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  1. Capture of cell culture-derived influenza virus by lectins: strain independent, but host cell dependent.

    Science.gov (United States)

    Opitz, Lars; Zimmermann, Anke; Lehmann, Sylvia; Genzel, Yvonne; Lübben, Holger; Reichl, Udo; Wolff, Michael W

    2008-12-01

    Strategies to control influenza outbreaks are focused mainly on prophylactic vaccination. Human influenza vaccines are trivalent blends of different virus subtypes. Therefore and due to frequent antigenic drifts, strain independent manufacturing processes are required for vaccine production. This study verifies the strain independency of a capture method based on Euonymus europaeus lectin-affinity chromatography (EEL-AC) for downstream processing of influenza viruses under various culture conditions propagated in MDCK cells. A comprehensive lectin binding screening was conducted for two influenza virus types from the season 2007/2008 (A/Wisconsin/67/2005, B/Malaysia/2506/2004) including a comparison of virus-lectin interaction by surface plasmon resonance technology. EEL-AC resulted in a reproducible high product recovery rate and a high degree of contaminant removal in the case of both MDCK cell-derived influenza virus types demonstrating clearly the general applicability of EEL-AC. In addition, host cell dependency of EEL-AC was studied with two industrial relevant cell lines: Vero and MDCK cells. However, the choice of the host cell lines is known to lead to different product glycosylation profiles. Hence, altered lectin specificities have been observed between the two cell lines, requiring process adaptations between different influenza vaccine production systems.

  2. Uniaxial cyclic strain enhances adipose-derived stem cell fusion with skeletal myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Jens Isak; Juhl, Morten; Nielsen, Thøger; Emmersen, Jeppe; Fink, Trine; Zachar, Vladimir; Pennisi, Cristian Pablo, E-mail: cpennisi@hst.aau.dk

    2014-07-25

    Highlights: • Uniaxial cyclic tensile strain (CTS) applied to ASCs alone or in coculture with myogenic precursors. • CTS promoted the formation of a highly ordered array of parallel ASCs. • Without biochemical supplements, CTS did not support advanced myogenic differentiation of ASCs. • Mechanical stimulation of cocultures boosted fusion of ASCs with skeletal myoblasts. - Abstract: Although adult muscle tissue possesses an exceptional capacity for regeneration, in the case of large defects, the restoration to original state is not possible. A well-known source for the de novo regeneration is the adipose-derived stem cells (ASCs), which can be readily isolated and have been shown to have a broad differentiation and regenerative potential. In this work, we employed uniaxial cyclic tensile strain (CTS), to mechanically stimulate human ASCs to participate in the formation skeletal myotubes in an in vitro model of myogenesis. The application of CTS for 48 h resulted in the formation of a highly ordered array of parallel ASCs, but failed to support skeletal muscle terminal differentiation. When the same stimulation paradigm was applied to cocultures with mouse skeletal muscle myoblasts, the percentage of ASCs contributing to the formation of myotubes significantly exceeded the levels reported in the literature hitherto. In perspective, the mechanical strain may be used to increase the efficiency of incorporation of ASCs in the skeletal muscles, which could be found useful in diverse traumatic or pathologic scenarios.

  3. Derivation of hybrid ES cell lines from two different strains of mice

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    Ho-Tak Lau

    2016-03-01

    Full Text Available Parental origin-dependent expression of the imprinted genes is essential for mammalian development. Zfp57 maintains genomic imprinting in mouse embryos and ES cells. To examine the allelic expression patterns of the imprinted genes in ES cells, we obtained multiple hybrid ES clones that were directly derived from the blastocysts generated from the cross between mice on two different genetic backgrounds. The blastocyst-derived ES clones displayed largely intact DNA methylation imprint at the tested imprinted regions. These hybrid ES clones will be useful for future studies to examine the allelic expression of the imprinted genes in ES cells and their differentiated progeny.

  4. Immune responses of mature chicken bone-marrow-derived dendritic cells infected with Newcastle disease virus strains with differing pathogenicity.

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    Xiang, Bin; Zhu, Wenxian; Li, Yaling; Gao, Pei; Liang, Jianpeng; Liu, Di; Ding, Chan; Liao, Ming; Kang, Yinfeng; Ren, Tao

    2018-06-01

    Infection of chickens with virulent Newcastle disease virus (NDV) is associated with severe pathology and increased morbidity and mortality. The innate immune response contributes to the pathogenicity of NDV. As professional antigen-presenting cells, dendritic cells (DCs) play a unique role in innate immunity. However, the contribution of DCs to NDV infection has not been investigated in chickens. In this study, we selected two representative NDV strains, i.e., the velogenic NDV strain Chicken/Guangdong/GM/2014 (GM) and the lentogenic NDV strain La Sota, to investigate whether NDVs could infect LPS-activated chicken bone-derived marrow DCs (mature chicken BM-DCs). We compared the viral titres and innate immune responses in mature chicken BM-DCs following infection with those strains. Both NDV strains could infect mature chicken BM-DC, but the GM strain showed stronger replication capacity than the La Sota strain in mature chicken BM-DCs. Gene expression profiling showed that MDA5, LGP2, TLR3, TLR7, IFN-α, IFN-β, IFN-γ, IL-1β, IL-6, IL-18, IL-8, CCL5, IL-10, IL-12, MHC-I, and MHC-II levels were altered in mature DCs after infection with NDVs at all evaluated times postinfection. Notably, the GM strain triggered stronger innate immune responses than the La Sota strain in chicken BM-DCs. However, both strains were able to suppress the expression of some cytokines, such as IL-6 and IFN-α, in mature chicken DCs at 24 hpi. These data provide a foundation for further investigation of the role of chicken DCs in NDV infection.

  5. Mechanical strain modulates age-related changes in the proliferation and differentiation of mouse adipose-derived stromal cells

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    Chiang Wen-Sheng

    2010-03-01

    Full Text Available Abstract Background Previous studies on the effects of aging in human and mouse mesenchymal stem cells suggest that a decline in the number and differentiation potential of stem cells may contribute to aging and aging-related diseases. In this report, we used stromal cells isolated from adipose tissue (ADSCs of young (8-10 weeks, adult (5 months, and old (21 months mice to test the hypothesis that mechanical loading modifies aging-related changes in the self-renewal and osteogenic and adipogenic differentiation potential of these cells. Results We show that aging significantly reduced the proliferation and increased the adipogenesis of ADSCs, while the osteogenic potential is not significantly reduced by aging. Mechanical loading (10% cyclic stretching, 0.5 Hz, 48 h increased the subsequent proliferation of ADSCs from mice of all ages. Although the number of osteogenic colonies with calcium deposition was increased in ADSCs subjected to pre-strain, it resulted from an increase in colony number rather than from an increase in osteogenic potential after strain. Pre-strain significantly reduced the number of oil droplets and the expression of adipogenic marker genes in adult and old ADSCs. Simultaneously subjecting ADSCs to mechanical loading and adipogenic induction resulted in a stronger inhibition of adipogenesis than that caused by pre-strain. The reduction of adipogenesis by mechanical strain was loading-magnitude dependent: loading with 2% strain only resulted in a partial inhibition, and loading with 0.5% strain could not inhibit adipogenesis in ADSCs. Conclusions We demonstrate that mechanical stretching counteracts the loss of self-renewal in aging ADSCs by enhancing their proliferation and, at the same time, reduces the heightened adipogenesis of old cells. These findings are important for the further study of stem cell control and treatment for a variety of aging related diseases.

  6. Cross-sensitivity of X-ray-hypersensitive cells derived from LEC strain rats to DNA-damaging agents

    International Nuclear Information System (INIS)

    Okui, T.; Endoh, D.; Arai, S.; Isogai, E.; Hayashi, M.

    1996-01-01

    The cross-sensitivity of X-ray-hypersensitive lung fibroblasts from LEC strain (LEC) rats to other DNA-damaging agents was examined. The LEC cells were 2- to 3-fold more sensitive to bleomycin (BLM) that induces DNA double-strand breaks, and to a cross-linking agent, mitomycin C, than the cells from WKAH strain (WKAH) rats, while they were slightly sensitive to alkylating agents, ethyl nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine, but not to UV-irradiation. Although no difference was observed in the initial yields of DNA double-strand breaks induced by BLM between LEC and WKAH cells, the repair process of DNA double-strand breaks was significantly slower in LEC cells than in WKAH cells

  7. Different Transcriptional Profiles of Human Monocyte-Derived Dendritic Cells Infected with Distinct Strains of Mycobacterium tuberculosis and Mycobacterium bovis Bacillus Calmette-Guérin

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    Nunzia Sanarico

    2011-01-01

    Full Text Available In order to analyze dendritic cells (DCs activation following infection with different mycobacterial strains, we studied the expression profiles of 165 genes of human monocyte-derived DCs infected with H37Rv, a virulent Mycobacterium tuberculosis (MTB laboratory strain, CMT97, a clinical MTB isolate, Mycobacterium bovis bacillus Calmette-Guérin (BCG, Aventis Pasteur, and BCG Japan, both employed as vaccine against tuberculosis. The analysis of the gene expression reveals that, despite a set of genes similarly modulated, DCs response resulted strain dependent. In particular, H37Rv significantly upregulated EBI3 expression compared with BCG Japan, while it was the only strain that failed to release a significant IL-10 amount. Of note, BCG Japan showed a marked increase in CCR7 and TNF-α expression regarding both MTB strains and it resulted the only strain failing in exponential intracellular growth. Our results suggest that DCs display the ability to elicit a tailored strain-specific immune response.

  8. Adipose-Derived-Stem-Cell-Seeded Fibrin Matrices for Periodontal Ligament Engineering: The Need for Dynamic Strain

    NARCIS (Netherlands)

    de Jong, Thijs; Oostendorp, Corien; Bakker, Astrid D.; van Kuppevelt, Toin H.; Smit, Theo H.

    2017-01-01

    Introduction: The periodontal ligament (PDL) connects the tooth to the alveolar bone. For PDL regeneration after tissue damage, we propose human adipose-derived stem cells (hASCs) embedded in fibrin. We showed previously that hASCs in fibrin extensively produce collagen, but in a non-functional,

  9. Multiple hypersensitivity to mutagens in a cell strain (46BR) derived from a patient with immuno-deficiencies

    International Nuclear Information System (INIS)

    Teo, I.A.; Arlett, C.F.; Harcourt, S.A.; Priestley, A.; Broughton, B.C.

    1983-01-01

    46BR is a fibroblast cell strain established from an individual with hypogammaglobulinaemia. The cells are unique in showing hypersensitivity to the lethal effects of a wide range of DNA-damaging agents. Thus they are hypersensitive to #betta#- and 254-nm UV-irradiation and show a limited capacity to repair potentially lethal #betta#-irradiation damage when compared with fibroblasts from normal individuals. A slight hypersensitivity to mitomycin C was also revealed but we were not able to discriminate 46BR from normals with 4-nitroquinoline oxide. The cells were hypersensitive to the alkylating agents, dimethyl sulphate, methyl methanesulphonate, ethyl methanesulphonate, N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea but not N-ethyl-N-nitrosourea. A consideration of the spectra of DNA lesions produced by these alkylating agents together with the sensitivity to ionising radiation and mitomycin C suggests that 46BR cells are defective in a repair step that is common to all agents. We suggest that the cells are defective in DNA polymerisation or ligation. Support for this suggestion comes from the absence of any hypersensitivity to N-ethyl-N-nitrosourea since its major reaction products are not removed by excision pathways that require polymerisation and ligation. (orig.)

  10. Interclonal variations in the molecular karyotype of Trypanosoma cruzi: chromosome rearrangements in a single cell-derived clone of the G strain.

    Science.gov (United States)

    Lima, Fabio Mitsuo; Souza, Renata Torres; Santori, Fábio Rinaldo; Santos, Michele Fernandes; Cortez, Danielle Rodrigues; Barros, Roberto Moraes; Cano, Maria Isabel; Valadares, Helder Magno Silva; Macedo, Andréa Mara; Mortara, Renato Arruda; da Silveira, José Franco

    2013-01-01

    Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure.

  11. Mitochondrial proteomics of the acetic acid – induced programmed cell death response in a highly tolerant Zygosaccharomyces bailii – derived hybrid strain

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    Joana F Guerreiro

    2016-01-01

    Full Text Available Very high concentrations of acetic acid at low pH induce programmed cell death (PCD in both the experimental model Saccharomyces cerevisiae and in Zygosaccharomyces bailii, the latter being considered the most problematic acidic food spoilage yeast due to its remarkable intrinsic resistance to this food preservative. However, while the mechanisms underlying S. cerevisiae PCD induced by acetic acid have been previously examined, the corresponding molecular players remain largely unknown in Z. bailii. Also, the reason why acetic acid concentrations known to be necrotic for S. cerevisiae induce PCD with an apoptotic phenotype in Z. bailii remains to be elucidated. In this study, a 2-DE-based expression mitochondrial proteomic analysis was explored to obtain new insights into the mechanisms involved in PCD in the Z. bailii derived hybrid strain ISA1307. This allowed the quantitative assessment of expression of protein species derived from each of the parental strains, with special emphasis on the processes taking place in the mitochondria known to play a key role in acetic acid – induced PCD. A marked decrease in the content of proteins involved in mitochondrial metabolism, in particular, in respiratory metabolism (Cor1, Rip1, Lpd1, Lat1 and Pdb1, with a concomitant increase in the abundance of proteins involved in fermentation (Pdc1, Ald4, Dld3 was registered. Other differentially expressed identified proteins also suggest the involvement of the oxidative stress response, protein translation, amino acid and nucleotide metabolism, among other processes, in the PCD response. Overall, the results strengthen the emerging concept of the importance of metabolic regulation of yeast PCD.

  12. Biotechnological methods for chalcone reduction using whole cells of Lactobacillus, Rhodococcus and Rhodotorula strains as a way to produce new derivatives.

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    Stompor, Monika; Kałużny, Mateusz; Żarowska, Barbara

    2016-10-01

    Microbial strains of the genera Dietzia, Micrococcus, Pseudomonas, Rhodococcus, Gordonia, Streptomyces, Pseudomonas, Bacillus, Penicillium, Rhodotorula and Lactobacillus were screened for the ability to convert chalcones. Synthesis of chalcones was performed by the Claisen-Schmidt reaction. There were three groups of chalcones obtained as the products, which included the derivatives containing 4-substituted chalcone, 2'-hydroxychalcone and 4'-methoxychalcone. The B ring of the chalcones was substituted in the para position with different groups, such as halide, hydroxyl, nitro, methyl, ethyl and ethoxy one. The structure-activity relationship of the tested chalcones in biotransformation processes was studied. It has been proven that Gram-positive bacterial strains Rhodococcus and Lactobacillus catalyzed reduction of C=C bond in the chalcones to give respective dihydrochalcones. The strain Rhodotorula rubra AM 82 transformed chalcones into dihydrochalcones and respective secondary alcohols. These results suggest that the probiotic strain of Lactobacillus can be used for biotransformations of chalcones, which has not been described before. The structure of new metabolites 14a and 15b were established as 4-ethoxy-4'-methoxydihydrochalcone and 3-(4-bromophenyl)-1-(4'-O-methylphenyl)-2-propan-1-ol, respectively, which was confirmed by (1)H NMR and (13)C NMR analysis.

  13. Human antigen-presenting cells respond differently to gut-derived probiotic bacteria but mediate similar strain-dependent NK and T cell activation

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Zeuthen, Louise Hjerrild; Ferlazzo, Guido

    2007-01-01

    The intestinal microbiota is essential for homeostasis of the local and systemic immune system, and particularly strains of lactic acid bacteria and Escherichia coli have been shown to have balancing effects on inflammatory conditions such as allergy and inflammatory bowel disease. However, in vi...

  14. Mechanical Stimulation of Adipose-Derived Stem Cells for Functional Tissue Engineering of the Musculoskeletal System via Cyclic Hydrostatic Pressure, Simulated Microgravity, and Cyclic Tensile Strain.

    Science.gov (United States)

    Nordberg, Rachel C; Bodle, Josie C; Loboa, Elizabeth G

    2018-01-01

    It is critical that human adipose stem cell (hASC) tissue-engineering therapies possess appropriate mechanical properties in order to restore function of the load bearing tissues of the musculoskeletal system. In an effort to elucidate the hASC response to mechanical stimulation and develop mechanically robust tissue engineered constructs, recent research has utilized a variety of mechanical loading paradigms including cyclic tensile strain, cyclic hydrostatic pressure, and mechanical unloading in simulated microgravity. This chapter describes methods for applying these mechanical stimuli to hASC to direct differentiation for functional tissue engineering of the musculoskeletal system.

  15. Response to high LET radiation 12C (LET, 295 keV/microm) in M5 cells, a radio resistant cell strain derived from Chinese hamster V79 cells.

    Science.gov (United States)

    Pathak, R; Sarma, A; Sengupta, B; Dey, S K; Khuda-Bukhsh, A R

    2007-01-01

    To study the effects of 12C-beam of 295 keV/microm (57.24 MeV) on M5 and Chinese hamster V79 cells by using cytogenetic assays like micronuclei (MN) induction, chromosomal aberrations (CA) and apoptosis. Additionally, the relative survival of these two cell lines was tested by the colony forming ability of the cells, with a view to understanding the mechanism of cellular damages that lead to difference in cell survival. Confluent cells were irradiated with 12C-beam at various doses using 15UD Pelletron accelerator. Cell survival was studied by the colony forming ability of cells. MN assay was done by fluorescent staining. Different types of chromosomal aberrations in metaphase cells were scored at 12 h after irradiation. Apoptosis was measured at different post irradiation times as detected by nuclear fragmentation and DNA ladder was prepared after 48 h of incubation. Dose-dependent decrease in surviving fractions was found in both the cell lines. However, the surviving fractions were higher in M5 cells in comparison to V79 cells when exposed to the same radiation doses. On the other hand, induced MN frequencies, CA frequencies and apoptosis percentages were less in M5 cells than V79 cells. Very good correlations between surviving fractions and induced MN frequencies or induced total CA or induced apoptosis percentages were obtained in this study. The cell strain M5 showed relatively more radio-resistance to 12C-beam compared to Chinese hamster V79 cells in this study. As the MN formation, CA and apoptosis induction were less in M5 cells as compared to parental V79 cells, the higher cell survival in the former could possibly be attributed to their better repairing ability leading to higher cell survival.

  16. Human-derived probiotic Lactobacillus reuteri strains differentially reduce intestinal inflammation.

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    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

  17. Cells as strain-cued automata

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    Cox, Brian N.; Snead, Malcolm L.

    2016-02-01

    We argue in favor of representing living cells as automata and review demonstrations that autonomous cells can form patterns by responding to local variations in the strain fields that arise from their individual or collective motions. An autonomous cell's response to strain stimuli is assumed to be effected by internally-generated, internally-powered forces, which generally move the cell in directions other than those implied by external energy gradients. Evidence of cells acting as strain-cued automata have been inferred from patterns observed in nature and from experiments conducted in vitro. Simulations that mimic particular cases of pattern forming share the idealization that cells are assumed to pass information among themselves solely via mechanical boundary conditions, i.e., the tractions and displacements present at their membranes. This assumption opens three mechanisms for pattern formation in large cell populations: wavelike behavior, kinematic feedback in cell motility that can lead to sliding and rotational patterns, and directed migration during invasions. Wavelike behavior among ameloblast cells during amelogenesis (the formation of dental enamel) has been inferred from enamel microstructure, while strain waves in populations of epithelial cells have been observed in vitro. One hypothesized kinematic feedback mechanism, "enhanced shear motility", accounts successfully for the spontaneous formation of layered patterns during amelogenesis in the mouse incisor. Directed migration is exemplified by a theory of invader cells that sense and respond to the strains they themselves create in the host population as they invade it: analysis shows that the strain fields contain positional information that could aid the formation of cell network structures, stabilizing the slender geometry of branches and helping govern the frequency of branch bifurcation and branch coalescence (the formation of closed networks). In simulations of pattern formation in

  18. Survey of radiosensitivity in a variety of human cell strains

    Energy Technology Data Exchange (ETDEWEB)

    Arlett, C.F.; Harcourt, S.A.

    1980-03-01

    Gamma-ray sensitivity for cell killing was assayed in 54 human cell strains, including some derived from individuals suffering from certain hereditary diseases. The overall range of Do values in this study was 38 to 180 rads, indicating a considerable range of variability in humans. The normal sensitivity was described by a range of Do values of 97 to 180 rads. All ten ataxia telangiectasia cell strains tested proved radiosensitive and gave a mean Do value of 57 +- 15 (S.E.) rads, and these represent the most radiosensitive human skin fibroblasts currently available. Representative cell strains from familial retinoblastoma, Fanconi's anemia, and Hutchinson-Gilford progeria occupied positions of intermediate sensitivity, as did one of two ataxia telangiectasia heterozygotes. Six xeroderma pigmentosum cell strains together with two Cockayne's syndrome cell strains (all known to be sensitive to ultraviolet light) fell into the normal range, indicating an absence of cross-sensitivity between ultraviolet light and gamma-irradiation.

  19. On strain and stress in living cells

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    Cox, Brian N.; Smith, David W.

    2014-11-01

    Recent theoretical simulations of amelogenesis and network formation and new, simple analyses of the basic multicellular unit (BMU) allow estimation of the order of magnitude of the strain energy density in populations of living cells in their natural environment. A similar simple calculation translates recent measurements of the force-displacement relation for contacting cells (cell-cell adhesion energy) into equivalent volume energy densities, which are formed by averaging the changes in contact energy caused by a cell's migration over the cell's volume. The rates of change of these mechanical energy densities (energy density rates) are then compared to the order of magnitude of the metabolic activity of a cell, expressed as a rate of production of metabolic energy per unit volume. The mechanical energy density rates are 4-5 orders of magnitude smaller than the metabolic energy density rate in amelogenesis or bone remodeling in the BMU, which involve modest cell migration velocities, and 2-3 orders of magnitude smaller for innervation of the gut or angiogenesis, where migration rates are among the highest for all cell types. For representative cell-cell adhesion gradients, the mechanical energy density rate is 6 orders of magnitude smaller than the metabolic energy density rate. The results call into question the validity of using simple constitutive laws to represent living cells. They also imply that cells need not migrate as inanimate objects of gradients in an energy field, but are better regarded as self-powered automata that may elect to be guided by such gradients or move otherwise. Thus Ġel=d/dt 1/2 >[(C11+C12)ɛ02+2μγ02]=(C11+C12)ɛ0ɛ˙0+2μγ0γ˙0 or Ġel=ηEɛ0ɛ˙0+η‧Eγ0γ˙0 with 1.4≤η≤3.4 and 0.7≤η‧≤0.8 for Poisson's ratio in the range 0.2≤ν≤0.4 and η=1.95 and η‧=0.75 for ν=0.3. The spatial distribution of shear strains arising within an individual cell as cells slide past one another during amelogenesis is not known

  20. Inhibition spectrum studies of microthecin and other anhydrofructose derivatives using selected strains of Gram-positive and –negative bacteria, yeast and moulds, and investigation of the cytotoxicity of microthecin to malignant blood cell lines

    DEFF Research Database (Denmark)

    Fiskesund, R.; Thomas, L.V.; Schobert, M.

    2009-01-01

    Aims: To prepare 1,5-anhydro-d-fructose (AF) derivatives, test their microbial inhibition spectrum, and to further examine the most effective AF derivative against Pseudomonas aeruginosa and malignant blood cell lines. Methods and Results: Microthecin and nine other AF derivatives were synthesized...... both GP and GN bacteria, including Ps. aeruginosa. Further tests with three malignant blood cell lines (Mutu, Ramos, Raji) and one normal cell line indicated that microthecin was a cell toxin, with a cell mortality >85% at 50 ppm. The other nine AF derivatives demonstrated low or no antimicrobial...... activity. Conclusions: Microthecin was active 100–2000 ppm against GP and GN bacteria including Ps. aeruginosa, but was inactive against yeasts and moulds. Microthecin was also a cytotoxin to some mammalian cell lines. Significance and Impact of the Study: Microthecin might have potential for development...

  1. Nonsense and sense suppression abilities of original and derivative Methanosarcina mazei pyrrolysyl-tRNA synthetase-tRNA(Pyl pairs in the Escherichia coli BL21(DE3 cell strain.

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    Keturah A Odoi

    Full Text Available Systematic studies of nonsense and sense suppression of the original and three derivative Methanosarcina mazei PylRS-tRNA(Pyl pairs and cross recognition between nonsense codons and various tRNA(Pyl anticodons in the Escherichia coli BL21(DE3 cell strain are reported. tRNA(CUA(Pyl is orthogonal in E. coli and able to induce strong amber suppression when it is co-expressed with pyrrolysyl-tRNA synthetase (PylRS and charged with a PylRS substrate, N(ε-tert-butoxycarbonyl-L-lysine (BocK. Similar to tRNA(CUA(Pyl, tRNA(UUA(Pyl is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although tRNA(UUA(Pyl is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS-tRNA(UUA(Pyl pair does not give rise to amber suppression that surpasses the basal amber suppression level in E. coli. E. coli itself displays a relatively high opal suppression level and tryptophan (Trp is incorporated at an opal mutation site. Although the PylRS-tRNA(UCA(Pyl pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. tRNA(CCU(Pyl fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli.

  2. Strain and Vibration in Mesenchymal Stem Cells

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    Brooke McClarren

    2018-01-01

    Full Text Available Mesenchymal stem cells (MSCs are multipotent cells capable of differentiating into any mesenchymal tissue, including bone, cartilage, muscle, and fat. MSC differentiation can be influenced by a variety of stimuli, including environmental and mechanical stimulation, scaffold physical properties, or applied loads. Numerous studies have evaluated the effects of vibration or cyclic tensile strain on MSCs towards developing a mechanically based method of differentiation, but there is no consensus between studies and each investigation uses different culture conditions, which also influence MSC fate. Here we present an overview of the response of MSCs to vibration and cyclic tension, focusing on the effect of various culture conditions and strain or vibration parameters. Our review reveals that scaffold type (e.g., natural versus synthetic; 2D versus 3D can influence cell response to vibration and strain to the same degree as loading parameters. Hence, in the efforts to use mechanical loading as a reliable method to differentiate cells, scaffold selection is as important as method of loading.

  3. Genomic, Transcriptomic and Metabolomic Studies of Two Well-Characterized, Laboratory-Derived Vancomycin-Intermediate Staphylococcus aureus Strains Derived from the Same Parent Strain

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    Dipti S. Hattangady

    2015-02-01

    Full Text Available Complete genome comparisons, transcriptomic and metabolomic studies were performed on two laboratory-selected, well-characterized vancomycin-intermediate Staphylococcus aureus (VISA derived from the same parent MRSA that have changes in cell wall composition and decreased autolysis. A variety of mutations were found in the VISA, with more in strain 13136p−m+V20 (vancomycin MIC = 16 µg/mL than strain 13136p−m+V5 (MIC = 8 µg/mL. Most of the mutations have not previously been associated with the VISA phenotype; some were associated with cell wall metabolism and many with stress responses, notably relating to DNA damage. The genomes and transcriptomes of the two VISA support the importance of gene expression regulation to the VISA phenotype. Similarities in overall transcriptomic and metabolomic data indicated that the VISA physiologic state includes elements of the stringent response, such as downregulation of protein and nucleotide synthesis, the pentose phosphate pathway and nutrient transport systems. Gene expression for secreted virulence determinants was generally downregulated, but was more variable for surface-associated virulence determinants, although capsule formation was clearly inhibited. The importance of activated stress response elements could be seen across all three analyses, as in the accumulation of osmoprotectant metabolites such as proline and glutamate. Concentrations of potential cell wall precursor amino acids and glucosamine were increased in the VISA strains. Polyamines were decreased in the VISA, which may facilitate the accrual of mutations. Overall, the studies confirm the wide variability in mutations and gene expression patterns that can lead to the VISA phenotype.

  4. Biological characterization of clones derived from the edmonston strain of measles virus in comparison with schwarz and CAM-70 vaccine strains

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    Maria Beatriz Junqueira Borges

    1996-08-01

    Full Text Available Four virus clones were derived from the Edmonston strain of measles virus by repeated plaque purification. These clones were compared with the vaccine strains Schwarz and CAM-70 in terms of biological activities including plaque formation, hemagglutination, hemolysis and replication in Vero cells and chick embryo fibroblasts (CEF. Two clones of intermediate plaque yielded mixed plaque populations on subcultivation whereas the other two, showing small and large plaque sizes, showed stable plaque phenotypes. The vaccine strains showed consistent homogeneous plaque populations. All the Edmonston clones showed agglutination of monkey erythrocytes in isotonic solution while both vaccine strains hemagglutinated only in the presence of high salt concentrations. Variation in the hemolytic activity was observed among the four clones but no hemolytic activity was detected for the vaccine virus strains. Vaccine strains replicated efficiently both in Vero cells and CEF. All four clones showed efficient replication in Vero cells but different replication profiles in CEF. Two of them replicated efficiently, one was of intermediate efficiency and the other showed no replication in CEF. Two of the clones showed characteristics similar to vaccine strains. One in terms of size and homogeneity of plaques, the other for a low hemolytic activity and both for the efficiency of propagation in CEF.

  5. Patient-Derived Antibody Targets Tumor Cells

    Science.gov (United States)

    An NCI Cancer Currents blog on an antibody derived from patients that killed tumor cells in cell lines of several cancer types and slowed tumor growth in mouse models of brain and lung cancer without evidence of side effects.

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

    Science.gov (United States)

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

  7. Solitary waves in morphogenesis: Determination fronts as strain-cued strain transformations among automatous cells

    Science.gov (United States)

    Cox, Brian N.; Landis, Chad M.

    2018-02-01

    We present a simple theory of a strain pulse propagating as a solitary wave through a continuous two-dimensional population of cells. A critical strain is assumed to trigger a strain transformation, while, simultaneously, cells move as automata to tend to restore a preferred cell density. We consider systems in which the strain transformation is a shape change, a burst of proliferation, or the commencement of growth (which changes the shape of the population sheet), and demonstrate isomorphism among these cases. Numerical and analytical solutions describe a strain pulse whose height does not depend on how the strain disturbance was first launched, or the rate at which the strain transformation is achieved, or the rate constant in the rule for the restorative cell motion. The strain pulse is therefore very stable, surviving the imposition of strong perturbations: it would serve well as a timing signal in development. The automatous wave formulation is simple, with few model parameters. A strong case exists for the presence of a strain pulse during amelogenesis. Quantitative analysis reveals a simple relationship between the velocity of the leading edge of the pulse in amelogenesis and the known speed of migration of ameloblast cells. This result and energy arguments support the depiction of wave motion as an automatous cell response to strain, rather than as a response to an elastic energy gradient. The theory may also contribute to understanding the determination front in somitogenesis, moving fronts of convergent-extension transformation, and mitotic wavefronts in the syncytial drosophila embryo.

  8. Ultrasonic Derivative Measurements of Bone Strain During Exercise, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — Luna Innovations, Inc., in collaboration with the SUNY Stony Brook, proposes to extend ultrasonic pulsed phase locked loop (PPLL) derivative measurements to the...

  9. Adipose-Derived Stem Cells

    DEFF Research Database (Denmark)

    Toyserkani, Navid Mohamadpour; Quaade, Marlene Louise; Sheikh, Søren Paludan

    2015-01-01

    , regulate the inflammatory process, and differentiate into multiple cell types makes them a potential ideal therapy for chronic wounds. The aim of this article was to review all preclinical trials using ASCs in problem wound models. A systematic search was performed and 12 studies were found where different...

  10. Adipose-Derived Stem Cells

    NARCIS (Netherlands)

    Gathier, WA; Türktas, Z; Duckers, HJ

    2015-01-01

    Until recently bone marrow was perceived to be the only significant reservoir of stem cells in the body. However, it is now recognized that there are other and perhaps even more abundant sources, which include adipose tissue. Subcutaneous fat is readily available in most patients, and can easily be

  11. Novel synthesis of a strained para-cyclophane derivative

    Institute of Scientific and Technical Information of China (English)

    Zhi Hao Shi; Yun Yang Wei

    2007-01-01

    A [P. Rajakumar, A.M.A. Rasheed, Tetrahedron 61(22) (2005) 5351] para-cyclophane derivative was synthesized via intramolecular esterification of a dipeptide surrogate containing asparagine to form tetrahydropyrimidinone ring. The structures of the product and intermediates were characterized by 1H NMR, 13C NMR and mass spectrum.

  12. In vitro virucidal activity of a styrylpyrone derivative against herpes simplex virus strain KOS-1

    Science.gov (United States)

    Moses, Micheal; Nor, Norefrina Shafinaz Md.; Ibrahim, Nazlina

    2014-09-01

    In this study, styrylpyrone derivative (SPD) extracted from Goniothalamus umbrosus root was tested against herpes simplex virus (HSV) strain KOS-1. Firstly, the cytotoxicity of SPD on Vero cells was tested and the value of cytotoxic concentration, CC50, was 44 μM (8.88 μg/mL), and the 50% Effective Concentration, EC50, was 3.35 μM (0.67 μg/mL). Selectivity index of SPD against HSV Kos-1 was more than 13 indicating potential as antiviral agent. Three treatments were used in the antiviral test; 1) post-treatment, 2) pre-treatment, and 3) virucidal. The results revealed that the post-treatment was more effective in inhibiting viral replication compared to pre-treatment. The findings indicated that the SPD from G. umbrosus has good potential for prospective nature-based antiviral drug.

  13. Mycobacterium tuberculosis strains exhibit differential and strain-specific molecular signatures in pulmonary epithelial cells.

    Science.gov (United States)

    Mvubu, Nontobeko Eunice; Pillay, Balakrishna; Gamieldien, Junaid; Bishai, William; Pillay, Manormoney

    2016-12-01

    Although pulmonary epithelial cells are integral to innate and adaptive immune responses during Mycobacterium tuberculosis infection, global transcriptomic changes in these cells remain largely unknown. Changes in gene expression induced in pulmonary epithelial cells infected with M. tuberculosis F15/LAM4/KZN, F11, F28, Beijing and Unique genotypes were investigated by RNA sequencing (RNA-Seq). The Illumina HiSeq 2000 platform generated 50 bp reads that were mapped to the human genome (Hg19) using Tophat (2.0.10). Differential gene expression induced by the different strains in infected relative to the uninfected cells was quantified and compared using Cufflinks (2.1.0) and MeV (4.0.9), respectively. Gene expression varied among the strains with the total number of genes as follows: F15/LAM4/KZN (1187), Beijing (1252), F11 (1639), F28 (870), Unique (886) and H37Rv (1179). A subset of 292 genes was commonly induced by all strains, where 52 genes were down-regulated while 240 genes were up-regulated. Differentially expressed genes were compared among the strains and the number of induced strain-specific gene signatures were as follows: F15/LAM4/KZN (138), Beijing (52), F11 (255), F28 (55), Unique (186) and H37Rv (125). Strain-specific molecular gene signatures associated with functional pathways were observed only for the Unique and H37Rv strains while certain biological functions may be associated with other strain signatures. This study demonstrated that strains of M. tuberculosis induce differential gene expression and strain-specific molecular signatures in pulmonary epithelial cells. Specific signatures induced by clinical strains of M. tuberculosis can be further explored for novel host-associated biomarkers and adjunctive immunotherapies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Distinct gut-derived lactic acid bacteria elicit divergent dendritic cell-mediated NK cell responses

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Zeuthen, Louise Hjerrild; Christensen, Hanne

    2007-01-01

    Lactic acid bacteria (LAB) are abundant in the gastrointestinal tract where they continuously regulate the immune system. NK cells are potently activated by dendritic cells (DCs) matured by inflammatory stimuli, and NK cells are present in the gut epithelium and in mesenteric lymph nodes......, but it is not known how NK-DC interactions are affected by the predominantly non-pathogenic LAB. We demonstrate that human DCs exposed to different strains of gut-derived LAB consistently induce proliferation, cytotoxicity and activation markers in autologous NK cells. On the contrary, strains of LAB differ greatly...... in their ability to induce DC-dependent IFN-gamma production by NK cells. This suggests that DCs stimulated by gut LAB may expand the pool of NK cells and increase their cytotoxic potential. Specific LAB, inducing high levels of IL-12 in DCs, may promote amplification of a type-1 response via potent stimulation...

  15. 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.)

  16. History of myeloid-derived suppressor cells.

    Science.gov (United States)

    Talmadge, James E; Gabrilovich, Dmitry I

    2013-10-01

    Tumour-induced granulocytic hyperplasia is associated with tumour vasculogenesis and escape from immunity via T cell suppression. Initially, these myeloid cells were identified as granulocytes or monocytes; however, recent studies have revealed that this hyperplasia is associated with populations of multipotent progenitor cells that have been identified as myeloid-derived suppressor cells (MDSCs). The study of MDSCs has provided a wealth of information regarding tumour pathobiology, has extended our understanding of neoplastic progression and has modified our approaches to immune adjuvant therapy. In this Timeline article, we discuss the history of MDSCs, their influence on tumour progression and metastasis, and the crosstalk between tumour cells, MDSCs and the host macroenvironment.

  17. 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.

  18. 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

  19. Metabolic engineering of a haploid strain derived from a triploid industrial yeast for producing cellulosic ethanol.

    Science.gov (United States)

    Kim, Soo Rin; Skerker, Jeffrey M; Kong, In Iok; Kim, Heejin; Maurer, Matthew J; Zhang, Guo-Chang; Peng, Dairong; Wei, Na; Arkin, Adam P; Jin, Yong-Su

    2017-03-01

    Many desired phenotypes for producing cellulosic biofuels are often observed in industrial Saccharomyces cerevisiae strains. However, many industrial yeast strains are polyploid and have low spore viability, making it difficult to use these strains for metabolic engineering applications. We selected the polyploid industrial strain S. cerevisiae ATCC 4124 exhibiting rapid glucose fermentation capability, high ethanol productivity, strong heat and inhibitor tolerance in order to construct an optimal yeast strain for producing cellulosic ethanol. Here, we focused on developing a general approach and high-throughput screening method to isolate stable haploid segregants derived from a polyploid parent, such as triploid ATCC 4124 with a poor spore viability. Specifically, we deleted the HO genes, performed random sporulation, and screened the resulting segregants based on growth rate, mating type, and ploidy. Only one stable haploid derivative (4124-S60) was isolated, while 14 other segregants with a stable mating type were aneuploid. The 4124-S60 strain inherited only a subset of desirable traits present in the parent strain, same as other aneuploids, suggesting that glucose fermentation and specific ethanol productivity are likely to be genetically complex traits and/or they might depend on ploidy. Nonetheless, the 4124-60 strain did inherit the ability to tolerate fermentation inhibitors. When additional genetic perturbations known to improve xylose fermentation were introduced into the 4124-60 strain, the resulting engineered strain (IIK1) was able to ferment a Miscanthus hydrolysate better than a previously engineered laboratory strain (SR8), built by making the same genetic changes. However, the IIK1 strain showed higher glycerol and xylitol yields than the SR8 strain. In order to decrease glycerol and xylitol production, an NADH-dependent acetate reduction pathway was introduced into the IIK1 strain. By consuming 2.4g/L of acetate, the resulting strain (IIK1A

  20. Establishment and characterization of a hypocatalasemic mouse cell strain

    International Nuclear Information System (INIS)

    Utsumi, Hiroshi; Tano, Keizo; Hashimoto, Mitsumasa W.; Kodama, Seiji; Watanabe, Hiromitsu

    1998-01-01

    Contact-inhibited catalase-deficient fibroblast cell strain has been established from the homozygous hypocatalasemic C3H/Cs b mutant mouse. This cell strain has low level of catalase enzyme activity and has normal level of enzyme activities of both glutathione peroxidase and superoxide dismutase. Catalase-deficient C3H/Cs b mutant cell strain is markedly more sensitive to the toxicity of hydrogen peroxide compared to wild-type C3H/Cs a cell strain. In addition, mutant cell strain is sensitive to X-rays and near-UV compared to wild-type cell strain, but shows the same sensitivities to topoisomerase II inhibitors, adriamycin and 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA), and the DNA cross-linking agents, cis-diamminedichloroplatinum (II) (cis-Pt) and trans-diamminedichloroplatinum (II) (trans-Pt). These cell strains will be of use in the study of the roles which catalase plays in the intracellular prevention of DNA damage induced by oxidative stress. (author)

  1. Strain preservation of experimental animals: vitrification of two-cell stage embryos for multiple mouse strains.

    Science.gov (United States)

    Eto, Tomoo; Takahashi, Riichi; Kamisako, Tsutomu

    2015-04-01

    Strain preservation of experimental animals is crucial for experimental reproducibility. Maintaining complete animal strains, however, is costly and there is a risk for genetic mutations as well as complete loss due to disasters or illness. Therefore, the development of effective vitrification techniques for cryopreservation of multiple experimental animal strains is important. We examined whether a vitrification method using cryoprotectant solutions, P10 and PEPeS, is suitable for preservation of multiple inbred and outbred mouse strains. First, we investigated whether our vitrification method using cryoprotectant solutions was suitable for two-cell stage mouse embryos. In vitro development of embryos exposed to the cryoprotectant solutions was similar to that of fresh controls. Further, the survival rate of the vitrified embryos was extremely high (98.1%). Next, we collected and vitrified two-cell stage embryos of 14 mouse strains. The average number of embryos obtained from one female was 7.3-33.3. The survival rate of vitrified embryos ranged from 92.8% to 99.1%, with no significant differences among mouse strains. In vivo development did not differ significantly between fresh controls and vitrified embryos of each strain. For strain preservation using cryopreserved embryos, two offspring for inbred lines and one offspring for outbred lines must be produced from two-cell stage embryos collected from one female. The expected number of surviving fetuses obtained from embryos collected from one female of either the inbred or outbred strains ranged from 2.9 to 19.5. The findings of the present study indicated that this vitrification method is suitable for strain preservation of multiple mouse strains. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Interaction of Coxiella burnetii Strains of Different Sources and Genotypes with Bovine and Human Monocyte-Derived Macrophages

    Directory of Open Access Journals (Sweden)

    Katharina Sobotta

    2018-01-01

    Full Text Available Most human Q fever infections originate from small ruminants. By contrast, highly prevalent shedding of Coxiella (C. burnetii by bovine milk rarely results in human disease. We hypothesized that primary bovine and human monocyte-derived macrophages (MDM represent a suitable in vitro model for the identification of strain-specific virulence properties at the cellular level. Twelve different C. burnetii strains were selected to represent different host species and multiple loci variable number of tandem repeat analysis (MLVA genotypes. Infection efficiency and replication of C. burnetii were monitored by cell culture re-titration and qPCR. Expression of immunoregulatory factors after MDM infection was measured by qRT-PCR and flow cytometry. Invasion, replication and MDM response differed between C. burnetii strains but not between MDMs of the two hosts. Strains isolated from ruminants were less well internalized than isolates from humans and rodents. Internalization of MLVA group I strains was lower compared to other genogroups. Replication efficacy of C. burnetii in MDM ranged from low (MLVA group III to high (MLVA group IV. Infected human and bovine MDM responded with a principal up-regulation of pro-inflammatory cytokines such as IL-1β, IL-12, and TNF-α. However, MLVA group IV strains induced a pronounced host response whereas infection with group I strains resulted in a milder response. C. burnetii infection marginally affected polarization of MDM. Only one C. burnetii strain of MLVA group IV caused a substantial up-regulation of activation markers (CD40, CD80 on the surface of bovine and human MDM. The study showed that replication of C. burnetii in MDM and the subsequent host cell response is genotype-specific rather than being determined by the host species pointing to a clear distinction in C. burnetii virulence between the genetic groups.

  3. Direct coordinate-free derivation of the compatibility equation for finite strains

    Science.gov (United States)

    Ryzhak, E. I.

    2014-07-01

    The compatibility equation for the Cauchy-Green tensor field (squared tensor of pure extensionwith respect to the reference configuration) is directly derived from the well-known relation expressing this tensor via the vector field determining the mapping (transformation) of the reference configuration into the actual one. The derivation is based on the use of the apparatus of coordinatefree tensor calculus and does not apply any notions and relations of Riemannian geometry at all. The method is illustrated by deriving the well-known compatibility equation for small strains. It is shown that when the obtained compatibility equation for finite strains is linearized, it becomes the compatibility equation for small strains which indirectly confirms its correctness.

  4. Derivatives of buckling loads and vibration frequencies with respect to stiffness and initial strain parameters

    Science.gov (United States)

    Haftka, Raphael T.; Cohen, Gerald A.; Mroz, Zenon

    1990-01-01

    A uniform variational approach to sensitivity analysis of vibration frequencies and bifurcation loads of nonlinear structures is developed. Two methods of calculating the sensitivities of bifurcation buckling loads and vibration frequencies of nonlinear structures, with respect to stiffness and initial strain parameters, are presented. A direct method requires calculation of derivatives of the prebuckling state with respect to these parameters. An adjoint method bypasses the need for these derivatives by using instead the strain field associated with the second-order postbuckling state. An operator notation is used and the derivation is based on the principle of virtual work. The derivative computations are easily implemented in structural analysis programs. This is demonstrated by examples using a general purpose, finite element program and a shell-of-revolution program.

  5. Cell Factory Stability and Genetic Circuits for Improved Strain Development

    DEFF Research Database (Denmark)

    Rugbjerg, Peter

    . However, all synthetic gene systems -­ including the target metabolic pathways themselves -­ represent a possible fitness burden to the cell and thus constitute a threat to strain stability. In this thesis, several studies served to develop genetic systems for optimizing cell factory development...... systems can challenge the stability of strain designs. A metabolite-­producing Escherichia coli strain was long-­term cultured to study production stability and the dynamic effects of mutations within the cell population. A genetic error landscape of pathway disruptions was identified including particular......Development of new chemical-­‐producing microbial cell factories is an iterative trial-­and-­error process, and to screen candidate cells at high throughput, genetic biosensor systems are appealing. Each biosensor has distinct biological parameters, making modular tuning networks attractive...

  6. Chimaeric Virus-Like Particles Derived from Consensus Genome Sequences of Human Rotavirus Strains Co-Circulating in Africa

    Science.gov (United States)

    Jere, Khuzwayo C.; O'Neill, Hester G.; Potgieter, A. Christiaan; van Dijk, Alberdina A.

    2014-01-01

    Rotavirus virus-like particles (RV-VLPs) are potential alternative non-live vaccine candidates due to their high immunogenicity. They mimic the natural conformation of native viral proteins but cannot replicate because they do not contain genomic material which makes them safe. To date, most RV-VLPs have been derived from cell culture adapted strains or common G1 and G3 rotaviruses that have been circulating in communities for some time. In this study, chimaeric RV-VLPs were generated from the consensus sequences of African rotaviruses (G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes) characterised directly from human stool samples without prior adaptation of the wild type strains to cell culture. Codon-optimised sequences for insect cell expression of genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBAC vector, which allowed simultaneous expression of up to four genes using the Bac-to-Bac Baculovirus Expression System (BEVS; Invitrogen). Several combinations of the genome segments originating from different field strains were cloned to produce double-layered RV-VLPs (dRV-VLP; VP2/6), triple-layered RV-VLPs (tRV-VLP; VP2/6/7 or VP2/6/7/4) and chimaeric tRV-VLPs. The RV-VLPs were produced by infecting Spodoptera frugiperda 9 and Trichoplusia ni cells with recombinant baculoviruses using multi-cistronic, dual co-infection and stepwise-infection expression strategies. The size and morphology of the RV-VLPs, as determined by transmission electron microscopy, revealed successful production of RV-VLPs. The novel approach of producing tRV-VLPs, by using the consensus insect cell codon-optimised nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed-up vaccine research and development by by-passing the need to adapt rotaviruses to cell culture. Other problems associated with cell culture adaptation, such as possible changes in epitopes, can also be circumvented

  7. No postnatal maternal effect on male aggressiveness in wild-derived strains of house mice

    Czech Academy of Sciences Publication Activity Database

    Ďureje, Ľudovít; Vošlajerová Bímová, Barbora; Piálek, Jaroslav

    2011-01-01

    Roč. 37, č. 1 (2011), s. 48-55 ISSN 0096-140X R&D Projects: GA AV ČR KJB600930701 Institutional research plan: CEZ:AV0Z60930519 Keywords : aggression * cross-fostering * wild-derived inbred strains * house mouse Subject RIV: EG - Zoology Impact factor: 2.630, year: 2011

  8. Four-point bending protocols to study the effects of dynamic strain in osteoblastic cells in vitro.

    Science.gov (United States)

    Galea, Gabriel L; Price, Joanna S

    2015-01-01

    Strain engendered within bone tissue by mechanical loading of the skeleton is a major influence on the processes of bone modeling and remodeling and so a critical determinant of bone mass and architecture. The cells best placed to respond to strain in bone tissue are the resident osteocytes and osteoblasts. To address the mechanisms of strain-related responses in osteoblast-like cells, our group uses both in vivo and in vitro approaches, including a system of four-point bending of the substrate on which cells are cultured. A range of cell lines can be studied using this system but we routinely compare their responses to those in primary cultures of osteoblast-like cells derived from explants of mouse long bones. These cells show a range of well-characterized responses to physiological levels of strain, including increased proliferation, which in vivo is a feature of the osteogenic response.

  9. Detection and Identification of Probiotic Lactobacillus plantarum Strains by Multiplex PCR Using RAPD-Derived Primers.

    Science.gov (United States)

    Galanis, Alex; Kourkoutas, Yiannis; Tassou, Chrysoula C; Chorianopoulos, Nikos

    2015-10-22

    Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are two lactic acid bacteria (LAB) strains that have been isolated from Feta cheese. Both display significant potential for the production of novel probiotic food products. The aim of the present study was the development of an accurate and efficient method for the molecular detection and identification of the above strains in a single reaction. A multiplex PCR assay was designed for each strain, based on specific primers derived from Random Amplified Polymorphic DNA (RAPD) Sequenced Characterized Amplified Region (SCAR) analysis. The specificity of the assay was tested with a total of 23 different LAB strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay was also successfully applied for the detection of the above cultures in yogurt samples prepared in our lab. The proposed methodology may be applied for monitoring the presence of these strains in food products, thus evaluating their probiotic character. Moreover, our strategy may be adapted for other novel LAB strains with probiotic potential, thus providing a powerful tool for molecular discrimination that could be invaluable to the food industry.

  10. Detection and Identification of Probiotic Lactobacillus plantarum Strains by Multiplex PCR Using RAPD-Derived Primers

    Directory of Open Access Journals (Sweden)

    Alex Galanis

    2015-10-01

    Full Text Available Lactobacillus plantarum 2035 and Lactobacillus plantarum ACA-DC 2640 are two lactic acid bacteria (LAB strains that have been isolated from Feta cheese. Both display significant potential for the production of novel probiotic food products. The aim of the present study was the development of an accurate and efficient method for the molecular detection and identification of the above strains in a single reaction. A multiplex PCR assay was designed for each strain, based on specific primers derived from Random Amplified Polymorphic DNA (RAPD Sequenced Characterized Amplified Region (SCAR analysis. The specificity of the assay was tested with a total of 23 different LAB strains, for L. plantarum 2035 and L. plantarum ACA-DC 2640. The multiplex PCR assay was also successfully applied for the detection of the above cultures in yogurt samples prepared in our lab. The proposed methodology may be applied for monitoring the presence of these strains in food products, thus evaluating their probiotic character. Moreover, our strategy may be adapted for other novel LAB strains with probiotic potential, thus providing a powerful tool for molecular discrimination that could be invaluable to the food industry.

  11. Complete Genome Sequences of emm111 Type Streptococcus pyogenes Strain GUR, with Antitumor Activity, and Its Derivative Strain GURSA1 with an Inactivated emm Gene

    DEFF Research Database (Denmark)

    Suvorova, Maria A; Tsapieva, Anna N; Bak, Emilie Glad

    2017-01-01

    We present here the complete genome sequence of Streptococcus pyogenes type emm111 strain GUR, a throat isolate from a scarlet fever patient, which has been used to treat cancer patients in the former Soviet Union. We also present the complete genome sequence of its derivative strain GURSA1...

  12. 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.

  13. Stretching of red blood cells at high strain rates

    Science.gov (United States)

    Mancuso, J. E.; Ristenpart, W. D.

    2017-10-01

    Most work on the mechanical behavior of red blood cells (RBCs) in flow has focused on simple shear flows. Relatively little work has examined RBC deformations in the physiologically important extensional flow that occurs at the entrance to a constriction. In particular, previous work suggests that RBCs rapidly stretch out and then retract upon entering the constriction, but to date no model predicts this behavior for the extremely high strain rates typically experienced there. In this Rapid Communication, we use high speed video to perform systematic measurements of the dynamic stretching behavior of RBCs as they enter a microfluidic constriction. We demonstrate that both the Kelvin-Voigt and Skalak viscoelastic models capture the observed stretching dynamics, up to strain rates as high as 2000 s-1. The results indicate that the effective elastic modulus of the RBC membrane at these strain rates is an order of magnitude larger than moduli measured by micropipette aspiration or other low strain rate techniques.

  14. Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells.

    Directory of Open Access Journals (Sweden)

    Rossana Domenis

    Full Text Available A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression, proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs. Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression.

  15. ES-cell derived hematopoietic cells induce transplantation tolerance.

    Directory of Open Access Journals (Sweden)

    Sabrina Bonde

    Full Text Available BACKGROUND: Bone marrow cells induce stable mixed chimerism under appropriate conditioning of the host, mediating the induction of transplantation tolerance. However, their strong immunogenicity precludes routine use in clinical transplantation due to the need for harsh preconditioning and the requirement for toxic immunosuppression to prevent rejection and graft-versus-host disease. Alternatively, embryonic stem (ES cells have emerged as a potential source of less immunogenic hematopoietic progenitor cells (HPCs. Up till now, however, it has been difficult to generate stable hematopoietic cells from ES cells. METHODOLOGY/PRINCIPAL FINDINGS: Here, we derived CD45(+ HPCs from HOXB4-transduced ES cells and showed that they poorly express MHC antigens. This property allowed their long-term engraftment in sublethally irradiated recipients across MHC barriers without the need for immunosuppressive agents. Although donor cells declined in peripheral blood over 2 months, low level chimerism was maintained in the bone marrow of these mice over 100 days. More importantly, chimeric animals were protected from rejection of donor-type cardiac allografts. CONCLUSIONS: Our data show, for the first time, the efficacy of ES-derived CD45(+ HPCs to engraft in allogenic recipients without the use of immunosuppressive agents, there by protecting cardiac allografts from rejection.

  16. Characterisation of the exopolysaccharide (EPS)-producing Lactobacillus paraplantarum BGCG11 and its non-EPS producing derivative strains as potential probiotics.

    Science.gov (United States)

    Nikolic, Milica; López, Patricia; Strahinic, Ivana; Suárez, Ana; Kojic, Milan; Fernández-García, María; Topisirovic, Ljubisa; Golic, Natasa; Ruas-Madiedo, Patricia

    2012-08-17

    Traditional fermented foods are the best source for the isolation of strains with specific traits to act as functional starters and to keep the biodiversity of the culture collections. Besides, these strains could be used in the formulation of foods claimed to promote health benefits, i.e. those containing probiotic microorganisms. For the rational selection of strains acting as probiotics, several in vitro tests have been proposed. In the current study, we have characterized the probiotic potential of the strain Lactobacillus paraplantarum BGCG11, isolated from a Serbian soft, white, homemade cheese, which is able to produce a "ropy" exopolysaccharide (EPS). Three novobiocin derivative strains, which have lost the ropy phenotype, were characterized as well in order to determine the putative role of the EPS in the probiotic potential. Under chemically gastrointestinal conditions, all strains were able to survive around 1-2% (10(6)-10(7)cfu/ml cultivable bacteria) only when they were included in a food matrix (1% skimmed milk). The strains were more resistant to acid conditions than to bile salts and gastric or pancreatic enzymes, which could be due to a pre-adaptation of the parental strain to acidic conditions in the cheese habitat. The ropy EPS did not improve the survival of the producing strain. On the contrary, the presence of an EPS layer surrounding the strain BGCG11 hindered its adhesion to the three epithelial intestinal cell lines tested, since the adhesion of the three non-ropy derivatives was higher than the parental one and also than that of the reference strain Lactobacillus rhamnosus GG. Aiming to propose a potential target application of these strains as probiotics, the cytokine production of peripheral blood mononuclear cells (PBMC) was analyzed. The EPS-producing L. paraplantarum BGCG11 strain showed an anti-inflammatory or immunosuppressor profile whereas the non-ropy derivative strains induced higher pro-inflammatory response. In addition, when

  17. Characterization Of Bovine Adipose-Derived Stem Cells

    OpenAIRE

    Daniel Cebo

    2017-01-01

    Bovine adipose-derived stem cells were obtained from the subcutaneous abdominal adipose tissue. The cells were cultured by the modified tissue-explants method developed in our laboratory and then analyzed using optical microscopy and flow cytometry. These cells were able to replicate in our cell culture conditions. cell Flow cytometry showed that bovine adipose-derived stem cells expressed mesenchymal stem cell markers CD73 and CD90. Meanwhile haematopoietic markers CD45 and CD34 are absent f...

  18. Lobophorin C and D, New Kijanimicin Derivatives from a Marine Sponge-Associated Actinomycetal Strain AZS17

    Directory of Open Access Journals (Sweden)

    Yong-Cheng Lin

    2011-03-01

    Full Text Available Marine sponge Hymeniacidon sp. was collected from coastal waters of the East China Sea to isolate symbiotic microorganisms. The resulting sponge-associated actinomycete, Streptomyces carnosus strain AZS17, was cultivated in a 20 L volume of medium for production of bioactive secondary metabolites. Bioassay-guided isolation and purification by varied chromatographic methods yielded two new compounds of kijanimicin derivatives, AS7-2 and AS9-12. Their structures were elucidated by spectroscopy and comparison with literatures. Results showed these two compounds were structurally similar to the previously reported compounds lobophorin A and B, yet differed in specific bond forms, stereochemistry and optical activities. The two novel compounds were named lobophorin C and D. In vitro cytotoxicity investigation by MTT assay indicated their selective activities. Lobophorin C displayed potent cytotoxic activity against the human liver cancer cell line 7402, while lobophorin D showed significant inhibitory effect on human breast cancer cells MDA-MB 435.

  19. Development and introduction of inactivated poliovirus vaccines derived from Sabin strains in Japan.

    Science.gov (United States)

    Shimizu, Hiroyuki

    2016-04-07

    During the endgame of global polio eradication, the universal introduction of inactivated poliovirus vaccines is urgently required to reduce the risk of vaccine-associated paralytic poliomyelitis and polio outbreaks due to wild and vaccine-derived polioviruses. In particular, the development of inactivated poliovirus vaccines (IPVs) derived from the attenuated Sabin strains is considered to be a highly favorable option for the production of novel IPV that reduce the risk of facility-acquired transmission of poliovirus to the communities. In Japan, Sabin-derived IPVs (sIPVs) have been developed and introduced for routine immunization in November 2012. They are the first licensed sIPVs in the world. Consequently, trivalent oral poliovirus vaccine was used for polio control in Japan for more than half a century but has now been removed from the list of vaccines licensed for routine immunization. This paper reviews the development, introduction, characterization, and global status of IPV derived from attenuated Sabin strains. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. A new human male diploid cell strain, TIG-7: its age-related changes and comparison with a matched female TIG-1 cell strain.

    Science.gov (United States)

    Yamamoto, K; Kaji, K; Kondo, H; Matsuo, M; Shibata, Y; Tasaki, Y; Utakoji, T; Ooka, H

    1991-01-01

    A new human diploid cell strain, TIG-7, which has the male karyotype, was established and characterized. Isozyme and histocompatibility typing of the cell strain was performed. The average in vitro life span of the cells is 73 population doublings. Changes in cell volume, doubling time, saturation density, the efficiency of cell attachment, plating efficiency, and relative DNA content were examined during in vitro cellular aging. Hydrocortisone slightly prolongs the life span of the cell strain when the hormone is administered to the cultures during middle passages. The age-related changes in the parameters of TIG-7 are not appreciably different from those of the previously established TIG-1 cell strain. These results show that this cell strain is useful for research on cellular aging; further profit is anticipated from research using a combination of these two sexually different cell strains.

  1. Invasion of Porphyromonas gingivalis strains into vascular cells and tissue

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    Ingar Olsen

    2015-08-01

    Full Text Available Porphyromonas gingivalis is considered a major pathogen in adult periodontitis and is also associated with multiple systemic diseases, for example, cardiovascular diseases. One of its most important virulence factors is invasion of host cells. The invasion process includes attachment, entry/internalization, trafficking, persistence, and exit. The present review discusses these processes related to P. gingivalis in cardiovascular cells and tissue. Although most P. gingivalis strains invade, the invasion capacity of strains and the mechanisms of invasion including intracellular trafficking among them differ. This is consistent with the fact that there are significant differences in the pathogenicity of P. gingivalis strains. P. gingivalis invasion mechanisms are also dependent on types of host cells. Although much is known about the invasion process of P. gingivalis, we still have little knowledge of its exit mechanisms. Nevertheless, it is intriguing that P. gingivalis can remain viable in human cardiovascular cells and atherosclerotic plaque and later exit and re-enter previously uninfected host cells.

  2. Derivatives of a vancomycin-resistant Staphylococcus aureus strain isolated at Hershey Medical Center.

    Science.gov (United States)

    Bozdogan, Bülent; Ednie, Lois; Credito, Kim; Kosowska, Klaudia; Appelbaum, Peter C

    2004-12-01

    Antimicrobial susceptibilities and genetic relatedness of the vancomycin-resistant Staphylococcus aureus strain (VRSA) isolated at Hershey, Pa. (VRSA Hershey), and its vancomycin-susceptible and high-level-resistant derivatives were studied and compared to 32 methicillin-resistant S. aureus strains (MRSA) isolated from patients and medical staff in contact with the VRSA patient. Derivatives of VRSA were obtained by subculturing six VRSA colonies from the original culture with or without vancomycin. Ten days of drug-free subculture caused the loss of vanA in two vancomycin-susceptible derivatives for which vancomycin MICs were 1 to 4 microg/ml. Multistep selection of three VRSA clones with vancomycin for 10 days increased vancomycin MICs from 32 to 1,024 to 2,048 microg/ml. MICs of teicoplanin, dalbavancin, and oritavancin were also increased from 4, 0.5, and 0.12 to 64, 1, and 32 microg/ml, respectively. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing analysis indicated that VRSA Hershey was the vanA-acquired variety of a common MRSA clone in our hospital with sequence type 5 (ST5). Three of five vancomycin-intermediate S. aureus strains tested from geographically different areas were also ST5, and the Michigan VRSA was ST371, a one-allele variant of ST5. Derivatives of VRSA Hershey had differences in PFGE profiles and the size of SmaI fragment that carries the vanA gene cluster, indicating instability of this cluster in VRSA Hershey. However induction with vancomycin increased glycopeptide MICs and stabilized the resistance.

  3. Work strain in decontamination of hot cells, 2

    International Nuclear Information System (INIS)

    Kinouchi, Nobuyuki; Ikezawa, Yoshio

    1991-01-01

    In decontamination of hot cells, the workers should wear suitable protective clothing to protect them from internal exposure and skin contamination. But such protective clothing causes some work strain, especially heat-stress. As a simple method to evaluate quantitative work strain, we used sweat rates of the wearers. In the previous paper, sweat rates for workers with two types of protective clothing were reported. In the present paper, sweat rates under severer working conditions are measured for three types: (1) pressure ventilated blouse; (2) full-face mask and polyethylene coverall; (3) full-face mask and vinyl anorak. The measured values for 65 subjects widely scatter from 0.2 to 2.5 l/h for all the protective clothing. Based on these values, the effects of protective clothing and working conditions (ambient temperature and humidity) on work strain are discussed. (author)

  4. Orthopoxvirus species and strain differences in cell entry

    Energy Technology Data Exchange (ETDEWEB)

    Bengali, Zain; Satheshkumar, P.S. [Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-3210 (United States); Moss, Bernard, E-mail: bmoss@nih.gov [Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-3210 (United States)

    2012-11-25

    Vaccinia virus (VACV) enters cells by a low pH endosomal route or by direct fusion with the plasma membrane. We previously found differences in entry properties of several VACV strains: entry of WR was enhanced by low pH, reduced by bafilomycin A1 and relatively unaffected by heparin, whereas entry of IHD-J, Copenhagen and Elstree were oppositely affected. Since binding and entry modes may have been selected by specific conditions of in vitro propagation, we now examined the properties of three distinct, recently isolated cowpox viruses and a monkeypox virus as well as additional VACV and cowpox virus strains. The recent isolates were more similar to WR than to other VACV strains, underscoring the biological importance of endosomal entry by orthopoxviruses. Sequence comparisons, gene deletions and gene swapping experiments indicated that viral determinants, other than or in addition to the A26 and A25 'fusion-suppressor' proteins, impact entry properties.

  5. 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.

  6. Virulent and avirulent strains of equine arteritis virus induce different quantities of TNF-α and other proinflammatory cytokines in alveolar and blood-derived equine macrophages

    International Nuclear Information System (INIS)

    Moore, Brian D.; Balasuriya, Udeni B.R.; Watson, Johanna L.; Bosio, Catharine M.; MacKay, Robert J.; MacLachlan, N. James

    2003-01-01

    Equine arteritis virus (EAV) infects endothelial cells (ECs) and macrophages in horses, and many of the clinical manifestations of equine viral arteritis (EVA) reflect vascular injury. To further evaluate the potential role of EAV-induced, macrophage-derived cytokines in the pathogenesis of EVA, we infected cultured equine alveolar macrophages (AMphi), blood monocyte-derived macrophages (BMphi), and pulmonary artery ECs with either a virulent (KY84) or an avirulent (CA95) strain of EAV. EAV infection of equine AMphi, BMphi, and ECs resulted in their activation with increased transcription of genes encoding proinflammatory mediators, including interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α. Furthermore, the virulent KY84 strain of EAV induced significantly higher levels of mRNA encoding proinflammatory cytokines in infected AMphi and BMphi than did the avirulent CA95 strain. Treatment of equine ECs with the culture supernatants of EAV-infected AMphi and BMphi also resulted in EC activation with cell surface expression of E-selectin, whereas infection of ECs with purified EAV alone caused only minimal expression of E-selectin. The presence of TNF-α in the culture supernatants of EAV-infected equine AMphi, BMphi, and ECs was confirmed by bioassay, and the virulent KY84 strain of EAV induced significantly more TNF-α in all cell types than did the avirulent CA95 strain. Thus, the data indicate that EAV-induced, macrophage-derived cytokines may contribute to the pathogenesis of EVA in horses, and that the magnitude of the cytokine response of equine AMphi, BMphi, and ECs to EAV infection reflects the virulence of the infecting virus strain

  7. Characterization of field isolates of Suid herpesvirus 1 (Aujeszky's disease virus) as derivatives of attenuated vaccine strains

    DEFF Research Database (Denmark)

    Christensen, Laurids Siig; Medveczky, I.; Strandbygaard, Bertel

    1992-01-01

    Field isolates of suid herpesvirus 1 (Aujeszky's disease virus) from Poland and Hungary were identified by restriction fragment pattern analysis as derivatives of attenuated vaccine strains. The Polish isolates were found to be related to the BUK-TK-900 strain (Suivac A) which is widely used...

  8. Adipose-derived stem cells for treatment of chronic ulcers

    DEFF Research Database (Denmark)

    Holm, Jens Selch; Toyserkani, Navid Mohamadpour; Sorensen, Jens Ahm

    2018-01-01

    Chronic ulcers remain a difficult challenge in healthcare systems. While treatment options are limited, stem cells may be a novel alternative. Adipose-derived stem cells (ADSC) have become increasingly popular compared with bone marrow-derived stem cells as they are far easier to harvest...

  9. 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

  10. Uranium uptake by immobilized cells of Pseudomonas strain EPS 5028

    International Nuclear Information System (INIS)

    Pons, M.P.; Fuste, M.C.

    1993-01-01

    Polyacrylamide-gel-immobilized cells of Pseudomonas strain EPS 5028 were effective in the removal of uranium (U) from synthetic effluents. Metal accumulation was performed in an open system in columns filled with immobilized cells that were challenged with continuous flows containing U. Possible variable of the system were studied. Uranium uptake by the immobilized cells of this microorganism was affected by pH but not by temperature or flow rate. In addition, U binding could be interpreted in terms of the Freundlich adsorption isotherm indicating single-layer adsorption. The feasibility of reusing the immobilized cells was suggested after the recovery of U with a solution of 0.1 M sodium carbonate. (orig.)

  11. Antigenic characterization of a formalin-inactivated poliovirus vaccine derived from live-attenuated Sabin strains.

    Science.gov (United States)

    Tano, Yoshio; Shimizu, Hiroyuki; Martin, Javier; Nishimura, Yorihiro; Simizu, Bunsiti; Miyamura, Tatsuo

    2007-10-10

    A candidate inactivated poliovirus vaccine derived from live-attenuated Sabin strains (sIPV), which are used in the oral poliovirus vaccine (OPV), was prepared in a large-production scale. The modification of viral antigenic epitopes during the formalin inactivation process was investigated by capture ELISA assays using type-specific and antigenic site-specific monoclonal antibodies (MoAbs). The major antigenic site 1 was modified during the formalin inactivation of Sabin 1. Antigenic sites 1-3 were slightly modified during the formalin inactivation of Sabin 2 strain. Sites 1 and 3 were altered on inactivated Sabin 3 virus. These alterations were different to those shown by wild-type Saukett strain, used in conventional IPV (cIPV). It has been previously reported that type 1 sIPV showed higher immunogenicity to type 1 cIPV whereas types 2 and 3 sIPV induced lower level of immunogenicity than their cIPV counterparts. Our results suggest that the differences in epitope structure after formalin inactivation may account, at least in part, for the observed differences in immunogenicity between Sabin and wild-type inactivated poliovaccines.

  12. Competitive accumulation of betaines by Escherichia coli K-12 and derivative strains lacking betaine porters.

    Science.gov (United States)

    Randall, K; Lever, M; Peddie, B A; Chambers, S T

    1995-08-17

    Escherichia coli was grown in hyperosmotic media containing both glycine betaine and one other betaine. E. coli K-12 derivative WG439 (putP- proP- proU-) did not accumulate any of 15 betaines. Strains WG445 (putP- proP- proU+), WG443 (putP- proP+ proU-) and the control strains all accumulated less betaine, (CH3)3N(+)-(CH2)n-COO-, when n was greater than 1. Accumulation was not detectable when n = 5. Both L- and D-isomers of alpha-substituted betaines were accumulated by both strains WG443 and WG445, the D-isomers more slowly. Hydroxylated alpha-substituted betaines were accumulated relatively more through the osmoregulated transport protein ProU than through ProP. In actively growing cultures glycine betaine appeared to be the preferred substrate for accumulation, but the proportion of the second accumulated betaine increased as cultures approached stationary phase.

  13. Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines

    OpenAIRE

    Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

    2016-01-01

    Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However,...

  14. Highly Efficient CRISPR/Cas9-Mediated Cloning and Functional Characterization of Gastric Cancer-Derived Epstein-Barr Virus Strains.

    Science.gov (United States)

    Kanda, Teru; Furuse, Yuki; Oshitani, Hitoshi; Kiyono, Tohru

    2016-05-01

    The Epstein-Barr virus (EBV) is etiologically linked to approximately 10% of gastric cancers, in which viral genomes are maintained as multicopy episomes. EBV-positive gastric cancer cells are incompetent for progeny virus production, making viral DNA cloning extremely difficult. Here we describe a highly efficient strategy for obtaining bacterial artificial chromosome (BAC) clones of EBV episomes by utilizing a CRISPR/Cas9-mediated strand break of the viral genome and subsequent homology-directed repair. EBV strains maintained in two gastric cancer cell lines (SNU719 and YCCEL1) were cloned, and their complete viral genome sequences were determined. Infectious viruses of gastric cancer cell-derived EBVs were reconstituted, and the viruses established stable latent infections in immortalized keratinocytes. While Ras oncoprotein overexpression caused massive vacuolar degeneration and cell death in control keratinocytes, EBV-infected keratinocytes survived in the presence of Ras expression. These results implicate EBV infection in predisposing epithelial cells to malignant transformation by inducing resistance to oncogene-induced cell death. Recent progress in DNA-sequencing technology has accelerated EBV whole-genome sequencing, and the repertoire of sequenced EBV genomes is increasing progressively. Accordingly, the presence of EBV variant strains that may be relevant to EBV-associated diseases has begun to attract interest. Clearly, the determination of additional disease-associated viral genome sequences will facilitate the identification of any disease-specific EBV variants. We found that CRISPR/Cas9-mediated cleavage of EBV episomal DNA enabled the cloning of disease-associated viral strains with unprecedented efficiency. As a proof of concept, two gastric cancer cell-derived EBV strains were cloned, and the infection of epithelial cells with reconstituted viruses provided important clues about the mechanism of EBV-mediated epithelial carcinogenesis. This

  15. A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

    Directory of Open Access Journals (Sweden)

    Anne-lie Ståhl

    2015-02-01

    Full Text Available Shiga toxin (Stx is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS, associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.

  16. Matrix production and organization by endothelial colony forming cells in mechanically strained engineered tissue constructs.

    Directory of Open Access Journals (Sweden)

    Nicky de Jonge

    Full Text Available AIMS: Tissue engineering is an innovative method to restore cardiovascular tissue function by implanting either an in vitro cultured tissue or a degradable, mechanically functional scaffold that gradually transforms into a living neo-tissue by recruiting tissue forming cells at the site of implantation. Circulating endothelial colony forming cells (ECFCs are capable of differentiating into endothelial cells as well as a mesenchymal ECM-producing phenotype, undergoing Endothelial-to-Mesenchymal-transition (EndoMT. We investigated the potential of ECFCs to produce and organize ECM under the influence of static and cyclic mechanical strain, as well as stimulation with transforming growth factor β1 (TGFβ1. METHODS AND RESULTS: A fibrin-based 3D tissue model was used to simulate neo-tissue formation. Extracellular matrix organization was monitored using confocal laser-scanning microscopy. ECFCs produced collagen and also elastin, but did not form an organized matrix, except when cultured with TGFβ1 under static strain. Here, collagen was aligned more parallel to the strain direction, similar to Human Vena Saphena Cell-seeded controls. Priming ECFC with TGFβ1 before exposing them to strain led to more homogenous matrix production. CONCLUSIONS: Biochemical and mechanical cues can induce extracellular matrix formation by ECFCs in tissue models that mimic early tissue formation. Our findings suggest that priming with bioactives may be required to optimize neo-tissue development with ECFCs and has important consequences for the timing of stimuli applied to scaffold designs for both in vitro and in situ cardiovascular tissue engineering. The results obtained with ECFCs differ from those obtained with other cell sources, such as vena saphena-derived myofibroblasts, underlining the need for experimental models like ours to test novel cell sources for cardiovascular tissue engineering.

  17. Graphene and its derivatives for cell biotechnology.

    Science.gov (United States)

    Yang, Mei; Yao, Jun; Duan, Yixiang

    2013-01-07

    Every few years, a novel material with salient and often unique properties emerges and attracts both academic and industrial interest from the scientific community. The latest blockbuster is graphene, an increasingly important nanomaterial with atomically thin sheets of carbon, which has become a shining star and has shown great promise in the field of material science and nanotechnology. In recent years, it has changed from being the exclusive domain of physicists to the new passion of chemists and biologists. Graphene and its derivatives are now at the forefront of nearly every rapidly developing field of science and engineering, including biochemistry, biomedicine and certain cutting-edge interdisciplines that have intense popularity. The aim of this review is, firstly, to provide readers with a comprehensive, systematic and in-depth prospective of graphene's band structure and properties, and secondly, to concentrate on the recent progress in producing graphene-based nanomaterials, including mechanical exfoliation, chemical vapor deposition, plasma enhanced chemical vapor deposition, chemical reduction of graphene oxide, total organic synthesis, electrochemical synthesis and other fabrication strategies widely accepted by research scientists. At the same time, important definitions related to graphene are also introduced. The focus of this Tutorial Review is to emphasize the current situation and significance of using this new kind of two-dimensional material in the hot and emerging fields that are closely related to human life quality, for instance, cell biochemistry, bioimaging along with other frontier areas. Finally, the latest developments and possible impact that affect the heart of the whole scientific community have been discussed. In addition, the future trends along with potential challenges of this rapidly rising layered carbon have been pointed out in this paper.

  18. Genetic variation of viral protein 1 genes of field strains of waterfowl parvoviruses and their attenuated derivatives.

    Science.gov (United States)

    Tsai, Hsiang-Jung; Tseng, Chun-hsien; Chang, Poa-chun; Mei, Kai; Wang, Shih-Chi

    2004-09-01

    To understand the genetic variations between the field strains of waterfowl parvoviruses and their attenuated derivatives, we analyzed the complete nucleotide sequences of the viral protein 1 (VP1) genes of nine field strains and two vaccine strains of waterfowl parvoviruses. Sequence comparison of the VP1 proteins showed that these viruses could be divided into goose parvovirus (GPV) related and Muscovy duck parvovirus (MDPV) related groups. The amino acid difference between GPV- and MDPV-related groups ranged from 13.1% to 15.8%, and the most variable region resided in the N terminus of VP2. The vaccine strains of GPV and MDPV exhibited only 1.2% and 0.3% difference in amino acid when compared with their parental field strains, and most of these differences resided in residues 497-575 of VP1, suggesting that these residues might be important for the attenuation of GPV and MDPV. When the GPV strains isolated in 1982 (the strain 82-0308) and in 2001 (the strain 01-1001) were compared, only 0.3% difference in amino acid was found, while MDPV strains isolated in 1990 (the strain 90-0219) and 1997 (the strain 97-0104) showed only 0.4% difference in amino acid. The result indicates that the genome of waterfowl parvovirus had remained highly stable in the field.

  19. Anterior cruciate ligament-derived cells have high chondrogenic potential.

    Science.gov (United States)

    Furumatsu, Takayuki; Hachioji, Motomi; Saiga, Kenta; Takata, Naoki; Yokoyama, Yusuke; Ozaki, Toshifumi

    2010-01-01

    Anterior cruciate ligament (ACL)-derived cells have a character different from medial collateral ligament (MCL)-derived cells. However, the critical difference between ACL and MCL is still unclear in their healing potential and cellular response. The objective of this study was to investigate the mesenchymal differentiation property of each ligament-derived cell. Both ligament-derived cells differentiated into adipogenic, osteogenic, and chondrogenic lineages. In chondrogenesis, ACL-derived cells had the higher chondrogenic property than MCL-derived cells. The chondrogenic marker genes, Sox9 and alpha1(II) collagen (Col2a1), were induced faster in ACL-derived pellets than in MCL-derived pellets. Sox9 expression preceded the increase of Col2a1 in both pellet-cultured cells. However, the expression level of Sox9 and a ligament/tendon transcription factor Scleraxis did not parallel the increase of Col2a1 expression along with chondrogenic induction. The present study demonstrates that the balance between Sox9 and Scleraxis have an important role in the chondrogenic differentiation of ligament-derived cells. Copyright 2009 Elsevier Inc. All rights reserved.

  20. 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...

  1. Phage-resistance linked to cell heterogeneity in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1.

    Science.gov (United States)

    Suárez, Viviana B; Maciel, Natalia; Guglielmotti, Daniela; Zago, Miriam; Giraffa, Giorgio; Reinheimer, Jorge

    2008-12-10

    The aim of this work was to study the relationship between the cell morphological heterogeneity and the phage-resistance in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1. Two morphological variants (named C and T) were isolated from this strain. Phage-resistant derivatives were isolated from them and the percentage of occurrence of confirmed phage-resistant cells was 0.001% of the total cellular population. Within these phage-resistant cell derivatives there were T (3 out of 4 total isolates) and C (1 out of 4 total isolates) variants. The study of some technological properties (e.g. proteolytic and acidifying activities) demonstrated that most of phage-resistant derivatives were not as good as the parental strain. However, for one derivative (a T variant), the technological properties were better than those of the parental strain. On the other hand, it was possible to determinate that the system of phage-resistance in the T variants was interference in adsorption step, with adsorption rates M.

  2. Breeding of a xylose-fermenting hybrid strain by mating genetically engineered haploid strains derived from industrial Saccharomyces cerevisiae.

    Science.gov (United States)

    Inoue, Hiroyuki; Hashimoto, Seitaro; Matsushika, Akinori; Watanabe, Seiya; Sawayama, Shigeki

    2014-12-01

    The industrial Saccharomyces cerevisiae IR-2 is a promising host strain to genetically engineer xylose-utilizing yeasts for ethanol fermentation from lignocellulosic hydrolysates. Two IR-2-based haploid strains were selected based upon the rate of xylulose fermentation, and hybrids were obtained by mating recombinant haploid strains harboring heterogeneous xylose dehydrogenase (XDH) (wild-type NAD(+)-dependent XDH or engineered NADP(+)-dependent XDH, ARSdR), xylose reductase (XR) and xylulose kinase (XK) genes. ARSdR in the hybrids selected for growth rates on yeast extract-peptone-dextrose (YPD) agar and YP-xylose agar plates typically had a higher activity than NAD(+)-dependent XDH. Furthermore, the xylose-fermenting performance of the hybrid strain SE12 with the same level of heterogeneous XDH activity was similar to that of a recombinant strain of IR-2 harboring a single set of genes, XR/ARSdR/XK. These results suggest not only that the recombinant haploid strains retain the appropriate genetic background of IR-2 for ethanol production from xylose but also that ARSdR is preferable for xylose fermentation.

  3. Control of RFM strain endogenous retrovirus in RFM mouse cells

    International Nuclear Information System (INIS)

    Tennant, R.W.; Otten, J.A.; Wang, T.W.; Liou, R.S.; Brown, A.; Yang, W.K.

    1983-01-01

    RFM/Un mice express an endogenous type C retrovirus throughout their life span in many tissues; primary or established embryo fibroblast cell cultures do not express a virus but can be induced by exposure to 5-iodo-2'-deoxyuridine. All of our sources yielded a single ecotropic virus (RFV) which appeared to be related more closely to the endogenous N-tropic virus (WN1802N) of BALB/c mice than to Gross leukemia virus on the basis of two-dimensional gel electropherograms of virion proteins. No xenotropic or recombinant viruses were isolated by cocultivation techniques. RFV is N-tropic, and RFM/Un cells possess the Fv-1/sup n/ allele, as indicated by restriction of B-tropic virus and susceptibility to Gross strain N-tropic virus. However, RFM cells are highly resistant to RFV and other endogenous N-tropic viruses. This resistance is expressed by two-hit titration kinetics and by inhibition of viral linear duplex DNA formation. This is similar to the effects of the Fv-1 locus, but preliminary work has shown no apparent genetic linkage between the two restrictions. The relative strength of the restriction, the presence of a single class of ecotropic virus, and the absence of recombinant viruses suggest that in RFM mice virus is expressed only in cells in which it is induced and not by cell-to-cell transmission

  4. DNA repair in a Fanconi's anemia fibroblast cell strain

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Little, J.B.; Weichselbaum, R.R.

    1979-01-01

    DNA repair and colony survival were measured in fibroblasts from a patient with Fanconi's anemia, HG 261, and from normal human donors after exposure to these cells to the cross-linking agent mitomycin C, X-rays or ultraviolet light. Survival was similar in HG 261 and normal cells after X-ray or ultraviolet radiation, but was reduced in the Fanconi's anemia cells after treatment with mitomycin C. The level of DNA cross-linking, as measured by the method of alkaline elution, was the same in both cell strains after exposure to various doses of mitomycin C. With incubation after drug treatment, a gradual decrease in the amount of cross-linking was observed, the rate of this apparent repair of cross-link damage was the same in both normal and HG 261 cells. The rejoining of DNA single strand breaks after X-irradiation and the production of excision breaks after ultraviolet radiation were also normal in HG 261 cells as determined by alkaline elution. (Auth.)

  5. DNA repair in a Fanconi's anemia fibroblast cell strain

    Energy Technology Data Exchange (ETDEWEB)

    Fornace, Jr, A J; Little, J B [Harvard School of Public Health, Boston, MA (USA); Weichselbaum, R R [Harvard Medical School, Boston, MA (USA)

    1979-01-26

    DNA repair and colony survival were measured in fibroblasts from a patient with Fanconi's anemia, HG 261, and from normal human donors after exposure to these cells to the cross-linking agent mitomycin C, X-rays or ultraviolet light. Survival was similar in HG 261 and normal cells after X-ray or ultraviolet radiation, but was reduced in the Fanconi's anemia cells after treatment with mitomycin C. The level of DNA cross-linking, as measured by the method of alkaline elution, was the same in both cell strains after exposure to various doses of mitomycin C. With incubation after drug treatment, a gradual decrease in the amount of cross-linking was observed, the rate of this apparent repair of cross-link damage was the same in both normal and HG 261 cells. The rejoining of DNA single strand breaks after X-irradiation and the production of excision breaks after ultraviolet radiation were also normal in HG 261 cells as determined by alkaline elution.

  6. Induced pluripotent stem cells-derived myeloid-derived suppressor cells regulate the CD8+ T cell response

    Directory of Open Access Journals (Sweden)

    Daniel Joyce

    2018-05-01

    Full Text Available Myeloid-derived suppressor cells (MDSCs are markedly increased in cancer patients and tumor-bearing mice and promote tumor growth and survival by inhibiting host innate and adaptive immunity. In this study, we generated and characterized MDSCs from murine-induced pluripotent stem cells (iPSCs. The iPSCs were co-cultured with OP9 cells, stimulated with GM-CSF, and became morphologically heterologous under co-culturing with hepatic stellate cells. Allogeneic and OVA-specific antigen stimulation demonstrated that iPS-MDSCs have a T-cell regulatory function. Furthermore, a popliteal lymph node assay and autoimmune hepatitis model showed that iPS-MDSCs also regulate immune responsiveness in vivo and have a therapeutic effect against hepatitis. Taken together, our results demonstrated a method of generating functional MDSCs from iPSCs and highlighted the potential of iPS-MDSCs as a key cell therapy resource for transplantation and autoimmune diseases. Keywords: Myeloid-derived suppressor cells, Induced pluripotent stem cells, T cell response

  7. A national reference for inactivated polio vaccine derived from Sabin strains in Japan.

    Science.gov (United States)

    Shirato, Haruko; Someya, Yuichi; Ochiai, Masaki; Horiuchi, Yoshinobu; Takahashi, Motohide; Takeda, Naokazu; Wakabayashi, Kengo; Ouchi, Yasumitsu; Ota, Yoshihiro; Tano, Yoshio; Abe, Shinobu; Yamazaki, Shudo; Wakita, Takaji

    2014-09-08

    As one aspect of its campaign to eradicate poliomyelitis, the World Health Organization (WHO) has encouraged development of the inactivated polio vaccine (IPV) derived from the Sabin strains (sIPV) as an option for an affordable polio vaccine, especially in low-income countries. The Japan Poliomyelitis Research Institute (JPRI) inactivated three serotypes of the Sabin strains and made sIPV preparations, including serotypes 1, 2 and 3 D-antigens in the ratio of 3:100:100. The National Institute of Infectious Diseases, Japan, assessed the immunogenic stability of these sIPV preparations in a rat potency test, according to an evaluation method recommended by the WHO. The immunogenicity of the three serotypes was maintained for at least 4 years when properly stored under -70°C. Based on these data, the sIPV preparations made by JPRI have been approved as national reference vaccines by the Japanese national control authority and used for the quality control of the tetracomponent sIPV-containing diphtheria-tetanus-acellular pertussis combination vaccines that were licensed for a routine polio immunization in Japan. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Inactive vaccine derived from velogenic strain of local Newcastle disease virus .

    Directory of Open Access Journals (Sweden)

    Darminto

    1996-03-01

    Full Text Available The objective of this research is to evaluate an application of an inactive Newcastle disease (ND vaccine derived from velogenic strain of local Newcastle disease virus (NDV. In this research . the Ira strain of velogenic ND virus was grown in specific pathogen free (SPF eggs and then was inactivated by formalin at a final concentration of 1 :1,000 at 4°C. The inactive antigen was then emulsified with an oil adjuvant or aluminium hydroxide gel before being administered for vaccination in layers and compared to a commercial inactive ND vaccine . Results indicated that application of these inactivated ND vaccines for booster vaccination following vaccination with an active lentogenic ND virus in pullets nearly producing eggs, resulted in high antibody titre which persisted for considerable long period of time and capable of protecting layers from sick of ND and from reducing egg production . Hence, it could be concluded that the inactivated vaccine emulsified in either oil-adjuvant (lanolin-paraffin or aluminium hydroxide gel were considered to be highly immunogenic and capable of protecting layers from sick of ND and from reducing egg production

  9. Adipose-derived mesenchymal stem cells and regenerative medicine.

    Science.gov (United States)

    Konno, Masamitsu; Hamabe, Atsushi; Hasegawa, Shinichiro; Ogawa, Hisataka; Fukusumi, Takahito; Nishikawa, Shimpei; Ohta, Katsuya; Kano, Yoshihiro; Ozaki, Miyuki; Noguchi, Yuko; Sakai, Daisuke; Kudoh, Toshihiro; Kawamoto, Koichi; Eguchi, Hidetoshi; Satoh, Taroh; Tanemura, Masahiro; Nagano, Hiroaki; Doki, Yuichiro; Mori, Masaki; Ishii, Hideshi

    2013-04-01

    Adipose tissue-derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β-cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow-derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  10. Whole exome sequencing of wild-derived inbred strains of mice improves power to link phenotype and genotype.

    Science.gov (United States)

    Chang, Peter L; Kopania, Emily; Keeble, Sara; Sarver, Brice A J; Larson, Erica; Orth, Annie; Belkhir, Khalid; Boursot, Pierre; Bonhomme, François; Good, Jeffrey M; Dean, Matthew D

    2017-10-01

    The house mouse is a powerful model to dissect the genetic basis of phenotypic variation, and serves as a model to study human diseases. Despite a wealth of discoveries, most classical laboratory strains have captured only a small fraction of genetic variation known to segregate in their wild progenitors, and existing strains are often related to each other in complex ways. Inbred strains of mice independently derived from natural populations have the potential to increase power in genetic studies with the addition of novel genetic variation. Here, we perform exome-enrichment and high-throughput sequencing (~8× coverage) of 26 wild-derived strains known in the mouse research community as the "Montpellier strains." We identified 1.46 million SNPs in our dataset, approximately 19% of which have not been detected from other inbred strains. This novel genetic variation is expected to contribute to phenotypic variation, as they include 18,496 nonsynonymous variants and 262 early stop codons. Simulations demonstrate that the higher density of genetic variation in the Montpellier strains provides increased power for quantitative genetic studies. Inasmuch as the power to connect genotype to phenotype depends on genetic variation, it is important to incorporate these additional genetic strains into future research programs.

  11. Emergence of hyper-resistant Escherichia coli MG1655 derivative strains after applying sub-inhibitory doses of individual constituents of essential oils

    Directory of Open Access Journals (Sweden)

    Beatriz eChueca

    2016-03-01

    Full Text Available The improvement of food preservation by using essential oils (EOs and their individual constituents (ICs is attracting enormous interest worldwide. Until now, researchers considered that treatments with such antimicrobial compounds did not induce bacterial resistance via a phenotypic (i.e. transient response. Nevertheless, the emergence of genotypic (i.e. stable resistance after treatment with these compounds had not been previously tested. Our results confirm that growth of Escherichia coli MG1655 in presence of sub-inhibitory concentrations of the ICs carvacrol, citral, and (+-limonene oxide do not increase resistance to further treatments with either the same IC (direct resistance or with other preservation treatments (cross-resistance such as heat or pulsed electric fields (PEF. Bacterial mutation frequency was likewise lower when those IC’s were applied; however, after 10 days of re-culturing cells in presence of sub-inhibitory concentrations of the ICs, we were able to isolate several derivative strains (i.e. mutants displaying an increased minimum inhibitory concentration to those ICs. Furthermore, when compared to the wild type (WT strain, they also displayed direct resistance and cross-resistance. Derivative strains selected with carvacrol and citral also displayed morphological changes involving filamentation along with cell counts at late-stationary growth phase that were lower than the WT strain. In addition, co-cultures of each derivative strain with the WT strain resulted in a predominance of the original strain in absence of ICs, indicating that mutants would not out-compete WT cells under optimal growth conditions. Nevertheless, growth in the presence of ICs facilitated the selection of these resistant mutants. Thus, as a result, subsequent food preservation treatments of these bacterial cultures might be less effective than expected for WT cultures. In conclusion, this study recommends that treatment with ICs at sub

  12. Emergence of Hyper-Resistant Escherichia coli MG1655 Derivative Strains after Applying Sub-Inhibitory Doses of Individual Constituents of Essential Oils.

    Science.gov (United States)

    Chueca, Beatriz; Berdejo, Daniel; Gomes-Neto, Nelson J; Pagán, Rafael; García-Gonzalo, Diego

    2016-01-01

    The improvement of food preservation by using essential oils (EOs) and their individual constituents (ICs) is attracting enormous interest worldwide. Until now, researchers considered that treatments with such antimicrobial compounds did not induce bacterial resistance via a phenotypic (i.e., transient) response. Nevertheless, the emergence of genotypic (i.e., stable) resistance after treatment with these compounds had not been previously tested. Our results confirm that growth of Escherichia coli MG1655 in presence of sub-inhibitory concentrations of the ICs carvacrol, citral, and (+)-limonene oxide do not increase resistance to further treatments with either the same IC (direct resistance) or with other preservation treatments (cross-resistance) such as heat or pulsed electric fields (PEF). Bacterial mutation frequency was likewise lower when those IC's were applied; however, after 10 days of re-culturing cells in presence of sub-inhibitory concentrations of the ICs, we were able to isolate several derivative strains (i.e., mutants) displaying an increased minimum inhibitory concentration to those ICs. Furthermore, when compared to the wild type (WT) strain, they also displayed direct resistance and cross-resistance. Derivative strains selected with carvacrol and citral also displayed morphological changes involving filamentation along with cell counts at late-stationary growth phase that were lower than the WT strain. In addition, co-cultures of each derivative strain with the WT strain resulted in a predominance of the original strain in absence of ICs, indicating that mutants would not out-compete WT cells under optimal growth conditions. Nevertheless, growth in the presence of ICs facilitated the selection of these resistant mutants. Thus, as a result, subsequent food preservation treatments of these bacterial cultures might be less effective than expected for WT cultures. In conclusion, this study recommends that treatment with ICs at sub

  13. Isolation and Characterization of Current Human Coronavirus Strains in Primary Human Epithelial Cell Cultures Reveal Differences in Target Cell Tropism

    Science.gov (United States)

    Dijkman, Ronald; Jebbink, Maarten F.; Koekkoek, Sylvie M.; Deijs, Martin; Jónsdóttir, Hulda R.; Molenkamp, Richard; Ieven, Margareta; Goossens, Herman; Thiel, Volker

    2013-01-01

    The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in nonhospitalized and hospitalized children. Studies of the fundamental aspects of these HCoV infections at the primary entry port, such as cell tropism, are seriously hampered by the lack of a universal culture system or suitable animal models. To expand the knowledge on fundamental virus-host interactions for all four HCoVs at the site of primary infection, we used pseudostratified HAE cell cultures to isolate and characterize representative clinical HCoV strains directly from nasopharyngeal material. Ten contemporary isolates were obtained, representing HCoV-229E (n = 1), HCoV-NL63 (n = 1), HCoV-HKU1 (n = 4), and HCoV-OC43 (n = 4). For each strain, we analyzed the replication kinetics and progeny virus release on HAE cell cultures derived from different donors. Surprisingly, by visualizing HCoV infection by confocal microscopy, we observed that HCoV-229E employs a target cell tropism for nonciliated cells, whereas HCoV-OC43, HCoV-HKU1, and HCoV-NL63 all infect ciliated cells. Collectively, the data demonstrate that HAE cell cultures, which morphologically and functionally resemble human airways in vivo, represent a robust universal culture system for isolating and comparing all contemporary HCoV strains. PMID:23427150

  14. Strain energy storage and dissipation rate in active cell mechanics

    Science.gov (United States)

    Agosti, A.; Ambrosi, D.; Turzi, S.

    2018-05-01

    When living cells are observed at rest on a flat substrate, they can typically exhibit a rounded (symmetric) or an elongated (polarized) shape. Although the cells are apparently at rest, the active stress generated by the molecular motors continuously stretches and drifts the actin network, the cytoskeleton of the cell. In this paper we theoretically compare the energy stored and dissipated in this active system in two geometric configurations of interest: symmetric and polarized. We find that the stored energy is larger for a radially symmetric cell at low activation regime, while the polar configuration has larger strain energy when the active stress is beyond a critical threshold. Conversely, the dissipation of energy in a symmetric cell is always larger than that of a nonsymmetric one. By a combination of symmetry arguments and competition between surface and bulk stress, we argue that radial symmetry is an energetically expensive metastable state that provides access to an infinite number of lower-energy states, the polarized configurations.

  15. Carbon-flux distribution within Streptomyces coelicolor metabolism: a comparison between the actinorhodin-producing strain M145 and its non-producing derivative M1146.

    Directory of Open Access Journals (Sweden)

    Fabien Coze

    Full Text Available Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two Streptomyces coelicolor A3 (2 strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and (13C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest.

  16. Gastrocnemius tendon strain in a dog treated with autologous mesenchymal stem cells and a custom orthosis.

    Science.gov (United States)

    Case, J Brad; Palmer, Ross; Valdes-Martinez, Alex; Egger, Erick L; Haussler, Kevin K

    2013-05-01

    To report clinical findings and outcome in a dog with gastrocnemius tendon strain treated with autologous mesenchymal stem cells and a custom orthosis. Clinical report. A 4-year-old spayed female Border Collie. Bone-marrow derived, autologous mesenchymal stem cells were transplanted into the tendon core lesion. A custom, progressive, dynamic orthosis was fit to the tarsus. Serial orthopedic examinations and ultrasonography as well as long-term force-plate gait analysis were utilized for follow up. Lameness subjectively resolved and peak vertical force increased from 43% to 92% of the contralateral pelvic limb. Serial ultrasonographic examinations revealed improved but incomplete restoration of normal linear fiber pattern of the gastrocnemius tendon. Findings suggest that autologous mesenchymal stem cell transplantation with custom, progressive, dynamic orthosis may be a viable, minimally invasive technique for treatment of calcaneal tendon injuries in dogs. © Copyright 2013 by The American College of Veterinary Surgeons.

  17. The Bone Marrow-Derived Stromal Cells

    DEFF Research Database (Denmark)

    Tencerova, Michaela; Kassem, Moustapha

    2016-01-01

    Bone marrow (BM) microenvironment represents an important compartment of bone that regulates bone homeostasis and the balance between bone formation and bone resorption depending on the physiological needs of the organism. Abnormalities of BM microenvironmental dynamics can lead to metabolic bone...... diseases. BM stromal cells (also known as skeletal or mesenchymal stem cells) [bone marrow stromal stem cell (BMSC)] are multipotent stem cells located within BM stroma and give rise to osteoblasts and adipocytes. However, cellular and molecular mechanisms of BMSC lineage commitment to adipocytic lineage...

  18. 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.

  19. Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone.

    Directory of Open Access Journals (Sweden)

    Rika Etchuuya

    Full Text Available Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain. In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as 'cell-to-cell transformation'. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ∼9 kDa and ∼30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10(-5-10(-6, suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria.

  20. Physiology of stem cell-derived cardiomyocytes

    NARCIS (Netherlands)

    Boer, T.P. de

    2007-01-01

    All chapters in this thesis revolve around the general theme, stem cells and their electrophysiological characteristics and capacity to induce pro-arrhythmia. The first part of this thesis focusses on key aspects that are relevant to possible pro-arrhythmic effects of stem cell transplantation. An

  1. 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.

  2. Senescence and quiescence in adipose-derived stromal cells

    DEFF Research Database (Denmark)

    Søndergaard, Rebekka Harary; Follin, Bjarke; Lund, Lisbeth Drozd

    2017-01-01

    Background aims. Adipose-derived stromal cells (ASCs) are attractive sources for cell-based therapies. The hypoxic niche of ASCs in vivo implies that cells will benefit from hypoxia during in vitro expansion. Human platelet lysate (hPL) enhances ASC proliferation rates, compared with fetal bovine...

  3. Actin and microtubule networks contribute differently to cell response for small and large strains

    Science.gov (United States)

    Kubitschke, H.; Schnauss, J.; Nnetu, K. D.; Warmt, E.; Stange, R.; Kaes, J.

    2017-09-01

    Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell’s response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small (≤5% deformation) and large strains (>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules.

  4. Staphylococcus aureus produces membrane-derived vesicles that induce host cell death.

    Directory of Open Access Journals (Sweden)

    Mamata Gurung

    Full Text Available Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs produced by gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.

  5. Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

    Science.gov (United States)

    Chaudhari, Pratik Rajeev; Charles, Silvania Emlit; D'Souza, Zinia Charlotte; Vaidya, Milind Murlidhar

    2017-11-15

    BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through β4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating β4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Skin appendage-derived stem cells: cell biology and potential for wound repair

    OpenAIRE

    Xie, Jiangfan; Yao, Bin; Han, Yutong; Huang, Sha; Fu, Xiaobing

    2016-01-01

    Stem cells residing in the epidermis and skin appendages are imperative for skin homeostasis and regeneration. These stem cells also participate in the repair of the epidermis after injuries, inducing restoration of tissue integrity and function of damaged tissue. Unlike epidermis-derived stem cells, comprehensive knowledge about skin appendage-derived stem cells remains limited. In this review, we summarize the current knowledge of skin appendage-derived stem cells, including their fundament...

  7. The performance of five fruit-derived and freeze-dried potentially probiotic Lactobacillus strains in apple, orange and grape juices.

    Science.gov (United States)

    Garcia, Estefânia Fernandes; de Oliveira Araújo, Amanda; Luciano, Winnie Alencar; de Albuquerque, Thatyane Mariano Rodrigues; de Oliveira Arcanjo, Narciza Maria; Madruga, Marta Suely; Dos Santos Lima, Marcos; Magnani, Marciane; Saarela, Maria; de Souza, Evandro Leite

    2018-03-30

    This study assessed the survival of the fruit-derived and freeze-dried L. plantarum 49, L. brevis 59, L. paracasei 108, L. fermentum 111 and L. pentosus 129 strains during frozen storage and when incorporated into apple, orange and grape juice stored under refrigeration. Physicochemical parameters of juices containing the freeze-dried Lactobacillus strains and the survival of the test strains in the fruit juices during in vitro digestion were also evaluated. No decreases in survival rates (log N/log N0) of the freeze-dried cells were observed up to 1 month of storage. The survival rates of the freeze-dried strains L. plantarum 49 and L. paracasei 108 were >0.75 up to 4 months of storage. All freeze-dried strains exhibited survival rates of >0.75 up to 2 weeks of storage in apple juice; only L. plantarum 49 and L. paracasei 108 showed similar survival rates in orange and grape juices up to 2 weeks of storage. The contents of the monitored organic acids or sugars during storage varied depending on the added strain and the type of fruit juice. At the end of the in vitro digestion, L. brevis 59, L. paracasei 108 and L. fermentum 111 showed survival rates of >0.80 in apple juice. Apple juice was as the best substrate to the survival of the tested freeze-dried Lactobacillus strains over time. L. paracasei 108 and L. plantarum 49 as the strains presenting the best performance for incorporation in potentially probiotic fruit juices. This article is protected by copyright. All rights reserved.

  8. Balancing Ethical Pros and Cons of Stem Cell Derived Gametes.

    Science.gov (United States)

    Segers, Seppe; Mertes, Heidi; de Wert, Guido; Dondorp, Wybo; Pennings, Guido

    2017-07-01

    In this review we aim to provide an overview of the most important ethical pros and cons of stem cell derived gametes (SCD-gametes), as a contribution to the debate about reproductive tissue engineering. Derivation of gametes from stem cells holds promising applications both for research and for clinical use in assisted reproduction. We explore the ethical issues connected to gametes derived from embryonic stem cells (both patient specific and non-patient specific) as well as those related to gametes derived from induced pluripotent stem cells. The technology of SCD-gametes raises moral concerns of how reproductive autonomy relates to issues of embryo destruction, safety, access, and applications beyond clinical infertility.

  9. 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

  10. 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 ...

  11. Structural phenotyping of stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Pasqualini, Francesco Silvio; Sheehy, Sean Paul; Agarwal, Ashutosh; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

    2015-03-10

    Structural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Positive selection of mutants with cell envelope defects of a Salmonella typhimurium strain hypersensitive to the products of genes hisF and hisH

    International Nuclear Information System (INIS)

    Anton, D.N.

    1979-01-01

    Strain SB564 and its derivative DA78 are hypersensitive to the inhibitory action of the proteins coded for by genes hisF and hisH on cell division. Transduction of hisO1242, a regulatory mutation that elicits a very high level of expression of the histidine operon, into these strains resulted in the production of long filamentous cells carrying large balloons and in growth failure. Forty-one hisO1242 derivatives that escaped inhibition were isolated. These strains showed a large variety of alterations, many of which were related to the cell envelope. The more-frequent alterations included: changes in cell shape, increased sensitivity to one or more of several drugs (deoxycholate, cycloserine, penicillin, novobiocin, acridine orange), increased autolytic activity in alkaline buffer, anomalous fermentation of maltose on eosin--methylene blue plates, and temperature-conditional cell division. The alterations are produced, in some of the strains, by pleiotropic mutations in gene envB. Strains affected in divC, divD, and rodA loci have also been identified. Genetic analaysis has shown that several strains carry more than one envelope mutation. It is assumed that envelope mutations are positively selected because they somehow alleviate the particularly severe inhibition of cell division caused, in strains SB564 and DA78, by the excessive synthesis of hisF and hisH gene products

  13. Isolation and Osteogenic Differentiation of Rat Periosteum-derived Cells

    OpenAIRE

    Declercq, Heidi Andrea; De Ridder, Leo Isabelle; Cornelissen, Maria Jozefa

    2005-01-01

    Selection of appropriate cultures having an osteogenic potential is a necessity if cell/biomaterial interactions are studied in long-term cultures. Osteoblastic cells derived from rat long bones or calvaria have the disadvantage of being in an advanced differentiation stage which results in terminal differentiation within 21 days. In this regard, less differentiated periosteum-derived osteoprogenitors could be more suitable.

  14. Sarcocyst Development in Raccoons (Procyon lotor) Inoculated with Different Strains of Sarcocystis neurona Culture-Derived Merozoites.

    Science.gov (United States)

    Dryburgh, E L; Marsh, A E; Dubey, J P; Howe, D K; Reed, S M; Bolten, K E; Pei, W; Saville, W J A

    2015-08-01

    Sarcocystis neurona is considered the major etiologic agent of equine protozoal myeloencephalitis (EPM), a neurological disease in horses. Raccoon ( Procyon lotor ) is considered the most important intermediate host in the life cycle of S. neurona in the United States; S. neurona sarcocysts do mature in raccoon muscles, and raccoons also develop clinical signs simulating EPM. The focus of this study was to determine if sarcocysts would develop in raccoons experimentally inoculated with different host-derived strains of in vitro-cultivated S. neurona merozoites. Four raccoons were inoculated with strains derived from a raccoon, a sea otter, a cat, and a horse. Raccoon tissues were fed to laboratory-raised opossums ( Didelphis virginiana ), the definitive host of S. neurona . Intestinal scraping revealed sporocysts in opossums who received muscle tissue from raccoons inoculated with the raccoon-derived or the sea otter-derived isolates. These results demonstrate that sarcocysts can mature in raccoons inoculated with in vitro-derived S. neurona merozoites. In contrast, the horse and cat-derived isolates did not produce microscopically or biologically detected sarcocysts. Immunoblot analysis revealed both antigenic and antibody differences when testing the inoculated raccoons. Immunohistochemical staining indicated differences in staining between the merozoite and sarcocyst stages. The successful infections achieved in this study indicates that the life cycle can be manipulated in the laboratory without affecting subsequent stage development, thereby allowing further purification of strains and artificial maintenance of the life cycle.

  15. Staphylococcus aureus α-toxin-dependent induction of host cell death by membrane-derived vesicles.

    Directory of Open Access Journals (Sweden)

    Bernard Thay

    Full Text Available Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs, which analogously to outer membrane vesicles (OMVs of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol-dependent fusion of S. aureus MVs with the plasma membrane represents a route for delivery of a key virulence factor, α-toxin (α-hemolysin; Hla to human cells. Most S. aureus strains produce this 33-kDa pore-forming protein, which can lyse a wide range of human cells, and induce apoptosis in T-lymphocytes. Our results revealed a tight association of biologically active α-toxin with membrane-derived vesicles isolated from S. aureus strain 8325-4. Concomitantly, α-toxin contributed to HeLa cell cytotoxicity of MVs, and was the main vesicle-associated protein responsible for erythrocyte lysis. In contrast, MVs obtained from an isogenic hla mutant were significantly attenuated with regards to both causing lysis of erythrocytes and death of HeLa cells. This is to our knowledge the first recognition of an S. aureus MV-associated factor contributing to host cell cytotoxicity.

  16. Cells derived from young bone marrow alleviate renal aging.

    Science.gov (United States)

    Yang, Hai-Chun; Rossini, Michele; Ma, Li-Jun; Zuo, Yiqin; Ma, Ji; Fogo, Agnes B

    2011-11-01

    Bone marrow-derived stem cells may modulate renal injury, but the effects may depend on the age of the stem cells. Here we investigated whether bone marrow from young mice attenuates renal aging in old mice. We radiated female 12-mo-old 129SvJ mice and reconstituted them with bone marrow cells (BMC) from either 8-wk-old (young-to-old) or 12-mo-old (old-to-old) male mice. Transfer of young BMC resulted in markedly decreased deposition of collagen IV in the mesangium and less β-galactosidase staining, an indicator of cell senescence. These changes paralleled reduced expression of plasminogen activator inhibitor-1 (PAI-1), PDGF-B (PDGF-B), the transdifferentiation marker fibroblast-specific protein-1 (FSP-1), and senescence-associated p16 and p21. Tubulointerstitial and glomerular cells derived from the transplanted BMC did not show β-galactosidase activity, but after 6 mo, there were more FSP-1-expressing bone marrow-derived cells in old-to-old mice compared with young-to-old mice. Young-to-old mice also exhibited higher expression of the anti-aging gene Klotho and less phosphorylation of IGF-1 receptor β. Taken together, these data suggest that young bone marrow-derived cells can alleviate renal aging in old mice. Direct parenchymal reconstitution by stem cells, paracrine effects from adjacent cells, and circulating anti-aging molecules may mediate the aging of the kidney.

  17. Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines.

    Science.gov (United States)

    Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

    2017-03-01

    Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.

  18. Technological characterization and survival of the exopolysaccharide-producing strain Lactobacillus delbrueckii subsp. lactis 193 and its bile-resistant derivative 193+ in simulated gastric and intestinal juices.

    Science.gov (United States)

    Burns, Patricia; Vinderola, Gabriel; Reinheimer, Jorge; Cuesta, Isabel; de Los Reyes-Gavilán, Clara G; Ruas-Madiedo, Patricia

    2011-08-01

    The capacity of lactic acid bacteria to produce exopolysaccharides (EPS) conferring microorganisms a ropy phenotype could be an interesting feature from a technological point of view. Progressive adaptation to bile salts might render some lactobacilli able to overcome physiological gut barriers but could also modify functional properties of the strain, including the production of EPS. In this work some technological properties and the survival ability in simulated gastrointestinal conditions of Lactobacillus delbrueckii subsp. lactis 193, and Lb. delbrueckii subsp. lactis 193+, a strain with stable bile-resistant phenotype derived thereof, were characterized in milk in order to know whether the acquisition of resistance to bile could modify some characteristics of the microorganism. Both strains were able to grow and acidify milk similarly; however the production of ethanol increased at the expense of the aroma compound acetaldehyde in milk fermented by the strain 193+, with respect to milk fermented by the strain 193. Both microorganisms produced a heteropolysaccharide composed of glucose and galactose, and were able to increase the viscosity of fermented milks. In spite of the higher production yield of EPS by the bile-resistant strain 193+, it displayed a lower ability to increase viscosity than Lb. delbrueckii subsp. lactis 193. Milk increased survival in simulated gastric juice; the presence of bile improved adhesion to the intestinal cell line HT29-MTX in both strains. However, the acquisition of a stable resistance phenotype did not improve survival in simulated gastric and intestinal conditions or the adhesion to the intestinal cell line HT29-MTX. Thus, Lb. delbrueckii subsp. lactis 193 presents suitable technological properties for the manufacture of fermented dairy products; the acquisition of a stable bile-resistant phenotype modified some properties of the microorganism. This suggests that the possible use of bile-resistant derivative strains should be

  19. Stem cell-derived vascular endothelial cells and their potential application in regenerative medicine

    Science.gov (United States)

    Although a 'vascular stem cell' population has not been identified or generated, vascular endothelial and mural cells (smooth muscle cells and pericytes) can be derived from currently known pluripotent stem cell sources, including human embryonic stem cells and induced pluripotent stem cells. We rev...

  20. Stromal cell-derived factor 1α (SDF-1α)

    DEFF Research Database (Denmark)

    Li, Dana; Bjørnager, Louise; Langkilde, Anne

    2016-01-01

    OBJECTIVES: Stromal cell-derived factor 1a (SDF-1α), is a chemokine and is able to home hematopoietic progenitor cells to injured areas of heart tissue for structural repair. Previous studies have found increased levels of SDF-1α in several cardiac diseases, but only few studies have investigated...

  1. Translational applications of adult stem cell-derived organoids

    NARCIS (Netherlands)

    Drost, Jarno; Clevers, Hans

    2017-01-01

    Adult stem cells from a variety of organs can be expanded long-term in vitro as three-dimensional organotypic structures termed organoids. These adult stem cell-derived organoids retain their organ identity and remain genetically stable over long periods of time. The ability to grow organoids from

  2. 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.

  3. Modeling neurodegenerative diseases with patient-derived induced pluripotent cells

    DEFF Research Database (Denmark)

    Poon, Anna; Zhang, Yu; Chandrasekaran, Abinaya

    2017-01-01

    patient-specific induced pluripotent stem cells (iPSCs) and isogenic controls generated using CRISPR-Cas9 mediated genome editing. The iPSCs are self-renewable and capable of being differentiated into the cell types affected by the diseases. These in vitro models based on patient-derived iPSCs provide...... the possibilities of generating three-dimensional (3D) models using the iPSCs-derived cells and compare their advantages and disadvantages to conventional two-dimensional (2D) models....

  4. Platelet-derived stromal cell-derived factor-1 is required for the transformation of circulating monocytes into multipotential cells.

    Directory of Open Access Journals (Sweden)

    Noriyuki Seta

    Full Text Available BACKGROUND: We previously described a primitive cell population derived from human circulating CD14(+ monocytes, named monocyte-derived multipotential cells (MOMCs, which are capable of differentiating into mesenchymal and endothelial lineages. To generate MOMCs in vitro, monocytes are required to bind to fibronectin and be exposed to soluble factor(s derived from circulating CD14(- cells. The present study was conducted to identify factors that induce MOMC differentiation. METHODS: We cultured CD14(+ monocytes on fibronectin in the presence or absence of platelets, CD14(- peripheral blood mononuclear cells, platelet-conditioned medium, or candidate MOMC differentiation factors. The transformation of monocytes into MOMCs was assessed by the presence of spindle-shaped adherent cells, CD34 expression, and the potential to differentiate in vitro into mesenchymal and endothelial lineages. RESULTS: The presence of platelets or platelet-conditioned medium was required to generate MOMCs from monocytes. A screening of candidate platelet-derived soluble factors identified stromal cell-derived factor (SDF-1 as a requirement for generating MOMCs. Blocking an interaction between SDF-1 and its receptor CXCR4 inhibited MOMC generation, further confirming SDF-1's critical role in this process. Finally, circulating MOMC precursors were found to reside in the CD14(+CXCR4(high cell population. CONCLUSION: The interaction of SDF-1 with CXCR4 is essential for the transformation of circulating monocytes into MOMCs.

  5. A New Acetylenic Compound and Other Bioactive Metabolites from a Shark Gill-derived Penicillium Strain

    Directory of Open Access Journals (Sweden)

    Yi Zhang

    2017-01-01

    Full Text Available Nine chiral compounds (1−9 were isolated from the static fermentation culture of a shark gill-derived fungus Penicillium polonicum AP2T1. These compounds include a new acetylenic aromatic ether (1 , (--WA , four alkaloids ( a urantiomide C ( 2 , fructigenine A (3, cyclopenin (4 and cyclopenol (5 and four oxygenated compounds ((R-penipratynolene (6, (3S,4S-3,4-dihydro-3,4,8-trihydroxyl-naphthalenone (7, verrucosidin (8 and norverrucosidin (9. Their structures were elucidated by MS, NMR , optical rotation and circular dichroism (CD . In antimicrobial tests , compounds 1–4, 6 and 8–9 showed weak antibacterial activity against Staphylococcus aureus, Bacillus subtilis, and/or Escherichia coli.Compounds 3, 8 and 9 also exhibited moderate toxicity against Artemia salina larva , and showed cytotoxicity against human colon cancer cell line HCT116.

  6. Tumor-derived circulating endothelial cell clusters in colorectal cancer.

    KAUST Repository

    Cima, Igor; Kong, Say Li; Sengupta, Debarka; Tan, Iain B; Phyo, Wai Min; Lee, Daniel; Hu, Min; Iliescu, Ciprian; Alexander, Irina; Goh, Wei Lin; Rahmani, Mehran; Suhaimi, Nur-Afidah Mohamed; Vo, Jess H; Tai, Joyce A; Tan, Joanna H; Chua, Clarinda; Ten, Rachel; Lim, Wan Jun; Chew, Min Hoe; Hauser, Charlotte; van Dam, Rob M; Lim, Wei-Yen; Prabhakar, Shyam; Lim, Bing; Koh, Poh Koon; Robson, Paul; Ying, Jackie Y; Hillmer, Axel M; Tan, Min-Han

    2016-01-01

    Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

  7. Tumor-derived circulating endothelial cell clusters in colorectal cancer.

    KAUST Repository

    Cima, Igor

    2016-06-29

    Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

  8. Xylanase production from marine derived Trichoderma pleuroticola 08ÇK001 strain isolated from Mediterranean coastal sediments.

    Science.gov (United States)

    Korkmaz, Melih N; Ozdemir, Sennur C; Uzel, Ataç

    2017-10-01

    Xylanases constitutes one the most important enzymes with diverse applications in different industries such as bioethanol production, animal feedstock production, production of xylo-oligosaccharides, baking industry, paper and pulp industry, xylitol production, fruit juice, and beer finishing, degumming, and agriculture. Currently, industrial xylanases are mainly produced by Aspergillus and Trichoderma members. Since the marine environments are less studied compared to terrestrial environments and harbors great microbial diversity we aimed to investigate the xylanase production of 88 marine-derived filamentous fungal strains. These strains are semi-quantitatively screened for their extracellular xylanase production and Trichoderma pleuroticola 08ÇK001 xylanase activity was further characterized. Optimum pH and temperature was determined as 5.0 and 50 °C, respectively. The enzyme preparation retained 53% of its activity at pH 5.0 after 1 h and have found resistant against several ions and compounds such as K + , Ba 2+ , Na + , β-mercaptoethanol, Triton X-100 and toluene. This study demonstrates that marine-derived fungal strains are prolific sources for xylanase production and presents the first report about the production and characterization of xylanase from a marine derived T. pleuroticola strain. The characteristics of T. pleuroticola 08ÇK001 xylanase activity indicate possible employment in some industrial processes such as animal feed, juice and wine industries or paper pulping applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Benzoxazole derivatives suppress lipopolysaccharide-induced mast cell activation.

    Science.gov (United States)

    Cho, Kyung-Ah; Park, Minhwa; Kim, Yu-Hee; Choo, Hea-Young Park; Lee, Kyung Ho

    2018-05-01

    Mast cells are central regulators of allergic inflammation that function by releasing various proallergic inflammatory mediators, including histamine, eicosanoids and proinflammatory cytokines. Occasionally, bacterial infections may initiate or worsen allergic inflammation. A number of studies have indicated that activation of lipoxygenase in mast cells positive regulates allergic inflammatory responses by generating leukotrienes and proinflammatory cytokines. In the present study, the effects of benzoxazole derivatives on the lipopolysaccharide (LPS)‑induced expression of proinflammatory cytokines, production of histamine and surface expression of co‑stimulatory molecules on bone marrow-derived mast cells (BMMCs) were studied. The benzoxazole derivatives significantly reduced the expression of interleukin (IL)‑1β, IL‑6, IL‑13, tumor necrosis factor‑α, perilipin (PLIN) 2, and PLIN3 in BMMCs treated with LPS. Furthermore, histamine production was suppressed in BMMCs treated with LPS, or treated with phorbol-12-myristate-13-acetate/ionomycin. Benzoxazole derivatives marginally affected the surface expression of cluster of differentiation (CD)80 and CD86 on BMMCs in the presence of LPS, although LPS alone did not increase the expression of those proteins. Therefore, benzoxazole derivatives inhibited the secretion of proinflammatory cytokines in mast cells and may be potential candidate anti‑allergic agents to suppress mast cell activation.

  10. Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures.

    Science.gov (United States)

    Tuomola, E M; Salminen, S J

    1998-05-05

    The adhesion of 12 different Lactobacillus strains was studied using Caco-2 cell line as an in vitro model for intestinal epithelium. Some of the strains tested have been used as probiotics, and most of them are used in the dairy and food industry. Human and bovine enterotoxigenic Escherichia coli strains were used as positive and negative control, respectively. Bacterial adhesion to Caco-2 cell cultures was quantitated using radiolabelled bacteria. The adherence of bacteria was also observed microscopically after Gram staining. Viability of bacteria prior to adhesion was verified using flow cytometry. Among the tested strains, L. casei (Fyos) was the most adhesive strain and L. casei var. rhamnosus (Lactophilus) was the least adhesive strain, approximately 14 and 3% of the added bacteria adhered to Caco-2 cell cultures, respectively. The corresponding values for positive and negative control E. coli strains were 14 and 4%, respectively. The Lactobacillus strains tested could not be divided into distinctly adhesive or non-adhesive strains, since there was a continuation of adhesion rates. The four most adhesive strains were L. casei (Fyos), L. acidophilus 1 (LC1), L. rhamnosus LC-705 and Lactobacillus GG (ATCC 53103). No significant differences in the percentage adhesion were observed between these strains. Adhesion of all the strains was dependent on the number of bacteria used, since an approximately constant number of Caco-2 cells was used, indicating that the Caco-2 cell binding sites were not saturated. Viability of bacteria was high since approximately 90% of the bacteria were viable with the exception of L. acidophilus 1 which was 74% viable. Microscopic evaluations agreed with the radiolabelled binding as evidenced by observing more bacteria in Gram-stained preparations of good adhering strains compared to poorly adhering strains.

  11. Role of adipose-derived stem cells in wound healing.

    Science.gov (United States)

    Hassan, Waqar Ul; Greiser, Udo; Wang, Wenxin

    2014-01-01

    Impaired wound healing remains a challenge to date and causes debilitating effects with tremendous suffering. Recent advances in tissue engineering approaches in the area of cell therapy have provided promising treatment options to meet the challenges of impaired skin wound healing such as diabetic foot ulcers. Over the last few years, stem cell therapy has emerged as a novel therapeutic approach for various diseases including wound repair and tissue regeneration. Several different types of stem cells have been studied in both preclinical and clinical settings such as bone marrow-derived stem cells, adipose-derived stem cells (ASCs), circulating angiogenic cells (e.g., endothelial progenitor cells), human dermal fibroblasts, and keratinocytes for wound healing. Adipose tissue is an abundant source of mesenchymal stem cells, which have shown an improved outcome in wound healing studies. ASCs are pluripotent stem cells with the ability to differentiate into different lineages and to secrete paracrine factors initiating tissue regeneration process. The abundant supply of fat tissue, ease of isolation, extensive proliferative capacities ex vivo, and their ability to secrete pro-angiogenic growth factors make them an ideal cell type to use in therapies for the treatment of nonhealing wounds. In this review, we look at the pathogenesis of chronic wounds, role of stem cells in wound healing, and more specifically look at the role of ASCs, their mechanism of action and their safety profile in wound repair and tissue regeneration. © 2014 by the Wound Healing Society.

  12. Adipose-Derived Stem Cells and Application Areas

    Directory of Open Access Journals (Sweden)

    Mujde Kivanc

    2015-09-01

    Full Text Available The use of stem cells derived from adipose tissue as an autologous and self-replenishing source for a variety of differentiated cell phenotypes, provides a great deal of promise for reconstructive surgery. The secret of the human body, stem cells are reserved. Stem cells are undifferentiated cells found in the human body placed in any body tissue characteristics that differentiate and win ever known to cross the tissue instead of more than 200 diseases and thus improve and, rejuvenates the tissues. So far, the cord blood of newborn babies are used as a source of stem cells, bone marrow, and twenty years after tooth stem cells in human adipose tissue, scientists studied more than other sources of stem cells in adipose tissue and discovered that. Increase in number of in vitro studies on adult stem cells, depending on many variables is that the stem cells directly to the desired soybean optimization can be performed.. We will conclude by assessing potential avenues for developing this incredibly promising field. The aim of this paper is to review the existing literature on applications of harvest, purification, characterization and cryopreservation of adipose-derived stem cells (ASCs. [Cukurova Med J 2015; 40(3.000: 399-408

  13. Evaluation of the use of various rat strains for immunogenic potency tests of Sabin-derived inactivated polio vaccines.

    Science.gov (United States)

    Someya, Yuichi; Ami, Yasushi; Takai-Todaka, Reiko; Fujimoto, Akira; Haga, Kei; Murakami, Kosuke; Fujii, Yoshiki; Shirato, Haruko; Oka, Tomoichiro; Shimoike, Takashi; Katayama, Kazuhiko; Wakita, Takaji

    2018-03-01

    Slc:Wistar rats have been the only strain used in Japan for purpose of evaluating a national reference vaccine for the Sabin-derived inactivated polio vaccine (sIPV) and the immunogenicity of sIPV-containing products. However, following the discovery that the Slc:Wistar strain was genetically related to the Fischer 344 strain, other "real" Wistar strains, such as Crlj:WI, that are available worldwide were tested in terms of their usefulness in evaluating the immunogenicity of the past and current lots of a national reference vaccine. The response of the Crlj:WI rats against the serotype 1 of sIPV was comparable to that of the Slc:Wistar rats, while the Crlj:WI rats exhibited a higher level of response against the serotypes 2 and 3. The immunogenic potency units of a national reference vaccine determined using the Slc:Wistar rats were reproduced on tests using the Crlj:WI rats. These results indicate that a titer of the neutralizing antibody obtained in response to a given dose of sIPV cannot be directly compared between these two rat strains, but that, more importantly, the potency units are almost equivalent for the two rat strains. Copyright © 2018 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  14. Large Scale Production of Stem Cells and Their Derivatives

    Science.gov (United States)

    Zweigerdt, Robert

    Stem cells have been envisioned to become an unlimited cell source for regenerative medicine. Notably, the interest in stem cells lies beyond direct therapeutic applications. They might also provide a previously unavailable source of valuable human cell types for screening platforms, which might facilitate the development of more efficient and safer drugs. The heterogeneity of stem cell types as well as the numerous areas of application suggests that differential processes are mandatory for their in vitro culture. Many of the envisioned applications would require the production of a high number of stem cells and their derivatives in scalable, well-defined and potentially clinical compliant manner under current good manufacturing practice (cGMP). In this review we provide an overview on recent strategies to develop bioprocesses for the expansion, differentiation and enrichment of stem cells and their progenies, presenting examples for adult and embryonic stem cells alike.

  15. Foetal stem cell derivation & characterization for osteogenic lineage

    Directory of Open Access Journals (Sweden)

    A Mangala Gowri

    2013-01-01

    Full Text Available Background & objectives: Mesencymal stem cells (MSCs derived from foetal tissues present a multipotent progenitor cell source for application in tissue engineering and regenerative medicine. The present study was carried out to derive foetal mesenchymal stem cells from ovine source and analyze their differentiation to osteogenic linage to serve as an animal model to predict human applications. Methods: Isolation and culture of sheep foetal bone marrow cells were done and uniform clonally derived MSC population was collected. The cells were characterized using cytochemical, immunophenotyping, biochemical and molecular analyses. The cells with defined characteristics were differentiated into osteogenic lineages and analysis for differentiated cell types was done. The cells were analyzed for cell surface marker expression and the gene expression in undifferentiated and differentiated osteoblast was checked by reverse transcriptase PCR (RT PCR analysis and confirmed by sequencing using genetic analyzer. Results: Ovine foetal samples were processed to obtain mononuclear (MNC cells which on culture showed spindle morphology, a characteristic oval body with the flattened ends. MSC population CD45 - /CD14 - was cultured by limiting dilution to arrive at uniform spindle morphology cells and colony forming units. The cells were shown to be positive for surface markers such as CD44, CD54, integrinβ1, and intracellular collagen type I/III and fibronectin. The osteogenically induced MSCs were analyzed for alkaline phosphatase (ALP activity and mineral deposition. The undifferentiated MSCs expressed RAB3B, candidate marker for stemness in MSCs. The osteogenically induced and uninduced MSCs expressed collagen type I and MMP13 gene in osteogenic induced cells. Interpretation & conclusions: The protocol for isolation of ovine foetal bone marrow derived MSCs was simple to perform, and the cultural method of obtaining pure spindle morphology cells was established

  16. Extracellular matrix-derived hydrogels for dental stem cell delivery

    OpenAIRE

    Viswanath, Aiswarya; Vanacker, Julie; Germain, Loic; Leprince, Julien G.; Diogenes, Anibal; Shakesheff, Kevin M.; White, Lisa J.; des Rieux, Anne

    2016-01-01

    Decellularised mammalian extracellular matrices (ECM) have been widely accepted as an ideal substrate for repair and remodelling of numerous tissues in clinical and pre-clinical studies. Recent studies have demonstrated the ability of ECM scaffolds derived from site-specific homologous tissues to direct cell differentiation. The present study investigated the suitability of hydrogels derived from different source tissues: bone, spinal cord and dentine, as suitable carriers to deliver human ap...

  17. Assessment of stress tolerance acquisition in the heat-tolerant derivative strains of Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus rhamnosus GG.

    Science.gov (United States)

    Aakko, J; Sánchez, B; Gueimonde, M; Salminen, S

    2014-07-01

    The purpose of this study was to investigate the heat-shock response at molecular level in Lactobacillus rhamnosus GG, Bifidobacterium animalis subsp. lactis BB-12 and their heat-tolerant derivatives and to characterize the changes that make the derivatives more robust in terms of heat stress. The study strains were exposed for 2 h to a heat-shock treatment, Bif. animalis subsp. lactis BB-12 and its derivative at 50°C and the Lact. rhamnosus GG and its derivative at 60°C. Protein synthesis before and after heat shock was examined using proteomics and RT-qPCR. The analysis revealed that the regulation of seven proteins in both strain pairs was modified as a response to heat or between the original and the derivative strain. The comparison of wild-type strains and the heat-tolerant derivatives suggests that the acquisition of heat tolerance in the Bif. animalis subsp. lactis BB-12 derivative is due to a slightly increased constitutive level of chaperones, while in Lact. rhamnosus GG derivative, the main reason seems to be a higher ability to induce the production of chaperones. This study revealed possible markers of heat tolerance in B. lactis and Lact. rhamnosus strains. This study increases our knowledge on how Lactobacillus and Bifidobacterium strains may acquire heat tolerance. These findings may be useful for improving the heat tolerance of existing probiotic strains as well as screening new heat-tolerant strains. © 2014 The Society for Applied Microbiology.

  18. Strain and culture medium optimization for production enhancement of prodiginines from marine-derived Streptomyces sp. GQQ-10

    Science.gov (United States)

    Li, Xueping; Zhang, Guojian; Zhu, Tianjiao; Li, Dehai; Gu, Qianqun

    2012-09-01

    A mutant (GQQ-M6) of a Sponge-Derived streptomyces sp. GQQ-10 obtained by UV-induced mutation was used for producing prodiginines (PGs). Single factor experiments and orthogonal array design (OAD) methods were employed for medium optimization. In the single factor method, the effects of soluble starch, glucose, soybean flour, yeast extract and sodium acetate on PGs production were investigated individually. In the subsequent OAD experiments, the concentrations of these 5 key nutritional components combined with salinity were further adjusted. The mutant strain GQQ-M6 gave a 2.2-fold higher PGs production than that of the parent strain; OAD experiments offered a PGs yield of 61mg L-1, which was 10 times higher than that of the initial GQQ-10 strain under the original cultivation mode.

  19. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy

    Science.gov (United States)

    Askari, Arman T.; Unzek, Samuel; Popovic, Zoran B.; Goldman, Corey K.; Forudi, Farhad; Kiedrowski, Matthew; Rovner, Aleksandr; Ellis, Stephen G.; Thomas, James D.; DiCorleto, Paul E.; hide

    2003-01-01

    BACKGROUND: Myocardial regeneration via stem-cell mobilisation at the time of myocardial infarction is known to occur, although the mechanism for stem-cell homing to infarcted tissue subsequently and whether this approach can be used for treatment of ischaemic cardiomyopathy are unknown. We investigated these issues in a Lewis rat model (ligation of the left anterior descending artery) of ischaemic cardiomyopathy. METHODS: We studied the effects of stem-cell mobilisation by use of granulocyte colony-stimulating factor (filgrastim) with or without transplantation of syngeneic cells. Shortening fraction and myocardial strain by tissue doppler imaging were quantified by echocardiography. FINDINGS: Stem-cell mobilisation with filgrastim alone did not lead to engraftment of bone-marrow-derived cells. Stromal-cell-derived factor 1 (SDF-1), required for stem-cell homing to bone marrow, was upregulated immediately after myocardial infarction and downregulated within 7 days. 8 weeks after myocardial infarction, transplantation into the peri-infarct zone of syngeneic cardiac fibroblasts stably transfected to express SDF-1 induced homing of CD117-positive stem cells to injured myocardium after filgrastim administration (control vs SDF-1-expressing cardiac fibroblasts mean 7.2 [SD 3.4] vs 33.2 [6.0] cells/mm2, n=4 per group, pcell homing to injured myocardium and suggest a strategy for directed stem-cell engraftment into injured tissues. Our findings also indicate that therapeutic strategies focused on stem-cell mobilisation for regeneration of myocardial tissue must be initiated within days of myocardial infarction unless signalling for stem-cell homing is re-established.

  20. Delivery of chemotherapeutic drugs in tumour cell-derived microparticles.

    Science.gov (United States)

    Tang, Ke; Zhang, Yi; Zhang, Huafeng; Xu, Pingwei; Liu, Jing; Ma, Jingwei; Lv, Meng; Li, Dapeng; Katirai, Foad; Shen, Guan-Xin; Zhang, Guimei; Feng, Zuo-Hua; Ye, Duyun; Huang, Bo

    2012-01-01

    Cellular microparticles are vesicular plasma membrane fragments with a diameter of 100-1,000 nanometres that are shed by cells in response to various physiological and artificial stimuli. Here we demonstrate that tumour cell-derived microparticles can be used as vectors to deliver chemotherapeutic drugs. We show that tumour cells incubated with chemotherapeutic drugs package these drugs into microparticles, which can be collected and used to effectively kill tumour cells in murine tumour models without typical side effects. We describe several mechanisms involved in this process, including uptake of drug-containing microparticles by tumour cells, synthesis of additional drug-packaging microparticles by these cells that contribute to the cytotoxic effect and the inhibition of drug efflux from tumour cells. This study highlights a novel drug delivery strategy with potential clinical application.

  1. Nestin expression in the cell lines derived from glioblastoma multiforme

    International Nuclear Information System (INIS)

    Veselska, Renata; Kuglik, Petr; Cejpek, Pavel; Svachova, Hana; Neradil, Jakub; Loja, Tomas; Relichova, Jirina

    2006-01-01

    Nestin is a protein belonging to class VI of intermediate filaments that is produced in stem/progenitor cells in the mammalian CNS during development and is consecutively replaced by other intermediate filament proteins (neurofilaments, GFAP). Down-regulated nestin may be re-expressed in the adult organism under certain pathological conditions (brain injury, ischemia, inflammation, neoplastic transformation). Our work focused on a detailed study of the nestin cytoskeleton in cell lines derived from glioblastoma multiforme, because re-expression of nestin together with down-regulation of GFAP has been previously reported in this type of brain tumor. Two cell lines were derived from the tumor tissue of patients treated for glioblastoma multiforme. Nestin and other cytoskeletal proteins were visualized using imunocytochemical methods: indirect immunofluorescence and immunogold-labelling. Using epifluorescence and confocal microscopy, we described the morphology of nestin-positive intermediate filaments in glioblastoma cells of both primary cultures and the derived cell lines, as well as the reorganization of nestin during mitosis. Our most important result came through transmission electron microscopy and provided clear evidence that nestin is present in the cell nucleus. Detailed information concerning the pattern of the nestin cytoskeleton in glioblastoma cell lines and especially the demonstration of nestin in the nucleus represent an important background for further studies of nestin re-expression in relationship to tumor malignancy and invasive potential

  2. 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.

  3. Neural stem cell-derived exosomes mediate viral entry

    Directory of Open Access Journals (Sweden)

    Sims B

    2014-10-01

    Full Text Available Brian Sims,1,2,* Linlin Gu,3,* Alexandre Krendelchtchikov,3 Qiana L Matthews3,4 1Division of Neonatology, Department of Pediatrics, 2Department of Cell, Developmental, and Integrative Biology, 3Division of Infectious Diseases, Department of Medicine, 4Center for AIDS Research, University of Alabama at Birmingham, Birmingham, AL, USA *These authors contributed equally to this work Background: Viruses enter host cells through interactions of viral ligands with cellular receptors. Viruses can also enter cells in a receptor-independent fashion. Mechanisms regarding the receptor-independent viral entry into cells have not been fully elucidated. Exosomal trafficking between cells may offer a mechanism by which viruses can enter cells.Methods: To investigate the role of exosomes on cellular viral entry, we employed neural stem cell-derived exosomes and adenovirus type 5 (Ad5 for the proof-of-principle study. Results: Exosomes significantly enhanced Ad5 entry in Coxsackie virus and adenovirus receptor (CAR-deficient cells, in which Ad5 only had very limited entry. The exosomes were shown to contain T-cell immunoglobulin mucin protein 4 (TIM-4, which binds phosphatidylserine. Treatment with anti-TIM-4 antibody significantly blocked the exosome-mediated Ad5 entry.Conclusion: Neural stem cell-derived exosomes mediated significant cellular entry of Ad5 in a receptor-independent fashion. This mediation may be hampered by an antibody specifically targeting TIM-4 on exosomes. This set of results will benefit further elucidation of virus/exosome pathways, which would contribute to reducing natural viral infection by developing therapeutic agents or vaccines. Keywords: neural stem cell-derived exosomes, adenovirus type 5, TIM-4, viral entry, phospholipids

  4. Curcumin Modulates Pancreatic Adenocarcinoma Cell-Derived Exosomal Function

    Science.gov (United States)

    Osterman, Carlos J. Diaz; Lynch, James C.; Leaf, Patrick; Gonda, Amber; Ferguson Bennit, Heather R.; Griffiths, Duncan; Wall, Nathan R.

    2015-01-01

    Pancreatic cancer has the highest mortality rates of all cancer types. One potential explanation for the aggressiveness of this disease is that cancer cells have been found to communicate with one another using membrane-bound vesicles known as exosomes. These exosomes carry pro-survival molecules and increase the proliferation, survival, and metastatic potential of recipient cells, suggesting that tumor-derived exosomes are powerful drivers of tumor progression. Thus, to successfully address and eradicate pancreatic cancer, it is imperative to develop therapeutic strategies that neutralize cancer cells and exosomes simultaneously. Curcumin, a turmeric root derivative, has been shown to have potent anti-cancer and anti-inflammatory effects in vitro and in vivo. Recent studies have suggested that exosomal curcumin exerts anti-inflammatory properties on recipient cells. However, curcumin’s effects on exosomal pro-tumor function have yet to be determined. We hypothesize that curcumin will alter the pro-survival role of exosomes from pancreatic cancer cells toward a pro-death role, resulting in reduced cell viability of recipient pancreatic cancer cells. The main objective of this study was to determine the functional alterations of exosomes released by pancreatic cancer cells exposed to curcumin compared to exosomes from untreated pancreatic cancer cells. We demonstrate, using an in vitro cell culture model involving pancreatic adenocarcinoma cell lines PANC-1 and MIA PaCa-2, that curcumin is incorporated into exosomes isolated from curcumin-treated pancreatic cancer cells as observed by spectral studies and fluorescence microscopy. Furthermore, curcumin is delivered to recipient pancreatic cancer cells via exosomes, promoting cytotoxicity as demonstrated by Hoffman modulation contrast microscopy as well as AlamarBlue and Trypan blue exclusion assays. Collectively, these data suggest that the efficacy of curcumin may be enhanced in pancreatic cancer cells through

  5. Coelomic epithelium-derived cells in visceral morphogenesis.

    Science.gov (United States)

    Ariza, Laura; Carmona, Rita; Cañete, Ana; Cano, Elena; Muñoz-Chápuli, Ramón

    2016-03-01

    Coelomic cavities of vertebrates are lined by a mesothelium which develops from the lateral plate mesoderm. During development, the coelomic epithelium is a highly active cell layer, which locally is able to supply mesenchymal cells that contribute to the mesodermal elements of many organs and provide signals which are necessary for their development. The relevance of this process of mesenchymal cell supply to the developing organs is becoming clearer because genetic lineage tracing techniques have been developed in recent years. Body wall, heart, liver, lungs, gonads, and gastrointestinal tract are populated by cells derived from the coelomic epithelium which contribute to their connective and vascular tissues, and sometimes to specialized cell types such as the stellate cells of the liver, the Cajal interstitial cells of the gut or the Sertoli cells of the testicle. In this review we collect information about the contribution of coelomic epithelium derived cells to visceral development, their developmental fates and signaling functions. The common features displayed by all these processes suggest that the epithelial-mesenchymal transition of the embryonic coelomic epithelium is an underestimated but key event of vertebrate development, and probably it is shared by all the coelomate metazoans. © 2015 Wiley Periodicals, Inc.

  6. [Cell-derived microparticles unveil their fibrinolytic and proteolytic function].

    Science.gov (United States)

    Doeuvre, Loïc; Angles-Cano, Eduardo

    2009-01-01

    Cell-derived microparticles (MP) are membrane microvesicles, 0.1-1 microm in size, shed by cells following activation or during apoptosis in a variety of pathological conditions. MPs released by blood cells or by vascular endothelial cells display molecular signatures that allow their identification and functional characterization. In addition, they provide tissue factor (TF) and a procoagulant phospholipid surface. Therefore, at present, the most strongly established applied research on MPs is their procoagulant activity as a determinant of thrombotic risk in various clinical conditions. Previous studies have indicated that MPs derived from malignant cells express matrix metalloproteinases, urokinase and its receptor (uPA/uPAR) that, in the presence of plasminogen, may act in concert to degrade extracellular matrix proteins. Recently, it was shown that MPs from TNFa-stimulated endothelial cells served as a surface for interaction with plasminogen and its conversion into plasmin by the uPA/uPAR system expressed at their surface. This capacity of MPs to promote plasmin generation confers them a new profibrinolytic and proteolytic function that may be of relevance in fibrinolysis, cell migration, angiogenesis, dissemination of malignant cells, cell detachment and apoptosis.

  7. Sensitivity to fuel diesel oil and cell wall structure of some Scenedesmus (Chlorococcales strains

    Directory of Open Access Journals (Sweden)

    Zbigniew Tukaj

    2014-01-01

    Full Text Available Sensitivity of three Scenedesmus strains exposed to aqueous fuel-oil extract (AFOE is strongly strain-dependent S. quadricauda is the most resistant, S. armatus moderately tolerant whereas the most sensitive appears to be S. microspina. The sensitivity of tested species increases parallel with decreasing of cell size and cell number in coenobium. The values of the cell surface/cell volumes ratios only partly explain the above relationships. Electron microscope investigations reveal that the sensitivity may depend on cell wall structure of the strains. Cell wall of all here investigated strains is built of two layers: the inner so-called cellulosic layer and the outer one showing a three-laminar structure (TLS. The latter contains an acetolysis-resistant biopolymer (ARB. These two layers are similar in thickness in the three strains tested, but the surface of Scenedesmus is covered with various epistructures that are characteristic of strains. Some of them as the tightly fitting warty layer of S. armatus and especially the loosely fitting reticulate layer of S. quadricauda may contribute to lower permeability of cell wall. The structure of the rosettes also appears to be correlated with the sensitivity of strains. Presence of invaginations of plasmalemma in areas under rosettes indicates their role in transport processes inside/outside the cells.

  8. Cell-derived microparticles in haemostasis and vascular medicine.

    Science.gov (United States)

    Burnier, Laurent; Fontana, Pierre; Kwak, Brenda R; Angelillo-Scherrer, Anne

    2009-03-01

    Considerable interest for cell-derived microparticles has emerged, pointing out their essential role in haemostatic response and their potential as disease markers, but also their implication in a wide range of physiological and pathological processes. They derive from different cell types including platelets - the main source of microparticles - but also from red blood cells, leukocytes and endothelial cells, and they circulate in blood. Despite difficulties encountered in analyzing them and disparities of results obtained with a wide range of methods, microparticle generation processes are now better understood. However, a generally admitted definition of microparticles is currently lacking. For all these reasons we decided to review the literature regarding microparticles in their widest definition, including ectosomes and exosomes, and to focus mainly on their role in haemostasis and vascular medicine.

  9. Pathologic bladder microenvironment attenuates smooth muscle differentiation of skin derived precursor cells: implications for tissue regeneration.

    Directory of Open Access Journals (Sweden)

    Cornelia Tolg

    Full Text Available Smooth muscle cell containing organs (bladder, heart, blood vessels are damaged by a variety of pathological conditions necessitating surgery or organ replacement. Currently, regeneration of contractile tissues is hampered by lack of functional smooth muscle cells. Multipotent skin derived progenitor cells (SKPs can easily be isolated from adult skin and can be differentiated in vitro into contractile smooth muscle cells by exposure to FBS. Here we demonstrate an inhibitory effect of a pathologic contractile organ microenvironment on smooth muscle cell differentiation of SKPs. In vivo, urinary bladder strain induces microenvironmental changes leading to de-differentiation of fully differentiated bladder smooth muscle cells. Co-culture of SKPs with organoids isolated from ex vivo stretched bladders or exposure of SKPs to diffusible factors released by stretched bladders (e.g. bFGF suppresses expression of smooth muscle markers (alpha SMactin, calponin, myocardin, myosin heavy chain as demonstrated by qPCR and immunofluorescent staining. Rapamycin, an inhibitor of mTOR signalling, previously observed to prevent bladder strain induced de-differentiation of fully differentiated smooth muscle cells in vitro, inhibits FBS-induced smooth muscle cell differentiation of undifferentiated SKPs. These results suggest that intended precursor cell differentiation may be paradoxically suppressed by the disease context for which regeneration may be required. Organ-specific microenvironment contexts, particularly prevailing disease, may play a significant role in modulating or attenuating an intended stem cell phenotypic fate, possibly explaining the variable and inefficient differentiation of stem cell constructs in in vivo settings. These observations must be considered in drafting any regeneration strategies.

  10. Effect of Uniaxial Tensile Cyclic Loading Regimes on Matrix Organization and Tenogenic Differentiation of Adipose-Derived Stem Cells Encapsulated within 3D Collagen Scaffolds

    Directory of Open Access Journals (Sweden)

    Gayathri Subramanian

    2017-01-01

    Full Text Available Adipose-derived mesenchymal stem cells have become a popular cell choice for tendon repair strategies due to their relative abundance, ease of isolation, and ability to differentiate into tenocytes. In this study, we investigated the solo effect of different uniaxial tensile strains and loading frequencies on the matrix directionality and tenogenic differentiation of adipose-derived stem cells encapsulated within three-dimensional collagen scaffolds. Samples loaded at 0%, 2%, 4%, and 6% strains and 0.1 Hz and 1 Hz frequencies for 2 hours/day over a 7-day period using a custom-built uniaxial tensile strain bioreactor were characterized in terms of matrix organization, cell viability, and musculoskeletal gene expression profiles. The results displayed that the collagen fibers of the loaded samples exhibited increased matrix directionality with an increase in strain values. Gene expression analyses demonstrated that ASC-encapsulated collagen scaffolds loaded at 2% strain and 0.1 Hz frequency showed significant increases in extracellular matrix genes and tenogenic differentiation markers. Importantly, no cross-differentiation potential to osteogenic, chondrogenic, and myogenic lineages was observed at 2% strain and 0.1 Hz frequency loading condition. Thus, 2% strain and 0.1 Hz frequency were identified as the appropriate mechanical loading regime to induce tenogenic differentiation of adipose-derived stem cells cultured in a three-dimensional environment.

  11. Effect of Uniaxial Tensile Cyclic Loading Regimes on Matrix Organization and Tenogenic Differentiation of Adipose-Derived Stem Cells Encapsulated within 3D Collagen Scaffolds.

    Science.gov (United States)

    Subramanian, Gayathri; Stasuk, Alexander; Elsaadany, Mostafa; Yildirim-Ayan, Eda

    2017-01-01

    Adipose-derived mesenchymal stem cells have become a popular cell choice for tendon repair strategies due to their relative abundance, ease of isolation, and ability to differentiate into tenocytes. In this study, we investigated the solo effect of different uniaxial tensile strains and loading frequencies on the matrix directionality and tenogenic differentiation of adipose-derived stem cells encapsulated within three-dimensional collagen scaffolds. Samples loaded at 0%, 2%, 4%, and 6% strains and 0.1 Hz and 1 Hz frequencies for 2 hours/day over a 7-day period using a custom-built uniaxial tensile strain bioreactor were characterized in terms of matrix organization, cell viability, and musculoskeletal gene expression profiles. The results displayed that the collagen fibers of the loaded samples exhibited increased matrix directionality with an increase in strain values. Gene expression analyses demonstrated that ASC-encapsulated collagen scaffolds loaded at 2% strain and 0.1 Hz frequency showed significant increases in extracellular matrix genes and tenogenic differentiation markers. Importantly, no cross-differentiation potential to osteogenic, chondrogenic, and myogenic lineages was observed at 2% strain and 0.1 Hz frequency loading condition. Thus, 2% strain and 0.1 Hz frequency were identified as the appropriate mechanical loading regime to induce tenogenic differentiation of adipose-derived stem cells cultured in a three-dimensional environment.

  12. Myeloid derived suppressor cells as therapeutic target in hematological malignancies

    Directory of Open Access Journals (Sweden)

    Kim eDe Veirman

    2014-12-01

    Full Text Available Myeloid derived suppressor cells (MDSC are a heterogeneous population of immature myeloid cells that accumulate during pathological conditions such as cancer and are associated with a poor clinical outcome. MDSC expansion hampers the host anti-tumor immune response by inhibition of T cell proliferation, cytokine secretion and recruitment of regulatory T cells. In addition, MDSC exert non-immunological functions including the promotion of angiogenesis, tumor invasion and metastasis. Recent years, MDSC are considered as a potential target in solid tumors and hematological malignancies to enhance the effects of currently used immune modulating agents. This review focuses on the characteristics, distribution, functions, cell-cell interactions and targeting of MDSC in hematological malignancies including multiple myeloma, lymphoma and leukemia.

  13. Characterization of goat inner cell mass derived cells in double kinase inhibition condition

    International Nuclear Information System (INIS)

    Wei, Qiang; Xi, Qihui; Liu, Xiaokun; Meng, Kai; Zhao, Xiaoe; Ma, Baohua

    2017-01-01

    The identification of small molecular inhibitors, which were reported to promote the derivation of mouse and human embryonic stem cells (ESCs), provides a potential strategy for the derivation of domesticated ungulate ESCs. In present study, goat inner cell mass (ICM) derived cells in the double inhibition (2i) condition, in which, mitogen-activated protein kinase kinase (MAP2K) and glycogen synthase kinase 3 (GSK3) were inhibited by PD0325901 and BIO respectively, were characterized. The results showed that goat ICM derived cells in 2i medium adding leukaemia inhibitor factor (LIF) possessed a mouse ES-like morphology. But these cells had much compromised proliferation capacity, resulting in difficulty in expansion. In 2i alone medium, goat ICM derived cells possessed primate ES-like morphology. These cells expressed pluripotent markers and could differentiate into derivatives of three germ layers in vitro. However, these cells could not be proliferated in long-term (persisted for 15 passages) because of spontaneously neural differentiation. Additionally, goat ICM derived cells could be inducing differentiated into neural lineage in vitro. Although goat ESCs could not be established in PD0325901 and BIO alone medium, this derivation condition provides a useful research system to find signaling molecular those regulate early embryonic development and pluripotency in goat. - Highlights: • Goat inner cell mass derived cells possessed finite pluripotency in 2i condition. • These cells could not be proliferated in long-term in 2i condition. • These cells could spontaneously and inductively differentiate into neural lineage.

  14. Exosomes Derived From Pancreatic Stellate Cells: MicroRNA Signature and Effects on Pancreatic Cancer Cells.

    Science.gov (United States)

    Takikawa, Tetsuya; Masamune, Atsushi; Yoshida, Naoki; Hamada, Shin; Kogure, Takayuki; Shimosegawa, Tooru

    2017-01-01

    Pancreatic stellate cells (PSCs) interact with pancreatic cancer cells in the tumor microenvironment. Cell constituents including microRNAs may be exported from cells within membranous nanovesicles termed exosomes. Exosomes might play a pivotal role in intercellular communication. This study aimed to clarify the microRNA signature of PSC-derived exosomes and their effects on pancreatic cancer cells. Exosomes were prepared from the conditioned medium of immortalized human PSCs. MicroRNAs were prepared from the exosomes and their source PSCs, and the microRNA expression profiles were compared by microarray. The effects of PSC-derived exosomes on proliferation, migration, and the mRNA expression profiles were examined in pancreatic cancer cells. Pancreatic stellate cell-derived exosomes contained a variety of microRNAs including miR-21-5p. Several microRNAs such as miR-451a were enriched in exosomes compared to their source PSCs. Pancreatic stellate cell-derived exosomes stimulated the proliferation, migration and expression of mRNAs for chemokine (C - X - C motif) ligands 1 and 2 in pancreatic cancer cells. The stimulation of proliferation, migration, and chemokine gene expression by the conditioned medium of PSCs was suppressed by GW4869, an exosome inhibitor. We clarified the microRNA expression profile in PSC-derived exosomes. Pancreatic stellate cell-derived exosomes might play a role in the interactions between PSCs and pancreatic cancer cells.

  15. Macrophages discriminate glycosylation patterns of apoptotic cell-derived microparticles.

    Science.gov (United States)

    Bilyy, Rostyslav O; Shkandina, Tanya; Tomin, Andriy; Muñoz, Luis E; Franz, Sandra; Antonyuk, Volodymyr; Kit, Yuriy Ya; Zirngibl, Matthias; Fürnrohr, Barbara G; Janko, Christina; Lauber, Kirsten; Schiller, Martin; Schett, Georg; Stoika, Rostyslav S; Herrmann, Martin

    2012-01-02

    Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.

  16. Bone marrow-derived CD13+ cells sustain tumor progression

    Science.gov (United States)

    Dondossola, Eleonora; Corti, Angelo; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata

    2014-01-01

    Non-malignant cells found within neoplastic lesions express alanyl (membrane) aminopeptidase (ANPEP, best known as CD13), and CD13-null mice exhibit limited tumor growth and angiogenesis. We have recently demonstrated that a subset of bone marrow-derived CD11b+CD13+ myeloid cells accumulate within neoplastic lesions in several murine models of transplantable cancer to promote angiogenesis. If these findings were confirmed in clinical settings, CD11b+CD13+ myeloid cells could become a non-malignant target for the development of novel anticancer regimens. PMID:25339996

  17. Predictive value of cardiac magnetic resonance imaging-derived myocardial strain for poor outcomes in patients with acute myocarditis

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Won; Jeong, Yeon Joo; Lee, Gee Won; Lee, Nam Kyung; Lee, Hye Won; Kim, Jin You [Pusan National University School of Medicine and Medical Research Institute, Pusan National University Hospital, Busan (Korea, Republic of); Choi, Bum Sung; Choo, Ki Seok [Pusan National University Yangsan Hospital, Yangsan (Korea, Republic of)

    2017-08-01

    To evaluate the utility of cardiovascular magnetic resonance (CMR)-derived myocardial strain measurement for the prediction of poor outcomes in patients with acute myocarditis We retrospectively analyzed data from 37 patients with acute myocarditis who underwent CMR. Left ventricular (LV) size, LV mass index, ejection fraction and presence of myocardial late gadolinium enhancement (LGE) were analyzed. LV circumferential strain (EccSAX), radial strain (ErrSAX) from mid-ventricular level short-axis cine views and LV longitudinal strain (EllLV), radial strain (ErrLax) measurements from 2-chamber long-axis views were obtained. In total, 31 of 37 patients (83.8%) underwent follow-up echocardiography. The primary outcome was major adverse cardiovascular event (MACE). Incomplete LV functional recovery was a secondary outcome. During an average follow-up of 41 months, 11 of 37 patients (29.7%) experienced MACE. Multivariable Cox proportional hazard regression analysis, which included LV mass index, LV ejection fraction, the presence of LGE, EccSAX, ErrSAX, EllLV, and ErrLax values, indicated that the presence of LGE (hazard ratio, 42.88; p = 0.014), together with ErrLax (hazard ratio, 0.77 per 1%, p = 0.004), was a significant predictor of MACE. Kaplan-Meier analysis demonstrated worse outcomes in patient with LGE and an ErrLax value ≤ 9.48%. Multivariable backward regression analysis revealed that ErrLax values were the only significant predictors of LV functional recovery (hazard ratio, 0.54 per 1%; p = 0.042). CMR-derived ErrLax values can predict poor outcomes, both MACE and incomplete LV functional recovery, in patients with acute myocarditis, while LGE is only a predictor of MACE.

  18. 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.

  19. Establishment of an immortalized mouse dermal papilla cell strain with optimized culture strategy

    Directory of Open Access Journals (Sweden)

    Haiying Guo

    2018-01-01

    Full Text Available Dermal papilla (DP plays important roles in hair follicle regeneration. Long-term culture of mouse DP cells can provide enough cells for research and application of DP cells. We optimized the culture strategy for DP cells from three dimensions: stepwise dissection, collagen I coating, and optimized culture medium. Based on the optimized culture strategy, we immortalized primary DP cells with SV40 large T antigen, and established several immortalized DP cell strains. By comparing molecular expression and morphologic characteristics with primary DP cells, we found one cell strain named iDP6 was similar with primary DP cells. Further identifications illustrate that iDP6 expresses FGF7 and α-SMA, and has activity of alkaline phosphatase. During the process of characterization of immortalized DP cell strains, we also found that cells in DP were heterogeneous. We successfully optimized culture strategy for DP cells, and established an immortalized DP cell strain suitable for research and application of DP cells.

  20. Mechanical stretch increases CCN2/CTGF expression in anterior cruciate ligament-derived cells

    International Nuclear Information System (INIS)

    Miyake, Yoshiaki; Furumatsu, Takayuki; Kubota, Satoshi; Kawata, Kazumi; Ozaki, Toshifumi; Takigawa, Masaharu

    2011-01-01

    Highlights: → CCN2/CTGF localizes to the ligament-to-bone interface, but is not to the midsubstance region of human anterior cruciate ligament (ACL). → Mechanical stretch induces higher increase of CCN2/CTGF gene expression and protein secretion in ACL interface cells compared with ACL midsubstance cells. → CCN2/CTGF treatment stimulates the proliferation of ACL interface cells. -- Abstract: Anterior cruciate ligament (ACL)-to-bone interface serves to minimize the stress concentrations that would arise between two different tissues. Mechanical stretch plays an important role in maintaining cell-specific features by inducing CCN family 2/connective tissue growth factor (CCN2/CTGF). We previously reported that cyclic tensile strain (CTS) stimulates α1(I) collagen (COL1A1) expression in human ACL-derived cells. However, the biological function and stress-related response of CCN2/CTGF were still unclear in ACL fibroblasts. In the present study, CCN2/CTGF was observed in ACL-to-bone interface, but was not in the midsubstance region by immunohistochemical analyses. CTS treatments induced higher increase of CCN2/CTGF expression and secretion in interface cells compared with midsubstance cells. COL1A1 expression was not influenced by CCN2/CTGF treatment in interface cells despite CCN2/CTGF stimulated COL1A1 expression in midsubstance cells. However, CCN2/CTGF stimulated the proliferation of interface cells. Our results suggest that distinct biological function of stretch-induced CCN2/CTGF might regulate region-specific phenotypes of ACL-derived cells.

  1. Mechanical stretch increases CCN2/CTGF expression in anterior cruciate ligament-derived cells

    Energy Technology Data Exchange (ETDEWEB)

    Miyake, Yoshiaki [Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama (Japan); Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama (Japan); Furumatsu, Takayuki, E-mail: matino@md.okayama-u.ac.jp [Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama (Japan); Kubota, Satoshi; Kawata, Kazumi [Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama (Japan); Ozaki, Toshifumi [Department of Orthopaedic Surgery, Science of Functional Recovery and Reconstruction, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama (Japan); Takigawa, Masaharu [Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama (Japan)

    2011-06-03

    Highlights: {yields} CCN2/CTGF localizes to the ligament-to-bone interface, but is not to the midsubstance region of human anterior cruciate ligament (ACL). {yields} Mechanical stretch induces higher increase of CCN2/CTGF gene expression and protein secretion in ACL interface cells compared with ACL midsubstance cells. {yields} CCN2/CTGF treatment stimulates the proliferation of ACL interface cells. -- Abstract: Anterior cruciate ligament (ACL)-to-bone interface serves to minimize the stress concentrations that would arise between two different tissues. Mechanical stretch plays an important role in maintaining cell-specific features by inducing CCN family 2/connective tissue growth factor (CCN2/CTGF). We previously reported that cyclic tensile strain (CTS) stimulates {alpha}1(I) collagen (COL1A1) expression in human ACL-derived cells. However, the biological function and stress-related response of CCN2/CTGF were still unclear in ACL fibroblasts. In the present study, CCN2/CTGF was observed in ACL-to-bone interface, but was not in the midsubstance region by immunohistochemical analyses. CTS treatments induced higher increase of CCN2/CTGF expression and secretion in interface cells compared with midsubstance cells. COL1A1 expression was not influenced by CCN2/CTGF treatment in interface cells despite CCN2/CTGF stimulated COL1A1 expression in midsubstance cells. However, CCN2/CTGF stimulated the proliferation of interface cells. Our results suggest that distinct biological function of stretch-induced CCN2/CTGF might regulate region-specific phenotypes of ACL-derived cells.

  2. Myeloid-Derived Suppressor Cells and Therapeutic Strategies in Cancer

    Directory of Open Access Journals (Sweden)

    Hiroshi Katoh

    2015-01-01

    Full Text Available Development of solid cancer depends on escape from host immunosurveillance. Various types of immune cells contribute to tumor-induced immune suppression, including tumor associated macrophages, regulatory T cells, type 2 NKT cells, and myeloid-derived suppressor cells (MDSCs. Growing body of evidences shows that MDSCs play pivotal roles among these immunosuppressive cells in multiple steps of cancer progression. MDSCs are immature myeloid cells that arise from myeloid progenitor cells and comprise a heterogeneous immune cell population. MDSCs are characterized by the ability to suppress both adaptive and innate immunities mainly through direct inhibition of the cytotoxic functions of T cells and NK cells. In clinical settings, the number of circulating MDSCs is associated with clinical stages and response to treatment in several cancers. Moreover, MDSCs are reported to contribute to chemoresistant phenotype. Collectively, targeting MDSCs could potentially provide a rationale for novel treatment strategies in cancer. This review summarizes recent understandings of MDSCs in cancer and discusses promissing clinical approaches in cancer patients.

  3. Rhodacyanine derivative selectively targets cancer cells and overcomes tamoxifen resistance.

    Directory of Open Access Journals (Sweden)

    John Koren

    Full Text Available MKT-077, a rhodacyanine dye, was shown to produce cancer specific cell death. However, complications prevented the use of this compound beyond clinical trials. Here we describe YM-1, a derivative of MKT-077. We found that YM-1 was more cytotoxic and localized differently than MKT-077. YM-1 demonstrated this cytotoxicity across multiple cancer cell lines. This toxicity was limited to cancer cell lines; immortalized cell models were unaffected. Brief applications of YM-1 were found to be non-toxic. Brief treatment with YM-1 restored tamoxifen sensitivity to a refractory tamoxifen-resistant MCF7 cell model. This effect is potentially due to altered estrogen receptor alpha phosphorylation, an outcome precipitated by selective reductions in Akt levels (Akt/PKB. Thus, modifications to the rhodocyanine scaffold could potentially be made to improve efficacy and pharmacokinetic properties. Moreover, the impact on tamoxifen sensitivity could be a new utility for this compound family.

  4. Recruitment of bone marrow derived cells during anti-angiogenic therapy in GBM : Bone marrow derived cell in GBM

    NARCIS (Netherlands)

    Boer, Jennifer C.; Walenkamp, Annemiek M. E.; den Dunnen, Wilfred F. A.

    2014-01-01

    Glioblastoma (GBM) is a highly vascular tumor characterized by rapid and invasive tumor growth, followed by oxygen depletion, hypoxia and neovascularization, which generate a network of disorganized, tortuous and permeable vessels. Recruitment of bone marrow derived cells (BMDC) is crucial for

  5. Derivation and characterization of monkey embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Wolf Don P

    2004-06-01

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

  6. Extracellular matrix-derived hydrogels for dental stem cell delivery.

    Science.gov (United States)

    Viswanath, Aiswarya; Vanacker, Julie; Germain, Loïc; Leprince, Julian G; Diogenes, Anibal; Shakesheff, Kevin M; White, Lisa J; des Rieux, Anne

    2017-01-01

    Decellularized mammalian extracellular matrices (ECM) have been widely accepted as an ideal substrate for repair and remodelling of numerous tissues in clinical and pre-clinical studies. Recent studies have demonstrated the ability of ECM scaffolds derived from site-specific homologous tissues to direct cell differentiation. The present study investigated the suitability of hydrogels derived from different source tissues: bone, spinal cord and dentine, as suitable carriers to deliver human apical papilla derived mesenchymal stem cells (SCAP) for spinal cord regeneration. Bone, spinal cord, and dentine ECM hydrogels exhibited distinct structural, mechanical, and biological characteristics. All three hydrogels supported SCAP viability and proliferation. However, only spinal cord and bone derived hydrogels promoted the expression of neural lineage markers. The specific environment of ECM scaffolds significantly affected the differentiation of SCAP to a neural lineage, with stronger responses observed with spinal cord ECM hydrogels, suggesting that site-specific tissues are more likely to facilitate optimal stem cell behavior for constructive spinal cord regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 319-328, 2017. © 2016 Wiley Periodicals, Inc.

  7. Myocardial regeneration potential of adipose tissue-derived stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Bai, Xiaowen, E-mail: baixw01@yahoo.com [Department of Molecular Pathology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe, Houston, TX 77030 (United States); Alt, Eckhard, E-mail: ealt@mdanderson.org [Department of Molecular Pathology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe, Houston, TX 77030 (United States)

    2010-10-22

    Research highlights: {yields} Various tissue resident stem cells are receiving tremendous attention from basic scientists and clinicians and hold great promise for myocardial regeneration. {yields} For practical reasons, human adipose tissue-derived stem cells are attractive stem cells for future clinical application in repairing damaged myocardium. {yields} This review summarizes the characteristics of cultured and freshly isolated stem cells obtained from adipose tissue, their myocardial regeneration potential and the, underlying mechanisms, and safety issues. -- Abstract: Various tissue resident stem cells are receiving attention from basic scientists and clinicians as they hold promise for myocardial regeneration. For practical reasons, adipose tissue-derived stem cells (ASCs) are attractive cells for clinical application in repairing damaged myocardium based on the following advantages: abundant adipose tissue in most patients and easy accessibility with minimally invasive lipoaspiration procedure. Several recent studies have demonstrated that both cultured and freshly isolated ASCs could improve cardiac function in animal model of myocardial infarction. The mechanisms underlying the beneficial effect of ASCs on myocardial regeneration are not fully understood. Growing evidence indicates that transplantation of ASCs improve cardiac function via the differentiation into cardiomyocytes and vascular cells, and through paracrine pathways. Paracrine factors secreted by injected ASCs enhance angiogenesis, reduce cell apoptosis rates, and promote neuron sprouts in damaged myocardium. In addition, Injection of ASCs increases electrical stability of the injured heart. Furthermore, there are no reported cases of arrhythmia or tumorigenesis in any studies regarding myocardial regeneration with ASCs. This review summarizes the characteristics of both cultured and freshly isolated stem cells obtained from adipose tissue, their myocardial regeneration potential, and the

  8. Myocardial regeneration potential of adipose tissue-derived stem cells

    International Nuclear Information System (INIS)

    Bai, Xiaowen; Alt, Eckhard

    2010-01-01

    Research highlights: → Various tissue resident stem cells are receiving tremendous attention from basic scientists and clinicians and hold great promise for myocardial regeneration. → For practical reasons, human adipose tissue-derived stem cells are attractive stem cells for future clinical application in repairing damaged myocardium. → This review summarizes the characteristics of cultured and freshly isolated stem cells obtained from adipose tissue, their myocardial regeneration potential and the, underlying mechanisms, and safety issues. -- Abstract: Various tissue resident stem cells are receiving attention from basic scientists and clinicians as they hold promise for myocardial regeneration. For practical reasons, adipose tissue-derived stem cells (ASCs) are attractive cells for clinical application in repairing damaged myocardium based on the following advantages: abundant adipose tissue in most patients and easy accessibility with minimally invasive lipoaspiration procedure. Several recent studies have demonstrated that both cultured and freshly isolated ASCs could improve cardiac function in animal model of myocardial infarction. The mechanisms underlying the beneficial effect of ASCs on myocardial regeneration are not fully understood. Growing evidence indicates that transplantation of ASCs improve cardiac function via the differentiation into cardiomyocytes and vascular cells, and through paracrine pathways. Paracrine factors secreted by injected ASCs enhance angiogenesis, reduce cell apoptosis rates, and promote neuron sprouts in damaged myocardium. In addition, Injection of ASCs increases electrical stability of the injured heart. Furthermore, there are no reported cases of arrhythmia or tumorigenesis in any studies regarding myocardial regeneration with ASCs. This review summarizes the characteristics of both cultured and freshly isolated stem cells obtained from adipose tissue, their myocardial regeneration potential, and the underlying

  9. Short communication: Antiproliferative effect of wild Lactobacillus strains isolated from fermented foods on HT-29 cells.

    Science.gov (United States)

    Tuo, Y F; Zhang, L W; Yi, H X; Zhang, Y C; Zhang, W Q; Han, X; Du, M; Jiao, Y H; Wang, S M

    2010-06-01

    In vitro studies, animal models, epidemiology, and human intervention studies provide evidence that some lactic acid bacteria can reduce the risk of certain cancers. In this study, heat-killed bacterial cells, genomic DNA, and cell wall of 7 wild Lactobacillus strains isolated from traditional fermented foods in western China were tested in vitro for cytotoxicity on colonic cancer cell line HT-29 by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The heat-killed bacterial cells, genomic DNA, and cell wall of the 7 strains exhibited direct antiproliferative activities against HT-29 cells. Among the strains, the cellular components of Lactobacillus coryniformis ssp. torquens T3L exerted marked antiproliferative activities against HT-29 cells. The maximum inhibition rates of HT-29 cells by the heat-killed bacterial cells (1x10(7) cfu/mL), cell wall (20 microg of protein/mL) and genomic DNA (100 microg/mL) of L. coryniformis ssp. torquens T3L were 30, 44.9, and 35.9%, respectively. The results indicate that the heat-killed bacterial cells, cell wall, and genomic DNA of the 7 wild Lactobacillus strains could inhibit the growth of HT-29 cells. 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  10. Development of a Probiotic Cheddar Cheese Containing Human-Derived Lactobacillus paracasei Strains

    Science.gov (United States)

    Gardiner, G.; Ross, R. P.; Collins, J. K.; Fitzgerald, G.; Stanton, C.

    1998-01-01

    Cheddar cheese was manufactured with either Lactobacillus salivarius NFBC 310, NFBC 321, or NFBC 348 or L. paracasei NFBC 338 or NFBC 364 as the dairy starter adjunct. These five strains had previously been isolated from the human small intestine and have been characterized extensively with respect to their probiotic potential. Enumeration of these strains in mature Cheddar cheese, however, was complicated by the presence of high numbers (>107 CFU/g of cheese) of nonstarter lactic acid bacteria, principally composed of lactobacilli which proliferate as the cheese ripens. Attempts to differentiate the adjunct lactobacilli from the nonstarter lactobacilli based on bile tolerance and growth temperature were unsuccessful. In contrast, the randomly amplified polymorphic DNA method allowed the generation of discrete DNA fingerprints for each strain which were clearly distinguishable from those generated from the natural flora of the cheeses. Using this approach, it was found that both L. paracasei strains grew and sustained high viability in cheese during ripening, while each of the L. salivarius species declined over the ripening period. These data demonstrate that Cheddar cheese can be an effective vehicle for delivery of some probiotic organisms to the consumer. PMID:9603834

  11. On the derivative of the stress-strain relation in a no-tension material

    Czech Academy of Sciences Publication Activity Database

    Padovani, C.; Šilhavý, Miroslav

    2017-01-01

    Roč. 22, č. 7 (2017), s. 1606-1618 ISSN 1081-2865 Institutional support: RVO:67985840 Keywords : no-tension material * stress-strain relation Subject RIV: BA - General Mathematics OBOR OECD: Applied mathematics Impact factor: 2.953, year: 2016 http:// journals .sagepub.com/doi/10.1177/1081286515571786

  12. Probing localized strain in solution-derived YB a2C u3O7 -δ nanocomposite thin films

    Science.gov (United States)

    Guzman, Roger; Gazquez, Jaume; Mundet, Bernat; Coll, Mariona; Obradors, Xavier; Puig, Teresa

    2017-07-01

    Enhanced pinning due to nanoscale strain is unique to the high-Tc cuprates, where pairing may be modified with lattice distortion. Therefore a comprehensive understanding of the defect landscape is required for a broad range of applications. However, determining the type and distribution of defects and their associated strain constitutes a critical task, and for this aim, real-space techniques for atomic resolution characterization are necessary. Here, we use scanning transmission electron microscopy (STEM) to study the atomic structure of individual defects of solution-derived YB a2C u3O7 (YBCO) nanocomposites, where the inclusion of incoherent secondary phase nanoparticles within the YBCO matrix dramatically increases the density of Y1B a2C u4O8 (Y124) intergrowths, the commonest defect in YBCO thin films. The formation of the Y124 is found to trigger a concatenation of strain-derived interactions with other defects and the concomitant nucleation of intrinsic defects, which weave a web of randomly distributed nanostrained regions that profoundly transform the vortex-pinning landscape of the YBCO nanocomposite thin films.

  13. Pleiotropy of Glycogen Synthase Kinase-3 Inhibition by CHIR99021 Promotes Self-Renewal of Embryonic Stem Cells from Refractory Mouse Strains

    Science.gov (United States)

    Ye, Shoudong; Tan, Li; Yang, Rongqing; Fang, Bo; Qu, Su; Schulze, Eric N.; Song, Houyan; Ying, Qilong; Li, Ping

    2012-01-01

    Background Inhibition of glycogen synthase kinase-3 (GSK-3) improves the efficiency of embryonic stem (ES) cell derivation from various strains of mice and rats, as well as dramatically promotes ES cell self-renewal potential. β-catenin has been reported to be involved in the maintenance of self-renewal of ES cells through TCF dependent and independent pathway. But the intrinsic difference between ES cell lines from different species and strains has not been characterized. Here, we dissect the mechanism of GSK-3 inhibition by CHIR99021 in mouse ES cells from refractory mouse strains. Methodology/Principal Findings We found that CHIR99021, a GSK-3 specific inhibitor, promotes self-renewal of ES cells from recalcitrant C57BL/6 (B6) and BALB/c mouse strains through stabilization of β-catenin and c-Myc protein levels. Stabilized β-catenin promoted ES self-renewal through two mechanisms. First, β-catenin translocated into the nucleus to maintain stem cell pluripotency in a lymphoid-enhancing factor/T-cell factor–independent manner. Second, β-catenin binds plasma membrane-localized E-cadherin, which ensures a compact, spherical morphology, a hallmark of ES cells. Further, elevated c-Myc protein levels did not contribute significantly to CH-mediated ES cell self-renewal. Instead, the role of c-Myc is dependent on its transformation activity and can be replaced by N-Myc but not L-Myc. β-catenin and c-Myc have similar effects on ES cells derived from both B6 and BALB/c mice. Conclusions/Significance Our data demonstrated that GSK-3 inhibition by CH promotes self-renewal of mouse ES cells with non-permissive genetic backgrounds by regulation of multiple signaling pathways. These findings would be useful to improve the availability of normally non-permissive mouse strains as research tools. PMID:22540008

  14. Fullerene derivatives as electron acceptors for organic photovoltaic cells.

    Science.gov (United States)

    Mi, Dongbo; Kim, Ji-Hoon; Kim, Hee Un; Xu, Fei; Hwang, Do-Hoon

    2014-02-01

    Energy is currently one of the most important problems humankind faces. Depletion of traditional energy sources such as coal and oil results in the need to develop new ways to create, transport, and store electricity. In this regard, the sun, which can be considered as a giant nuclear fusion reactor, represents the most powerful source of energy available in our solar system. For photovoltaic cells to gain widespread acceptance as a source of clean and renewable energy, the cost per watt of solar energy must be decreased. Organic photovoltaic cells, developed in the past two decades, have potential as alternatives to traditional inorganic semiconductor photovoltaic cells, which suffer from high environmental pollution and energy consumption during production. Organic photovoltaic cells are composed of a blended film of a conjugated-polymer donor and a soluble fullerene-derivative acceptor sandwiched between a poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate)-coated indium tin oxide positive electrode and a low-work-function metal negative electrode. Considerable research efforts aim at designing and synthesizing novel fullerene derivatives as electron acceptors with up-raised lowest unoccupied molecular orbital energy, better light-harvesting properties, higher electron mobility, and better miscibility with the polymer donor for improving the power conversion efficiency of the organic photovoltaic cells. In this paper, we systematically review novel fullerene acceptors synthesized through chemical modification for enhancing the photovoltaic performance by increasing open-circuit voltage, short-circuit current, and fill factor, which determine the performance of organic photovoltaic cells.

  15. Identification of rabbit annulus fibrosus-derived stem cells.

    Directory of Open Access Journals (Sweden)

    Chen Liu

    Full Text Available Annulus fibrosus (AF injuries can lead to substantial deterioration of intervertebral disc (IVD which characterizes degenerative disc disease (DDD. However, treatments for AF repair/regeneration remain challenging due to the intrinsic heterogeneity of AF tissue at cellular, biochemical, and biomechanical levels. In this study, we isolated and characterized a sub-population of cells from rabbit AF tissue which formed colonies in vitro and could self-renew. These cells showed gene expression of typical surface antigen molecules characterizing mesenchymal stem cells (MSCs, including CD29, CD44, and CD166. Meanwhile, they did not express negative markers of MSCs such as CD4, CD8, and CD14. They also expressed Oct-4, nucleostemin, and SSEA-4 proteins. Upon induced differentiation they showed typical osteogenesis, chondrogenesis, and adipogenesis potential. Together, these AF-derived colony-forming cells possessed clonogenicity, self-renewal, and multi-potential differentiation capability, the three criteria characterizing MSCs. Such AF-derived stem cells may potentially be an ideal candidate for DDD treatments using cell therapies or tissue engineering approaches.

  16. The potential of induced pluripotent stem cell derived hepatocytes.

    Science.gov (United States)

    Hannoun, Zara; Steichen, Clara; Dianat, Noushin; Weber, Anne; Dubart-Kupperschmitt, Anne

    2016-07-01

    Orthotopic liver transplantation remains the only curative treatment for liver disease. However, the number of patients who die while on the waiting list (15%) has increased in recent years as a result of severe organ shortages; furthermore the incidence of liver disease is increasing worldwide. Clinical trials involving hepatocyte transplantation have provided encouraging results. However, transplanted cell function appears to often decline after several months, necessitating liver transplantation. The precise aetiology of the loss of cell function is not clear, but poor engraftment and immune-mediated loss appear to be important factors. Also, primary human hepatocytes (PHH) are not readily available, de-differentiate, and die rapidly in culture. Hepatocytes are available from other sources, such as tumour-derived human hepatocyte cell lines and immortalised human hepatocyte cell lines or porcine hepatocytes. However, all these cells suffer from various limitations such as reduced or differences in functions or risk of zoonotic infections. Due to their significant potential, one possible inexhaustible source of hepatocytes is through the directed differentiation of human induced pluripotent stem cells (hiPSCs). This review will discuss the potential applications and existing limitations of hiPSC-derived hepatocytes in regenerative medicine, drug screening, in vitro disease modelling and bioartificial livers. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  17. Apple derived cellulose scaffolds for 3D mammalian cell culture.

    Directory of Open Access Journals (Sweden)

    Daniel J Modulevsky

    Full Text Available There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment.

  18. Circulating cell-derived microparticles in women with pregnancy loss.

    Science.gov (United States)

    Alijotas-Reig, Jaume; Palacio-Garcia, Carles; Farran-Codina, Immaculada; Zarzoso, Cristina; Cabero-Roura, Luis; Vilardell-Tarres, Miquel

    2011-09-01

    To analyze cell-derived microparticles (cMP) in pregnancy loss (PL), both recurrent miscarriages (RM) and unexplained fetal loss (UFL). Non-matched case-control study was performed at Vall d'Hebron Hospital. Cell-derived microparticles of 53 PL cases, 30 with RM, 16 with UFL, and 7 (RM + UFL), were compared to 38 healthy pregnant women. Twenty healthy non-pregnant women act as controls. Cell-derived microparticles were analyzed through flow cytometry. Results are given as total annexin (A5+), endothelial-(CD144+/CD31+ CD41-), platelet-(CD41+), leukocyte-(CD45+) and CD41- c-MP/μL of plasma. Antiphospholipid antibodies (aPLA) were analyzed according to established methods. Comparing PL versus healthy pregnant, we observed a significant endothelial cMP decrease in PL. When comparing RM subgroup with controls, we observed significant decreases in endothelial cMP. When comparing the PL positive for aPLA versus PL-aPLA-negative, no cMP numbering differences were seen. Pregnancy loss seems to be related to endothelial cell activation and/or consumption. A relationship between aPLA and cMP could not be demonstrated. © 2011 John Wiley & Sons A/S.

  19. Abnormal sensitivity of some Cockayne's syndrome cell strains to UV- and γ-rays

    International Nuclear Information System (INIS)

    Deschavanne, P.J.; Chavaudra, N.; Fertil, B.; Malaise, E.P.

    1984-01-01

    Cockayne's syndrome (CS) is a rare autosomal recessive genetic disease. Using a colony assay in vitro, we studied the sensitivity of 5 CS cell strains and two normal ones to UV- and γ-irradiation. The 5 CS strains appear to be UV-hypersensitive but the sensitivity varies widely from one strain to another. Hypersensitivity to γ-rays has been reported for 4 out of the 5 CS cell strains investigated. Repair of potentially lethal damage (PLD) after UV- and γ-irradiation was investigated by using unfed plateau cell cultures. Under these conditions, control cells show a great capacity to repair PLD. The two CS strains, which are hypersensitive to both UV- and γ-irradiation, exhibit no or only little PLD repair after treatment. The response of CS cell strains after γ-irradiation suggests a genetic heterogeneity. Three complementation groups are described in CS cells when dealing with UV radiosensitivity. However, variations in γ-ray sensitivity are reported for cells within the same UV complementation group. (Auth.)

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

    Science.gov (United States)

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

  1. BCG strain S4-Jena: An early BCG strain is capable to reduce the proliferation of bladder cancer cells by induction of apoptosis

    Directory of Open Access Journals (Sweden)

    Hermann Inge-Marie

    2010-06-01

    Full Text Available Abstract Background Intravesical immunotherapy with Mycobacterium bovis bacillus Calmette-Guérin has been established as the most effective adjuvant treatment for high risk non-muscle-invasive bladder cancer (NMIBC. We investigated the differences between the S4-Jena BCG strain and commercially available BCG strains. We tested the genotypic varieties between S4-Jena and other BCG strains and analysed the effect of the BCG strains TICE and S4-Jena on two bladder cancer cell lines. Results In contrast to commercially available BCG strains the S4-Jena strain shows genotypic differences. Spoligotyping verifies the S4-Jena strain as a BCG strain. Infection with viable S4-Jena or TICE decreased proliferation in the T24 cell line. Additionally, hallmarks of apoptosis were detectable. In contrast, Cal29 cells showed only a slightly decreased proliferation with TICE. Cal29 cells infected with S4-Jena, though, showed a significantly decreased proliferation in contrast to TICE. Concordantly with these results, infection with TICE had no effect on the morphology and hallmarks of apoptosis of Cal29 cells. However, S4-Jena strain led to clearly visible morphological changes and caspases 3/7 activation and PS flip. Conclusions S4-Jena strain has a direct influence on bladder cancer cell lines as shown by inhibition of cell proliferation and induction of apoptosis. The data implicate that the T24 cells are responder for S4-Jena and TICE BCG. However, the Cal29 cells are only responder for S4-Jena and they are non-responder for TICE BCG. S4-Jena strain may represent an effective therapeutic agent for NMIBC.

  2. Phylogenetic Analysis of Shewanella Strains by DNA Relatedness Derived from Whole Genome Microarray DNA-DNA Hybridization and Comparisons with Other Methods

    International Nuclear Information System (INIS)

    Wu, Liyou; Yi, T.Y.; Van Nostrand, Joy; Zhou, Jizhong

    2010-01-01

    Phylogenetic analyses were done for the Shewanella strains isolated from Baltic Sea (38 strains), US DOE Hanford Uranium bioremediation site (Hanford Reach of the Columbia River (HRCR), 11 strains), Pacific Ocean and Hawaiian sediments (8 strains), and strains from other resources (16 strains) with three out group strains, Rhodopseudomonas palustris, Clostridium cellulolyticum, and Thermoanaerobacter ethanolicus X514, using DNA relatedness derived from WCGA-based DNA-DNA hybridizations, sequence similarities of 16S rRNA gene and gyrB gene, and sequence similarities of 6 loci of Shewanella genome selected from a shared gene list of the Shewanella strains with whole genome sequenced based on the average nucleotide identity of them (ANI). The phylogenetic trees based on 16S rRNA and gyrB gene sequences, and DNA relatedness derived from WCGA hybridizations of the tested Shewanella strains share exactly the same sub-clusters with very few exceptions, in which the strains were basically grouped by species. However, the phylogenetic analysis based on DNA relatedness derived from WCGA hybridizations dramatically increased the differentiation resolution at species and strains level within Shewanella genus. When the tree based on DNA relatedness derived from WCGA hybridizations was compared to the tree based on the combined sequences of the selected functional genes (6 loci), we found that the resolutions of both methods are similar, but the clustering of the tree based on DNA relatedness derived from WMGA hybridizations was clearer. These results indicate that WCGA-based DNA-DNA hybridization is an idea alternative of conventional DNA-DNA hybridization methods and it is superior to the phylogenetics methods based on sequence similarities of single genes. Detailed analysis is being performed for the re-classification of the strains examined.

  3. Phylogenetic Analysis of Shewanella Strains by DNA Relatedness Derived from Whole Genome Microarray DNA-DNA Hybridization and Comparison with Other Methods

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Liyou; Yi, T. Y.; Van Nostrand, Joy; Zhou, Jizhong

    2010-05-17

    Phylogenetic analyses were done for the Shewanella strains isolated from Baltic Sea (38 strains), US DOE Hanford Uranium bioremediation site [Hanford Reach of the Columbia River (HRCR), 11 strains], Pacific Ocean and Hawaiian sediments (8 strains), and strains from other resources (16 strains) with three out group strains, Rhodopseudomonas palustris, Clostridium cellulolyticum, and Thermoanaerobacter ethanolicus X514, using DNA relatedness derived from WCGA-based DNA-DNA hybridizations, sequence similarities of 16S rRNA gene and gyrB gene, and sequence similarities of 6 loci of Shewanella genome selected from a shared gene list of the Shewanella strains with whole genome sequenced based on the average nucleotide identity of them (ANI). The phylogenetic trees based on 16S rRNA and gyrB gene sequences, and DNA relatedness derived from WCGA hybridizations of the tested Shewanella strains share exactly the same sub-clusters with very few exceptions, in which the strains were basically grouped by species. However, the phylogenetic analysis based on DNA relatedness derived from WCGA hybridizations dramatically increased the differentiation resolution at species and strains level within Shewanella genus. When the tree based on DNA relatedness derived from WCGA hybridizations was compared to the tree based on the combined sequences of the selected functional genes (6 loci), we found that the resolutions of both methods are similar, but the clustering of the tree based on DNA relatedness derived from WMGA hybridizations was clearer. These results indicate that WCGA-based DNA-DNA hybridization is an idea alternative of conventional DNA-DNA hybridization methods and it is superior to the phylogenetics methods based on sequence similarities of single genes. Detailed analysis is being performed for the re-classification of the strains examined.

  4. Paradoxical binding levels of vasoactive amines to cultured cerebral microvessel derived endothelial cells

    International Nuclear Information System (INIS)

    Robinson, R.A.; TenEyck, C.J.; Linthicum, D.S.; Hart, M.N.

    1986-01-01

    Vascular sensitization to vasoactive amines (VAA) may be critical for the development of experimental autoimmune encephalitis as well as other autoimmune diseases. Some inbred stains of mice such as SJL/J are particularly sensitive to the effects of VAA while others (BALB/c) are not. This study was performed to determine if the differing response to VAA in vivo is due to differing levels of binding of VAA to cultured brain endothelial (En) cells in vitro. Cells were isolated, grown to confluence, washed twice with binding buffer and incubated with either 3 H-histamine, 3 H-mepyramine or 3 H-5 hydroxytryptamine (5HT) for 1 hour at 37 0 C. Results showed that the BALB derived En cells specifically bound approximately twice as much mepyramine and three times as much 5-HT as the SJL derived En cells. The relative low binding of VAA to SJL En cells may reflect the extreme in vivo sensitivity that this mouse strain displays toward VAA. These seemingly paradoxical levels of VAA binding in the cultured cerebral endothelium may be due to genetic factors and may give insight into diseases that affect the blood brain barrier

  5. 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.

  6. Derivation of corneal endothelial cell-like cells from rat neural crest cells in vitro.

    Directory of Open Access Journals (Sweden)

    Chengqun Ju

    Full Text Available The aim of this study was to investigate the feasibility of inducing rat neural crest cells (NCC to differentiate to functional corneal endothelial cell (CEC-like cells in vitro. Rat NCC were induced with adult CEC-derived conditioned medium. Immunofluorescence, flow cytometry and real time RT-PCR assay were used to detect expression of the corneal endothelium differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2. CFDA SE-labeled CEC-like cells were transplanted to the corneal endothelium of a rat corneal endothelium deficiency model, and an eye-down position was maintained for 24 hours to allow cell attachment. The animals were observed for as long as 2 months after surgery and underwent clinical and histological examination. Spindle-like NCC turned to polygonal CEC-like after induction and expressed N-cadherin, FoxC1, Pitx2, zonula occludens-1 and sodium-potassium pump Na(+/K(+ ATPase. The corneas of the experimental group were much clearer than those of the control group and the mean corneal thickness in the experimental group was significantly less than in the control group7, 14, 21 and 28 days after surgery. Confocal microscopy through focusing and histological analysis confirmed that green fluorescence-positive CEC-like cells formed a monolayer covering the Descemet's membrane in the experimental group. In conclusion, CEC-like cells derived from NCCs displayed characters of native CEC, and the induction protocol provides guidance for future human CEC induction from NCC.

  7. Prion strain discrimination based on rapid in vivo amplification and analysis by the cell panel assay.

    Directory of Open Access Journals (Sweden)

    Yervand Eduard Karapetyan

    Full Text Available Prion strain identification has been hitherto achieved using time-consuming incubation time determinations in one or more mouse lines and elaborate neuropathological assessment. In the present work, we make a detailed study of the properties of PrP-overproducing Tga20 mice. We show that in these mice the four prion strains examined are rapidly and faithfully amplified and can subsequently be discriminated by a cell-based procedure, the Cell Panel Assay.

  8. Role of bone marrow-derived stem cells, renal progenitor cells and ...

    African Journals Online (AJOL)

    It remains the leading cause of late allograft loss. Bone marrow derived stem cells are undifferentiated cells typically characterized by their capacity for self renewal, ability to give rise to multiple differentiated cellular population, including hematopoietic (HSCs) and mesenchymal stem cells (MSCs). Characterization of HSCs ...

  9. 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

  10. 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

  11. Transfection of bone marrow derived cells with immunoregulatory proteins.

    Science.gov (United States)

    Khantakova, Julia N; Silkov, Alexander N; Tereshchenko, Valeriy P; Gavrilova, Elena V; Maksyutov, Rinat A; Sennikov, Sergey V

    2018-03-23

    In vitro electroporation gene transfer was first performed in 1982. Today, this technology has become one of the major vehicles for non-viral transfection of cells. All non-viral transfections, such as calcium phosphate precipitation, lipofection, and magnetic transfection, have been shown to achieve a transfection efficiency of up to 70% in commonly used cell lines, but not in primary cells. Here we describe the use of electroporation to transfect primary mouse bone marrow-derived cells, such as macrophages (Mφ) and dendritic cells (DCs) with high efficiencies (45%-72%) and minimal cell death. The transfection efficiencies and cell death varied depending on the culture duration of the DCs and Mφ. Moreover, the electroporation efficiency was increased when conditioning medium was used for culturing the cells. Furthermore, we demonstrated that measuring the plasmid-encoded secreted proteins is a highly sensitive method for determining the transfection efficiency. In summary, electroporation with plasmid vectors is an efficient method for producing DCs and Mφ with transient expression of immunoregulatory proteins. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. Myeloid-derived suppressor cells in breast cancer.

    Science.gov (United States)

    Markowitz, Joseph; Wesolowski, Robert; Papenfuss, Tracey; Brooks, Taylor R; Carson, William E

    2013-07-01

    Myeloid-derived suppressor cells (MDSCs) are a population of immature myeloid cells defined by their suppressive actions on immune cells such as T cells, dendritic cells, and natural killer cells. MDSCs typically are positive for the markers CD33 and CD11b but express low levels of HLADR in humans. In mice, MDSCs are typically positive for both CD11b and Gr1. These cells exert their suppressive activity on the immune system via the production of reactive oxygen species, arginase, and cytokines. These factors subsequently inhibit the activity of multiple protein targets such as the T cell receptor, STAT1, and indoleamine-pyrrole 2,3-dioxygenase. The numbers of MDSCs tend to increase with cancer burden while inhibiting MDSCs improves disease outcome in murine models. MDSCs also inhibit immune cancer therapeutics. In light of the poor prognosis of metastatic breast cancer in women and the correlation of increasing levels of MDSCs with increasing disease burden, the purposes of this review are to (1) discuss why MDSCs may be important in breast cancer, (2) describe model systems used to study MDSCs in vitro and in vivo, (3) discuss mechanisms involved in MDSC induction/function in breast cancer, and (4) present pre-clinical and clinical studies that explore modulation of the MDSC-immune system interaction in breast cancer. MDSCs inhibit the host immune response in breast cancer patients and diminishing MDSC actions may improve therapeutic outcomes.

  13. Cell-derived microparticles promote coagulation after moderate exercise.

    Science.gov (United States)

    Sossdorf, Maik; Otto, Gordon P; Claus, Ralf A; Gabriel, Holger H W; Lösche, Wolfgang

    2011-07-01

    Cell-derived procoagulant microparticles (MP) might be able to contribute to exercise-induced changes in blood hemostasis. This study aimed to examine (i) the concentration and procoagulant activity of cell-derived MP after a moderate endurance exercise and (ii) the differences in the release, clearance, and activity of MP before and after exercise between trained and untrained individuals. All subjects performed a single bout of physical exercise on a bicycle ergometer for 90 min at 80% of their individual anaerobic threshold. MP were identified and quantified by flow cytometry measurements. Procoagulant activity of MP was measured by a prothrombinase activity assay as well as tissue factor-induced fibrin formation in MP-containing plasma. At baseline, no differences were observed for the absolute number and procoagulant activities of MP between trained and untrained subjects. However, trained individuals had a lower number of tissue factor-positive monocyte-derived MP compared with untrained individuals. In trained subjects, exercise induced a significant increase in the number of MP derived from platelets, monocytes, and endothelial cells, with maximum values at 45 min after exercise and returned to basal levels at 2 h after exercise. Untrained subjects revealed a similar increase in platelet-derived MP, but their level was still increased at 2 h after exercise, indicating a reduced clearance compared with trained individuals. Procoagulant activities of MP were increased immediately after exercise and remained elevated up to 2 h after exercise. We conclude that increased levels of MP were found in healthy individuals after an acute bout of exercise, that the amount of circulating MP contributes to an exercise-induced increase of hemostatic potential, and that there were differences in kinetic and dynamic characteristics between trained and untrained individuals.

  14. Tangent moduli of the Hencky material model derived from the stored energy function at finite strains

    Czech Academy of Sciences Publication Activity Database

    Poživilová, Alena; Plešek, Jiří

    2005-01-01

    Roč. 482, č. 482 (2005), s. 327-330 ISSN 0255-5476 R&D Projects: GA ČR(CZ) GA101/03/0331; GA ČR(CZ) GP106/03/D038 Institutional research plan: CEZ:AV0Z20760514 Keywords : acoustoelasticity * large strains * third order elastic moduli Subject RIV: JG - Metallurgy Impact factor: 0.399, year: 2005

  15. Patulin and secondary metabolite production by marine-derived Penicillium strains

    DEFF Research Database (Denmark)

    Vansteelandt, Marieke; Kerzaon, Isabelle; Blanchet, Elodie

    2012-01-01

    )–mass spectrometry (MS)/MS. Each strain was grown on six different culture media to enhance the number of observable metabolites.Thirty-two secondary metabolites were detected in crude extracts with twenty first observations for studied species. Patulin, a major mycotoxin, was classically detected in extracts...... of these fungi in shellfish farming areas.Patulin induced acute neurotoxicity on Diptera larvae, indicating the interest of this bioassay as an additional tool for detection of this major mycotoxin in crude extracts....

  16. Capsid proteins from field strains of foot-and-mouth disease virus confer a pathogenic phenotype in cattle on an attenuated, cell-culture-adapted virus

    DEFF Research Database (Denmark)

    Bøtner, Anette; Kakker, Naresh K.; Barbezange, Cyril

    2011-01-01

    Chimeric foot-and-mouth disease viruses (FMDVs) have been generated from plasmids containing full-length FMDV cDNAs and characterized. The parental virus cDNA was derived from the cell-culture-adapted O1Kaufbeuren B64 (O1K B64) strain. Chimeric viruses, containing capsid coding sequences derived...... cells than the rescued parental O1K B64 virus. The two chimeric viruses displayed the expected antigenicity in serotype-specific antigen ELISAs. Following inoculation of each virus into cattle, the rescued O1K B64 strain proved to be attenuated whereas, with each chimeric virus, typical clinical signs...... region within the O1K B64 strain that inhibits replication in cattle. These chimeric infectious cDNA plasmids provide a basis for the analysis of FMDV pathogenicity and characterization of receptor utilization in vivo....

  17. Raspberry wine fermentation with suspended and immobilized yeast cells of two strains of Saccharomyces cerevisiae.

    Science.gov (United States)

    Djordjević, Radovan; Gibson, Brian; Sandell, Mari; de Billerbeck, Gustavo M; Bugarski, Branko; Leskošek-Čukalović, Ida; Vunduk, Jovana; Nikićević, Ninoslav; Nedović, Viktor

    2015-01-01

    The objectives of this study were to assess the differences in fermentative behaviour of two different strains of Saccharomyces cerevisiae (EC1118 and RC212) and to determine the differences in composition and sensory properties of raspberry wines fermented with immobilized and suspended yeast cells of both strains at 15 °C. Analyses of aroma compounds, glycerol, acetic acid and ethanol, as well as the kinetics of fermentation and a sensory evaluation of the wines, were performed. All fermentations with immobilized yeast cells had a shorter lag phase and faster utilization of sugars and ethanol production than those fermented with suspended cells. Slower fermentation kinetics were observed in all the samples that were fermented with strain RC212 (suspended and immobilized) than in samples fermented with strain EC1118. Significantly higher amounts of acetic acid were detected in all samples fermented with strain RC212 than in those fermented with strain EC1118 (0.282 and 0.602 g/l, respectively). Slightly higher amounts of glycerol were observed in samples fermented with strain EC1118 than in those fermented with strain RC212. Copyright © 2014 John Wiley & Sons, Ltd.

  18. Genetic control of eosinophilia in mice: gene(s) expressed in bone marrow-derived cells control high responsiveness

    International Nuclear Information System (INIS)

    Vadas, M.A.

    1982-01-01

    A heterogeneity in the capacity of strains of mice to mount eosinophilia is described. BALB/c and C3H are eosinophil high responder strains (EO-HR) and CBA and A/J are eosinophil low responder strains (EO-LR), judged by the response of blood eosinophils to Ascaris suum, and the response of blood, bone marrow, and spleen eosinophils to keyhole limpet hemocyanin given 2 days after 150 mg/kg cyclophosphamide. Some of the gene(s) for high responsiveness appear to be dominant because (EO-HR x EO-LR)F 1 mice were intermediate to high responders. This gene is expressed in bone marrow-derived cells because radiation chimeras of the type EO-HR→F 1 were high responders and EO-LR→F 1 were low responders. This description of a genetic control of eosinophilia in mice may be useful in understanding the role of this cell in parasite immunity and allergy

  19. Inferring time derivatives including cell growth rates using Gaussian processes

    Science.gov (United States)

    Swain, Peter S.; Stevenson, Keiran; Leary, Allen; Montano-Gutierrez, Luis F.; Clark, Ivan B. N.; Vogel, Jackie; Pilizota, Teuta

    2016-12-01

    Often the time derivative of a measured variable is of as much interest as the variable itself. For a growing population of biological cells, for example, the population's growth rate is typically more important than its size. Here we introduce a non-parametric method to infer first and second time derivatives as a function of time from time-series data. Our approach is based on Gaussian processes and applies to a wide range of data. In tests, the method is at least as accurate as others, but has several advantages: it estimates errors both in the inference and in any summary statistics, such as lag times, and allows interpolation with the corresponding error estimation. As illustrations, we infer growth rates of microbial cells, the rate of assembly of an amyloid fibril and both the speed and acceleration of two separating spindle pole bodies. Our algorithm should thus be broadly applicable.

  20. Stem-cell-derived products: an FDA update.

    Science.gov (United States)

    Moos, Malcolm

    2008-12-01

    The therapeutic potential of products derived from stem cells of various types has prompted increasing research and development and public attention. Initiation of human clinical trials in the not-too-distant future is now a realistic possibility. It is, therefore, important to weigh the potential benefits against known, theoretical and totally unsuspected risks in light of current knowledge to ensure that subjects participating in these trials are afforded the most reasonable balance possible between potential risks and potential benefits. There are no apparent differences in fundamental, qualitative biological characteristics between stem-cell-derived products and other cellular therapies regulated by the United States Food and Drug Administration (FDA). Existing authorities can, therefore, be applied. Nevertheless, these products do have properties that require careful evaluation.

  1. Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism.

    Science.gov (United States)

    Zhao, Hongyun; Yang, Lifeng; Baddour, Joelle; Achreja, Abhinav; Bernard, Vincent; Moss, Tyler; Marini, Juan C; Tudawe, Thavisha; Seviour, Elena G; San Lucas, F Anthony; Alvarez, Hector; Gupta, Sonal; Maiti, Sourindra N; Cooper, Laurence; Peehl, Donna; Ram, Prahlad T; Maitra, Anirban; Nagrath, Deepak

    2016-02-27

    Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells. Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described dependence on oncogenic-Kras signaling. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions.

  2. Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism

    Science.gov (United States)

    Zhao, Hongyun; Yang, Lifeng; Baddour, Joelle; Achreja, Abhinav; Bernard, Vincent; Moss, Tyler; Marini, Juan C; Tudawe, Thavisha; Seviour, Elena G; San Lucas, F Anthony; Alvarez, Hector; Gupta, Sonal; Maiti, Sourindra N; Cooper, Laurence; Peehl, Donna; Ram, Prahlad T; Maitra, Anirban; Nagrath, Deepak

    2016-01-01

    Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells. Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described dependence on oncogenic-Kras signaling. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions. DOI: http://dx.doi.org/10.7554/eLife.10250.001 PMID:26920219

  3. Characterization and modulation of canine mast cell derived eicosanoids

    Science.gov (United States)

    Lin, Tzu-Yin; London, Cheryl A.

    2013-01-01

    Mast cells play an important role in both innate and acquired immunity as well as several pathological conditions including allergy, arthritis and neoplasia. They influence these processes by producing a variety of mediators including cytokines, chemokines and eicosanoids. Very little is currently known about the spectrum of inflammatory mediators, particularly eicosanoids (prostaglandins and leukotrienes), produced by canine mast cells. This is important since modulating mast cell derived eicosanoids may help in the treatment of autoimmune and inflammatory disorders. The purpose of this study was to investigate the spectrum of eicosanoids produced by normal canine mast cells and to evaluate the effects of cytokines and non-steroidal anti-inflammatory mediators (NSAIDS) on eicosanoid production and release. Canine bone marrow derived cultured mast cells (cBMCMCs) expressed COX-1, COX-2, and 5-LOX and synthesized and released PGD2, PGE2, LTB4, and LTC4 following activation by a variety of stimuli. The selective COX-2 NSAIDs carprofen (Rimadyl®) and deracoxib (Deramaxx®) inhibited PGD2 and PGE2 production but only slightly inhibited LTB4 and LTC4. The mixed COX-1/COX-2 inhibitor piroxicam blocked PGD2 and PGE2 production, but upregulated LTC4 following treatment while tepoxilan (Zubrin®), a pan COX/LOX inhibitor, markedly reduced the production of all eicosanoids. The LOX inhibitor nordihydroguaiaretic acid (NDGA) prevented LTB4/LTC4 release and BMBMC degranulation. Pre-incubation of cBMCMCs with IL-4 and SCF sensitized these cells to degranulation in response to substance P. In conclusion, canine BMCMCs produce an array of eicosanoids similar to those produced by mast cells from other species. Tepoxilan appeared to be the most effective NSAID for blocking eicosanoid production and thus may be useful for modulating mast cell mediated responses in dogs. PMID:20036014

  4. Elliptical posts allow for detailed control of non-equibiaxial straining of cell cultures

    DEFF Research Database (Denmark)

    Olesen, Christian Gammelgaard; Pennisi, Cristian Pablo; de Zee, Mark

    2013-01-01

    tissue cells in vivo are subjected to a range of mechanical deformations including shear strain caused by activities of daily living. Shear strains are suspected to play an important role in tissue necrosis. Method The Flexcell system was redesigned using a finite element model in order to obtain large...... areas of the membrane in a controlled, uniform non-equibiaxial strain state. Results The redesign was manufactured and the resulting strains were experimentally validated by means of image analysis methods. The results showed that the system could be used for experiments varying the shear strain....... Conclusion The result allows scientists and experimentalists to apply detailed control of the strain tensor applied to tissue samples in two dimensions....

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Monocyte-Derived Signals Activate Human Natural Killer Cells in Response to Leishmania Parasites

    Science.gov (United States)

    Messlinger, Helena; Sebald, Heidi; Heger, Lukas; Dudziak, Diana; Bogdan, Christian; Schleicher, Ulrike

    2018-01-01

    Activated natural killer (NK) cells release interferon (IFN)-γ, which is crucial for the control of intracellular pathogens such as Leishmania. In contrast to experimental murine leishmaniasis, the human NK cell response to Leishmania is still poorly characterized. Here, we investigated the interaction of human blood NK cells with promastigotes of different Leishmania species (Leishmania major, Leishmania mexicana, Leishmania infantum, and Leishmania donovani). When peripheral blood mononuclear cells or purified NK cells and monocytes (all derived from healthy blood donors from Germany without a history of leishmaniasis) were exposed to promastigotes, NK cells showed increased surface expression of the activation marker CD69. The extent of this effect varied depending on the Leishmania species; differences between dermotropic and viscerotropic L. infantum strains were not observed. Upregulation of CD69 required direct contact between monocytes and Leishmania and was partly inhibitable by anti-interleukin (IL)-18. Unexpectedly, IL-18 was undetectable in most of the supernatants (SNs) of monocyte/parasite cocultures. Confocal fluorescence microscopy of non-permeabilized cells revealed that Leishmania-infected monocytes trans-presented IL-18 to NK cells. Native, but not heat-treated SNs of monocyte/Leishmania cocultures also induced CD69 on NK cells, indicating the involvement of a soluble heat-labile factor other than IL-18. A role for the NK cell-activating cytokines IL-1β, IL-2, IL-12, IL-15, IL-21, and IFN-α/β was excluded. The increase of CD69 was not paralleled by NK cell IFN-γ production or enhanced cytotoxicity. However, prior exposure of NK cells to Leishmania parasites synergistically increased their IFN-γ release in response to IL-12, which was dependent on endogenous IL-18. CD1c+ dendritic cells were identified as possible source of Leishmania-induced IL-12. Finally, we observed that direct contact between Leishmania and NK cells reduced the

  7. Monocyte-Derived Signals Activate Human Natural Killer Cells in Response to Leishmania Parasites

    Directory of Open Access Journals (Sweden)

    Helena Messlinger

    2018-01-01

    Full Text Available Activated natural killer (NK cells release interferon (IFN-γ, which is crucial for the control of intracellular pathogens such as Leishmania. In contrast to experimental murine leishmaniasis, the human NK cell response to Leishmania is still poorly characterized. Here, we investigated the interaction of human blood NK cells with promastigotes of different Leishmania species (Leishmania major, Leishmania mexicana, Leishmania infantum, and Leishmania donovani. When peripheral blood mononuclear cells or purified NK cells and monocytes (all derived from healthy blood donors from Germany without a history of leishmaniasis were exposed to promastigotes, NK cells showed increased surface expression of the activation marker CD69. The extent of this effect varied depending on the Leishmania species; differences between dermotropic and viscerotropic L. infantum strains were not observed. Upregulation of CD69 required direct contact between monocytes and Leishmania and was partly inhibitable by anti-interleukin (IL-18. Unexpectedly, IL-18 was undetectable in most of the supernatants (SNs of monocyte/parasite cocultures. Confocal fluorescence microscopy of non-permeabilized cells revealed that Leishmania-infected monocytes trans-presented IL-18 to NK cells. Native, but not heat-treated SNs of monocyte/Leishmania cocultures also induced CD69 on NK cells, indicating the involvement of a soluble heat-labile factor other than IL-18. A role for the NK cell-activating cytokines IL-1β, IL-2, IL-12, IL-15, IL-21, and IFN-α/β was excluded. The increase of CD69 was not paralleled by NK cell IFN-γ production or enhanced cytotoxicity. However, prior exposure of NK cells to Leishmania parasites synergistically increased their IFN-γ release in response to IL-12, which was dependent on endogenous IL-18. CD1c+ dendritic cells were identified as possible source of Leishmania-induced IL-12. Finally, we observed that direct contact between Leishmania and NK cells

  8. Aging enhances the vulnerability of mesenchymal stromal cells to uniaxial tensile strain-induced apoptosis.

    Science.gov (United States)

    McKayed, Katey; Prendergast, Patrick J; Campbell, Veronica A

    2016-02-08

    Mechanical priming can be employed in tissue engineering strategies to control the fate and differentiation pattern of mesenchymal stromal cells. This is relevant to regenerative medicine whereby mechanical cues can promote the regeneration of a specific tissue type from mesenchymal precursors. The ability of cells to respond to mechanical forces is dependent upon mechanotransduction pathways that involve membrane-associated proteins, such as integrins. During the aging process changes in the mechanotransduction machinery may influence how cells from aged individuals respond to mechanical priming. In this study mesenchymal stromal cells were prepared from young adult and aged rats and exposed to uniaxial tensile strain at 5% and 10% for 3 days, or 2.5% for 7 days. Application of 5% tensile strain had no impact on cell viability. In contrast, application of 10% tensile strain evoked apoptosis and the strain-induced apoptosis was significantly higher in the mesenchymal stromal cells prepared from the aged rats. In parallel to the age-related difference in cellular responsiveness to strain, an age-related decrease in expression of α2 integrin and actin, and enhanced lipid peroxidation was observed. This study demonstrates that mesenchymal stem cells from aged animals have an altered membrane environment, are more vulnerable to the pro-apoptotic effects of 10% tensile strain and less responsive to the pro-osteogenic effects of 2.5% tensile strain. Thus, it is essential to consider how aged cells respond to mechanical stimuli in order to identify optimal mechanical priming strategies that minimise cell loss, particularly if this approach is to be applied to an aged population. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Novel image analysis methods for quantification of in situ 3-D tendon cell and matrix strain.

    Science.gov (United States)

    Fung, Ashley K; Paredes, J J; Andarawis-Puri, Nelly

    2018-01-23

    Macroscopic tendon loads modulate the cellular microenvironment leading to biological outcomes such as degeneration or repair. Previous studies have shown that damage accumulation and the phases of tendon healing are marked by significant changes in the extracellular matrix, but it remains unknown how mechanical forces of the extracellular matrix are translated to mechanotransduction pathways that ultimately drive the biological response. Our overarching hypothesis is that the unique relationship between extracellular matrix strain and cell deformation will dictate biological outcomes, prompting the need for quantitative methods to characterize the local strain environment. While 2-D methods have successfully calculated matrix strain and cell deformation, 3-D methods are necessary to capture the increased complexity that can arise due to high levels of anisotropy and out-of-plane motion, particularly in the disorganized, highly cellular, injured state. In this study, we validated the use of digital volume correlation methods to quantify 3-D matrix strain using images of naïve tendon cells, the collagen fiber matrix, and injured tendon cells. Additionally, naïve tendon cell images were used to develop novel methods for 3-D cell deformation and 3-D cell-matrix strain, which is defined as a quantitative measure of the relationship between matrix strain and cell deformation. The results support that these methods can be used to detect strains with high accuracy and can be further extended to an in vivo setting for observing temporal changes in cell and matrix mechanics during degeneration and healing. Copyright © 2017. Published by Elsevier Ltd.

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

    Science.gov (United States)

    Wakayama, Sayaka; Wakayama, Teruhiko

    2010-01-01

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

  11. Skin appendage-derived stem cells: cell biology and potential for wound repair.

    Science.gov (United States)

    Xie, Jiangfan; Yao, Bin; Han, Yutong; Huang, Sha; Fu, Xiaobing

    2016-01-01

    Stem cells residing in the epidermis and skin appendages are imperative for skin homeostasis and regeneration. These stem cells also participate in the repair of the epidermis after injuries, inducing restoration of tissue integrity and function of damaged tissue. Unlike epidermis-derived stem cells, comprehensive knowledge about skin appendage-derived stem cells remains limited. In this review, we summarize the current knowledge of skin appendage-derived stem cells, including their fundamental characteristics, their preferentially expressed biomarkers, and their potential contribution involved in wound repair. Finally, we will also discuss current strategies, future applications, and limitations of these stem cells, attempting to provide some perspectives on optimizing the available therapy in cutaneous repair and regeneration.

  12. 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.

  13. Genome Sequences of Industrially Relevant Saccharomyces cerevisiae Strain M3707, Isolated from a Sample of Distillers Yeast and Four Haploid Derivatives

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Steven D.; Klingeman, Dawn M.; Johnson, Courtney M.; Clum, Alicia; Aerts, Andrea; Salamov, Asaf; Sharma, Aditi; Zane, Matthew; Barry, Kerrie; Grigoriev, Igor V.; Davison, Brian H.; Lynd, Lee R.; Gilna, Paul; Hau, Heidi; Hogsett, David A.; Froehlich, Allan C.

    2013-04-19

    Saccharomyces cerevisiae strain M3707 was isolated from a sample of commercial distillers yeast, and its genome sequence together with the genome sequences for the four derived haploid strains M3836, M3837, M3838, and M3839 has been determined. Yeasts have potential for consolidated bioprocessing (CBP) for biofuel production, and access to these genome sequences will facilitate their development.

  14. Defective recoveryfrom potentially lethal damage in some human fibroblast cell strains

    International Nuclear Information System (INIS)

    Arlett, C.F.; Priestley, A.

    1983-01-01

    The repair of potentially lethal damage following treatment by gamma radiation was investigated in human fibroblasts held in a non-cycling state by maintenance in a medium containing 0.5 per cent foetal calf serum. Variation in their capacity to repair PLD was noted between three normal cell strains. A failure to repair PLD in ataxia-telangiectasia cells (AT5BI) was confirmed. In three cell strains which were intermediate between normals and A-T cells in their sensitivity, XP3BR, 46BR and GB1142, a limited capacity for the repair of PLD was observed. Two other cell strains, 47BR and 67BR, which showed little if any hypersensitivity could be clearly distinguished from normals after a 24 hour period for the repair of PLD. Thus the technique might permit better discrimination between cell strains. One other cell strain, H15617, could be distinguished from normals by proving hypersensitive under all conditions. Here, however, the repair of PLD appeared to be normal. (author)

  15. Finite-Element Modeling of Viscoelastic Cells During High-Frequency Cyclic Strain

    Directory of Open Access Journals (Sweden)

    David W. Holdsworth

    2012-03-01

    Full Text Available Mechanotransduction refers to the mechanisms by which cells sense and respond to local loads and forces. The process of mechanotransduction plays an important role both in maintaining tissue viability and in remodeling to repair damage; moreover, it may be involved in the initiation and progression of diseases such as osteoarthritis and osteoporosis. An understanding of the mechanisms by which cells respond to surrounding tissue matrices or artificial biomaterials is crucial in regenerative medicine and in influencing cellular differentiation. Recent studies have shown that some cells may be most sensitive to low-amplitude, high-frequency (i.e., 1–100 Hz mechanical stimulation. Advances in finite-element modeling have made it possible to simulate high-frequency mechanical loading of cells. We have developed a viscoelastic finite-element model of an osteoblastic cell (including cytoskeletal actin stress fibers, attached to an elastomeric membrane undergoing cyclic isotropic radial strain with a peak value of 1,000 µstrain. The results indicate that cells experience significant stress and strain amplification when undergoing high-frequency strain, with peak values of cytoplasmic strain five times higher at 45 Hz than at 1 Hz, and peak Von Mises stress in the nucleus increased by a factor of two. Focal stress and strain amplification in cells undergoing high-frequency mechanical stimulation may play an important role in mechanotransduction.

  16. The 6-chromanol derivate SUL-109 enables prolonged hypothermic storage of adipose tissue-derived stem cells

    NARCIS (Netherlands)

    Hajmousa, Ghazaleh; Vogelaar, Pieter; Brouwer, Linda A.; Graaf, Adrianus Cornelis van der; Henning, Robert H.; Krenning, Guido

    Encouraging advances in cell therapy research with adipose derived stem cells (ASC) require an effective short-term preservation method that provides time for quality control and transport of cells from their manufacturing facility to their clinical destination. Hypothermic storage of cells in their

  17. Comparative study of adipose-derived stem cells and bone marrow-derived stem cells in similar microenvironmental conditions

    Energy Technology Data Exchange (ETDEWEB)

    Guneta, Vipra [Division of Materials Technology, School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore); Tan, Nguan Soon [School of Biological Science, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); KK Research Centre, KK Women' s and Children Hospital, 100 Bukit Timah Road, Singapore 229899 (Singapore); Institute of Molecular and Cell Biology, Agency for Science Technology & Research - A*STAR, 61 Biopolis Drive, Proteos, Singapore 138673 (Singapore); Chan, Soon Kiat Jeremy [School of Biological Science, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Tanavde, Vivek [Bioinformatics Institute, Agency for Science Technology & Research - A*STAR, 30 Biopolis Street, Matrix, Singapore 138671 (Singapore); Lim, Thiam Chye [Division of Plastic, Reconstructive and Aesthetic Surgery, Department of Surgery, National University Hospital (NUH) and National University of Singapore (NUS), Kent Ridge Wing, Singapore 119074 (Singapore); Wong, Thien Chong Marcus [Plastic, Reconstructive and Aesthetic Surgery Section, Tan Tock Seng Hospital (TTSH), 11, Jalan Tan Tock Seng, Singapore 308433 (Singapore); Choong, Cleo, E-mail: cleochoong@ntu.edu.sg [Division of Materials Technology, School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore); KK Research Centre, KK Women' s and Children Hospital, 100 Bukit Timah Road, Singapore 229899 (Singapore)

    2016-11-01

    Mesenchymal stem cells (MSCs), which were first isolated from the bone marrow, are now being extracted from various other tissues in the body, including the adipose tissue. The current study presents systematic evidence of how the adipose tissue-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (Bm-MSCs) behave when cultured in specific pro-adipogenic microenvironments. The cells were first characterized and identified as MSCs in terms of their morphology, phenotypic expression, self-renewal capabilities and multi-lineage potential. Subsequently, the proliferation and gene expression profiles of the cell populations cultured on two-dimensional (2D) adipose tissue extracellular matrix (ECM)-coated tissue culture plastic (TCP) and in three-dimensional (3D) AlgiMatrix® microenvironments were analyzed. Overall, it was found that adipogenesis was triggered in both cell populations due to the presence of adipose tissue ECM. However, in 3D microenvironments, ASCs and Bm-MSCs were predisposed to the adipogenic and osteogenic lineages respectively. Overall, findings from this study will contribute to ongoing efforts in adipose tissue engineering as well as provide new insights into the role of the ECM and cues provided by the immediate microenvironment for stem cell differentiation. - Highlights: • Native adipose tissue ECM coated on 2D TCP triggers adipogenesis in both ASCs and Bm-MSCs. • A 3D microenvironment with similar stiffness to adipose tissue induces adipogenic differentiation of ASCs. • ASCs cultured in 3D alginate scaffolds exhibit predisposition to adipogenesis. • Bm-MSCs cultured in 3D alginate scaffolds exhibit predisposition to osteogenesis. • The native microenvironment of the cells affects their differentiation behaviour in vitro.

  18. Isolation of Mature (Peritoneum-Derived Mast Cells and Immature (Bone Marrow-Derived Mast Cell Precursors from Mice.

    Directory of Open Access Journals (Sweden)

    Steffen K Meurer

    Full Text Available Mast cells (MCs are a versatile cell type playing key roles in tissue morphogenesis and host defence against bacteria and parasites. Furthermore, they can enhance immunological danger signals and are implicated in inflammatory disorders like fibrosis. This granulated cell type originates from the myeloid lineage and has similarities to basophilic granulocytes, both containing large quantities of histamine and heparin. Immature murine mast cells mature in their destination tissue and adopt either the connective tissue (CTMC or mucosal (MMC type. Some effector functions are executed by activation/degranulation of MCs which lead to secretion of a typical set of MC proteases (MCPT and of the preformed or newly synthesized mediators from its granules into the local microenvironment. Due to the potential accumulation of mutations in key signalling pathway components of corresponding MC cell-lines, primary cultured MCs are an attractive mean to study general features of MC biology and aspects of MC functions relevant to human disease. Here, we describe a simple protocol for the simultaneous isolation of mature CTMC-like murine MCs from the peritoneum (PMCs and immature MC precursors from the bone marrow (BM. The latter are differentiated in vitro to yield BM-derived MCs (BMMC. These cells display the typical morphological and phenotypic features of MCs, express the typical MC surface markers, and can be propagated and kept in culture for several weeks. The provided protocol allows simple amplification of large quantities of homogenous, non-transformed MCs from the peritoneum and bone marrow-derived mast cells for cell- and tissue-based biomedical research.

  19. Comparative study of adipose-derived stem cells and bone marrow-derived stem cells in similar microenvironmental conditions

    International Nuclear Information System (INIS)

    Guneta, Vipra; Tan, Nguan Soon; Chan, Soon Kiat Jeremy; Tanavde, Vivek; Lim, Thiam Chye; Wong, Thien Chong Marcus; Choong, Cleo

    2016-01-01

    Mesenchymal stem cells (MSCs), which were first isolated from the bone marrow, are now being extracted from various other tissues in the body, including the adipose tissue. The current study presents systematic evidence of how the adipose tissue-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (Bm-MSCs) behave when cultured in specific pro-adipogenic microenvironments. The cells were first characterized and identified as MSCs in terms of their morphology, phenotypic expression, self-renewal capabilities and multi-lineage potential. Subsequently, the proliferation and gene expression profiles of the cell populations cultured on two-dimensional (2D) adipose tissue extracellular matrix (ECM)-coated tissue culture plastic (TCP) and in three-dimensional (3D) AlgiMatrix® microenvironments were analyzed. Overall, it was found that adipogenesis was triggered in both cell populations due to the presence of adipose tissue ECM. However, in 3D microenvironments, ASCs and Bm-MSCs were predisposed to the adipogenic and osteogenic lineages respectively. Overall, findings from this study will contribute to ongoing efforts in adipose tissue engineering as well as provide new insights into the role of the ECM and cues provided by the immediate microenvironment for stem cell differentiation. - Highlights: • Native adipose tissue ECM coated on 2D TCP triggers adipogenesis in both ASCs and Bm-MSCs. • A 3D microenvironment with similar stiffness to adipose tissue induces adipogenic differentiation of ASCs. • ASCs cultured in 3D alginate scaffolds exhibit predisposition to adipogenesis. • Bm-MSCs cultured in 3D alginate scaffolds exhibit predisposition to osteogenesis. • The native microenvironment of the cells affects their differentiation behaviour in vitro.

  20. Interaction between adipose tissue-derived mesenchymal stem cells and regulatory T-cells

    OpenAIRE

    Engela, Anja; Baan, Carla; Peeters, Anna; Weimar, Willem; Hoogduijn, Martin

    2013-01-01

    textabstractMesenchymal stem cells (MSCs) exhibit immunosuppressive capabilities, which have evoked interest in their application as cell therapy in transplant patients. So far it has been unclear whether allogeneic MSCs and host regulatory T-cells (Tregs) functionally influence each other. We investigated the interaction between both cell types using perirenal adipose tissue-derived MSCs (ASCs) from kidney donors and Tregs from blood bank donors or kidney recipients 6 months after transplant...

  1. Human umbilical cord blood stem cells and brain-derived neurotrophic factor for optic nerve injury: a biomechanical evaluation

    Directory of Open Access Journals (Sweden)

    Zhong-jun Zhang

    2015-01-01

    Full Text Available Treatment for optic nerve injury by brain-derived neurotrophic factor or the transplantation of human umbilical cord blood stem cells has gained progress, but analysis by biomechanical indicators is rare. Rabbit models of optic nerve injury were established by a clamp. At 7 days after injury, the vitreous body received a one-time injection of 50 μg brain-derived neurotrophic factor or 1 × 10 6 human umbilical cord blood stem cells. After 30 days, the maximum load, maximum stress, maximum strain, elastic limit load, elastic limit stress, and elastic limit strain had clearly improved in rabbit models of optical nerve injury after treatment with brain-derived neurotrophic factor or human umbilical cord blood stem cells. The damage to the ultrastructure of the optic nerve had also been reduced. These findings suggest that human umbilical cord blood stem cells and brain-derived neurotrophic factor effectively repair the injured optical nerve, improve biomechanical properties, and contribute to the recovery after injury.

  2. Antiplasmodial activity of new 4-aminoquinoline derivatives against chloroquine resistant strain.

    Science.gov (United States)

    Sinha, Manish; Dola, Vasanth R; Agarwal, Pooja; Srivastava, Kumkum; Haq, Wahajul; Puri, Sunil K; Katti, Seturam B

    2014-07-15

    Emergence and spread of multidrug resistant strains of Plasmodium falciparum has severely limited the antimalarial chemotherapeutic options. In order to overcome the obstacle, a set of new side-chain modified 4-aminoquinolines were synthesized and screened against chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of P. falciparum. The key feature of the designed molecules is the use of methylpiperazine linked α, β(3)- and γ-amino acids to generate novel side chain modified 4-aminoquinoline analogues. Among the evaluated compounds, 20c and 30 were found more potent than CQ against K1 and displayed a four-fold and a three-fold higher activity respectively, with a good selectivity index (SI=5846 and 11,350). All synthesized compounds had resistance index between 1.06 and >14.13 as against 47.2 for chloroquine. Biophysical studies suggested that this series of compounds act on heme polymerization target. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Subsidence and current strain patterns on Tenerife Island (Canary Archipelago, Spain) derived from continuous GNSS time series (2008-2015)

    Science.gov (United States)

    Sánchez-Alzola, A.; Martí, J.; García-Yeguas, A.; Gil, A. J.

    2016-11-01

    In this paper we present the current crustal deformation model of Tenerife Island derived from daily CGPS time series processing (2008-2015). Our results include the position time series, a global velocity estimation and the current crustal deformation on the island in terms of strain tensors. We detect a measurable subsidence of 1.5-2 mm/yr. in the proximities of the Cañadas-Teide-Pico Viejo (CTPV) complex. These values are higher in the central part of the complex and could be explained by a lateral spreading of the elastic lithosphere combined with the effect of the drastic descent of the water table in the island experienced during recent decades. The results show that the Anaga massif is stable in both its horizontal and vertical components. The strain tensor analysis shows a 70 nstrain/yr. E-W compression in the central complex, perpendicular to the 2004 sismo-volcanic area, and 50 nstrain/yr. SW-NE extension towards the Northeast ridge. The residual velocity and strain patterns coincide with a decline in volcanic activity since the 2004 unrest.

  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. Generation of induced pluripotent stem cell-derived mice by reprogramming of a mature NKT cell.

    Science.gov (United States)

    Ren, Yue; Dashtsoodol, Nyambayar; Watarai, Hiroshi; Koseki, Haruhiko; Quan, Chengshi; Taniguchi, Masaru

    2014-10-01

    NKT cells are characterized by their expression of an NKT-cell-specific invariant antigen-receptor α chain encoded by Vα14Jα18 gene segments. These NKT cells bridge the innate and acquired immune systems to mediate effective and augmented responses; however, the limited number of NKT cells in vivo hampers their analysis. Here, two lines of induced pluripotent stem cell-derived mice (NKT-iPSC-derived mice) were generated by reprogramming of mature NKT cells, where one harbors both rearranged Vα14Jα18 and Vβ7 genes and the other carries rearranged Vα14Jα18 on both alleles but germline Vβ loci. The analysis of NKT-iPSC-derived mice showed a significant increase in NKT cell numbers with relatively normal frequencies of functional subsets, but significantly enhanced in some cases, and acquired functional NKT cell maturation in peripheral lymphoid organs. NKT-iPSC-derived mice also showed normal development of other immune cells except for the absence of γδT cells and disturbed development of conventional CD4 αβT cells. These results suggest that the NKT-iPSC-derived mice are a better model for NKT cell development and function study rather than transgenic mouse models reported previously and also that the presence of a pre-rearranged Vα14Jα18 in the natural chromosomal context favors the developmental fate of NKT cells. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society for Immunology.

  6. Adipose-derived mesenchymal stem cells promote cell proliferation and invasion of epithelial ovarian cancer

    Energy Technology Data Exchange (ETDEWEB)

    Chu, Yijing; Tang, Huijuan; Guo, Yan; Guo, Jing; Huang, Bangxing; Fang, Fang; Cai, Jing, E-mail: caijingmmm@hotmail.com; Wang, Zehua, E-mail: zehuawang@163.net

    2015-09-10

    Adipose-derived mesenchymal stem cell (ADSC) is an important component of tumor microenvironment. However, whether ADSCs have a hand in ovarian cancer progression remains unclear. In this study, we investigated the impact of human ADSCs derived from the omentum of normal donors on human epithelial ovarian cancer (EOC) cells in vitro and in vivo. Direct and indirect co-culture models including ADSCs and human EOC cell lines were established and the effects of ADSCs on EOC cell proliferation were evaluated by EdU incorporation and flow cytometry. Transwell migration assays and detection of MMPs were performed to assess the invasion activity of EOC cells in vitro. Mouse models were established by intraperitoneal injection of EOC cells with or without concomitant ADSCs to investigate the role of ADSCs in tumor progression in vivo. We found that ADSCs significantly promoted proliferation and invasion of EOC cells in both direct and indirect co-culture assays. In addition, after co-culture with ADSCs, EOC cells secreted higher levels of matrix metalloproteinases (MMPs), and inhibition of MMP2 and MMP9 partially relieved the tumor-promoting effects of ADSCs in vitro. In mouse xenograft models, we confirmed that ADSCs promoted EOC growth and metastasis and elevated the expression of MMP2 and MMP9. Our findings indicate that omental ADSCs play a promotive role during ovarian cancer progression. - Highlights: • Omental adipose derived stem cells enhanced growth and invasion properties of ovarian cancer cells. • Adipose derived stem cells promoted the growth and metastasis of ovarian cancer in mice models. • Adipose derived stem cells promoted MMPs expression and secretion of ovarian cancer cells. • Elevated MMPs mediated the tumor promoting effects of ADSCs.

  7. Adipose-derived mesenchymal stem cells promote cell proliferation and invasion of epithelial ovarian cancer

    International Nuclear Information System (INIS)

    Chu, Yijing; Tang, Huijuan; Guo, Yan; Guo, Jing; Huang, Bangxing; Fang, Fang; Cai, Jing; Wang, Zehua

    2015-01-01

    Adipose-derived mesenchymal stem cell (ADSC) is an important component of tumor microenvironment. However, whether ADSCs have a hand in ovarian cancer progression remains unclear. In this study, we investigated the impact of human ADSCs derived from the omentum of normal donors on human epithelial ovarian cancer (EOC) cells in vitro and in vivo. Direct and indirect co-culture models including ADSCs and human EOC cell lines were established and the effects of ADSCs on EOC cell proliferation were evaluated by EdU incorporation and flow cytometry. Transwell migration assays and detection of MMPs were performed to assess the invasion activity of EOC cells in vitro. Mouse models were established by intraperitoneal injection of EOC cells with or without concomitant ADSCs to investigate the role of ADSCs in tumor progression in vivo. We found that ADSCs significantly promoted proliferation and invasion of EOC cells in both direct and indirect co-culture assays. In addition, after co-culture with ADSCs, EOC cells secreted higher levels of matrix metalloproteinases (MMPs), and inhibition of MMP2 and MMP9 partially relieved the tumor-promoting effects of ADSCs in vitro. In mouse xenograft models, we confirmed that ADSCs promoted EOC growth and metastasis and elevated the expression of MMP2 and MMP9. Our findings indicate that omental ADSCs play a promotive role during ovarian cancer progression. - Highlights: • Omental adipose derived stem cells enhanced growth and invasion properties of ovarian cancer cells. • Adipose derived stem cells promoted the growth and metastasis of ovarian cancer in mice models. • Adipose derived stem cells promoted MMPs expression and secretion of ovarian cancer cells. • Elevated MMPs mediated the tumor promoting effects of ADSCs

  8. Strain differences in the response of mouse testicular stem cells to fractionated radiation

    International Nuclear Information System (INIS)

    Meistrich, M.L.; Finch, M.; Lu, C.C.; de Ruiter-Bootsma, A.L.; de Rooij, D.G.; Davids, J.A.G.

    1984-01-01

    The survival of spermatogonial stem cells in CBA and C3H mice after single and split-dose (24-hr interval) irradiation with fission neutrons and gamma rays was compared. The first doses of the fractionated regimes were either 150 rad (neutrons) or 600 rad (gamma). For both strains the neutron survival curves were exponential. The D 0 value of stem cells in CBA decreased from 83 to 25 rad upon fractionation; that of C3H stem cells decreased only from 54 to 36 rad. The survival curves for gamma irradiation, which all showed shoulders, indicated that C3H stem cells had larger repair capacities than CBA stem cells. However, the most striking difference between the two strains in response to gamma radiation was in the slopes of the second-dose curves. Whereas C3H stem cells showed a small increase of the D 0 upon fractionation (from 196 to 218 rad), CBA stem cells showed a marked decrease (from 243 to 148 rad). The decreases in D 0 upon fractionation, observed in both strains with neutron irradiation and also with gamma irradiation in CBA, are most likely the result of recruitment or progression of radioresistant survivors to a more sensitive state of proliferation or cell cycle phase. It may be that the survivng stem cells in C3H mice are recruited less rapidly and synchronously into active cycle than in CBA mice. Thus, it appears that the strain differences may be quantitative, rather than qualitative

  9. Adhesion of Two Lactobacillus gasseri Probiotic Strains on Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Mojca Narat

    2003-01-01

    Full Text Available Previous in vitro and in vivo studies showed that two human isolates of Lactobacillus gasseri, LF221 and K7 are able to survive the passage through the gastrointestinal tract and to colonise intestines of pigs at least temporarily. The aim of this study was to examine the adhesion ability of LF221 and K7 strains to Caco-2 cells. Adhesion of lactobacilli from early stationary growth phase was examined at two pH values of DMEM buffer (4.5 and 7. Lactobacillus rhamnosus GG, a widely used strain with clinical evidences of its efficiency, served as a positive control. The number of lactobacilli added to each well was found to be crucial in the adhesion assay. When added, lactobacilli were in range of 2.5 · 106 to 2.5 · 108 cfu/well, the linear correlation between the number of adhered cells (log cfu and the number of added cells (log cfu was found for all three strains (R2 > 0.99 at both pH values (4.5 and 7. At the highest concentration of added K7 and GG cells tested (app. 109 cfu/well, the efficiency of adhesion was reduced. pH value of the medium strongly affected the adhesion, which was promoted in acidic conditions (pH=4.5. The adhesion of K7 strain was slightly weaker compared to GG strain at both pH values, while at pH=4.5 the adhesion of LF221 strain was even better than GG adhesion, at least at lower concentration of lactobacilli. The direct comparison of these strains was possible by regression analysis. At lower concentration of lactobacilli (2.5 · 106, the best efficiency of adhesion (% of adhered bacteria was observed for the strain LF221, reaching the values of 7.8 and 1.9 % at pH=4.5 and 7, respectively, while at higher lactobacilli concentration the ration of adhesion was higher for GG strain (3.3 % at pH=4.5. In conclusion, strains LF221 and K7 were demonstrated to be adhesive, especially in acidic conditions. The level of adhesion of K7 and GG strains positively correlates with the number of added lactobacilli only up to the

  10. N,N'-disubstituted cinnamamide derivatives potentiate ciprofloxacin activity against overexpressing NorA efflux pump Staphylococcus aureus 1199B strains.

    Science.gov (United States)

    Radix, Sylvie; Jordheim, Anne Doléans; Rocheblave, Luc; N'Digo, Serge; Prignon, Anne-Laure; Commun, Carine; Michalet, Serge; Dijoux-Franca, Marie-Geneviève; Mularoni, Angélique; Walchshofer, Nadia

    2018-04-25

    A multi-step procedure has been described which afforded satisfactory yields of N,N'-disubstituted cinnamamides derived from N-Boc-protected amino acids (Boc-Gly, Boc-Val, Boc-Phe). The key step of this synthesis was a regioselective RedAl reduction of an amide function in presence of a carbamate group. Next, these cinnamamides were evaluated in co-admnistration with ciprofloxacin as efflux pump inhibitors against two S. aureus strains, NorA overexpressing SA1199B and wild type SA1199. In parallel, their intrinsic toxicity was appreciated on human lung fibroblast MRC5 cells. Therefore, the cinnamamide combining both carbamate and indol-3-yl groups, was found to be the most active and one of the less toxic EPI and constituted a promising hit. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  11. Adipose tissue-derived stem cells in neural regenerative medicine.

    Science.gov (United States)

    Yeh, Da-Chuan; Chan, Tzu-Min; Harn, Horng-Jyh; Chiou, Tzyy-Wen; Chen, Hsin-Shui; Lin, Zung-Sheng; Lin, Shinn-Zong

    2015-01-01

    Adipose tissue-derived stem cells (ADSCs) have two essential characteristics with regard to regenerative medicine: the convenient and efficient generation of large numbers of multipotent cells and in vitro proliferation without a loss of stemness. The implementation of clinical trials has prompted widespread concern regarding safety issues and has shifted research toward the therapeutic efficacy of stem cells in dealing with neural degeneration in cases such as stroke, amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, cavernous nerve injury, and traumatic brain injury. Most existing studies have reported that cell therapies may be able to replenish lost cells and promote neuronal regeneration, protect neuronal survival, and play a role in overcoming permanent paralysis and loss of sensation and the recovery of neurological function. The mechanisms involved in determining therapeutic capacity remain largely unknown; however, this concept can still be classified in a methodical manner by citing current evidence. Possible mechanisms include the following: 1) the promotion of angiogenesis, 2) the induction of neuronal differentiation and neurogenesis, 3) reductions in reactive gliosis, 4) the inhibition of apoptosis, 5) the expression of neurotrophic factors, 6) immunomodulatory function, and 7) facilitating neuronal integration. In this study, several human clinical trials using ADSCs for neuronal disorders were investigated. It is suggested that ADSCs are one of the choices among various stem cells for translating into clinical application in the near future.

  12. 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.

  13. Sepsis puerperalis caused by a genotypically proven cat-derived Pasteurella multocida strain.

    Science.gov (United States)

    Voss, A; van Zwam, Y H; Meis, J F; Melchers, W; Steegers, E A

    1998-01-01

    We report a disseminated intrauterine Pasteurella multocida infection in a puerperal woman who could not remember any traumatic exposure to her cat. An oral swab taken from the cat, just 2 days after the patient's admission, grew Pasteurella multocida, with an PCR-fingerprinting pattern identical to the patient's isolate. Hand-washing after every contact with cats and dogs and if feasible separation of in-house pets from mother and infant should be applied to prevent this uncommon but serious occurrence of post-partum infections. To our knowledge this is the first case of Pasteurella multocida 'child-bed fever', with a genotypically identical strain isolated from the in-house cat.

  14. 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.

  15. Bacillus megaterium strains derived from water and soil exhibit differential responses to the herbicide mesotrione.

    Science.gov (United States)

    Dobrzanski, Tatiane; Gravina, Fernanda; Steckling, Bruna; Olchanheski, Luiz R; Sprenger, Ricardo F; Espírito Santo, Bruno C; Galvão, Carolina W; Reche, Péricles M; Prestes, Rosilene A; Pileggi, Sônia A V; Campos, Francinete R; Azevedo, Ricardo A; Sadowsky, Michael J; Beltrame, Flávio L; Pileggi, Marcos

    2018-01-01

    The intense use of herbicides for weed control in agriculture causes selection pressure on soil microbiota and water ecosystems, possibly resulting in changes to microbial processes, such as biogeochemical cycles. These xenobiotics may increase the production of reactive oxygen species and consequently affect the survival of microorganisms, which need to develop strategies to adapt to these conditions and maintain their ecological functionality. This study analyzed the adaptive responses of bacterial isolates belonging to the same species, originating from two different environments (water and soil), and subjected to selection pressure by herbicides. The effects of herbicide Callisto and its active ingredient, mesotrione, induced different adaptation strategies on the cellular, enzymatic, and structural systems of two Bacillus megaterium isolates obtained from these environments. The lipid saturation patterns observed may have affected membrane permeability in response to this herbicide. Moreover, this may have led to different levels of responses involving superoxide dismutase and catalase activities, and enzyme polymorphisms. Due to these response systems, the strain isolated from water exhibited higher growth rates than did the soil strain, in evaluations made in oligotrophic culture media, which would be more like that found in semi-pristine aquatic environments. The influence of the intracellular oxidizing environments, which changed the mode of degradation of mesotrione in our experimental model and produced different metabolites, can also be observed in soil and water at sites related to agriculture. Since the different metabolites may present different levels of toxicity, we suggest that this fact should be considered in studies on the fate of agrochemicals in different environments.

  16. The SGBS cell strain as a model for the in vitro study of obesity and cancer.

    LENUS (Irish Health Repository)

    Allott, Emma H

    2012-10-01

    The murine adipocyte cell line 3T3-L1 is well characterised and used widely, while the human pre-adipocyte cell strain, Simpson-Golabi-Behmel Syndrome (SGBS), requires validation for use in human studies. Obesity is currently estimated to account for up to 41 % of the worldwide cancer burden. A human in vitro model system is required to elucidate the molecular mechanisms for this poorly understood association. This work investigates the relevance of the SGBS cell strain for obesity and cancer research in humans.

  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. Mechanical strain stimulates vasculogenesis and expression of angiogenesis guidance molecules of embryonic stem cells through elevation of intracellular calcium, reactive oxygen species and nitric oxide generation.

    Science.gov (United States)

    Sharifpanah, Fatemeh; Behr, Sascha; Wartenberg, Maria; Sauer, Heinrich

    2016-12-01

    Differentiation of embryonic stem (ES) cells may be regulated by mechanical strain. Herein, signaling molecules underlying mechanical stimulation of vasculogenesis and expression of angiogenesis guidance cues were investigated in ES cell-derived embryoid bodies. Treatment of embryoid bodies with 10% static mechanical strain using a Flexercell strain system significantly increased CD31-positive vascular structures and the angiogenesis guidance molecules plexinB1, ephrin B2, neuropilin1 (NRP1), semaphorin 4D (sem4D) and robo4 as well as vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-BB (PDGF-BB) as evaluated by Western blot and real time RT-PCR. In contrast ephrin type 4 receptor B (EphB4) expression was down-regulated upon mechanical strain, indicating an arterial-type differentiation. Robo1 protein expression was modestly increased with no change in mRNA expression. Mechanical strain increased intracellular calcium as well as reactive oxygen species (ROS) and nitric oxide (NO). Mechanical strain-induced vasculogenesis was abolished by the NOS inhibitor L-NAME, the NADPH oxidase inhibitor VAS2870, upon chelation of intracellular calcium by BAPTA as well as upon siRNA inactivation of ephrin B2, NRP1 and robo4. BAPTA blunted the strain-induced expression of angiogenic growth factors, the increase in NO and ROS as well as the expression of NRP1, sem4D and plexinB1, whereas ephrin B2, EphB4 as well as robo1 and robo4 expression were not impaired. Mechanical strain stimulates vasculogenesis of ES cells by the intracellular messengers ROS, NO and calcium as well as by upregulation of angiogenesis guidance molecules and the angiogenic growth factors VEGF, FGF-2 and PDGF-BB. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Adipose-Derived Stem Cells Respond to Increased Osmolarities.

    Directory of Open Access Journals (Sweden)

    Urška Potočar

    Full Text Available Cell therapies present a feasible option for the treatment of degenerated cartilaginous and intervertebral disc (IVD tissues. Microenvironments of these tissues are specific and often differ from the microenvironment of cells that, could be potentially used for therapy, e.g. human adipose-derived stem cells (hASC. To ensure safe and efficient implantation of hASC, it is important to evaluate how microenvironmental conditions at the site of implantation affect the implanted cells. This study has demonstrated that cartilaginous tissue-specific osmolarities ranging from 400-600 mOsm/L affected hASC in a dose- and time-dependent fashion in comparison to 300 mOsm/L. Increased osmolarities resulted in transient (nuclear DNA and actin reorganisation and non-transient, long-term morphological changes (vesicle formation, increase in cell area, and culture morphology, as well as reduced proliferation in monolayer cultures. Increased osmolarities diminished acid proteoglycan production and compactness of chondrogenically induced pellet cultures, indicating decreased chondrogenic potential. Viability of hASC was strongly dependent on the type of culture, with hASC in monolayer culture being more tolerant to increased osmolarity compared to hASC in suspension, alginate-agarose hydrogel, and pellet cultures, thus emphasizing the importance of choosing relevant in vitro conditions according to the specifics of clinical application.

  20. Immune Privilege and Eye-Derived T-Regulatory Cells

    Directory of Open Access Journals (Sweden)

    Hiroshi Keino

    2018-01-01

    Full Text Available Certain cellular components of the eye, such as neural retina, are unable to regenerate and replicate after destructive inflammation. Ocular immune privilege provides the eye with immune protection against intraocular inflammation in order to minimize the risk to vision integrity. The eye and immune system use strategies to maintain the ocular immune privilege by regulating the innate and adaptive immune response, which includes immunological ignorance, peripheral tolerance to eye-derived antigens, and intraocular immunosuppressive microenvironment. In this review, we summarize current knowledge regarding the molecular mechanism responsible for the development and maintenance of ocular immune privilege via regulatory T cells (Tregs, which are generated by the anterior chamber-associated immune deviation (ACAID, and ocular resident cells including corneal endothelial (CE cells, ocular pigment epithelial (PE cells, and aqueous humor. Furthermore, we examined the therapeutic potential of Tregs generated by RPE cells that express transforming growth factor beta (TGF-β, cytotoxic T lymphocyte-associated antigen-2 alpha (CTLA-2α, and retinoic acid for autoimmune uveoretinitis and evaluated a new strategy using human RPE-induced Tregs for clinical application in inflammatory ocular disease. We believe that a better understanding of the ocular immune privilege associated with Tregs might offer a new approach with regard to therapeutic interventions for ocular autoimmunity.

  1. Evaluation of cyanobacteria cell count detection derived from ...

    Science.gov (United States)

    Inland waters across the United States (US) are at potential risk for increased outbreaks of toxic cyanobacteria (Cyano) harmful algal bloom (HAB) events resulting from elevated water temperatures and extreme hydrologic events attributable to climate change and increased nutrient loadings associated with intensive agricultural practices. Current monitoring efforts are limited in scope due to resource limitations, analytical complexity, and data integration efforts. The goals of this study were to validate a new ocean color algorithm for satellite imagery that could potentially be used to monitor CyanoHAB events in near real-time to provide a compressive monitoring capability for freshwater lakes (>100 ha). The algorithm incorporated narrow spectral bands specific to the European Space Agency’s (ESA’s) MEdium Resolution Imaging Spectrometer (MERIS) instrument that were optimally oriented at phytoplankton pigment absorption features including phycocyanin at 620 nm. A validation of derived Cyano cell counts was performed using available in situ data assembled from existing monitoring programs across eight states in the eastern US over a 39-month period (2009–2012). Results indicated that MERIS provided robust estimates for Low (10,000–109,000 cells/mL) and Very High (>1,000,000 cells/mL) cell enumeration ranges (approximately 90% and 83%, respectively). However, the results for two intermediate ranges (110,000–299,000 and 300,000–1,000,000 cells/mL)

  2. Large-scale generation of cell-derived nanovesicles

    Science.gov (United States)

    Jo, W.; Kim, J.; Yoon, J.; Jeong, D.; Cho, S.; Jeong, H.; Yoon, Y. J.; Kim, S. C.; Gho, Y. S.; Park, J.

    2014-09-01

    Exosomes are enclosed compartments that are released from cells and that can transport biological contents for the purpose of intercellular communications. Research into exosomes is hindered by their rarity. In this article, we introduce a device that uses centrifugal force and a filter with micro-sized pores to generate a large quantity of cell-derived nanovesicles. The device has a simple polycarbonate structure to hold the filter, and operates in a common centrifuge. Nanovesicles are similar in size and membrane structure to exosomes. Nanovesicles contain intracellular RNAs ranging from microRNA to mRNA, intracellular proteins, and plasma membrane proteins. The quantity of nanovesicles produced using the device is 250 times the quantity of naturally secreted exosomes. Also, the quantity of intracellular contents in nanovesicles is twice that in exosomes. Nanovesicles generated from murine embryonic stem cells can transfer RNAs to target cells. Therefore, this novel device and the nanovesicles that it generates are expected to be used in exosome-related research, and can be applied in various applications such as drug delivery and cell-based therapy.

  3. Immune Privilege and Eye-Derived T-Regulatory Cells.

    Science.gov (United States)

    Keino, Hiroshi; Horie, Shintaro; Sugita, Sunao

    2018-01-01

    Certain cellular components of the eye, such as neural retina, are unable to regenerate and replicate after destructive inflammation. Ocular immune privilege provides the eye with immune protection against intraocular inflammation in order to minimize the risk to vision integrity. The eye and immune system use strategies to maintain the ocular immune privilege by regulating the innate and adaptive immune response, which includes immunological ignorance, peripheral tolerance to eye-derived antigens, and intraocular immunosuppressive microenvironment. In this review, we summarize current knowledge regarding the molecular mechanism responsible for the development and maintenance of ocular immune privilege via regulatory T cells (Tregs), which are generated by the anterior chamber-associated immune deviation (ACAID), and ocular resident cells including corneal endothelial (CE) cells, ocular pigment epithelial (PE) cells, and aqueous humor. Furthermore, we examined the therapeutic potential of Tregs generated by RPE cells that express transforming growth factor beta (TGF- β ), cytotoxic T lymphocyte-associated antigen-2 alpha (CTLA-2 α ), and retinoic acid for autoimmune uveoretinitis and evaluated a new strategy using human RPE-induced Tregs for clinical application in inflammatory ocular disease. We believe that a better understanding of the ocular immune privilege associated with Tregs might offer a new approach with regard to therapeutic interventions for ocular autoimmunity.

  4. Entrance and Survival of Brucella pinnipedialis Hooded Seal Strain in Human Macrophages and Epithelial Cells

    Science.gov (United States)

    Briquemont, Benjamin; Sørensen, Karen K.; Godfroid, Jacques

    2013-01-01

    Marine mammal Brucella spp. have been isolated from pinnipeds (B. pinnipedialis) and cetaceans (B. ceti) from around the world. Although the zoonotic potential of marine mammal brucellae is largely unknown, reports of human disease exist. There are few studies of the mechanisms of bacterial intracellular invasion and multiplication involving the marine mammal Brucella spp. We examined the infective capacity of two genetically different B. pinnipedialis strains (reference strain; NTCT 12890 and a hooded seal isolate; B17) by measuring the ability of the bacteria to enter and replicate in cultured phagocytes and epithelial cells. Human macrophage-like cells (THP-1), two murine macrophage cell lines (RAW264.7 and J774A.1), and a human malignant epithelial cell line (HeLa S3) were challenged with bacteria in a gentamicin protection assay. Our results show that B. pinnipedialis is internalized, but is then gradually eliminated during the next 72 – 96 hours. Confocal microscopy revealed that intracellular B. pinnipedialis hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of B. pinnipedialis hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of B. pinnipedialis hooded seal strain in human macrophages was significantly reduced by 65.8 % (± 17.3), suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded by B. pinnipedialis do not release nitric oxide (NO) and intracellular bacterial presence does not induce cell death. In summary, B. pinnipedialis hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of B. pinnipedialis hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. Brucella pinnipedialis does not multiply or survive for prolonged periods intracellulary. PMID:24376851

  5. Induced Pluripotent Stem Cell Derived Mesenchymal Stem Cells for Attenuating Age-Related Bone Loss

    Science.gov (United States)

    2012-07-01

    Mesenchymal stem cell (MSC) differentiation towards the bone forming osteoblastic lineage decreases as a function of age and may contribute to age-related...problem of age-related reduced availability of MSC we propose to examine the bone anabolic potential of induced pluripotent stem cell (iPS) derived MSC

  6. Strain elastography in the characterization of renal cell carcinoma and angiomyolipoma

    Science.gov (United States)

    Keskin, Suat; Güven, Selçuk; Keskin, Zeynep; Özbiner, Hüseyin; Kerimoğlu, Ülkü; Yeşildağ, Ahmet

    2015-01-01

    Introduction: We evaluate the diagnostic performance of strain elastography to differentiate renal cell carcinoma (RCC) from angiomyolipoma (AML). Methods: Strain elastography was performed in 65 patients (mean age 55.5 years; range: 32–81) who had renal lesions (24 AMLs and 41 RCCs) prospectively. Lesions were classified according to lesion size and histological subtypes. The strain ratios of the RCCs and AMLs were evaluated by a radiologist. The area under the curve and the cut-off point were used to assess diagnostic performance. Sensitivity, specificity, and positive and negative predictive values were obtained. Results: In assessing the mean strain ratio, we divided the groups in 3 according to size: (1) 40-mm lesions; the respective mean strain ratios were: 1.5 ± 0.5 (range: 0.06–5.92), 2.8 ± 0.4 (range: 0.17–9.92), 2.7 ± 0.3 (range: 0.08–6.15). When RCCs and AMLs were compared, there was a statistically significant difference in the strain ratio among the 3 groups divided per lesion size (p < 0.01). For the strain ratio, the mean ± standard deviation was 1.1 ± 0.1 for AMLs and 3.4 ± 0.3 for RCCs (p < 0.01). When lesion subtypes were compared, there was a statistically significant difference in the strain ratio between the AML and clear cell RCC (p < 0.01). Conclusions: For assessing renal lesions, strain elastography and strain ratio values may be useful in differentiating RCCs from AMLs. PMID:25737764

  7. Autologous Pluripotent Stem Cell-Derived β-Like Cells for Diabetes Cellular Therapy.

    Science.gov (United States)

    Millman, Jeffrey R; Pagliuca, Felicia W

    2017-05-01

    Development of stem cell technologies for cell replacement therapy has progressed rapidly in recent years. Diabetes has long been seen as one of the first applications for stem cell-derived cells because of the loss of only a single cell type-the insulin-producing β-cell. Recent reports have detailed strategies that overcome prior hurdles to generate functional β-like cells from human pluripotent stem cells in vitro, including from human induced pluripotent stem cells (hiPSCs). Even with this accomplishment, addressing immunological barriers to transplantation remains a major challenge for the field. The development of clinically relevant hiPSC derivation methods from patients and demonstration that these cells can be differentiated into β-like cells presents a new opportunity to treat diabetes without immunosuppression or immunoprotective encapsulation or with only targeted protection from autoimmunity. This review focuses on the current status in generating and transplanting autologous β-cells for diabetes cell therapy, highlighting the unique advantages and challenges of this approach. © 2017 by the American Diabetes Association.

  8. Radioresistant cell strain of human fibrosarcoma cells obtained after long-term exposure to X-rays

    International Nuclear Information System (INIS)

    Wei, K.; Kodym, R.

    1998-01-01

    A radioresistant cell strain from human fibrosarcoma HT1080 has been obtained after prolonged exposure to x-rays for 7 months (2 Gy per day, 5 days per week). This new strain, HT1080R, differs from HT1080 in a significantly increased ability of clonogenical survival, with coefficient α decreasing from 0.161 to 0.123 Gy -1 and coefficient β decreasing from 0.0950 to 0.0565 Gy -2 . Furthermore, the radioresistance of HT1080R proved to be stable in long-term passaged cultures as well as in frozen samples. Differences between the two cell lines are also observed in the G-banded karyotype; the new cell line shows monosomy of chromosome 17 and loss of 5p + and 11q + present in the parental cells. These data suggest that the radioresistance may have been caused by radiation-induced cell mutation and that the resistant cells may have been selected by repeated irradiations. In order to characterize this new strain, the ability of the cells to rejoin DNA double-strand breaks, the cell cycle distribution and the amount of apoptosis after irradiation have been estimated; however, no differences are observed between these two cell strains. Although the mechanism of the elevated radioresistance remains unknown, this pair of cell strains can provide a new model system for further investigations with regard to the mechanisms of cellular radioresistance. The results also show that any type of irradiation similar to the schedules used in radiotherapy can lead to the formation and selection of more radioresistant cell clones in vitro, a phenomenon with possible implications for radiotherapy. (orig.)

  9. Cytopathogenic effects in enterocytelike Caco-2 cells differentiate virulent from avirulent Listeria strains.

    OpenAIRE

    Pine, L; Kathariou, S; Quinn, F; George, V; Wenger, J D; Weaver, R E

    1991-01-01

    We have developed a simple test that differentiates between virulent and avirulent Listeria species as defined by the mouse 50% lethal doses (LD50S). The assay is based on trypan blue-revealed cytopathogenic effects that are produced during the infection of the human enterocytelike cell line Caco-2. These effects were elicited only by Listeria strains that had an intraperitoneal mouse LD50 less than 10(8) and were not produced by nonhemolytic, avirulent strains of Listeria monocytogenes gener...

  10. Adipose-derived mesenchymal stromal cells for chronic myocardial ischemia (MyStromalCell Trial)

    DEFF Research Database (Denmark)

    Qayyum, Abbas Ali; Haack-Sørensen, Mandana; Mathiasen, Anders Bruun

    2012-01-01

    Adipose tissue represents an abundant, accessible source of multipotent adipose-derived stromal cells (ADSCs). Animal studies have suggested that ADSCs have the potential to differentiate in vivo into endothelial cells and cardiomyocytes. This makes ADSCs a promising new cell source...... for regenerative therapy to replace injured tissue by creating new blood vessels and cardiomyocytes in patients with chronic ischemic heart disease. The aim of this special report is to review the present preclinical data leading to clinical stem cell therapy using ADSCs in patients with ischemic heart disease....... In addition, we give an introduction to the first-in-man clinical trial, MyStromalCell Trial, which is a prospective, randomized, double-blind, placebo-controlled study using culture-expanded ADSCs obtained from adipose-derived cells from abdominal adipose tissue and stimulated with VEGF-A(165) the week...

  11. Potential differentiation of islet-like cells from pregnant cow-derived placental stem cells.

    Science.gov (United States)

    Peng, Shao-Yu; Chou, Chien-Wen; Kuo, Yu-Hsuan; Shen, Perng-Chih; Shaw, S W Steven

    2017-06-01

    Type 1 diabetes is an autoimmune disease that destroys islet cells and results in insufficient insulin secretion by pancreatic β-cells. Islet transplantation from donors is an approach used for treating patients with diabetes; however, this therapy is difficult to implement because of the lack of donors. Nevertheless, several stem cells have the potential to differentiate from islet-like cells and enable insulin secretion for treating diabetes in animal models. For example, placenta is considered a waste material and can be harvested noninvasively during delivery without ethical or moral concerns. To date, the differentiation of islet-like cells from cow-derived placental stem cells (CPSCs) has yet to be demonstrated. The investigation of potential differentiation of islet-like cells from CPSCs was conducted by supplementation with nicotinamide, exendin-4, glucose, and poly-d-lysine and was detected through reverse transcription polymerase chain reaction, dithizone staining, and immunocytochemical methods. Our results indicated that CPSCs are established and express mesenchymal stem cell surface antigen markers, such as CD73, CD166, β-integrin, and Oct-4, but not hematopoietic stem cell surface antigen markers, such as CD45. After induction, the CPSCs successfully differentiated into islet-like cells. The CPSC-derived islet-like cells expressed islet cell development-related genes, such as insulin, glucagon, pax-4, Nkx6.1, pax-6, and Fox. Moreover, CPSC-derived islet-like cells can be stained with zinc ions, which are widely distributed in the islet cells and enable insulin secretion. Altogether, islet-like cells have the potential to be differentiated from CPSCs without gene manipulation, and can be used in diabetic animal models in the future for preclinical and drug testing trial investigations. Copyright © 2017. Published by Elsevier B.V.

  12. [Thiamine and its derivatives in the regulation of cell metabolism].

    Science.gov (United States)

    Tylicki, Adam; Siemieniuk, Magdalena

    2011-07-06

    For over 70 years thiamine (vitamin B1) has aroused the interest of biologists, biochemists and medical doctors because of its multilateral participation in key biochemical and physiological processes. The thiamine molecule is composed of pyrimidine and thiazole rings which are linked by a methylene bridge. It is synthesized by microorganisms, fungi and plants, whereas animals and humans have to obtain it from food. There are several known forms of vitamin B1 inside cells: free thiamine, three phosphate esters (mono-, di-, and triphosphate), and the recently found adenosine thiamine triphosphate. Thiamine has a dual, coenzymatic and non-coenzymatic role. First of all, it is a precursor of thiamin diphosphate, which is a coenzyme for over 20 characterized enzymes which are involved in cell bioenergetic processes leading to the synthesis of ATP. Moreover, these enzymes take part in the biosynthesis of pentose (required for the synthesis of nucleotides), amino acids and other organic compounds of cell metabolism. On the other hand, recent discoveries show the non-coenzymatic role of thiamine derivatives in the process of regulation of gene expression (riboswitches in microorganisms and plants), the stress response, and perhaps so far unknown signal transduction pathways associated with adverse environmental conditions, or transduction of nerve signals with participation of thiamine triphosphate and adenosine thiamine triphosphate. From the clinical point of view thiamine deficiency is related to beri-beri, Parkinson disease, Alzheimer disease, Wernicke-Korsakoff syndrome and other pathologies of the nervous system, and it is successfully applied in medical practice. On the other hand, identifying new synthetic analogues of thiamine which could be used as cytostatics, herbicides or agents preventing deficiency of vitamin B1 is currently the major goal of the research. In this paper we present the current state of knowledge of thiamine and its derivatives, indicating

  13. Induced Pluripotent Stem Cell-Derived Neural Cells Survive and Mature in the Nonhuman Primate Brain

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    Marina E. Emborg

    2013-03-01

    Full Text Available The generation of induced pluripotent stem cells (iPSCs opens up the possibility for personalized cell therapy. Here, we show that transplanted autologous rhesus monkey iPSC-derived neural progenitors survive for up to 6 months and differentiate into neurons, astrocytes, and myelinating oligodendrocytes in the brains of MPTP-induced hemiparkinsonian rhesus monkeys with a minimal presence of inflammatory cells and reactive glia. This finding represents a significant step toward personalized regenerative therapies.

  14. Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories

    DEFF Research Database (Denmark)

    Zhou, Yongjin J.; Buijs, Nicolaas A; Zhu, Zhiwei

    2016-01-01

    Sustainable production of oleochemicals requires establishment of cell factory platform strains. The yeast Saccharomyces cerevisiae is an attractive cell factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level p...

  15. Endothelial cell-derived microparticles induce plasmacytoid dendritic cell maturation: potential implications in inflammatory diseases.

    Science.gov (United States)

    Angelot, Fanny; Seillès, Estelle; Biichlé, Sabeha; Berda, Yael; Gaugler, Béatrice; Plumas, Joel; Chaperot, Laurence; Dignat-George, Françoise; Tiberghien, Pierre; Saas, Philippe; Garnache-Ottou, Francine

    2009-11-01

    Increased circulating endothelial microparticles, resulting from vascular endothelium dysfunction, and plasmacytoid dendritic cell activation are both encountered in common inflammatory disorders. The aim of our study was to determine whether interactions between endothelial microparticles and plasmacytoid dendritic cells could contribute to such pathologies. Microparticles generated from endothelial cell lines, platelets or activated T cells were incubated with human plasmacytoid dendritic cells sorted from healthy donor blood or with monocyte-derived dendritic cells. Dendritic cell maturation was evaluated by flow cytometry, cytokine secretion as well as naive T-cell activation and polarization. Labeled microparticles were also used to study cellular interactions. Endothelial microparticles induced plasmacytoid dendritic cell maturation. In contrast, conventional dendritic cells were resistant to endothelial microparticle-induced maturation. In addition to upregulation of co-stimulatory molecules, endothelial microparticle-matured plasmacytoid dendritic cells secreted inflammatory cytokines (interleukins 6 and 8, but no interferon-alpha) and also induced allogeneic naive CD4(+) T cells to proliferate and to produce type 1 cytokines such as interferon-gamma and tumor necrosis factor-alpha. Endothelial microparticle endocytosis by plasmacytoid dendritic cells appeared to be required for plasmacytoid dendritic cell maturation. Importantly, the ability of endothelial microparticles to induce plasmacytoid dendritic cells to mature was specific as microparticles derived from activated T cells or platelets (the major source of circulating microparticules in healthy subjects) did not induce such plasmacytoid dendritic cell maturation. Our data show that endothelial microparticles specifically induce plasmacytoid dendritic cell maturation and production of inflammatory cytokines. This novel activation pathway may be implicated in various inflammatory disorders and

  16. Exosomes derived from mesenchymal non-small cell lung cancer cells promote chemoresistance.

    Science.gov (United States)

    Lobb, Richard J; van Amerongen, Rosa; Wiegmans, Adrian; Ham, Sunyoung; Larsen, Jill E; Möller, Andreas

    2017-08-01

    Non-small cell lung cancer (NSCLC) is the most common lung cancer type and the most common cause of mortality in lung cancer patients. NSCLC is often associated with resistance to chemotherapeutics and together with rapid metastatic spread, results in limited treatment options and poor patient survival. NSCLCs are heterogeneous, and consist of epithelial and mesenchymal NSCLC cells. Mesenchymal NSCLC cells are thought to be responsible for the chemoresistance phenotype, but if and how this phenotype can be transferred to other NSCLC cells is currently not known. We hypothesised that small extracellular vesicles, exosomes, secreted by mesenchymal NSCLC cells could potentially transfer the chemoresistance phenotype to surrounding epithelial NSCLC cells. To explore this possibility, we used a unique human bronchial epithelial cell (HBEC) model in which the parental cells were transformed from an epithelial to mesenchymal phenotype by introducing oncogenic alterations common in NSCLC. We found that exosomes derived from the oncogenically transformed, mesenchymal HBECs could transfer chemoresistance to the parental, epithelial HBECs and increase ZEB1 mRNA, a master EMT transcription factor, in the recipient cells. Additionally, we demonstrate that exosomes from mesenchymal, but not epithelial HBECs contain the ZEB1 mRNA, thereby providing a potential mechanism for the induction of a mesenchymal phenotype in recipient cells. Together, this work demonstrates for the first time that exosomes derived from mesenchymal, oncogenically transformed lung cells can transfer chemoresistance and mesenchymal phenotypes to recipient cells, likely via the transfer of ZEB1 mRNA in exosomes. © 2017 UICC.

  17. Adipose tissue-derived stem cells promote pancreatic cancer cell proliferation and invasion

    International Nuclear Information System (INIS)

    Ji, S.Q.; Cao, J.; Zhang, Q.Y.; Li, Y.Y.; Yan, Y.Q.; Yu, F.X.

    2013-01-01

    To explore the effects of adipose tissue-derived stem cells (ADSCs) on the proliferation and invasion of pancreatic cancer cells in vitro and the possible mechanism involved, ADSCs were cocultured with pancreatic cancer cells, and a cell counting kit (CCK-8) was used to detect the proliferation of pancreatic cancer cells. ELISA was used to determine the concentration of stromal cell-derived factor-1 (SDF-1) in the supernatants. RT-PCR was performed to detect the expression of the chemokine receptor CXCR4 in pancreatic cancer cells and ADSCs. An in vitro invasion assay was used to measure invasion of pancreatic cancer cells. SDF-1 was detected in the supernatants of ADSCs, but not in pancreatic cancer cells. Higher CXCR4 mRNA levels were detected in the pancreatic cancer cell lines compared with ADSCs (109.3±10.7 and 97.6±7.6 vs 18.3±1.7, respectively; P<0.01). In addition, conditioned medium from ADSCs promoted the proliferation and invasion of pancreatic cancer cells, and AMD3100, a CXCR4 antagonist, significantly downregulated these growth-promoting effects. We conclude that ADSCs can promote the proliferation and invasion of pancreatic cancer cells, which may involve the SDF-1/CXCR4 axis

  18. Adipose tissue-derived stem cells promote pancreatic cancer cell proliferation and invasion

    Energy Technology Data Exchange (ETDEWEB)

    Ji, S.Q.; Cao, J. [Department of Liver Surgery I, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai (China); Zhang, Q.Y.; Li, Y.Y. [Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Wenzhou Medical College, Wenzhou (China); Yan, Y.Q. [Department of Liver Surgery I, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai (China); Yu, F.X. [Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Wenzhou Medical College, Wenzhou (China)

    2013-09-27

    To explore the effects of adipose tissue-derived stem cells (ADSCs) on the proliferation and invasion of pancreatic cancer cells in vitro and the possible mechanism involved, ADSCs were cocultured with pancreatic cancer cells, and a cell counting kit (CCK-8) was used to detect the proliferation of pancreatic cancer cells. ELISA was used to determine the concentration of stromal cell-derived factor-1 (SDF-1) in the supernatants. RT-PCR was performed to detect the expression of the chemokine receptor CXCR4 in pancreatic cancer cells and ADSCs. An in vitro invasion assay was used to measure invasion of pancreatic cancer cells. SDF-1 was detected in the supernatants of ADSCs, but not in pancreatic cancer cells. Higher CXCR4 mRNA levels were detected in the pancreatic cancer cell lines compared with ADSCs (109.3±10.7 and 97.6±7.6 vs 18.3±1.7, respectively; P<0.01). In addition, conditioned medium from ADSCs promoted the proliferation and invasion of pancreatic cancer cells, and AMD3100, a CXCR4 antagonist, significantly downregulated these growth-promoting effects. We conclude that ADSCs can promote the proliferation and invasion of pancreatic cancer cells, which may involve the SDF-1/CXCR4 axis.

  19. Phenotypic and genotypic characteristics of novel mouse cell line (NIH/3T3-adapted human enterovirus 71 strains (EV71:TLLm and EV71:TLLmv.

    Directory of Open Access Journals (Sweden)

    Carla Bianca Luena Victorio

    Full Text Available Since its identification in 1969, Enterovirus 71 (EV71 has been causing periodic outbreaks of infection in children worldwide and most prominently in the Asia-Pacific Region. Understanding the pathogenesis of Enterovirus 71 (EV71 is hampered by the virus's inability to infect small animals and replicate in their derived in vitro cultured cells. This manuscript describes the phenotypic and genotypic characteristics of two selected EV71 strains (EV71:TLLm and EV71:TLLmv, which have been adapted to replicate in mouse-derived NIH/3T3 cells, in contrast to the original parental virus which is only able to replicate in primate cell lines. The EV71:TLLm strain exhibited productive infection in all primate and rodent cell lines tested, while EV71:TLLmv exhibited greater preference for mouse cell lines. EV71:TLLmv displayed higher degree of adaptation and temperature adaptability in NIH/3T3 cells than in Vero cells, suggesting much higher fitness in NIH/3T3 cells. In comparison with the parental EV71:BS strain, the adapted strains accumulated multiple adaptive mutations in the genome resulting in amino acid substitutions, most notably in the capsid-encoding region (P1 and viral RNA-dependent RNA polymerase (3D. Two mutations, E167D and L169F, were mapped to the VP1 canyon that binds the SCARB2 receptor on host cells. Another two mutations, S135T and K140I, were located in the VP2 neutralization epitope spanning amino acids 136-150. This is the first report of human EV71 with the ability to productively infect rodent cell lines in vitro.

  20. Gold nanoparticles cellular toxicity and recovery: adipose Derived Stromal cells.

    Science.gov (United States)

    Mironava, Tatsiana; Hadjiargyrou, Michael; Simon, Marcia; Rafailovich, Miriam H

    2014-03-01

    Gold nanoparticles (AuNPs) are currently used in numerous medical applications. Herein, we describe their in vitro impact on human adipose-derived stromal cells (ADSCs) using 13 nm and 45 nm citrate-coated AuNPs. In their non-differentiated state, ADSCs were penetrated by the AuNPs and stored in vacuoles. The presence of the AuNPs in ADSCs resulted in increased population doubling times, decreased cell motility and cell-mediated collagen contraction. The degree to which the cells were impacted was a function of particle concentration, where the smaller particles required a sevenfold higher concentration to have the same effect as the larger ones. Furthermore, AuNPs reduced adipogenesis as measured by lipid droplet accumulation and adiponectin secretion. These effects correlated with transient increases in DLK1 and with relative reductions in fibronectin. Upon removal of exogenous AuNPs, cellular NP levels decreased and normal ADSC functions were restored. As adiponectin helps regulate energy metabolism, local fluctuations triggered by AuNPs can lead to systemic changes. Hence, careful choice of size, concentration and clinical application duration of AuNPs is warranted.

  1. Characterization of mesenchymal stem cells derived from equine adipose tissue

    Directory of Open Access Journals (Sweden)

    A.M. Carvalho

    2013-08-01

    Full Text Available Stem cell therapy has shown promising results in tendinitis and osteoarthritis in equine medicine. The purpose of this work was to characterize the adipose-derived mesenchymal stem cells (AdMSCs in horses through (1 the assessment of the capacity of progenitor cells to perform adipogenic, osteogenic and chondrogenic differentiation; and (2 flow cytometry analysis using the stemness related markers: CD44, CD90, CD105 and MHC Class II. Five mixed-breed horses, aged 2-4 years-old were used to collect adipose tissue from the base of the tail. After isolation and culture of AdMSCs, immunophenotypic characterization was performed through flow cytometry. There was a high expression of CD44, CD90 and CD105, and no expression of MHC Class II markers. The tri-lineage differentiation was confirmed by specific staining: adipogenic (Oil Red O, osteogenic (Alizarin Red, and chondrogenic (Alcian Blue. The equine AdMSCs are a promising type of adult progenitor cell for tissue engineering in veterinary medicine.

  2. Global seasonal strain and stress models derived from GRACE loading, and their impact on seismicity

    Science.gov (United States)

    Chanard, K.; Fleitout, L.; Calais, E.; Craig, T. J.; Rebischung, P.; Avouac, J. P.

    2017-12-01

    Loading by continental water, atmosphere and oceans deforms the Earth at various spatio-temporal scales, inducing crustal and mantelic stress perturbations that may play a role in earthquake triggering.Deformation of the Earth by this surface loading is observed in GNSS position time series. While various models predict well vertical observations, explaining horizontal displacements remains challenging. We model the elastic deformation induced by loading derived from GRACE for coefficients 2 and higher. We estimate the degree-1 deformation field by comparison between predictions of our model and IGS-repro2 solutions at a globally distributed network of 700 GNSS sites, separating the horizontal and vertical components to avoid biases between components. The misfit between model and data is reduced compared to previous studies, particularly on the horizontal component. The associated geocenter motion time series are consistent with results derived from other datasets. We also discuss the impact on our results of systematic errors in GNSS geodetic products, in particular of the draconitic error.We then compute stress tensors time series induced by GRACE loads and discuss the potential link between large scale seasonal mass redistributions and seismicity. Within the crust, we estimate hydrologically induced stresses in the intraplate New Madrid Seismic Zone, where secular stressing rates are unmeasurably low. We show that a significant variation in the rate of micro-earthquakes at annual and multi-annual timescales coincides with stresses induced by hydrological loading in the upper Mississippi embayment, with no significant phase-lag, directly modulating regional seismicity. We also investigate pressure variations in the mantle transition zone and discuss potential correlations between the statistically significant observed seasonality of deep-focus earthquakes, most likely due to mineralogical transformations, and surface hydrological loading.

  3. 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.

  4. Exosomes derived from pancreatic cancer cells induce activation and profibrogenic activities in pancreatic stellate cells.

    Science.gov (United States)

    Masamune, Atsushi; Yoshida, Naoki; Hamada, Shin; Takikawa, Tetsuya; Nabeshima, Tatsuhide; Shimosegawa, Tooru

    2018-01-01

    Pancreatic cancer cells (PCCs) interact with pancreatic stellate cells (PSCs), which play a pivotal role in pancreatic fibrogenesis, to develop the cancer-conditioned tumor microenvironment. Exosomes are membrane-enclosed nanovesicles, and have been increasingly recognized as important mediators of cell-to-cell communications. The aim of this study was to clarify the effects of PCC-derived exosomes on cell functions in PSCs. Exosomes were isolated from the conditioned medium of Panc-1 and SUIT-2 PCCs. Human primary PSCs were treated with PCC-derived exosomes. PCC-derived exosomes stimulated the proliferation, migration, activation of ERK and Akt, the mRNA expression of α-smooth muscle actin (ACTA2) and fibrosis-related genes, and procollagen type I C-peptide production in PSCs. Ingenuity pathway analysis of the microarray data identified transforming growth factor β1 and tumor necrosis factor as top upstream regulators. PCCs increased the expression of miR-1246 and miR-1290, abundantly contained in PCC-derived exosomes, in PSCs. Overexpression of miR-1290 induced the expression of ACTA2 and fibrosis-related genes in PSCs. In conclusion, PCC-derived exosomes stimulate activation and profibrogenic activities in PSCs. Exosome-mediated interactions between PSCs and PCCs might play a role in the development of the tumor microenvironment. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Effect of chondrocyte-derived early extracellular matrix on chondrogenesis of placenta-derived mesenchymal stem cells.

    Science.gov (United States)

    Park, Yong-Beom; Seo, Sinji; Kim, Jin-A; Heo, Jin-Chul; Lim, Young-Cheol; Ha, Chul-Won

    2015-06-24

    The extracellular matrix (ECM) surrounding cells contains a variety of proteins that provide structural support and regulate cellular functions. Previous studies have shown that decellularized ECM isolated from tissues or cultured cells can be used to improve cell differentiation in tissue engineering applications. In this study we evaluated the effect of decellularized chondrocyte-derived ECM (CDECM) on the chondrogenesis of human placenta-derived mesenchymal stem cells (hPDMSCs) in a pellet culture system. After incubation with or without chondrocyte-derived ECM in chondrogenic medium for 1 or 3 weeks, the sizes and wet masses of the cell pellets were compared with untreated controls (hPDMSCs incubated in chondrogenic medium without chondrocyte-derived ECM). In addition, histologic analysis of the cell pellets (Safranin O and collagen type II staining) and quantitative reverse transcription-PCR analysis of chondrogenic markers (aggrecan, collagen type II, and SOX9) were carried out. Our results showed that the sizes and masses of hPDMSC pellets incubated with chondrocyte-derived ECM were significantly higher than those of untreated controls. Differentiation of hPDMSCs (both with and without chondrocyte-derived ECM) was confirmed by Safranin O and collagen type II staining. Chondrogenic marker expression and glycosaminoglycan (GAG) levels were significantly higher in hPDMSC pellets incubated with chondrocyte-derived ECM compared with untreated controls, especially in cells precultured with chondrocyte-derived ECM for 7 d. Taken together, these results demonstrate that chondrocyte-derived ECM enhances the chondrogenesis of hPDMSCs, and this effect is further increased by preculture with chondrocyte-derived ECM. This preculture method for hPDMSC chondrogenesis represents a promising approach for cartilage tissue engineering.

  6. Production of high titer attenuated poliovirus strains on the serum-free PER.C6(®) cell culture platform for the generation of safe and affordable next generation IPV.

    Science.gov (United States)

    Sanders, Barbara P; Oakes, Isabel de los Rios; van Hoek, Vladimir; Liu, Ying; Marissen, Wilfred; Minor, Philip D; Wimmer, Eckard; Schuitemaker, Hanneke; Custers, Jerome H H V; Macadam, Andrew; Cello, Jeronimo; Edo-Matas, Diana

    2015-11-27

    As poliovirus eradication draws closer, alternative Inactivated Poliovirus Vaccines (IPV) are needed to overcome the risks associated with continued use of the Oral Poliovirus Vaccine and of neurovirulent strains used during manufacture of conventional (c) IPV. We have previously demonstrated the susceptibility of the PER.C6(®) cell line to cIPV strains; here we investigated the suspension cell culture platform for growth of attenuated poliovirus strains. We examined attenuated Sabin strain productivity on the PER.C6(®) cell platform compared to the conventional Vero cell platform. The suitability of the suspension cell platform for propagation of rationally-attenuated poliovirus strains (stabilized Sabin type 3 S19 derivatives and genetically attenuated and stabilized MonoCre(X) strains), was also assessed. Yields were quantified by infectious titer determination and D-antigen ELISA using either serotype-specific polyclonal rabbit sera for Sabin strains or monoclonal cIPV-strain-specific antibodies for cIPV, S19 and MonoCre(X) strains. PER.C6(®) cells supported the replication of Sabin strains to yields of infectious titers that were in the range of cIPV strains at 32.5°C. Sabin strains achieved 30-fold higher yields (pSabin strain productivity on the PER.C6(®) cell platform was maintained at 10l scale. Yields of infectious titers of S19 and MonoCre(X) strains were 0.5-1 log10 lower than seen for cIPV strains, whereas D-antigen yield and productivities in doses/ml using rationally-attenuated strains were in line with yields reported for cIPV strains. Sabin and rationally-attenuated polioviruses can be grown to high infectious titers and D-antigen yields. Sabin strain infection shows increased productivity on the PER.C6(®) cell platform as compared to the conventional Vero cell platform. Novel cell platforms with the potential for higher yields could contribute to increased affordability of a next generation of IPV vaccines needed for achieving and

  7. Establishing clonal cell lines with endothelial-like potential from CD9(hi, SSEA-1(- cells in embryonic stem cell-derived embryoid bodies.

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    Qizhou Lian

    Full Text Available BACKGROUND: Differentiation of embryonic stem cells (ESCs into specific cell types with minimal risk of teratoma formation could be efficiently directed by first reducing the differentiation potential of ESCs through the generation of clonal, self-renewing lineage-restricted stem cell lines. Efforts to isolate these stem cells are, however, mired in an impasse where the lack of purified lineage-restricted stem cells has hindered the identification of defining markers for these rare stem cells and, in turn, their isolation. METHODOLOGY/PRINCIPAL FINDINGS: We describe here a method for the isolation of clonal lineage-restricted cell lines with endothelial potential from ESCs through a combination of empirical and rational evidence-based methods. Using an empirical protocol that we have previously developed to generate embryo-derived RoSH lines with endothelial potential, we first generated E-RoSH lines from mouse ESC-derived embryoid bodies (EBs. Despite originating from different mouse strains, RoSH and E- RoSH lines have similar gene expression profiles (r(2 = 0.93 while that between E-RoSH and ESCs was 0.83. In silico gene expression analysis predicted that like RoSH cells, E-RoSH cells have an increased propensity to differentiate into vasculature. Unlike their parental ESCs, E-RoSH cells did not form teratomas and differentiate efficiently into endothelial-like cells in vivo and in vitro. Gene expression and FACS analysis revealed that RoSH and E-RoSH cells are CD9(hi, SSEA-1(- while ESCs are CD9(lo, SSEA-1(+. Isolation of CD9(hi, SSEA-1(- cells that constituted 1%-10% of EB-derived cultures generated an E-RoSH-like culture with an identical E-RoSH-like gene expression profile (r(2 = 0.95 and a propensity to differentiate into endothelial-like cells. CONCLUSIONS: By combining empirical and rational evidence-based methods, we identified definitive selectable surface antigens for the isolation and propagation of lineage-restricted stem cells

  8. Mesenchymal Stem Cell-Derived Factors Restore Function to Human Frataxin-Deficient Cells.

    Science.gov (United States)

    Kemp, Kevin; Dey, Rimi; Cook, Amelia; Scolding, Neil; Wilkins, Alastair

    2017-08-01

    Friedreich's ataxia is an inherited neurological disorder characterised by mitochondrial dysfunction and increased susceptibility to oxidative stress. At present, no therapy has been shown to reduce disease progression. Strategies being trialled to treat Friedreich's ataxia include drugs that improve mitochondrial function and reduce oxidative injury. In addition, stem cells have been investigated as a potential therapeutic approach. We have used siRNA-induced knockdown of frataxin in SH-SY5Y cells as an in vitro cellular model for Friedreich's ataxia. Knockdown of frataxin protein expression to levels detected in patients with the disorder was achieved, leading to decreased cellular viability, increased susceptibility to hydrogen peroxide-induced oxidative stress, dysregulation of key anti-oxidant molecules and deficiencies in both cell proliferation and differentiation. Bone marrow stem cells are being investigated extensively as potential treatments for a wide range of neurological disorders, including Friedreich's ataxia. The potential neuroprotective effects of bone marrow-derived mesenchymal stem cells were therefore studied using our frataxin-deficient cell model. Soluble factors secreted by mesenchymal stem cells protected against cellular changes induced by frataxin deficiency, leading to restoration in frataxin levels and anti-oxidant defences, improved survival against oxidative stress and stimulated both cell proliferation and differentiation down the Schwann cell lineage. The demonstration that mesenchymal stem cell-derived factors can restore cellular homeostasis and function to frataxin-deficient cells further suggests that they may have potential therapeutic benefits for patients with Friedreich's ataxia.

  9. Clinical benefits of probiotic canine-derived Bifidobacterium animalis strain AHC7 in dogs with acute idiopathic diarrhea.

    Science.gov (United States)

    Kelley, R L; Minikhiem, Debbie; Kiely, Barry; O'Mahony, Liam; O'Sullivan, David; Boileau, Tom; Park, Jean Soon

    2009-01-01

    This study evaluated the effect of supplementation with canine-derived probiotic Bifidobacterium animalis strain AHC7 (lams Prostora, Procter & Gamble Pet Care) on the resolution rate of acute idiopathic diarrhea in dogs randomly assigned to receive a placebo (n=18) or the probiotic (n=13). Nutritional management with the probiotic fed at 2 x 10(10) CFU/day significantly reduced the time to resolution (3.9 +/- 2.3 versus 6.6 +/- 2.7 days; P dogs that were administered metronidazole (38.5% versus 50.0%) compared with placebo. Probiotic B. animalis AHC7 may provide veterinarians another tool for management of acute diarrhea in dogs.

  10. Pericytes derived from adipose-derived stem cells protect against retinal vasculopathy.

    Directory of Open Access Journals (Sweden)

    Thomas A Mendel

    Full Text Available Retinal vasculopathies, including diabetic retinopathy (DR, threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy.We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR, ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area. ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction. Treatment of ASCs with transforming growth factor beta (TGF-β1 enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection.ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated

  11. Apheresis platelet concentrates contain platelet-derived and endothelial cell-derived microparticles.

    Science.gov (United States)

    Rank, A; Nieuwland, R; Liebhardt, S; Iberer, M; Grützner, S; Toth, B; Pihusch, R

    2011-02-01

    Microparticles (MP) are membrane vesicles with thrombogenic and immunomodulatory properties. We determined MP subgroups from resting platelets, activated platelets and endothelial cells in donors and apheresis platelet concentrates (PC). MP were double stained with annexin V and CD61 (platelet-derived MP; PMP), P-selectin or CD63 (MP from activated platelets) and CD144 plus E-selectin (endothelial cell-derived MP; EMP) and detected by flow cytometry in platelet donors (n=36) and apheresis PC (n=11; Trima™). PC contained MP, mainly from resting platelets [93% (90-95)], and minor fractions of PMP from activated platelets [P-selectin(+) or CD63(+); 4·8% (3·2-7·7) and 2·6% (2·0-4·0)]. Compared to donors, levels of annexin V+ MP, PMP, P-selectin(+) and CD63(+) MP were 1·7-, 2·3-, 8·6- and 3·1-fold higher in PC (all P<0·05). During storage (1-5 days), levels of annexin V+ MP and PMP did not increase, although small increases in the fraction of P-selectin(+) or CD63(+) MP occurred (both P<0·05). PC also contained EMP, which were 2·6- to 3·7-fold enriched in PC compared to donors (P<0·05). Transfusion of apheresis PC also results in transfusion of HLA-carrying PMP and EMP. This might counteract the aim of reducing transfused HLA load by leucodepletion. The increases in PMP exposing P-selectin or CD63 reflect mild platelet activation during storage. We conclude that in leucodepleted platelet apheresis using fluidized particle bed technology, MP are harvested mainly from the donor by apheresis. Improvement in apheresis technology might reduce MP load. © 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.

  12. Comprehensive characterization of genomic instability in pluripotent stem cells and their derived neuroprogenitor cell lines

    Directory of Open Access Journals (Sweden)

    Nestor Luis Lopez Corrales

    2012-12-01

    Full Text Available The genomic integrity of two human pluripotent stem cells and their derived neuroprogenitor cell lines was studied, applying a combination of high-resolution genetic methodologies. The usefulness of combining array-comparative genomic hybridization (aCGH and multiplex fluorescence in situ hybridization (M-FISH techniques should be delineated to exclude/detect a maximum of possible genomic structural aberrations. Interestingly, in parts different genomic imbalances at chromosomal and subchromosomal levels were detected in pluripotent stem cells and their derivatives. Some of the copy number variations were inherited from the original cell line, whereas other modifications were presumably acquired during the differentiation and manipulation procedures. These results underline the necessity to study both pluripotent stem cells and their differentiated progeny by as many approaches as possible in order to assess their genomic stability before using them in clinical therapies.

  13. 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.

  14. Umbilical Cord-Derived Mesenchymal Stem Cells for Hematopoietic Stem Cell Transplantation

    Directory of Open Access Journals (Sweden)

    Yu-Hua Chao

    2012-01-01

    Full Text Available Hematopoietic stem cell transplantation (HSCT is becoming an effective therapeutic modality for a variety of diseases. Mesenchymal stem cells (MSCs can be used to enhance hematopoietic engraftment, accelerate lymphocyte recovery, reduce the risk of graft failure, prevent and treat graft-versus-host disease, and repair tissue damage in patients receiving HSCT. Till now, most MSCs for human clinical application have been derived from bone marrow. However, acquiring bone-marrow-derived MSCs involves an invasive procedure. Umbilical cord is rich with MSCs. Compared to bone-marrow-derived MSCs, umbilical cord-derived MSCs (UCMSCs are easier to obtain without harm to the donor and can proliferate faster. No severe adverse effects were noted in our previous clinical application of UCMSCs in HSCT. Accordingly, application of UCMSCs in humans appears to be feasible and safe. Further studies are warranted.

  15. Safety and immune regulatory properties of canine induced pluripotent stem cell-derived mesenchymal stem cells.

    Science.gov (United States)

    Chow, Lyndah; Johnson, Valerie; Regan, Dan; Wheat, William; Webb, Saiphone; Koch, Peter; Dow, Steven

    2017-12-01

    Mesenchymal stem cells (MSCs) exhibit broad immune modulatory activity in vivo and can suppress T cell proliferation and dendritic cell activation in vitro. Currently, most MSC for clinical usage are derived from younger donors, due to ease of procurement and to the superior immune modulatory activity. However, the use of MSC from multiple unrelated donors makes it difficult to standardize study results and compare outcomes between different clinical trials. One solution is the use of MSC derived from induced pluripotent stem cells (iPSC); as iPSC-derived MSC have nearly unlimited proliferative potential and exhibit in vitro phenotypic stability. Given the value of dogs as a spontaneous disease model for pre-clinical evaluation of stem cell therapeutics, we investigated the functional properties of canine iPSC-derived MSC (iMSC), including immune modulatory properties and potential for teratoma formation. We found that canine iMSC downregulated expression of pluripotency genes and appeared morphologically similar to conventional MSC. Importantly, iMSC retained a stable phenotype after multiple passages, did not form teratomas in immune deficient mice, and did not induce tumor formation in dogs following systemic injection. We concluded therefore that iMSC were phenotypically stable, immunologically potent, safe with respect to tumor formation, and represented an important new source of cells for therapeutic modulation of inflammatory disorders. Copyright © 2017. Published by Elsevier B.V.

  16. Phenotype and Function of CD209+ Bovine Blood Dendritic Cells, Monocyte-Derived-Dendritic Cells and Monocyte-Derived Macrophages.

    Directory of Open Access Journals (Sweden)

    Kun Taek Park

    Full Text Available Phylogenic comparisons of the mononuclear phagocyte system (MPS of humans and mice demonstrate phenotypic divergence of dendritic cell (DC subsets that play similar roles in innate and adaptive immunity. Although differing in phenotype, DC can be classified into four groups according to ontogeny and function: conventional DC (cDC1 and cDC2, plasmacytoid DC (pDC, and monocyte derived DC (MoDC. DC of Artiodactyla (pigs and ruminants can also be sub-classified using this system, allowing direct functional and phenotypic comparison of MoDC and other DC subsets trafficking in blood (bDC. Because of the high volume of blood collections required to study DC, cattle offer the best opportunity to further our understanding of bDC and MoDC function in an outbred large animal species. As reported here, phenotyping DC using a monoclonal antibody (mAb to CD209 revealed CD209 is expressed on the major myeloid population of DC present in blood and MoDC, providing a phenotypic link between these two subsets. Additionally, the present study demonstrates that CD209 is also expressed on monocyte derived macrophages (MoΦ. Functional analysis revealed each of these populations can take up and process antigens (Ags, present them to CD4 and CD8 T cells, and elicit a T-cell recall response. Thus, bDC, MoDC, and MoΦ pulsed with pathogens or candidate vaccine antigens can be used to study factors that modulate DC-driven T-cell priming and differentiation ex vivo.

  17. Effects of Lactobacillus strains on cancer cell proliferation and oxidative stress in vitro.

    Science.gov (United States)

    Choi, S S; Kim, Y; Han, K S; You, S; Oh, S; Kim, S H

    2006-05-01

    The objective of this study was to assess in vitro, whether heat-killed (HK) lactic acid bacteria cells and fractionations of HK cells could suppress the viability of human cancer cells and inhibit the cytotoxicity associated with oxidative stress. Among the strains, the HK cells of Lactobacillus acidophilus 606 and Lactobacillus casei ATCC 393 exhibited the most profound inhibitory activity in all of the tested cell lines. HK cells of L. acidophilus 606 were determined to be less toxic to healthy human embryo fibroblasts (hEF cells) than were HK cells of L. casei ATCC 393. The soluble polysaccharides from L. acidophilus 606 evidenced the most effective anticancer activity, but inhibited hEF cell growth by only 20%. The soluble polysaccharides from L. acidophilus 606 were partly observed to induce apoptosis in the HT-29 cells by DNA fragmentation and propidium iodine staining. Both the HK cells of L. acidophilus 606 and the soluble polysaccharide components of this strain also exhibited potent antioxidative activity. Our findings suggest that the soluble polysaccharide fraction from L. acidophilus 606 may constitute a novel anticancer agent, which manifests a high degree of selectivity for human cancer cells and antioxidative agent in the food industry. These soluble polysaccharide components from Lactobacillus may be applied to various foods, and used as adjuncts for cancer therapy and prevention.

  18. 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.

  19. Cyclic strain-induced endothelial MMP-2: role in vascular smooth muscle cell migration

    International Nuclear Information System (INIS)

    Sweeney, Nicholas von Offenberg; Cummins, Philip M.; Birney, Yvonne A.; Redmond, Eileen M.; Cahill, Paul A.

    2004-01-01

    Matrix metalloproteinases (MMPs) play a vital role in vasculature response to hemodynamic stimuli via the degradation of extracellular matrix substrates. In this study, we investigated the putative role of cyclic strain-induced endothelial MMP-2 (and MMP-9) expression and release in modulating bovine aortic smooth muscle cell (BASMC) migration in vitro. Equibiaxial cyclic strain of bovine aortic endothelial cells (BAECs) leads to elevation in cellular MMP-2 (and MMP-9) expression, activity, and secretion into conditioned media, events which were time- and force-dependent. Subsequent incubation of BASMCs with conditioned media from chronically strained BAECs (5%, 24 h) significantly reduces BASMC migration (38 ± 6%), an inhibitory effect which could be completely reversed by targeted siRNA 'knock-down' of MMP-2 (but not MMP-9) expression and activity in BAECs. Moreover, inhibition of strain-mediated MMP-2 expression in BAECs by protein tyrosine kinase (PTK) blockade with genistein (50 μM) was also found to completely reverse this inhibitory effect on BASMC migration. Finally, direct supplementation of recombinant MMP-2 into the BASMC migration assay was found to have no significant effect on migration. However, the effect on BASMC migration of MMP-2 siRNA transfection in BAECs could be reversed by supplementation of recombinant MMP-2 into BAEC media prior to (and for the duration of) strain. These findings reveal a potentially novel role for strain-induced endothelial MMP-2 in regulating vascular SMC migration

  20. Marrow-derived mesenchymal stem cells: role in epithelial tumor cell determination.

    Science.gov (United States)

    Fierro, Fernando A; Sierralta, Walter D; Epuñan, Maria J; Minguell, José J

    2004-01-01

    Marrow stroma represents an advantageous environment for development of micrometastatic cells. Within the cellular structure of marrow stroma, mesenchymal stem cells (MSC) have been postulated as an interacting target for disseminated cancer cells. The studies reported here were performed to gain more information on the interaction of the human breast cancer cell line MCF-7 with human bone marrow-derived MSC cells and to investigate whether this interaction affects tumor cell properties. The results showed that after co-culture with MSC, changes were detected in the morphology, proliferative capacity and aggregation pattern of MCF-7 cells, but these parameters were not affected after the co-culture of MSC cells with a non-tumorigenic breast epithelial cell line, MCF-10. Since the indirect culture of MCF-7 with MSC or its products also resulted in functional changes in the tumor cells, we evaluated whether these effects could be attributed to growth factors produced by MSC cells. It was found that VEGF and IL-6 mimic the effects produced by MSC or its products on the proliferation and aggregation properties of MCF-7, cells, respectively. Thus, it seems that after entry of disseminated tumor cells into the marrow space, their proliferative and morphogenetic organization patterns are modified after interaction with distinct stromal cells and/or with specific signals from the marrow microenvironment.

  1. Potential of laryngeal muscle regeneration using induced pluripotent stem cell-derived skeletal muscle cells.

    Science.gov (United States)

    Dirja, Bayu Tirta; Yoshie, Susumu; Ikeda, Masakazu; Imaizumi, Mitsuyoshi; Nakamura, Ryosuke; Otsuki, Koshi; Nomoto, Yukio; Wada, Ikuo; Hazama, Akihiro; Omori, Koichi

    2016-01-01

    Conclusion Induced pluripotent stem (iPS) cells may be a new potential cell source for laryngeal muscle regeneration in the treatment of vocal fold atrophy after recurrent laryngeal nerve paralysis. Objectives Unilateral vocal fold paralysis can lead to degeneration, atrophy, and loss of force of the thyroarytenoid muscle. At present, there are some treatments such as thyroplasty, arytenoid adduction, and vocal fold injection. However, such treatments cannot restore reduced mass of the thyroarytenoid muscle. iPS cells have been recognized as supplying a potential resource for cell transplantation. The aim of this study was to assess the effectiveness of the use of iPS cells for the regeneration of laryngeal muscle through the evaluation of both in vitro and in vivo experiments. Methods Skeletal muscle cells were generated from tdTomato-labeled iPS cells using embryoid body formation. Differentiation into skeletal muscle cells was analyzed by gene expression and immunocytochemistry. The tdTomato-labeled iPS cell-derived skeletal muscle cells were transplanted into the left atrophied thyroarytenoid muscle. To evaluate the engraftment of these cells after transplantation, immunohistochemistry was performed. Results The tdTomato-labeled iPS cells were successfully differentiated into skeletal muscle cells through an in vitro experiment. These cells survived in the atrophied thyroarytenoid muscle after transplantation.

  2. Adipose-derived stromal cells inhibit prostate cancer cell proliferation inducing apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Takahara, Kiyoshi [Department of Urology, Faculty of Medicine, Osaka Medical College, Osaka (Japan); Ii, Masaaki, E-mail: masaii@art.osaka-med.ac.jp [Department of Pharmacology, Faculty of Medicine, Osaka Medical College, Osaka (Japan); Inamoto, Teruo; Komura, Kazumasa; Ibuki, Naokazu; Minami, Koichiro; Uehara, Hirofumi; Hirano, Hajime; Nomi, Hayahito; Kiyama, Satoshi [Department of Urology, Faculty of Medicine, Osaka Medical College, Osaka (Japan); Asahi, Michio [Department of Pharmacology, Faculty of Medicine, Osaka Medical College, Osaka (Japan); Azuma, Haruhito [Department of Urology, Faculty of Medicine, Osaka Medical College, Osaka (Japan)

    2014-04-18

    Highlights: • AdSC transplantation exhibits inhibitory effect on tumor progressions of PCa cells. • AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway. • High expression of the TGF-β1 gene in AdSCs. - Abstract: Mesenchymal stem cells (MSCs) have generated a great deal of interest in the field of regenerative medicine. Adipose-derived stromal cells (AdSCs) are known to exhibit extensive proliferation potential and can undergo multilineage differentiation, sharing similar characteristics to bone marrow-derived MSCs. However, as the effect of AdSCs on tumor growth has not been studied sufficiently, we assessed the degree to which AdSCs affect the proliferation of prostate cancer (PCa) cell. Human AdSCs exerted an inhibitory effect on the proliferation of androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) human PCa cells, while normal human dermal fibroblasts (NHDFs) did not, and in fact promoted PCa cell proliferation to a degree. Moreover, AdSCs induced apoptosis of LNCaP cells and PC3 cells, activating the caspase3/7 signaling pathway. cDNA microarray analysis suggested that AdSC-induced apoptosis in both LNCaP and PC3 cells was related to the TGF-β signaling pathway. Consistent with our in vitro observations, local transplantation of AdSCs delayed the growth of tumors derived from both LNCaP- and PC3-xenografts in immunodeficient mice. This is the first preclinical study to have directly demonstrated that AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway, irrespective of androgen-responsiveness. Since autologous AdSCs can be easily isolated from adipose tissue without any ethical concerns, we suggest that therapy with these cells could be a novel approach for patients with PCa.

  3. Characterisation of insulin-producing cells differentiated from tonsil derived mesenchymal stem cells.

    Science.gov (United States)

    Kim, So-Yeon; Kim, Ye-Ryung; Park, Woo-Jae; Kim, Han Su; Jung, Sung-Chul; Woo, So-Youn; Jo, Inho; Ryu, Kyung-Ha; Park, Joo-Won

    2015-01-01

    Tonsil-derived (T-) mesenchymal stem cells (MSCs) display mutilineage differentiation potential and self-renewal capacity and have potential as a banking source. Diabetes mellitus is a prevalent disease in modern society, and the transplantation of pancreatic progenitor cells or various stem cell-derived insulin-secreting cells has been suggested as a novel therapy for diabetes. The potential of T-MSCs to trans-differentiate into pancreatic progenitor cells or insulin-secreting cells has not yet been investigated. We examined the potential of human T-MSCs to trans-differentiate into pancreatic islet cells using two different methods based on β-mercaptoethanol and insulin-transferin-selenium, respectively. First, we compared the efficacy of the two methods for inducing differentiation into insulin-producing cells. We demonstrated that the insulin-transferin-selenium method is more efficient for inducing differentiation into insulin-secreting cells regardless of the source of the MSCs. Second, we compared the differentiation potential of two different MSC types: T-MSCs and adipose-derived MSCs (A-MSCs). T-MSCs had a differentiation capacity similar to that of A-MSCs and were capable of secreting insulin in response to glucose concentration. Islet-like clusters differentiated from T-MSCs had lower synaptotagmin-3, -5, -7, and -8 levels, and consequently lower secreted insulin levels than cells differentiated from A-MSCs. These results imply that T-MSCs can differentiate into functional pancreatic islet-like cells and could provide a novel, alternative cell therapy for diabetes mellitus. Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  4. Adipose-derived stromal cells inhibit prostate cancer cell proliferation inducing apoptosis

    International Nuclear Information System (INIS)

    Takahara, Kiyoshi; Ii, Masaaki; Inamoto, Teruo; Komura, Kazumasa; Ibuki, Naokazu; Minami, Koichiro; Uehara, Hirofumi; Hirano, Hajime; Nomi, Hayahito; Kiyama, Satoshi; Asahi, Michio; Azuma, Haruhito

    2014-01-01

    Highlights: • AdSC transplantation exhibits inhibitory effect on tumor progressions of PCa cells. • AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway. • High expression of the TGF-β1 gene in AdSCs. - Abstract: Mesenchymal stem cells (MSCs) have generated a great deal of interest in the field of regenerative medicine. Adipose-derived stromal cells (AdSCs) are known to exhibit extensive proliferation potential and can undergo multilineage differentiation, sharing similar characteristics to bone marrow-derived MSCs. However, as the effect of AdSCs on tumor growth has not been studied sufficiently, we assessed the degree to which AdSCs affect the proliferation of prostate cancer (PCa) cell. Human AdSCs exerted an inhibitory effect on the proliferation of androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) human PCa cells, while normal human dermal fibroblasts (NHDFs) did not, and in fact promoted PCa cell proliferation to a degree. Moreover, AdSCs induced apoptosis of LNCaP cells and PC3 cells, activating the caspase3/7 signaling pathway. cDNA microarray analysis suggested that AdSC-induced apoptosis in both LNCaP and PC3 cells was related to the TGF-β signaling pathway. Consistent with our in vitro observations, local transplantation of AdSCs delayed the growth of tumors derived from both LNCaP- and PC3-xenografts in immunodeficient mice. This is the first preclinical study to have directly demonstrated that AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway, irrespective of androgen-responsiveness. Since autologous AdSCs can be easily isolated from adipose tissue without any ethical concerns, we suggest that therapy with these cells could be a novel approach for patients with PCa

  5. Murine adipose tissue-derived stromal cell apoptosis and susceptibility to oxidative stress in vitro are regulated by genetic background.

    Directory of Open Access Journals (Sweden)

    Robert Pazdro

    Full Text Available Adipose tissue-derived stromal cells (ADSCs are of interest for regenerative medicine as they are isolated easily and can differentiate into multiple cell lineages. Studies of their in vitro proliferation, survival, and differentiation are common; however, genetic effects on these phenotypes remain unknown. To test if these phenotypes are genetically regulated, ADSCs were isolated from three genetically diverse inbred mouse strains--C57BL/6J (B6, BALB/cByJ (BALB, and DBA/2J (D2--in which genetic regulation of hematopoietic stem function is well known. ADSCs from all three strains differentiated into osteogenic and chondrogenic lineages in vitro. ADSCs from BALB grew least well in vitro, probably due to apoptotic cell death after several days in culture. BALB ADSCs were also the most susceptible to the free radical inducers menadione and H2O2. ADSCs from the three possible F1 hybrids were employed to further define genetic regulation of ADSC phenotypes. D2, but not B6, alleles stimulated ADSC expansion in BALB cells. In contrast, B6, but not D2, alleles rescued BALB H2O2 resistance. We conclude that low oxidative stress resistance does not limit BALB ADSC growth in vitro, as these phenotypes are genetically regulated independently. In addition, ADSCs from these strains are an appropriate model system to investigate genetic regulation of ADSC apoptosis and stress resistance in future studies. Such investigations are essential to optimize cell expansion and differentiation and thus, potential for regenerative medicine.

  6. Mast cells enhance T cell activation: Importance of mast cell-derived TNF

    Science.gov (United States)

    Nakae, Susumu; Suto, Hajime; Kakurai, Maki; Sedgwick, Jonathon D.; Tsai, Mindy; Galli, Stephen J.

    2005-05-01

    Mast cells are not only important effector cells in immediate hypersensitivity reactions and immune responses to pathogens but also can contribute to T cell-mediated disorders. However, the mechanisms by which mast cells might influence T cells in such settings are not fully understood. We find that mast cells can enhance proliferation and cytokine production in multiple T cell subsets. Mast cell-dependent enhancement of T cell activation can be promoted by FcRI-dependent mast cell activation, TNF production by both mast cells and T cells, and mast cell-T cell contact. However, at high concentrations of cells, mast cells can promote T cell activation independent of IgE or TNF. Finally, mast cells also can promote T cell activation by means of soluble factors. These findings identify multiple mechanisms by which mast cells can influence T cell proliferation and cytokine production. allergy | asthma | autoimmunity | cytokines | immune response

  7. HIV-derived vectors for gene therapy targeting dendritic cells.

    Science.gov (United States)

    Rossetti, Maura; Cavarelli, Mariangela; Gregori, Silvia; Scarlatti, Gabriella

    2013-01-01

    Human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LV) have the potential to mediate stable therapeutic gene transfer. However, similarly to other viral vectors, their benefit is compromised by the induction of an immune response toward transgene-expressing cells that closely mimics antiviral immunity. LV share with the parental HIV the ability to activate dendritic cells (DC), while lack the peculiar ability of subverting DC functions, which is responsible for HIV immune escape. Understanding the interaction between LV and DC, with plasmacytoid and myeloid DC playing fundamental and distinct roles, has paved the way to novel approaches aimed at regulating transgene-specific immune responses. Thanks to the ability to target either DC subsets LV might be a powerful tool to induce immunity (i.e., gene therapy of cancer), cell death (i.e., in HIV/AIDS infection), or tolerance (i.e., gene therapy strategies for monogenic diseases). In this chapter, similarities and differences between the LV-mediated and HIV-mediated induction of immune responses, with specific focus on their interactions with DC, are discussed.

  8. Mesenchymal stem cell-derived microparticles: a promising therapeutic strategy.

    Science.gov (United States)

    Tan, Xi; Gong, Yong-Zhen; Wu, Ping; Liao, Duan-Fang; Zheng, Xi-Long

    2014-08-18

    Mesenchymal stem cells (MSCs) are multipotent stem cells that give rise to various cell types of the mesodermal germ layer. Because of their unique ability to home in on injured and cancerous tissues, MSCs are of great potential in regenerative medicine. MSCs also contribute to reparative processes in different pathological conditions, including cardiovascular diseases and cancer. However, many studies have shown that only a small proportion of transplanted MSCs can actually survive and be incorporated into host tissues. The effects of MSCs cannot be fully explained by their number. Recent discoveries suggest that microparticles (MPs) derived from MSCs may be important for the physiological functions of their parent. Though the physiological role of MSC-MPs is currently not well understood, inspiring results indicate that, in tissue repair and anti-cancer therapy, MSC-MPs have similar pro-regenerative and protective properties as their cellular counterparts. Thus, MSC-MPs represent a promising approach that may overcome the obstacles and risks associated with the use of native or engineered MSCs.

  9. Production of cell culture (MDCK) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process.

    Science.gov (United States)

    George, Meena; Farooq, Masiha; Dang, Thi; Cortes, Bernadette; Liu, Jonathan; Maranga, Luis

    2010-08-15

    The majority of influenza vaccines are manufactured using embryonated hens' eggs. The potential occurrence of a pandemic outbreak of avian influenza might reduce or even eliminate the supply of eggs, leaving the human population at risk. Also, the egg-based production technology is intrinsically cumbersome and not easily scalable to provide a rapid worldwide supply of vaccine. In this communication, the production of a cell culture (Madin-Darby canine kidney (MDCK)) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process using a novel Single Use Bioreactor (SUB) is presented. The cell culture and virus infection was maintained in a disposable stirred tank reactor with PID control of pH, DO, agitation, and temperature, similar to traditional glass or stainless steel bioreactors. The application of this technology was tested using MDCK cells grown on microcarriers in proprietary serum free medium and infection with 2006/2007 seasonal LAIV strains at 25-30 L scale. The MDCK cell growth was optimal at the agitation rate of 100 rpm. Optimization of this parameter allowed the cells to grow at a rate similar to that achieved in the conventional 3 L glass stirred tank bioreactors. Influenza vaccine virus strains, A/New Caledonia/20/99 (H1N1 strain), A/Wisconsin/67/05 (H3N2 strain), and B/Malaysia/2506/04 (B strain) were all successfully produced in SUB with peak virus titers > or =8.6 log(10) FFU/mL. This result demonstrated that more than 1 million doses of vaccine can be produced through one single run of a small bioreactor at the scale of 30 L and thus provided an alternative to the current vaccine production platform with fast turn-around and low upfront facility investment, features that are particularly useful for emerging and developing countries and clinical trial material production.

  10. Aggregative adherent strains of Corynebacterium pseudodiphtheriticum enter and survive within HEp-2 epithelial cells

    Directory of Open Access Journals (Sweden)

    Monica Cristina de Souza

    2012-06-01

    Full Text Available Corynebacterium pseudodiphtheriticum is a well-known human pathogen that mainly causes respiratory disease and is associated with high mortality in compromised hosts. Little is known about the virulence factors and pathogenesis of C. pseudodiphtheriticum. In this study, cultured human epithelial (HEp-2 cells were used to analyse the adherence pattern, internalisation and intracellular survival of the ATCC 10700 type strain and two additional clinical isolates. These microorganisms exhibited an aggregative adherence-like pattern to HEp-2 cells characterised by clumps of bacteria with a "stacked-brick" appearance. The differences in the ability of these microorganisms to invade and survive within HEp-2 cells and replicate in the extracellular environment up to 24 h post infection were evaluated. The fluorescent actin staining test demonstrated that actin polymerisation is involved in the internalisation of the C. pseudodiphtheriticum strains. The depolymerisation of microfilaments by cytochalasin E significantly reduced the internalisation of C. pseudodiphtheriticum by HEp-2 cells. Bacterial internalisation and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 h. These characteristics may explain how some C. pseudodiphtheriticum strains cause severe infection in human patients.

  11. Effects of furan derivatives and phenolic compounds on electricity generation in microbial fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Catal, Tunc [Department of Biological and Ecological Engineering, Oregon State University, 116 Gilmore Hall, Corvallis, OR 97331 (United States); Department of Wood Science and Engineering, Oregon State University, 102 97331, Corvallis, OR (United States); Department of Molecular Biology and Genetics, Istanbul Technical University, 34469-Maslak, Istanbul (Turkey); Fan, Yanzhen; Liu, Hong [Department of Biological and Ecological Engineering, Oregon State University, 116 Gilmore Hall, Corvallis, OR 97331 (United States); Li, Kaichang [Department of Wood Science and Engineering, Oregon State University, 102 97331, Corvallis, OR (United States); Bermek, Hakan [Department of Molecular Biology and Genetics, Istanbul Technical University, 34469-Maslak, Istanbul (Turkey)

    2008-05-15

    Lignocellulosic biomass is an attractive fuel source for MFCs due to its renewable nature and ready availability. Furan derivatives and phenolic compounds could be potentially formed during the pre-treatment process of lignocellulosic biomass. In this study, voltage generation from these compounds and the effects of these compounds on voltage generation from glucose in air-cathode microbial fuel cells (MFCs) were examined. Except for 5-hydroxymethyl furfural (5-HMF), all the other compounds tested were unable to be utilized directly for electricity production in MFCs in the absence of other electron donors. One furan derivate, 5-HMF and two phenolic compounds, trans-cinnamic acid and 3,5-dimethoxy-4-hydroxy-cinnamic acid did not affect electricity generation from glucose at a concentration up to 10 mM. Four phenolic compounds, including syringaldeyhde, vanillin, trans-4-hydroxy-3-methoxy, and 4-hydroxy cinnamic acids inhibited electricity generation at concentrations above 5 mM. Other compounds, including 2-furaldehyde, benzyl alcohol and acetophenone, inhibited the electricity generation even at concentrations less than 0.2 mM. This study suggests that effective electricity generation from the hydrolysates of lignocellulosic biomass in MFCs may require the employment of the hydrolysis methods with low furan derivatives and phenolic compounds production, or the removal of some strong inhibitors prior to the MFC operation, or the improvement of bacterial tolerance against these compounds through the enrichment of new bacterial cultures or genetic modification of the bacterial strains. (author)

  12. Effects of furan derivatives and phenolic compounds on electricity generation in microbial fuel cells

    Science.gov (United States)

    Catal, Tunc; Fan, Yanzhen; Li, Kaichang; Bermek, Hakan; Liu, Hong

    Lignocellulosic biomass is an attractive fuel source for MFCs due to its renewable nature and ready availability. Furan derivatives and phenolic compounds could be potentially formed during the pre-treatment process of lignocellulosic biomass. In this study, voltage generation from these compounds and the effects of these compounds on voltage generation from glucose in air-cathode microbial fuel cells (MFCs) were examined. Except for 5-hydroxymethyl furfural (5-HMF), all the other compounds tested were unable to be utilized directly for electricity production in MFCs in the absence of other electron donors. One furan derivate, 5-HMF and two phenolic compounds, trans-cinnamic acid and 3,5-dimethoxy-4-hydroxy-cinnamic acid did not affect electricity generation from glucose at a concentration up to 10 mM. Four phenolic compounds, including syringaldeyhde, vanillin, trans-4-hydroxy-3-methoxy, and 4-hydroxy cinnamic acids inhibited electricity generation at concentrations above 5 mM. Other compounds, including 2-furaldehyde, benzyl alcohol and acetophenone, inhibited the electricity generation even at concentrations less than 0.2 mM. This study suggests that effective electricity generation from the hydrolysates of lignocellulosic biomass in MFCs may require the employment of the hydrolysis methods with low furan derivatives and phenolic compounds production, or the removal of some strong inhibitors prior to the MFC operation, or the improvement of bacterial tolerance against these compounds through the enrichment of new bacterial cultures or genetic modification of the bacterial strains.

  13. Three-dimensional development of tensile pre-strained annulus fibrosus cells for tissue regeneration: An in-vitro study

    Energy Technology Data Exchange (ETDEWEB)

    Chuah, Yon Jin [School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459 (Singapore); Lee, Wu Chean [University Hospital Conventry & Warwickshire NHS Trust, Clifford Bridge Road, West Midlands CV2, 2DX (United Kingdom); Wong, Hee Kit [Department of Orthopedic Surgery, National University Health System, NUHS Tower Block Level 11, 1E Kent Ridge Road, Singapore 119228 (Singapore); Kang, Yuejun, E-mail: yuejun.kang@ntu.edu.sg [School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459 (Singapore); Hee, Hwan Tak, E-mail: HTHee@ntu.edu.sg [School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459 (Singapore); Pinnacle Spine & Scoliosis Centre, 3 Mount Elizabeth, Mount Elizabeth Medical Centre, #04-07, Singapore 228510 (Singapore); School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637459 (Singapore)

    2015-02-01

    Prior research has investigated the immediate response after application of tensile strain on annulus fibrosus (AF) cells for the past decade. Although mechanical strain can produce either catabolic or anabolic consequences to the cell monolayer, little is known on how to translate these findings into further tissue engineering applications. Till to date, the application and effect of tensile pre-strained cells to construct a three-dimensional (3D) AF tissue remains unknown. This study aims to investigate the effect of tensile pre-strained exposure of 1 to 24 h on the development of AF pellet culture for 3 weeks. Equibiaxial cyclic tensile strain was applied on AF monolayer cells over a period of 24 h, which was subsequently developed into a cell pellet. Investigation on cellular proliferation, phenotypic gene expression, and histological changes revealed that tensile pre-strain for 24 h had significant and lasting effect on the AF tissue development, with enhanced cell proliferation, and up-regulation of collagen type I, II, and aggrecan expression. Our results demonstrated the regenerative ability of AF cell pellets subjected to 24 h tensile pre-straining. Knowledge on the effects of tensile pre-strain exposure is necessary to optimize AF development for tissue reconstruction. Moreover, the tensile pre-strained cells may further be utilized in either cell therapy to treat mild disc degeneration disease, or the development of a disc construct for total disc replacement. - Highlights: • Establishment of tensile pre-strained cell line population for annulus development. • Tensile strain limits collagen gene expression declination in monolayer culture. • Tensile pre-strained cells up-regulate their matrix protein in 3D pellet culture.

  14. Bone marrow-derived mesenchymal stem cells versus adipose-derived mesenchymal stem cells for peripheral nerve regeneration

    Directory of Open Access Journals (Sweden)

    Marcela Fernandes

    2018-01-01

    Full Text Available Studies have confirmed that bone marrow-derived mesenchymal stem cells (MSCs can be used for treatment of several nervous system diseases. However, isolation of bone marrow-derived MSCs (BMSCs is an invasive and painful process and the yield is very low. Therefore, there is a need to search for other alterative stem cell sources. Adipose-derived MSCs (ADSCs have phenotypic and gene expression profiles similar to those of BMSCs. The production of ADSCs is greater than that of BMSCs, and ADSCs proliferate faster than BMSCs. To compare the effects of venous grafts containing BMSCs or ADSCs on sciatic nerve injury, in this study, rats were randomly divided into four groups: sham (only sciatic nerve exposed, Matrigel (MG; sciatic nerve injury + intravenous transplantation of MG vehicle, ADSCs (sciatic nerve injury + intravenous MG containing ADSCs, and BMSCs (sciatic nerve injury + intravenous MG containing BMSCs groups. Sciatic functional index was calculated to evaluate the function of injured sciatic nerve. Morphologic characteristics of nerves distal to the lesion were observed by toluidine blue staining. Spinal motor neurons labeled with Fluoro-Gold were quantitatively assessed. Compared with sham-operated rats, sciatic functional index was lower, the density of small-diameter fibers was significantly increased, and the number of motor neurons significantly decreased in rats with sciatic nerve injury. Neither ADSCs nor BMSCs significantly improved the sciatic nerve function of rats with sciatic nerve injury, increased fiber density, fiber diameters, axonal diameters, myelin sheath thickness, and G ratios (axonal diameter/fiber diameter ratios in the sciatic nerve distal to the lesion site. There was no significant difference in the number of spinal motor neurons among ADSCs, BMSCs and MG groups. These results suggest that neither BMSCs nor ADSCs provide satisfactory results for peripheral nerve repair when using MG as the conductor for

  15. Defect in negative selection in lpr donor-derived T cells differentiating in non-lpr host thymus

    International Nuclear Information System (INIS)

    Matsumoto, K.; Yoshikai, Y.; Asano, T.; Himeno, K.; Iwasaki, A.; Nomoto, K.

    1991-01-01

    Transplantation of bone marrow cells of lpr/lpr mice into irradiated normal mice fails to develop massive lymphadenopathy or autoimmunity but causes severe graft-vs.-host-like syndrome. To elucidate an abnormality of lpr/lpr bone marrow-derived T cells, we transplanted bone marrow cells of Mlsb lpr/lpr mice into H-2-compatible Mlsa non-lpr mice. Although lpr/lpr T cell precursors repopulated the host thymus as well as +/+ cells, a proportion of CD4+CD8+ cells decreased, and that of both CD4- and CD8- single-positive cells increased compared with those of +/+ recipients. Notably, in MRL/lpr----AKR and C3H/lpr----AKR chimeras, CD4 single-positive thymocytes contained an increased number of V beta 6+ cells in spite of potentially deleting alleles of Mlsa, whereas V beta 6+ mature T cells were deleted in the MRL/+ ----AKR and C3H/+ ----AKR chimeras. There was no difference between MRL/+ ----AKR and MRL/lpr----AKR chimeras in their proportion of V beta 3+ cells because both host and donor strain lack the deleting alleles. Interleukin 2 receptor expression of mature T cells, in the thymus and lymph node, was obviously higher in the MRL/lpr----AKR chimeras, in particular in the forbidden V beta 6+ subset. Moreover, lpr donor-derived peripheral T cells showed vigorous anti-CD3 response. These results indicate that lpr-derived T cells escape not only tolerance-related clonal deletion but also some induction of unresponsiveness in the non-lpr thymus

  16. Efficient infectious cell culture systems of the hepatitis C virus (HCV) prototype strains HCV-1 and H77.

    Science.gov (United States)

    Li, Yi-Ping; Ramirez, Santseharay; Mikkelsen, Lotte; Bukh, Jens

    2015-01-01

    The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5'UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3'UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 10(4.0) focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 10(3.5) and 10(4.4) FFU/ml, respectively. Hepatitis C virus (HCV) was discovered in 1989 with

  17. Experimental infection of Leishmania (L. chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae

    Directory of Open Access Journals (Sweden)

    Felio J Bello

    2005-10-01

    Full Text Available The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5 cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6% and on day 4 in the J774 cells (51%. This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

  18. 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

  19. 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

  20. 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.

  1. Bone marrow-derived stromal cells are more beneficial cell sources for tooth regeneration compared with adipose-derived stromal cells.

    Science.gov (United States)

    Ye, Lanfeng; Chen, Lin; Feng, Fan; Cui, Junhui; Li, Kaide; Li, Zhiyong; Liu, Lei

    2015-10-01

    Tooth loss is presently a global epidemic and tooth regeneration is thought to be a feasible and ideal treatment approach. Choice of cell source is a primary concern in tooth regeneration. In this study, the odontogenic differentiation potential of two non-dental-derived stem cells, adipose-derived stromal cells (ADSCs) and bone marrow-derived stromal cells (BMSCs), were evaluated both in vitro and in vivo. ADSCs and BMSCs were induced in vitro in the presence of tooth germ cell-conditioned medium (TGC-CM) prior to implantation into the omentum majus of rats, in combination with inactivated dentin matrix (IDM). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression levels of odontogenic-related genes. Immunofluorescence and immunohistochemical assays were used to detect the protein levels of odontogenic-specific genes, such as DSP and DMP-1 both in vitro and in vivo. The results suggest that both ADSCs and BMSCs have odontogenic differentiation potential. However, the odontogenic potential of BMSCs was greater compared with ADSCs, showing that BMSCs are a more appropriate cell source for tooth regeneration. © 2015 International Federation for Cell Biology.

  2. Cell culture isolation and sequence analysis of genetically diverse US porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene.

    Science.gov (United States)

    Oka, Tomoichiro; Saif, Linda J; Marthaler, Douglas; Esseili, Malak A; Meulia, Tea; Lin, Chun-Ming; Vlasova, Anastasia N; Jung, Kwonil; Zhang, Yan; Wang, Qiuhong

    2014-10-10

    The highly contagious and deadly porcine epidemic diarrhea virus (PEDV) first appeared in the US in April 2013. Since then the virus has spread rapidly nationwide and to Canada and Mexico causing high mortality among nursing piglets and significant economic losses. Currently there are no efficacious preventive measures or therapeutic tools to control PEDV in the US. The isolation of PEDV in cell culture is the first step toward the development of an attenuated vaccine, to study the biology of PEDV and to develop in vitro PEDV immunoassays, inactivation assays and screen for PEDV antivirals. In this study, nine of 88 US PEDV strains were isolated successfully on Vero cells with supplemental trypsin and subjected to genomic sequence analysis. They differed genetically mainly in the N-terminal S protein region as follows: (1) strains (n=7) similar to the highly virulent US PEDV strains; (2) one similar to the reportedly US S INDEL PEDV strain; and (3) one novel strain most closely related to highly virulent US PEDV strains, but with a large (197aa) deletion in the S protein. Representative strains of these three genetic groups were passaged serially and grew to titers of ∼5-6log10 plaque forming units/mL. To our knowledge, this is the first report of the isolation in cell culture of an S INDEL PEDV strain and a PEDV strain with a large (197aa) deletion in the S protein. We also designed primer sets to detect these genetically diverse US PEDV strains. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Cell-based lipid flippase assay employing fluorescent lipid derivatives

    DEFF Research Database (Denmark)

    Jensen, Maria Stumph; Costa, Sara; Günther-Pomorski, Thomas

    2016-01-01

    P-type ATPases in the P4 subfamily (P4-ATPases) are transmembrane proteins unique for eukaryotes that act as lipid flippases, i.e., to translocate phospholipids from the exofacial to the cytofacial monolayer of cellular membranes. While initially characterized as aminophospholipid translocases, s...... flippase activities in the plasma membrane of cells, using yeast as an example.......P-type ATPases in the P4 subfamily (P4-ATPases) are transmembrane proteins unique for eukaryotes that act as lipid flippases, i.e., to translocate phospholipids from the exofacial to the cytofacial monolayer of cellular membranes. While initially characterized as aminophospholipid translocases......, studies of individual P4-ATPase family members from fungi, plants, and animals show that P4-ATPases differ in their substrate specificities and mediate transport of a broader range of lipid substrates. Here, we describe an assay based on fluorescent lipid derivatives to monitor and characterize lipid...

  4. Enrichment of skin-derived neural precursor cells from dermal cell populations by altering culture conditions.

    Science.gov (United States)

    Bayati, Vahid; Gazor, Rohoullah; Nejatbakhsh, Reza; Negad Dehbashi, Fereshteh

    2016-01-01

    As stem cells play a critical role in tissue repair, their manipulation for being applied in regenerative medicine is of great importance. Skin-derived precursors (SKPs) may be good candidates for use in cell-based therapy as the only neural stem cells which can be isolated from an accessible tissue, skin. Herein, we presented a simple protocol to enrich neural SKPs by monolayer adherent cultivation to prove the efficacy of this method. To enrich neural SKPs from dermal cell populations, we have found that a monolayer adherent cultivation helps to increase the numbers of neural precursor cells. Indeed, we have cultured dermal cells as monolayer under serum-supplemented (control) and serum-supplemented culture, followed by serum free cultivation (test) and compared. Finally, protein markers of SKPs were assessed and compared in both experimental groups and differentiation potential was evaluated in enriched culture. The cells of enriched culture concurrently expressed fibronectin, vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors as compared to control culture. In addition, they possessed a multipotential capacity to differentiate into neurogenic, glial, adipogenic, osteogenic and skeletal myogenic cell lineages. It was concluded that serum-free adherent culture reinforced by growth factors have been shown to be effective on proliferation of skin-derived neural precursor cells (skin-NPCs) and drive their selective and rapid expansion.

  5. Thrombin-specific inactivation of endothelial cell derived plasminogen activator

    International Nuclear Information System (INIS)

    Highsmith, R.F.; Gallaher, M.J.

    1986-01-01

    Although thrombin (T) has diverse functions in the overall hemostatic mechanism, relatively little is known about its direct effect on components of the fibrinolytic enzyme system. The authors have investigated the interaction of T with plasminogen activators (PA) derived from bovine aortic endothelial cells (EC) in culture (2-5th passage, preconfluent monolayers). Varying concentrations of purified bovine or human thrombin were added to EC-conditioned media (CM). CM + T mixtures were assayed at various times for PA activity using purified plasminogen and a sensitive 125 I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface

  6. Thrombin-specific inactivation of endothelial cell derived plasminogen activator

    Energy Technology Data Exchange (ETDEWEB)

    Highsmith, R.F.; Gallaher, M.J.

    1986-03-05

    Although thrombin (T) has diverse functions in the overall hemostatic mechanism, relatively little is known about its direct effect on components of the fibrinolytic enzyme system. The authors have investigated the interaction of T with plasminogen activators (PA) derived from bovine aortic endothelial cells (EC) in culture (2-5th passage, preconfluent monolayers). Varying concentrations of purified bovine or human thrombin were added to EC-conditioned media (CM). CM + T mixtures were assayed at various times for PA activity using purified plasminogen and a sensitive /sup 125/I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface.

  7. Induced Pluripotent Stem Cell-Derived Endothelial Cells in Insulin Resistance and Metabolic Syndrome.

    Science.gov (United States)

    Carcamo-Orive, Ivan; Huang, Ngan F; Quertermous, Thomas; Knowles, Joshua W

    2017-11-01

    Insulin resistance leads to a number of metabolic and cellular abnormalities including endothelial dysfunction that increase the risk of vascular disease. Although it has been particularly challenging to study the genetic determinants that predispose to abnormal function of the endothelium in insulin-resistant states, the possibility of deriving endothelial cells from induced pluripotent stem cells generated from individuals with detailed clinical phenotyping, including accurate measurements of insulin resistance accompanied by multilevel omic data (eg, genetic and genomic characterization), has opened new avenues to study this relationship. Unfortunately, several technical barriers have hampered these efforts. In the present review, we summarize the current status of induced pluripotent stem cell-derived endothelial cells for modeling endothelial dysfunction associated with insulin resistance and discuss the challenges to overcoming these limitations. © 2017 American Heart Association, Inc.

  8. Influence of red blood cell-derived microparticles upon vasoregulation.

    Science.gov (United States)

    Said, Ahmed S; Doctor, Allan

    2017-10-01

    Here we review recent data and the evolving understanding of the role of red blood cell-derived microparticles (RMPs) in normal physiology and in disease progression. Microparticles (MPs) are small membrane vesicles derived from various parent cell types. MPs are produced in response to a variety of stimuli through several cytoskeletal and membrane phospholipid changes. MPs have been investigated as potential biomarkers for multiple disease processes and are thought to have biological effects, most notably in: promotion of coagulation, production and handling of reactive oxygen species, immune modulation, angiogenesis, and in apoptosis. Specifically, RMPs are produced normally during RBC maturation and their production is accelerated during processing and storage for transfusion. Several factors during RBC storage are known to trigger RMP production, including: increased intracellular calcium, increased potassium leakage, and energy failure with ATP depletion. Of note, RMP composition differs from that of intact RBCs, and the nature and composition of RMP components are affected by both storage duration and the character of storage solutions. Recognised RMP bioactivities include: promotion of coagulation, immune modulation, and promotion of endothelial adhesion, as well as influence upon vasoregulation via nitric oxide (NO) scavenging. Of particular relevance, RMPs are more avid NO scavengers than intact RBCs and this feature has been proposed as a mechanism for the impaired oxygen delivery homeostasis that has been observed following transfusion. Preliminary human studies demonstrate that circulating RMP abundance increases with RBC transfusion and is associated with altered plasma vasoactivity and abnormal vasoregulation. In summary, RMPs are submicron particles released from stored RBCs, with demonstrated vasoactive properties that appear to disturb oxygen delivery homeostasis. The clinical impact of RMPs in transfusion recipients is an area of continued

  9. Red cell-derived microparticles (RMP) as haemostatic agent.

    Science.gov (United States)

    Jy, Wenche; Johansen, Max E; Bidot, Carlos; Horstman, Lawrence L; Ahn, Yeon S

    2013-10-01

    Among circulating cell-derived microparticles, those derived from red cells (RMP) have been least well investigated. To exploit potential haemostatic benefit of RMP, we developed a method of producing them in quantity, and here report on their haemostatic properties. High-pressure extrusion of washed RBC was employed to generate RMP. RMP were identified and enumerated by flow cytometry. Their size distribution was assessed by Doppler electrophoretic light scattering analysis (DELSA). Interaction with platelets was studied by platelet aggregometry, and shear-dependent adhesion by Diamed IMPACT-R. Thrombin generation and tissue factor (TF) expression was also measured. The effect of RMP on blood samples of patients with bleeding disorders was investigated ex vivo by thromboelastography (TEG). Haemostatic efficacy in vivo was assessed by measuring reduction of blood loss and bleeding time in rats and rabbits. RMP have mean diameter of 0.45 µm and 50% of them exhibit annexin V binding, a proxy for procoagulant phospholipids (PL). No TF could be detected by flow cytometry. At saturating concentrations of MPs, RMP generated thrombin robustly but after longer delay compared to PMP and EMP. RMP enhanced platelet adhesion and aggregation induced by low-dose ADP or AA. In TEG study, RMP corrected or improved haemostatic defects in blood of patients with platelet and coagulation disorders. RMP reduced bleeding time and blood loss in thrombocytopenic rabbits (busulfan-treated) and in Plavix-treated rats. In conclusion, RMP has broad haemostatic activity, enhancing both primary (platelet) and secondary (coagulation) haemostasis, suggesting potential use as haemostatic agent for treatment of bleeding.

  10. Strain-specific helper T cell profile in the gut-associated lymphoid tissue.

    Science.gov (United States)

    Stanisavljević, Suzana; Đedović, Neda; Vujičić, Milica; Saksida, Tamara; Jevtić, Bojan; Milovanović, Boško; Momčilović, Miljana; Miljković, Đorđe; Stojanović, Ivana

    2017-10-01

    C57BL/6, BALB/c and NOD mice are among the most frequently used strains in autoimmunity research. NOD mice spontaneously develop type 1 diabetes (T1D) and they are prone to induction of experimental autoimmune encephalomyelitis (EAE). Both diseases can be routinely induced in C57BL/6 mice, but not in BALB/c mice. Also, C57BL/6 mice are generally considered T helper (Th)1-biased and BALB/c Th2-biased mice. Having in mind increasingly appreciated role of gut associated lymphoid tissue (GALT) cells in autoimmunity, especially in relation to gut Th17 and regulatory T (Treg) cells, our aim was to determine if there are differences in proportion of CD4 + T cell populations in mesenteric lymph nodes and Peyer's patches of these mouse strains. Lower proportion of Treg was observed in NOD PP, Th2 cells dominated in BALB/c mice in mesenteric lymph nodes (MLN) and Peyer's patches (PP), while Th1 cells prevailed in C57BL/6 MLN. Intradermal immunization of mice with complete Freund's adjuvant resulted in significant difference in Th cell distribution in GALT of NOD mice. Differences were less pronounced in C57BL/6 mice, while GALT of BALB/c mice was almost unresponsive to the immunization. The observed strain- and tissue-dependent changes in Treg proportion after the immunization was probably a consequence of different CCR2 or CCR6-related migration patterns and/or in situ Treg proliferation. In conclusion, NOD, a highly autoimmunity-prone mouse strain, exhibits more profound GALT-related immune response upon immunization compared to the strains that are less prone to autoimmunity. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  11. 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.

  12. 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.

  13. The differentiation potential of adipose tissue-derived mesenchymal stem cells into cell lineage related to male germ cells

    Directory of Open Access Journals (Sweden)

    P. Bräunig

    Full Text Available ABSTRACT The adipose tissue is a reliable source of Mesenchymal stem cells (MSCs showing a higher plasticity and transdifferentiation potential into multilineage cells. In the present study, adipose tissue-derived mesenchymal stem cells (AT-MSCs were isolated from mice omentum and epididymis fat depots. The AT-MSCs were initially compared based on stem cell surface markers and on the mesodermal trilineage differentiation potential. Additionally, AT-MSCs, from both sources, were cultured with differentiation media containing retinoic acid (RA and/or testicular cell-conditioned medium (TCC. The AT-MSCs expressed mesenchymal surface markers and differentiated into adipogenic, chondrogenic and osteogenic lineages. Only omentum-derived AT-MSCs expressed one important gene marker related to male germ cell lineages, after the differentiation treatment with RA. These findings reaffirm the importance of adipose tissue as a source of multipotent stromal-stem cells, as well as, MSCs source regarding differentiation purpose.

  14. Effects of Saponins against Clinical E. coli Strains and Eukaryotic Cell Line

    Science.gov (United States)

    Arabski, Michał; Węgierek-Ciuk, Aneta; Czerwonka, Grzegorz; Lankoff, Anna; Kaca, Wiesław

    2012-01-01

    Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 μg/mL and in the range of 12–50 μg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 μg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed. PMID:22500084

  15. Effects of Saponins against Clinical E. coli Strains and Eukaryotic Cell Line

    Directory of Open Access Journals (Sweden)

    Michał Arabski

    2012-01-01

    Full Text Available Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 μg/mL and in the range of 12–50 μg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 μg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed.

  16. Regulation of thrombomodulin expression and release in human aortic endothelial cells by cyclic strain.

    Directory of Open Access Journals (Sweden)

    Fiona A Martin

    Full Text Available Thrombomodulin (TM, an integral membrane glycoprotein expressed on the lumenal surface of vascular endothelial cells, promotes anti-coagulant and anti-inflammatory properties. Release of functional TM from the endothelium surface into plasma has also been reported. Much is still unknown however about how endothelial TM is regulated by physiologic hemodynamic forces (and particularly cyclic strain intrinsic to endothelial-mediated vascular homeostasis.This study employed human aortic endothelial cells (HAECs to investigate the effects of equibiaxial cyclic strain (7.5%, 60 cycles/min, 24 hrs, and to a lesser extent, laminar shear stress (10 dynes/cm2, 24 hrs, on TM expression and release. Time-, dose- and frequency-dependency studies were performed.Our initial studies demonstrated that cyclic strain strongly downregulated TM expression in a p38- and receptor tyrosine kinase-dependent manner. This was in contrast to the upregulatory effect of shear stress. Moreover, both forces significantly upregulated TM release over a 48 hr period. With continuing focus on the cyclic strain-induced TM release, we noted both dose (0-7.5% and frequency (0.5-2.0 Hz dependency, with no attenuation of strain-induced TM release observed following inhibition of MAP kinases (p38, ERK-1/2, receptor tyrosine kinase, or eNOS. The concerted impact of cyclic strain and inflammatory mediators on TM release from HAECs was also investigated. In this respect, both TNFα (100 ng/ml and ox-LDL (10-50 µg/ml appeared to potentiate strain-induced TM release. Finally, inhibition of neither MMPs (GM6001 nor rhomboids (3,4-dichloroisocoumarin had any effect on strain-induced TM release. However, significantly elevated levels (2.1 fold of TM were observed in isolated microparticle fractions following 7.5% strain for 24 hrs.A preliminary in vitro investigation into the effects of cyclic strain on TM in HAECs is presented. Physiologic cyclic strain was observed to downregulate TM

  17. The microstructural origin of strain hardening in two-dimensional open-cell metal foams

    NARCIS (Netherlands)

    Mangipudi, K. R.; van Buuren, S. W.; Onck, P. R.

    2010-01-01

    This paper aims at elucidating the microstructural origin of strain hardening in open-cell metal foams. We have developed a multiscale model that allows to study the development of plasticity at two length scales: (i) the development of plastic zones inside individual struts (microscopic scale) and

  18. Interaction between the Bacterium Pseudomonas fluorescens strain CHA0, its genetic derivatives and vermiculite: Effects on chemical, mineralogical and mechanical properties of vermiculite

    Science.gov (United States)

    Mueller, Barbara

    2016-04-01

    Using bacteria of the strain Pseudomonas fluorescens wild type CHA0 and its genetic derivative strains CHA77, CHA89, CHA400, CHA631 and CHA661 (which differ in one gene only) the changes in chemical, mineralogical and rheological properties of the clay mineral vermiculite affected by microbial activity were studied in order to test whether the individually different production of metabolites by the genetically engineered strains may alter the clay mineral vermiculite in distinct ways. With the novel strategy of working with living wild type bacteria, their genetic derivatives and clay, the following properties of the mineral altered by the various strains of Pseudomonas fluorescens were determined: grain size, X-Ray diffraction pattern, intercrystalline swelling with glycerol, layer charge, CEC, BET surface and uptake of trace elements. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to determine the changes in major, minor and trace elements of the clay vermiculite affected by microbial activity. Among all analyzed trace elements, Fe, Mn and Cu are the most interesting. Fe and Mn are taken up from the clay mineral by all bacterial strains whereas Cu is only removed from vermiculite by strains CHA0, CHA77, CHA400 and CHA661. The latter mentioned strains all produce the antibiotics 2,4-diacetylphloroglucinol and monoacetylphloroglucinol which can complex Cu efficiently. Therefore the alteration of only one gene of the bacteria is causing significant effects on the clay mineral.

  19. VUV treatment combined with mechanical strain of stretchable polymer foils resulting in cell alignment

    Energy Technology Data Exchange (ETDEWEB)

    Barb, R.-A. [Institute of Applied Physics, Johannes Kepler University Linz (Austria); Magnus, B. [Innovacell Biotechnologie AG, Innsbruck (Austria); Innerbichler, S. [Innerbichler GmbH, Breitenbach am Inn (Austria); Greunz, T. [CDL-MS-MACH, Johannes Kepler University Linz (Austria); Wiesbauer, M. [Institute of Applied Physics, Johannes Kepler University Linz (Austria); Marksteiner, R. [Innovacell Biotechnologie AG, Innsbruck (Austria); Stifter, D. [CDL-MS-MACH, Johannes Kepler University Linz (Austria); Heitz, J., E-mail: johannes.heitz@jku.at [Institute of Applied Physics, Johannes Kepler University Linz (Austria)

    2015-01-15

    Highlights: • Elastic polyurethane (PU) foils were exposed to the vacuum-UV in reactive atmosphere. • The photomodification resulted in improved cytocompatibilty. • Parallel microgrooves formed on the irradiated PU surfaces after strong elongation. • Cells seeded onto microgrooves aligned their shapes in the direction of the grooves. • Elongation occurred also for cells on PU subjected to cyclic mechanical stretching. - Abstract: Cell-alignment along a defined direction can have a direct effect on the cell functionality and differentiation. Oriented micro- or nanotopographic structures on cell culture substrates can induce cell-alignment. Surface chemistry, wettability, and stiffness of the substrate are also important material features as they strongly influence the cell–substrate interactions. For improved bio-compatibility, highly elastic polyurethane (PU) foils were exposed to the vacuum-UV (VUV) light of a Xe{sub 2}{sup *} excimer lamp at 172 nm in a nitrogen containing atmosphere (N{sub 2} or NH{sub 3}). The irradiation resulted in a change in the chemical surface composition. Additionally, the formation of regular parallel microgrooves was observed on the irradiated surfaces after strong uni-axial deformation (i.e., more than about 50% strain) of the photo-modified PU foils. Cell seeding experiments demonstrated that the VUV modified polymer foils strongly enhance cell adhesion and proliferation. Cells seeded onto microgrooves aligned their shapes and elongated in the direction of the grooves. A similar effect was observed for cells seeded on photo-modified PU foils subjected to cyclic mechanical stretching at lower strain levels (i.e., typically 10% strain) without groove-formation. The cells had also here an elongated shape, however they not always align in a defined direction relative to the stretching.

  20. Norcantharidin, Derivative of Cantharidin, for Cancer Stem Cells

    Directory of Open Access Journals (Sweden)

    Chen-Hsi Hsieh

    2013-01-01

    Full Text Available Cancer stem cells (CSCs existing in human cancers have been demonstrated to be a major cause of cancer treatment resistance, invasion, metastasis, and relapse. Self-renewal pathways, Wnt/β-catenin, Sonic hedgehog (Shh, and the Notch signaling pathway play critical roles in developing CSCs and lead to angiogenesis, migration, invasion, and metastasis. Multidrug resistance (MDR is an unfavorable factor causing the failure of treatments against cancer cells. The most important and thoroughly studied mechanism involved in MDR is the active efflux of chemotherapeutic agents through membrane drug transporters. There is growing evidence that Norcantharidin (NCTD, a water-soluble synthetic small molecule derivative of naturally occurring cantharidin from the medicinal insect blister beetle (Mylabris phalerata Pallas, is capable of chemoprevention and tumor inhibition. We summarize investigations into the modulation of self-renewal pathways and MDR in CSCs by NCTD. This review may aid in further investigation of using NCTD to develop more effective strategies for cancer treatment to reduce resistance and recurrence.

  1. Generation of insulin-producing cells from gnotobiotic porcine skin-derived stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Ji Hoon; Lee, Sung Ho; Heo, Young Tae [Department of Bioscience and Biotechnology, Bio-Organ Research Center, Konkuk University, Seoul 143-701 (Korea, Republic of); Uhm, Sang Jun [Department of Animal Biotechnology, Bio-Organ Research Center, Konkuk University, Seoul 143-701 (Korea, Republic of); Lee, Hoon Taek, E-mail: htl3675@konkuk.ac.kr [Department of Animal Biotechnology, Bio-Organ Research Center, Konkuk University, Seoul 143-701 (Korea, Republic of)

    2010-07-09

    A major problem in the treatment of type 1 diabetes mellitus is the limited availability of alternative sources of insulin-producing cells for islet transplantation. In this study, we investigated the effect of bone morphogenetic protein 4 (BMP-4) treatments of gnotobiotic porcine skin-derived stem cells (gSDSCs) on their reprogramming and subsequent differentiation into insulin-producing cells (IPCs). We isolated SDSCs from the ear skin of a gnotobiotic pig. During the proliferation period, the cells expressed stem-cell markers Oct-4, Sox-2, and CD90; nestin expression also increased significantly. The cells could differentiate into IPCs after treatments with activin-A, glucagon-like peptide-1 (GLP-1), and nicotinamide. After 15 days in the differentiation medium, controlled gSDSCs began expressing endocrine progenitor genes and proteins (Ngn3, Neuro-D, PDX-1, NKX2.2, NKX6.1, and insulin). The IPCs showed increased insulin synthesis after glucose stimulation. The results indicate that stem cells derived from the skin of gnotobiotic pigs can differentiate into IPCs under the appropriate conditions in vitro. Our three-stage induction protocol could be applied without genetic modification to source IPCs from stem cells in the skin of patients with diabetes for autologous transplantation.

  2. Generation of insulin-producing cells from gnotobiotic porcine skin-derived stem cells

    International Nuclear Information System (INIS)

    Yang, Ji Hoon; Lee, Sung Ho; Heo, Young Tae; Uhm, Sang Jun; Lee, Hoon Taek

    2010-01-01

    A major problem in the treatment of type 1 diabetes mellitus is the limited availability of alternative sources of insulin-producing cells for islet transplantation. In this study, we investigated the effect of bone morphogenetic protein 4 (BMP-4) treatments of gnotobiotic porcine skin-derived stem cells (gSDSCs) on their reprogramming and subsequent differentiation into insulin-producing cells (IPCs). We isolated SDSCs from the ear skin of a gnotobiotic pig. During the proliferation period, the cells expressed stem-cell markers Oct-4, Sox-2, and CD90; nestin expression also increased significantly. The cells could differentiate into IPCs after treatments with activin-A, glucagon-like peptide-1 (GLP-1), and nicotinamide. After 15 days in the differentiation medium, controlled gSDSCs began expressing endocrine progenitor genes and proteins (Ngn3, Neuro-D, PDX-1, NKX2.2, NKX6.1, and insulin). The IPCs showed increased insulin synthesis after glucose stimulation. The results indicate that stem cells derived from the skin of gnotobiotic pigs can differentiate into IPCs under the appropriate conditions in vitro. Our three-stage induction protocol could be applied without genetic modification to source IPCs from stem cells in the skin of patients with diabetes for autologous transplantation.

  3. Different levels of immunogenicity of two strains of Fowlpox virus as recombinant vaccine vectors eliciting T-cell responses in heterologous prime-boost vaccination strategies.

    Science.gov (United States)

    Cottingham, Matthew G; van Maurik, Andre; Zago, Manola; Newton, Angela T; Anderson, Richard J; Howard, M Keith; Schneider, Jörg; Skinner, Michael A

    2006-07-01

    The FP9 strain of F has been described as a more immunogenic recombinant vaccine vector than the Webster FPV-M (FPW) strain (R. J. Anderson et al., J. Immunol. 172:3094-3100, 2004). This study expands the comparison to include two separate recombinant antigens and multiple, rather than single, independent viral clones derived from the two strains. Dual-poxvirus heterologous prime-boost vaccination regimens using individual clones of recombinant FP9 or FPW in combination with recombinant modified V Ankara expressing the same antigen were evaluated for their ability to elicit T-cell responses against recombinant antigens from Plasmodium berghei (circumsporozoite protein) or human immunodeficiency virus type 1 (a Gag-Pol-Nef fusion protein). Gamma interferon enzyme-linked immunospot assay and fluorescence-activated cell sorting assays of the responses to specific epitopes confirmed the approximately twofold-greater cellular immunogenicity of FP9 compared to FPW, when given as the priming or boosting immunization. Equality of transgene expression in mouse cells infected with the two strains in vitro was verified by Western blotting. Directed partial sequence analysis and PCR analysis of FPW and comparison to available whole-genome sequences revealed that many loci that are mutated in the highly attenuated and culture-adapted FP9 strain are wild type in FPW, including the seven multikilobase deletions. These "passage-specific" alterations are hypothesized to be involved in determining the immunogenicity of fowlpox virus as a recombinant vaccine vector.

  4. Single-Cell Functional Analysis of Stem-Cell Derived Cardiomyocytes on Micropatterned Flexible Substrates

    NARCIS (Netherlands)

    Kijlstra, Jan David; Hu, Dongjian; van der Meer, Peter; Domian, Ibrahim J

    2017-01-01

    Human pluripotent stem-cell derived cardiomyocytes (hPSC-CMs) hold great promise for applications in human disease modeling, drug discovery, cardiotoxicity screening, and, ultimately, regenerative medicine. The ability to study multiple parameters of hPSC-CM function, such as contractile and

  5. Effects of everolimus on macrophage-derived foam cell behavior

    International Nuclear Information System (INIS)

    Hsu, Steven; Koren, Eugen; Chan, Yen; Koscec, Mirna; Sheehy, Alexander; Kolodgie, Frank; Virmani, Renu; Feder, Debra

    2014-01-01

    Purpose: The purpose of this study was to investigate the effects of everolimus on foam cell (FC) viability, mRNA levels, and inflammatory cytokine production to better understand its potential inhibitory effects on atheroma progression. Methods and materials: Human THP1 macrophage-derived FC were formed using acetylated LDL (acLDL, 100 μg/mL) for 72 hours, followed by everolimus treatment (10 -5 –10 -11 M) for 24 hours. FC viability was quantified using fluorescent calcein AM/DAPI staining. FC lysates and media supernatants were analyzed for apoptosis and necrosis using a Cell Death ELISA PLUS assay. FC lysates and media supernatants were also analyzed for inflammatory cytokine (IL1β, IL8, MCP1, TNFα) mRNA levels and protein expression using quantitative reverse transcription real-time polymerase chain reaction (QPCR) and a Procarta® immunoassay, respectively. mRNA levels of autophagy (MAP1LC3), apoptosis (survivin, clusterin), and matrix degradation (MMP1, MMP9) markers were evaluated by Quantigene® Plex assay and verified with QPCR. Additionally, hypercholesterolemic rabbits received everolimus-eluting stents (EES) for 28 or 60 days. RAM-11 immunohistochemical staining was performed to compare %RAM-11 positive area between stented sections and unstented proximal sections. Statistical significance was calculated using one-way ANOVA (p ≤ 0.05). Results: Calcein AM/DAPI staining showed that FC exposed to everolimus (10 -5 M) had significantly decreased viability compared to control. FC apoptosis was significantly increased at a high dose of everolimus (10 -5 M), with no necrotic effects at any dose tested. Everolimus did not affect endothelial (HUVEC) and smooth muscle (HCASMC) cell apoptosis or necrosis. Everolimus (10 -5 M) significantly increased MAP1LC3, caused an increased trend in clusterin (p = 0.10), and significantly decreased survivin and MMP1 mRNA levels in FC. MCP1 cytokine mRNA levels and secreted protein expression was significantly decreased

  6. Effects of everolimus on macrophage-derived foam cell behavior

    Energy Technology Data Exchange (ETDEWEB)

    Hsu, Steven, E-mail: steven.hsu@av.abbott.com [Abbott Vascular, 3200 Lakeside Drive, Santa Clara, CA 95054 (United States); Koren, Eugen; Chan, Yen; Koscec, Mirna; Sheehy, Alexander [Abbott Vascular, 3200 Lakeside Drive, Santa Clara, CA 95054 (United States); Kolodgie, Frank; Virmani, Renu [CVPath Institute, Inc., 19 Firstfield Road, Gaithersburg, MD 20878 (United States); Feder, Debra [Abbott Vascular, 3200 Lakeside Drive, Santa Clara, CA 95054 (United States)

    2014-07-15

    Purpose: The purpose of this study was to investigate the effects of everolimus on foam cell (FC) viability, mRNA levels, and inflammatory cytokine production to better understand its potential inhibitory effects on atheroma progression. Methods and materials: Human THP1 macrophage-derived FC were formed using acetylated LDL (acLDL, 100 μg/mL) for 72 hours, followed by everolimus treatment (10{sup -5}–10{sup -11} M) for 24 hours. FC viability was quantified using fluorescent calcein AM/DAPI staining. FC lysates and media supernatants were analyzed for apoptosis and necrosis using a Cell Death ELISA{sup PLUS} assay. FC lysates and media supernatants were also analyzed for inflammatory cytokine (IL1β, IL8, MCP1, TNFα) mRNA levels and protein expression using quantitative reverse transcription real-time polymerase chain reaction (QPCR) and a Procarta® immunoassay, respectively. mRNA levels of autophagy (MAP1LC3), apoptosis (survivin, clusterin), and matrix degradation (MMP1, MMP9) markers were evaluated by Quantigene® Plex assay and verified with QPCR. Additionally, hypercholesterolemic rabbits received everolimus-eluting stents (EES) for 28 or 60 days. RAM-11 immunohistochemical staining was performed to compare %RAM-11 positive area between stented sections and unstented proximal sections. Statistical significance was calculated using one-way ANOVA (p ≤ 0.05). Results: Calcein AM/DAPI staining showed that FC exposed to everolimus (10{sup -5} M) had significantly decreased viability compared to control. FC apoptosis was significantly increased at a high dose of everolimus (10{sup -5} M), with no necrotic effects at any dose tested. Everolimus did not affect endothelial (HUVEC) and smooth muscle (HCASMC) cell apoptosis or necrosis. Everolimus (10{sup -5} M) significantly increased MAP1LC3, caused an increased trend in clusterin (p = 0.10), and significantly decreased survivin and MMP1 mRNA levels in FC. MCP1 cytokine mRNA levels and secreted protein

  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. Differentiation and Application of Induced Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells.

    Science.gov (United States)

    Maguire, Eithne Margaret; Xiao, Qingzhong; Xu, Qingbo

    2017-11-01

    Vascular smooth muscle cells (VSMCs) play a role in the development of vascular disease, for example, neointimal formation, arterial aneurysm, and Marfan syndrome caused by genetic mutations in VSMCs, but little is known about the mechanisms of the disease process. Advances in induced pluripotent stem cell technology have now made it possible to derive VSMCs from several different somatic cells using a selection of protocols. As such, researchers have set out to delineate key signaling processes involved in triggering VSMC gene expression to grasp the extent of gene regulatory networks involved in phenotype commitment. This technology has also paved the way for investigations into diseases affecting VSMC behavior and function, which may be treatable once an identifiable culprit molecule or gene has been repaired. Moreover, induced pluripotent stem cell-derived VSMCs are also being considered for their use in tissue-engineered blood vessels as they may prove more beneficial than using autologous vessels. Finally, while several issues remains to be clarified before induced pluripotent stem cell-derived VSMCs can become used in regenerative medicine, they do offer both clinicians and researchers hope for both treating and understanding vascular disease. In this review, we aim to update the recent progress on VSMC generation from stem cells and the underlying molecular mechanisms of VSMC differentiation. We will also explore how the use of induced pluripotent stem cell-derived VSMCs has changed the game for regenerative medicine by offering new therapeutic avenues to clinicians, as well as providing researchers with a new platform for modeling of vascular disease. © 2017 American Heart Association, Inc.

  9. Comparative Survival Rates of Human-Derived Probiotic Lactobacillus paracasei and L. salivarius Strains during Heat Treatment and Spray Drying

    Science.gov (United States)

    Gardiner, G. E.; O'Sullivan, E.; Kelly, J.; Auty, M. A. E.; Fitzgerald, G. F.; Collins, J. K.; Ross, R. P.; Stanton, C.

    2000-01-01

    Spray drying of skim milk was evaluated as a means of preserving Lactobacillus paracasei NFBC 338 and Lactobacillus salivarius UCC 118, which are human-derived strains with probiotic potential. Our initial experiments revealed that NFBC 338 is considerably more heat resistant in 20% (wt/vol) skim milk than UCC 118 is; the comparable decimal reduction times were 11.1 and 1.1 min, respectively, at 59°C. An air outlet temperature of 80 to 85°C was optimal for spray drying; these conditions resulted in powders with moisture contents of 4.1 to 4.2% and viable counts of 3.2 × 109 CFU/g for NFBC 338 and 5.2 × 107 CFU/g for UCC 118. Thus, L. paracasei NFBC 338 survived better than L. salivarius UCC 118 during spray drying; similar results were obtained when we used confocal scanning laser microscopy and LIVE/DEAD BacLight viability staining. In addition, confocal scanning laser microscopy revealed that the probiotic lactobacilli were located primarily in the powder particles. Although both spray-dried cultures appeared to be stressed, as shown by increased sensitivity to NaCl, bacteriocin production by UCC 118 was not affected by the process, nor was the activity of the bacteriocin peptide. The level of survival of NFBC 338 remained constant at ∼1 × 109 CFU/g during 2 months of powder storage at 4°C, while a decline in the level of survival of approximately 1 log (from 7.2 × 107 to 9.5 × 106 CFU/g) was observed for UCC 118 stored under the same conditions. However, survival of both Lactobacillus strains during powder storage was inversely related to the storage temperature. Our data demonstrate that spray drying may be a cost-effective way to produce large quantities of some probiotic cultures. PMID:10831444

  10. Polymorphism in hybrid male sterility in wild-derived Mus musculus musculus strains on proximal chromosome 17.

    Science.gov (United States)

    Vyskocilová, Martina; Prazanová, Gabriela; Piálek, Jaroslav

    2009-02-01

    The hybrid sterility-1 (Hst1) locus at Chr 17 causes male sterility in crosses between the house mouse subspecies Mus musculus domesticus (Mmd) and M. m. musculus (Mmm). This locus has been defined by its polymorphic variants in two laboratory strains (Mmd genome) when mated to PWD/Ph mice (Mmm genome): C57BL/10 (carrying the sterile allele) and C3H (fertile allele). The occurrence of sterile and/or fertile (wild Mmm x C57BL)F1 males is evidence that polymorphism for this trait also exists in natural populations of Mmm; however, the nature of this polymorphism remains unclear. Therefore, we derived two wild-origin Mmm strains, STUS and STUF, that produce sterile and fertile males, respectively, in crosses with C57BL mice. To determine the genetic basis underlying male fertility, the (STUS x STUF)F1 females were mated to C57BL/10 J males. About one-third of resulting hybrid males (33.8%) had a significantly smaller epididymis and testes than parental animals and lacked spermatozoa due to meiotic arrest. A further one-fifth of males (20.3%) also had anomalous reproductive traits but produced some spermatozoa. The remaining fertile males (45.9%) displayed no deviation from values found in parental individuals. QTL analysis of the progeny revealed strong associations of male fitness components with the proximal end of Chr 17, and a significant effect of the central section of Chr X on testes mass. The data suggest that genetic incompatibilities associated with male sterility have evolved independently at the proximal end of Chr 17 and are polymorphic within both Mmd and Mmm genomes.

  11. X-ray sensitive strains of CHO cells show decreased frequency of stable transfection

    International Nuclear Information System (INIS)

    Jeggo, P.; Smith, J.

    1987-01-01

    Six X-ray sensitive (xrs) strains of the Chinese hamster ovary cell line have previously been isolated and shown to have a defect in double strand break rejoining. In this study, these strains have been investigated for their ability to take up and integrate foreign DNA. All the xrs strains investigated so far have shown a decreased frequency of stable transfectants compared to their parent line, in experiments using the plasmid pSV2gpt, which contains the selectable bacterial gene, guanine phosphoribosyl transferase. This decreased frequency is observed over a wide range of DNA concentrations (0.1 to 20 μg DNA) but is more pronounced at higher DNA concentrations. In contrast, these xrs strains show the same level of transfection proficiency as the wild type parent using a transient transfection system with a plasmid containing the bacterial CAT (chloramphenicol acetyl transferase) gene. Since the level of CAT activity does not depend on integration of foreign DNA, this suggests that the xrs strains are able to take up the same amount of DNA as the parent strains, but have a defect in the integration of foreign DNA. Since this integration of foreign DNA probably occurs by non-homologous recombination, this may indicate a role of the xrs gene product in this process

  12. Transport of curcumin derivatives in Caco-2 cell monolayers.

    Science.gov (United States)

    Zeng, Zhen; Shen, Zhe L; Zhai, Shuo; Xu, Jia L; Liang, Hui; Shen, Qin; Li, Qing Y

    2017-08-01

    Curcumin (Cur) is a strong natural antioxidant, who can prevent multiple diseases such as anti-cancer, anti-inflammatory, have a resistance to alzheimer's disease and various malignant diseases. But it has poor oral bioavailability due to its poor aqueous solubility, as well as instability. While its novel derivatives (CB and FE), showed better anti-tumor activity, better anti-oxidant activity and better stability than the original drug (Cur). The aim of this study was to study the intestinal transport of Cur, CB and FE using an in vitro Caco-2 cell monolayer model. The results showed that Cur had a lower permeability coefficient (1.13×10 -6 ±0.11×10 -6 cm/s) for apical-to-basolated (AP-BL) transport at 25μM, while the transport rate for AP to BL flux of CB (3.18×10 -6 ±0.31×10 -6 cm/s) and FE (5.28×10 -6 ±0.83×10 -6 cm/s) were significantly greater than that of Cur. The efflux ratio (ER) value at the concentration of 25μM was 1.31 for Cur, 1.26 for CB and 1.33 for FE, suggesting there was no active efflux involved in the translocation across the Caco-2 cell monolayers for the three compounds. Furthermore, the transport flux of CB and FE was in a concentration dependent manner, suggesting the intestinal transport mechanism in them was passive transport. In summary, the results demonstrated that both the intestinal permeability of CB and FE across Caco-2 cell monolayers was significantly improved compare to Cur. Thus they might show a higher oral bioavailability in vivo, and show the potential application in clinic or nutraceutical. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Donor lung derived myeloid and plasmacytoid dendritic cells differentially regulate T cell proliferation and cytokine production

    Directory of Open Access Journals (Sweden)

    Benson Heather L

    2012-03-01

    Full Text Available Abstract Background Direct allorecognition, i.e., donor lung-derived dendritic cells (DCs stimulating recipient-derived T lymphocytes, is believed to be the key mechanism of lung allograft rejection. Myeloid (cDCs and plasmacytoid (pDCs are believed to have differential effects on T cell activation. However, the roles of each DC type on T cell activation and rejection pathology post lung transplantation are unknown. Methods Using transgenic mice and antibody depletion techniques, either or both cell types were depleted in lungs of donor BALB/c mice (H-2d prior to transplanting into C57BL/6 mice (H-2b, followed by an assessment of rejection pathology, and pDC or cDC-induced proliferation and cytokine production in C57BL/6-derived mediastinal lymph node T cells (CD3+. Results Depleting either DC type had modest effect on rejection pathology and T cell proliferation. In contrast, T cells from mice that received grafts depleted of both DCs did not proliferate and this was associated with significantly reduced acute rejection scores compared to all other groups. cDCs were potent inducers of IFNγ, whereas both cDCs and pDCs induced IL-10. Both cell types had variable effects on IL-17A production. Conclusion Collectively, the data show that direct allorecognition by donor lung pDCs and cDCs have differential effects on T cell proliferation and cytokine production. Depletion of both donor lung cDC and pDC could prevent the severity of acute rejection episodes.

  14. Biodegradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1.

    Science.gov (United States)

    Tallur, Preeti N; Mulla, Sikandar I; Megadi, Veena B; Talwar, Manjunatha P; Ninnekar, Harichandra Z

    2015-01-01

    Pyrethroid pesticide cypermethrin is a environmental pollutant because of its widespread use, toxicity and persistence. Biodegradation of such chemicals by microorganisms may provide an cost-effective method for their detoxification. We have investigated the degradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1 in various matrices such as, polyurethane foam (PUF), polyacrylamide, sodium alginate and agar. The optimum temperature and pH for the degradation of cypermethrin by immobilized cells of Micrococcus sp. were found to be 30 °C and 7.0, respectively. The rate of degradation of 10 and 20 mM of cypermethrin by freely suspended cells were compared with that of immobilized cells in batches and semi-continuous with shaken cultures. PUF-immobilized cells showed higher degradation of cypermethrin (10 mM and 20 mM) than freely suspended cells and cells immobilized in other matrices. The PUF-immobilized cells of Micrococcus sp. strain CPN 1 were retain their degradation capacity. Thus, they can be reused for more than 32 cycles, without losing their degradation capacity. Hence, the PUF-immobilized cells of Micrococcus sp. could potentially be used in the bioremediation of cypermethrin contaminated water.

  15. Enhanced degradation of 2-nitrotoluene by immobilized cells of Micrococcus sp. strain SMN-1.

    Science.gov (United States)

    Mulla, Sikandar I; Talwar, Manjunatha P; Bagewadi, Zabin K; Hoskeri, Robertcyril S; Ninnekar, Harichandra Z

    2013-02-01

    Nitrotoluenes are the toxic pollutants of the environment because of their large scale use in the production of explosives. Biodegradation of such chemicals by microorganisms may provide an effective method for their detoxification. We have studied the degradation of 2-nitrotoluene by cells of Micrococcus sp. strain SMN-1 immobilized in various matrices such as polyurethane foam (PUF), sodium alginate (SA), sodium alginate-polyvinyl alcohol (SA-PVA), agar and polyacrylamide. The rate of degradation of 15 and 30 mM 2-nitrotoluene by freely suspended cells and immobilized cells in batches and fed-batch with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 15 and 30 mM 2-nitrotoluene than freely suspended cells and the cells immobilized in SA-PVA, polyacrylamide, SA and agar. The PUF-immobilized cells could be reused more than 24 cycles without loosing their degradation capacity and showed more tolerance to pH and temperature changes than freely suspended cells. These results revealed the enhanced rate of degradation of 2-nitrotoluene by PUF-immobilized cells of Micrococcus sp. strain SMN-1. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Biodegradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1

    Directory of Open Access Journals (Sweden)

    Preeti N. Tallur

    2015-09-01

    Full Text Available Pyrethroid pesticide cypermethrin is a environmental pollutant because of its widespread use, toxicity and persistence. Biodegradation of such chemicals by microorganisms may provide an cost-effective method for their detoxification. We have investigated the degradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1 in various matrices such as, polyurethane foam (PUF, polyacrylamide, sodium alginate and agar. The optimum temperature and pH for the degradation of cypermethrin by immobilized cells of Micrococcus sp. were found to be 30 °C and 7.0, respectively. The rate of degradation of 10 and 20 mM of cypermethrin by freely suspended cells were compared with that of immobilized cells in batches and semi-continuous with shaken cultures. PUF-immobilized cells showed higher degradation of cypermethrin (10 mM and 20 mM than freely suspended cells and cells immobilized in other matrices. The PUF-immobilized cells of Micrococcus sp. strain CPN 1 were retain their degradation capacity. Thus, they can be reused for more than 32 cycles, without losing their degradation capacity. Hence, the PUF-immobilized cells of Micrococcus sp. could potentially be used in the bioremediation of cypermethrin contaminated water.

  17. Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos

    International Nuclear Information System (INIS)

    Fang, Zhen F.; Gai, Hui; Huang, You Z.; Li, Shan G.; Chen, Xue J.; Shi, Jian J.; Wu, Li; Liu, Ailian; Xu, Ping; Sheng, Hui Z.

    2006-01-01

    Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ES cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines

  18. A modified method of insulin producing cells' generation from bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Czubak, Paweł; Bojarska-Junak, Agnieszka; Tabarkiewicz, Jacek; Putowski, Lechosław

    2014-01-01

    Type 1 diabetes mellitus is a result of autoimmune destruction of pancreatic insulin producing β-cells and so far it can be cured only by insulin injection, by pancreas transplantation, or by pancreatic islet cells' transplantation. The methods are, however, imperfect and have a lot of disadvantages. Therefore new solutions are needed. The best one would be the use of differentiated mesenchymal stem cells (MSCs). In the present study, we investigated the potential of the bone marrow-derived MSCs line for in vitro differentiation into insulin producing cells (IPSs). We applied an 18-day protocol to differentiate MSCs. Differentiating cells formed cell clusters some of which resembled pancreatic islet-like cells. Using dithizone we confirmed the presence of insulin in the cells. What is more, the expression of proinsulin C-peptide in differentiated IPCs was analyzed by flow cytometry. For the first time, we investigated the influence of growth factors' concentration on IPCs differentiation efficiency. We have found that an increase in the concentration of growth factors up to 60 ng/mL of β-FGF/EGF and 30 ng/mL of activin A/β-cellulin increases the percentage of IPCs. Further increase of growth factors does not show any increase of the percentage of differentiated cells. Our findings suggest that the presented protocol can be adapted for differentiation of insulin producing cells from stem cells.

  19. Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion

    International Nuclear Information System (INIS)

    Robinson, Claire; Kolb, Andreas F.

    2009-01-01

    The transcriptional regulation of a gene is best analysed in the context of its normal chromatin surroundings. However, most somatic cells, in contrast to embryonic stem cells, are refractory to accurate modification by homologous recombination. We show here that it is possible to introduce precise genomic modifications in ES cells and to analyse the phenotypic consequences in differentiated cells by using a combination of gene targeting, site-specific recombination and somatic cell fusion. To provide a proof of principle, we have analysed the regulation of the casein gene locus in mammary gland cells derived from modified murine ES cells by somatic cell fusion. A β-galactosidase reporter gene was inserted in place of the β-casein gene and the modified ES cells, which do not express the reporter gene, were fused with the mouse mammary gland cell line HC11. The resulting cell clones expressed the β-galactosidase gene to a similar extent and with similar hormone responsiveness as the endogenous gene. However, a reporter gene under the control of a minimal β-casein promoter (encompassing the two consensus STAT5 binding sites which mediate the hormone response of the casein genes) was unable to replicate expression levels or hormone responsiveness of the endogenous gene when inserted into the same site of the casein locus. As expected, these results implicate sequences other than the STAT5 sites in the regulation of the β-casein gene

  20. Derivation of keratinocytes from chicken embryonic stem cells: Establishment and characterization of differentiated proliferative cell populations

    Directory of Open Access Journals (Sweden)

    Mathilde Couteaudier

    2015-03-01

    Full Text Available A common challenge in avian cell biology is the generation of differentiated cell-lines, especially in the keratinocyte lineage. Only a few avian cell-lines are available and very few of them show an interesting differentiation profile. During the last decade, mammalian embryonic stem cell-lines were shown to differentiate into almost all lineages, including keratinocytes. Although chicken embryonic stem cells had been obtained in the 1990s, few differentiation studies toward the ectodermal lineage were reported. Consequently, we explored the differentiation of chicken embryonic stem cells toward the keratinocyte lineage by using a combination of stromal induction, ascorbic acid, BMP4 and chicken serum. During the induction period, we observed a downregulation of pluripotency markers and an upregulation of epidermal markers. Three homogenous cell populations were derived, which were morphologically similar to chicken primary keratinocytes, displaying intracellular lipid droplets in almost every pavimentous cell. These cells could be serially passaged without alteration of their morphology and showed gene and protein expression profiles of epidermal markers similar to chicken primary keratinocytes. These cells represent an alternative to the isolation of chicken primary keratinocytes, being less cumbersome to handle and reducing the number of experimental animals used for the preparation of primary cells.

  1. A newly isolated probiotic Enterococcus faecalis strain from vagina microbiota enhances apoptosis of human cancer cells.

    Science.gov (United States)

    Nami, Y; Abdullah, N; Haghshenas, B; Radiah, D; Rosli, R; Yari Khosroushahi, A

    2014-08-01

    This study aimed to describe probiotic properties and bio-therapeutic effects of newly isolated Enterococcus faecalis from the human vaginal tract. The Enterococcus faecalis strain was originally isolated from the vaginal microbiota of Iranian women and was molecularly identified using 16SrDNA gene sequencing. Some biochemical methodologies were preliminarily used to characterize the probiotic potential of Ent. faecalis, including antibiotic susceptibility, antimicrobial activity, as well as acid and bile resistance. The bio-therapeutic effects of this strain's secreted metabolites on four human cancer cell lines (AGS, HeLa, MCF-7 and HT-29) and one normal cell line (HUVEC) were evaluated by cytotoxicity assay and apoptosis scrutiny. The characterization results demonstrated into the isolated bacteria strain revealed probiotic properties, such as antibiotic susceptibility, antimicrobial activity and resistance under conditions similar to those in the gastrointestinal tract. Results of bio-therapeutic efficacy assessments illustrated acceptable apoptotic effects on four human cancer cell lines and negligible side effects on assayed normal cell line. Our findings revealed that the apoptotic effect of secreted metabolites mainly depended on proteins secreted by Ent. faecalis on different cancer cells. These proteins can induce the apoptosis of cancer cells. The metabolites produced by this vaginal Ent. faecalis strain can be used as alternative pharmaceutical compounds with promising therapeutic indices because they are not cytotoxic to normal mammalian cells. Accordingly, the physicochemical, structural and functional properties of the secreted anticancer substances should be further investigated before using them as anticancer therapeutics. This study aim to screen total bacterial secreted metabolites as a wealthy source to find the new active compounds to introduce as anticancer therapeutics in the future. © 2014 The Society for Applied Microbiology.

  2. 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...

  3. Functional paralysis of GM-CSF-derived bone marrow cells productively infected with ectromelia virus.

    Directory of Open Access Journals (Sweden)

    Lidia Szulc-Dąbrowska

    Full Text Available Ectromelia virus (ECTV is an orthopoxvirus responsible for mousepox, a lethal disease of certain strains of mice that is similar to smallpox in humans, caused by variola virus (VARV. ECTV, similar to VARV, exhibits a narrow host range and has co-evolved with its natural host. Consequently, ECTV employs sophisticated and host-specific strategies to control the immune cells that are important for induction of antiviral immune response. In the present study we investigated the influence of ECTV infection on immune functions of murine GM-CSF-derived bone marrow cells (GM-BM, comprised of conventional dendritic cells (cDCs and macrophages. Our results showed for the first time that ECTV is able to replicate productively in GM-BM and severely impaired their innate and adaptive immune functions. Infected GM-BM exhibited dramatic changes in morphology and increased apoptosis during the late stages of infection. Moreover, GM-BM cells were unable to uptake and process antigen, reach full maturity and mount a proinflammatory response. Inhibition of cytokine/chemokine response may result from the alteration of nuclear translocation of NF-κB, IRF3 and IRF7 transcription factors and down-regulation of many genes involved in TLR, RLR, NLR and type I IFN signaling pathways. Consequently, GM-BM show inability to stimulate proliferation of purified allogeneic CD4+ T cells in a primary mixed leukocyte reaction (MLR. Taken together, our data clearly indicate that ECTV induces immunosuppressive mechanisms in GM-BM leading to their functional paralysis, thus compromising their ability to initiate downstream T-cell activation events.

  4. Induced pluripotent stem cell-derived neural cells survive and mature in the nonhuman primate brain.

    Science.gov (United States)

    Emborg, Marina E; Liu, Yan; Xi, Jiajie; Zhang, Xiaoqing; Yin, Yingnan; Lu, Jianfeng; Joers, Valerie; Swanson, Christine; Holden, James E; Zhang, Su-Chun

    2013-03-28

    The generation of induced pluripotent stem cells (iPSCs) opens up the possibility for personalized cell therapy. Here, we show that transplanted autologous rhesus monkey iPSC-derived neural progenitors survive for up to 6 months and differentiate into neurons, astrocytes, and myelinating oligodendrocytes in the brains of MPTP-induced hemiparkinsonian rhesus monkeys with a minimal presence of inflammatory cells and reactive glia. This finding represents a significant step toward personalized regenerative therapies. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  5. 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

  6. Indirubin inhibits cell proliferation, migration, invasion and angiogenesis in tumor-derived endothelial cells

    Directory of Open Access Journals (Sweden)

    Li Z

    2018-05-01

    Full Text Available Zhuohong Li, Chaofu Zhu, Baiping An, Yu Chen, Xiuyun He, Lin Qian, Lan Lan, Shijie Li Department of Oncology, The Affiliated Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China Purpose: Hepatocellular carcinoma is one of the most predominant malignancies with high fatality rate and its incidence is rising at an alarming rate because of its resistance to radio- and chemotherapy. Indirubin is the major active anti-tumor ingredient of a traditional Chinese herbal medicine. The present study aimed to analyze the effects of indirubin on cell proliferation, migration, invasion, and angiogenesis of tumor-derived endothelial cells (Td-EC. Methods: Td-EC were derived from human umbilical vein endothelial cells (HUVEC by treating HUVEC with the conditioned medium of human liver cancer cell line HepG2. Cell proliferation, migration, invasion, and angiogenesis were assessed by MTT, wound healing, in vitro cell invasion, and in vitro tube formation assay. Results: Td-EC were successfully obtained from HUVEC cultured with 50% culture supernatant from serum-starved HepG2 cells. Indirubin significantly inhibited Td-EC proliferation in a dose- and time-dependent manner. Indirubin also inhibited Td-EC migration, invasion, and angiogenesis. However, indirubin’s effects were weaker on HUVEC than Td-EC. Conclusion: Indirubin significantly inhibited Td-EC proliferation, migration, invasion, and angiogenesis. Keywords: indirubin, Td-EC, proliferation, migration, invasion, angiogenesis

  7. Role of bone marrow-derived stem cells, renal progenitor cells and stem cell factor in chronic renal allograft nephropathy

    OpenAIRE

    Hayam Abdel Meguid El Aggan; Mona Abdel Kader Salem; Nahla Mohamed Gamal Farahat; Ahmad Fathy El-Koraie; Ghaly Abd Al-Rahim Mohammed Kotb

    2013-01-01

    Introduction: Chronic allograft nephropathy (CAN) is a poorly understood clinico-pathological entity associated with chronic allograft loss due to immunologic and non-immunologic causes. It remains the leading cause of late allograft loss. Bone marrow derived stem cells are undifferentiated cells typically characterized by their capacity for self renewal, ability to give rise to multiple differentiated cellular population, including hematopoietic (HSCs) and mesenchymal stem cells (MSCs). Char...

  8. 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.

  9. 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.

  10. 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.

  11. Human Adipose Derived Stem Cells Induced Cell Apoptosis and S Phase Arrest in Bladder Tumor

    Directory of Open Access Journals (Sweden)

    Xi Yu

    2015-01-01

    Full Text Available The aim of this study was to determine the effect of human adipose derived stem cells (ADSCs on the viability and apoptosis of human bladder cancer cells. EJ and T24 cells were cocultured with ADSCs or cultured with conditioned medium of ADSCs (ADSC-CM, respectively. The cell counting and colony formation assay showed ADSCs inhibited the proliferation of EJ and T24 cells. Cell viability assessment revealed that the secretions of ADSCs, in the form of conditioned medium, were able to decrease cancer cell viability. Wound-healing assay suggested ADSC-CM suppressed migration of T24 and EJ cells. Moreover, the results of the flow cytometry indicated that ADSC-CM was capable of inducing apoptosis of T24 cells and inducing S phase cell cycle arrest. Western blot revealed ADSC-CM increased the expression of cleaved caspase-3 and cleaved PARP, indicating that ADSC-CM induced apoptosis in a caspase-dependent way. PTEN/PI3K/Akt pathway and Bcl-2 family proteins were involved in the mechanism of this reaction. Our study indicated that ADSCs may provide a promising and practicable manner for bladder tumor therapy.

  12. Adipose Tissue-Derived Mesenchymal Stem Cells as a New Host Cell in Latent Leishmaniasis

    Science.gov (United States)

    Allahverdiyev, Adil M.; Bagirova, Melahat; Elcicek, Serhat; Koc, Rabia Cakir; Baydar, Serap Yesilkir; Findikli, Necati; Oztel, Olga N.

    2011-01-01

    Some protozoan infections such as Toxoplasma, Cryptosporidium, and Plasmodium can be transmitted through stem cell transplantations. To our knowledge, so far, there is no study about transmission of Leishmania parasites in stem cell transplantation and interactions between parasites and stem cells in vitro. Therefore, the aim of this study was to investigate the interaction between different species of Leishmania parasites and adipose tissue-derived mesenchymal stem cells (ADMSCs). ADMSCs have been isolated, cultured, characterized, and infected with different species of Leishmania parasites (L. donovani, L. major, L. tropica, and L. infantum). Infectivity was examined by Giemsa staining, microculture, and polymerase chain reaction methods. As a result, infectivity of ADMSCs by Leishmania parasites has been determined for the first time in this study. According to our findings, it is very important that donors are screened for Leishmania parasites before stem cell transplantations in regions where leishmaniasis is endemic. PMID:21896818

  13. 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...

  14. Establishing porcine monocyte-derived macrophage and dendritic cell systems for studying the interaction with PRRSV-1

    Directory of Open Access Journals (Sweden)

    Helen eSingleton

    2016-06-01

    Full Text Available Monocyte-derived macrophages (MoMØ and monocyte-derived dendritic cells (MoDC are two model systems well established in human and rodent systems that can be used to study the interaction of pathogens with host cells. Porcine reproductive and respiratory syndrome virus (PRRSV is known to infect myeloid cells, such as macrophages (MØ and dendritic cells (DC. Therefore, this study aimed to establish systems for the differentiation and characterization of MoMØ and MoDC for subsequent infection with PRRSV-1. M-CSF differentiated monocyte-derived macrophages (MoMØ were stimulated with activators for classical (M1 or alternative (M2 activation. GM-CSF and IL-4 generated monocyte-derived dendritic cells (MoDC were activated with the well established maturation cocktail containing PAMPs and cytokines. In addition, MoMØ and MoDC were treated with dexamethasone and IL-10, which are known immuno-suppressive reagents. Cells were characterized by morphology, phenotype and function and porcine MØ subsets highlighted some divergence from described human counterparts, while MoDC, appeared more similar to mouse and human DCs. The infection with PRRSV-1 strain Lena demonstrated different replication kinetics between MoMØ and MoDC and within subsets of each cell type. While MoMØ susceptibility was significantly increased by dexamethasone and IL-10 with an accompanying increase in CD163/CD169 expression, MoDC supported only a minimal replication of PRRSV These findings underline the high variability in the susceptibility of porcine myeloid cells towards PRRSV-1 infection.

  15. Notch signaling is required for maintaining stem-cell features of neuroprogenitor cells derived from human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Chung Hyung-Min

    2009-08-01

    Full Text Available Abstract Background Studies have provided important findings about the roles of Notch signaling in neural development. Unfortunately, however, most of these studies have investigated the neural stem cells (NSCs of mice or other laboratory animals rather than humans, mainly owing to the difficulties associated with obtaining human brain samples. It prompted us to focus on neuroectodermal spheres (NESs which are derived from human embryonic stem cell (hESC and densely inhabited by NSCs. We here investigated the role of Notch signaling with the hESC-derived NESs. Results From hESCs, we derived NESs, the in-vitro version of brain-derived neurospheres. NES formation was confirmed by increased levels of various NSC marker genes and the emergence of rosette structures in which neuroprogenitors are known to reside. We found that Notch signaling, which maintains stem cell characteristics of in-vivo-derived neuroprogenitors, is active in these hESC-derived NESs, similar to their in-vivo counterpart. Expression levels of Notch signaling molecules such as NICD, DLLs, JAG1, HES1 and HES5 were increased in the NESs. Inhibition of the Notch signaling by a γ-secretase inhibitor reduced rosette structures, expression levels of NSC marker genes and proliferation potential in the NESs, and, if combined with withdrawal of growth factors, triggered differentiation toward neurons. Conclusion Our results indicate that the hESC-derived NESs, which share biochemical features with brain-derived neurospheres, maintain stem cell characteristics mainly through Notch signaling, which suggests that the hESC-derived NESs could be an in-vitro model for in-vivo neurogenesis.

  16. Blastema from rabbit ear contains progenitor cells comparable to marrow derived mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Mohamadreza Baghaban Eslaminejad

    2012-09-01

    Full Text Available Rabbits have the capacity to regenerate holes in their ears by forming a blastema, a tissue that is made up of a group of undifferentiated cells. The purpose of the present study was to isolate and characterize blastema progenitor cells and compare them with marrow mesenchymal stem cells (MSCs. Five New Zealand white male rabbits were used in the present study. A 2-mm hole was created in the animal ears. After 4 days, the blastema ring formed in the periphery of the hole was removed and cultivated. The cells were expanded through several subcultures and compared with the MSCs derived from the marrow of same animal in terms of in vitro differentiation capacity, growth kinetics and culture requirements for optimal proliferation. The primary cultures from both cells tended to be heterogeneous. Fibroblastic cells became progressively dominant with advancing passages. Similar to MSCs blastema passaged-3 cells succeeded to differentiate into bone, cartilage and adipose cell lineages. Even lineage specific genes tended to express in higher level in blastema cells compared to MSCs (p < 0.05. Moreover blastema cells appeared more proliferative; producing more colonies (p < 0.05. While blastema cells showed extensive proliferation in 15% fetal bovine serum (FBS, MSCs displayed higher expansion rate at 10% FBS. In conclusion, blastema from rabbit ear contains a population of fibroblastic cells much similar in characteristic to bone marrow mesenchymal stem cells. However, the two cells were different in the level of lineage-specific gene expression, the growth curve characteristics and the culture requirements.

  17. Characterization and comparison of osteoblasts derived from mouse embryonic stem cells and induced pluripotent stem cells.

    Science.gov (United States)

    Ma, Ming-San; Kannan, Vishnu; de Vries, Anneriek E; Czepiel, Marcin; Wesseling, Evelyn M; Balasubramaniyan, Veerakumar; Kuijer, Roel; Vissink, Arjan; Copray, Sjef C V M; Raghoebar, Gerry M

    2017-01-01

    New developments in stem cell biology offer alternatives for the reconstruction of critical-sized bone defects. One of these developments is the use of induced pluripotent stem (iPS) cells. These stem cells are similar to embryonic stem (ES) cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Proper characterization of iPS-derived osteoblasts is important for future development of safe clinical applications of these cells. For this reason, we differentiated mouse ES and iPS cells toward osteoblasts using osteogenic medium and compared their functionality. Immunocytochemical analysis showed significant expression of bone markers (osteocalcin and collagen type I) in osteoblasts differentiated from ES and iPS cells on days 7 and 30. An in vitro mineralization assay confirmed the functionality of osteogenically differentiated ES and iPS cells. Gene expression arrays focusing on osteogenic differentiation were performed in order to compare the gene expression pattern in both differentiated and undifferentiated ES cells and iPS cells. We observed a significant upregulation of osteogenesis-related genes such as Runx2, osteopontin, collagen type I, Tnfsf11, Csf1, and alkaline phosphatase upon osteogenic differentiation of the ES and iPS cells. We further validated the expression of key osteogenic genes Runx2, osteopontin, osteocalcin, collagen type I, and osterix in both differentiated and undifferentiated ES and iPS cells by means of quantified real-time polymerase chain reaction. We conclude that ES and iPS cells are similar in their osteogenic differentiation capacities, as well as in their gene expression patterns.

  18. Cell kinetics, DNA integrity, differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells.

    Science.gov (United States)

    Barretto, Letícia Siqueira de Sá; Lessio, Camila; Sawaki e Nakamura, Ahy Natally; Lo Turco, Edson Guimarães; da Silva, Camila Gonzaga; Zambon, João Paulo; Gozzo, Fábio César; Pilau, Eduardo Jorge; de Almeida, Fernando Gonçalves

    2014-10-01

    Human adipose tissue has been described as a potential alternative reservoir for stem cells. Although studies have been performed in rabbits using autologous adipose-derived stem cells (ADSC), these cells have not been well characterized. The primary objectives of this study were to demonstrate the presence of adipose-derived stem cells isolated from rabbit inguinal fat pads and to characterize them through osteogenic and adipogenic in vitro differentiation and lipid fingerprinting analysis. The secondary objective was to evaluate cell behavior through growth kinetics, cell viability, and DNA integrity. Rabbit ADSCs were isolated to determine the in vitro growth kinetics and cell viability. DNA integrity was assessed by an alkaline Comet assay in passages 0 and 5. The osteogenic differentiation was evaluated by Von Kossa, and Alizarin Red S staining and adipogenic differentiation were assessed by Oil Red O staining. Lipid fingerprinting analyses of control, adipogenic, and osteogenic differentiated cells were performed by MALDI-TOF/MS. We demonstrate that rabbit ADSC have a constant growth rate at the early passages, with increased DNA fragmentation at or after passage 5. Rabbit ADSC viability was similar in passages 2 and 5 (90.7% and 86.6%, respectively), but there was a tendency to decreased cellular growth rate after passage 3. The ADSC were characterized by the expression of surface markers such as CD29 (67.4%) and CD44 (89.4%), using CD 45 (0.77%) as a negative control. ADSC from rabbits were successfully isolated form the inguinal region. These cells were capable to differentiate into osteogenic and adipogenic tissue when they were placed in inductive media. After each passage, there was a trend towards decreased cell growth. On the other hand, DNA fragmentation increased at each passage. ADSC had a different lipid profile when placed in control, adipogenic, or osteogenic media.

  19. Evaluation of a dental pulp-derived cell sheet cultured on amniotic membrane substrate.

    Science.gov (United States)

    Honjo, Ken-ichi; Yamamoto, Toshiro; Adachi, Tetsuya; Amemiya, Takeshi; Mazda, Osam; Kanamura, Narisato; Kita, Masakazu

    2015-01-01

    Mesenchymal stem cells (MSC) are transplanted for periodontal tissue regeneration, and the periodontal ligament (PDL) is regenerated using a cultured cell sheet. This cultured cell sheet is prepared using PDL-derived cells, growth factors, and amniotic membrane (AM). Dental pulp (DP)-derived cells can be easily obtained from extracted wisdom teeth, proliferate rapidly, and are less susceptible to bacterial infection than PDL-derived cells. Thus, to prepare a novel cell sheet, DP-derived cells were cultured on AM as a culture substrate for immunohistochemical examination. Wisdom teeth extracted from three adults were cut along the cement-enamel border. DP tissue was collected, minced, and primarily cultured. After three or four passage cultures, DP-derived cells were cultured on AM, followed by hematoxylin-eosin (H-E) and immunofluorescence staining. DP-derived cells cultured on AM formed a layered structure. Cells positive for vimentin, Ki-67, ZO-1, desmoplakin, CD29, 44, 105 or 146, STRO-1, collagen IV or VII or laminin 5 or α5 chain were localized. DP-derived cells proliferated on AM, while retaining the properties of DP, which allowed the cultured cell sheet to be prepared. In addition, the cultured cell sheet contained MSC, which suggests its potential application in periodontal tissue regeneration.

  20. Design and construction of a strain gage compression load cell to measure rolling forces

    International Nuclear Information System (INIS)

    Schoeffer, L.; Borchardt, I.G.; Carvalho, L.F.A.

    1978-05-01

    A complete detailed mechanical desion of a strain gauge compression load cell is presented. This cell was specialy designed to measure rolling forces at conventional duo or trio industrial roughing stands. The stands, in general, have little space (height) to adjust to the cells. Moreover the contact stands surfaces are very rough. Do to this facts, load cells of elastic cilindrical geometries are not recommended for accuracies better than 8%. This work describes the complete design and the construction of a circular (membrane) steel plate load cell. A prototype of 300 KN (approximately 30t) capacity, with 2% accuracies and with a height of 6 cm was constructed and tested. The design proposed is a general one and permits the construction of small load cells to measure any compression load [pt

  1. Continent-Wide Estimates of Antarctic Strain Rates from Landsat 8-Derived Velocity Grids and Their Application to Ice Shelf Studies

    Science.gov (United States)

    Alley, K. E.; Scambos, T.; Anderson, R. S.; Rajaram, H.; Pope, A.; Haran, T.

    2017-12-01

    Strain rates are fundamental measures of ice flow used in a wide variety of glaciological applications including investigations of bed properties, calculations of basal mass balance on ice shelves, application to Glen's flow law, and many other studies. However, despite their extensive application, strain rates are calculated using widely varying methods and length scales, and the calculation details are often not specified. In this study, we compare the results of nominal and logarithmic strain-rate calculations based on a satellite-derived velocity field of the Antarctic ice sheet generated from Landsat 8 satellite data. Our comparison highlights the differences between the two commonly used approaches in the glaciological literature. We evaluate the errors introduced by each code and their impacts on the results. We also demonstrate the importance of choosing and specifying a length scale over which strain-rate calculations are made, which can have large local impacts on other derived quantities such as basal mass balance on ice shelves. We present strain-rate data products calculated using an approximate viscous length-scale with satellite observations of ice velocity for the Antarctic continent. Finally, we explore the applications of comprehensive strain-rate maps to future ice shelf studies, including investigations of ice fracture, calving patterns, and stability analyses.

  2. Chitosan derived co-spheroids of neural stem cells and mesenchymal stem cells for neural regeneration.

    Science.gov (United States)

    Han, Hao-Wei; Hsu, Shan-Hui

    2017-10-01

    Chitosan has been considered as candidate biomaterials for neural applications. The effective treatment of neurodegeneration or injury to the central nervous system (CNS) is still in lack nowadays. Adult neural stem cells (NSCs) represents a promising cell source to treat the CNS diseases but they are limited in number. Here, we developed the core-shell spheroids of NSCs (shell) and mesenchymal stem cells (MSCs, core) by co-culturing cells on the chitosan surface. The NSCs in chitosan derived co-spheroids displayed a higher survival rate than those in NSC homo-spheroids. The direct interaction of NSCs with MSCs in the co-spheroids increased the Notch activity and differentiation tendency of NSCs. Meanwhile, the differentiation potential of MSCs in chitosan derived co-spheroids was significantly enhanced toward neural lineages. Furthermore, NSC homo-spheroids and NSC/MSC co-spheroids derived on chitosan were evaluated for their in vivo efficacy by the embryonic and adult zebrafish brain injury models. The locomotion activity of zebrafish receiving chitosan derived NSC homo-spheroids or NSC/MSC co-spheroids was partially rescued in both models. Meanwhile, the higher survival rate was observed in the group of adult zebrafish implanted with chitosan derived NSC/MSC co-spheroids as compared to NSC homo-spheroids. These evidences indicate that chitosan may provide an extracellular matrix-like environment to drive the interaction and the morphological assembly between NSCs and MSCs and promote their neural differentiation capacities, which can be used for neural regeneration. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Functional Differences in Engineered Myocardium from Embryonic Stem Cell-Derived versus Neonatal Cardiomyocytes

    NARCIS (Netherlands)

    Feinberg, Adam W.; Ripplinger, Crystal M.; van der Meer, Peter; Sheehy, Sean P.; Domian, Ibrahim; Chien, Kenneth R.; Parker, Kevin Kit

    2013-01-01

    Stem cell-derived cardiomyocytes represent unique tools for cell-and tissue-based regenerative therapies, drug discovery and safety, and studies of fundamental heart-failure mechanisms. However, the degree to which stem cell-derived cardiomyocytes compare to mature cardiomyocytes is often debated.

  4. Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds—Present and Future Strain Construction Strategies

    Directory of Open Access Journals (Sweden)

    Nils J. H. Averesch

    2018-03-01

    Full Text Available The aromatic nature of shikimate pathway intermediates gives rise to a wealth of potential bio-replacements for commonly fossil fuel-derived aromatics, as well as naturally produced secondary metabolites. Through metabolic engineering, the abundance of certain intermediates may be increased, while draining flux from other branches off the pathway. Often targets for genetic engineering lie beyond the shikimate pathway, altering flux deep in central metabolism. This has been extensively used to develop microbial production systems for a variety of compounds valuable in chemical industry, including aromatic and non-aromatic acids like muconic acid, para-hydroxybenzoic acid, and para-coumaric acid, as well as aminobenzoic acids and aromatic α-amino acids. Further, many natural products and secondary metabolites that are valuable in food- and pharma-industry are formed outgoing from shikimate pathway intermediates. (Reconstruction of such routes has been shown by de novo production of resveratrol, reticuline, opioids, and vanillin. In this review, strain construction strategies are compared across organisms and put into perspective with requirements by industry for commercial viability. Focus is put on enhancing flux to and through shikimate pathway, and engineering strategies are assessed in order to provide a guideline for future optimizations.

  5. Anti-inflammatory effects of secondary metabolites isolated from the marine-derived fungal strain Penicillium sp. SF-5629.

    Science.gov (United States)

    Ngan, Nguyen Thi Thanh; Quang, Tran Hong; Kim, Kwan-Woo; Kim, Hye Jin; Sohn, Jae Hak; Kang, Dae Gill; Lee, Ho Sub; Kim, Youn-Chul; Oh, Hyuncheol

    2017-03-01

    After the chemical investigation of the ethyl acetate extract of the marine-derived fungal strain Penicillium sp. SF-5629, the isolation and structural elucidation of eight secondary metabolites, including (3R,4S)-6,8-dihydroxy-3,4,7-trimethylisocoumarin (1), (3S,4S)-sclerotinin A (2), penicitrinone A (3), citrinin H1 (4), emodin (5), ω-hydroxyemodin (6), 8-hydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate (7), and 3,8-dihydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate (8) were carried out. Evaluation of the anti-inflammatory activity of these metabolites showed that 4 inhibited nitric oxide and prostaglandin E2 production in lipopolysaccharide-stimulated BV2 microglia, with IC 50 values of 8.1 ± 1.9 and 8.0 ± 2.8 μM, respectively. The inhibitory function of 4 was confirmed based on decreases in inducible nitric oxide synthesis and cyclooxygenase-2 gene expression. In addition, 4 was found to suppress the phosphorylation of inhibitor kappa B-α, interrupt the nuclear translocation of nuclear factor kappa B, and decrease the activation of p38 mitogen-activated protein kinase.

  6. Evaluation of left ventricular function by tissue Doppler and speckle-derived strain rate echocardiography after percutaneous ductus closure.

    Science.gov (United States)

    Amoogzar, Hamid; Shakiba, Ali Mohammad; Derakhshan, Dorna; Ajami, Gholamhossein; Cheriki, Sirous; Borzouee, Mohammad; Edraki, Mohammad Reza; Mehdizadegan, Nima

    2015-01-01

    The aim of this study was to evaluate the left ventricular systolic and diastolic function before and after transcatheter percutaneous patent ductus arteriosus (PDA) closure. 21 children (age >6 months old) diagnosed with hemodynamically significant PDA underwent percutaneous PDA closure. Conventional, Doppler and tissue Doppler imaging and speckled-derived strain rate echocardiography were done at pre-closure, 1 day (early) and 1 month (late) post-closure. Mean age of the patients (female/male: 1.3) was 17.54 ± 24.7 months with the mean PDA diameter of 3.6 ± 0.8 mm. Systolic measures (ejection fraction, shortening fraction) reduced significantly early after PDA closure (P closure status. Early and late diastolic flow velocities of mitral (E M and A M) reduced considerably in early and late post-closure time (P closure. After 1 month, E'M increased considerably. (P = 0.01) but E'M/A'M had an insignificant rise (P > 0.05). E M/E'M ratio did not change in early post-closure but it had a considerable reduction in the subsequent month compared with the pre- and early post-closure (P closure (P closure causes a significant decrease in left ventricular performance early after PDA closure which recovers completely within 1 month. Also PDA size can affect post-closure left ventricular function.

  7. 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.

  8. Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.

    Science.gov (United States)

    Witek, Rafal P; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S; Cheong, Yeiwon; Fearing, Caitlin M; Agboola, Kolade M; Chen, Wei; Diehl, Anna Mae

    2009-01-01

    Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). MF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Platelet-derived growth factor-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.

  9. Induced Pluripotent Stem Cell-Derived Red Blood Cells and Platelet Concentrates: From Bench to Bedside.

    Science.gov (United States)

    Focosi, Daniele; Amabile, Giovanni

    2017-12-27

    Red blood cells and platelets are anucleate blood components indispensable for oxygen delivery and hemostasis, respectively. Derivation of these blood elements from induced pluripotent stem (iPS) cells has the potential to develop blood donor-independent and genetic manipulation-prone products to complement or replace current transfusion banking, also minimizing the risk of alloimmunization. While the production of erythrocytes from iPS cells has challenges to overcome, such as differentiation into adult-type phenotype that functions properly after transfusion, platelet products are qualitatively and quantitatively approaching a clinically-applicable level owing to advances in expandable megakaryocyte (MK) lines, platelet-producing bioreactors, and novel reagents. Guidelines that assure the quality of iPS cells-derived blood products for clinical application represent a novel challenge for regulatory agencies. Considering the minimal risk of tumorigenicity and the expected significant demand of such products, ex vivo production of iPS-derived blood components can pave the way for iPS translation into the clinic.

  10. Cytoprotective dibenzoylmethane derivatives protect cells from oxidative stress-induced necrotic cell death.

    Science.gov (United States)

    Hegedűs, Csaba; Lakatos, Petra; Kiss-Szikszai, Attila; Patonay, Tamás; Gergely, Szabolcs; Gregus, Andrea; Bai, Péter; Haskó, György; Szabó, Éva; Virág, László

    2013-06-01

    Screening of a small in-house library of 1863 compounds identified 29 compounds that protected Jurkat cells from hydrogen peroxide-induced cytotoxicity. From the cytoprotective compounds eleven proved to possess antioxidant activity (ABTS radical scavenger effect) and two were found to inhibit poly(ADP-ribosyl)ation (PARylation), a cytotoxic pathway operating in severely injured cells. Four cytoprotective dibenzoylmethane (DBM) derivatives were investigated in more detail as they did not scavenge hydrogen peroxide nor did they inhibit PARylation. These compounds protected cells from necrotic cell death while caspase activation, a parameter of apoptotic cell death was not affected. Hydrogen peroxide activated extracellular signal regulated kinase (ERK1/2) and p38 MAP kinases but not c-Jun N-terminal kinase (JNK). The cytoprotective DBMs suppressed the activation of Erk1/2 but not that of p38. Cytoprotection was confirmed in another cell type (A549 lung epithelial cells), indicating that the cytoprotective effect is not cell type specific. In conclusion we identified DBM analogs as a novel class of cytoprotective compounds inhibiting ERK1/2 kinase and protecting from necrotic cell death by a mechanism independent of poly(ADP-ribose) polymerase inhibition. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Adipose derived stem cells in radiotherapy injury: a new frontier

    Directory of Open Access Journals (Sweden)

    Lipi eShukla

    2015-01-01

    Full Text Available Radiotherapy is increasingly used to treat numerous human malignancies. In addition to the beneficial anti-cancer effects, there are a series of undesirable effects on normal host tissues surrounding the target tumour. Whilst the early effects of radiotherapy (desquamation, erythema and hair loss typically resolve, the chronic effects persist as unpredictable and often troublesome sequelae of cancer treatment, long after oncological treatment has been completed. Plastic surgeons are often called upon to treat the problems subsequently arising in irradiated tissues, such as recurrent infection, impaired healing, fibrosis, contracture and/or lymphoedema. Recently, it was anecdotally noted - then validated in more robust animal and human studies - that fat grafting can ameliorate some of these chronic tissue effects. Despite the widespread usage of fat grafting, the mechanism of its action remains poorly understood. This review provides an overview of the current understanding of (i mechanisms of chronic radiation injury and its clinical manifestations; (ii biological properties of fat grafts and their key constituent, Adipose-Derived Stem Cells (ADSCs; (iii the role of ADSCs in radiotherapy-induced soft-tissue injury.

  12. Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses.

    Science.gov (United States)

    Furusawa, Tadashi; Ohkoshi, Katsuhiro; Kimura, Koji; Matsuyama, Shuichi; Akagi, Satoshi; Kaneda, Masahiro; Ikeda, Mitsumi; Hosoe, Misa; Kizaki, Keiichiro; Tokunaga, Tomoyuki

    2013-08-01

    Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages.

  13. Proteomic Profiling of Mesenchymal Stem Cell Responses to Mechanical Strain and TGF-B1

    Energy Technology Data Exchange (ETDEWEB)

    Kurpinski, Kyle; Chu, Julia; Wang, Daojing; Li, Song

    2009-10-12

    Mesenchymal stem cells (MSCs) are a potential source of smooth muscle cells (SMCs) for constructing tissue-engineered vascular grafts. However, the details of how specific combinations of vascular microenvironmental factors regulate MSCs are not well understood. Previous studies have suggested that both mechanical stimulation with uniaxial cyclic strain and chemical stimulation with transforming growth factor {beta}1 (TGF-{beta}1) can induce smooth muscle markers in MSCs. In this study, we investigated the combined effects of uniaxial cyclic strain and TGF-{beta}1 stimulation on MSCs. By using a proteomic analysis, we found differential regulation of several proteins and genes, such as the up-regulation of TGF-{beta}1-induced protein ig-h3 (BGH3) protein levels by TGF-{beta}1 and up-regulation of calponin 3 protein level by cyclic strain. At the gene expression level, BGH3 was induced by TGF-{beta}1, but calponin 3 was not significantly regulated by mechanical strain or TGF-{beta}1, which was in contrast to the synergistic up-regulation of calponin 1 gene expression by cyclic strain and TGF-{beta}1. Further experiments with cycloheximide treatment suggested that the up-regulation of calponin 3 by cyclic strain was at post-transcriptional level. The results in this study suggest that both mechanical stimulation and TGF-{beta}1 signaling play unique and important roles in the regulation of MSCs at both transcriptional and post-transcriptional levels, and that a precise combination of microenvironmental cues may promote MSC differentiation.

  14. A novel podoplanin-GFPCre mouse strain for gene deletion in lymphatic endothelial cells.

    Science.gov (United States)

    Gil, Hyea Jin; Ma, Wanshu; Oliver, Guillermo

    2018-04-01

    The lymphatic vascular system is a one-direction network of thin-walled capillaries and larger vessels covered by a continuous layer of endothelial cells responsible for maintaining fluid homeostasis. Some of the main functions of the lymphatic vasculature are to drain fluid from the extracellular spaces and return it back to the blood circulation, lipid absorption from the intestinal tract, and transport of immune cells to lymphoid organs. A number of genes controlling the development of the mammalian lymphatic vasculature have been identified in the last few years, and their functional roles started to be characterized using gene inactivation approaches in mice. Unfortunately, only few mouse Cre strains relatively specific for lymphatic endothelial cells (LECs) are currently available. In this article, we report the generation of a novel Podoplanin (Pdpn) GFPCre transgenic mouse strain using its 5' regulatory region. Pdpn encodes a transmembrane mucin-type O-glycoprotein that is expressed on the surface of embryonic and postnatal LECs, in addition to few other cell types. Our detailed characterization of this novel strain indicates that it will be a valuable additional genetic tool for the analysis of gene function in LECs. © 2018 Wiley Periodicals, Inc.

  15. Optimization of in vitro cell labeling methods for human umbilical cord-derived mesenchymal stem cells.

    Science.gov (United States)

    Tao, R; Sun, T-J; Han, Y-Q; Xu, G; Liu, J; Han, Y-F

    2014-01-01

    Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) are a novel source of seed cells for cell therapy and tissue engineering. However, in vitro labeling methods for hUCMSCs need to be optimized for better detection of transplanted cells. To identify the most stable and efficient method for labeling hUCMSCs in vitro. hUCMSCs were isolated using a modified enzymatic digestion procedure and cultured. hUCMSCs of passage three (P3) were then labeled with BrdU, PKH26, or lentivirus-GFP and passaged further. Cells from the first labeled passage (LP1), the fourth labeled passage (LP4) and later passages were observed using a fluorescence microscope. The differentiation potential of LP4 cells was assessed by induction with adipogenic and osteogenic medium. Flow cytometry was used to measure the percentage of labeled cells and the percentage of apoptotic or dead cells. The labeling efficiencies of the three hUCMSC-labeling methods were compared in vitro. BrdU, PKH26, and lentivirus-GFP all labeled LP1 cells with high intensity and clarity. However, the BrdU labeling of the LP4 cells was vague and not localized to the cell nuclei; LP9 cells were not detected under a fluorescence microscope. There was also a significant decrease in the fluorescence intensity of PKH26-labeled LP4 cells, and LP11 cells were not detected under a fluorescence microscope. However, the fluorescence of LP4 cells labeled with lentivirus-GFP remained strong, and cells labeled with lentivirus-GFP were detected up to LP14 under a fluorescence microscope. Statistical analyses indicated that percentages of LP1 cells labeled with PKH26 and lentivirus-GFP were significantly higher than that of cells labeled with BrdU (p 0.05) was observed between the death rates of labeled and unlabeled cells. Lentivirus-GFP is a valid method for long-term in vitro labeling, and it may be used as a long-term hUCMSC tracker following transplantation in vivo.

  16. Characterization of cell cultures derived from Lutzomyia spinicrassa (Diptera: Psychodidae) and their susceptibility to infection with Leishmania (Viannia) braziliensis.

    Science.gov (United States)

    Zapata Lesmes, Angela Cristina; Cárdenas Castro, Estrella; Bello, Felio

    2005-12-01

    The sand fly Lutzomyia spinicrassa (Morales, Osorno-Mesa, Osorno & de Hoyos, 1969) is a vector of Leishmania (Viannia) braziliensis, an etiological agent of cutaneous leishmaniasis in Colombia. The present article describes, for the first time, the morphological, karyotypical, and isozymatic characteristics of cell cultures derived from L. Spinicrassa embryonic tissues as well as the interaction of L. Braziliensis with these cell cultures. L. Spinicrassa embryonated eggs and neonate larvae were taken for tissue explants. These were seeded in Grace, L-15, Grace/L-15, MM/VP12, and MK/VP12 culture media. The pH range in these media was 6.7 to 6.9 and the cultures were incubated at 28 degrees C. The MHOM/CO/86/CL250 strain of L. Braziliensis was used for experimental infection of cell cultures of L. Spinicrassa. Cell growth was achieved in L-15 medium and a confluent monolayer was obtained 180 days after the embryonated eggs were explanted. The cell morphology of the primary cell cultures was initially heterogeneous, but in the confluent monolayer of these cell cultures and in the subcultures the predominant cell types were later fibroblast-like and epithelial-like. Cultured cells were predominantly diploid (2n=8); however, significant percentages of aneuploids were also recorded. The cell culture isozyme patterns of L. Spinicrassa coincided with pupae samples from the same species. Promastigote forms of L. Braziliensis could invade cells and transform into amastigote-like forms inside them. The characteristics of cell cultures derived from L. Spinicrassa embryonic tissues were determined. These cultures emerge as a new model to study the life-cycle of L. Braziliensis.

  17. Brain immune cell composition and functional outcome after cerebral ischemia: Comparison of two mouse strains

    Directory of Open Access Journals (Sweden)

    Hyun Ah eKim

    2014-11-01

    Full Text Available Inflammatory cells may contribute to secondary brain injury following cerebral ischemia. The C57Bl/6 mouse strain is known to exhibit a T helper 1-prone, pro-inflammatory type response to injury, whereas the FVB strain is relatively T helper 2-prone, or anti-inflammatory, in its immune response. We tested whether stroke outcome is more severe in C57Bl/6 than FVB mice. Male mice of each strain underwent sham surgery or 1 h occlusion of the middle cerebral artery followed by 23 h of reperfusion. Despite no difference in infarct size, C57Bl/6 mice displayed markedly greater functional deficits than FVB mice after stroke, as assessed by neurological scoring and hanging wire test. Total numbers of CD45+ leukocytes tended to be larger in the brains of C57Bl/6 than FVB mice after stroke, but there were marked differences in leukocyte composition between the two mouse strains. The inflammatory response in C57Bl/6 mice primarily involved T and B lymphocytes, whereas neutrophils, monocytes and macrophages were more prominent in FVB mice. Our data are consistent with the concept that functional outcome after stroke is dependent on the immune cell composition which develops following ischemic brain injury.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-08-15

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

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

    International Nuclear Information System (INIS)

    Horiuchi, Rie; Akimoto, Takayuki; Hong, Zhang; Ushida, Takashi

    2012-01-01

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

  20. Synovium-derived stem cells: a tissue-specific stem cell for cartilage engineering and regeneration.

    Science.gov (United States)

    Jones, Brendan A; Pei, Ming

    2012-08-01

    Articular cartilage is difficult to heal once injury or disease occurs. Autologous chondrocyte transplantation is a biological treatment with good prognosis, but donor site morbidity and limited cell source are disadvantages. Currently, mesenchymal stem cells (MSCs) are a promising approach for cartilage regeneration. Despite there being various sources, the best candidate for cartilage regeneration is the one with the greatest chondrogenic potential and the least hypertrophic differentiation. These properties are able to insure that the regenerated tissue is hyaline cartilage of high quality. This review article will summarize relevant literature to justify synovium-derived stem cells (SDSCs) as a tissue-specific stem cell for chondrogenesis by comparing synovium and cartilage with respect to anatomical location and functional structure, comparing the growth characterization and chondrogenic capacity of SDSCs and MSCs, evaluating the application of SDSCs in regenerative medicine and diseases, and discussing potential future directions.

  1. Activated T cells sustain myeloid-derived suppressor cell-mediated immune suppression

    Science.gov (United States)

    Damuzzo, Vera; Francescato, Samuela; Pozzuoli, Assunta; Berizzi, Antonio; Mocellin, Simone; Rossi, Carlo Riccardo; Bronte, Vincenzo; Mandruzzato, Susanna

    2016-01-01

    The expansion of myeloid derived suppressor cells (MDSCs), a suppressive population able to hamper the immune response against cancer, correlates with tumor progression and overall survival in several cancer types. We have previously shown that MDSCs can be induced in vitro from precursors present in the bone marrow and observed that these cells are able to actively proliferate in the presence of activated T cells, whose activation level is critical to drive the suppressive activity of MDSCs. Here we investigated at molecular level the mechanisms involved in the interplay between MDSCs and activated T cells. We found that activated T cells secrete IL-10 following interaction with MDSCs which, in turn, activates STAT3 phosphorylation on MDSCs then leading to B7-H1 expression. We also demonstrated that B7-H1+ MDSCs are responsible for immune suppression through a mechanism involving ARG-1 and IDO expression. Finally, we show that the expression of ligands B7-H1 and MHC class II both on in vitro-induced MDSCs and on MDSCs in the tumor microenvironment of cancer patients is paralleled by an increased expression of their respective receptors PD-1 and LAG-3 on T cells, two inhibitory molecules associated with T cell dysfunction. These findings highlight key molecules and interactions responsible for the extensive cross-talk between MDSCs and activated T cells that are at the basis of immune suppression. PMID:26700461

  2. 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.

  3. Bone marrow-derived fibrocytes promote stem cell-like properties of lung cancer cells.

    Science.gov (United States)

    Saijo, Atsuro; Goto, Hisatsugu; Nakano, Mayuri; Mitsuhashi, Atsushi; Aono, Yoshinori; Hanibuchi, Masaki; Ogawa, Hirohisa; Uehara, Hisanori; Kondo, Kazuya; Nishioka, Yasuhiko

    2018-05-01

    Cancer stem cells (CSCs) represent a minor population that have clonal tumor initiation and self-renewal capacity and are responsible for tumor initiation, metastasis, and therapeutic resistance. CSCs reside in niches, which are composed of diverse types of stromal cells and extracellular matrix components. These stromal cells regulate CSC-like properties by providing secreted factors or by physical contact. Fibrocytes are differentiated from bone marrow-derived CD14 + monocytes and have features of both macrophages and fibroblasts. Accumulating evidence has suggested that stromal fibrocytes might promote cancer progression. However, the role of fibrocytes in the CSC niches has not been revealed. We herein report that human fibrocytes enhanced the CSC-like properties of lung cancer cells through secreted factors, including osteopontin, CC-chemokine ligand 18, and plasminogen activator inhibitor-1. The PIK3K/AKT pathway was critical for fibrocytes to mediate the CSC-like functions of lung cancer cells. In human lung cancer specimens, the number of tumor-infiltrated fibrocytes was correlated with high expression of CSC-associated protein in cancer cells. These results suggest that fibrocytes may be a novel cell population that regulates the CSC-like properties of lung cancer cells in the CSC niches. Copyright © 2018. Published by Elsevier B.V.

  4. Cell-derived micro-environment helps dental pulp stem cells promote dental pulp regeneration.

    Science.gov (United States)

    Zhang, Xuexin; Li, Hui; Sun, Jingjing; Luo, Xiangyou; Yang, Hefeng; Xie, Li; Yang, Bo; Guo, Weihua; Tian, Weidong

    2017-10-01

    The function of the dental pulp is closely connected to the extracellular matrix (ECM) structure, and ECM has received significant attention due to its biological functions for regulating cells. As such, the interaction between the ECM niche and cells is worth exploring for potential clinical uses. In this study, dental pulp stem cell (DPSC)-derived ECM (DPM) was prepared through cell culture and decellularization to function as the cell niche, and changes in DPSC behaviour and histological analysis of dental pulp tissue regeneration were evaluated following the DPM culture. DPM promoted the replication of DPSCs and exhibited retention of their mineralization. Then, the DPM-based culture strategy under odontogenic culture medium was further investigated, and the mineralization-related markers showed that DPSCs were regulated towards odontogenic differentiation. Dental pulp-like tissue with well-arranged ECM was harvested after a 2-month subcutaneous implantation in nude mice with DPM application. Additionally, DPSCs cultured on the plastic culture surface showed the up-regulation of mineralization makers in vitro, but there was a disorder in matrix formation and mineralization when the cells were cultured in vivo. DPM-based cultivation could serve as a cell niche and modulate DPSC behaviour, and this method also provided an alternative to harvest tissue-specific ECM and provided a strategy for ECM-cell interaction. © 2017 John Wiley & Sons Ltd.

  5. Isolation and expansion of adipose-derived stem cells for tissue engineering

    DEFF Research Database (Denmark)

    Fink, Trine; Rasmussen, Jeppe Grøndahl; Lund, Pia

    2011-01-01

    For treatment of cardiac failure with bone marrow-derived mesenchymal stem cells, several clinical trials are ongoing. However, more attention is gathering on the use of adipose tissue-derived stem cells (ASCs). This paper describes the optimization of isolation and propagation of ASCs for subseq......For treatment of cardiac failure with bone marrow-derived mesenchymal stem cells, several clinical trials are ongoing. However, more attention is gathering on the use of adipose tissue-derived stem cells (ASCs). This paper describes the optimization of isolation and propagation of ASCs...

  6. Appearance of differentiated cells derived from polar body nuclei in the silkworm, Bombyx mori

    Directory of Open Access Journals (Sweden)

    Hiroki eSakai

    2013-09-01

    Full Text Available AbstractIn Bombyx mori, polar body nuclei are observed until 9h after egg lying, however, the fate of polar body nuclei remains unclear. To examine the fate of polar body nuclei, we employed a mutation of serosal cell pigmentation, pink-eyed white egg (pe. The heterozygous pe/+pe females produced black serosal cells in white eggs, while pe/pe females did not produce black serosal cells in white eggs. These results suggest that the appearance of black serosal cells in white eggs depends on the genotype (pe/ +pe of the mother. Because the polar body nuclei had +pe genes in the white eggs laid by a pe/ +pe female, polar body nuclei participate in development and differentiate into functional cell (serosal cells. Analyses of serosal cells pigmentation indicated that approximately 30% of the eggs contained polar-body-nucleus-derived cells. These results demonstrate that polar-body-nucleus-derived cells appeared at a high frequency under natural conditions. Approximately 80% of polar-body-nucleus-derived cells appeared near the anterior pole and the dorsal side, which is opposite to where embryogenesis occurs. The number of cells derived from the polar body nuclei was very low. Approximately 26 % of these eggs contained only one black serosal cell. PCR-based analysis revealed that the polar-body-nucleus-derived cells disappeared in late embryonic stages (stage 25. Overall, polar-body-nuclei-derived cells were unlikely to contribute to embryos.

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

    Directory of Open Access Journals (Sweden)

    Kathryn L McCabe

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

  8. Induced pluripotent stem cells (iPSC)-derived retinal cells in disease modeling and regenerative medicine.

    Science.gov (United States)

    Rathod, Reena; Surendran, Harshini; Battu, Rajani; Desai, Jogin; Pal, Rajarshi

    2018-02-12

    Retinal degenerative disorders are a leading cause of the inherited, irreversible and incurable vision loss. While various rodent model systems have provided crucial information in this direction, lack of disease-relevant tissue availability and species-specific differences have proven to be a major roadblock. Human induced pluripotent stem cells (iPSC) have opened up a whole new avenue of possibilities not just in understanding the disease mechanism but also potential therapeutic approaches towards a cure. In this review, we have summarized recent advances in the methods of deriving retinal cell types from iPSCs which can serve as a renewable source of disease-relevant cell population for basic as well as translational studies. We also provide an overview of the ongoing efforts towards developing a suitable in vitro model for modeling retinal degenerative diseases. This basic understanding in turn has contributed to advances in translational goals such as drug screening and cell-replacement therapies. Furthermore we discuss gene editing approaches for autologous repair of genetic disorders and allogeneic transplantation of stem cell-based retinal derivatives for degenerative disorders with an ultimate goal to restore vision. It is pertinent to note however, that these exciting new developments throw up several challenges that need to be overcome before their full clinical potential can be realized. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells

    International Nuclear Information System (INIS)

    Yamagishi, Naoko; Teshima-Kondo, Shigetada; Masuda, Kiyoshi; Nishida, Kensei; Kuwano, Yuki; Dang, Duyen T; Dang, Long H; Nikawa, Takeshi; Rokutan, Kazuhito

    2013-01-01

    Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. Our findings suggest that chronic inhibition of tumor cell-derived VEGF

  10. Generation of Dopamine-Secreting Cells from Human Adipose Tissue-Derived Stem Cells In Vitro.

    Science.gov (United States)

    Soheilifar, Mohammad Hasan; Javeri, Arash; Amini, Hossein; Taha, Masoumeh Fakhr

    2018-03-12

    Several studies have demonstrated the differentiation of human adipose tissue-derived stem cells (hADSCs) to neuronal and glial phenotypes, but directing the fate of these cells toward dopaminergic neurons has not been frequently reported. The aim of this study was to investigate dopaminergic specification of hADSCs in vitro. ADSCs were isolated from subcutaneous abdominal adipose tissue and were characterized. For dopaminergic differentiation, a cocktail of sonic hedgehog, fibroblast growth factor 8, basic fibroblast growth factor, and brain-derived neurotrophic factor were used under a low serum condition. As the control group, the ADSCs were cultured under the same low serum condition without the dopaminergic cocktail. At the end of differentiation period, the cells expressed neuron-specific markers, NES, NSE, and NEFL, and dopaminergic markers, EN1, NURR1, PITX3, VMAT2, TH, and GIRK2 genes. TH, NURR1, and EN1 mRNAs were upregulated in the dopaminergic group compared with the control group. NEFL and TH proteins were also expressed in the differentiated cells. A total of 27.9% of the cells differentiated in dopaminergic induction medium showed positive staining for TH protein. Based on reversed-phase high-performance liquid chromatography analysis, the differentiated cells released a significant amount of dopamine in response to KCl-induced depolarization. In conclusion, results of this study indicate that hADSCs can be induced by a growth factor cocktail to produce dopamine secreting cells with possible applications for future cell replacement therapy of Parkinson's disease.

  11. 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.

  12. Comparison of neurosphere-like cell clusters derived from dental follicle precursor cells and retinal Müller cells

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Petersen, Jørgen; Felthaus, Oliver

    2011-01-01

    Unrelated cells such as dental follicle precursor cells (DFPCs) and retinal Müller cells (MCs) make spheres after cultivation in serum-replacement medium (SRM). Until today, the relation and molecular processes of sphere formation from different cell types remain undescribed. Thus in this study we...... compared proteomes of spheres derived from MCs and DFPCs. 73% of 676 identified proteins were similar expressed in both cell types and many of them are expressed in the brain (55%). Moreover proteins are overrepresented that are associated with pathways for neural diseases such as Huntington disease...... or Alzheimer disease. Interestingly up-regulated proteins in DFPCs are involved in the biosynthesis of glycosphingolipids. These lipids are components of gangliosides such as GD3, which is a novel neural stem cell marker. In conclusion spheres from different types of cells have highly similar proteomes...

  13. Sox17-Mediated XEN Cell Conversion Identifies Dynamic Networks Controlling Cell-Fate Decisions in Embryo-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Angela C.H. McDonald

    2014-10-01

    Full Text Available Little is known about the gene regulatory networks (GRNs distinguishing extraembryonic endoderm (ExEn stem (XEN cells from those that maintain the extensively characterized embryonic stem cell (ESC. An intriguing network candidate is Sox17, an essential transcription factor for XEN derivation and self-renewal. Here, we show that forced Sox17 expression drives ESCs toward ExEn, generating XEN cells that contribute to ExEn when placed back into early mouse embryos. Transient Sox17 expression is sufficient to drive this fate change during which time cells transit through distinct intermediate states prior to the generation of functional XEN-like cells. To orchestrate this conversion process, Sox17 acts in autoregulatory and feedforward network motifs, regulating dynamic GRNs directing cell fate. Sox17-mediated XEN conversion helps to explain the regulation of cell-fate changes and reveals GRNs regulating lineage decisions in the mouse embryo.

  14. Amniotic fluid-derived mesenchymal stem cells as a novel ...

    African Journals Online (AJOL)

    CLEMENTINA

    2012-06-28

    Jun 28, 2012 ... stem cells (AFMSCs) have many advantages over other stem cells: avoiding much ethical controversy ... showed that induced pluripotent stem cells (iPSCs) have ... disadvantages of ESCs, BM-MSCs and iPSCs have.

  15. Epigenetic and phenotypic profile of fibroblasts derived from induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Kyle J Hewitt

    2011-02-01

    Full Text Available Human induced pluripotent stem (hiPS cells offer a novel source of patient-specific cells for regenerative medicine. However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES cells remain unclear. We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK and their hES-derived counterparts (EDK showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts. Array-based methylation analysis was performed for EDK, iPDK and their parental hES and iPS cell lines, and hierarchical clustering revealed that EDK and iPDK had closely-related methylation profiles. DNA methylation analysis of promoter regions associated with extracellular matrix (ECM-production (COL1A1 by iPS- and hESC-derived fibroblasts and fibroblast lineage commitment (PDGFRβ, revealed promoter demethylation linked to their expression, and patterns of transcription and methylation of genes related to the functional properties of mature stromal cells were seen in both hiPS- and hES-derived fibroblasts. iPDK cells also showed functional properties analogous to those of hES-derived and mature fibroblasts, as seen by their capacity to direct the morphogenesis of engineered human skin equivalents. Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.

  16. 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.

  17. Effect of feeding tannin degrading bacterial culture (Streptococcus gallolyticus strain TDGB 406) on nutrient utilization, urinary purine derivatives and growth performance of goats fed on Quercus semicarpifolia leaves.

    Science.gov (United States)

    Kumar, K; Chaudhary, L C; Agarwal, N; Kamra, D N

    2014-10-01

    To study the effect of supplementation of tannin degrading bacterial culture (Streptococcus gallolyticus strain TDGB 406) on growth performance, nutrient utilization and urinary purine derivatives of goats fed on oak (Quercus semicarpifolia) leaves. For growth study, eighteen billy goats (4 month old, average body weight 9.50 ± 1.50 kg) were distributed into three groups of six animals each. The animals of group 1 served as control while animals of groups 2 (T1) and 3 (T2) were given (@ 5 ml/kg live weight) autoclaved and live culture of isolate TDGB 406 (10(6) cells/ml) respectively. The animals were fed measured quantity of dry oak leaves as the main roughage source and ad libitum maize hay along with fixed quantity of concentrate mixture. The feeding of live culture of isolate TDGB 406 (probiotic) did not affect dry matter intake and digestibility of nutrients except that of dry matter and crude protein, which was higher in T2 group as compared to control. All the animals were in positive nitrogen balance. There was no significant effect of feeding isolate TDGB 406 on urinary purine derivatives (microbial protein production) in goats. The body weight gain and average live weight gain was significantly higher (p = 0.071) in T2 group as compared to control. Feed conversion efficiency was also better in the goats fed on live culture of TDGB 406 (T2). The feeding of tannin degrading bacterial isolate TDGB 406 as probiotic resulted in improved growth performance and feed conversion ratio in goats fed on oak leaves as one of the main roughage source. Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH.

  18. Lethal and mutagenic effects of radiation and alkylating agents on two strains of mouse L5178Y cells

    International Nuclear Information System (INIS)

    Evans, H.H.; Horng, M.; Beer, J.Z.

    1986-01-01

    The two closely related strains of L5178Y (LY) mouse lymphoma cells, LY-R and LY-S, have been shown to differ in their sensitivity to UV and ionizing radiation. In the present work, the lethal and mutagenic effects of ethyl methanesulfonate (EMS), methyl nitrosourea (MNU) and UV radiation (254 nm) were compared in the two strains. Mutability at the Na + /K + -ATPase locus as well as the HGPRT locus was determined. The authors found strain LY-S to be more resistant than strain LY-R to the lethal effects of UV radiation. In contrast, strain LY-S was more sensitive to the cytotoxic effects of the two alkylating agents. In spite of these differences in sensitivity, the authors found strain LY-S to be less mutable than strain LY-R by all 3 agents at the HGPRT locus. At the Na + /K + -ATPase locus, strain LY-S was also less mutable than strain LY-R by equal concentrations of EMS and UV radiation and by equitoxic concentrations of MNU. However, the difference between the strains was much more pronounced at the HGPRT locus than at the Na + /K + -ATPase locus. The authors have suggested that the interaction of unrepaired lesions in strain LY-S tends to cause an excess of deletions and multilocus effects, which in turn result in a locus-dependent decrease in the recovery of viable LY-S mutant cells. (Auth.)

  19. Skeletal Muscle-derived Hematopoietic Stem Cells: Muscular Dystrophy Therapy by Bone Marrow Transplantation

    OpenAIRE

    Asakura, Atsushi

    2012-01-01

    For postnatal growth and regeneration of skeletal muscle, satellite cells, a self-renewing pool of muscle stem cells, give rise to daughter myogenic precursor cells that contribute to the formation of new muscle fibers. In addition to this key myogenic cell class, adult skeletal muscle also contains hematopoietic stem cell and progenitor cell populations which can be purified as a side population (SP) fraction or as a hematopoietic marker CD45-positive cell population. These muscle-derived he...

  20. Tissue Source and Cell Expansion Condition Influence Phenotypic Changes of Adipose-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Lauren H. Mangum

    2017-01-01

    Full Text Available Stem cells derived from the subcutaneous adipose tissue of debrided burned skin represent an appealing source of adipose-derived stem cells (ASCs for regenerative medicine. Traditional tissue culture uses fetal bovine serum (FBS, which complicates utilization of ASCs in human medicine. Human platelet lysate (hPL is one potential xeno-free, alternative supplement for use in ASC culture. In this study, adipogenic and osteogenic differentiation in media supplemented with 10% FBS or 10% hPL was compared in human ASCs derived from abdominoplasty (HAP or from adipose associated with debrided burned skin (BH. Most (95–99% cells cultured in FBS were stained positive for CD73, CD90, CD105, and CD142. FBS supplementation was associated with increased triglyceride content and expression of adipogenic genes. Culture in hPL significantly decreased surface staining of CD105 by 31% and 48% and CD142 by 27% and 35% in HAP and BH, respectively (p<0.05. Culture of BH-ASCs in hPL also increased expression of markers of osteogenesis and increased ALP activity. These data indicate that application of ASCs for wound healing may be influenced by ASC source as well as culture conditions used to expand them. As such, these factors must be taken into consideration before ASCs are used for regenerative purposes.

  1. Tissue Source and Cell Expansion Condition Influence Phenotypic Changes of Adipose-Derived Stem Cells

    Science.gov (United States)

    Mangum, Lauren H.; Stone, Randolph; Wrice, Nicole L.; Larson, David A.; Florell, Kyle F.; Christy, Barbara A.; Herzig, Maryanne C.; Cap, Andrew P.

    2017-01-01

    Stem cells derived from the subcutaneous adipose tissue of debrided burned skin represent an appealing source of adipose-derived stem cells (ASCs) for regenerative medicine. Traditional tissue culture uses fetal bovine serum (FBS), which complicates utilization of ASCs in human medicine. Human platelet lysate (hPL) is one potential xeno-free, alternative supplement for use in ASC culture. In this study, adipogenic and osteogenic differentiation in media supplemented with 10% FBS or 10% hPL was compared in human ASCs derived from abdominoplasty (HAP) or from adipose associated with debrided burned skin (BH). Most (95–99%) cells cultured in FBS were stained positive for CD73, CD90, CD105, and CD142. FBS supplementation was associated with increased triglyceride content and expression of adipogenic genes. Culture in hPL significantly decreased surface staining of CD105 by 31% and 48% and CD142 by 27% and 35% in HAP and BH, respectively (p < 0.05). Culture of BH-ASCs in hPL also increased expression of markers of osteogenesis and increased ALP activity. These data indicate that application of ASCs for wound healing may be influenced by ASC source as well as culture conditions used to expand them. As such, these factors must be taken into consideration before ASCs are used for regenerative purposes. PMID:29138638

  2. Characterization of the myeloid-derived suppressor cell subset regulated by NK cells in malignant lymphoma.

    Science.gov (United States)

    Sato, Yusuke; Shimizu, Kanako; Shinga, Jun; Hidaka, Michihiro; Kawano, Fumio; Kakimi, Kazuhiro; Yamasaki, Satoru; Asakura, Miki; Fujii, Shin-Ichiro

    2015-03-01

    Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population with the ability to suppress immune responses and are currently classified into three distinct MDSC subsets: monocytic, granulocytic and non-monocytic, and non-granulocytic MDSCs. Although NK cells provide an important first-line defense against newly transformed cancer cells, it is unknown whether NK cells can regulate MDSC populations in the context of cancer. In this study, we initially found that the frequency of MDSCs in non-Hodgkin lymphoma (NHL) patients was increased and inversely correlated with that of NK cells, but not that of T cells. To investigate the regulation of MDSC subsets by NK cells, we used an EL4 murine lymphoma model and found the non-monocytic and non-granulocytic MDSC subset, i.e., Gr1 + CD11b + Ly6G med Ly6C med MDSC, is increased after NK cell depletion. The MDSC population that expresses MHC class II, CD80, CD124, and CCR2 is regulated mainly by CD27 + CD11b + NK cells. In addition, this MDSC subset produces some immunosuppressive cytokines, including IL-10 but not nitric oxide (NO) or arginase. We also examined two subsets of MDSCs (CD14 + HLA-DR - and CD14 - HLA-DR - MDSC) in NHL patients and found that higher IL-10-producing CD14 + HLA-DR - MDSC subset can be seen in lymphoma patients with reduced NK cell frequency in peripheral blood. Our analyses of MDSCs in this study may enable a better understanding of how MDSCs manipulate the tumor microenvironment and are regulated by NK cells in patients with lymphoma.

  3. A Bilayer Construct Controls Adipose-Derived Stem Cell Differentiation into Endothelial Cells and Pericytes without Growth Factor Stimulation

    Science.gov (United States)

    2011-01-01

    A Bilayer Construct Controls Adipose-Derived Stem Cell Differentiation into Endothelial Cells and Pericytes Without Growth Factor Stimulation...Ph.D.3 This work describes the differentiation of adipose-derived mesenchymal stem cells (ASC) in a composite hy- drogel for use as a vascularized...tissue from a single population of ASC. This work underscores the importance of the extracellular matrix in controlling stem cell phenotype. It is our

  4. Assessment of Myocardial Contractile Function Using Global and Segmental Circumferential Strain following Intracoronary Stem Cell Infusion after Myocardial Infarction: MRI Feature Tracking Feasibility Study

    International Nuclear Information System (INIS)

    Bhatti, Sabha; Al-Khalidi, Hussein; Hor, Kan; Hakeem, Abdul; Taylor, Michael; Quyyumi, Arshed A.; Oshinski, John; Pecora, Andrew L.; Kereiakes, Dean; Chung, Eugene; Pedrizzetti, Gianni; Miszalski-Jamka, Tomasz; Mazur, Wojciech

    2012-01-01

    Background. Magnetic resonance imaging (MRI) strain analysis is a sensitive method to assess myocardial function. Our objective was to define the feasibility of MRI circumferential strain (ε cc ) analysis in assessing subtle changes in myocardial function following stem cell therapy. Methods and Results. Patients in the Amorcyte Phase I trial were randomly assigned to treatment with either autologous bone-marrow-derived stem cells infused into the infarct-related artery 5 to 11 days following primary PCI or control. MRI studies were obtained at baseline, 3, and 6 months. ε cc was measured in the short axis views at the base, mid and apical slices of the left ventricle (LV) for each patient (13 treatments and 10 controls). Mid-anterior LV ε cc improved between baseline −18.5 ± 8.6 and 3 months −22.6 ± 7.0, P = 0.03. There were no significant changes in ε cc at 3 months and 6 months compared to baseline for other segments. There was excellent intraobserver and interobserver agreement for basal and mid circumferential strain. Conclusion. MRI segmental strain analysis is feasible in assessment of regional myocardial function following cell therapy with excellent intra- and inter-observer variability's. Using this method, a modest interval change in segmental ε cc was detected in treatment group

  5. The Pericytic Phenotype of Adipose Tissue-Derived Stromal Cells Is Promoted by NOTCH2

    NARCIS (Netherlands)

    Terlizzi, Vincenzo; Kolibabka, Matthias; Burgess, Janette Kay; Hammes, Hans Peter; Harmsen, Martin Conrad

    Long-term diabetes leads to macrovascular and microvascular complication. In diabetic retinopathy (DR), persistent hyperglycemia causes permanent loss of retinal pericytes and aberrant proliferation of microvascular endothelial cells (ECs). Adipose tissue-derived stromal cells (ASCs) may serve to

  6. Adipose tissue-derived stem cells in oral mucosa tissue engineering ...

    African Journals Online (AJOL)

    Jane

    2011-10-10

    Oct 10, 2011 ... stem cells (ADSCs) may play an important role in this field. In this research ..... Adipose tissue is derived from embryonic mesodermal precursors and .... Clonogenic multipotent stem cells in human adipose tissue differentiate ...

  7. Targeting of cancer neoantigens with donor-derived T cell receptor repertoires

    DEFF Research Database (Denmark)

    Strønen, Erlend; Toebes, Mireille; Kelderman, Sander

    2016-01-01

    Accumulating evidence suggests that clinically efficacious cancer immunotherapies are driven by T cell reactivity against DNA mutation-derived neoantigens. However, among the large number of predicted neoantigens, only a minority is recognized by autologous patient T cells, and strategies...

  8. Application of stem cell derived neuronal cells to evaluate neurotoxic chemotherapy

    Directory of Open Access Journals (Sweden)

    Claudia Wing

    2017-07-01

    Full Text Available The generation of induced pluripotent stem cells (iPSCs and differentiation to cells composing major organs has opened up the possibility for a new model system to study adverse toxicities associated with chemotherapy. Therefore, we used human iPSC-derived neurons to study peripheral neuropathy, one of the most common adverse effects of chemotherapy and cause for dose reduction. To determine the utility of these neurons in investigating the effects of neurotoxic chemotherapy, we measured morphological differences in neurite outgrowth, cell viability as determined by ATP levels and apoptosis through measures of caspase 3/7 activation following treatment with clinically relevant concentrations of platinating agents (cisplatin, oxaliplatin and carboplatin, taxanes (paclitaxel, docetaxel and nab-paclitaxel, a targeted proteasome inhibitor (bortezomib, an antiangiogenic compound (thalidomide, and 5-fluorouracil, a chemotherapeutic that does not cause neuropathy. We demonstrate differential sensitivity of neurons to mechanistically distinct classes of chemotherapeutics. We also show a dose-dependent reduction of electrical activity as measured by mean firing rate of the neurons following treatment with paclitaxel. We compared neurite outgrowth and cell viability of iPSC-derived cortical (iCell® Neurons and peripheral (Peri.4U neurons to cisplatin, paclitaxel and vincristine. Goshajinkigan, a Japanese herbal neuroprotectant medicine, was protective against paclitaxel-induced neurotoxicity but not oxaliplatin as measured by morphological phenotypes. Thus, we have demonstrated the utility of human iPSC-derived neurons as a useful model to distinguish drug class differences and for studies of a potential neuroprotectant for the prevention of chemotherapy-induced peripheral neuropathy.

  9. HEK293 cell culture media study towards bioprocess optimization: Animal derived component free and animal derived component containing platforms.

    Science.gov (United States)

    Liste-Calleja, Leticia; Lecina, Martí; Cairó, Jordi Joan

    2014-04-01

    The increasing demand for biopharmaceuticals produced in mammalian cells has lead industries to enhance bioprocess volumetric productivity through different strategies. Among those strategies, cell culture media development is of major interest. In the present work, several commercially available culture media for Human Embryonic Kidney cells (HEK293) were evaluated in terms of maximal specific growth rate and maximal viable cell concentration supported. The main objective was to provide different cell culture platforms which are suitable for a wide range of applications depending on the type and the final use of the product obtained. Performing simple media supplementations with and without animal derived components, an enhancement of cell concentration from 2 × 10(6) cell/mL to 17 × 10(6) cell/mL was achieved in batch mode operation. Additionally, the media were evaluated for adenovirus production as a specific application case of HEK293 cells. None of the supplements interfered significantly with the adenovirus infection although some differences were encountered in viral productivity. To the best of our knowledge, the high cell density achieved in the work presented has never been reported before in HEK293 batch cell cultures and thus, our results are greatly promising to further study cell culture strategies in bioreactor towards bioprocess optimization. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  10. The Phenotypic Fate of Bone Marrow-Derived Stem Cells in Acute Kidney Injury

    Directory of Open Access Journals (Sweden)

    Guowei Feng

    2013-11-01

    Full Text Available Background: Despite increasing attention on the role of bone marrow derived stem cells in repair or rejuvenation of tissues and organs, cellular mechanisms of such cell-based therapy remain poorly understood. Methods: We reconstituted hematopoiesis in recipient C57BL/6J mice by transplanting syngeneic GFP+ bone marrow (BM cells. Subsequently, the recipients received subcutaneous injection of granulocyte-colony stimulating factor (G-CSF and were subjected to acute renal ischemic injury. Flow cytometry and immunostaining were performed at various time points to assess engraftment and phenotype of BM derived stem cells. Results: Administration of G-CSF increased the release of BM derived stem cells into circulation and enhanced the ensuing recruitment of BM derived stem cells into injured kidney. During the second month post injury, migrated BM derived stem cells lost hematopoietic phenotype (CD45 but maintained the expression of other markers (Sca-1, CD133 and CD44, suggesting their potential of transdifferentiation into renal stem cells. Moreover, G-CSF treatment enhanced the phenotypic conversion. Conclusion: Our work depicted a time-course dependent transition of phenotypic characteristics of BM derived stem cells, demonstrated the existence of BM derived stem cells in damaged kidney and revealed the effects of G-CSF on cell transdifferentiation.

  11. Circulating angiogenic cells can be derived from cryopreserved peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Tanja Sofrenovic

    Full Text Available Cell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreserving peripheral blood mononuclear cells (PBMCs for the subsequent in vitro generation of their derived therapeutic population, circulating angiogenic cells (CACs.PBMCs were isolated from healthy human donors. Freshly isolated cells were either analyzed immediately or cryopreserved in media containing 6% plasma serum and 5% dimethyl sulfoxide. PBMCs were thawed after being frozen for 1 (early thaw or 28 (late thaw days and analyzed, or cultured for 4 days to generate CACs. Analysis of the cells consisted of flow cytometry for viability and phenotype, as well as functional assays for their adhesion and migration potential, cytokine secretion, and in vivo angiogenic potential.The viability of PBMCs and CACs as well as their adhesion and migration properties did not differ greatly after cryopreservation. Phenotypic changes did occur in PBMCs and to a lesser extent in CACs after freezing; however the potent CD34(+VEGFR2(+CD133(+ population remained unaffected. The derived CACs, while exhibiting changes in inflammatory cytokine secretion, showed no changes in the secretion of important regenerative and chemotactic cytokines, nor in their ability to restore perfusion in ischemic muscle.Overall, it appears that changes do occur in cryopreserved PBMCs and their generated CACs; however, the CD34(+VEGFR2(+CD133(+ progenitor population, the secretion of pro-vasculogenic factors, and the in vivo angiogenic potential of CACs remain unaffected by cryopreservation.

  12. Preclinical Studies of Induced Pluripotent Stem Cell-Derived Astrocyte Transplantation in ALS

    Science.gov (United States)

    2012-10-01

    Pluripotent Stem Cell -Derived Astrocyte Transplantation in ALS PRINCIPAL INVESTIGATOR: Nicholas J. Maragakis, M.D...Pluripotent Stem Cell -Derived Astrocyte Transplantation in ALS 5b. GRANT NUMBER W81XWH-10-1-0520 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d...into astrocytes following transplantation. 15. SUBJECT TERMS Stem Cells , iPS cells, astrocytes, familial ALS 16. SECURITY CLASSIFICATION OF

  13. Distinct Effects of Adipose-Derived Stem Cells and Adipocytes on Normal and Cancer Cell Hierarchy.

    Science.gov (United States)

    Anjanappa, Manjushree; Burnett, Riesa; Zieger, Michael A; Merfeld-Clauss, Stephanie; Wooden, William; March, Keith; Tholpady, Sunil; Nakshatri, Harikrishna

    2016-07-01

    Adipose-derived stem cells (ASC) have received considerable attention in oncology because of the known direct link between obesity and cancer as well as the use of ASCs in reconstructive surgery after tumor ablation. Previous studies have documented how cancer cells commandeer ASCs to support their survival by altering extracellular matrix composition and stiffness, migration, and metastasis. This study focused on delineating the effects of ASCs and adipocytes on the self-renewal of stem/progenitor cells and hierarchy of breast epithelial cells. The immortalized breast epithelial cell line MCF10A, ductal carcinoma in situ (DCIS) cell lines MCF10DCIS.com and SUM225, and MCF10A-overexpressing SRC oncogene were examined using a mammosphere assay and flow cytometry for the effects of ASCs on their self-renewal and stem-luminal progenitor-differentiated cell surface marker profiles. Interestingly, ASCs promoted the self-renewal of all cell types except SUM225. ASC coculture or treatment with ASC conditioned media altered the number of CD49f(high)/EpCAM(low) basal/stem-like and CD49f(medium)/EpCAM(medium) luminal progenitor cells. Among multiple factors secreted by ASCs, IFNγ and hepatocyte growth factor (HGF) displayed unique actions on epithelial cell hierarchy. IFNγ increased stem/progenitor-like cells while simultaneously reducing the size of mammospheres, whereas HGF increased the size of mammospheres with an accompanying increase in luminal progenitor cells. ASCs expressed higher levels of HGF, whereas adipocytes expressed higher levels of IFNγ. As luminal progenitor cells are believed to be prone for transformation, IFNγ and HGF expression status of ASCs may influence susceptibility for developing breast cancer as well as on outcomes of autologous fat transplantation on residual/dormant tumor cells. This study suggests that the ratio of ASCs to adipocytes influences cancer cell hierarchy, which may impact incidence and progression. Mol Cancer Res; 14(7); 660

  14. [Ability of Staphylococcus cohnii strains to adhere to epithelial cells and solid surfaces in the hospital environment].

    Science.gov (United States)

    Waldon, Edyta; Szewczyk, Eligia M

    2002-01-01

    Presented study describes abilities of staphylococci to adhere to exfoliated cheek and uroepithelial epithelium cells and to various surfaces such as plastics, glass and steel. The subject of the study were strains of Staphylococcus cohnii ssp. cohnii and Staphylococcus cohnii ssp. urealyticus isolated from Intensive Care Unit of Pediatric Hospital. Staphylococcus cohnii ssp.cohnii adhered in great number to epithelial cells. However, the adhesion differed by individual strains. We did not find relationship between slime production and adherence to epithelial cell. Most of investigated strains adhered closely to surfaces--especially of plastics and glass. This phenomenon was stronger in the presence of culture medium and phosphate buffer.

  15. Environmental and nutritional factors that affect growth and metabolism of the pneumococcal serotype 2 strain D39 and its nonencapsulated derivative strain R6.

    Directory of Open Access Journals (Sweden)

    Sandra M Carvalho

    Full Text Available Links between carbohydrate metabolism and virulence in Streptococcus pneumoniae have been recurrently established. To investigate these links further we developed a chemically defined medium (CDM and standardized growth conditions that allowed for high growth yields of the related pneumococcal strains D39 and R6. The utilization of the defined medium enabled the evaluation of different environmental and nutritional factors on growth and fermentation patterns under controlled conditions of pH, temperature and gas atmosphere. The same growth conditions impacted differently on the nonencapsulated R6, and its encapsulated progenitor D39. A semi-aerobic atmosphere and a raised concentration of uracil, a fundamental component of the D39 capsule, improved considerably D39 growth rate and biomass. In contrast, in strain R6, the growth rate was enhanced by strictly anaerobic conditions and uracil had no effect on biomass. In the presence of oxygen, the difference in the growth rates was mainly attributed to a lower activity of pyruvate oxidase in strain D39. Our data indicate an intricate connection between capsule production in strain D39 and uracil availability. In this study, we have also successfully applied the in vivo NMR technique to study sugar metabolism in S. pneumoniae R6. Glucose consumption, end-products formation and evolution of intracellular metabolite pools were monitored online by (13C-NMR. Additionally, the pools of NTP and inorganic phosphate were followed by (31P-NMR after a pulse of glucose. These results represent the first metabolic profiling data obtained non-invasively for S. pneumoniae, and pave the way to a better understanding of regulation of central metabolism.

  16. IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Daniela Lehnen

    2017-10-01

    Full Text Available Human pluripotent stem cell (hPSC-derived mesencephalic dopaminergic (mesDA neurons can relieve motor deficits in animal models of Parkinson's disease (PD. Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP+ mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA neurons in vitro. Intrastriatal transplantation of IAP+ cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP+ mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies.

  17. IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells.

    Science.gov (United States)

    Lehnen, Daniela; Barral, Serena; Cardoso, Tiago; Grealish, Shane; Heuer, Andreas; Smiyakin, Andrej; Kirkeby, Agnete; Kollet, Jutta; Cremer, Harold; Parmar, Malin; Bosio, Andreas; Knöbel, Sebastian

    2017-10-10

    Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP + mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP + cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP + mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies. Copyright © 2017 Miltenyi Biotec GmbH. Published by Elsevier Inc. All rights reserved.

  18. From Human Mesenchymal Stem Cells to Insulin-Producing Cells: Comparison between Bone Marrow- and Adipose Tissue-Derived Cells.

    Science.gov (United States)

    Gabr, Mahmoud M; Zakaria, Mahmoud M; Refaie, Ayman F; Abdel-Rahman, Engy A; Reda, Asmaa M; Ali, Sameh S; Khater, Sherry M; Ashamallah, Sylvia A; Ismail, Amani M; Ismail, Hossam El-Din A; El-Badri, Nagwa; Ghoneim, Mohamed A

    2017-01-01

    The aim of this study is to compare human bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs), for their differentiation potentials to form insulin-producing cells. BM-MSCs were obtained during elective orthotopic surgery and AT-MSCs from fatty aspirates during elective cosmetics procedures. Following their expansion, cells were characterized by phenotyping, trilineage differentiation ability, and basal gene expression of pluripotency genes and for their metabolic characteristics. Cells were differentiated according to a Trichostatin-A based protocol. The differentiated cells were evaluated by immunocytochemistry staining for insulin and c-peptide. In addition the expression of relevant pancreatic endocrine genes was determined. The release of insulin and c-peptide in response to a glucose challenge was also quantitated. There were some differences in basal gene expression and metabolic characteristics. After differentiation the proportion of the resulting insulin-producing cells (IPCs), was comparable among both cell sources. Again, there were no differences neither in the levels of gene expression nor in the amounts of insulin and c-peptide release as a function of glucose challenge. The properties, availability, and abundance of AT-MSCs render them well-suited for applications in regenerative medicine. Conclusion . BM-MSCs and AT-MSCs are comparable regarding their differential potential to form IPCs. The availability and properties of AT-MSCs render them well-suited for applications in regenerative medicine.

  19. From Human Mesenchymal Stem Cells to Insulin-Producing Cells: Comparison between Bone Marrow- and Adipose Tissue-Derived Cells

    Directory of Open Access Journals (Sweden)

    Mahmoud M. Gabr

    2017-01-01

    Full Text Available The aim of this study is to compare human bone marrow-derived mesenchymal stem cells (BM-MSCs and adipose tissue-derived mesenchymal stem cells (AT-MSCs, for their differentiation potentials to form insulin-producing cells. BM-MSCs were obtained during elective orthotopic surgery and AT-MSCs from fatty aspirates during elective cosmetics procedures. Following their expansion, cells were characterized by phenotyping, trilineage differentiation ability, and basal gene expression of pluripotency genes and for their metabolic characteristics. Cells were differentiated according to a Trichostatin-A based protocol. The differentiated cells were evaluated by immunocytochemistry staining for insulin and c-peptide. In addition the expression of relevant pancreatic endocrine genes was determined. The release of insulin and c-peptide in response to a glucose challenge was also quantitated. There were some differences in basal gene expression and metabolic characteristics. After differentiation the proportion of the resulting insulin-producing cells (IPCs, was comparable among both cell sources. Again, there were no differences neither in the levels of gene expression nor in the amounts of insulin and c-peptide release as a function of glucose challenge. The properties, availability, and abundance of AT-MSCs render them well-suited for applications in regenerative medicine. Conclusion. BM-MSCs and AT-MSCs are comparable regarding their differential potential to form IPCs. The availability and properties of AT-MSCs render them well-suited for applications in regenerative medicine.

  20. Plasma Rich in Growth Factors Induces Cell Proliferation, Migration, Differentiation, and Cell Survival of Adipose-Derived Stem Cells.

    Science.gov (United States)

    Mellado-López, Maravillas; Griffeth, Richard J; Meseguer-Ripolles, Jose; Cugat, Ramón; García, Montserrat; Moreno-Manzano, Victoria

    2017-01-01

    Adipose-derived stem cells (ASCs) are a promising therapeutic alternative for tissue repair in various clinical applications. However, restrictive cell survival, differential tissue integration, and undirected cell differentiation after transplantation in a hostile microenvironment are complications that require refinement. Plasma rich in growth factors (PRGF) from platelet-rich plasma favors human and canine ASC survival, proliferation, and delaying human ASC senescence and autophagocytosis in comparison with serum-containing cultures. In addition, canine and human-derived ASCs efficiently differentiate into osteocytes, adipocytes, or chondrocytes in the presence of PRGF. PRGF treatment induces phosphorylation of AKT preventing ASC death induced by lethal concentrations of hydrogen peroxide. Indeed, AKT inhibition abolished the PRGF apoptosis prevention in ASC exposed to 100  μ M of hydrogen peroxide. Here, we show that canine ASCs respond to PRGF stimulus similarly to the human cells regarding cell survival and differentiation postulating the use of dogs as a suitable translational model. Overall, PRGF would be employed as a serum substitute for mesenchymal stem cell amplification to improve cell differentiation and as a preconditioning agent to prevent oxidative cell death.

  1. Plasma Rich in Growth Factors Induces Cell Proliferation, Migration, Differentiation, and Cell Survival of Adipose-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Maravillas Mellado-López

    2017-01-01

    Full Text Available Adipose-derived stem cells (ASCs are a promising therapeutic alternative for tissue repair in various clinical applications. However, restrictive cell survival, differential tissue integration, and undirected cell differentiation after transplantation in a hostile microenvironment are complications that require refinement. Plasma rich in growth factors (PRGF from platelet-rich plasma favors human and canine ASC survival, proliferation, and delaying human ASC senescence and autophagocytosis in comparison with serum-containing cultures. In addition, canine and human-derived ASCs efficiently differentiate into osteocytes, adipocytes, or chondrocytes in the presence of PRGF. PRGF treatment induces phosphorylation of AKT preventing ASC death induced by lethal concentrations of hydrogen peroxide. Indeed, AKT inhibition abolished the PRGF apoptosis prevention in ASC exposed to 100 μM of hydrogen peroxide. Here, we show that canine ASCs respond to PRGF stimulus similarly to the human cells regarding cell survival and differentiation postulating the use of dogs as a suitable translational model. Overall, PRGF would be employed as a serum substitute for mesenchymal stem cell amplification to improve cell differentiation and as a preconditioning agent to prevent oxidative cell death.

  2. 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.

  3. A quantitative risk assessment of exposure to adventitious agents in a cell culture-derived subunit influenza vaccine.

    Science.gov (United States)

    Gregersen, Jens-Peter

    2008-06-19

    A risk-assessment model has demonstrated the ability of a new cell culture-based vaccine manufacturing process to reduce the level of any adventitious agent to a million-fold below infectious levels. The cell culture-derived subunit influenza vaccine (OPTAFLU), Novartis Vaccines and Diagnostics) is produced using Madin-Darby canine kidney (MDCK) cells to propagate seasonal viral strains, as an alternative to embryonated chicken-eggs. As only a limited range of mammalian viruses can grow in MDCK cells, similar to embryonated eggs, MDCK cells can act as an effective filter for a wide range of adventitious agents that might be introduced during vaccine production. However, the introduction of an alternative cell substrate (for example, MDCK cells) into a vaccine manufacturing process requires thorough investigations to assess the potential for adventitious agent risk in the final product, in the unlikely event that contamination should occur. The risk assessment takes into account the entire manufacturing process, from initial influenza virus isolation, through to blending of the trivalent subunit vaccine and worst-case residual titres for the final vaccine formulation have been calculated for >20 viruses or virus families. Maximum residual titres for all viruses tested were in the range of 10(-6) to 10(-16) infectious units per vaccine dose. Thus, the new cell culture-based vaccine manufacturing process can reduce any adventitious agent to a level that is unable to cause infection.

  4. Flocculation in ale brewing strains of Saccharomyces cerevisiae: re-evaluation of the role of cell surface charge and hydrophobicity.

    Science.gov (United States)

    Holle, Ann Van; Machado, Manuela D; Soares, Eduardo V

    2012-02-01

    Flocculation is an eco-friendly process of cell separation, which has been traditionally exploited by the brewing industry. Cell surface charge (CSC), cell surface hydrophobicity (CSH) and the presence of active flocculins, during the growth of two (NCYC 1195 and NCYC 1214) ale brewing flocculent strains, belonging to the NewFlo phenotype, were examined. Ale strains, in exponential phase of growth, were not flocculent and did not present active flocculent lectins on the cell surface; in contrast, the same strains, in stationary phase of growth, were highly flocculent (>98%) and presented a hydrophobicity of approximately three to seven times higher than in exponential phase. No relationship between growth phase, flocculation and CSC was observed. For comparative purposes, a constitutively flocculent strain (S646-1B) and its isogenic non-flocculent strain (S646-8D) were also used. The treatment of ale brewing and S646-1B strains with pronase E originated a loss of flocculation and a strong reduction of CSH; S646-1B pronase E-treated cells displayed a similar CSH as the non-treated S646-8D cells. The treatment of the S646-8D strain with protease did not reduce CSH. In conclusion, the increase of CSH observed at the onset of flocculation of ale strains is a consequence of the presence of flocculins on the yeast cell surface and not the cause of yeast flocculation. CSH and CSC play a minor role in the auto-aggregation of the ale strains since the degree of flocculation is defined, primarily, by the presence of active flocculins on the yeast cell wall.

  5. 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.

  6. Culture supernatants from V. cholerae O1 El Tor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity.

    Science.gov (United States)

    Vidal, Jorge E; Enríquez-Rincón, Fernando; Giono-Cerezo, Silvia; Ribas-Aparicio, Rosa María; Figueroa-Arredondo, Paula

    2009-01-01

    To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.

  7. Comparative analysis of conventional natural killer cell responses to acute infection with Toxoplasma gondii strains of different virulence

    Directory of Open Access Journals (Sweden)

    Daria L Ivanova

    2016-09-01

    Full Text Available Conventional Natural Killer Cells (cNK, members of group 1 innate lymphoid cells, are a diverse cell subpopulation based on surface receptor expression, maturation and functional potential. cNK cells are critical for early immunity to T. gondii via IFNγ production. Acute cNK cell responses to infection with different strains of T. gondii have not yet been characterized in detail. Here we comprehensively performed this analysis with Type I virulent RH, and Type II avirulent ME49 and fully attenuated Type I cps1-1 strains. In response to these three parasite strains, murine cNK cells produce IFNγ, become cytotoxic and polyfunctional (IFNγ+CD107a+ at the site of infection. In contrast to virulent RH and avirulent ME49 T. gondii strains, attenuated cps1-1 induced only local cNK cell responses. Infections with RH and ME49 parasites significantly decreased cNK cell frequency and numbers in spleen 5 days post infection compared to cps1-1 parasites. cNK cell subsets expressing activating receptors Ly49H, Ly49D, NKG2D and inhibitory receptors Ly49I and NKG2A/CD94 were similar when compared between the strains and at 5 days post infection. cNK cells were not proliferating (Ki67- 5 days post infection with any of the strains. cNK cell maturation as measured by CD27, CD11b and KLRG1 was affected after infection with different parasite strains. RH and ME49 infection significantly reduced mature cNK cell frequency and increased immature cNK cell populations compared to cps1-1 infection. Interestingly, KLRG1 was highly expressed on immature cNK cells after RH infection. After RH and ME49 infections, CD69+ cNK cells were present at higher numbers than after cps1-1 infection, which may correlate with loss of the mature cNK cell population. Cytokine multiplex analysis indicated cNK cell responses correlated with peritoneal exudate cell (PEC, spleen and serum proinflammatory cytokine levels including IL-12. qPCR analysis of parasite-specific B1 gene revealed

  8. Evaluation of the significance of cell wall polymers in flax infected with a pathogenic strain of Fusarium oxysporum.

    Science.gov (United States)

    Wojtasik, Wioleta; Kulma, Anna; Dymińska, Lucyna; Hanuza, Jerzy; Czemplik, Magdalena; Szopa, Jan

    2016-03-22

    Fusarium oxysporum infection leads to Fusarium-derived wilt, which is responsible for the greatest losses in flax (Linum usitatissimum) crop yield. Plants infected by Fusarium oxysporum show severe symptoms of dehydration due to the growth of the fungus in vascular tissues. As the disease develops, vascular browning and leaf yellowing can be observed. In the case of more virulent strains, plants die. The pathogen's attack starts with secretion of enzymes degrading the host cell wall. The main aim of the study was to evaluate the role of the cell wall polymers in the flax plant response to the infection in order to better understand the process of resistance and develop new ways to protect plants against infection. For this purpose, the expression of genes involved in cell wall polymer metabolism and corresponding polymer levels were investigated in flax seedlings after incubation with Fusarium oxysporum. This analysis was facilitated by selecting two groups of genes responding differently to the infection. The first group comprised genes strongly affected by the infection and activated later (phenylalanine ammonia lyase and glucosyltransferase). The second group comprised genes which are slightly affected (up to five times) and their expression vary as the infection progresses. Fusarium oxysporum infection did not affect the contents of cell wall polymers, but changed their structure. The results suggest that the role of the cell wall polymers in the plant response to Fusarium oxysporum infection is manifested through changes in expression of their genes and rearrangement of the cell wall polymers. Our studies provided new information about the role of cellulose and hemicelluloses in the infection process, the change of their structure and the expression of genes participating in their metabolism during the pathogen infection. We also confirmed the role of pectin and lignin in this process, indicating the major changes at the mRNA level of lignin metabolism genes

  9. Transcriptional and Cell Cycle Alterations Mark Aging of Primary Human Adipose-Derived Stem Cells.

    Science.gov (United States)

    Shan, Xiaoyin; Roberts, Cleresa; Kim, Eun Ji; Brenner, Ariana; Grant, Gregory; Percec, Ivona

    2017-05-01

    Adult stem cells play a critical role in the maintenance of tissue homeostasis and prevention of aging. While the regenerative potential of stem cells with low cellular turnover, such as adipose-derived stem cells (ASCs), is increasingly recognized, the study of chronological aging in ASCs is technically difficult and remains poorly understood. Here, we use our model of chronological aging in primary human ASCs to examine genome-wide transcriptional networks. We demonstrate first that the transcriptome of aging ASCs is distinctly more stable than that of age-matched fibroblasts, and further, that age-dependent modifications in cell cycle progression and translation initiation specifically characterize aging ASCs in conjunction with increased nascent protein synthesis and a distinctly shortened G1 phase. Our results reveal novel chronological aging mechanisms in ASCs that are inherently different from differentiated cells and that may reflect an organismal attempt to meet the increased demands of tissue and organ homeostasis during aging. Stem Cells 2017;35:1392-1401. © 2017 AlphaMed Press.

  10. Differentiation of hepatocytes from induced pluripotent stem cells derived from human hair follicle mesenchymal stem cells.

    Science.gov (United States)

    Shi, Xu; Lv, Shuang; He, Xia; Liu, Xiaomei; Sun, Meiyu; Li, Meiying; Chi, Guangfan; Li, Yulin

    2016-10-01

    Due to the limitations of organ donors and immune rejection in severe liver diseases, stem cell-based therapy presents a promising application for tissue repair and regeneration. As a novel cell source, mesenchymal stem cells separated from human hair follicles (HF-MSCs) are convenient to obtain and have no age limit. To date, the differentiation of HF-MSCs into hepatocytes has not been reported. In this study, we explored whether HF-MSCs and HF-MSC-derived-induced pluripotent stem cells (HF-iPS) could differentiate into hepatocytes in vitro. Flow cytometry, Oil Red O stain and Alizarin Red stain were used to identify the characteristics of HF-MSCs. The expression of liver-specific gene was detected by immunofluorescence and Quantitative Polymerase Chain Reaction. Periodic Acid-Schiff stain, Indocyanine Green stain and Low-Density Lipoprotein stain were performed to evaluate the functions of induced hepatocyte-like cells (HLCs). HF-MSCs were unable to differentiate into HLCs using previously reported procedures for MSCs from other tissues. However, HF-iPS efficiently induced the generation of HLCs that expressed hepatocyte markers and drug metabolism-related genes. HF-iPS can be used as novel and alternative cellular tools for inducing hepatocytes in vitro, simultaneously benefiting from utilizing HF-MSCs as a noninvasive and convenient cell source for reprogramming.

  11. Stem cell-derived kidney cells and organoids: Recent breakthroughs and emerging applications.

    Science.gov (United States)

    Chuah, Jacqueline Kai Chin; Zink, Daniele

    The global rise in the numbers of kidney patients and the shortage in transplantable organs have led to an increasing interest in kidney-specific regenerative therapies, renal disease modelling and bioartificial kidneys. Sources for large quantities of high-quality renal cells and tissues would be required, also for applications in in vitro platforms for compound safety and efficacy screening. Stem cell-based approaches for the generation of renal-like cells and tissues would be most attractive, but such methods were not available until recently. This situation has drastically changed since 2013, and various protocols for the generation of renal-like cells and precursors from pluripotent stem cells (PSC) have been established. The most recent breakthroughs were related to the establishment of various protocols for the generation of PSC-derived kidney organoids. In combination with recent advances in genome editing, bioprinting and the establishment of predictive renal screening platforms this results in exciting new possibilities. This review will give a comprehensive overview over current PSC-based protocols for the generation of renal-like cells, precursors and organoids, and their current and potential applications in regenerative medicine, compound screening, disease modelling and bioartificial organs. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Abnormal G1 arrest in the cell lines from LEC strain rats after X-irradiation

    International Nuclear Information System (INIS)

    Hayashi, M.; Uehara, K.; Kirisawa, R.; Endoh, D.; Arai, S.; Okui, T.

    1997-01-01

    The effect of X-irradiation of cell lines from LEC and WKAH strain rats on a progression o cell cycle was investigated. When WKAH rat ells were exposed to 5 Gy of X-rays and their cell cycle distribution was determined by a flow cytometer, the proportion of S-phase cells decrease and that of G2/M-phase cells in creased at 8 hr post-irradiation. At 18 and 24 hr post-irradiation, approximately 80% of the cells appeared in the G1 phase. On the contrary, the proportion of S-phase cells increased and that of G1-phase cells decreased in LEC rats during 8-24 hr post-irradiation, compared with that at 0 hr post-irradiation. Thus, radiation-induced delay in the progression from the G1 phase to S phase (G1 arrest) was observed inWKAH rat cells but not in LEC rat cells. In the case of WKAH rat cells, the intensities of the bands of p53 protein increased at 1 and 2 hr after X-irradiation at 5 Gy, compared with those of un-irradiated cells and at 0 hr post-irradiation. In contrast, the intensities of the bands were faint and did not significantly increase in LEC rat ells during 0-6 hr incubation after X-irradiation. Present results suggested that the radioresistant DNA synthesis in LEC rat cells is thought to be due to the abnormal G1 arrest following X-irradiation

  13. [Expression of embryonic markers in pterygium derived mesenchymal cells].

    Science.gov (United States)

    Pascual, G; Montes, M A; Pérez-Rico, C; Pérez-Kohler, B; Bellón, J M; Buján, J

    2010-12-01

    Destruction of the limbal epithelium barrier is the most important mechanism of pterygium formation (conjunctiva proliferation, encroaching onto the cornea). It is thought to arise from activated and proliferating limbal epithelial stem cells. The objective of this study is to evaluate the presence of undifferentiated mesenchymal cells (stem cells) in cultured cells extracted from human pterygium. Cells from 6 human pterygium were isolated by explantation and placed in cultures with amniomax medium. Once the monolayer was reached the cells were seeded onto 24 well microplates. The cells were studied in the second sub-culture. The immunohistochemical expression of different embryonic stem cell markers, OCT3/4 and CD9, was analysed. The differentiated phenotypes were characterised with the monoclonal antibodies anti-CD31, α-actin and vimentin. All the cell populations obtained from pterygium showed vimentin expression. Less than 1% of the cells were positive for CD31 and α-actin markers. The majority of the cell population was positive for OCT3/4 and CD9. The cell population obtained from pterygium expressed mesenchymal cell phenotype and embryonic markers, such us OCT3/4 and CD9. This undifferentiated population could be involved in the large recurrence rate of this type of tissue after surgery. Copyright © 2010 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

  14. Significance of adipose tissue-derived stem cells regulate CD4+ T cell immune in the treatment of multiple sclerosis

    Directory of Open Access Journals (Sweden)

    Yong-lin XIE

    2014-10-01

    Full Text Available Adipose tissue-derived stem cells (ADSCs are genetically engineered seed cells with immunomodulatory effects, widely used in the treatment of autoimmune diseases. This article focuses on the immunomodulatory effects of adipose tissue-derived stem cells on CD4+ T cell subsets, including T helper cell (Th 1, 2, 17 and regulatory T cell (Treg, and its clinical significance in the treatment of multiple sclerosis. doi: 10.3969/j.issn.1672-6731.2014.10.005

  15. Cyclic mechanical strain-induced proliferation and migration of human airway smooth muscle cells: role of EMMPRIN and MMPs.

    Science.gov (United States)

    Hasaneen, Nadia A; Zucker, Stanley; Cao, Jian; Chiarelli, Christian; Panettieri, Reynold A; Foda, Hussein D

    2005-09-01

    Airway smooth muscle (ASM) proliferation and migration are major components of airway remodeling in asthma. Asthmatic airways are exposed to mechanical strain, which contributes to their remodeling. Matrix metalloproteinase (MMP) plays an important role in remodeling. In the present study, we examined if the mechanical strain of human ASM (HASM) cells contributes to their proliferation and migration and the role of MMPs in this process. HASM were exposed to mechanical strain using the FlexCell system. HASM cell proliferation, migration and MMP release, activation, and expression were assessed. Our results show that cyclic strain increased the proliferation and migration of HASM; cyclic strain increased release and activation of MMP-1, -2, and -3 and membrane type 1-MMP; MMP release was preceded by an increase in extracellular MMP inducer; Prinomastat [a MMP inhibitor (MMPI)] significantly decreased cyclic strain-induced proliferation and migration of HASM; and the strain-induced increase in the release of MMPs was accompanied by an increase in tenascin-C release. In conclusion, cyclic mechanical strain plays an important role in HASM cell proliferation and migration. This increase in proliferation and migration is through an increase in MMP release and activation. Pharmacological MMPIs should be considered in the pursuit of therapeutic options for airway remodeling in asthma.

  16. Selected Lactobacillus strains isolated from sugary and milk kefir reduce Salmonella infection of epithelial cells in vitro.

    Science.gov (United States)

    Zavala, L; Golowczyc, M A; van Hoorde, K; Medrano, M; Huys, G; Vandamme, P; Abraham, A G

    2016-09-01

    The isolation of potentially probiotic strains and the subsequent study of their properties are very important steps to gain insight in the health benefits ascribed to sugary and milk kefir. The aim of the present study was to characterise fifteen Lactobacillus strains isolated from these beverages by determining some surface properties and their ability to antagonise enterocyte cell damage after Salmonella infection in vitro. Lactobacillus surface properties were determined by hydrophobicity, autoaggregation, and coaggregation assays with Salmonella. In addition, lactobacilli adhesion to Caco-2/TC-7 cells and the effect on Salmonella invasion were evaluated. Finally, the disassembly of F-actin cytoskeleton on intestinal epithelial cells was assayed in vitro when Salmonella infection was performed in the presence of selected Lactobacillus strains. Ten out of the 15 strains showed a high adhesion capacity to Caco-2/TC-7 cells. Most of the strains were hydrophilic and non-autoaggregating. Strains isolated from sugary kefir were non-coaggregating with Salmonella, while strains Lactobacillus paracasei CIDCA 83120, 83121, 83123, 83124, 8339, 83102 isolated from milk kefir were able to coaggregate after 1 h. L. paracasei CIDCA 8339 and Lactobacillus kefiri CIDCA 83102 were able to diminish Salmonella invasion to the enterocytes. An antagonistic effect on cytoskeleton disruption elicited by the pathogen was also demonstrated. Our results suggest that both strains isolated from milk kefir could be considered as appropriate probiotic candidates.

  17. Stem Cell-Derived Exosome in Cardiovascular Diseases: Macro Roles of Micro Particles.

    Science.gov (United States)

    Yuan, Ye; Du, Weijie; Liu, Jiaqi; Ma, Wenya; Zhang, Lai; Du, Zhimin; Cai, Benzhi

    2018-01-01

    The stem cell-based therapy has emerged as the promising therapeutic strategies for cardiovascular diseases (CVDs). Recently, increasing evidence suggest stem cell-derived active exosomes are important communicators among cells in the heart via delivering specific substances to the adjacent/distant target cells. These exosomes and their contents such as certain proteins, miRNAs and lncRNAs exhibit huge beneficial effects on preventing heart damage and promoting cardiac repair. More importantly, stem cell-derived exosomes are more effective and safer than stem cell transplantation. Therefore, administration of stem cell-derived exosomes will expectantly be an alternative stem cell-based therapy for the treatment of CVDs. Furthermore, modification of stem cell-derived exosomes or artificial synthesis of exosomes will be the new therapeutic tools for CVDs in the future. In addition, stem cell-derived exosomes also have been implicated in the diagnosis and prognosis of CVDs. In this review, we summarize the current advances of stem cell-derived exosome-based treatment and prognosis for CVDs, including their potential benefits, underlying mechanisms and limitations, which will provide novel insights of exosomes as a new tool in clinical therapeutic translation in the future.

  18. 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.

  19. Hypoxia Enhances Differentiation of Adipose Tissue-Derived Stem Cells toward the Smooth Muscle Phenotype

    Directory of Open Access Journals (Sweden)

    Fang Wang

    2018-02-01

    Full Text Available Smooth muscle differentiated adipose tissue-derived stem cells are a valuable resource for regeneration of gastrointestinal tissues, such as the gut and sphincters. Hypoxia has been shown to promote adipose tissue-derived stem cells proliferation and maintenance of pluripotency, but the influence of hypoxia on their smooth myogenic differentiation remains unexplored. This study investigated the phenotype and contractility of adipose-derived stem cells differentiated toward the smooth myogenic lineage under hypoxic conditions. Oxygen concentrations of 2%, 5%, 10%, and 20% were used during differentiation of adipose tissue-derived stem cells. Real time reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of smooth muscle cells-specific markers, including early marker smooth muscle alpha actin, middle markers calponin, caldesmon, and late marker smooth muscle myosin heavy chain. The specific contractile properties of cells were verified with both a single cell contraction assay and a gel contraction assay. Five percent oxygen concentration significantly increased the expression levels of α-smooth muscle actin, calponin, and myosin heavy chain in adipose-derived stem cell cultures after 2 weeks of induction (p < 0.01. Cells differentiated in 5% oxygen conditions showed greater contraction effect (p < 0.01. Hypoxia influences differentiation of smooth muscle cells from adipose stem cells and 5% oxygen was the optimal condition to generate smooth muscle cells that contract from adipose stem cells.

  20. Derivation of porcine pluripotent stem cells for biomedical research.

    Science.gov (United States)

    Shiue, Yow-Ling; Yang, Jenn-Rong; Liao, Yu-Jing; Kuo, Ting-Yung; Liao, Chia-Hsin; Kang, Ching-Hsun; Tai, Chein; Anderson, Gary B; Chen, Lih-Ren

    2016-07-01

    Pluripotent stem cells including embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced pluripotent stem cells (iPSCs) are capable of self-renew and limitlessly proliferating in vitro with undifferentiated characteristics. They are able to differentiate in vitro, spontaneously or responding to suitable signals, into cells of all three primary germ layers. Consequently, these pluripotent stem cells will be valuable sources for cell replacement therapy in numerous disorders. However, the promise of human ESCs and EGCs is cramped by the ethical argument about destroying embryos and fetuses for cell line creation. Moreover, there are still carcinogenic risks existing toward the goal of clinical application for human ESCs, EGCs, and iPSCs. Therefore, a suitable animal model for stem cell research will benefit the further development of human stem cell technology. The pigs, on the basis of their similarity in anatomy, immunology, physiology, and biochemical properties, have been wide used as model animals in the study of various human diseases. The development of porcine pluripotent stem cell lines will hold the opportunity to provide an excellent material for human counterpart to the transplantation in biomedical research and further development of cell-based therapeutic strategy. Copyright © 2016 Elsevier Inc. All rights reserved.