WorldWideScience

Sample records for display derived human

  1. Affinity isolation of antigen-specific circulating B cells for generation of phage display-derived human monoclonal antibodies

    DEFF Research Database (Denmark)

    Ditzel, Henrik

    2009-01-01

    A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries generated from...... peripheral blood lymphocytes caused by the low abundance of antigen-specific B cells in the circulation. The preselection of B cells is based on the specificity of the surface Ig receptor and is accomplished using the antigen of interest conjugated to magnetic beads. This method should significantly increase...... the frequency of antibody phage particles of interest in the library and allow for efficient isolation monoclonal antibodies with the predefined specificity....

  2. Potato virus X displaying the E7 peptide derived from human papillomavirus type 16: a novel position for epitope presentation

    Czech Academy of Sciences Publication Activity Database

    Vaculík, Petr; Plchová, Helena; Moravec, Tomáš; Hoffmeisterová, Hana; Čeřovská, Noemi; Šmahel, M.

    2015-01-01

    Roč. 120, č. 2 (2015), s. 671-680 ISSN 0167-6857 R&D Projects: GA ČR(CZ) GAP501/12/1761 Grant - others:European Regional Development Fund(XE) CZ.2.16/3.1.00/24014 Institutional support: RVO:61389030 Keywords : Potato virus X * Human papillomavirus * Nicotiana benthamiana Subject RIV: EE - Microbiology, Virology Impact factor: 2.390, year: 2015

  3. Human Pluripotent Stem Cells and Derived Neuroprogenitors Display Differential Degrees of Susceptibility to BH3 Mimetics ABT-263, WEHI-539 and ABT-199.

    Directory of Open Access Journals (Sweden)

    Carolina Paola García

    Full Text Available Human embryonic stem cells (hESCs are hypersensitive to genotoxic stress and display lower survival ability relative to their differentiated progeny. Herein, we attempted to investigate the source of this difference by comparing the DNA damage responses triggered by the topoisomerase I inhibitor camptothecin, in hESCs, human induced pluripotent stem cells (hiPSCs and hESCs-derived neuroprogenitors (NP. We observed that upon camptothecin exposure pluripotent stem cells underwent apoptosis more swiftly and at a higher rate than differentiated cells. However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types. Importantly, we observed that hESCs and hiPSCs express lower levels of the anti-apoptotic protein Bcl-2 than NP. To assess whether Bcl-2 abundance could account for this differential response we treated cells with ABT-263, WEHI-539 and ABT-199, small molecules that preferentially target the BH3-binding pocket of Bcl-xL and/or Bcl-2 and reduce their ability to sequester pro-apoptotic factors. We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs. Conversely, all tested cell types appeared to be highly resistant to the Bcl-2 specific inhibitor, ABT-199. However, in all cases we determined that ABT-263 or WEHI-539 treatment exacerbated camptothecin-induced apoptosis. Importantly, similar responses were observed after siRNA-mediated down-regulation of Bcl-xL or Bcl-2. Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells. The emerging knowledge of the relative dependence of pluripotent and progenitor cells on Bcl-2 and Bcl-xL activities may help

  4. Human Factors Military Lexicon: Auditory Displays

    National Research Council Canada - National Science Library

    Letowski, Tomasz

    2001-01-01

    .... In addition to definitions specific to auditory displays, speech communication, and audio technology, the lexicon includes several terms unique to military operational environments and human factors...

  5. The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

    Science.gov (United States)

    Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai

    2013-01-01

    Abstract Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest. PMID:23941674

  6. Interactive displays natural human-interface technologies

    CERN Document Server

    Bhowmik, Achintya K

    2014-01-01

    One of the first books to provide an in-depth discussion of the technologies, applications and trends in the rapidly emerging field of interactive displays (touch, gesture & voice) The book will cover the technologies, applications and trends in the field of interactive displays, namely interfaces based on touch, gesture and voice and those using a combination of these technologies. The book will be split into 4 main parts with each being dedicated to a specific user interface. Part 1 ''Touch Interfaces'' will provide a review of the currently deployed touch-screen technologies and applications. It will also cover the recent developments towards achieving thinner, lightweight and cost-reduced touch screen panels in the future via integration of touch functionalities. Part 2 ''Gesture Interfaces'' will examine techniques and applications in stereoscopic 3D computer vision, structured-light 3D computer vision and time-of-flight 3D computer vision in gesture interfaces. Part 3 ''Voice Interfaces'' will revie...

  7. Phage Display-Derived Cross-Reactive Neutralizing Antibody against Enterovirus 71 and Coxsackievirus A16.

    Science.gov (United States)

    Zhang, Xiao; Sun, Chunyun; Xiao, Xiangqian; Pang, Lin; Shen, Sisi; Zhang, Jie; Cen, Shan; Yang, Burton B; Huang, Yuming; Sheng, Wang; Zeng, Yi

    2016-01-01

    Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are members of the Picornaviridae family and are considered the main causative agents of hand, foot and mouth disease (HFMD). In recent decades large HFMD outbreaks caused by EV71 and CVA16 have become significant public health concerns in the Asia-Pacific region. Vaccines and antiviral drugs are unavailable to prevent EV71 and CVA16 infection. In the current study, a chimeric antibody targeting a highly conserved peptide in the EV71 VP4 protein was isolated by using a phage display technique. The antibody showed cross-neutralizing capability against EV71 and CVA16 in vitro. The results suggest that this phage display-derived antibody will have great potential as a broad neutralizing antibody against EV71 and CVA16 after affinity maturation and humanization.

  8. Generation of human antibody fragments against Streptococcus mutans using a phage display chain shuffling approach

    Directory of Open Access Journals (Sweden)

    Barth Stefan

    2005-01-01

    Full Text Available Abstract Background Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials. Results The antibody derivatives were generated using a chain-shuffling approach based on human antibody variable gene phage-display libraries. Like the parent antibody, these derivatives bound specifically to SAI/II, the surface adhesin of the oral pathogen S. mutans. Conclusions Humanization of murine antibodies can be easily achieved using phage display libraries. The human antibody fragments bind the antigen as well as the causative agent of dental caries. In addition the human diabody derivative is capable of aggregating S. mutans in vitro, making it a useful candidate passive immunotherapeutic agent for oral diseases.

  9. Development of exosome surface display technology in living human cells

    Energy Technology Data Exchange (ETDEWEB)

    Stickney, Zachary, E-mail: zstickney@scu.edu; Losacco, Joseph, E-mail: jlosacco@scu.edu; McDevitt, Sophie, E-mail: smmcdevitt@scu.edu; Zhang, Zhiwen, E-mail: zzhang@scu.edu; Lu, Biao, E-mail: blu2@scu.edu

    2016-03-25

    Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell–cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated the successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy.

  10. Development of exosome surface display technology in living human cells

    International Nuclear Information System (INIS)

    Stickney, Zachary; Losacco, Joseph; McDevitt, Sophie; Zhang, Zhiwen; Lu, Biao

    2016-01-01

    Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell–cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated the successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy.

  11. The endogenous hallucinogen and trace amine N,N-dimethyltryptamine (DMT displays potent protective effects against hypoxia via sigma-1 receptor activation in human primary iPSC-derived cortical neurons and microglia-like immune cells

    Directory of Open Access Journals (Sweden)

    Attila Szabo

    2016-09-01

    Full Text Available N,N-dimethyltryptamine (DMT is a potent endogenous hallucinogen present in the brain of humans and other mammals. Despite extensive research, its physiological role remains largely unknown. Recently, DMT has been found to activate the sigma-1 receptor (Sig-1R, an intracellular chaperone fulfilling an interface role between the endoplasmic reticulum (ER and mitochondria. It ensures the correct transmission of ER stress into the nucleus resulting in the enhanced production of antistress and antioxidant proteins. Due to this function, the activation of Sig-1R can mitigate the outcome of hypoxia or oxidative stress. In this paper we aimed to test the hypothesis that DMT plays a neuroprotective role in the brain by activating the Sig-1R. We tested whether DMT can mitigate hypoxic stress in in vitro cultured human cortical neurons (derived from induced pluripotent stem cells, and in monocyte-derived macrophages and dendritic cells. Here we report that DMT robustly increases the survival of these cell types in severe hypoxia (0.5% O2 through the Sig-1R. Furthermore, this phenomenon is associated with the decreased expression and function of the alpha subunit of the hypoxia-inducible factor 1 (HIF-1 suggesting that DMT-mediated Sig-1R activation may alleviate hypoxia-induced cellular stress and increase survival in a HIF-1-independent manner. Our results reveal a novel and important role of DMT in human cellular physiology. We postulate that this compound may be endogenously generated in situations of stress, ameliorating the adverse effects of hypoxic/ischemic insult to the brain.

  12. The Endogenous Hallucinogen and Trace Amine N,N-Dimethyltryptamine (DMT) Displays Potent Protective Effects against Hypoxia via Sigma-1 Receptor Activation in Human Primary iPSC-Derived Cortical Neurons and Microglia-Like Immune Cells.

    Science.gov (United States)

    Szabo, Attila; Kovacs, Attila; Riba, Jordi; Djurovic, Srdjan; Rajnavolgyi, Eva; Frecska, Ede

    2016-01-01

    N,N-dimethyltryptamine (DMT) is a potent endogenous hallucinogen present in the brain of humans and other mammals. Despite extensive research, its physiological role remains largely unknown. Recently, DMT has been found to activate the sigma-1 receptor (Sig-1R), an intracellular chaperone fulfilling an interface role between the endoplasmic reticulum (ER) and mitochondria. It ensures the correct transmission of ER stress into the nucleus resulting in the enhanced production of antistress and antioxidant proteins. Due to this function, the activation of Sig-1R can mitigate the outcome of hypoxia or oxidative stress. In this paper, we aimed to test the hypothesis that DMT plays a neuroprotective role in the brain by activating the Sig-1R. We tested whether DMT can mitigate hypoxic stress in in vitro cultured human cortical neurons (derived from induced pluripotent stem cells, iPSCs), monocyte-derived macrophages (moMACs), and dendritic cells (moDCs). Results showed that DMT robustly increases the survival of these cell types in severe hypoxia (0.5% O2) through the Sig-1R. Furthermore, this phenomenon is associated with the decreased expression and function of the alpha subunit of the hypoxia-inducible factor 1 (HIF-1) suggesting that DMT-mediated Sig-1R activation may alleviate hypoxia-induced cellular stress and increase survival in a HIF-1-independent manner. Our results reveal a novel and important role of DMT in human cellular physiology. We postulate that this compound may be endogenously generated in situations of stress, ameliorating the adverse effects of hypoxic/ischemic insult to the brain.

  13. Human Engineering of Space Vehicle Displays and Controls

    Science.gov (United States)

    Whitmore, Mihriban; Holden, Kritina L.; Boyer, Jennifer; Stephens, John-Paul; Ezer, Neta; Sandor, Aniko

    2010-01-01

    Proper attention to the integration of the human needs in the vehicle displays and controls design process creates a safe and productive environment for crew. Although this integration is critical for all phases of flight, for crew interfaces that are used during dynamic phases (e.g., ascent and entry), the integration is particularly important because of demanding environmental conditions. This panel addresses the process of how human engineering involvement ensures that human-system integration occurs early in the design and development process and continues throughout the lifecycle of a vehicle. This process includes the development of requirements and quantitative metrics to measure design success, research on fundamental design questions, human-in-the-loop evaluations, and iterative design. Processes and results from research on displays and controls; the creation and validation of usability, workload, and consistency metrics; and the design and evaluation of crew interfaces for NASA's Crew Exploration Vehicle are used as case studies.

  14. Conformal displays: human factor analysis of innovative landing aids

    Science.gov (United States)

    Schmerwitz, Sven; Lueken, Thomas; Doehler, Hans-Ullrich; Peinecke, Niklas; Ernst, Johannes M.; da Silva Rosa, David L.

    2017-05-01

    In the past couple of years, research on display content for helicopter operations headed in a new direction. The already reached goals could evolve into a paradigm change for information visualization. Technology advancements allow implementing three-dimensional and conformal content on a helmet-mounted see-through device. This superimposed imagery inherits the same optical flow as the environment. It is supposed to ease switching between display information and environmental cues. The concept is neither pathbreaking nor new, but it has not been successfully established in aviation yet. Nevertheless, there are certainly some advantages to expect-at least from perspective of a human-centered system design. Within the following pages, the next generation displays will be presented and discussed with a focus on human factors. Beginning with recalling some human factor related research facts, an experiment comparing the former two-dimensional research displays will be presented. Before introducing the DLR conformal symbol set and the three experiments about an innovative drift, indication related research activities toward conformal symbol sets will be addressed.

  15. Human factors guidelines for large-screen displays

    International Nuclear Information System (INIS)

    Collier, Steve

    2005-09-01

    Any control-room project (including upgrades or evolutionary improvements to existing control-rooms) is well advised at the outset first to gather and update related background material for the design. This information-gathering exercise should also take into account experience from similar projects and operating experience. For these reasons, we decided to use our research, and experience in large-screen display design with several clients to update human factors guidance for large-screen displays, to take into account new ergonomics guidelines, operating experience, and work from similar projects. To write the updated guidelines, we drew on much of our experience across several departments at IFE, including research funded by the HRP programme, and experience with individual clients. Guidance here is accordingly focused mainly on recent areas of technical and human innovations in the man-machine interface. One particular area of focus was on the increasing use of large-screen display systems in modern control-rooms, and on how guidelines could be adapted and supplemented for their design. Guidance or reference to recommended sources is also given for control suite arrangement and layout, control-room layout, workstation layout, design of displays and controls, and design of the work environment, especially insofar as these ergonomic issues interact with the effectiveness of modern displays, in particular large screen displays. The work shows that there can be synergy between HRP research and bilateral activities: the one side offers a capability to develop tools and guidelines, while the other side gives an opportunity to test and refine these in practice, to the benefit of both parties. (Author)

  16. Human factors guidelines for large-screen displays

    Energy Technology Data Exchange (ETDEWEB)

    Collier, Steve

    2005-09-15

    Any control-room project (including upgrades or evolutionary improvements to existing control-rooms) is well advised at the outset first to gather and update related background material for the design. This information-gathering exercise should also take into account experience from similar projects and operating experience. For these reasons, we decided to use our research, and experience in large-screen display design with several clients to update human factors guidance for large-screen displays, to take into account new ergonomics guidelines, operating experience, and work from similar projects. To write the updated guidelines, we drew on much of our experience across several departments at IFE, including research funded by the HRP programme, and experience with individual clients. Guidance here is accordingly focused mainly on recent areas of technical and human innovations in the man-machine interface. One particular area of focus was on the increasing use of large-screen display systems in modern control-rooms, and on how guidelines could be adapted and supplemented for their design. Guidance or reference to recommended sources is also given for control suite arrangement and layout, control-room layout, workstation layout, design of displays and controls, and design of the work environment, especially insofar as these ergonomic issues interact with the effectiveness of modern displays, in particular large screen displays. The work shows that there can be synergy between HRP research and bilateral activities: the one side offers a capability to develop tools and guidelines, while the other side gives an opportunity to test and refine these in practice, to the benefit of both parties. (Author)

  17. Display analysis with the optimal control model of the human operator. [pilot-vehicle display interface and information processing

    Science.gov (United States)

    Baron, S.; Levison, W. H.

    1977-01-01

    Application of the optimal control model of the human operator to problems in display analysis is discussed. Those aspects of the model pertaining to the operator-display interface and to operator information processing are reviewed and discussed. The techniques are then applied to the analysis of advanced display/control systems for a Terminal Configured Vehicle. Model results are compared with those obtained in a large, fixed-base simulation.

  18. 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.

  19. The human factors of CRT displays for nuclear power plant control

    International Nuclear Information System (INIS)

    Danchak, M.M.

    1984-01-01

    This chapter attempts to show how the Cathode Ray Tube (CRT) can be used to effectively present information to the operator rather than just data. The capabilities of the human as a sensing and information processing subsystem are discussed with CRT displays in mind. The display system is described in terms of its hardware and functioning. The interface between the two is examined by providing substantive guidelines for the effective design of CRT displays for nuclear power plant control. Alphanumeric displays, graphic displays, and representational displays are treated. The design of CRT displays for nuclear power plant control requires an extensive knowledge of cognitive psychology, computer display systems and the process being controlled

  20. Identification of human embryonic progenitor cell targeting peptides using phage display.

    Directory of Open Access Journals (Sweden)

    Paola A Bignone

    Full Text Available Human pluripotent stem (hPS cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken

  1. Pro-region engineering for improved yeast display and secretion of brain derived neurotrophic factor.

    Science.gov (United States)

    Burns, Michael L; Malott, Thomas M; Metcalf, Kevin J; Puguh, Arthya; Chan, Jonah R; Shusta, Eric V

    2016-03-01

    Brain derived neurotrophic factor (BDNF) is a promising therapeutic candidate for a variety of neurological diseases. However, it is difficult to produce as a recombinant protein. In its native mammalian context, BDNF is first produced as a pro-protein with subsequent proteolytic removal of the pro-region to yield mature BDNF protein. Therefore, in an attempt to improve yeast as a host for heterologous BDNF production, the BDNF pro-region was first evaluated for its effects on BDNF surface display and secretion. Addition of the wild-type pro-region to yeast BDNF production constructs improved BDNF folding both as a surface-displayed and secreted protein in terms of binding its natural receptors TrkB and p75, but titers remained low. Looking to further enhance the chaperone-like functions provided by the pro-region, two rounds of directed evolution were performed, yielding mutated pro-regions that further improved the display and secretion properties of BDNF. Subsequent optimization of the protease recognition site was used to control whether the produced protein was in pro- or mature BDNF forms. Taken together, we have demonstrated an effective strategy for improving BDNF compatibility with yeast protein engineering and secretion platforms. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Human factors considerations in the design and evaluation of flight deck displays and controls

    Science.gov (United States)

    2013-11-01

    The objective of this effort is to have a single source document for human factors regulatory and guidance material for flight deck displays and controls, in the interest of improving aviation safety. This document identifies guidance on human factor...

  3. Human Visual Performance and Flat Panel Display Image Quality

    Science.gov (United States)

    1980-07-01

    visible light energy can be described in electro- magnetic energy space as that portion of the electromagnetic wavelength (or frequency) domain to...temporal frequency domain much as one analyzes spatial information in the spatial frequency domain. As the content of a complex but periodic sound ...briefly with the more intepretive aspects of information display, those pertaining to information encoding and the design problems and parame- ters

  4. 28-mer Fragment Derived from Enterocin CRL35 Displays an Unexpected Bactericidal Effect on Listeria Cells.

    Science.gov (United States)

    Masias, Emilse; Sanches, Paulo R S; Dupuy, Fernando G; Acuna, Leonardo; Bellomio, Augusto; Cilli, Eduardo; Saavedra, Lucila; Minahk, Carlos

    2015-01-01

    Two shorter peptides derived from enterocin CRL35, a 43-mer bacteriocin, were synthesized i.e. the N-terminal fragment spanning from residues 1 to 15, and a 28-mer fragment that represents the C-terminal of enterocin CRL35, the residues 16 to 43. The separate peptides showed no activity when combined. On one hand, the 28-mer peptide displayed an unpredicted antimicrobial activity. On the other, 15- mer peptide had no consistent anti-Listeria effect. The dissociation constants calculated from experimental data indicated that all peptides could bind at similar extent to the sensitive cells. However, transmembrane electrical potential was not dissipated to the same level by the different peptides; whereas the full-length and the C-terminal 28-mer fragment induced almost full dissipation, 15-mer fragment produced only a slow and incomplete effect. Furthermore, a different interaction of each peptide with membranes was demonstrated based on studies carried out with liposomes, which led us to conclude that activity was related to structure rather than to net positive charges. These results open up the possibility of designing new peptides based on the 28-mer fragment with enhanced activity, which would represent a promising approach for combating Listeria and other pathogens.

  5. Design and Development of Functionally Effective Human-Machine Interfaces for Firing Room Displays

    Science.gov (United States)

    Cho, Henry

    2013-01-01

    This project involves creating software for support equipment used on the Space Launch System (SLS). The goal is to create applications and displays that will be used to remotely operate equipment from the firing room and will continue to support the SLS launch vehicle to the extent of its program. These displays include design practices that help to convey information effectively, such as minimizing distractions at normal operating state and displaying intentional distractions during a warning or alarm state. The general practice for creating an operator display is to reduce the detail of unimportant aspects of the display and promote focus on data and dynamic information. These practices include using minimalist design, using muted tones for background colors, using a standard font at a readable text size, displaying alarms visible for immediate attention, grouping data logically, and displaying data appropriately varying on the type of data. Users of these displays are more likely to stay focused on operating for longer periods by using design practices that reduce eye strain and fatigue. Effective operator displays will improve safety by reducing human errors during operation, which will help prevent catastrophic accidents. This report entails the details of my work on developing remote displays for the Hypergolic fuel servicing system. Before developing a prototype display, the design and requirements of the system are outlined and compiled into a document. Then each subsystem has schematic representations drawn that meet the specifications detailed in the document. The schematics are then used as the outline to create display representations of each subsystem. Each display is first tested individually. Then the displays are integrated with a prototype of the master system, and they are tested in a simulated environment then retested in the real environment. Extensive testing is important to ensure the displays function reliably as intended.

  6. Human factors considerations for the use of color in display systems

    Science.gov (United States)

    Demars, S. A.

    1975-01-01

    Identified and assessed are those human factor considerations impacting an operator's ability to perform when information is displayed in color as contrasted to monochrome (black and white only). The findings provide valuable guidelines for the assessment of the advantages (and disadvantages) of using a color display system. The use of color provides an additional sensory channel (color perception) which is not available with black and white. The degree to which one can exploit the use of this channel is highly dependent on available display technology, mission information display requirements, and acceptable operational modes.

  7. Human factors engineering in the design of colour-graphic displays

    International Nuclear Information System (INIS)

    Fenton, E.F.

    1985-01-01

    The operator interface for Ontario Hydro's Darlington Nuclear Generating Station will rely extensively on the use of coloured graphic display formats. These are used for the presentation of both control and monitoring information. The displays are organized in a hierarchical relationship and a simple interactive selection method using light pens has been implemented. The application of human factors engineering principles has been a major factor in all aspects of the design. This paper describes the system and the human factors engineering function

  8. Termini of human chromosomes display elevated rates of mitotic recombination.

    Science.gov (United States)

    Cornforth, M N; Eberle, R L

    2001-01-01

    The strand-specific in situ hybridization technique of CO-FISH was used to probe telomeres of human mitotic cells in order to determine the spontaneous frequency of crossover. This approach allowed the detection of recombinational crossovers occurring anywhere along the length of individual chromosomes, including reciprocal events taking place between sister chromatids. Although the process of sister chromatid exchange (SCE) is the most prominent type of recombination in somatic mammalian cells, our results show that SCEs accounted for less than a third of the recombinational events revealed by CO-FISH. It is concluded that chromosomal regions near the termini of chromosome arms undergo extraordinarily high rates of spontaneous recombination, producing terminal crossovers whose small size precludes detection by standard cytogenetic methods. That similar results were observed for transformed epithelial cells, as well as primary fibroblasts, suggests that the phenomenon is a common characteristic of human cells. These findings are noteworthy because, although telomeric and subtelomeric DNA is known to be preferentially involved in certain types of recombination, the tips of somatic mammalian chromosomes have not previously been identified as preferred sites for crossover. Implications of these results are discussed in terms of limitations imposed on CO-FISH for its proposed use in directional hybridization mapping.

  9. Techniques for optimizing human-machine information transfer related to real-time interactive display systems

    Science.gov (United States)

    Granaas, Michael M.; Rhea, Donald C.

    1989-01-01

    In recent years the needs of ground-based researcher-analysts to access real-time engineering data in the form of processed information has expanded rapidly. Fortunately, the capacity to deliver that information has also expanded. The development of advanced display systems is essential to the success of a research test activity. Those developed at the National Aeronautics and Space Administration (NASA), Western Aeronautical Test Range (WATR), range from simple alphanumerics to interactive mapping and graphics. These unique display systems are designed not only to meet basic information display requirements of the user, but also to take advantage of techniques for optimizing information display. Future ground-based display systems will rely heavily not only on new technologies, but also on interaction with the human user and the associated productivity with that interaction. The psychological abilities and limitations of the user will become even more important in defining the difference between a usable and a useful display system. This paper reviews the requirements for development of real-time displays; the psychological aspects of design such as the layout, color selection, real-time response rate, and interactivity of displays; and an analysis of some existing WATR displays.

  10. 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.

  11. HCMV Displays a Unique Transcriptome of Immunomodulatory Genes in Primary Monocyte-Derived Cell Types.

    Directory of Open Access Journals (Sweden)

    Ellen Van Damme

    Full Text Available Human cytomegalovirus (HCMV is a betaherpesvirus which rarely presents problems in healthy individuals, yet may result in severe morbidity in immunocompromised patients and in immune-naïve neonates. HCMV has a large 235 kb genome with a coding capacity of at least 165 open reading frames (ORFs. This large genome allows complex gene regulation resulting in different sets of transcripts during lytic and latent infection. While latent virus mainly resides within monocytes and CD34+ progenitor cells, reactivation to lytic infection is driven by differentiation towards terminally differentiated myeloid dendritic cells and macrophages. Consequently, it has been suggested that macrophages and dendritic cells contribute to viral spread in vivo. Thus far only limited knowledge is available on the expression of HCMV genes in terminally differentiated myeloid primary cells and whether or not the virus exhibits a different set of lytic genes in primary cells compared with lytic infection in NHDF fibroblasts. To address these questions, we used Illumina next generation sequencing to determine the HCMV transcriptome in macrophages and dendritic cells during lytic infection and compared it to the transcriptome in NHDF fibroblasts. Here, we demonstrate unique expression profiles in macrophages and dendritic cells which significantly differ from the transcriptome in fibroblasts mainly by modulating the expression of viral transcripts involved in immune modulation, cell tropism and viral spread. In a head to head comparison between macrophages and dendritic cells, we observed that factors involved in viral spread and virion composition are differentially regulated suggesting that the plasticity of the virion facilitates the infection of surrounding cells. Taken together, this study provides the full transcript expression analysis of lytic HCMV genes in monocyte-derived type 1 and type 2 macrophages as well as in monocyte-derived dendritic cells. Thereby

  12. What is 3D good for? A review of human performance on stereoscopic 3D displays

    Science.gov (United States)

    McIntire, John P.; Havig, Paul R.; Geiselman, Eric E.

    2012-06-01

    This work reviews the human factors-related literature on the task performance implications of stereoscopic 3D displays, in order to point out the specific performance benefits (or lack thereof) one might reasonably expect to observe when utilizing these displays. What exactly is 3D good for? Relative to traditional 2D displays, stereoscopic displays have been shown to enhance performance on a variety of depth-related tasks. These tasks include judging absolute and relative distances, finding and identifying objects (by breaking camouflage and eliciting perceptual "pop-out"), performing spatial manipulations of objects (object positioning, orienting, and tracking), and navigating. More cognitively, stereoscopic displays can improve the spatial understanding of 3D scenes or objects, improve memory/recall of scenes or objects, and improve learning of spatial relationships and environments. However, for tasks that are relatively simple, that do not strictly require depth information for good performance, where other strong cues to depth can be utilized, or for depth tasks that lie outside the effective viewing volume of the display, the purported performance benefits of 3D may be small or altogether absent. Stereoscopic 3D displays come with a host of unique human factors problems including the simulator-sickness-type symptoms of eyestrain, headache, fatigue, disorientation, nausea, and malaise, which appear to effect large numbers of viewers (perhaps as many as 25% to 50% of the general population). Thus, 3D technology should be wielded delicately and applied carefully; and perhaps used only as is necessary to ensure good performance.

  13. Human engineering guidelines for the evaluation and assessment of Video Display Units

    International Nuclear Information System (INIS)

    Gilmore, W.E.

    1985-07-01

    This report provides the Nuclear Regulatory Commission with a single source that documents known guidelines for conducting formal Human Factors evaluations of Video Display Units (VDUs). The handbook is a ''cookbook'' of acceptance guidelines for the reviewer faced with the task of evaluating VDUs already designed or planned for service in the control room. The areas addressed are video displays, controls, control/display integration, and workplace layout. Guidelines relevant to each of those areas are presented. The existence of supporting research is also indicated for each guideline. A Comment section and Method for Assessment section are provided for each set of guidelines

  14. Construction of human antibody gene libraries and selection of antibodies by phage display.

    Science.gov (United States)

    Frenzel, André; Kügler, Jonas; Wilke, Sonja; Schirrmann, Thomas; Hust, Michael

    2014-01-01

    Antibody phage display is the most commonly used in vitro selection technology and has yielded thousands of useful antibodies for research, diagnostics, and therapy.The prerequisite for successful generation and development of human recombinant antibodies using phage display is the construction of a high-quality antibody gene library. Here, we describe the methods for the construction of human immune and naive scFv gene libraries.The success also depends on the panning strategy for the selection of binders from these libraries. In this article, we describe a panning strategy that is high-throughput compatible and allows parallel selection in microtiter plates.

  15. Human factors review of CFMS displays for Ulchin Nuclear Power Unit 3 and 4

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yong Hee; Baek, Seung Min; Kim, Jung Taek; Park, Jae Chang; Lee, Jung Woon; Oh, In Suk; Lee, Joon Whan; Jung, Kwang Tae; Cha, Hye Young [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

    1996-11-01

    This report describes the human factors review of CFMS displays for Ulchin 3 and 4 by the following four subjects; At first, by reviewing issues regarding the design process of present CFMS, human factors engineering program plan (HFEPP) and human factors verification and validation plan Were proposed to accomplish the completeness of design word; Secondly, researches and developments were integrated into the review results at the point of suitability of CFMS design concept and basic function; For the third, availability and suitability were assessed according to human factors evaluation criteria on the CFMS display design, and overall effectiveness was also evaluated in parts; For the fourth, recommendations were made to human factors problems in accordance with their importance and an implementation plan was suggested for the resolution of problems. 54 refs., 34 tabs., 42 figs. (author)

  16. Derivatives Trading, Climate Science and Human Rights

    DEFF Research Database (Denmark)

    Haigh, Matthew

    for capital flows associated with climate management. Media communications and decision making theories are used to interpret data drawn from participant observation and interviews with climate scientists, policy makers and institutional investors. Findings - The framework suggests a digital divide between...... the heaviest polluters as contributing to carbon-minimised investment portfolios. Assets owned by privately managed pension funds have remained materially exposed to risks posed by climate change. In public finance, a narrow range of financial instruments centred on derivatives trading has entrenched global...... between human rights, climate change, and the stability of private pensions provision. Originality/value - Provides policy sciences useful assessments of communication media and financial instruments used in climate management. Establishes bases for theoretical and applied communications research...

  17. Human engineering design considerations for the use of signal color enhancement in ASW displays

    Energy Technology Data Exchange (ETDEWEB)

    Banks, W.W.

    1990-11-01

    The Lawrence Livermore National Laboratory (LLNL) was requested to examine and define man-machine limits as part of the Office of Naval Technology's High Gain Initiative program (HGI). As an initial investigative area, LLNL's Systems and Human Performance effort focused upon color display interfaces and the use of color enhancement techniques to define human and system interface limits in signal detection and discrimination tasks. The knowledgeable and prudent use of color in different types of display is believed to facilitate human visual detection, discrimination and recognition in complex visual tasks. The consideration and understanding of the complex set of interacting variables associated with the prudent use of color is essential to optimize human performance, especially in the ASW community. The designers of advanced display technology and signal processing algorithms may be eventually called upon to present pre-processed information to ASW operators and researchers using the latest color enhancement techniques. These techniques, however, may be limited if one does not understand the complexity and limits of human information processing which reflects the assessed state of knowledge relevant to the use of color in displays. The initial sections of this report discuss various aspects of color presentation and the problems typically encountered, while the last section deals with a specific research proposal required to further our understanding and proper use of color enhancement methods.

  18. Human factors engineering design review acceptance criteria for the safety parameter display

    International Nuclear Information System (INIS)

    McGevna, V.; Peterson, L.R.

    1981-01-01

    This report contains human factors engineering design review acceptance criteria developed by the Human Factors Engineering Branch (HFEB) of the Nuclear Regulatory Commission (NRC) to use in evaluating designs of the Safety Parameter Display System (SPDS). These criteria were developed in response to the functional design criteria for the SPDS defined in NUREG-0696, Functional Criteria for Emergency Response Facilities. The purpose of this report is to identify design review acceptance criteria for the SPDS installed in the control room of a nuclear power plant. Use of computer driven cathode ray tube (CRT) displays is anticipated. General acceptance criteria for displays of plant safety status information by the SPDS are developed. In addition, specific SPDS review criteria corresponding to the SPDS functional criteria specified in NUREG-0696 are established

  19. 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

  20. Phage-display libraries of murine and human antibody Fab fragments

    DEFF Research Database (Denmark)

    Engberg, J; Andersen, P S; Nielsen, L K

    1996-01-01

    We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab...

  1. A recombinant, fully human monoclonal antibody with antitumor activity constructed from phage-displayed antibody fragments

    NARCIS (Netherlands)

    Huls, GA; Heijnen, IAFM; Cuomo, ME; Koningsberger, JC; Boel, E; de Vries, ARV; Loyson, SAJ; Helfrich, W; Henegouwen, GPV; van Meijer, M; de Kruif, J; Logtenberg, T

    A single-chain Fv antibody fragment specific for the tumor-associated Ep-CAM molecule was isolated from a semisynthetic phage display library and converted into an intact, fully human IgG1 monoclonal antibody (huMab), The purified huMab had an affinity of 5 nM and effectively mediated tumor cell

  2. The Effect of Information Analysis Automation Display Content on Human Judgment Performance in Noisy Environments

    OpenAIRE

    Bass, Ellen J.; Baumgart, Leigh A.; Shepley, Kathryn Klein

    2012-01-01

    Displaying both the strategy that information analysis automation employs to makes its judgments and variability in the task environment may improve human judgment performance, especially in cases where this variability impacts the judgment performance of the information analysis automation. This work investigated the contribution of providing either information analysis automation strategy information, task environment information, or both, on human judgment performance in a domain where noi...

  3. Effects of Viewing Displays from Different Distances on Human Visual System

    Directory of Open Access Journals (Sweden)

    Mohamed Z. Ramadan

    2017-11-01

    Full Text Available The current stereoscopic 3D displays have several human-factor issues including visual-fatigue symptoms such as eyestrain, headache, fatigue, nausea, and malaise. The viewing time and viewing distance are factors that considerably affect the visual fatigue associated with 3D displays. Hence, this study analyzes the effects of display type (2D vs. 3D and viewing distance on visual fatigue during a 60-min viewing session based on electroencephalogram (EEG relative beta power, and alpha/beta power ratio. In this study, twenty male participants watched four videos. The EEGs were recorded at two occipital lobes (O1 and O2 of each participant in the pre-session (3 min, post-session (3 min, and during a 60-min viewing session. The results showed that the decrease in relative beta power of the EEG and the increase in the alpha/beta ratio from the start until the end of the viewing session were significantly higher when watching the 3D display. When the viewing distance was increased from 1.95 m to 3.90 m, the visual fatigue was decreased in the case of the 3D-display, whereas the fatigue was increased in the case of the 2D-display. Moreover, there was approximately the same level of visual fatigue when watching videos in 2D or 3D from a long viewing distance (3.90 m.

  4. The display of multiple images derived from emission computed assisted tomography (ECAT)

    International Nuclear Information System (INIS)

    Jackson, P.C.; Davies, E.R.; Goddard, P.R.; Wilde, R.P.H.

    1983-01-01

    In emission computed assisted tomography, a technique has been developed to display the multiple sections of an organ within a single image, such that three dimensional appreciation of the organ can be obtained, whilst also preserving functional information. The technique when tested on phantoms showed no obvious deterioration in resolution and when used clinically gave satisfactory visual results. Such a method should allow easier appreciation of the extent of a lesion through an organ and thus allow dimensions to be obtained by direct measurement. (U.K.)

  5. Development of HANARO human factors management plan and evaluation of BCS display

    International Nuclear Information System (INIS)

    Oh, I. S.; Lee, J. W.; Lee, Y. H.

    2004-01-01

    In this study, human factors evaluation of BCS display design was performed. We adopted the suitability of design elements of BCS display as human factors evaluation measure. And, we also adopted guideline based evaluation, field survey and expert evaluation as evaluation method. The checklist was utilized for the evaluation, and the results of evaluation were well arranged in the evaluation format. We did not find out the HED (Human Engineering Discrepancy) impede safety of HANARO, except some necessary items to improve during short periods. We also provide some items of improvement for the enhancement of safety and operator's performance in the aspect of long periods. If the proposed improvement items were completely fulfilled, the more improved safety of HANARO will be secured

  6. Despite differential gene expression profiles pediatric MDS derived mesenchymal stromal cells display functionality in vitro

    Directory of Open Access Journals (Sweden)

    F.G.J. Calkoen

    2015-03-01

    An altered mRNA expression profile, associated with cell survival and malignant transformation, of MSC derived from children with MDS strengthens the hypothesis that the micro-environment is of importance in this disease. Our data support the understanding that pediatric and adult MDS are two different diseases. Further evaluation of the pathways involved might reveal additional therapy targets.

  7. Human-scale interaction for virtual model displays: a clear case for real tools

    Science.gov (United States)

    Williams, George C.; McDowall, Ian E.; Bolas, Mark T.

    1998-04-01

    We describe a hand-held user interface for interacting with virtual environments displayed on a Virtual Model Display. The tool, constructed entirely of transparent materials, is see-through. We render a graphical counterpart of the tool on the display and map it one-to-one with the real tool. This feature, combined with a capability for touch- sensitive, discrete input, results in a useful spatial input device that is visually versatile. We discuss the tool's design and interaction techniques it supports. Briefly, we look at the human factors issues and engineering challenges presented by this tool and, in general, by the class of hand-held user interfaces that are see-through.

  8. Impact of Advanced Alarm Systems and Information Displays on Human Reliability in the Digital Control Room of Nuclear Power Plants

    International Nuclear Information System (INIS)

    Kim, Jong Hyun; Dang, Vinh N

    2011-01-01

    This paper discusses the potential impacts of two advanced features of digital control rooms, alarm systems and information display systems, on the Human Reliability Analysis (HRA) in nuclear power plants. Although the features of digital control rooms have already been implemented in new or upgraded nuclear power plants, HRAs have so far not taken much credit for these features. In this circumstance, this paper aims at examining the potential effects of these features on human performance and discussing how these effects can be addressed with existing HRA methods. A conclusion derivable from past experimental studies is that those features are supportive in the severe conditions such as complex scenarios and knowledge-based works. However, in the less complex scenarios and rule-based work, they may have no difference with or sometimes negative impacts on operator performance. The discussion about the impact on the HRA is provided on the basis on the THERP method

  9. Large Displacement in Relaxor Ferroelectric Terpolymer Blend Derived Actuators Using Al Electrode for Braille Displays

    Science.gov (United States)

    Lu, S. G.; Chen, X.; Levard, T.; Diglio, P. J.; Gorny, L. J.; Rahn, C. D.; Zhang, Q. M.

    2015-06-01

    Poly(vinylidene fluoride) (PVDF) based polymers are attractive for applications for artificial muscles, high energy density storage devices etc. Recently these polymers have been found great potential for being used as actuators for refreshable full-page Braille displays for visually impaired people in terms of light weight, miniaturized size, and larger displacement, compared with currently used lead zirconate titanate ceramic actuators. The applied voltages of published polymer actuators, however, cannot be reduced to meet the requirements of using city power. Here, we report the polymer actuator generating quite large displacement and blocking force at a voltage close to the city power. Our embodiments also show good self-healing performance and disuse of lead-containing material, which makes the Braille device safer, more reliable and more environment-friendly.

  10. The Effect of Information Analysis Automation Display Content on Human Judgment Performance in Noisy Environments

    Science.gov (United States)

    Bass, Ellen J.; Baumgart, Leigh A.; Shepley, Kathryn Klein

    2014-01-01

    Displaying both the strategy that information analysis automation employs to makes its judgments and variability in the task environment may improve human judgment performance, especially in cases where this variability impacts the judgment performance of the information analysis automation. This work investigated the contribution of providing either information analysis automation strategy information, task environment information, or both, on human judgment performance in a domain where noisy sensor data are used by both the human and the information analysis automation to make judgments. In a simplified air traffic conflict prediction experiment, 32 participants made probability of horizontal conflict judgments under different display content conditions. After being exposed to the information analysis automation, judgment achievement significantly improved for all participants as compared to judgments without any of the automation's information. Participants provided with additional display content pertaining to cue variability in the task environment had significantly higher aided judgment achievement compared to those provided with only the automation's judgment of a probability of conflict. When designing information analysis automation for environments where the automation's judgment achievement is impacted by noisy environmental data, it may be beneficial to show additional task environment information to the human judge in order to improve judgment performance. PMID:24847184

  11. The Effect of Information Analysis Automation Display Content on Human Judgment Performance in Noisy Environments.

    Science.gov (United States)

    Bass, Ellen J; Baumgart, Leigh A; Shepley, Kathryn Klein

    2013-03-01

    Displaying both the strategy that information analysis automation employs to makes its judgments and variability in the task environment may improve human judgment performance, especially in cases where this variability impacts the judgment performance of the information analysis automation. This work investigated the contribution of providing either information analysis automation strategy information, task environment information, or both, on human judgment performance in a domain where noisy sensor data are used by both the human and the information analysis automation to make judgments. In a simplified air traffic conflict prediction experiment, 32 participants made probability of horizontal conflict judgments under different display content conditions. After being exposed to the information analysis automation, judgment achievement significantly improved for all participants as compared to judgments without any of the automation's information. Participants provided with additional display content pertaining to cue variability in the task environment had significantly higher aided judgment achievement compared to those provided with only the automation's judgment of a probability of conflict. When designing information analysis automation for environments where the automation's judgment achievement is impacted by noisy environmental data, it may be beneficial to show additional task environment information to the human judge in order to improve judgment performance.

  12. Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF

    Directory of Open Access Journals (Sweden)

    Blewett Ann

    2008-12-01

    Full Text Available Abstract Background To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme. Results Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 μM, and the Ki was established at 420 μM with respect to the mixed type of inhibition against D-Ala-D-Ala. Conclusion MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development.

  13. Despite differential gene expression profiles pediatric MDS derived mesenchymal stromal cells display functionality in vitro.

    Science.gov (United States)

    Calkoen, F G J; Vervat, C; van Pel, M; de Haas, V; Vijfhuizen, L S; Eising, E; Kroes, W G M; 't Hoen, P A C; van den Heuvel-Eibrink, M M; Egeler, R M; van Tol, M J D; Ball, L M

    2015-03-01

    Pediatric myelodysplastic syndrome (MDS) is a heterogeneous disease covering a spectrum ranging from aplasia (RCC) to myeloproliferation (RAEB(t)). In adult-type MDS there is increasing evidence for abnormal function of the bone-marrow microenvironment. Here, we extensively studied the mesenchymal stromal cells (MSCs) derived from children with MDS. MSCs were expanded from the bone-marrow of 17 MDS patients (RCC: n=10 and advanced MDS: n=7) and pediatric controls (n=10). No differences were observed with respect to phenotype, differentiation capacity, immunomodulatory capacity or hematopoietic support. mRNA expression analysis by Deep-SAGE revealed increased IL-6 expression in RCC- and RAEB(t)-MDS. RCC-MDS MSC expressed increased levels of DKK3, a protein associated with decreased apoptosis. RAEB(t)-MDS revealed increased CRLF1 and decreased DAPK1 expressions. This pattern has been associated with transformation in hematopoietic malignancies. Genes reported to be differentially expressed in adult MDS-MSC did not differ between MSC of pediatric MDS and controls. An altered mRNA expression profile, associated with cell survival and malignant transformation, of MSC derived from children with MDS strengthens the hypothesis that the micro-environment is of importance in this disease. Our data support the understanding that pediatric and adult MDS are two different diseases. Further evaluation of the pathways involved might reveal additional therapy targets. Copyright © 2015. Published by Elsevier B.V.

  14. Performance evaluation of phage-displayed synthetic human single-domain antibody libraries: A retrospective analysis.

    Science.gov (United States)

    Henry, Kevin A; Tanha, Jamshid

    2018-05-01

    Fully human synthetic single-domain antibodies (sdAbs) are desirable therapeutic molecules but their development is a considerable challenge. Here, using a retrospective analysis of in-house historical data, we examined the parameters that impact the outcome of screening phage-displayed synthetic human sdAb libraries to discover antigen-specific binders. We found no evidence for a differential effect of domain type (V H or V L ), library randomization strategy, incorporation of a stabilizing disulfide linkage or sdAb display format (monovalent vs. multivalent) on the probability of obtaining any antigen-binding human sdAbs, instead finding that the success of library screens was primarily related to properties of target antigens, especially molecular mass. The solubility and binding affinity of sdAbs isolated from successful screens depended both on properties of the sdAb libraries (primarily domain type) and the target antigens. Taking attrition of sdAbs with major manufacturability concerns (aggregation; low expression) and sdAbs that do not recognize native cell-surface antigens as independent probabilities, we calculate the overall likelihood of obtaining ≥1 antigen-binding human sdAb from a single library-target screen as ~24%. Successful library-target screens should be expected to yield ~1.3 human sdAbs on average, each with average binding affinity of ~2 μM. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Integrated multimodal human-computer interface and augmented reality for interactive display applications

    Science.gov (United States)

    Vassiliou, Marius S.; Sundareswaran, Venkataraman; Chen, S.; Behringer, Reinhold; Tam, Clement K.; Chan, M.; Bangayan, Phil T.; McGee, Joshua H.

    2000-08-01

    We describe new systems for improved integrated multimodal human-computer interaction and augmented reality for a diverse array of applications, including future advanced cockpits, tactical operations centers, and others. We have developed an integrated display system featuring: speech recognition of multiple concurrent users equipped with both standard air- coupled microphones and novel throat-coupled sensors (developed at Army Research Labs for increased noise immunity); lip reading for improving speech recognition accuracy in noisy environments, three-dimensional spatialized audio for improved display of warnings, alerts, and other information; wireless, coordinated handheld-PC control of a large display; real-time display of data and inferences from wireless integrated networked sensors with on-board signal processing and discrimination; gesture control with disambiguated point-and-speak capability; head- and eye- tracking coupled with speech recognition for 'look-and-speak' interaction; and integrated tetherless augmented reality on a wearable computer. The various interaction modalities (speech recognition, 3D audio, eyetracking, etc.) are implemented a 'modality servers' in an Internet-based client-server architecture. Each modality server encapsulates and exposes commercial and research software packages, presenting a socket network interface that is abstracted to a high-level interface, minimizing both vendor dependencies and required changes on the client side as the server's technology improves.

  16. Increased androgenic sensitivity in the hind limb muscular system marks the evolution of a derived gestural display.

    Science.gov (United States)

    Mangiamele, Lisa A; Fuxjager, Matthew J; Schuppe, Eric R; Taylor, Rebecca S; Hödl, Walter; Preininger, Doris

    2016-05-17

    Physical gestures are prominent features of many species' multimodal displays, yet how evolution incorporates body and leg movements into animal signaling repertoires is unclear. Androgenic hormones modulate the production of reproductive signals and sexual motor skills in many vertebrates; therefore, one possibility is that selection for physical signals drives the evolution of androgenic sensitivity in select neuromotor pathways. We examined this issue in the Bornean rock frog (Staurois parvus, family: Ranidae). Males court females and compete with rivals by performing both vocalizations and hind limb gestural signals, called "foot flags." Foot flagging is a derived display that emerged in the ranids after vocal signaling. Here, we show that administration of testosterone (T) increases foot flagging behavior under seminatural conditions. Moreover, using quantitative PCR, we also find that adult male S. parvus maintain a unique androgenic phenotype, in which androgen receptor (AR) in the hind limb musculature is expressed at levels ∼10× greater than in two other anuran species, which do not produce foot flags (Rana pipiens and Xenopus laevis). Finally, because males of all three of these species solicit mates with calls, we accordingly detect no differences in AR expression in the vocal apparatus (larynx) among taxa. The results show that foot flagging is an androgen-dependent gestural signal, and its emergence is associated with increased androgenic sensitivity within the hind limb musculature. Selection for this novel gestural signal may therefore drive the evolution of increased AR expression in key muscles that control signal production to support adaptive motor performance.

  17. 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

  18. 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 ...

  19. Human factors considerations in the design and evaluation of flight deck displays and controls : version 2.0

    Science.gov (United States)

    2016-12-01

    The objective of this effort is to have a single source reference document for human factors regulatory and guidance material for flight deck displays and controls, in the interest of improving aviation safety. This document identifies guidance on hu...

  20. Prazosin Displays Anticancer Activity against Human Prostate Cancers: Targeting DNA, Cell Cycle

    Directory of Open Access Journals (Sweden)

    Ssu-Chia Lin

    2007-10-01

    Full Text Available Quinazoline-based α1,-adrenoceptor antagonists, in particular doxazosin, terazosin, are suggested to display antineoplastic activity against prostate cancers. However, there are few studies elucidating the effect of prazosin. In this study, prazosin displayed antiproliferative activity superior to that of other α1-blockers, including doxazosin, terazosin, tamsulosin, phentolamine. Prazosin induced G2 checkpoint arrest, subsequent apoptosis in prostate cancer PC-3, DU-145, LNCaP cells. In p53-null PC-3 cells, prazosin induced an increase in DNA str, breaks, ATM/ATR checkpoint pathways, leading to the activation of downstream signaling cascades, including Cdc25c phosphorylation at Ser216, nuclear export of Cdc25c, cyclin-dependent kinase (Cdk 1 phosphorylation at Tyr15. The data, together with sustained elevated cyclin A levels (other than cyclin B1 levels, suggested that Cdki activity was inactivated by prazosin. Moreover, prazosin triggered mitochondria-mediated, caspaseexecuted apoptotic pathways in PC-3 cells. The oral administration of prazosin significantly reduced tumor mass in PC-3-derived cancer xenografts in nude mice. In summary, we suggest that prazosin is a potential antitumor agent that induces cell apoptosis through the induction of DNA damage stress, leading to Cdki inactivation, G2 checkpoint arrest. Subsequently, mitochondriamediated caspase cascades are triggered to induce apoptosis in PC-3 cells.

  1. Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display

    Directory of Open Access Journals (Sweden)

    Zu-Quan Hu

    2012-06-01

    Full Text Available Fusarium verticillioides is the primary causal agent of Fusarium ear and kernel rot in maize, producing fumonisin mycotoxins that are toxic to humans and domestic animals. Rapid detection and monitoring of fumonisin-producing fungi are pivotally important for the prevention of mycotoxins from entering into food/feed products. Chicken-derived single-chain variable fragments (scFvs against cell wall-bound proteins from F. verticillioides were isolated from an immunocompetent phage display library. Comparative phage enzyme-linked immunosorbant assays (ELISAs and sequencing analyses identified four different scFv antibodies with high sensitivity. Soluble antibody ELISAs identified two highly sensitive scFv antibodies, FvCA3 and FvCA4, with the latter being slightly more sensitive. Three-dimensional modeling revealed that the FvCA4 may hold a better overall structure with CDRH3, CDRL1 and CDRL3 centered in the core region of antibody surface compared with that of other scFvs. Immunofluorescence labeling revealed that the binding of FvCA4 antibody was localized to the cell walls of conidiospores and hyphae of F. verticillioides, confirming the specificity of this antibody for a surface target. This scFv antibody was able to detect the fungal mycelium as low as 10−2 μg/mL and contaminating mycelium at a quantity of 10−2 mg/g maize. This is the first report that scFv antibodies derived from phage display have a wide application for rapid and accurate detection and monitoring of fumonisin-producing pathogens in agricultural samples.

  2. A compact phage display human scFv library for selection of antibodies to a wide variety of antigens

    Directory of Open Access Journals (Sweden)

    Kristensen Peter

    2009-01-01

    Full Text Available Abstract Background Phage display technology is a powerful new tool for making antibodies outside the immune system, thus avoiding the use of experimental animals. In the early days, it was postulated that this technique would eventually replace hybridoma technology and animal immunisations. However, since this technology emerged more than 20 years ago, there have only been a handful reports on the construction and application of phage display antibody libraries world-wide. Results Here we report the simplest and highly efficient method for the construction of a highly useful human single chain variable fragment (scFv library. The least number of oligonucleotide primers, electroporations and ligation reactions were used to generate a library of 1.5 × 108 individual clones, without generation of sub-libraries. All possible combinations of heavy and light chains, among all immunoglobulin isotypes, were included by using a mixture of primers and overlapping extension PCR. The key difference from other similar libraries was the highest diversity of variable gene repertoires, which was derived from 140 non-immunized human donors. A wide variety of antigens were successfully used to affinity select specific binders. These included pure recombinant proteins, a hapten and complex antigens such as viral coat proteins, crude snake venom and cancer cell surface antigens. In particular, we were able to use standard bio-panning method to isolate antibody that can bind to soluble Aflatoxin B1, when using BSA-conjugated toxin as a target, as demonstrated by inhibition ELISA. Conclusion These results suggested that by using an optimized protocol and very high repertoire diversity, a compact and efficient phage antibody library can be generated. This advanced method could be adopted by any molecular biology laboratory to generate both naïve or immunized libraries for particular targets as well as for high-throughput applications.

  3. USING OLFACTORY DISPLAYS AS A NONTRADITIONAL INTERFACE IN HUMAN COMPUTER INTERACTION

    Directory of Open Access Journals (Sweden)

    Alper Efe

    2017-07-01

    Full Text Available Smell has its limitations and disadvantages as a display medium, but it also has its strengths and many have recognized its potential. At present, in communications and virtual technologies, smell is either forgotten or improperly stimulated, because non controlled odorants present in the physical space surrounding the user. Nonetheless a controlled presentation of olfactory information can give advantages in various application fields. Therefore, two enabling technologies, electronic noses and especially olfactory displays are reviewed. Scenarios of usage are discussed together with relevant psycho-physiological issues. End-to-end systems including olfactory interfaces are quantitatively characterised under many respects. Recent works done by the authors on field are reported. The article will touch briefly on the control of scent emissions; an important factor to consider when building scented computer systems. As a sample application SUBSMELL system investigated. A look at areas of human computer interaction where olfaction output may prove useful will be presented. The article will finish with some brief conclusions and discuss some shortcomings and gaps of the topic. In particular, the addition of olfactory cues to a virtual environment increased the user's sense of presence and memory of the environment. Also, this article discusses the educational aspect of the subsmell systems.

  4. Isolation of Osteosarcoma-Associated Human Antibodies from a Combinatorial Fab Phage Display Library

    Directory of Open Access Journals (Sweden)

    Carmela Dantas-Barbosa

    2009-01-01

    Full Text Available Osteosarcoma, a highly malignant disease, is the most common primary bone tumor and is frequently found in children and adolescents. In order to isolate antibodies against osteosarcoma antigens, a combinatorial osteosarcoma Fab library displayed on the surface of phages was used. After three rounds of selection on the surface of tumor cells, several osteosarcoma-reactive Fabs were detected. From these Fabs, five were better characterized, and despite having differences in their VH (heavy chain variable domain and Vκ (kappa chain variable domain regions, they all bound to a protein with the same molecular mass. Further analysis by cell ELISA and immunocytochemistry suggested that the Fabs recognize a membrane-associated tumor antigen expressed in higher amounts in neoplasic cells than in normal tissue. These results suggest that the human Fabs selected in this work are a valuable tool for the study of this neoplasia.

  5. Translating chimpanzee personality to humans: Investigating the transportability of chimpanzee-derived personality scales to humans.

    Science.gov (United States)

    Latzman, Robert D; Sauvigné, Katheryn C; Hopkins, William D

    2016-06-01

    There is a growing interest in the study of personality in chimpanzees with repeated findings of a similar structure of personality in apes to that found in humans. To date, however, the direct translational value of instruments used to assess chimpanzee personality to humans has yet to be explicitly tested. As such, in the current study we sought to determine the transportability of factor analytically-derived chimpanzee personality scales to humans in a large human sample (N = 301). Human informants reporting on target individuals they knew well completed chimpanzee-derived and human-derived measures of personality from the two most widely studied models of human personality: Big Five and Big Three. The correspondence between informant-reported chimpanzee- and human-derived personality scales was then investigated. Results indicated high convergence for corresponding scales across most chimpanzee- and human-derived personality scales. Findings from the current study provide evidence that chimpanzee-derived scales translate well to humans and operate quite similarly to the established human-derived personality scales in a human sample. This evidence of transportability lends support to the translational nature of chimpanzee personality research suggesting clear relevance of this growing literature to humans. Am. J. Primatol. 78:601-609, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  6. Genes expressed in specific areas of the human fetal cerebral cortex display distinct patterns of evolution.

    Directory of Open Access Journals (Sweden)

    Nelle Lambert

    2011-03-01

    Full Text Available The developmental mechanisms through which the cerebral cortex increased in size and complexity during primate evolution are essentially unknown. To uncover genetic networks active in the developing cerebral cortex, we combined three-dimensional reconstruction of human fetal brains at midgestation and whole genome expression profiling. This novel approach enabled transcriptional characterization of neurons from accurately defined cortical regions containing presumptive Broca and Wernicke language areas, as well as surrounding associative areas. We identified hundreds of genes displaying differential expression between the two regions, but no significant difference in gene expression between left and right hemispheres. Validation by qRTPCR and in situ hybridization confirmed the robustness of our approach and revealed novel patterns of area- and layer-specific expression throughout the developing cortex. Genes differentially expressed between cortical areas were significantly associated with fast-evolving non-coding sequences harboring human-specific substitutions that could lead to divergence in their repertoires of transcription factor binding sites. Strikingly, while some of these sequences were accelerated in the human lineage only, many others were accelerated in chimpanzee and/or mouse lineages, indicating that genes important for cortical development may be particularly prone to changes in transcriptional regulation across mammals. Genes differentially expressed between cortical regions were also enriched for transcriptional targets of FoxP2, a key gene for the acquisition of language abilities in humans. Our findings point to a subset of genes with a unique combination of cortical areal expression and evolutionary patterns, suggesting that they play important roles in the transcriptional network underlying human-specific neural traits.

  7. Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

    Science.gov (United States)

    Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

    1995-07-01

    Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

  8. Guidelines for the use of vibro-tactile displays in human computer interaction

    NARCIS (Netherlands)

    Erp, J.B.F. van

    2002-01-01

    Vibro-tactile displays convey messages by presenting vibration to the user's skin. In recent years, the interest in and application of vibro-tactile displays is growing. Vibratory displays are introduced in mobile devices, desktop applications and even in aircraft [1]. Despite the growing interest,

  9. Internal epithelia in Drosophila display rudimentary competence to form cytoplasmic networks of transgenic human vimentin.

    Science.gov (United States)

    Gullmets, Josef; Torvaldson, Elin; Lindqvist, Julia; Imanishi, Susumu Y; Taimen, Pekka; Meinander, Annika; Eriksson, John E

    2017-12-01

    Cytoplasmic intermediate filaments (cIFs) are found in all eumetazoans, except arthropods. To investigate the compatibility of cIFs in arthropods, we expressed human vimentin (hVim), a cIF with filament-forming capacity in vertebrate cells and tissues, transgenically in Drosophila Transgenic hVim could be recovered from whole-fly lysates by using a standard procedure for intermediate filament (IF) extraction. When this procedure was used to test for the possible presence of IF-like proteins in flies, only lamins and tropomyosin were observed in IF-enriched extracts, thereby providing biochemical reinforcement to the paradigm that arthropods lack cIFs. In Drosophila , transgenic hVim was unable to form filament networks in S2 cells and mesenchymal tissues; however, cage-like vimentin structures could be observed around the nuclei in internal epithelia, which suggests that Drosophila retains selective competence for filament formation. Taken together, our results imply that although the filament network formation competence is partially lost in Drosophila , a rudimentary filament network formation ability remains in epithelial cells. As a result of the observed selective competence for cIF assembly in Drosophila , we hypothesize that internal epithelial cIFs were the last cIFs to disappear from arthropods.-Gullmets, J., Torvaldson, E., Lindqvist, J., Imanishi, S. Y., Taimen, P., Meinander, A., Eriksson, J. E. Internal epithelia in Drosophila display rudimentary competence to form cytoplasmic networks of transgenic human vimentin. © FASEB.

  10. Differentiation and molecular profiling of human embryonic stem cell-derived corneal epithelial cells.

    Science.gov (United States)

    Brzeszczynska, J; Samuel, K; Greenhough, S; Ramaesh, K; Dhillon, B; Hay, D C; Ross, J A

    2014-06-01

    It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular, the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory, the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel, provides a robust model of human development and in the future, may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally, we demonstrate that following continued cell culture, stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore, also offer an in vitro model of disease.

  11. Modular Construction of Large Non-Immune Human Antibody Phage-Display Libraries from Variable Heavy and Light Chain Gene Cassettes.

    Science.gov (United States)

    Lee, Nam-Kyung; Bidlingmaier, Scott; Su, Yang; Liu, Bin

    2018-01-01

    Monoclonal antibodies and antibody-derived therapeutics have emerged as a rapidly growing class of biological drugs for the treatment of cancer, autoimmunity, infection, and neurological diseases. To support the development of human antibodies, various display techniques based on antibody gene repertoires have been constructed over the last two decades. In particular, scFv-antibody phage display has been extensively utilized to select lead antibodies against a variety of target antigens. To construct a scFv phage display that enables efficient antibody discovery, and optimization, it is desirable to develop a system that allows modular assembly of highly diverse variable heavy chain and light chain (Vκ and Vλ) repertoires. Here, we describe modular construction of large non-immune human antibody phage-display libraries built on variable gene cassettes from heavy chain and light chain repertoires (Vκ- and Vλ-light can be made into independent cassettes). We describe utility of such libraries in antibody discovery and optimization through chain shuffling.

  12. Screening of synthetic phage display scFv libraries yields competitive ligands of human leptin receptor.

    Science.gov (United States)

    Molek, Peter; Vodnik, Miha; Strukelj, Borut; Bratkovič, Tomaž

    2014-09-26

    Initially considered the main endogenous anorexigenic factor, fat-derived leptin turned out to be a markedly pleiotropic hormone, influencing diverse physiological processes. Moreover, hyperleptinemia in obese individuals has been linked to the onset or progression of serious disorders, such as cancer, autoimmune diseases, and atherosclerosis, and antagonizing peripheral leptin's signalization has been shown to improve these conditions. To develop an antibody-based leptin antagonist we have devised a tailored panning procedure and screened two phage display libraries of single chain variable antibody fragments (scFvs) against recombinant leptin receptor. One of the scFvs was expressed in Escherichia coli and its interaction with leptin receptor was characterized in more detail. It was found to recognize a discontinuous epitope and to compete with leptin for receptor binding with IC50 and Kd values in the nanomolar range. The reported scFv represents a lead for development of leptin antagonists that may ultimately find use in therapy of various hyperleptinemia-related disorders. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. 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.

  14. 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

  15. 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.

  16. Neurite outgrowth in human iPSC-derived neurons

    Science.gov (United States)

    Data on morphology of rat and human neurons in cell cultureThis dataset is associated with the following publication:Druwe, I., T. Freudenrich , K. Wallace , T. Shafer , and W. Mundy. Comparison of Human Induced PluripotentStem Cell-Derived Neurons and Rat Primary CorticalNeurons as In Vitro Models of Neurite Outgrowth. Applied In vitro Toxicology. Mary Ann Liebert, Inc., Larchmont, NY, USA, 2(1): 26-36, (2016).

  17. A Comparison between Human Selected, Derived and System ...

    African Journals Online (AJOL)

    admpather

    Using a prototype implementation of our scheme, we compare the results of human-selected, derived passwords and system generated to reveal the practical viability of our approach in terms of results achieved, ease of implementation and use. Keywords: Security, Biometric, Behavioral, Keystroke dynamics, Password.

  18. A Comparison between Human Selected, Derived and System ...

    African Journals Online (AJOL)

    Using a prototype implementation of our scheme, we compare the results of human-selected, derived passwords and system generated to reveal the practical viability of our approach in terms of results achieved, ease of implementation and use. Keywords: Security, Biometric, Behavioral, Keystroke dynamics, Password ...

  19. Derivation of the human embryonic stem cell line RCM1

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  20. P1-32: Response of Human Visual System to Paranormal Stimuli Appearing in Three-Dimensional Display

    Directory of Open Access Journals (Sweden)

    Jisoo Hong

    2012-10-01

    Full Text Available Three-dimensional (3D display became one of indispensable features of commercial TVs in recent years. However, the 3D content displayed by 3D display may contain the abrupt change of depth when the scene changes, which might be considered as a paranormal stimulus. Because the human visual system is not accustomed to such paranormal stimuli in natural conditions, they can cause unexpected responses which usually induce discomfort. Following the change of depth expressed by 3D display, the eyeballs rotate to match the convergence to the new 3D image position. The amount of rotation varies according to the initial longitudinal location and depth displacement of 3D image. Because the change of depth is abrupt, there is delay in human visual system following the change and such delay can be a source of discomfort. To guarantee the safety in watching 3D TV, the acceptable level of displacement in the longitudinal direction should be revealed quantitatively. Additionally, the artificially generated scenes also can provide paranormal stimuli such as periodic depth variations. In the presentation, we investigate the response of human visual system to such paranormal stimuli given by 3D display system. Using the result of investigation, we can give guideline to creating the 3D content to minimize the discomfort coming from the paranormal stimuli.

  1. Coupling Retinal Scanning Displays to the Human Visual System: Visual System Response and Engineering Considerations

    National Research Council Canada - National Science Library

    Turner, Stuart

    2002-01-01

    A retinal scanning display (RSD) is a visual display that presents an image to an observer via a modulated beam of light that is directed through the eye's pupil and rapidly scanned in a raster-like pattern across the retina...

  2. Canine and human gastrointestinal stromal tumors display similar mutations in c-KIT exon 11

    International Nuclear Information System (INIS)

    Gregory-Bryson, Emmalena; Bartlett, Elizabeth; Kiupel, Matti; Hayes, Schantel; Yuzbasiyan-Gurkan, Vilma

    2010-01-01

    Gastrointestinal stromal tumors (GISTs) are common mesenchymal neoplasms in the gastrointestinal tract of humans and dogs. Little is known about the pathogenesis of these tumors. This study evaluated the role of c-KIT in canine GISTs; specifically, we investigated activating mutations in exons 8, 9, 11, 13, and 17 of c-KIT and exons 12, 14, and 18 of platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), all of which have been implicated in human GISTs. Seventeen canine GISTs all confirmed to be positive for KIT immunostaining were studied. Exons 8, 9, 11, 13 and 17 of c-KIT and exons 12, 14, and 18 of PDGFRA, were amplified from DNA isolated from formalin-fixed paraffin-embedded samples. Of these seventeen cases, six amplicons of exon 11 of c-KIT showed aberrant bands on gel electrophoresis. Sequencing of these amplicons revealed heterozygous in-frame deletions in six cases. The mutations include two different but overlapping six base pair deletions. Exons 8, 9, 13, and 17 of c-KIT and exons 12, 14, and 18 of PDGFRA had no abnormalities detected by electrophoresis and sequencing did not reveal any mutations, other than synonymous single nucleotide polymorphisms (SNPs) found in exon 11 of c-KIT and exons 12 and 14 of PDGFRA. The deletion mutations detected in canine GISTs are similar to those previously found in the juxtamembrane domain of c-KIT in canine cutaneous mast cell tumors in our laboratory as well as to those reported in human GISTs. Interestingly, none of the other c-KIT or PDGFRA exons showed any abnormalities in our cases. This finding underlines the critical importance of c-KIT in the pathophysiology of canine GISTs. The expression of KIT and the identification of these activating mutations in c-KIT implicate KIT in the pathogenesis of these tumors. Our results indicate that mutations in c-KIT may be of prognostic significance and that targeting KIT may be a rational approach to treatment of these malignant tumors. This study further

  3. Functional displays

    International Nuclear Information System (INIS)

    Angelis De, F.; Haentjens, J.

    1995-01-01

    The Functional Displays are directly derived from the Man-Machine Design key document: Function-Based Task Analysis. The presentation defines and describes the goals-means structure of the plant function along with applicable control volumes and parameters of interest. The purpose of the subject is to show, through an example of a preliminary design, what the main parts of a function are. (3 figs.)

  4. Identification of human candidate genes for male infertility by digital differential display.

    Science.gov (United States)

    Olesen, C; Hansen, C; Bendsen, E; Byskov, A G; Schwinger, E; Lopez-Pajares, I; Jensen, P K; Kristoffersson, U; Schubert, R; Van Assche, E; Wahlstroem, J; Lespinasse, J; Tommerup, N

    2001-01-01

    Evidence for the importance of genetic factors in male fertility is accumulating. In the literature and the Mendelian Cytogenetics Network database, 265 cases of infertile males with balanced reciprocal translocations have been described. The candidacy for infertility of 14 testis-expressed transcripts (TETs) were examined by comparing their chromosomal mapping position to the position of balanced reciprocal translocation breakpoints found in the 265 infertile males. The 14 TETs were selected by using digital differential display (electronic subtraction) to search for apparently testis-specific transcripts in the TIGR database. The testis specificity of the 14 TETs was further examined by reverse transcription-polymerase chain reaction (RT-PCR) on adult and fetal tissues showing that four TETs (TET1 to TET4) were testis-expressed only, six TETs (TET5 to TET10) appeared to be differentially expressed and the remaining four TETs (TET11 to TET14) were ubiquitously expressed. Interestingly, the two tesis expressed-only transcripts, TET1 and TET2, mapped to chromosomal regions where seven and six translocation breakpoints have been reported in infertile males respectively. Furthermore, one ubiquitously, but predominantly testis-expressed, transcript, TET11, mapped to 1p32-33, where 13 translocation breakpoints have been found in infertile males. Interestingly, the mouse mutation, skeletal fusions with sterility, sks, maps to the syntenic region in the mouse genome. Another transcript, TET7, was the human homologue of rat Tpx-1, which functions in the specific interaction of spermatogenic cells with Sertoli cells. TPX-1 maps to 6p21 where three cases of chromosomal breakpoints in infertile males have been reported. Finally, TET8 was a novel transcript which in the fetal stage is testis-specific, but in the adult is expressed in multiple tissues, including testis. We named this novel transcript fetal and adult testis-expressed transcript (FATE).

  5. Experimental study on display-control stereotype and development of human factors guidelines

    International Nuclear Information System (INIS)

    Lee, Jung Woon; Lee, Yong Hee; Oh, In Seok; Lee, Hyun Chul; Cha, Woo Chang

    2003-01-01

    It is very important to develop the design guidelines which can be applicable for Korean operators for the purpose of designing the KSNP more safely. The objective of this project is to provide the standards, guidelines and bases applicable for HF-010 through the within-subject experiment for obtaining Korean operators' population stereotype for direction-of-movement of controls associated with displays on the control panels. Through the survey of researches on display compatibility and the classification of types of displays and controls in the main control room of Uljin units 3 and 4, methods for an experiment on the stereotype were established. Experimental interface prototypes for a total of 108 combinations of display and control types were implemented. Experimental data collection and analysis system was built in association with the interface prototypes. The experiment was performed with participation of 250 students as subjects. About 20 guideline items were developed based on the results obtained from our analysis of experimental data

  6. Profiling of benzophenone derivatives using fish and human estrogen receptor-specific in vitro bioassays

    International Nuclear Information System (INIS)

    Molina-Molina, Jose-Manuel; Escande, Aurelie; Pillon, Arnaud; Gomez, Elena; Pakdel, Farzad; Cavailles, Vincent; Olea, Nicolas; Ait-Aissa, Selim; Balaguer, Patrick

    2008-01-01

    Benzophenone (BP) derivatives, BP1 (2,4-dihydroxybenzophenone), BP2 (2,2',4,4'-tetrahydroxybenzophenone), BP3 (2-hydroxy-4-methoxybenzophenone), and THB (2,4,4'-trihydroxybenzophenone) are UV-absorbing chemicals widely used in pharmaceutical, cosmetics, and industrial applications, such as topical sunscreens in lotions and hair sprays to protect skin and hair from UV irradiation. Studies on their endocrine disrupting properties have mostly focused on their interaction with human estrogen receptor alpha (hERα), and there has been no comprehensive analysis of their potency in a system allowing comparison between hERα and hERβ activities. The objective of this study was to provide a comprehensive ER activation profile of BP derivatives using ER from human and fish origin in a battery of in vitro tests, i.e., competitive binding, reporter gene based assays, vitellogenin (Vtg) induction in isolated rainbow trout hepatocytes, and proliferation based assays. The ability to induce human androgen receptor (hAR)-mediated reporter gene expression was also examined. All BP derivatives tested except BP3 were full hERα and hERβ agonists (BP2 > THB > BP1) and displayed a stronger activation of hERβ compared with hERα, the opposite effect to that of estradiol (E 2 ). Unlike E 2 , BPs were more active in rainbow trout ERα (rtERα) than in hERα assay. All four BP derivatives showed anti-androgenic activity (THB > BP2 > BP1 > BP3). Overall, the observed anti-androgenic potencies of BP derivatives, together with their proposed greater effect on ERβ versus ERα activation, support further investigation of their role as endocrine disrupters in humans and wildlife

  7. Surface Immobilization of Human Arginase-1 with an Engineered Ice Nucleation Protein Display System in E. coli.

    Directory of Open Access Journals (Sweden)

    Zhen Zhang

    Full Text Available Ice nucleation protein (INP is frequently used as a surface anchor for protein display in gram-negative bacteria. Here, MalE and TorA signal peptides, and three charged polypeptides, 6×Lys, 6×Glu and 6×Asp, were anchored to the N-terminus of truncated INP (InaK-N to improve its surface display efficiency for human Arginase1 (ARG1. Our results indicated that the TorA signal peptide increased the surface translocation of non-protein fused InaK-N and human ARG1 fused InaK-N (InaK-N/ARG1 by 80.7% and 122.4%, respectively. Comparably, the MalE signal peptide decreased the display efficiencies of both the non-protein fused InaK-N and InaK-N/ARG1. Our results also suggested that the 6×Lys polypeptide significantly increased the surface display efficiency of K6-InaK-N/ARG1 by almost 2-fold, while also practically abolishing the surface translocation of non-protein fused InaK-N, indicating the interesting roles of charged polypeptides in bacteria surface display systems. Cell surface-immobilized K6-InaK-N/ARG1 presented an arginase activity of 10.7 U/OD600 under the optimized conditions of 40°C, pH 10.0 and 1 mM Mn2+, which could convert more than 95% of L-Arginine (L-Arg to L-Ornithine (L-Orn in 16 hours. The engineered InaK-Ns expanded the INP surface display system, which aided in the surface immobilization of human ARG1 in E. coli cells.

  8. Purification of human platelet-derived growth factor

    International Nuclear Information System (INIS)

    Raines, E.W.; Ross, R.

    1985-01-01

    The paper describes a method for purification of human platelet-derived growth factor (PDGF) from outdated platelet-rich plasma (PRP) using commonly available laboratory reagents and yielding a mitogen purified 800,000-fold over the starting material. [ 3 H]thymidine incorporation into DNA of cultured cells responsive to PDGF represents the most readily available method to follow its purification and define the biological activity of a purified preparation. Other assays to quantitate PDGF include radioreceptor assay and radioimmunoassay

  9. Tumorigenicity studies for human pluripotent stem cell-derived products.

    Science.gov (United States)

    Kuroda, Takuya; Yasuda, Satoshi; Sato, Yoji

    2013-01-01

    Human pluripotent stem cells (hPSCs), i.e. human embryonic stem cells and human induced pluripotent stem cells, are able to self-renew and differentiate into multiple cell types. Because of these abilities, numerous attempts have been made to utilize hPSCs in regenerative medicine/cell therapy. hPSCs are, however, also tumorigenic, that is, they can give rise to the progressive growth of tumor nodules in immunologically unresponsive animals. Therefore, assessing and managing the tumorigenicity of all final products is essential in order to prevent ectopic tissue formation, tumor development, and/or malignant transformation elicited by residual pluripotent stem cells after implantation. No detailed guideline for the tumorigenicity testing of hPSC-derived products has yet been issued for regenerative medicine/cell therapy, despite the urgent necessity. Here, we describe the current situations and issues related to the tumorigenicity testing of hPSC-derived products and we review the advantages and disadvantages of several types of tumorigenicity-associated tests. We also refer to important considerations in the execution and design of specific studies to monitor the tumorigenicity of hPSC-derived products.

  10. Anticancer activities of bovine and human lactoferricin-derived peptides.

    Science.gov (United States)

    Arias, Mauricio; Hilchie, Ashley L; Haney, Evan F; Bolscher, Jan G M; Hyndman, M Eric; Hancock, Robert E W; Vogel, Hans J

    2017-02-01

    Lactoferrin (LF) is a mammalian host defense glycoprotein with diverse biological activities. Peptides derived from the cationic region of LF possess cytotoxic activity against cancer cells in vitro and in vivo. Bovine lactoferricin (LFcinB), a peptide derived from bovine LF (bLF), exhibits broad-spectrum anticancer activity, while a similar peptide derived from human LF (hLF) is not as active. In this work, several peptides derived from the N-terminal regions of bLF and hLF were studied for their anticancer activities against leukemia and breast-cancer cells, as well as normal peripheral blood mononuclear cells. The cyclized LFcinB-CLICK peptide, which possesses a stable triazole linkage, showed improved anticancer activity, while short peptides hLF11 and bLF10 were not cytotoxic to cancer cells. Interestingly, hLF11 can act as a cell-penetrating peptide; when combined with the antimicrobial core sequence of LFcinB (RRWQWR) through either a Pro or Gly-Gly linker, toxicity to Jurkat cells increased. Together, our work extends the library of LF-derived peptides tested for anticancer activity, and identified new chimeric peptides with high cytotoxicity towards cancerous cells. Additionally, these results support the notion that short cell-penetrating peptides and antimicrobial peptides can be combined to create new adducts with increased potency.

  11. The diterpenoid 7-keto-sempervirol, derived from Lycium chinense, displays anthelmintic activity against both Schistosoma mansoni and Fasciola hepatica.

    Directory of Open Access Journals (Sweden)

    Jennifer Edwards

    2015-03-01

    integrity and spine loss. CONCLUSIONS/ SIGNIFICANCE: 7-keto-sempervirol negatively affects the viability and phenotype of two related pathogenic trematodes responsible for significant human and animal infectious diseases. This plant-derived, natural product is also active against both larval and adult developmental forms. As such, the data collectively indicate that 7-keto-sempervirol is an important starting point for anthelmintic drug development. Medicinal chemistry optimisation of more potent 7-keto-sempervirol analogues could lead to the identification of novel chemical entities useful for future combinatorial or replacement anthelmintic control.

  12. Identification and purification of human induced pluripotent stem cell-derived atrial-like cardiomyocytes based on sarcolipin expression.

    Directory of Open Access Journals (Sweden)

    Rebecca Josowitz

    Full Text Available The use of human stem cell-derived cardiomyocytes to study atrial biology and disease has been restricted by the lack of a reliable method for stem cell-derived atrial cell labeling and purification. The goal of this study was to generate an atrial-specific reporter construct to identify and purify human stem cell-derived atrial-like cardiomyocytes. We have created a bacterial artificial chromosome (BAC reporter construct in which fluorescence is driven by expression of the atrial-specific gene sarcolipin (SLN. When purified using flow cytometry, cells with high fluorescence specifically express atrial genes and display functional calcium handling and electrophysiological properties consistent with atrial cardiomyocytes. Our data indicate that SLN can be used as a marker to successfully monitor and isolate hiPSC-derived atrial-like cardiomyocytes. These purified cells may find many applications, including in the study of atrial-specific pathologies and chamber-specific lineage development.

  13. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine

    Energy Technology Data Exchange (ETDEWEB)

    Tomar, Geetanjali B.; Srivastava, Rupesh K.; Gupta, Navita; Barhanpurkar, Amruta P.; Pote, Satish T. [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India); Jhaveri, Hiral M. [Department of Periodontics and Oral Implantology, Dr. D.Y. Patil Dental College and Hospital, Pune (India); Mishra, Gyan C. [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India); Wani, Mohan R., E-mail: mohanwani@nccs.res.in [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India)

    2010-03-12

    Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into multiple cell lineages. Presently, bone marrow is considered as a prime source of MSCs; however, there are some drawbacks and limitations in use of these MSCs for cell therapy. In this study, we demonstrate that human gingival tissue-derived MSCs have several advantages over bone marrow-derived MSCs. Gingival MSCs are easy to isolate, homogenous and proliferate faster than bone marrow MSCs without any growth factor. Importantly, gingival MSCs display stable morphology and do not loose MSC characteristic at higher passages. In addition, gingival MSCs maintain normal karyotype and telomerase activity in long-term cultures, and are not tumorigenic. Thus, we reveal that human gingiva is a better source of MSCs than bone marrow, and large number of functionally competent clinical grade MSCs can be generated in short duration for cell therapy in regenerative medicine and tissue engineering.

  14. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine

    International Nuclear Information System (INIS)

    Tomar, Geetanjali B.; Srivastava, Rupesh K.; Gupta, Navita; Barhanpurkar, Amruta P.; Pote, Satish T.; Jhaveri, Hiral M.; Mishra, Gyan C.; Wani, Mohan R.

    2010-01-01

    Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into multiple cell lineages. Presently, bone marrow is considered as a prime source of MSCs; however, there are some drawbacks and limitations in use of these MSCs for cell therapy. In this study, we demonstrate that human gingival tissue-derived MSCs have several advantages over bone marrow-derived MSCs. Gingival MSCs are easy to isolate, homogenous and proliferate faster than bone marrow MSCs without any growth factor. Importantly, gingival MSCs display stable morphology and do not loose MSC characteristic at higher passages. In addition, gingival MSCs maintain normal karyotype and telomerase activity in long-term cultures, and are not tumorigenic. Thus, we reveal that human gingiva is a better source of MSCs than bone marrow, and large number of functionally competent clinical grade MSCs can be generated in short duration for cell therapy in regenerative medicine and tissue engineering.

  15. Experimental study on display-control stereotype and development of human factors guidelines

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jung Woon; Lee, Yong Hee; Oh, In Seok; Lee, Hyun Chul [KAERI, Taejon (Korea, Republic of); Cha, Woo Chang [Kumoh National Univ. of Technolgy, Gumi (Korea, Republic of)

    2003-01-01

    It is very important to develop the design guidelines which can be applicable for Korean operators for the purpose of designing the KSNP more safely. The objective of this project is to provide the standards, guidelines and bases applicable for HF-010 through the within-subject experiment for obtaining Korean operators' population stereotype for direction-of-movement of controls associated with displays on the control panels. Through the survey of researches on display compatibility and the classification of types of displays and controls in the main control room of Uljin units 3 and 4, methods for an experiment on the stereotype were established. Experimental interface prototypes for a total of 108 combinations of display and control types were implemented. Experimental data collection and analysis system was built in association with the interface prototypes. The experiment was performed with participation of 250 students as subjects. About 20 guideline items were developed based on the results obtained from our analysis of experimental data.

  16. Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries.

    Science.gov (United States)

    El Zoeiby, Ahmed; Sanschagrin, François; Darveau, André; Brisson, Jean-Robert; Levesque, Roger C

    2003-03-01

    The machinery of peptidoglycan biosynthesis is an ideal site at which to look for novel antimicrobial targets. Phage display was used to develop novel peptide inhibitors for MurC, an essential enzyme involved in the early steps of biosynthesis of peptidoglycan monomer. We cloned and overexpressed the murA, -B and -C genes from Pseudomonas aeruginosa in the pET expression vector, adding a His-tag to their C termini. The three proteins were overproduced in Escherichia coli and purified to homogeneity in milligram quantities. MurA and -B were combinatorially used to synthesize the MurC substrate UDP-N-acetylmuramate, the identity of which was confirmed by mass spectrometry and nuclear magnetic resonance analysis. Two phage-display libraries were screened against MurC in order to identify peptide ligands to the enzyme. Three rounds of biopanning were carried out, successively increasing elution specificity from round 1 to 3. The third round was accomplished with both non-specific elution and competitive elution with each of the three MurC substrates, UDP-N-acetylmuramic acid (UNAM), ATP and L-alanine. The DNA of 10 phage, selected randomly from each group, was extracted and sequenced, and consensus peptide sequences were elucidated. Peptides were synthesized and tested for inhibition of the MurC-catalysed reaction, and two peptides were shown to be inhibitors of MurC activity with IC(50)s of 1.5 and 0.9 mM, respectively. The powerful selection technique of phage display allowed us to identify two peptide inhibitors of the essential bacterial enzyme MurC. The peptide sequences represent the basis for the synthesis of inhibitory peptidomimetic molecules.

  17. Curcuma longa Is Able to Induce Apoptotic Cell Death of Pterygium-Derived Human Keratinocytes.

    Science.gov (United States)

    Sancilio, Silvia; Di Staso, Silvio; Sebastiani, Stefano; Centurione, Lucia; Di Girolamo, Nick; Ciancaglini, Marco; Di Pietro, Roberta

    2017-01-01

    Pterygium is a relatively common eye disease that can display an aggressive clinical behaviour. To evaluate the in vitro effects of Curcuma longa on human pterygium-derived keratinocytes, specimens of pterygium from 20 patients undergoing pterygium surgical excision were collected. Pterygium explants were put into culture and derived keratinocytes were treated with an alcoholic extract of 1.3% Curcuma longa in 0.001% Benzalkonium Chloride for 3, 6, and 24 h. Cultured cells were examined for CAM5.2 (anti-cytokeratin antibody) and CD140 (anti-fibroblast transmembrane glycoprotein antibody) expression between 3th and 16th passage to assess cell homogeneity. TUNEL technique and Annexin-V/PI staining in flow cytometry were used to detect keratinocyte apoptosis. We showed that Curcuma longa exerts a proapoptotic effect on pterygium-derived keratinocytes already after 3 h treatment. Moreover, after 24 h treatment, Curcuma longa induces a significant increase in TUNEL as well as Annexin-V/PI positive cells in comparison to untreated samples. Our study confirms previous observations highlighting the expression, in pterygium keratinocytes, of nuclear VEGF and gives evidence for the first time to the expression of nuclear and cytoplasmic VEGF-R1. All in all, these findings suggest that Curcuma longa could have some therapeutic potential in the treatment and prevention of human pterygium.

  18. Curcuma longa Is Able to Induce Apoptotic Cell Death of Pterygium-Derived Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    Silvia Sancilio

    2017-01-01

    Full Text Available Pterygium is a relatively common eye disease that can display an aggressive clinical behaviour. To evaluate the in vitro effects of Curcuma longa on human pterygium-derived keratinocytes, specimens of pterygium from 20 patients undergoing pterygium surgical excision were collected. Pterygium explants were put into culture and derived keratinocytes were treated with an alcoholic extract of 1.3% Curcuma longa in 0.001% Benzalkonium Chloride for 3, 6, and 24 h. Cultured cells were examined for CAM5.2 (anti-cytokeratin antibody and CD140 (anti-fibroblast transmembrane glycoprotein antibody expression between 3th and 16th passage to assess cell homogeneity. TUNEL technique and Annexin-V/PI staining in flow cytometry were used to detect keratinocyte apoptosis. We showed that Curcuma longa exerts a proapoptotic effect on pterygium-derived keratinocytes already after 3 h treatment. Moreover, after 24 h treatment, Curcuma longa induces a significant increase in TUNEL as well as Annexin-V/PI positive cells in comparison to untreated samples. Our study confirms previous observations highlighting the expression, in pterygium keratinocytes, of nuclear VEGF and gives evidence for the first time to the expression of nuclear and cytoplasmic VEGF-R1. All in all, these findings suggest that Curcuma longa could have some therapeutic potential in the treatment and prevention of human pterygium.

  19. Production of iPS-Derived Human Retinal Organoids for Use in Transgene Expression Assays

    NARCIS (Netherlands)

    Quinn, Peter M; Buck, Thilo M; Ohonin, Charlotte; Mikkers, Harald M M; Wijnholds, J.

    2018-01-01

    In vitro retinal organoid modeling from human pluripotent stem cells is becoming more common place in many ophthalmic laboratories worldwide. These organoids mimic human retinogenesis through formation of organized layered retinal structures that display markers for typical retinal cell types.

  20. Electrophysiological properties of neurosensory progenitors derived from human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Karina Needham

    2014-01-01

    Full Text Available In severe cases of sensorineural hearing loss where the numbers of auditory neurons are significantly depleted, stem cell-derived neurons may provide a potential source of replacement cells. The success of such a therapy relies upon producing a population of functional neurons from stem cells, to enable precise encoding of sound information to the brainstem. Using our established differentiation assay to produce sensory neurons from human stem cells, patch-clamp recordings indicated that all neurons examined generated action potentials and displayed both transient sodium and sustained potassium currents. Stem cell-derived neurons reliably entrained to stimuli up to 20 pulses per second (pps, with 50% entrainment at 50 pps. A comparison with cultured primary auditory neurons indicated similar firing precision during low-frequency stimuli, but significant differences after 50 pps due to differences in action potential latency and width. The firing properties of stem cell-derived neurons were also considered relative to time in culture (31–56 days and revealed no change in resting membrane potential, threshold or firing latency over time. Thus, while stem cell-derived neurons did not entrain to high frequency stimulation as effectively as mammalian auditory neurons, their electrical phenotype was stable in culture and consistent with that reported for embryonic auditory neurons.

  1. Semisynthetic Lipopeptides Derived from Nisin Display Antibacterial Activity and Lipid II Binding on Par with That of the Parent Compound

    NARCIS (Netherlands)

    Koopmans, Timo; Wood, Thomas M.; 't Hart, Peter; Kleijn, Laurens H. J.; Hendrickx, Antoni P. A.; Willems, Rob J. L.; Breukink, Eefjan; Martin, Nathaniel I.

    2015-01-01

    The lipid II-binding N-terminus of nisin, comprising the so-called A/B ring system, was synthetically modified to provide antibacterially active and proteolytically stable derivatives. A variety of lipids were coupled to the C-terminus of the nisin A/B ring system to generate semisynthetic

  2. Human Engineering Data Base for Design and Selection of Cathode Ray Tube and Other Display Systems

    Science.gov (United States)

    1984-07-01

    on a display. Several studies (Ericksen, 195’f; Garner & Creelman , ige’f) have reported improved ability to discriminate among objects when size and...strength (directly related to target echo signal-to-noise ratio) and the psychological state of the operator. Single- scan-detection probability as a...information analysis of verbal and motor responses to symbolic and conventional arable numerals. Journal of Applied Psychology , 1958, ^, 79-8^^. Allusi, E

  3. Chemical Synthesis and In Vitro Evaluation of a Phage Display-Derived Peptide Active against Infectious Salmon Anemia Virus.

    Science.gov (United States)

    Ojeda, Nicolás; Cárdenas, Constanza; Guzmán, Fanny; Marshall, Sergio H

    2016-04-01

    Infectious salmon anemia virus (ISAV) is the etiological agent of the disease by the same name and causes major losses in the salmon industry worldwide. Epizootic ISAV outbreaks have occurred in Norway and, to a lesser degree, in Canada. In 2007, an ISAV outbreak in Chile destroyed most of the seasonal production and endangered the entire Chilean salmon industry. None of the existing prophylactic approaches have demonstrated efficacy in providing absolute protection from or even a palliative effect on ISAV proliferation. Sanitary control measures for ISAV, based on molecular epidemiology data, have proven insufficient, mainly due to high salmon culture densities and a constant presence of a nonpathogenic strain of the virus. This report describes an alternative treatment approach based on interfering peptides selected from a phage display library. The screening of a phage display heptapeptide library resulted in the selection of a novel peptide with significant in vitro antiviral activity against ISAV. This peptide specifically interacted with the viral hemagglutinin-esterase protein, thereby impairing virus binding, with plaque reduction assays showing a significant reduction in viral yields. The identified peptide acts at micromolar concentrations against at least two different pathogenic strains of the virus, without detectable cytotoxic effects on the tested fish cells. Therefore, antiviral peptides represent a novel alternative for controlling ISAV and, potentially, other fish pathogens. Identifying novel methods for the efficient control of infectious diseases is imperative for the future of global aquaculture. The present study used a phage display heptapeptide library to identify a peptide with interfering activity against a key protein of the infectious salmon anemia virus (ISAV). A piscine orthomyxovirus, ISAV is a continuous threat to the commercial sustainability of cultured salmon production worldwide. The complex epidemiological strategy of this

  4. Isolation and characterization of human umbilical cord-derived endothelial colony-forming cells

    Science.gov (United States)

    Zhang, Hao; Tao, Yanling; Ren, Saisai; Liu, Haihui; Zhou, Hui; Hu, Jiangwei; Tang, Yongyong; Zhang, Bin; Chen, Hu

    2017-01-01

    Endothelial colony-forming cells (ECFCs) are a population of endothelial progenitor cells (EPCs) that display robust proliferative potential and vessel-forming capability. Previous studies have demonstrated that a limited number of ECFCs may be obtained from adult bone marrow, peripheral blood and umbilical cord (UC) blood. The present study describes an effective method for isolating ECFCs from human UC. The ECFCs derived from human UC displayed the full properties of EPCs. Analysis of the growth kinetics, cell cycle and colony-forming ability of the isolated human UC-ECFCs indicated that the cells demonstrated properties of stem cells, including relative stability and rapid proliferation in vitro. Gene expression of Fms related tyrosine kinase 1, kinase insert domain receptor, vascular endothelial cadherin, cluster of differentiation (CD)31, CD34, epidermal growth factor homology domains-2, von Willebrand factor and endothelial nitric oxide synthase was assessed by reverse transcription-polymerase chain reaction. The cells were positive for CD34, CD31, CD73, CD105 and vascular endothelial growth factor receptor-2, and negative for CD45, CD90 and human leukocyte antigen-antigen D related protein according to flow cytometry. 1,1′-dioctadecyl-3,3,3′,3′-tetra-methyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein and fluorescein isothiocyanate-Ulex europaeus-l were used to verify the identity of the UC-ECFCs. Matrigel was used to investigate tube formation capability. The results demonstrated that the reported technique is a valuable method for isolating human UC-ECFCs, which have potential for use in vascular regeneration. PMID:29067104

  5. A simple signaling rule for variable life-adjusted display derived from an equivalent risk-adjusted CUSUM chart.

    Science.gov (United States)

    Wittenberg, Philipp; Gan, Fah Fatt; Knoth, Sven

    2018-04-17

    The variable life-adjusted display (VLAD) is the first risk-adjusted graphical procedure proposed in the literature for monitoring the performance of a surgeon. It displays the cumulative sum of expected minus observed deaths. It has since become highly popular because the statistic plotted is easy to understand. But it is also easy to misinterpret a surgeon's performance by utilizing the VLAD, potentially leading to grave consequences. The problem of misinterpretation is essentially caused by the variance of the VLAD's statistic that increases with sample size. In order for the VLAD to be truly useful, a simple signaling rule is desperately needed. Various forms of signaling rules have been developed, but they are usually quite complicated. Without signaling rules, making inferences using the VLAD alone is difficult if not misleading. In this paper, we establish an equivalence between a VLAD with V-mask and a risk-adjusted cumulative sum (RA-CUSUM) chart based on the difference between the estimated probability of death and surgical outcome. Average run length analysis based on simulation shows that this particular RA-CUSUM chart has similar performance as compared to the established RA-CUSUM chart based on the log-likelihood ratio statistic obtained by testing the odds ratio of death. We provide a simple design procedure for determining the V-mask parameters based on a resampling approach. Resampling from a real data set ensures that these parameters can be estimated appropriately. Finally, we illustrate the monitoring of a real surgeon's performance using VLAD with V-mask. Copyright © 2018 John Wiley & Sons, Ltd.

  6. Embryonic stem cells derived from in vivo or in vitro-generated murine blastocysts display similar transcriptome and differentiation potential.

    Directory of Open Access Journals (Sweden)

    Rhodel K Simbulan

    Full Text Available The use of assisted reproductive technologies (ART such as in vitro fertilization (IVF has resulted in the birth of more than 5 million children. While children conceived by these technologies are generally healthy, there is conflicting evidence suggesting an increase in adult-onset complications like glucose intolerance and high blood pressure in IVF children. Animal models indicate similar potential risks. It remains unclear what molecular mechanisms may be operating during in vitro culture to predispose the embryo to these diseases. One of the limitations faced by investigators is the paucity of the material in the preimplantation embryo to test for molecular analysis. To address this problem, we generated mouse embryonic stem cells (mESC from blastocysts conceived after natural mating (mESCFB or after IVF, using optimal (KSOM + 5% O2; mESCKAA and suboptimal (Whitten's Medium, + 20% O2, mESCWM conditions. All three groups of embryos showed similar behavior during both derivation and differentiation into their respective mESC lines. Unsupervised hierarchical clustering of microarray data showed that blastocyst culture does not affect the transcriptome of derived mESCs. Transcriptomic changes previously observed in the inner cell mass (ICM of embryos derived in the same conditions were not present in mESCs, regardless of method of conception or culture medium, suggesting that mESC do not fully maintain a memory of the events occurring prior to their derivation. We conclude that the fertilization method or culture media used to generate blastocysts does not affect differentiation potential, morphology and transcriptome of mESCs.

  7. 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

  8. Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

    Science.gov (United States)

    Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

    2004-09-01

    Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

  9. Human fibroblasts display a differential focal adhesion phenotype relative to chimpanzee.

    Science.gov (United States)

    Advani, Alexander S; Chen, Annie Y; Babbitt, Courtney C

    2016-01-01

    There are a number of documented differences between humans and our closest relatives in responses to wound healing and in disease susceptibilities, suggesting a differential cellular response to certain environmental factors. In this study, we sought to look at a specific cell type, fibroblasts, to examine differences in cellular adhesion between humans and chimpanzees in visualized cells and in gene expression. We have found significant differences in the number of focal adhesions between primary human and chimpanzee fibroblasts. Additionally, we see that adhesion related gene ontology categories are some of the most differentially expressed between human and chimpanzee in normal fibroblast cells. These results suggest that human and chimpanzee fibroblasts may have somewhat different adhesive properties, which could play a role in differential disease phenotypes and responses to external factors. © The Author(s) 2016. Published by Oxford University Press on behalf of the Foundation for Evolution, Medicine, and Public Health.

  10. Human engineering evaluation of Hamaoka No. 3 advanced control room CRT display system

    International Nuclear Information System (INIS)

    Okajima, Hiroyuki; Ito, Takahiko; Noguchi, Yoshihiro; Kabasawa, Yoshihiko

    1985-01-01

    As the central control board of Hamaoka No. 3 plant, a new type control board was adopted for the purpose of powerfully supporting the general judgement function of operators by processing enormous plant data in the normal operation and at the time of an accident with a computer, and displaying the results on CRT screens in the summarized form. Recently, the evaluation of this new control board was carried out by ergonomical techniques, and it was confirmed that the initial objective has been attained. Besides, the criteria for the ergonomical evaluation of central control boards mainly for CRT display were determined, and the contents of design were able to be systematically evaluated and verified. As the ergonomical design of the central control board, the easy distinction of the function of switches, the easy confirmation of system constitution by mimics, the use of CRTs, the easy identification of operation pilot lamps, alarms, instruments and so on were considered. The central control board consists of the main control board, auxiliary control boards and vertical boards, and 12 CRTs are installed. (Kako, I.)

  11. Human health and ecological toxicity potentials due to heavy metal content in waste electronic devices with flat panel displays

    International Nuclear Information System (INIS)

    Lim, Seong-Rin; Schoenung, Julie M.

    2010-01-01

    Display devices such as cathode-ray tube (CRT) televisions and computer monitors are known to contain toxic substances and have consequently been banned from disposal in landfills in the State of California and elsewhere. New types of flat panel display (FPD) devices, millions of which are now purchased each year, also contain toxic substances, but have not previously been systematically studied and compared to assess the potential impact that could result from their ultimate disposal. In the current work, the focus is on the evaluation of end-of-life toxicity potential from the heavy metal content in select FPD devices with the intent to inform material selection and design-for-environment (DfE) decisions. Specifically, the metals antimony, arsenic, barium, beryllium, cadmium, chromium, cobalt, copper, lead, mercury, molybdenum, nickel, selenium, silver, vanadium, and zinc in plasma TVs, LCD (liquid crystal display) TVs, LCD computer monitors and laptop computers are considered. The human health and ecotoxicity potentials are evaluated through a life cycle assessment perspective by combining data on the respective heavy metal contents, the characterization factors in the U.S. EPA Tool for the Reduction and Assessment of Chemical and other environmental Impacts (TRACI), and a pathway and impact model. Principal contributors to the toxicity potentials are lead, arsenic, copper, and mercury. Although the heavy metal content in newer flat panel display devices creates less human health toxicity potential than that in CRTs, for ecological toxicity, the new devices are worse, especially because of the mercury in LCD TVs and the copper in plasma TVs.

  12. Production and evaluation of antibodies and phage display-derived peptide ligands for immunomagnetic separation of Mycobacterium bovis.

    Science.gov (United States)

    Stewart, Linda D; McNair, James; McCallan, Lyanne; Thompson, Suzan; Kulakov, Leonid A; Grant, Irene R

    2012-05-01

    This study describes the development and optimization of an immunomagnetic separation (IMS) method to isolate Mycobacterium bovis cells from lymph node tissues. Gamma-irradiated whole M. bovis AF2122/97 cells and ethanol-extracted surface antigens of such cells were used to produce M. bovis-specific polyclonal and monoclonal antibodies in rabbits and mice. They were also used to generate M. bovis-specific peptide ligands by phage display biopanning. The various antibodies and peptide ligands obtained were used to coat MyOne tosyl-activated Dynabeads (Life Technologies), singly or in combination, and evaluated for IMS. Initially, M. bovis capture from Middlebrook 7H9 broth suspensions (concentration range, 10 to 10(5) CFU/ml) was evaluated by IMS combined with an M. bovis-specific touchdown PCR. IMS-PCR results and, subsequently, IMS-culture results indicated that the beads with greatest immunocapture capability for M. bovis in broth were those coated simultaneously with a monoclonal antibody and a biotinylated 12-mer peptide. These dually coated beads exhibited minimal capture (mean of 0.36% recovery) of 12 other Mycobacterium spp. occasionally encountered in veterinary tuberculosis (TB) diagnostic laboratories. When the optimized IMS method was applied to various M. bovis-spiked lymph node matrices, it demonstrated excellent detection sensitivities (50% limits of detection of 3.16 and 57.7 CFU/ml of lymph node tissue homogenate for IMS-PCR and IMS-culture, respectively). The optimized IMS method therefore has the potential to improve isolation of M. bovis from lymph nodes and hence the diagnosis of bovine tuberculosis.

  13. Antimicrobial Effects of Helix D-derived Peptides of Human Antithrombin III*

    Science.gov (United States)

    Papareddy, Praveen; Kalle, Martina; Bhongir, Ravi K. V.; Mörgelin, Matthias; Malmsten, Martin; Schmidtchen, Artur

    2014-01-01

    Antithrombin III (ATIII) is a key antiproteinase involved in blood coagulation. Previous investigations have shown that ATIII is degraded by Staphylococcus aureus V8 protease, leading to release of heparin binding fragments derived from its D helix. As heparin binding and antimicrobial activity of peptides frequently overlap, we here set out to explore possible antibacterial effects of intact and degraded ATIII. In contrast to intact ATIII, the results showed that extensive degradation of the molecule yielded fragments with antimicrobial activity. Correspondingly, the heparin-binding, helix d-derived, peptide FFFAKLNCRLYRKANKSSKLV (FFF21) of human ATIII, was found to be antimicrobial against particularly the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Fluorescence microscopy and electron microscopy studies demonstrated that FFF21 binds to and permeabilizes bacterial membranes. Analogously, FFF21 was found to induce membrane leakage of model anionic liposomes. In vivo, FFF21 significantly reduced P. aeruginosa infection in mice. Additionally, FFF21 displayed anti-endotoxic effects in vitro. Taken together, our results suggest novel roles for ATIII-derived peptide fragments in host defense. PMID:25202017

  14. Antimicrobial effects of helix D-derived peptides of human antithrombin III.

    Science.gov (United States)

    Papareddy, Praveen; Kalle, Martina; Bhongir, Ravi K V; Mörgelin, Matthias; Malmsten, Martin; Schmidtchen, Artur

    2014-10-24

    Antithrombin III (ATIII) is a key antiproteinase involved in blood coagulation. Previous investigations have shown that ATIII is degraded by Staphylococcus aureus V8 protease, leading to release of heparin binding fragments derived from its D helix. As heparin binding and antimicrobial activity of peptides frequently overlap, we here set out to explore possible antibacterial effects of intact and degraded ATIII. In contrast to intact ATIII, the results showed that extensive degradation of the molecule yielded fragments with antimicrobial activity. Correspondingly, the heparin-binding, helix D-derived, peptide FFFAKLNCRLYRKANKSSKLV (FFF21) of human ATIII, was found to be antimicrobial against particularly the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Fluorescence microscopy and electron microscopy studies demonstrated that FFF21 binds to and permeabilizes bacterial membranes. Analogously, FFF21 was found to induce membrane leakage of model anionic liposomes. In vivo, FFF21 significantly reduced P. aeruginosa infection in mice. Additionally, FFF21 displayed anti-endotoxic effects in vitro. Taken together, our results suggest novel roles for ATIII-derived peptide fragments in host defense. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. 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.

  16. Apoptotic effect of chalcone derivatives of 2-acetylthiophene in human breast cancer cells.

    Science.gov (United States)

    Fogaça, Tatiana B; Martins, Rosiane M; Begnini, Karine R; Carapina, Caroline; Ritter, Marina; de Pereira, Claudio M P; Seixas, Fabiana K; Collares, Tiago

    2017-02-01

    A variety of chalcones have demonstrated cytotoxic activity toward several cancer cell lines. This study aimed to investigate the cytotoxicity of four chalcones derivatives of 2-acetylthiophene in human breast cancer cell lines. MCF-7 and MDA-MB-231 cells were treated with synthesized chalcones and the cytotoxicity was evaluated by tetrazolium dye (MTT), live/dead, and DAPI assays. Chalcones significantly decreased MCF-7 and MDA-MB-231 cells viability in vitro in a dose dependent manner. After 48h treatment, the IC 50 values ranging from 5.52 to 34.23μM. Chalcone 3c displayed the highest cytotoxic activity from all the tested compounds. Cytotoxic effects of compounds were confirmed in the live/dead assay. In addition, DAPI staining revealed that these compounds induce death by apoptosis. The data speculate that chalcone derivatives of 2-acetylthiophene may represent a source of therapeutic agents for human breast cancer. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  17. Human risk relationships derived from epidemiology and laboratory studies

    International Nuclear Information System (INIS)

    Cuddihy, R.G.; Boecker, B.B.; Hahn, F.F.; McClellan, R.O.

    1983-01-01

    Proven techniques are needed for incorporating the results of laboratory toxicology studies into human risk assessments. Two sample calculations of lung cancer risk factors for inhaled radioactive particles and diesel engine exhaust are given here to illustrate a toxicology information matrix approach. This approach combines the results of epidemiology and laboratory animal studies of the substance or agent of principal concern, along with similar information on other surrogate substances. Beyond the estimates of lung cancer risk factors derived by using this approach, an additional advantage is gained by having estimates of uncertainty that can be obtained by incorporating all available toxicology information into the analysis. This approach is recommended for both risk assessment and in designing follow-on toxicology studies to improve preliminary assessments for new potentially harmful agents entering our environment

  18. Derivation of Two New Human Embryonic Stem Cell Lines from Nonviable Human Embryos

    Directory of Open Access Journals (Sweden)

    Svetlana Gavrilov

    2011-01-01

    Full Text Available We report the derivation and characterization of two new human embryonic stem cells (hESC lines (CU1 and CU2 from embryos with an irreversible loss of integrated organismic function. In addition, we analyzed retrospective data of morphological progression from embryonic day (ED 5 to ED6 for 2480 embryos not suitable for clinical use to assess grading criteria indicative of loss of viability on ED5. Our analysis indicated that a large proportion of in vitro fertilization (IVF embryos not suitable for clinical use could be used for hESC derivation. Based on these combined findings, we propose that criteria commonly used in IVF clinics to determine optimal embryos for uterine transfer can be employed to predict the potential for hESC derivation from poor quality embryos without the destruction of vital human embryos.

  19. Derivation of novel genetically diverse human embryonic stem cell lines.

    Science.gov (United States)

    Stefanova, Valentina T; Grifo, James A; Hansis, Christoph

    2012-06-10

    Human embryonic stem cells (hESCs) have the potential to revolutionize many biomedical fields ranging from basic research to disease modeling, regenerative medicine, drug discovery, and toxicity testing. A multitude of hESC lines have been derived worldwide since the first 5 lines by Thomson et al. 13 years ago, but many of these are poorly characterized, unavailable, or do not represent desired traits, thus making them unsuitable for application purposes. In order to provide the scientific community with better options, we have derived 12 new hESC lines at New York University from discarded genetically normal and abnormal embryos using the latest techniques. We examined the genetic status of the NYUES lines in detail as well as their molecular and cellular features and DNA fingerprinting profile. Furthermore, we differentiated our hESCs into the tissues most affected by a specific condition or into clinically desired cell types. To our knowledge, a number of characteristics of our hESCs have not been previously reported, for example, mutation for alpha thalassemia X-linked mental retardation syndrome, linkage to conditions with a genetic component such as asthma or poor sperm morphology, and novel combinations of ethnic backgrounds. Importantly, all of our undifferentiated euploid female lines tested to date did not show X chromosome inactivation, believed to result in superior potency. We continue to derive new hESC lines and add them to the NIH registry and other registries. This should facilitate the use of our hESCs and lead to advancements for patient-benefitting applications.

  20. Physiological characterisation of human iPS-derived dopaminergic neurons.

    Directory of Open Access Journals (Sweden)

    Elizabeth M Hartfield

    Full Text Available Human induced pluripotent stem cells (hiPSCs offer the potential to study otherwise inaccessible cell types. Critical to this is the directed differentiation of hiPSCs into functional cell lineages. This is of particular relevance to research into neurological disease, such as Parkinson's disease (PD, in which midbrain dopaminergic neurons degenerate during disease progression but are unobtainable until post-mortem. Here we report a detailed study into the physiological maturation over time of human dopaminergic neurons in vitro. We first generated and differentiated hiPSC lines into midbrain dopaminergic neurons and performed a comprehensive characterisation to confirm dopaminergic functionality by demonstrating dopamine synthesis, release, and re-uptake. The neuronal cultures include cells positive for both tyrosine hydroxylase (TH and G protein-activated inward rectifier potassium channel 2 (Kir3.2, henceforth referred to as GIRK2, representative of the A9 population of substantia nigra pars compacta (SNc neurons vulnerable in PD. We observed for the first time the maturation of the slow autonomous pace-making (<10 Hz and spontaneous synaptic activity typical of mature SNc dopaminergic neurons using a combination of calcium imaging and electrophysiology. hiPSC-derived neurons exhibited inositol tri-phosphate (IP3 receptor-dependent release of intracellular calcium from the endoplasmic reticulum in neuronal processes as calcium waves propagating from apical and distal dendrites, and in the soma. Finally, neurons were susceptible to the dopamine neuron-specific toxin 1-methyl-4-phenylpyridinium (MPP+ which reduced mitochondrial membrane potential and altered mitochondrial morphology. Mature hiPSC-derived dopaminergic neurons provide a neurophysiologically-defined model of previously inaccessible vulnerable SNc dopaminergic neurons to bridge the gap between clinical PD and animal models.

  1. Production of a Human Antibody Library in the Phage-Display Vector pSEX81.

    Science.gov (United States)

    Welschof, M; Little, M; Dörsam, H

    1998-01-01

    Human monoclonal antibodies (MAbs) are more suitable than MAbs of animal origin for clinical applications because of lower hypersensitivity reactions, less formation of circulating immune complexes and lower anti-immunoglobulin responses The classical production of human MAbs via the hybridoma technique or Epstein-Barr virus (EBV) transformation is limited by the instability of cell lines, low antibody production, and the problems of imununizing humans with certain antigens (1,2). A promising alternative 1s the production of human recombinant antibodies (3). Recombinant DNA technology has made it possible to clone human antibody genes in vectors and to generate antibody expression libraries (4-7). One approach has been to amplify and recombine the IgG repertoire of an "immunized" donor. This has been used to isolate several antibodies related to diseases (8,9). In order to obtain more universal antibody libraries the naive IgM repertoire of several "unimmunized" donors were pooled (10,12). The complexity of the combinatorial libraries has been further increased by creating the so-called "semisynthetic" antibody libraries (22-14).

  2. Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulates rejuvenation of human skin.

    Science.gov (United States)

    Kim, Yoon-Jin; Yoo, Sae Mi; Park, Hwan Hee; Lim, Hye Jin; Kim, Yu-Lee; Lee, Seunghee; Seo, Kwang-Won; Kang, Kyung-Sun

    2017-11-18

    Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) play an important role in cutaneous wound healing, and recent studies suggested that MSC-derived exosomes activate several signaling pathways, which are conducive in wound healing and cell growth. In this study, we investigated the roles of exosomes that are derived from USC-CM (USC-CM Exos) in cutaneous collagen synthesis and permeation. We found that USC-CM has various growth factors associated with skin rejuvenation. Our in vitro results showed that USC-CM Exos integrate in Human Dermal Fibroblasts (HDFs) and consequently promote cell migration and collagen synthesis of HDFs. Moreover, we evaluated skin permeation of USC-CM Exos by using human skin tissues. Results showed that Exo-Green labeled USC-CM Exos approached the outermost layer of the epidermis after 3 h and gradually approached the epidermis after 18 h. Moreover, increased expressions of Collagen I and Elastin were found after 3 days of treatment on human skin. The results showed that USC-CM Exos is absorbed into human skin, it promotes Collagen I and Elastin synthesis in the skin, which are essential to skin rejuvenation and shows the potential of USC-CM integration with the cosmetics or therapeutics. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. 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.

  4. Photodynamic toxicity of hematoporphyrin derivatives to human keratinocytes in culture.

    Science.gov (United States)

    Kappus, H; Reinhold, C; Artuc, M

    Human keratinocytes in culture were able to take up hematoporphyrin derivatives (HPDs) used during photodynamic chemotherapy of tumors. In the absence of light, HPDs showed no cytotoxic effects to keratinocytes. However, after irradiation with visible light, HPDs induced immediate cytotoxicity as measured by the neutral red uptake assay. On the other hand, cell attachment as measured by protein estimation was not affected. When the cells treated with HPDs and irradiated with light were cultured for a further 72 h, they partially lost their ability to attach to the collagen surface. Most of the cells remaining attached after 72 h were no longer viable following treatment with HPDs and light. All parameters measured depended on the intracellular concentration of HPDs used (7-50 ng/10(5) cells) and the time of irradiation (0-30 min). These results suggest that human keratinocytes are a good model to study cytotoxic effects of photodynamically active drugs. Further, keratinocytes were unable to recover after damage caused by HPDs and light.

  5. Oogenesis in cultures derived from adult human ovaries

    Directory of Open Access Journals (Sweden)

    Caudle Michael R

    2005-05-01

    Full Text Available Abstract Ten years ago, we reported that in adult human females the ovarian surface epithelium (OSE is a source of germ cells. Recently, we also demonstrated that new primary follicles are formed by assembly of oocytes with nests of primitive granulosa cells in the ovarian cortex. The components of the new primary follicles, primitive granulosa and germ cells, differentiated sequentially from the OSE, which arises from cytokeratin positive mesenchymal progenitor cells residing in the ovarian tunica albuginea. In the present study, we investigated the possibility that the oocytes and granulosa cells may differentiate in cultures derived from adult human ovaries. Cells were scrapped from the surface of ovaries and cultured for 5 to 6 days, in the presence or absence of estrogenic stimuli [phenol red (PhR]. The OSE cells cultured in the medium without PhR differentiated into small (15 micron cells of granulosa phenotype, and epithelial, neural, and mesenchymal type cells. In contrast, OSE cells cultured in the presence of PhR differentiated directly into large (180 micron cells of the oocyte phenotype. Such cells exhibited germinal vesicle breakdown, expulsion of the polar body, and surface expression of zona pellucida proteins, i.e. characteristics of secondary oocytes. These in vitro studies confirm our in vivo observations that in adult human ovaries, the OSE is a bipotent source of oocytes and granulosa cells. Development of numerous mature oocytes from adult ovarian stem cells in vitro offers new strategies for the egg preservation, IVF utilization, and treatment of female infertility. In addition, other clinical applications aiming to utilize stem cells, and basic stem cell research as well, may employ totipotent embryonic stem cells developing from fertilized oocytes.

  6. Sensible biological models to be exposed to VDT (Video Display Terminal) radiations in human male reproduction

    International Nuclear Information System (INIS)

    Tritto, J.; North, M.-O.; Laverdure, A.M.; Surbeck, J.

    1999-01-01

    Temperature and environmental effects, particularly endocrine disrupters and EMF radiations, are actively investigated in human and non-human reproduction experimental models. Sensitivity and specificity of the different cell types of the testes seminiferous tubules in animals and in human are evaluated, showing a specific responsiveness of spermatogonia (SPG) and resting pachytene spermatocytes (SPC). At 32 o C the 24 h short-term cultures of biopsies of normal human testis show an expected low occurrence of apoptotic SPG (1 %) that increases to 3,4 % in peer samples exposed to VDT for the same period, with the appearance of apoptotic SPC (4,6 %). In samples from a thermically-impaired testis of the same subject the apoptotic occurrence of SPG is 2,6 % with 15,4 % for SPC after 24 h cultures. After 24 h exposure to VDT the apoptotic score is 7,6 % for SPG and 18,5 % for SPC in thermically impaired peer samples. With EMF-bioshields the apoptotic score for SPG is 0,8 % in normal 2,2 % for SPG and 13,8 % for SPC in T-impaired peer-samples. NMRS of the cultures fluids show a proportional production of lactate, corresponding to the different degrees of histopathological impairment of the samples. IVOS (Integrated Visual Optic System) analysis of sperm samples from thermically-impaired, not-repaired and repaired testes exposed to VDT shows sensible variations on straightness (STR), linearity (LIN) and lateral head displacement (LHD) parameters. To evaluate the thermic and non-thermic potential bioeffects of VDT on human spermatogenesis the specificity, the sensitivity and the reproducibility of the biological models on one side and the specificity of the methodologies on the other side must be provided. (author)

  7. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific...... Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively....

  8. Goats display audience-dependent human-directed gazing behaviour in a problem-solving task.

    Science.gov (United States)

    Nawroth, Christian; Brett, Jemma M; McElligott, Alan G

    2016-07-01

    Domestication is an important factor driving changes in animal cognition and behaviour. In particular, the capacity of dogs to communicate in a referential and intentional way with humans is considered a key outcome of how domestication as a companion animal shaped the canid brain. However, the lack of comparison with other domestic animals makes general conclusions about how domestication has affected these important cognitive features difficult. We investigated human-directed behaviour in an 'unsolvable problem' task in a domestic, but non-companion species: goats. During the test, goats experienced a forward-facing or an away-facing person. They gazed towards the forward-facing person earlier and for longer and showed more gaze alternations and a lower latency until the first gaze alternation when the person was forward-facing. Our results provide strong evidence for audience-dependent human-directed visual orienting behaviour in a species that was domesticated primarily for production, and show similarities with the referential and intentional communicative behaviour exhibited by domestic companion animals such as dogs and horses. This indicates that domestication has a much broader impact on heterospecific communication than previously believed. © 2016 The Author(s).

  9. Bacterial metabolism of human polymorphonuclear leukocyte-derived arachidonic acid.

    Science.gov (United States)

    Sorrell, T C; Muller, M; Sztelma, K

    1992-05-01

    Evidence for transcellular bacterial metabolism of phagocyte-derived arachidonic acid was sought by exposing human blood polymorphonuclear leukocytes, prelabelled with [3H]arachidonic acid, to opsonized, stationary-phase Pseudomonas aeruginosa (bacteria-to-phagocyte ratio of 50:1) for 90 min at 37 degrees C. Control leukocytes were stimulated with the calcium ionophore A23187 (5 microM) for 5 min. Radiochromatograms of arachidonic acid metabolites, extracted from A23187-stimulated cultures and then separated by reverse-phase high-performance liquid chromatography, revealed leukotriene B4, its omega-oxidation products, and 5-hydroxy-eicosatetraenoic acid. In contrast, two major metabolite peaks, distinct from known polymorphonuclear leukocyte arachidonic acid products by high-performance liquid chromatography or by thin-layer chromatography, were identified in cultures of P. aeruginosa with [3H]arachidonic acid-labelled polymorphonuclear leukocytes. Respective chromatographic characteristics of these novel products were identical to those of two major metabolite peaks produced by incubation of stationary-phase P. aeruginosa with [3H]arachidonic acid. Production of the metabolites was dependent upon pseudomonal viability. UV spectral data were consistent with a conjugated diene structure. Metabolism of arachidonic acid by P. aeruginosa was not influenced by the presence of catalase, superoxide dismutase, nordihydroguaiaretic acid, ethanol, dimethyl sulfoxide, or ferrous ions but was inhibited by carbon monoxide, ketoconazole, and 1,2-epoxy-3,3,3-trichloropropane. Our data suggest that pseudomonal metabolism of polymorphonuclear leukocyte-derived arachidonic acid occurs during phagocytosis, probably by enzymatic epoxidation and hydroxylation via an oxygenase. By this means, potential proinflammatory effects of arachidonic acid or its metabolites may be modulated by P. aeruginosa at sites of infection in vivo.

  10. Preclinical and first-in-human evaluation of PRX-105, a PEGylated, plant-derived, recombinant human acetylcholinesterase-R

    International Nuclear Information System (INIS)

    Atsmon, Jacob; Brill-Almon, Einat; Nadri-Shay, Carmit; Chertkoff, Raul; Alon, Sari; Shaikevich, Dimitri; Volokhov, Inna; Haim, Kirsten Y.; Bartfeld, Daniel; Shulman, Avidor; Ruderfer, Ilya; Ben-Moshe, Tehila; Shilovitzky, Orit; Soreq, Hermona; Shaaltiel, Yoseph

    2015-01-01

    PRX-105 is a plant-derived recombinant version of the human ‘read-through’ acetylcholinesterase splice variant (AChE-R). Its active site structure is similar to that of the synaptic variant, and it displays the same affinity towards organophosphorus (OP) compounds. As such, PRX-105 may serve as a bio-scavenger for OP pesticides and chemical warfare agents. To assess its potential use in prophylaxis and treatment of OP poisoning we conducted several preliminary tests, reported in this paper. Intravenous (IV) PRX-105 was administered to mice either before or after exposure to an OP toxin. All mice who received an IV dose of 50 nmol/kg PRX-105, 2 min before being exposed to 1.33 × LD 50 and 1.5 × LD 50 of toxin and 10 min after exposure to 1.5 × LD 50 survived. The pharmacokinetic and toxicity profiles of PRX-105 were evaluated in mice and mini-pigs. Following single and multiple IV doses (50 to 200 mg/kg) no deaths occurred and no significant laboratory and histopathological changes were observed. The overall elimination half-life (t ½ ) in mice was 994 (± 173) min. Additionally, a first-in-human study, to assess the safety, tolerability and pharmacokinetics of the compound, was conducted in healthy volunteers. The t ½ in humans was substantially longer than in mice (average 26.7 h). Despite the small number of animals and human subjects who were assessed, the fact that PRX-105 exerts a protective and therapeutic effect following exposure to lethal doses of OP, its favorable safety profile and its relatively long half-life, renders it a promising candidate for treatment and prophylaxis against OP poisoning and warrants further investigation. - Highlights: • PRX-105 is a PEGylated plant-derived recombinant human acetylcholinesterase-R. • PRX-105 is a promising bio-scavenger for organophosphorous toxins at lethal doses. • PRX-105 was shown to protect animals both prophylactically and post-poisoning. • First-in-human study exhibited its safety

  11. Preclinical and first-in-human evaluation of PRX-105, a PEGylated, plant-derived, recombinant human acetylcholinesterase-R

    Energy Technology Data Exchange (ETDEWEB)

    Atsmon, Jacob [Clinical Research Center, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University (Israel); Brill-Almon, Einat; Nadri-Shay, Carmit; Chertkoff, Raul; Alon, Sari [Protalix Biotherapeutics, Science Park, Carmiel (Israel); Shaikevich, Dimitri; Volokhov, Inna; Haim, Kirsten Y. [Clinical Research Center, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University (Israel); Bartfeld, Daniel [Protalix Biotherapeutics, Science Park, Carmiel (Israel); Shulman, Avidor, E-mail: avidors@protalix.com [Protalix Biotherapeutics, Science Park, Carmiel (Israel); Ruderfer, Ilya; Ben-Moshe, Tehila; Shilovitzky, Orit [Protalix Biotherapeutics, Science Park, Carmiel (Israel); Soreq, Hermona [Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem (Israel); Shaaltiel, Yoseph [Protalix Biotherapeutics, Science Park, Carmiel (Israel)

    2015-09-15

    PRX-105 is a plant-derived recombinant version of the human ‘read-through’ acetylcholinesterase splice variant (AChE-R). Its active site structure is similar to that of the synaptic variant, and it displays the same affinity towards organophosphorus (OP) compounds. As such, PRX-105 may serve as a bio-scavenger for OP pesticides and chemical warfare agents. To assess its potential use in prophylaxis and treatment of OP poisoning we conducted several preliminary tests, reported in this paper. Intravenous (IV) PRX-105 was administered to mice either before or after exposure to an OP toxin. All mice who received an IV dose of 50 nmol/kg PRX-105, 2 min before being exposed to 1.33 × LD{sub 50} and 1.5 × LD{sub 50} of toxin and 10 min after exposure to 1.5 × LD{sub 50} survived. The pharmacokinetic and toxicity profiles of PRX-105 were evaluated in mice and mini-pigs. Following single and multiple IV doses (50 to 200 mg/kg) no deaths occurred and no significant laboratory and histopathological changes were observed. The overall elimination half-life (t{sub ½}) in mice was 994 (± 173) min. Additionally, a first-in-human study, to assess the safety, tolerability and pharmacokinetics of the compound, was conducted in healthy volunteers. The t{sub ½} in humans was substantially longer than in mice (average 26.7 h). Despite the small number of animals and human subjects who were assessed, the fact that PRX-105 exerts a protective and therapeutic effect following exposure to lethal doses of OP, its favorable safety profile and its relatively long half-life, renders it a promising candidate for treatment and prophylaxis against OP poisoning and warrants further investigation. - Highlights: • PRX-105 is a PEGylated plant-derived recombinant human acetylcholinesterase-R. • PRX-105 is a promising bio-scavenger for organophosphorous toxins at lethal doses. • PRX-105 was shown to protect animals both prophylactically and post-poisoning. • First-in-human study

  12. Merkel cells and Meissner's corpuscles in human digital skin display Piezo2 immunoreactivity.

    Science.gov (United States)

    García-Mesa, Y; García-Piqueras, J; García, B; Feito, J; Cabo, R; Cobo, J; Vega, J A; García-Suárez, O

    2017-12-01

    The transformation of mechanical energy into electrical signals is the first step in mechanotransduction in the peripheral sensory nervous system and relies on the presence of mechanically gated ion channels within specialized sensory organs called mechanoreceptors. Piezo2 is a vertebrate stretch-gated ion channel necessary for mechanosensitive channels in mammalian cells. Functionally, it is related to light touch, which has been detected in murine cutaneous Merkel cell-neurite complexes, Meissner-like corpuscles and lanceolate nerve endings. To the best of our knowledge, the occurrence of Piezo2 in human cutaneous mechanoreceptors has never been investigated. Here, we used simple and double immunohistochemistry to investigate the occurrence of Piezo2 in human digital glabrous skin. Piezo2 immunoreactivity was detected in approximately 80% of morphologically and immunohistochemically characterized (cytokeratin 20 + , chromogranin A + and synaptophisin + ) Merkel cells. Most of them were in close contact with Piezo2 - nerve fibre profiles. Moreover, the axon, but not the lamellar cells, of Meissner's corpuscles was also Piezo2 + , but other mechanoreceptors, i.e. Pacinian or Ruffini's corpuscles, were devoid of immunoreactivity. Piezo2 was also observed in non-nervous tissue, especially the basal keratinocytes, endothelial cells and sweat glands. The present results demonstrate the occurrence of Piezo2 in cutaneous sensory nerve formations that functionally work as slowly adapting (Merkel cells) and rapidly adapting (Meissner's corpuscles) low-threshold mechanoreceptors and are related to fine and discriminative touch but not to vibration or hard touch. These data offer additional insight into the molecular basis of mechanosensing in humans. © 2017 Anatomical Society.

  13. Human Digital Meissner Corpuscles Display Immunoreactivity for the Multifunctional Ion Channels Trpc6 and Trpv4.

    Science.gov (United States)

    Alonso-González, Paula; Cabo, Roberto; San José, Isabel; Gago, Angel; Suazo, Iván C; García-Suárez, Olivia; Cobo, Juan; Vega, José A

    2017-06-01

    Ion channels are at the basis of the sensory processes including mechanosensing. Some members of the transient receptor potential (TRP) ion channel superfamily have been proposed as mechanosensors, but their putative role in mechanotransduction is controversial. Among them there are TRP canonical 6 (TRPC6) and TRP vanilloid 4 (TRPV4) ion channels, which are known to cooperate in mechanical hyperalgesia. Here, we investigated the occurrence, distribution, and possible colocalization of TRPC6 and TRPV4 in human digital Meissner sensory corpuscles using immunohistochemistry and double immunofluorescence (associate with markers for specific corpuscular constituents). TRPC6 immunoreactivity was restricted to the axon of Meissner corpuscles, whereas TRPV4 was detected in the axon but also in the lamellar cells. Moreover, axonal colocalization of TRPV4 and TRPC6 was found in the digital Meissner corpuscles. Present results demonstrate for the first time the occurrence and colocalization of two ion channels candidates to mechanosensors in human cutaneous mechanoreceptors. The functional significance of these ion channels in that place remains to be clarified, but should be related to different properties of mechanosensitivity. Anat Rec, 300:1022-1031, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

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    Vanesa Alonso-Camino

    2013-01-01

    Full Text Available A human single-chain variable fragment (scFv antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs. The repertoire was fused to a first-generation T cell receptor ζ (TCRζ-based chimeric antigen receptor (CAR. We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2 bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR and the selection context (cell synapse, which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.

  15. Penetratin and derivatives acting as antifungal agents

    NARCIS (Netherlands)

    Masman, Marcelo F.; Rodriguez, Ana M.; Raimondi, Marcela; Zacchino, Susana A.; Luiten, Paul G. M.; Somlai, Csaba; Kortvelyesi, Tamas; Penke, Botond; Enriz, Ricardo D.

    The synthesis, in vitro evaluation, and conformational study of RQIKTWFQNRRMKWKK-NH(2) (penetratin) and related derivatives acting as antifungal agents are reported. Penetratin and some of its derivatives displayed antifungal activity against the human opportunistic pathogenic standardized ATCC

  16. Interaction of glucocorticoids and progesterone derivatives with human serum albumin.

    Science.gov (United States)

    Abboud, Rola; Akil, Mohammad; Charcosset, Catherine; Greige-Gerges, Hélène

    2017-10-01

    Glucocorticoids (GCs) and progesterone derivatives (PGDs) are steroid hormones with well-known biological activities. Their interaction with human serum albumin (HSA) may control their distribution. Their binding to albumin is poorly studied in literature. This paper deals with the interaction of a series of GCs (cortisol, cortisone, prednisolone, prednisone, 6-methylprednisolone and 9-fluorocortisol acetate) and PGDs (progesterone, hydroxylated PGDs, methylated PGDs and dydrogesterone) with HSA solution (pH 7.4) at molar ratios steroid to HSA varying from 0 to 10. Similar titrations were conducted using Trp aqueous solution. Fluorescence titration method and Fourier transform infrared spectroscopy (FTIR) are used. PGDs (except dydrogesterone), cortisone and 9-fluorocortisol acetate affected weakly the fluorescence of Trp in buffer solution while they decreased in a dose-dependent manner that of HSA. Their binding constants to HSA were then calculated. Moreover, displacement experiment was performed using bilirubin as a site marker. The binding constant of bilirubin to albumin was determined in the absence and presence of a steroid at a molar ratio steroid to HSA of 1. The results indicate that the steroids bind to HSA at site I in a pocket different from that of bilirubin. Furthermore, the peak positions of amide I and amide II bands of HSA were shifted in the presence of progesterone, dydrogesterone and GCs. Also a variation was observed in amide I region indicating the formation of hydrogen bonding between albumin and steroids. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Deriving human resource requirements for new nuclear plants

    International Nuclear Information System (INIS)

    Goodnight, Ch.T.

    2007-01-01

    For those contemplating the deployment of a new nuclear plant, critical issues include the development of the operational staff and its organization. This paper will discuss the key elements of deriving an appropriate staff plan for steady state operations. There are five areas that must be analyzed, and each area has unique requirements. These key areas are 1) Operations, 2) Engineering, 3) Maintenance, 4) Regulatory/Oversight, and 5) Site Support. After the analysis for each area is complete, and the human resource requirements are identified, an organizational structure must be developed to support the necessary management, potential centralization, and appropriate functional alignments for effective and safe plant operation. Four organizational design principals have been defined. The first design principal relates to the organizational structure: no more than 7 layers of management between an individual contributor and the senior nuclear manager in the NPP. The second design principal relates to the grouping of activities in order to ensure appropriate management of related or supporting activities. The third design principal relates to out-sourcing support activities when these activities comply with particular conditions for instance when they are not mission critical to day-to day plant operations. The fourth design principal relates to the centralization of some activities when there is more than one NPP operated by the same parent company

  18. Zika Virus Strains Potentially Display Different Infectious Profiles in Human Neural Cells

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    Yannick Simonin

    2016-10-01

    Full Text Available The recent Zika virus (ZIKV epidemic has highlighted the poor knowledge on its physiopathology. Recent studies showed that ZIKV of the Asian lineage, responsible for this international outbreak, causes neuropathology in vitro and in vivo. However, two African lineages exist and the virus is currently found circulating in Africa. The original African strain was also suggested to be neurovirulent but its laboratory usage has been criticized due to its multiple passages. In this study, we compared the French Polynesian (Asian ZIKV strain to an African strain isolated in Central African Republic and show a difference in infectivity and cellular response between both strains in human neural stem cells and astrocytes. Consistently, this African strain led to a higher infection rate and viral production, as well as stronger cell death and anti-viral response. Our results highlight the need to better characterize the physiopathology and predict neurological impairment associated with African ZIKV.

  19. Predicting the Impacts of Intravehicular Displays on Driving Performance with Human Performance Modeling

    Science.gov (United States)

    Mitchell, Diane Kuhl; Wojciechowski, Josephine; Samms, Charneta

    2012-01-01

    A challenge facing the U.S. National Highway Traffic Safety Administration (NHTSA), as well as international safety experts, is the need to educate car drivers about the dangers associated with performing distraction tasks while driving. Researchers working for the U.S. Army Research Laboratory have developed a technique for predicting the increase in mental workload that results when distraction tasks are combined with driving. They implement this technique using human performance modeling. They have predicted workload associated with driving combined with cell phone use. In addition, they have predicted the workload associated with driving military vehicles combined with threat detection. Their technique can be used by safety personnel internationally to demonstrate the dangers of combining distracter tasks with driving and to mitigate the safety risks.

  20. Hypoxia Epigenetically Confers Astrocytic Differentiation Potential on Human Pluripotent Cell-Derived Neural Precursor Cells

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    Tetsuro Yasui

    2017-06-01

    Full Text Available Human neural precursor cells (hNPCs derived from pluripotent stem cells display a high propensity for neuronal differentiation, but they require long-term culturing to differentiate efficiently into astrocytes. The mechanisms underlying this biased fate specification of hNPCs remain elusive. Here, we show that hypoxia confers astrocytic differentiation potential on hNPCs through epigenetic gene regulation, and that this was achieved by cooperation between hypoxia-inducible factor 1α and Notch signaling, accompanied by a reduction of DNA methylation level in the promoter region of a typical astrocyte-specific gene, Glial fibrillary acidic protein. Furthermore, we found that this hypoxic culture condition could be applied to rapid generation of astrocytes from Rett syndrome patient-derived hNPCs, and that these astrocytes impaired neuronal development. Thus, our findings shed further light on the molecular mechanisms regulating hNPC differentiation and provide attractive tools for the development of therapeutic strategies for treating astrocyte-mediated neurological disorders.

  1. Unique biological properties of catalytic domain directed human anti-CAIX antibodies discovered through phage-display technology.

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    Chen Xu

    2010-03-01

    Full Text Available Carbonic anhydrase IX (CAIX, gene G250/MN-encoded transmembrane protein is highly expressed in various human epithelial tumors such as renal clear cell carcinoma (RCC, but absent from the corresponding normal tissues. Besides the CA signal transduction activity, CAIX may serve as a biomarker in early stages of oncogenesis and also as a reliable marker of hypoxia, which is associated with tumor resistance to chemotherapy and radiotherapy. Although results from preclinical and clinical studies have shown CAIX as a promising target for detection and therapy for RCC, only a limited number of murine monoclonal antibodies (mAbs and one humanized mAb are available for clinical testing and development. In this study, paramagnetic proteoliposomes of CAIX (CAIX-PMPLs were constructed and used for anti-CAIX antibody selection from our 27 billion human single-chain antibody (scFv phage display libraries. A panel of thirteen human scFvs that specifically recognize CAIX expressed on cell surface was identified, epitope mapped primarily to the CA domain, and affinity-binding constants (KD determined. These human anti-CAIX mAbs are diverse in their functions including induction of surface CAIX internalization into endosomes and inhibition of the carbonic anhydrase activity, the latter being a unique feature that has not been previously reported for anti-CAIX antibodies. These human anti-CAIX antibodies are important reagents for development of new immunotherapies and diagnostic tools for RCC treatment as well as extending our knowledge on the basic structure-function relationships of the CAIX molecule.

  2. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    International Nuclear Information System (INIS)

    Holers, V.M.; Kotzin, B.L.

    1985-01-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases

  3. Human umbilical cord blood-derived stem cells and brain-derived neurotrophic factor protect injured optic nerve: viscoelasticity characterization

    Directory of Open Access Journals (Sweden)

    Xue-man Lv

    2016-01-01

    Full Text Available The optic nerve is a viscoelastic solid-like biomaterial. Its normal stress relaxation and creep properties enable the nerve to resist constant strain and protect it from injury. We hypothesized that stress relaxation and creep properties of the optic nerve change after injury. More-over, human brain-derived neurotrophic factor or umbilical cord blood-derived stem cells may restore these changes to normal. To validate this hypothesis, a rabbit model of optic nerve injury was established using a clamp approach. At 7 days after injury, the vitreous body re-ceived a one-time injection of 50 µg human brain-derived neurotrophic factor or 1 × 106 human umbilical cord blood-derived stem cells. At 30 days after injury, stress relaxation and creep properties of the optic nerve that received treatment had recovered greatly, with patho-logical changes in the injured optic nerve also noticeably improved. These results suggest that human brain-derived neurotrophic factor or umbilical cord blood-derived stem cell intervention promotes viscoelasticity recovery of injured optic nerves, and thereby contributes to nerve recovery.

  4. Immunotoxicity assessment of rice-derived recombinant human serum albumin using human peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Kai Fu

    Full Text Available Human serum albumin (HSA is extensively used in clinics to treat a variety of diseases, such as hypoproteinemia, hemorrhagic shock, serious burn injuries, cirrhotic ascites and fetal erythroblastosis. To address supply shortages and high safety risks from limited human donors, we recently developed recombinant technology to produce HSA from rice endosperm. To assess the risk potential of HSA derived from Oryza sativa (OsrHSA before a First-in-human (FIH trial, we compared OsrHSA and plasma-derived HSA (pHSA, evaluating the potential for an immune reaction and toxicity using human peripheral blood mononuclear cells (PBMCs. The results indicated that neither OsrHSA nor pHSA stimulated T cell proliferation at 1x and 5x dosages. We also found no significant differences in the profiles of the CD4(+ and CD8(+ T cell subsets between OsrHSA- and pHSA-treated cells. Furthermore, the results showed that there were no significant differences between OsrHSA and pHSA in the production of cytokines such as interferon-gamma (IFN-γ, tumor necrosis factor-alpha (TNF-α, interleukin (IL-10 and IL-4. Our results demonstrated that OsrHSA has equivalent immunotoxicity to pHSA when using the PBMC model. Moreover, this ex vivo system could provide an alternative approach to predict potential risks in novel biopharmaceutical development.

  5. Use of human factors in the integration of a safety parameter display system (SPDS) and emergency response facility (ERF) capabilities for the James A. Fitzpatrick nuclear power plant

    International Nuclear Information System (INIS)

    Fish, H.C. Jr.; Gutierrez, R.

    1987-01-01

    In 1987, New York Power Authority's Emergency and Process Information Computer (EPIC) System will be operational. The EPIC system was designed to perform two distinct functions: to serve as an advanced replacement for the existing plant process computer and to assist in providing the JAFNPP operations staff with information during plant emergency conditions. Besides meeting the operational and regulatory goals of NYPA, EPIC was designed to meet NYPA human factors criteria. Using human factors literature, industry standards and guidelines, a Human Factors Criteria document was prepared for the EPIC project. This document served as the bases for all hardware, display, and documentation design applicable to EPIC. The major tasks of the EPIC human factors plan include integration of past and present applicable human factors information, establishment of a user definition using data gathered from control room observations (startup and shutdown of JAFNPP), survey of computer programs used by the operators, and obtaining input from licensed operators. These major tasks played a role in the design of the design of the control room configuration, the design and development of computer generated displays, and the format in which the displayed and hard copy information would be presented to the user. This paper presents an overview of the human factors work performed and the documents researched or developed in the design of EPIC displays, software, and hardware. The location and operation of this new computer based information system, like the displays was based on the systematic application of operations and human factors principles

  6. Wnt5a Regulates the Assembly of Human Adipose Derived Stromal Vascular Fraction-Derived Microvasculatures.

    Directory of Open Access Journals (Sweden)

    Venkat M Ramakrishnan

    Full Text Available Human adipose-derived stromal vascular fraction (hSVF cells are an easily accessible, heterogeneous cell system that can spontaneously self-assemble into functional microvasculatures in vivo. However, the mechanisms underlying vascular self-assembly and maturation are poorly understood, therefore we utilized an in vitro model to identify potential in vivo regulatory mechanisms. We utilized passage one (P1 hSVF because of the rapid UEA1+ endothelium (EC loss at even P2 culture. We exposed hSVF cells to a battery of angiogenesis inhibitors and found that the pan-Wnt inhibitor IWP2 produced the most significant hSVF-EC networking decrease (~25%. To determine which Wnt isoform(s and receptor(s may be involved, hSVF was screened by PCR for isoforms associated with angiogenesis, with only WNT5A and its receptor, FZD4, being expressed for all time points observed. Immunocytochemistry confirmed Wnt5a protein expression by hSVF. To see if Wnt5a alone could restore IWP2-induced EC network inhibition, recombinant human Wnt5a (0-150 ng/ml was added to IWP2-treated cultures. The addition of rhWnt5a significantly increased EC network area and significantly decreased the ratio of total EC network length to EC network area compared to untreated controls. To determine if Wnt5a mediates in vivo microvascular self-assembly, 3D hSVF constructs containing an IgG isotype control, anti-Wnt5a neutralizing antibody or rhWnt5a were implanted subcutaneously for 2w in immune compromised mice. Compared to IgG controls, anti-Wnt5a treatment significantly reduced vessel length density by ~41%, while rhWnt5a significantly increased vessel length density by ~62%. However, anti-Wnt5a or rhWnt5a did not significantly affect the density of segments and nodes, both of which measure vascular complexity. Taken together, this data demonstrates that endogenous Wnt5a produced by hSVF plays a regulatory role in microvascular self-assembly in vivo. These findings also suggest that

  7. Engineering bacterial surface displayed human norovirus capsid proteins: A novel system to explore interaction between norovirus and ligands

    Directory of Open Access Journals (Sweden)

    Mengya eNiu

    2015-12-01

    Full Text Available Human noroviruses (HuNoVs are major contributors to acute nonbacterial gastroenteritis outbreaks. Many aspects of HuNoVs are poorly understood due to both the current inability to culture HuNoVs, and the lack of efficient small animal models. Surrogates for HuNoVs, such as recombinant viral like particles (VLPs expressed in eukaryotic system or P particles expressed in prokaryotic system, have been used for studies in immunology and interaction between the virus and its receptors. However, it is difficult to use VLPs or P particles to collect or isolate potential ligands binding to these recombinant capsid proteins. In this study, a new strategy was used to collect HuNoVs binding ligands through the use of ice nucleation protein (INP to display recombinant capsid proteins of HuNoVs on bacterial surfaces. The viral protein-ligand complex could be easily separated by a low speed centrifugation step. This system was also used to explore interaction between recombinant capsid proteins of HuNoVs and their receptors. In this system, the VP1 capsid encoding gene (ORF2 and the protruding domain (P domain encoding gene (3’ terminal fragment of ORF2 of HuNoVs GI.1 and GII.4 were fused with 5’ terminal fragment of ice nucleation protein encoding gene (inaQn. The results demonstrated that the recombinant VP1 and P domains of HuNoVs were expressed and anchored on the surface of Escherichia coli BL21 cells after the bacteria were transformed with the corresponding plasmids. Both cell surface displayed VP1 and P domains could be recognized by HuNoVs specific antibodies and interact with the viral histo-blood group antigens receptors. In both cases, displayed P domains had better binding abilities than VP1. This new strategy of using displayed HuNoVs capsid proteins on the bacterial surface could be utilized to separate HuNoVs binding components from complex samples, to investigate interaction between the virus and its receptors, as well as to develop an

  8. Functional and Pharmacological Analysis of Cardiomyocytes Differentiated from Human Peripheral Blood Mononuclear-Derived Pluripotent Stem Cells

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    Michael Riedel

    2014-07-01

    Full Text Available Advances in induced pluripotent stem cell (iPSC technology have set the stage for routine derivation of patient- and disease-specific human iPSC-cardiomyocyte (CM models for preclinical drug screening and personalized medicine approaches. Peripheral blood mononuclear cells (PBMCs are an advantageous source of somatic cells because they are easily obtained and readily amenable to transduction. Here, we report that the electrophysiological properties and pharmacological responses of PBMC-derived iPSC CM are generally similar to those of iPSC CM derived from other somatic cells, using patch-clamp, calcium transient, and multielectrode array (MEA analyses. Distinct iPSC lines derived from a single patient display similar electrophysiological features and pharmacological responses. Finally, we demonstrate that human iPSC CMs undergo acute changes in calcium-handling properties and gene expression in response to rapid electrical stimulation, laying the foundation for an in-vitro-tachypacing model system for the study of human tachyarrhythmias.

  9. Anticancer activities of bovine and human lactoferricin-derived peptides

    NARCIS (Netherlands)

    Arias, M.; Hilchie, A.L.; Haney, E.F.; Bolscher, J.G.M.; Hyndman, M.E.; Hancock, R.E.W.; Vogel, H.J.

    2017-01-01

    Lactoferrin (LF) is a mammalian host defense glycoprotein with diverse biological activities. Peptides derived from the cationic region of LF possess cytotoxic activity against cancer cells in vitro and in vivo. Bovine lactoferricin (LFcinB), a peptide derived from bovine LF (bLF), exhibits

  10. T lymphocytes derived from human cord blood provide effective antitumor immunotherapy against a human tumor

    Directory of Open Access Journals (Sweden)

    Kim Tae-Sik

    2011-06-01

    Full Text Available Abstract Background Although the graft-versus-tumor (GVT effect of donor-derived T cells after allogeneic hematopoietic stem cell transplantation has been used as an effective adoptive immunotherapy, the antitumor effects of cord blood (CB transplantation have not been well studied. Methods We established the animal model by transplantation of CB mononuclear cells and/or tumor cells into NOD/SCID mice. The presence of CB derived T cells in NOD/SCID mice or tumor tissues were determined by flow cytometric and immunohistochemical analysis. The anti-tumor effects of CB derived T cells against tumor was determined by tumor size and weight, and by the cytotoxicity assay and ELISPOT assay of T cells. Results We found dramatic tumor remission following transfer of CB mononuclear cells into NOD/SCID mice with human cervical tumors with a high infiltration of CD3+ T cells in tumors. NOD/SCID mice that receive neonatal CB transplants have reconstituted T cells with significant antitumor effects against human cervical and lung tumors, with a high infiltration of CD3+ T cells showing dramatic induction of apoptotic cell death. We also confirmed that T cells showed tumor specific antigen cytotoxicity in vitro. In adoptive transfer of CD3+ T cells into mice with pre-established tumors, we observed much higher antitumor effects of HPV-specific T cells by ELISPOT assays. Conclusions Our results show that CB derived T lymphocytes will be useful for novel immunotherapeutic candidate cells for therapy of several tumors in clinic.

  11. SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

    Directory of Open Access Journals (Sweden)

    Oliinyk O. S.

    2014-02-01

    Full Text Available Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, and 6 unique restriction patterns were found. Single-chain antibodies were expressed in Escherichia coli XL1-blue. The recombinant proteins were characterized by immunoblotting of bacterial extracts and detection with an anti-E-tag antibody. The toxin B-subunit-binding function of the single-chain antibody was shown by ELISA. The affinity constants for different clones were found to be from 106 to 108 М–1. Due to the fact, that these antibody fragments recognized epitopes in the receptor-binding Bsubunit of diphtheria toxin, further studies are interesting to evaluate their toxin neutralization properties and potential for therapeutic applications. Obtained scFv-antibodies can also be used for detection and investigation of biological properties of diphtheria toxin.

  12. Human isolates of Salmonella enterica serovar Typhimurium from Taiwan displayed significantly higher levels of antimicrobial resistance than those from Denmark.

    Science.gov (United States)

    Torpdahl, Mia; Lauderdale, Tsai-Ling; Liang, Shiu-Yun; Li, Ishien; Wei, Sung-Hsi; Chiou, Chien-Shun

    2013-02-01

    Salmonella enterica serovar Typhimurium is a major zoonotic pathogen with a high prevalence of antimicrobial resistance. This pathogen can disseminate across borders and spread far distances via the food trade and international travel. In this study, we compared the genotypes and antimicrobial resistance of 378 S. Typhimurium isolates collected in Taiwan and Denmark between 2009 and 2010. Genotyping revealed that many S. Typhimurium strains were concurrently circulating in Taiwan, Denmark and other countries in 2009 and 2010. When compared to the isolates collected from Denmark, the isolates from Taiwan displayed a significantly higher level of resistance to 11 of the 12 tested antimicrobials. Seven genetic clusters (A-G) were designated for the isolates. A high percentage of the isolates in genetic clusters C, F and G were multidrug-resistant. Of the isolates in cluster C, 79.2% were ASSuT-resistant, characterized by resistance to ampicillin, streptomycin, sulfamethoxazole, and tetracycline. In cluster F, 84.1% of the isolates were ACSSuT-resistant (resistant to ASSuT and chloramphenicol). Cluster G was unique to Taiwan and characterized in most isolates by the absence of three VNTRs (ST20, ST30 and STTR6) as well as a variety of multidrug resistance profiles. This cluster exhibited very high to extremely high levels of resistance to several first-line drugs, and among the seven clusters, it displayed the highest levels of resistance to cefotaxime and ceftazidime, ciprofloxacin and gentamicin. The high prevalence of antimicrobial resistance in S. Typhimurium from Taiwan highlights the necessity to strictly regulate the use of antimicrobials in the agriculture and human health care sectors. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Growth, development, reproduction, physiological and behavioural studies on living organisms, human adults and children exposed to radiation from video displays

    International Nuclear Information System (INIS)

    Laverdure, A.M.; Surbeck, J.; North, M.O.; Tritto, J.

    2001-01-01

    Various living organisms, human workers and children were tested for any biological action resulting from exposure to radiation from video display terminals (VDTs). VDTs were powered by a 50-Hz alternating voltage of 220 V. Measured electric and magnetic fields were 13 V/M and 50 nT, respectively. Living organisms were maintained under their normal breeding conditions and control values were obtained before switching on the VDT. Various effects related to the irradiation time were demonstrated, i.e. growth delay in algae and Drosophila, a body weight deficiency in rats, abnormal peaks of mortality in Daphnia and Drosophila, teratological effects in chick embryos and behavioural disturbances in rats. The embryonic and neonatal periods showed a high sensitivity to the VDT radiation. In humans, after 4 h of working in front of a VDT screen, an increase in tiredness and a decrease in the resistance of the immune system were observed in workers. In prepubertal children, 20 min of exposure were sufficient to induce neuropsychological disturbances; pre-pubertal young people appear to be particularly sensitive to the effect of the radiation. In human testicular biopsies cultured in vitro for 24 h in front of a VDT screen, mitotic and meiotic disturbances, the appearance of degeneration in some aspects of the cells and significant disorganisation of the seminiferous tubules were demonstrated and related to modification of the metabolism of the sample. An experimental apparatus has been developed and tested that aims to prevent the harm from VDT radiation. Known commercially as the 'emf-Bioshield', it ensures effective protection against harmful biological effects of VDT radiation. (author)

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

    DEFF Research Database (Denmark)

    Harkness, Linda; Kassem, Moustapha

    2012-01-01

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

  15. Generation and characterization of human smooth muscle cell lines derived from atherosclerotic plaque.

    Science.gov (United States)

    Bonin, L R; Madden, K; Shera, K; Ihle, J; Matthews, C; Aziz, S; Perez-Reyes, N; McDougall, J K; Conroy, S C

    1999-03-01

    The study of atherogenesis in humans has been restricted by the limited availability and brief in vitro life span of plaque smooth muscle cells (SMCs). We describe plaque SMC lines with extended life spans generated by the expression of the human papillomavirus (HPV)-16 E6 and E7 genes, which has been shown to extend the life span of normal adult human aortic SMCs. Resulting cell lines (pdSMC1A and 2) demonstrated at least 10-fold increases in life span; pdSMC1A became immortal. The SMC identity of both pdSMC lines was confirmed by SM22 mRNA expression. pdSMC2 were generally diploid but with various structural and numerical alterations; pdSMC1A demonstrated several chromosomal abnormalities, most commonly -Y, +7, -13, anomalies previously reported in both primary pdSMCs and atherosclerotic tissue. Confluent pdSMC2 appeared grossly similar to HPV-16 E6/E7-expressing normal adult aortic SMCs (AASMCs), exhibiting typical SMC morphology/growth patterns; pdSMC1A displayed irregular cell shape/organization with numerous mitotic figures. Dedifferentiation to a synthetic/proliferative phenotype has been hypothesized as a critical step in atherogenesis, because rat neonatal SMCs and adult intimal SMCs exhibit similar gene expression patterns. To confirm that our pdSMC lines likewise express this apparent plaque phenotype, osteopontin, platelet-derived growth factor B, and elastin mRNA levels were determined in pdSMC1A, pdSMC2, and AASMCs. However, no significant increases in osteopontin or platelet-derived growth factor B expression levels were observed in either pdSMC compared with AASMCs. pdSMC2 alone expressed high levels of elastin mRNA. Lower levels of SM22 mRNA in pdSMC1A suggested greater dedifferentiation and/or additional population doublings in pdSMC1A relative to pdSMC2. Both pdSMC lines (particularly 1A) demonstrated high message levels for matrix Gla protein, previously reported to be highly expressed by human neointimal SMCs in vitro. These results describe 2

  16. Human Adipose-Derived Stem Cells on Rapid Prototyped Three-Dimensional Hydroxyapatite/Beta-Tricalcium Phosphate Scaffold.

    Science.gov (United States)

    Canciani, Elena; Dellavia, Claudia; Ferreira, Lorena Maria; Giannasi, Chiara; Carmagnola, Daniela; Carrassi, Antonio; Brini, Anna Teresa

    2016-05-01

    In the study, we assess a rapid prototyped scaffold composed of 30/70 hydroxyapatite (HA) and beta-tricalcium-phosphate (β-TCP) loaded with human adipose-derived stem cells (hASCs) to determine cell proliferation, differentiation toward osteogenic lineage, adhesion and penetration on/into the scaffold.In this in vitro study, hASCs isolated from fat tissue discarded after plastic surgery were expanded, characterized, and then loaded onto the scaffold. Cells were tested for: viability assay (Alamar Blue at days 3, 7 and Live/Dead at day 32), differentiation index (alkaline phosphatase activity at day 14), scaffold adhesion (standard error of the mean analysis at days 5 and 18), and penetration (ground sections at day 32).All the hASC populations displayed stemness markers and the ability to differentiate toward adipogenic and osteogenic lineages.Cellular vitality increased between 3 and 7 days, and no inhibitory effect by HA/β-TCP was observed. Under osteogenic stimuli, scaffold increased alkaline phosphatase activity of +243% compared with undifferentiated samples. Human adipose-derived stem cells adhered on HA/β-TCP surface through citoplasmatic extensions that occupied the macropores and built networks among them. Human adipose derived stem cells were observed in the core of HA/β-TCP. The current combination of hASCs and HA/β-TCP scaffold provided encouraging results. If authors' data will be confirmed in preclinical models, the present engineering approach could represent an interesting tool in treating large bone defects.

  17. Characterization of Human Knee and Chin Adipose-Derived Stromal Cells

    Directory of Open Access Journals (Sweden)

    Magali Kouidhi

    2015-01-01

    Full Text Available Animal study findings have revealed that individual fat depots are not functionally equivalent and have different embryonic origins depending on the anatomic location. Mouse bone regeneration studies have also shown that it is essential to match the Hox code of transplanted cells and host tissues to achieve correct repair. However, subcutaneous fat depots from any donor site are often used in autologous fat grafting. Our study was thus carried out to determine the embryonic origins of human facial (chin and limb (knee fat depots and whether they had similar features and molecular matching patterns. Paired chin and knee fat depots were harvested from 11 subjects and gene expression profiles were determined by DNA microarray analyses. Adipose-derived stromal cells (ASCs from both sites were isolated and analyzed for their capacity to proliferate, form clones, and differentiate. Chin and knee fat depots expressed a different HOX code and could have different embryonic origins. ASCs displayed a different phenotype, with chin-ASCs having the potential to differentiate into brown-like adipocytes, whereas knee-ASCs differentiated into white adipocytes. These results highlighted different features for these two fat sites and indicated that donor site selection might be an important factor to be considered when applying adipose tissue in cell-based therapies.

  18. Graphene Sheet-Induced Global Maturation of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Wang, Jiaxian; Cui, Chang; Nan, Haiyan; Yu, Yuanfang; Xiao, Yini; Poon, Ellen; Yang, Gang; Wang, Xijie; Wang, Chenchen; Li, Lingsong; Boheler, Kenneth Richard; Ma, Xu; Cheng, Xin; Ni, Zhenhua; Chen, Minglong

    2017-08-09

    Human induced pluripotent stem cells (hiPSCs) can proliferate infinitely. Their ability to differentiate into cardiomyocytes provides abundant sources for disease modeling, drug screening and regenerative medicine. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) display a low degree of maturation and fetal-like properties. Current in vitro differentiation methods do not mimic the structural, mechanical, or physiological properties of the cardiogenesis niche. Recently, we present an efficient cardiac maturation platform that combines hiPSCs monolayer cardiac differentiation with graphene substrate, which is a biocompatible and superconductive material. The hiPSCs lines were successfully maintained on the graphene sheets and were able to differentiate into functional cardiomyocytes. This strategy markedly increased the myofibril ultrastructural organization, elevated the conduction velocity, and enhanced both the Ca 2+ handling and electrophysiological properties in the absence of electrical stimulation. On the graphene substrate, the expression of connexin 43 increased along with the conduction velocity. Interestingly, the bone morphogenetic proteins signaling was also significantly activated during early cardiogenesis, confirmed by RNA sequencing analysis. Here, we reasoned that graphene substrate as a conductive biomimetic surface could facilitate the intrinsic electrical propagation, mimicking the microenvironment of the native heart, to further promote the global maturation of hiPSC-CMs. Our findings highlight the capability of electrically active substrates to influence cardiomyocyte development. We believe that application of graphene sheets will be useful for simple, fast, and scalable maturation of regenerated cardiomyocytes.

  19. Automated Annotation of Microbial and Human Flavonoid-Derived Metabolites

    NARCIS (Netherlands)

    Mihaleva, V.V.; Ünlü, F.; Vervoort, J.J.M.; Ridder, L.O.

    2015-01-01

    Flavonoids are a class of natural compounds essentially produced by plants that are part of animal and human diets and have assumed health-promoting benefits. Upon human consumption, these flavonoids are to a modest extent absorbed in the small intestines. The major part arrives in the colon where

  20. 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.

  1. Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

    Science.gov (United States)

    Bidlingmaier, Scott; Su, Yang; Liu, Bin

    2015-01-01

    Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

  2. Human Induced Pluripotent Stem Cell-Derived Macrophages for Unraveling Human Macrophage Biology.

    Science.gov (United States)

    Zhang, Hanrui; Reilly, Muredach P

    2017-11-01

    Despite a substantial appreciation for the critical role of macrophages in cardiometabolic diseases, understanding of human macrophage biology has been hampered by the lack of reliable and scalable models for cellular and genetic studies. Human induced pluripotent stem cell (iPSC)-derived macrophages (IPSDM), as an unlimited source of subject genotype-specific cells, will undoubtedly play an important role in advancing our understanding of the role of macrophages in human diseases. In this review, we summarize current literature in the differentiation and characterization of IPSDM at phenotypic, functional, and transcriptomic levels. We emphasize the progress in differentiating iPSC to tissue resident macrophages, and in understanding the ontogeny of in vitro differentiated IPSDM that resembles primitive hematopoiesis, rather than adult definitive hematopoiesis. We review the application of IPSDM in modeling both Mendelian genetic disorders and host-pathogen interactions. Finally, we highlighted the potential areas of research using IPSDM in functional validation of coronary artery disease loci in genome-wide association studies, functional genomic analyses, drug testing, and cell therapeutics in cardiovascular diseases. © 2017 American Heart Association, Inc.

  3. Neurite outgrowth in human iPSC-derived neurons

    Data.gov (United States)

    U.S. Environmental Protection Agency — Data on morphology of rat and human neurons in cell culture. This dataset is associated with the following publication: Druwe, I., T. Freudenrich , K. Wallace , T....

  4. Transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into rat spinal cord injuries does not cause harm.

    Science.gov (United States)

    Cloutier, Frank; Siegenthaler, Monica M; Nistor, Gabriel; Keirstead, Hans S

    2006-07-01

    Demyelination contributes to loss of function following spinal cord injury. We have shown previously that transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into adult rat 200 kD contusive spinal cord injury sites enhances remyelination and promotes recovery of motor function. Previous studies using oligodendrocyte lineage cells have noted a correlation between the presence of demyelinating pathology and the survival and migration rate of the transplanted cells. The present study compared the survival and migration of human embryonic stem cell-derived oligodendrocyte progenitors injected 7 days after a 200 or 50 kD contusive spinal cord injury, as well as the locomotor outcome of transplantation. Our findings indicate that a 200 kD spinal cord injury induces extensive demyelination, whereas a 50 kD spinal cord injury induces no detectable demyelination. Cells transplanted into the 200 kD injury group survived, migrated, and resulted in robust remyelination, replicating our previous studies. In contrast, cells transplanted into the 50 kD injury group survived, exhibited limited migration, and failed to induce remyelination as demyelination in this injury group was absent. Animals that received a 50 kD injury displayed only a transient decline in locomotor function as a result of the injury. Importantly, human embryonic stem cell-derived oligodendrocyte progenitor transplants into the 50 kD injury group did not cause a further decline in locomotion. Our studies highlight the importance of a demyelinating pathology as a prerequisite for the function of transplanted myelinogenic cells. In addition, our results indicate that transplantation of human embryonic stem cell-derived oligodendrocyte progenitor cells into the injured spinal cord is not associated with a decline in locomotor function.

  5. Diploid, but not haploid, human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

    Science.gov (United States)

    Fan, Yong; Li, Rong; Huang, Jin; Yu, Yang; Qiao, Jie

    2013-01-01

    Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells. PMID:23255130

  6. Circulating Red Cell–derived Microparticles in Human Malaria

    Science.gov (United States)

    Nantakomol, Duangdao; Dondorp, Arjen M.; Krudsood, Srivicha; Udomsangpetch, Rachanee; Pattanapanyasat, Kovit; Combes, Valery; Grau, Georges E.; White, Nicholas J.; Viriyavejakul, Parnpen; Day, Nicholas P.J.

    2011-01-01

    In patients with falciparum malaria, plasma concentrations of cell-derived microparticles correlate with disease severity. Using flow cytometry, we quantified red blood cell–derived microparticles (RMPs) in patients with malaria and identified the source and the factors associated with production. RMP concentrations were increased in patients with Plasmodium falciparum (n = 29; median, 457 RMPs/μL [range, 13–4,342 RMPs/μL]), Plasmodium vivax (n = 5; median, 409 RMPs/μL [range, 281–503/μL]), and Plasmodium malariae (n = 2; median, 163 RMPs/μL [range, 127–200 RMPs/μL]) compared with those in healthy subjects (n = 11; median, 8 RMPs/μL [range, 3–166 RMPs/μL]; P = .01). RMP concentrations were highest in patients with severe falciparum malaria (P = .01). Parasitized red cells produced >10 times more RMPs than did unparasitized cells, but the overall majority of RMPs still derived from uninfected red blood cells (URBCs). In cultures, RMP production increased as the parasites matured. Hemin and parasite products induced RMP production in URBCs, which was inhibited by N-acetylcysteine, suggesting heme-mediated oxidative stress as a pathway for the generation of RMPs. PMID:21282195

  7. Circulating red cell-derived microparticles in human malaria.

    Science.gov (United States)

    Nantakomol, Duangdao; Dondorp, Arjen M; Krudsood, Srivicha; Udomsangpetch, Rachanee; Pattanapanyasat, Kovit; Combes, Valery; Grau, Georges E; White, Nicholas J; Viriyavejakul, Parnpen; Day, Nicholas P J; Chotivanich, Kesinee

    2011-03-01

    In patients with falciparum malaria, plasma concentrations of cell-derived microparticles correlate with disease severity. Using flow cytometry, we quantified red blood cell-derived microparticles (RMPs) in patients with malaria and identified the source and the factors associated with production. RMP concentrations were increased in patients with Plasmodium falciparum (n = 29; median, 457 RMPs/μL [range, 13-4,342 RMPs/μL]), Plasmodium vivax (n = 5; median, 409 RMPs/μL [range, 281-503/μL]), and Plasmodium malariae (n = 2; median, 163 RMPs/μL [range, 127-200 RMPs/μL]) compared with those in healthy subjects (n = 11; median, 8 RMPs/μL [range, 3-166 RMPs/μL]; P = .01). RMP concentrations were highest in patients with severe falciparum malaria (P = .01). Parasitized red cells produced >10 times more RMPs than did unparasitized cells, but the overall majority of RMPs still derived from uninfected red blood cells (URBCs). In cultures, RMP production increased as the parasites matured. Hemin and parasite products induced RMP production in URBCs, which was inhibited by N-acetylcysteine, suggesting heme-mediated oxidative stress as a pathway for the generation of RMPs.

  8. Generation of a transplantable erythropoietin-producer derived from human mesenchymal stem cells.

    Science.gov (United States)

    Yokoo, Takashi; Fukui, Akira; Matsumoto, Kei; Ohashi, Toya; Sado, Yoshikazu; Suzuki, Hideaki; Kawamura, Tetsuya; Okabe, Masataka; Hosoya, Tatsuo; Kobayashi, Eiji

    2008-06-15

    Differentiation of autologous stem cells into functional transplantable tissue for organ regeneration is a promising regenerative therapeutic approach for cancer, diabetes, and many human diseases. Yet to be established, however, is differentiation into tissue capable of producing erythropoietin (EPO), which has a critical function in anemia. We report a novel EPO-producing organ-like structure (organoid) derived from human mesenchymal stem cells. Using our previously established relay culture system, a human mesenchymal stem cell-derived, human EPO-competent organoid was established in rat omentum. The organoid-derived levels of human EPO increased in response to anemia induced by rapid blood withdrawal. In addition, the presence of an organoid in rats suppressed for native (rat) EPO production enhanced recovery from anemia when compared with control animals lacking the organoid. Together these results confirmed the generation of a stem cell-derived organoid that is capable of producing EPO and sensitive to physiological regulation.

  9. Preliminary Investigation of Visual Attention to Human Figures in Photographs: Potential Considerations for the Design of Aided AAC Visual Scene Displays

    Science.gov (United States)

    Wilkinson, Krista M.; Light, Janice

    2011-01-01

    Purpose: Many individuals with complex communication needs may benefit from visual aided augmentative and alternative communication systems. In visual scene displays (VSDs), language concepts are embedded into a photograph of a naturalistic event. Humans play a central role in communication development and might be important elements in VSDs.…

  10. An anti-tumor protein produced by Trichinella spiralis and identified by screening a T7 phage display library, induces apoptosis in human hepatoma H7402 cells

    Science.gov (United States)

    Trichinella spiralis infection confers effective resistance to tumor cell expansion. In this study, a T7 phage cDNA display library was constructed to express genes encoded by T. spiralis. Organic phase multi-cell screening was used to sort through candidate proteins in a transfected human chronic m...

  11. Bacterial surface-displayed GII.4 human norovirus capsid proteins bound to surface of Romaine lettuce through HBGA-like molecules

    Science.gov (United States)

    Human Noroviruses (HuNoVs) are the main cause of nonbacterial gastroenteritis. Contaminated produce is a main vehicle for dissemination of HuNoVs. In this study, we used an ice nucleation protein (INP) mediated surface display system to present the protruding domain of GII.4 HuNoV capsid protein (G...

  12. Mice with missense and nonsense NF1 mutations display divergent phenotypes compared with human neurofibromatosis type I

    Directory of Open Access Journals (Sweden)

    Kairong Li

    2016-07-01

    Full Text Available Neurofibromatosis type 1 (NF1 is a common genetic disorder characterized by the occurrence of nerve sheath tumors and considerable clinical heterogeneity. Some translational studies have been limited by the lack of animal models available for assessing patient-specific mutations. In order to test therapeutic approaches that might restore function to the mutated gene or gene product, we developed mice harboring NF1 patient-specific mutations including a nonsense mutation (c.2041C>T; p.Arg681* and a missense mutation (c.2542G>C; p.Gly848Arg. The latter is associated with the development of multiple plexiform neurofibromas along spinal nerve roots. We demonstrate that the human nonsense NF1Arg681* and missense NF1Gly848Arg mutations have different effects on neurofibromin expression in the mouse and each recapitulates unique aspects of the NF1 phenotype, depending upon the genetic context when assessed in the homozygous state or when paired with a conditional knockout allele. Whereas the missense Nf1Gly848Arg mutation fails to produce an overt phenotype in the mouse, animals homozygous for the nonsense Nf1Arg681* mutation are not viable. Mice with one Nf1Arg681* allele in combination with a conditional floxed Nf1 allele and the DhhCre transgene (Nf14F/Arg681*; DhhCre display disorganized nonmyelinating axons and neurofibromas along the spinal column, which leads to compression of the spinal cord and paralysis. This model will be valuable for preclinical testing of novel nonsense suppression therapies using drugs to target in-frame point mutations that create premature termination codons in individuals with NF1.

  13. Anti-aging effects of vitamin C on human pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Kim, Yoon Young; Ku, Seung-Yup; Huh, Yul; Liu, Hung-Ching; Kim, Seok Hyun; Choi, Young Min; Moon, Shin Yong

    2013-10-01

    Human pluripotent stem cells (hPSCs) have arisen as a source of cells for biomedical research due to their developmental potential. Stem cells possess the promise of providing clinicians with novel treatments for disease as well as allowing researchers to generate human-specific cellular metabolism models. Aging is a natural process of living organisms, yet aging in human heart cells is difficult to study due to the ethical considerations regarding human experimentation as well as a current lack of alternative experimental models. hPSC-derived cardiomyocytes (CMs) bear a resemblance to human cardiac cells and thus hPSC-derived CMs are considered to be a viable alternative model to study human heart cell aging. In this study, we used hPSC-derived CMs as an in vitro aging model. We generated cardiomyocytes from hPSCs and demonstrated the process of aging in both human embryonic stem cell (hESC)- and induced pluripotent stem cell (hiPSC)-derived CMs. Aging in hESC-derived CMs correlated with reduced membrane potential in mitochondria, the accumulation of lipofuscin, a slower beating pattern, and the downregulation of human telomerase RNA (hTR) and cell cycle regulating genes. Interestingly, the expression of hTR in hiPSC-derived CMs was not significantly downregulated, unlike in hESC-derived CMs. In order to delay aging, vitamin C was added to the cultured CMs. When cells were treated with 100 μM of vitamin C for 48 h, anti-aging effects, specifically on the expression of telomere-related genes and their functionality in aging cells, were observed. Taken together, these results suggest that hPSC-derived CMs can be used as a unique human cardiomyocyte aging model in vitro and that vitamin C shows anti-aging effects in this model.

  14. Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions

    Directory of Open Access Journals (Sweden)

    Sandy Azzi

    2015-06-01

    Full Text Available Intrarenal interleukin-15 (IL-15 participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15 isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC to peritumoral (ptumTEC, tumoral (RCC, and cancer stem cells (CSC/CD105+. RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15 isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα. This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105+, where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105+ from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, “apparently normal” ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral “preneoplastic” environment committed to favor tumor progression.

  15. Data of methylome and transcriptome derived from human dilated cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Bong-Seok Jo

    2016-12-01

    Full Text Available Alterations in DNA methylation and gene expression have been implicated in the development of human dilated cardiomyopathy (DCM. Differentially methylated probes (DMPs and differentially expressed genes (DEGs were identified between the left ventricle (LV, a pathological locus for DCM and the right ventricle (RV, a proxy for normal hearts. The data in this DiB are for supporting our report entitled “Methylome analysis reveals alterations in DNA methylation in the regulatory regions of left ventricle development genes in human dilated cardiomyopathy” (Bong-Seok Jo, In-Uk Koh, Jae-Bum Bae, Ho-Yeong Yu, Eun-Seok Jeon, Hae-Young Lee, Jae-Joong Kim, Murim Choi, Sun Shim Choi, 2016 [1].

  16. SLS-Derived Lab: Precursor to Deep Space Human Exploration

    Science.gov (United States)

    Griffin, Brand; Lewis, Ruthan; Eppler, Dean; Smitherman, David

    2014-01-01

    Plans to send humans to Mars are in work and the launch system is being built. Are we ready? Robotic missions have successfully demonstrated transportation, entry, landing and surface operations but for human missions there are significant, potentially show-stopping issues. These issues, called Strategic Knowledge Gaps (SKGs) are the unanswered questions concerning long-duration exploration beyond low-earth-orbit. The gaps represent a risk of loss of life or mission and because they require extended exposure to the weightless environment outside earth's protective geo-magnetic field they cannot be resolved on the earth or on the International Space Station (ISS). Placing a laboratory at the relatively close and stable lunar Distant Retrograde Orbit (DRO) provides an accessible location with the requisite environmental conditions for conducting SKG research and testing mitigation solutions. Configurations comprised of multiple 3 meter and 4.3 meter diameter modules have been studied but the most attractive solution uses elements of the human Mars launch vehicle or Space Launch System (SLS) for a Mars proving ground laboratory. A shortened version of an SLS hydrogen propellant tank creates a Skylab-like pressure vessel that flies fully outfitted on a single launch. This not only offers significant savings by incorporating SLS pressure vessel development costs but avoids the expensive ISS approach using many launches with substantial on-orbit assembly before becoming operational. One of the most challenging SKGs is crew radiation protection; this is why SKG laboratory research is combined with Mars transit Habitat systems development. Fundamentally, the two cannot be divorced because using the habitat systems for protection requires actual hardware geometry and material properties intended to contribute to shielding effectiveness. The SKGs are difficult problems, solutions are not obvious, and require integrated, iterative, and multi-disciplinary development. A lunar

  17. Development of human factors experimental evaluation techniques - Human factors design and evaluation of the main control room of atomic power plants using visual display terminals

    Energy Technology Data Exchange (ETDEWEB)

    Han, Sung Ho; Chung, Min Kyun; Choi, Kyung Lim; Song, Young Woong; Eoh, Hong Joon; Lee, In Suk; Kim, Bom Soo; Yeo, Yun Shin; Lee, Haeo Sun [Pohang University of Science and Technology, Pohang (Korea, Republic of)

    1995-07-01

    This study was conducted to build a prototype of the operator interface on a visual display terminal, and to provide a framework for using and applying prototyping techniques. A typical subsystem of an MCR was prototyped and validated by operator testing. Lessons learned during the development as well as prototyping techniques were described in this report for an efficient development. In addition, human factors experimental plans were surveyed and summarized for evaluating new design alternatives as well as the current design of the operator interface. The major results of this study are listed as follow: A method for designing an operator interface prototype on a VDT, A prototype of the operator interface of a typical subsystem, Guidelines for applying prototyping techniques, Characteristics and major considerations of experimental plans, Guidelines for applying prototyping techniques, Characteristics and major considerations of experimental plans, Guidelines for analyzing experimental data, A paradigm for human factors experimentation including experimental designs, procedures, and data analyses. 27 refs., 60 tabs., 47 figs. (author)

  18. Handbook of Visual Display Technology

    CERN Document Server

    Cranton, Wayne; Fihn, Mark

    2012-01-01

    The Handbook of Visual Display Technology is a unique work offering a comprehensive description of the science, technology, economic and human interface factors associated with the displays industry. An invaluable compilation of information, the Handbook will serve as a single reference source with expert contributions from over 150 international display professionals and academic researchers. All classes of display device are covered including LCDs, reflective displays, flexible solutions and emissive devices such as OLEDs and plasma displays, with discussion of established principles, emergent technologies, and particular areas of application. The wide-ranging content also encompasses the fundamental science of light and vision, image manipulation, core materials and processing techniques, display driving and metrology.

  19. Isotope derivative assay of human serum bile acids

    International Nuclear Information System (INIS)

    Pageaux, J.F.; Duperray, B.; Dubois, M.; Pacheco, H.

    1981-01-01

    A new method for the selective determination of the main serum bile acids has been developed. Serum samples with added 14 C-labeled bile acid were submitted to deproteinization, alkaline hydrolysis, methylation, and were then chromatographed on alumina before acetylation with 2 microliters of [ 3 H]acetic anhydride. Excess reagent was eliminated by evaporation; elimination of residual tritiated contaminants and separation of the doubly labeled bile acid derivatives were obtained by thin-layer chromatography, column chromatography on Lipidex 5000, and crystallization. The sensitivity of the method is about 10 pmol of each bile acid. Analyses of seven sera with normal or elevated concentration of bile acids by the proposed method and gas-liquid chromatography showed a close correlation

  20. An Experimental Investigation of Improving Human Problem-Solving Performance by Guiding Attention and Adaptively Proving Details on Information Displays

    National Research Council Canada - National Science Library

    Narayanan, N. H

    2007-01-01

    .... Results showed that various display strategies for augmenting information presented based on knowledge about both the viewer's gaze patterns and the problem solving procedure he or she is employing could indeed improve problem-solving performance.

  1. An Experimental Investigation of Improving Human Problem-Solving Performance by Guiding Attention and Adaptively Providing Details on Information Displays

    National Research Council Canada - National Science Library

    Narayanan, N. H

    2007-01-01

    .... Results showed that various display strategies for augmenting information presented based on knowledge about both the viewer's gaze patterns and the problem solving procedure he or she is employing could indeed improve problem-solving performance.

  2. An Experimental Investigation of Improving Human Problem-Solving Performance by Guiding Attention and Adaptively Providing Details on Information Displays

    National Research Council Canada - National Science Library

    Narayanan, N. H

    2007-01-01

    ... in real-time in response to the viewer's gaze patterns. Designs of these displays were based on a cognitive model of multimodal information comprehension developed in a previous ONR project (N00014-96-1-1187...

  3. An Experimental Investigation of Improving Human Problem-Solving Performance by Guiding Attention and Adaptively Proving Details on Information Displays

    National Research Council Canada - National Science Library

    Narayanan, N. H

    2007-01-01

    ... in real-time in response to the viewer's gaze patterns. Designs of these displays were based on a cognitive model of multimodal information comprehension developed in a previous ONR project (N0001496l1187...

  4. SLS-Derived Lab- Precursor to Deep Space Human Exploration

    Science.gov (United States)

    Griffin, Brand M.; Lewis, Ruthan; Eppler, Dean; Smitherman, David

    2015-01-01

    Plans to send humans to Mars are in the works and the launch system is being built. Are we ready? Transportation, entry, landing, and surface operations have been successfully demonstrated for robotic missions. However, for human missions, there are significant, potentially show-stopping issues. These issues, called Strategic Knowledge Gaps (SKGs), are the unanswered questions concerning long duration exploration Beyond low Earth Orbit (BEO). The gaps represent a risk of loss of life or mission and because they require extended exposure to the weightless environment outside of earth's protective geo-magnetic field, they cannot be resolved on Earth or on the International Space Station (ISS). Placing a laboratory at a relatively close and stable lunar Distant Retrograde Orbit (DRO) provides an accessible location with the requisite environmental conditions for conducting SKG research and testing mitigation solutions. Configurations comprised of multiple 3 m and 4.3 m diameter modules have been studied but the most attractive solution uses elements of the human Mars launch vehicle or Space Launch System (SLS) for a Mars proving ground laboratory. A shortened version of an SLS hydrogen propellant tank creates a Skylab-like pressure vessel that flies fully outfitted on a single launch. This not only offers significant savings by incorporating SLS pressure vessel development costs but avoids the expensive ISS approach using many launches with substantial on-orbit assembly before becoming operational. One of the most challenging SKGs is crew radiation protection; this is why SKG laboratory research is combined with Mars transit habitat systems development. Fundamentally, the two cannot be divorced because using the habitat systems for protection requires actual hardware geometry and material properties intended to contribute to shielding effectiveness. The SKGs are difficult problems. The solutions to these problems are not obvious; they require integrated, iterative

  5. Interactive exploration of the vulnerability of the human infrastructure: an approach using simultaneous display of similar locations

    Science.gov (United States)

    Ceré, Raphaël; Kaiser, Christian

    2015-04-01

    Currently, three quarters of the Swiss population is living in urban areas. The total population is still increasing, and urbanized space is increasing event faster. Consequently, the intensity of use has decreased but the exposure of the urban space to natural events has grown along with the cost related to the impact of hazards. In line with this fact, during the 20th century there has been a noticeable increase of natural disasters accompanied by the rapid increase of the world population, leading to higher costs. Additionally to the fact that more people are exposed to natural hazards, the value of goods globally has increased more than proportionally. Consequently, the vulnerability of urban space is, more than ever before, a major issue for socio-economic development. Here, vulnerability is defined as the potential human loss or loss of infrastructure caused by a hazardous event. It encompasses factors of urban infrastructure, population and the environment, which increase the susceptibility of a location to the impact of hazards. This paper describes a novel method for improving the interactive use of exploratory data analysis in the context of minimizing vulnerability and disaster risk by prevention or mitigation. This method is used to assess the similarity between different locations with respect to several characteristics relevant to vulnerability at different scales, allowing for automatic display of multiple locations similar to the one under investigation by an expert. Visualizing vulnerability simultaneously for several locations allows for analyzing and comparing of metric characteristics between multiple places and at different scales. The interactivity aspect is also useful for understanding vulnerability patterns and it facilitates disaster risk management and decisions on global preventive measures in urban spaces. Metrics for vulnerability assessment can be extracted from extensive geospatial datasets such as high-resolution digital elevation

  6. Benzofuroxan derivatives N-Br and N-I induce intrinsic apoptosis in melanoma cells by regulating AKT/BIM signaling and display anti metastatic activity in vivo.

    Science.gov (United States)

    Farias, C F; Massaoka, M H; Girola, N; Azevedo, R A; Ferreira, A K; Jorge, S D; Tavares, L C; Figueiredo, C R; Travassos, L R

    2015-10-27

    Malignant melanoma is an aggressive type of skin cancer, and despite recent advances in treatment, the survival rate of the metastatic form remains low. Nifuroxazide analogues are drugs based on the substitution of the nitrofuran group by benzofuroxan, in view of the pharmacophore similarity of the nitro group, improving bioavailability, with higher intrinsic activity and less toxicity. Benzofuroxan activity involves the intracellular production of free-radical species. In the present work, we evaluated the antitumor effects of different benzofuroxan derivatives in a murine melanoma model. B16F10-Nex2 melanoma cells were used to investigate the antitumor effects of Benzofuroxan derivatives in vitro and in a syngeneic melanoma model in C57Bl/6 mice. Cytotoxicity, morphological changes and reactive oxygen species (ROS) were assessed by a diphenyltetrasolium reagent, optical and fluorescence microscopy, respectively. Annexin-V binding and mitochondrial integrity were analyzed by flow cytometry. Western blotting and colorimetry identified cell signaling proteins. Benzofuroxan N-Br and N-I derivatives were active against murine and human tumor cell lines, exerting significant protection against metastatic melanoma in a syngeneic model. N-Br and N-I induce apoptosis in melanoma cells, evidenced by specific morphological changes, DNA condensation and degradation, and phosphatidylserine translocation in the plasma membrane. The intrinsic mitochondrial pathway in B16F10-Nex2 cells is suggested owing to reduced outer membrane potential in mitochondria, followed by caspase -9, -3 activation and cleavage of PARP. The cytotoxicity of N-Br and N-I in B16F10-Nex2 cells is mediated by the generation of ROS, inhibited by pre-incubation of the cells with N-acetylcysteine (NAC). The induction of ROS by N-Br and N-I resulted in the inhibition of AKT activation, an important molecule related to tumor cell survival, followed by upregulation of BIM. We conclude that N-Br and N-I are

  7. Benzofuroxan derivatives N-Br and N-I induce intrinsic apoptosis in melanoma cells by regulating AKT/BIM signaling and display anti metastatic activity in vivo

    International Nuclear Information System (INIS)

    Farias, C. F.; Massaoka, M. H.; Girola, N.; Azevedo, R. A.; Ferreira, A. K.; Jorge, S. D.; Tavares, L. C.; Figueiredo, C. R.; Travassos, L. R.

    2015-01-01

    Malignant melanoma is an aggressive type of skin cancer, and despite recent advances in treatment, the survival rate of the metastatic form remains low. Nifuroxazide analogues are drugs based on the substitution of the nitrofuran group by benzofuroxan, in view of the pharmacophore similarity of the nitro group, improving bioavailability, with higher intrinsic activity and less toxicity. Benzofuroxan activity involves the intracellular production of free-radical species. In the present work, we evaluated the antitumor effects of different benzofuroxan derivatives in a murine melanoma model. B16F10-Nex2 melanoma cells were used to investigate the antitumor effects of Benzofuroxan derivatives in vitro and in a syngeneic melanoma model in C57Bl/6 mice. Cytotoxicity, morphological changes and reactive oxygen species (ROS) were assessed by a diphenyltetrasolium reagent, optical and fluorescence microscopy, respectively. Annexin-V binding and mitochondrial integrity were analyzed by flow cytometry. Western blotting and colorimetry identified cell signaling proteins. Benzofuroxan N-Br and N-I derivatives were active against murine and human tumor cell lines, exerting significant protection against metastatic melanoma in a syngeneic model. N-Br and N-I induce apoptosis in melanoma cells, evidenced by specific morphological changes, DNA condensation and degradation, and phosphatidylserine translocation in the plasma membrane. The intrinsic mitochondrial pathway in B16F10-Nex2 cells is suggested owing to reduced outer membrane potential in mitochondria, followed by caspase −9, −3 activation and cleavage of PARP. The cytotoxicity of N-Br and N-I in B16F10-Nex2 cells is mediated by the generation of ROS, inhibited by pre-incubation of the cells with N-acetylcysteine (NAC). The induction of ROS by N-Br and N-I resulted in the inhibition of AKT activation, an important molecule related to tumor cell survival, followed by upregulation of BIM. We conclude that N-Br and N-I are

  8. S100B protein, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor in human milk.

    Directory of Open Access Journals (Sweden)

    Ruisong Li

    Full Text Available Human milk contains a wide variety of nutrients that contribute to the fulfillment of its functions, which include the regulation of newborn development. However, few studies have investigated the concentrations of S100B protein, brain-derived neurotrophic factor (BDNF, and glial cell line-derived neurotrophic factor (GDNF in human milk. The associations of the concentrations of S100B protein, BDNF, and GDNF with maternal factors are not well explored.To investigate the concentrations of S100B protein, BDNF, and GDNF in human milk and characterize the maternal factors associated with their levels in human milk, human milk samples were collected at days 3, 10, 30, and 90 after parturition. Levels of S100B protein, BDNF, and GDNF, and their mRNAs in the samples were detected. Then, these concentrations were compared with lactation and other maternal factors. S100B protein levels in human milk samples collected at 3, 10, 30, and 90 d after parturition were 1249.79±398.10, 1345.05±539.16, 1481.83±573.30, and 1414.39±621.31 ng/L, respectively. On the other hand, the BDNF concentrations in human milk samples were 10.99±4.55, 13.01±5.88, 13.35±6.43, and 2.83±5.47 µg/L, while those of GDNF were 10.90±1.65, 11.38±1., 11.29±3.10, and 11.40±2.21 g/L for the same time periods. Maternal post-pregnancy body mass index was positively associated with S100B levels in human milk (r = 0.335, P = 0.030<0.05. In addition, there was a significant correlation between the levels of S100B protein and BDNF (z = 2.09, P = 0.037<0.05. Delivery modes were negatively associated with the concentration of GDNF in human milk.S100B protein, BDNF, and GDNF are present in all samples of human milk, and they may be responsible for the long term effects of breast feeding.

  9. Chondrogenesis of human adipose derived stem cells for future microtia repair using co-culture technique.

    Science.gov (United States)

    Goh, Bee See; Che Omar, Siti Nurhadis; Ubaidah, Muhammad Azhan; Saim, Lokman; Sulaiman, Shamsul; Chua, Kien Hui

    2017-04-01

    In conclusion, these result showed HADSCs could differentiate into chondrocytes-like cells, dependent on signaling induced by TGF-β3 and chondrocytes. This is a promising result and showed that HADSCs is a potential source for future microtia repair. The technique of co-culture is a positive way forward to assist the microtia tissue. Reconstructive surgery for the repair of microtia still remains the greatest challenge among the surgeons. Its repair is associated with donor-site morbidity and the degree of infection is inevitable when using alloplastic prosthesis with uncertain long-term durability. Thus, human adipose derived stem cells (HADSCs) can be an alternative cell source for cartilage regeneration. This study aims to evaluate the chondrogenic potential of HADSCs cultured with transforming growth factor-beta (TGF-β) and interaction of auricular chondrocytes with HADSCs for new cartilage generation. Multi-lineages differentiation features of HADSCs were monitored by Alcian Blue, Alizarin Red, and Oil Red O staining for chondrogenic, adipogenic, and osteogenic differentiation capacity, respectively. Further, HADSCs alone were culture in medium added with TGF-β3; and human auricular chondrocytes were interacted indirectly in the culture with and without TGF-βs for up to 21 days, respectively. Cell morphology and chondrogenesis were monitored by inverted microscope. For cell viability, Alamar Blue assay was used to measure the cell viability and the changes in gene expression of auricular chondrocyte markers were determined by real-time polymerase chain reaction analysis. For the induction of chondrogenic differentiation, HADSCs showed a feature of aggregation and formed a dense matrix of proteoglycans. Staining results from Alizirin Red and Oil Red O indicated the HADSCs also successfully differentiated into adipogenic and osteogenic lineages after 21 days. According to a previous study, HADSCs were strongly positive for the mesenchymal markers CD90, CD73

  10. Human iPSC-derived neurons and lymphoblastoid cells for personalized medicine research in neuropsychiatric disorders.

    Science.gov (United States)

    Gurwitz, David

    2016-09-01

    The development and clinical implementation of personalized medicine crucially depends on the availability of high-quality human biosamples; animal models, although capable of modeling complex human diseases, cannot reflect the large variation in the human genome, epigenome, transcriptome, proteome, and metabolome. Although the biosamples available from public biobanks that store human tissues and cells may represent the large human diversity for most diseases, these samples are not always sufficient for developing biomarkers for patient-tailored therapies for neuropsychiatric disorders. Postmortem human tissues are available from many biobanks; nevertheless, collections of neuronal human cells from large patient cohorts representing the human diversity remain scarce. Two tools are gaining popularity for personalized medicine research on neuropsychiatric disorders: human induced pluripotent stem cell-derived neurons and human lymphoblastoid cell lines. This review examines and contrasts the advantages and limitations of each tool for personalized medicine research.

  11. 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

  12. Fibroblast-Derived Extracellular Matrix Induces Chondrogenic Differentiation in Human Adipose-Derived Mesenchymal Stromal/Stem Cells in Vitro

    Directory of Open Access Journals (Sweden)

    Kevin Dzobo

    2016-08-01

    Full Text Available Mesenchymal stromal/stem cells (MSCs represent an area being intensively researched for tissue engineering and regenerative medicine applications. MSCs may provide the opportunity to treat diseases and injuries that currently have limited therapeutic options, as well as enhance present strategies for tissue repair. The cellular environment has a significant role in cellular development and differentiation through cell–matrix interactions. The aim of this study was to investigate the behavior of adipose-derived MSCs (ad-MSCs in the context of a cell-derived matrix so as to model the in vivo physiological microenvironment. The fibroblast-derived extracellular matrix (fd-ECM did not affect ad-MSC morphology, but reduced ad-MSC proliferation. Ad-MSCs cultured on fd-ECM displayed decreased expression of integrins α2 and β1 and subsequently lost their multipotency over time, as shown by the decrease in CD44, Octamer-binding transcription factor 4 (OCT4, SOX2, and NANOG gene expression. The fd-ECM induced chondrogenic differentiation in ad-MSCs compared to control ad-MSCs. Loss of function studies, through the use of siRNA and a mutant Notch1 construct, revealed that ECM-mediated ad-MSCs chondrogenesis requires Notch1 and β-catenin signaling. The fd-ECM also showed anti-senescence effects on ad-MSCs. The fd-ECM is a promising approach for inducing chondrogenesis in ad-MSCs and chondrogenic differentiated ad-MSCs could be used in stem cell therapy procedures.

  13. Kojyl cinnamate ester derivatives promote adiponectin production during adipogenesis in human adipose tissue-derived mesenchymal stem cells.

    Science.gov (United States)

    Rho, Ho Sik; Hong, Soo Hyun; Park, Jongho; Jung, Hyo-Il; Park, Young-Ho; Lee, John Hwan; Shin, Song Seok; Noh, Minsoo

    2014-05-01

    The subcutaneous fat tissue mass gradually decreases with age, and its regulation is a strategy to develop anti-aging compounds to ameliorate the photo-aging of human skin. The adipogenesis of human adipose tissue-mesenchymal stem cells (hAT-MSCs) can be used as a model to discover novel anti-aging compounds. Cinnamomum cassia methanol extracts were identified as adipogenesis-promoting agents by natural product library screening. Cinnamates, the major chemical components of Cinnamomum cassia extracts, promoted adipogenesis in hAT-MSCs. We synthesized kojyl cinnamate ester derivatives to improve the pharmacological activity of cinnamates. Structure-activity studies of kojyl cinnamate derivatives showed that both the α,β-unsaturated carbonyl ester group and the kojic acid moiety play core roles in promoting adiponectin production during adipogenesis in hAT-MSCs. We conclude that kojyl cinnamate ester derivatives provide novel pharmacophores that can regulate adipogenesis in hAT-MSCs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Antimicrobial activity of synthetic cationic peptides and lipopeptides derived from human lactoferricin against Pseudomonas aeruginosa planktonic cultures and biofilms.

    Science.gov (United States)

    Sánchez-Gómez, Susana; Ferrer-Espada, Raquel; Stewart, Philip S; Pitts, Betsey; Lohner, Karl; Martínez de Tejada, Guillermo

    2015-07-07

    Infections by Pseudomonas aeruginosa constitute a serious health threat because this pathogen -particularly when it forms biofilms - can acquire resistance to the majority of conventional antibiotics. This study evaluated the antimicrobial activity of synthetic peptides based on LF11, an 11-mer peptide derived from human lactoferricin against P. aeruginosa planktonic and biofilm-forming cells. We included in this analysis selected N-acylated derivatives of the peptides to analyze the effect of acylation in antimicrobial activity. To assess the efficacy of compounds against planktonic bacteria, microdilution assays to determine the minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill studies were conducted. The anti-biofilm activity of the agents was assessed on biofilms grown under static (on microplates) and dynamic (in a CDC-reactor) flow regimes. The antimicrobial activity of lipopeptides differed from that of non-acylated peptides in their killing mechanisms on planktonic and biofilm-forming cells. Thus, acylation enhanced the bactericidal activity of the parental peptides and resulted in lipopeptides that were uniformly bactericidal at their MIC. In contrast, acylation of the most potent anti-biofilm peptides resulted in compounds with lower anti-biofilm activity. Both peptides and lipopeptides displayed very rapid killing kinetics and all of them required less than 21 min to reduce 1,000 times the viability of planktonic cells when tested at 2 times their MBC. The peptides, LF11-215 (FWRIRIRR) and LF11-227 (FWRRFWRR), displayed the most potent anti-biofilm activity causing a 10,000 fold reduction in cell viability after 1 h of treatment at 10 times their MIC. At that concentration, these two compounds exhibited low citotoxicity on human cells. In addition to its bactericidal activity, LF11-227 removed more that 50 % of the biofilm mass in independent assays. Peptide LF11-215 and two of the shortest and least

  15. Role of Human Na,K-ATPase alpha 4 in Sperm Function, Derived from Studies in Transgenic Mice

    Science.gov (United States)

    McDermott, Jeffrey; Sánchez, Gladis; Nangia, Ajay K.; Blanco, Gustavo

    2014-01-01

    SUMMARY Most of our knowledge on the biological role of the testis-specific Na,K-ATPase alpha 4 isoform derives from studies performed in non-human species. Here, we studied the function of human Na,K-ATPase alpha 4 after its expression in transgenic mice. Using a bacterial artificial chromosome (BAC) construct, containing the human ATP1A4 gene locus, we obtained expression of the human α4 transgene specifically in mouse sperm, enriched in the sperm flagellum. The expressed, human alpha 4 was active, and compared to wild-type sperm, those from transgenic mice displayed higher Na,K-ATPase alpha 4 activity and greater binding of fluorescently labeled ouabain, which is typical of the alpha 4 isoform. The expression and activity of endogenous alpha 4 and the other Na,K-ATPase alpha isoform present in sperm, alpha 1, remained unchanged. Male mice expressing the human ATP1A4 transgene exhibited similar testis size and morphology, normal sperm number and shape, and no changes in overall fertility compared to wild-type mice. Sperm carrying the human transgene exhibited enhanced total motility and an increase in multiple parameters of sperm movement, including higher sperm hyperactive motility. In contrast, no statistically significant changes in sperm membrane potential, protein tyrosine phosphorylation, or spontaneous acrosome reaction were found between wild-type and transgenic mice. Altogether, these results provide new genetic evidence for an important role of human Na,K-ATPase alpha 4 in sperm motility and hyperactivation, and establishes a new animal model for future studies of this isoform. PMID:25640246

  16. Molecular characterisation of stromal populations derived from human embryonic stem cells

    DEFF Research Database (Denmark)

    Harkness, L.; Twine, N. A.; Abu Dawud, R.

    2015-01-01

    Human bone marrow-derived stromal (skeletal) stem cells (BM-hMSC) are being employed in an increasing number of clinical trials for tissue regeneration. A limiting factor for their clinical use is the inability to obtain sufficient cell numbers. Human embryonic stem cells (hESC) can provide an un...

  17. Functional Characterization of Preadipocytes Derived from Human Periaortic Adipose Tissue

    Directory of Open Access Journals (Sweden)

    Diana Vargas

    2017-01-01

    Full Text Available Adipose tissue can affect the metabolic control of the cardiovascular system, and its anatomic location can affect the vascular function differently. In this study, biochemical and phenotypical characteristics of adipose tissue from periaortic fat were evaluated. Periaortic and subcutaneous adipose tissues were obtained from areas surrounding the ascending aorta and sternotomy incision, respectively. Adipose tissues were collected from patients undergoing myocardial revascularization or mitral valve replacement surgery. Morphological studies with hematoxylin/eosin and immunohistochemical assay were performed in situ to quantify adipokine expression. To analyze adipogenic capacity, adipokine expression, and the levels of thermogenic proteins, adipocyte precursor cells were isolated from periaortic and subcutaneous adipose tissues and induced to differentiation. The precursors of adipocytes from the periaortic tissue accumulated less triglycerides than those from the subcutaneous tissue after differentiation and were smaller than those from subcutaneous adipose tissue. The levels of proteins involved in thermogenesis and energy expenditure increased significantly in periaortic adipose tissue. Additionally, the expression levels of adipokines that affect carbohydrate metabolism, such as FGF21, increased significantly in mature adipocytes induced from periaortic adipose tissue. These results demonstrate that precursors of periaortic adipose tissue in humans may affect cardiovascular events and might serve as a target for preventing vascular diseases.

  18. Recombinant human monoclonal autoantibodies specific for citrulline-containing peptides from phage display libraries derived from patients with rheumatoid arthritis.

    NARCIS (Netherlands)

    Raats, J.M.H.; Wijnen, E.M.; Pruijn, G.J.M.; Hoogen, F.H.J. van den; Venrooij, W.J.W. van

    2003-01-01

    OBJECTIVE: To isolate and characterize monoclonal autoantibodies (Mab) directed to citrullinated antigens from patients with rheumatoid arthritis (RA). METHODS: Using lymphocytes from bone marrow or peripheral blood from RA patients, we constructed antibody fragment libraries representing the

  19. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts.

    Science.gov (United States)

    Kakudo, Natsuko; Minakata, Tatsuya; Mitsui, Toshihito; Kushida, Satoshi; Notodihardjo, Frederik Zefanya; Kusumoto, Kenji

    2008-11-01

    This study evaluated changes in platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets by platelet-rich plasma activation, and the proliferation potential of activated platelet-rich plasma and platelet-poor plasma on human adipose-derived stem cells and human dermal fibroblasts. Platelet-rich plasma was prepared using a double-spin method, with the number of platelets counted in each preparation stage. Platelet-rich and platelet-poor plasma were activated with autologous thrombin and calcium chloride, and levels of platelet-released PDGF-AB and TGF-beta1 were determined by enzyme-linked immunosorbent assay. Cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 5% whole blood plasma, nonactivated platelet-rich plasma, nonactivated platelet-poor plasma, activated platelet-rich plasma, or activated platelet-poor plasma. In parallel, these cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 1%, 5%, 10%, or 20% activated platelet-rich plasma. The cultured human adipose-derived stem cells and human dermal fibroblasts were assayed for proliferation. Platelet-rich plasma contained approximately 7.9 times as many platelets as whole blood, and its activation was associated with the release of large amounts of PDGF-AB and TGF-beta1. Adding activated platelet-rich or platelet-poor plasma significantly promoted the proliferation of human adipose-derived stem cells and human dermal fibroblasts. Adding 5% activated platelet-rich plasma to the medium maximally promoted cell proliferation, but activated platelet-rich plasma at 20% did not promote it. Platelet-rich plasma can enhance the proliferation of human adipose-derived stem cells and human dermal fibroblasts. These results support clinical platelet-rich plasma application for cell-based, soft-tissue engineering and wound healing.

  20. The effect of short-chain fatty acids on human monocyte-derived dendritic cells

    DEFF Research Database (Denmark)

    Nastasi, Claudia; Candela, Marco; Bonefeld, Charlotte Menné

    2015-01-01

    negligible effects, while both butyrate and propionate strongly modulated gene expression in both immature and mature human monocyte-derived DC. An Ingenuity pathway analysis based on the differentially expressed genes suggested that propionate and butyrate modulate leukocyte trafficking, as SCFA strongly......The gut microbiota is essential for human health and plays an important role in the pathogenesis of several diseases. Short-chain fatty acids (SCFA), such as acetate, butyrate and propionate, are end-products of microbial fermentation of macronutrients that distribute systemically via the blood....... The aim of this study was to investigate the transcriptional response of immature and LPS-matured human monocyte-derived DC to SCFA. Our data revealed distinct effects exerted by each individual SCFA on gene expression in human monocyte-derived DC, especially in the mature ones. Acetate only exerted...

  1. A heptadecanuclear Mn(III)9Dy(III)8 cluster derived from triethanolamine with two edge sharing supertetrahedra as the core and displaying SMM behaviour.

    Science.gov (United States)

    Langley, Stuart K; Moubarakia, Boujemaa; Murray, Keith S

    2010-06-07

    A heterometallic, heptadecanuclear cluster of formula [Mn(III)9Dy(III)8O8(OH)8(tea)2(teaH)2(teaH2)4(Ac)4(NO3)2(H2O)4](NO3)7·8H2O (1) is reported. The core of 1 displays two edge sharing Mn(III)5Dy(III)5 supertetrahedra and represents one of the largest Mn/4f cluster compound so far reported. Magnetic studies show that 1 displays probable SMM behaviour as observed via non-zero values in the χM''vs T plot.

  2. Predators or prey? Spatio-temporal discrimination of human-derived risk by brown bears.

    Science.gov (United States)

    Ordiz, Andrés; Støen, Ole-Gunnar; Delibes, Miguel; Swenson, Jon E

    2011-05-01

    Prey usually adjust anti-predator behavior to subtle variations in perceived risk. However, it is not clear whether adult large carnivores that are virtually free of natural predation adjust their behavior to subtle variations in human-derived risk, even when living in human-dominated landscapes. As a model, we studied resting-site selection by a large carnivore, the brown bear (Ursus arctos), under different spatial and temporal levels of human activity. We quantified horizontal and canopy cover at 440 bear beds and 439 random sites at different distances from human settlements, seasons, and times of the day. We hypothesized that beds would be more concealed than random sites and that beds would be more concealed in relation to human-derived risk. Although human densities in Scandinavia are the lowest within bear ranges in Western Europe, we found an effect of human activity; bears chose beds with higher horizontal and canopy cover during the day (0700-1900 hours), especially when resting closer to human settlements, than at night (2200-0600 hours). In summer/fall (the berry season), with more intensive and dispersed human activity, including hunting, bears rested further from human settlements during the day than in spring (pre-berry season). Additionally, day beds in the summer/fall were the most concealed. Large carnivores often avoid humans at a landscape scale, but total avoidance in human-dominated areas is not possible. Apparently, bears adjust their behavior to avoid human encounters, which resembles the way prey avoid their predators. Bears responded to fine-scale variations in human-derived risk, both on a seasonal and a daily basis.

  3. Preliminary study of gaze toward humans in photographs by individuals with autism, Down syndrome, or other intellectual disabilities: implications for design of visual scene displays.

    Science.gov (United States)

    Wilkinson, Krista M; Light, Janice

    2014-06-01

    Visual scene displays (VSDs) are a form of augmentative and alternative communication display in which language concepts are embedded into an image of a naturalistic event. VSDs are based on the theory that language learning occurs through interactions with other people, and recommendations for VSD design have emphasized using images of these events that include humans. However, many VSDs also include other items that could potentially be distracting. We examined gaze fixation in 18 school-aged participants with and without severe intellectual/developmental disabilities (i.e., individuals with typical development, autism, Down syndrome and other intellectual disabilities) while they viewed photographs with human figures of various sizes and locations in the image, appearing alongside other interesting, and potentially distracting items. In all groups, the human figures attracted attention rapidly (within 1.5 seconds). The proportions of each participant's own fixation time spent on the human figures were similar across all groups, as were the proportions of total fixations made to the human figures. Although the findings are preliminary, this initial evidence supports the inclusion of humans in VSD images.

  4. Display hardware

    International Nuclear Information System (INIS)

    Myers, D.R.

    1983-01-01

    To appreciate the limitations and possibilities of computer graphics it is necessary to have some acquaintance with the available technology. The aim of this chapter is to mention briefly the different display types and their 'ball-park' price ranges. It must be stressed that prices change rapidly, and so those quoted here are only intended to give an idea of the cost at the time of writing.

  5. CULTURAL DISPLAY RULES DRIVE EYE GAZE DURING THINKING.

    Science.gov (United States)

    McCarthy, Anjanie; Lee, Kang; Itakura, Shoji; Muir, Darwin W

    2006-11-01

    The authors measured the eye gaze displays of Canadian, Trinidadian, and Japanese participants as they answered questions for which they either knew, or had to derive, the answers. When they knew the answers, Trinidadians maintained the most eye contact, whereas Japanese maintained the least. When thinking about the answers to questions, Canadians and Trinidadians looked up, whereas Japanese looked down. Thus, for humans, gaze displays while thinking are at least in part culturally determined.

  6. Full-color wide field-of-view holographic helmet-mounted display for pilot/vehicle interface development and human factors studies

    Science.gov (United States)

    Burley, James R., II; LaRussa, Joseph A.

    1990-10-01

    A Helmet-Mounted Display (HMD) which utilizes highly efficient trichromatic holographic elements has been designed to support pilot vechicle interface development and human factors studies at the NASA-Langley Research Center. While the optics are fully color corrected, the miniature CRT's are monochromatic. This design provides an upgrade path to full-color when miniature display technology matures to color. The optical design conforms to the helmet shape and provides a 50 degree field-of-view (FOV) to each eye. Built-in adjustments allow each ocular to be independently moved so that the overall horizontal FOV may be varied from 50 degrees to 100 degrees with a corresponding change in the stereo overlap region. The helmet design and interpupillary adjustment allow for the 5th through 95th percentile male and female wearer. Total head-borne weight is approximately 4.2 pounds. The high-resolution monochromatic CRTs are driven by a set of multisync electronics with a maximum video bandwidth of 88 Mhz and supports bith raster and stroke modes. The electronics are designed to be compatiable with the Silicon Graphics IRIS 4D graphics workstations and the ADAGE 340 stroke graphics computer. A Polhemus magnetic tracking device is used to determine the helmet line-of-sight. The helmet will be used to develop innovative new display concepts for the F- 1 8 High-Alpha Research Vehicle (HARV) which make use of the unique display properties of the HMD. Pictorial displays, which convey the appropriate information intuitively, are envisioned. Human factors studies are also planned to evaluate the utility of stereopsis and determine the FOV requirements for different tasks. Concepts proven in the simulator will be carried to flight test in 1993 with a lighter weight, "hardened" version of this HMD design.

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

    Directory of Open Access Journals (Sweden)

    Bandi Sriram

    2008-01-01

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

  8. 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.

  9. Cardiotoxicity evaluation using human embryonic stem cells and induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Zhao, Qi; Wang, Xijie; Wang, Shuyan; Song, Zheng; Wang, Jiaxian; Ma, Jing

    2017-03-09

    Cardiotoxicity remains an important concern in drug discovery. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become an attractive platform to evaluate cardiotoxicity. However, the consistency between human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in prediction of cardiotoxicity has yet to be elucidated. Here we screened the toxicities of four representative drugs (E-4031, isoprenaline, quinidine, and haloperidol) using both hESC-CMs and hiPSC-CMs, combined with an impedance-based bioanalytical method. It showed that both hESC-CMs and hiPSC-CMs can recapitulate cardiotoxicity and identify the effects of well-characterized compounds. The combined platform of hPSC-CMs and an impedance-based bioanalytical method could improve preclinical cardiotoxicity screening, holding great potential for increasing drug development accuracy.

  10. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    International Nuclear Information System (INIS)

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M.

    1990-01-01

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures

  11. In vitro chondrogenic differentiation of human adipose-derived stem cells with silk scaffolds

    Directory of Open Access Journals (Sweden)

    Hyeon Joo Kim

    2012-12-01

    Full Text Available Human adipose-derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with natural and synthetic biomaterials. In the present study, we hypothesized that porous aqueous-derived silk protein scaffolds would be suitable for chondrogenic differentiation of human adipose-derived stem cells. Human adipose-derived stem cells were cultured up to 6 weeks, and cell proliferation and chondrogenic differentiation were investigated and compared with those in conventional micromass culture. Cell proliferation, glycosaminoglycan, and collagen levels in aqueous-derived silk scaffolds were significantly higher than in micromass culture. Transcript levels of SOX9 and type II collagen were also upregulated in the cell–silk constructs at 6 weeks. Histological examination revealed that the pores of the silk scaffolds were filled with cells uniformly distributed. In addition, chondrocyte-specific lacunae formation was evident and distributed in the both groups. The results suggest the biodegradable and biocompatible three-dimensional aqueous-derived silk scaffolds provided an improved environment for chondrogenic differentiation compared to micromass culture.

  12. In vitro fermentation of alginate and its derivatives by human gut microbiota.

    Science.gov (United States)

    Li, Miaomiao; Li, Guangsheng; Shang, Qingsen; Chen, Xiuxia; Liu, Wei; Pi, Xiong'e; Zhu, Liying; Yin, Yeshi; Yu, Guangli; Wang, Xin

    2016-06-01

    Alginate (Alg) has a long history as a food ingredient in East Asia. However, the human gut microbes responsible for the degradation of alginate and its derivatives have not been fully understood yet. Here, we report that alginate and the low molecular polymer derivatives of mannuronic acid oligosaccharides (MO) and guluronic acid oligosaccharides (GO) can be completely degraded and utilized at various rates by fecal microbiota obtained from six Chinese individuals. However, the derivative of propylene glycol alginate sodium sulfate (PSS) was not hydrolyzed. The bacteria having a pronounced ability to degrade Alg, MO and GO were isolated from human fecal samples and were identified as Bacteroides ovatus, Bacteroides xylanisolvens, and Bacteroides thetaiotaomicron. Alg, MO and GO can increase the production level of short chain fatty acids (SCFA), but GO generates the highest level of SCFA. Our data suggest that alginate and its derivatives could be degraded by specific bacteria in the human gut, providing the basis for the impacts of alginate and its derivates as special food additives on human health. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Derivation and characterization of human fetal MSCs: an alternative cell source for large-scale production of cardioprotective microparticles.

    Science.gov (United States)

    Lai, Ruenn Chai; Arslan, Fatih; Tan, Soon Sim; Tan, Betty; Choo, Andre; Lee, May May; Chen, Tian Sheng; Teh, Bao Ju; Eng, John Kun Long; Sidik, Harwin; Tanavde, Vivek; Hwang, Wei Sek; Lee, Chuen Neng; El Oakley, Reida Menshawe; Pasterkamp, Gerard; de Kleijn, Dominique P V; Tan, Kok Hian; Lim, Sai Kiang

    2010-06-01

    The therapeutic effects of mesenchymal stem cells (MSCs) transplantation are increasingly thought to be mediated by MSC secretion. We have previously demonstrated that human ESC-derived MSCs (hESC-MSCs) produce cardioprotective microparticles in pig model of myocardial ischemia/reperfusion (MI/R) injury. As the safety and availability of clinical grade human ESCs remain a concern, MSCs from fetal tissue sources were evaluated as alternatives. Here we derived five MSC cultures from limb, kidney and liver tissues of three first trimester aborted fetuses and like our previously described hESC-derived MSCs; they were highly expandable and had similar telomerase activities. Each line has the potential to generate at least 10(16-19) cells or 10(7-10) doses of cardioprotective secretion for a pig model of MI/R injury. Unlike previously described fetal MSCs, they did not express pluripotency-associated markers such as Oct4, Nanog or Tra1-60. They displayed a typical MSC surface antigen profile and differentiated into adipocytes, osteocytes and chondrocytes in vitro. Global gene expression analysis by microarray and qRT-PCR revealed a typical MSC gene expression profile that was highly correlated among the five fetal MSC cultures and with that of hESC-MSCs (r(2)>0.90). Like hESC-MSCs, they produced secretion that was cardioprotective in a mouse model of MI/R injury. HPLC analysis of the secretion revealed the presence of a population of microparticles with a hydrodynamic radius of 50-65 nm. This purified population of microparticles was cardioprotective at approximately 1/10 dosage of the crude secretion. (c) 2009 Elsevier Ltd. All rights reserved.

  14. 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

  15. 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

  16. Generation of functional islets from human umbilical cord and placenta derived mesenchymal stem cells.

    Science.gov (United States)

    Kadam, Sachin; Govindasamy, Vijayendran; Bhonde, Ramesh

    2012-01-01

    Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been used for allogeneic application in tissue engineering but have certain drawbacks. Therefore, mesenchymal stem cells (MSCs) derived from other adult tissue sources have been considered as an alternative. The human umbilical cord and placenta are easily available noncontroversial sources of human tissue, which are often discarded as biological waste, and their collection is noninvasive. These sources of MSCs are not subjected to ethical constraints, as in the case of embryonic stem cells. MSCs derived from umbilical cord and placenta are multipotent and have the ability to differentiate into various cell types crossing the lineage boundary towards endodermal lineage. The aim of this chapter is to provide a detailed reproducible cookbook protocol for the isolation, propagation, characterization, and differentiation of MSCs derived from human umbilical cord and placenta with special reference to harnessing their potential towards pancreatic/islet lineage for utilization as a cell therapy product. We show here that mesenchymal stromal cells can be extensively expanded from umbilical cord and placenta of human origin retaining their multilineage differentiation potential in vitro. Our report indicates that postnatal tissues obtained as delivery waste represent a rich source of mesenchymal stromal cells, which can be differentiated into functional islets employing three-stage protocol developed by our group. These islets could be used as novel in vitro model for screening hypoglycemics/insulin secretagogues, thus reducing animal experimentation for this purpose and for the future human islet transplantation programs to treat diabetes.

  17. Human multipotent adipose-derived stem cells differentiate into functional brown adipocytes

    DEFF Research Database (Denmark)

    Elabd, Christian; Chiellini, Chiara; Carmona, Mamen

    2009-01-01

    adipose-derived stem (hMADS) cells exhibit a normal karyotype and high self-renewal ability; they are known to differentiate into cells that exhibit the key properties of human white adipocytes, that is, uncoupling protein two expression, insulin-stimulated glucose uptake, lipolysis in response to beta......In contrast to the earlier contention, adult humans have been shown recently to possess active brown adipose tissue with a potential of being of metabolic significance. Up to now, brown fat precursor cells have not been available for human studies. We have shown previously that human multipotent......-agonists and atrial natriuretic peptide, and release of adiponectin and leptin. Herein, we show that, upon chronic exposure to a specific PPARgamma but not to a PPARbeta/delta or a PPARalpha agonist, hMADS cell-derived white adipocytes are able to switch to a brown phenotype by expressing both uncoupling protein one...

  18. Extracellular Matrix-Mediated Maturation of Human Pluripotent Stem Cell-Derived Cardiac Monolayer Structure and Electrophysiological Function.

    Science.gov (United States)

    Herron, Todd J; Rocha, Andre Monteiro Da; Campbell, Katherine F; Ponce-Balbuena, Daniela; Willis, B Cicero; Guerrero-Serna, Guadalupe; Liu, Qinghua; Klos, Matt; Musa, Hassan; Zarzoso, Manuel; Bizy, Alexandra; Furness, Jamie; Anumonwo, Justus; Mironov, Sergey; Jalife, José

    2016-04-01

    Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electric impulse propagation velocity and immature action potential profiles. Here, we have identified an optimal extracellular matrix for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal extracellular matrix combination have impulse propagation velocities ≈2× faster than previously reported (43.6±7.0 cm/s; n=9) and have mature cardiomyocyte action potential profiles, including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s; n=5 monolayers). In addition, the optimal extracellular matrix promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1, and connexin43) and myofilament markers (cardiac troponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of β1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase activation prevented structural maturation. Maturation of human stem cell-derived cardiomyocyte monolayers is achieved in a 1-week period by plating cardiomyocytes on PDMS (polydimethylsiloxane) coverslips rather than on conventional 2-dimensional cell culture formats, such as glass coverslips or plastic dishes. Activation of integrin signaling and focal adhesion kinase is essential for significant maturation of human cardiac monolayers. © 2016 American Heart Association, Inc.

  19. Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells.

    Science.gov (United States)

    Kang, N-H; Hwang, K-A; Kim, S U; Kim, Y-B; Hyun, S-H; Jeung, E-B; Choi, K-C

    2012-08-01

    As stem cells are capable of self-renewal and can generate differentiated progenies for organ development, they are considered as potential source for regenerative medicine and tissue replacement after injury or disease. Along with this capacity, stem cells have the therapeutic potential for treating human diseases including cancers. According to the origins, stem cells are broadly classified into two types: embryonic stem cells (ESCs) and adult stem cells. In terms of differentiation potential, ESCs are pluripotent and adult stem cells are multipotent. Amnion, which is a membranous sac that contains the fetus and amniotic fluid and functions in protecting the developing embryo during gestation, is another stem cell source. Amnion-derived stem cells are classified as human amniotic membrane-derived epithelial stem cells, human amniotic membrane-derived mesenchymal stem cells and human amniotic fluid-derived stem cells. They are in an intermediate stage between pluripotent ESCs and lineage-restricted adult stem cells, non-tumorigenic, and contribute to low immunogenicity and anti-inflammation. Furthermore, they are easily available and do not cause any controversial issues in their recovery and applications. Not only are amnion-derived stem cells applicable in regenerative medicine, they have anticancer capacity. In non-engineered stem cells transplantation strategies, amnion-derived stem cells effectively target the tumor and suppressed the tumor growth by expressing cytotoxic cytokines. Additionally, they also have a potential as novel delivery vehicles transferring therapeutic genes to the cancer formation sites in gene-directed enzyme/prodrug combination therapy. Owing to their own advantageous properties, amnion-derived stem cells are emerging as a new candidate in anticancer therapy.

  20. Adipogenic human adenovirus Ad-36 induces commitment, differentiation, and lipid accumulation in human adipose-derived stem cells

    DEFF Research Database (Denmark)

    Pasarica, Magdalena; Mashtalir, Nazar; McAllister, Emily J

    2008-01-01

    Human adenovirus Ad-36 is causatively and correlatively linked with animal and human obesity, respectively. Ad-36 enhances differentiation of rodent preadipocytes, but its effect on adipogenesis in humans is unknown. To indirectly assess the role of Ad-36-induced adipogenesis in human obesity......, the effect of the virus on commitment, differentiation, and lipid accumulation was investigated in vitro in primary human adipose-derived stem/stromal cells (hASC). Ad-36 infected hASC in a time- and dose-dependent manner. Even in the presence of osteogenic media, Ad-36-infected hASC showed significantly...... greater lipid accumulation, suggestive of their commitment to the adipocyte lineage. Even in the absence of adipogenic inducers, Ad-36 significantly increased hASC differentiation, as indicated by a time-dependent expression of genes within the adipogenic cascade-CCAAT/Enhancer binding protein...

  1. Tumor tropism of intravenously injected human-induced pluripotent stem cell-derived neural stem cells and their gene therapy application in a metastatic breast cancer model.

    Science.gov (United States)

    Yang, Jing; Lam, Dang Hoang; Goh, Sally Sallee; Lee, Esther Xingwei; Zhao, Ying; Tay, Felix Chang; Chen, Can; Du, Shouhui; Balasundaram, Ghayathri; Shahbazi, Mohammad; Tham, Chee Kian; Ng, Wai Hoe; Toh, Han Chong; Wang, Shu

    2012-05-01

    Human pluripotent stem cells can serve as an accessible and reliable source for the generation of functional human cells for medical therapies. In this study, we used a conventional lentiviral transduction method to derive human-induced pluripotent stem (iPS) cells from primary human fibroblasts and then generated neural stem cells (NSCs) from the iPS cells. Using a dual-color whole-body imaging technology, we demonstrated that after tail vein injection, these human NSCs displayed a robust migratory capacity outside the central nervous system in both immunodeficient and immunocompetent mice and homed in on established orthotopic 4T1 mouse mammary tumors. To investigate whether the iPS cell-derived NSCs can be used as a cellular delivery vehicle for cancer gene therapy, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected through tail vein into 4T1 tumor-bearing mice. The transduced NSCs were effective in inhibiting the growth of the orthotopic 4T1 breast tumor and the metastatic spread of the cancer cells in the presence of ganciclovir, leading to prolonged survival of the tumor-bearing mice. The use of iPS cell-derived NSCs for cancer gene therapy bypasses the sensitive ethical issue surrounding the use of cells derived from human fetal tissues or human embryonic stem cells. This approach may also help to overcome problems associated with allogeneic transplantation of other types of human NSCs. Copyright © 2012 AlphaMed Press.

  2. Broad T-cell receptor repertoire in T-lymphocytes derived from human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Chia-Wei Chang

    Full Text Available Human induced pluripotent stem cells (hiPSCs have enormous potential for the treatment of inherited and acquired disorders. Recently, antigen-specific T lymphocytes derived from hiPSCs have been reported. However, T lymphocyte populations with broad T cell receptor (TCR diversity have not been generated. We report that hiPSCs derived from skin biopsy are capable of producing T lymphocyte populations with a broad TCR repertoire. In vitro T cell differentiation follows a similar developmental program as observed in vivo, indicated by sequential expression of CD7, intracellular CD3 and surface CD3. The γδ TCR locus is rearranged first and is followed by rearrangement of the αβ locus. Both γδ and αβ T cells display a diverse TCR repertoire. Upon activation, the cells express CD25, CD69, cytokines (TNF-α, IFN-γ, IL-2 and cytolytic proteins (Perforin and Granzyme-B. These results suggest that most, if not all, mechanisms required to generate functional T cells with a broad TCR repertoire are intact in our in vitro differentiation protocol. These data provide a foundation for production of patient-specific T cells for the treatment of acquired or inherited immune disorders and for cancer immunotherapy.

  3. Detection and quantification of microcystins (cyanobacterial hepatotoxins) with recombinant antibody fragments isolated from a naïve human phage display library.

    Science.gov (United States)

    McElhiney, J; Lawton, L A; Porter, A J

    2000-12-01

    Single-chain antibody fragments against the cyanobacterial hepatotoxin microcystin-LR were isolated from a naive human phage display library and expressed in Escherichia coli. In competition enzyme-linked immunosorbent assay (ELISA), the most sensitive antibody clone selected from the library detected free microcystin-LR with an IC(50) value of 4 microM. It was found to cross react with three other microcystin variants - microcystin-RR, microcystin-LW and microcystin-LF - and detected microcystins in extracts of the cyanobacterium Microcystis aeruginosa, found to contain the toxins by high-performance liquid chromatography (HPLC). The quantification of microcystins in these extracts by ELISA and HPLC showed good correlation. Although the antibody isolated in this study was considerably less sensitive than the polyclonal and monoclonal antibodies already available for microcystin detection, phage display technology represents a cheaper, more rapid alternative for the production of anti-microcystin antibodies than the methods currently in use.

  4. In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells

    International Nuclear Information System (INIS)

    Kadivar, Mehdi; Khatami, Shohreh; Mortazavi, Yousef; Shokrgozar, Mohammad Ali; Taghikhani, Mohammad; Soleimani, Masoud

    2006-01-01

    Cardiomyocyte loss in the ischemically injured human heart often leads to irreversible defects in cardiac function. Recently, cellular cardiomyoplasty with mesenchymal stem cells, which are multipotent cells with the ability to differentiate into specialized cells under appropriate stimuli, has emerged as a new approach for repairing damaged myocardium. In the present study, the potential of human umbilical cord-derived mesenchymal stem cells to differentiate into cells with characteristics of cardiomyocyte was investigated. Mesenchymal stem cells were isolated from endothelial/subendothelial layers of the human umbilical cords using a method similar to that of human umbilical vein endothelial cell isolation. Isolated cells were characterized by transdifferentiation ability to adipocytes and osteoblasts, and also with flow cytometry analysis. After treatment with 5-azacytidine, the human umbilical cord-derived mesenchymal stem cells were morphologically transformed into cardiomyocyte-like cells and expressed cardiac differentiation markers. During the differentiation, cells were monitored by a phase contrast microscope and their morphological changes were demonstrated. Immunostaining of the differentiated cells for sarcomeric myosin (MF20), desmin, cardiac troponin I, and sarcomeric α-actinin was positive. RT-PCR analysis showed that these differentiated cells express cardiac-specific genes. Transmission electron microscopy revealed a cardiomyocyte-like ultrastructure and typical sarcomers. These observations confirm that human umbilical cord-derived mesenchymal stem cells can be chemically transformed into cardiomyocytes and can be considered as a source of cells for cellular cardiomyoplasty

  5. Electrophysiological analysis of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) using multi-electrode arrays (MEAs)

    NARCIS (Netherlands)

    Sala, Luca; Ward-van Oostwaard, Dorien; Tertoolen, Leon G.J.; Mummery, Christine L.; Bellin, Milena

    2017-01-01

    Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes.

  6. Gene expression profiling and secretome analysis differentiate adult-derived human liver stem/progenitor cells and human hepatic stellate cells.

    Directory of Open Access Journals (Sweden)

    Silvia Berardis

    Full Text Available Adult-derived human liver stem/progenitor cells (ADHLSC are obtained after primary culture of the liver parenchymal fraction. The cells are of fibroblastic morphology and exhibit a hepato-mesenchymal phenotype. Hepatic stellate cells (HSC derived from the liver non-parenchymal fraction, present a comparable morphology as ADHLSC. Because both ADHLSC and HSC are described as liver stem/progenitor cells, we strived to extensively compare both cell populations at different levels and to propose tools demonstrating their singularity. ADHLSC and HSC were isolated from the liver of four different donors, expanded in vitro and followed from passage 5 until passage 11. Cell characterization was performed using immunocytochemistry, western blotting, flow cytometry, and gene microarray analyses. The secretion profile of the cells was evaluated using Elisa and multiplex Luminex assays. Both cell types expressed α-smooth muscle actin, vimentin, fibronectin, CD73 and CD90 in accordance with their mesenchymal origin. Microarray analysis revealed significant differences in gene expression profiles. HSC present high expression levels of neuronal markers as well as cytokeratins. Such differences were confirmed using immunocytochemistry and western blotting assays. Furthermore, both cell types displayed distinct secretion profiles as ADHLSC highly secreted cytokines of therapeutic and immuno-modulatory importance, like HGF, interferon-γ and IL-10. Our study demonstrates that ADHLSC and HSC are distinct liver fibroblastic cell populations exhibiting significant different expression and secretion profiles.

  7. Transcriptomic analysis of monocytes and macrophages derived from CLL patients which display differing abilities to respond to therapeutic antibody immune complexes

    Directory of Open Access Journals (Sweden)

    M. Burgess

    2016-03-01

    Full Text Available Chronic lymphocytic leukemia (CLL is the most common adult leukemia. While therapeutic antibodies show clinical activity in CLL patients, resistance inevitably develops resulting in treatment failure. Identifying mechanisms of antibody resistance and methods to reduce resistance would be valuable in managing CLL. Monocyte derived cells (MDCs, also known as nurse like cells (NLCs in CLL [1,2], are known to be crucial components of the CLL microenvironment network and following “maturation” in in vitro culture systems are able to provide support for the survival of the malignant B cells from CLL patients. In addition to their protective role, MDCs are key effector cells in mediating responses to therapeutic antibody therapies [3]. We have determined that macrophages from patients with early stable CLL are able to elicit superior cytotoxic response to therapeutic antibodies than macrophages derived from patients with progressive CLL. We have exploited this unique finding to gain insight into antibody resistance. Thus, we have profiled monocytes on day 0 and MDCs on day 7 from antibody sensitive and antibody resistant CLL patients (GEO accession number GEO: GSE71409. We show that there are no significant differences in transcriptomes from the monocytes or MDCs derived from sensitive or resistant patient samples. However, we show that MDCs acquire an M2-like macrophage transcriptomic signature following 7 days culture regardless of whether they were derived from sensitive or resistant patient samples. Keywords: Chronic lymphocytic leukemia, Monocyte derived cells, Antibody resistance, Microarray

  8. Human heart disease : lessons from human pluripotent stem cell-derived cardiomyocytes

    NARCIS (Netherlands)

    Giacomelli, E.; Mummery, C.L.; Bellin, M.

    2017-01-01

    Technical advances in generating and phenotyping cardiomyocytes from human pluripotent stem cells (hPSC-CMs) are now driving their wider acceptance as in vitro models to understand human heart disease and discover therapeutic targets that may lead to new compounds for clinical use. Current

  9. 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...

  10. A genome-wide comparison of mesenchymal stem cells derived from human placenta and umbilical cord

    Directory of Open Access Journals (Sweden)

    Sen-Wen Teng

    2017-10-01

    Conclusion: We identified the consistence and specific DEGs of human placenta and umbilical cord based on the genome-wide comparison. Our results indicated that hMSCs derived from umbilical cord and placenta have different gene expression patterns, and most of specific genes are involved in the cell cycle, cell division, cell death, and cell developmental processes.

  11. Expanding the Tissue Toolbox : Deriving Colon Tissue from Human Pluripotent Stem Cells

    NARCIS (Netherlands)

    Bruens, Lotte; Snippert, Hugo J.G.

    2017-01-01

    Organoid technology holds great potential for disease modeling and regenerative medicine. In this issue of Cell Stem Cell, Múnera et al. (2017) establish the generation of pluripotent stem cell-derived colon organoids that upon transplantation in mice, resembling human colon to a large extent,

  12. Functional Studies of Missense TREM2 Mutations in Human Stem Cell-Derived Microglia

    Directory of Open Access Journals (Sweden)

    Philip W. Brownjohn

    2018-04-01

    Full Text Available Summary: The derivation of microglia from human stem cells provides systems for understanding microglial biology and enables functional studies of disease-causing mutations. We describe a robust method for the derivation of human microglia from stem cells, which are phenotypically and functionally comparable with primary microglia. We used stem cell-derived microglia to study the consequences of missense mutations in the microglial-expressed protein triggering receptor expressed on myeloid cells 2 (TREM2, which are causal for frontotemporal dementia-like syndrome and Nasu-Hakola disease. We find that mutant TREM2 accumulates in its immature form, does not undergo typical proteolysis, and is not trafficked to the plasma membrane. However, in the absence of plasma membrane TREM2, microglia differentiate normally, respond to stimulation with lipopolysaccharide, and are phagocytically competent. These data indicate that dementia-associated TREM2 mutations have subtle effects on microglia biology, consistent with the adult onset of disease in individuals with these mutations. : Brownjohn and colleagues report methods to generate microglia from induced pluripotent human stem cells, which they demonstrate are highly similar to cultured primary human microglia. Microglia differentiated from patient-derived stem cells carrying neurological disease-causing mutations in the TREM2 receptor differentiate normally and respond appropriately to pathogenic stimuli, despite the absence of functional TREM2 receptor on the plasma membrane. Keywords: dementia, microglia, TREM2, Nasu-Hakola disease, frontotemporal dementia, iPSC-microglia, neuroinflammation

  13. Human obesity associated with an intronic SNP in the brain-derived neurotrophic factor locus

    Science.gov (United States)

    Brain-derived neurotrophic factor (BDNF) plays a key role in energy balance. In population studies, SNPs of the BDNF locus have been linked to obesity, but the mechanism by which these variants cause weight gain is unknown. Here, we examined human hypothalamic BDNF expression in association with 44 ...

  14. Brain-derived neurotrophic factor in human subjects with function-altering melanocortin-4 receptor variants

    Science.gov (United States)

    In rodents, hypothalamic brain-derived neurotrophic factor (BDNF) expression appears to be regulated by melanocortin-4 receptor (MC4R) activity. The impact of MC4R genetic variation on circulating BDNF in humans is unknown. The objective of this study is to compare BDNF concentrations of subjects wi...

  15. Guidance document on the derivation of maximum permissible risk levels for human intake of soil contaminants

    NARCIS (Netherlands)

    Janssen PJCM; Speijers GJA; CSR

    1997-01-01

    This report contains a basic step-to-step description of the procedure followed in the derivation of the human-toxicological Maximum Permissible Risk (MPR ; in Dutch: Maximum Toelaatbaar Risico, MTR) for soil contaminants. In recent years this method has been applied for a large number of compounds

  16. Embryonic stem cell-like cells derived from adult human testis

    NARCIS (Netherlands)

    Mizrak, S. C.; Chikhovskaya, J. V.; Sadri-Ardekani, H.; van Daalen, S.; Korver, C. M.; Hovingh, S. E.; Roepers-Gajadien, H. L.; Raya, A.; Fluiter, K.; de Reijke, Th M.; de la Rosette, J. J. M. C. H.; Knegt, A. C.; Belmonte, J. C.; van der Veen, F.; de rooij, D. G.; Repping, S.; van Pelt, A. M. M.

    2010-01-01

    Given the significant drawbacks of using human embryonic stem (hES) cells for regenerative medicine, the search for alternative sources of multipotent cells is ongoing. Studies in mice have shown that multipotent ES-like cells can be derived from neonatal and adult testis. Here we report the

  17. Sequential use of human-derived medium supplements favours cardiovascular tissue engineering

    NARCIS (Netherlands)

    Riem Vis, P.W.; Sluijter, J.P.G.; Soekhradj - Soechit, R.S.; Herwerden, van L.A.; Kluin, J.; Bouten, C.V.C.

    2012-01-01

    For clinical application of tissue engineering strategies, the use of animal-derived serum in culture medium is not recommended, because it can evoke immune responses in patients. We previously observed that human platelet-lysate (PL) is favourable for cell expansion, but generates weaker tissue as

  18. Activated human mast cells induce LOX-1-specific scavenger receptor expression in human monocyte-derived macrophages.

    Directory of Open Access Journals (Sweden)

    Mervi Alanne-Kinnunen

    Full Text Available Activated mast cells in atherosclerotic lesions degranulate and release bioactive compounds capable of regulating atherogenesis. Here we examined the ability of activated human primary mast cells to regulate the expression of the major scavenger receptors in cultured human primary monocyte-derived macrophages (HMDMs.Components released by immunologically activated human primary mast cells induced a transient expression of lectin-like oxidized LDL receptor (LOX-1 mRNA in HMDMs, while the expression of two other scavenger receptors, MSR1 and CD36, remained unaffected. The LOX-1-inducing secretory components were identified as histamine, tumor necrosis factor alpha (TNF-α, and transforming growth factor beta (TGF-β1, which exhibited a synergistic effect on LOX-1 mRNA expression. Histamine induced a transient expression of LOX-1 protein. Mast cell -induced increase in LOX-1 expression was not associated with increased uptake of oxidized LDL by the macrophages.Mast cell-derived histamine, TNF-α, and TGF-β1 act in concert to induce a transient increase in LOX-1 expression in human primary monocyte-derived macrophages. The LOX-1-inducing activity potentially endows mast cells a hitherto unrecognized role in the regulation of innate immune reactions in atherogenesis.

  19. Chondrogenic potential of physically treated bovine cartilage matrix derived porous scaffolds on human dermal fibroblast cells.

    Science.gov (United States)

    Moradi, Ali; Ataollahi, Forough; Sayar, Katayoun; Pramanik, Sumit; Chong, Pan-Pan; Khalil, Alizan Abdul; Kamarul, Tunku; Pingguan-Murphy, Belinda

    2016-01-01

    Extracellular matrices have drawn attention in tissue engineering as potential biomaterials for scaffold fabrication because of their bioactive components. Noninvasive techniques of scaffold fabrication and cross-linking treatments are believed to maintain the integrity of bioactive molecules while providing proper architectural and mechanical properties. Cartilage matrix derived scaffolds are designed to support the maintenance of chondrocytes and provide proper signals for differentiation of chondroinducible cells. Chondroinductive potential of bovine articular cartilage matrix derived porous scaffolds on human dermal fibroblasts and the effect of scaffold shrinkage on chondrogenesis were investigated. An increase in sulfated glycosaminoglycans production along with upregulation of chondrogenic genes confirmed that physically treated cartilage matrix derived scaffolds have chondrogenic potential on human dermal fibroblasts. © 2015 Wiley Periodicals, Inc.

  20. PreproVIP-derived peptides in the human female genital tract: expression and biological function

    DEFF Research Database (Denmark)

    Bredkjoer, H E; Palle, C; Ekblad, E

    1997-01-01

    The aim of the study was to elucidate the localization, distribution, colocalization and biological effect of preproVIP-derived peptides in the human female genital tract. Radioimmunoassays applying antisera against the five functional domains of the VIP precursor in combination with immunohistoc......The aim of the study was to elucidate the localization, distribution, colocalization and biological effect of preproVIP-derived peptides in the human female genital tract. Radioimmunoassays applying antisera against the five functional domains of the VIP precursor in combination...... with immunohistochemistry were used. The effect of preproVIP 22-79, preproVIP 111-122 and preproVIP 156-170 on genital smooth muscle activity in the Fallopian tube was investigated in vitro and compared to that of VIP. All the preproVIP-derived peptides were expressed throughout the genital tract in neuronal elements...

  1. Investigation of Virtual Digital Human and Robotic Device Technology Merger Complimented by Haptics and Autostereoscopic Displays, Phase II

    Data.gov (United States)

    National Aeronautics and Space Administration — As expected, the STTR Phase I investigation confirmed that the Digital Virtual Human (DVH) and Robonaut technologies can be merged, and that haptic and...

  2. Investigation of Virtual Digital Human and Robotic Device Technology Merger Complimented by Haptics and Autostereoscopic Displays, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — The proposed innovations conform precisely to the technology needs described in Subtopic T5.02, Robotics and Virtual Digital Human Technologies. ?Two potential areas...

  3. Human-derived nanoparticles and vascular response to injury in rabbit carotid arteries: Proof of principle

    Directory of Open Access Journals (Sweden)

    Maria A K Schwartz

    2008-06-01

    Full Text Available Maria A K Schwartz1, John C Lieske2, Vivek Kumar2, Gerard Farell-Baril2, Virginia M Miller1,31Departments of Physiology and Biomedical Engineering, Internal Medicine; 2Division of Nephrology, and 3Surgery, Mayo Clinic College of Medicine, Rochester, MN, USAAbstract: Self-calcifying, self-replicating nanoparticles have been isolated from calcified human tissues. However, it is unclear if these nanoparticles participate in disease processes. Therefore, this study was designed to preliminarily test the hypothesis that human-derived nanoparticles are causal to arterial disease processes. One carotid artery of 3 kg male rabbits was denuded of endothelium; the contralateral artery remained unoperated as a control. Each rabbit was injected intravenously with either saline, calcified, or decalcified nanoparticles cultured from calcified human arteries or kidney stones. After 35 days, both injured and control arteries were removed for histological examination. Injured arteries from rabbits injected with saline showed minimal, eccentric intimal hyperplasia. Injured arteries from rabbits injected with calcified kidney stone- and arterial-derived nanoparticles occluded, sometimes with canalization. The calcified kidney stone-derived nanoparticles caused calcifications within the occlusion. Responses to injury in rabbits injected with decalcified kidney stone-derived nanoparticles were similar to those observed in saline-injected animals. However, decalcified arterial-derived nanoparticles produced intimal hyperplasia that varied from moderate to occlusion with canalization and calcifi cation. This study offers the first evidence that there may be a causal relationship between human-derived nanoparticles and response to injury including calcification in arteries with damaged endothelium.Keywords: arterial calcification, endothelial injury, intimal hyperplasia

  4. Characterization of Induced Pluripotent Stem Cell-derived Human Serotonergic Neurons

    Directory of Open Access Journals (Sweden)

    Lining Cao

    2017-05-01

    Full Text Available In the brain, the serotonergic neurons located in the raphe nucleus are the unique resource of the neurotransmitter serotonin, which plays a pivotal role in the regulation of brain development and functions. Dysfunction of the serotonin system is present in many psychiatric disorders. Lack of in vitro functional human model limits the understanding of human central serotonergic system and its related diseases and clinical applications. Previously, we have developed a method generating human serotonergic neurons from induced pluripotent stem cells (iPSCs. In this study, we analyzed the features of these human iPSCs-derived serotonergic neurons both in vitro and in vivo. We found that these human serotonergic neurons are sensitive to the selective neurotoxin 5, 7-Dihydroxytryptamine (5,7-DHT in vitro. After being transplanted into newborn mice, the cells not only expressed their typical molecular markers, but also showed the migration and projection to the host’s cerebellum, hindbrain and spinal cord. The data demonstrate that these human iPSCs-derived neurons exhibit the typical features as the serotonergic neurons in the brain, which provides a solid foundation for studying on human serotonin system and its related disorders.

  5. Elucidation of the binding mechanism of coumarin derivatives with human serum albumin.

    Directory of Open Access Journals (Sweden)

    Archit Garg

    Full Text Available Coumarin is a benzopyrone which is widely used as an anti-coagulant, anti-oxidant, anti-cancer and also to cure arthritis, herpes, asthma and inflammation. Here, we studied the binding of synthesized coumarin derivatives with human serum albumin (HSA at physiological pH 7.2 by using fluorescence spectroscopy, circular dichroism spectroscopy, molecular docking and molecular dynamics simulation studies. By addition of coumarin derivatives to HSA the maximum fluorescence intensity was reduced due to quenching of intrinsic fluorescence upon binding of coumarin derivatives to HSA. The binding constant and free energy were found to be 1.957±0.01×10(5 M(-1, -7.175 Kcal M(-1 for coumarin derivative (CD enamide; 0.837±0.01×10(5 M(-1, -6.685 Kcal M(-1 for coumarin derivative (CD enoate, and 0.606±0.01×10(5 M(-1, -6.49 Kcal M(-1 for coumarin derivative methylprop (CDM enamide. The CD spectroscopy showed that the protein secondary structure was partially unfolded upon binding of coumarin derivatives. Further, the molecular docking studies showed that coumarin derivatives were binding to HSA at sub-domain IB with the hydrophobic interactions and also with hydrogen bond interactions. Additionally, the molecular dynamics simulations studies contributed in understanding the stability of protein-drug complex system in the aqueous solution and the conformational changes in HSA upon binding of coumarin derivatives. This study will provide insights into designing of the new inspired coumarin derivatives as therapeutic agents against many life threatening diseases.

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

    DEFF Research Database (Denmark)

    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....

  7. The effect of magnesium ion implantation into alumina upon the adhesion of human bone derived cells

    International Nuclear Information System (INIS)

    Howlett, C.R.; Zreiqat, H.; O'Dell, R.; Noorman, J.; Evans, P.; Dalton, B.A.; McFarland, C.; Steele, J.G.

    1994-01-01

    Our group is investigating the potential of modifying the surface atomic layers of biomaterials by ion beam implantation in order to stimulate adhesion of bone cells to these treated biomaterials. In this study alumina that had been implanted with magnesium ions (Mg)-(Al 2 O 3 ), was compared to unmodified alumina (Al 2 O 3 ) for the adhesion of cells cultured from explanted human bone. The attachment and spreading of cultured human bone derived cells onto (Mg)-(Al 2 O 3 ) was significantly enhanced as compared to Al 2 O 3 . The role of adsorption of serum adhesive glycoproteins firbronectin (Fn) and vitronectin (Vn) in the adhesion of human bone derived cells to (Mg)-(Al 2 O 3 ) was determined. (Author)

  8. Neuromuscular junction formation between human stem cell-derived motoneurons and human skeletal muscle in a defined system.

    Science.gov (United States)

    Guo, Xiufang; Gonzalez, Mercedes; Stancescu, Maria; Vandenburgh, Herman H; Hickman, James J

    2011-12-01

    Functional in vitro models composed of human cells will constitute an important platform in the next generation of system biology and drug discovery. This study reports a novel human-based in vitro Neuromuscular Junction (NMJ) system developed in a defined serum-free medium and on a patternable non-biological surface. The motoneurons and skeletal muscles were derived from fetal spinal stem cells and skeletal muscle stem cells. The motoneurons and skeletal myotubes were completely differentiated in the co-culture based on morphological analysis and electrophysiology. NMJ formation was demonstrated by phase contrast microscopy, immunocytochemistry and the observation of motoneuron-induced muscle contractions utilizing time-lapse recordings and their subsequent quenching by d-Tubocurarine. Generally, functional human based systems would eliminate the issue of species variability during the drug development process and its derivation from stem cells bypasses the restrictions inherent with utilization of primary human tissue. This defined human-based NMJ system is one of the first steps in creating functional in vitro systems and will play an important role in understanding NMJ development, in developing high information content drug screens and as test beds in preclinical studies for spinal or muscular diseases/injuries such as muscular dystrophy, Amyotrophic lateral sclerosis and spinal cord repair. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Augmenting digital displays with computation

    Science.gov (United States)

    Liu, Jing

    As we inevitably step deeper and deeper into a world connected via the Internet, more and more information will be exchanged digitally. Displays are the interface between digital information and each individual. Naturally, one fundamental goal of displays is to reproduce information as realistically as possible since humans still care a lot about what happens in the real world. Human eyes are the receiving end of such information exchange; therefore it is impossible to study displays without studying the human visual system. In fact, the design of displays is rather closely coupled with what human eyes are capable of perceiving. For example, we are less interested in building displays that emit light in the invisible spectrum. This dissertation explores how we can augment displays with computation, which takes both display hardware and the human visual system into consideration. Four novel projects on display technologies are included in this dissertation: First, we propose a software-based approach to driving multiview autostereoscopic displays. Our display algorithm can dynamically assign views to hardware display zones based on multiple observers' current head positions, substantially reducing crosstalk and stereo inversion. Second, we present a dense projector array that creates a seamless 3D viewing experience for multiple viewers. We smoothly interpolate the set of viewer heights and distances on a per-vertex basis across the arrays field of view, reducing image distortion, crosstalk, and artifacts from tracking errors. Third, we propose a method for high dynamic range display calibration that takes into account the variation of the chrominance error over luminance. We propose a data structure for enabling efficient representation and querying of the calibration function, which also allows user-guided balancing between memory consumption and the amount of computation. Fourth, we present user studies that demonstrate that the ˜ 60 Hz critical flicker fusion

  10. Generation, purification and transplantation of photoreceptors derived from human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Deepak A Lamba

    2010-01-01

    Full Text Available Inherited and acquired retinal degenerations are frequent causes of visual impairment and photoreceptor cell replacement therapy may restore visual function to these individuals. To provide a source of new retinal neurons for cell based therapies, we developed methods to derive retinal progenitors from human ES cells.In this report we have used a similar method to direct induced pluripotent stem cells (iPS from human fibroblasts to a retinal progenitor fate, competent to generate photoreceptors. We also found we could purify the photoreceptors derived from the iPS cells using fluorescence activated cell sorting (FACS after labeling photoreceptors with a lentivirus driving GFP from the IRBP cis-regulatory sequences. Moreover, we found that when we transplanted the FACS purified iPSC derived photoreceptors, they were able to integrate into a normal mouse retina and express photoreceptor markers.This report provides evidence that enriched populations of human photoreceptors can be derived from iPS cells.

  11. Exosomes Derived from Human Bone Marrow Mesenchymal Stem Cells Promote Tumor Growth Through Hedgehog Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Jin Qi

    2017-08-01

    Full Text Available Background/Aims: Mesenchymal stem/stromal cells (MSCs are known to home to sites of tumor microenvironments where they participate in the formation of the tumor microenvironment and to interplay with tumor cells. However, the potential functional effects of MSCs on tumor cell growth are controversial. Here, we, from the view of bone marrow MSC-derived exosomes, study the molecular mechanism of MSCs on the growth of human osteosarcoma and human gastric cancer cells. Methods: MSCs derived from human bone marrow (hBMSCs were isolated and cultured in complete DMEM/F12 supplemented with 10% exosome-depleted fetal bovine serum and 1% penicillin-streptomycin, cell culture supernatants containing exosomes were harvested and exosome purification was performed by ultracentrifugation. Osteosarcoma (MG63 and gastric cancer (SGC7901 cells, respectively, were treated with hBMSC-derived exosomes in the presence or absence of a small molecule inhibitor of Hedgehog pathway. Cell viability was measured by transwell invasion assay, scratch migration assay and CCK-8 test. The expression of the signaling molecules Smoothened, Patched-1, Gli1 and the ligand Shh were tested by western blot and RT-PCR. Results: In this study, we found that hBMSC-derived exosomes promoted MG63 and SGC7901 cell growth through the activation of Hedgehog signaling pathway. Inhibition of Hedgehog signaling pathway significantly suppressed the process of hBMSC-derived exosomes on tumor growth. Conclusion: Our findings demonstrated the new roles of hedgehog signaling pathway in the hBMSCs-derived exosomes induced tumor progression.

  12. Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV

    Directory of Open Access Journals (Sweden)

    Hust Michael

    2008-09-01

    Full Text Available Abstract Background Venezuelan equine encephalitis virus (VEEV belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV. Results In this work, human anti-VEEV single chain Fragments variable (scFv were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV and Eastern equine encephalitis virus (EEEV antigenic complex, nor did they react with Chikungunya virus (CHIKV, if they were used as detection reagent. Conclusion For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak.

  13. Human platelet lysate improves human cord blood derived ECFC survival and vasculogenesis in three dimensional (3D) collagen matrices.

    Science.gov (United States)

    Kim, Hyojin; Prasain, Nutan; Vemula, Sasidhar; Ferkowicz, Michael J; Yoshimoto, Momoko; Voytik-Harbin, Sherry L; Yoder, Mervin C

    2015-09-01

    Human cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Since diminished ECFC survival is known to dampen the vasculogenic response in vivo, we tested how long implanted ECFC survive and generate vessels in three-dimensional (3D) type I collagen matrices in vitro and in vivo. We hypothesized that human platelet lysate (HPL) would promote cell survival and enhance vasculogenesis in the 3D collagen matrices. We report that the percentage of ECFC co-cultured with HPL that were alive was significantly enhanced on days 1 and 3 post-matrix formation, compared to ECFC alone containing matrices. Also, co-culture of ECFC with HPL displayed significantly more vasculogenic activity compared to ECFC alone and expressed significantly more pro-survival molecules (pAkt, p-Bad and Bcl-xL) in the 3D collagen matrices in vitro. Treatment with Akt1 inhibitor (A-674563), Akt2 inhibitor (CCT128930) and Bcl-xL inhibitor (ABT-263/Navitoclax) significantly decreased the cell survival and vasculogenesis of ECFC co-cultured with or without HPL and implicated activation of the Akt1 pathway as the critical mediator of the HPL effect on ECFC in vitro. A significantly greater average vessel number and total vascular area of human CD31(+) vessels were present in implants containing ECFC and HPL, compared to the ECFC alone implants in vivo. We conclude that implantation of ECFC with HPL in vivo promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Derivation of the clinical grade human embryonic stem cell line RCe016-A (RC-12

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe016-A (RC-12 was derived under quality assured compliance with UK regulations, EU Directives and International guidance for tissue procurement, processing and storage according to good manufacturing practice (GMP standards. The cell line was derived from a cryopreserved blastocyst stage embryo voluntarily donated as surplus to fertility requirements following informed consent. RCe016-A (RC-12 shows normal pluripotency marker expression and differentiation to three germ layers in vitro. Karyology revealed a mixed male karyotype at early passage (P15, which resolved as normal 46XY by passage 33. Microsatellite PCR identity, HLA and blood group typing data is available.

  15. Ketamine Causes Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Neurons

    Science.gov (United States)

    Ito, Hiroyuki; Uchida, Tokujiro; Makita, Koshi

    2015-01-01

    Purpose Ketamine toxicity has been demonstrated in nonhuman mammalian neurons. To study the toxic effect of ketamine on human neurons, an experimental model of cultured neurons from human induced pluripotent stem cells (iPSCs) was examined, and the mechanism of its toxicity was investigated. Methods Human iPSC-derived dopaminergic neurons were treated with 0, 20, 100 or 500 μM ketamine for 6 and 24 h. Ketamine toxicity was evaluated by quantification of caspase 3/7 activity, reactive oxygen species (ROS) production, mitochondrial membrane potential, ATP concentration, neurotransmitter reuptake activity and NADH/NAD+ ratio. Mitochondrial morphological change was analyzed by transmission electron microscopy and confocal microscopy. Results Twenty-four-hour exposure of iPSC-derived neurons to 500 μM ketamine resulted in a 40% increase in caspase 3/7 activity (P ketamine (100 μM) decreased the ATP level (22%, P ketamine concentration, which suggests that mitochondrial dysfunction preceded ROS generation and caspase activation. Conclusions We established an in vitro model for assessing the neurotoxicity of ketamine in iPSC-derived neurons. The present data indicate that the initial mitochondrial dysfunction and autophagy may be related to its inhibitory effect on the mitochondrial electron transport system, which underlies ketamine-induced neural toxicity. Higher ketamine concentration can induce ROS generation and apoptosis in human neurons. PMID:26020236

  16. Derivation and characterization of human embryonic stem cell lines from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhao Wu; Huimin Dai; Lei Qian; Qing Tian; Lei Xiao; Xiaojun Tan; Hui Li; Lingjun Rao; Lixiazi He; Lei Bao; Jing Liao; Chun Cui; Zhenyu Zuo; Qiao Li

    2011-01-01

    Human embryonic stem cells (hESCs) can self-renew indefinitely and differentiate into all cell types in the human body. Therefore, they are valuable in regenerative medicine, human developmental biology and drug discovery. A number of hESC lines have been derived from the Chinese population,but limited of them are available for research purposes. Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin. These hESCs express alkaline phosphatase and hESC-specific markers, including Oct4, Nanog, SSEA-3, SSEA-4,TRA-1-60 and TRA-1-81. They also have high levels of telomerase activity and normal karyotypes. These cells can form embryoid body in vitro and can be differentiated into all three germ layers in vivo by teratoma formation. The newly established hESCs will be distributed for research purposes.The availability of hESC lines from the Chinese population will facilitate studies on the differences in hESCs from different ethnic groups.

  17. Replication of human noroviruses in stem cell-derived human enteroids

    Science.gov (United States)

    The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the leading cause of gastroenteritis worldwide. We report successful cultivation of multiple HuNoV strains in...

  18. Mechanism of transfer of LDL-derived free cholesterol to HDL subfractions in human plasma

    International Nuclear Information System (INIS)

    Miida, T.; Fielding, C.J.; Fielding, P.E.

    1990-01-01

    The transfer of [ 3 H]cholesterol in low-density lipoprotein (LDL) to different high-density lipoprotein (HDL) species in native human plasma was determined by using nondenaturing two-dimensional electrophoresis. Transfer from LDL had a t 1/2 at 37 degree C of 51 ± 8 min and an activation energy of 18.0 kCal mol -1 . There was unexpected specificity among HDL species as acceptors of LDL-derived labeled cholesterol. The largest fraction of the major α-migrating class (HDL 2b ) was the major initial acceptor of LDL-derived cholesterol. Kinetic analysis indicated a rapid secondary transfer from HDL 2b to smaller αHDL (particularly HDL 3 ) driven enzymatically by the lecithin-cholesterol acyltransferase reaction. Rates of transfer among αHDL were most rapid from the largest αHDL fraction (HDL 2b ), suggesting possible protein-mediated facilitation. Simultaneous measurements of the transport of LDL-derived and cell-derived isotopic cholesterol indicated that the former preferably utilized the αHDL pathyway, with little label in pre-βHDL. The same experiments confirmed earlier data that cell-derived cholesterol is preferentially channeled through pre-βHDL. The authors suggest that the functional heterogeneity of HDL demonstrated here includes the ability to independently process cell- and LDL-derived free cholesterol

  19. Caspase-1 from Human Myeloid-Derived Suppressor Cells Can Promote T Cell-Independent Tumor Proliferation.

    Science.gov (United States)

    Zeng, Qi; Fu, Juan; Korrer, Michael; Gorbounov, Mikhail; Murray, Peter J; Pardoll, Drew; Masica, David L; Kim, Young J

    2018-05-01

    Immunosuppressive myeloid-derived suppressive cells (MDSCs) are characterized by their phenotypic and functional heterogeneity. To better define their T cell-independent functions within the tumor, sorted monocytic CD14 + CD11b + HLA-DR low/- MDSCs (mMDSC) from squamous cell carcinoma patients showed upregulated caspase-1 activity, which was associated with increased IL1β and IL18 expression. In vitro studies demonstrated that mMDSCs promoted caspase-1-dependent proliferation of multiple squamous carcinoma cell lines in both human and murine systems. In vivo , growth rates of B16, MOC1, and Panc02 were significantly blunted in chimeric mice adoptively transferred with caspase-1 null bone marrow cells under T cell-depleted conditions. Adoptive transfer of wild-type Gr-1 + CD11b + MDSCs from tumor-bearing mice reversed this antitumor response, whereas caspase-1 inhibiting thalidomide-treated MDSCs phenocopied the antitumor response found in caspase-1 null mice. We further hypothesized that MDSC caspase-1 activity could promote tumor-intrinsic MyD88-dependent carcinogenesis. In mice with wild-type caspase-1, MyD88-silenced tumors displayed reduced growth rate, but in chimeric mice with caspase-1 null bone marrow cells, MyD88-silenced tumors did not display differential tumor growth rate. When we queried the TCGA database, we found that caspase-1 expression is correlated with overall survival in squamous cell carcinoma patients. Taken together, our findings demonstrated that caspase-1 in MDSCs is a direct T cell-independent mediator of tumor proliferation. Cancer Immunol Res; 6(5); 566-77. ©2018 AACR . ©2018 American Association for Cancer Research.

  20. Essential oils of culinary herbs and spices display agonist and antagonist activities at human aryl hydrocarbon receptor AhR.

    Science.gov (United States)

    Bartoňková, Iveta; Dvořák, Zdeněk

    2018-01-01

    Essential oils (EOs) of culinary herbs and spices are used to flavor, color and preserve foods and drinks. Dietary intake of EOs is significant, deserving an attention of toxicologists. We examined the effects of 31 EOs of culinary herbs and spices on the transcriptional activity of human aryl hydrocarbon receptor (AhR), which is a pivotal xenobiotic sensor, having also multiple roles in human physiology. Tested EOs were sorted out into AhR-inactive ones (14 EOs) and AhR-active ones, including full agonists (cumin, jasmine, vanilla, bay leaf), partial agonists (cloves, dill, thyme, nutmeg, oregano) and antagonists (tarragon, caraway, turmeric, lovage, fennel, spearmint, star anise, anise). Major constituents (>10%) of AhR-active EOs were studied in more detail. We identified AhR partial agonists (carvacrol, ligustilide, eugenol, eugenyl acetate, thymol, ar-turmerone) and antagonists (trans-anethole, butylidine phtalide, R/S-carvones, p-cymene), which account for AhR-mediated activities of EOs of fennel, anise, star anise, caraway, spearmint, tarragon, cloves, dill, turmeric, lovage, thyme and oregano. We also show that AhR-mediated effects of some individual constituents of EOs differ from those manifested in mixtures. In conclusion, EOs of culinary herbs and spices are agonists and antagonists of human AhR, implying a potential for food-drug interactions and interference with endocrine pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. cGAS Senses Human Cytomegalovirus and Induces Type I Interferon Responses in Human Monocyte-Derived Cells.

    Directory of Open Access Journals (Sweden)

    Jennifer Paijo

    2016-04-01

    Full Text Available Human cytomegalovirus (HCMV infections of healthy individuals are mostly unnoticed and result in viral latency. However, HCMV can also cause devastating disease, e.g., upon reactivation in immunocompromised patients. Yet, little is known about human immune cell sensing of DNA-encoded HCMV. Recent studies indicated that during viral infection the cyclic GMP/AMP synthase (cGAS senses cytosolic DNA and catalyzes formation of the cyclic di-nucleotide cGAMP, which triggers stimulator of interferon genes (STING and thus induces antiviral type I interferon (IFN-I responses. We found that plasmacytoid dendritic cells (pDC as well as monocyte-derived DC and macrophages constitutively expressed cGAS and STING. HCMV infection further induced cGAS, whereas STING expression was only moderately affected. Although pDC expressed particularly high levels of cGAS, and the cGAS/STING axis was functional down-stream of STING, as indicated by IFN-I induction upon synthetic cGAMP treatment, pDC were not susceptible to HCMV infection and mounted IFN-I responses in a TLR9-dependent manner. Conversely, HCMV infected monocyte-derived cells synthesized abundant cGAMP levels that preceded IFN-I production and that correlated with the extent of infection. CRISPR/Cas9- or siRNA-mediated cGAS ablation in monocytic THP-1 cells and primary monocyte-derived cells, respectively, impeded induction of IFN-I responses following HCMV infection. Thus, cGAS is a key sensor of HCMV for IFN-I induction in primary human monocyte-derived DC and macrophages.

  2. Human induced pluripotent stem cell (hiPSC)-derived neurons respond to convulsant drugs when co-cultured with hiPSC-derived astrocytes.

    Science.gov (United States)

    Ishii, Misawa Niki; Yamamoto, Koji; Shoji, Masanobu; Asami, Asano; Kawamata, Yuji

    2017-08-15

    Accurate risk assessment for drug-induced seizure is expected to be performed before entering clinical studies because of its severity and fatal damage to drug development. Induced pluripotent stem cell (iPSC) technology has allowed the use of human neurons and glial cells in toxicology studies. Recently, several studies showed the advantage of co-culture system of human iPSC (hiPSC)-derived neurons with rodent/human primary astrocytes regarding neuronal functions. However, the application of hiPSC-derived neurons for seizure risk assessment has not yet been fully addressed, and not at all when co-cultured with hiPSC-derived astrocytes. Here, we characterized hiPSC-derived neurons co-cultured with hiPSC-derived astrocytes to discuss how hiPSC-derived neurons are useful to assess seizure risk of drugs. First, we detected the frequency of spikes and synchronized bursts hiPSC-derived neurons when co-cultured with hiPSC-derived astrocytes for 8 weeks. This synchronized burst was suppressed by the treatment with 6-cyano-7-nitroquinoxaline-2,3-dione, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, and D-(-)-2-amino-5-phosphonopentanoic acid, an N-Methyl-d-aspartate (NMDA) receptor antagonist. These data suggested that co-cultured hiPSC-derived neurons formed synaptic connections mediated by AMPA and NMDA receptors. We also demonstrated that co-cultured hiPSC-derived neurons showed epileptiform activity upon treatment with gabazine or kaliotoxin. Finally, we performed single-cell transcriptome analysis in hiPSC-derived neurons and found that hiPSC-derived astrocytes activated the pathways involved in the activities of AMPA and NMDA receptor functions, neuronal polarity, and axon guidance in hiPSC-derived neurons. These data suggested that hiPSC-derived astrocytes promoted the development of action potential, synaptic functions, and neuronal networks in hiPSC-derived neurons, and then these functional alterations result in the epileptiform

  3. Detection of Volatile Metabolites Derived from Garlic (Allium sativum in Human Urine

    Directory of Open Access Journals (Sweden)

    Laura Scheffler

    2016-12-01

    Full Text Available The metabolism and excretion of flavor constituents of garlic, a common plant used in flavoring foods and attributed with several health benefits, in humans is not fully understood. Likewise, the physiologically active principles of garlic have not been fully clarified to date. It is possible that not only the parent compounds present in garlic but also its metabolites are responsible for the specific physiological properties of garlic, including its influence on the characteristic body odor signature of humans after garlic consumption. Accordingly, the aim of this study was to investigate potential garlic-derived metabolites in human urine. To this aim, 14 sets of urine samples were obtained from 12 volunteers, whereby each set comprised one sample that was collected prior to consumption of food-relevant concentrations of garlic, followed by five to eight subsequent samples after garlic consumption that covered a time interval of up to 26 h. The samples were analyzed chemo-analytically using gas chromatography-mass spectrometry/olfactometry (GC-MS/O, as well as sensorially by a trained human panel. The analyses revealed three different garlic-derived metabolites in urine, namely allyl methyl sulfide (AMS, allyl methyl sulfoxide (AMSO and allyl methyl sulfone (AMSO2, confirming our previous findings on human milk metabolite composition. The excretion rates of these metabolites into urine were strongly time-dependent with distinct inter-individual differences. These findings indicate that the volatile odorant fraction of garlic is heavily biotransformed in humans, opening up a window into substance circulation within the human body with potential wider ramifications in view of physiological effects of this aromatic plant that is appreciated by humans in their daily diet.

  4. Development of humanized culture medium with plant-derived serum replacement for human pluripotent stem cells

    Czech Academy of Sciences Publication Activity Database

    Kunová, M.; Matulka, K.; Eiselleová, L.; Trčková, P.; Hampl, Aleš; Dvořák, Petr

    2010-01-01

    Roč. 21, - (2010), s. 676-686 ISSN 1472-6483 Grant - others:GA MŠk(CZ) LC06077; EC FP6(XE) LSHG-CT-2006-018739 Program:LC Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z50390703 Keywords : animal protein-free culture * high-density culture * human embryonic stem cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.285, year: 2010

  5. Derivation of the human embryonic stem cell line RCe014-A (RC-10

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe014-A (RC-10 was derived from a fresh oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XY and 47XY +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  6. Derivation of the human embryonic stem cell line RCe006-A (RC-2

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe006-A (RC-2 was derived from a frozen and thawed blastocyst voluntarily donated as surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line exhibits expression of expected pluripotency markers and in vitro differentiation potential to three germinal lineage representative cell populations. It has a male trisomy 12 karyotype (47XY, +12. Microsatellite DNA marker identity and HLA and blood group typing data are available.

  7. Derivation of the human embryonic stem cell line RCe010-A (RC-6

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe010-A (RC-6 was derived from a frozen and thawed blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  8. Derivation of the human embryonic stem cell line RCe012-A (RC-8

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe012-A (RC-8 was derived from a frozen and thawed day 5 embryo cultivated to the blastocyst stage. The embryo was voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  9. 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.

  10. CULTIVATION OF HUMAN LIVER CELLS AND ADIPOSE-DERIVED MESENCHYMAL STROMAL CELLS IN PERFUSION BIOREACTOR

    Directory of Open Access Journals (Sweden)

    Yu. В. Basok

    2018-01-01

    Full Text Available Aim: to show the progress of the experiment of cultivation of human liver cells and adipose-derived mesenchymal stromal cells in perfusion bioreactor.Materials and methods. The cultivation of a cell-engineered construct, consisting of a biopolymer microstructured collagen-containing hydrogel, human liver cells, adipose-derived mesenchymal stromal cells, and William’s E Medium, was performed in a perfusion bioreactor.Results. On the 7th day large cells with hepatocyte morphology – of a polygonal shape and a centrally located round nucleus, – were present in the culture chambers of the bioreactor. The metabolic activity of hepatocytes in cell-engineered constructs was confi rmed by the presence of urea in the culture medium on the seventh day of cultivation in the bioreactor and by the resorption of a biopolymer microstructured collagen-containing hydrogel.

  11. Physics-based and human-derived information fusion for analysts

    Science.gov (United States)

    Blasch, Erik; Nagy, James; Scott, Steve; Okoth, Joshua; Hinman, Michael

    2017-05-01

    Recent trends in physics-based and human-derived information fusion (PHIF) have amplified the capabilities of analysts; however with the big data opportunities there is a need for open architecture designs, methods of distributed team collaboration, and visualizations. In this paper, we explore recent trends in the information fusion to support user interaction and machine analytics. Challenging scenarios requiring PHIF include combing physics-based video data with human-derived text data for enhanced simultaneous tracking and identification. A driving effort would be to provide analysts with applications, tools, and interfaces that afford effective and affordable solutions for timely decision making. Fusion at scale should be developed to allow analysts to access data, call analytics routines, enter solutions, update models, and store results for distributed decision making.

  12. Derivation of main drivers affecting the possibility of human errors during low power and shutdown operation

    International Nuclear Information System (INIS)

    Kim, Ar Ryum; Seong, Poong Hyun; Park, Jin Kyun; Kim, Jae Whan

    2016-01-01

    are commonly called as performance shaping factors (PSFs) are aspects of the human's individual characteristics, environment, organization, or task that specifically decrements or improves human performance, thus respectively increasing or decreasing the likelihood of human errors. As a result of reviewing eight literatures and performing event report analysis, four main drivers were derived, including procedure, experience level, workload/stress, and training. Since there was less attention paid to the risks during LPSD operations, there have been insufficient human performance data. In this aspect, even though there is a limitation due to insufficient data from operating experience, it is believed that this research may be a reasonable starting point in the study of main drivers in the development of comprehensive HRA methods for LPSD operation

  13. Derivation of main drivers affecting the possibility of human errors during low power and shutdown operation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ar Ryum; Seong, Poong Hyun [KAIST, Daejeon (Korea, Republic of); Park, Jin Kyun; Kim, Jae Whan [KAERI, Daejeon (Korea, Republic of)

    2016-05-15

    which are commonly called as performance shaping factors (PSFs) are aspects of the human's individual characteristics, environment, organization, or task that specifically decrements or improves human performance, thus respectively increasing or decreasing the likelihood of human errors. As a result of reviewing eight literatures and performing event report analysis, four main drivers were derived, including procedure, experience level, workload/stress, and training. Since there was less attention paid to the risks during LPSD operations, there have been insufficient human performance data. In this aspect, even though there is a limitation due to insufficient data from operating experience, it is believed that this research may be a reasonable starting point in the study of main drivers in the development of comprehensive HRA methods for LPSD operation.

  14. Deriving Dorsal Spinal Sensory Interneurons from Human Pluripotent Stem Cells

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    Sandeep Gupta

    2018-02-01

    Full Text Available Summary: Cellular replacement therapies for neurological conditions use human embryonic stem cell (hESC- or induced pluripotent stem cell (hiPSC-derived neurons to replace damaged or diseased populations of neurons. For the spinal cord, significant progress has been made generating the in-vitro-derived motor neurons required to restore coordinated movement. However, there is as yet no protocol to generate in-vitro-derived sensory interneurons (INs, which permit perception of the environment. Here, we report on the development of a directed differentiation protocol to derive sensory INs for both hESCs and hiPSCs. Two developmentally relevant factors, retinoic acid in combination with bone morphogenetic protein 4, can be used to generate three classes of sensory INs: the proprioceptive dI1s, the dI2s, and mechanosensory dI3s. Critical to this protocol is the competence state of the neural progenitors, which changes over time. This protocol will facilitate developing cellular replacement therapies to reestablish sensory connections in injured patients. : In this article, Gupta and colleagues describe a robust protocol to derive spinal dorsal sensory interneurons from human pluripotent stem cells using the sequential addition of RA and BMP4. They find that neural progenitors must be in the correct competence state to respond to RA/BMP4 as dorsalizing signals. This competence state changes over time and determines the efficiency of the protocol. Keywords: spinal cord, neurons, sensory interneurons, proprioception, mechanosensation, human embryonic stem cells, induced pluripotent stem cells, directed differentiation, primate spinal cord, mouse spinal cord

  15. Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure

    NARCIS (Netherlands)

    Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C.; van Meer, Berend; Ward-van Oostwaard, Dorien; Passier, Robert; Tertoolen, Leon G. J.; Mummery, Christine L.; Casini, Simona

    2015-01-01

    One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes

  16. 99mTc-HYNIC-TNF analogues (WH701) derived from phage display peptide libraries for imaging TNF-receptor-positive ovarian carcinoma: Preclinical evaluation

    International Nuclear Information System (INIS)

    Xia, J.S.; Wu, H.; Xiang, Y.; Xia, T.; Li, H.

    2002-01-01

    Aim: In this investigation, 99m Tc-hydrazinonicotinyl-TNF analogs (WH701) was labeled using ethylenediaminediacetic acid (EDDA) as coligand(A number of TNF analogs had been selected and synthesized using random phage-display peptides library in our lab ) and Pharmacokinetics and feasibility studies were performed for its potential use as diagnostic radiopharmaceutical. Material and Methods: The peptide was radiolabeled with 99mTc using HYNIC as a bifunctional chelator and EDDA as coligand. The complexes were characterized by HPLC. The in vitro stability of the radiolabeled peptide WH701 in serum and in phosphate buffer were examined simultaneity. Biodistribution studies were conducted to determine the in vivo characteristics of the complexes. The tumor uptake and image were also conducted in HOC8 tumor-bearing nude mice. Results: The peptide analog permitted efficient incorporation of 99mTc. The preparation of 99mTc-WH701 was stable in vitro. HPLC analysis of the urine samples collected after injection of 99mTc-WH701 showed that the radioactivity elution profile and Rt of the peak were similar to those of the preparation injected. Studies in vivo suggested that the biological activity of the peptide was not compromised. The agent cleared rapidly from the blood. The labeled peptide was shown in the mouse model to localize rapidly and specifically in site of tumor. Images of diagnostic quality could be obtained within 30 min post-administration in all studies. Conclusion: The TNF analogue peptide WH701 can be radiolabeled with 99mTc by HYNIC using EDDA as coligand without loss of affinity, and the 99mTc -WH701 is not degraded in serum and shows radiochemical stability for an extended period of time in vitro. The high specific tumor uptake, rapid blood clearance, and predominantly renal excretion make 99mTc -WH701 a promising candidate for tumor imaging. This agent is worthy of further investigation

  17. A mammalian conserved element derived from SINE displays enhancer properties recapitulating Satb2 expression in early-born callosal projection neurons.

    Directory of Open Access Journals (Sweden)

    Kensuke Tashiro

    Full Text Available Short interspersed repetitive elements (SINEs are highly repeated sequences that account for a significant proportion of many eukaryotic genomes and are usually considered "junk DNA". However, we previously discovered that many AmnSINE1 loci are evolutionarily conserved across mammalian genomes, suggesting that they may have acquired significant functions involved in controlling mammalian-specific traits. Notably, we identified the AS021 SINE locus, located 390 kbp upstream of Satb2. Using transgenic mice, we showed that this SINE displays specific enhancer activity in the developing cerebral cortex. The transcription factor Satb2 is expressed by cortical neurons extending axons through the corpus callosum and is a determinant of callosal versus subcortical projection. Mouse mutants reveal a crucial function for Sabt2 in corpus callosum formation. In this study, we compared the enhancer activity of the AS021 locus with Satb2 expression during telencephalic development in the mouse. First, we showed that the AS021 enhancer is specifically activated in early-born Satb2(+ neurons. Second, we demonstrated that the activity of the AS021 enhancer recapitulates the expression of Satb2 at later embryonic and postnatal stages in deep-layer but not superficial-layer neurons, suggesting the possibility that the expression of Satb2 in these two subpopulations of cortical neurons is under genetically distinct transcriptional control. Third, we showed that the AS021 enhancer is activated in neurons projecting through the corpus callosum, as described for Satb2(+ neurons. Notably, AS021 drives specific expression in axons crossing through the ventral (TAG1(-/NPY(+ portion of the corpus callosum, confirming that it is active in a subpopulation of callosal neurons. These data suggest that exaptation of the AS021 SINE locus might be involved in enhancement of Satb2 expression, leading to the establishment of interhemispheric communication via the corpus callosum

  18. MUTZ-3 derived Langerhans cells in human skin equivalents show differential migration and phenotypic plasticity after allergen or irritant exposure

    NARCIS (Netherlands)

    Kosten, I.J.; Spiekstra, S.W.; de Gruijl, T.D.; Gibbs, S.

    2015-01-01

    After allergen or irritant exposure, Langerhans cells (LC) undergo phenotypic changes and exit the epidermis. In this study we describe the unique ability of MUTZ-3 derived Langerhans cells (MUTZ-LC) to display similar phenotypic plasticity as their primary counterparts when incorporated into a

  19. The effects of physical activity and exercise on brain-derived neurotrophic factor in healthy humans

    DEFF Research Database (Denmark)

    Huang, T; Larsen, K T; Ried-Larsen, M

    2014-01-01

    The purpose of this study was to summarize the effects of physical activity and exercise on peripheral brain-derived neurotrophic factor (BDNF) in healthy humans. Experimental and observational studies were identified from PubMed, Web of Knowledge, Scopus, and SPORT Discus. A total of 32 articles...... studies suggested an inverse relationship between the peripheral BDNF level and habitual physical activity or cardiorespiratory fitness. More research is needed to confirm the findings from the observational studies....

  20. 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.

  1. Human iPSC-Derived Endothelial Cells and Microengineered Organ-Chip Enhance Neuronal Development

    Directory of Open Access Journals (Sweden)

    Samuel Sances

    2018-04-01

    Full Text Available Summary: Human stem cell-derived models of development and neurodegenerative diseases are challenged by cellular immaturity in vitro. Microengineered organ-on-chip (or Organ-Chip systems are designed to emulate microvolume cytoarchitecture and enable co-culture of distinct cell types. Brain microvascular endothelial cells (BMECs share common signaling pathways with neurons early in development, but their contribution to human neuronal maturation is largely unknown. To study this interaction and influence of microculture, we derived both spinal motor neurons and BMECs from human induced pluripotent stem cells and observed increased calcium transient function and Chip-specific gene expression in Organ-Chips compared with 96-well plates. Seeding BMECs in the Organ-Chip led to vascular-neural interaction and specific gene activation that further enhanced neuronal function and in vivo-like signatures. The results show that the vascular system has specific maturation effects on spinal cord neural tissue, and the use of Organ-Chips can move stem cell models closer to an in vivo condition. : Sances et al. combine Organ-Chip technology with human induced pluripotent stem cell-derived spinal motor neurons to study the maturation effects of Organ-Chip culture. By including microvascular cells also derived from the same patient line, the authors show enhancement of neuronal function, reproduction of vascular-neuron pathways, and specific gene activation that resembles in vivo spinal cord development. Keywords: organ-on-chip, spinal cord, iPSC, disease modeling, amyotrophic lateral sclerosis, microphysiological system, brain microvascular endothelial cells, spinal motor neurons, vasculature, microfluidic device

  2. Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture

    International Nuclear Information System (INIS)

    Wei, Yan; Li, Yuan; Chen, Chao; Stoelzel, Katharina; Kaufmann, Andreas M.; Albers, Andreas E.

    2011-01-01

    Human skeletal muscle contains an accessible adult stem-cell compartment in which differentiated myofibers are maintained and replaced by a self-renewing stem cell pool. Previously, studies using mouse models have established a critical role for resident stem cells in skeletal muscle, but little is known about this paradigm in human muscle. Here, we report the reproducible isolation of a population of cells from human skeletal muscle that is able to proliferate for extended periods of time as floating clusters of rounded cells, termed 'myospheres' or myosphere-derived progenitor cells (MDPCs). The phenotypic characteristics and functional properties of these cells were determined using reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry and immunocytochemistry. Our results showed that these cells are clonogenic, express skeletal progenitor cell markers Pax7, ALDH1, Myod, and Desmin and the stem cell markers Nanog, Sox2, and Oct3/4 significantly elevated over controls. They could be maintained proliferatively active in vitro for more than 20 weeks and passaged at least 18 times, despite an average donor-age of 63 years. Individual clones (4.2%) derived from single cells were successfully expanded showing clonogenic potential and sustained proliferation of a subpopulation in the myospheres. Myosphere-derived cells were capable of spontaneous differentiation into myotubes in differentiation media and into other mesodermal cell lineages in induction media. We demonstrate here that direct culture and expansion of stem cells from human skeletal muscle is straightforward and reproducible with the appropriate technique. These cells may provide a viable resource of adult stem cells for future therapies of disease affecting skeletal muscle or mesenchymal lineage derived cell types.

  3. Inhibition of the differentiation of monocyte-derived dendritic cells by human gingival fibroblasts.

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    Sylvie Séguier

    Full Text Available We investigated whether gingival fibroblasts (GFs can modulate the differentiation and/or maturation of monocyte-derived dendritic cells (DCs and analyzed soluble factors that may be involved in this immune modulation. Experiments were performed using human monocytes in co-culture with human GFs in Transwell® chambers or using monocyte cultures treated with conditioned media (CM from GFs of four donors. The four CM and supernatants from cell culture were assayed by ELISA for cytokines involved in the differentiation of dendritic cells, such as IL-6, VEGF, TGFβ1, IL-13 and IL-10. The maturation of monocyte-derived DCs induced by LPS in presence of CM was also studied. Cell surface phenotype markers were analyzed by flow cytometry. In co-cultures, GFs inhibited the differentiation of monocyte-derived DCs and the strength of this blockade correlated with the GF/monocyte ratio. Conditioned media from GFs showed similar effects, suggesting the involvement of soluble factors produced by GFs. This inhibition was associated with a lower stimulatory activity in MLR of DCs generated with GFs or its CM. Neutralizing antibodies against IL-6 and VEGF significantly (P<0.05 inhibited the inhibitory effect of CM on the differentiation of monocytes-derived DCs and in a dose dependent manner. Our data suggest that IL-6 is the main factor responsible for the inhibition of DCs differentiation mediated by GFs but that VEGF is also involved and constitutes an additional mechanism.

  4. Low antigenicity of hematopoietic progenitor cells derived from human ES cells

    Directory of Open Access Journals (Sweden)

    Eun-Mi Kim

    2010-02-01

    Full Text Available Eun-Mi Kim1, Nicholas Zavazava1,21Department of Internal Medicine, University of Iowa and Veterans Affairs Medical Center, Iowa City, Iowa, USA; 2Immunology Graduate Program, University of Iowa, Iowa City, Iowa, USAAbstract: Human embryonic stem (hES cells are essential for improved understanding of diseases and our ability to probe new therapies for use in humans. Currently, bone marrow cells and cord blood cells are used for transplantation into patients with hematopoietic malignancies, immunodeficiencies and in some cases for the treatment of autoimmune diseases. However, due to the high immunogenicity of these hematopoietic cells, toxic regimens of drugs are required for preconditioning and prevention of rejection. Here, we investigated the efficiency of deriving hematopoietic progenitor cells (HPCs from the hES cell line H13, after co-culturing with the murine stromal cell line OP9. We show that HPCs derived from the H13 ES cells poorly express major histocompatibility complex (MHC class I and no detectable class II antigens (HLA-DR. These characteristics make hES cell-derived hematopoietic cells (HPCs ideal candidates for transplantation across MHC barriers under minimal immunosuppression.Keywords: human embryonic stem cells, H13, hematopoiesis, OP9 stromal cells, immunogenicity

  5. Distinct p53 genomic binding patterns in normal and cancer-derived human cells

    Energy Technology Data Exchange (ETDEWEB)

    Botcheva K.; McCorkle S. R.; McCombie W. R.; Dunn J. J.; Anderson C. W.

    2011-12-15

    We report here genome-wide analysis of the tumor suppressor p53 binding sites in normal human cells. 743 high-confidence ChIP-seq peaks representing putative genomic binding sites were identified in normal IMR90 fibroblasts using a reference chromatin sample. More than 40% were located within 2 kb of a transcription start site (TSS), a distribution similar to that documented for individually studied, functional p53 binding sites and, to date, not observed by previous p53 genome-wide studies. Nearly half of the high-confidence binding sites in the IMR90 cells reside in CpG islands, in marked contrast to sites reported in cancer-derived cells. The distinct genomic features of the IMR90 binding sites do not reflect a distinct preference for specific sequences, since the de novo developed p53 motif based on our study is similar to those reported by genome-wide studies of cancer cells. More likely, the different chromatin landscape in normal, compared with cancer-derived cells, influences p53 binding via modulating availability of the sites. We compared the IMR90 ChIPseq peaks to the recently published IMR90 methylome1 and demonstrated that they are enriched at hypomethylated DNA. Our study represents the first genome-wide, de novo mapping of p53 binding sites in normal human cells and reveals that p53 binding sites reside in distinct genomic landscapes in normal and cancer-derived human cells.

  6. T3 Regulates a Human Macrophage-Derived TSH-β Splice Variant: Implications for Human Bone Biology.

    Science.gov (United States)

    Baliram, R; Latif, R; Morshed, S A; Zaidi, M; Davies, T F

    2016-09-01

    TSH and thyroid hormones (T3 and T4) are intimately involved in bone biology. We have previously reported the presence of a murine TSH-β splice variant (TSH-βv) expressed specifically in bone marrow-derived macrophages and that exerted an osteoprotective effect by inducing osteoblastogenesis. To extend this observation and its relevance to human bone biology, we set out to identify and characterize a TSH-β variant in human macrophages. Real-time PCR analyses using human TSH-β-specific primers identified a 364-bp product in macrophages, bone marrow, and peripheral blood mononuclear cells that was sequence verified and was homologous to a human TSH-βv previously reported. We then examined TSH-βv regulation using the THP-1 human monocyte cell line matured into macrophages. After 4 days, 46.1% of the THP-1 cells expressed the macrophage markers CD-14 and macrophage colony-stimulating factor and exhibited typical morphological characteristics of macrophages. Real-time PCR analyses of these cells treated in a dose-dependent manner with T3 showed a 14-fold induction of human TSH-βv mRNA and variant protein. Furthermore, these human TSH-βv-positive cells, induced by T3 exposure, had categorized into both M1 and M2 macrophage phenotypes as evidenced by the expression of macrophage colony-stimulating factor for M1 and CCL-22 for M2. These data indicate that in hyperthyroidism, bone marrow resident macrophages have the potential to exert enhanced osteoprotective effects by oversecreting human TSH-βv, which may exert its local osteoprotective role via osteoblast and osteoclast TSH receptors.

  7. Induction of Skin-Derived Precursor Cells from Human Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Sugiyama-Nakagiri, Yoriko; Fujimura, Tsutomu; Moriwaki, Shigeru

    2016-01-01

    The generation of full thickness human skin from dissociated cells is an attractive approach not only for treating skin diseases, but also for treating many systemic disorders. However, it is currently not possible to obtain an unlimited number of skin dermal cells. The goal of this study was to develop a procedure to produce skin dermal stem cells from induced pluripotent stem cells (iPSCs). Skin-derived precursor cells (SKPs) were isolated as adult dermal precursors that could differentiate into both neural and mesodermal progenies and could reconstitute the dermis. Thus, we attempted to generate SKPs from iPSCs that could reconstitute the skin dermis. Human iPSCs were initially cultured with recombinant noggin and SB431542, an inhibitor of activin/nodal and TGFβ signaling, to induce neural crest progenitor cells. Those cells were then treated with SKP medium that included CHIR99021, a WNT signal activator. The induction efficacy from neural crest progenitor cells to SKPs was more than 97%. No other modifiers tested were able to induce those cells. Those human iPSC-derived SKPs (hiPSC-SKPs) showed a similar gene expression signature to SKPs isolated from human skin dermis. Human iPSC-SKPs differentiated into neural and mesodermal progenies, including adipocytes, skeletogenic cell types and Schwann cells. Moreover, they could be induced to follicular type keratinization when co-cultured with human epidermal keratinocytes. We here provide a new efficient protocol to create human skin dermal stem cells from hiPSCs that could contribute to the treatment of various skin disorders.

  8. Lung-Derived Microscaffolds Facilitate Diabetes Reversal after Mouse and Human Intraperitoneal Islet Transplantation.

    Science.gov (United States)

    Abualhassan, Nasser; Sapozhnikov, Lena; Pawlick, Rena L; Kahana, Meygal; Pepper, Andrew R; Bruni, Antonio; Gala-Lopez, Boris; Kin, Tatsuya; Mitrani, Eduardo; Shapiro, A M James

    2016-01-01

    There is a need to develop three-dimensional structures that mimic the natural islet tissue microenvironment. Endocrine micro-pancreata (EMPs) made up of acellular organ-derived micro-scaffolds seeded with human islets have been shown to express high levels of key beta-cell specific genes and secrete quantities of insulin per cell similar to freshly isolated human islets in a glucose-regulated manner for more than three months in vitro. The aim of this study was to investigate the capacity of EMPs to restore euglycemia in vivo after transplantation of mouse or human islets in chemically diabetic mice. We proposed that the organ-derived EMPs would restore the extracellular components of the islet microenvironment, generating favorable conditions for islet function and survival. EMPs seeded with 500 mouse islets were implanted intraperitoneally into streptozotocin-induced diabetic mice and reverted diabetes in 67% of mice compared to 13% of controls (p = 0.018, n = 9 per group). Histological analysis of the explanted grafts 60 days post-transplantation stained positive for insulin and exhibited increased vascular density in a collagen-rich background. EMPs were also seeded with human islets and transplanted into the peritoneal cavity of immune-deficient diabetic mice at 250 islet equivalents (IEQ), 500 IEQ and 1000 IEQ. Escalating islet dose increased rates of normoglycemia (50% of the 500 IEQ group and 75% of the 1000 IEQ group, n = 3 per group). Human c-peptide levels were detected 90 days post-transplantation in a dose-response relationship. Herein, we report reversal of diabetes in mice by intraperitoneal transplantation of human islet seeded on EMPs with a human islet dose as low as 500 IEQ.

  9. Subfractions of enamel matrix derivative differentially influence cytokine secretion from human oral fibroblasts

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    Oscar Villa

    2015-03-01

    Full Text Available Enamel matrix derivative is used to promote periodontal regeneration during the corrective phase of the treatment of periodontal defects. Our main goal was to analyze the bioactivity of different molecular weight fractions of enamel matrix derivative. Enamel matrix derivative, a complex mixture of proteins, was separated into 13 fractions using size-exclusion chromatography and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and liquid chromatography–electrospray ionization–tandem mass spectrometry. Human periodontal ligament fibroblasts were treated with either enamel matrix derivative or the different fractions. Proliferation and cytokine secretion to the cell culture medium were measured and compared to untreated cells. The liquid chromatography–electrospray ionization–tandem mass spectrometry analyses revealed that the most abundant peptides were amelogenin and leucine-rich amelogenin peptide related. The fractions containing proteins above 20 kDa induced an increase in vascular endothelial growth factor and interleukin-6 secretion, whereas lower molecular weight fractions enhanced proliferation and secretion of interleukin-8 and monocyte chemoattractant protein-1 and reduced interleukin-4 release. The various molecular components in the enamel matrix derivative formulation might contribute to reported effects on tissue regeneration through their influence on vascularization, the immune response, and chemotaxis.

  10. Human embryonic stem cell derived mesenchymal progenitors express cardiac markers but do not form contractile cardiomyocytes.

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    Christophe M Raynaud

    Full Text Available Mesenchymal progenitors or stromal cells have shown promise as a therapeutic strategy for a range of diseases including heart failure. In this context, we explored the growth and differentiation potential of mesenchymal progenitors (MPs derived in vitro from human embryonic stem cells (hESCs. Similar to MPs isolated from bone marrow, hESC derived MPs (hESC-MPs efficiently differentiated into archetypical mesenchymal derivatives such as chondrocytes and adipocytes. Upon treatment with 5-Azacytidine or TGF-β1, hESC-MPs modified their morphology and up-regulated expression of key cardiac transcription factors such as NKX2-5, MEF2C, HAND2 and MYOCD. Nevertheless, NKX2-5+ hESC-MP derivatives did not form contractile cardiomyocytes, raising questions concerning the suitability of these cells as a platform for cardiomyocyte replacement therapy. Gene profiling experiments revealed that, although hESC-MP derived cells expressed a suite of cardiac related genes, they lacked the complete repertoire of genes associated with bona fide cardiomyocytes. Our results suggest that whilst agents such as TGF-β1 and 5-Azacytidine can induce expression of cardiac related genes, but treated cells retain a mesenchymal like phenotype.

  11. A comparative study of the structural organization of spheres derived from the adult human subventricular zone and glioblastoma biopsies

    International Nuclear Information System (INIS)

    Vik-Mo, Einar Osland; Sandberg, Cecilie; Joel, Mrinal; Stangeland, Biljana; Watanabe, Yasuhiro; Mackay-Sim, Alan; Moe, Morten Carstens; Murrell, Wayne; Langmoen, Iver Arne

    2011-01-01

    Sphere forming assays have been useful to enrich for stem like cells in a range of tumors. The robustness of this system contrasts the difficulties in defining a stem cell population based on cell surface markers. We have undertaken a study to describe the cellular and organizational composition of tumorspheres, directly comparing these to neurospheres derived from the adult human subventricular zone (SVZ). Primary cell cultures from brain tumors were found to contain variable fractions of cells positive for tumor stem cell markers (CD133 (2-93%)/SSEA1 (3-15%)/CXCR4 (1-72%)). All cultures produced tumors upon xenografting. Tumorspheres contained a heterogeneous population of cells, but were structurally organized with stem cell markers present at the core of spheres, with markers of more mature glial progenitors and astrocytes at more peripheral location. Ultrastructural studies showed that tumorspheres contained a higher fraction of electron dense cells in the core than the periphery (36% and 19%, respectively). Neurospheres also contained a heterogeneous cell population, but did not have an organization similar to tumorspheres. Although tumorspheres clearly display irregular and neoplastic cells, they establish an organized structure with an outward gradient of differentiation. We suggest that this organization is central in maintaining the tumor stem cell pool.

  12. Prospectively Isolated Human Bone Marrow Cell-Derived MSCs Support Primitive Human CD34-Negative Hematopoietic Stem Cells.

    Science.gov (United States)

    Matsuoka, Yoshikazu; Nakatsuka, Ryusuke; Sumide, Keisuke; Kawamura, Hiroshi; Takahashi, Masaya; Fujioka, Tatsuya; Uemura, Yasushi; Asano, Hiroaki; Sasaki, Yutaka; Inoue, Masami; Ogawa, Hiroyasu; Takahashi, Takayuki; Hino, Masayuki; Sonoda, Yoshiaki

    2015-05-01

    Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) niche, which consists of osteoblasts, endothelial cells, and a variety of mesenchymal stem/stromal cells (MSCs). However, precisely what types of MSCs support human HSCs in the BM remain to be elucidated because of their heterogeneity. In this study, we succeeded in prospectively isolating/establishing three types of MSCs from human BM-derived lineage- and CD45-negative cells, according to their cell surface expression of CD271 and stage-specific embryonic antigen (SSEA)-4. Among them, the MSCs established from the Lineage(-) CD45(-) CD271(+) SSEA-4(+) fraction (DP MSC) could differentiate into osteoblasts and chondrocytes, but they lacked adipogenic differentiation potential. The DP MSCs expressed significantly higher levels of well-characterized HSC-supportive genes, including IGF-2, Wnt3a, Jagged1, TGFβ3, nestin, CXCL12, and Foxc1, compared with other MSCs. Interestingly, these osteo-chondrogenic DP MSCs possessed the ability to support cord blood-derived primitive human CD34-negative severe combined immunodeficiency-repopulating cells. The HSC-supportive actions of DP MSCs were partially carried out by soluble factors, including IGF-2, Wnt3a, and Jagged1. Moreover, contact between DP MSCs and CD34-positive (CD34(+) ) as well as CD34-negative (CD34(-) ) HSCs was important for the support/maintenance of the CD34(+/-) HSCs in vitro. These data suggest that DP MSCs might play an important role in the maintenance of human primitive HSCs in the BM niche. Therefore, the establishment of DP MSCs provides a new tool for the elucidation of the human HSC/niche interaction in vitro as well as in vivo. © 2014 AlphaMed Press.

  13. Human Breast Adipose-Derived Stem Cells Transfected with the Stromal Cell-Derived Factor-1 Receptor CXCR4 Exhibit Enhanced Viability in Human Autologous Free Fat Grafts

    Directory of Open Access Journals (Sweden)

    Fang-tian Xu

    2014-11-01

    Full Text Available Background: The main complication of autologous free fat tissue transplantation is fat resorption and calcification due to the ischemic necrosis of fat. The promotion of transplant neovascularization soon after autologous free fat grafts may reduce these outcomes. In adulthood, stromal cell-derived factor-1 (SDF-1 and its membrane receptor C-X-C chemokine receptor type 4 (CXCR4 are involved in the homing and migration of multiple stem cell types, neovascularization, and cell proliferation. We hypothesized that CXCR4 may improve the long-term survival of free fat tissue transplants by recruiting endothelial progenitor cells (EPCs and may therefore improve graft revascularization. In this study, we aimed to determine the effect of human breast adipose-derived stem cells (HBASCs transfected with the CXCR4 gene on the survival rate of human autologous free fat transplants in nude mice. Methods: Human breast adipose-derived stem cells (HBASCs were expanded ex vivo for 3 passages, labeled with green fluorescent protein (GFP and transfected with CXCR4 or left untransfected. Autologous fat tissues were mixed with the GFP-labeled, CXCR4-transfected HBASCs (group A, GFP-labeled HBASCs (group B, the known vascularization-promoting agent VEGF (group C, or medium (group D and then injected subcutaneously into 32 nude mice at 4 spots in a random fashion. Six months later, the transplanted tissue volume and histology were evaluated, and neo-vascularization was quantified by counting the capillaries. CXCR4 and SDF-1α mRNA expression in the transplants was determined using real-time quantitative PCR analysis (qPCR. Results: The data revealed that the control (group D transplant volume survival was 28.3 ± 4.5%. Mixing CXCR4-transfected (group A and untransfected (group B HBASCs significantly increased transplant volume survival (79.5 ± 8.3% and 67.2 ± 5.9%, respectively, whereas VEGF-transfected HBASCs (group C were less effective (41.2 ± 5.1%. Histological

  14. Perception of human-derived risk influences choice at top of the food chain.

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    Bogdan Cristescu

    Full Text Available On human-used landscapes, animal behavior is a trade-off between maximizing fitness and minimizing human-derived risk. Understanding risk perception in wildlife can allow mitigation of anthropogenic risk, with benefits to long-term animal fitness. Areas where animals choose to rest should minimize risk from predators, which for large carnivores typically equate to humans. We hypothesize that high human activity leads to selection for habitat security, whereas low activity enables trading security for forage. We investigated selection of resting (bedding sites by GPS radiocollared adult grizzly bears (n = 10 in a low density population on a multiple-use landscape in Canada. We compared security and foods at resting and random locations while accounting for land use, season, and time of day. On reclaimed mines with low human access, bears selected high horizontal cover far from trails, but did not avoid open (herbaceous areas, resting primarily at night. In protected areas bears also bedded at night, in areas with berry shrubs and Hedysarum spp., with horizontal cover selected in the summer, during high human access. On public lands with substantial human recreation, bears bedded at day, selected resting sites with high horizontal cover in the summer and habitat edges, with bedding associated with herbaceous foods. These spatial and temporal patterns of selection suggest that bears perceive human-related risk differentially in relation to human activity level, season and time of day, and employ a security-food trade-off strategy. Although grizzly bears are presently not hunted in Alberta, their perceived risks associated with humans influence resting-site selection.

  15. Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation.

    Science.gov (United States)

    Hamid, Adila A; Idrus, Ruszymah Bt Hj; Saim, Aminuddin Bin; Sathappan, Somasumdaram; Chua, Kien-Hui

    2012-01-01

    Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction. Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction. Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN) was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction. Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adipose-derived stem cells was most prominent after one week of chondrogenic induction.

  16. Human Superior Temporal Gyrus Organization of Spectrotemporal Modulation Tuning Derived from Speech Stimuli.

    Science.gov (United States)

    Hullett, Patrick W; Hamilton, Liberty S; Mesgarani, Nima; Schreiner, Christoph E; Chang, Edward F

    2016-02-10

    The human superior temporal gyrus (STG) is critical for speech perception, yet the organization of spectrotemporal processing of speech within the STG is not well understood. Here, to characterize the spatial organization of spectrotemporal processing of speech across human STG, we use high-density cortical surface field potential recordings while participants listened to natural continuous speech. While synthetic broad-band stimuli did not yield sustained activation of the STG, spectrotemporal receptive fields could be reconstructed from vigorous responses to speech stimuli. We find that the human STG displays a robust anterior-posterior spatial distribution of spectrotemporal tuning in which the posterior STG is tuned for temporally fast varying speech sounds that have relatively constant energy across the frequency axis (low spectral modulation) while the anterior STG is tuned for temporally slow varying speech sounds that have a high degree of spectral variation across the frequency axis (high spectral modulation). This work illustrates organization of spectrotemporal processing in the human STG, and illuminates processing of ethologically relevant speech signals in a region of the brain specialized for speech perception. Considerable evidence has implicated the human superior temporal gyrus (STG) in speech processing. However, the gross organization of spectrotemporal processing of speech within the STG is not well characterized. Here we use natural speech stimuli and advanced receptive field characterization methods to show that spectrotemporal features within speech are well organized along the posterior-to-anterior axis of the human STG. These findings demonstrate robust functional organization based on spectrotemporal modulation content, and illustrate that much of the encoded information in the STG represents the physical acoustic properties of speech stimuli. Copyright © 2016 the authors 0270-6474/16/362014-13$15.00/0.

  17. Beneficial effects of non-alcoholic grape-derived products on human health: A literature review

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    Di Lorenzo Chiara

    2015-01-01

    Full Text Available Vine is widely cultivated due to the economic value of wine and other grape derivatives. The grape berry is character- ized by the presence of a wide variety of flavonoids, which have been investigated for their health promoting properties. Several epidemiological studies have shown that a moderate consumption of wine is associated with a J-shaped effect on some risk fac- tors for chronic diseases. On the other hand, the wine market has shown a decreasing trend due to the frequent abuse of alcoholic beverages also by young people, as denounced by WHO. Accordingly, the scientific research in the field of non-alcoholic grape products has been further stimulated. The aim of this paper was a preliminary collection of data on human studies supporting the beneficial properties of unfermented grape products. The most convincing positive effects, observed in humans, consisted in the reduction of risk factors for cardiovascular diseases, such as hypertension and oxidative stress. Other human trials have been published in the area of: immune system, diabetes, cognitive functions, oral health, and cancer. Generally speaking, the findings listed in this review support the use of non-alcoholic grape derivatives, as a source of beneficial compounds for the human diet, even though further studies are necessary.

  18. In utero transplantation of human bone marrow-derived multipotent mesenchymal stem cells in mice.

    Science.gov (United States)

    Chou, Shiu-Huey; Kuo, Tom K; Liu, Ming; Lee, Oscar K

    2006-03-01

    Mesenchymal stem cells (MSCs) are multipotent cells that can be isolated from human bone marrow and possess the potential to differentiate into progenies of embryonic mesoderm. However, current evidence is based predominantly on in vitro experiments. We used a murine model of in utero transplantation (IUT) to study the engraftment capabilities of human MSCs. MSCs were obtained from bone marrow by negative immunoselection and limiting dilution, and were characterized by flow cytometry and by in vitro differentiation into osteoblasts, chondrocytes, and adipocytes. MSCs were transplanted into fetal mice at a gestational age of 14 days. Engraftment of human MSCs was determined by flow cytometry, polymerase chain reaction, and fluorescence in situ hybridization (FISH). MSCs engrafted into tissues originating from all three germ layers and persisted for up to 4 months or more after delivery, as evidenced by the expression of the human-specific beta-2 microglobulin gene and by FISH for donor-derived cells. Donor-derived CD45+ cells were detectable in the peripheral blood of recipients, suggesting the participation of MSCs in hematopoiesis at the fetal stage. This model can further serve to evaluate possible applications of MSCs. Copyright 2006 Orthopaedic Research Society.

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

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    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.

  20. Mast cell-deficient Kit(W-sh) "Sash" mutant mice display aberrant myelopoiesis leading to the accumulation of splenocytes that act as myeloid-derived suppressor cells.

    Science.gov (United States)

    Michel, Anastasija; Schüler, Andrea; Friedrich, Pamela; Döner, Fatma; Bopp, Tobias; Radsak, Markus; Hoffmann, Markus; Relle, Manfred; Distler, Ute; Kuharev, Jörg; Tenzer, Stefan; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Schild, Hansjörg; Schmitt, Edgar; Becker, Marc; Stassen, Michael

    2013-06-01

    Mast cell-deficient Kit(W-sh) "sash" mice are widely used to investigate mast cell functions. However, mutations of c-Kit also affect additional cells of hematopoietic and nonimmune origin. In this study, we demonstrate that Kit(W-sh) causes aberrant extramedullary myelopoiesis characterized by the expansion of immature lineage-negative cells, common myeloid progenitors, and granulocyte/macrophage progenitors in the spleen. A consistent feature shared by these cell types is the reduced expression of c-Kit. Populations expressing intermediate and high levels of Ly6G, a component of the myeloid differentiation Ag Gr-1, are also highly expanded in the spleen of sash mice. These cells are able to suppress T cell responses in vitro and phenotypically and functionally resemble myeloid-derived suppressor cells (MDSC). MDSC typically accumulate in tumor-bearing hosts and are able to dampen immune responses. Consequently, transfer of MDSC from naive sash mice into line 1 alveolar cell carcinoma tumor-bearing wild-type littermates leads to enhanced tumor progression. However, although it can also be observed in sash mice, accelerated growth of transplanted line 1 alveolar cell carcinoma tumors is a mast cell-independent phenomenon. Thus, the Kit(W-sh) mutation broadly affects key steps in myelopoiesis that may have an impact on mast cell research.

  1. In Vitro Generation of IL-35-expressing Human Wharton's Jelly-derived Mesenchymal Stem Cells Using Lentiviral Vector.

    Science.gov (United States)

    Amari, Afshin; Ebtekar, Massoumeh; Moazzeni, Seyed Mohammad; Soleimani, Masoud; Mohammadi Amirabad, Leila; Tahoori, Mohammad Taher; Massumi, Mohammad

    2015-08-01

    Human Wharton's Jelly-derived Mesenchymal Stem Cells (hWJ-MSCs) are easily available cells without transplant rejection problems or ethical concerns compared to bone-marrow-derived MSCs for prospective clinical applications. These cells display immunosuppressive properties and may be able to play an important role in autoimmune disorders. Regulatory T-cells (Treg) are important to prevent autoimmune disease development. Interleukin 35 (IL-35) induces the proliferation of Treg cell populations and reduces the activity of T helper 17 (Th17) and T helper 1 (Th1) cells, which play a central role in initiation of inflammation and autoimmune disease. Recent studies identified IL-35 as a new inhibitory cytokine required for the suppressive function of Treg cells. We created IL-35-producing hWJ-MSCs as a good vehicle for reduction of inflammation and autoimmune diseases. We isolated hWJ-MSCs based on explant culture. HWJ-MSCs were transduced at MOI=50 (Multiplicity of Infection) with lentiviral particles harboring murine Interleukin 35 (mIL-35). Expression of IL-35 in hWJ-MSCs was quantified by an IL-35 ELISA kit. IL-35 bioactivity was analyzed by inhibiting the proliferation of mouse splenocytes using CFSE cell proliferation kit. Frequency of CD4+CD25+CD127 low/neg Foxp3+ Treg cells was measured by flow cytometry. There was an up to 85% GFP positive transduction rate, and the cells successfully released a high level of mIL-35 protein (750 ng/ml). IL-35 managed to inhibit CD4+ T cell proliferation with PHA, and improved the frequency of Treg cells. Our data suggest that transduced hWJ-MSCs overexpressing IL-35 may provide a useful approach for basic research on gene therapy for autoimmune disorders.

  2. Technetium-99m sestamibi uptake in human breast carcinoma cell lines displaying glutathione-associated drug-resistance

    International Nuclear Information System (INIS)

    Kabasakal, L.; Oezker, K.; Hayward, M.; Akansel, G.; Griffith, O.; Isitman, A.T.; Hellman, R.; Collier, D.

    1996-01-01

    An in vitro study was designed to evaluate the uptake of sestamibi (MIBI) in P-glycoprotein (Pgp) and glutathione-associated (GSH) multidrug-resistant (MDR) cell lines. MIBI uptake was studied in various human breast carcinoma cell lines, i.e. in wild-type (MCF7/wt) cells, in adriamycin-resistant (MCF7/adr) cells which express Pgp and in melphalan-resistant (MCF7/mph) cells with increased levels of GSH. The effects of buthiomine sulphoximine (BSO) and verapamil on MIBI uptake were also studied in the MCF7/mph and MCF7/adr cells respectively. The cells were incubated for 1 h with a dose of 0.1 MBq thallium-201 and technetium-99m MIBI. Both BIBI and 201 Tl uptakes were higher for MCF7/mph cells than for the other cells studied. The mean MIBI uptake in MCF7/adr cells was significantly lower than that in MCF7/wt cells (1.9%±0.5% vs 3.1%.0.6%; P 0.1). This study suggests that the uptake of MIBI is not diminished by glutathione-associated drug resistance and that MIBI uptake in a tumour sample does not necessarly indicate that a cancer is sensitive to drugs. (orig.)

  3. Differentiation, Evaluation, and Application of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.

    Science.gov (United States)

    Lin, Yang; Gil, Chang-Hyun; Yoder, Mervin C

    2017-11-01

    The emergence of induced pluripotent stem cell (iPSC) technology paves the way to generate large numbers of patient-specific endothelial cells (ECs) that can be potentially delivered for regenerative medicine in patients with cardiovascular disease. In the last decade, numerous protocols that differentiate EC from iPSC have been developed by many groups. In this review, we will discuss several common strategies that have been optimized for human iPSC-EC differentiation and subsequent studies that have evaluated the potential of human iPSC-EC as a cell therapy or as a tool in disease modeling. In addition, we will emphasize the importance of using in vivo vessel-forming ability and in vitro clonogenic colony-forming potential as a gold standard with which to evaluate the quality of human iPSC-EC derived from various protocols. © 2017 American Heart Association, Inc.

  4. The N-Terminus of Human Lactoferrin Displays Anti-biofilm Activity on Candida parapsilosis in Lumen Catheters.

    Science.gov (United States)

    Fais, Roberta; Di Luca, Mariagrazia; Rizzato, Cosmeri; Morici, Paola; Bottai, Daria; Tavanti, Arianna; Lupetti, Antonella

    2017-01-01

    Candida parapsilosis is a major cause of hospital-acquired infection, often related to parenteral nutrition administered via catheters and hand colonization of health care workers, and its peculiar biofilm formation ability on plastic surfaces. The mortality rate of 30% points to the pressing need for new antifungal drugs. The present study aimed at analyzing the inhibitory activity of the N-terminal lactoferrin-derived peptide, further referred to as hLF 1-11, against biofilms produced by clinical isolates of C. parapsilosis characterized for their biofilm forming ability and fluconazole susceptibility. hLF 1-11 anti-biofilm activity was assessed in terms of reduction of biofilm biomass, metabolic activity, and observation of sessile cell morphology on polystyrene microtiter plates and using an in vitro model of catheter-associated C. parapsilosis biofilm production. Moreover, fluctuation in transcription levels of genes related to cell adhesion, hyphal development and extracellular matrix production upon peptide exposure were evaluated by quantitative real time RT-PCR. The results revealed that hLF 1-11 exhibits an inhibitory effect on biofilm formation by all the C. parapsilosis isolates tested, in a dose-dependent manner, regardless of their fluconazole susceptibility. In addition, hLF 1-11 induced a statistically significant dose-dependent reduction of preformed-biofilm cellular density and metabolic activity at high peptide concentrations only. Interestingly, when assessed in a catheter lumen, hLF 1-11 was able to induce a 2-log reduction of sessile cell viability at both the peptide concentrations used in RPMI diluted in NaPB. A more pronounced anti-biofilm effect was observed (3.5-log reduction) when a 10% glucose solution was used as experimental condition on both early and preformed C. parapsilosis biofilm. Quantitative real time RT-PCR experiments confirmed that hLF 1-11 down-regulates key biofilm related genes. The overall findings suggest hLF 1-11 as a

  5. Mucopolysaccharidosis enzyme production by bone marrow and dental pulp derived human mesenchymal stem cells.

    Science.gov (United States)

    Jackson, Matilda; Derrick Roberts, Ainslie; Martin, Ellenore; Rout-Pitt, Nathan; Gronthos, Stan; Byers, Sharon

    2015-04-01

    Mucopolysaccharidoses (MPS) are inherited metabolic disorders that arise from a complete loss or a reduction in one of eleven specific lysosomal enzymes. MPS children display pathology in multiple cell types leading to tissue and organ failure and early death. Mesenchymal stem cells (MSCs) give rise to many of the cell types affected in MPS, including those that are refractory to current treatment protocols such as hematopoietic stem cell (HSC) based therapy. In this study we compared multiple MPS enzyme production by bone marrow derived (hBM) and dental pulp derived (hDP) MSCs to enzyme production by HSCs. hBM MSCs produce significantly higher levels of MPS I, II, IIIA, IVA, VI and VII enzyme than HSCs, while hDP MSCs produce significantly higher levels of MPS I, IIIA, IVA, VI and VII enzymes. Higher transfection efficiency was observed in MSCs (89%) compared to HSCs (23%) using a lentiviral vector. Over-expression of four different lysosomal enzymes resulted in up to 9303-fold and up to 5559-fold greater levels in MSC cell layer and media respectively. Stable, persistent transduction of MSCs and sustained over-expression of MPS VII enzyme was observed in vitro. Transduction of MSCs did not affect the ability of the cells to differentiate down osteogenic, adipogenic or chondrogenic lineages, but did partially delay differentiation down the non-mesodermal neurogenic lineage. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Immunogenicity of adenovirus-derived porcine parvovirus-like particles displaying B and T cell epitopes of foot-and-mouth disease.

    Science.gov (United States)

    Pan, Qunxing; Wang, Hui; Ouyang, Wei; Wang, Xiaoli; Bi, Zhenwei; Xia, Xingxia; Wang, Yongshan; He, Kongwang

    2016-01-20

    Virus-like particles (VLPs) vaccines combine many of the advantages of whole-virus vaccines and recombinant subunit vaccines, integrating key features that underlay their immunogenicity, safety and protective potential. We have hypothesized here the effective insertion of the VP1 epitopes (three amino acid residues 21-40, 141-160 and 200-213 in VP1, designated VPe) of foot-and-mouth disease (FMDV) within the external loops of PPV VP2 could be carried out without altering assembly based on structural and antigenic data. To investigate the possibility, development of two recombinant adenovirus rAd-PPV:VP2-FMDV:VPe a or rAd-PPV:VP2-FMDV:VPe b were expressed in HEK-293 cells. Out of the two insertion strategies tested, one of them tolerated an insert of 57 amino acids in one of the four external loops without disrupting the VLPs assembly. Mice were inoculated with the two recombinant adenoviruses, and an immunogenicity study showed that the highest levels of FMDV-specific humoral responses and T cell proliferation could be induced by rAd-PPV:VP2-FMDV:VPe b expressing hybrid PPV:VLPs (FMDV) in the absence of an adjuvant. Then, the protective efficacy of inoculating swine with rAd-PPV:VP2-FMDV:VPe b was tested. All pigs inoculated with rAd-PPV:VP2-FMDV:VPe b were protected from viral challenge, meanwhile the neutralizing antibody titers were significantly higher than those in the group inoculated with swine FMD type O synthetic peptide vaccine. Our results clearly demonstrate the potential usefulness of adenovirus-derived PPV VLPs as a vaccine strategy in prevention of FMDV. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Ribosome display: next-generation display technologies for production of antibodies in vitro.

    Science.gov (United States)

    He, Mingyue; Khan, Farid

    2005-06-01

    Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.

  8. Human decidual macrophages and NK cells differentially express Toll-like receptors and display distinct cytokine profiles upon TLR stimulation.

    Directory of Open Access Journals (Sweden)

    Marion eDuriez

    2014-07-01

    Full Text Available Maternofetal pathogen transmission is partially controlled at the level of the maternal uterine mucosa at the fetal implantation site (the decidua basalis, where maternal and fetal cells are in close contact. Toll-like receptors (TLRs may play an important role in initiating rapid immune responses against pathogens in the decidua basalis, however the tolerant microenvironment should be preserved in order to allow fetal development. Here we investigated the expression and functionality of TLRs expressed by decidual macrophages (dMs and NK cells (dNKs, the major decidual immune cell populations.We report for the first time that both human dMs and dNK cells express mRNAs encoding TLRs 1-9, albeit with a higher expression level in dMs. TLR2, TLR3 and TLR4 protein expression checked by flow cytometry was positive for both dMs and dNK cells. In vitro treatment of primary dMs and dNK cells with specific TLR2, TLR3, TLR4, TLR7/8 and TLR9 agonists enhanced their secretion of pro- and anti-inflammatory cytokines, as well as cytokines and chemokines involved in immune cell crosstalk. Only dNK cells released IFN-γ, whereas only dMs released IL-1β, IL-10 and IL-12. TLR9 activation of dMs resulted in a distinct pattern of cytokine expression compared to the other TLRs. The cytokine profiles expressed by dMs and dNK cells upon TLR activation are compatible with maintenance of the fetotolerant immune environment during initiation of immune responses to pathogens at the maternofetal interface.

  9. The use of /sup 125/I recombinant DNA/sub 125/ derived human erythropoietin (R-HuEPO) as a replacement for /sup 125/I human urinary epo as tracer antigen in a radioimmunoassay for human epo

    International Nuclear Information System (INIS)

    Cotes, P.M.; Tam, R.C.; GainesDas, R.E.

    1987-01-01

    This paper represents evidence that in a radioimmunoassay for human erythropoietin, recombinant DNA derived human erythropoietin can replace highly purified human urinary erythropoietin in the preparation of radioiodinated tracer antigen

  10. One-step derivation of mesenchymal stem cell (MSC-like cells from human pluripotent stem cells on a fibrillar collagen coating.

    Directory of Open Access Journals (Sweden)

    Yongxing Liu

    Full Text Available Controlled differentiation of human embryonic stem cells (hESCs and induced pluripotent stem cells (iPSCs into cells that resemble adult mesenchymal stem cells (MSCs is an attractive approach to obtain a readily available source of progenitor cells for tissue engineering. The present study reports a new method to rapidly derive MSC-like cells from hESCs and hiPSCs, in one step, based on culturing the cells on thin, fibrillar, type I collagen coatings that mimic the structure of physiological collagen. Human H9 ESCs and HDFa-YK26 iPSCs were singly dissociated in the presence of ROCK inhibitor Y-27632, plated onto fibrillar collagen coated plates and cultured in alpha minimum essential medium (alpha-MEM supplemented with 10% fetal bovine serum, 50 uM magnesium L-ascorbic acid phosphate and 100 nM dexamethasone. While fewer cells attached on the collagen surface initially than standard tissue culture plastic, after culturing for 10 days, resilient colonies of homogenous spindle-shaped cells were obtained. Flow cytometric analysis showed that a high percentage of the derived cells expressed typical MSC surface markers including CD73, CD90, CD105, CD146 and CD166 and were negative as expected for hematopoietic markers CD34 and CD45. The MSC-like cells derived from pluripotent cells were successfully differentiated in vitro into three different lineages: osteogenic, chondrogenic, and adipogenic. Both H9 hES and YK26 iPS cells displayed similar morphological changes during the derivation process and yielded MSC-like cells with similar properties. In conclusion, this study demonstrates that bioimimetic, fibrillar, type I collagen coatings applied to cell culture plates can be used to guide a rapid, efficient derivation of MSC-like cells from both human ES and iPS cells.

  11. Rotator cuff repair using cell sheets derived from human rotator cuff in a rat model.

    Science.gov (United States)

    Harada, Yoshifumi; Mifune, Yutaka; Inui, Atsuyuki; Sakata, Ryosuke; Muto, Tomoyuki; Takase, Fumiaki; Ueda, Yasuhiro; Kataoka, Takeshi; Kokubu, Takeshi; Kuroda, Ryosuke; Kurosaka, Masahiro

    2017-02-01

    To achieve biological regeneration of tendon-bone junctions, cell sheets of human rotator-cuff derived cells were used in a rat rotator cuff injury model. Human rotator-cuff derived cells were isolated, and cell sheets were made using temperature-responsive culture plates. Infraspinatus tendons in immunodeficient rats were resected bilaterally at the enthesis. In right shoulders, infraspinatus tendons were repaired by the transosseous method and covered with the cell sheet (sheet group), whereas the left infraspinatus tendons were repaired in the same way without the cell sheet (control group). Histological examinations (safranin-O and fast green staining, isolectin B4, type II collagen, and human-specific CD31) and mRNA expression (vascular endothelial growth factor; VEGF, type II collagen; Col2, and tenomodulin; TeM) were analyzed 4 weeks after surgery. Biomechanical tests were performed at 8 weeks. In the sheet group, proteoglycan at the enthesis with more type II collagen and isolectin B4 positive cells were seen compared with in the control group. Human specific CD31-positive cells were detected only in the sheet group. VEGF and Col2 gene expressions were higher and TeM gene expression was lower in the sheet group than in the control group. In mechanical testing, the sheet group showed a significantly higher ultimate failure load than the control group at 8 weeks. Our results indicated that the rotator-cuff derived cell sheet could promote cartilage regeneration and angiogenesis at the enthesis, with superior mechanical strength compared with the control. Treatment for rotator cuff injury using cell sheets could be a promising strategy for enthesis of tendon tissue engineering. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:289-296, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  12. Composition and function of macroencapsulated human embryonic stem cell-derived implants: comparison with clinical human islet cell grafts.

    Science.gov (United States)

    Motté, Evi; Szepessy, Edit; Suenens, Krista; Stangé, Geert; Bomans, Myriam; Jacobs-Tulleneers-Thevissen, Daniel; Ling, Zhidong; Kroon, Evert; Pipeleers, Daniel

    2014-11-01

    β-Cells generated from large-scale sources can overcome current shortages in clinical islet cell grafts provided that they adequately respond to metabolic variations. Pancreatic (non)endocrine cells can develop from human embryonic stem (huES) cells following in vitro derivation to pancreatic endoderm (PE) that is subsequently implanted in immune-incompetent mice for further differentiation. Encapsulation of PE increases the proportion of endocrine cells in subcutaneous implants, with enrichment in β-cells when they are placed in TheraCyte-macrodevices and predominantly α-cells when they are alginate-microencapsulated. At posttransplant (PT) weeks 20-30, macroencapsulated huES implants presented higher glucose-responsive plasma C-peptide levels and a lower proinsulin-over-C-peptide ratio than human islet cell implants under the kidney capsule. Their ex vivo analysis showed the presence of single-hormone-positive α- and β-cells that exhibited rapid secretory responses to increasing and decreasing glucose concentrations, similar to isolated human islet cells. However, their insulin secretory amplitude was lower, which was attributed in part to a lower cellular hormone content; it was associated with a lower glucose-induced insulin biosynthesis, but not with lower glucagon-induced stimulation, which together is compatible with an immature functional state of the huES-derived β-cells at PT weeks 20-30. These data support the therapeutic potential of macroencapsulated huES implants but indicate the need for further functional analysis. Their comparison with clinical-grade human islet cell grafts sets references for future development and clinical translation. Copyright © 2014 the American Physiological Society.

  13. Comparison of human adipose-derived stem cells and bone marrow-derived stem cells in a myocardial infarction model

    DEFF Research Database (Denmark)

    Rasmussen, Jeppe; Frøbert, Ole; Holst-Hansen, Claus

    2014-01-01

    Background: Treatment of myocardial infarction with bone marrow-derived mesenchymal stem cells and recently also adipose-derived stem cells has shown promising results. In contrast to clinical trials and their use of autologous bone marrow-derived cells from the ischemic patient, the animal...... myocardial infarction models are often using young donors and young, often immune-compromised, recipient animals. Our objective was to compare bone marrow-derived mesenchymal stem cells with adipose-derived stem cells from an elderly ischemic patient in the treatment of myocardial infarction, using a fully...... grown non-immunecompromised rat model. Methods: Mesenchymal stem cells were isolated from adipose tissue and bone marrow and compared with respect to surface markers and proliferative capability. To compare the regenerative potential of the two stem cell populations, male Sprague-Dawley rats were...

  14. Human detection and discrimination of tactile repeatability, mechanical backlash, and temporal delay in a combined tactile-kinesthetic haptic display system.

    Science.gov (United States)

    Doxon, Andrew J; Johnson, David E; Tan, Hong Z; Provancher, William R

    2013-01-01

    Many of the devices used in haptics research are over-engineered for the task and are designed with capabilities that go far beyond human perception levels. Designing devices that more closely match the limits of human perception will make them smaller, less expensive, and more useful. However, many device-centric perception thresholds have yet to be evaluated. To this end, three experiments were conducted, using one degree-of-freedom contact location feedback device in combination with a kinesthetic display, to provide a more explicit set of specifications for similar tactile-kinesthetic haptic devices. The first of these experiments evaluated the ability of humans to repeatedly localize tactile cues across the fingerpad. Subjects could localize cues to within 1.3 mm and showed bias toward the center of the fingerpad. The second experiment evaluated the minimum perceptible difference of backlash at the tactile element. Subjects were able to discriminate device backlash in excess of 0.46 mm on low-curvature models and 0.93 mm on high-curvature models. The last experiment evaluated the minimum perceptible difference of system delay between user action and device reaction. Subjects were able to discriminate delays in excess of 61 ms. The results from these studies can serve as the maximum (i.e., most demanding) device specifications for most tactile-kinesthetic haptic systems.

  15. Influence of mechanical stimulation on human dermal fibroblasts derived from different body sites.

    Science.gov (United States)

    Kuang, Ruixia; Wang, Zhiguo; Xu, Quanchen; Liu, Su; Zhang, Weidong

    2015-01-01

    Mechanical stimulation is highly associated with pathogenesis of human hypertrophic scar. Although much work has focused on the influence of mechanical stress on fibroblast populations from various tissues and organs in the human body, their effects on cultured dermal fibroblasts by the area of the body have not been as well studied. In this study, cultures of skin fibroblasts from two different body sites were subjected to cyclic mechanical stimulation with a 10% stretching amplitude at a frequency of 0.1 Hz for 24, 36 and 48 hours, respectively, and thereafter harvested for experimental assays. Fibroblasts from scapular upper back skin, subjected to mechanical loads for 36 and 48 hours, respectively, were observed to proliferate at a higher rate and reach confluent more rapidly during in vitro culturing, had higher expression levels of mRNA and protein production of integrin β1, p130Cas and TGF β1 versus those from medial side of upper arm. These data indicate that skin fibroblasts, with regard to originated body sites studied in the experiments, display a diversity of mechanotransduction properties and biochemical reactions in response to applied mechanical stress, which contributes to the increased susceptibility to hypertrophic scars formation at certain areas of human body characterized by higher skin and muscle tension.

  16. Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support

    Directory of Open Access Journals (Sweden)

    Anita Muraglia

    2017-11-01

    Full Text Available Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i an heparin-free human platelet lysate (PL devoid of serum or plasma components (v-PL and (ii an heparin-free human serum derived from plasma devoid of PL components (Pl-s and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment, but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79 regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype.

  17. 2-Arylbenzo[b]furan derivatives as potent human lipoxygenase inhibitors.

    Science.gov (United States)

    Lang, Li; Dong, Ningning; Wu, Deyan; Yao, Xue; Lu, Weiqiang; Zhang, Chen; Ouyang, Ping; Zhu, Jin; Tang, Yun; Wang, Wei; Li, Jian; Huang, Jin

    2016-01-01

    Human lipoxygenases (LOXs) have been emerging as effective therapeutic targets for inflammatory diseases. In this study, we found that four natural 2-arylbenzo[b]furan derivatives isolated from Artocarpus heterophyllus exhibited potent inhibitory activities against human LOXs, including moracin C (1), artoindonesianin B-1 (2), moracin D (3), moracin M (4). In our in vitro experiments, compound 1 was identified as the most potent LOX inhibitor and the moderate subtype selective inhibitor of 12-LOX. Compounds 1 and 2 act as competitive inhibitors of LOXs. Moreover, 1 significantly inhibits LTB4 production and chemotactic capacity of neutrophils, and is capable of protecting vascular barrier from plasma leakage in vivo. In addition, the preliminary structure-activity relationship analysis was performed based on the above four naturally occurring (1-4) and six additional synthetic 2-arylbenzo[b]furan derivatives. Taken together, these 2-arylbenzo[b]furan derivatives, as LOXs inhibitors, could represent valuable leads for the future development of therapeutic agents for inflammatory diseases.

  18. Generation and characterisation of human umbilical cord derived mesenchymal stem cells by explant method.

    Science.gov (United States)

    Yusoff, Z; Maqbool, M; George, E; Hassan, R; Ramasamy, R

    2016-06-01

    Mesenchymal stem cells (MSCs) derived from human umbilical cord (UC) have been considered as an important tool for treating various malignancies, tissue repair and organ regeneration. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are better alternative to MSCs that derived from bone marrow (BM-MSCs) as they are regarded as medical waste with little ethical concern for research and easily culture-expanded. In this present study, the foetal distal end of human UC was utilised to generate MSC by explant method. Upon in vitro culture, adherent cells with fibroblastic morphology were generated with rapid growth kinetics. Under the respective inductive conditions, these cells were capable of differentiating into adipocytes and osteocytes; express an array of standard MSC's surface markers CD29, CD73, CD90, CD106 and MHC-class I. Further assessment of immunosuppression activity revealed that MSCs generated from UC had profoundly inhibited the proliferation of mitogen-activated T lymphocytes in a dosedependent manner. The current laboratory findings have reinforced the application of explant method to generate UCMSCs thus, exploring an ideal platform to fulfil the increasing demand of MSCs for research and potential clinical use.

  19. Immunomodulatory Effects of Diterpene Quinone Derivatives from the Roots of Horminum pyrenaicum in Human PBMC

    Directory of Open Access Journals (Sweden)

    K. Becker

    2018-01-01

    Full Text Available Several phytochemicals were shown to interfere with redox biology in the human system. Moreover, redox biochemistry is crucially involved in the orchestration of immunological cascades. When screening for immunomodulatory compounds, the two interferon gamma- (IFN-γ- dependent immunometabolic pathways of tryptophan breakdown via indoleamine 2,3-dioxygenase-1 (IDO-1 and neopterin formation by GTP-cyclohydrolase 1 (GTP-CH-I represent prominent targets, as IFN-γ-related signaling is strongly sensitive to oxidative triggers. Herein, the analysis of these pathway activities in human peripheral mononuclear cells was successfully applied in a bioactivity-guided fractionation strategy to screen for anti-inflammatory substances contained in the root of Horminum (H. pyrenaicum L. (syn. Dragon’s mouth, the only representative of the monophyletic genus Horminum. Four abietane diterpene quinone derivatives (horminone, 7-O-acetylhorminone, inuroyleanol and its 15,16-dehydro-derivative, a novel natural product, two nor-abietane diterpene quinones (agastaquinone and 3-deoxyagastaquinone and two abeo 18 (4 → 3 abietane diterpene quinones (agastol and its 15,16-dehydro-derivative could be identified. These compounds were able to dose-dependently suppress the above mentioned pathways with different potency. Beside the description of new active compounds, this study demonstrates the feasibility of integrating IDO-1 and GTP-CH-I activity in the search for novel anti-inflammatory compounds, which can then be directed towards a more detailed mode of action analysis.

  20. Radiopharmacology of iminodiacetic acid N-derivatives analysis in biological models and comparison to human beings

    International Nuclear Information System (INIS)

    Canellas, C.O.; Arguelles, M.G.; Mitta, A.E.A.

    1987-01-01

    It was studied the influence of chemical structures and molecular weight in the distribution of several iminodiacetic acid N-derivatives and to determine the potential use of these radiopharmaceuticals in humans. The study was performed with the following derivatives: N-(2,6 dimetyphenylcarbamoylmethy) iminodiacetic acid, N(2.6 dietylphenyl-carbamoylmethy) iminodiacetic acid, N-(2,6 diisopropylphenylcarbamoylmethy) iminodiacetic acid and the previously unknown N-derivative N-(2,6 diisopropyl, phenylcarbamoylethyl) iminodiacetic aced. These were sinthesized by a modified procedure by MITTA et al. and controlled by NMR, mass spectrometry, elemental composition and also toxicity pirogens, lethal dose and the chelate's radiochemical dose were determined. Liver gallbladder, intestinal and renal kinetics were studied in mice. In order to evaluate the metabolic pathways of the radiopharmaceuticals, the content of gallbladder and the urine were reinjected. Plasma kinetics and the plasmatic half life was determined by extracorporeal circulation in Wistar rats. For the use in human beings, test were carried out in different branches of nuclear medicine, in normal volunteers and carriers of different pathologic disorders. The patients were divided into four groups: acute and chronic cholecystitis, cirrhosis and jaundice. It was obtained the liver/heart activity ratio and estimated the appearance times of the intrahepatic ducts, gallbladder, duodenum and renal persistence. (M.E.L.) [es

  1. Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

    Science.gov (United States)

    Zou, Li; Kidwai, Fahad K.; Kopher, Ross A.; Motl, Jason; Kellum, Cory A.; Westendorf, Jennifer J.; Kaufman, Dan S.

    2015-01-01

    Summary We generated a RUNX2-yellow fluorescent protein (YFP) reporter system to study osteogenic development from human embryonic stem cells (hESCs). Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development. PMID:25680477

  2. Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Li Zou

    2015-02-01

    Full Text Available We generated a RUNX2-yellow fluorescent protein (YFP reporter system to study osteogenic development from human embryonic stem cells (hESCs. Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development.

  3. Sex Differences in Maturation of Human Embryonic Stem Cell-Derived β Cells in Mice.

    Science.gov (United States)

    Saber, Nelly; Bruin, Jennifer E; O'Dwyer, Shannon; Schuster, Hellen; Rezania, Alireza; Kieffer, Timothy J

    2018-04-01

    Pancreatic progenitors derived from human embryonic stem cells (hESCs) are now in clinical trials for insulin replacement in patients with type 1 diabetes. Animal studies indicate that pancreatic progenitor cells can mature into a mixed population of endocrine cells, including glucose-responsive β cells several months after implantion. However, it remains unclear how conditions in the recipient may influence the maturation and ultimately the function of these hESC-derived cells. Here, we investigated the effects of (1) pregnancy on the maturation of human stage 4 (S4) pancreatic progenitor cells and (2) the impact of host sex on both S4 cells and more mature stage 7 (S7) pancreatic endocrine cells implanted under the kidney capsule of immunodeficient SCID-beige mice. Pregnancy led to increased proliferation of endogenous pancreatic β cells, but did not appear to affect proliferation or maturation of S4 cells at midgestation. Interestingly, S4 and S7 cells both acquired glucose-stimulated C-peptide secretion in females before males. Moreover, S4 cells lowered fasting blood glucose levels in females sooner than in males, whereas the responses with S7 cells were similar. These data indicate that the host sex may impact the maturation of hESC-derived cells in vivo and that this effect can be minimized by more advanced differentiation of the cells before implantation.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adipose-derived stem cells.

    Science.gov (United States)

    Wang, Chuan; Xiao, Ran; Cao, Yi-Lin; Yin, Hong-Yu

    2017-09-09

    Cryopreservation provides an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs). Dimethylsulfoxide (DMSO) and fetal bovine serum (FBS) are frequently used as cryoprotectants for this purpose. However, the use of DMSO can result in adverse effects and toxic reactions and FBS can introduce risks of viral, prion, zoonose contaminations and evoke immune responses after injection. It is therefore crucial to reduce DMSO concentrations and use serum-free solution in the cryopreservation process. Human platelet lysate (PL) is a promising candidate for use as an alternative to DMSO and FBS. Therefore, in this study, with an aim to identify a cryoprotective agent for ASC cryopreservation, we determined the viability, proliferation potential, phenotype, and differentiation potential of fresh ASCs and ASCs cryopreserved using different combinations of three cryoprotective agents: fetal bovine serum (FBS), dimethylsulfoxide (DMSO), and human platelet lysate (PL). The viability of the ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO was >80%, and the proliferation potentials, cell phenotypes, and differentiation potentials of these groups were similar to those of fresh ASCs. Together, our findings suggest that a combination of 97% PL and 3% DMSO is an ideal cryoprotective agent for the efficient cryopreservation of human ASCs. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. PrPC displays an essential protective role from oxidative stress in an astrocyte cell line derived from PrPC knockout mice

    International Nuclear Information System (INIS)

    Bertuchi, Fernanda R.; Bourgeon, Dominique M.G.; Landemberger, Michele C.; Martins, Vilma R.; Cerchiaro, Giselle

    2012-01-01

    Highlights: ► PrP C in solution acts as a radical scavenger. ► PrP C reduces hydrogen peroxide toxicity in astrocytes. ► Increase in ROS disrupted the cell cycle in the PrP C -knockout astrocytes. ► PrP C prevents the cell death independently of an SOD-like activity. -- Abstract: The PrP C protein, which is especially present in the cellular membrane of nervous system cells, has been extensively studied for its controversial antioxidant activity. In this study, we elucidated the free radical scavenger activity of purified murine PrP C in solution and its participation as a cell protector in astrocytes that were subjected to treatment with an oxidant. In vitro and using an EPR spin-trapping technique, we observed that PrP C decreased the oxidation of the DMPO trap in a Fenton reaction system (Cu 2+ /ascorbate/H 2 O 2 ), which was demonstrated by approximately 70% less DMPO/OH · . In cultured PrP C -knockout astrocytes from mice, the absence of PrP C caused an increase in intracellular ROS (reactive oxygen species) generation during the first 3 h of H 2 O 2 treatment. This rapid increase in ROS disrupted the cell cycle in the PrP C -knockout astrocytes, which increased the population of cells in the sub-G1 phase when compared with cultured wild-type astrocytes. We conclude that PrP C in solution acts as a radical scavenger, and in astrocytes, it is essential for protection from oxidative stress caused by an external chemical agent, which is a likely condition in human neurodegenerative CNS disorders and pathological conditions such as ischemia.

  7. No Relationship between Embryo Morphology and Successful Derivation of Human Embryonic Stem Cell Lines

    Science.gov (United States)

    Ström, Susanne; Rodriguez-Wallberg, Kenny; Holm, Frida; Bergström, Rosita; Eklund, Linda; Strömberg, Anne-Marie; Hovatta, Outi

    2010-01-01

    Background The large number (30) of permanent human embryonic stem cell (hESC) lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002–2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation. Methods We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines. Results Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM) was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN) zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line. Conclusion Even very poor quality embryos with few cells in the ICM can give origin to hESC lines. PMID:21217828

  8. Towards Investigating Global Warming Impact on Human Health Using Derivatives of Photoplethysmogram Signals.

    Science.gov (United States)

    Elgendi, Mohamed; Norton, Ian; Brearley, Matt; Fletcher, Richard R; Abbott, Derek; Lovell, Nigel H; Schuurmans, Dale

    2015-10-14

    Recent clinical studies show that the contour of the photoplethysmogram (PPG) wave contains valuable information for characterizing cardiovascular activity. However, analyzing the PPG wave contour is difficult; therefore, researchers have applied first or higher order derivatives to emphasize and conveniently quantify subtle changes in the filtered PPG contour. Our hypothesis is that analyzing the whole PPG recording rather than each PPG wave contour or on a beat-by-beat basis can detect heat-stressed subjects and that, consequently, we will be able to investigate the impact of global warming on human health. Here, we explore the most suitable derivative order for heat stress assessment based on the energy and entropy of the whole PPG recording. The results of our study indicate that the use Int. J. Environ. Res. Public Health 2015, 7 12777 of the entropy of the seventh derivative of the filtered PPG signal shows promising results in detecting heat stress using 20-second recordings, with an overall accuracy of 71.6%. Moreover, the combination of the entropy of the seventh derivative of the filtered PPG signal with the root mean square of successive differences, or RMSSD (a traditional heart rate variability index of heat stress), improved the detection of heat stress to 88.9% accuracy.

  9. In vitro effects of three blood derivatives on human corneal epithelial cells.

    Science.gov (United States)

    Freire, Vanesa; Andollo, Noelia; Etxebarria, Jaime; Durán, Juan A; Morales, María-Celia

    2012-08-15

    We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.

  10. Towards Investigating Global Warming Impact on Human Health Using Derivatives of Photoplethysmogram Signals

    Directory of Open Access Journals (Sweden)

    Mohamed Elgendi

    2015-10-01

    Full Text Available Recent clinical studies show that the contour of the photoplethysmogram (PPG wave contains valuable information for characterizing cardiovascular activity. However, analyzing the PPG wave contour is difficult; therefore, researchers have applied first or higher order derivatives to emphasize and conveniently quantify subtle changes in the filtered PPG contour. Our hypothesis is that analyzing the whole PPG recording rather than each PPG wave contour or on a beat-by-beat basis can detect heat-stressed subjects and that, consequently, we will be able to investigate the impact of global warming on human health. Here, we explore the most suitable derivative order for heat stress assessment based on the energy and entropy of the whole PPG recording. The results of our study indicate that the use of the entropy of the seventh derivative of the filtered PPG signal shows promising results in detecting heat stress using 20-second recordings, with an overall accuracy of 71.6%. Moreover, the combination of the entropy of the seventh derivative of the filtered PPG signal with the root mean square of successive differences, or RMSSD (a traditional heart rate variability index of heat stress, improved the detection of heat stress to 88.9% accuracy.

  11. Towards Investigating Global Warming Impact on Human Health Using Derivatives of Photoplethysmogram Signals

    Science.gov (United States)

    Elgendi, Mohamed; Norton, Ian; Brearley, Matt; Fletcher, Richard R.; Abbott, Derek; Lovell, Nigel H.; Schuurmans, Dale

    2015-01-01

    Recent clinical studies show that the contour of the photoplethysmogram (PPG) wave contains valuable information for characterizing cardiovascular activity. However, analyzing the PPG wave contour is difficult; therefore, researchers have applied first or higher order derivatives to emphasize and conveniently quantify subtle changes in the filtered PPG contour. Our hypothesis is that analyzing the whole PPG recording rather than each PPG wave contour or on a beat-by-beat basis can detect heat-stressed subjects and that, consequently, we will be able to investigate the impact of global warming on human health. Here, we explore the most suitable derivative order for heat stress assessment based on the energy and entropy of the whole PPG recording. The results of our study indicate that the use of the entropy of the seventh derivative of the filtered PPG signal shows promising results in detecting heat stress using 20-second recordings, with an overall accuracy of 71.6%. Moreover, the combination of the entropy of the seventh derivative of the filtered PPG signal with the root mean square of successive differences, or RMSSD (a traditional heart rate variability index of heat stress), improved the detection of heat stress to 88.9% accuracy. PMID:26473907

  12. Effect of Wasabi Component 6-(Methylsulfinylhexyl Isothiocyanate and Derivatives on Human Pancreatic Cancer Cells

    Directory of Open Access Journals (Sweden)

    Yu-Jen Chen

    2014-01-01

    Full Text Available The naturally occurring compound 6-(methylsulfinylhexyl isothiocyanate (6-MITC was isolated from Wasabia japonica (Wasabi, a pungent spice used in Japanese food worldwide. The synthetic derivatives 6-(methylsulfenylhexyl isothiocyanate (I7447 and 6-(methylsulfonylhexyl isothiocyanate (I7557 are small molecule compounds derived from 6-MITC. This study aimed to evaluate the effect of these compounds on human pancreatic cancer cells. Human pancreatic cancer cell lines PANC-1 and BxPC-3 were used to perform an MTT assay for cell viability and Liu’s stain for morphological observation. The cell cycle was analyzed by DNA histogram. Aldehyde dehydrogenase (ALDH activity was used as a marker for cancer stem cells (CSC. Western blotting was performed for the expression of proteins related to CSC signaling. The results showed that compounds 6-MITC and I7557, but not I7447, inhibited viability of both PANC-1 and BxPC-3 cells. Morphological observation showed mitotic arrest and apoptosis in 6-MITC- and I7557-treated cells. These two compounds induced G2/M phase arrest and hypoploid population. Percentages of ALDH-positive PANC-1 cells were markedly reduced by 6-MITC and I7557 treatment. The expression of CSC signaling molecule SOX2, but not NOTCH1, ABCG2, Sonic hedgehog, or OCT4, was inhibited by 6-MITC and I7557. In conclusion, wasabi compounds 6-MITC and I7557 may possess activity against the growth and CSC phenotypes of human pancreatic cancer cells.

  13. Efficacy of Human Adipose Tissue-Derived Stem Cells on Neonatal Bilirubin Encephalopathy in Rats.

    Science.gov (United States)

    Amini, Naser; Vousooghi, Nasim; Hadjighassem, Mahmoudreza; Bakhtiyari, Mehrdad; Mousavi, Neda; Safakheil, Hosein; Jafari, Leila; Sarveazad, Arash; Yari, Abazar; Ramezani, Sara; Faghihi, Faezeh; Joghataei, Mohammad Taghi

    2016-05-01

    Kernicterus is a neurological syndrome associated with indirect bilirubin accumulation and damages to the basal ganglia, cerebellum and brain stem nuclei particularly the cochlear nucleus. To mimic haemolysis in a rat model such that it was similar to what is observed in a preterm human, we injected phenylhydrazine in 7-day-old rats to induce haemolysis and then infused sulfisoxazole into the same rats at day 9 to block bilirubin binding sites in the albumin. We have investigated the effectiveness of human adiposity-derived stem cells as a therapeutic paradigm for perinatal neuronal repair in a kernicterus animal model. The level of total bilirubin, indirect bilirubin, brain bilirubin and brain iron was significantly increased in the modelling group. There was a significant decreased in all severity levels of the auditory brainstem response test in the two modelling group. Akinesia, bradykinesia and slip were significantly declined in the experience group. Apoptosis in basal ganglia and cerebellum were significantly decreased in the stem cell-treated group in comparison to the vehicle group. All severity levels of the auditory brainstem response tests were significantly decreased in 2-month-old rats. Transplantation results in the substantial alleviation of walking impairment, apoptosis and auditory dysfunction. This study provides important information for the development of therapeutic strategies using human adiposity-derived stem cells in prenatal brain damage to reduce potential sensori motor deficit.

  14. Colonizing the embryonic zebrafish gut with anaerobic bacteria derived from the human gastrointestinal tract.

    Science.gov (United States)

    Toh, Michael C; Goodyear, Mara; Daigneault, Michelle; Allen-Vercoe, Emma; Van Raay, Terence J

    2013-06-01

    The zebrafish has become increasingly popular for microbiological research. It has been used as an infection model for a variety of pathogens, and is also emerging as a tool for studying interactions between a host and its resident microbial communities. The mouse microbiota has been transplanted into the zebrafish gut, but to our knowledge, there has been no attempt to introduce a bacterial community derived from the human gut. We explored two methods for colonizing the developing gut of 5-day-old germ-free zebrafish larvae with a defined anaerobic microbial community derived from a single human fecal sample. Both environmental exposure (static immersion) and direct microinjection into the gut resulted in the establishment of two species-Lactobacillus paracasei and Eubacterium limosum-from a community of 30 strains consisting of 22 anaerobic species. Of particular interest is E. limosum, which, as a strict anaerobe, represents a group of bacteria which until now have not been shown to colonize the developing zebrafish gut. Our success here indicates that further investigation of zebrafish as a tool for studying human gut microbial communities is warranted.

  15. Purification of human induced pluripotent stem cell-derived neural precursors using magnetic activated cell sorting.

    Science.gov (United States)

    Rodrigues, Gonçalo M C; Fernandes, Tiago G; Rodrigues, Carlos A V; Cabral, Joaquim M S; Diogo, Maria Margarida

    2015-01-01

    Neural precursor (NP) cells derived from human induced pluripotent stem cells (hiPSCs), and their neuronal progeny, will play an important role in disease modeling, drug screening tests, central nervous system development studies, and may even become valuable for regenerative medicine treatments. Nonetheless, it is challenging to obtain homogeneous and synchronously differentiated NP populations from hiPSCs, and after neural commitment many pluripotent stem cells remain in the differentiated cultures. Here, we describe an efficient and simple protocol to differentiate hiPSC-derived NPs in 12 days, and we include a final purification stage where Tra-1-60+ pluripotent stem cells (PSCs) are removed using magnetic activated cell sorting (MACS), leaving the NP population nearly free of PSCs.

  16. Human Pluripotent Stem Cell-Derived Cardiomyocytes as Research and Therapeutic Tools

    Directory of Open Access Journals (Sweden)

    Ivana Acimovic

    2014-01-01

    Full Text Available Human pluripotent stem cells (hPSCs, namely, embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs, with their ability of indefinite self-renewal and capability to differentiate into cell types derivatives of all three germ layers, represent a powerful research tool in developmental biology, for drug screening, disease modelling, and potentially cell replacement therapy. Efficient differentiation protocols that would result in the cell type of our interest are needed for maximal exploitation of these cells. In the present work, we aim at focusing on the protocols for differentiation of hPSCs into functional cardiomyocytes in vitro as well as achievements in the heart disease modelling and drug testing on the patient-specific iPSC-derived cardiomyocytes (iPSC-CMs.

  17. Neuron-derived orphan receptor 1 promoted human pulmonary artery smooth muscle cells proliferation.

    Science.gov (United States)

    Wang, Chang-Guo; Lei, Wei; Li, Chang; Zeng, Da-Xiong; Huang, Jian-An

    2015-05-01

    As a transcription factor of the nuclear receptor superfamily, neuron-derived orphan receptor 1 (NOR1) is induced rapidly in response to various extracellular stimuli. But, it is still unclear its role in pulmonary artery smooth muscle cells proliferation. Human PASMCs were cultured in vitro and stimulated by serum. The special antisense oligodeoxynucleotides (AS-ODNs) were used to knockdown human NOR1 gene expression. Real-time PCR and Western-blot were used to evaluate the gene expression and protein levels. Fetal bovine serum (FBS) induced human PASMCs proliferation in a dose dependent manner. Furthermore, FBS promoted NOR1 gene expression in a dose dependent manner and a time dependent manner. 10% FBS induced a maximal NOR1 mRNA levels at 2 h. FBS also induced a significant higher NOR1 protein levels as compared with control. The NOR1 over-expressed plasmid significantly promoted DNA synthesis and cells proliferation. Moreover, the special AS-ODNs against human NOR1 not only prevented NOR1 expression but also inhibited DNA synthesis and cells proliferation significantly. The NOR1 over-expression plasmid could up-regulate cyclin D1 expression markedly, but the AS-ODNs inhibited cyclin D1 expression significantly. So, we concluded that NOR1 could promote human PASMCs proliferation. Cyclin D1 might be involved in this process.

  18. A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells.

    Directory of Open Access Journals (Sweden)

    Romeo Cecchelli

    Full Text Available The human blood brain barrier (BBB is a selective barrier formed by human brain endothelial cells (hBECs, which is important to ensure adequate neuronal function and protect the central nervous system (CNS from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.

  19. A potential pro-anagogic cell therapy with human placenta-derived mesenchymal cells

    International Nuclear Information System (INIS)

    Nishishita, Toshihide; Ouchi, Kunie; Zhang, Xiaohong; Inoue, Mariko; Inazawa, Takeshi; Yoshiura, Kenta; Kuwabara, Koichiro; Nakaoka, Takashi; Watanabe, Nobukazu; Igura, Koichi; Takahashi, Tsuneo A.; Yamashita, Naohide

    2004-01-01

    Recently several strategies to treat ischemic diseases have been proposed but the ideal way has to be determined. We explored whether human placenta-derived mesenchymal cells (hPDMCs) can be used for this purpose because placenta is very rich in vessels. First, production of human vascular endothelial growth factor (hVEGF) from hPDMCs was examined. The amount of hVEGF secreted by hPDMCs was similar to the amount produced by HeLa cells. hVEGF was barely detected in human umbilical vein endothelial cells (hUVECs) or human peripheral blood mononuclear cells. hVEGF secreted from hPDMCs stimulated the proliferation of hUVECs, indicating its biological activity. Transplantation of hPDMCs to the ischemic limbs of NOD/Shi-scid mice significantly improved the blood flow of the affected limbs. Blood vessel formation was more prominently observed in the limbs of treated mice as compared to the control mice. Real-time RT-PCR revealed that hPDMCs produced hVEGF for at least 7 days after transplantation. Thus, transplantation of hPDMCs could potentially be a promising treatment for human ischemic diseases

  20. Modeling chemotherapeutic neurotoxicity with human induced pluripotent stem cell-derived neuronal cells.

    Directory of Open Access Journals (Sweden)

    Heather E Wheeler

    Full Text Available There are no effective agents to prevent or treat chemotherapy-induced peripheral neuropathy (CIPN, the most common non-hematologic toxicity of chemotherapy. Therefore, we sought to evaluate the utility of human neuron-like cells derived from induced pluripotent stem cells (iPSCs as a means to study CIPN. We used high content imaging measurements of neurite outgrowth phenotypes to compare the changes that occur to iPSC-derived neuronal cells among drugs and among individuals in response to several classes of chemotherapeutics. Upon treatment of these neuronal cells with the neurotoxic drug paclitaxel, vincristine or cisplatin, we identified significant differences in five morphological phenotypes among drugs, including total outgrowth, mean/median/maximum process length, and mean outgrowth intensity (P < 0.05. The differences in damage among drugs reflect differences in their mechanisms of action and clinical CIPN manifestations. We show the potential of the model for gene perturbation studies by demonstrating decreased expression of TUBB2A results in significantly increased sensitivity of neurons to paclitaxel (0.23 ± 0.06 decrease in total neurite outgrowth, P = 0.011. The variance in several neurite outgrowth and apoptotic phenotypes upon treatment with one of the neurotoxic drugs is significantly greater between than within neurons derived from four different individuals (P < 0.05, demonstrating the potential of iPSC-derived neurons as a genetically diverse model for CIPN. The human neuron model will allow both for mechanistic studies of specific genes and genetic variants discovered in clinical studies and for screening of new drugs to prevent or treat CIPN.

  1. Human Adipose Tissue Derived Extracellular Matrix and Methylcellulose Hydrogels Augments and Regenerates the Paralyzed Vocal Fold.

    Directory of Open Access Journals (Sweden)

    Dong Wook Kim

    Full Text Available Vocal fold paralysis results from various etiologies and can induce voice changes, swallowing complications, and issues with aspiration. Vocal fold paralysis is typically managed using injection laryngoplasty with fat or synthetic polymers. Injection with autologous fat has shown excellent biocompatibility. However, it has several disadvantages such as unpredictable resorption rate, morbidities associated with liposuction procedure which has to be done in operating room under general anesthesia. Human adipose-derived extracellular matrix (ECM grafts have been reported to form new adipose tissue and have greater biostability than autologous fat graft. Here, we present an injectable hydrogel that is constructed from adipose tissue derived soluble extracellular matrix (sECM and methylcellulose (MC for use in vocal fold augmentation. Human sECM derived from adipose tissue was extracted using two major steps-ECM was isolated from human adipose tissue and was subsequently solubilized. Injectable sECM/MC hydrogels were prepared by blending of sECM and MC. Sustained vocal fold augmentation and symmetric vocal fold vibration were accomplished by the sECM/MC hydrogel in paralyzed vocal fold which were confirmed by laryngoscope, histology and a high-speed imaging system. There were increased number of collagen fibers and fatty granules at the injection site without significant inflammation or fibrosis. Overall, these results indicate that the sECM/MC hydrogel can enhance vocal function in paralyzed vocal folds without early resorption and has potential as a promising material for injection laryngoplasty for stable vocal fold augmentation which can overcome the shortcomings of autologous fat such as unpredictable duration and morbidity associated with the fat harvest.

  2. Human Adipose Tissue Derived Extracellular Matrix and Methylcellulose Hydrogels Augments and Regenerates the Paralyzed Vocal Fold.

    Science.gov (United States)

    Kim, Dong Wook; Kim, Eun Ji; Kim, Eun Na; Sung, Myung Whun; Kwon, Tack-Kyun; Cho, Yong Woo; Kwon, Seong Keun

    2016-01-01

    Vocal fold paralysis results from various etiologies and can induce voice changes, swallowing complications, and issues with aspiration. Vocal fold paralysis is typically managed using injection laryngoplasty with fat or synthetic polymers. Injection with autologous fat has shown excellent biocompatibility. However, it has several disadvantages such as unpredictable resorption rate, morbidities associated with liposuction procedure which has to be done in operating room under general anesthesia. Human adipose-derived extracellular matrix (ECM) grafts have been reported to form new adipose tissue and have greater biostability than autologous fat graft. Here, we present an injectable hydrogel that is constructed from adipose tissue derived soluble extracellular matrix (sECM) and methylcellulose (MC) for use in vocal fold augmentation. Human sECM derived from adipose tissue was extracted using two major steps-ECM was isolated from human adipose tissue and was subsequently solubilized. Injectable sECM/MC hydrogels were prepared by blending of sECM and MC. Sustained vocal fold augmentation and symmetric vocal fold vibration were accomplished by the sECM/MC hydrogel in paralyzed vocal fold which were confirmed by laryngoscope, histology and a high-speed imaging system. There were increased number of collagen fibers and fatty granules at the injection site without significant inflammation or fibrosis. Overall, these results indicate that the sECM/MC hydrogel can enhance vocal function in paralyzed vocal folds without early resorption and has potential as a promising material for injection laryngoplasty for stable vocal fold augmentation which can overcome the shortcomings of autologous fat such as unpredictable duration and morbidity associated with the fat harvest.

  3. Pharmacological Characterisation of Nicotinic Acetylcholine Receptors Expressed in Human iPSC-Derived Neurons.

    Directory of Open Access Journals (Sweden)

    Anna Chatzidaki

    Full Text Available Neurons derived from human induced pluripotent stem cells (iPSCs represent a potentially valuable tool for the characterisation of neuronal receptors and ion channels. Previous studies on iPSC-derived neuronal cells have reported the functional characterisation of a variety of receptors and ion channels, including glutamate receptors, γ-aminobutyric acid (GABA receptors and several voltage-gated ion channels. In the present study we have examined the expression and functional properties of nicotinic acetylcholine receptors (nAChRs in human iPSC-derived neurons. Gene expression analysis indicated the presence of transcripts encoding several nAChR subunits, with highest levels detected for α3-α7, β1, β2 and β4 subunits (encoded by CHRNA3-CHRNA7, CHRNB1, CHRNB2 and CHRNB4 genes. In addition, similarly high transcript levels were detected for the truncated dupα7 subunit transcript, encoded by the partially duplicated gene CHRFAM7A, which has been associated with psychiatric disorders such as schizophrenia. The functional properties of these nAChRs have been examined by calcium fluorescence and by patch-clamp recordings. The data obtained suggest that the majority of functional nAChRs expressed in these cells have pharmacological properties typical of α7 receptors. Large responses were induced by a selective α7 agonist (compound B, in the presence of the α7-selective positive allosteric modulator (PAM PNU-120596, which were blocked by the α7-selective antagonist methyllycaconitine (MLA. In addition, a small proportion of the neurons express nAChRs with properties typical of heteromeric (non-α7 containing nAChR subtypes. These cells therefore represent a great tool to advance our understanding of the properties of native human nAChRs, α7 in particular.

  4. Xenotransplantation of human adipose-derived stem cells in zebrafish embryos.

    Directory of Open Access Journals (Sweden)

    Jin Li

    Full Text Available Zebrafish is a widely used animal model with well-characterized background in developmental biology. The fate of human adipose-derived stem cells (ADSCs after their xenotransplantation into the developing embryos of zebrafish is unknown. Therefore, human ADSCs were firstly isolated, and then transduced with lentiviral vector system carrying a green fluorescent protein (GFP reporter gene, and followed by detection of their cell viability and the expression of cell surface antigens. These GFP-expressing human ADSCs were transplanted into the zebrafish embryos at 3.3-4.3 hour post-fertilization (hpf. Green fluorescent signal, the proliferation and differentiation of human ADSCs in recipient embryos were respectively examined using fluorescent microscopy and immunohistochemical staining. The results indicated that human ADSCs did not change their cell viability and the expression levels of cell surface antigens after GFP transduction. Microscopic examination demonstrated that green fluorescent signals of GFP expressed in the transplanted cells were observed in the embryos and larva fish at post-transplantation. The positive staining of Ki-67 revealed the survival and proliferation of human ADSCs in fish larvae after transplantation. The expression of CD105 was observable in the xenotransplanted ADSCs, but CD31 expression was undetectable. Therefore, our results indicate that human ADSCs xenotransplanted in the zebrafish embryos not only can survive and proliferate at across-species circumstance, but also seem to maintain their undifferentiation status in a short term. This xenograft model of zebrafish embryos may provide a promising and useful technical platform for the investigation of biology and physiology of stem cells in vivo.

  5. Regenerative Medicine, Disease Modelling, and Drug Discovery in Human Pluripotent Stem Cell-Derived Kidney Tissue

    Directory of Open Access Journals (Sweden)

    Navin Gupta

    2017-08-01

    Full Text Available The multitude of research clarifying critical factors in embryonic organ development has been instrumental in human stem cell research. Mammalian organogenesis serves as the archetype for directed differentiation protocols, subdividing the process into a series of distinct intermediate stages that can be chemically induced and monitored for the expression of stage-specific markers. Significant advances over the past few years include established directed differentiation protocols of human embryonic stem cells and human induced pluripotent stem cells (hiPSC into human kidney organoids in vitro. Human kidney tissue in vitro simulates the in vivo response when subjected to nephrotoxins, providing a novel screening platform during drug discovery to facilitate identification of lead candidates, reduce developmental expenditures, and reduce future rates of drug-induced acute kidney injury. Patient-derived hiPSC, which bear naturally occurring DNA mutations, may allow for modelling of human genetic diseases to enable determination of pathological mechanisms and screening for novel therapeutics. In addition, recent advances in genome editing with clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 enable the generation of specific mutations to study genetic disease, with non-mutated lines serving as an ideal isogenic control. The growing population of patients with end-stage kidney disease is a worldwide healthcare problem, with high morbidity and mortality rates, that warrants the discovery of novel forms of renal replacement therapy. Coupling the outlined advances in hiPSC research with innovative bioengineering techniques, such as decellularised kidney and three-dimensional printed scaffolds, may contribute to the development of bioengineered transplantable human kidney tissue as a means of renal replacement therapy.

  6. Prevention of Human Lymphoproliferative Tumor Formation in Ovarian Cancer Patient-Derived Xenografts

    Directory of Open Access Journals (Sweden)

    Kristina A. Butler

    2017-08-01

    Full Text Available Interest in preclinical drug development for ovarian cancer has stimulated development of patient-derived xenograft (PDX or tumorgraft models. However, the unintended formation of human lymphoma in severe combined immunodeficiency (SCID mice from Epstein-Barr virus (EBV–infected human lymphocytes can be problematic. In this study, we have characterized ovarian cancer PDXs which developed human lymphomas and explore methods to suppress lymphoproliferative growth. Fresh human ovarian tumors from 568 patients were transplanted intraperitoneally in SCID mice. A subset of PDX models demonstrated atypical patterns of dissemination with mediastinal masses, hepatosplenomegaly, and CD45-positive lymphoblastic atypia without ovarian tumor engraftment. Expression of human CD20 but not CD3 supported a B-cell lineage, and EBV genomes were detected in all lymphoproliferative tumors. Immunophenotyping confirmed monoclonal gene rearrangements consistent with B-cell lymphoma, and global gene expression patterns correlated well with other human lymphomas. The ability of rituximab, an anti-CD20 antibody, to suppress human lymphoproliferation from a patient's ovarian tumor in SCID mice and prevent growth of an established lymphoma led to a practice change with a goal to reduce the incidence of lymphomas. A single dose of rituximab during the primary tumor heterotransplantation process reduced the incidence of CD45-positive cells in subsequent PDX lines from 86.3% (n = 117 without rituximab to 5.6% (n = 160 with rituximab, and the lymphoma rate declined from 11.1% to 1.88%. Taken together, investigators utilizing PDX models for research should routinely monitor for lymphoproliferative tumors and consider implementing methods to suppress their growth.

  7. Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium

    DEFF Research Database (Denmark)

    Marthin, June Kehlet; Stevens, Elizabeth Munkebjerg; Larsen, Lars Allan

    2017-01-01

    BACKGROUND: Three-dimensional explant spheroid formation is an ex vivo technique previously used in studies of airway epithelial ion and water transport. Explanted cells and sheets of nasal epithelium form fully differentiated spheroids enclosing a partly fluid-filled lumen with the ciliated apical...... surface facing the outside and accessible for analysis of ciliary function. METHODS: We performed a two-group comparison study of ciliary beat pattern and ciliary beat frequency in spheroids derived from nasal airway epithelium in patients with primary ciliary dyskinesia (PCD) and in healthy controls...... in the investigation of pathophysiological aspects and drug effects in human nasal airway epithelium....

  8. Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

    Science.gov (United States)

    Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

    2013-12-01

    Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained

  9. The proteomic dataset for bone marrow derived human mesenchymal stromal cells: Effect of in vitro passaging

    Directory of Open Access Journals (Sweden)

    Samuel T. Mindaye

    2015-12-01

    Full Text Available Bone-marrow derived mesenchymal stromal cells (BMSCs have been in clinical trials for therapy. One major bottleneck in the advancement of BMSC-based products is the challenge associated with cell isolation, characterization, and ensuring cell fitness over the course of in vitro cell propagation steps. The data in this report is part of publications that explored the proteomic changes following in vitro passaging of BMSCs [4] and the molecular heterogeneity in cultures obtained from different human donors [5,6].The methodological details involving cell manufacturing, proteome harvesting, protein identification and quantification as well as the bioinformatic analyses were described to ensure reproducibility of the results.

  10. Genetically modified human bone marrow derived mesenchymal stem cells for improving the outcome of human islet transplantation.

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    Vaibhav Mundra

    Full Text Available The objective of this study was to determine the potential of human bone marrow derived mesenchymal stem cells (hBMSCs as gene carriers for improving the outcome of human islet transplantation. hBMSCs were characterized for the expression of phenotypic markers and transduced with Adv-hVEGF-hIL-1Ra to overexpress human vascular endothelial growth factor (hVEGF and human interleukin-1 receptor antagonist (hIL-1Ra. Human islets were co-cultured with hBMSCs overexpressing hVEGF and hIL-1Ra. Islet viability was determined by membrane fluorescent method and glucose stimulation test. Transduced hBMSCs and human islets were co-transplanted under the kidney capsule of NOD.Cg-Prkdc(scid Il2rg(tm1Wjl /SzJ (NSG diabetic mice and blood glucose levels were measured over time to demonstrate the efficacy of genetically modified hBMSCs. At the end of study, immunofluorescent staining of kidney section bearing islets was performed for insulin and von Willebrand Factor (vWF. hBMSCs were positive for the expression of CD73, CD90, CD105, CD146 and Stro-1 surface markers as determined by flow cytometry. Transduction of hBMSCs with adenovirus did not affect their stemness and differentiation potential as confirmed by mRNA levels of stem cell markers and adipogenic differentiation of transduced hBMSCs. hBMSCs were efficiently transduced with Adv-hVEGF-hIL-1Ra to overexpress hVEGF and hIL-1Ra. Live dead cell staining and glucose stimulation test have shown that transduced hBMSCs improved the viability of islets against cytokine cocktail. Co-transplantation of human islets with genetically modified hBMSCs improved the glycemic control of diabetic NSG mice as determined by mean blood glucose levels and intraperitoneal glucose tolerance test. Immunofluorescent staining of kidney sections was positive for human insulin and vWF. In conclusion, our results have demonstrated that hBMSCs may be used as gene carriers and nursing cells to improve the outcome of islet

  11. Broad in vitro efficacy of plant-derived betulinic acid against cell lines derived from the most prevalent human cancer types

    NARCIS (Netherlands)

    Kessler, Jan H.; Mullauer, Franziska B.; de Roo, Guido M.; Medema, Jan Paul

    2007-01-01

    Betulinic acid (BA) is a widely available plant-derived triterpene with reported activity against cancer cells of neuroectodermal origin and leukaemias. Treatment with BA was shown to protect mice against transplanted human melanoma and led to tumor regression. In contrast, cells from healthy

  12. 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.

  13. The stability of human, bovine and avian tuberculin purified protein derivative (PPD).

    Science.gov (United States)

    Maes, Mailis; Giménez, José Francisco; D'Alessandro, Adriana; De Waard, Jacobus H

    2011-11-15

    Guidelines recommend storing tuberculin purified protein derivative (PPD) refrigerated. However, especially in developing countries, maintaining the product refrigerated under field conditions can be difficult, limiting its use. Here we determine the effect of prolonged exposure to high temperatures on the potency of human, bovine and avian tuberculin PPD. Human, bovine and avian tuberculin PPD were stored for several weeks exposed to temperatures ranging from 37º to 100ºC. The potency was evaluated in vivo, in sensitized or naturally infected animals. Most test situations didn't affect the biological activity of the tuberculin PPDs and only very long and extreme incubations (several days at 100 °C) compromised the potency. Tuberculin PPD is very stable and can be stored or transported for long periods without refrigeration. 

  14. Structural Analysis of Three-dimensional Human Neural Tissue derived from Induced Pluripotent Stem Cells

    DEFF Research Database (Denmark)

    Terrence Brooks, Patrick; Rasmussen, Mikkel Aabech; Hyttel, Poul

    2016-01-01

    Objective: The present study aimed at establishing a method for production of a three-dimensional (3D) human neural tissue derived from induced pluripotent stem cells (iPSCs) and analyzing the outcome by a combination of tissue ultrastructure and expression of neural markers. Methods: A two......-step cell culture procedure was implemented by subjecting human iPSCs to a 3D scaffoldbased neural differentiation protocol. First, neural fate-inducing small molecules were used to create a neuroepithelial monolayer. Second, the monolayer was trypsinized into single cells and seeded into a porous...... polystyrene scaffold and further cultured to produce a 3D neural tissue. The neural tissue was characterized by a combination of immunohistochemistry and transmission electron microscopy (TEM). Results: iPSCs developed into a 3D neural tissue expressing markers for neural progenitor cells, early neural...

  15. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Afford New Opportunities in Inherited Cardiovascular Disease Modeling

    Directory of Open Access Journals (Sweden)

    Daniel R. Bayzigitov

    2016-01-01

    Full Text Available Fundamental studies of molecular and cellular mechanisms of cardiovascular disease pathogenesis are required to create more effective and safer methods of their therapy. The studies can be carried out only when model systems that fully recapitulate pathological phenotype seen in patients are used. Application of laboratory animals for cardiovascular disease modeling is limited because of physiological differences with humans. Since discovery of induced pluripotency generating induced pluripotent stem cells has become a breakthrough technology in human disease modeling. In this review, we discuss a progress that has been made in modeling inherited arrhythmias and cardiomyopathies, studying molecular mechanisms of the diseases, and searching for and testing drug compounds using patient-specific induced pluripotent stem cell-derived cardiomyocytes.

  16. Tissue-Engineered Vascular Rings from Human iPSC-Derived Smooth Muscle Cells

    Directory of Open Access Journals (Sweden)

    Biraja C. Dash

    2016-07-01

    Full Text Available There is an urgent need for an efficient approach to obtain a large-scale and renewable source of functional human vascular smooth muscle cells (VSMCs to establish robust, patient-specific tissue model systems for studying the pathogenesis of vascular disease, and for developing novel therapeutic interventions. Here, we have derived a large quantity of highly enriched functional VSMCs from human induced pluripotent stem cells (hiPSC-VSMCs. Furthermore, we have engineered 3D tissue rings from hiPSC-VSMCs using a facile one-step cellular self-assembly approach. The tissue rings are mechanically robust and can be used for vascular tissue engineering and disease modeling of supravalvular aortic stenosis syndrome. Our method may serve as a model system, extendable to study other vascular proliferative diseases for drug screening. Thus, this report describes an exciting platform technology with broad utility for manufacturing cell-based tissues and materials for various biomedical applications.

  17. 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.

  18. 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...

  19. Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells

    International Nuclear Information System (INIS)

    Timper, Katharina; Seboek, Dalma; Eberhardt, Michael; Linscheid, Philippe; Christ-Crain, Mirjam; Keller, Ulrich; Mueller, Beat; Zulewski, Henryk

    2006-01-01

    Mesenchymal stem cells (MSC) from mouse bone marrow were shown to adopt a pancreatic endocrine phenotype in vitro and to reverse diabetes in an animal model. MSC from human bone marrow and adipose tissue represent very similar cell populations with comparable phenotypes. Adipose tissue is abundant and easily accessible and could thus also harbor cells with the potential to differentiate in insulin producing cells. We isolated human adipose tissue-derived MSC from four healthy donors. During the proliferation period, the cells expressed the stem cell markers nestin, ABCG2, SCF, Thy-1 as well as the pancreatic endocrine transcription factor Isl-1. The cells were induced to differentiate into a pancreatic endocrine phenotype by defined culture conditions within 3 days. Using quantitative PCR a down-regulation of ABCG2 and up-regulation of pancreatic developmental transcription factors Isl-1, Ipf-1, and Ngn3 were observed together with induction of the islet hormones insulin, glucagon, and somatostatin

  20. Alpha-amidated peptides derived from pro-opiomelanocortin in human pituitary tumours

    DEFF Research Database (Denmark)

    Fenger, M; Johnsen, A H

    1988-01-01

    Human pituitary tumours, obtained at surgery for Cushing's disease and Nelson's syndrome, were extracted and the content and molecular forms of pro-opiomelanocortin (POMC)-derived peptides determined by radioimmunoassay, gel chromatography, reversed-phase high-performance liquid chromatography....... In conclusion, all the molecular forms of the amidated peptides detected in tumours from patients with Cushing's disease and Nelson's syndrome were similar to the molecular forms found in the normal human pituitary. The main difference between the tumours and the normal pituitary was the greater amount...... (HPLC) and sequence analysis. In the tumours from patients with Cushing's disease the mean concentrations of amidated peptides relative to the total amount of POMC were as follows: alpha-MSH, 1.7%; amidated gamma-MSH (gamma 1-MSH), 8.5% and the peptide linking gamma-MSH and ACTH in the precursor (hinge...

  1. MUTZ-3 derived Langerhans cells in human skin equivalents show differential migration and phenotypic plasticity after allergen or irritant exposure

    Energy Technology Data Exchange (ETDEWEB)

    Kosten, Ilona J.; Spiekstra, Sander W. [Department of Dermatology, VU University Medical Center, Amsterdam (Netherlands); Gruijl, Tanja D. de [Department of Dermatology Medical Oncology, VU University Medical Center, Amsterdam (Netherlands); Gibbs, Susan, E-mail: s.gibbs@acta.nl [Department of Dermatology, VU University Medical Center, Amsterdam (Netherlands); Department of Oral Cell Biology, Academic Center for Dentistry (ACTA), Amsterdam (Netherlands)

    2015-08-15

    After allergen or irritant exposure, Langerhans cells (LC) undergo phenotypic changes and exit the epidermis. In this study we describe the unique ability of MUTZ-3 derived Langerhans cells (MUTZ-LC) to display similar phenotypic plasticity as their primary counterparts when incorporated into a physiologically relevant full-thickness skin equivalent model (SE-LC). We describe differences and similarities in the mechanisms regulating LC migration and plasticity upon allergen or irritant exposure. The skin equivalent consisted of a reconstructed epidermis containing primary differentiated keratinocytes and CD1a{sup +} MUTZ-LC on a primary fibroblast-populated dermis. Skin equivalents were exposed to a panel of allergens and irritants. Topical exposure to sub-toxic concentrations of allergens (nickel sulfate, resorcinol, cinnamaldehyde) and irritants (Triton X-100, SDS, Tween 80) resulted in LC migration out of the epidermis and into the dermis. Neutralizing antibody to CXCL12 blocked allergen-induced migration, whereas anti-CCL5 blocked irritant-induced migration. In contrast to allergen exposure, irritant exposure resulted in cells within the dermis becoming CD1a{sup −}/CD14{sup +}/CD68{sup +} which is characteristic of a phenotypic switch of MUTZ-LC to a macrophage-like cell in the dermis. This phenotypic switch was blocked with anti-IL-10. Mechanisms previously identified as being involved in LC activation and migration in native human skin could thus be reproduced in the in vitro constructed skin equivalent model containing functional LC. This model therefore provides a unique and relevant research tool to study human LC biology in situ under controlled in vitro conditions, and will provide a powerful tool for hazard identification, testing novel therapeutics and identifying new drug targets. - Highlights: • MUTZ-3 derived Langerhans cells integrated into skin equivalents are fully functional. • Anti-CXCL12 blocks allergen-induced MUTZ-LC migration.

  2. Phenotypic and Proteomic Characteristics of Human Dental Pulp Derived Mesenchymal Stem Cells from a Natal, an Exfoliated Deciduous, and an Impacted Third Molar Tooth

    Directory of Open Access Journals (Sweden)

    Gurler Akpinar

    2014-01-01

    Full Text Available The level of heterogeneity among the isolated stem cells makes them less valuable for clinical use. The purpose of this study was to understand the level of heterogeneity among human dental pulp derived mesenchymal stem cells by using basic cell biology and proteomic approaches. The cells were isolated from a natal (NDPSCs, an exfoliated deciduous (stem cells from human exfoliated deciduous (SHED, and an impacted third molar (DPSCs tooth of three different donors. All three stem cells displayed similar features related to morphology, proliferation rates, expression of various cell surface markers, and differentiation potentials into adipocytes, osteocytes, and chondrocytes. Furthermore, using 2DE approach coupled with MALDI-TOF/TOF, we have generated a common 2DE profile for all three stem cells. We found that 62.3±7% of the protein spots were conserved among the three mesenchymal stem cell lines. Sixty-one of these conserved spots were identified by MALDI-TOF/TOF analysis. Classification of the identified proteins based on biological function revealed that structurally important proteins and proteins that are involved in protein folding machinery are predominantly expressed by all three stem cell lines. Some of these proteins may hold importance in understanding specific properties of human dental pulp derived mesenchymal stem cells.

  3. Three-dimensional bioprinting of stem-cell derived tissues for human regenerative medicine.

    Science.gov (United States)

    Skeldon, Gregor; Lucendo-Villarin, Baltasar; Shu, Wenmiao

    2018-07-05

    Stem cell technology in regenerative medicine has the potential to provide an unlimited supply of cells for drug testing, medical transplantation and academic research. In order to engineer a realistic tissue model using stem cells as an alternative to human tissue, it is essential to create artificial stem cell microenvironment or niches. Three-dimensional (3D) bioprinting is a promising tissue engineering field that offers new opportunities to precisely place stem cells within their niches layer-by-layer. This review covers bioprinting technologies, the current development of 'bio-inks' and how bioprinting has already been applied to stem-cell culture, as well as their applications for human regenerative medicine. The key considerations for bioink properties such as stiffness, stability and biodegradation, biocompatibility and printability are highlighted. Bioprinting of both adult and pluriopotent stem cells for various types of artificial tissues from liver to brain has been reviewed. 3D bioprinting of stem-cell derived tissues for human regenerative medicine is an exciting emerging area that represents opportunities for new research, industries and products as well as future challenges in clinical translation.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'. © 2018 The Author(s).

  4. The effect of physical activity on the brain derived neurotrophic factor: from animal to human studies.

    Science.gov (United States)

    Zoladz, J A; Pilc, A

    2010-10-01

    It is well documented that physical activity can induce a number of various stimuli which are able to enhance the strength and endurance performance of muscles. Moreover, regular physical activity can preserve or delay the appearance of several metabolic disorders in the human body. Physical exercise is also known to enhance the mood and cognitive functions of active people, although the physiological backgrounds of these effects remain unclear. In recent years, since the pioneering study in the past showed that physical activity increases the expression of the brain derived neurothophic factor (BDNF) in the rat brain, a number of studies were undertaken in order to establish the link between that neurothrophin and post-exercise enhancement of mood and cognitive functions in humans. It was recently demonstrated that physical exercise can increase plasma and/or serum BDNF concentration in humans. It was also reported that physical exercise or electrical stimulation can increase the BDNF expression in the skeletal muscles. In the present review, we report the current state of research concerning the effect of a single bout of exercise and training on the BDNF expression in the brain, in both the working muscles as well as on its concentrations in the blood. We have concluded that there may be potential benefits of the exercise-induced enhancement of the BDNF expression and release in the brain as well as in the peripheral tissues, resulting in the improvement of the functioning of the body, although this effect, especially in humans, requires more research.

  5. Interleukin-3 Does Not Affect the Differentiation of Mast Cells Derived from Human Bone Marrow Progenitors

    Science.gov (United States)

    Shimizu, Yuji; Matsumoto, Kenji; Okayama, Yoshimichi; Kentaro, Sakai; Maeno, Toshitaka; Suga, Tatsuo; Miura, Toru; Takai, Shinji; Kurabayashi, Masahiko; Saito, Hirohisa

    2008-01-01

    Although IL-3 is commonly used for culture of human progenitor-derived mast cells together with Stem cell factor (SCF) and IL-6, the effect of IL-3 on human mast cell differentiation has not been well elucidated. Human bone marrow CD34+ progenitors were cultured for up to 12 weeks in the presence of rhSCF and rhIL-6 either with rhIL-3 (IL-3 (+)) or without rhIL-3 (IL-3 (−)) for the initial 1-week of culture. Total cell number increased at 2 weeks in IL-3 (+), as compared to IL-3 (−), but changes in the appearance of mast cells were delayed. When IL-3 was present for the initial 1-week culture, granules looked more mature with IL-3 than without IL-3. However, tryptase and chymase contents, and surface antigen expression (CD18, CD51, CD54, and CD117) were not altered by IL-3. Surface expression and mRNA level of FcεRIα and histamine release by crosslinking of FcεRIα did not differ from one preparation to the next. GeneChip analysis revealed that no significant differences were observed between IL-3 (+) and IL-3 (−) cells either when inactivated or activated by aggregation of FcεRIα. These findings indicate that initial incubation of human bone marrow CD34+ progenitors with IL-3 does not affect the differentiation of mast cells. PMID:18214796

  6. The Protein Content of Extracellular Vesicles Derived from Expanded Human Umbilical Cord Blood-Derived CD133+ and Human Bone Marrow-Derived Mesenchymal Stem Cells Partially Explains Why both Sources are Advantageous for Regenerative Medicine.

    Science.gov (United States)

    Angulski, Addeli B B; Capriglione, Luiz G; Batista, Michel; Marcon, Bruna H; Senegaglia, Alexandra C; Stimamiglio, Marco A; Correa, Alejandro

    2017-04-01

    Adult stem cells have beneficial effects when exposed to damaged tissue due, at least in part, to their paracrine activity, which includes soluble factors and extracellular vesicles (EVs). Given the multiplicity of signals carried by these vesicles through the horizontal transfer of functional molecules, human mesenchymal stem cell (hMSCs) and CD133 + cell-derived EVs have been tested in various disease models and shown to recover damaged tissues. In this study, we profiled the protein content of EVs derived from expanded human CD133 + cells and bone marrow-derived hMSCs with the intention of better understanding the functions performed by these vesicles/cells and delineating the most appropriate use of each EV in future therapeutic procedures. Using LC-MS/MS analysis, we identified 623 proteins for expanded CD133 + -EVs and 797 proteins for hMSCs-EVs. Although the EVs from both origins were qualitatively similar, when protein abundance was considered, hMSCs-EVs and CD133 + -EVs were different. Gene Ontology (GO) enrichment analysis in CD133 + -EVs revealed proteins involved in a variety of angiogenesis-related functions as well proteins related to the cytoskeleton and highly implicated in cell motility and cellular activation. In contrast, when overrepresented proteins in hMSCs-EVs were analyzed, a GO cluster of immune response-related genes involved with immune response-regulating factors acting on phagocytosis and innate immunity was identified. Together our data demonstrate that from the point of view of protein content, expanded CD133 + -EVs and hMSCs-EVs are in part similar but also sufficiently different to reflect the main beneficial paracrine effects widely reported in pre-clinical studies using expanded CD133 + cells and/or hBM-MSCs.

  7. Oral administration of human papillomavirus type 16 E7 displayed on Lactobacillus casei induces E7-specific antitumor effects in C57/BL6 mice.

    Science.gov (United States)

    Poo, Haryoung; Pyo, Hyun-Mi; Lee, Tae-Young; Yoon, Sun-Woo; Lee, Jong-Soo; Kim, Chul-Joong; Sung, Moon-Hee; Lee, Seung-Hoon

    2006-10-01

    The mounting of a specific immune response against the human papillomavirus type 16 E7 protein (HPV16 E7) is important for eradication of HPV16 E7-expressing cancer cells from the cervical mucosa. To induce a mucosal immune response by oral delivery of the E7 antigen, we expressed the HPV16 E7 antigen on the surface of Lactobacillus casei by employing a novel display system in which the poly-gamma-glutamic acid (gamma-PGA) synthetase complex A (PgsA) from Bacillus subtilis (chungkookjang) was used as an anchoring motif. After surface expression of the HPV16 E7 protein was confirmed by Western blot, flow cytometry and immunofluorescence microscopy, mice were orally inoculated with L. casei-PgsA-E7. E7-specific serum IgG and mucosal IgA productions were enhanced after oral administration and significantly enhanced after boosting. Systemic and local cellular immunities were significantly increased after boosting, as shown by increased counts of lymphocytes (SI = 9.7 +/- 1.8) and IFN-gamma secreting cells [510 +/- 86 spot-forming cells/10(6)cells] among splenocytes and increased IFN-gamma in supernatants of vaginal lymphocytes. Furthermore, in an E7-based mouse tumor model, animals receiving orally administered L. casei-PgsA-E7 showed reduced tumor size and increased survival rate versus mice receiving control (L. casei-PgsA) immunization. These results collectively indicate that the oral administration of E7 displayed on lactobacillus induces cellular immunity and antitumor effects in mice. Copyright 2006 Wiley-Liss, Inc.

  8. Abundant Rodent Furan-Derived Urinary Metabolites Are Associated with Tobacco Smoke Exposure in Humans.

    Science.gov (United States)

    Grill, Alex E; Schmitt, Thaddeus; Gates, Leah A; Lu, Ding; Bandyopadhyay, Dipankar; Yuan, Jian-Min; Murphy, Sharon E; Peterson, Lisa A

    2015-07-20

    Furan, a possible human carcinogen, is found in heat treated foods and tobacco smoke. Previous studies have shown that humans are capable of converting furan to its reactive metabolite, cis-2-butene-1,4-dial (BDA), and therefore may be susceptible to furan toxicity. Human risk assessment of furan exposure has been stymied because of the lack of mechanism-based exposure biomarkers. Therefore, a sensitive LC-MS/MS assay for six furan metabolites was applied to measure their levels in urine from furan-exposed rodents as well as in human urine from smokers and nonsmokers. The metabolites that result from direct reaction of BDA with lysine (BDA-N(α)-acetyllysine) and from cysteine-BDA-lysine cross-links (N-acetylcysteine-BDA-lysine, N-acetylcysteine-BDA-N(α)-acetyllysine, and their sulfoxides) were targeted in this study. Five of the six metabolites were identified in urine from rodents treated with furan by gavage. BDA-N(α)-acetyllysine, N-acetylcysteine-BDA-lysine, and its sulfoxide were detected in most human urine samples from three different groups. The levels of N-acetylcysteine-BDA-lysine sulfoxide were more than 10 times higher than that of the corresponding sulfide in many samples. The amount of this metabolite was higher in smokers relative to that in nonsmokers and was significantly reduced following smoking cessation. Our results indicate a strong relationship between BDA-derived metabolites and smoking. Future studies will determine if levels of these biomarkers are associated with adverse health effects in humans.

  9. Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

    Science.gov (United States)

    Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik

    2014-04-01

    Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Synthesis and biological evaluation of lycorine derivatives as dual inhibitors of human acetylcholinesterase and butyrylcholinesterase

    Directory of Open Access Journals (Sweden)

    Wang Yue-Hu

    2012-09-01

    Full Text Available Abstract Background Alzheimer’s disease (AD is a neurologically degenerative disorder that affects more than 20 million people worldwide. The selective butyrylcholinesterase (BChE inhibitors and bivalent cholinesterase (ChE inhibitors represent new treatments for AD. Findings A series of lycorine derivatives (1–10 were synthesized and evaluated for anti-cholinesterase activity. Result showed that the novel compound 2-O-tert-butyldimethylsilyl-1-O-(methylthiomethyllycorine (7 was a dual inhibitor of human acetylcholinesterase (hAChE and butyrylcholinesterase (hBChE with IC50 values of 11.40 ± 0.66 μM and 4.17 ± 0.29 μM, respectively. The structure-activity relationships indicated that (i the 1-O-(methylthiomethyl substituent in lycorine was better than the 1-O-acetyl group for the inhibition of cholinesterase; (ii the acylated or etherified derivatives of lycorine and lycorin-2-one were more potent against hBChE than hAChE; and (iii the oxidation of lycorine at C-2 decreases the activity. Conclusion Acylated or etherified derivatives of lycorine are potential dual inhibitors of hBChE and hAChE. Hence, further study on the modification of lycorine for ChE inhibition is necessary.

  11. Human reliability under sleep deprivation: Derivation of performance shaping factor multipliers from empirical data

    International Nuclear Information System (INIS)

    Griffith, Candice D.; Mahadevan, Sankaran

    2015-01-01

    This paper develops a probabilistic approach that could use empirical data to derive values of performance shaping factor (PSF) multipliers for use in quantitative human reliability analysis (HRA). The proposed approach is illustrated with data on sleep deprivation effects on performance. A review of existing HRA methods reveals that sleep deprivation is not explicitly included at present, and expert opinion is frequently used to inform HRA model multipliers. In this paper, quantitative data from empirical studies regarding the effect of continuous hours of wakefulness on performance measures (reaction time, accuracy, and number of lapses) are used to develop a method to derive PSF multiplier values for sleep deprivation, in the context of the SPAR-H model. Data is extracted from the identified studies according to the meta-analysis research synthesis method and used to investigate performance trends and error probabilities. The error probabilities in test and control conditions are compared, and the resulting probability ratios are suggested for use in informing the selection of PSF multipliers in HRA methods. Although illustrated for sleep deprivation, the proposed methodology is general, and can be applied to other performance shaping factors. - Highlights: • Method proposed to derive performance shaping factor multipliers from empirical data. • Studies reporting the effect of sleep deprivation on performance are analyzed. • Test data using psychomotor vigilance tasks are analyzed. • Error probability multipliers computed for reaction time, lapses, and accuracy measures.

  12. Surface chemical functionalities affect the behavior of human adipose-derived stem cells in vitro

    International Nuclear Information System (INIS)

    Liu, Xujie; Feng, Qingling; Bachhuka, Akash; Vasilev, Krasimir

    2013-01-01

    This study examines the effect of surface chemical functionalities on the behavior of human adipose-derived stem cells (hASCs) in vitro. Plasma polymerized films rich in amine (-NH 2 ), carboxyl (-COOH) and methyl (-CH 3 ), were generated on hydroxyapatite (HAp) substrates. The surface chemical functionalities were characterized by X-ray photoelectron spectroscopy (XPS). The ability of different substrates to absorb proteins was evaluated. The results showed that substrates modified with hydrophilic functional group (-COOH and -NH 2 ) can absorb more proteins than these modified with more hydrophobic functional group (-CH 3 ). The behavior of human adipose-derived stem cells (hASCs) cultured on different substrates was investigated in vitro: cell counting kit-8 (CCK-8) analysis was used to characterize cell proliferation, scanning electronic microscopy (SEM) analysis was used to characterize cell morphology and alkaline phosphatase (ALP) activity analysis was used to account for differentiation. The results of this study demonstrated that the -NH 2 modified surfaces encourage osteogenic differentiation; the -COOH modified surfaces promote cell adhesion and spreading and the -CH 3 modified surfaces have the lowest ability to induce osteogenic differentiation. These findings confirmed that the surface chemical states of biomaterials can affect the behavior of hASCs in vitro.

  13. Surface chemical functionalities affect the behavior of human adipose-derived stem cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xujie [State key laboratory of new ceramics and fine processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Feng, Qingling, E-mail: biomater@mail.tsinghua.edu.cn [State key laboratory of new ceramics and fine processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Bachhuka, Akash [Mawson Institute, University of South Australia, Mawson Lakes 5095 (Australia); Vasilev, Krasimir [Mawson Institute, University of South Australia, Mawson Lakes 5095 (Australia); School of Advanced Manufacturing, University of South Australia, Mawson Lakes 5095 (Australia)

    2013-04-01

    This study examines the effect of surface chemical functionalities on the behavior of human adipose-derived stem cells (hASCs) in vitro. Plasma polymerized films rich in amine (-NH{sub 2}), carboxyl (-COOH) and methyl (-CH{sub 3}), were generated on hydroxyapatite (HAp) substrates. The surface chemical functionalities were characterized by X-ray photoelectron spectroscopy (XPS). The ability of different substrates to absorb proteins was evaluated. The results showed that substrates modified with hydrophilic functional group (-COOH and -NH{sub 2}) can absorb more proteins than these modified with more hydrophobic functional group (-CH{sub 3}). The behavior of human adipose-derived stem cells (hASCs) cultured on different substrates was investigated in vitro: cell counting kit-8 (CCK-8) analysis was used to characterize cell proliferation, scanning electronic microscopy (SEM) analysis was used to characterize cell morphology and alkaline phosphatase (ALP) activity analysis was used to account for differentiation. The results of this study demonstrated that the -NH{sub 2} modified surfaces encourage osteogenic differentiation; the -COOH modified surfaces promote cell adhesion and spreading and the -CH{sub 3} modified surfaces have the lowest ability to induce osteogenic differentiation. These findings confirmed that the surface chemical states of biomaterials can affect the behavior of hASCs in vitro.

  14. Disease Modeling Using 3D Organoids Derived from Human Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Ho, Beatrice Xuan; Pek, Nicole Min Qian; Soh, Boon-Seng

    2018-03-21

    The rising interest in human induced pluripotent stem cell (hiPSC)-derived organoid culture has stemmed from the manipulation of various combinations of directed multi-lineage differentiation and morphogenetic processes that mimic organogenesis. Organoids are three-dimensional (3D) structures that are comprised of multiple cell types, self-organized to recapitulate embryonic and tissue development in vitro. This model has been shown to be superior to conventional two-dimensional (2D) cell culture methods in mirroring functionality, architecture, and geometric features of tissues seen in vivo. This review serves to highlight recent advances in the 3D organoid technology for use in modeling complex hereditary diseases, cancer, host-microbe interactions, and possible use in translational and personalized medicine where organoid cultures were used to uncover diagnostic biomarkers for early disease detection via high throughput pharmaceutical screening. In addition, this review also aims to discuss the advantages and shortcomings of utilizing organoids in disease modeling. In summary, studying human diseases using hiPSC-derived organoids may better illustrate the processes involved due to similarities in the architecture and microenvironment present in an organoid, which also allows drug responses to be properly recapitulated in vitro.

  15. The functional performance of microencapsulated human pancreatic islet-derived precursor cells.

    Science.gov (United States)

    Montanucci, Pia; Pennoni, Ilaria; Pescara, Teresa; Blasi, Paolo; Bistoni, Giovanni; Basta, Giuseppe; Calafiore, Riccardo

    2011-12-01

    We have examined long-term cultured, human islet-derived stem/precursor cells (hIPC). Whole human islets (HI) were obtained by multi-enzymatic digestion of cadaveric donor pancreases, plated on tissue flasks, and allowed to adhere and expand for several in vitro passages, in order to obtain hIPC. We detected specific stem cell markers (Oct-4, Sox-2, Nanog, ABCG2, Klf-4, CD117) in both intact HI and hIPC. Moreover, hIPC while retaining the expression of Glut-2, Pdx-1, CK-19, and ICA-512, started re-expressing Ngn3, thereby indicating acquisition of a specific pancreatic islet beta cell-oriented phenotype identity. The intrinsic plasticity of hIPC was documented by their ability to differentiate into various germ layer-derived cell phenotypes (ie, osteocytic, adipocytic and neural), including endocrine cells associated with insulin secretory capacity. To render hIPC suitable for transplantation we have enveloped them within our highly purified, alginate-based microcapsules. Upon intraperitoneal graft in NOD/SCID mice we have observed that the microcapsules acted as three-dimensional niches favouring post-transplant hIPC differentiation and acquisition of beta cell-like functional competence. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Functional Properties of Human Stem Cell-Derived Neurons in Health and Disease

    Directory of Open Access Journals (Sweden)

    Jason P. Weick

    2016-01-01

    Full Text Available Stem cell-derived neurons from various source materials present unique model systems to examine the fundamental properties of central nervous system (CNS development as well as the molecular underpinnings of disease phenotypes. In order to more accurately assess potential therapies for neurological disorders, multiple strategies have been employed in recent years to produce neuronal populations that accurately represent in vivo regional and transmitter phenotypes. These include new technologies such as direct conversion of somatic cell types into neurons and glia which may accelerate maturation and retain genetic hallmarks of aging. In addition, novel forms of genetic manipulations have brought human stem cells nearly on par with those of rodent with respect to gene targeting. For neurons of the CNS, the ultimate phenotypic characterization lies with their ability to recapitulate functional properties such as passive and active membrane characteristics, synaptic activity, and plasticity. These features critically depend on the coordinated expression and localization of hundreds of ion channels and receptors, as well as scaffolding and signaling molecules. In this review I will highlight the current state of knowledge regarding functional properties of human stem cell-derived neurons, with a primary focus on pluripotent stem cells. While significant advances have been made, critical hurdles must be overcome in order for this technology to support progression toward clinical applications.

  17. Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis

    Science.gov (United States)

    Ong, Catherine W. M.; Elkington, Paul T.; Brilha, Sara; Ugarte-Gil, Cesar; Tome-Esteban, Maite T.; Tezera, Liku B.; Pabisiak, Przemyslaw J.; Moores, Rachel C.; Sathyamoorthy, Tarangini; Patel, Vimal; Gilman, Robert H.; Porter, Joanna C.; Friedland, Jon S.

    2015-01-01

    Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease. PMID:25996154

  18. Overexpressed human heme Oxygenase-1 decreases adipogenesis in pigs and porcine adipose-derived stem cells.

    Science.gov (United States)

    Park, Eun Jung; Koo, Ok Jae; Lee, Byeong Chun

    2015-11-27

    Adipose-derived mesenchymal stem cells (ADSC) are multipotent, which means they are able to differentiate into several lineages in vivo and in vitro under proper conditions. This indicates it is possible to determine the direction of differentiation of ADSC by controlling the microenvironment. Heme oxygenase 1 (HO-1), a type of antioxidant enzyme, attenuates adipogenicity and obesity. We produced transgenic pigs overexpressing human HO-1 (hHO-1-Tg), and found that these animals have little fatty tissue when autopsied. To determine whether overexpressed human HO-1 suppresses adipogenesis in pigs, we analyzed body weight increases of hHO-1-Tg pigs and wild type (WT) pigs of the same strain, and induced adipogenic differentiation of ADSC derived from WT and hHO-1-Tg pigs. The hHO-1-Tg pigs had lower body weights than WT pigs from 16 weeks of age until they died. In addition, hHO-1-Tg ADSC showed reduced adipogenic differentiation and expression of adipogenic molecular markers such as PPARγ and C/EBPα compared to WT ADSC. These results suggest that HO-1 overexpression reduces adipogenesis both in vivo and in vitro, which could support identification of therapeutic targets of obesity and related metabolic diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis.

    Science.gov (United States)

    Ong, Catherine W M; Elkington, Paul T; Brilha, Sara; Ugarte-Gil, Cesar; Tome-Esteban, Maite T; Tezera, Liku B; Pabisiak, Przemyslaw J; Moores, Rachel C; Sathyamoorthy, Tarangini; Patel, Vimal; Gilman, Robert H; Porter, Joanna C; Friedland, Jon S

    2015-05-01

    Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease.

  20. Parkin Mutations Reduce the Complexity of Neuronal Processes in iPSC-derived Human Neurons

    Science.gov (United States)

    Ren, Yong; Jiang, Houbo; Hu, Zhixing; Fan, Kevin; Wang, Jun; Janoschka, Stephen; Wang, Xiaomin; Ge, Shaoyu; Feng, Jian

    2015-01-01

    Parkinson’s disease (PD) is characterized by the degeneration of nigral dopaminergic (DA) neurons and non-DA neurons in many parts of the brain. Mutations of parkin, an E3 ubiquitin ligase that strongly binds to microtubules, are the most frequent cause of recessively inherited Parkinson’s disease. The lack of robust PD phenotype in parkin knockout mice suggests a unique vulnerability of human neurons to parkin mutations. Here, we show that the complexity of neuronal processes as measured by total neurite length, number of terminals, number of branch points and Sholl analysis, was greatly reduced in induced pluripotent stem cell (iPSC)-derived TH+ or TH− neurons from PD patients with parkin mutations. Consistent with these, microtubule stability was significantly decreased by parkin mutations in iPSC-derived neurons. Overexpression of parkin, but not its PD-linked mutant nor GFP, restored the complexity of neuronal processes and the stability of microtubules. Consistent with these, the microtubule-depolymerizing agent colchicine mimicked the effect of parkin mutations by decreasing neurite length and complexity in control neurons while the microtubule-stabilizing drug taxol mimicked the effect of parkin overexpression by enhancing the morphology of parkin-deficient neurons. The results suggest that parkin maintains the morphological complexity of human neurons by stabilizing microtubules. PMID:25332110

  1. Osteogenic differentiation of human adipose-derived mesenchymal stem cells on gum tragacanth hydrogel.

    Science.gov (United States)

    Haeri, Seyed Mohammad Jafar; Sadeghi, Yousef; Salehi, Mohammad; Farahani, Reza Masteri; Mohsen, Nourozian

    2016-05-01

    Currently, natural polymer based hydrogels has attracted great attention of orthopedic surgeons for application in bone tissue engineering. With this aim, osteoinductive capacity of Gum Tragacanth (GT) based hydrogel was compared to collagen hydrogel and tissue culture plate (TCPS). For this purpose, adipose-derived mesenchymal stem cells (AT-MSCs) was cultured on the hydrogels and TCPS and after investigating the biocompatibility of hydrogels using MTT assay, osteoinductivity of hydrogels were evaluated using pan osteogenic markers such as Alizarin red staining, alkaline phosphatase (ALP) activity, calcium content and osteo-related genes. Increasing proliferation trend of AT-MSCs on GT hydrogel demonstrated that TG has no-cytotoxicity and can even be better than the other groups i.e., highest proliferation at day 5. GT hydrogel displayed highest ALP activity and mineralization when compared to the collagen hydrogel and TCPS. Relative gene expression levels have demonstrated that highest expression of Runx2, osteonectin and osteocalcin in the cells cultured GT hydrogel but the expression of collagen type-1 remains constant in hydrogels. Above results demonstrate that GT hydrogel could be an appropriate scaffold for accelerating and supporting the adhesion, proliferation and osteogenic differentiation of stem cells which further can be used for orthopedic applications. Copyright © 2016. Published by Elsevier Ltd.

  2. A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display.

    Science.gov (United States)

    Baskar, Sivasubramanian; Suschak, Jessica M; Samija, Ivan; Srinivasan, Ramaprasad; Childs, Richard W; Pavletic, Steven Z; Bishop, Michael R; Rader, Christoph

    2009-11-12

    Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only potentially curative treatment available for patients with B-cell chronic lymphocytic leukemia (B-CLL). Here, we show that post-alloHSCT antibody repertoires can be mined for the discovery of fully human monoclonal antibodies to B-CLL cell-surface antigens. Sera collected from B-CLL patients at defined times after alloHSCT showed selective binding to primary B-CLL cells. Pre-alloHSCT sera, donor sera, and control sera were negative. To identify post-alloHSCT serum antibodies and subsequently B-CLL cell-surface antigens they recognize, we generated a human antibody-binding fragment (Fab) library from post-alloHSCT peripheral blood mononuclear cells and selected it on primary B-CLL cells by phage display. A panel of Fab with B-CLL cell-surface reactivity was strongly enriched. Selection was dominated by highly homologous Fab predicted to bind the same antigen. One Fab was converted to immunoglobulin G1 and analyzed for reactivity with peripheral blood mononuclear cells from B-CLL patients and healthy volunteers. Cell-surface antigen expression was restricted to primary B cells and up-regulated in primary B-CLL cells. Mining post-alloHSCT antibody repertoires offers a novel route to discover fully human monoclonal antibodies and identify antigens of potential therapeutic relevance to B-CLL and possibly other cancers. Trials described herein were registered at www.clinicaltrials.gov as nos. NCT00055744 and NCT00003838.

  3. Characterization of energy and neurotransmitter metabolism in cortical glutamatergic neurons derived from human induced pluripotent stem cells

    DEFF Research Database (Denmark)

    Aldana, Blanca I; Zhang, Yu; Lihme, Maria Fog

    2017-01-01

    pathways in neurons derived from human induced pluripotent stem cells (hiPSC). With this aim, cultures of hiPSC-derived neurons were incubated with [U-(13)C]glucose, [U-(13)C]glutamate or [U-(13)C]glutamine. Isotopic labeling in metabolites was determined using gas chromatography coupled to mass...

  4. Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting.

    Science.gov (United States)

    Ma, Xuanyi; Qu, Xin; Zhu, Wei; Li, Yi-Shuan; Yuan, Suli; Zhang, Hong; Liu, Justin; Wang, Pengrui; Lai, Cheuk Sun Edwin; Zanella, Fabian; Feng, Gen-Sheng; Sheikh, Farah; Chien, Shu; Chen, Shaochen

    2016-02-23

    The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. Major liver functions are tightly linked to the 3D assembly of hepatocytes, with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture. The application of rapid, digital 3D bioprinting to tissue engineering has allowed 3D patterning of multiple cell types in a predefined biomimetic manner. Here we present a 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adipose-derived stem cells in a microscale hexagonal architecture. In comparison with 2D monolayer culture and a 3D HPC-only model, our 3D triculture model shows both phenotypic and functional enhancements in the hiPSC-HPCs over weeks of in vitro culture. Specifically, we find improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction. The application of bioprinting technology in tissue engineering enables the development of a 3D biomimetic liver model that recapitulates the native liver module architecture and could be used for various applications such as early drug screening and disease modeling.

  5. The anti-oxidant effects of melatonin derivatives on human gingival fibroblasts.

    Science.gov (United States)

    Phiphatwatcharaded, Chawapon; Puthongking, Ploenthip; Chaiyarit, Ponlatham; Johns, Nutjaree Pratheepawanit; Sakolchai, Sumon; Mahakunakorn, Pramote

    2017-07-01

    Aim of this in vitro study was to evaluate the anti-oxidant activity of indole ring modified melatonin derivatives as compared with melatonin in primary human gingival fibroblast (HGF) cells. Anti-oxidant activity of melatonin (MLT), acetyl-melatonin (AMLT) and benzoyl-melatonin (BMLT) was evaluated by5 standard methods as follows: 2, 2-diphenyl-1-picrylhydrazyl (DPPH); ferric ion reducing antioxidant power (FRAP); superoxide anion scavenging; nitric oxide (NO) scavenging; and thiobarbituric acid reactive substances (TBARs).Evaluation of cellular antioxidant activity (CAA) and protectivity against H 2 O 2 induced cellular damage was performed via MTT assay in HGF cells. According to the standard anti-oxidant assays, the antioxidant power of AMLT and BMLT were slightly less than MLT in FRAP and superoxide scavenging assays. In the NO scavenging and TBARs assays, BMLT and AMLT were more potent than MLT, whereas DPPH assays demonstrated that MLT was more potent than others. BMLT and AMLT had more potent anti-oxidant and protective activities against H 2 O 2 in HGF cells as compared with MLT. MLT derivatives demonstrated different anti-oxidant activities as compared with MLT, depending upon assays. These findings imply that N-indole substitution of MLT may help to improve hydrogen atom transfer to free radicals but electron transfer property is slightly decreased. Anti-oxidant and protective effects of melatonin derivatives (AMLT and BMLT) on human gingival fibroblasts imply the potential use of these molecules as alternative therapeutics for chronic inflammatory oral diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

    International Nuclear Information System (INIS)

    Yang, Yingbin; Cai, Shaoxi; Yang, Li; Yu, Shuhui; Jiang, Jiahuan; Yan, Xiaoqing; Zhang, Haoxing; Liu, Lan; Liu, Qun; Du, Jun; Cai, Shaohui; Sung, K.L. Paul

    2010-01-01

    Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

  7. Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yingbin [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); School of Life Science, Southwest University, Chongqing 400715 (China); Cai, Shaoxi, E-mail: sxcai@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Yang, Li [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); College of Pharmacy, Jinan University, Guangzhou 510632 (China); Yu, Shuhui [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Library of Southwest University, Chongqing 400715 (China); Jiang, Jiahuan; Yan, Xiaoqing [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Zhang, Haoxing [School of Life Science, Southwest University, Chongqing 400715 (China); Liu, Lan [Department of Laboratory of Medicine, Children' s Hospital of Chongqin Medical University, Chongqing 400014 (China); Liu, Qun [College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041 (China); Du, Jun [Center of Microbiology, Biochemistry, and Pharmacology, School of Pharmaceutical Science, Sun Yat-Sen University, Guangzhou 510080 (China); Cai, Shaohui [College of Pharmacy, Jinan University, Guangzhou 510632 (China); Sung, K.L. Paul [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Departments of Orthopaedic Surgery and Bioengineering, University of California, SD 0412 (United States)

    2010-12-10

    Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

  8. 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.

  9. Safety of recombinant human platelet-derived growth factor-BB in Augment® Bone Graft

    Directory of Open Access Journals (Sweden)

    Luis A Solchaga

    2012-12-01

    Full Text Available This article discusses nonclinical and clinical data regarding the safety of recombinant human platelet-derived growth factor-BB as a component of the Augment® Bone Graft (Augment. Augment is a bone graft substitute intended to be used as an alternative to autologous bone graft in the fusion of hindfoot and ankle joints. Nonclinical studies included assessment of the pharmacokinetic profile of intravenously administered recombinant human platelet-derived growth factor-BB in rat and dog, effects of intravenous administration of recombinant human platelet-derived growth factor-BB in a reproductive and development toxicity study in rats, and chronic toxicity and carcinogenicity of Augment in a 12-month implantation model. These studies showed that systemic exposure was brief and clearance was rapid. No signs of toxicity, carcinogenicity, or tumor promotion were observed even with doses far exceeding the maximum clinical dose. Results of clinical trials (605 participants and commercial use of recombinant human platelet-derived growth factor-BB containing products indicate that these products are not associated with increased incidence of adverse events or cancer. The safety data presented provide evidence that recombinant human platelet-derived growth factor-BB is a safe therapeutic when used in combination products as a single administration during surgical procedures for bone repair and fusion. There is no evidence associating use of recombinant human platelet-derived growth factor-BB in Augment with chronic toxicity, carcinogenicity, or tumor promotion.

  10. SYNTHESIS AND CYTOTOXIC ACTIVITY OF CHALCONE DERIVATIVES ON HUMAN BREAST CANCER CELL LINES

    Directory of Open Access Journals (Sweden)

    Nuraini Harmastuti

    2012-12-01

    Full Text Available Chalcone, an α,β-unsaturated ketone, has been shown have many biological activities such as anticancer and antifungi. This research was conducted to synthesize the chalcone derivatives and to obtain their cytotoxic activity on human cervix cancer cell lines. Synthesis of chalcone and its derivatives, 4II-methylchalcone, 4II-methoxychalcone, and 3II,4II-dichlorochalcone was carried out using starting materials of benzaldehide and acetofenon, p-methylacetophenone, p-methoxyacetophenone, as well as m,p-dichloroacetophenone through Claisen Schmidt condensation catalized by NaOH in ethanol at 15 °C. The purity of synthesized compounds were analyzed by thin layer chromatography, melting range, and gas chromatography. Structure elucidations were conducted by UV spectrophotometer, IR spectrometer, 1H-NMR spectrometer, as well as mass spectrometer. Cytotoxic activities were determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT microculture tetrazolium viability assay. The results showed that chalcone and derivatives compounds have been able to be synthesized and purified and had the same structure as a predicted structure. Chalcone had highest cytotoxic activity compared to that of its derivatives, with the IC50 values of chalcone, 4II-methylchalcone, 4II-methoxychalcone, and 3II,4II-dichlorochalcone were 9.49, 14.79, 11.48, and 24.26 µg/mL respectively. It was concluded that methyl, methoxy as well as chlorine substitution at 3 II and 4II position decrease the cytotoxic activity of chalcone.

  11. Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries.

    Science.gov (United States)

    Henry, Kevin A; Kim, Dae Young; Kandalaft, Hiba; Lowden, Michael J; Yang, Qingling; Schrag, Joseph D; Hussack, Greg; MacKenzie, C Roger; Tanha, Jamshid

    2017-01-01

    Human autonomous V H /V L single-domain antibodies (sdAbs) are attractive therapeutic molecules, but often suffer from suboptimal stability, solubility and affinity for cognate antigens. Most commonly, human sdAbs have been isolated from in vitro display libraries constructed via synthetic randomization of rearranged V H /V L domains. Here, we describe the design and characterization of three novel human V H /V L sdAb libraries through a process of: (i) exhaustive biophysical characterization of 20 potential V H /V L sdAb library scaffolds, including assessment of expression yield, aggregation resistance, thermostability and tolerance to complementarity-determining region (CDR) substitutions; (ii) in vitro randomization of the CDRs of three V H /V L sdAb scaffolds, with tailored amino acid representation designed to promote solubility and expressibility; and (iii) systematic benchmarking of the three V H /V L libraries by panning against five model antigens. We isolated ≥1 antigen-specific human sdAb against four of five targets (13 V H s and 7 V L s in total); these were predominantly monomeric, had antigen-binding affinities ranging from 5 nM to 12 µM (average: 2-3 µM), but had highly variable expression yields (range: 0.1-19 mg/L). Despite our efforts to identify the most stable V H /V L scaffolds, selection of antigen-specific binders from these libraries was unpredictable (overall success rate for all library-target screens: ~53%) with a high attrition rate of sdAbs exhibiting false positive binding by ELISA. By analyzing V H /V L sdAb library sequence composition following selection for monomeric antibody expression (binding to protein A/L followed by amplification in bacterial cells), we found that some V H /V L sdAbs had marked growth advantages over others, and that the amino acid composition of the CDRs of this set of sdAbs was dramatically restricted (bias toward Asp and His and away from aromatic and hydrophobic residues). Thus, CDR sequence

  12. SL-01, an oral derivative of gemcitabine, inhibited human breast cancer growth through induction of apoptosis

    International Nuclear Information System (INIS)

    Li, Yuan-Yuan; Qin, Yi-Zhuo; Wang, Rui-Qi; Li, Wen-Bao; Qu, Xian-Jun

    2013-01-01

    Highlights: •SL-01 is an oral derivative of gemcitabine. •SL-01 possessed activity against human breast cancer growth via apoptotic induction. •SL-01’s activity was more potently than that of gemcitabine. •SL-01 inhibited cancer growth without toxicity to mice. -- Abstract: SL-01 is an oral derivative of gemcitabine that was synthesized by introducing the moiety of 3-(dodecyloxycarbonyl) pyrazine-2-carbonyl at N4-position on cytidine ring of gemcitabine. We aimed to evaluate the efficacy of SL-01 on human breast cancer growth. SL-01 significantly inhibited MCF-7 proliferation as estimated by colorimetric assay. Flow cytometry assay indicated the apoptotic induction and cell cycle arrest in G1 phase. SL-01 modulated the expressions of p-ATM, p53 and p21 and decrease of cyclin D1 in MCF-7 cells. Further experiments were performed in a MCF-7 xenografts mouse model. SL-01 by oral administration strongly inhibited MCF-7 xenografts growth. This effect of SL-01 might arise from its roles in the induction of apoptosis. Immunohistochemistry assay showed the increase of TUNEL staining cells. Western blotting indicated the modulation of apoptotic proteins in SL-01-treated xenografts. During the course of study, there was no evidence of toxicity to mice. In contrast, the decrease of neutrophil cells in peripheral and increase of AST and ALT levels in serum were observed in the gemcitabine-treated mice. Conclusion: SL-01 possessed similar activity against human breast cancer growth with gemcitabine, whereas, with lower toxicity to gemcitabine. SL-01 is a potent oral agent that may supplant the use of gemcitabine

  13. Efflux protein expression in human stem cell-derived retinal pigment epithelial cells.

    Directory of Open Access Journals (Sweden)

    Kati Juuti-Uusitalo

    Full Text Available Retinal pigment epithelial (RPE cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP, the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC and RPE derived from the hESC (hESC-RPE. Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE-derived diseases, drug testing and targeted drug therapy.

  14. Phage display selection of fully human antibody fragments to inhibit growth-promoting effects of glycine-extended gastrin 17 on human colorectal cancer cells.

    Science.gov (United States)

    Khajeh, Shirin; Tohidkia, Mohammad Reza; Aghanejad, Ayuob; Mehdipour, Tayebeh; Fathi, Farzaneh; Omidi, Yadollah

    2018-06-09

    Glycine-extended gastrin 17 (G17-Gly), a dominant processing intermediate of gastrin gene, has been implicated in the development or maintenance of colorectal cancers (CRCs). Hence, neutralizing G17-Gly activity by antibody entities can provide a potential therapeutic strategy in the patients with CRCs. To this end, we isolated fully human antibody fragments from a phage antibody library through biopanning against different epitopes of G17-Gly in order to obtain the highest possible antibody diversity. ELISA screening and sequence analysis identified 2 scFvs and 4 V L antibody fragments. Kinetic analysis of the antibody fragments by SPR revealed K D values to be in the nanomolar range (87.9-334 nM). The selected anti-G17-Gly antibody fragments were analyzed for growth inhibition and apoptotic assays in a CRC cell line, HCT-116, which is well-characterized for expressing gastrin intermediate species but not amidated gastrin. The antibody fragments exhibited significant inhibition of HCT-116 cells proliferation ranging from 36.5 to 73% of controls. Further, Annexin V/PI staining indicated that apoptosis rates of scFv H8 and V L G8 treated cells were 45.8 and 63%, respectively. Based on these results, we for the first time, demonstrated the isolation of anti-G17-Gly human scFv and V L antibodies with potential therapeutic applications in G17-Gly-responsive tumors.

  15. Interferon-β lipofection I. Increased efficacy of chemotherapeutic drugs on human tumor cells derived monolayers and spheroids.

    Science.gov (United States)

    Villaverde, M S; Gil-Cardeza, M L; Glikin, G C; Finocchiaro, L M E

    2012-07-01

    We evaluated the effect of hIFNβ gene transfer alone or in combination with different antineoplastic drugs commonly used in cancer treatment. Five human tumor-derived cell lines were cultured as monolayers and spheroids. Four cell lines (Ewing sarcomas EW7 and COH, melanoma M8 and mammary carcinoma MCF-7) were sensitive to hIFNβ gene lipofection. Although this effect appeared in both culture configurations, spheroids showed a relative multicellular resistance (insensitive colon carcinoma HT-29 excluded). EW7 and M8 hIFNβ-expressing cells were exposed to different concentrations of bleomycin, bortezomib, carboplatin, doxorubicin, etoposide, methotrexate, paclitaxel and vincristine in both configuration models. In chemotherapy-sensitive EW7 monolayers, the combination of hIFNβ gene and antineoplastic drugs displayed only additive or counteractive (methotrexate) effects, suggesting that cytotoxic mechanisms triggered by hIFNβ gene lipofection could be saturating the signaling pathways. Conversely, in chemotherapy-resistant EW7 spheroids or M8 cells, the combination of hIFNβ with drugs that mainly operate at the genotoxic level (doxorubicin, methotrexate and paclitaxel) presented only additive effects. However, drugs that also increase pro-oxidant species can complement the antitumor efficacy of the hIFNβ gene and clearly caused potentiated effects (bleomycin, bortezomib, carboplatin, etoposide and vincristine). The great bystander effect induced by hIFNβ gene lipofection could be among the main causes of its effectiveness, because only 1 or 2% of EW7 or M8 hIFNβ-expressing cells killed more than 60 or 80% of cell population, respectively.

  16. Acute stress reduces wound-induced activation of microbicidal potential of ex vivo isolated human monocyte-derived macrophages.

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    Ulrike Kuebler

    Full Text Available BACKGROUND: Psychological stress delays wound healing but the precise underlying mechanisms are unclear. Macrophages play an important role in wound healing, in particular by killing microbes. We hypothesized that (a acute psychological stress reduces wound-induced activation of microbicidal potential of human monocyte-derived macrophages (HMDM, and (b that these reductions are modulated by stress hormone release. METHODS: Fourty-one healthy men (mean age 35 ± 13 years were randomly assigned to either a stress or stress-control group. While the stress group underwent a standardized short-term psychological stress task after catheter-induced wound infliction, stress-controls did not. Catheter insertion was controlled. Assessing the microbicidal potential, we investigated PMA-activated superoxide anion production by HMDM immediately before and 1, 10 and 60 min after stress/rest. Moreover, plasma norepinephrine and epinephrine and salivary cortisol were repeatedly measured. In subsequent in vitro studies, whole blood was incubated with norepinephrine in the presence or absence of phentolamine (norepinephrine blocker before assessing HMDM microbicidal potential. RESULTS: Compared with stress-controls, HMDM of the stressed subjects displayed decreased superoxide anion-responses after stress (p's <.05. Higher plasma norepinephrine levels statistically mediated lower amounts of superoxide anion-responses (indirect effect 95% CI: 4.14-44.72. Norepinephrine-treated HMDM showed reduced superoxide anion-production (p<.001. This effect was blocked by prior incubation with phentolamine. CONCLUSIONS: Our results suggest that acute psychological stress reduces wound-induced activation of microbicidal potential of HMDM and that this reduction is mediated by norepinephrine. This might have implications for stress-induced impairment in wound healing.

  17. In vivo effects of human adipose-derived stem cells reseeding on acellular bovine pericardium in nude mice.

    Science.gov (United States)

    Wu, Qingkai; Dai, Miao; Xu, Peirong; Hou, Min; Teng, Yincheng; Feng, Jie

    2016-01-01

    Tissue-engineered biologic products may be a viable option in the reconstruction of pelvic organ prolapse (POP). This study was based on the hypothesis that human adipose-derived stem cells (hASCs) are viable in acellular bovine pericardium (ABP), when reseeded by two different techniques, and thus, aid in the reconstruction. To investigate the reseeding of hASCs on ABP grafts by using non-invasive bioluminescence imaging (BLI), and to identify the effective hASCs-scaffold combinations that enabled regeneration. Thirty female athymic nude mice were randomly divided into three groups: In the VIVO group, ABPs were implanted in the subcutaneous pockets and enhanced green fluorescent protein luciferase (eGFP·Luc)-hASCs (1 × 10(6) cells/50 µL) were injected on the ABP at the same time. In the VITRO group, the mice were implanted with grafts that ABP were co-cultured with eGFP·Luc-hASCs in vitro. The BLANK group mice were implanted with ABP only. The eGFP·Luc-hASCs reseeded on ABP were analyzed by BLI, histology, and immunohistochemistry. The eGFP·Luc-hASCs reseeded on ABP could be visualized at 12 weeks in vivo. Histology revealed that the VIVO group displayed the highest cell ingrowths, small vessels, and percent of collagen content per unit area. Desmin and α-smooth muscle actin were positive at the same site in the VIVO group cells. However, few smooth muscles were observed in the VITRO and BLANK groups. These results suggest that hASCs reseeded on ABP in vivo during surgery may further enhance the properties of ABP and may promote regeneration at the recipient site, resulting in a promising treatment option for POP. © 2016 by the Society for Experimental Biology and Medicine.

  18. Comparison of human platelet lysate alternatives using expired and freshly isolated platelet concentrates for adipose-derived stromal cell expansion.

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    Dessels, Carla; Durandt, Chrisna; Pepper, Michael S

    2018-03-19

    Pooled human platelet lysate (pHPL) has been used to expand adipose-derived stromal cells (ASCs) and can be formulated using fresh or expired buffy coats (BCs) which are then resuspended in either plasma or an additive solution. Not much is known about the effects that expired products and additive solutions have on ASC expansion, and the need for quality control and release criteria has been expressed. This pilot study compared proliferation, cell size, morphology and immunophenotype of ASCs expanded in the different pHPL alternatives versus foetal bovine serum (FBS). Quality control criteria were assessed prior to and during the manufacture of the pHPL alternatives. ASCs were then expanded in 1%, 2.5%, 5% or 10% of the different pHPL alternatives or in 10% FBS. Cell size, morphology, cell number and immunophenotype were measured using microscopy and flow cytometry. The majority of the pHPL alternatives were within the recommended ranges for the quality control criteria. ASCs expanded in the pHPL alternatives were smaller in size, displayed a tighter spindle-shaped morphology, increased cell growth and had a similar immunophenotype (with the exception of CD34 and CD36) when compared to ASCs expanded in FBS. Here we report on the effects that expired BC products and additive solutions have on ASC expansion. When taken together, our findings indicate that all of the pHPL alternatives can be considered to be suitable replacements for FBS for ASC expansion, and that expired BC products can be used as an alternative to fresh BC products.

  19. Skeletal myogenic differentiation of human urine-derived cells as a potential source for skeletal muscle regeneration.

    Science.gov (United States)

    Chen, Wei; Xie, Minkai; Yang, Bin; Bharadwaj, Shantaram; Song, Lujie; Liu, Guihua; Yi, Shanhong; Ye, Gang; Atala, Anthony; Zhang, Yuanyuan

    2017-02-01

    Stem cells are regarded as possible cell therapy candidates for skeletal muscle regeneration. However, invasive harvesting of those cells can cause potential harvest-site morbidity. The goal of this study was to assess whether human urine-derived stem cells (USCs), obtained through non-invasive procedures, can differentiate into skeletal muscle linage cells (Sk-MCs) and potentially be used for skeletal muscle regeneration. In this study, USCs were harvested from six healthy individuals aged 25-55. Expression profiles of cell-surface markers were assessed by flow cytometry. To optimize the myogenic differentiation medium, we selected two from four different types of myogenic differentiation media to induce the USCs. Differentiated USCs were identified with myogenic markers by gene and protein expression. USCs were implanted into the tibialis anterior muscles of nude mice for 1 month. The results showed that USCs displayed surface markers with positive staining for CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133, CD146, SSEA-4 and STRO-1, and negative staining for CD14, CD31, CD34 and CD45. After myogenic differentiation, a change in morphology was observed from 'rice-grain'-like cells to spindle-shaped cells. The USCs expressed specific Sk-MC transcripts and protein markers (myf5, myoD, myosin, and desmin) after being induced with different myogenic culture media. Implanted cells expressed Sk-MC markers stably in vivo. Our findings suggest that USCs are able to differentiate into the Sk-MC lineage in vitro and after being implanted in vivo. Thus, they might be a potential source for cell injection therapy in the use of skeletal muscle regeneration. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  20. Patch-Clamp Recording from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Improving Action Potential Characteristics through Dynamic Clamp

    Science.gov (United States)

    Veerman, Christiaan C.; Zegers, Jan G.; Mengarelli, Isabella; Bezzina, Connie R.

    2017-01-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current (IK1) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in “ventricular-like” and “atrial-like” hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial IK1 (computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic IK1 through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon IK1 injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic IK1. In conclusion, the dynamic clamp-based approach of IK1 injection has broad applicability for detailed AP measurements in hiPSC-CMs. PMID:28867785

  1. [Primary study on fluro [ 19F] berberine derivative for human hepatocellular carcinoma targetting in vitro].

    Science.gov (United States)

    Zhang, Tong; Wu, Xiaoai; Cai, Huawei; Liang, Meng; Fan, Chengzhong

    2017-04-01

    [ 18 F]HX-01, a Fluorine-18 labeled berberine derivative, is a potential positron emission tomography (PET) tumor imaging agent, while [ 19 F]HX-01 is a nonradioactive reference substance with different energy state and has the same physical and chemical properties. In order to collect data for further study of [ 18 F]HX-01 PET imaging of hepatocellular carcinoma in vivo , this study compared the uptake of [ 19 F]HX-01 by human hepatocellular carcinoma and normal hepatocytes in vitro . The target compound, [ 19 F]HX-01, was synthesized in one step using berberrubine and 3-fluoropropyl 4-methylbenzenesulfonate. Cellular uptake and localization of [ 19 F]HX-01 were performed by a fluorescence microscope in human hepatocellular carcinoma HepG2, SMMC-7721 and human normal hepatocyte HL-7702. Cellular proliferation inhibition and cell cytotoxicity assay of the [ 19 F]HX-01 were conducted using cell counting kit-8 (CCK-8) on HepG2, SMMC-7721 and HL-7702 cells. Fluorescent microscopy showed that the combining ability of [ 19 F]HX-01 to the carcinoma SMMC-7721 and HepG2 was higher than that to the normal HL-7702. Cellular proliferation inhibition assay demonstrated that [ 19 F]HX-01 leaded to a dose-dependent inhibition on SMMC-7721, HepG2, and HL-7702 proliferation. Cell cytotoxicity assay presented that the cytotoxicity of [ 19 F]HX-01 to SMMC-7721 and HepG2 was obviously higher than that to HL-7702. This in vitro study showed that [ 19 F]HX-01 had a higher selectivity on human hepatocellular carcinoma cells (SMMC-7721, HepG2) but has less toxicity to normal hepatocytes (HL-7702). This could set up the idea that the radioactive reference substance [ 18 F]HX-01 may be worthy of further development as a potential molecular probe targeting human hepatocellular carcinoma using PET.

  2. Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

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    Ingrid R. Cordeiro

    2015-09-01

    Full Text Available Human adipose-derived stromal cells (hADSC are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1 regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues.

  3. Characterization of a receptor for human monocyte-derived neutrophil chemotactic factor/interleukin-8

    International Nuclear Information System (INIS)

    Grob, P.M.; David, E.; Warren, T.C.; DeLeon, R.P.; Farina, P.R.; Homon, C.A.

    1990-01-01

    Monocyte-derived neutrophil chemotactic factor/interleukin-8 (MDNCF/IL-8) is an 8,000-dalton protein produced by monocytes which exhibits activity as a chemoattractant for neutrophils with maximal activity achieved at a concentration of 50 ng/ml. This polypeptide has been iodinated by chloramine-T methodology (350 Ci/mM), and specific receptors for MDNCF/IL-8 have been detected on human neutrophils, U937 cells, THP-1 cells, and dimethyl sulfoxide-differentiated HL-60 cells. The binding of MDNCF/IL-8 to human neutrophils is not inhibited by interleukin-1 alpha, tumor necrosis factor-alpha, insulin, or epidermal growth factor. In addition, chemoattractants such as C5a, fMet-Leu-Phe, leukotriene B4, and platelet-activating factor fail to inhibit binding, suggesting that MDNCF/IL-8 utilizes a unique receptor. The receptor for MDNCF/IL-8 is apparently glycosylated since ligand binding is inhibited by the presence of wheat germ agglutinin, a lectin with a binding specificity for N-acetylglucosamine and neuraminic acid. Steady state binding experiments indicate Kd values of 4 and 0.5 nM and receptor numbers of 75,000 and 7,400 for human neutrophils and differentiated HL-60 cells, respectively. 125I-MDNCF/IL-8 bound to human neutrophils is rapidly internalized and subsequently released from cells as trichloroacetic acid-soluble radioactivity. Affinity labeling experiments suggest that the human neutrophil MDNCF/IL-8 receptor exhibits a mass of approximately 58,000 daltons

  4. Interactions between resin monomers and commercial composite resins with human saliva derived esterases.

    Science.gov (United States)

    Jaffer, F; Finer, Y; Santerre, J P

    2002-04-01

    Cholesterol esterase (CE) and pseudocholinesterase (PCE) have been reported to degrade commercial and model composite resins containing bisphenylglycidyl dimethacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA) or the latter in combination with urethane modified BisGMA monomer systems. In addition, human saliva has been shown to contain esterase like activities similar to CE and PCE. Hence, it was the aim of the current study to determine to what extent human saliva could degrade two common commercial composite resins (Z250 from 3M Inc. and Spectrum TPH from L.D. Caulk) which contain the above monomer systems. Saliva samples from different volunteers were collected, processed, pooled, and freeze-dried. TEGDMA and BisGMA monomers were incubated with human saliva derived esterase activity (HSDEA) and their respective hydrolysis was monitored using high performance liquid chromatography (HPLC). Both monomers were completely hydrolyzed within 25 h by HSDEA. Photopolymerized composites were incubated with buffer or human saliva (pH 7.0 and 37 C) for 2, 8 and 16 days. The incubation solutions were analyzed using HPLC and mass spectrometry. Surface morphology characterization was carried out using scanning electron microscopy. Upon biodegradation, the Z250 composite yielded higher amounts of BisGMA and TEGDMA related products relative to the TPH composite. However, there were higher amounts of ethoxylated bis-phenol A released from the TPH material. In terms of total mass of products released, human saliva demonstrated a greater ability to degrade Z250. In summary, HSDEA has been shown to contain esterase activities that can readily catalyze the biodegradation of current commercial composite resins.

  5. Thiamine status in humans and content of phosphorylated thiamine derivatives in biopsies and cultured cells.

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    Marjorie Gangolf

    Full Text Available BACKGROUND: Thiamine (vitamin B1 is an essential molecule for all life forms because thiamine diphosphate (ThDP is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP, thiamine triphosphate (ThTP and adenosine thiamine triphosphate (AThTP, present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines. METHODOLOGY AND PRINCIPAL FINDINGS: Thiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ∼60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP]/([Thiamine]+[ThMP] ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines. CONCLUSIONS AND SIGNIFICANCE: The high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells

  6. 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

  7. Human pluripotent stem cell-derived erythropoietin-producing cells ameliorate renal anemia in mice.

    Science.gov (United States)

    Hitomi, Hirofumi; Kasahara, Tomoko; Katagiri, Naoko; Hoshina, Azusa; Mae, Shin-Ichi; Kotaka, Maki; Toyohara, Takafumi; Rahman, Asadur; Nakano, Daisuke; Niwa, Akira; Saito, Megumu K; Nakahata, Tatsutoshi; Nishiyama, Akira; Osafune, Kenji

    2017-09-27

    The production of erythropoietin (EPO) by the kidneys, a principal hormone for the hematopoietic system, is reduced in patients with chronic kidney disease (CKD), eventually resulting in severe anemia. Although recombinant human EPO treatment improves anemia in patients with CKD, returning to full red blood cell production without fluctuations does not always occur. We established a method to generate EPO-producing cells from human induced pluripotent stem cells (hiPSCs) by modifying previously reported hepatic differentiation protocols. These cells showed increased EPO expression and secretion in response to low oxygen conditions, prolyl hydroxylase domain-containing enzyme inhibitors, and insulin-like growth factor 1. The EPO protein secreted from hiPSC-derived EPO-producing (hiPSC-EPO) cells induced the erythropoietic differentiation of human umbilical cord blood progenitor cells in vitro. Furthermore, transplantation of hiPSC-EPO cells into mice with CKD induced by adenine treatment improved renal anemia. Thus, hiPSC-EPO cells may be a useful tool for clarifying the mechanisms of EPO production and may be useful as a therapeutic strategy for treating renal anemia. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  8. Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo.

    Science.gov (United States)

    Saravanan, Rathi; Adav, Sunil S; Choong, Yeu Khai; van der Plas, Mariena J A; Petrlova, Jitka; Kjellström, Sven; Sze, Siu Kwan; Schmidtchen, Artur

    2017-10-13

    The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major contributor to wound healing impairments. Proteolytic peptide patterns could therefore correlate and "report" healing activity and infection. This work describes a proof of principle delineating a strategy by which peptides from a selected protein, human thrombin, are detected and attributed to proteolytic actions. With a particular focus on thrombin-derived C-terminal peptides (TCP), we show that distinct peptide patterns are generated in vitro by the human S1 peptidases human neutrophil elastase and cathepsin G, and the bacterial M4 peptidases Pseudomonas aeruginosa elastase and Staphylococcus aureus aureolysin, respectively. Corresponding peptide sequences were identified in wound fluids from acute and non-healing ulcers, and notably, one peptide, FYT21 (FYTHVFRLKKWIQKVIDQFGE), was only present in wound fluid from non-healing ulcers colonized by P. aeruginosa and S. aureus. Our result is a proof of principle pointing at the possibility of defining peptide biomarkers reporting distinct proteolytic activities, of potential implication for improved diagnosis of wound healing and infection.

  9. Human Gut-Derived Commensal Bacteria Suppress CNS Inflammatory and Demyelinating Disease.

    Science.gov (United States)

    Mangalam, Ashutosh; Shahi, Shailesh K; Luckey, David; Karau, Melissa; Marietta, Eric; Luo, Ningling; Choung, Rok Seon; Ju, Josephine; Sompallae, Ramakrishna; Gibson-Corley, Katherine; Patel, Robin; Rodriguez, Moses; David, Chella; Taneja, Veena; Murray, Joseph

    2017-08-08

    The human gut is colonized by a large number of microorganisms (∼10 13 bacteria) that support various physiologic functions. A perturbation in the healthy gut microbiome might lead to the development of inflammatory diseases, such as multiple sclerosis (MS). Therefore, gut commensals might provide promising therapeutic options for treating MS and other diseases. We report the identification of human gut-derived commensal bacteria, Prevotella histicola, which can suppress experimental autoimmune encephalomyelitis (EAE) in a human leukocyte antigen (HLA) class II transgenic mouse model. P. histicola suppresses disease through the modulation of systemic immune responses. P. histicola challenge led to a decrease in pro-inflammatory Th1 and Th17 cells and an increase in the frequencies of CD4 + FoxP3 + regulatory T cells, tolerogenic dendritic cells, and suppressive macrophages. Our study provides evidence that the administration of gut commensals may regulate a systemic immune response and may, therefore, have a possible role in treatment strategies for MS. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  10. Effects of murine and human bone marrow-derived mesenchymal stem cells on cuprizone induced demyelination.

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    Jasmin Nessler

    Full Text Available For the treatment of patients with multiple sclerosis there are no regenerative approaches to enhance remyelination. Mesenchymal stem cells (MSC have been proposed to exert such regenerative functions. Intravenous administration of human MSC reduced the clinical severity of experimental autoimmune encephalomyelitis (EAE, an animal model mimicking some aspects of multiple sclerosis. However, it is not clear if this effect was achieved by systemic immunomodulation or if there is an active neuroregeneration in the central nervous system (CNS. In order to investigate remyelination and regeneration in the CNS we analysed the effects of intravenously and intranasally applied murine and human bone marrow-derived MSC on cuprizone induced demyelination, a toxic animal model which allows analysis of remyelination without the influence of the peripheral immune system. In contrast to EAE no effects of MSC on de- and remyelination and glial cell reactions were found. In addition, neither murine nor human MSC entered the lesions in the CNS in this toxic model. In conclusion, MSC are not directed into CNS lesions in the cuprizone model where the blood-brain-barrier is intact and thus cannot provide support for regenerative processes.

  11. Human Skin Constructs with Spatially Controlled Vasculature Using Primary and iPSC-Derived Endothelial Cells.

    Science.gov (United States)

    Abaci, Hasan E; Guo, Zongyou; Coffman, Abigail; Gillette, Brian; Lee, Wen-Han; Sia, Samuel K; Christiano, Angela M

    2016-07-01

    Vascularization of engineered human skin constructs is crucial for recapitulation of systemic drug delivery and for their long-term survival, functionality, and viable engraftment. In this study, the latest microfabrication techniques are used and a novel bioengineering approach is established to micropattern spatially controlled and perfusable vascular networks in 3D human skin equivalents using both primary and induced pluripotent stem cell (iPSC)-derived endothelial cells. Using 3D printing technology makes it possible to control the geometry of the micropatterned vascular networks. It is verified that vascularized human skin equivalents (vHSEs) can form a robust epidermis and establish an endothelial barrier function, which allows for the recapitulation of both topical and systemic delivery of drugs. In addition, the therapeutic potential of vHSEs for cutaneous wounds on immunodeficient mice is examined and it is demonstrated that vHSEs can both promote and guide neovascularization during wound healing. Overall, this innovative bioengineering approach can enable in vitro evaluation of topical and systemic drug delivery as well as improve the potential of engineered skin constructs to be used as a potential therapeutic option for the treatment of cutaneous wounds. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

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    Yu Takahashi

    2018-01-01

    Full Text Available Gut epithelial organoids are routinely used to investigate intestinal biology; however, current culture methods are not amenable to genetic manipulation, and it is difficult to generate sufficient numbers for high-throughput studies. Here, we present an improved culture system of human induced pluripotent stem cell (iPSC-derived intestinal organoids involving four methodological advances. (1 We adopted a lentiviral vector to readily establish and optimize conditioned medium for human intestinal organoid culture. (2 We obtained intestinal organoids from human iPSCs more efficiently by supplementing WNT3A and fibroblast growth factor 2 to induce differentiation into definitive endoderm. (3 Using 2D culture, followed by re-establishment of organoids, we achieved an efficient transduction of exogenous genes in organoids. (4 We investigated suspension organoid culture without scaffolds for easier harvesting and assays. These techniques enable us to develop, maintain, and expand intestinal organoids readily and quickly at low cost, facilitating high-throughput screening of pathogenic factors and candidate treatments for gastrointestinal diseases.

  13. Pleomorphic Structures in Human Blood Are Red Blood Cell-Derived Microparticles, Not Bacteria.

    Science.gov (United States)

    Mitchell, Adam J; Gray, Warren D; Schroeder, Max; Yi, Hong; Taylor, Jeannette V; Dillard, Rebecca S; Ke, Zunlong; Wright, Elizabeth R; Stephens, David; Roback, John D; Searles, Charles D

    2016-01-01

    Red blood cell (RBC) transfusions are a common, life-saving therapy for many patients, but they have also been associated with poor clinical outcomes. We identified unusual, pleomorphic structures in human RBC transfusion units by negative-stain electron microscopy that appeared identical to those previously reported to be bacteria in healthy human blood samples. The presence of viable, replicating bacteria in stored blood could explain poor outcomes in transfusion recipients and have major implications for transfusion medicine. Here, we investigated the possibility that these structures were bacteria. Flow cytometry, miRNA analysis, protein analysis, and additional electron microscopy studies strongly indicated that the pleomorphic structures in the supernatant of stored RBCs were RBC-derived microparticles (RMPs). Bacterial 16S rDNA PCR amplified from these samples were sequenced and was found to be highly similar to species that are known to commonly contaminate laboratory reagents. These studies suggest that pleomorphic structures identified in human blood are RMPs and not bacteria, and they provide an example in which laboratory contaminants may can mislead investigators.

  14. Molecular cloning of the human eosinophil-derived neurotoxin: A member of the ribonuclease gene family

    International Nuclear Information System (INIS)

    Rosenberg, H.F.; Tenen, D.G.; Ackerman, S.J.

    1989-01-01

    The authors have isolated a 725-base-pair cDNA clone for human eosinophil-derived neurotoxin (EDN). EDN is a distinct cationic protein of the eosinophil's large specific granule known primarily for its ability to induce ataxia, paralysis, and central nervous system cellular degeneration in experimental animals (Gordon phenomenon). The open reading frame encodes a 134-amino acid mature polypeptide with a molecular mass of 15.5 kDa and a 27-residue amino-terminal hydrophobic leader sequence. The sequence of the mature polypeptide is identical to that reported for human urinary ribonuclease, and to the amino-terminal sequence of human liver ribonuclease; the cDNA encodes a tryptophan in position 7. Both EDN and the related granule protein, eosinophil cationic protein, have ribonucleolytic activity; sequence similarities among EDN, eosinophil cationic protein, ribonucleases from liver, urine, and pancreas, and angiogenin define a ribonuclease multigene family. mRNA encoding EDN was detected in uninduced HL-60 cells and was up-regulated in cells induced toward eosinophilic differentiation with B-cell growth factor 2/interleukin 5 and toward neutrophilic differentiation with dimethyl sulfoxide. EDN mRNA was detected in mature neutrophils even though EDN-like neurotoxic activity is not found neutrophil extracts. These results suggest that neutrophils contain a protein that is closely related or identical to EDN

  15. Performance of NCAP projection displays

    Science.gov (United States)

    Jones, Philip J.; Tomita, Akira; Wartenberg, Mark

    1991-08-01

    Prototypes of projection displays based on dispersions of liquid crystal in polymer matrices are beginning to appear. The principle of operation depends on electrically switchable light scattering. They are potentially much brighter than current cathode ray tube (CRT) or twisted nematic liquid crystal (TN LC) cell based displays. Comparisons of efficacy and efficiency show this. The contrast and brightness of such displays depend on a combination of the f- number of the projection system and the scattering characteristics of the light valve. Simplified equations can be derived to show these effects. The degree of scattering of current NCAP formulations is sufficient to produce good contrast projection displays, at convenient voltages, that are around three times brighter than TN LC projectors because of the lack of polarizers in the former.

  16. Human amnion-derived mesenchymal stem cells protect against UVA irradiation-induced human dermal fibroblast senescence, in vitro

    Science.gov (United States)

    Zhang, Chunli; Yuchi, Haishen; Sun, Lu; Zhou, Xiaoli; Lin, Jinde

    2017-01-01

    The aim of the present study was to determine if human amnion-derived mesenchymal stem cells (HAMSCs) exert a protective effect on ultraviolet A (UVA) irradiation-induced human dermal fibroblast (HDF) senescence. A senescence model was constructed as follows: HDFs (104–106 cells/well) were cultured in a six-well plate in vitro and then exposed to UVA irradiation at 9 J/cm2 for 30 min. Following the irradiation period, HDFs were co-cultured with HAMSCs, which were seeded on transwells. A total of 72 h following the co-culturing, senescence-associated β-galactosidase staining was performed and reactive oxygen species (ROS) content and mitochondrial membrane potential (Δψm) were detected in the HDFs via flow cytometric analysis. The results demonstrated that the percentage of HDFs, detected via staining with X-gal, were markedly decreased when co-cultured with human HAMSCs, compared with the group that were not co-cultured. The ROS content was decreased and the mitochondrial membrane potential (Δψm) recovered in cells treated with UVA and HAMSCs, compared with that of cells treated with UVA alone. Reverse transcription-quantitative polymerase chain reaction revealed the significant effects of HAMSCs on the HDF senescence marker genes p53 and matrix metalloproteinase-1 mRNA expression. In addition to this, western blot analysis verified the effects of HAMSCs on UVA induced senescence, providing a foundation for novel regenerative therapeutic methods. Furthermore, the results suggested that activation of the extracellular-signal regulated kinase 1/2 mitogen activated protein kinase signal transduction pathway, is essential for the HAMSC-mediated UVA protective effects. The decrease in ROS content additionally indicated that HAMSCs may exhibit the potential to treat oxidative stress-mediated UVA skin senescence in the future. PMID:28627622

  17. Platelet-derived growth factor receptors in the human central nervous system : autoradiographic distribution and receptor densities in multiple sclerosis

    NARCIS (Netherlands)

    De Keyser, J; Wilczak, N

    1997-01-01

    Platelet derived growth factor (PDGF) receptors were studied in postmortem adult human brain and cervical spinal cord using autoradiography with human recombinant I-125-PDGF-BB. PDGF-BB binds to the three different dimers of PDGF receptors (alpha alpha, alpha beta and beta beta) PDGF receptors were

  18. Human Induced Pluripotent Stem Cell-Derived Macrophages Share Ontogeny with MYB-Independent Tissue-Resident Macrophages

    Directory of Open Access Journals (Sweden)

    Julian Buchrieser

    2017-02-01

    Full Text Available Tissue-resident macrophages, such as microglia, Kupffer cells, and Langerhans cells, derive from Myb-independent yolk sac (YS progenitors generated before the emergence of hematopoietic stem cells (HSCs. Myb-independent YS-derived resident macrophages self-renew locally, independently of circulating monocytes and HSCs. In contrast, adult blood monocytes, as well as infiltrating, gut, and dermal macrophages, derive from Myb-dependent HSCs. These findings are derived from the mouse, using gene knockouts and lineage tracing, but their applicability to human development has not been formally demonstrated. Here, we use human induced pluripotent stem cells (iPSCs as a tool to model human hematopoietic development. By using a CRISPR-Cas9 knockout strategy, we show that human iPSC-derived monocytes/macrophages develop in an MYB-independent, RUNX1-, and SPI1 (PU.1-dependent fashion. This result makes human iPSC-derived macrophages developmentally related to and a good model for MYB-independent tissue-resident macrophages, such as alveolar and kidney macrophages, microglia, Kupffer cells, and Langerhans cells.

  19. A Novel Dual HDAC6 and Tubulin Inhibitor, MPT0B451, Displays Anti-tumor Ability in Human Cancer Cells in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Yi-Wen Wu

    2018-03-01

    Full Text Available The combination cancer therapy is a new strategy to circumvent drug resistance for the treatment of high metastasis and advanced malignancies. Herein, we developed a synthesized compound MPT0B451 that display inhibitory effect against histone deacetylase (HDAC 6 and tubulin assembly. Our data demonstrated that MPT0B451 significantly inhibited cancer cell growths in HL-60 and PC-3 cells due to inhibition of HDAC activity. MPT0B451 also markedly increased caspase-mediated apoptosis in these cells. The cell cycle analysis showed mitotic arrest induced by MPT0B451 with enhanced expression of G2/M transition proteins. Moreover, molecular docking analysis supported MPT0B451 as a dual HDAC6 and tubulin inhibitor. Finally, MPT0B451 led to tumor growth inhibition (TGI in HL-60 and PC-3 xenograft models. These findings indicated that MPT0B451 has dual inhibition effects for HDAC6 and tubulin, and also contributed to G2/M arrest followed by apoptotic induction. Together, our results suggested that MPT0B451 may serve as a potent anti-cancer treatment regimen in human prostate cancer and acute myeloid leukemia.

  20. Human SGT interacts with Bag-6/Bat-3/Scythe and cells with reduced levels of either protein display persistence of few misaligned chromosomes and mitotic arrest

    International Nuclear Information System (INIS)

    Winnefeld, Marc; Grewenig, Annabel; Schnoelzer, Martina; Spring, Herbert; Knoch, Tobias A.; Gan, Eugene C.; Rommelaere, Jean; Cziepluch, Celina

    2006-01-01

    The human small glutamine-rich TPR-containing protein (hSGT) is essential for cell division since RNA-interference-mediated strong reduction of hSGT protein levels causes mitotic arrest (M. Winnefeld, J. Rommelaere, and C. Cziepluch, The human small glutamine-rich TPR-containing protein is required for progress through cell division, Exp. Cell Res. 293 (2004), 43-57). Analysis of HeLa cells expressing a histone 2A-YFP fusion protein revealed the continuous presence of few mislocalized chromosomes close to the spindle poles as possible cause for hSGT depletion-dependent prometaphase arrest. Cells unable to rescue these mislocalized chromosomes into the metaphase plate died at this stage through apoptosis. In order to address hSGT function at the molecular level, mass spectrometry analysis of proteins which co-immunoprecipitated with Flag-tagged hSGT was performed. Thereby, Hsp70 and Bag-6/Bat-3/Scythe were identified as novel hSGT interaction partners while interaction with Hsc70 was confirmed. Results obtained with truncated versions of the hSGT protein revealed that Bag-6/Bat-3/Scythe and Hsp70 or Hsc70 were independently able to form complexes with hSGT. Interaction of hSGT with Hsc70, Hsp70 or Bag-6/Bat-3/Scythe was demonstrated in prometaphase, thereby suggesting a possible role for complexes containing hSGT and distinct (co)-chaperones during mitosis. Finally, cells from populations with reduced levels of Bag-6/Bat-3/Scythe also displayed persistence of mislocalized chromosomes and mitotic arrest, which strongly indicated that hSGT-Bag-6/Bat-3/Scythe complexes could be directly or indirectly required for complete chromosome congression

  1. Signaling via class IA Phosphoinositide 3-kinases (PI3K in human, breast-derived cell lines.

    Directory of Open Access Journals (Sweden)

    Veronique Juvin

    Full Text Available We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K in human breast-derived MCF10a (and iso-genetic derivatives and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, β, δ and γ and p50-101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5-trisphosphate (PtdIns(3,4,5P3 that can activate effectors, eg protein kinase B (PKB, and responses, eg migration. The PtdIns(3,4,5P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110β>>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not β- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not β- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K, basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN(-/- cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110β, but not α- or δ- activity; in PTEN(-/- MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, via acutely

  2. Comparison of Gene Expression in Human Embryonic Stem Cells, hESC-Derived Mesenchymal Stem Cells and Human Mesenchymal Stem Cells

    OpenAIRE

    Romain Barbet; Isabelle Peiffer; Antoinette Hatzfeld; Pierre Charbord; Jacques A. Hatzfeld

    2011-01-01

    We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs ...

  3. Generation of Functional Lentoid Bodies From Human Induced Pluripotent Stem Cells Derived From Urinary Cells.

    Science.gov (United States)

    Fu, Qiuli; Qin, Zhenwei; Jin, Xiuming; Zhang, Lifang; Chen, Zhijian; He, Jiliang; Ji, Junfeng; Yao, Ke

    2017-01-01

    The pathological mechanisms underlying cataract formation remain largely unknown on account of the lack of appropriate in vitro cellular models. The aim of this study is to develop a stable in vitro system for human lens regeneration using pluripotent stem cells. Isolated human urinary cells were infected with four Yamanaka factors to generate urinary human induced pluripotent stem cells (UiPSCs), which were induced to differentiate into lens progenitor cells and lentoid bodies (LBs). The expression of lens-specific markers was examined by real-time PCR, immunostaining, and Western blotting. The structure and magnifying ability of LBs were investigated using transmission electron microscopy and observing the magnification of the letter "X," respectively. We developed a "fried egg" differentiation method to generate functional LBs from UiPSCs. The UiPSC-derived LBs exhibited crystalline lens-like morphology and a transparent structure and expressed lens-specific markers αA-, αB-, β-, and γ-crystallin and MIP. During LB differentiation, the placodal markers SIX1, EYA1, DLX3, PAX6, and the specific early lens markers SOX1, PROX1, FOXE3, αA-, and αB-crystallin were observed at certain time points. Microscopic examination revealed the presence of lens epithelial cells adjacent to the lens capsule as well as both immature and mature fiber-like cells. Optical analysis further demonstrated the magnifying ability (1.7×) of the LBs generated from UiPSCs. Our study provides the first evidence toward generating functional LBs from UiPSCs, thereby establishing an in vitro system that can be used to study human lens development and cataractogenesis and perhaps even be useful for drug screening.

  4. Antimicrobial Nisin Acts Against Saliva Derived Multi-Species Biofilms without Cytotoxicity to Human Oral Cells

    Directory of Open Access Journals (Sweden)

    Yvonne Lorraine Kapila

    2015-06-01

    Full Text Available Objectives: Nisin is a lantibiotic widely used for the preservation of food and beverages. Recently, investigators have reported that nisin may have clinical applications for treating bacterial infections. The aim of this study was to investigate the effects of ultra pure food grade Nisin ZP (> 95% purity on taxonomically diverse bacteria common to the human oral cavity and saliva derived multi-species oral biofilms, and to discern the toxicity of nisin against human cells relevant to the oral cavity. Methods: The MICs and MBCs of taxonomically distinct oral bacteria were determined using agar and broth dilution methods. To assess the effects of nisin on biofilms, two model systems were utilized: a static and a controlled flow microfluidic system. Biofilms were inoculated with pooled human saliva and fed filter-sterilized saliva for 20-22 h at 37°C. Nisin effects on cellular apoptosis and proliferation were evaluated using acridine orange/ethidium bromide fluorescent nuclear staining and lactate dehydrogenase activity assays. Results: Nisin inhibited planktonic growth of oral bacteria at low concentrations (2.5 – 50 μg/ml. Nisin also retarded development of multi-species biofilms at concentrations ≥ 1 μg/ml. Specifically, under biofilm model conditions, nisin interfered with biofilm development and reduced biofilm biomass and thickness in a dose-dependent manner. The treatment of pre-formed biofilms with nisin resulted in dose- and time-dependent disruption of the biofilm architecture along with decreased bacterial viability. Human cells relevant to the oral cavity were unaffected by the treatment of nisin at anti-biofilm concentrations and showed no signs of apoptotic changes unless treated with much higher concentrations (> 200 μg/ml. Conclusions: This work highlights the potential therapeutic value of high purity food grade nisin to inhibit the growth of oral bacteria and the development of biofilms relevant to oral diseases.

  5. Human induced pluripotent stem cell-derived beating cardiac tissues on paper.

    Science.gov (United States)

    Wang, Li; Xu, Cong; Zhu, Yujuan; Yu, Yue; Sun, Ning; Zhang, Xiaoqing; Feng, Ke; Qin, Jianhua

    2015-11-21

    There is a growing interest in using paper as a biomaterial scaffold for cell-based applications. In this study, we made the first attempt to fabricate a paper-based array for the culture, proliferation, and direct differentiation of human induced pluripotent stem cells (hiPSCs) into functional beating cardiac tissues and create "a beating heart on paper." This array was simply constructed by binding a cured multi-well polydimethylsiloxane (PDMS) mold with common, commercially available paper substrates. Three types of paper material (print paper, chromatography paper and nitrocellulose membrane) were tested for adhesion, proliferation and differentiation of human-derived iPSCs. We found that hiPSCs grew well on these paper substrates, presenting a three-dimensional (3D)-like morphology with a pluripotent property. The direct differentiation of human iPSCs into functional cardiac tissues on paper was also achieved using our modified differentiation approach. The cardiac tissue retained its functional activities on the coated print paper and chromatography paper with a beating frequency of 40-70 beats per min for up to three months. Interestingly, human iPSCs could be differentiated into retinal pigment epithelium on nitrocellulose membrane under the conditions of cardiac-specific induction, indicating the potential roles of material properties and mechanical cues that are involved in regulating stem cell differentiation. Taken together, these results suggest that different grades of paper could offer great opportunities as bioactive, low-cost, and 3D in vitro platforms for stem cell-based high-throughput drug testing at the tissue/organ level and for tissue engineering applications.

  6. Large-scale production and properties of human plasma-derived activated Factor VII concentrate.

    Science.gov (United States)

    Tomokiyo, K; Yano, H; Imamura, M; Nakano, Y; Nakagaki, T; Ogata, Y; Terano, T; Miyamoto, S; Funatsu, A

    2003-01-01

    An activated Factor VII (FVIIa) concentrate, prepared from human plasma on a large scale, has to date not been available for clinical use for haemophiliacs with antibodies against FVIII and FIX. In the present study, we attempted to establish a large-scale manufacturing process to obtain plasma-derived FVIIa concentrate with high recovery and safety, and to characterize its biochemical and biological properties. FVII was purified from human cryoprecipitate-poor plasma, by a combination of anion exchange and immunoaffinity chromatography, using Ca2+-dependent anti-FVII monoclonal antibody. To activate FVII, a FVII preparation that was nanofiltered using a Bemberg Microporous Membrane-15 nm was partially converted to FVIIa by autoactivation on an anion-exchange resin. The residual FVII in the FVII and FVIIa mixture was completely activated by further incubating the mixture in the presence of Ca2+ for 18 h at 10 degrees C, without any additional activators. For preparation of the FVIIa concentrate, after dialysis of FVIIa against 20 mm citrate, pH 6.9, containing 13 mm glycine and 240 mm NaCl, the FVIIa preparation was supplemented with 2.5% human albumin (which was first pasteurized at 60 degrees C for 10 h) and lyophilized in vials. To inactivate viruses contaminating the FVIIa concentrate, the lyophilized product was further heated at 65 degrees C for 96 h in a water bath. Total recovery of FVII from 15 000 l of plasma was approximately 40%, and the FVII preparation was fully converted to FVIIa with trace amounts of degraded products (FVIIabeta and FVIIagamma). The specific activity of the FVIIa was approximately 40 U/ micro g. Furthermore, virus-spiking tests demonstrated that immunoaffinity chromatography, nanofiltration and dry-heating effectively removed and inactivated the spiked viruses in the FVIIa. These results indicated that the FVIIa concentrate had both high specific activity and safety. We established a large-scale manufacturing process of human plasma-derived

  7. Accumulation and altered localization of telomere-associated protein TRF2 in immortally transformed and tumor-derived human breast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nijjar, Tarlochan; Bassett, Ekaterina; Garbe, James; Takenaka, Yasuhiro; Stampfer, Martha R.; Gilley, David; Yaswen, Paul

    2004-12-23

    We have used cultured human mammary epithelial cells (HMEC) and breast tumor-derived lines to gain information on defects that occur during breast cancer progression. HMEC immortalized by a variety of agents (the chemical carcinogen benzo(a)pyrene, oncogenes c-myc and ZNF217, and/or dominant negative p53 genetic suppressor element GSE22) displayed marked up regulation (10-15 fold) of the telomere binding protein, TRF2. Up-regulation of TRF2 protein was apparently due to differences in post-transcriptional regulation, as mRNA levels remained comparable in finite life span and immortal HMEC. TRF2 protein was not up-regulated by the oncogenic agents alone in the absence of immortalization, nor by expression of exogenously introduced hTERT genes. We found TRF2 levels to be at least 2-fold higher than in control cells in 11/15 breast tumor cell lines, suggesting that elevated TRF2 levels are a frequent occurrence during the transformation of breast tumor cells in vivo. The dispersed distribution of TRF2 throughout the nuclei in some immortalized and tumor-derived cells indicated that not all the TRF2 was associated with telomeres in these cells. The process responsible for accumulation of TRF2 in immortalized HMEC and breast tumor-derived cell lines may promote tumorigenesis by contributing to the cells ability to maintain an indefinite life span.

  8. The Use of Recombinant Human Platelet-Derived Growth Factor for Maxillary Sinus Augmentation.

    Science.gov (United States)

    Kubota, Atsushi; Sarmiento, Hector; Alqahtani, Mohammed Saad; Llobell, Arturo; Fiorellini, Joseph P

    The maxillary sinus augmentation procedure has become a predictable treatment to regenerate bone for implant placement. The purpose of this study was to evaluate the effect of recombinant human platelet-derived growth factor BB (rhPDGF-BB) combined with a deproteinized cancellous bovine bone graft for sinus augmentation. The lateral window approach was used for maxillary sinuses with minimal residual bone. After a healing period of 4 months, dental implants were placed and then restored following a 2-month osseointegration period. The result demonstrated increased bone height and ISQ values and a 100% survival rate. This study indicates that the addition of rhPDGF-BB to deproteinized cancellous bovine bone accelerated the healing period in maxillary sinuses with minimal native bone.

  9. Generation of a Motor Nerve Organoid with Human Stem Cell-Derived Neurons

    Directory of Open Access Journals (Sweden)

    Jiro Kawada

    2017-11-01

    Full Text Available During development, axons spontaneously assemble into a fascicle to form nerves and tracts in the nervous system as they extend within a spatially constrained path. However, understanding of the axonal fascicle has been hampered by lack of an in vitro model system. Here, we report generation of a nerve organoid composed of a robust fascicle of axons extended from a spheroid of human stem cell-derived motor neurons within our custom-designed microdevice. The device is equipped with a narrow channel providing a microenvironment that facilitates the growing axons to spontaneously assemble into a unidirectional fascicle. The fascicle was specifically made with axons. We found that it was electrically active and elastic and could serve as a model to evaluate degeneration of axons in vitro. This nerve organoid model should facilitate future studies on the development of the axonal fascicle and drug screening for diseases affecting axon fascicles.

  10. Human adipose-derived stromal/stem cell isolation, culture, and osteogenic differentiation.

    Science.gov (United States)

    Qureshi, Ammar T; Chen, Cong; Shah, Forum; Thomas-Porch, Caasy; Gimble, Jeffrey M; Hayes, Daniel J

    2014-01-01

    Annually, more than 200,000 elective liposuction procedures are performed in the United States and over a million worldwide. The ease of harvest and abundance make human adipose-derived stromal/stem cells (hASCs) isolated from lipoaspirates an attractive, readily available source of adult stem cells that have become increasingly popular for use in many studies. Here, we describe common methods for hASC culture, preservation, and osteogenic differentiation. We introduce methods of ceramic, polymer, and composite scaffold synthesis with a description of morphological, chemical, and mechanical characterization techniques. Techniques for scaffold loading are compared, and methods for determining cell loading efficiency and proliferation are described. Finally, we provide both qualitative and quantitative techniques for in vitro assessment of hASC osteogenic differentiation. © 2014 Elsevier Inc. All rights reserved.

  11. Atomic force microscope observation of branching in single transcript molecules derived from human cardiac muscle

    International Nuclear Information System (INIS)

    Reed, Jason; Hsueh, Carlin; Gimzewski, James K; Mishra, Bud

    2008-01-01

    We have used an atomic force microscope to examine a clinically derived sample of single-molecule gene transcripts, in the form of double-stranded cDNA, (c: complementary) obtained from human cardiac muscle without the use of polymerase chain reaction (PCR) amplification. We observed a log-normal distribution of transcript sizes, with most molecules being in the range of 0.4-7.0 kilobase pairs (kb) or 130-2300 nm in contour length, in accordance with the expected distribution of mRNA (m: messenger) sizes in mammalian cells. We observed novel branching structures not previously known to exist in cDNA, and which could have profound negative effects on traditional analysis of cDNA samples through cloning, PCR and DNA sequencing

  12. Taurine Promotes the Cartilaginous Differentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Vitro.

    Science.gov (United States)

    Yao, Xiuhua; Huang, Huiling; Li, Zhou; Liu, Xiaohua; Fan, Weijia; Wang, Xinping; Sun, Xuelian; Zhu, Jianmin; Zhou, Hongrui; Wei, Huaying

    2017-08-01

    Taurine has been reported to influence osteogenic differentiation, but the role of taurine on cartilaginous differentiation using human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) remains unclear. In this study, we investigated the effect of taurine (0, 1, 5 and 10 mM) on the proliferation and chondrogenesis of hUC-MSCs by analyzing cell proliferation, accumulation of glycosaminoglycans and expression of cartilage specific mRNA. The results show though taurine did not affected the proliferation of hUC-MSCs, 5 mM of taurine is sufficient to enhanced the accumulation of glycosaminoglycans and up-regulate cartilage specific mRNA expression, namely collagen type II, aggrecan and SOX9. Taurine also inhibits chondrocyte dedifferentiation by reducing expression of collagen type I mRNA. Taken together, our study reveals that taurine promotes and maintains the chondrogenesis of hUC-MSCs.

  13. DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

    International Nuclear Information System (INIS)

    Yaki, T.

    1982-01-01

    DNA repair in mouse cells derived from embryos of 3 inbred strains were investigated in comparison with that in human cells. The levels of unscheduled DNA synthesis after UV irradiation appeared to change at different passages, but capacities of host-cell reactivation of UV-irradiated herpes simplex virus were always reduced to the same levels as those in xeroderma pigmentosum cells. This implied that mouse cells are reduced in excision-repair capacities and that the apparently high levels of unscheduled DNA synthesis at certain passages are not quantitatively related to high levels of cell survival. Essentially no differences in DNA repair were noted among 3 strains - BALB/c, C3H/He and C57BL/10. (orig.)

  14. Fibroblast growth factor-2 stimulates adipogenic differentiation of human adipose-derived stem cells

    International Nuclear Information System (INIS)

    Kakudo, Natsuko; Shimotsuma, Ayuko; Kusumoto, Kenji

    2007-01-01

    Adipose-derived stem cells (ASCs) have demonstrated a capacity for differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and factors. It is acknowledged that fibroblast growth factor-2 (FGF-2) promotes chondrogenic and inhibits osteogenic differentiation of ASCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate at the cellular and molecular levels the effect of FGF-2 on adipogenic differentiation of ASCs, as induced by an adipogenic hormonal cocktail consisting of 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and indomethacin. FGF-2 significantly enhances the adipogenic differentiation of human ASCs. Furthermore, in cultures receiving FGF-2 before adipogenic induction, mRNA expression of peroxisome proliferator-activated receptor γ2 (PPARγ2), a key transcription factor in adipogenesis, was upregulated. The results of FGF-2 supplementation suggest the potential applications of FGF-2 and ASCs in adipose tissue regeneration

  15. Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation

    Directory of Open Access Journals (Sweden)

    Adila A Hamid

    2012-01-01

    Full Text Available OBJECTIVES: Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction. MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction. RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction. CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adiposederived stem cells was most prominent after one week of chondrogenic induction.

  16. A Highly Efficient Human Pluripotent Stem Cell Microglia Model Displays a Neuronal-Co-culture-Specific Expression Profile and Inflammatory Response

    Directory of Open Access Journals (Sweden)

    Walther Haenseler

    2017-06-01

    Full Text Available Microglia are increasingly implicated in brain pathology, particularly neurodegenerative disease, with many genes implicated in Alzheimer's, Parkinson's, and motor neuron disease expressed in microglia. There is, therefore, a need for authentic, efficient in vitro models to study human microglial pathological mechanisms. Microglia originate from the yolk sac as MYB-independent macrophages, migrating into the developing brain to complete differentiation. Here, we recapitulate microglial ontogeny by highly efficient differentiation of embryonic MYB-independent iPSC-derived macrophages then co-culture them with iPSC-derived cortical neurons. Co-cultures retain neuronal maturity and functionality for many weeks. Co-culture microglia express key microglia-specific markers and neurodegenerative disease-relevant genes, develop highly dynamic ramifications, and are phagocytic. Upon activation they become more ameboid, releasing multiple microglia-relevant cytokines. Importantly, co-culture microglia downregulate pathogen-response pathways, upregulate homeostatic function pathways, and promote a more anti-inflammatory and pro-remodeling cytokine response than corresponding monocultures, demonstrating that co-cultures are preferable for modeling authentic microglial physiology.

  17. Generation of glucose-responsive, insulin-producing cells from human umbilical cord blood-derived mesenchymal stem cells.

    Science.gov (United States)

    Prabakar, Kamalaveni R; Domínguez-Bendala, Juan; Molano, R Damaris; Pileggi, Antonello; Villate, Susana; Ricordi, Camillo; Inverardi, Luca

    2012-01-01

    We sought to assess the potential of human cord blood-derived mesenchymal stem cells (CB-MSCs) to derive insulin-producing, glucose-responsive cells. We show here that differentiation protocols based on stepwise culture conditions initially described for human embryonic stem cells (hESCs) lead to differentiation of cord blood-derived precursors towards a pancreatic endocrine phenotype, as assessed by marker expression and in vitro glucose-regulated insulin secretion. Transplantation of these cells in immune-deficient animals shows human C-peptide production in response to a glucose challenge. These data suggest that human cord blood may be a promising source for regenerative medicine approaches for the treatment of diabetes mellitus.

  18. Human cardiac-derived adherent proliferating cells reduce murine acute Coxsackievirus B3-induced myocarditis.

    Directory of Open Access Journals (Sweden)

    Kapka Miteva

    Full Text Available BACKGROUND: Under conventional heart failure therapy, inflammatory cardiomyopathy typically has a progressive course, indicating a need for alternative therapeutic strategies to improve long-term outcomes. We recently isolated and identified novel cardiac-derived cells from human cardiac biopsies: cardiac-derived adherent proliferating cells (CAPs. They have similarities with mesenchymal stromal cells, which are known for their anti-apoptotic and immunomodulatory properties. We explored whether CAPs application could be a novel strategy to improve acute Coxsackievirus B3 (CVB3-induced myocarditis. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the safety of our approach, we first analyzed the expression of the coxsackie- and adenovirus receptor (CAR and the co-receptor CD55 on CAPs, which are both required for effective CVB3 infectivity. We could demonstrate that CAPs only minimally express both receptors, which translates to minimal CVB3 copy numbers, and without viral particle release after CVB3 infection. Co-culture of CAPs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis and viral progeny release. In addition, CAPs reduced CD4 and CD8 T cell proliferation. All CAPs-mediated protective effects were nitric oxide- and interleukin-10-dependent and required interferon-γ. In an acute murine model of CVB3-induced myocarditis, application of CAPs led to a decrease of cardiac apoptosis, cardiac CVB3 viral load and improved left ventricular contractility parameters. This was associated with a decline in cardiac mononuclear cell activity, an increase in T regulatory cells and T cell apoptosis, and an increase in left ventricular interleukin-10 and interferon-γ mRNA expression. CONCLUSIONS: We conclude that CAPs are a unique type of cardiac-derived cells and promising tools to improve acute CVB3-induced myocarditis.

  19. Human-derived probiotic Lactobacillus reuteri strains differentially reduce intestinal inflammation.

    Science.gov (United States)

    Liu, Yuying; Fatheree, Nicole Y; Mangalat, Nisha; Rhoads, Jon Marc

    2010-11-01

    Lactobacillus reuteri (L. reuteri) is a probiotic that inhibits the severity of enteric infections and modulates the immune system. Human-derived L. reuteri strains DSM17938, ATCC PTA4659, ATCC PTA 5289, and ATCC PTA 6475 have demonstrated strain-specific immunomodulation in cultured monocytoid cells, but information about how these strains affect inflammation in intestinal epithelium is limited. We determined the effects of the four different L. reuteri strains on lipopolysaccharide (LPS)-induced inflammation in small intestinal epithelial cells and in the ileum of newborn rats. IPEC-J2 cells (derived from the jejunal epithelium of a neonatal piglet) and IEC-6 cells (derived from the rat crypt) were treated with L. reuteri. Newborn rat pups were gavaged cow milk formula supplemented with L. reuteri strains in the presence or absence of LPS. Protein and mRNA levels of cytokines and histological changes were measured. We demonstrate that even though one L. reuteri strain (DSM 17938) did not inhibit LPS-induced IL-8 production in cultured intestinal cells, all strains significantly reduced intestinal mucosal levels of KC/GRO (∼IL-8) and IFN-γ when newborn rat pups were fed formula containing LPS ± L. reuteri. Intestinal histological damage produced by LPS plus cow milk formula was also significantly reduced by all four strains. Cow milk formula feeding (without LPS) produced mild gut inflammation, evidenced by elevated mucosal IFN-γ and IL-13 levels, a process that could be suppressed by strain 17938. Other cytokines and chemokines were variably affected by the different strains, and there was no toxic effect of L. reuteri on intestinal cells or mucosa. In conclusion, L. reuteri strains differentially modulate LPS-induced inflammation. Probiotic interactions with both epithelial and nonepithelial cells in vivo must be instrumental in modulating intrinsic anti-inflammatory effects in the intestine. We suggest that the terms anti- and proinflammatory be used only

  20. Human Urine-Derived Renal Progenitors for Personalized Modeling of Genetic Kidney Disorders.

    Science.gov (United States)

    Lazzeri, Elena; Ronconi, Elisa; Angelotti, Maria Lucia; Peired, Anna; Mazzinghi, Benedetta; Becherucci, Francesca; Conti, Sara; Sansavini, Giulia; Sisti, Alessandro; Ravaglia, Fiammetta; Lombardi, Duccio; Provenzano, Aldesia; Manonelles, Anna; Cruzado, Josep M; Giglio, Sabrina; Roperto, Rosa Maria; Materassi, Marco; Lasagni, Laura; Romagnani, Paola

    2015-08-01

    The critical role of genetic and epigenetic factors in the pathogenesis of kidney disorders is gradually becoming clear, and the need for disease models that recapitulate human kidney disorders in a personalized manner is paramount. In this study, we describe a method to select and amplify renal progenitor cultures from the urine of patients with kidney disorders. Urine-derived human renal progenitors exhibited phenotype and functional properties identical to those purified from kidney tissue, including the capacity to differentiate into tubular cells and podocytes, as demonstrated by confocal microscopy, Western blot analysis of podocyte-specific proteins, and scanning electron microscopy. Lineage tracing studies performed with conditional transgenic mice, in which podocytes are irreversibly tagged upon tamoxifen treatment (NPHS2.iCreER;mT/mG), that were subjected to doxorubicin nephropathy demonstrated that renal progenitors are the only urinary cell population that can be amplified in long-term culture. To validate the use of these cells for personalized modeling of kidney disorders, renal progenitors were obtained from (1) the urine of children with nephrotic syndrome and carrying potentially pathogenic mutations in genes encoding for podocyte proteins and (2) the urine of children without genetic alterations, as validated by next-generation sequencing. Renal progenitors obtained from patients carrying pathogenic mutations generated podocytes that exhibited an abnormal cytoskeleton structure and functional abnormalities compared with those obtained from patients with proteinuria but without genetic mutations. The results of this study demonstrate that urine-derived patient-specific renal progenitor cultures may be an innovative research tool for modeling of genetic kidney disorders. Copyright © 2015 by the American Society of Nephrology.

  1. The suitability of human adipose-derived stem cells for the engineering of ligament tissue.

    Science.gov (United States)

    Eagan, Michael J; Zuk, Patricia A; Zhao, Ke-Wei; Bluth, Benjamin E; Brinkmann, Elyse J; Wu, Benjamin M; McAllister, David R

    2012-10-01

    Rupture of the anterior cruciate ligament (ACL) is the one of the most common sports-related injuries. With its poor healing capacity, surgical reconstruction using either autografts or allografts is currently required to restore function. However, serious complications are associated with graft reconstructions and the number of such reconstructions has steadily risen over the years, necessitating the search for an alternative approach to ACL repair. Such an approach may likely be tissue engineering. Recent engineering approaches using ligament-derived fibroblasts have been promising, but the slow growth rate of such fibroblasts in vitro may limit their practical application. More promising results are being achieved using bone marrow mesenchymal stem cells (MSCs). The adipose-derived stem cell (ASC) is often proposed as an alternative choice to the MSC and, as such, may be a suitable stem cell for ligament engineering. However, the use of ASCs in ligament engineering still remains relatively unexplored. Therefore, in this study, the potential use of human ASCs in ligament tissue engineering was initially explored by examining their ability to express several ligament markers under growth factor treatment. ASC populations treated for up to 4 weeks with TGFβ1 or IGF1 did not show any significant and consistent upregulation in the expression of collagen types 1 and 3, tenascin C and scleraxis. While treatment with EGF or bFGF resulted in increased tenascin C expression, increased expression of collagens 1 and 3 were never observed. Therefore, simple in vitro treatment of human ASC populations with growth factors may not stimulate their ligament differentiative potential. Copyright © 2011 John Wiley & Sons, Ltd.

  2. Human fibrocyte-derived exosomes accelerate wound healing in genetically diabetic mice

    Energy Technology Data Exchange (ETDEWEB)

    Geiger, Adolf, E-mail: ageiger@dreirosen-pharma.com; Walker, Audrey, E-mail: awalker@dreirosen-pharma.com; Nissen, Erwin, E-mail: enissen@dreirosen-pharma.com

    2015-11-13

    Diabetic ulcers represent a substantial societal and healthcare burden worldwide and scarcely respond to current treatment strategies. This study was addressed to evaluate the therapeutic potential of exosomes secreted by human circulating fibrocytes, a population of mesenchymal progenitors involved in normal wound healing via paracrine signaling. The exosomes released from cells sequentially stimulated with platelet-derived growth factor-BB and transforming growth factor-β1, in the presence of fibroblast growth factor 2, did not show potential immunogenicity. These exosomes exhibited in-vitro proangiogenic properties, activated diabetic dermal fibroblasts, induced the migration and proliferation of diabetic keratinocytes, and accelerated wound closure in diabetic mice in vivo. Important components of the exosomal cargo were heat shock protein-90α, total and activated signal transducer and activator of transcription 3, proangiogenic (miR-126, miR-130a, miR-132) and anti-inflammatory (miR124a, miR-125b) microRNAs, and a microRNA regulating collagen deposition (miR-21). This proof-of-concept study demonstrates the feasibility of the use of fibrocytes-derived exosomes for the treatment of diabetic ulcers. - Highlights: • Fibrocytes have shown potent wound healing properties in vitro and in vivo. • Their clinical use is precluded by low numbers and antigen-presenting function. • We isolated exosomes with no immunogenicity potential from human fibrocytes. • Their cargo included microRNAs and proteins that are known healing promoters. • They accelerated wound closure in diabetic mice in a dose-dependent manner.

  3. Cyclosporin A promotes mineralization by human cementoblastoma-derived cells in culture.

    Science.gov (United States)

    Arzate, Higinio; Alvarez, Marco A; Narayanan, A Sampath

    2005-06-01

    The immunosuppressive drug cyclosporin A has been shown to induce cementum deposition in vivo in experimental animals. Using cementoblastoma-derived cells, we have studied whether this drug will be useful to study cementum mineralization and differentiation in vitro. Human cementoblastoma cells and gingival fibroblasts (controls) were cultured and treated with 0.5, 1.0 and 5.0 microg/ml of cyclosporin A. Cell proliferation was evaluated by MTT (tetrazolium) assay and cell number, and cell viability was assessed by trypan blue dye exclusion. Induction of mineralization was evaluated by alizarin red S staining to detect mineralized nodules and by reverse transcription-polymerase chain reaction (RT-PCR) to assess the expression of bone differentiation markers alkaline phosphatase, osteocalcin, bone sialoprotein and core-binding factor a1 (Cbfa1). Cyclosporin A at 5.0 microg/ml concentration reduced significantly the increase in the number of cementoblastoma cells. A dose-dependent increase in the number of mineralized nodules occurred in cultures of cementoblastoma-derived cells treated with cyclosporin A, and RT-PCR analyses showed significantly higher levels of expression of alkaline phosphatase, bone sialoprotein, type I collagen, matrix metalloproteinase-1, osteocalcin, osteopontin, and Cbfa1. Human gingival fibroblast proliferation and cell number were not affected. Mineralized nodules were not detected in gingival fibroblasts and bone specific proteins were not expressed. Presence of cyclosporin A during 14-day culture period appears to suppress the proliferation of cementoblastoma cells and induce the formation mineralized-like tissue by these cells.

  4. Cell surface glycopeptides from human intestinal epithelial cell lines derived from normal colon and colon adenocarcinomas

    International Nuclear Information System (INIS)

    Youakim, A.; Herscovics, A.

    1985-01-01

    The cell surface glycopeptides from an epithelial cell line (CCL 239) derived from normal human colon were compared with those from three cell lines (HCT-8R, HCT-15, and CaCo-2) derived independently from human colonic adenocarcinomas. Cells were incubated with D-[2- 3 H]mannose or L-[5,6- 3 H]fucose for 24 h and treated with trypsin to release cell surface components which were then digested exhaustively with Pronase and fractionated on Bio-Gel P-6 before and after treatment with endo-beta-N-acetylglucosaminidase H. The most noticeable difference between the labeled glycopeptides from the tumor and CCL 239 cells was the presence in the former of an endo-beta-N-acetylglucosaminidase H-resistant high molecular weight glycopeptide fraction which was eluted in the void volume of Bio-Gel P-6. This fraction was obtained with both labeled mannose and fucose as precursors. However, acid hydrolysis of this fraction obtained after incubation with [2- 3 H]mannose revealed that as much as 60-90% of the radioactivity was recovered as fucose. Analysis of the total glycopeptides (cell surface and cell pellet) obtained after incubation with [2- 3 H]mannose showed that from 40-45% of the radioactivity in the tumor cells and less than 10% of the radioactivity in the CCL 239 cells was recovered as fucose. After incubation of the HCT-8R cells with D-[1,6- 3 H]glucosamine and L-[1- 14 C]fucose, strong acid hydrolysis of the labeled glycopeptide fraction excluded from Bio-Gel P-6 produced 3 H-labeled N-acetylglucosamine and N-acetylgalactosamine

  5. Suppression of human monocyte tissue factor induction by red wine phenolics and synthetic derivatives of resveratrol.

    Science.gov (United States)

    Kaur, Gurjeet; Roberti, Marinella; Raul, Francis; Pendurthi, Usha R

    2007-01-01

    Prevention of cardiovascular disease through nutritional supplements is growing in popularity throughout the world. Multiple epidemiologic studies found that moderate consumption of alcohol, particularly red wine, lowers mortality rates from coronary heart diseases (CHD). Chronic inflammation and atherosclerosis associated with CHD culminate in aberrant intravascular expression of tissue factor (TF), which triggers blood coagulation leading to thrombosis, a major cause for heart attack. We showed earlier that two red wine phenolics, resveratrol and quercetin, suppressed TF induction in endothelial cells. In the present study, we investigated efficacy of seven resveratrol derivatives, which were shown to be effective in regulating cancer cell growth in vitro at much lower concentrations than the parent compound resveratrol, in inhibiting TF induction in peripheral blood mononuclear cells (PBMCs). We also tested possible synergistic effects of resveratrol and quercetin with the other major red wine phenolics in suppression of lipopolysaccharide-induced TF expression in human PBMCs. We found that several resveratrol derivatives were 2- to 10-fold more efficient than resveratrol in inhibiting TF induction. Our study found no evidence for synergism among red wine polyphenolics. These data suggest that structural alterations of resveratrol can be effective in producing potent antithrombotic agents that will have therapeutic potential in the improvement of cardiovascular health and prevention of CHD. Among major red wine phenolics, quercetin appears to be the predominant suppressor of TF induction.

  6. Suppression of human monocyte tissue factor induction by red wine phenolics and synthetic derivatives of resveratrol

    Science.gov (United States)

    Kaur, Gurjeet; Roberti, Marinella; Raul, Francis; Pendurthi, Usha R.

    2010-01-01

    Prevention of cardiovascular disease through nutritional supplements is growing in popularity throughout the world. Multiple epidemiologic studies found that moderate consumption of alcohol, particularly red wine, lowers mortality rates from coronary heart diseases (CHD). Chronic inflammation and atherosclerosis associated with CHD culminate in aberrant intravascular expression of tissue factor (TF), which triggers blood coagulation leading to thrombosis, a major cause for heart attack. We showed earlier that two red wine phenolics, resveratrol and quercetin, suppressed TF induction in endothelial cells. In the present study, we investigated efficacy of seven resveratrol derivatives, which were shown to be effective in regulating cancer cell growth in vitro at much lower concentrations than the parent compound resveratrol, in inhibiting TF induction in peripheral blood mononuclear cells (PBMCs). We also tested possible synergistic effects of resveratrol and quercetin with the other major red wine phenolics in suppression of lipopolysaccharide-induced TF expression in human PBMCs. We found that several resveratrol derivatives were 2- to 10-fold more efficient than resveratrol in inhibiting TF induction. Our study found no evidence for synergism among red wine polyphenolics. These data suggest that structural alterations of resveratrol can be effective in producing potent antithrombotic agents that will have therapeutic potential in the improvement of cardiovascular health and prevention of CHD. Among major red wine phenolics, quercetin appears to be the predominant suppressor of TF induction. PMID:16507316

  7. Transplantation of Human Chorion-Derived Cholinergic Progenitor Cells: a Novel Treatment for Neurological Disorders.

    Science.gov (United States)

    Mohammadi, Alireza; Maleki-Jamshid, Ali; Sanooghi, Davood; Milan, Peiman Brouki; Rahmani, Arash; Sefat, Farshid; Shahpasand, Koorosh; Soleimani, Mansoureh; Bakhtiari, Mehrdad; Belali, Rafie; Faghihi, Faezeh; Joghataei, Mohammad Taghi; Perry, George; Mozafari, Masoud

    2018-03-16

    A neurological disorder is any disorder or abnormality in the nervous system. Among different neurological disorders, Alzheimer's disease (AD) is recognized as the sixth leading cause of death globally. Considerable research has been conducted to find pioneer treatments for this devastating disorder among which cell therapy has attracted remarkable attentions over the last decade. Up to now, targeted differentiation into specific desirable cell types has remained a major obstacle to clinical application of cell therapy. Also, potential risks including uncontrolled growth of stem cells could be disastrous. In our novel protocol, we used basal forebrain cholinergic progenitor cells (BFCN) derived from human chorion-derived mesenchymal stem cells (hC-MSCs) which made it possible to obtain high-quality population of cholinergic neurons and in vivo in much shorter time period than previous established methods. Remarkably, the transplanted progenitors fully differentiated to cholinergic neurons which in turn integrated in higher cortical networks of host brains, resulting in significant improvement in cognitive assessments. This method may have profound implications in cell therapies for any other neurodegenerative disorders. Graphical Abstract ᅟ.

  8. Derivation of Skeletal Myogenic Precursors from Human Pluripotent Stem Cells Using Conditional Expression of PAX7.

    Science.gov (United States)

    Darabi, Radbod; Perlingeiro, Rita C R

    2016-01-01

    Cell-based therapies are considered as one of the most promising approaches for the treatment of degenerating pathologies including muscle disorders and dystrophies. Advances in the approach of reprogramming somatic cells into induced pluripotent stem (iPS) cells allow for the possibility of using the patient's own pluripotent cells to generate specific tissues for autologous transplantation. In addition, patient-specific tissue derivatives have been shown to represent valuable material for disease modeling and drug discovery. Nevertheless, directed differentiation of pluripotent stem cells into a specific lineage is not a trivial task especially in the case of skeletal myogenesis, which is generally poorly recapitulated during the in vitro differentiation of pluripotent stem cells.Here, we describe a practical and efficient method for the derivation of skeletal myogenic precursors from differentiating human pluripotent stem cells using controlled expression of PAX7. Flow cytometry (FACS) purified myogenic precursors can be expanded exponentially and differentiated in vitro into myotubes, enabling researchers to use these cells for disease modeling as well as therapeutic purposes.

  9. Human umbilical cord blood-derived f-macrophages retain pluripotentiality after thrombopoietin expansion

    International Nuclear Information System (INIS)

    Zhao Yong; Mazzone, Theodore

    2005-01-01

    We have previously characterized a new type of stem cell from human peripheral blood, termed fibroblast-like macrophage (f-MΦ). Here, using umbilical cord blood as a source, we identified cells with similar characteristics including expression of surface markers (CD14, CD34, CD45, CD117, and CD163), phagocytosis, and proliferative capacity. Further, thrombopoietin (TPO) significantly stimulated the proliferation of cord blood-derived f-MΦ (CB f-MΦ) at low dosage without inducing a megakaryocytic phenotype. Additional experiments demonstrated that TPO-expanded cord blood-derived f-MΦ (TCB f-MΦ) retained their surface markers and differentiation ability. Treatment with vascular endothelial cell growth factor (VEGF) gave rise to endothelial-like cells, expressing Flt-1, Flk-1, von Willebrand Factor (vWF), CD31, acetylated low density lipoprotein internalization, and the ability to form endothelial-like cell chains. In the presence of lipopolyssacharide (LPS) and 25 mM glucose, the TCB f-MΦ differentiated to express insulin mRNA, C-peptide, and insulin. In vitro functional analysis demonstrated that these insulin-positive cells could release insulin in response to glucose and other secretagogues. These findings demonstrate a potential use of CB f-MΦ and may lead to develop new therapeutic strategy for treating dominant disease

  10. Human iPSC-derived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening.

    Science.gov (United States)

    Smith, Alec S T; Macadangdang, Jesse; Leung, Winnie; Laflamme, Michael A; Kim, Deok-Ho

    Improved methodologies for modeling cardiac disease phenotypes and accurately screening the efficacy and toxicity of potential therapeutic compounds are actively being sought to advance drug development and improve disease modeling capabilities. To that end, much recent effort has been devoted to the development of novel engineered biomimetic cardiac tissue platforms that accurately recapitulate the structure and function of the human myocardium. Within the field of cardiac engineering, induced pluripotent stem cells (iPSCs) are an exciting tool that offer the potential to advance the current state of the art, as they are derived from somatic cells, enabling the development of personalized medical strategies and patient specific disease models. Here we review different aspects of iPSC-based cardiac engineering technologies. We highlight methods for producing iPSC-derived cardiomyocytes (iPSC-CMs) and discuss their application to compound efficacy/toxicity screening and in vitro modeling of prevalent cardiac diseases. Special attention is paid to the application of micro- and nano-engineering techniques for the development of novel iPSC-CM based platforms and their potential to advance current preclinical screening modalities. Published by Elsevier Inc.

  11. Design, Synthesis and Evaluation of N13-Substituted Evodiamine Derivatives against Human Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Senchuan Song

    2013-12-01

    Full Text Available Attempting to improve the anticancer activity and solubility of evodiamine in simulated gastric fluid (SGF and simulated intestinal fluid (SIF solutions, thirty-eight N13-substituted evodiamine derivatives were designed, synthesized and tested for antitumor activities against six kinds of human cancer cell lines, namely prostate cancer (DU-145 and PC-3, lung cancer (H460, breast cancer (MCF-7, colon cancer (HCT-5 and glioblastoma (SF-268. The solubility of these compounds in SGF and SIF solutions was evaluated, and apoptosis induced by 2-2, 2-3, 2-16 and 3-2 was determined. The results showed: (1 among all compounds examined, 2-16 showed the highest antitumor activity and a broader spectrum of activity, with IC50 values ranging from 1–2 µM; (2 their solubility was obviously improved; (3 2-3, 2-16 and 3-2 had a significant impact inducing apoptosis in some cancer cell lines. The preliminary structure-activity relationships of these derivatives were discussed.

  12. Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Solmi, Rossella [Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna (Italy); Montroni, Isacco [Dipartimento Emergenza/Urgenza, Chirurgia Generale e dei Trapianti, Università di Bologna, Bologna (Italy); Mattei, Gabriella [Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna (Italy); Taffurelli, Mario [Dipartimento Emergenza/Urgenza, Chirurgia Generale e dei Trapianti, Università di Bologna, Bologna (Italy); Santini, Donatella [Dipartimento di Patologia, Università di Bologna, Bologna (Italy); Pezzetti, Furio [Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna (Italy); Ruggeri, Alessandro [Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell' Apparato Locomotore, Università di Bologna, Bologna (Italy); Castellani, Gastone [Centro Interdipartimentale L. Galvani, Università di Bologna, Bologna (Italy); DIMORFIPA, Università di Bologna, Bologna (Italy); Guidotti, Lia [Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna (Italy); Coppola, Domenico [H. Lee Moffit Cancer Center and Research Institute, University of South Florida, Tampa, FL (United States); Strippoli, Pierluigi; Lauriola, Mattia [Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna (Italy); Francesconi, Mirko [Centro Interdipartimentale L. Galvani, Università di Bologna, Bologna (Italy); DIMORFIPA, Università di Bologna, Bologna (Italy); Martini, Désirée [Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell' Apparato Locomotore, Università di Bologna, Bologna (Italy); Voltattorni, Manuela [Laboratori di Biotecnologie, Via Beverara 123, Bologna (Italy); Ceccarelli, Claudio [Dipartimento di Patologia, Università di Bologna, Bologna (Italy); Ugolini, Giampaolo; Rosati, Giancarlo; Zanotti, Simone [Dipartimento Emergenza/Urgenza, Chirurgia Generale e dei Trapianti, Università di Bologna, Bologna (Italy)

    2008-08-08

    EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF. Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A. Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases. This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination

  13. Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines

    Directory of Open Access Journals (Sweden)

    Pezzetti Furio

    2008-08-01

    Full Text Available Abstract Background EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF. Methods Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A. Results Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2, possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases. Conclusion This is the first study to have systematically investigated

  14. Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines

    International Nuclear Information System (INIS)

    Solmi, Rossella; Montroni, Isacco; Mattei, Gabriella; Taffurelli, Mario; Santini, Donatella; Pezzetti, Furio; Ruggeri, Alessandro; Castellani, Gastone; Guidotti, Lia; Coppola, Domenico; Strippoli, Pierluigi; Lauriola, Mattia; Francesconi, Mirko; Martini, Désirée; Voltattorni, Manuela; Ceccarelli, Claudio; Ugolini, Giampaolo; Rosati, Giancarlo; Zanotti, Simone

    2008-01-01

    EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF. Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A. Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases. This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination

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

    Directory of Open Access Journals (Sweden)

    Tetsuya Ishii

    2014-10-01

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

  16. Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Contribute to Chondrogenesis in Coculture with Chondrocytes.

    Science.gov (United States)

    Li, Xingfu; Duan, Li; Liang, Yujie; Zhu, Weimin; Xiong, Jianyi; Wang, Daping

    2016-01-01

    Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have been shown as the most potential stem cell source for articular cartilage repair. In this study, we aimed to develop a method for long-term coculture of human articular chondrocytes (hACs) and hUCB-MSCs at low density in vitro to determine if the low density of hACs could enhance the hUCB-MSC chondrogenic differentiation as well as to determine the optimal ratio of the two cell types. Also, we compared the difference between direct coculture and indirect coculture at low density. Monolayer cultures of hUCB-MSCs and hACs were investigated at different ratios, at direct cell-cell contact groups for 21 days. Compared to direct coculture, hUCB-MSCs and hACs indirect contact culture significantly increased type II collagen (COL2) and decreased type I collagen (COL1) protein expression levels. SRY-box 9 (SOX9) mRNA levels and protein expression were highest in indirect coculture. Overall, these results indicate that low density direct coculture induces fibrocartilage. However, indirect coculture in conditioned chondrocyte cell culture medium can increase expression of chondrogenic markers and induce hUCB-MSCs differentiation into mature chondrocytes. This work demonstrates that it is possible to promote chondrogenesis of hUCB-MSCs in combination with hACs, further supporting the concept of novel coculture strategies for tissue engineering.

  17. Automated grouping of action potentials of human embryonic stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Gorospe, Giann; Zhu, Renjun; Millrod, Michal A; Zambidis, Elias T; Tung, Leslie; Vidal, Rene

    2014-09-01

    Methods for obtaining cardiomyocytes from human embryonic stem cells (hESCs) are improving at a significant rate. However, the characterization of these cardiomyocytes (CMs) is evolving at a relatively slower rate. In particular, there is still uncertainty in classifying the phenotype (ventricular-like, atrial-like, nodal-like, etc.) of an hESC-derived cardiomyocyte (hESC-CM). While previous studies identified the phenotype of a CM based on electrophysiological features of its action potential, the criteria for classification were typically subjective and differed across studies. In this paper, we use techniques from signal processing and machine learning to develop an automated approach to discriminate the electrophysiological differences between hESC-CMs. Specifically, we propose a spectral grouping-based algorithm to separate a population of CMs into distinct groups based on the similarity of their action potential shapes. We applied this method to a dataset of optical maps of cardiac cell clusters dissected from human embryoid bodies. While some of the nine cell clusters in the dataset are presented with just one phenotype, the majority of the cell clusters are presented with multiple phenotypes. The proposed algorithm is generally applicable to other action potential datasets and could prove useful in investigating the purification of specific types of CMs from an electrophysiological perspective.

  18. Predicting human splicing branchpoints by combining sequence-derived features and multi-label learning methods.

    Science.gov (United States)

    Zhang, Wen; Zhu, Xiaopeng; Fu, Yu; Tsuji, Junko; Weng, Zhiping

    2017-12-01

    Alternative splicing is the critical process in a single gene coding, which removes introns and joins exons, and splicing branchpoints are indicators for the alternative splicing. Wet experiments have identified a great number of human splicing branchpoints, but many branchpoints are still unknown. In order to guide wet experiments, we develop computational methods to predict human splicing branchpoints. Considering the fact that an intron may have multiple branchpoints, we transform the branchpoint prediction as the multi-label learning problem, and attempt to predict branchpoint sites from intron sequences. First, we investigate a variety of intron sequence-derived features, such as sparse profile, dinucleotide profile, position weight matrix profile, Markov motif profile and polypyrimidine tract profile. Second, we consider several multi-label learning methods: partial least squares regression, canonical correlation analysis and regularized canonical correlation analysis, and use them as the basic classification engines. Third, we propose two ensemble learning schemes which integrate different features and different classifiers to build ensemble learning systems for the branchpoint prediction. One is the genetic algorithm-based weighted average ensemble method; the other is the logistic regression-based ensemble method. In the computational experiments, two ensemble learning methods outperform benchmark branchpoint prediction methods, and can produce high-accuracy results on the benchmark dataset.

  19. ECM microenvironment unlocks brown adipogenic potential of adult human bone marrow-derived MSCs.

    Science.gov (United States)

    Lee, Michelle H; Goralczyk, Anna G; Kriszt, Rókus; Ang, Xiu Min; Badowski, Cedric; Li, Ying; Summers, Scott A; Toh, Sue-Anne; Yassin, M Shabeer; Shabbir, Asim; Sheppard, Allan; Raghunath, Michael

    2016-02-17

    Key to realizing the diagnostic and therapeutic potential of human brown/brite adipocytes is the identification of a renewable, easily accessible and safe tissue source of progenitor cells, and an efficacious in vitro differentiation protocol. We show that macromolecular crowding (MMC) facilitates brown adipocyte differentiation in adult human bone marrow mesenchymal stem cells (bmMSCs), as evidenced by substantially upregulating uncoupling protein 1 (UCP1) and uncoupled respiration. Moreover, MMC also induced 'browning' in bmMSC-derived white adipocytes. Mechanistically, MMC creates a 3D extracellular matrix architecture enshrouding maturing adipocytes in a collagen IV cocoon that is engaged by paxillin-positive focal adhesions also at the apical side of cells, without contact to the stiff support structure. This leads to an enhanced matrix-cell signaling, reflected by increased phosphorylation of ATF2, a key transcription factor in UCP1 regulation. Thus, tuning the dimensionality of the microenvironment in vitro can unlock a strong brown potential dormant in bone marrow.

  20. Hyperglycemia induces mixed M1/M2 cytokine profile in primary human monocyte-derived macrophages.

    Science.gov (United States)

    Moganti, Kondaiah; Li, Feng; Schmuttermaier, Christina; Riemann, Sarah; Klüter, Harald; Gratchev, Alexei; Harmsen, Martin C; Kzhyshkowska, Julia

    2017-10-01

    Hyperglycaemia is a key factor in diabetic pathology. Macrophages are essential regulators of inflammation which can be classified into two major vectors of polarisation: classically activated macrophages (M1) and alternatively activated macrophages (M2). Both types of macrophages play a role in diabetes, where M1 and M2-produced cytokines can have detrimental effects in development of diabetes-associated inflammation and diabetic vascular complications. However, the effect of hyperglycaemia on differentiation and programming of primary human macrophages was not systematically studied. We established a unique model to assess the influence of hyperglycaemia on M1 and M2 differentiation based on primary human monocyte-derived macrophages. The effects of hyperglycaemia on the gene expression and secretion of prototype M1 cytokines TNF-alpha and IL-1beta, and prototype M2 cytokines IL-1Ra and CCL18 were quantified by RT-PCR and ELISA. Hyperglycaemia stimulated production of TNF-alpha, IL-1beta and IL-1Ra during macrophage differentiation. The effect of hyperglycaemia on TNF-alpha was acute, while the stimulating effect on IL-1beta and IL-1Ra was constitutive. Expression of CCL18 was supressed in M2 macrophages by hyperglycaemia. However the secreted levels remained to be biologically significant. Our data indicate that hyperglycaemia itself, without additional metabolic factors induces mixed M1/M2 cytokine profile that can support of diabetes-associated inflammation and development of vascular complications. Copyright © 2016 Elsevier GmbH. All rights reserved.

  1. The transcriptome of Legionella pneumophila-infected human monocyte-derived macrophages.

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    Christopher T D Price

    Full Text Available Legionella pneumophila is an intracellular bacterial pathogen that invades and replicates within alveolar macrophages through injection of ∼ 300 effector proteins by its Dot/Icm type IV translocation apparatus. The bona fide F-box protein, AnkB, is a nutritional virulence effector that triggers macrophages to generate a surplus of amino acids, which is essential for intravacuolar proliferation. Therefore, the ankB mutant represents a novel genetic tool to determine the transcriptional response of human monocyte-derived macrophages (hMDMs to actively replicating L. pneumophila.Here, we utilized total human gene microarrays to determine the global transcriptional response of hMDMs to infection by wild type or the ankB mutant of L. pneumophila. The transcriptomes of hMDMs infected with either actively proliferating wild type or non-replicative ankB mutant bacteria were remarkably similar. The transcriptome of infected hMDMs was predominated by up-regulation of inflammatory pathways (IL-10 anti-inflammatory, interferon signaling and amphoterin signaling, anti-apoptosis, and down-regulation of protein synthesis pathways. In addition, L. pneumophila modulated diverse metabolic pathways, particularly those associated with bio-active lipid metabolism, and SLC amino acid transporters expression.Taken together, the hMDM transcriptional response to L. pneumophila is independent of intra-vacuolar replication of the bacteria and primarily involves modulation of the immune response and metabolic as well as nutritional pathways.

  2. Endocrine gland derived-VEGF is down-regulated in human pituitary adenoma.

    Science.gov (United States)

    Raica, Marius; Coculescu, Mihail; Cimpean, Anca Maria; Ribatti, Domenico

    2010-10-01

    Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is an angiogenic molecule restricted to endocrine glands and, particularly, to steroid-secreting cells. The expression of EG-VEGF and its significance in human adenohypophysis in physiological and pathological conditions is still unknown. In this study, we investigated by immunohistochemistry the expression of EG-VEGF in 2 samples of normal adenohypophysis and 43 bioptic samples of pituitary adenoma. Moreover, the expression of growth hormone (GH), prolactin (PRL), follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH) and adrenocorticoprophic hormone (ACTH) were also estimated. The results of this study for the first time demonstrate a down-regulation of EG-VEGF expression in human pituitary adenoma as compared to normal adenohypophysis, suggesting an impaired function of the neoplastic cells in terms of hormone release in the blood stream, as a consequence of impaired tumor angiogenesis in the tumor. On the basis of our data showing a marked decrease in the expression of EG-VEGF in pituitary adenoma, with the exception of LH-secreting adenomas, we suggest that LH might be involved in the induction of EG-VEGF secretion.

  3. Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Relieve Acute Myocardial Ischemic Injury

    Directory of Open Access Journals (Sweden)

    Yuanyuan Zhao

    2015-01-01

    Full Text Available This study is aimed at investigating whether human umbilical cord mesenchymal stem cell- (hucMSC- derived exosomes (hucMSC-exosomes have a protective effect on acute myocardial infarction (AMI. Exosomes were characterized under transmission electron microscopy and the particles of exosomes were further examined through nanoparticle tracking analysis. Exosomes (400 μg protein were intravenously administrated immediately following ligation of the left anterior descending (LAD coronary artery in rats. Cardiac function was evaluated by echocardiography and apoptotic cells were counted using TUNEL staining. The cardiac fibrosis was assessed using Masson’s trichrome staining. The Ki67 positive cells in ischemic myocardium were determined using immunohistochemistry. The effect of hucMSC-exosomes on blood vessel formation was evaluated through tube formation and migration of human umbilical vein endothelial cells (EA.hy926 cells. The results indicated that ligation of the LAD coronary artery reduced cardiac function and induced cardiomyocyte apoptosis. Administration of hucMSC-exosomes significantly improved cardiac systolic function and reduced cardiac fibrosis. Moreover, hucMSC-exosomes protected myocardial cells from apoptosis and promoted the tube formation and migration of EA.hy926 cells. It is concluded that hucMSC-exosomes improved cardiac systolic function by protecting myocardial cells from apoptosis and promoting angiogenesis. These effects of hucMSC-exosomes might be associated with regulating the expression of Bcl-2 family.

  4. Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats.

    Science.gov (United States)

    Lund, Raymond D; Wang, Shaomei; Klimanskaya, Irina; Holmes, Toby; Ramos-Kelsey, Rebeca; Lu, Bin; Girman, Sergej; Bischoff, N; Sauvé, Yves; Lanza, Robert

    2006-01-01

    Embryonic stem cells promise to provide a well-characterized and reproducible source of replacement tissue for human clinical studies. An early potential application of this technology is the use of retinal pigment epithelium (RPE) for the treatment of retinal degenerative diseases such as macular degeneration. Here we show the reproducible generation of RPE (67 passageable cultures established from 18 different hES cell lines); batches of RPE derived from NIH-approved hES cells (H9) were tested and shown capable of extensive photoreceptor rescue in an animal model of retinal disease, the Royal College of Surgeons (RCS) rat, in which photoreceptor loss is caused by a defect in the adjacent retinal pigment epithelium. Improvement in visual performance was 100% over untreated controls (spatial acuity was approximately 70% that of normal nondystrophic rats) without evidence of untoward pathology. The use of somatic cell nuclear transfer (SCNT) and/or the creation of banks of reduced complexity human leucocyte antigen (HLA) hES-RPE lines could minimize or eliminate the need for immunosuppressive drugs and/or immunomodulatory protocols.

  5. Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Contribute to Chondrogenesis in Coculture with Chondrocytes

    Directory of Open Access Journals (Sweden)

    Xingfu Li

    2016-01-01

    Full Text Available Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs have been shown as the most potential stem cell source for articular cartilage repair. In this study, we aimed to develop a method for long-term coculture of human articular chondrocytes (hACs and hUCB-MSCs at low density in vitro to determine if the low density of hACs could enhance the hUCB-MSC chondrogenic differentiation as well as to determine the optimal ratio of the two cell types. Also, we compared the difference between direct coculture and indirect coculture at low density. Monolayer cultures of hUCB-MSCs and hACs were investigated at different ratios, at direct cell-cell contact groups for 21 days. Compared to direct coculture, hUCB-MSCs and hACs indirect contact culture significantly increased type II collagen (COL2 and decreased type I collagen (COL1 prot