ CD45- BCC subpopulation recreated BCC tumor growth in vivo with typical histological architecture and expression of sonic hedgehog-regulated genes. Reproducible in vivo BCC growth was achieved with as few as 10,000 CD200+ CD45- cells, representing ~1500-fold enrichment. The methods used to identify and purify CD200+ CD45- BCC cells, as well as characterize gene expression, are described herein.

  10. Interactions between human mesenchymal stem cells and natural killer cells.

    Science.gov (United States)

    Sotiropoulou, Panagiota A; Perez, Sonia A; Gritzapis, Angelos D; Baxevanis, Constantin N; Papamichail, Michael

    2006-01-01

    Mesenchymal stem cells (MSCs) are multipotent progenitor cells representing an attractive therapeutic tool for regenerative medicine. They possess unique immunomodulatory properties, being capable of suppressing T-cell responses and modifying dendritic cell differentiation, maturation, and function, whereas they are not inherently immunogenic, failing to induce alloreactivity to T cells and freshly isolated natural killer (NK) cells. To clarify the generation of host immune responses to implanted MSCs in tissue engineering and their potential use as immunosuppressive elements, the effect of MSCs on NK cells was investigated. We demonstrate that at low NK-to-MSC ratios, MSCs alter the phenotype of NK cells and suppress proliferation, cytokine secretion, and cyto-toxicity against HLA-class I- expressing targets. Some of these effects require cell-to-cell contact, whereas others are mediated by soluble factors, including transforming growth factor-beta1 and prostaglandin E2, suggesting the existence of diverse mechanisms for MSC-mediated NK-cell suppression. On the other hand, MSCs are susceptible to lysis by activated NK cells. Overall, these data improve our knowledge of interactions between MSCs and NK cells and consequently of their effect on innate immune responses and their contribution to the regulation of adaptive immunity, graft rejection, and cancer immunotherapy.

  11. Preferential radiosensitization of human prostatic carcinoma cells by mild hyperthermia

    International Nuclear Information System (INIS)

    Ryu, Samuel; Brown, Stephen L.; Kim, Sang-Hie; Khil, Mark S.; Kim, Jae Ho

    1996-01-01

    Purpose: Recent cell culture studies by us and others suggest that some human carcinoma cells are more sensitive to heat than are rodent cells following mild hyperthermia. In studying the cellular mechanism of enhanced thermosensitivity of human tumor cells to hyperthermia, prostatic carcinoma cells of human origin were found to be more sensitive to mild hyperthermia than other human cancer cells. The present study was designed to determine the magnitude of radiosensitization of human prostatic carcinoma cells by mild hyperthermia and to examine whether the thermal radiosensitization is related to the intrinsic thermosensitivity of cancer cells. Methods and Materials: Two human prostatic carcinoma cell lines (DU-145 and PC-3) and other carcinoma cells of human origin, in particular, colon (HT-29), breast (MCF-7), lung (A-549), and brain (U-251) were exposed to temperatures of 40-41 deg. C. Single acute dose rate radiation and fractionated radiation were combined with mild hyperthermia to determine thermal radiosensitization. The end point of the study was the colony-forming ability of single-plated cells. Results: DU-145 and PC-3 cells were found to be exceedingly thermosensitive to 41 deg. C for 24 h, relative to other cancer cell lines. Ninety percent of the prostatic cancer cells were killed by a 24 h heat exposure. Prostatic carcinoma cells exposed to a short duration of heating at 41 deg. C for 2 h resulted in a substantial enhancement of radiation-induced cytotoxicity. The thermal enhancement ratios (TERs) of single acute dose radiation following heat treatment 41 deg. C for 2 h were 2.0 in DU-145 cells and 1.4 in PC-3 cells. The TERs of fractionated irradiation combined with continuous heating at 40 deg. C were similarly in the range of 2.1 to 1.4 in prostate carcinoma cells. No significant radiosensitization was observed in MCF-7 and HT-29 cells under the same conditions. Conclusion: The present data suggest that a significant radiosensitization of

  12. Cloning the interleukin 1 receptor from human T cells

    International Nuclear Information System (INIS)

    Sims, J.E.; Acres, R.B.; Grubin, C.E.; McMahan, C.J.; Wignall, J.M.; March, C.J.; Dower, S.K.

    1989-01-01

    cDNA clones of the interleukin 1 (IL-1) receptor expressed in a human T-cell clone have been isolated by using a murine IL-1 receptor cDNA as a probe. The human and mouse receptors show a high degree of sequence conservation. Both are integral membrane proteins possessing a single membrane-spanning segment. Similar to the mouse receptor, the human IL-1 receptor contains a large cytoplasmic region and an extracellular, IL-1 binding portion composed of three immunoglobulin-like domains. When transfected into COS cells, the human IL-1 receptor cDNA clone leads to expression of two different affinity classes of receptors, with K a values indistinguishable from those determined for IL-1 receptors in the original T-cell clone. An IL-1 receptor expressed in human dermal fibroblasts has also been cloned and sequenced and found to be identical to the IL-1 receptor expressed in T cells

  13. Radiation response characteristics of human cell in vitro

    International Nuclear Information System (INIS)

    Hall, E.J.

    1987-01-01

    Improvements in tissue culture techniques and growth media have made it possible to culture a range of cells of human origin, both normal and malignant. The most recent addition to the list are endothelial cells. Interesting results have been obtained, some of which may have implications in Radiation Therapy. (i) Repair of Potentially Lethal Damage (PLDR) has been observed in all cell lines investigated; cells of normal origin repair PLD at least as well as malignant cells, which makes clinical trials of PLDR inhibitors of doubtful usefulness. (ii) PLD in fibroblasts of human origin appears to have a component that is repaired rapidly, in a matter of minutes, as well as a slower component that takes hours to repair. (iii) Sublethal damage repair, manifest by a dose-rate effect, has also been observed in all human cell lines tested. Cells of normal tissue origin, including fibroblasts and endothelial cells, exhibit a dose-rate effect that is intermediate between that for cells from traditionally resistant tumors (melanoma and osteosarcoma) and cells from more sensitive tumors (neuroblastoma and breast). (iv) Fibroblasts from patients with Ataxia Telangectasia (AT) are much more sensitive to x-rays, with a D/sub o/ about half that for normal human fibroblasts. Nevertheless repair of both PLD and SLD can be demonstrated in these cells

  14. Derivation of novel human ground state naive pluripotent stem cells.

    Science.gov (United States)

    Gafni, Ohad; Weinberger, Leehee; Mansour, Abed AlFatah; Manor, Yair S; Chomsky, Elad; Ben-Yosef, Dalit; Kalma, Yael; Viukov, Sergey; Maza, Itay; Zviran, Asaf; Rais, Yoach; Shipony, Zohar; Mukamel, Zohar; Krupalnik, Vladislav; Zerbib, Mirie; Geula, Shay; Caspi, Inbal; Schneir, Dan; Shwartz, Tamar; Gilad, Shlomit; Amann-Zalcenstein, Daniela; Benjamin, Sima; Amit, Ido; Tanay, Amos; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

    2013-12-12

    Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation

  15. Concise Review: Kidney Generation with Human Pluripotent Stem Cells.

    Science.gov (United States)

    Morizane, Ryuji; Miyoshi, Tomoya; Bonventre, Joseph V

    2017-11-01

    Chronic kidney disease (CKD) is a worldwide health care problem, resulting in increased cardiovascular mortality and often leading to end-stage kidney disease, where patients require kidney replacement therapies such as hemodialysis or kidney transplantation. Loss of functional nephrons contributes to the progression of CKD, which can be attenuated but not reversed due to inability to generate new nephrons in human adult kidneys. Human pluripotent stem cells (hPSCs), by virtue of their unlimited self-renewal and ability to differentiate into cells of all three embryonic germ layers, are attractive sources for kidney regenerative therapies. Recent advances in stem cell biology have identified key signals necessary to maintain stemness of human nephron progenitor cells (NPCs) in vitro, and led to establishment of protocols to generate NPCs and nephron epithelial cells from human fetal kidneys and hPSCs. Effective production of large amounts of human NPCs and kidney organoids will facilitate elucidation of developmental and pathobiological pathways, kidney disease modeling and drug screening as well as kidney regenerative therapies. We summarize the recent studies to induce NPCs and kidney cells from hPSCs, studies of NPC expansion from mouse and human embryonic kidneys, and discuss possible approaches in vivo to regenerate kidneys with cell therapies and the development of bioengineered kidneys. Stem Cells 2017;35:2209-2217. © 2017 AlphaMed Press.

  16. Chemical Conversion of Human Fibroblasts into Functional Schwann Cells

    Directory of Open Access Journals (Sweden)

    Eva C. Thoma

    2014-10-01

    Full Text Available Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.

  17. Rho GTPase expression in human myeloid cells.

    Directory of Open Access Journals (Sweden)

    Suzanne F G van Helden

    Full Text Available Myeloid cells are critical for innate immunity and the initiation of adaptive immunity. Strict regulation of the adhesive and migratory behavior is essential for proper functioning of these cells. Rho GTPases are important regulators of adhesion and migration; however, it is unknown which Rho GTPases are expressed in different myeloid cells. Here, we use a qPCR-based approach to investigate Rho GTPase expression in myeloid cells.We found that the mRNAs encoding Cdc42, RhoQ, Rac1, Rac2, RhoA and RhoC are the most abundant. In addition, RhoG, RhoB, RhoF and RhoV are expressed at low levels or only in specific cell types. More differentiated cells along the monocyte-lineage display lower levels of Cdc42 and RhoV, while RhoC mRNA is more abundant. In addition, the Rho GTPase expression profile changes during dendritic cell maturation with Rac1 being upregulated and Rac2 downregulated. Finally, GM-CSF stimulation, during macrophage and osteoclast differentiation, leads to high expression of Rac2, while M-CSF induces high levels of RhoA, showing that these cytokines induce a distinct pattern. Our data uncover cell type specific modulation of the Rho GTPase expression profile in hematopoietic stem cells and in more differentiated cells of the myeloid lineage.

  18. Immune surveillance properties of human NK cell-derived exosomes.

    Science.gov (United States)

    Lugini, Luana; Cecchetti, Serena; Huber, Veronica; Luciani, Francesca; Macchia, Gianfranco; Spadaro, Francesca; Paris, Luisa; Abalsamo, Laura; Colone, Marisa; Molinari, Agnese; Podo, Franca; Rivoltini, Licia; Ramoni, Carlo; Fais, Stefano

    2012-09-15

    Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.

  19. Bacterial Feeders, the Nematode Caenorhabditis elegans and the Flagellate Cercomonas longicauda, have different Effects on Outcome of Competition among the Pseudomonas Biocontrol Strains CHA0 and DSS73

    DEFF Research Database (Denmark)

    Pedersen, Annette; Nybroe, Ole; Winding, Anne

    2009-01-01

    How bacterial feeding fauna affects colonization and survival of bacteria in soil is not well understood, which constrains the applicability of bacterial inoculants in agriculture. This study aimed to unravel how food quality of bacteria and bacterial feeders with different feeding habits (the......50090 or one of two biocontrol strains P. fluorescens CHA0 or Pseudomonas sp. DSS73) or combinations of two bacterial strains. DSM50090 is a suitable food bacterium, DSS73 is of intermediate food quality, and CHA0 is inedible to the bacterial feeders. Bacterial and protozoan cell numbers were measured...... predation pressure. Hence, the results suggested that the outcome of competition among bacteria depended on their ability to cope with the prevailing bacterial predator....

  20. Cell sources for in vitro human liver cell culture models

    Science.gov (United States)

    Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

    2016-01-01

    In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro. However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro. Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. PMID:27385595

  1. Calorimetric signatures of human cancer cells and their nuclei

    Energy Technology Data Exchange (ETDEWEB)

    Todinova, S. [Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113 (Bulgaria); Stoyanova, E. [Department of Molecular Immunology, Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, Tzarigradsko shose Blvd. 73, Sofia 1113 (Bulgaria); Krumova, S., E-mail: sakrumo@gmail.com [Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113 (Bulgaria); Iliev, I. [Institute of Experimental Morphology, Pathology and Anthropology with Museum, Acad. G. Bonchev Str., Bl. 25, Sofia 1113 (Bulgaria); Taneva, S.G. [Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113 (Bulgaria)

    2016-01-10

    Graphical abstract: - Highlights: • Two temperature ranges are distinguished in the thermograms of cells/nuclei. • Different thermodynamic properties of cancer and normal human cells/nuclei. • Dramatic reduction of the enthalpy of the low-temperature range in cancer cells. • Oxaliplatin and 5-FU affect the nuclear matrix proteins and the DNA stability. - Abstract: The human cancer cell lines HeLa, JEG-3, Hep G2, SSC-9, PC-3, HT-29, MCF7 and their isolated nuclei were characterized by differential scanning calorimetry. The calorimetric profiles differed from normal human fibroblast (BJ) cells in the two well distinguished temperature ranges—the high-temperature range (H{sub T}, due to DNA-containing structures) and the low-temperature range (L{sub T}, assigned to the nuclear matrix and cellular proteins). The enthalpy of the L{sub T} range, and, respectively the ratio of the enthalpies of the L{sub T}- vs. H{sub T}-range, ΔH{sub L}/ΔH{sub H}, is strongly reduced for all cancer cells compared to normal fibroblasts. On the contrary, for most of the cancer nuclei this ratio is higher compared to normal nuclei. The HT-29 human colorectal cancer cells/nuclei differed most drastically from normal human fibroblast cells/nuclei. Our data also reveal that the treatment of HT-29 cancer cells with cytostatic drugs affects not only the DNA replication but also the cellular proteome.

  2. Stem cell markers in the heart of the human newborn

    Directory of Open Access Journals (Sweden)

    Armando Faa

    2016-07-01

    Full Text Available The identification of cardiac progenitor cells in mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. Several recent studies now show that the different cell types that characterize the adult human heart arise from a common ancestor. Human cardiac stem cells differentiate into cardiomyocytes, and, in lesser extent, into smooth muscle and endothelial cells. The characterization of human cardiac stem cells (CSCs has important clinical implications. In recent years, CD117 (c-kit has been reported to mark a subtype of stem/progenitor cells in the human heart, with stem cell-like properties, including the ability to self-renewal and clonogenicity multipotentiality. Proceedings of the 2nd International Course on Perinatal Pathology (part of the 11th International Workshop on Neonatology · October 26th-31st, 2015 · Cagliari (Italy · October 31st, 2015 · Stem cells: present and future Guest Editors: Gavino Faa, Vassilios Fanos, Antonio Giordano

  3. Hurdles to clinical translation of human induced pluripotent stem cells

    OpenAIRE

    Neofytou, Evgenios; O’Brien, Connor Galen; Couture, Larry A.; Wu, Joseph C.

    2015-01-01

    Human pluripotent stem cells are known to have the capacity to renew indefinitely, being intrinsically able to differentiate into many different cell types. These characteristics have generated tremendous enthusiasm about the potential applications of these cells in regenerative medicine. However, major challenges remain with the development and testing of novel experimental stem cell therapeutics in the field. In this Review, we focus on the nature of the preclinical challenges and discuss p...

  4. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin.

    Science.gov (United States)

    Dorn, Isabel; Klich, Katharina; Arauzo-Bravo, Marcos J; Radstaak, Martina; Santourlidis, Simeon; Ghanjati, Foued; Radke, Teja F; Psathaki, Olympia E; Hargus, Gunnar; Kramer, Jan; Einhaus, Martin; Kim, Jeong Beom; Kögler, Gesine; Wernet, Peter; Schöler, Hans R; Schlenke, Peter; Zaehres, Holm

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential. Copyright© Ferrata Storti Foundation.

  5. Generation of Spinal Motor Neurons from Human Pluripotent Stem Cells.

    Science.gov (United States)

    Santos, David P; Kiskinis, Evangelos

    2017-01-01

    Human embryonic stem cells (ESCs) are characterized by their unique ability to self-renew indefinitely, as well as to differentiate into any cell type of the human body. Induced pluripotent stem cells (iPSCs) share these salient characteristics with ESCs and can easily be generated from any given individual by reprogramming somatic cell types such as fibroblasts or blood cells. The spinal motor neuron (MN) is a specialized neuronal subtype that synapses with muscle to control movement. Here, we present a method to generate functional, postmitotic, spinal motor neurons through the directed differentiation of ESCs and iPSCs by the use of small molecules. These cells can be utilized to study the development and function of human motor neurons in healthy and disease states.

  6. Rhein Induces Apoptosis in Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ching-Yao Chang

    2012-01-01

    Full Text Available Human breast cancers cells overexpressing HER2/neu are more aggressive tumors with poor prognosis, and resistance to chemotherapy. This study investigates antiproliferation effects of anthraquinone derivatives of rhubarb root on human breast cancer cells. Of 7 anthraquinone derivatives, only rhein showed antiproliferative and apoptotic effects on both HER2-overexpressing MCF-7 (MCF-7/HER2 and control vector MCF-7 (MCF-7/VEC cells. Rhein induced dose- and time-dependent manners increase in caspase-9-mediated apoptosis correlating with activation of ROS-mediated activation of NF-κB- and p53-signaling pathways in both cell types. Therefore, this study highlighted rhein as processing anti-proliferative activity against HER2 overexpression or HER2-basal expression in breast cancer cells and playing important roles in apoptotic induction of human breast cancer cells.

  7. Comparative mutagenesis of human cells in vivo and in vitro

    International Nuclear Information System (INIS)

    Thilly, W.G.

    1990-01-01

    Our goal is to develop the tools of mutational spectrometry in order to discover the cause(s) of genetic change in somatic and germinal cells in humans. Our study of the spectrum of point mutations in human mitochrondrial DNA sequences has revealed that there are multiple point mutation hotspots in each of four separate sequences in the mitochrondrial genome. These spectra were revealed by a combination of high fidelity PCR (modified T 7 polymerase) and denaturing gradient gel electrophoresis which has a limit of detection of about 10 -3 . There appear to be identical hotspot mutations in both cultured B cell and fresh human blood T cell samples

  8. Cardiac Progenitor Cell Extraction from Human Auricles

    KAUST Repository

    Di Nardo, Paolo

    2017-02-22

    For many years, myocardial tissue has been considered terminally differentiated and, thus, incapable of regenerating. Recent studies have shown, instead, that cardiomyocytes, at least in part, are slowly substituted by new cells originating by precursor cells mostly embedded into the heart apex and in the atria. We have shown that an elective region of progenitor cell embedding is represented by the auricles, non-contractile atria appendages that can be easily sampled without harming the patient. The protocol here reported describes how from auricles a population of multipotent, cardiogenic cells can be isolated, cultured, and differentiated. Further studies are needed to fully exploit this cell population, but, sampling auricles, it could be possible to treat cardiac patients using their own cells circumventing rejection or organ shortage limitations.

  9. Immune and Inflammatory Cell Composition of Human Lung Cancer Stroma.

    Directory of Open Access Journals (Sweden)

    G-Andre Banat

    Full Text Available Recent studies indicate that the abnormal microenvironment of tumors may play a critical role in carcinogenesis, including lung cancer. We comprehensively assessed the number of stromal cells, especially immune/inflammatory cells, in lung cancer and evaluated their infiltration in cancers of different stages, types and metastatic characteristics potential. Immunohistochemical analysis of lung cancer tissue arrays containing normal and lung cancer sections was performed. This analysis was combined with cyto-/histomorphological assessment and quantification of cells to classify/subclassify tumors accurately and to perform a high throughput analysis of stromal cell composition in different types of lung cancer. In human lung cancer sections we observed a significant elevation/infiltration of total-T lymphocytes (CD3+, cytotoxic-T cells (CD8+, T-helper cells (CD4+, B cells (CD20+, macrophages (CD68+, mast cells (CD117+, mononuclear cells (CD11c+, plasma cells, activated-T cells (MUM1+, B cells, myeloid cells (PD1+ and neutrophilic granulocytes (myeloperoxidase+ compared with healthy donor specimens. We observed all of these immune cell markers in different types of lung cancers including squamous cell carcinoma, adenocarcinoma, adenosquamous cell carcinoma, small cell carcinoma, papillary adenocarcinoma, metastatic adenocarcinoma, and bronchioloalveolar carcinoma. The numbers of all tumor-associated immune cells (except MUM1+ cells in stage III cancer specimens was significantly greater than those in stage I samples. We observed substantial stage-dependent immune cell infiltration in human lung tumors suggesting that the tumor microenvironment plays a critical role during lung carcinogenesis. Strategies for therapeutic interference with lung cancer microenvironment should consider the complexity of its immune cell composition.

  10. Manufacture and test of seismic bellows for ITER magnet feeder

    International Nuclear Information System (INIS)

    Liu, Chen; Lu, Kun; Sheng, Liang; Song, Yuntao; Su, Jinjin; Su, Man; Gung, Chenyu

    2016-01-01

    Highlights: • The design of the double bellows was iterated with the results of analysis based on the Expansion Joint Manufacturers Association (EJMA) standard. • The seismic bellows was tested with cyclic pressurization of the interlayer space to 2 bars absolute pressure for 5 cycles. • 200 cycles of tensile fatigue test with 90 mm of stretching from the nominal design length. • A full tensile test with 315 mm of stretching from its nominal length was conducted. • The prototype bellows was qualified for its leak tightness (less than 1 × 10 −9 Pam 3 /s of helium) at all time during the qualification test. - Abstract: This paper presents the key manufacturing and testing processes of the prototype ITER feeder seismic bellows. The design of the double bellows was iterated with the results of analysis based on the Expansion Joint Manufacturers Association (EJMA) standard. Each inner and outer bellows was supported in dedicated molds and formed by a hydraulic pressure machine rated at 800 tons. The double bellows were constructed by welding individual collars to the end flanges. The seismic bellows was tested with cyclic pressurization of the interlayer space to 2 bars absolute pressure for 5 cycles. This was followed by 200 cycles of tensile fatigue test with 90 mm of stretching from the nominal design length. After the mechanical fatigue test, a full tensile test with 315 mm of stretching from its nominal length was conducted. Helium leak tests, with the sensitivity of the helium leak detector set to 1 × 10 −9 Pa m 3 /s of helium, were performed at different stages of pressure and mechanical tests. The prototype bellows was qualified for its leak tightness at all time during the qualification test.

  11. Manufacture and test of seismic bellows for ITER magnet feeder

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chen, E-mail: liuchen@ipp.ac.cn [Institute of Plasma Physics Chinese Academy of Sciences, ShuShanhu Road No. 350, Hefei (China); Lu, Kun [Institute of Plasma Physics Chinese Academy of Sciences, ShuShanhu Road No. 350, Hefei (China); Sheng, Liang [AEROSUN-TOLA Expansion Joint Co., Ltd., Jiangjun Road No. 199, Nanjing (China); Song, Yuntao; Su, Jinjin [Institute of Plasma Physics Chinese Academy of Sciences, ShuShanhu Road No. 350, Hefei (China); Su, Man; Gung, Chenyu [ITER Organization, Route de Vinon-sur-Verdon, CS 90 046, 13067 St. Paul-lez-Durance (France)

    2016-11-01

    Highlights: • The design of the double bellows was iterated with the results of analysis based on the Expansion Joint Manufacturers Association (EJMA) standard. • The seismic bellows was tested with cyclic pressurization of the interlayer space to 2 bars absolute pressure for 5 cycles. • 200 cycles of tensile fatigue test with 90 mm of stretching from the nominal design length. • A full tensile test with 315 mm of stretching from its nominal length was conducted. • The prototype bellows was qualified for its leak tightness (less than 1 × 10{sup −9} Pam{sup 3}/s of helium) at all time during the qualification test. - Abstract: This paper presents the key manufacturing and testing processes of the prototype ITER feeder seismic bellows. The design of the double bellows was iterated with the results of analysis based on the Expansion Joint Manufacturers Association (EJMA) standard. Each inner and outer bellows was supported in dedicated molds and formed by a hydraulic pressure machine rated at 800 tons. The double bellows were constructed by welding individual collars to the end flanges. The seismic bellows was tested with cyclic pressurization of the interlayer space to 2 bars absolute pressure for 5 cycles. This was followed by 200 cycles of tensile fatigue test with 90 mm of stretching from the nominal design length. After the mechanical fatigue test, a full tensile test with 315 mm of stretching from its nominal length was conducted. Helium leak tests, with the sensitivity of the helium leak detector set to 1 × 10{sup −9} Pa m{sup 3}/s of helium, were performed at different stages of pressure and mechanical tests. The prototype bellows was qualified for its leak tightness at all time during the qualification test.

  12. 5-Fluorouracil-radiation interactions in human colon adenocarcinoma cells

    International Nuclear Information System (INIS)

    Buchholz, Daniel J.; Lepek, Katherine J.; Rich, Tyvin A.; Murray, David

    1995-01-01

    Purpose: To determine the effect of cellular proliferation and cell cycle stage on the ability of postirradiation 5-fluorouracil (5-FU) to radiosensitize cultured human colon adenocarcinoma Clone A cells. Methods and Materials: Cell survival curves were generated for irradiated: (a) log- and plateau-phase Clone A cells; and (b) Clone A cells separated by centrifugal elutriation into the various phases of the cell cycle; with and without postirradiation treatment with 100 μg/ml 5-FU. Results: Postirradiation treatment with 5-FU sensitized proliferating cells to a greater degree than it sensitized cells growing in plateau phase. The β component of cell kill in log-phase cells was increased by a factor of 1.5 with a sensitizer enhancement ratio of 1.21 at the 0.01 survival level. Plateau-phase cells showed less radiosensitization (sensitizer enhancement ratio of 1.13 at the 0.01 survival level); however, there was a mild increase in both α and β kill in plateau-phase cells. Elutriated G 1 cells were the most radiosensitive, independent of treatment with 5-FU. The phase of the cell cycle had little effect on the ability of fluorouracil to radiosensitize Clone A cells. Conclusion: Proliferating cells are more susceptible to radiosensitization with 5-FU than plateau-phase cells are, but this effect appears to be independent of the phase of the cell cycle

  13. The contribution of human/non-human animal chimeras to stem cell research

    Directory of Open Access Journals (Sweden)

    Sonya Levine

    2017-10-01

    Full Text Available Chimeric animals are made up of cells from two separate zygotes. Human/non-human animal chimeras have been used for a number of research purposes, including human disease modeling. Pluripotent stem cell (PSC research has relied upon the chimera approach to examine the developmental potential of stem cells, to determine the efficacy of cell replacement therapies, and to establish a means of producing human organs. Based on ethical issues, this work has faced pushback from various sources including funding agencies. We discuss here the essential role these studies have played, from gaining a better understanding of human biology to providing a stepping stone to human disease treatments. We also consider the major ethical issues, as well as the current status of support for this work in the United States.

  14. The contribution of human/non-human animal chimeras to stem cell research.

    Science.gov (United States)

    Levine, Sonya; Grabel, Laura

    2017-10-01

    Chimeric animals are made up of cells from two separate zygotes. Human/non-human animal chimeras have been used for a number of research purposes, including human disease modeling. Pluripotent stem cell (PSC) research has relied upon the chimera approach to examine the developmental potential of stem cells, to determine the efficacy of cell replacement therapies, and to establish a means of producing human organs. Based on ethical issues, this work has faced pushback from various sources including funding agencies. We discuss here the essential role these studies have played, from gaining a better understanding of human biology to providing a stepping stone to human disease treatments. We also consider the major ethical issues, as well as the current status of support for this work in the United States. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Human pituitary and placental hormones control human insulin-like growth factor II secretion in human granulosa cells

    International Nuclear Information System (INIS)

    Ramasharma, K.; Li, C.H.

    1987-01-01

    Human granulosa cells cultured with calf serum actively proliferated for 18-20 generations and secreted progesterone into the medium; progesterone levels appeared to decline with increase in generation number. Cells cultured under serum-free conditions secreted significant amounts of progesterone and insulin-like growth factor II (IGF-II). The progesterone secretion was enhanced by the addition of human follitropin, lutropin, and chorionic gonadotropin but not by growth hormone. These cells, when challenged to varying concentrations of human growth hormone, human chorionic somatomammotropin, human prolactin, chorionic gonadotropin, follitropin, and lutropin, secreted IGF-II into the medium as measured by specific IGF-II RIA. Among these human hormones, chorionic gonadotropin, follitropin, and lutropin were most effective in inducing IGF-II secretion from these cells. When synthetic lutropin-releasing hormone and α-inhibin-92 were tested, only lutropin-releasing hormone was effective in releasing IGF-II. The results described suggest that cultured human granulosa cells can proliferate and actively secrete progesterone and IGF-II into the medium. IGF-II production in human granulosa cells was influenced by a multi-hormonal complex including human growth hormone, human chorionic somatomammotropin, and prolactin

  16. Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells.

    Science.gov (United States)

    Bhise, Nupura S; Wahlin, Karl J; Zack, Donald J; Green, Jordan J

    2013-01-01

    Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts. A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling. 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents, including Lipofectamine® 2000, FuGENE® HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester) reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups. This

  17. Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

    Directory of Open Access Journals (Sweden)

    José J. Gaforio

    2011-10-01

    Full Text Available Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A or breast cancer cells (MDA-MB-231 and MCF7. We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

  18. Induction of apoptosis by eugenol in human breast cancer cells

    International Nuclear Information System (INIS)

    Vidhya, N.; Niranjali Devaraj, S.

    2011-01-01

    In the present study, potential anticancer effect of eugenol on inhibition of cell proliferation and induction of apoptosis in human MCF-7 breast cancer cells was investigated. Induction of cell death by eugenol was evaluated following MTT assay and monitoring lactate dehydrogenase released into the culture medium for cell viability and cytotoxicity, giemsa staining for morphological alterations, fluorescence microscopy analysis of cells using ethidium bromide and acridine orange and quantitation of DNA fragments for induction of apoptosis. Effect of eugenol on intracellular redox status of the human breast cancer cells was assessed by determining the level of glutathione and lipid peroxidation products (TBARS). Eugenol treatment inhibited the growth and proliferation of human MCF-7 breast cancer cells through induction of cell death, which was dose and time dependent. Microscopic examination of eugenol treated cells showed cell shrinkage, membrane blebbing and apoptotic body formation. Further, eugenol treatment also depleted the level of intracellular glutathione and increased the level of lipid peroxidation. The dose dependent increase in the percentage of apoptotic cells and DNA fragments suggested that apoptosis was involved in eugenol induced cell death and apoptosis might have played a role in the chemopreventive action of eugenol. (author)

  19. Inhibition of fatty acid metabolism reduces human myeloma cells proliferation.

    Directory of Open Access Journals (Sweden)

    José Manuel Tirado-Vélez

    Full Text Available Multiple myeloma is a haematological malignancy characterized by the clonal proliferation of plasma cells. It has been proposed that targeting cancer cell metabolism would provide a new selective anticancer therapeutic strategy. In this work, we tested the hypothesis that inhibition of β-oxidation and de novo fatty acid synthesis would reduce cell proliferation in human myeloma cells. We evaluated the effect of etomoxir and orlistat on fatty acid metabolism, glucose metabolism, cell cycle distribution, proliferation, cell death and expression of G1/S phase regulatory proteins in myeloma cells. Etomoxir and orlistat inhibited β-oxidation and de novo fatty acid synthesis respectively in myeloma cells, without altering significantly glucose metabolism. These effects were associated with reduced cell viability and cell cycle arrest in G0/G1. Specifically, etomoxir and orlistat reduced by 40-70% myeloma cells proliferation. The combination of etomoxir and orlistat resulted in an additive inhibitory effect on cell proliferation. Orlistat induced apoptosis and sensitized RPMI-8226 cells to apoptosis induction by bortezomib, whereas apoptosis was not altered by etomoxir. Finally, the inhibitory effect of both drugs on cell proliferation was associated with reduced p21 protein levels and phosphorylation levels of retinoblastoma protein. In conclusion, inhibition of fatty acid metabolism represents a potential therapeutic approach to treat human multiple myeloma.

  20. X-ray sensitivity of human tumor cells in vitro

    International Nuclear Information System (INIS)

    Weichselbaum, R.R.; Nove, J.; Little, J.B.

    1980-01-01

    Clonally-derived cells from ten human malignant tumors considered radiocurable (breast, neuroblastoma, medulloblastoma) or non-radiocurable (osteosarcoma, hypernephroma, glioblastoma, melanoma) were studied in cell culture and their in vitro x-ray survival curve parameters determined (anti n, D 0 ). There were no significant differences among the tumor cell lines suggesting that survival parameters in vitro do not explain differences in clinical radiocurability. Preliminary investigation with density inhibited human tumor cells indicate that such an approach may yield information regarding inherent cellular differences in radiocurability

  1. Radiosensitivity of normal human epidermal cells in culture

    International Nuclear Information System (INIS)

    Dover, R.; Potten, C.S.

    1983-01-01

    Using an in vitro culture system the authors have derived #betta#-radiation survival curves over a dose range 0-8 Gy for the clonogenic cells of normal human epidermis. The culture system used allows the epidermal cells to stratify and form a multi-layered sheet of keratinizing cells. The cultures appear to be a very good model for epidermis in vivo. The survival curves show a population which is apparently more sensitive than murine epidermis in vivo. It remains unclear whether this is an intrinsic difference between the species or is a consequence of the in vitro cultivation of the human cells. (author)

  2. Modeling human neurological disorders with induced pluripotent stem cells.

    Science.gov (United States)

    Imaizumi, Yoichi; Okano, Hideyuki

    2014-05-01

    Human induced pluripotent stem (iPS) cells obtained by reprogramming technology are a source of great hope, not only in terms of applications in regenerative medicine, such as cell transplantation therapy, but also for modeling human diseases and new drug development. In particular, the production of iPS cells from the somatic cells of patients with intractable diseases and their subsequent differentiation into cells at affected sites (e.g., neurons, cardiomyocytes, hepatocytes, and myocytes) has permitted the in vitro construction of disease models that contain patient-specific genetic information. For example, disease-specific iPS cells have been established from patients with neuropsychiatric disorders, including schizophrenia and autism, as well as from those with neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. A multi-omics analysis of neural cells originating from patient-derived iPS cells may thus enable investigators to elucidate the pathogenic mechanisms of neurological diseases that have heretofore been unknown. In addition, large-scale screening of chemical libraries with disease-specific iPS cells is currently underway and is expected to lead to new drug discovery. Accordingly, this review outlines the progress made via the use of patient-derived iPS cells toward the modeling of neurological disorders, the testing of existing drugs, and the discovery of new drugs. The production of human induced pluripotent stem (iPS) cells from the patients' somatic cells and their subsequent differentiation into specific cells have permitted the in vitro construction of disease models that contain patient-specific genetic information. Furthermore, innovations of gene-editing technologies on iPS cells are enabling new approaches for illuminating the pathogenic mechanisms of human diseases. In this review article, we outlined the current status of neurological diseases-specific iPS cell research and described recently obtained

  3. Using Human Induced Pluripotent Stem Cells to Model Skeletal Diseases.

    Science.gov (United States)

    Barruet, Emilie; Hsiao, Edward C

    2016-01-01

    Musculoskeletal disorders affecting the bones and joints are major health problems among children and adults. Major challenges such as the genetic origins or poor diagnostics of severe skeletal disease hinder our understanding of human skeletal diseases. The recent advent of human induced pluripotent stem cells (human iPS cells) provides an unparalleled opportunity to create human-specific models of human skeletal diseases. iPS cells have the ability to self-renew, allowing us to obtain large amounts of starting material, and have the potential to differentiate into any cell types in the body. In addition, they can carry one or more mutations responsible for the disease of interest or be genetically corrected to create isogenic controls. Our work has focused on modeling rare musculoskeletal disorders including fibrodysplasia ossificans progressive (FOP), a congenital disease of increased heterotopic ossification. In this review, we will discuss our experiences and protocols differentiating human iPS cells toward the osteogenic lineage and their application to model skeletal diseases. A number of critical challenges and exciting new approaches are also discussed, which will allow the skeletal biology field to harness the potential of human iPS cells as a critical model system for understanding diseases of abnormal skeletal formation and bone regeneration.

  4. Human natural killer cell committed thymocytes and their relation to the T cell lineage

    NARCIS (Netherlands)

    Sánchez, M. J.; Spits, H.; Lanier, L. L.; Phillips, J. H.

    1993-01-01

    Recent studies have demonstrated that mature natural killer (NK) cells can be grown from human triple negative (TN; CD3-, CD4-, CD8-) thymocytes, suggesting that a common NK/T cell precursor exists within the thymus that can give rise to both NK cells and T cells under appropriate conditions. In the

  5. Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus.

    Science.gov (United States)

    Soares, Filipa A C; Pedersen, Roger A; Vallier, Ludovic

    2016-01-01

    This protocol describes the efficient isolation of peripheral blood mononuclear cells from circulating blood via density gradient centrifugation and subsequent generation of integration-free human induced pluripotent stem cells. Peripheral blood mononuclear cells are cultured for 9 days to allow expansion of the erythroblast population. The erythroblasts are then used to derive human induced pluripotent stem cells using Sendai viral vectors, each expressing one of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc.

  6. Cell membrane softening in human breast and cervical cancer cells

    Science.gov (United States)

    Händel, Chris; Schmidt, B. U. Sebastian; Schiller, Jürgen; Dietrich, Undine; Möhn, Till; Kießling, Tobias R.; Pawlizak, Steve; Fritsch, Anatol W.; Horn, Lars-Christian; Briest, Susanne; Höckel, Michael; Zink, Mareike; Käs, Josef A.

    2015-08-01

    Biomechanical properties are key to many cellular functions such as cell division and cell motility and thus are crucial in the development and understanding of several diseases, for instance cancer. The mechanics of the cellular cytoskeleton have been extensively characterized in cells and artificial systems. The rigidity of the plasma membrane, with the exception of red blood cells, is unknown and membrane rigidity measurements only exist for vesicles composed of a few synthetic lipids. In this study, thermal fluctuations of giant plasma membrane vesicles (GPMVs) directly derived from the plasma membranes of primary breast and cervical cells, as well as breast cell lines, are analyzed. Cell blebs or GPMVs were studied via thermal membrane fluctuations and mass spectrometry. It will be shown that cancer cell membranes are significantly softer than their non-malignant counterparts. This can be attributed to a loss of fluid raft forming lipids in malignant cells. These results indicate that the reduction of membrane rigidity promotes aggressive blebbing motion in invasive cancer cells.

  7. Fraction against Human Cancer Cell Lines

    African Journals Online (AJOL)

    fraction of A. sieberi against seven cancer cell lines (Colo20, HCT116, DLD, MCF7, Jurkat, HepG2 and ... The morphology of the HepG2 cell nucleus was investigated by Hoechst 33342, ..... Gong F, Liang Y, Xie P, Chau F. Information theory.

  8. Cardiac Progenitor Cell Extraction from Human Auricles

    KAUST Repository

    Di Nardo, Paolo; Pagliari, Francesca

    2017-01-01

    by precursor cells mostly embedded into the heart apex and in the atria. We have shown that an elective region of progenitor cell embedding is represented by the auricles, non-contractile atria appendages that can be easily sampled without harming the patient

  9. Transcriptional profiling of putative human epithelial stem cells

    Directory of Open Access Journals (Sweden)

    Koçer Salih S

    2008-07-01

    Full Text Available Abstract Background Human interfollicular epidermis is sustained by the proliferation of stem cells and their progeny, transient amplifying cells. Molecular characterization of these two cell populations is essential for better understanding of self renewal, differentiation and mechanisms of skin pathogenesis. The purpose of this study was to obtain gene expression profiles of alpha 6+/MHCI+, transient amplifying cells and alpha 6+/MHCI-, putative stem cells, and to compare them with existing data bases of gene expression profiles of hair follicle stem cells. The expression of Major Histocompatibility Complex (MHC class I, previously shown to be absent in stem cells in several tissues, and alpha 6 integrin were used to isolate MHCI positive basal cells, and MHCI low/negative basal cells. Results Transcriptional profiles of the two cell populations were determined and comparisons made with published data for hair follicle stem cell gene expression profiles. We demonstrate that presumptive interfollicular stem cells, alpha 6+/MHCI- cells, are enriched in messenger RNAs encoding surface receptors, cell adhesion molecules, extracellular matrix proteins, transcripts encoding members of IFN-alpha family proteins and components of IFN signaling, but contain lower levels of transcripts encoding proteins which take part in energy metabolism, cell cycle, ribosome biosynthesis, splicing, protein translation, degradation, DNA replication, repair, and chromosome remodeling. Furthermore, our data indicate that the cell signaling pathways Notch1 and NF-κB are downregulated/inhibited in MHC negative basal cells. Conclusion This study demonstrates that alpha 6+/MHCI- cells have additional characteristics attributed to stem cells. Moreover, the transcription profile of alpha 6+/MHCI- cells shows similarities to transcription profiles of mouse hair follicle bulge cells known to be enriched for stem cells. Collectively, our data suggests that alpha 6+/MHCI- cells

  10. DNA amplification is rare in normal human cells

    International Nuclear Information System (INIS)

    Wright, J.A.; Watt, F.M.; Hudson, D.L.; Stark, G.R.; Smith, H.S.; Hancock, M.C.

    1990-01-01

    Three types of normal human cells were selected in tissue culture with three drugs without observing a single amplification event from a total of 5 x 10 8 cells. No drug-resistant colonies were observed when normal foreskin keratinocytes were selected with N-(phosphonacetyl)-L-aspartate or with hydroxyurea or when normal mammary epithelial cells were selected with methotrexate. Some slightly resistant colonies with limited potential for growth were obtained when normal diploid fibroblast cells derived from fetal lung were selected with methotrexate or hydroxyurea but careful copy-number analysis of the dihydrofolate reductase and ribonucleotide reductase genes revealed no evidence of amplification. The rarity of DNA amplification in normal human cells contrasts strongly with the situation in tumors and in established cell lines, where amplification of onogenes and of genes mediating drug resistance is frequent. The results suggest that tumors and cell lines have acquired the abnormal ability to amplify DNA with high frequency

  11. Toona Sinensis Extracts Induced Cell Cycle Arrest and Apoptosis in the Human Lung Large Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Cheng-Yuan Wang

    2010-02-01

    Full Text Available Toona sinensis extracts have been shown to exhibit anti-cancer effects in human ovarian cancer cell lines, human promyelocytic leukemia cells and human lung adenocarcinoma. Its safety has also been confirmed in animal studies. However, its anti-cancer properties in human lung large cell carcinoma have not been studied. Here, we used a powder obtained by freeze-drying the super-natant of centrifuged crude extract from Toona sinensis leaves (TSL-1 to treat the human lung carcinoma cell line H661. Cell viability was evaluated by the 3-(4-,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide assay. Flow cytometry analysis revealed that TSL-1 blocked H661 cell cycle progression. Western blot analysis showed decreased expression of cell cycle proteins that promote cell cycle progression, including cyclin-dependent kinase 4 and cyclin D1, and increased the expression of proteins that inhibit cell cycle progression, including p27. Furthermore, flow cytometry analysis showed that TSL-1 induced H661 cell apoptosis. Western blot analysis showed that TSL-1 reduced the expression of the anti-apoptotic protein B-cell lymphoma 2, and degraded the DNA repair protein, poly(ADP-ribose polymerase. TSL-1 shows potential as a novel therapeutic agent or for use as an adjuvant for treating human lung large cell carcinoma.

  12. Culture of human cell lines by a pathogen-inactivated human platelet lysate.

    Science.gov (United States)

    Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

    2016-08-01

    Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

  13. Agonist-induced desensitization of human β3-adrenoceptors expressed in human embryonic kidney cells

    NARCIS (Netherlands)

    Michel-Reher, Martina B.; Michel, Martin C.

    2013-01-01

    β3-Adrenoceptors are resistant to agonist-induced desensitization in some cell types but susceptible in others including transfected human embryonic kidney (HEK) cells. Therefore, we have studied cellular and molecular changes involved in agonist-induced β3-adrenoceptor desensitization in HEK cells.

  14. Cell shape regulates global histone acetylation in human mammaryepithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Le Beyec, Johanne; Xu, Ren; Lee, Sun-Young; Nelson, Celeste M.; Rizki, Aylin; Alcaraz, Jordi; Bissell, Mina J.

    2007-02-28

    Extracellular matrix (ECM) regulates cell morphology and gene expression in vivo; these relationships are maintained in three-dimensional (3D) cultures of mammary epithelial cells. In the presence of laminin-rich ECM (lrECM), mammary epithelial cells round up and undergo global histone deacetylation, a process critical for their functional differentiation. However, it remains unclear whether lrECM-dependent cell rounding and global histone deacetylation are indeed part of a common physical-biochemical pathway. Using 3D cultures as well as nonadhesive and micropatterned substrata, here we showed that the cell 'rounding' caused by lrECM was sufficient to induce deacetylation of histones H3 and H4 in the absence of biochemical cues. Microarray and confocal analysis demonstrated that this deacetylation in 3D culture is associated with a global increase in chromatin condensation and a reduction in gene expression. Whereas cells cultured on plastic substrata formed prominent stress fibers, cells grown in 3D lrECM or on micropatterns lacked these structures. Disruption of the actin cytoskeleton with cytochalasin D phenocopied the lrECM-induced cell rounding and histone deacetylation. These results reveal a novel link between ECM-controlled cell shape and chromatin structure, and suggest that this link is mediated by changes in the actin cytoskeleton.

  15. The development of human mast cells. An historical reappraisal

    Energy Technology Data Exchange (ETDEWEB)

    Ribatti, Domenico, E-mail: domenico.ribatti@uniba.it

    2016-03-15

    The understanding of mast cell (MC) differentiation is derived mainly from in vitro studies of different stages of stem and progenitor cells. The hematopoietic lineage development of human MCs is unique compared to other myeloid-derived cells. Human MCs originate from CD34{sup +}/CD117{sup +}/CD13{sup +}multipotent hematopoietic progenitors, which undergo transendothelial recruitment into peripheral tissues, where they complete differentiation. Stem cell factor (SCF) is a major chemotactic factor for MCs and their progenitors. SCF also elicits cell-cell and cell-substratum adhesion, facilitates the proliferation, and sustains the survival, differentiation, and maturation, of MCs. Because MC maturation is influenced by local microenvironmental factors, different MC phenotypes can develop in different tissues and organs. - Highlights: • Human mast cells originate from CD34/CD117/CD13 positive multipotent hematopoietic progenitors. • Stem cell factor is a major chemotactic factor for mast cells and their progenitors. • Different mast cell phenotypes can develop in different tissues and organs.

  16. Generation and purification of human stem cell-derived cardiomyocytes

    NARCIS (Netherlands)

    Schwach, Verena; Passier, Robert

    2016-01-01

    © 2016 International Society of Differentiation Efficient and reproducible generation and purification of human stem cell-derived cardiomyocytes (CMs) is crucial for regenerative medicine, disease modeling, drug screening and study of developmental events during cardiac specification. Established

  17. Research on human placenta-derived mesenchymal stem cells ...

    African Journals Online (AJOL)

    Research on human placenta-derived mesenchymal stem cells transfected with pIRES2-EGFP-VEGF165 using liposome. ... African Journal of Biotechnology. Journal Home · ABOUT THIS JOURNAL · Advanced Search · Current Issue ...

  18. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  19. Chimeric animal models in human stem cell biology.

    Science.gov (United States)

    Glover, Joel C; Boulland, Jean-Luc; Halasi, Gabor; Kasumacic, Nedim

    2009-01-01

    The clinical use of stem cells for regenerative medicine is critically dependent on preclinical studies in animal models. In this review we examine some of the key issues and challenges in the use of animal models to study human stem cell biology-experimental standardization, body size, immunological barriers, cell survival factors, fusion of host and donor cells, and in vivo imaging and tracking. We focus particular attention on the various imaging modalities that can be used to track cells in living animals, comparing their strengths and weaknesses and describing technical developments that are likely to lead to new opportunities for the dynamic assessment of stem cell behavior in vivo. We then provide an overview of some of the most commonly used animal models, their advantages and disadvantages, and examples of their use for xenotypic transplantation of human stem cells, with separate reviews of models involving rodents, ungulates, nonhuman primates, and the chicken embryo. As the use of human somatic, embryonic, and induced pluripotent stem cells increases, so too will the range of applications for these animal models. It is likely that increasingly sophisticated uses of human/animal chimeric models will be developed through advances in genetic manipulation, cell delivery, and in vivo imaging.

  20. Human airway xenograft models of epithelial cell regeneration

    Directory of Open Access Journals (Sweden)

    Puchelle Edith

    2000-10-01

    Full Text Available Abstract Regeneration and restoration of the airway epithelium after mechanical, viral or bacterial injury have a determinant role in the evolution of numerous respiratory diseases such as chronic bronchitis, asthma and cystic fibrosis. The study in vivo of epithelial regeneration in animal models has shown that airway epithelial cells are able to dedifferentiate, spread, migrate over the denuded basement membrane and progressively redifferentiate to restore a functional respiratory epithelium after several weeks. Recently, human tracheal xenografts have been developed in immunodeficient severe combined immunodeficiency (SCID and nude mice. In this review we recall that human airway cells implanted in such conditioned host grafts can regenerate a well-differentiated and functional human epithelium; we stress the interest in these humanized mice in assaying candidate progenitor and stem cells of the human airway mucosa.

  1. Studies on catecholamine function in human fat cells

    OpenAIRE

    Hellström, Lena

    1996-01-01

    Catecholamine function in human fat cells Lena Hellström, Centre for Metabolism and Endocrinology, Department of Medicine, Huddinge University Hospital, Karolinska Institute, S-141 86 Huddinge, Sweden Human adipose tissue is a heterogeneous organ as regards metabolism. The effects of catecholamines, the main lipolytic hormones in man vary considerably in different regions. Fat cell lipolysis also changes in a number of physiological and pathophysiological states...

  2. Dissection of a stem cell hierarchy in the human breast

    DEFF Research Database (Denmark)

    Rubner Fridriksdottir, Agla Jael

    and apoptosis during each menstrual cycle. These changes are most prominent during pregnancy, lactation and involution after breast feeding. These highly dynamic changes are thought to rely on the presence of a breast epithelial stem cell population (reviewed in (Fridriksdottir et al. 2005)). Nevertheless......, cellular pathways that contribute to adult human breast gland architecture and cell lineages have not been described. Here, I identify a candidate stem cell niche in ducts, and zones containing progenitor cells in lobules (Villadsen and Fridriksdottir et al. 2007). Putative stem cells residing in ducts......-rich extracellular matrix gel. Staining for the epithelial lineage markers, cytokeratins K14 and K19, further reveals multipotent cells in the stem cell zone and three lineage- restricted cell types outside this zone. Multiparameter cell sorting and functional characterization with reference to anatomical sites...

  3. Engineering antigen-specific T cells from genetically modified human hematopoietic stem cells in immunodeficient mice.

    Directory of Open Access Journals (Sweden)

    Scott G Kitchen

    Full Text Available There is a desperate need for effective therapies to fight chronic viral infections. The immune response is normally fastidious at controlling the majority of viral infections and a therapeutic strategy aimed at reestablishing immune control represents a potentially powerful approach towards treating persistent viral infections. We examined the potential of genetically programming human hematopoietic stem cells to generate mature CD8+ cytotoxic T lymphocytes that express a molecularly cloned, "transgenic" human anti-HIV T cell receptor (TCR. Anti-HIV TCR transduction of human hematopoietic stem cells directed the maturation of a large population of polyfunctional, HIV-specific CD8+ cells capable of recognizing and killing viral antigen-presenting cells. Thus, through this proof-of-concept we propose that genetic engineering of human hematopoietic stem cells will allow the tailoring of effector T cell responses to fight HIV infection or other diseases that are characterized by the loss of immune control.

  4. FOXL2-induced follistatin attenuates activin A-stimulated cell proliferation in human granulosa cell tumors

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Jung-Chien; Chang, Hsun-Ming; Qiu, Xin; Fang, Lanlan; Leung, Peter C.K., E-mail: peter.leung@ubc.ca

    2014-01-10

    Highlights: •Activin A stimulates cell proliferation in KGN human granulosa cell tumor-derived cell line. •Cyclin D2 mediates activin A-induced KGN cell proliferation. •FOXL2 induces follistatin expression in KGN cells. •FOXL2-induced follistatin attenuates activin A-stimulated KGN cell proliferation. -- Abstract: Human granulosa cell tumors (GCTs) are rare, and their etiology remains largely unknown. Recently, the FOXL2 402C > G (C134W) mutation was found to be specifically expressed in human adult-type GCTs; however, its function in the development of human GCTs is not fully understood. Activins are members of the transforming growth factor-beta superfamily, which has been shown to stimulate normal granulosa cell proliferation; however, little is known regarding the function of activins in human GCTs. In this study, we examined the effect of activin A on cell proliferation in the human GCT-derived cell line KGN. We show that activin A treatment stimulates KGN cell proliferation. Treatment with the activin type I receptor inhibitor SB431542 blocks activin A-stimulated cell proliferation. In addition, our results show that cyclin D2 is induced by treatment with activin A and is involved in activin A-stimulated cell proliferation. Moreover, the activation of Smad signaling is required for activin A-induced cyclin D2 expression. Finally, we show that the overexpression of the wild-type FOXL2 but not the C134W mutant FOXL2 induced follistatin production. Treatment with exogenous follistatin blocks activin A-stimulated cell proliferation, and the overexpression of wild-type FOXL2 attenuates activin A-stimulated cell proliferation. These results suggest that FOXL2 may act as a tumor suppressor in human adult-type GCTs by inducing follistatin expression, which subsequently inhibits activin-stimulated cell proliferation.

  5. Morphological characterization of a human glioma cell l ine.

    Science.gov (United States)

    Machado, Camila Ml; Schenka, André; Vassallo, José; Tamashiro, Wirla Msc; Gonçalves, Estela M; Genari, Selma C; Verinaud, Liana

    2005-05-10

    A human malignant continuous cell line, named NG97, was recently established in our laboratory. This cell line has been serially subcultured over 100 times in standard culture media presenting no sign of cell senescence. The NG97 cell line has a doubling time of about 24 h. Immunocytochemical analysis of glial markers demonstrated that cells are positive for glial fibrillary acidic protein (GFAP) and S-100 protein, and negative for vimentin. Under phase-contrast microscope, cultures of NG97 showed cells with variable morphological features, such as small rounded cells, fusiform cells (fibroblastic-like cells), and dendritic-like cells. However, at confluence just small rounded and fusiform cells can be observed. At scanning electron microscopy (SEM) small rounded cells showed heterogeneous microextentions, including blebs and filopodia. Dendritic-like cells were flat and presented extensive prolongations, making several contacts with small rounded cells, while fusiform cells presented their surfaces dominated by microvilli.We believe that the knowledge about NG97 cell line may be useful for a deeper understanding of biological and immunological characteristics of gliomas.

  6. A defined and xeno-free culture method enabling the establishment of clinical-grade human embryonic, induced pluripotent and adipose stem cells.

    Directory of Open Access Journals (Sweden)

    Kristiina Rajala

    2010-04-01

    Full Text Available The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.Here, we report the development of a fully defined xeno-free medium (RegES, capable of supporting the expansion of human embryonic stem cells (hESC, induced pluripotent stem cells (iPSC and adipose stem cells (ASC. We describe the use of the xeno-free medium in the derivation and long-term (>80 passages culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS, while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific

  7. Guidelines for human embryonic stem cell research

    National Research Council Canada - National Science Library

    Committee on Guidelines for Human Embryonic Stem Cell Research; National Research Council; Board on Health Sciences Policy; Institute of Medicine; Division on Earth and Life Studies; National Research Council

    2005-01-01

    .... Given limited federal involvement, privately funded hES cell research has thus far been carried out under a patchwork of existing regulations, many of which were not designed with this research specifically in mind...

  8. Ethacrynic acid: a novel radiation enhancer in human carcinoma cells

    International Nuclear Information System (INIS)

    Khil, Mark S.; Sang, Hie Kim; Pinto, John T.; Jae, Ho Kim

    1996-01-01

    Purpose: Because agents that interfere with thiol metabolism and glutathione S-transferase (GST) functions have been shown to enhance antitumor effects of alkylating agents in vitro and in vivo, the present study was conceived on the basis that an inhibitor of GST would enhance the radiation response of some selected human carcinoma cells. Ethacrynic acid (EA) was chosen for the study because it is an effective inhibitor of GST and is a well known diuretic in humans. Methods and Materials: Experiments were carried out with well-established human tumor cells in culture growing in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS). Cell lines used were MCF-7, MCF-7 adriamycin resistant (AR) cells (breast carcinoma), HT-29 cells (colon carcinoma), DU-145 cells (prostate carcinoma), and U-373 cells (malignant glioma). Cell survival following the exposure of cells to drug alone, radiation alone, and a combined treatment was assayed by determining the colony-forming ability of single plated cells in culture to obtain dose-survival curves. The drug enhancement ratio was correlated with levels of GST. Results: The cytotoxicity of EA was most pronounced in MCF-7, U-373, and DU-145 cells compared to MCF-7 AR and HT-29 cells. The levels of GST activity were found to be lower in those EA-sensitive cells. A significant radiation enhancement was obtained with EA-sensitive cells exposed to nontoxic concentrations of the drug immediately before or after irradiation. The sensitizer enhancement ratio (SER) of MCF-7 cells was 1.55 with EA (20 μg/ml), while the SER of MCF-7 AR was less than 1.1. Based on five different human tumor cells, a clear inverse relationship was demonstrated between the magnitude of SER and GST levels of tumor cells prior to the combined treatment. Conclusion: The present results suggest that EA, which acts as both a reversible and irreversible inhibitor of GST activity, could significantly enhance the radiation response of

  9. Isolating human DNA repair genes using rodent-cell mutants

    International Nuclear Information System (INIS)

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-01-01

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab

  10. Peptidomic analysis of human cell lines

    Science.gov (United States)

    Gelman, Julia S.; Sironi, Juan; Castro, Leandro M.; Ferro, Emer S.; Fricker, Lloyd D.

    2011-01-01

    Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEK293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the P1 position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells. PMID:21204522

  11. Development of a feeder device for logging residue for a drum chipper; Hakkurin syoetoen kehittaeminen energiapuulle

    Energy Technology Data Exchange (ETDEWEB)

    Leinonen, T.; Kemppainen, J P [Oulu Univ. (Finland). Dept. of Mechanical Engineering

    1997-12-01

    The aim of this project is to increase the productivity of a large drum chipper by developing a feeder device to be used especially when chipping logging residue. Logging residue from regeneration areas is the most important reserve of forest biomass for the production of renewable energy in Finland. It is possible ecologically and economically to use about 5 million m{sup 3} of logging residue yearly for energy purposes. Currently both large lorry based chippers and smaller forwarder based chippers are used for chipping logging residue. However even the latest versions of these chippers are not well equipped for chipping logging residue. The ball like shape of residue piles causes blockades, if too large amounts of residue are fed into the chipper. Keeping the material flow even is difficult. In this project a prototype of a feeder device will be designed and manufactured to exactly find out the problems, that occur when feeding logging residue to a chipper. The designed prototype is based on a tubular chassis, which supports a conveyor made of steel plates. Prototype also has a 2,5 m wide feeding table and a feeder roll, which is located above the conveyor just before the chipper opening. Both the conveyor and the feeder roll are powered by hydraulic motors. The maximum feeding force is 20 kN. For research purposes both hydraulic pressure and flow can be adjusted individually for both motors. During 1996 activities included designing and building the prototype feeder device. In 1997 the prototype will be tested to find out possible problems, which need to be cured. After problems are solved and other improvements are made, the feeder device will be installed to an existing chipper and then tested again to determine the productivity of the new feeder device compared to the old unit. (orig.)

  12. Curcumin affects cell survival and cell volume regulation in human renal and intestinal cells

    Science.gov (United States)

    Kössler, Sonja; Nofziger, Charity; Jakab, Martin; Dossena, Silvia; Paulmichl, Markus

    2012-01-01

    Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1E,6E-heptadiene-3,5-dione or diferuloyl methane) is a polyphenol derived from the Curcuma longa plant, commonly known as turmeric. This substance has been used extensively in Ayurvedic medicine for centuries for its anti-oxidant, analgesic, anti-inflammatory and antiseptic activity. More recently curcumin has been found to possess anti-cancer properties linked to its pro-apoptotic and anti-proliferative actions. The underlying mechanisms of these diverse effects are complex, not fully elucidated and subject of intense scientific debate. Despite increasing evidence indicating that different cation channels can be a molecular target for curcumin, very little is known about the effect of curcumin on chloride channels. Since, (i) the molecular structure of curcumin indicates that the substance could potentially interact with chloride channels, (ii) chloride channels play a role during the apoptotic process and regulation of the cell volume, and (iii) apoptosis is a well known effect of curcumin, we set out to investigate whether or not curcumin could (i) exert a modulatory effect (direct or indirect) on the swelling activated chloride current IClswell in a human cell system, therefore (ii) affect cell volume regulation and (iii) ultimately modulate cell survival. The IClswell channels, which are essential for regulating the cell volume after swelling, are also known to be activated under isotonic conditions as an early event in the apoptotic process. Here we show that long-term exposure of a human kidney cell line to extracellular 0.1–10 μM curcumin modulates IClswell in a dose-dependent manner (0.1 μM curcumin is ineffective, 0.5–5.0 μM curcumin increase, while 10 μM curcumin decrease the current), and short-term exposure to micromolar concentrations of curcumin does not affect IClswell neither if applied from the extracellular nor from the intracellular side – therefore, a direct effect of curcumin on

  13. Curcumin affects cell survival and cell volume regulation in human renal and intestinal cells

    International Nuclear Information System (INIS)

    Kössler, Sonja; Nofziger, Charity; Jakab, Martin; Dossena, Silvia; Paulmichl, Markus

    2012-01-01

    Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1E,6E-heptadiene-3,5-dione or diferuloyl methane) is a polyphenol derived from the Curcuma longa plant, commonly known as turmeric. This substance has been used extensively in Ayurvedic medicine for centuries for its anti-oxidant, analgesic, anti-inflammatory and antiseptic activity. More recently curcumin has been found to possess anti-cancer properties linked to its pro-apoptotic and anti-proliferative actions. The underlying mechanisms of these diverse effects are complex, not fully elucidated and subject of intense scientific debate. Despite increasing evidence indicating that different cation channels can be a molecular target for curcumin, very little is known about the effect of curcumin on chloride channels. Since, (i) the molecular structure of curcumin indicates that the substance could potentially interact with chloride channels, (ii) chloride channels play a role during the apoptotic process and regulation of the cell volume, and (iii) apoptosis is a well known effect of curcumin, we set out to investigate whether or not curcumin could (i) exert a modulatory effect (direct or indirect) on the swelling activated chloride current ICl swell in a human cell system, therefore (ii) affect cell volume regulation and (iii) ultimately modulate cell survival. The ICl swell channels, which are essential for regulating the cell volume after swelling, are also known to be activated under isotonic conditions as an early event in the apoptotic process. Here we show that long-term exposure of a human kidney cell line to extracellular 0.1–10 μM curcumin modulates ICl swell in a dose-dependent manner (0.1 μM curcumin is ineffective, 0.5–5.0 μM curcumin increase, while 10 μM curcumin decrease the current), and short-term exposure to micromolar concentrations of curcumin does not affect ICl swell neither if applied from the extracellular nor from the intracellular side – therefore, a direct effect of curcumin on ICl

  14. Studies on a role of XRCC4 in human cells

    International Nuclear Information System (INIS)

    Mori, M.; Itsukaichi, H.; Kanda, R.; Nakamura, A.; Shiomi, N.; Aizawa, S.; Shiomi, T.

    2003-01-01

    Full text: Ionizing radiation produces a variety of lesions in DNA including single-strand breaks, double-strand breaks and base damage. The repair of DNA double-strand breaks is essential for the maintenance of genomic integrity. Failure to repair DNA double-strand breaks result in loss of genetic information, chromosome translocations, carcinogenesis and cell death. XRCC4 is a member of non-homologous end-joining proteins that functioned in DNA double-strand break repair in eukaryote including human. XRCC4 is a DNA ligase IV accessory factor and required for the rejoining of DNA double-strand breaks. Both XRCC4 and DNA ligase IV deficient mice have been generated. Both deficient mice are not viable because of neuronal degeneration caused by p53-induced apoptosis. Cells obtained from XRCC4 or DNA ligase IV deficient embryo are viable, but show reduced cell proliferation and hypersensitivity to ionizing radiation. To study the role of XRCC4 in human cells, we tried to inactivate XRCC4 gene by using gene targeting technology in human colon cancer cell line, HCT116. We have succeeded to disrupt both alleles of XRCC4 gene. Heterozygous (XRCC4 +/-) cells showed reduced cell proliferation but normal X ray-sensitivity, indicating haploinsufficiency in cell proliferation but not in X ray-sensitivity. Homozygous (XRCC4 -/-) cells show reduced cell proliferation and increased chromosome aberrations, and are highly sensitive to X rays

  15. Protein dynamics in individual human cells: experiment and theory.

    Directory of Open Access Journals (Sweden)

    Ariel Aharon Cohen

    Full Text Available A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle-dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell-cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell-cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells.

  16. Formation of human hepatocyte-like cells with different cellular phenotypes by human umbilical cord blood-derived cells in the human-rat chimeras

    International Nuclear Information System (INIS)

    Sun, Yan; Xiao, Dong; Zhang, Ruo-Shuang; Cui, Guang-Hui; Wang, Xin-Hua; Chen, Xi-Gu

    2007-01-01

    We took advantage of the proliferative and permissive environment of the developing pre-immune fetus to develop a noninjury human-rat xenograft small animal model, in which the in utero transplantation of low-density mononuclear cells (MNCs) from human umbilical cord blood (hUCB) into fetal rats at 9-11 days of gestation led to the formation of human hepatocyte-like cells (hHLCs) with different cellular phenotypes, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), cytokeratin 19 (CK19), cytokeratin 8 (CK8), cytokeratin 18 (CK18), and albumin (Alb), and with some animals exhibiting levels as high as 10.7% of donor-derived human cells in the recipient liver. More interestingly, donor-derived human cells stained positively for CD34 and CD45 in the liver of 2-month-old rat. Human hepatic differentiation appeared to partially follow the process of hepatic ontogeny, as evidenced by the expression of AFP gene at an early stage and albumin gene at a later stage. Human hepatocytes generated in this model retained functional properties of normal hepatocytes. In this xenogeneic system, the engrafted donor-derived human cells persisted in the recipient liver for at least 6 months after birth. Taken together, these findings suggest that the donor-derived human cells with different cellular phenotypes are found in the recipient liver and hHLCs hold biological activity. This humanized small animal model, which offers an in vivo environment more closely resembling the situations in human, provides an invaluable approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future

  17. effects of septrin administration on blood cells parameters in humans

    African Journals Online (AJOL)

    honey

    2014-03-31

    Mar 31, 2014 ... RESEARCH PAPER. EFFECTS OF SEPTRIN ADMINISTRATION ON BLOOD CELLS PARAMETERS IN. HUMANS. *1Onyebuagu P.C., 2Kiridi K. and 1Pughikumo D.T.. 1Department of Human Physiology, Niger Delta University, Bayelsa, Nigeria. 2Department of Radiology, Niger. Delta University, Bayelsa ...

  18. Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells

    OpenAIRE

    Merkle, Florian T.; Maroof, Asif; Wataya, Takafumi; Sasai, Yoshiki; Studer, Lorenz; Eggan, Kevin; Schier, Alexander F.

    2015-01-01

    Hypothalamic neurons orchestrate many essential physiological and behavioral processes via secreted neuropeptides, and are relevant to human diseases such as obesity, narcolepsy and infertility. We report the differentiation of human pluripotent stem cells into many of the major types of neuropeptidergic hypothalamic neurons, including those producing pro-opiolemelanocortin, agouti-related peptide, hypocretin/orexin, melanin-concentrating hormone, oxytocin, arginine vasopressin, corticotropin...

  19. Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization

    OpenAIRE

    Jin, Muzi; Wu, Asga; Dorzhin, Sergei; Yue, Qunhua; Ma, Yuzhen; Liu, Dongjun

    2012-01-01

    Although isolation and characterization of embryonic stem cells have been successful in cattle, maintenance of bovine embryonic stem cells in culture remains difficult. In this study, we compared different methods of cell passaging, feeder cell layers and medium conditions for bovine embryonic stem cell-like cells. We found that a murine embryonic fibroblast feeder layer is more suitable for embryonic stem cell-like cells than bovine embryonic fibroblasts. When murine embryonic fibroblasts we...

  20. Lentivirus display: stable expression of human antibodies on the surface of human cells and virus particles.

    Directory of Open Access Journals (Sweden)

    Ran Taube

    Full Text Available BACKGROUND: Isolation of human antibodies using current display technologies can be limited by constraints on protein expression, folding and post-translational modifications. Here we describe a discovery platform that utilizes self-inactivating (SIN lentiviral vectors for the surface display of high-affinity single-chain variable region (scFv antibody fragments on human cells and lentivirus particles. METHODOLOGY/PRINCIPAL FINDINGS: Bivalent scFvFc human antibodies were fused in frame with different transmembrane (TM anchoring moieties to allow efficient high-level expression on human cells and the optimal TM was identified. The addition of an eight amino acid HIV-1 gp41 envelope incorporation motif further increased scFvFc expression on human cells and incorporation into lentiviral particles. Both antibody-displaying human cells and virus particles bound antigen specifically. Sulfation of CDR tyrosine residues, a property recently shown to broaden antibody binding affinity and antigen recognition was also demonstrated. High level scFvFc expression and stable integration was achieved in human cells following transduction with IRES containing bicistronic SIN lentivectors encoding ZsGreen when scFvFc fusion proteins were expressed from the first cassette. Up to 10(6-fold enrichment of antibody expressing cells was achieved with one round of antigen coupled magnetic bead pre-selection followed by FACS sorting. Finally, the scFvFc displaying human cells could be used directly in functional biological screens with remarkable sensitivity. CONCLUSIONS/SIGNIFICANCE: This antibody display platform will complement existing technologies by virtue of providing properties unique to lentiviruses and antibody expression in human cells, which, in turn, may aid the discovery of novel therapeutic human mAbs.

  1. NKT cell depletion in humans during early HIV infection.

    Science.gov (United States)

    Fernandez, Caroline S; Kelleher, Anthony D; Finlayson, Robert; Godfrey, Dale I; Kent, Stephen J

    2014-08-01

    Natural killer T (NKT) cells bridge across innate and adaptive immune responses and have an important role in chronic viral infections such as human immunodeficiency virus (HIV). NKT cells are depleted during chronic HIV infection, but the timing, drivers and implications of this NKT cell depletion are poorly understood. We studied human peripheral blood NKT cell levels, phenotype and function in 31 HIV-infected subjects not on antiretroviral treatment from a mean of 4 months to 2 years after HIV infection. We found that peripheral CD4(+) NKT cells were substantially depleted and dysfunctional by 4 months after HIV infection. The depletion of CD4(+) NKT cells was more marked than the depletion of total CD4(+) T cells. Further, the early depletion of NKT cells correlated with CD4(+) T-cell decline, but not HIV viral levels. Levels of activated CD4(+) T cells correlated with the loss of NKT cells. Our studies suggest that the early loss of NKT cells is associated with subsequent immune destruction during HIV infection.

  2. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.

    Science.gov (United States)

    D'Amour, Kevin A; Bang, Anne G; Eliazer, Susan; Kelly, Olivia G; Agulnick, Alan D; Smart, Nora G; Moorman, Mark A; Kroon, Evert; Carpenter, Melissa K; Baetge, Emmanuel E

    2006-11-01

    Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.

  3. Long-term maintenance of human induced pluripotent stem cells by automated cell culture system.

    Science.gov (United States)

    Konagaya, Shuhei; Ando, Takeshi; Yamauchi, Toshiaki; Suemori, Hirofumi; Iwata, Hiroo

    2015-11-17

    Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells.

  4. New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

    Science.gov (United States)

    O'Brien, Carmel M; Chy, Hun S; Zhou, Qi; Blumenfeld, Shiri; Lambshead, Jack W; Liu, Xiaodong; Kie, Joshua; Capaldo, Bianca D; Chung, Tung-Liang; Adams, Timothy E; Phan, Tram; Bentley, John D; McKinstry, William J; Oliva, Karen; McMurrick, Paul J; Wang, Yu-Chieh; Rossello, Fernando J; Lindeman, Geoffrey J; Chen, Di; Jarde, Thierry; Clark, Amander T; Abud, Helen E; Visvader, Jane E; Nefzger, Christian M; Polo, Jose M; Loring, Jeanne F; Laslett, Andrew L

    2017-03-01

    The study and application of human pluripotent stem