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Sample records for cancer cell-derived microvesicles

  1. Breast cancer exosome-like microvesicles and salivary gland cells interplay alters salivary gland cell-derived exosome-like microvesicles in vitro.

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    Chang S Lau

    Full Text Available Saliva is a useful biofluid for the early detection of disease, but how distal tumors communicate with the oral cavity and create disease-specific salivary biomarkers remains unclear. Using an in vitro breast cancer model, we demonstrated that breast cancer-derived exosome-like microvesicles are capable of interacting with salivary gland cells, altering the composition of their secreted exosome-like microvesicles. We found that the salivary gland cells secreted exosome-like microvesicles encapsulating both protein and mRNA. We also showed that the interaction with breast cancer-derived exosome-like microvesicles communicated and activated the transcriptional machinery of the salivary gland cells. Thus, the interaction altered the composition of the salivary gland cell-derived exosome-like microvesicles on both the transcriptomically and proteomically.

  2. Colorectal cancer cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells

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    Kim Yoon-Keun

    2009-11-01

    Full Text Available Abstract Background Various cancer cells, including those of colorectal cancer (CRC, release microvesicles (exosomes into surrounding tissues and peripheral circulation. These microvesicles can mediate communication between cells and affect various tumor-related processes in their target cells. Results We present potential roles of CRC cell-derived microvesicles in tumor progression via a global comparative microvesicular and cellular transcriptomic analysis of human SW480 CRC cells. We first identified 11,327 microvesicular mRNAs involved in tumorigenesis-related processes that reflect the physiology of donor CRC cells. We then found 241 mRNAs enriched in the microvesicles above donor cell levels, of which 27 were involved in cell cycle-related processes. Network analysis revealed that most of the cell cycle-related microvesicle-enriched mRNAs were associated with M-phase activities. The integration of two mRNA datasets showed that these M-phase-related mRNAs were differentially regulated across CRC patients, suggesting their potential roles in tumor progression. Finally, we experimentally verified the network-driven hypothesis by showing a significant increase in proliferation of endothelial cells treated with the microvesicles. Conclusion Our study demonstrates that CRC cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells, suggesting that microvesicles of cancer cells can be involved in tumor growth and metastasis by facilitating angiogenesis-related processes. This information will help elucidate the pathophysiological functions of tumor-derived microvesicles, and aid in the development of cancer diagnostics, including colorectal cancer.

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

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    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.

  4. Rapid and specific detection of cell-derived microvesicles using a magnetoresistive biochip

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    Cherré, Solène; Fernandes, Elisabete; Germano, José

    2017-01-01

    Microvesicles (MVs) are a promising source of diagnostic biomarkers which have gained a wide interest in the biomedical and biosensing field. They can be interpreted as a "fingerprint" of various diseases. Nonetheless, MVs implementation into clinical settings has been hampered by the lack of tec...

  5. Rapid and specific detection of cell-derived microvesicles using a magnetoresistive biochip.

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    Cherré, Solène; Fernandes, Elisabete; Germano, José; Dias, Tomás; Cardoso, Susana; Piedade, Moisés S; Rozlosnik, Noemi; Oliveira, Marta I; Freitas, Paulo P

    2017-03-13

    Microvesicles (MVs) are a promising source of diagnostic biomarkers which have gained a wide interest in the biomedical and biosensing field. They can be interpreted as a "fingerprint" of various diseases. Nonetheless, MVs implementation into clinical settings has been hampered by the lack of technologies to accurately characterize, detect and quantify them. Here, we report the specific sensing and quantification of MVs from endothelial cells using a portable magnetoresistive (MR) biochip platform, in less than one hour and within physiologically relevant concentrations (1 × 10(8) MVs per ml). MVs were isolated from both endothelial and epithelial cells undergoing apoptosis, and characterized by atomic force microscopy (AFM) and nanoparticle tracking analysis (NTA), which revealed similar MV sizes. Importantly, our results showed that the two distinct MV populations could be discriminated with the MR biochip platform, with over a 5-fold capture efficiency of endothelial MVs in comparison to the control (epithelial MVs). Also, unspecific binding of MVs to BSA was less than 1% of the specific signal. The detection strategy was based on a sandwich immunoassay, where MVs were labelled with magnetic nanoparticles (MNPs) functionalized with Annexin V and then captured by anti-CD31 antibodies previously immobilized on the surface of the sensor. Results suggest that this approach allows the detection of specific MVs from complex samples such as serum, and highlight the potential of this technology to become a suitable tool for MVs detection as a complementary method of diagnosis.

  6. Mesenchymal Stromal Cell-Derived Microvesicles Regulate an Internal Pro-Inflammatory Program in Activated Macrophages

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    Juan S. Henao Agudelo

    2017-07-01

    Full Text Available Mesenchymal stromal cells (MSCs are multipotent cells with abilities to exert immunosuppressive response promoting tissue repair. Studies have shown that MSCs can secrete extracellular vesicles (MVs-MSCs with similar regulatory functions to the parental cells. Furthermore, strong evidence suggesting that MVs-MSCs can modulate several immune cells (i.e., Th1, Th17, and Foxp3+ T cells. However, their precise effect on macrophages (Mϕs remains unexplored. We investigated the immunoregulatory effect of MVs-MSCs on activated M1-Mϕs in vitro and in vivo using differentiated bone marrow Mϕs and an acute experimental model of thioglycollate-induced peritonitis, respectively. We observed that MVs-MSCs shared surface molecules with MSCs (CD44, CD105, CD90, CD73 and expressed classical microvesicle markers (Annexin V and CD9. The in vitro treatment with MVs-MSCs exerted a regulatory-like phenotype in M1-Mϕs, which showed higher CD206 level and reduced CCR7 expression. This was associated with decreased levels of inflammatory molecules (IL-1β, IL-6, nitric oxide and increased immunoregulatory markers (IL-10 and Arginase in M1-Mϕs. In addition, we detected that MVs-MSCs promoted the downregulation of inflammatory miRNAs (miR-155 and miR-21, as well as, upregulated its predicted target gene SOCS3 in activated M1-Mϕs. In vivo MVs-MSCs treatment reduced the Mϕs infiltrate in the peritoneal cavity inducing a M2-like regulatory phenotype in peritoneal Mϕs (higher arginase activity and reduced expression of CD86, iNOS, IFN-γ, IL-1β, TNF-α, IL-1α, and IL-6 molecules. This in vivo immunomodulatory effect of MVs-MSCs on M1-Mϕs was partially associated with the upregulation of CX3CR1 in F4/80+/Ly6C+/CCR2+ Mϕs subsets. In summary, our findings indicate that MVs-MSCs can modulate an internal program in activated Mϕs establishing an alternative regulatory-like phenotype.

  7. Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche

    National Research Council Canada - National Science Library

    Grange, Cristina; Tapparo, Marta; Collino, Federica; Vitillo, Loriana; Damasco, Christian; Deregibus, Maria Chiara; Tetta, Ciro; Bussolati, Benedetta; Camussi, Giovanni

    2011-01-01

    Recent studies suggest that tumor-derived microvesicles (MV) act as a vehicle for exchange of genetic information between tumor and stromal cells, engendering a favorable microenvironment for cancer development...

  8. Hepatic Stellate Cell-Derived Microvesicles Prevent Hepatocytes from Injury Induced by APAP/H2O2

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    Renwei Huang

    2016-01-01

    Full Text Available Hepatic stellate cells (HSCs, previously described for liver-specific mesenchymal stem cells (MSCs, appear to contribute to liver regeneration. Microvesicles (MVs are nanoscale membrane fragments, which can regulate target cell function by transferring contents from their parent cells. The aim of this study was to investigate the effect of HSC-derived MVs on xenobiotic-induced liver injury. Rat and human hepatocytes, BRL-3A and HL-7702, were used to build hepatocytes injury models by n-acetyl-p-aminophenol n-(APAP or H2O2 treatment. MVs were prepared from human and rat HSCs, LX-2, and HST-T6 and, respectively, added to injured BRL-3A and HL-7702 hepatocytes. MTT assay was utilized to determine cell proliferation. Cell apoptosis was analyzed by flow cytometry and hoechst33258 staining. Western blot was used for analyzing the expression of activated caspase-3. Liver injury indicators, alanine aminotransferase (ALT, aspartate aminotransferase (AST, and lactate dehydrogenase (LDH in culture medium were also assessed. Results showed that (1 HSC-MVs derived from LX-2 and HST-T6 were positive to CD90 and annexin V surface markers; (2 HSC-MVs dose-dependently improved the viability of hepatocytes in both injury models; (3 HSC-MVs dose-dependently inhibited the APAP/H2O2 induced hepatocytes apoptosis and activated caspase-3 expression and leakage of LDH, ALT, and AST. Our results demonstrate that HSC-derived MVs protect hepatocytes from toxicant-induced injury.

  9. Signal Factors Secreted by 2D and Spheroid Mesenchymal Stem Cells and by Cocultures of Mesenchymal Stem Cells Derived Microvesicles and Retinal Photoreceptor Neurons.

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    Xie, Lili; Mao, Mao; Zhou, Liang; Zhang, Lusi; Jiang, Bing

    2017-01-01

    We aim to identify levels of signal factors secreted by MSCs cultured in 2D monolayers (2D-MSCs), spheroids (spheroids MSCs), and cocultures of microvesicles (MVs) derived from 2D-MSCs or spheroid MSCs and retinal photoreceptor neurons. We seeded 2D-MSCs, spheroid MSCs, and cells derived from spheroids MSCs at equal numbers. MVs isolated from all 3 culture conditions were incubated with 661W cells. Levels of 51 signal factors in conditioned medium from those cultured conditions were quantified with bead-based assay. We found that IL-8, IL-6, and GROα were the top three most abundant signal factors. Moreover, compared to 2D-MSCs, levels of 11 cytokines and IL-2Rα were significantly increased in conditioned medium from spheroid MSCs. Finally, to test if enhanced expression of these factors reflects altered immunomodulating activities, we assessed the effect of 2D-MSC-MVs and 3D-MSC-MVs on CD14+ cell chemoattraction. Compared to 2D-MSC-MVs, 3D-MSC-MVs significantly decreased the chemotactic index of CD14+ cells. Our results suggest that spheroid culture conditions improve the ability of MSCs to selectively secrete signal factors. Moreover, 3D-MSC-MVs also possessed an enhanced capability to promote signal factors secretion compared to 2D-MSC-MVs and may possess enhanced immunomodulating activities and might be a better regenerative therapy for retinal degenerative diseases.

  10. Platelet microvesicles in health and disease.

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    Melki, Imene; Tessandier, Nicolas; Zufferey, Anne; Boilard, Eric

    2017-05-01

    Interest in cell-derived extracellular vesicles and their physiological and pathological implications is constantly growing. Microvesicles, also known as microparticles, are small extracellular vesicles released by cells in response to activation or apoptosis. Among the different microvesicles present in the blood of healthy individuals, platelet-derived microvesicles (PMVs) are the most abundant. Their characterization has revealed a heterogeneous cargo that includes a set of adhesion molecules. Similarly to platelets, PMVs are also involved in thrombosis through support of the coagulation cascade. The levels of circulatory PMVs are altered during several disease manifestations such as coagulation disorders, rheumatoid arthritis, systemic lupus erythematosus, cancers, cardiovascular diseases, and infections, pointing to their potential contribution to disease and their development as a biomarker. This review highlights recent findings in the field of PMV research and addresses their contribution to both healthy and diseased states.

  11. Evolution of Cancer Stem-like Cells in Endocrine-Resistant Metastatic Breast Cancers Is Mediated by Stromal Microvesicles.

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    Sansone, Pasquale; Berishaj, Marjan; Rajasekhar, Vinagolu K; Ceccarelli, Claudio; Chang, Qing; Strillacci, Antonio; Savini, Claudia; Shapiro, Lauren; Bowman, Robert L; Mastroleo, Chiara; De Carolis, Sabrina; Daly, Laura; Benito-Martin, Alberto; Perna, Fabiana; Fabbri, Nicola; Healey, John H; Spisni, Enzo; Cricca, Monica; Lyden, David; Bonafé, Massimiliano; Bromberg, Jacqueline

    2017-04-15

    The hypothesis that microvesicle-mediated miRNA transfer converts noncancer stem cells into cancer stem cells (CSC) leading to therapy resistance remains poorly investigated. Here we provide direct evidence supporting this hypothesis, by demonstrating how microvesicles derived from cancer-associated fibroblasts (CAF) transfer miR-221 to promote hormonal therapy resistance (HTR) in models of luminal breast cancer. We determined that CAF-derived microvesicles horizontally transferred miR-221 to tumor cells and, in combination with hormone therapy, activated an ER(lo)/Notch(hi) feed-forward loop responsible for the generation of CD133(hi) CSCs. Importantly, microvesicles from patients with HTR metastatic disease expressed high levels of miR-221. We further determined that the IL6-pStat3 pathway promoted the biogenesis of onco-miR-221(hi) CAF microvesicles and established stromal CSC niches in experimental and patient-derived breast cancer models. Coinjection of patient-derived CAFs from bone metastases led to de novo HTR tumors, which was reversed with IL6R blockade. Finally, we generated patient-derived xenograft (PDX) models from patient-derived HTR bone metastases and analyzed tumor cells, stroma, and microvesicles. Murine and human CAFs were enriched in HTR tumors expressing high levels of CD133(hi) cells. Depletion of murine CAFs from PDX restored sensitivity to HT, with a concurrent reduction of CD133(hi) CSCs. Conversely, in models of CD133(neg), HT-sensitive cancer cells, both murine and human CAFs promoted de novo HT resistance via the generation of CD133(hi) CSCs that expressed low levels of estrogen receptor alpha. Overall, our results illuminate how microvesicle-mediated horizontal transfer of genetic material from host stromal cells to cancer cells triggers the evolution of therapy-resistant metastases, with potentially broad implications for their control. Cancer Res; 77(8); 1927-41. ©2017 AACR. ©2017 American Association for Cancer Research.

  12. Microvesicles released constitutively from prostate cancer cells differ biochemically and functionally to stimulated microvesicles released through sublytic C5b-9

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    Stratton, Dan; Moore, Colin; Antwi-Baffour, Samuel [Cellular and Molecular Immunology Research Centre, London Metropolitan University (United Kingdom); Lange, Sigrun [University College London School of Pharmacy, London UK (United Kingdom); Inal, Jameel, E-mail: j.inal@londonmet.ac.uk [Cellular and Molecular Immunology Research Centre, London Metropolitan University (United Kingdom)

    2015-05-08

    We have classified microvesicles into two subtypes: larger MVs released upon stimulation of prostate cancer cells, sMVs, and smaller cMVs, released constitutively. cMVs are released as part of cell metabolism and sMVs, released at 10-fold higher levels, produced upon activation, including sublytic C5b-9. From electron microscopy, nanosight tracking analysis, dynamic light scattering and flow cytometry, cMVs (194–210 nm in diameter) are smaller than sMVs (333–385 nm). Furthermore, using a Quartz Crystal Microbalance measuring changes in resonant frequency (Δf) that equate to mass deposited on a sensor, an sMV and a cMV are estimated at 0.267 and 0.241 pg, respectively. sMVs carry more calcium and protein, express higher levels of lipid rafts, GPI-anchored CD55 and phosphatidylserine including deposited C5b-9 compared to cMVs. This may allude to biological differences such as increased bound C4BP on sMVs inhibiting complement more effectively. - Highlights: • Prostate cells release microvesicles constitutively (cMVs) or upon stimulus (sMVs). • sMVs are larger than cMVs and carry more protein, lipid rafts and surface PstSer. • sMVs inhibit complement more effectively than cMVs.

  13. Comparison of microRNA expression profiles in K562-cells-derived microvesicles and parental cells, and analysis of their roles in leukemia.

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    Chen, Xiaomei; Xiong, Wei; Li, Huiyu

    2016-12-01

    Microvesicles (MVs) are 30-1,000-nm extracellular vesicles that are released from a multitude of cell types and perform diverse cellular functions, including intercellular communication, antigen presentation, and transfer of proteins, messenger RNA and microRNA (also known as miR). MicroRNAs have been demonstrated to be aberrantly expressed in leukemia, and the overall microRNA expression profile may differentiate normal blood cells vs. leukemia cells. MVs containing microRNAs may enable intercellular cross-talk in vivo. This prompted us to investigate specific variations of microRNA expression patterns in MVs derived from leukemia cells. The present study examined the microRNA expression profile of MVs from chronic myeloid leukemia K562 cells and that of MVs from normal human volunteers' peripheral blood cells. The potential targets of the differentially expressed microRNAs were predicted using computational searches. Bioinformatic analyses of the predicted target genes were performed for further evaluation. The present study analyzed microRNAs of MVs derived from leukemia and normal cells, and characterized specific microRNAs expression. The results revealed that MVs derived from K562 cells expressed 181 microRNAs of the 888 microRNAs assessed. Further analysis revealed that 16 microRNAs were downregulated, while 7 were upregulated in these MVs. In addition, significant differences in microRNA expression profiles between MVs derived from K562 cells and K562 cells were identified. The present results revealed that 77 and 122 microRNAs were only expressed in MVs derived from K562 cells and in K562 cells, respectively. There were 104 microRNAs co-expressed in MVs derived from K562 cells and in K562 cells. Target gene-related pathway analyses demonstrated that the majority of the dysregulated microRNAs were involved in pathways associated with leukemia, particularly the mitogen-activated protein kinase (MAPK) and the p53 signaling pathways. By further conducting

  14. Chemotherapy and anti-angiogenic drugs affect composition and coagulant phenotype of cell-derived vesicles in cancer patients

    NARCIS (Netherlands)

    Kleinjan, A.; Verhoeff, J.; Berckmans, R.; Kunst, P.; Van Doormaal, F.; Di Nisio, M.; Richel, D.; Kamphuisen, P.W.; Büller, H.R.; Nieuwland, R.

    Background: The relationship between chemotherapy and circulating microparticles in patients with cancer is complex. First, release of cancer cell-derived microparticles may contribute to resistance of cancer cells to chemotherapy. Second, chemotherapy and angiogenesis inhibiting agents promote a

  15. Metabolic and Signaling Functions of Cancer Cell-Derived Extracellular Vesicles.

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    Fonseca, P; Vardaki, I; Occhionero, A; Panaretakis, T

    2016-01-01

    Extracellular vesicles have gained tremendous attention in the recent years as a novel mechanism of cell to cell communication. There are several types of extracellular vesicles, including exosomes, microvesicles, exosome, like vesicles, apoptotic bodies that differ mainly in the mechanism of biogenesis and secretion. The most well studied type of extracellular vesicles are the exosomes which are endosome-derived vesicles with a diameter of 50-150nm and enriched in ESCRT proteins including Alix, TSG101, Hsp70, and tetraspanins. It is now well established that exosomes promote tumor growth, alter the tumor microenvironment, facilitate the dissemination of cancer cells in an organotropic manner, modulate immune responses, and mediate resistance to therapy. Exosomes have also been recently implicated in an emerging hallmark of cancer, the cancer cell metabolism. The metabolic state of the cell defines, to a certain extent, both the rate of secretion and the molecular content of tumor-derived exosomes. Furthermore, exosomes have been shown to possess intrinsic metabolic activity since they can synthesize ATP by glycolysis. It follows that exosomes carry a number of metabolic enzymes and metabolites, including lactate, PGE, LDH isoforms, pyruvate, and monocarboxylate transporters. Last but not the least, exosomes are implicated in fatty acid synthesis and cholesterol metabolism and are thought to be crucial for the transcellular metabolism procedure. Uptake of exosomes is thought to alter the intracellular metabolic state of the cell. In summary, we describe the state of the art on the role of metabolism in the secretion, uptake, and the biological effects of exosomes in the metabolism of recipient cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Cancer Cell-Derived Extracellular Vesicles Are Associated with Coagulopathy Causing Ischemic Stroke via Tissue Factor-Independent Way: The OASIS-CANCER Study.

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    Bang, Oh Young; Chung, Jong-Won; Lee, Mi Ji; Kim, Suk Jae; Cho, Yeon Hee; Kim, Gyeong-Moon; Chung, Chin-Sang; Lee, Kwang Ho; Ahn, Myung-Ju; Moon, Gyeong Joon

    2016-01-01

    Cancer and stroke, which are known to be associated with one another, are the most common causes of death in the elderly. However, the pathomechanisms that lead to stroke in cancer patients are not well known. Circulating extracellular vesicles (EVs) play a role in cancer-associated thrombosis and tumor progression. Therefore, we hypothesized that cancer cell-derived EVs cause cancer-related coagulopathy resulting in ischemic stroke. Serum levels of D-dimer and EVs expressing markers for cancer cells (epithelial cell adhesion molecule [CD326]), tissue factor (TF [CD142]), endothelial cells (CD31+CD42b-), and platelets (CD62P) were measured using flow cytometry in (a) 155 patients with ischemic stroke and active cancer (116 - cancer-related, 39 - conventional stroke mechanisms), (b) 25 patients with ischemic stroke without cancer, (c) 32 cancer patients without stroke, and (d) 101 healthy subjects. The levels of cancer cell-derived EVs correlated with the levels of D-dimer and TF+ EVs. The levels of cancer cell-derived EVs (CD326+ and CD326+CD142+) were higher in cancer-related stroke than in other groups (P<0.05 in all the cases). Path analysis showed that cancer cell-derived EVs are related to stroke via coagulopathy as measured by D-dimer levels. Poor correlation was observed between TF+ EV and D-dimer, and path analysis demonstrated that cancer cell-derived EVs may cause cancer-related coagulopathy independent of the levels of TF+ EVs. Our findings suggest that cancer cell-derived EVs mediate coagulopathy resulting in ischemic stroke via TF-independent mechanisms.

  17. The Impact of Lipoprotein-Associated Oxidative Stress on Cell-Specific Microvesicle Release in Patients with Familial Hypercholesterolemia

    DEFF Research Database (Denmark)

    Nielsen, Morten Hjuler; Irvine, H; Vedel, S

    2016-01-01

    Objective. Microvesicles (MVs) are small cell-derived particles shed upon activation. Familial hypercholesterolemia (FH) particularly when associated with Achilles tendon xanthomas (ATX) predisposes to atherosclerosis, possibly through oxLDL-C interaction with the CD36 receptor. To investigate...

  18. Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

    OpenAIRE

    Nakles, Rebecca E.; Millman, Sarah L.; Cabrera, M. Carla; Johnson, Peter; Mueller, Susette; Hoppe, Philipp S.; Schroeder, Timm; Furth, Priscilla A.

    2013-01-01

    Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without...

  19. Dynamic microvesicle release and clearance within the cardiovascular system: triggers and mechanisms.

    Science.gov (United States)

    Ayers, Lisa; Nieuwland, Rienk; Kohler, Malcolm; Kraenkel, Nicolle; Ferry, Berne; Leeson, Paul

    2015-12-01

    Interest in cell-derived microvesicles (or microparticles) within cardiovascular diagnostics and therapeutics is rapidly growing. Microvesicles are often measured in the circulation at a single time point. However, it is becoming clear that microvesicle levels both increase and decrease rapidly in response to certain stimuli such as hypoxia, acute cardiac stress, shear stress, hypertriglyceridaemia and inflammation. Consequently, the levels of circulating microvesicles will reflect the balance between dynamic mechanisms for release and clearance. The present review describes the range of triggers currently known to lead to microvesicle release from different cellular origins into the circulation. Specifically, the published data are used to summarize the dynamic impact of these triggers on the degree and rate of microvesicle release. Secondly, a summary of the current understanding of microvesicle clearance via different cellular systems, including the endothelial cell and macrophage, is presented, based on reported studies of clearance in experimental models and clinical scenarios, such as transfusion or cardiac stress. Together, this information can be used to provide insights into potential underlying biological mechanisms that might explain the increases or decreases in circulating microvesicle levels that have been reported and help to design future clinical studies. © 2015 Authors; published by Portland Press Limited.

  20. Cancer cell-derived lymphotoxin mediates reciprocal tumour-stromal interactions in human ovarian cancer by inducing CXCL11 in fibroblasts.

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    Lau, Tat-San; Chung, Tony Kwok-Hung; Cheung, Tak-Hong; Chan, Loucia Kit-Ying; Cheung, Leonard Wing-Hong; Yim, So-Fan; Siu, Nelson Shing-Shun; Lo, Kwok-Wai; Yu, May Mei-Yung; Kulbe, Hagen; Balkwill, Frances R; Kwong, Joseph

    2014-01-01

    We have investigated the role of cytokine lymphotoxin in tumour-stromal interactions in human ovarian cancer. We found that lymphotoxin overexpression is commonly shared by the cancer cells of various ovarian cancer subtypes, and lymphotoxin-beta receptor (LTBR) is expressed ubiquitously in both the cancer cells and cancer-associated fibroblasts (CAFs). In monoculture, we showed that ovarian cancer cells are not the major lymphotoxin-responsive cells. On the other hand, our co-culture studies demonstrated that the cancer cell-derived lymphotoxin induces chemokine expression in stromal fibroblasts through LTBR-NF-κB signalling. Amongst the chemokines being produced, we found that fibroblast-secreted CXCL11 promotes proliferation and migration of ovarian cancer cells via the chemokine receptor CXCR3. CXCL11 is highly expressed in CAFs in ovarian cancer biopsies, while CXCR3 is found in malignant cells in primary ovarian tumours. Additionally, the overexpression of CXCR3 is significantly associated with the tumour grade and lymph node metastasis of ovarian cancer, further supporting the role of CXCR3, which interacts with CXCL11, in promoting growth and metastasis of human ovarian cancer. Taken together, these results demonstrated that cancer-cell-derived lymphotoxin mediates reciprocal tumour-stromal interactions in human ovarian cancer by inducing CXCL11 in fibroblasts. Our findings suggest that lymphotoxin-LTBR and CXCL11-CXCR3 signalling represent therapeutic targets in ovarian cancer. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  1. A novel role for peptidylarginine deiminases in microvesicle release reveals therapeutic potential of PAD inhibition in sensitizing prostate cancer cells to chemotherapy

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    Sharad Kholia

    2015-06-01

    Full Text Available Introduction: Protein deimination, defined as the post-translational conversion of protein-bound arginine to citrulline, is carried out by a family of 5 calcium-dependent enzymes, the peptidylarginine deiminases (PADs and has been linked to various cancers. Cellular microvesicle (MV release, which is involved in cancer progression, and deimination have not been associated before. We hypothesize that elevated PAD expression, observed in cancers, causes increased MV release in cancer cells and contributes to cancer progression. Background: We have previously reported that inhibition of MV release sensitizes cancer cells to chemotherapeutic drugs. PAD2 and PAD4, the isozymes expressed in patients with malignant tumours, can be inhibited with the pan-PAD-inhibitor chloramidine (Cl-am. We sought to investigate whether Cl-am can inhibit MV release and whether this pathway could be utilized to further increase the sensitivity of cancer cells to drug-directed treatment. Methods: Prostate cancer cells (PC3 were induced to release high levels of MVs upon BzATP stimulation of P2X7 receptors. Western blotting with the pan-protein deimination antibody F95 was used to detect a range of deiminated proteins in cells stimulated to microvesiculate. Changes in deiminated proteins during microvesiculation were revealed by immunoprecipitation and immunoblotting, and mass spectrometry identified deiminated target proteins with putative roles in microvesiculation. Conclusion: We report for the first time a novel function of PADs in the biogenesis of MVs in cancer cells. Our results reveal that during the stimulation of prostate cancer cells (PC3 to microvesiculate, PAD2 and PAD4 expression levels and the deimination of cytoskeletal actin are increased. Pharmacological inhibition of PAD enzyme activity using Cl-am significantly reduced MV release and abrogated the deimination of cytoskeletal actin. We demonstrated that combined Cl-am and methotrexate (MTX treatment of

  2. Adult stromal cells derived from human adipose tissue provoke pancreatic cancer cell death both in vitro and in vivo.

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    Beatrice Cousin

    Full Text Available BACKGROUND: Normal tissue homeostasis is maintained by dynamic interactions between epithelial cells and their microenvironment. Disrupting this homeostasis can induce aberrant cell proliferation, adhesion, function and migration that might promote malignant behavior. Indeed, aberrant stromal-epithelial interactions contribute to pancreatic ductal adenocarcinoma (PDAC spread and metastasis, and this raises the possibility that novel stroma-targeted therapies represent additional approaches for combating this malignant disease. The aim of the present study was to determine the effect of human stromal cells derived from adipose tissue (ADSC on pancreatic tumor cell proliferation. PRINCIPAL FINDINGS: Co-culturing pancreatic tumor cells with ADSC and ADSC-conditioned medium sampled from different donors inhibited cancer cell viability and proliferation. ADSC-mediated inhibitory effect was further extended to other epithelial cancer-derived cell lines (liver, colon, prostate. ADSC conditioned medium induced cancer cell necrosis following G1-phase arrest, without evidence of apoptosis. In vivo, a single intra-tumoral injection of ADSC in a model of pancreatic adenocarcinoma induced a strong and long-lasting inhibition of tumor growth. CONCLUSION: These data indicate that ADSC strongly inhibit PDAC proliferation, both in vitro and in vivo and induce tumor cell death by altering cell cycle progression. Therefore, ADSC may constitute a potential cell-based therapeutic alternative for the treatment of PDAC for which no effective cure is available.

  3. PlGF Knockdown Decreases Tumorigenicity and Stemness Properties of Spheroid Body Cells Derived from Gastric Cancer Cells.

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    Mahmoodi, Fatemeh; Akrami, Hassan

    2017-04-01

    Placental growth factor (PlGF) a member of the vascular endothelial growth factor family regulates some cell processes such as survival, growth of vascular endothelial cells, invasiveness, and also involves in pathological angiogenesis and metastasis in most cancers. Cancer stem cells are believed to be the main reason for the tumor relapse and resistance to therapy. These cells have various characteristics as same as normal tissue-specific adult stem cells including self-renewability and potent to differentiate into various cell types. However, the function of PlGF in gastric cancer stem cells is not well understood. We have investigated the effect of PlGF knockdown on the tumorigenicity and stem cell properties of spheroid body cells derived from two human gastric cancer cell lines. In this study, we isolated spheroid body cells which have stemness properties from MKN-45 and AGS without using growth factors. Validation of spheroid body cells was confirmed by various methods. Then the effects of PlGF knockdown were investigated on in vitro tumorigenicity, differentiation, migration, angiogenesis, and transcription levels of stemness markers of spheroid body cells. Our findings indicated that isolation of spheroid body cells from MKN-45 and AGS cells without using growth factors is an easy and inexpensive method to isolate cancer stem cells and knockdown of PlGF in spheroid body cells reduced in vitro tumorigenicity and stemness properties of spheroid body cells such as Self-renewal ability, colony forming, migratory, and MMPs activities and decreased ability to differentiation and angiogenesis. J. Cell. Biochem. 118: 851-859, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. Optimization of the tissue source, malignancy, and initial substrate of tumor cell-derived matrices to increase cancer cell chemoresistance against 5-fluorouracil.

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    Hoshiba, Takashi; Tanaka, Masaru

    2015-02-13

    The low chemoresistance of in vitro cancer cells inhibits the development of new anti-cancer drugs. Thus, development of a new in vitro culture system is required to increase the chemoresistance of in vitro cancer cells. Tumor cell-derived matrices have been reported to increase the chemoresistance of in vitro cancer cells. However, it remains unclear how tissue sources and the malignancy of cells used for the preparation of matrices affect the chemoresistance of tumor cell-derived matrices. Moreover, it remains unclear how the initial substrates used for the preparation of matrices affect the chemoresistance. In this study, we compared the effects of tissue sources and the malignancy of tumor cells, as well as the effect of the initial substrates on chemoresistance against 5-fluorouracil (5-FU). The chemoresistance of breast and colon cancer cells against 5-FU increased on matrices prepared with cells derived from the corresponding original tissues with higher malignancy. Moreover, the chemoresistance against 5-FU was altered on matrices prepared using different initial substrates that exhibited different characteristics of protein adsorption. Taken together, these results indicated that the appropriate selection of tissue sources, malignancy of tumor cells, and initial substrates used for matrix preparation is important for the preparation of tumor cell-derived matrices for chemoresistance assays. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Treatment of distant metastases from follicular cell-derived thyroid cancer.

    Science.gov (United States)

    Schlumberger, Martin; Leboulleux, Sophie

    2015-01-01

    Distant metastases from thyroid cancer of follicular origin are uncommon. Treatment includes levothyroxine administration at suppressive doses, focal treatment modalities with surgery, external radiation therapy and thermal ablation, and radioiodine in patients with uptake of (131)I in their metastases. Two thirds of distant metastases will become refractory to radioiodine at some point, and when there is a significant tumor burden and documented progression on imaging, a treatment with a kinase inhibitor may provide benefits.

  6. Stromal Cells Derived from Visceral and Obese Adipose Tissue Promote Growth of Ovarian Cancers.

    Directory of Open Access Journals (Sweden)

    Yan Zhang

    Full Text Available Obesity, and in particular visceral obesity, has been associated with an increased risk of developing cancers as well as higher rates of mortality following diagnosis. The impact of obesity on adipose-derived stromal cells (ASC, which contribute to the formation of tumor stroma, is unknown. Here we hypothesized that visceral source and diet-induced obesity (DIO changes the ASC phenotype, contributing to the tumor promoting effects of obesity. We found that ASC isolated from subcutaneous (SC-ASC and visceral (V-ASC white adipose tissue(WAT of lean(Le and obese(Ob mice exhibited similar mesenchymal cell surface markers expression, and had comparable effects on ovarian cancer cell proliferation and migration. Obese and visceral derived ASC proliferated slower and exhibited impaired differentiation into adipocytes and osteocytes in vitro as compared to ASC derived from subcutaneous WAT of lean mice. Intraperitoneal co-injection of ovarian cancer cells with obese or visceral derived ASC, but not lean SC-ASC, increased growth of intraperitoneal ID8 tumors as compared to controls. Obese and V-ASC increased stromal infiltration of inflammatory cells, including CD3+ T cells and F4/80+ macrophages. Obese and visceral derived ASC, but not lean SC-ASC, increased expression of chemotactic factors IL-6, MIP-2, and MCP-1 when cultured with tumor cells. Overall, these results demonstrate that obese and V-ASC have a unique phenotype, with more limited proliferation and differentiation capacity but enhanced expression of chemotactic factors in response to malignant cells which support infiltration of inflammatory cells and support tumor growth and dissemination.

  7. Stromal Cells Derived from Visceral and Obese Adipose Tissue Promote Growth of Ovarian Cancers.

    Science.gov (United States)

    Zhang, Yan; Nowicka, Aleksandra; Solley, Travis N; Wei, Caimiao; Parikh, Aaroh; Court, Laurence; Burks, Jared K; Andreeff, Michael; Woodward, Wendy A; Dadbin, Ali; Kolonin, Mikhail G; Lu, Karen H; Klopp, Ann H

    2015-01-01

    Obesity, and in particular visceral obesity, has been associated with an increased risk of developing cancers as well as higher rates of mortality following diagnosis. The impact of obesity on adipose-derived stromal cells (ASC), which contribute to the formation of tumor stroma, is unknown. Here we hypothesized that visceral source and diet-induced obesity (DIO) changes the ASC phenotype, contributing to the tumor promoting effects of obesity. We found that ASC isolated from subcutaneous (SC-ASC) and visceral (V-ASC) white adipose tissue(WAT) of lean(Le) and obese(Ob) mice exhibited similar mesenchymal cell surface markers expression, and had comparable effects on ovarian cancer cell proliferation and migration. Obese and visceral derived ASC proliferated slower and exhibited impaired differentiation into adipocytes and osteocytes in vitro as compared to ASC derived from subcutaneous WAT of lean mice. Intraperitoneal co-injection of ovarian cancer cells with obese or visceral derived ASC, but not lean SC-ASC, increased growth of intraperitoneal ID8 tumors as compared to controls. Obese and V-ASC increased stromal infiltration of inflammatory cells, including CD3+ T cells and F4/80+ macrophages. Obese and visceral derived ASC, but not lean SC-ASC, increased expression of chemotactic factors IL-6, MIP-2, and MCP-1 when cultured with tumor cells. Overall, these results demonstrate that obese and V-ASC have a unique phenotype, with more limited proliferation and differentiation capacity but enhanced expression of chemotactic factors in response to malignant cells which support infiltration of inflammatory cells and support tumor growth and dissemination.

  8. B-cell-derived lymphotoxin promotes castration-resistant prostate cancer.

    Science.gov (United States)

    Ammirante, Massimo; Luo, Jun-Li; Grivennikov, Sergei; Nedospasov, Sergei; Karin, Michael

    2010-03-11

    Prostate cancer (CaP) progresses from prostatic intraepithelial neoplasia through locally invasive adenocarcinoma to castration-resistant metastatic carcinoma. Although radical prostatectomy, radiation and androgen ablation are effective therapies for androgen-dependent CaP, metastatic castration-resistant CaP is a major complication with high mortality. Androgens stimulate growth and survival of prostate epithelium and early CaP. Although most patients initially respond to androgen ablation, many develop castration-resistant CaP within 12-18 months. Despite extensive studies, the mechanisms underlying the emergence of castration-resistant CaP remain poorly understood and their elucidation is critical for developing improved therapies. Curiously, castration-resistant CaP remains androgen-receptor dependent, and potent androgen-receptor antagonists induce tumour regression in castrated mice. The role of inflammation in castration-resistant CaP has not been addressed, although it was reported that intrinsic NF-kappaB activation supports its growth. Inflammation is a localized protective reaction to injury or infection, but it also has a pathogenic role in many diseases, including cancer. Whereas acute inflammation is critical for host defence, chronic inflammation contributes to tumorigenesis and metastatic progression. The inflammation-responsive IkappaB kinase (IKK)-beta and its target NF-kappaB have important tumour-promoting functions within malignant cells and inflammatory cells. The latter, including macrophages and lymphocytes, are important elements of the tumour microenvironment, but the mechanisms underlying their recruitment remain obscure, although they are thought to depend on chemokine and cytokine production. We found that CaP progression is associated with inflammatory infiltration and activation of IKK-alpha, which stimulates metastasis by an NF-kappaB-independent, cell autonomous mechanism. Here we show that androgen ablation causes infiltration of

  9. Differentiating sepsis from non-infectious systemic inflammation based on microvesicle-bacteria aggregation

    Science.gov (United States)

    Herrmann, I. K.; Bertazzo, S.; O'Callaghan, D. J. P.; Schlegel, A. A.; Kallepitis, C.; Antcliffe, D. B.; Gordon, A. C.; Stevens, M. M.

    2015-08-01

    Sepsis is a severe medical condition and a leading cause of hospital mortality. Prompt diagnosis and early treatment has a significant, positive impact on patient outcome. However, sepsis is not always easy to diagnose, especially in critically ill patients. Here, we present a conceptionally new approach for the rapid diagnostic differentiation of sepsis from non-septic intensive care unit patients. Using advanced microscopy and spectroscopy techniques, we measure infection-specific changes in the activity of nano-sized cell-derived microvesicles to bind bacteria. We report on the use of a point-of-care-compatible microfluidic chip to measure microvesicle-bacteria aggregation and demonstrate rapid (sepsis diagnosis and introduces microvesicle-bacteria aggregation as a potentially useful parameter for making early clinical management decisions.Sepsis is a severe medical condition and a leading cause of hospital mortality. Prompt diagnosis and early treatment has a significant, positive impact on patient outcome. However, sepsis is not always easy to diagnose, especially in critically ill patients. Here, we present a conceptionally new approach for the rapid diagnostic differentiation of sepsis from non-septic intensive care unit patients. Using advanced microscopy and spectroscopy techniques, we measure infection-specific changes in the activity of nano-sized cell-derived microvesicles to bind bacteria. We report on the use of a point-of-care-compatible microfluidic chip to measure microvesicle-bacteria aggregation and demonstrate rapid (sepsis diagnosis and introduces microvesicle-bacteria aggregation as a potentially useful parameter for making early clinical management decisions. Electronic supplementary information (ESI) available: Fig. S1: Markers of inflammation and microvesicle characteristics in patient plasma samples, Fig. S2: Experimental sepsis model, Table S1: Patient characteristics. Table S2: Inclusion/exclusion criteria. See DOI: 10.1039/c5nr01851j

  10. Microvesicles derived from human Wharton's jelly mesenchymal stem cells promote human renal cancer cell growth and aggressiveness through induction of hepatocyte growth factor.

    Directory of Open Access Journals (Sweden)

    Tao Du

    Full Text Available In our previous study, microvesicles (MVs released from human Wharton's jelly mesenchymal stem cells (hWJ-MSCs retard the growth of bladder cancer cells. We would like to know if MVs have a similar effect on human renal cell carcinoma (RCC. By use of cell culture and the BALB/c nu/nu mice xeno-graft model, the influence of MVs upon the growth and aggressiveness of RCC (786-0 was assessed. Cell counting kit-8 (CCK-8 assay, incidence of tumor, tumor size, Ki-67 or TUNEL staining was used to evaluate tumor cell growth in vitro or in vivo. Flow cytometry assay (in vitro or examination of cyclin D1 expression (in vivo was carried out to determine the alteration of cell cycle. The aggressiveness was analyzed by Wound Healing Assay (in vitro or MMP-2 and MMP-9 expression (in vivo. AKT/p-AKT, ERK1/2/p-ERK1/2 or HGF/c-MET expression was detected by real-time PCR or western blot. Our data demonstrated that MVs promote the growth and aggressiveness of RCC both in vitro and in vivo. In addition, MVs facilitated the progression of cell cycle from G0/1 to S. HGF expression in RCC was greatly induced by MVs, associated with activation of AKT and ERK1/2 signaling pathways. RNase pre-treatment abrogated all effects of MVs. In summary, induction of HGF synthesis via RNA transferred by MVs activating AKT and ERK1/2 signaling is one of crucial contributors to the pro-tumor effect.

  11. In Vivo Efficacy of Umbilical Cord Blood Stem Cell-Derived NK Cells in the Treatment of Metastatic Colorectal Cancer.

    Science.gov (United States)

    Veluchamy, John P; Lopez-Lastra, Silvia; Spanholtz, Jan; Bohme, Fenna; Kok, Nina; Heideman, Daniëlle A M; Verheul, Henk M W; Di Santo, James P; de Gruijl, Tanja D; van der Vliet, Hans J

    2017-01-01

    Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) act by inhibiting EGFR downstream signaling and by eliciting a natural killer (NK) cell-mediated antitumor response. The IgG1 mAb cetuximab has been used for treatment of RAS(wt) metastatic colorectal cancer (mCRC) patients, showing limited efficacy. In the present study, we address the potential of adoptive NK cell therapy to overcome these limitations investigating two allogeneic NK cell products, i.e., allogeneic activated peripheral blood NK cells (A-PBNK) and umbilical cord blood stem cell-derived NK cells (UCB-NK). While cetuximab monotherapy was not effective against EGFR(-) RAS(wt), EGFR(+) RAS(mut), and EGFR(+) BRAF(mut) cells, A-PBNK were able to initiate lysis of EGFR(+) colon cancer cells irrespective of RAS or BRAF status. Cytotoxic effects of A-PBNK (but not UCB-NK) were further potentiated significantly by coating EGFR(+) colon cancer cells with cetuximab. Of note, a significantly higher cytotoxicity was induced by UCB-NK in EGFR(-)RAS(wt) (42 ± 8 versus 67 ± 7%), EGFR(+) RAS(mut) (20 ± 2 versus 37 ± 6%), and EGFR(+) BRAF(mut) (23 ± 3 versus 43 ± 7%) colon cancer cells compared to A-PBNK and equaled the cytotoxic efficacy of the combination of A-PBNK and cetuximab. The antitumor efficacy of UCB-NK cells against cetuximab-resistant human EGFR(+) RAS(mut) colon cancer cells was further confirmed in an in vivo preclinical mouse model where UCB-NK showed enhanced antitumor cytotoxicity against colon cancer independent of EGFR and RAS status. As UCB-NK have been proven safe in a recently conducted phase I clinical trial in acute myeloid leukemia, a fast translation into clinical proof of concept for mCRC could be considered.

  12. Time-lapse imaging of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer.

    Science.gov (United States)

    Nakles, Rebecca E; Millman, Sarah L; Cabrera, M Carla; Johnson, Peter; Mueller, Susette; Hoppe, Philipp S; Schroeder, Timm; Furth, Priscilla A

    2013-02-08

    Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 μm x 700 μm fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

  13. Extracellular Disposal of Tumor-Suppressor miRs-145 and -34a via Microvesicles and 5-FU Resistance of Human Colon Cancer Cells

    Directory of Open Access Journals (Sweden)

    Yukihiro Akao

    2014-01-01

    Full Text Available The dysregulation of microRNA (miRNA expression causes various kinds of diseases. Especially, alterations in miRNA expression levels are frequently observed in human tumor cells and are associated with cancer pathogenesis. Earlier we established Fluorouracil (5-FU-resistant human colon cancer DLD-1 cells (DLD-1/5FU from parental 5-FU- sensitive DLD-1 cells. In the present study, we examined the expression of miRNA in each cell line and in its extracellular microvesicles (MVs before and after treatment with 5-FU. The nascent RNAs of anti-oncogenic miR-34a and -145 labeled with EU in both cells were proved to be transferred into MVs in both cell lines. The levels of miR-34a and -145 in the cells and in their MVs were not largely different in the two cell lines, and a substantial amount of both miRNAs was secreted by both cell lines even in the steady-state condition. The exposure of both cell lines to 5-FU significantly increased the intracellular levels of miR-145 and miR-34a in the 5-FU-sensitive DLD-1 cells, whereas the level of neither miR was elevated in the DLD-1/5FU cells. Interestingly, the amount of miR-145 detected in the small MVs shed into the medium of the parental cells was reduced after the treatment with 5-FU. On the other hand, the intracellular expression of miR-34a in the DLD-1/5FU cells was down-regulated compared with that in the parental DLD-1 cells even in the steady-state condition. As to the miR-34a secreted into MVs, the increase in the level in DLD-1/5FU cells was greater than that in the parental DLD-1 cells after the treatment with 5-FU. Thus, the intra- and extracellular miR-145 and -34a were closely associated with 5-FU resistance, and the resistance was in part due to the enhanced secretion of miR-145 and -34a via MVs, resulting in low intracellular levels of both miRNAs.

  14. Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases*

    Science.gov (United States)

    Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J.; Nie, Guangjun

    2016-01-01

    Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. PMID:26895960

  15. Therapeutic effect of human iPS-cell-derived myeloid cells expressing IFN-β against peritoneally disseminated cancer in xenograft models.

    Science.gov (United States)

    Koba, Chihiro; Haruta, Miwa; Matsunaga, Yusuke; Matsumura, Keiko; Haga, Eriko; Sasaki, Yuko; Ikeda, Tokunori; Takamatsu, Koutaro; Nishimura, Yasuharu; Senju, Satoru

    2013-01-01

    We recently developed a method to generate myeloid cells with proliferation capacity from human iPS cells. iPS-ML (iPS-cell-derived myeloid/macrophage line), generated by introducing proliferation and anti-senescence factors into iPS-cell-derived myeloid cells, grew continuously in an M-CSF-dependent manner. A large number of cells exhibiting macrophage-like properties can be readily obtained by using this technology. In the current study, we evaluated the possible application of iPS-ML in anti-cancer therapy. We established a model of peritoneally disseminated gastric cancer by intraperitoneally injecting NUGC-4 human gastric cancer cells into SCID mice. When iPS-ML were injected intraperitoneally into the mice with pre-established peritoneal NUGC-4 tumors, iPS-ML massively accumulated and infiltrated into the tumor tissues. iPS-ML expressing IFN-β (iPS-ML/IFN-β) significantly inhibited the intra-peritoneal growth of NUGC-4 cancer. Furthermore, iPS-ML/IFN-β also inhibited the growth of human pancreatic cancer MIAPaCa-2 in a similar model. iPS-ML are therefore a promising treatment agent for peritoneally disseminated cancers, for which no standard treatment is currently available.

  16. Expression of cancer cell-derived IgG and extra domain A-containing fibronectin in salivary adenoid cystic carcinoma.

    Science.gov (United States)

    Lv, Wan-Qi; Peng, Jing; Wang, Hai-Cheng; Chen, De-Ping; Yang, Yue; Zhao, Yang; Qiu, Xiao-Yan; Jiang, Jiu-Hui; Li, Cui-Ying

    2017-09-01

    Cancer-IgG is a newly-discovered molecule, mainly derived from epithelial carcinoma cells and is significantly correlated with differentiation, metastasis, local invasion, and poor prognosis of many cancers. In our previous study we detected IgG expression in oral epithelial carcinoma, including salivary adenoid cystic carcinoma (SACC), using an IgG-specific commercial antibody. Here, we explored the correlation between cancer-IgG and clinicopathological features of SACC. A total of 68 human SACC tissue specimens and 2 siRNAs were used to analyze the correlation between cancer-IgG and extra domain A (EDA(+))-containing fibronectin using the cancer-IgG-specific monoclonal antibody, RP215. We found an unexpected correlation between cancer-IgG and EDA(+) fibronectin, both of which showed aberrant expression in SACC tissue samples. Both were highly expressed in SACC with nerve invasion. In our previous study, EDA(+) fibronectin overexpression in SACC cells decreased N-cadherin expression. In the present study, we used SACC-83 cells, wherein EDA(+) fibronectin is overexpressed and cancer-IgG is knocked down. EDA(+) fibronectin expression was reduced with cancer-IgG knockdown, while cancer-IgG expression did not affect EDA(+) fibronectin overexpression. Furthermore, knockdown of non-B cell-derived IgG in SACC cells decreased cellular motility (PIgG potentially regulates EDA(+) fibronectin expression, thereby suggesting possible new therapeutic approaches for SACC. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. The effect of corn trypsin inhibitor, anti-tissue factor pathway inhibitor antibodies and phospholipids on microvesicle-associated thrombin generation in patients with pancreatic cancer and healthy controls.

    Science.gov (United States)

    Hellum, Marit; Franco-Lie, Isabel; Øvstebø, Reidun; Hauge, Truls; Henriksson, Carola E

    2017-01-01

    Circulating microvesicles (MVs) are suggested to be important contributors to cancer-associated thrombosis due to the presence of surface-bound procoagulant molecules like tissue factor (TF) and phosphatidylserine (PS). Pancreatic cancer is considered to be one of the most prothrombotic malignancies. The aim of this study was to describe the impact of analytical variables on MV-associated thrombin generation in patients with pancreatic cancer and in healthy controls. MVs were isolated from citrated plasma and added to pooled normal plasma (PNP). Thrombin generation was measured by the calibrated automated thrombogram. The impact of corn trypsin inhibitor (CTI), anti-tissue factor pathway inhibitor (TFPI) antibodies and phospholipids was described. Antibodies against TF were used to assess TF-dependency, and MV-bound PS activity was measured with the Zymuphen MP-activity kit. MVs from the pancreatic cancer patients displayed higher thrombin generation and higher PS-activity than MVs from the healthy control group, while TF-dependency was observed in only 1 out of 13 patient samples. Adequate thrombin generation-curves were only achieved when CTI was omitted and anti-TFPI antibodies were added to PNP prepared in low contact-activating tubes. Addition of phospholipids reduced the significant differences between the two groups, and should be omitted. This modified thrombin generation assay could be useful for measurement of procoagulant circulating MVs, allowing the contribution from MVs affecting both the intrinsic and the extrinsic pathway to be measured.

  18. Characterization of membrane-shed micro-vesicles from cytokine-stimulated beta-cells using proteomics strategies

    DEFF Research Database (Denmark)

    Palmisano, Giuseppe; Jensen, Soren Skov; Le Bihan, Marie Catherine

    2012-01-01

    Micro-particles and exosomes are two of the most well characterized membrane-derived micro-vesicles released either directly from the plasma membrane or released through the fusion of intracellular multi-vesicular bodies with the plasma membrane, respectively. They are thought to be involved...... in many significant biological processes such as cell-to-cell communication, rescue from apoptosis and immunological responses. Here we report for the first time a quantitative study of proteins from beta-cell-derived micro-vesicles generated after cytokine induced apoptosis using stable-isotope labeled...... amino acids in cell culture (SILAC) combined with mass spectrometry. We identified and quantified a large number of beta-cell specific proteins and proteins previously described in micro-vesicles from other cell types in addition to new proteins located to these vesicles. In addition, we quantified...

  19. Release of toxic microvesicles by Actinobacillus actinomycetemcomitans.

    OpenAIRE

    Nowotny, A; Behling, U H; Hammond, B.; Lai, C H; Listgarten, M; Pham, P H; Sanavi, F

    1982-01-01

    Oral isolates of Actinobacillus actinomycetemcomitans (strain Y4) release spherical microvesicles in large numbers during normal growth. The biological activities of these products were studied, and it was estimated that approximately 1/10 of their dry weight was made up of heat- and proteolysis-resistant endotoxin. The chicken embryo lethality and bone-resorbing activity of the microvesicles were heat stable but proteolysis sensitive. Other laboratories have reported the presence of a heat- ...

  20. Susceptibility to cytotoxic T cell lysis of cancer stem cells derived from cervical and head and neck tumor cell lines.

    Science.gov (United States)

    Liao, Tian; Kaufmann, Andreas M; Qian, Xu; Sangvatanakul, Voramon; Chen, Chao; Kube, Tina; Zhang, Guoyou; Albers, Andreas E

    2013-01-01

    To explore cancer stem cell susceptibility to a host's cytotoxic T lymphocyte (CTL)-mediated immune response. We compared the susceptibility of putative CSC generated from cancer cell lines to immunologic recognition and killing by alloantigen-specific CD8(+) CTL. CSC-enriched spheroid culture-derived cells (SDC) exhibited higher expression of ALDH, ICAM1 and of stem/progenitor cell markers on all 3 tumor cell lines investigated and lower MHC class I on the cervical cancer cell line as compared to their monolayer-derived cells (MDC). The expression of ICAM1 and MHCI was upregulated by IFN-γ treatment. CSC populations were less sensitive to MHC class I-restricted alloantigen-specific CD8(+) CTL lysis as compared to matched MDC. IFN-γ pretreatment resulted in over-proportionally enhanced lysis of SDC. Finally, the subset of ALDH(high) expressing SDC presented more sensitivity toward CD8(+) CTL killing than the ALDH(low) SDC. Tumor therapy resistance has been attributed to cancer stem cells (CSC). We show in vitro susceptibility of CSC to CTL-mediated lysis. Immunotherapy targeting of ALDH(+) CSC may therefore be a promising approach. Our results and method may be helpful for the development and optimization of adjuvants, as here exemplified for INF-γ, for CSC-targeted vaccines, independent of the availability of CSC-specific antigens.

  1. Gene expression changes after ionizing radiation in endothelial cells derived from human endometrial cancer-preliminary outcomes.

    Science.gov (United States)

    Liu, Ting; Du, Xuelian; Sheng, Xiugui

    2014-06-01

    Accumulating evidence has demonstrated that death of microvascular endothelial cells plays a decisive role in the tumor response against radiotherapy. Nevertheless, radiation-induced gene alterations on cancer-associated endothelial cells of human endometrial carcinoma remain poorly understood. The purpose of this study was to elucidate the gene expression changes after X-ray radiation in human endometrial carcinoma vascular endothelial cells and to provide new targets for combined treatment of radiation and anti-angiogenesis in human endometrial carcinoma. Endometrial cancer-derived endothelial cells, which obtained before and 4 h after 400 cGy X-ray radiation from four endometrial carcinomas, were analyzed by gene expression profile. The selected meaningful genes from gene microarray experiments were validated by real-time quantitative PCR. Microarray analyses showed 49 significantly changed genes which were common to all the microarray experiments. There into, 14 genes were found to be in persistent up-regulation and 14 in persistent down-regulation 4 h after X-ray radiation when compared with the control group. These genes were involved in cell cycle and growth regulation, cell-apoptosis, chemokine, cell signaling, cellular stress response, angiogenesis, DNA synthesis and repair and cell adhesion. Eight randomly selected genes were validated by real-time PCR. The genes of cancer-derived endothelial cells regulated by X-ray radiation as well as their related signal pathways, which obtained from gene expression profiling data, were relevant to radiosensitivity of endometrial cancer. This study shows that the identified genes and their related signaling pathways are candidated biomarkers for radiation and anti-angiogenesis of human endometrial carcinoma.

  2. Hybrid cells derived from breast epithelial cell/breast cancer cell fusion events show a differential RAF-AKT crosstalk

    Directory of Open Access Journals (Sweden)

    Özel Cem

    2012-04-01

    Full Text Available Abstract Background The biological phenomenon of cell fusion has been linked to several characteristics of tumour progression, including an enhanced metastatogenic capacity and an enhanced drug resistance of hybrid cells. We demonstrated recently that M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics spontaneously fused with MDA-MB-435-Hyg breast cancer cells, thereby giving rise to stable M13MDA435 hybrid cells, which are characterised by a unique gene expression profile and migratory behaviour. Here we investigated the involvement of the PLC-β/γ1, PI3K/AKT and RAS-RAF-ERK signal transduction cascades in the EGF and SDF-1α induced migration of two M13MDA435 hybrid cell clones in comparison to their parental cells. Results Analysis of the migratory behaviour by using the three-dimensional collagen matrix migration assay showed that M13SV1-EGFP-Neo cells as well as M13MDA435 hybrid cells, but not the breast cancer cell line, responded to EGF stimulation with an increased locomotory activity. By contrast, SDF-1α solely stimulated the migration of M13SV1-EGFP-Neo cells, whereas the migratory activity of the other cell lines was blocked. Analysis of signal transduction cascades revealed a putative differential RAF-AKT crosstalk in M13MDA435-1 and -3 hybrid cell clones. The PI3K inhibitor Ly294002 effectively blocked the EGF induced migration of M13MDA435-3 hybrid cells, whereas the EGF induced locomotion of M13MDA435-1 hybrid cells was markedly increased. Analysis of RAF-1 S259 phosphorylation, being a major mediator of the negative regulation of RAF-1 by AKT, showed decreased pRAF-1 S259 levels in LY294002 treated M13MDA435-1 hybrid cells. By contrast, pRAF-1 S259 levels remained unaltered in the other cell lines. Inhibition of PI3K/AKT signalling by Ly294002 relieves the AKT mediated phosphorylation of RAF-1, thereby restoring MAPK signalling. Conclusions Here we show that hybrid cells could evolve exhibiting a

  3. Label-free real-time acoustic sensing of microvesicle release from prostate cancer (PC3) cells using a Quartz Crystal Microbalance

    Energy Technology Data Exchange (ETDEWEB)

    Stratton, Dan [Cellular and Molecular Immunology Research Centre, School of Human Sciences, London Metropolitan University, London (United Kingdom); Lange, Sigrun [University College London School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX (United Kingdom); Kholia, Sharad; Jorfi, Samireh; Antwi-Baffour, Samuel [Cellular and Molecular Immunology Research Centre, School of Human Sciences, London Metropolitan University, London (United Kingdom); Inal, Jameel, E-mail: j.inal@londonmet.ac.uk [Cellular and Molecular Immunology Research Centre, School of Human Sciences, London Metropolitan University, London (United Kingdom)

    2014-10-24

    Highlights: • Microvesiculating cells record loss of mass on a Quartz Crystal Microbalance. • Using the Quartz Crystal Microbalance microvesicles are measured at 0.24 pg. • The QCM-D reveals loss in viscoelastic properties in microvesiculating cells. - Abstract: Using a Quartz Crystal Microbalance with dissipation monitoring, QCM-D (label-free system) measuring changes in resonant frequency (Δf) that equate to mass deposited on a sensor, we showed the attachment, over a 60 min period, of a monolayer of PC3 cells to the gold electrodes of the quartz crystal sensor, which had been rendered hydrophilic. That MVs were released upon BzATP stimulation of cells was confirmed by NTA analysis (average 250 nm diameter), flow cytometry, showing high phosphatidylserine exposition and by fluorescent (Annexin V Alexa Fluor® 488-positive) and electron microscopy. Over a period of 1000s (16.7 min) during which early apoptosis increased from 4% plateauing at 10% and late apoptosis rose to 2%, the Δf increased 20 Hz, thereupon remaining constant for the last 1000s of the experiment. Using the Sauerbrey equation, the loss in mass, which corresponded to the release of 2.36 × 10{sup 6} MVs, was calculated to be 23 ng. We therefore estimated the mass of an MV to be 0.24 pg. With the deposition on the QCM-D of 3.5 × 10{sup 7} MVs over 200s, the decrease in Δf (Hz) gave an estimate of 0.235 pg per MV.

  4. Historically aggressive types of follicular cell-derived thyroid cancer often have radioactive avid distant metastases: a study of 314 patients with distant metastases at a single institution

    Energy Technology Data Exchange (ETDEWEB)

    Tala, H.P.; Rondeau, G.; Fagin, J.A.; Tuttle, R.M. [Endocrinology Division, Department of Medicine, Nuclear Medicine Division, Memorial Sloan Kettering Cancer Center, New-York (United States); Ghossein, R.A. [Pathology Department, Nuclear Medecine Division, Memorial Sloan Kettering Cancer Center, New-York (United States); Grewal, R.K.; Larson, S.M. [Radiology Department, Nuclear Medicine Division, Memorial Sloan Kettering Cancer Center, New-York (United States)

    2012-07-01

    Radioactive iodine (RAI) remains one of the primary treatment options for metastatic, follicular cell derived thyroid cancers. The aim of this study was to determine the likelihood that metastatic lesions arising from one of the aggressive thyroid cancer histologies [tall cell variant of papillary thyroid carcinoma (TCV-PTC), poorly differentiated thyroid carcinoma (PDTC) and Hurthle cell carcinoma (HCC)] would demonstrate sufficient RAI avidity for visualization on RAI scanning and therefore could potentially benefit from RAI therapy. The study shows that in patients selected for RAI scanning or therapy at our center, RAI avid lesions can be identified in more than two thirds of the patients with distant metastases arising in the setting of C-PTC, WD-FTC, FV-PTC, TCV-PTC, or PDTC primary tumors. While RAI avidity on a post-therapy scan does not always correlate with clinically significant tumor killing activity, it is likely that some of these patients with RAI avid metastatic disease did obtain a clinical benefit

  5. Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions.

    Science.gov (United States)

    de Lourdes Mora-García, María; García-Rocha, Rosario; Morales-Ramírez, Omar; Montesinos, Juan José; Weiss-Steider, Benny; Hernández-Montes, Jorge; Ávila-Ibarra, Luis Roberto; Don-López, Christian Azucena; Velasco-Velázquez, Marco Antonio; Gutiérrez-Serrano, Vianey; Monroy-García, Alberto

    2016-10-26

    In recent years, immunomodulatory mechanisms of mesenchymal stem/stromal cells (MSCs) from bone marrow and other "classic" sources have been described. However, the phenotypic and functional properties of tumor MSCs are poorly understood. The aim of this study was to analyze the immunosuppressive capacity of cervical cancer-derived MSCs (CeCa-MSCs) on effector T lymphocytes through the purinergic pathway. We determined the expression and functional activity of the membrane-associated ectonucleotidases CD39 and CD73 on CeCa-MSCs and normal cervical tissue-derived MSCs (NCx-MSCs). We also analyzed their immunosuppressive capacity to decrease proliferation, activation and effector cytotoxic T (CD8+) lymphocyte function through the generation of adenosine (Ado). We detected that CeCa-MSCs express higher levels of CD39 and CD73 ectonucleotidases in cell membranes compared to NCx-MSCs, and that this feature was associated with the ability to strongly suppress the proliferation, activation and effector functions of cytotoxic T-cells through the generation of large amounts of Ado from the hydrolysis of ATP, ADP and AMP nucleotides. This study suggests that CeCa-MSCs play an important role in the suppression of the anti-tumor immune response in CeCa through the purinergic pathway.

  6. Phosphoproteome Characterization of Human Colorectal Cancer SW620 Cell-Derived Exosomes and New Phosphosite Discovery for C-HPP.

    Science.gov (United States)

    Guo, Jiahui; Cui, Yizhi; Yan, Ziqi; Luo, Yanzhang; Zhang, Wanling; Deng, Suyuan; Tang, Shengquan; Zhang, Gong; He, Qing-Yu; Wang, Tong

    2016-11-04

    Identification of all phosphorylation forms of known proteins is a major goal of the Chromosome-Centric Human Proteome Project (C-HPP). Recent studies have found that certain phosphoproteins can be encapsulated in exosomes and function as key regulators in tumor microenvironment, but no deep coverage phosphoproteome of human exosomes has been reported to date, which makes the exosome a potential source for the new phosphosite discovery. In this study, we performed highly optimized MS analyses on the exosomal and cellular proteins isolated from human colorectal cancer SW620 cells. With stringent data quality control, 313 phosphoproteins with 1091 phosphosites were confidently identified from the SW620 exosome, from which 202 new phosphosites were detected. Exosomal phosphoproteins were significantly enriched in the 11q12.1-13.5 region of chromosome 11 and had a remarkably high level of tyrosine-phosphorylated proteins (6.4%), which were functionally relevant to ephrin signaling pathway-directed cytoskeleton remodeling. In conclusion, we here report the first high-coverage phosphoproteome of human cell-secreted exosomes, which leads to the identification of new phosphosites for C-HPP. Our findings provide insights into the exosomal phosphoprotein systems that help to understand the signaling language being delivered by exosomes in cell-cell communications. The mass spectrometry proteomics data have been deposited to the ProteomeXchange consortium with the data set identifier PXD004079, and iProX database (accession number: IPX00076800).

  7. Cisplatin targets the stromal cell-derived factor-1-CXC chemokine receptor type 4 axis to suppress metastasis and invasion of ovarian cancer-initiating cells.

    Science.gov (United States)

    Yu, Zhi-hua; Liu, Te; Zhao, Yan-hui; Huang, Yong-yi; Gao, Yong-tao

    2014-05-01

    In ovarian cancer, CD44+/CD117+ stem cells, also known as cancer-initiating cells (CICs), are highly proliferative and invasive. Therefore, the CD44+/CD117+ subpopulation is thought to be an important target for novel therapeutic strategies. In this study, we investigated the effects of cisplatin (CDDP) on metastasis and invasion suppression of ovarian CICs by targeting the CXC chemokine receptor-4 (CXCR4) signaling pathway in vitro and in vivo. CD44+/CD117+ ovarian CICs were enriched from human primary ovarian tumor tissues and confirmed by flow cytometry sorting. A 3-(4,5-dimethylthiazol-2-yl)-2.5-dipheny-tetrazolium bromide (MTT) assay revealed significant inhibition of proliferation of ovarian CICs with increasing CDDP drug concentrations. Moreover, colony formation and transwell migration assays indicated that CDDP significantly suppressed the invasive capacity of ovarian CICs in vitro. The expression levels of stromal cell-derived factor (SDF)-1, CXCR4, matrix metalloproteinase (MMP) 2, and MMP9 mRNA and protein levels were significantly reduced in CDDP-treated cells compared to untreated ovarian CICs. Furthermore, xenograft experiments confirmed that CDDP suppressed the growth of xenograft tumors formed by ovarian CICs in vivo. In addition, CXCR4 agonist (diprotin A) treatment of ovarian CICs weakened the effects of CDDP and enhanced SDF-1-CXCR4 axis expression in ovarian CICs. Thus, the SDF-1-CXCR4 axis is an important mediator of proliferation and invasion in CXCR4-overexpressing ovarian cancer-initiating cells (OCICs). Furthermore, CDDP inhibits invasion and metastasis of OCICs by targeting SDF-1-CXCR4 axis expression.

  8. A Safety Checkpoint to Eliminate Cancer Risk of the Immune Evasive Cells Derived from Human Embryonic Stem Cells.

    Science.gov (United States)

    He, Jingjin; Rong, Zhili; Fu, Xuemei; Xu, Yang

    2017-05-01

    Human embryonic stem cells (hESCs) hold great promise in the regenerative therapy of many currently untreatable human diseases. One of the key bottlenecks is the immune rejection of hESC-derived allografts by the recipient. To overcome this challenge, we have established new approaches to induce immune protection of hESC-derived allografts through the coexpression of immune suppressive molecules CTLA4-Ig and PD-L1. However, this in turn raises a safety concern of cancer risk because these hESC-derived cells can evade immune surveillance. To address this safety concern, we developed a safety checkpoint so that the immune evasive hESC-derived cells in the graft can be effectively eliminated if any cellular transformation is detected. In this context, we knock-in the suicidal gene herpes simplex virus thymidine kinase (HSVTK) into the constitutive HPRT locus of CP hESCs (knock-in hESCs expressing CTLA4-Ig and PD-L1), denoted CPTK hESCs. Employing humanized mice (Hu-mice) reconstituted with human immune system, we demonstrated that the CPTK hESC-derived cells are protected from immune rejection. In addition, CPTK hESC-derived cells can be efficiently eliminated in vitro and in vivo with FDA approved TK-targeting drug ganciclovir. Therefore, this new safety checkpoint improves the feasibility to use the immune evasive hESC-derived cells for regenerative medicine. Stem Cells 2017;35:1154-1161. © 2017 AlphaMed Press.

  9. CXC chemokine ligand 12/stromal cell-derived factor-1 regulates cell adhesion in human colon cancer cells by induction of intercellular adhesion molecule-1.

    Science.gov (United States)

    Tung, Shui-Yi; Chang, Shun-Fu; Chou, Ming-Hui; Huang, Wen-Shih; Hsieh, Yung-Yu; Shen, Chien-Heng; Kuo, Hsing-Chun; Chen, Cheng-Nan

    2012-10-25

    The CXC chemokine ligand 12 (CXCL12)/stromal cell-derived factor-1 (SDF-1) and CXC receptor 4 (CXCR4) axis is involved in human colorectal cancer (CRC) carcinogenesis and can promote the progression of CRC. Interaction between CRC cells and endothelium is a key event in tumor progression. The aim of this study was to investigate the effect of SDF-1 on the adhesion of CRC cells. Human CRC DLD-1 cells were used to study the effect of SDF-1 on intercellular adhesion molecule-1 (ICAM-1) expression and cell adhesion to endothelium. SDF-1 treatment induced adhesion of DLD-1 cells to the endothelium and increased the expression level of the ICAM-1. Inhibition of ICAM-1 by small interfering RNA (siRNA) and neutralizing antibody inhibited SDF-1-induced cell adhesion. By using specific inhibitors and short hairpin RNA (shRNA), we demonstrated that the activation of ERK, JNK and p38 pathways is critical for SDF-1-induced ICAM-1 expression and cell adhesion. Promoter activity and transcription factor ELISA assays showed that SDF-1 increased Sp1-, C/EBP-β- and NF-κB-DNA binding activities in DLD-1 cells. Inhibition of Sp1, C/EBP-β and NF-κB activations by specific siRNA blocked the SDF-1-induced ICAM-1 promoter activity and expression. The effect of SDF-1 on cell adhesion was mediated by the CXCR4. Our findings support the hypothesis that ICAM-1 up-regulation stimulated by SDF-1 may play an active role in CRC cell adhesion.

  10. Epithelial cell-derived periostin functions as a tumor suppressor in gastric cancer through stabilizing p53 and E-cadherin proteins via the Rb/E2F1/p14ARF/Mdm2 signaling pathway.

    Science.gov (United States)

    Lv, Hongjun; Liu, Rui; Fu, Jiao; Yang, Qi; Shi, Jing; Chen, Pu; Ji, Meiju; Shi, Bingyin; Hou, Peng

    2014-01-01

    Periostin is usually considered as an oncogene in diverse human cancers, including breast, prostate, colon, esophagus, and pancreas cancers, whereas it acts as a tumor suppressor in bladder cancer. In gastric cancer, it has been demonstrated that periglandular periostin expression is decreased whereas stromal periostin expression is significantly increased as compared with normal gastric tissues. Moreover, periostin produced by stromal myofibroblasts markedly promotes gastric cancer cell growth. These observations suggest that periostin derived from different types of cells may play distinct biological roles in gastric tumorigenesis. The aim of this study was to explore the biological functions and related molecular mechanisms of epithelial cell-derived periostin in gastric cancer. Our data showed that periglandular periostin was significantly down-regulated in gastric cancer tissues as compared with matched normal gastric mucosa. In addition, its expression in metastatic lymph nodes was significantly lower than that in their primary cancer tissues. Our data also demonstrated that periglandular periostin expression was negatively associated with tumor stage. More importantly, restoration of periostin expression in gastric cancer cells dramatically suppressed cell growth and invasiveness. Elucidation of the mechanisms involved revealed that periostin restoration enhanced Rb phosphorylation and sequentially activated the transcription of E2F1 target gene p14(ARF), leading to Mdm2 inactivation and the stabilization of p53 and E-cadherin proteins. Strikingly, these effects of periostin were abolished upon Rb deletion. Collectively, we have for the first time demonstrated that epithelial cell-derived periostin exerts tumor-suppressor activities in gastric cancer through stabilizing p53 and E-cadherin proteins via the Rb/E2F1/p14(ARF)/Mdm2 signaling pathway.

  11. Prostate cancer cells stimulated by calcium-mediated activation of protein kinase C undergo a refractory period before re-releasing calcium-bearing microvesicles.

    Science.gov (United States)

    Stratton, Dan; Moore, Colin; Zheng, Lei; Lange, Sigrun; Inal, Jameel

    2015-05-08

    MVs are released in response to several stress agents, in an attempt to prevent continued cellular damage. After an initial stimulus of prostate cancer cells with sublytic C5b-9 and activation of MV release through PKC, cells take at least 20 min to fully recover their ability to microvesiculate. This release of MVs through activation of sublytic C5b-9 was inhibited by the PKC inhibitor bisindoylmaleimide I but not the Rho kinase inhibitor, Y27632. After stimulus there is a rise of 79 nMs(-1) over 11 s, reaching a peak [Ca(2+)]i of 920 nM. The concentration of cytosolic calcium then falls steadily at 2.4 nMs(-1) over 109 s reaching baseline levels (50-100 nM) within 10-15 min. In PC3 cells the rate of release of MVs from stimulated cells also reaches a minimum within 10-15 min. Using fura-2 AM-loaded cells, upon stimulation, cells were found to release MVs with a concentration of intravesicular calcium estimated at ∼ 430 nM. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Role of Platelet-derived Microvesicles as Crosstalk Mediators in Atherothrombosis and Future Pharmacology Targets: a Link between Inflammation, Atherosclerosis and Thrombosis

    Directory of Open Access Journals (Sweden)

    Lina Badimon

    2016-08-01

    Full Text Available Reports in the last decade have suggested that the role of platelets in atherosclerosis and its thrombotic complications may be mediated, in part, by local secretion of platelet-derived microvesicles (pMVs, small cell blebs released during the platelet activation process. MVs are the most abundant cell-derived microvesicle subtype in the circulation. High concentrations of circulating MVs have been reported in patients with atherosclerosis, acute vascular syndromes, and/or diabetes mellitus, suggesting a potential correlation between the quantity of microvesicles and the clinical severity of the atherosclerotic disease. pMVs are considered to be biomarkers of disease but new information indicates that pMVs are also involved in signaling functions. pMVs evoke or promote haemostatic and inflammatory responses, neovascularization, cell survival and apoptosis, processes involved in the pathophysiology of cardiovascular disease. This review is focused on the complex cross-talk between platelet-derived microvesicles, inflammatory cells and vascular elements and their relevance in the development of the atherosclerotic disease and its clinical outcomes, providing an updated state-of-the art of pMV involvement in atherothrombosis and pMV potential use as therapeutic agent influencing cardiovascular biomedicine in the future.

  13. Platelet-Derived Microvesicles in Cardiovascular Diseases

    Directory of Open Access Journals (Sweden)

    Maria T. K. Zaldivia

    2017-11-01

    Full Text Available Microvesicles (MVs circulating in the blood are small vesicles (100–1,000 nm in diameter derived from membrane blebs of cells such as activated platelets, endothelial cells, and leukocytes. A growing body of evidence now supports the concept that platelet-derived microvesicles (PMVs, the most abundant MVs in the circulation, are important regulators of hemostasis, inflammation, and angiogenesis. Compared with healthy individuals, a large increase of circulating PMVs has been observed, particularly in patients with cardiovascular diseases. As observed in MVs from other parent cells, PMVs exert their biological effects in multiple ways, such as triggering various intercellular signaling cascades and by participating in transcellular communication by the transfer of their “cargo” of cytoplasmic components and surface receptors to other cell types. This review describes our current understanding of the potential role of PMVs in mediating hemostasis, inflammation, and angiogenesis and their consequences on the pathogenesis of cardiovascular diseases, such as atherosclerosis, myocardial infarction, and venous thrombosis. Furthermore, new developments of the therapeutic potential of PMVs for the treatment of cardiovascular diseases will be discussed.

  14. Microvesicles in the venom of Crotalus durissus terrificus (Serpentes, Viperidae).

    Science.gov (United States)

    Carneiro, Sylvia Mendes; Fernandes, Wilson; Sant'Anna, Sávio Stefanini; Yamanouye, Norma

    2007-01-01

    Microvesicles with electron-dense content are consistently observed by transmission electron microscopy on the luminal face of secretory cells of venom glands of viperid snakes. In this work, we evaluated their presence in Crotalus durissus terrificus venom glands and also in freshly collected venom. Microvesicles were found in the venom glands mainly in regions of exocytosis. They ranged from 40 to 80 nm in diameter. Freeze-fracture replicas of the glands revealed particles on the cytoplasmic leaflet (P-face) of these vesicles, suggesting that they carry transmembrane proteins. Vesicles separated by ultracentrifugation from cell-free venom were similar in size and structure to the microvesicles observed in the glands. A fine fuzzy coat surrounded each microvesicle. The function of these venom vesicles is still unknown, but they may contribute to inactivation of stored venom components, or their activation after the venom is released.

  15. Resistin-induced stromal cell-derived factor-1 expression through Toll-like receptor 4 and activation of p38 MAPK/ NFκB signaling pathway in gastric cancer cells.

    Science.gov (United States)

    Hsieh, Yung-Yu; Shen, Chien-Heng; Huang, Wen-Shih; Chin, Chih-Chien; Kuo, Yi-Hung; Hsieh, Meng Chiao; Yu, Hong-Ren; Chang, Te-Sheng; Lin, Tseng-Hsi; Chiu, Yung-Wei; Chen, Cheng-Nan; Kuo, Hsing-Chun; Tung, Shui-Yi

    2014-06-14

    Stromal cell-derived factor-1 (SDF-1) (CXC chemokine ligand-12)/CXC chemokine receptor 4 (CXCR4) is involved in the carcinogenesis of human gastric cancer, where it stimulates angiogenesis and favors metastasis of tumor cells to distant organs. In addition, resistin is suggested to be an important link between obesity and the development of gastric cancer. Resistin has identified as an important player in inflammatory responses, and emerged as a mediator in inflammation-associated cancer. A limited number of studies have investigated the association of resistin and SDF-1 with gastric cancer. Herein, we investigated the molecular mechanisms by which resistin influences the expression of SDF-1 in gastric carcinoma cells. Human gastric cancer cell lines were exposed to doses of resistin; SDF-1 expression and secretion levels were then determined. Real-time polymerase chain reaction and western blotting analyses were performed to clarify molecular changes. Inhibition of Toll-like receptor 4 (TLR4) by a competitive antagonist inhibited resistin-induced SDF-1 expression. Pharmacological inhibitors and small interfering RNA (siRNA) demonstrated that activation of the p38 mitogen-activated protein kinase (MAPK) pathway is critical for resistin-induced SDF-1 expression mediated by TLR4. The promoter activity and transcription factor enzyme-linked immunosorbent assay revealed that resistin induced expression of SDF-1 mediated by NF-κB in gastric cancer cells. Inhibition of p38 MARK activation blocked the SDF-1-induced expression and the SDF-1 promoter activity in the cancer gastric cells. Chromatin immunoprecipitation assay revealed that inhibition of p38 MARK activation also blocked the resistin-increased NF-κB-DNA-binding activity. Resistin-induced SDF-1 upregulation by activation of TLR4, p38 MARK and NF-κB may explain a new role of resistin in the link of obesity and gastric cancer.

  16. Therapy of primary and metastatic liver cancer by human iPS cell-derived myeloid cells producing interferon-β.

    Science.gov (United States)

    Sakisaka, Masataka; Haruta, Miwa; Komohara, Yoshihiro; Umemoto, Satoshi; Matsumura, Keiko; Ikeda, Tokunori; Takeya, Motohiro; Inomata, Yukihiro; Nishimura, Yasuharu; Senju, Satoru

    2017-02-01

    iPS-ML are myeloid lineage cells with a proliferative capacity derived from induced pluripotent stem (iPS) cells. This study aimed to examine therapeutic effect of iPS-ML producing interferon-β (iPS-ML/IFN-β) towards primary and metastatic liver cancer and investigate the mechanism of that effect. We established a xenograft model of liver metastasis by injecting the spleen of SCID mice with MKN-45 human gastric cancer cells and also a primary liver cancer model by injecting SK-HEP-1 human hepatocellular carcinoma cells into the liver. After cancer lesions were established, iPS-ML/IFN-β was administered by intraperitoneal injection, and therapeutic effect was evaluated. The i.p. injection of iPS-ML/IFN-β resulted in a significant retardation of cancer progression and prolonged mouse survival. The infiltration of i.p. administered iPS-ML into tumor lesions located below the liver capsule was observed, suggesting tumor-directed migration and penetration of the liver capsule by iPS-ML. The IFN-β concentration in the liver was maintained at levels sufficient to exert an anti-cancer effect for at least 3 days post-injection, accounting for the potent therapeutic effect obtained by injection two to three times per week. This study demonstrates the therapeutic potential of the iPS-ML/IFN-β in patients with liver cancer. © 2017 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

  17. Microvesicles Contribute to the Bystander Effect of DNA Damage.

    Science.gov (United States)

    Lin, Xiaozeng; Wei, Fengxiang; Major, Pierre; Al-Nedawi, Khalid; Al Saleh, Hassan A; Tang, Damu

    2017-04-07

    Genotoxic treatments elicit DNA damage response (DDR) not only in cells that are directly exposed but also in cells that are not in the field of treatment (bystander cells), a phenomenon that is commonly referred to as the bystander effect (BE). However, mechanisms underlying the BE remain elusive. We report here that etoposide and ultraviolet (UV) exposure stimulate the production of microvesicles (MVs) in DU145 prostate cancer cells. MVs isolated from UV-treated DU145 and A431 epidermoid carcinoma cells as well as etoposide-treated DU145 cells induced phosphorylation of ataxia-telangiectasia mutated (ATM) at serine 1981 (indicative of ATM activation) and phosphorylation of histone H2AX at serine 139 (γH2AX) in naïve DU145 cells. Importantly, neutralization of MVs derived from UV-treated cells with annexin V significantly reduced the MV-associated BE activities. Etoposide and UV are known to induce DDR primarily through the ATM and ATM- and Rad3-related (ATR) pathways, respectively. In this regard, MV is likely a common source for the DNA damage-induced bystander effect. However, pre-treatment of DU145 naïve cells with an ATM (KU55933) inhibitor does not affect the BE elicited by MVs isolated from etoposide-treated cells, indicating that the BE is induced upstream of ATM actions. Taken together, we provide evidence supporting that MVs are a source of the DNA damage-induced bystander effect.

  18. Ovarian cancer-derived lysophosphatidic acid stimulates secretion of VEGF and stromal cell-derived factor-1 alpha from human mesenchymal stem cells.

    Science.gov (United States)

    Jeon, Eun Su; Heo, Soon Chul; Lee, Il Hwan; Choi, Yoon Ji; Park, Ji Hye; Choi, Kyung Un; Park, Do Youn; Suh, Dong Soo; Yoon, Man Soo; Kim, Jae Ho

    2010-04-30

    Lysophosphatidic acid (LPA) stimulates growth and invasion of ovarian cancer cells and tumor angiogenesis. Cancer-derived LPA induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) to alpha-smooth muscle actin (alpha-SMA)-positive cancer-associated fibroblasts. Presently, we explored whether cancer-derived LPA regulates secretion of pro-angiogenic factors from hASCs. Conditioned medium (CM) from the OVCAR-3 and SKOV3 ovarian cancer cell lines stimulated secretion angiogenic factors such as stromal-derived factor-1 alpha (SDF-1 alpha) and VEGF from hASCs. Pretreatment with the LPA receptor inhibitor Ki16425 or short hairpin RNA lentiviral silencing of the LPA((1)) receptor abrogated the cancer CM-stimulated expression of alpha-SMA, SDF-1, and VEGF from hASCs. LPA induced expression of myocardin and myocardin-related transcription factor-A, transcription factors involved in smooth muscle differentiation, in hASCs. siRNA-mediated depletion of endogenous myocardin and MRTF-A abrogated the expression of alpha-SMA, but not SDF-1 and VEGF. LPA activated RhoA in hASCs and pretreatment with the Rho kinase inhibitor Y27632 completely abrogated the LPA-induced expression of alpha-SMA, SDF-1, and VEGF in hASCs. Moreover, LPA-induced alpha-SMA expression was abrogated by treatment with the ERK inhibitor U0126 or the phosphoinositide-3-kinase inhibitor LY294002, but not the PLC inhibitor U73122. LPA-induced VEGF secretion was inhibited by LY294002, whereas LPA-induced SDF-1 secretion was markedly attenuated by U0126, U73122, and LY294002. These results suggest that cancer-secreted LPA induces differentiation of hASCs to cancer-associated fibroblasts through multiple signaling pathways involving Rho kinase, ERK, PLC, and phosphoinositide-3-kinase.

  19. Dynamic flux of microvesicles modulate parasite-host cell interaction of Trypanosoma cruzi in eukaryotic cells.

    Science.gov (United States)

    Ramirez, M I; Deolindo, P; de Messias-Reason, I J; Arigi, Emma A; Choi, H; Almeida, I C; Evans-Osses, I

    2017-04-01

    Extracellular vesicles released from pathogens may alter host cell functions. We previously demonstrated the involvement of host cell-derived microvesicles (MVs) during early interaction between Trypanosoma cruzi metacyclic trypomastigote (META) stage and THP-1 cells. Here, we aim to understand the contribution of different parasite stages and their extracellular vesicles in the interaction with host cells. First, we observed that infective host cell-derived trypomastigote (tissue culture-derived trypomastigote [TCT]), META, and noninfective epimastigote (EPI) stages were able to induce different levels of MV release from THP-1 cells; however, only META and TCT could increase host cell invasion. Fluorescence resonance energy transfer microscopy revealed that THP-1-derived MVs can fuse with parasite-derived MVs. Furthermore, MVs derived from the TCT-THP-1 interaction showed a higher fusogenic capacity than those from META- or EPI-THP-1 interaction. However, a higher presence of proteins from META (25%) than TCT (12%) or EPI (5%) was observed in MVs from parasite-THP-1 interaction, as determined by proteomics. Finally, sera from patients with chronic Chagas disease at the indeterminate or cardiac phase differentially recognized antigens in THP-1-derived MVs resulting only from interaction with infective stages. The understanding of intracellular trafficking and the effect of MVs modulating the immune system may provide important clues about Chagas disease pathophysiology. © 2016 John Wiley & Sons Ltd.

  20. Distinct functions of macrophage-derived and cancer cell-derived cathepsin Z combine to promote tumor malignancy via interactions with the extracellular matrix.

    Science.gov (United States)

    Akkari, Leila; Gocheva, Vasilena; Kester, Jemila C; Hunter, Karen E; Quick, Marsha L; Sevenich, Lisa; Wang, Hao-Wei; Peters, Christoph; Tang, Laura H; Klimstra, David S; Reinheckel, Thomas; Joyce, Johanna A

    2014-10-01

    During the process of tumor progression, cancer cells can produce the requisite growth- and invasion-promoting factors and can also rely on noncancerous cells in the tumor microenvironment as an alternative, cell-extrinsic source. However, whether the cellular source influences the function of such tumor-promoting factors remains an open question. Here, we examined the roles of the cathepsin Z (CtsZ) protease, which is provided by both cancer cells and macrophages in pancreatic neuroendocrine tumors in humans and mice. We found that tumor proliferation was exclusively regulated by cancer cell-intrinsic functions of CtsZ, whereas tumor invasion required contributions from both macrophages and cancer cells. Interestingly, several of the tumor-promoting functions of CtsZ were not dependent on its described catalytic activity but instead were mediated via the Arg-Gly-Asp (RGD) motif in the enzyme prodomain, which regulated interactions with integrins and the extracellular matrix. Together, these results underscore the complexity of interactions within the tumor microenvironment and indicate that cellular source can indeed impact molecular function. © 2014 Akkari et al.; Published by Cold Spring Harbor Laboratory Press.

  1. Cisplatin-resistant lung cancer cell-derived exosomes increase cisplatin resistance of recipient cells in exosomal miR-100-5p-dependent manner.

    Science.gov (United States)

    Qin, Xiaobing; Yu, Shaorong; Zhou, Leilei; Shi, Meiqi; Hu, Yong; Xu, Xiaoyue; Shen, Bo; Liu, Siwen; Yan, Dali; Feng, Jifeng

    2017-01-01

    Exosomes derived from lung cancer cells confer cisplatin (DDP) resistance to other cancer cells. However, the underlying mechanism is still unknown. A549 resistance to DDP (A549/DDP) was established. Microarray was used to analyze microRNA (miRNA) expression profiles of A549 cells, A549/DDP cells, A549 exosomes, and A549/DDP exosomes. There was a strong correlation of miRNA profiles between exosomes and their maternal cells. A total of 11 miRNAs were significantly upregulated both in A549/DDP cells compared with A549 cells and in exosomes derived from A549/DDP cells in contrast to exosomes from A549 cells. A total of 31 downregulated miRNAs were also observed. miR-100-5p was the most prominent decreased miRNA in DDP-resistant exosomes compared with the corresponding sensitive ones. Downregulated miR-100-5p was proved to be involved in DDP resistance in A549 cells, and mammalian target of rapamycin (mTOR) expression was reverse regulated by miR-100-5p. Exosomes confer recipient cells' resistance to DDP in an exosomal miR-100-5p-dependent manner with mTOR as its potential target both in vitro and in vivo. Exosomes from DDP-resistant lung cancer cells A549 can alter other lung cancer cells' sensitivity to DDP in exosomal miR-100-5p-dependent manner. Our study provides new insights into the molecular mechanism of DDP resistance in lung cancer.

  2. Announcing , the Official Journal of the American Society for Exosomes and Microvesicles

    Directory of Open Access Journals (Sweden)

    Stephen J. Gould

    2013-01-01

    Full Text Available This editorial article introduces the new scientific journal Exosomes and Microvesicles (EXMV , the official journal of the American Society for Exosomes and Microvesicles (ASEMV, and describes its editorial line and mission in relation to the role of the Society, the state of the art of the study of exosomes and microvesicles, and the overall approach of the publication.

  3. Stromal cell-derived factor-1 (SDF-1) enhances cells invasion by αvβ6 integrin-mediated signaling in ovarian cancer.

    Science.gov (United States)

    Xue, Baoyao; Wu, Weiguang; Huang, Kan; Xie, Tingting; Xu, Xiaobo; Zhang, Hongju; Qi, Chunlei; Ge, Junli; Yu, Yuecheng

    2013-08-01

    Ovarian carcinoma is a common gynecological malignancy and a great threat to health as a result of metastasis. The chemokine stromal-derived factor (SDF-1) plays multiple roles in tumor pathogenesis. However, the precise molecular mechanism underlying SDF-1-induced ovarian cancer cell invasion is still undefined. αvβ6 integrin is an important factor in tumor progression. Therefore, we speculate that SDF-1-enhanced ovarian cancer cell invasion is related to αvβ6 integrin-mediated signaling. After culturing with SDF-1, an obvious time- and dose-dependent increase in αvβ6 integrin was demonstrated. Furthermore, CXC receptor 4 (CXCR4) was responsible for SDF-1-induced αvβ6 integrin expression. Simultaneously, SDF-1 was found to dramatically enhance extracellular matrix degradation via urokinase-type plasminogen activator (uPA) expression and cell invasion by αvβ6 integrin expression; these reinforce failed to be increased when pretreatment was performed with the CXCR4 inhibitor AMD3100 or anti-αvβ6 integrin antibody, respectively. In addition, αvβ6 integrin induced the phosphorylation of p38 MAPK and PI3 K/Akt, contributing to the up-regulation of uPA, as treatment with the specific inhibitor for p38 mitogen-activated protein kinases (MAPK) (SB203580) or phosphatidylinositol 3-kinase (PI3 K)/Akt (LY294002) strikingly abrogated uPA expression. Taken together, these results demonstrated that SDF-1 enhanced ovarian cancer cell invasion through αvβ6 integrin-mediated uPA expression via the p38 MAPK and PI3 K/Akt pathway. Consequently, our findings will provide a new explanation about how SDF-1 aggravates the pathogenesis of ovarian cancer.

  4. Hybrid Cells Derived from Human Breast Cancer Cells and Human Breast Epithelial Cells Exhibit Differential TLR4 and TLR9 Signaling

    Directory of Open Access Journals (Sweden)

    Songül Tosun

    2016-05-01

    Full Text Available TLRs are important receptors of cells of the innate immune system since they recognize various structurally conserved molecular patterns of different pathogens as well as endogenous ligands. In cancer, the role of TLRs is still controversial due to findings that both regression and progression of tumors could depend on TLR signaling. In the present study, M13SV1-EGFP-Neo human breast epithelial cells, MDA-MB-435-Hyg human breast cancer cells and two hybrids M13MDA435-1 and -3 were investigated for TLR4 and TLR9 expression and signaling. RT-PCR data revealed that LPS and CpG-ODN induced the expression of pro-inflammatory cytokines, like IFN-β, TNF-α, IL-1β and IL-6 in hybrid cells, but not parental cells. Interestingly, validation of RT-PCR data by Western blot showed detectable protein levels solely after LPS stimulation, suggesting that regulatory mechanisms are also controlled by TLR signaling. Analysis of pAKT and pERK1/2 levels upon LPS and CpG-ODN stimulation revealed a differential phosphorylation pattern in all cells. Finally, the migratory behavior of the cells was investigated showing that both LPS and CpG-ODN potently blocked the locomotory activity of the hybrid cells in a dose-dependent manner. In summary, hybrid cells exhibit differential TLR4 and TLR9 signaling.

  5. Characterization of cancer stem-like cells derived from a side population of a human gallbladder carcinoma cell line, SGC-996.

    Science.gov (United States)

    Li, Xin-xing; Wang, Jian; Wang, Hao-lu; Wang, Wei; Yin, Xiao-bin; Li, Qi-wei; Chen, Yu-ying; Yi, Jing

    2012-03-23

    The cancer stem cell (CSC) hypothesis proposes that CSCs, which can renew themselves proliferate infinitely, and escape chemotherapy, become the root of recurrence and metastasis. Previous studies have verified that side population (SP) cells, characterized by their ability to efflux lipophilic substrate Hoechst 33342, to share many characteristics of CSCs in multiplying solid tumors. The purpose of this study was to sort SP cells from a human gallbladder carcinoma cell line, SGC-996 and to preliminarily identify the biological characteristics of SP cells from the cell line. Using flow cytometry we effectively sorted SP cells from the cell line SGC-996. SP cells not only displayed higher proliferative, stronger clonal-generating, more migratory and more invasive capacities, but showed stronger resistance. Furthermore, our experiments demonstrated that SP cells were more tumorigenic than non-SP counterparts in vivo. Real-time PCR analysis and immunocytochemistry showed that the expression of ATP-binding cassette subfamily G member 2 (ABCG2) was significantly higher in SP cells. Hence, these results collectively suggest that SP cells are progenitor/stem-like cells and ABCG2 might be a candidate marker for SP cells in human gallbladder cancer. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer

    Science.gov (United States)

    Pospichalova, Vendula; Svoboda, Jan; Dave, Zankruti; Kotrbova, Anna; Kaiser, Karol; Klemova, Dobromila; Ilkovics, Ladislav; Hampl, Ales; Crha, Igor; Jandakova, Eva; Minar, Lubos; Weinberger, Vit; Bryja, Vitezslav

    2015-01-01

    Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80–200 nm, microvesicles: ~200–1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for

  7. Two-dimensional electrophoretic comparison of metastatic and non-metastatic human breast tumors using in vitro cultured epithelial cells derived from the cancer tissues

    Directory of Open Access Journals (Sweden)

    Buršíková Eva

    2008-04-01

    Full Text Available Abstract Background Breast carcinomas represent a heterogeneous group of tumors diverse in behavior, outcome, and response to therapy. Identification of proteins resembling the tumor biology can improve the diagnosis, prediction, treatment selection, and targeting of therapy. Since the beginning of the post-genomic era, the focus of molecular biology gradually moved from genomes to proteins and proteomes and to their functionality. Proteomics can potentially capture dynamic changes in protein expression integrating both genetic and epigenetic influences. Methods We prepared primary cultures of epithelial cells from 23 breast cancer tissue samples and performed comparative proteomic analysis. Seven patients developed distant metastases within three-year follow-up. These samples were included into a metastase-positive group, the others formed a metastase-negative group. Two-dimensional electrophoretical (2-DE gels in pH range 4–7 were prepared. Spot densities in 2-DE protein maps were subjected to statistical analyses (R/maanova package and data-mining analysis (GUHA. For identification of proteins in selected spots, liquid chromatography-tandem mass spectrometry (LC-MS/MS was employed. Results Three protein spots were significantly altered between the metastatic and non-metastatic groups. The correlations were proven at the 0.05 significance level. Nucleophosmin was increased in the group with metastases. The levels of 2,3-trans-enoyl-CoA isomerase and glutathione peroxidase 1 were decreased. Conclusion We have performed an extensive proteomic study of mammary epithelial cells from breast cancer patients. We have found differentially expressed proteins between the samples from metastase-positive and metastase-negative patient groups.

  8. Cavin-1/PTRF alters prostate cancer cell-derived extracellular vesicle content and internalization to attenuate extracellular vesicle-mediated osteoclastogenesis and osteoblast proliferation

    Directory of Open Access Journals (Sweden)

    Kerry L. Inder

    2014-06-01

    Full Text Available Background: Tumour-derived extracellular vesicles (EVs play a role in tumour progression; however, the spectrum of molecular mechanisms regulating EV secretion and cargo selection remain to be fully elucidated. We have reported that cavin-1 expression in prostate cancer PC3 cells reduced the abundance of a subset of EV proteins, concomitant with reduced xenograft tumour growth and metastasis. Methods: We examined the functional outcomes and mechanisms of cavin-1 expression on PC3-derived EVs (PC3-EVs. Results: PC3-EVs were internalized by osteoclast precursor RAW264.7 cells and primary human osteoblasts (hOBs in vitro, stimulating osteoclastogenesis 37-fold and hOB proliferation 1.5-fold, respectively. Strikingly, EVs derived from cavin-1-expressing PC3 cells (cavin-1-PC3-EVs failed to induce multinucleate osteoblasts or hOB proliferation. Cavin-1 was not detected in EVs, indicating an indirect mechanism of action. EV morphology, size and quantity were also not affected by cavin-1 expression, suggesting that cavin-1 modulated EV cargo recruitment rather than release. While cavin-1-EVs had no osteoclastogenic function, they were internalized by RAW264.7 cells but at a reduced efficiency compared to control EVs. EV surface proteins are required for internalization of PC3-EVs by RAW264.7 cells, as proteinase K treatment abolished uptake of both control and cavin-1-PC3-EVs. Removal of sialic acid modifications by neuraminidase treatment increased the amount of control PC3-EVs internalized by RAW264.7 cells, without affecting cavin-1-PC3-EVs. This suggests that cavin-1 expression altered the glycosylation modifications on PC3-EV surface. Finally, cavin-1 expression did not affect EV in vivo tissue targeting as both control and cavin-1-PC3-EVs were predominantly retained in the lung and bone 24 hours after injection into mice. Discussion: Taken together, our results reveal a novel pathway for EV cargo sorting, and highlight the potential of utilizing

  9. Smart blood cell and microvesicle-based Trojan horse drug delivery: Merging expertise in blood transfusion and biomedical engineering in the field of nanomedicine.

    Science.gov (United States)

    Wu, Yu-Wen; Goubran, Hadi; Seghatchian, Jerard; Burnouf, Thierry

    2016-04-01

    Therapeutic and diagnostic applications of nanomedicine are playing increasingly important roles in human health. Various types of synthetic nanoparticles, including liposomes, micelles, and other nanotherapeutic platforms and conjugates, are being engineered to encapsulate or carry drugs for treating diseases such as cancer, cardiovascular disorders, neurodegeneration, and inflammations. Nanocarriers are designed to increase the half-life of drugs, decrease their toxicity and, ideally, target pathological sites. Developing smart carriers with the capacity to deliver drugs specifically to the microenvironment of diseased cells with minimum systemic toxicity is the goal. Blood cells, and potentially also the liposome-like micro- and nano-vesicles they generate, may be regarded as ideally suited to perform such specific targeting with minimum immunogenic risks. Blood cell membranes are "decorated" with complex physiological receptors capable of targeting and communicating with other cells and tissues and delivering their content to the surrounding pathological microenvironment. Blood cells, such as erythrocytes, have been developed as permeable carriers to release drugs to diseased tissues or act as biofactory allowing enzymatic degradation of a pathological substrate. Interestingly, attempts are also being made to improve the targeting capacity of synthetic nanoparticles by "decorating" their surface with blood cell membrane receptor-like biochemical structures. Research is needed to further explore the benefits that blood cell-derived microvesicles, as a Trojan horse delivery systems, can bring to the arsenal of therapeutic micro- and nanotechnologies. This short review focuses on the therapeutic roles that red blood cells and platelets can play as smart drug-delivery systems, and highlights the benefits that blood transfusion expertise can bring to this exciting and novel biomedical engineering field. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Breast-cancer extracellular vesicles induce platelet activation and aggregation by tissue factor-independent and -dependent mechanisms.

    Science.gov (United States)

    Gomes, Fausto G; Sandim, Vanessa; Almeida, Vitor H; Rondon, Araci M R; Succar, Barbara B; Hottz, Eugenio D; Leal, Ana Carolina; Verçoza, Brunno Renato F; Rodrigues, Juliany Cola F; Bozza, Patrícia T; Zingali, Russolina B; Monteiro, Robson Q

    2017-11-01

    Cancer-associated thrombosis is one of the major causes of worse prognosis among tumor-bearing patients. Extracellular vesicles derived from cancer cells, which can be divided mainly into microvesicles and exosomes, can participate in several tumor progression phenomena. Tumor-derived microvesicles positive for tissue factor (TF) have been associated with thrombotic risk in certain cancer types. Cancer cell-derived exosomes, however, have not. In this study we evaluated the capacity of extracellular vesicles (EVs, containing both microvesicles and exosomes) derived from breast-cancer cell lines in promoting platelet activation, aggregation and plasma coagulation, in experiments that access both TF-dependent and -independent activities. EVs were isolated from the conditioned media of two human mammary carcinoma cell lines: MDA-MB-231 (highly invasive) and MCF-7 (less invasive). TF-independent EV/platelet interaction, platelet P-selectin exposure and aggregation were evaluated. Western blotting, plasma clotting and platelet aggregation in the presence of plasma were performed for the measurement of TF-dependent activity in EVs. Interaction between MDA-MB-231 EVs and washed platelets led to increased platelet P-selectin exposure and platelet aggregation compared to MCF-7 EVs. MDA-MB-231 EVs had higher TF protein levels and TF-dependent procoagulant activity than MCF-7 EVs. Consequently, TF-dependent platelet aggregation was also induced by MDA-MB-231 EVs, but not by MCF-7 EVs. Our results suggest that MDA-MB-231 EVs induce TF-independent platelet activation and aggregation, as well as TF-dependent plasma clotting and platelet aggregation by means of thrombin generation. In this context, aggressive breast cancer-derived EVs may contribute to cancer-associated thrombosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. The Impact of Lipoprotein-Associated Oxidative Stress on Cell-Specific Microvesicle Release in Patients with Familial Hypercholesterolemia

    DEFF Research Database (Denmark)

    Nielsen, M H; Irvine, H; Vedel, S

    2016-01-01

    Objective. Microvesicles (MVs) are small cell-derived particles shed upon activation. Familial hypercholesterolemia (FH) particularly when associated with Achilles tendon xanthomas (ATX) predisposes to atherosclerosis, possibly through oxLDL-C interaction with the CD36 receptor. To investigate...... the hypothesis that MVs derived from cells involved in atherosclerosis are increased in FH and that CD36 expressing MVs (CD36+ MVs) may be markers of oxLDL-C-induced cell activation, cell-specific MVs were measured in FH patients with and without ATX and their association with atherogenic lipid profile...... was studied. Approach and Results. Thirty FH patients with and without ATX and twenty-three controls were included. Plasma concentrations of MVs and CD36+ MVs derived from platelets (PMVs), erythrocytes (ErytMVs), monocytes (MMVs), and endothelial cells (EMVs), as well as tissue factor-positive cells (TF+ MVs...

  12. Cell-derived microparticles and the lung.

    Science.gov (United States)

    Nieri, Dario; Neri, Tommaso; Petrini, Silvia; Vagaggini, Barbara; Paggiaro, Pierluigi; Celi, Alessandro

    2016-09-01

    Cell-derived microparticles are small (0.1-1 μm) vesicles shed by most eukaryotic cells upon activation or during apoptosis. Microparticles carry on their surface, and enclose within their cytoplasm, molecules derived from the parental cell, including proteins, DNA, RNA, microRNA and phospholipids. Microparticles are now considered functional units that represent a disseminated storage pool of bioactive effectors and participate both in the maintenance of homeostasis and in the pathogenesis of diseases. The mechanisms involved in microparticle generation include intracellular calcium mobilisation, cytoskeleton rearrangement, kinase phosphorylation and activation of the nuclear factor-κB. The role of microparticles in blood coagulation and inflammation, including airway inflammation, is well established in in vitro and animal models. The role of microparticles in human pulmonary diseases, both as pathogenic determinants and biomarkers, is being actively investigated. Microparticles of endothelial origin, suggestive of apoptosis, have been demonstrated in the peripheral blood of patients with emphysema, lending support to the hypothesis that endothelial dysfunction and apoptosis are involved in the pathogenesis of the disease and represent a link with cardiovascular comorbidities. Microparticles also have potential roles in patients with asthma, diffuse parenchymal lung disease, thromboembolism, lung cancer and pulmonary arterial hypertension. Copyright ©ERS 2016.

  13. Expression of osteoprotegerin, receptor activator of nuclear factor kappa-B ligand, tumor necrosis factor-related apoptosis-inducing ligand, stromal cell-derived factor-1 and their receptors in epithelial metastatic breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Labovsky Vivian

    2012-06-01

    Full Text Available Abstract Background While breast cancer (BC is the major cause of death among women worldwide, there is no guarantee of better patient survival because many of these patients develop primarily metastases, despite efforts to detect it in its early stages. Bone metastasis is a common complication that occurs in 65-80 % of patients with disseminated disease, but the molecular basis underlying dormancy, dissemination and establishment of metastasis is not understood. Our objective has been to evaluate simultaneously osteoprotegerin (OPG, receptor activator of nuclear factor kappa B ligand (RANKL, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, stromal cell-derived factor-1 (SDF-1, and their receptors (R in 2 human BC cell lines, MDA-MB-231 and MCF-7. Methods OPG, RANKL, TRAIL and SDF-1 expression and release, in addition to the expression of their receptors has been investigated using immunofluorescence, immunocytochemistry and ELISA analyses. Results MCF-7 cells released higher levels of OPG in conditioned media (CM than MDA-MB-231 cells; 100 % of both types of cell expressed OPG, RANKL, TRAIL and SDF-1. Moreover, 100 % in both lines expressed membrane RANKL and RANK, whereas only 50 % expressed CXCR4. Furthermore, 100 % expressed TRAIL-R1 and R4, 30-50 % TRAIL-R2, and 40-55 % TRAIL-R3. Conclusions MCF-7 and MDA-MB-231 cells not only released OPG, but expressed RANKL, TRAIL and SDF-1. The majority of the cells also expressed RANK, CXCR4 and TRAIL-R. Since these ligands and their receptors are implicated in the regulation of proliferation, survival, migration and future bone metastasis during breast tumor progression, assessment of these molecules in tumor biopsies of BC patients could be useful in identifying patients with more aggressive tumors that are also at risk of bone metastasis, which may thus improve the available options for therapeutic intervention.

  14. Efficacy Against Human Prostate Cancer by Prostate-specific Membrane Antigen-specific, Transforming Growth Factor-β Insensitive Genetically Targeted CD8+T-cells Derived from Patients with Metastatic Castrate-resistant Disease.

    Science.gov (United States)

    Zhang, Qiang; Helfand, Brian T; Carneiro, Benedito A; Qin, Weijun; Yang, Ximing J; Lee, Chung; Zhang, Weipeng; Giles, Francis J; Cristofanilli, Massimo; Kuzel, Timothy M

    2017-12-21

    Current immunotherapy has limited efficacy on metastatic castrate-resistant prostate cancer (mCRPC). We therefore sought to improve the antitumor ability of mCRPC patient-derived CD8 + T-cells by the endowment of specificity to prostate-specific membrane antigen (PSMA) and insensitivity to immunosuppressant molecule transforming growth factor-β (TGF-ß) under the control of herpes simplex virus-1 thymidine kinase. CD8 + T-cells were collected by leukapheresis and cultured in a Food and Drug Administration-approved Cell Processing Work Station. We developed a chimeric antigen receptor retroviral construct using an anti-PSMA chimeric immunoglobulin-T-cell receptor(ζ) gene (PZ1) and dominant negative TGF-ß type II receptor (TßRIIDN), that could induce CD8 + T-cells to be PSMA reactive and insensitive to TGF-ß. Cr 51 release assay was performed on PC-3 and PC-3-PSMA. The further antitumor functions of PSMA-specific, TGF-ß insensitive CD8 + T-cells was evaluated using an immunodeficient RAG-1 -/- mouse model. We found PSMA-specific, TGF-ß insensitive CD8 + T-cells from mCRPC were expanded with strong expression of PZ1 and thymidine kinase genes, and their growth was not suppressed by TGF-ß. The survival of these cells decreased sharply after treatment with ganciclovir. Treatment of PSMA-specific TGF-ß, insensitive CD8 + T-cells was associated with 61.58% specific lysis on PC-3-PSMA, and significantly suppressed PC3-PSMA tumor compared with the PC3 tumor. A large amount of tumor apoptosis and CD8 + T-cell infiltration were found only in the PC3-PSMA tumor. This study verified that PSMA-specific, TGF-ß insensitive CD8 + T-cells derived from mCRPC patients could be successfully expanded and used to overcome the immunosuppressive effects of the tumor microenvironment to control PSMA-expressing PC in vitro and in vivo. This may provide a promising approach for men with mCRPC who fail androgen deprivation therapy. We investigated the role of a novel chimeric antigen

  15. Fluorescent Immortalized Human Adipose Derived Stromal Cells (hASCs-TS/GFP+) for Studying Cell Drug Delivery Mediated by Microvesicles.

    Science.gov (United States)

    Cocce, Valentina; Balducci, Luigi; Falchetti, Maria L; Pascucci, Luisa; Ciusani, Emilio; Brini, Anna T; Sisto, Francesca; Piovani, Giovanna; Alessandri, Giulio; Parati, Eugenio; Cabeza, Laura; Pessina, Augusto

    2017-11-24

    A new tool for the drug delivery is based on the use of Mesenchymal Stromal Cells (MSCs) loaded in vitro with anti-cancer drugs. Unfortunately, the restricted lifespan of MSCs represents a significant limitation to produce them in high amounts and for long time studies. Immortalized MSCs from adipose tissue (hASCs) have been generated as good source of cells with stable features. These cells could improve the development of standardized procedures for both in vitro and preclinical studies. Furthermore they facilitate procedures for preparing large amounts of secretome containing microvesicles (MVs). We used human adipose tissue derived MSCs immortalized with hTERT+SV40 (TS) genes and transfected with GFP (hASCs-TS/GFP+). This line was investigated for its ability to uptake and release anticancer drugs. Microvesicles associated to paclitaxel (MVs/PTX) were isolated, quantified, and tested on pancreatic cancer cells. The line hASCs-TS/GFP+ maintained the main mesenchymal characters and was able to uptake and release, in active form, both paclitaxel and gemcitabine. From paclitaxel loaded hASCs-TS/GFP+ cells were isolated microvesicles in sufficient amount to inhibit "in vitro" the proliferation of pancreatic tumor cells. Our study suggests that human immortalized MSCs could be used for a large scale production of cells for mediated drug delivery. Moreover, the secretion of drug-associated MVs could represent a new way for producing new drug formulation by "biogenesis". In the context of the "advanced cell therapy procedure", the MVs/PTX production would use less resource and time and it could possibly contribute to simplification of GMP procedures. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Plasma C4d+ Endothelial Microvesicles Increase in Acute Antibody-Mediated Rejection.

    Science.gov (United States)

    Tower, Cindy M; Reyes, Morayma; Nelson, Karen; Leca, Nicolae; Kieran, Niamh; Muczynski, Kimberly; Jefferson, Jonathan A; Blosser, Christopher; Kukla, Aleksandra; Maurer, David; Chandler, Wayne; Najafian, Behzad

    2017-09-01

    Antibody-mediated rejection (AMR) is a major cause of kidney allograft loss. Currently, AMR diagnosis relies on biopsy which is an invasive procedure. A noninvasive biomarker of acute AMR could lead to early diagnosis and treatment of this condition and improve allograft outcome. Microvesicles are membrane-bound vesicles released from the cell surface after injury. We hypothesized that because AMR is associated with allograft endothelial injury and C4d deposition, plasma microvesicles positive for endothelial (CD144) marker and C4d are increased in this condition. We studied microvesicle concentration in the plasma of 95 kidney transplant patients with allograft dysfunction and compared with 23 healthy volunteers. Biopsy diagnosis and scoring was performed using Banff classification. In the 28 subjects with AMR, the density of C4d+/CD144+ microvesicles was on average 11-fold (P = 0.002) higher than transplant recipients with no AMR and 24-fold (P = 0.008) than healthy volunteers. Densities of C4d+ and C4d+/annexin V+ (C4d+/AVB+) microvesicles were also increased in AMR patients compared with no AMR and healthy subjects. C4d+/AVB+ microvesicles correlated with AMR biopsy severity. Nine patients with acute AMR that received treatment showed a mean 72% decrease (P = 0.01) in C4d+/CD144+ microvesicle concentration compared with pretreatment values. Quantification of plasma C4d+ microvesicles provides information about presence of AMR, its severity and response to treatment in transplant patients.

  17. Microvesicles of women with gestational hypertension and preeclampsia affect human trophoblast fate and endothelial function.

    Science.gov (United States)

    Shomer, Einat; Katzenell, Sarah; Zipori, Yaniv; Sammour, Rami N; Isermann, Berend; Brenner, Benjamin; Aharon, Anat

    2013-11-01

    Microvesicles shedding from cell membrane affect inflammation, apoptosis, and angiogenesis. We hypothesize that microvesicles of women with gestational vascular complications reflect pathophysiological state of the patients and affect their endothelial and trophoblast cell function. Microvesicles of healthy pregnant women, women with gestational hypertension, mild, or severe preeclampsia/toxemia, were characterized, and their effects on early-stage or term trophoblasts and endothelial cells were evaluated using apoptosis, migration, and tube formation assays. Patient subgroups differed significantly only in proteinuria levels, therefore their microvesicles were assessed as 1 group, demonstrating higher levels of inflammatory and angiogenic proteins compared with those of healthy pregnant women. In endothelial cells, microvesicles of healthy pregnant women reduced caspase 3/7 activity, increased migration, and induced tube formation. These processes were suppressed by microvesicles of women with gestational vascular complications. In early-stage trophoblasts, microvesicles of healthy pregnant women decreased apoptosis compared with untreated cells (6±5% versus 13.8±5.8%; Papoptosis compared with cells exposed to microvesicles of healthy pregnant women (15.1±3.3% versus 6.5±2.1%; P<0.001) and inhibited early-stage trophoblasts migration (21.4±18.5 versus 39.7±10.1 mm2; P<0.001). In conclusion, microvesicle content and effects on endothelial and trophoblast cells vary according to the physiological/pathological state of a pregnant woman. Microvesicles seem to play a pivotal role in the course of pregnancy, which could potentially result in gestational vascular complications.

  18. Plasma microvesicle analysis identifies microRNA 129-5p as a biomarker of heart failure in univentricular heart disease.

    Science.gov (United States)

    Ramachandran, Sweta; Lowenthal, Alexander; Ritner, Carissa; Lowenthal, Shiri; Bernstein, Harold S

    2017-01-01

    Biomarkers of heart failure in adults have been extensively studied. However, biomarkers to monitor the progression of heart failure in children with univentricular physiology are less well understood. We proposed that as mediators of diverse pathophysiology, miRNAs contained within circulating microvesicles could serve as biomarkers for the presence and progression of heart failure in univentricular patients. To test this, we studied the association of heart failure with elevations in specific miRNAs isolated from circulating microvesicles in a cohort of children with univentricular heart disease and heart failure. We conducted a single site cross-sectional observational study of 71 children aged 1 month-7 years with univentricular heart disease and heart failure. We demonstrated that levels of miR129-5p isolated from plasma microvesicles were inversely related to the degree of clinical heart failure as assessed by Ross score. We then showed that miR129-5p levels are downregulated in HL1 cells and human embryonic stem cell-derived cardiomyocytes exposed to oxidative stress. We demonstrated that bone morphogenetic protein receptor 2, which has been implicated in the development of pulmonary vascular disease, is a target of miR129-5p, and conversely regulated in response to oxidative stress in cell culture. Levels of miR129-5p were inversely related to the degree of clinical heart failure in patients with univentricular heart disease. This study demonstrates that miR129-5p is a sensitive and specific biomarker for heart failure in univentricular heart disease independent of ventricular morphology or stage of palliation. Further study is warranted to understand the targets affected by miR129-5p with the development of heart failure in patients with univentricular physiology.

  19. Plasma microvesicle analysis identifies microRNA 129-5p as a biomarker of heart failure in univentricular heart disease.

    Directory of Open Access Journals (Sweden)

    Sweta Ramachandran

    Full Text Available Biomarkers of heart failure in adults have been extensively studied. However, biomarkers to monitor the progression of heart failure in children with univentricular physiology are less well understood. We proposed that as mediators of diverse pathophysiology, miRNAs contained within circulating microvesicles could serve as biomarkers for the presence and progression of heart failure in univentricular patients. To test this, we studied the association of heart failure with elevations in specific miRNAs isolated from circulating microvesicles in a cohort of children with univentricular heart disease and heart failure. We conducted a single site cross-sectional observational study of 71 children aged 1 month-7 years with univentricular heart disease and heart failure. We demonstrated that levels of miR129-5p isolated from plasma microvesicles were inversely related to the degree of clinical heart failure as assessed by Ross score. We then showed that miR129-5p levels are downregulated in HL1 cells and human embryonic stem cell-derived cardiomyocytes exposed to oxidative stress. We demonstrated that bone morphogenetic protein receptor 2, which has been implicated in the development of pulmonary vascular disease, is a target of miR129-5p, and conversely regulated in response to oxidative stress in cell culture. Levels of miR129-5p were inversely related to the degree of clinical heart failure in patients with univentricular heart disease. This study demonstrates that miR129-5p is a sensitive and specific biomarker for heart failure in univentricular heart disease independent of ventricular morphology or stage of palliation. Further study is warranted to understand the targets affected by miR129-5p with the development of heart failure in patients with univentricular physiology.

  20. RNA expression patterns in serum microvesicles from patients with glioblastoma multiforme and controls

    Directory of Open Access Journals (Sweden)

    Noerholm Mikkel

    2012-01-01

    Full Text Available Abstract Background RNA from exosomes and other microvesicles contain transcripts of tumour origin. In this study we sought to identify biomarkers of glioblastoma multiforme in microvesicle RNA from serum of affected patients. Methods Microvesicle RNA from serum from patients with de-novo primary glioblastoma multiforme (N = 9 and normal controls (N = 7 were analyzed by microarray analysis. Samples were collected according to protocols approved by the Institutional Review Board. Differential expressions were validated by qRT-PCR in a separate set of samples (N = 10 in both groups. Results Expression profiles of microvesicle RNA correctly separated individuals in two groups by unsupervised clustering. The most significant differences pertained to down-regulated genes (121 genes > 2-fold down in the glioblastoma multiforme patient microvesicle RNA, validated by qRT-PCR on several genes. Overall, yields of microvesicle RNA from patients was higher than from normal controls, but the additional RNA was primarily of size Conclusions Serum microvesicle RNA from patients with glioblastoma multiforme has significantly down-regulated levels of RNAs coding for ribosome production, compared to normal healthy controls, but a large overabundance of RNA of unknown origin with size

  1. Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells.

    Directory of Open Access Journals (Sweden)

    Anders Waldenström

    Full Text Available BACKGROUND: Shedding microvesicles are membrane released vesicles derived directly from the plasma membrane. Exosomes are released membrane vesicles of late endosomal origin that share structural and biochemical characteristics with prostasomes. Microvesicles/exosomes can mediate messages between cells and affect various cell-related processes in their target cells. We describe newly detected microvesicles/exosomes from cardiomyocytes and depict some of their biological functions. METHODOLOGY/PRINCIPAL FINDINGS: Microvesicles/exosomes from media of cultured cardiomyocytes derived from adult mouse heart were isolated by differential centrifugation including preparative ultracentrifugation and identified by transmission electron microscopy and flow cytometry. They were surrounded by a bilayered membrane and flow cytometry revealed presence of both caveolin-3 and flotillin-1 while clathrin and annexin-2 were not detected. Microvesicle/exosome mRNA was identified and out of 1520 detected mRNA, 423 could be directly connected in a biological network. Furthermore, by a specific technique involving TDT polymerase, 343 different chromosomal DNA sequences were identified in the microvesicles/exosomes. Microvesicle/exosomal DNA transfer was possible into target fibroblasts, where exosomes stained for DNA were seen in the fibroblast cytosol and even in the nuclei. The gene expression was affected in fibroblasts transfected by microvesicles/exosomes and among 333 gene expression changes there were 175 upregulations and 158 downregulations compared with controls. CONCLUSIONS/SIGNIFICANCE: Our study suggests that microvesicles/exosomes released from cardiomyocytes, where we propose that exosomes derived from cardiomyocytes could be denoted "cardiosomes", can be involved in a metabolic course of events in target cells by facilitating an array of metabolism-related processes including gene expression changes.

  2. Leukocyte-derived microvesicles dock on glomerular endothelial cells: stardust in the kidney.

    Science.gov (United States)

    Mack, Matthias

    2017-01-01

    Microvesicles are released from the plasma membrane of various cell types, can be taken up by other cells, and can transport membrane proteins and cytosolic contents between cells. Kahn et al. demonstrate that leukocyte-derived microvesicles bearing B1-kinin receptors are enriched in the plasma of vasculitis patients and dock on endothelial cells in the glomerulus. Cell culture experiments suggest that B1-receptors transferred by these microvesicles are functionally active on acceptor cells. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  3. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer

    Czech Academy of Sciences Publication Activity Database

    Pospíchalová, V.; Svoboda, Jan; Dave, Z.; Kotrbová, A.; Kaiser, K.; Klemová, D.; Ilkovics, L.; Hampl, A.; Crha, I.; Jandáková, E.; Minář, L.; Weinberger, V.; Bryja, Vítězslav

    2015-01-01

    Roč. 4, March 31 (2015), s. 25530 ISSN 2001-3078 Institutional support: RVO:61388971 ; RVO:68081707 Keywords : exosomes * microvesicles * extracellular vesicles Subject RIV: EE - Microbiology, Virology

  4. The Impact of Lipoprotein-Associated Oxidative Stress on Cell-Specific Microvesicle Release in Patients with Familial Hypercholesterolemia

    Directory of Open Access Journals (Sweden)

    M. H. Nielsen

    2016-01-01

    Full Text Available Objective. Microvesicles (MVs are small cell-derived particles shed upon activation. Familial hypercholesterolemia (FH particularly when associated with Achilles tendon xanthomas (ATX predisposes to atherosclerosis, possibly through oxLDL-C interaction with the CD36 receptor. To investigate the hypothesis that MVs derived from cells involved in atherosclerosis are increased in FH and that CD36 expressing MVs (CD36+ MVs may be markers of oxLDL-C-induced cell activation, cell-specific MVs were measured in FH patients with and without ATX and their association with atherogenic lipid profile was studied. Approach and Results. Thirty FH patients with and without ATX and twenty-three controls were included. Plasma concentrations of MVs and CD36+ MVs derived from platelets (PMVs, erythrocytes (ErytMVs, monocytes (MMVs, and endothelial cells (EMVs, as well as tissue factor-positive cells (TF+ MVs, were measured by flow cytometry. Total MVs, MMVs, EMVs, ErytMVs, and TF+ MVs were significantly increased in FH patients, compared to controls. CD36+ MVs derived from endothelial cells and monocytes were significantly higher in FH patients and oxLDL-C predicted all the investigated cell-specific CD36+ MVs in FH patients with ATX. Conclusions. MVs derived from cells involved in atherosclerosis were increased in FH and may contribute to elevated atherothrombosis risk. The increased cell-specific CD36+ MVs observed in FH may represent markers of oxLDL-C-induced cell activation.

  5. Post - prandial rise of microvesicles in peripheral blood of healthy human donors

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    Mam Keriya

    2011-03-01

    Full Text Available Abstract Background Microvesicles isolated from body fluids are membrane - enclosed fragments of cell interior which carry information on the status of the organism. It is yet unclear how metabolism affects the number and composition of microvesicles in isolates from the peripheral blood. Aim To study the post - prandial effect on microvesicles in isolates from the peripheral blood of 21 healthy donors, in relation to blood cholesterol and blood glucose concentrations. Results The average number of microvesicles in the isolates increased 5 hours post - prandially by 52%; the increase was statistically significant (p = 0.01 with the power P = 0.68, while the average total blood cholesterol concentration, average low density lipoprotein cholesterol concentration (LDL-C and average high density lipoprotein cholesterol concentration (HDL-C all remained within 2% of their fasting values. We found an 11% increase in triglycerides (p = 0.12 and a 6% decrease in blood glucose (p Conclusions In a population of healthy human subjects the number of microvesicles in isolates from peripheral blood increased in the post - prandial state. The increase in the number of microvesicles was affected by the fasting concentration of cholesterol and correlated with the decrease in blood glucose.

  6. Role of Exosomes/Microvesicles in the Nervous System and Use in Emerging Therapies

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    Charles Pin-Kuang Lai

    2012-06-01

    Full Text Available Extracellular membrane vesicles (EMVs are nanometer sized vesicles, including exosomes and microvesicles capable of transferring DNAs, mRNAs, microRNAs, non-coding RNAs, proteins and lipids among cells without direct cell-to-cell contact, thereby representing a novel form of intercellular communication. Many cells in the nervous system have been shown to release EMVs, implicating their active roles in development, function and pathologies of this system. While substantial progress has been made in understanding the biogenesis, biophysical properties and involvement of EMVs in diseases, relatively less information is known about their biological function in the normal nervous system. In addition, since EMVs are endogenous vehicles with low immunogenicity, they have also been actively investigated for the delivery of therapeutic genes/molecules in treatment of cancer and neurological diseases. The present review summarizes current knowledge about EMV functions in the nervous system under both physiological and pathological conditions, as well as emerging EMV-based therapies that could be applied to the nervous system in the foreseeable future.

  7. Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

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    Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo; Giavaresi, Gianluca; Alessandro, Riccardo

    2015-01-01

    Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient's sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes. PMID:25944696

  8. Microvesicles in vascular homeostasis and diseases Position Paper of the European Society of Cardiology (ESC) Working Group on Atherosclerosis and Vascular Biology

    NARCIS (Netherlands)

    Ridger, Victoria C.; Boulanger, Chantal M.; Angelillo-Scherrer, Anne; Badimon, Lina; Blanc-Brude, Olivier; Bochaton-Piallat, Marie-Luce; Boilard, Eric; Buzas, Edit I.; Caporali, Andreas; Dignat-George, Françoise; Evans, Paul C.; Lacroix, Romaric; Lutgens, Esther; Ketelhuth, Daniel F. J.; Nieuwland, Rienk; Toti, Florence; Tunon, Jose; Weber, Christian; Hoefer, Imo E.

    2017-01-01

    Microvesicles are members of the family of extracellular vesicles shed from the plasma membrane of activated or apoptotic cells. Microvesicles were initially characterised by their pro-coagulant activity and described as "microparticles". There is mounting evidence revealing a role for microvesicles

  9. Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells

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    Haqqani Arsalan S

    2013-01-01

    Full Text Available Abstract Background In addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes and ectosomes (originating from direct budding/shedding of plasma membranes. Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit. Methods To study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed. Results A total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially

  10. Cellular transfer of magnetic nanoparticles via cell microvesicles: impact on cell tracking by magnetic resonance imaging.

    Science.gov (United States)

    Silva, Amanda K Andriola; Wilhelm, Claire; Kolosnjaj-Tabi, Jelena; Luciani, Nathalie; Gazeau, Florence

    2012-05-01

    Cell labeling with magnetic nanoparticles can be used to monitor the fate of transplanted cells in vivo by magnetic resonance imaging. However, nanoparticles initially internalized in administered cells might end up in other cells of the host organism. We investigated a mechanism of intercellular cross-transfer of magnetic nanoparticles to different types of recipient cells via cell microvesicles released under cellular stress. Three cell types (mesenchymal stem cells, endothelial cells and macrophages) were labeled with 8-nm iron oxide nanoparticles. Then cells underwent starvation stress, during which they produced microvesicles that were subsequently transferred to unlabeled recipient cells. The analysis of the magnetophoretic mobility of donor cells indicated that magnetic load was partially lost under cell stress. Microvesicles shed by stressed cells participated in the release of magnetic label. Moreover, such microvesicles were uptaken by naïve cells, resulting in cellular redistribution of nanoparticles. Iron load of recipient cells allowed their detection by MRI. Cell microvesicles released under stress may be disseminated throughout the organism, where they can be uptaken by host cells. The transferred cargo may be sufficient to allow MRI detection of these secondarily labeled cells, leading to misinterpretations of the effectiveness of transplanted cells.

  11. A liposome-based size calibration method for measuring microvesicles by flow cytometry.

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    Simonsen, J B

    2016-01-01

    ESSENTIALS: A gold standard to determine the sizes of microvesicles by flow cytometry is needed. We used fluorescently labeled liposomes to estimate the size of microvesicles with flow cytometry. We suggest that liposomes are more accurate size calibrators than the commonly used polystyrene beads. The liposome-based size calibrators improve the size assessment of microvesicle made with flow cytometry. During the past years, the need for a gold standard to determine the sizes of extracellular vesicles including microvesicles by flow cytometry has been emphasized. This work suggests that artificial vesicles can be used as calibrators to estimate the size of microvesicles from the side scattering (SSC) measured with flow cytometry. We prepared fluorescently labeled liposomes with different maximum sizes defined by the pore size (200, 400, 800, and 1000 nm) of the membrane used for the extrusion. The fluorescence strengths from the largest liposomes pertaining to each pore size enabled us to verify the correlation between the SSC from a liposome and the corresponding size. This study indicates that artificial vesicles are more accurate size calibrators compared to the commonly used polystyrene calibrator beads illustrated by the SSC from 110 nm polystyrene beads corresponds to the scattering from ~400 nm vesicle-like particles. We also show that this method of size assessment based on SSC has a low resolution that is roughly estimated to be between 60 and 200 nm, dependent on the vesicle size. © 2015 International Society on Thrombosis and Haemostasis.

  12. Microvesicle Cargo of Tumor-Associated MUC1 to Dendritic Cells Allows Cross-presentation and Specific Carbohydrate Processing

    DEFF Research Database (Denmark)

    Rughetti, Aurelia; Rahimi, Hassan; Belleudi, Francesca

    2014-01-01

    . Here, we show that the form of the MUC1 antigen, i.e., soluble or as microvesicle cargo, influences MUC1 processing in dendritic cells. In fact, MUC1 carried by microvesicles translocates from the endolysosomal/HLA-II to the HLA-I compartment and is presented by dendritic cells to MUC1-specific CD8...

  13. Elevated levels of procoagulant plasma microvesicles in dialysis patients.

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    James O Burton

    Full Text Available Cardiovascular (CV death remains the largest cause of mortality in dialysis patients, unexplained by traditional risk factors. Endothelial microvesicles (EMVs are elevated in patients with traditional CV risk factors and acute coronary syndromes while platelet MVs (PMVs are associated with atherosclerotic disease states. This study compared relative concentrations of circulating MVs from endothelial cells and platelets in two groups of dialysis patients and matched controls and investigated their relative thromboembolic risk. MVs were isolated from the blood of 20 haemodialysis (HD, 17 peritoneal dialysis (PD patients and 20 matched controls. Relative concentrations of EMVs (CD144(+ ve and PMVs (CD42b(+ ve were measured by Western blotting and total MV concentrations were measured using nanoparticle-tracking analysis. The ability to support thrombin generation was measured by reconstituting the MVs in normal plasma, using the Continuous Automated Thrombogram assay triggered with 1µM tissue factor. The total concentration of MVs as well as the measured sub-types was higher in both patient groups compared to controls (p0.3. Dialysis patients have higher levels of circulating procoagulant MVs than healthy controls. This may represent a novel and potentially modifiable mediator or predictor of occlusive cardiovascular events in these patients.

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

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    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

  15. Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse

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    Choudhuri, Kaushik; Llodrá, Jaime; Roth, Eric W.; Tsai, Jones; Gordo, Susana; Wucherpfennig, Kai W.; Kam, Lance C.; Stokes, David L.; Dustin, Michael L.

    2014-03-01

    The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen-presenting cells (APCs). T-cell signalling is initiated at these contacts when surface-expressed T-cell receptors (TCRs) recognize peptide fragments (antigens) of pathogens bound to major histocompatibility complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft. T-cell recognition of pMHC and the adhesion ligand intercellular adhesion molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse, which is characterized by central accumulation of TCRs, adjacent to a secretory domain, both surrounded by an adhesive ring. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function, this domain is itself largely devoid of TCR signalling activity, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic immunological synapse periphery. Here we show that centrally accumulated TCRs are located on the surface of extracellular microvesicles that bud at the immunological synapse centre. Tumour susceptibility gene 101 (TSG101) sorts TCRs for inclusion in microvesicles, whereas vacuolar protein sorting 4 (VPS4) mediates scission of microvesicles from the T-cell plasma membrane. The human immunodeficiency virus polyprotein Gag co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCRs from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These

  16. [The origin and possible role of microvesicles in olfactory receptor cells].

    Science.gov (United States)

    Bakhtin, E K

    1975-08-01

    Microvesicles and spherical particles have been described in the bulbs of receptor olfactory cells of Acipenser ruthenus. Two pathways of the origin of the above vesicles have been followed. These structures derive at the stage of differentiation from non-ciliary to ciliary cell type. The first of the pathways involves the autolysis of microfibril bundles produced during the regression of microvilli. The other one includes micropinocytosis induced on the basis of regressing microvilli. Taking into account the genesis of the microvesicles of the receptor cell bulb, it is concluded that they cannot contain a mediator able to modify membrane ion permeability in response to the specific stimulus of the odorant.

  17. Characteristics of erythrocyte-derived microvesicles and its relation with atherosclerosis.

    Science.gov (United States)

    Li, Kai-Yin; Zheng, Lei; Wang, Qian; Hu, Yan-Wei

    2016-12-01

    Microvesicles are formed under many circumstances, especially in atheromatous plaques. Erythrocyte-derived microvesicles (ErMVs) have been proved to promote atherosclerosis by promoting hypercoagulation, mediating inflammation and inducing cell adhesion. Several clinical studies have reported potential roles of ErMVs in cardiovascular disease diagnosis, but the current understanding of ErMVs remains insufficient. In this paper, we will review current research on the formation and degradation of ErMVs and the possible effects of ErMVs in atherosclerosis, discuss potential clinical applications in cardiovascular disease, and hope to raise awareness of the relation with atherosclerosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. MicroRNA-100 shuttled by mesenchymal stem cell-derived exosomes suppresses in vitro angiogenesis through modulating the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells.

    Science.gov (United States)

    Pakravan, Katayoon; Babashah, Sadegh; Sadeghizadeh, Majid; Mowla, Seyed Javad; Mossahebi-Mohammadi, Majid; Ataei, Farangis; Dana, Nasim; Javan, Mohammad

    2017-10-01

    Human mesenchymal stem cells (MSCs) have been shown to be involved in the formation and modulation of tumor stroma and in interacting with tumor cells, partly through their secretome. Exosomes are nano-sized intraluminal multi-vesicular bodies secreted by most types of cells and have been found to mediate intercellular communication through the transfer of genetic information via coding and non-coding RNAs to recipient cells. Since exosomes are considered as protective and enriched sources of shuttle microRNAs (miRNAs), we hypothesized that exosomal transfer of miRNAs from MSCs may affect tumor cell behavior, particularly angiogenesis. Exosomes derived from MSCs were isolated and characterized by scanning electron microscopy analyses, dynamic light scattering measurements, and Western blotting. Fold changes in miR-100 expression levels were calculated in exosomes and their corresponding donor cells by qRT-PCR. The effects of exosomal transfer of miR-100 from MSCs were assessed by qRT-PCR and Western blotting of the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells. The quantification of secreted VEGF protein was determined by enzyme-linked immunosorbent assay. The putative paracrine effects of MSC-derived exosomes on tumor angiogenesis were explored by in vitro angiogenesis assays including endothelial cell proliferation, migration and tube formation assays. We found that MSC-derived exosomes induce a significant and dose-dependent decrease in the expression and secretion of vascular endothelial growth factor (VEGF) through modulating the mTOR/HIF-1α signaling axis in breast cancer-derived cells. We also found that miR-100 is enriched in MSC-derived exosomes and that its transfer to breast cancer-derived cells is associated with the down-regulation of VEGF in a time-dependent manner. The putative role of exosomal miR-100 transfer in regulating VEGF expression was substantiated by the ability of anti-miR-100 to rescue the inhibitory effects of MSC

  19. Microvesicles Correlated with Components of Metabolic Syndrome in Men with Type 2 Diabetes Mellitus and Lowered Testosterone Levels But Were Unaltered by Testosterone Therapy

    DEFF Research Database (Denmark)

    Botha, Jaco; Velling Magnussen, Line; Nielsen, Morten Hjuler

    2017-01-01

    not correlate with any microvesicle phenotypes. Microvesicle levels were unaffected by testosterone therapy. Conclusions. Metabolic syndrome components and hepatic fat accumulation correlated with microvesicle phenotypes, supporting the involvement of especially CD36 on monocytes in metabolic syndrome......Aims. To investigate how circulating microvesicle phenotypes correlate with insulin sensitivity, body composition, plasma lipids, and hepatic fat accumulation. We hypothesized that changes elicited by testosterone replacement therapy are reflected in levels of microvesicles. Methods. Thirty......-nine type 2 diabetic males with lowered testosterone levels were assigned to either testosterone replacement therapy or placebo and evaluated at baseline and after 24 weeks. Microvesicles were analysed by flow cytometry and defined as lactadherin-binding particles within the 0.1-1.0 μm gate. Microvesicles...

  20. Multiple Sclerosis Treatments Affect Monocyte-Derived Microvesicle Production

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    Maria Blonda

    2017-08-01

    Full Text Available Microvesicles (MVs are released by immune cells especially of the myeloid lineage upon stimulation with ATP on its cognate receptor P2X7, both in physiological and pathological conditions. In multiple sclerosis (MS the role of MVs remains little investigated. We aimed to compare the release of MVs in peripheral blood monocytes from MS patients with healthy donors (HDs and to see how current MS treatment may affect such a production. We also assessed the treatment effect on M1 and M2 monocyte polarization and on the inflammasome components. Spectrophotometric quantification was performed to compare monocyte-derived MVs from 20 untreated relapsing-remitting MS patients and 20 HDs and to evaluate the effect of different treatments. Subgroups of nine interferon-beta and of five teriflunomide-treated MS patients were evaluated at baseline and after 2, 6, and 12 months of treatment. Six MS patients taking Fingolimod, after switching from a first-line therapy, were included in the study and analyzed only at 12 months of treatment. MVs analysis revealed that monocytes from MS patients produced vesicles in higher amounts than controls. All treatments reduced vesicle production but only teriflunomide was associated with a downregulation of purinergic P2X7 receptor and inflammasome components expression. The therapies modulated mRNA expression of both M1 and M2 monocyte markers. Our results, suggesting new molecular targets for drugs currently used in MS, may potentially provide useful novel evidence to approach the disease.

  1. Levels of procoagulant microvesicles are elevated after traumatic injury and platelet microvesicles are negatively correlated with mortality

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    Nicola Curry

    2014-10-01

    Full Text Available Background: Microvesicles (MV have been implicated in the development of thrombotic disease, such as acute respiratory distress syndrome (ARDS and multiple organ failure (MOF. Trauma patients are at increased risk of late thrombotic events, particularly those who receive a major transfusion. The aims of this study were: (a to determine whether there were increased numbers of pro-coagulant MV following injury; (b to determine their cellular origin; and (c to explore the effects of MV with clinical outcomes; in particular red cell transfusion requirements and death. Methods: Trauma patients were recruited at a Level 1 trauma centre. The presence of MV procoagulant phospholipid (PPL was assessed using 2 activity assays (PPL and thrombin generation. Enumeration and MV cellular origin was assessed using 2 colour flow cytometry. Results: Fifty consecutive patients were recruited; median age 38 (IQR: 24–55, median ISS 18 (IQR: 9–27. Circulating procoagulant MV, rich in phospholipid, were significantly elevated following traumatic injury relative to controls and remained elevated at 72 h post-injury. Red cell/AnnV+ and platelet/AnnV+ MV numbers were 6-fold and 2-fold higher than controls, respectively. Patients who died (n=9, 18% had significantly fewer CD41/AnnV+ MV and lower endogenous thrombin potential relative to patients who survived. Conclusions: MV are elevated following traumatic injury and may be implicated in the increased risk of trauma patients to pro-thrombotic states such as MOF and ARDS. Lower levels of procoagulant MV are associated with mortality and further investigation of this association is warranted.

  2. Impact of biofluid viscosity on size and sedimentation efficiency of the isolated microvesicles

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    Fatemeh eMomen-Heravi

    2012-05-01

    Full Text Available Microvesicles are nano-sized lipid vesicles released by all cells in vivo and in vitro. They are released physiologically under normal conditions but their rate of release is higher under pathological conditions such as tumors. Once released they end up in the systemic circulation and have been found and characterized in all biofluids such as plasma, serum, cerebrospinal fluid (CSF, breast milk, ascites, and urine. Microvesicles represent the status of the donor cell they are released from and they are currently under intense investigation as a potential source for disease biomarkers. Currently, the gold standard for isolating microvesicles is ultracentrifugation, although alternative techniques such as affinity purification have been explored. Viscosity is the resistance of a fluid to a deforming force by either shear or tensile stress. The different chemical and molecular compositions of biofluids have an effect on its viscosity and this could affect movements of the particles inside the fluid. In this manuscript we addressed the issue of whether viscosity has an effect on sedimentation efficiency of microvesicles using ultracentrifugation. We used different biofluids and spiked them with polystyrene beads and assessed their recovery using the Nanoparticle Tracking Analysis. We demonstrate that MVs recovery inversely correlates with viscosity and as a result, sample dilutions should be considered prior to ultracentifugation when processing any biofluids.

  3. Expression of mucinous ovarian-cancer antigen in hybrid-cells derived by fusing a malignantly transformed bloom-syndrome cell-line (bs-shi-4m ovc-mu) and mouse L-cell-line.

    Science.gov (United States)

    Shiraishi, Y; Taguchi, T; Seguchi, H

    1995-12-01

    The expression of mucinous ovarian cancer (OVC) antigen by hybrid cell lines derived by fusing a malignantly transformed Bloom syndrome (BS) cell line (BS-SHI-4M OVC-MU) and mouse L cells has been studied. Cell surfaces of 16 hybrids which grew 12-40 days post-fusion have been analysed by using a number of sera from mucinous OVC patients and indirect membrane immunofluorescence (IF). Three hybrids which appeared at a late stage (30-40 days in 24-well culture plates) were clearly positive in almost 100% of the hybrid cell population, while all of those which appeared earlier (in 12 days) were completely negative. Cytogenetic analysis showed that these three hybrids with positive mucinous OVC antigen contained human chromosome 22 or 22q-, though no metaphases from antigen negative population had 22 or 22q-. Therefore, we assign the gene for mucinous OVC antigen to the chromosome 22. Identification of chromosome 22 in hybrid cells was confirmed by the fluorescence in situ hybridization (FISH) analysis using chromosome 22 painting probe.

  4. Microvesicles/exosomes as potential novel biomarkers of metabolic diseases

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    Müller G

    2012-08-01

    Full Text Available Günter MüllerDepartment of Biology 1, Genetics, Ludwig-Maximilians University Munich, Biocenter, Munich, GermanyAbstract: Biomarkers are of tremendous importance for the prediction, diagnosis, and observation of the therapeutic success of common complex multifactorial metabolic diseases, such as type II diabetes and obesity. However, the predictive power of the traditional biomarkers used (eg, plasma metabolites and cytokines, body parameters is apparently not sufficient for reliable monitoring of stage-dependent pathogenesis starting with the healthy state via its initiation and development to the established disease and further progression to late clinical outcomes. Moreover, the elucidation of putative considerable differences in the underlying pathogenetic pathways (eg, related to cellular/tissue origin, epigenetic and environmental effects within the patient population and, consequently, the differentiation between individual options for disease prevention and therapy – hallmarks of personalized medicine – plays only a minor role in the traditional biomarker concept of metabolic diseases. In contrast, multidimensional and interdependent patterns of genetic, epigenetic, and phenotypic markers presumably will add a novel quality to predictive values, provided they can be followed routinely along the complete individual disease pathway with sufficient precision. These requirements may be fulfilled by small membrane vesicles, which are so-called exosomes and microvesicles (EMVs that are released via two distinct molecular mechanisms from a wide variety of tissue and blood cells into the circulation in response to normal and stress/pathogenic conditions and are equipped with a multitude of transmembrane, soluble and glycosylphosphatidylinositol-anchored proteins, mRNAs, and microRNAs. Based on the currently available data, EMVs seem to reflect the diverse functional and dysfunctional states of the releasing cells and tissues along the

  5. Characterization of Microvesicles Released from Human Red Blood Cells

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    Duc Bach Nguyen

    2016-03-01

    Full Text Available Background/Aims: Extracellular vesicles (EVs are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Methods: Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS, scanning electron microscopy (SEM, atomic force microscopy (AFM and dynamic light scattering (DLS. The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Results: Treatment of RBCs with 4-bromo-A23187 (positive control, lysophosphatidic acid (LPA, or phorbol-12 myristate-13 acetate (PMA in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 m

  6. CD9-positive microvesicles mediate the transfer of molecules to Bovine Spermatozoa during epididymal maturation.

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    Julieta N Caballero

    Full Text Available Acquisition of fertilization ability by spermatozoa during epididymal transit occurs in part by the transfer of molecules from membranous vesicles called epididymosomes. Epididymosomes are heterogeneous in terms of both size and molecular composition. Exosomes and other related small membranous vesicles (30-120 nm containing tetraspanin proteins on their surface are found in many biological fluids. In this study, we demonstrate that these vesicles are present in bovine cauda epididymal fluid as a subpopulation of epididymosomes. They contain tetraspanin CD9 in addition to other proteins involved in sperm maturation such as P25b, GliPr1L1, and MIF. In order to study the mechanism of protein transfer to sperm, DilC12-labeled unfractionated epididymosomes or CD9-positive microvesicles were coincubated with epididymal spermatozoa, and their transfer was evaluated by flow cytometry. CD9-positive microvesicles from epididymal fluid specifically transferred molecules to spermatozoa, whereas those prepared from blood were unable to do so. The CD9-positive microvesicles transferred molecules to the same sperm regions (acrosome and midpiece as epididymosomes, with the same kinetics; however, the molecules were preferentially transferred to live sperm and, in contrast to epididymosomes, Zn(2+ did not demonstrate potentiated transfer. Tetraspanin CD9 was associated with other proteins on the membrane surface of CD9-positive microvesicles according to coimmunoprecipitation experiments. CD26 cooperated with CD9 in the molecular transfer to sperm since the amount of molecules transferred was significantly reduced in the presence of specific antibodies. In conclusion, CD9-positive microvesicles are present in bovine cauda epididymal fluid and transfer molecules to live maturing sperm in a tissue-specific manner that involves CD9 and CD26.

  7. CD9-positive microvesicles mediate the transfer of molecules to Bovine Spermatozoa during epididymal maturation.

    Science.gov (United States)

    Caballero, Julieta N; Frenette, Gilles; Belleannée, Clémence; Sullivan, Robert

    2013-01-01

    Acquisition of fertilization ability by spermatozoa during epididymal transit occurs in part by the transfer of molecules from membranous vesicles called epididymosomes. Epididymosomes are heterogeneous in terms of both size and molecular composition. Exosomes and other related small membranous vesicles (30-120 nm) containing tetraspanin proteins on their surface are found in many biological fluids. In this study, we demonstrate that these vesicles are present in bovine cauda epididymal fluid as a subpopulation of epididymosomes. They contain tetraspanin CD9 in addition to other proteins involved in sperm maturation such as P25b, GliPr1L1, and MIF. In order to study the mechanism of protein transfer to sperm, DilC12-labeled unfractionated epididymosomes or CD9-positive microvesicles were coincubated with epididymal spermatozoa, and their transfer was evaluated by flow cytometry. CD9-positive microvesicles from epididymal fluid specifically transferred molecules to spermatozoa, whereas those prepared from blood were unable to do so. The CD9-positive microvesicles transferred molecules to the same sperm regions (acrosome and midpiece) as epididymosomes, with the same kinetics; however, the molecules were preferentially transferred to live sperm and, in contrast to epididymosomes, Zn(2+) did not demonstrate potentiated transfer. Tetraspanin CD9 was associated with other proteins on the membrane surface of CD9-positive microvesicles according to coimmunoprecipitation experiments. CD26 cooperated with CD9 in the molecular transfer to sperm since the amount of molecules transferred was significantly reduced in the presence of specific antibodies. In conclusion, CD9-positive microvesicles are present in bovine cauda epididymal fluid and transfer molecules to live maturing sperm in a tissue-specific manner that involves CD9 and CD26.

  8. A Meta-Analysis of Circulating Microvesicles in Patients with Myocardial Infarction.

    Science.gov (United States)

    Wang, Zhida; Cai, Wang; Hu, Shaolan; Xia, Yufei; Wang, Yao; Zhang, Qi; Chen, Liming

    2017-07-10

    Cell-derived microvesicles (MVs) are vesicles released from activated or apoptotic cells. However, the levels of MVs in myocardial infarction have been found inconsistent in researches. To assess the association between MVs and myocardial infarction by conducting a meta-analysis. A systematic literature search on PubMed, Embase, Cochran, Google Scholar electronic database was conducted. Comparison of the MVs levels between myocardial infarction patients and healthy persons were included in our study. Standard Mean Difference (SMD) and 95% confidence interval (CI) in groups were calculated and meta-analyzed. 11 studies with a total of 436 participants were included. Compared with the health persons, AMVs [SMD = 3.65, 95% CI (1.03, 6.27)], PMVs [SMD = 2.88, 95% CI (1.82, 3.93),] and EMVs [SMD = 2.73, 95% CI (1.13, 4.34)], levels were higher in patients with myocardial infarction. However, LMVs levels [SMD = 0.73, 95% CI (-0.57, 2.03)] were not changed significantly in patients with myocardial infarction. AMVs, PMVs and EMVs might be potential biomarkers for myocardial infarction. As microvesículas derivadas de células (MVs) são vesículas liberadas de células ativadas ou apoptóticas. No entanto, os níveis de MVs no infarto do miocárdio foram encontrados inconsistentes nas pesquisas. Avaliar a associação entre MV e infarto do miocárdio por meio de uma meta-análise. Foi realizada uma pesquisa sistemática na literatura em PubMed, Embase, Cochran e no banco de dados eletrônico do Google Scholar. Uma comparação dos níveis de MV entre pacientes com infarto do miocárdio e pessoas saudáveis foi incluída no nosso estudo. A Diferença Média Padrão (DMP) e o intervalo de confiança (IC) de 95% nos grupos foram calculadas e meta-analisadas. Foram incluídos 11 estudos com um total de 436 participantes. Em comparação com as pessoas saudáveis, as MVA [DMP = 3,65, IC 95% (1,03, 6,27)], MVPs [DMP = 2,88, IC 95% (1,82, 3,93)] e MVEs [DMP = 2,73, IC 95% (1

  9. Microvesicles Derived From Human Mesenchymal Stem Cells Restore Alveolar Fluid Clearance in Human Lungs Rejected for Transplantation.

    Science.gov (United States)

    Gennai, S; Monsel, A; Hao, Q; Park, J; Matthay, M A; Lee, J W

    2015-09-01

    The need to increase the donor pool for lung transplantation is a major public health issue. We previously found that administration of mesenchymal stem cells "rehabilitated" marginal donor lungs rejected for transplantation using ex vivo lung perfusion. However, the use of stem cells has some inherent limitation such as the potential for tumor formation. In the current study, we hypothesized that microvesicles, small anuclear membrane fragments constitutively released from mesenchymal stem cells, may be a good alternative to using stem cells. Using our well established ex vivo lung perfusion model, microvesicles derived from human mesenchymal stem cells increased alveolar fluid clearance (i.e. ability to absorb pulmonary edema fluid) in a dose-dependent manner, decreased lung weight gain following perfusion and ventilation, and improved airway and hemodynamic parameters compared to perfusion alone. Microvesicles derived from normal human lung fibroblasts as a control had no effect. Co-administration of microvesicles with anti-CD44 antibody attenuated these effects, suggesting a key role of the CD44 receptor in the internalization of the microvesicles into the injured host cell and its effect. In summary, microvesicles derived from human mesenchymal stem cells were as effective as the parent mesenchymal stem cells in rehabilitating marginal donor human lungs. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

  10. Human pancreatic tumors grown in mice release tissue factor-positive microvesicles that increase venous clot size.

    Science.gov (United States)

    Hisada, Y; Ay, C; Auriemma, A C; Cooley, B C; Mackman, N

    2017-11-01

    Essentials Tumor-bearing mice have larger venous clots than controls. Human tissue factor is present in clots in tumor-bearing mice. Inhibition of human tissue factor reduces clot size in tumor-bearing mice. This new mouse model may be useful to study mechanisms of cancer-associated thrombosis. Background Pancreatic cancer patients have a high rate of venous thromboembolism. Human pancreatic tumors and cell lines express high levels of tissue factor (TF), and release TF-positive microvesicles (TF+ MVs). In pancreatic cancer patients, tumor-derived TF+ MVs are present in the blood, and increased levels are associated with venous thromboembolism and decreased survival. Previous studies have shown that mice with orthotopic human or murine pancreatic tumors have circulating tumor-derived TF+ MVs, an activated clotting system, and increased incidence and mean clot weight in an inferior vena cava stenosis model. These results suggest that TF+ MVs contribute to thrombosis. However, the specific role of tumor-derived TF+ MVs in venous thrombosis in mice has not been determined. Objectives To test the hypothesis that tumor-derived TF+ MVs enhance thrombosis in mice. Methods We determined the contribution of TF+ MVs derived from human pancreatic tumors grown orthotopically in nude mice to venous clot formation by using an anti-human TF mAb. We used an inferior vena cava stasis model of venous thrombosis. Results Tumor-bearing mice had significantly larger clots than control mice. Clots from tumor-bearing mice contained human TF, suggesting the incorporation of tumor-derived MVs. Importantly, administration of an anti-human TF mAb reduced clot size in tumor-bearing mice but did not affect clot size in control mice. Conclusions Our results indicate that TF+ MVs released from orthotopic pancreatic tumors increase venous thrombosis in mice. This new model may be useful for evaluating the roles of different factors in cancer-associated thrombosis. © 2017 International Society on

  11. A liposome-based size calibration method for measuring microvesicles by flow cytometry

    DEFF Research Database (Denmark)

    Simonsen, Jens Bæk

    2016-01-01

    BACKGROUND: Over the last years the need for a gold standard to determine the sizes of extracellular vesicles including microvesicles by flow cytometry has been emphasized. METHODS: This work suggests to use artificial vesicles as calibrators to ascertain the size of microvesicles from the side...... to verify the correlation between the SSC from a liposome and the corresponding size. CONCLUSIONS: We show that artificial vesicles are more accurate size calibrators compared to the commonly used polystyrene calibrator beads illustrated by the SSC from 110 nm polystyrene beads corresponds to the scattering...... from ~400 nm sized vesicle-like particles. We also show that this method of size assessment based on SSC has a low resolution that is roughly estimated to be between 60 to 200 nm dependent on the vesicle size. This article is protected by copyright. All rights reserved....

  12. A Novel Role for Pro-Coagulant Microvesicles in the Early Host Defense against Streptococcus pyogenes

    OpenAIRE

    Oehmcke, Sonja; Westman, Johannes; Malmstr?m, Johan; M?rgelin, Matthias; Olin, Anders I.; Kreikemeyer, Bernd; Herwald, Heiko

    2013-01-01

    Previous studies have shown that stimulation of whole blood or peripheral blood mononuclear cells with bacterial virulence factors results in the sequestration of pro-coagulant microvesicles (MVs). These particles explore their clotting activity via the extrinsic and intrinsic pathway of coagulation; however, their pathophysiological role in infectious diseases remains enigmatic. Here we describe that the interaction of pro-coagulant MVs with bacteria of the species Streptococcus pyogenes is ...

  13. Do Neural Cells Communicate with Endothelial Cells via Secretory Exosomes and Microvesicles?

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    Neil R. Smalheiser

    2009-01-01

    Full Text Available Neurons, glial, cells, and brain tumor cells tissues release small vesicles (secretory exosomes and microvesicles, which may represent a novel mechanism by which neuronal activity could influence angiogenesis within the embryonic and mature brain. If CNS-derived vesicles can enter the bloodstream as well, they may communicate with endothelial cells in the peripheral circulation and with cells concerned with immune surveillance.

  14. Mechanisms of infectivity and evasion derived from microvesicles cargo produced by Trypanosoma cruzi

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    Bruna Cristina Borges

    2016-11-01

    Full Text Available Cell invasion by the intracellular protozoans requires interaction of proteins from both the host and the parasite. Many parasites establish chronic infections, showing they have the potential to escape the immune system; for example, Trypanosoma cruzi is an intracellular parasite that causes Chagas disease. Parasite internalization into host cell requires secreted and surface molecules, such as microvesicles. The release of microvesicles and other vesicles, such as exosomes, by different eukaryotic organisms was first observed in the late 20th century. The characterization and function of these vesicles have recently been the focus of several investigations. In this review, we discuss the release of microvesicles by T. cruzi. The molecular content of these vesicles is composed of several molecules that take place during parasite-host cell interaction and contribute to the parasite-driven mechanism of evasion from the host immune system. These new findings appear to have a profound impact on the comprehension of T. cruzi biology and highlight novel potential strategies for developing more efficient therapeutic approaches.

  15. Microvesicles derived from adult human bone marrow and tissue specific mesenchymal stem cells shuttle selected pattern of miRNAs.

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    Federica Collino

    Full Text Available BACKGROUND: Cell-derived microvesicles (MVs have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs and liver resident stem cells (HLSCs were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs. METHODOLOGY/PRINCIPAL FINDINGS: MVs were isolated from MSCs and HLSCs. The presence in MVs of selected ribonucleoproteins involved in the traffic and stabilization of RNA was evaluated. We observed that MVs contained TIA, TIAR and HuR multifunctional proteins expressed in nuclei and stress granules, Stau1 and 2 implicated in the transport and stability of mRNA and Ago2 involved in miRNA transport and processing. RNA extracted from MVs and cells of origin was profiled for 365 known human mature miRNAs by real time PCR. Hierarchical clustering and similarity analysis of miRNAs showed 41 co-expressed miRNAs in MVs and cells. Some miRNAs were accumulated within MVs and absent in the cells after MV release; others were retained within the cells and not secreted in MVs. Gene ontology analysis of predicted and validated targets showed that the high expressed miRNAs in cells and MVs could be involved in multi-organ development, cell survival and differentiation. Few selected miRNAs shuttled by MVs were also associated with the immune system regulation. The highly expressed miRNAs in MVs were transferred to target cells after MV incorporation. CONCLUSIONS: This study demonstrated that MVs contained ribonucleoproteins involved in the intracellular traffic of RNA and selected pattern of miRNAs, suggesting a dynamic regulation of RNA compartmentalization in MVs. The observation that MV-highly expressed miRNAs were transferred to target cells, rises the possibility that the biological effect of stem

  16. Cell-derived vesicles exposing coagulant tissue factor in saliva

    NARCIS (Netherlands)

    Berckmans, René J.; Sturk, Auguste; van Tienen, Laurens M.; Schaap, Marianne C. L.; Nieuwland, Rienk

    2011-01-01

    On vascular damage, coagulation is initiated by extravascular tissue factor (TF). Intravascular TF, which is present on circulating cell-derived vesicles, is non-coagulant under physiologic conditions but prothrombotic under pathologic conditions. Human saliva triggers coagulation, but the mechanism

  17. 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...

  18. 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

  19. Comprehensive proteomic characterization of stem cell-derived extracellular matrices.

    Science.gov (United States)

    Ragelle, Héloïse; Naba, Alexandra; Larson, Benjamin L; Zhou, Fangheng; Prijić, Miralem; Whittaker, Charles A; Del Rosario, Amanda; Langer, Robert; Hynes, Richard O; Anderson, Daniel G

    2017-06-01

    In the stem-cell niche, the extracellular matrix (ECM) serves as a structural support that additionally provides stem cells with signals that contribute to the regulation of stem-cell function, via reciprocal interactions between cells and components of the ECM. Recently, cell-derived ECMs have emerged as in vitro cell culture substrates to better recapitulate the native stem-cell microenvironment outside the body. Significant changes in cell number, morphology and function have been observed when mesenchymal stem cells (MSC) were cultured on ECM substrates as compared to standard tissue-culture polystyrene (TCPS). As select ECM components are known to regulate specific stem-cell functions, a robust characterization of cell-derived ECM proteomic composition is critical to better comprehend the role of the ECM in directing cellular processes. Here, we characterized and compared the protein composition of ECM produced in vitro by bone marrow-derived MSC, adipose-derived MSC and neonatal fibroblasts from different donors, employing quantitative proteomic methods. Each cell-derived ECM displayed a specific and unique matrisome signature, yet they all shared a common set of proteins. We evaluated the biological response of cells cultured on the different matrices and compared them to cells on standard TCPS. The matrices lead to differential survival and gene-expression profiles among the cell types and as compared to TCPS, indicating that the cell-derived ECMs influence each cell type in a different manner. This general approach to understanding the protein composition of different tissue-specific and cell-derived ECM will inform the rational design of defined systems and biomaterials that recapitulate critical ECM signals for stem-cell culture and tissue engineering. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Stem Cell-Derived Extracellular Vesicles and Immune-Modulation.

    Science.gov (United States)

    Burrello, Jacopo; Monticone, Silvia; Gai, Chiara; Gomez, Yonathan; Kholia, Sharad; Camussi, Giovanni

    2016-01-01

    Extra-cellular vesicles (EVs) are bilayer membrane structures enriched with proteins, nucleic acids, and other active molecules and have been implicated in many physiological and pathological processes over the past decade. Recently, evidence suggests EVs to play a more dichotomic role in the regulation of the immune system, whereby an immune response may be enhanced or supressed by EVs depending on their cell of origin and its functional state. EVs derived from antigen (Ag)-presenting cells for instance, have been involved in both innate and acquired (or adaptive) immune responses, as Ag carriers or presenters, or as vehicles for delivering active signaling molecules. On the other hand, tumor and stem cell derived EVs have been identified to exert an inhibitory effect on immune responses by carrying immuno-modulatory effectors, such as transcriptional factors, non-coding RNA (Species), and cytokines. In addition, stem cell-derived EVs have also been reported to impair dendritic cell maturation and to regulate the activation, differentiation, and proliferation of B cells. They have been shown to control natural killer cell activity and to suppress the innate immune response (IIR). Studies reporting the role of EVs on T lymphocyte modulation are controversial. Discrepancy in literature may be due to stem cell culture conditions, methods of EV purification, EV molecular content, and functional state of both parental and target cells. However, mesenchymal stem cell-derived EVs were shown to play a more suppressive role by shifting T cells from an activated to a T regulatory phenotype. In this review, we will discuss how stem cell-derived EVs may contribute toward the modulation of the immune response. Collectively, stem cell-derived EVs mainly exhibit an inhibitory effect on the immune system.

  1. STEM CELL-DERIVED EXTRACELLULAR VESICLES AND IMMUNE-MODULATION

    Directory of Open Access Journals (Sweden)

    Jacopo Burrello

    2016-08-01

    Full Text Available Extra-cellular vesicles (EVs are bilayer membrane structures enriched with proteins, nucleic acids and other active molecules and have been implicated in many physiological and pathological processes over the past decade. Recently, evidence suggests EVs to play a more dichotomic role in the regulation of the immune system, whereby an immune response may be enhanced or supressed by EVs depending on their cell of origin and its functional state. EVs derived from antigen (Ag-presenting cells for instance, have been involved in both innate and acquired (or adaptive immune responses, as Ag carriers or presenters, or as vehicles for delivering active signalling molecules. On the other hand, tumor and stem cell derived EVs have been identified to exert an inhibitory effect on immune responses by carrying immuno-modulatory effectors, such as transcriptional factors, non-coding RNA (Species and cytokines. In addition, stem cell-derived EVs have also been reported to impair dendritic cell maturation and to regulate the activation, differentiation and proliferation of B cells. They have been shown to control natural killer cell activity and to suppress the innate immune response. Studies reporting the role of EVs on T lymphocyte modulation are controversial. Discrepancy in literature may be due to stem cell culture conditions, methods of EV purification, EV molecular content and functional state of both parental and target cells. However, mesenchymal stem cell-derived EVs were shown to play a more suppressive role by shifting T cells from an activated to a T regulatory phenotype. In this review we will discuss how stem cell-derived EVs may contribute towards the modulation of the immune response. Collectively, stem cell-derived EVs mainly exhibit an inhibitory effect on the immune system.

  2. Lung epithelial cell-derived extracellular vesicles activate macrophage-mediated inflammatory responses via ROCK1 pathway.

    Science.gov (United States)

    Moon, H-G; Cao, Y; Yang, J; Lee, J H; Choi, H S; Jin, Y

    2015-12-10

    Despite decades of research, the pathogenesis of acute respiratory distress syndrome (ARDS) remains poorly understood, thus impeding the development of effective treatment. Diffuse alveolar damage (DAD) and lung epithelial cell death are prominent features of ARDS. Lung epithelial cells are the first line of defense after inhaled stimuli, such as in the case of hyperoxia. We hypothesized that lung epithelial cells release 'messenger' or signaling molecules to adjacent or distant macrophages, thereby initiating or propagating inflammatory responses after noxious insult. We found that, after hyperoxia, a large amount of extracellular vesicles (EVs) were generated and released into bronchoalveolar lavage fluid (BALF). These hyperoxia-induced EVs were mainly derived from live lung epithelial cells as the result of hyperoxia-associated endoplasmic reticulum (ER) stress. These EVs were remarkably different from epithelial 'apoptotic bodies', as reflected by the significantly smaller size and differentially expressed protein markers. These EVs fall mainly in the size range of the exosomes and smaller microvesicles (MVs) (50-120 nm). The commonly featured protein markers of apoptotic bodies were not found in these EVs. Treating alveolar macrophages with hyperoxia-induced, epithelial cell-derived EVs led to an increased secretion of pro-inflammatory cytokines and macrophage inflammatory protein 2 (MIP-2). Robustly increased macrophage and neutrophil influx was found in the lung tissue of the mice intranasally treated with hyperoxia-induced EVs. It was determined that EV-encapsulated caspase-3 was largely responsible for the alveolar macrophage activation via the ROCK1 pathway. Caspase-3-deficient EVs induced less cytokine/MIP-2 release, reduced cell counts in BALF, less neutrophil infiltration and less inflammation in lung parenchyma, both in vitro and in vivo. Furthermore, the serum circulating EVs were increased and mainly derived from lung epithelial cells after

  3. Micropost arrays for measuring stem cell-derived cardiomyocyte contractility.

    Science.gov (United States)

    Beussman, Kevin M; Rodriguez, Marita L; Leonard, Andrea; Taparia, Nikita; Thompson, Curtis R; Sniadecki, Nathan J

    2016-02-01

    Stem cell-derived cardiomyocytes have the potential to be used to study heart disease and maturation, screen drug treatments, and restore heart function. Here, we discuss the procedures involved in using micropost arrays to measure the contractile forces generated by stem cell-derived cardiomyocytes. Cardiomyocyte contractility is needed for the heart to pump blood, so measuring the contractile forces of cardiomyocytes is a straightforward way to assess their function. Microfabrication and soft lithography techniques are utilized to create identical arrays of flexible, silicone microposts from a common master. Micropost arrays are functionalized with extracellular matrix protein to allow cardiomyocytes to adhere to the tips of the microposts. Live imaging is used to capture videos of the deflection of microposts caused by the contraction of the cardiomyocytes. Image analysis code provides an accurate means to quantify these deflections. The contractile forces produced by a beating cardiomyocyte are calculated by modeling the microposts as cantilever beams. We have used this assay to assess techniques for improving the maturation and contractile function of stem cell-derived cardiomyocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Mast cell-derived mediators promote murine neutrophil effector functions.

    Science.gov (United States)

    Doener, Fatma; Michel, Anastasija; Reuter, Sebastian; Friedrich, Pamela; Böhm, Livia; Relle, Manfred; Codarri, Laura; Tenzer, Stefan; Klein, Matthias; Bopp, Tobias; Schmitt, Edgar; Schild, Hansjörg; Radsak, Markus Philipp; Taube, Christian; Stassen, Michael; Becker, Marc

    2013-10-01

    Mast cells are able to trigger life-saving immune responses in murine models for acute inflammation. In such settings, several lines of evidence indicate that the rapid and protective recruitment of neutrophils initiated by the release of mast cell-derived pro-inflammatory mediators is a key element of innate immunity. Herein, we investigate the impact of mast cells on critical parameters of neutrophil effector function. In the presence of activated murine bone marrow-derived mast cells, neutrophils freshly isolated from bone marrow rapidly lose expression of CD62L and up-regulate CD11b, the latter being partly driven by mast cell-derived TNF and GM-CSF. Mast cells also strongly enhance neutrophil phagocytosis and generation of reactive oxygen species. All these phenomena partly depend on mast cell-derived TNF and to a greater extend on GM-CSF. Furthermore, spontaneous apoptosis of neutrophils is greatly diminished due to the ability of mast cells to deliver antiapoptotic GM-CSF. Finally, we show in a murine model for acute lung inflammation that neutrophil phagocytosis is impaired in mast cell-deficient Kit (W-sh) /Kit (W-sh) mice but can be restored upon mast cell engraftment. Thus, a previously underrated feature of mast cells is their ability to boost neutrophil effector functions in immune responses.

  5. Cell-derived vesicles exposing coagulant tissue factor in saliva.

    Science.gov (United States)

    Berckmans, René J; Sturk, Auguste; van Tienen, Laurens M; Schaap, Marianne C L; Nieuwland, Rienk

    2011-03-17

    On vascular damage, coagulation is initiated by extravascular tissue factor (TF). Intravascular TF, which is present on circulating cell-derived vesicles, is noncoagulant under physiologic conditions but prothrombotic under pathologic conditions. Human saliva triggers coagulation, but the mechanism and physiologic relevance are unknown. Because saliva is known to contain TF, we hypothesized that this TF may also be associated with cell-derived vesicles to facilitate coagulation when saliva directly contacts blood. The saliva-induced shortening of the clotting time of autologous plasma and whole blood from healthy subjects (n = 10) proved TF-dependent. This TF was associated with various types of cell-derived vesicles, including microparticles and exosomes. The physiologic function was shown by adding saliva to human pericardial wound blood collected from patients undergoing cardiac surgery. Addition of saliva shortened the clotting time from 300 ± 96 to 186 ± 24 seconds (P = .03). Our results show that saliva triggers coagulation, thereby reducing blood loss and the risk of pathogens entering the blood. We postulate that our reflex to lick a wound may be a mechanism to enable TF-exposing vesicles, present in saliva, to aid in the coagulation process and thus protect the organism from entering pathogens. This unique compartmentalization may be highly conserved because also animals lick their wounds.

  6. Cryptococcus neoformans-derived microvesicles enhance the pathogenesis of fungal brain infection.

    Directory of Open Access Journals (Sweden)

    Sheng-He Huang

    Full Text Available Cryptococcal meningoencephalitis is the most common fungal disease in the central nervous system. The mechanisms by which Cryptococcus neoformans invades the brain are largely unknown. In this study, we found that C. neoformans-derived microvesicles (CnMVs can enhance the traversal of the blood-brain barrier (BBB by C. neoformans invitro. The immunofluorescence imaging demonstrates that CnMVs can fuse with human brain microvascular endothelial cells (HBMECs, the constituents of the BBB. This activity is presumably due to the ability of the CnMVs to activate HBMEC membrane rafts and induce cell fusogenic activity. CnMVs also enhanced C. neoformans infection of the brain, found in both infected brains and cerebrospinal fluid. In infected mouse brains, CnMVs are distributed inside and around C. neoformans-induced cystic lesions. GFAP (glial fibrillary acidic protein-positive astrocytes were found surrounding the cystic lesions, overlapping with the 14-3-3-GFP (14-3-3-green fluorescence protein fusion signals. Substantial changes could be observed in areas that have a high density of CnMV staining. This is the first demonstration that C. neoformans-derived microvesicles can facilitate cryptococcal traversal across the BBB and accumulate at lesion sites of C. neoformans-infected brains. Results of this study suggested that CnMVs play an important role in the pathogenesis of cryptococcal meningoencephalitis.

  7. Ovine placental eluate immunoglobulins recognise isologous and third party acid-treated trophoblast microvesicle antigens in vitro

    Directory of Open Access Journals (Sweden)

    C.A. Omwandho

    2006-06-01

    Full Text Available Placental microvesicles were prepared from ovine placentae and immunoglobulins eluted with 0.5 M glycine buffer pH 2.5. The ability of eluate immunoglobulins to re-associate with isologous (self and third party acidified microvesicles was tested by ELISA. Ovine placental immunoglobulins re-associated with isologous and third party acidified microvesicles suggesting that at least 2 types of antigenic epitopes I and II may be expressed on the ovine placentae. Type I antigensmaybe present on placentae of all ovines while type II epitopes may be paternally derived, hence unique to each pregnancy. Analysis by SDS PAGE revealed the heavy and light chains of IgG at 57 and 27 kDa, respectively, together giving a relative molecular weight of 158 kDa. Results suggest that immunoglobulins produced to placental microvesicle antigens may be directed to some but not all antigenic epitopes expressed on the trophoblast, possibly defining a mechanism by which the foetus evades maternal immunological rejection.

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

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    Jessica R Risner-Janiczek

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

  9. Matrix metalloproteinase-3 in odontoblastic cells derived from ips cells: unique proliferation response as odontoblastic cells derived from ES cells.

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    Taiki Hiyama

    Full Text Available We previously reported that matrix metalloproteinase (MMP-3 accelerates wound healing following dental pulp injury. In addition, we reported that a proinflammatory cytokine mixture (tumor necrosis factor-α, interleukin (IL-1β and interferon-γ induced MMP-3 activity in odontoblast-like cells derived from mouse embryonic stem (ES cells, suggesting that MMP-3 plays a potential unique physiological role in wound healing and regeneration of dental pulp in odontoblast-like cells. In this study, we tested the hypothesis that upregulation of MMP-3 activity by IL-1β promotes proliferation and apoptosis of purified odontoblast-like cells derived from induced pluripotent stem (iPS and ES cells. Each odontoblast-like cell was isolated and incubated with different concentrations of IL-1β. MMP-3 mRNA and protein expression were assessed using RT-PCR and western blotting, respectively. MMP-3 activity was measured using immunoprecipitation and a fluorescence substrate. Cell proliferation and apoptosis were determined using ELISA for BrdU and DNA fragmentation, respectively. siRNA was used to reduce MMP-3 transcripts in these cells. Treatment with IL-1β increased MMP-3 mRNA and protein levels, and MMP-3 activity in odontoblast-like cells. Cell proliferation was found to markedly increase with no changes in apoptosis. Endogenous tissue inhibitor of metalloproteinase (TIMP-1 and TIMP-2 were constitutively expressed during all experiments. The exocytosis inhibitor, Exo1, potently suppressed the appearance of MMP-3 in the conditioned medium. Treatment with siRNA against MMP-3 suppressed an IL-1β-induced increase in MMP-3 expression and activity, and also suppressed cell proliferation, but unexpectedly increased apoptosis in these cells (P<0.05. Exogenous MMP-3 was found to induce cell proliferation in odontoblast-like cells derived from iPS cells and ES cells. This siRNA-mediated increase in apoptosis could be reversed with exogenous MMP-3 stimulation (P<0

  10. Functions of pancreatic stellate cell-derived soluble factors in the microenvironment of pancreatic ductal carcinoma.

    Science.gov (United States)

    Wu, Qi; Tian, Ying; Zhang, Jingqiu; Zhang, Hongpeng; Gu, Fengming; Lu, Yongdie; Zou, Shengnan; Chen, Yuji; Sun, Pengxiang; Xu, Mengyue; Sun, Xiaoming; Xia, Chao; Chi, Hao; Ying Zhu, A; Tang, Dong; Wang, Daorong

    2017-11-24

    Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer with poor prognosis because it is highly resistant to traditional chemotherapy and radiotherapy and it has a low rate of surgical resection eligibility. Pancreatic stellate cells (PSC) have become a research hotspot in recent years, and play a vital role in PDAC microenvironment by secreting soluble factors such as transforming growth factor β, interleukin-6, stromal cell-derived factor-1, hepatocyte growth factor and galectin-1. These PSC-derived cytokines and proteins contribute to PSC activation, participating in PDAC cell proliferation, migration, fibrosis, angiogenesis, immunosuppression, epithelial-mesenchymal transition, and chemoradiation resistance, leading to malignant outcome. Consequently, targeting these cytokines and proteins or their downstream signaling pathways is promising for treating PDAC.

  11. Coxsackievirus B exits the host cell in shed microvesicles displaying autophagosomal markers.

    Directory of Open Access Journals (Sweden)

    Scott M Robinson

    2014-04-01

    Full Text Available Coxsackievirus B3 (CVB3, a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, "fluorescent timer" protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3 following transfection in HeLa cells. "Fluorescent timer" protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs, and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of "fluorescent timer" protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. "Fluorescent timer" protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs containing matured "fluorescent timer" protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic

  12. Coxsackievirus B exits the host cell in shed microvesicles displaying autophagosomal markers.

    Science.gov (United States)

    Robinson, Scott M; Tsueng, Ginger; Sin, Jon; Mangale, Vrushali; Rahawi, Shahad; McIntyre, Laura L; Williams, Wesley; Kha, Nelson; Cruz, Casey; Hancock, Bryan M; Nguyen, David P; Sayen, M Richard; Hilton, Brett J; Doran, Kelly S; Segall, Anca M; Wolkowicz, Roland; Cornell, Christopher T; Whitton, J Lindsay; Gottlieb, Roberta A; Feuer, Ralph

    2014-04-01

    Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, "fluorescent timer" protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. "Fluorescent timer" protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of "fluorescent timer" protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. "Fluorescent timer" protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured "fluorescent timer" protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol

  13. Stem cell-derived systems in toxicology assessment.

    Science.gov (United States)

    Suter-Dick, Laura; Alves, Paula M; Blaauboer, Bas J; Bremm, Klaus-Dieter; Brito, Catarina; Coecke, Sandra; Flick, Burkhard; Fowler, Paul; Hescheler, Jürgen; Ingelman-Sundberg, Magnus; Jennings, Paul; Kelm, Jens M; Manou, Irene; Mistry, Pratibha; Moretto, Angelo; Roth, Adrian; Stedman, Donald; van de Water, Bob; Beilmann, Mario

    2015-06-01

    Industrial sectors perform toxicological assessments of their potential products to ensure human safety and to fulfill regulatory requirements. These assessments often involve animal testing, but ethical, cost, and time concerns, together with a ban on it in specific sectors, make appropriate in vitro systems indispensable in toxicology. In this study, we summarize the outcome of an EPAA (European Partnership of Alternatives to Animal Testing)-organized workshop on the use of stem cell-derived (SCD) systems in toxicology, with a focus on industrial applications. SCD systems, in particular, induced pluripotent stem cell-derived, provide physiological cell culture systems of easy access and amenable to a variety of assays. They also present the opportunity to apply the vast repository of existing nonclinical data for the understanding of in vitro to in vivo translation. SCD systems from several toxicologically relevant tissues exist; they generally recapitulate many aspects of physiology and respond to toxicological and pharmacological interventions. However, focused research is necessary to accelerate implementation of SCD systems in an industrial setting and subsequent use of such systems by regulatory authorities. Research is required into the phenotypic characterization of the systems, since methods and protocols for generating terminally differentiated SCD cells are still lacking. Organotypical 3D culture systems in bioreactors and microscale tissue engineering technologies should be fostered, as they promote and maintain differentiation and support coculture systems. They need further development and validation for their successful implementation in toxicity testing in industry. Analytical measures also need to be implemented to enable compound exposure and metabolism measurements for in vitro to in vivo extrapolation. The future of SCD toxicological tests will combine advanced cell culture technologies and biokinetic measurements to support regulatory and

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-07-12

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

  15. Activation of endothelial pro-resolving anti-inflammatory pathways by circulating microvesicles from non-muscular myosin light chain kinase-deficient mice

    Directory of Open Access Journals (Sweden)

    Abderahim Gaceb

    2016-09-01

    Full Text Available Microvesicles, small membrane vesicles released from cells, have beneficial and/or deleterious effects in sepsis. We previously reported that non-muscle myosin light chain kinase (nmMLCK deletion protects mice against endotoxic shock by reducing inflammation. Here, we have evaluated the consequences of nmMLCK deletion on microvesicles phenotypes and their effects on mouse aortic endothelial cells in association with vascular inflammation and endothelial dysfunction during endotoxic shock induced by lipopolysaccharide in mice. Treatment with lipopolysaccharide induced an increase in levels of circulating microvesicles in wild type but not in nmMLCK-deficient mice. Microvesicles from nmMLCK-deficient mice (MVsnmMLCK-/- prevented the inflammatory effects of lipopolysaccharide with concomitant increase of anti- inflammatory and reduction of pro-inflammatory secretome in mouse aortic endothelial cells. In addition, MVsnmMLCK-/- reduced the efficacy of lipopolysaccharide to increase aortic oxidative and nitrosative stresses as well as macrophage infiltration in the aorta. Moreover, MVsnmMLCK-/- prevented ex vivo endothelial dysfunction, vascular hyporeactivity and in vivo overproduction of nitric oxide in heart and liver in response to lipopolysaccharide. Altogether, these findings provide evidence that nmMLCK deletion generates circulating microvesicles displaying protective effects by activating endothelial pro-resolving anti-inflammatory pathways allowing the effective down-regulation of oxidative and nitrative stresses associated with endotoxic shock. Thus, nmMLCK plays a pivotal role in susceptibility to sepsis via the control of cellular activation and release of circulating microvesicles.

  16. Malignant cell-derived extracellular vesicles express different chromogranin epitopes compared to prostasomes.

    Science.gov (United States)

    Dubois, Louise; Stridsberg, Mats; Kharaziha, Pedram; Chioureas, Dimitris; Meersman, Niels; Panaretakis, Theocharis; Ronquist, K Göran

    2015-07-01

    Prostasomes are nanosized extracellular vesicles exocytosed by prostate epithelial cells. They have been assigned many roles propitious to sperm in favor of fertilization. Prostatic cancer cells can also produce and secrete extracellular vesicles. We assessed using ELISA, the surface expression of chromogranin proproteins on prostasomes and malignant extracellular vesicles of four different prostate cancer cell-lines, two hormone sensitive and two hormone refractory. We used a panel of chromogranin A and chromogranin B antibodies against peptides in-between hypothetical cleavage sites along the proproteins. A diverging pattern of chromogranin peptides was apparent when comparing prostasomes and malignant extracellular vesicles indicating a phenotypical change. We also compared western blot patterns (prostasomes and malignant extracellular vesicles) for selected antibodies that displayed high absorbances in the ELISA. Western blot analyses revealed various cleavage patterns of those proproteins that were analyzed in prostasomes and extracellular vesicles. Chromogranins are constituents of not only prostasomes but also of malignant prostate cell-derived extracellular vesicles with different amino acid sequences exposed at the membrane surface giving rise to a mosaic pattern. These findings may be of relevance for designing new assays for detection or even possible treatment of prostate cancers. © 2015 Wiley Periodicals, Inc.

  17. Matrix Metalloproteinase-3 in Odontoblastic Cells Derived from Ips Cells: Unique Proliferation Response as Odontoblastic Cells Derived from ES Cells

    Science.gov (United States)

    Hiyama, Taiki; Ozeki, Nobuaki; Mogi, Makio; Yamaguchi, Hideyuki; Kawai, Rie; Nakata, Kazuhiko; Kondo, Ayami; Nakamura, Hiroshi

    2013-01-01

    We previously reported that matrix metalloproteinase (MMP)-3 accelerates wound healing following dental pulp injury. In addition, we reported that a proinflammatory cytokine mixture (tumor necrosis factor-α, interleukin (IL)-1β and interferon-γ) induced MMP-3 activity in odontoblast-like cells derived from mouse embryonic stem (ES) cells, suggesting that MMP-3 plays a potential unique physiological role in wound healing and regeneration of dental pulp in odontoblast-like cells. In this study, we tested the hypothesis that upregulation of MMP-3 activity by IL-1β promotes proliferation and apoptosis of purified odontoblast-like cells derived from induced pluripotent stem (iPS) and ES cells. Each odontoblast-like cell was isolated and incubated with different concentrations of IL-1β. MMP-3 mRNA and protein expression were assessed using RT-PCR and western blotting, respectively. MMP-3 activity was measured using immunoprecipitation and a fluorescence substrate. Cell proliferation and apoptosis were determined using ELISA for BrdU and DNA fragmentation, respectively. siRNA was used to reduce MMP-3 transcripts in these cells. Treatment with IL-1β increased MMP-3 mRNA and protein levels, and MMP-3 activity in odontoblast-like cells. Cell proliferation was found to markedly increase with no changes in apoptosis. Endogenous tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were constitutively expressed during all experiments. The exocytosis inhibitor, Exo1, potently suppressed the appearance of MMP-3 in the conditioned medium. Treatment with siRNA against MMP-3 suppressed an IL-1β-induced increase in MMP-3 expression and activity, and also suppressed cell proliferation, but unexpectedly increased apoptosis in these cells (PiPS cells and ES cells. This siRNA-mediated increase in apoptosis could be reversed with exogenous MMP-3 stimulation (PiPS and ES cells. PMID:24358294

  18. Loss of signalling via Gα13 in germinal centre B-cell-derived lymphoma.

    Science.gov (United States)

    Muppidi, Jagan R; Schmitz, Roland; Green, Jesse A; Xiao, Wenming; Larsen, Adrien B; Braun, Sterling E; An, Jinping; Xu, Ying; Rosenwald, Andreas; Ott, German; Gascoyne, Randy D; Rimsza, Lisa M; Campo, Elias; Jaffe, Elaine S; Delabie, Jan; Smeland, Erlend B; Braziel, Rita M; Tubbs, Raymond R; Cook, J R; Weisenburger, Dennis D; Chan, Wing C; Vaidehi, Nagarajan; Staudt, Louis M; Cyster, Jason G

    2014-12-11

    Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined. Work in mice showed that sphingosine-1-phosphate receptor-2 (S1PR2), a Gα12 and Gα13 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA13 (encoding Gα13) and S1PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S1PR2 frequently disrupt the receptor's Akt and migration inhibitory functions. Gα13-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S1P, and Gα13-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Gα13, but not S1PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Gα13 effector ARHGEF1, and Arhgef1 deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Gα13- and S1PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt's lymphoma, also represses germinal centre B-cell growth and promotes confinement via Gα13. These findings identify a Gα13-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma.

  19. Exosomes or microvesicles? Two kinds of extracellular vesicles with different routes to modify protozoan-host cell interaction.

    Science.gov (United States)

    Evans-Osses, Ingrid; Reichembach, Luis H; Ramirez, Marcel I

    2015-10-01

    Parasite-host cell interaction can be modulated by a dynamic communication between extracellular vesicles (EVs). They should play key roles in cell-cell communications transferring biomolecules (miRNA, proteins, soluble factors) from one cell to another cell. While many names have been used to denominate EVs, a better comprehension to understand these vesicles is raised when we classify it according to biogenesis: originated from multivesicular bodies, named exosomes, and from plasmatic membranes, denominated microvesicles. Here, we have reviewed EV participation during the protozoan-host cell interaction and reinforced the differences and similarities between exosomes and microvesicles, suggesting different intracellular routes and functions. We also discussed perspectives to study EVs and the role of EVs in diagnosis and chemotherapies of infectious diseases.

  20. Bystander Effect Induced by Electroporation is Possibly Mediated by Microvesicles and Dependent on Pulse Amplitude, Repetition Frequency and Cell Type.

    Science.gov (United States)

    Prevc, Ajda; Bedina Zavec, Apolonija; Cemazar, Maja; Kloboves-Prevodnik, Veronika; Stimac, Monika; Todorovic, Vesna; Strojan, Primoz; Sersa, Gregor

    2016-10-01

    Bystander effect, a known phenomenon in radiation biology, where irradiated cells release signals which cause damage to nearby, unirradiated cells, has not been explored in electroporated cells yet. Therefore, our aim was to determine whether bystander effect is present in electroporated melanoma cells in vitro, by determining viability of non-electroporated cells exposed to medium from electroporated cells and by the release of microvesicles as potential indicators of the bystander effect. Here, we demonstrated that electroporation of cells induces bystander effect: Cells exposed to electric pulses mediated their damage to the non-electroporated cells, thus decreasing cell viability. We have shown that shedding microvesicles may be one of the ways used by the cells to mediate the death signals to the neighboring cells. The murine melanoma B16F1 cell line was found to be more electrosensitive and thus more prone to bystander effect than the canine melanoma CMeC-1 cell line. In B16F1 cell line, bystander effect was present above the level of electropermeabilization of the cells, with the threshold at 800 V/cm. Furthermore, with increasing electric field intensities and the number of pulses, the bystander effect also increased. In conclusion, electroporation can induce bystander effect which may be mediated by microvesicles, and depends on pulse amplitude, repetition frequency and cell type.

  1. 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.

  2. Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules.

    Science.gov (United States)

    Heijnen, H F; Schiel, A E; Fijnheer, R; Geuze, H J; Sixma, J J

    1999-12-01

    Platelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and alpha-granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or alpha-thrombin: microvesicles of 100 nm to 1 microm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and alpha-granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains alpha(IIb)-beta(3) and beta(1), GPIbalpha, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63(+) internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.

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

    Science.gov (United States)

    Said, Ahmed S; Doctor, Allan

    2017-05-15

    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

  4. 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.

  5. Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC

    Science.gov (United States)

    Besse, Benjamin; Charrier, Mélinda; Lapierre, Valérie; Dansin, Eric; Lantz, Olivier; Planchard, David; Le Chevalier, Thierry; Livartoski, Alain; Barlesi, Fabrice; Laplanche, Agnès; Ploix, Stéphanie; Vimond, Nadège; Peguillet, Isabelle; Théry, Clotilde; Lacroix, Ludovic; Zoernig, Inka; Dhodapkar, Kavita; Dhodapkar, Madhav; Viaud, Sophie; Soria, Jean-Charles; Reiners, Katrin S.; Pogge von Strandmann, Elke; Vély, Frédéric; Rusakiewicz, Sylvie; Eggermont, Alexander; Pitt, Jonathan M.; Zitvogel, Laurence; Chaput, Nathalie

    2016-01-01

    ABSTRACT Dendritic cell-derived exosomes (Dex) are small extracellular vesicles secreted by viable dendritic cells. In the two phase-I trials that we conducted using the first generation of Dex (IFN-γ-free) in end-stage cancer, we reported that Dex exerted natural killer (NK) cell effector functions in patients. A second generation of Dex (IFN-γ-Dex) was manufactured with the aim of boosting NK and T cell immune responses. We carried out a phase II clinical trial testing the clinical benefit of IFN-γ-Dex loaded with MHC class I- and class II-restricted cancer antigens as maintenance immunotherapy after induction chemotherapy in patients bearing inoperable non-small cell lung cancer (NSCLC) without tumor progression. The primary endpoint was to observe at least 50% of patients with progression-free survival (PFS) at 4 mo after chemotherapy cessation. Twenty-two patients received IFN-γ-Dex. One patient exhibited a grade three hepatotoxicity. The median time to progression was 2.2 mo and median overall survival (OS) was 15 mo. Seven patients (32%) experienced stabilization of >4 mo. The primary endpoint was not reached. An increase in NKp30-dependent NK cell functions were evidenced in a fraction of these NSCLC patients presenting with defective NKp30 expression. Importantly, MHC class II expression levels of the final IFN-γ-Dex product correlated with expression levels of the NKp30 ligand BAG6 on Dex, and with NKp30-dependent NK functions, the latter being associated with longer progression-free survival. This phase II trial confirmed the capacity of Dex to boost the NK cell arm of antitumor immunity in patients with advanced NSCLC. PMID:27141373

  6. Lactobacillus casei BL23 Produces Microvesicles Carrying Proteins That Have Been Associated with Its Probiotic Effect

    Directory of Open Access Journals (Sweden)

    A. Paula Domínguez Rubio

    2017-09-01

    Full Text Available Archaea, bacteria, and eukarya secrete membrane microvesicles (MVs as a mechanism for intercellular communication. We report the isolation and characterization of MVs from the probiotic strain Lactobacillus casei BL23. MVs were characterized using analytical high performance techniques, DLS, AFM and TEM. Similar to what has been described for other Gram-positive bacteria, MVs were on the nanometric size range (30–50 nm. MVs carried cytoplasmic components such as DNA, RNA and proteins. Using a proteomic approach (LC-MS, we identified a total of 103 proteins; 13 exclusively present in the MVs. The MVs content included cell envelope associated and secretory proteins, heat and cold shock proteins, several metabolic enzymes, proteases, structural components of the ribosome, membrane transporters, cell wall-associated hydrolases and phage related proteins. In particular, we identified proteins described as mediators of Lactobacillus’ probiotic effects such as p40, p75 and the product of LCABL_31160, annotated as an adhesion protein. The presence of these proteins suggests a role for the MVs in the bacteria-gastrointestinal cells interface. The expression and further encapsulation of proteins into MVs of GRAS (Generally Recognized as Safe bacteria could represent a scientific novelty, with applications in food, nutraceuticals and clinical therapies.

  7. Microvesicles from the plasma of elderly subjects and from senescent endothelial cells promote vascular calcification.

    Science.gov (United States)

    Alique, Matilde; Ruíz-Torres, María Piedad; Bodega, Guillermo; Noci, María Victoria; Troyano, Nuria; Bohórquez, Lourdes; Luna, Carlos; Luque, Rafael; Carmona, Andrés; Carracedo, Julia; Ramírez, Rafael

    2017-03-08

    Vascular calcification is commonly seen in elderly people, though it can also appear in middle-aged subjects affected by premature vascular aging. The aim of this work is to test the involvement of microvesicles (MVs) produced by senescent endothelial cells (EC) and from plasma of elderly people in vascular calcification. The present work shows that MVs produced by senescent cultured ECs, plus those found in the plasma of elderly subjects, promote calcification in vascular smooth muscle cells. Only MVs from senescent ECs, and from elderly subjects' plasma, induced calcification. This ability correlated with these types of MVs' carriage of: a) increased quantities of annexins (which might act as nucleation sites for calcification), b) increased quantities of bone-morphogenic protein, and c) larger Ca contents. The MVs of senescent, cultured ECs, and those present in the plasma of elderly subjects, promote vascular calcification. The present results provide mechanistic insights into the observed increase in vascular calcification-related diseases in the elderly, and in younger patients with premature vascular aging, paving the way towards novel therapeutic strategies.

  8. Immune cell activation by trophoblast-derived microvesicles is mediated by syncytin 1.

    Science.gov (United States)

    Holder, Beth S; Tower, Clare L; Forbes, Karen; Mulla, Melissa J; Aplin, John D; Abrahams, Vikki M

    2012-06-01

    Envelope glycoproteins of human endogenous retrovirus (HERV), such as syncytin 1 (HERV-W), are highly expressed in the placenta and some family members have immunomodulatory properties. Placental microvesicles (MV), which are shed into the maternal circulation during pregnancy, have been demonstrated to induce immune cell activation. Therefore, the aim of this study was to investigate the immunological properties of the highly expressed placental HERV-W protein, syncytin 1, and its potential involvement in placental MV modulation of immune cell activity. The MV shed from first trimester, normal term and pre-eclamptic term placentas, and from the BeWo trophoblast cell line, all contain syncytin 1. Recombinant syncytin 1 and syncytin 1-positive BeWo trophoblast MV both induced peripheral blood mononuclear cell (PBMC) activation, indicated through production of cytokines and chemokines. Reducing syncytin 1 content in BeWo MV inhibited PBMC activation. Recombinant syncytin 1 and syncytin-1-positive BeWo MV dampened PBMC responses to lipopolysaccharide challenge. Our findings suggest that syncytin 1 is shed from the placenta into the maternal circulation in association with MV, and modulates immune cell activation and the responses of immune cells to subsequent lipopolysaccharide stimulation. These studies implicate placental MV-associated HERV in fetal regulation of the maternal immune system. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.

  9. Heightened pro-inflammatory effect of preeclamptic placental microvesicles on peripheral blood immune cells in humans.

    Science.gov (United States)

    Holder, Beth S; Tower, Clare L; Jones, Carolyn J P; Aplin, John D; Abrahams, Vikki M

    2012-04-01

    Normal pregnancy is associated with the presence of circulating placental microvesicles (MVs). Increased MV shedding and altered immune activation are seen in patients with preeclampsia, suggesting that placental MVs may play a role in the pathophysiology of this disease. Therefore, the aim of this study was to investigate the activation of peripheral blood mononuclear cells (PBMCs) by MVs shed by first-trimester, normal term, and preeclamptic term placenta. First-trimester and preeclamptic term, but not normal term, placental-derived MVs activated PBMCs, as evidenced by elevated IL1B. Significant changes were also seen with several other cytokines and chemokines, and in general when compared to normal term MVs, preeclamptic MVs induced a greater pro-inflammatory response in PBMCs. Pretreatment of PBMCs with first-trimester or normal term placental MVs resulted in a dampened IL1B response to a subsequent lipopolysaccharide (LPS) challenge. In contrast, treatment of PBMCs with preeclamptic term placental MVs exacerbated the LPS response. This was also the case for several other cytokines and chemokines. These studies suggest that placental MVs can modulate basal peripheral immune cell activation and responsiveness to LPS during normal pregnancy, and that in preeclampsia this effect is exacerbated.

  10. Microvesicle formulations used in topical drugs and cosmetics affect product efficiency, performance and allergenicity.

    Science.gov (United States)

    Madsen, Jakob Torp; Andersen, Klaus Ejner

    2010-01-01

    Attempts to improve the formulations of topical products are continuing processes (ie, to increase cosmetic performance, enhance effects, and protect ingredients from degradation). The development of micro- and nanovesicular systems has led to the marketing of topical drugs and cosmetics that use these technologies. Several articles have reported improved clinical efficacy by the encapsulation of pharmaceuticals in vesicular systems, and the numbers of publications and patents are rising. Some vesicular systems may deliver the drug deeper in the skin as compared to conventional vehicles, or even make transdermal delivery more efficient for a number of drugs. Vesicular systems may also allow a more precise drug delivery to the site of action (ie, the hair follicles) and thereby minimize the applied drug concentration, reducing potential side effects. On the other hand, this may increase the risk of other side effects. Few case reports have suggested that microvesicle formulations may affect the allergenicity of topical products. This article gives an overview of the current knowledge about the topical use of microvesicular systems and the dermatoallergologic aspects.

  11. Mechanisms and modulation of microvesicle uptake in a model of alveolar cell communication.

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    Schneider, Daniel J; Speth, Jennifer M; Penke, Loka R; Wettlaufer, Scott H; Swanson, Joel A; Peters-Golden, Marc

    2017-11-03

    Extracellular vesicles (EVs), including exosomes and shed microvesicles (MVs), can be internalized by recipient cells to modulate function. Although the mechanism by which EVs are internalized is incompletely characterized, it is generally regarded to involve endocytosis and an initial surface binding event. Furthermore, modulation of uptake by microenvironmental factors is largely unstudied. Here we used flow cytometry, confocal microscopy, and pharmacologic and molecular targeting to address these gaps in knowledge in a model of pulmonary alveolar cell-cell communication. Alveolar macrophage-derived MVs were fully internalized by alveolar epithelial cells in a time-, dose-, and temperature-dependent manner. Uptake was dependent on dynamin and actin polymerization. However, it was neither saturable nor dependent on clathrin or receptor binding. Internalization was enhanced by extracellular proteins, but was inhibited by cigarette smoke extract via oxidative disruption of actin polymerization. We conclude that MV internalization occurs via a pathway more consistent with fluid-phase than receptordependent endocytosis, and is subject to bidirectional modulation by relevant pathologic perturbations. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  12. Analysis of synaptic-like microvesicle exocytosis of B-cells using a live imaging technique.

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    Aurélie Bergeron

    Full Text Available Pancreatic β-cells play central roles in blood glucose homeostasis. Beside insulin, these cells release neurotransmitters and other signaling molecules stored in synaptic-like microvesicles (SLMVs. We monitored SLMV exocytosis by transfecting a synaptophysin-pHluorin construct and by visualizing the cells by Total Internal Reflection Fluorescence (TIRF microscopy. SLMV fusion was elicited by 20 mM glucose and by depolarizing K(+ concentrations with kinetics comparable to insulin secretion. SLMV exocytosis was prevented by Tetanus and Botulinum-C neurotoxins indicating that the fusion machinery of these organelles includes VAMP-2/-3 and Syntaxin-1, respectively. Sequential visualization of SLMVs by TIRF and epifluorescence microscopy showed that after fusion the vesicle components are rapidly internalized and the organelles re-acidified. Analysis of single fusion episodes revealed the existence of two categories of events. While under basal conditions transient fusion events prevailed, long-lasting episodes were more frequent upon secretagogue exposure. Our observations unveiled similarities between the mechanism of exocytosis of insulin granules and SLMVs. Thus, diabetic conditions characterized by defective insulin secretion are most probably associated also with inappropriate release of molecules stored in SLMVs. The assessment of the contribution of SLMV exocytosis to the manifestation of the disease will be facilitated by the use of the imaging approach described in this study.

  13. Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures

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    Guescini, M. [Department of Biomolecular Sciences, University of Urbino ' Carlo Bo' , 61029 Urbino (Italy); Leo, G.; Genedani, S. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); Carone, C. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); IRCCS San Camillo Lido, Venezia (Italy); Pederzoli, F. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); Ciruela, F. [Departament Patologia i Terapeutica Experimental, Universitat de Barcelona (Spain); Guidolin, D. [Department of Human Anatomy and Physiology, University of Padua (Italy); Stocchi, V.; Mantuano, M. [Department of Biomolecular Sciences, University of Urbino ' Carlo Bo' , 61029 Urbino (Italy); Borroto-Escuela, D.O.; Fuxe, K. [Department of Neuroscience, Karolinska Institutet, Stockholm (Sweden); Agnati, L.F., E-mail: luigiagnati@tin.it [IRCCS San Camillo Lido, Venezia (Italy)

    2012-03-10

    Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A{sub 2A} and D{sub 2} receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D{sub 2}R-CFP or A{sub 2A}R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D{sub 2}R-CFP and A{sub 2A}R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A{sub 2A}R positive MVs were treated with the adenosine A{sub 2A} receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A{sub 2A}Rs were functionally competent in target cells. These findings demonstrate that A{sub 2A} receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

  14. Tumor-Derived Microvesicles Modulate Antigen Cross-Processing via Reactive Oxygen Species-Mediated Alkalinization of Phagosomal Compartment in Dendritic Cells

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    Federico Battisti

    2017-09-01

    Full Text Available Dendritic cells (DCs are the only antigen-presenting cells able to prime naïve T cells and cross-prime antigen-specific CD8+ T cells. Their functionality is a requirement for the induction and maintenance of long-lasting cancer immunity. Albeit intensively investigated, the in vivo mechanisms underlying efficient antigen cross-processing and presentation are not fully understood. Several pieces of evidence indicate that antigen transfer to DCs mediated by microvesicles (MVs enhances antigen immunogenicity. This mechanism is also relevant for cross-presentation of those tumor-associated glycoproteins such as MUC1 that are blocked in HLA class II compartment when internalized by DCs as soluble molecules. Here, we present pieces of evidence that the internalization of tumor-derived MVs modulates antigen-processing machinery of DCs. Employing MVs derived from ovarian cancer ascites fluid and established tumor cell lines, we show that MV uptake modifies DC phagosomal microenvironment, triggering reactive oxygen species (ROS accumulation and early alkalinization. Indeed, tumor MVs carry radical species and the MV uptake by DCs counteracts the chemically mediated acidification of the phagosomal compartment. Further pieces of evidence suggest that efficacious antigen cross-priming of the MUC1 antigen carried by the tumor MVs results from the early signaling induced by MV internalization and the function of the antigen-processing machinery of DCs. These results strongly support the hypothesis that tumor-derived MVs impact antigen immunogenicity by tuning the antigen-processing machinery of DCs, besides being carrier of tumor antigens. Furthermore, these findings have important implications for the exploitation of MVs as antigenic cell-free immunogen for DC-based therapeutic strategies.

  15. Induced pluripotent stem cell-derived mesenchymal stem cells: A leap toward personalized therapies.

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    Whitt, Jason; Vallabhaneni, Krishna C; Penfornis, Patrice; Pochampally, Radhika

    2016-01-01

    Mesenchymal Stem/stromal cell (MSCs) transplantation procedures have been used since the 1960's to treat leukemia and other diseases, but due to the risks involved only patients with life threatening illnesses were typically subjected to the transplantation procedure until the last decade. Recent advancements in transplantation techniques have made it more feasible to use it for non-life-threatening diseases. However, the potential uses for stem cells are still limited by their rarity, and, in the case of allogeneic transplants, graft-vs.-host complications. An evolving alternative to conventional stem cell therapies is induced pluripotent stem-cell derived mesenchymal stem/stromal cells (iPSC- MSCs), which have a multi-lineage potential comparable to conventionally acquired MSCs with the added benefit of being less immunoreactive. However there are still many hurdles left to be overcome before they can be used regularly for personalized therapies. This review will focus on recent advancements that have been made regarding the role MSCs play in tumor development and the potential uses iPSC-MSCs may have in future cancer treatment.

  16. Human induced pluripotent stem cell-derived hepatocytes for toxicology testing.

    Science.gov (United States)

    Mann, David A

    2015-01-01

    The need for more predictive in vitro toxicity models is a critical deficit in current preclinical pipeline safety evaluations. Current models employing tumor-derived cancer cell lines and isolated primary human hepatocytes (PHHs) afford an approximation of overt cytotoxicity but do not provide hepatotoxicity prediction owing to liabilities in metabolic activity along with phenotypic variability and instability in culture. Induced pluripotent stem cell-derived hepatocytes (iPSC-HCs) offer a long-term solution to accessing liver tissue from representative diverse as well as idiosyncratic patient populations and can be sourced indefinitely. iPSC-HCs are currently being evaluated as potential replacements for the existing cell models, but they have yet to prove superiority. It is acknowledged that iPSC-HCs are not functionally equivalent to PHHs and are somewhat mixed in terms of their gene expression profile, simultaneously displaying mature and immature markers in vitro. Combining iPSC-HCs with organotypic culture systems affords an opportunity to maximize the potential of both technologies where the cells benefit from more complex culture conditions while unlocking the potential of the culture systems by affording stability and reproducibility to provide the future of predictive in vitro toxicity models.

  17. Stretch Injury of Human Induced Pluripotent Stem Cell Derived Neurons in a 96 Well Format

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    Sherman, Sydney A.; Phillips, Jack K.; Costa, J. Tighe; Cho, Frances S.; Oungoulian, Sevan R.; Finan, John D.

    2016-01-01

    Traumatic brain injury (TBI) is a major cause of mortality and morbidity with limited therapeutic options. Traumatic axonal injury (TAI) is an important component of TBI pathology. It is difficult to reproduce TAI in animal models of closed head injury, but in vitro stretch injury models reproduce clinical TAI pathology. Existing in vitro models employ primary rodent neurons or human cancer cell line cells in low throughput formats. This in vitro neuronal stretch injury model employs human induced pluripotent stem cell-derived neurons (hiPSCNs) in a 96 well format. Silicone membranes were attached to 96 well plate tops to create stretchable, culture substrates. A custom-built device was designed and validated to apply repeatable, biofidelic strains and strain rates to these plates. A high content approach was used to measure injury in a hypothesis-free manner. These measurements are shown to provide a sensitive, dose-dependent, multi-modal description of the response to mechanical insult. hiPSCNs transition from healthy to injured phenotype at approximately 35% Lagrangian strain. Continued development of this model may create novel opportunities for drug discovery and exploration of the role of human genotype in TAI pathology. PMID:27671211

  18. Modeling trastuzumab-related cardiotoxicity in vitro using human stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Kurokawa, Yosuke K; Shang, Michael R; Yin, Rose T; George, Steven C

    2018-03-15

    Trastuzumab (Herceptin ® ), a monoclonal antibody against the ErbB2 (HER2) receptor, has significantly improved clinical outcomes for HER2 + breast cancer patients. However, the drug also has known cardiotoxic side effects through mechanisms that are not fully understood. Here we utilized human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) to model trastuzumab-related cardiotoxicity in vitro. We demonstrate that cardiotoxic effects of ErbB2 inhibition by trastuzumab can be recapitulated only when the cardioprotective effects of ErbB2/4 signaling is observed. We observed no cardioprotective effects of ErbB2/4 signaling without cellular stress (doxorubicin exposure in this study). In addition to neuregulin-1 (NRG-1), we show that heparin-binding epidermal growth factor-like growth factor (HB-EGF) also provides cardioprotective effects for iPS-CMs. Finally, we demonstrate a simple, high-throughput co-culture platform utilizing iPS-CMs and endothelial cells that is capable of detecting trastuzumab-related cardiotoxicity. We conclude that iPS-CMs can recapitulate trastuzumab-related cardiotoxicity, and may be used to elucidate additional modes of toxicity of trastuzumab and related compounds. Copyright © 2018. Published by Elsevier B.V.

  19. Vesicle-MaNiA: extracellular vesicles in liquid biopsy and cancer.

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    Torrano, Veronica; Royo, Felix; Peinado, Héctor; Loizaga-Iriarte, Ana; Unda, Miguel; Falcón-Perez, Juan M; Carracedo, Arkaitz

    2016-08-01

    Normal and tumor cells shed vesicles to the environment. Within the large family of extracellular vesicles, exosomes and microvesicles have attracted much attention in the recent years. Their interest ranges from mediators of cancer progression, inflammation, immune regulation and metastatic niche regulation, to non-invasive biomarkers of disease. In this respect, the procedures to purify and analyze extracellular vesicles have quickly evolved and represent a source of variability for data integration in the field. In this review, we provide an updated view of the potential of exosomes and microvesicles as biomarkers and the available technologies for their isolation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources

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    Reka A. Haraszti

    2016-11-01

    Full Text Available Extracellular vesicles (EVs, including exosomes and microvesicles (MVs, are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs. We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types.

  1. Up-Regulated Expression of Matrix Metalloproteinases in Endothelial Cells Mediates Platelet Microvesicle-Induced Angiogenesis.

    Science.gov (United States)

    Sun, Cheng; Feng, Shi-Bin; Cao, Zheng-Wang; Bei, Jun-Jie; Chen, Qiang; Zhao, Wei-Bo; Xu, Xian-Jie; Zhou, Zhou; Yu, Zheng-Ping; Hu, Hou-Yuan

    2017-01-01

    Platelet microvesicles (PMVs) contribute to angiogenesis and vasculogenesis, but the mechanisms underlying these contributions have not been fully elucidated. In the present study, we investigated whether PMVs regulate the angiogenic properties of endothelial cells (ECs) via mechanisms extending beyond the transport of angiogenic regulators from platelets. In vitro Matrigel tube formation assay and in vivo Matrigel plug assay were used to evaluate the pro-angiogenic activity of PMVs. The effects of PMVs on the migration of human umbilical vein endothelial cells (HUVECs) were detected by transwell assay and wound-healing assay. Real-time PCR and western blot were conducted to examine mRNA and protein expression of pro-angiogenic factors in HUVECs. Matrix metalloproteinase (MMP) activity was assayed by gelatin zymography. Moreover, the effects of specific MMP inhibitors were tested. PMVs promoted HUVEC capillary-like network formation in a dose-dependent manner. Meanwhile, PMVs dose-dependently facilitated HUVEC migration. Levels of MMP-2 and MMP-9 expression and activity were up-regulated in HUVECs stimulated with PMVs. Inhibition of MMPs decreased their pro-angiogenic and pro-migratory effects on HUVECs. Moreover, we confirmed the pro-angiogenic activity of PMVs in vivo in mice with subcutaneous implantation of Matrigel, and demonstrated that blockade of MMPs attenuated PMV-induced angiogenesis. The findings of our study indicate that PMVs promote angiogenesis by up-regulating MMP expression in ECs via mechanism extending beyond the direct delivery of angiogenic factors. © 2017 The Author(s). Published by S. Karger AG, Basel.

  2. A novel role for pro-coagulant microvesicles in the early host defense against streptococcus pyogenes.

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    Sonja Oehmcke

    Full Text Available Previous studies have shown that stimulation of whole blood or peripheral blood mononuclear cells with bacterial virulence factors results in the sequestration of pro-coagulant microvesicles (MVs. These particles explore their clotting activity via the extrinsic and intrinsic pathway of coagulation; however, their pathophysiological role in infectious diseases remains enigmatic. Here we describe that the interaction of pro-coagulant MVs with bacteria of the species Streptococcus pyogenes is part of the early immune response to the invading pathogen. As shown by negative staining electron microscopy and clotting assays, pro-coagulant MVs bind in the presence of plasma to the bacterial surface. Fibrinogen was identified as a linker that, through binding to the M1 protein of S. pyogenes, allows the opsonization of the bacteria by MVs. Surface plasmon resonance analysis revealed a strong interaction between pro-coagulant MVs and fibrinogen with a KD value in the nanomolar range. When performing a mass-spectrometry-based strategy to determine the protein quantity, a significant up-regulation of the fibrinogen-binding integrins CD18 and CD11b on pro-coagulant MVs was recorded. Finally we show that plasma clots induced by pro-coagulant MVs are able to prevent bacterial dissemination and possess antimicrobial activity. These findings were confirmed by in vivo experiments, as local treatment with pro-coagulant MVs dampens bacterial spreading to other organs and improved survival in an invasive streptococcal mouse model of infection. Taken together, our data implicate that pro-coagulant MVs play an important role in the early response of the innate immune system in infectious diseases.

  3. Epididymosomes: a heterogeneous population of microvesicles with multiple functions in sperm maturation and storage

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    Robert Sullivan

    2015-01-01

    Full Text Available Extracellular microvesicles present in the epididymal fluid have been named epididymosomes. Many epididymosome-associated proteins are transferred to spermatozoa during their maturation in the excurrent duct. Epididymosomes are heterogeneous, with their size varying between 50 and 250 nm. Two distinct population of epididymosomes characterized by different protein compositions and diameters have been isolated from the bovine epididymal fluid using different centrifugation protocols. One subpopulation of epididymosomes was characterized by CD9 and other tetraspanin partners. Transfer of proteins from these epididymosomes to maturing spermatozoa in co-incubation experiments was inhibited by antibodies against tetraspanin proteins. This suggests that this subpopulation of epididymosomes is involved in the acquisition of proteins involved in maturation by spermatozoa in the epididymis. The other population of epididymosomes was characterized by ELSPBP1 (epididymal sperm binding protein 1, known for its affinity for the phospholipid choline group. Flow cytometric analyses showed that ELSPBP1-positive epididymosomes only interacted with dying or dead epididymal spermatozoa in a Zn 2 + -dependent manner. BLVRA (biliverdin reductase was identified as a partner of ELSPBP1. This enzyme reduces biliverdin to bilirubin: two molecules with powerful anti-oxidant properties. We hypothesize that BLVRA is involved in an ROS-scavenging mechanism protecting live epididymal spermatozoa against detrimental molecules (ROS released by dying cells. Therefore, it appears that there are at least two epididymosome population with distinct functions: targeting specific proteins to transiting spermatozoa by tetraspanin-mediated membrane fusion, and protection of epididymal spermatozoa against ROS released from dying cells. Further work is needed to understand functions of epididymosomes in epididymal physiology and sperm maturation and storage.

  4. A novel role for pro-coagulant microvesicles in the early host defense against streptococcus pyogenes.

    Science.gov (United States)

    Oehmcke, Sonja; Westman, Johannes; Malmström, Johan; Mörgelin, Matthias; Olin, Anders I; Kreikemeyer, Bernd; Herwald, Heiko

    2013-01-01

    Previous studies have shown that stimulation of whole blood or peripheral blood mononuclear cells with bacterial virulence factors results in the sequestration of pro-coagulant microvesicles (MVs). These particles explore their clotting activity via the extrinsic and intrinsic pathway of coagulation; however, their pathophysiological role in infectious diseases remains enigmatic. Here we describe that the interaction of pro-coagulant MVs with bacteria of the species Streptococcus pyogenes is part of the early immune response to the invading pathogen. As shown by negative staining electron microscopy and clotting assays, pro-coagulant MVs bind in the presence of plasma to the bacterial surface. Fibrinogen was identified as a linker that, through binding to the M1 protein of S. pyogenes, allows the opsonization of the bacteria by MVs. Surface plasmon resonance analysis revealed a strong interaction between pro-coagulant MVs and fibrinogen with a KD value in the nanomolar range. When performing a mass-spectrometry-based strategy to determine the protein quantity, a significant up-regulation of the fibrinogen-binding integrins CD18 and CD11b on pro-coagulant MVs was recorded. Finally we show that plasma clots induced by pro-coagulant MVs are able to prevent bacterial dissemination and possess antimicrobial activity. These findings were confirmed by in vivo experiments, as local treatment with pro-coagulant MVs dampens bacterial spreading to other organs and improved survival in an invasive streptococcal mouse model of infection. Taken together, our data implicate that pro-coagulant MVs play an important role in the early response of the innate immune system in infectious diseases.

  5. Extracellular microvesicle production by human eosinophils activated by “inflammatory” stimuli

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    Praveen Akuthota

    2016-10-01

    Full Text Available A key function of human eosinophils is to secrete cytokines, chemokines and cationic proteins, trafficking and releasing these mediators for roles in inflammation and other immune responses. Eosinophil activation leads to secretion of pre-synthesized granule-stored mediators through different mechanisms, but the ability of eosinophils to secrete extracellular vesicles (EVs, very small vesicles with preserved membrane topology, is still poorly understood. In the present work, we sought to identify and characterize EVs released from human eosinophils during different conditions: after a culturing period or after isolation and stimulation with inflammatory stimuli, which are known to induce eosinophil activation and secretion: CCL11 (eotaxin-1 and tumor necrosis factor alpha (TNF-α. EV production was investigated by nanoscale flow cytometry, conventional transmission electron microscopy (TEM and pre-embedding immunonanogold EM. The tetraspanins CD63 and CD9 were used as EV biomarkers for both flow cytometry and ultrastructural immunolabeling. Nanoscale flow cytometry showed that human eosinophils produce EVs in culture and that a population of EVs expressed detectable CD9, while CD63 was not consistently detected. When eosinophils were stimulated immediately after isolation and analyzed by TEM, EVs were clearly identified as microvesicles (MVsoutwardly budding off the plasma membrane. Both CCL11 and TNF-α induced significant increases of MVs compared to unstimulated cells.TNF-α induced amplified release of MVs more than CCL11. Eosinophil MV diameters varied from 20-1000 nm. Immunonanogold EM revealed clear immunolabeling for CD63 and CD9 on eosinophil MVs, although not all MVs were labeled. Altogether, we identified, for the first time, that human eosinophils secrete MVs and that this production increases in response to inflammatory stimuli. This is important to understand the complex secretory activities of eosinophils underlying immune

  6. Argonaute 2 complexes selectively protect the circulating microRNAs in cell-secreted microvesicles.

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    Limin Li

    Full Text Available Cell-secreted miRNAs are highly stable and can serve as biomarkers for various diseases and signaling molecules in intercellular communication. The mechanism underlying the stability of circulating miRNAs, however, remains incompletely understood. Here we show that Argonaute 2 (Ago2 complexes and microvesicles (MVs provide specific and non-specific protection for miRNA in cell-secreted MVs, respectively. First, the resistance of MV-encapsulated miRNAs to RNaseA was both depended on intact vesicular structure of MVs and protease-sensitive. Second, an immunoprecipitation assay using a probe complementary to human miR-16, a miRNA primarily located in the MVs and showed a strong, protease-sensitive resistance to RNaseA, identified Ago2 as a major miR-16-associated protein. Compared with protein-free miR-16, Ago2-associated miR-16 was resistant to RNaseA in a dose- and time-dependent fashion. Third, when the miR-16/Ago2 complex was disrupted by trypaflavine, the resistance of miR-16 to RNaseA was decreased. In contrast, when the association of miR-16 with the Ago2 complexes was increased during cell apoptosis, although the total amount of miR-16 and Ago2 remained unchanged, the resistance of miR-16 to RNaseA in the MVs was enhanced. A similar correlation between the increase of miR-223/Ago2 association and the resistance of miR-223 against RNaseA was observed during all trans retinoic acid (ATRA-induced cell differentiation of promyelocytic HL60 cells. In conclusion, the association of miRNAs with Ago2 complexes, an event that is linked to cell functional status, plays a critical role in stabilizing the circulating miRNAs in cell-secreted MVs.

  7. Lactation-Related MicroRNA Expression in Microvesicles of Human Umbilical Cord Blood.

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    Wang, De-Jing; Wang, Chen-Meiyi; Wang, Yi-Ting; Qiao, Hai; Fang, Liao-Qiong; Wang, Zhi-Biao

    2016-11-24

    BACKGROUND The complex process by which lactation is initiated upon neonate delivery remains incompletely understood. Microvesicles (MVs) can transmit microRNAs (miRNAs) into recipient cells to influence cell function, and recent studies have identified miRNAs essential for mammary gland development and lactation. This study aimed to investigate the expression of lactation-related miRNAs in MVs isolated from human umbilical cord blood immediately after delivery. MATERIAL AND METHODS Umbilical cord blood samples were collected from 70 healthy pregnant women, and MVs were isolated through differential centrifugation and characterized by transmission electron microscopy, Western blotting, and nanoparticle tracking analysis. Lactation-related miRNAs were screened using bioinformatics tools for miRNA target prediction, gene ontology, and signaling pathway analyses. miRNA PCR arrays were used for miRNA expression analysis, and the results were validated by real-time PCR. Upon exposure of HBL-100 human mammary epithelial cells to MVs, MV uptake was examined by fluorescence confocal microscopy and b-casein secretion was detected by ELISA. RESULTS Spherical MVs extracted from umbilical cord blood expressed CD63 and had an average diameter of 167.0±77.1 nm. We profiled 337 miRNAs in human umbilical cord blood MVs and found that 85 were related to lactation by bioinformatics analysis. The 25 most differentially expressed lactation-related miRNAs were validated by real-time PCR. MV uptake by HBL-100 cells was after 4 h in culture, and significantly increased secretion of β-casein was observed after 96 h from cells exposed to MVs (Plactation-related miRNAs and can induce β-casein production by HBL-100 cells in vitro. Thus, umbilical cord blood MVs may mediate secretion of β-casein through miRNAs, thereby playing an important role in fetal-maternal crosstalk.

  8. Improved Methods to Generate Spheroid Cultures from Tumor Cells, Tumor Cells & Fibroblasts or Tumor-Fragments: Microenvironment, Microvesicles and MiRNA.

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    Zheng Lao

    Full Text Available Diagnostic and prognostic indicators are key components to achieve the goal of personalized cancer therapy. Two distinct approaches to this goal include predicting response by genetic analysis and direct testing of possible therapies using cultures derived from biopsy specimens. Optimally, the latter method requires a rapid assessment, but growing xenograft tumors or developing patient-derived cell lines can involve a great deal of time and expense. Furthermore, tumor cells have much different responses when grown in 2D versus 3D tissue environments. Using a modification of existing methods, we show that it is possible to make tumor-fragment (TF spheroids in only 2-3 days. TF spheroids appear to closely model characteristics of the original tumor and may be used to assess critical therapy-modulating features of the microenvironment such as hypoxia. A similar method allows the reproducible development of spheroids from mixed tumor cells and fibroblasts (mixed-cell spheroids. Prior literature reports have shown highly variable development and properties of mixed-cell spheroids and this has hampered the detailed study of how individual tumor-cell components interact. In this study, we illustrate this approach and describe similarities and differences using two tumor models (U87 glioma and SQ20B squamous-cell carcinoma with supporting data from additional cell lines. We show that U87 and SQ20B spheroids predict a key microenvironmental factor in tumors (hypoxia and that SQ20B cells and spheroids generate similar numbers of microvesicles. We also present pilot data for miRNA expression under conditions of cells, tumors, and TF spheroids.

  9. Microvesicles derived from human umbilical cord Wharton's jelly mesenchymal stem cells attenuate bladder tumor cell growth in vitro and in vivo.

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    Shuai Wu

    Full Text Available Several studies suggest that mesenchymal stem cells (MSCs possess antitumor properties; however, the exact mechanisms remain unclear. Recently, microvesicles (MVs are considered as a novel avenue intercellular communication, which may be a mediator in MSCs-related antitumor effect. In the present study, we evaluated whether MVs derived from human umbilical cord Wharton's jelly mesenchymal stem cells (hWJMSCs may inhibit bladder tumor T24 cells growth using cell culture and the BALB/c nu/nu mice xenograft model. CCK-8 assay and Ki-67 immunostaining were performed to estimate cell proliferation in vitro and in vivo. Flow cytometry and TUNEL assay were used to assess cell cycle and apoptosis. To study the conceivable mechanism by which hWJMSC-MVs attenuate bladder tumor T24 cells, we estimated the expression of Akt/p-Akt, p-p53, p21 and cleaved Caspase 3 by Western blot technique after exposing T24 cells to hWJMSC-MVs for 24, 48 and 72h. Our data indicated that hWJMSC-MVs can inhibit T24 cells proliferative viability via cell cycle arrest and induce apoptosis in T24 cells in vitro and in vivo. This study showed that hWJMSC-MVs down-regulated phosphorylation of Akt protein kinase and up-regulated cleaved Caspase 3 during the process of anti-proliferation and pro-apoptosis in T24 cells. These results demonstrate that hWJMSC-MVs play a vital role in hWJMSC-induced antitumor effect and may be a novel tool for cancer therapy as a new mechanism of cell-to-cell communication.

  10. Microvesicles Derived from Human Umbilical Cord Wharton’s Jelly Mesenchymal Stem Cells Attenuate Bladder Tumor Cell Growth In Vitro and In Vivo

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    Du, Tao; Zhu, Ying-Jian; Liu, Guo-Hua

    2013-01-01

    Several studies suggest that mesenchymal stem cells (MSCs) possess antitumor properties; however, the exact mechanisms remain unclear. Recently, microvesicles (MVs) are considered as a novel avenue intercellular communication, which may be a mediator in MSCs-related antitumor effect. In the present study, we evaluated whether MVs derived from human umbilical cord Wharton’s jelly mesenchymal stem cells (hWJMSCs) may inhibit bladder tumor T24 cells growth using cell culture and the BALB/c nu/nu mice xenograft model. CCK-8 assay and Ki-67 immunostaining were performed to estimate cell proliferation in vitro and in vivo. Flow cytometry and TUNEL assay were used to assess cell cycle and apoptosis. To study the conceivable mechanism by which hWJMSC-MVs attenuate bladder tumor T24 cells, we estimated the expression of Akt/p-Akt, p-p53, p21 and cleaved Caspase 3 by Western blot technique after exposing T24 cells to hWJMSC-MVs for 24, 48 and 72h. Our data indicated that hWJMSC-MVs can inhibit T24 cells proliferative viability via cell cycle arrest and induce apoptosis in T24 cells in vitro and in vivo. This study showed that hWJMSC-MVs down-regulated phosphorylation of Akt protein kinase and up-regulated cleaved Caspase 3 during the process of anti-proliferation and pro-apoptosis in T24 cells. These results demonstrate that hWJMSC-MVs play a vital role in hWJMSC-induced antitumor effect and may be a novel tool for cancer therapy as a new mechanism of cell-to-cell communication. PMID:23593475

  11. Kupffer Cell-Derived Tnf Triggers Cholangiocellular Tumorigenesis through JNK due to Chronic Mitochondrial Dysfunction and ROS.

    Science.gov (United States)

    Yuan, Detian; Huang, Shan; Berger, Emanuel; Liu, Lei; Gross, Nina; Heinzmann, Florian; Ringelhan, Marc; Connor, Tracy O; Stadler, Mira; Meister, Michael; Weber, Julia; Öllinger, Rupert; Simonavicius, Nicole; Reisinger, Florian; Hartmann, Daniel; Meyer, Rüdiger; Reich, Maria; Seehawer, Marco; Leone, Valentina; Höchst, Bastian; Wohlleber, Dirk; Jörs, Simone; Prinz, Marco; Spalding, Duncan; Protzer, Ulrike; Luedde, Tom; Terracciano, Luigi; Matter, Matthias; Longerich, Thomas; Knolle, Percy; Ried, Thomas; Keitel, Verena; Geisler, Fabian; Unger, Kristian; Cinnamon, Einat; Pikarsky, Eli; Hüser, Norbert; Davis, Roger J; Tschaharganeh, Darjus F; Rad, Roland; Weber, Achim; Zender, Lars; Haller, Dirk; Heikenwalder, Mathias

    2017-06-12

    Intrahepatic cholangiocarcinoma (ICC) is a highly malignant, heterogeneous cancer with poor treatment options. We found that mitochondrial dysfunction and oxidative stress trigger a niche favoring cholangiocellular overgrowth and tumorigenesis. Liver damage, reactive oxygen species (ROS) and paracrine tumor necrosis factor (Tnf) from Kupffer cells caused JNK-mediated cholangiocellular proliferation and oncogenic transformation. Anti-oxidant treatment, Kupffer cell depletion, Tnfr1 deletion, or JNK inhibition reduced cholangiocellular pre-neoplastic lesions. Liver-specific JNK1/2 deletion led to tumor reduction and enhanced survival in Akt/Notch- or p53/Kras-induced ICC models. In human ICC, high Tnf expression near ICC lesions, cholangiocellular JNK-phosphorylation, and ROS accumulation in surrounding hepatocytes are present. Thus, Kupffer cell-derived Tnf favors cholangiocellular proliferation/differentiation and carcinogenesis. Targeting the ROS/Tnf/JNK axis may provide opportunities for ICC therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Generating and characterizing the mechanical properties of cell-derived matrices using atomic force microscopy.

    Science.gov (United States)

    Tello, Marta; Spenlé, Caroline; Hemmerlé, Joseph; Mercier, Luc; Fabre, Roxane; Allio, Guillaume; Simon-Assmann, Patricia; Goetz, Jacky G

    2016-02-01

    Mechanical interaction between cells and their surrounding extracellular matrix (ECM) controls key processes such as proliferation, differentiation and motility. For many years, two-dimensional (2D) models were used to better understand the interactions between cells and their surrounding ECM. More recently, variation of the mechanical properties of tissues has been reported to play a major role in physiological and pathological scenarios such as cancer progression. The 3D architecture of the ECM finely tunes cellular behavior to perform physiologically relevant tasks. Technical limitations prevented scientists from obtaining accurate assessment of the mechanical properties of physiologically realistic matrices. There is therefore a need for combining the production of high-quality cell-derived 3D matrices (CDMs) and the characterization of their topographical and mechanical properties. Here, we describe methods that allow to accurately measure the young modulus of matrices produced by various cellular types. In the first part, we will describe and review several protocols for generating CDMs matrices from endothelial, epithelial, fibroblastic, muscle and mesenchymal stem cells. We will discuss tools allowing the characterization of the topographical details as well as of the protein content of such CDMs. In a second part, we will report the methodologies that can be used, based on atomic force microscopy, to accurately evaluate the stiffness properties of the CDMs through the quantification of their young modulus. Altogether, such methodologies allow characterizing the stiffness and topography of matrices deposited by the cells, which is key for the understanding of cellular behavior in physiological conditions. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Macrophage-derived microvesicles promote proliferation and migration of Schwann cell on peripheral nerve repair

    Energy Technology Data Exchange (ETDEWEB)

    Zhan, Chuan, E-mail: zhchuansy@163.com; Ma, Cheng-bin; Yuan, Hong-mou; Cao, Bao-yuan; Zhu, Jia-jun

    2015-12-04

    Background: Macrophages have been implicated in peripheral nerve regeneration. However, whether macrophages-derived microvesicles (MVs) are involved in this process remains unknown. In the present study, the effects of macrophages-derived MVs on proliferation and migration of Schwann cells (SCs) were evaluated in both in vitro and in vivo. Methods: Human monocytic leukaemia cell line (THP-1) was successfully driven to M1 and M2 phenotypes by delivery of either IFN-γ or IL-4, respectively. SCs incubated with M1 or M2 macrophages-derived MVs, the cell migration and proliferation were assessed, and expression levels of nerve growth factor (NGF) and Laminin were measured. A rat model of sciatic nerve was established and the effects of macrophages-derived MVs on nerve regeneration were investigated. Results: M2-derived MVs elevated migration, proliferation, NFG and Laminin protein levels of SCs compared with M1-or M0-derived MVs. The relative expression levels of miR-223 were also increased in M2 macrophages and M2-derived MVs. Transfected M2 macrophages with miR-223 inhibitor then co-incubated with SCs, an inhibition of cell migration and proliferation and a down-regulated levels of NFG and Laminin protein expression were observed. In vivo, M2-derived MVs significantly increased the infiltration and axon number of SCs. Conclusion: M2-derived MVs promoted proliferation and migration of SCs in vitro and in vivo, which provided a therapeutic strategy for nerve regeneration. - Highlights: • M2 macrophages-derived MVs elevated migration and proliferation of SCs. • M2 macrophages-derived MVs up-regulated NFG and Laminin expression of SCs. • MiR-223 expression was increased in M2 macrophages-derived MVs. • MiR-223 inhibitor reduced migration and proliferation of SCs co-incubated with MVs. • MiR-223 inhibitor down-regulated NFG and Laminin levels of SCs co-incubated with MVs.

  14. Microvesicle phenotypes are associated with transfusion requirements and mortality in subjects with severe injuries

    Directory of Open Access Journals (Sweden)

    Nena Matijevic

    2015-12-01

    Full Text Available Background: Severe injury often results in substantial bleeding and mortality. Injury provokes cellular activation and release of extracellular vesicles. Circulating microvesicles (MVs are predominantly platelet-derived and highly procoagulant. They support hemostasis and vascular function. The roles of MVs in survival after severe injury are largely unknown. We hypothesized that altered MV phenotypes would be associated with transfusion requirements and poor outcomes. Methods: This single-centre study was approved by the Institutional Review Board. The study cohort consisted of patients with major trauma requiring blood product transfusion and 26 healthy controls. Plasma samples for MVs were collected upon admission to the emergency department (n=169 and post-resuscitation (n=42, and analysed by flow cytometry for MV counts and cellular origin: platelet (PMV, erythrocyte (RMV, leukocyte (LMV, endothelial (EMV, tissue factor (TFMV, and annexin V (AVMV. Twenty-four hour mortality is the outcome measurement used to classify survivors versus non-survivors. Data were compared over time and analysed with demographic and clinical data. Results: The median age was 34 (IQR 23, 51, 72% were male, Injury Severity Score was 29 (IQR 19, 36, and 24 h mortality was 13%. MV levels and phenotypes differed between patients and controls. Elevated admission EMVs were found both in survivors (409/µL and non-survivors (393/µL compared to controls (23/µL, p<0.001 and persisted over time. Admission levels of PMV, AVMV, RMV, and TFMV were significantly lower in patients who died compared to survivors, but were not independently associated with the 24 h mortality rate. Patients with low MV levels at admission received the most blood products within the first 24 h. AVMV and PMV levels either increased over time or stabilized in survivors but decreased in non-survivors, resulting in significantly lower levels at intensive care unit admission in non-survivors (1,048 vs

  15. Microvesicles Derived from Inflammation-Challenged Endothelial Cells Modulate Vascular Smooth Muscle Cell Functions.

    Science.gov (United States)

    Pan, Qunwen; Liu, Hua; Zheng, Chunyan; Zhao, Yuhui; Liao, Xiaorong; Wang, Yan; Chen, Yanfang; Zhao, Bin; Lazartigues, Eric; Yang, Yi; Ma, Xiaotang

    2016-01-01

    Purpose: Microvesicles (MV) can modulate the function of recipient cells by transferring their contents. Our previous study highlighted that MV released from tumor necrosis factor-α (TNF-α) plus serum deprivation (SD)-stimulated endothelial progenitor cells, induce detrimental effects on endothelial cells. In this study, we investigated the potential effects of endothelial MV (EMV) on proliferation, migration, and apoptosis of human brain vascular smooth cells (HBVSMC). Methods: EMV were prepared from human brain microvascular endothelial cells (HBMEC) cultured in a TNF-α plus SD medium. RNase-EMV were made by treating EMV with RNase A for RNA depletion. The proliferation, apoptosis and migration abilities of HBVSMC were determined after co-culture with EMV or RNase-EMV. The Mek1/2 inhibitor, PD0325901, was used for pathway analysis. Western blot was used for analyzing the proteins of Mek1/2, Erk1/2, phosphorylation Erk1/2, activated caspase-3 and Bcl-2. The level of miR-146a-5p was measured by qRT-PCR. Results: (1) EMV significantly promoted the proliferation and migration of HBVSMC. The effects were accompanied by an increase in Mek1/2 and p-Erk1/2, which could be abolished by PD0325901; (2) EMV decreased the apoptotic rate of HBVSMC by approximately 35%, which was accompanied by cleaved caspase-3 down-regulation and Bcl-2 up-regulation; (3) EMV increased miR-146a-5p level in HBVSMC by about 2-folds; (4) RNase-treated EMV were less effective than EMV on HBVSMC activities and miR-146a-5p expression. Conclusion: EMV generated under inflammation challenge can modulate HBVSMC function and fate via their carried RNA. This is associated with activation of theMek1/2/Erk1/2 pathway and caspase-3/Bcl-2 regulation, during which miR-146a-5p may play an important role. The data suggest that EMV derived from inflammation-challenged endothelial cells are detrimental to HBVSMC homeostatic functions, highlighting potential novel therapeutic targets for vascular diseases.

  16. Melanoma cell-derived exosomes promote epithelial-mesenchymal transition in primary melanocytes through paracrine/autocrine signaling in the tumor microenvironment

    Science.gov (United States)

    Xiao, Deyi; Barry, Samantha; Kmetz, Daniel; Egger, Michael; Pan, Jianmin; Rai, Shesh N; Qu, Jifu; McMasters, Kelly M.; Hao, Hongying

    2016-01-01

    The tumor microenvironment is abundant with exosomes that are secreted by the cancer cells themselves. Exosomes are nanosized, organelle-like membranous structures that are increasingly being recognized as major contributors in the progression of malignant neoplasms. A critical element in melanoma progression is its propensity to metastasize, but little is known about how melanoma cell-derived exosomes modulate the microenvironment to optimize conditions for tumor progression and metastasis. Here, we provide evidence that melanoma cell-derived exosomes promote phenotype switching in primary melanocytes through paracrine/autocrine signaling. We found that the mitogen-activated protein kinase (MAPK) signaling pathway was activated during the exosome-mediated epithelial-to-mesenchymal transition (EMT)-resembling process, which promotes metastasis. Let-7i, an miRNA modulator of EMT, was also involved in this process. We further defined two other miRNA modulators of EMT (miR-191 and let-7a) in serum exosomes for differentiating stage I melanoma patients from non-melanoma subjects. These results provide the first strong molecular evidence that melanoma cell-derived exosomes promote the EMT-resembling process in the tumor microenvironment. Thus, novel strategies targeting EMT and modulating the tumor microenvironment may emerge as important approaches for the treatment of metastatic melanoma. PMID:27063098

  17. Melanoma cell-derived exosomes promote epithelial-mesenchymal transition in primary melanocytes through paracrine/autocrine signaling in the tumor microenvironment.

    Science.gov (United States)

    Xiao, Deyi; Barry, Samantha; Kmetz, Daniel; Egger, Michael; Pan, Jianmin; Rai, Shesh N; Qu, Jifu; McMasters, Kelly M; Hao, Hongying

    2016-07-01

    The tumor microenvironment is abundant with exosomes that are secreted by the cancer cells themselves. Exosomes are nanosized, organelle-like membranous structures that are increasingly being recognized as major contributors in the progression of malignant neoplasms. A critical element in melanoma progression is its propensity to metastasize, but little is known about how melanoma cell-derived exosomes modulate the microenvironment to optimize conditions for tumor progression and metastasis. Here, we provide evidence that melanoma cell-derived exosomes promote phenotype switching in primary melanocytes through paracrine/autocrine signaling. We found that the mitogen-activated protein kinase (MAPK) signaling pathway was activated during the exosome-mediated epithelial-to-mesenchymal transition (EMT)-resembling process, which promotes metastasis. Let-7i, an miRNA modulator of EMT, was also involved in this process. We further defined two other miRNA modulators of EMT (miR-191 and let-7a) in serum exosomes for differentiating stage I melanoma patients from non-melanoma subjects. These results provide the first strong molecular evidence that melanoma cell-derived exosomes promote the EMT-resembling process in the tumor microenvironment. Thus, novel strategies targeting EMT and modulating the tumor microenvironment may emerge as important approaches for the treatment of metastatic melanoma. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Microvesicle-mediated release of soluble LH/hCG receptor (LHCGR from transfected cells and placenta explants

    Directory of Open Access Journals (Sweden)

    Randeva Harpal

    2011-05-01

    Full Text Available Abstract Placental hCG and pitutary LH transduce signals in target tissues through a common receptor (LHCGR. We demonstrate that recombinant LHCGR proteins which include the hormone-binding domain are secreted from transfected cells and that natural LHCGR is also secreted from human placental explants. LHCGR recombinant proteins representing varying lengths of the N-terminal extracellular domain were expressed in Chinese Hamster Ovary cells in suspension culture. Secretion was minimal up to 72h but by 96h 24-37% of the LHCGR had been released into the culture medium. The secreted proteins were folded and sensitive to glycosidases suggesting N-linked glycosylation. Secretion was independent of recombinant size and was mediated via structurally defined membrane vesicles (50-150nm. Similarly cultured human early pregnancy placental explants also released LHCGR via microvesicles. These studies provide the first experimental evidence of the possible mechanistic basis of the secretion of LHCGR.

  19. Analysis of Cell-Derived Microparticles with Highly Precise Nanotechnological Methods

    DEFF Research Database (Denmark)

    Cherré, Solène; Østergaard, Ole; Heegaard, Niels H.H.

    2014-01-01

    Cell-derived microparticles have gained a broad interest in the past years. Being released by blood cells upon activation or induction of apoptosis, they have a great potential as novel diagnostic markers and their investigation can bring new knowledge into the pathogenesis of various diseases....... However, new analysis methods are required to correctly and reliably investigate these small biological particles (between 50 and 1000 nm). In this work, we aimed at developing an in vitro population of cell-derived microparticles from endothelial cells. The size of the microparticles was analysed...

  20. Data on importance of hematopoietic cell derived Lipocalin 2 against gut inflammation

    Directory of Open Access Journals (Sweden)

    Piu Saha

    2016-09-01

    Neutralization of Lcn2 in WT mice resulted in exacerbated DSS-induced colitis. Notably, mice lacking Lcn2 exhibited 100% mortality whereas only 20% mortality was observed in WT mice upon DSS challenge. Further, data from bone marrow chimera showed that immune cell-derived Lcn2 is the major contributor in conferring protection against colitis.

  1. Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future.

    Science.gov (United States)

    Luo, Mingyue; Chen, Youxin

    2018-01-01

    As a constituent of blood-retinal barrier and retinal outer segment (ROS) scavenger, retinal pigmented epithelium (RPE) is fundamental to normal function of retina. Malfunctioning of RPE contributes to the onset and advance of retinal degenerative diseases. Up to date, RPE replacement therapy is the only possible method to completely reverse retinal degeneration. Transplantation of human RPE stem cell-derived RPE (hRPESC-RPE) has shown some good results in animal models. With promising results in terms of safety and visual improvement, human embryonic stem cell-derived RPE (hESC-RPE) can be expected in clinical settings in the near future. Despite twists and turns, induced pluripotent stem cell-derived RPE (iPSC-RPE) is now being intensely investigated to overcome genetic and epigenetic instability. By far, only one patient has received iPSC-RPE transplant, which is a hallmark of iPSC technology development. During follow-up, no major complications such as immunogenicity or tumorigenesis have been observed. Future trials should keep focusing on the safety of stem cell-derived RPE (SC-RPE) especially in long period, and better understanding of the nature of stem cell and the molecular events in the process to generate SC-RPE is necessary to the prosperity of SC-RPE clinical application.

  2. Stromal cell-derived factor-1 stimulates cell recruitment, vascularization and osteogenic differentiation

    NARCIS (Netherlands)

    Eman, Rhandy M; Oner, F Cumhur; Kruyt, Moyo C; Dhert, Wouter J A; Alblas, Jacqueline

    The use of growth factors in osteogenic constructs to promote recruitment of bone forming endogenous cells is not clear, while the advantage of circumventing cell seeding techniques before implantation is highly recognized. Therefore, the additive effect of the chemokine stromal cell-derived

  3. Two Polymorphisms in the Epithelial Cell-Derived Neutrophil-Activating Peptide (ENA-78 Gene

    Directory of Open Access Journals (Sweden)

    Mahsa M. Amoli

    2005-01-01

    Full Text Available Increased expression of epithelial cell-derived neutrophil-activating peptide (ENA-78 has been reported in several immune and inflammatory conditions suggesting its role in inflammatory response. We have identified two single nucleotide polymorphisms in the promoter and exon 2 of the ENA-78 gene by scanning the full length gene using DHPLC DNA fragment analysis and DNA sequencing.

  4. Chemokine stromal cell-derived factor 1alpha activates basophils by means of CXCR4

    DEFF Research Database (Denmark)

    Jinquan, T; Jacobi, H H; Jing, C

    2000-01-01

    The CXC chemokine receptor 4 (CXCR4) is predominantly expressed on inactivated naive T lymphocytes, B lymphocytes, dendritic cells, and endothelial cells. CXC chemokine stromal cell-derived factor 1alpha (SDF-1alpha) is the only known ligand for CXCR4. To date, the CXCR4 expression and function o...

  5. 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...

  6. Molecular signature of primary retinal pigment epithelium and stem-cell-derived RPE cells

    Science.gov (United States)

    Liao, Jo-Ling; Yu, Juehua; Huang, Kevin; Hu, Jane; Diemer, Tanja; Ma, Zhicheng; Dvash, Tamar; Yang, Xian-Jie; Travis, Gabriel H.; Williams, David S.; Bok, Dean; Fan, Guoping

    2010-01-01

    Age-related macular degeneration (AMD) is characterized by the loss or dysfunction of retinal pigment epithelium (RPE) and is the most common cause of vision loss among the elderly. Stem-cell-based strategies, using human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs), may provide an abundant donor source for generating RPE cells in cell replacement therapies. Despite a significant amount of research on deriving functional RPE cells from various stem cell sources, it is still unclear whether stem-cell-derived RPE cells fully mimic primary RPE cells. In this report, we demonstrate that functional RPE cells can be derived from multiple lines of hESCs and hiPSCs with varying efficiencies. Stem-cell-derived RPE cells exhibit cobblestone-like morphology, transcripts, proteins and phagocytic function similar to human fetal RPE (fRPE) cells. In addition, we performed global gene expression profiling of stem-cell-derived RPE cells, native and cultured fRPE cells, undifferentiated hESCs and fibroblasts to determine the differentiation state of stem-cell-derived RPE cells. Our data indicate that hESC-derived RPE cells closely resemble human fRPE cells, whereas hiPSC-derived RPE cells are in a unique differentiation state. Furthermore, we identified a set of 87 signature genes that are unique to human fRPE and a majority of these signature genes are shared by stem-cell-derived RPE cells. These results establish a panel of molecular markers for evaluating the fidelity of human pluripotent stem cell to RPE conversion. This study contributes to our understanding of the utility of hESC/hiPSC-derived RPE in AMD therapy. PMID:20709808

  7. Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model

    DEFF Research Database (Denmark)

    Tønnesen, Jan; Parish, Clare L; Sørensen, Andreas T

    2011-01-01

    Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson's disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral...

  8. Stromal cell-derived factor-1 (SDF-1)/CXCR4 axis enhances cellular invasion in ovarian carcinoma cells via integrin β1 and β3 expressions.

    Science.gov (United States)

    Yu, Yuecheng; Shi, Xiaoyan; Shu, Zhen; Xie, Tingting; Huang, Kan; Wei, Li; Song, Hui; Zhang, Wei; Xue, Xiaochang

    2013-01-01

    Accumulating evidence has showed that stromal cell-derived factor-1 (SDF-1/CXCR4 axis played important roles in cancer metastases, but the detailed function in ovarian cancer is still largely unknown. In the present study, we determined the location of CXCR4 and lipid rafts, a specialized structure on cell membrane, in ovarian cancer tissues and ovarian cancer cell line SKOV3 cells by immunofluorescence. To analyze the role of SDF-1/CXCR4 and lipid rafts in tumor cell migration and invasion, Transwell assay and wound healing assay were also performed. Cytoflowmetry was carried out to determine the participation of integrins. Our data showed that CXCR4 and GM1 (marker of lipid rafts) were expressed in both ovarian cancer tissue and SKOV3 cells, and SDF-1 promoted the invasion and migration of SKOV3 cells, which was mediated by complete lipid rafts. Further studies uncovered that SDF-1 upregulated the expression of integrin β1 and β3, two molecules closely related with cancer metastasis. These results indicated that SDF-1 might promote the invasion and metastasis of ovarian cancer by regulating these two integrin molecules.

  9. Classification of follicular cell-derived thyroid cancer by global RNA profiling

    DEFF Research Database (Denmark)

    Rossing, Maria

    2013-01-01

    classification will not only contribute to our biological insight but also improve clinical and pathological examinations, thus advancing thyroid tumour diagnosis and ultimately preventing superfluous surgery. This review evaluates the status of classification and biological insights gained from molecular...... classifiers that may differentiate malignant from benign thyroid nodules. Molecular classification models based on global RNA profiles from fine-needle aspirations are currently being evaluated; results are preliminary and lack validation in prospective clinical trials. There is no doubt that molecular...

  10. Concise review: the relevance of human stem cell-derived organoid models for epithelial translational medicine.

    Science.gov (United States)

    Hynds, Robert E; Giangreco, Adam

    2013-03-01

    Epithelial organ remodeling is a major contributing factor to worldwide death and disease, costing healthcare systems billions of dollars every year. Despite this, most fundamental epithelial organ research fails to produce new therapies and mortality rates for epithelial organ diseases remain unacceptably high. In large part, this failure in translating basic epithelial research into clinical therapy is due to a lack of relevance in existing preclinical models. To correct this, new models are required that improve preclinical target identification, pharmacological lead validation, and compound optimization. In this review, we discuss the relevance of human stem cell-derived, three-dimensional organoid models for addressing each of these challenges. We highlight the advantages of stem cell-derived organoid models over existing culture systems, discuss recent advances in epithelial tissue-specific organoids, and present a paradigm for using organoid models in human translational medicine. Copyright © 2012 AlphaMed Press.

  11. Production of Single Contracting Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Matrigel Mattress Technique.

    Science.gov (United States)

    Cadar, Adrian G; Feaster, Tromondae K; Durbin, Matthew D; Hong, Charles C

    2017-08-14

    This unit describes the published Matrigel mattress method. Briefly, we describe the preparation of the mattress, replating of the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) on the Matrigel mattress, and hiPSC-CM mattress maintenance. Adherence to this protocol will yield individual, robustly shortening hiPSC-CMs, which can be used for downstream applications. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

  12. Side Population Cells Derived from Adult Human Liver Generate Hepatocyte-like Cells In Vitro

    OpenAIRE

    HUSSAIN, SUNNY ZAHEED; Strom, Stephen C.; Kirby, Martha R.; Burns, Sean; Langemeijer, Saskia; Ueda, Takahiro; HSIEH, MATTHEW; Tisdale, John F.

    2005-01-01

    We sought to determine whether hepatic side population (SP) cells derived from adult human liver possess the potential of a novel candidate hepatic stem cell. Human cadaveric donor liver was subjected to collagenase perfusion and hepatocytes were separated from nonparenchymal cells by differential centrifugation. SP cells were isolated from the nonparenchymal portion after Hoechst 33342 staining. Since CD45 is a panleukocyte antigen, CD45-negative SP cells were separated from the vast majorit...

  13. Side Population Cells Derived from Adult Human Liver Generate Hepatocyte-like Cells In Vitro

    Science.gov (United States)

    HUSSAIN, SUNNY ZAHEED; STROM, STEPHEN C.; KIRBY, MARTHA R.; BURNS, SEAN; LANGEMEIJER, SASKIA; UEDA, TAKAHIRO; HSIEH, MATTHEW; TISDALE, JOHN F.

    2009-01-01

    We sought to determine whether hepatic side population (SP) cells derived from adult human liver possess the potential of a novel candidate hepatic stem cell. Human cadaveric donor liver was subjected to collagenase perfusion and hepatocytes were separated from nonparenchymal cells by differential centrifugation. SP cells were isolated from the nonparenchymal portion after Hoechst 33342 staining. Since CD45 is a panleukocyte antigen, CD45-negative SP cells were separated from the vast majority of CD45-positive SP cells (90%), and hepatic growth medium was used to culture both groups. Both CD45-negative and CD45-positive hepatic SP cells generated colonies in the hepatic growth medium in 2–3 weeks. The colonies yielded large cells morphologically consistent with human hepatocytes, demonstrating granule-rich cytoplasm, dense, often double nuclei, and intracellular lipofuscin pigment. The cultured cells from both sources were positive for markers of human hepatocytes: HepPar, cytokeratin 8 (CK8), and human albumin. Reverse transcriptase–polymerase chain reaction (RT-PCR) performed on both groups demonstrated positivity for additional liver markers including human albumin, CK18, α-1 anti-trypsin, and the human cytochrome P450 enzyme CYP2B6. Double immunostaining (CD45 and HepPar) and RT-PCR confirmed that the hepatocyte-like cells derived from the CD45-negative SP cells acquired HepPar positivity but had no detectable CD45 antigen expression. In contrast, the cultured cells derived from the CD45-positive SP cells also acquired HepPar positivity, but only a minimal fraction expressed the CD45 antigen. We conclude that hepatic SP cells derived from the nonparenchymal portion of human liver are a potential source of human hepatocytes irrespective of their CD45 status, and further animal studies will be required to assess their regenerative potential. PMID:16187169

  14. A Stem Cell-Derived Platform for Studying Single Synaptic Vesicles in Dopaminergic Synapses

    OpenAIRE

    Gu, Haigang; Lazarenko, Roman M.; Koktysh, Dmitry; Iacovitti, Lorraine; Zhang, Qi

    2015-01-01

    For the use of stem cell-derived neurons in clinical applications, improved differentiation efficiency and more careful characterization of resultant cells are needed. The authors refined a procedure for differentiation of mouse embryonic stem cells into functional dopamine neurons. This preparation provides a high yield of dopaminergic cells that are morphologically and functionally similar to cultured midbrain dopamine neurons and can be used as a platform for thorough investigation of the ...

  15. Functional neuromuscular junctions formed by embryonic stem cell-derived motor neurons.

    Directory of Open Access Journals (Sweden)

    Joy A Umbach

    Full Text Available A key objective of stem cell biology is to create physiologically relevant cells suitable for modeling disease pathologies in vitro. Much progress towards this goal has been made in the area of motor neuron (MN disease through the development of methods to direct spinal MN formation from both embryonic and induced pluripotent stem cells. Previous studies have characterized these neurons with respect to their molecular and intrinsic functional properties. However, the synaptic activity of stem cell-derived MNs remains less well defined. In this study, we report the development of low-density co-culture conditions that encourage the formation of active neuromuscular synapses between stem cell-derived MNs and muscle cells in vitro. Fluorescence microscopy reveals the expression of numerous synaptic proteins at these contacts, while dual patch clamp recording detects both spontaneous and multi-quantal evoked synaptic responses similar to those observed in vivo. Together, these findings demonstrate that stem cell-derived MNs innervate muscle cells in a functionally relevant manner. This dual recording approach further offers a sensitive and quantitative assay platform to probe disorders of synaptic dysfunction associated with MN disease.

  16. Human iPS cell-derived dopaminergic neurons function in a primate Parkinson's disease model.

    Science.gov (United States)

    Kikuchi, Tetsuhiro; Morizane, Asuka; Doi, Daisuke; Magotani, Hiroaki; Onoe, Hirotaka; Hayashi, Takuya; Mizuma, Hiroshi; Takara, Sayuki; Takahashi, Ryosuke; Inoue, Haruhisa; Morita, Satoshi; Yamamoto, Michio; Okita, Keisuke; Nakagawa, Masato; Parmar, Malin; Takahashi, Jun

    2017-08-30

    Induced pluripotent stem cells (iPS cells) are a promising source for a cell-based therapy to treat Parkinson's disease (PD), in which midbrain dopaminergic neurons progressively degenerate. However, long-term analysis of human iPS cell-derived dopaminergic neurons in primate PD models has never been performed to our knowledge. Here we show that human iPS cell-derived dopaminergic progenitor cells survived and functioned as midbrain dopaminergic neurons in a primate model of PD (Macaca fascicularis) treated with the neurotoxin MPTP. Score-based and video-recording analyses revealed an increase in spontaneous movement of the monkeys after transplantation. Histological studies showed that the mature dopaminergic neurons extended dense neurites into the host striatum; this effect was consistent regardless of whether the cells were derived from patients with PD or from healthy individuals. Cells sorted by the floor plate marker CORIN did not form any tumours in the brains for at least two years. Finally, magnetic resonance imaging and positron emission tomography were used to monitor the survival, expansion and function of the grafted cells as well as the immune response in the host brain. Thus, this preclinical study using a primate model indicates that human iPS cell-derived dopaminergic progenitors are clinically applicable for the treatment of patients with PD.

  17. Stem Cell-Derived Exosomes: A Potential Alternative Therapeutic Agent in Orthopaedics

    Directory of Open Access Journals (Sweden)

    John Burke

    2016-01-01

    Full Text Available Within the field of regenerative medicine, many have sought to use stem cells as a promising way to heal human tissue; however, in the past few years, exosomes (packaged vesicles released from cells have shown more exciting promise. Specifically, stem cell-derived exosomes have demonstrated great ability to provide therapeutical benefits. Exosomal products can include miRNA, other genetic products, proteins, and various factors. They are released from cells in a paracrine fashion in order to combat local cellular stress. Because of this, there are vast benefits that medicine can obtain from stem cell-derived exosomes. If exosomes could be extracted from stem cells in an efficient manner and packaged with particular regenerative products, then diseases such as rheumatoid arthritis, osteoarthritis, bone fractures, and other maladies could be treated with cell-free regenerative medicine via exosomes. Many advances must be made to get to this point, and the following review highlights the current advances of stem cell-derived exosomes with particular attention to regenerative medicine in orthopaedics.

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

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    Siebler Mario

    2009-08-01

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

  19. Gene Expression Changes Induced by Trypanosoma cruzi Shed Microvesicles in Mammalian Host Cells: Relevance of tRNA-Derived Halves

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    Maria R. Garcia-Silva

    2014-01-01

    Full Text Available At present, noncoding small RNAs are recognized as key players in novel forms of posttranscriptional gene regulation in most eukaryotes. However, canonical small RNA pathways seem to be lost or excessively simplified in some unicellular organisms including Trypanosoma cruzi which lack functional RNAi pathways. Recently, we reported the presence of alternate small RNA pathways in T. cruzi mainly represented by homogeneous populations of tRNA- and rRNA-derived small RNAs, which are secreted to the extracellular medium included in extracellular vesicles. Extracellular vesicle cargo could be delivered to other parasites and to mammalian susceptible cells promoting metacyclogenesis and conferring susceptibility to infection, respectively. Here we analyzed the changes in gene expression of host HeLa cells induced by extracellular vesicles from T. cruzi. As assessed by microarray assays a large set of genes in HeLa cells were differentially expressed upon incorporation of T. cruzi-derived extracellular vesicles. The elicited response modified mainly host cell cytoskeleton, extracellular matrix, and immune responses pathways. Some genes were also modified by the most abundant tRNA-derived small RNAs included in extracellular vesicles. These data suggest that microvesicles secreted by T. cruzi could be relevant players in early events of the T. cruzi host cell interplay.

  20. Cancerous epithelial cell lines shed extracellular vesicles with a bimodal size distribution that is sensitive to glutamine inhibition

    Science.gov (United States)

    Santana, Steven Michael; Antonyak, Marc A.; Cerione, Richard A.; Kirby, Brian J.

    2014-12-01

    Extracellular shed vesicles (ESVs) facilitate a unique mode of cell-cell communication wherein vesicle uptake can induce a change in the recipient cell's state. Despite the intensity of ESV research, currently reported data represent the bulk characterization of concentrated vesicle samples with little attention paid to heterogeneity. ESV populations likely represent diversity in mechanisms of formation, cargo and size. To better understand ESV subpopulations and the signaling cascades implicated in their formation, we characterize ESV size distributions to identify subpopulations in normal and cancerous epithelial cells. We have discovered that cancer cells exhibit bimodal ESV distributions, one small-diameter and another large-diameter population, suggesting that two mechanisms may govern ESV formation, an exosome population and a cancer-specific microvesicle population. Altered glutamine metabolism in cancer is thought to fuel cancer growth but may also support metastatic niche formation through microvesicle production. We describe the role of a glutaminase inhibitor, compound 968, in ESV production. We have discovered that inhibiting glutamine metabolism significantly impairs large-diameter microvesicle production in cancer cells.

  1. Association of mast cell-derived VEGF and proteases in Dengue shock syndrome.

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    Takahisa Furuta

    Full Text Available BACKGROUND: Recent in-vitro studies have suggested that mast cells are involved in Dengue virus infection. To clarify the role of mast cells in the development of clinical Dengue fever, we compared the plasma levels of several mast cell-derived mediators (vascular endothelial cell growth factor [VEGF], soluble VEGF receptors [sVEGFRs], tryptase, and chymase and -related cytokines (IL-4, -9, and -17 between patients with differing severity of Dengue fever and healthy controls. METHODOLOGY/PRINCIPAL FINDINGS: The study was performed at Children's Hospital No. 2, Ho Chi Minh City, and Vinh Long Province Hospital, Vietnam from 2002 to 2005. Study patients included 103 with Dengue fever (DF, Dengue hemorrhagic fever (DHF, and Dengue shock syndrome (DSS, as diagnosed by the World Health Organization criteria. There were 189 healthy subjects, and 19 febrile illness patients of the same Kinh ethnicity. The levels of mast cell-derived mediators and -related cytokines in plasma were measured by ELISA. VEGF and sVEGFR-1 levels were significantly increased in DHF and DSS compared with those of DF and controls, whereas sVEGFR-2 levels were significantly decreased in DHF and DSS. Significant increases in tryptase and chymase levels, which were accompanied by high IL-9 and -17 concentrations, were detected in DHF and DSS patients. By day 4 of admission, VEGF, sVEGFRs, and proteases levels had returned to similar levels as DF and controls. In-vitro VEGF production by mast cells was examined in KU812 and HMC-1 cells, and was found to be highest when the cells were inoculated with Dengue virus and human Dengue virus-immune serum in the presence of IL-9. CONCLUSIONS: As mast cells are an important source of VEGF, tryptase, and chymase, our findings suggest that mast cell activation and mast cell-derived mediators participate in the development of DHF. The two proteases, particularly chymase, might serve as good predictive markers of Dengue disease severity.

  2. Reduced immunogenicity of induced pluripotent stem cells derived from Sertoli cells.

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    Xiaoying Wang

    Full Text Available Sertoli cells constitute the structural framework in testis and provide an immune-privileged environment for germ cells. Induced pluripotent stem cells (iPS cells resemble embryonic stem cells (ES cells and are generated from somatic cells by expression of specific reprogramming transcription factors. Here, we used C57BL/6 (B6 Sertoli cells to generate iPS cells (Ser-iPS cells and compared the immunogenicity of Ser-iPS cells with iPS cells derived from mouse embryonic fibroblast (MEF-iPS cells. Ser-iPS cells were injected into syngeneic mice to test for their in vivo immunogenicity in teratoma assay. Teratoma assay allows assessing in vivo immunogenicity of iPS cells and of their differentiated progeny simultaneously. We observed that early-passage Ser-iPS cells formed more teratomas with less immune cell infiltration and tissue damage and necrosis than MEF-iPS cells. Differentiating Ser-iPS cells in embryoid bodies (EBs showed reduced T cell activation potential compared to MEF-iPS cells, which was similar to syngeneic ES cells. However, Ser-iPS cells lost their reduced immunogenicity in vivo after extended passaging in vitro and late-passage Ser-iPS cells exhibited an immunogenicity similar to MEF-iPS cells. These findings indicate that early-passage Ser-iPS cells retain some somatic memory of Sertoli cells that impacts on immunogenicity of iPS cells and iPS cell-derived cells in vivo and in vitro. Our data suggest that immune-privileged Sertoli cells might represent a preferred source for iPS cell generation, if it comes to the use of iPS cell-derived cells for transplantation.

  3. Role of Lymphocyte Subsets in the Immune Response to Primary B Cell-Derived Exosomes.

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    Saunderson, Sarah C; McLellan, Alexander D

    2017-10-01

    Exosomes are lipid nanovesicles released after fusion of the endosomal limiting membrane with the plasma membrane. In this study, we investigated the requirement for CD4 T cells, B cells, and NK cells to provide help for CD8 T cell-mediated response to B cell-derived exosomes. CTL responses to Ag-loaded exosomes were dependent on host MHC class I, with a critical role for splenic langerin(+) CD8α(+) dendritic cells (DCs) in exosomal Ag cross-presentation. In addition, there was an absolute dependence on the presence of CD4 T cells, CD8 T cells, and NK cells, where the loss of any one of these subsets led to a complete loss of CTL response. Interestingly, NK cell depletion experiments demonstrated a critical cutoff point for depletion efficacy, with low-level residual NK cells providing sufficient help to allow optimal CD8 T cell proliferative responses to exosomal protein. Despite the potential role for B cells in the response to B cell-derived exosomal proteins, B cell depletion did not alter the exosome-induced CTL response. Similarly, a possible role for the BCR or circulating Ab in mediating CTL responses to B cell-derived exosomes was ruled out using DHLMP2A mice, which lack secreted and membrane-bound Ab, yet harbor marginal zone and follicular B cells. In contrast, CTL responses to DC-derived exosomes were significantly inhibited within Ab-deficient DHLMP2A mice compared with wild-type mice. However, this response was not restored upon serum transfer, implicating a role for the BCR, but not circulating Ab, in DC-derived exosome responses. Copyright © 2017 by The American Association of Immunologists, Inc.

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

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    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.

  5. Human embryos from induced pluripotent stem cell-derived gametes: ethical and quality considerations.

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    Ilic, Dusko; Ogilvie, Caroline; Noli, Laila; Kolundzic, Nikola; Khalaf, Yacoub

    2017-09-01

    Protocols for successful differentiation of male and female gametes from induced pluripotent stem cells have been published. Although culture of precursor cells in a natural microenvironment remains necessary to achieve terminal differentiation, the creation of human preimplantation embryos from induced pluripotent stem cell-derived gametes is technically feasible. Such embryos could provide a solution to the scarcity of human cleavage-stage embryos donated for research. Here, we discuss current technology, major research-related ethical concerns and propose the norms that would assure the quality and reliability of such embryos.

  6. Role of glial-cell-derived neurotrophic factor in salivary gland stem cell response to irradiation

    DEFF Research Database (Denmark)

    Peng, Xiaohong; Varendi, Kärt; Maimets, Martti

    2017-01-01

    Background and purpose Recently, stem cell therapy has been proposed to allow regeneration of radiation damaged salivary glands. It has been suggested that glial-cell-derived neurotrophic factor (GDNF) promotes survival of mice salivary gland stem cells (mSGSCs). The purpose of this study...... was to investigate the role of GDNF in the modulation of mSGSC response to irradiation and subsequent salivary gland regeneration. Methods Salivary gland sphere derived cells of Gdnf hypermorphic (Gdnfwt/hyper) and wild type mice (Gdnfwt/wt) were irradiated (IR) with γ-rays at 0, 1, 2, 4 and 8 Gy. mSGSC survival...

  7. Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

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    Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

    2013-12-01

    Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained

  8. A distribution analysis of action potential parameters obtained from patch-clamped human stem cell-derived cardiomyocytes

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    Fernando López-Redondo

    2016-06-01

    Full Text Available We investigated electrophysiological properties of human induced-pluripotent-stem-cell-derived and embryonic-stem-cell-derived cardiomyocytes, and analyzed action potential parameters by plotting their frequency distributions. In the both cell lines, the distribution analysis revealed that histograms of maximum upstroke velocity showed two subpopulations with similar intersection values. Sub-populations with faster maximum upstroke velocity showed significant prolongation of action potential durations by application of E-4031, whereas others did not, which may be partly due to shallower maximum diastolic potentials. We described electrophysiological and pharmacological properties of stem-cell-derived cardiomyocytes in the respective sub-populations, which provides a way to characterize diverse electrical properties of stem-cell-derived cardiomyocytes systematically.

  9. Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice.

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    Boylan, Brendan T; Moreira, Fernando R; Carlson, Tim W; Bernard, Kristen A

    2017-02-01

    Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and Zika viruses, infect humans by way of a bite from an infected mosquito. This infectious inoculum is insect cell-derived giving the virus particles distinct qualities not present in secondary infectious virus particles produced by infected vertebrate host cells. The insect cell-derived particles differ in the glycosylation of virus structural proteins and the lipid content of the envelope, as well as their induction of cytokines. Thus, in order to accurately mimic the inoculum delivered by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito cells and produced replicon particles at high concentration, allowing us to mimic mosquito cell-derived viral inoculum. These particles were mature with similar genome equivalents-to-infectious units as full-length West Nile virus. We then compared the mosquito cell-derived particles to mammalian cell-derived particles in mice. Both replicon particles infected skin at the inoculation site and the draining lymph node by 3 hours post-inoculation. The mammalian cell-derived replicon particles spread from the site of inoculation to the spleen and contralateral lymph nodes significantly more than the particles derived from mosquito cells. This in vivo difference in spread of West Nile replicons in the inoculum demonstrates the importance of using arthropod cell-derived particles to model early events in arboviral infection and highlights the value of these novel arthropod cell-derived replicon particles for studying the earliest virus-host interactions for arboviruses.

  10. Translating the microRNA signature of microvesicles derived from human coronary artery smooth muscle cells in patients with familial hypercholesterolemia and coronary artery disease.

    Science.gov (United States)

    de Gonzalo-Calvo, David; Cenarro, Ana; Garlaschelli, Katia; Pellegatta, Fabio; Vilades, David; Nasarre, Laura; Camino-Lopez, Sandra; Crespo, Javier; Carreras, Francesc; Leta, Rubén; Catapano, Alberico Luigi; Norata, Giuseppe Danilo; Civeira, Fernando; Llorente-Cortes, Vicenta

    2017-05-01

    To analyze the impact of atherogenic lipoproteins on the miRNA signature of microvesicles derived from human coronary artery smooth muscle cells (CASMC) and to translate these results to familial hypercholesterolemia (FH) and coronary artery disease (CAD) patients. Conditioned media was collected after exposure of CASMC to atherogenic lipoproteins. Plasma samples were collected from two independent populations of diagnosed FH patients and matched normocholesterolemic controls (Study population 1, N=50; Study population 2, N=24) and a population of patients with suspected CAD (Study population 3, N=50). Extracellular vesicles were isolated and characterized using standard techniques. A panel of 30 miRNAs related to vascular smooth muscle cell (VSMC) (patho-)physiology was analyzed using RT-qPCR. Atherogenic lipoproteins significantly reduced levels of miR-15b-5p, -24-3p, -29b-3p, -130a-3p, -143-3p, -146a-3p, -222-3p, -663a levels (P<0.050) in microvesicles (0.1μm-1μm in diameter) released by CASMC. Two of these miRNAs, miR-24-3p and miR-130a-3p, were reduced in circulating microvesicles from FH patients compared with normocholesterolemic controls in a pilot study (Study population 1) and in different validation studies (Study populations 1 and 2) (P<0.050). Supporting these results, plasma levels of miR-24-3p and miR-130a-3p were also downregulated in FH patients compared to controls (P<0.050). In addition, plasma levels of miR-130a-3p were inversely associated with coronary atherosclerosis in a cohort of suspected CAD patients (Study population 3) (P<0.050). Exposure to atherogenic lipoproteins modifies the miRNA profile of CASMC-derived microvesicles and these alterations are reflected in patients with FH. Circulating miR-130a-3p emerges as a potential biomarker for coronary atherosclerosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Exosomes derived from gastric cancer cells activate NF-κB pathway in macrophages to promote cancer progression.

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    Wu, Lijun; Zhang, Xu; Zhang, Bin; Shi, Hui; Yuan, Xiao; Sun, Yaoxiang; Pan, Zhaoji; Qian, Hui; Xu, Wenrong

    2016-09-01

    Exosomes are nano-sized membrane vesicles secreted by both normal and cancer cells. Emerging evidence indicates that cancer cells derived exosomes contribute to cancer progression through the modulation of tumor microenvironment. However, the effects of exosomes derived from gastric cancer cells on macrophages are not well understood. In this study, we investigated the biological role of gastric cancer cells derived exosomes in the activation of macrophages. We demonstrated that gastric cancer cells derived exosomes activated macrophages to express increased levels of proinflammatory factors, which in turn promoted tumor cell proliferation and migration. In addition, gastric cancer cells derived exosomes remarkably upregulated the phosphorylation of NF-κB in macrophages. Inhibiting the activation of NF-κB reversed the upregulation of proinflammatory factors in macrophages and blocked their promoting effects on gastric cancer cells. Moreover, we found that gastric cancer cells derived exosomes could also activate macrophages from human peripheral blood monocytes through the activation of NF-κB. In conclusion, our results suggest that gastric cancer cells derived exosomes stimulate the activation of NF-κB pathway in macrophages to promote cancer progression, which provides a potential therapeutic approach for gastric cancer by interfering with the interaction between exosomes and macrophages in tumor microenvironment.

  12. IL-33 stimulates the release of procoagulant microvesicles from human monocytes and differentially increases tissue factor in human monocyte subsets.

    Science.gov (United States)

    Stojkovic, Stefan; Thulin, Åsa; Hell, Lena; Thaler, Barbara; Rauscher, Sabine; Baumgartner, Johanna; Gröger, Marion; Ay, Cihan; Demyanets, Svitlana; Neumayer, Christoph; Huk, Ihor; Spittler, Andreas; Huber, Kurt; Wojta, Johann; Siegbahn, Agneta; Åberg, Mikael

    2017-06-28

    Monocytes and monocyte-derived microvesicles (MVs) are the main source of circulating tissue factor (TF). Increased monocyte TF expression and increased circulating levels of procoagulant MVs contribute to the formation of a prothrombotic state in patients with cardiovascular disease. Interleukin (IL)-33 is a pro-inflammatory cytokine involved in atherosclerosis and other inflammatory diseases, but its role in regulating thrombosis is still unclear. The aim of the present study was to investigate the effects of IL-33 on the procoagulant properties of human monocytes and monocyte-derived MVs. IL-33 induced a time- and concentration-dependent increase of monocyte TF mRNA and protein levels via binding to the ST2-receptor and activation of the NF-κB-pathway. The IL-33 treated monocytes also released CD14+TF+ MVs and IL-33 was found to increase the TF activity of both the isolated monocytes and monocyte-derived MVs. The monocytes were classified into subsets according to their CD14 and CD16 expression. Intermediate monocytes (IM) showed the highest ST2 receptor expression, followed by non-classical monocytes (NCM), and classical monocytes (CM). IL-33 induced a significant increase of TF only in the IM (p<0.01), with a tendency in NCM (p=0.06), but no increase was observed in CM. Finally, plasma levels of IL-33 were positively correlated with CD14+TF+ MVs in patients undergoing carotid endarterectomy (r=0.480; p=0.032; n=20). We hereby provide novel evidence that the proinflammatory cytokine IL-33 induces differential TF expression and activity in monocyte subsets, as well as the release of procoagulant MVs. In this manner, IL-33 may contribute to the formation of a prothrombotic state characteristic for cardiovascular disease.

  13. Procoagulant and platelet-derived microvesicle absolute counts determined by flow cytometry correlates with a measurement of their functional capacity

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    Lisa Ayers

    2014-09-01

    Full Text Available Background: Flow cytometry is the most commonly used technology to measure microvesicles (MVs. Despite reported limitations of this technique, MV levels obtained using conventional flow cytometry have yielded many clinically relevant findings, such as associations with disease severity and ability to predict clinical outcomes. This study aims to determine if MV enumeration by flow cytometry correlates with a measurement of their functional capacity, as this may explain how flow cytometry generates clinically relevant results. Methods: One hundred samples from healthy individuals and patients with obstructive sleep apnoea were analysed by conventional flow cytometry (FACSCalibur and by three functional MV assays: Zymuphen MP-activity in which data were given as phosphatidylserine equivalent, STA® Phospholipid Procoag Assay expressed as clotting time and Endogenous Thrombin Potential (ETP reflecting in vitro thrombin generation. Correlations were determined by Spearman correlation. Results: Absolute counts of lactadherin+ procoagulant MVs generated by flow cytometry weakly correlated with the results obtained from the Zymuphen MP-activity (r=0.5370, p<0.0001; correlated with ETP (r=0.7444, p<0.0001; negatively correlated with STA® Phospholipid Procoag Assay clotting time (−0.7872, p<0.0001, reflecting a positive correlation between clotting activity and flow cytometry. Levels of Annexin V+ procoagulant and platelet-derived MVs were also associated with functional assays. Absolute counts of MVs derived from other cell types were not correlated with the functional results. Conclusions: Quantitative results of procoagulant and platelet-derived MVs from conventional flow cytometry are associated with the functional capability of the MVs, as defined by three functional MV assays. Flow cytometry is a valuable technique for the quantification of MVs from different cellular origins; however, a combination of several analytical techniques may give the

  14. Procoagulant and platelet-derived microvesicle absolute counts determined by flow cytometry correlates with a measurement of their functional capacity.

    Science.gov (United States)

    Ayers, Lisa; Harrison, Paul; Kohler, Malcolm; Ferry, Berne

    2014-01-01

    Flow cytometry is the most commonly used technology to measure microvesicles (MVs). Despite reported limitations of this technique, MV levels obtained using conventional flow cytometry have yielded many clinically relevant findings, such as associations with disease severity and ability to predict clinical outcomes. This study aims to determine if MV enumeration by flow cytometry correlates with a measurement of their functional capacity, as this may explain how flow cytometry generates clinically relevant results. ONE HUNDRED SAMPLES FROM HEALTHY INDIVIDUALS AND PATIENTS WITH OBSTRUCTIVE SLEEP APNOEA WERE ANALYSED BY CONVENTIONAL FLOW CYTOMETRY (FACSCALIBUR) AND BY THREE FUNCTIONAL MV ASSAYS: Zymuphen MP-activity in which data were given as phosphatidylserine equivalent, STA(®) Phospholipid Procoag Assay expressed as clotting time and Endogenous Thrombin Potential (ETP) reflecting in vitro thrombin generation. Correlations were determined by Spearman correlation. Absolute counts of lactadherin+ procoagulant MVs generated by flow cytometry weakly correlated with the results obtained from the Zymuphen MP-activity (r=0.5370, pflow cytometry. Levels of Annexin V+ procoagulant and platelet-derived MVs were also associated with functional assays. Absolute counts of MVs derived from other cell types were not correlated with the functional results. Quantitative results of procoagulant and platelet-derived MVs from conventional flow cytometry are associated with the functional capability of the MVs, as defined by three functional MV assays. Flow cytometry is a valuable technique for the quantification of MVs from different cellular origins; however, a combination of several analytical techniques may give the most comprehensive information on the role of MVs in health and disease.

  15. Vesicle-MaNiA: extracellular vesicles in liquid biopsy and cancer

    OpenAIRE

    Torrano, Veronica; Royo, Felix; Peinado, Héctor; Loizaga-Iriarte, Ana; Unda, Miguel; Falcón-Perez, Juan M.; Carracedo, Arkaitz

    2016-01-01

    Normal and tumor cells shed vesicles to the environment. Within the large family of extracellular vesicles, exosomes and microvesicles have attracted much attention in the recent years. Their interest ranges from mediators of cancer progression, inflammation, immune regulation and metastatic niche regulation, to non-invasive biomarkers of disease. In this respect, the procedures to purify and analyze extracellular vesicles have quickly evolved and represent a source of variability for data in...

  16. Differential Uptake Mechanisms of Fluorescent Substrates into Stem-Cell-Derived Serotonergic Neurons.

    Science.gov (United States)

    Matthaeus, Friederike; Schloss, Patrick; Lau, Thorsten

    2015-12-16

    The actions of the neurotransmitters serotonin, dopamine, and norepinephrine are partly terminated by diffusion and in part by their uptake into neurons via the selective, high-affinity transporters for serotonin (SERT), dopamine (DAT), and norepinephrine (NET), respectively. There is also growing evidence that all three monoamines are taken up into neurons by low-affinity, high-capacity organic cation transporters (OCT) and the plasma membrane monoamine transporter (PMAT). Pharmacological characterization of these low-affinity recombinant transporter proteins in heterologous expression systems has revealed that they are not antagonized by classical inhibitors of SERT, DAT, or NET but that decynium-22 (D22) antagonizes OCT3 and PMAT, whereas corticosterone and progesterone selectively inhibit OCT3. Here, we show that SERT, PMAT, and OCT3, but not OCT1 and OCT2, are coexpressed in murine stem cell-derived serotonergic neurons. Using selective antagonists, we provide evidence that uptake of the fluorescent substrates FFN511, ASP+, and 5-HT into stem cell-derived serotonergic neurons is mediated differentially by these transporters and also involves an as yet unknown transport mechanism.

  17. The case for induced pluripotent stem cell-derived cardiomyocytes in pharmacological screening

    Science.gov (United States)

    Khan, Jaffar M; Lyon, Alexander R; Harding, Sian E

    2013-01-01

    The current drug screening models are deficient, particularly in detecting cardiac side effects. Human stem cell-derived cardiomyocytes could aid both early cardiotoxicity detection and novel drug discovery. Work over the last decade has generated human embryonic stem cells as potentially accurate sources of human cardiomyocytes, but ethical constraints and poor efficacy in establishing cell lines limit their use. Induced pluripotent stem cells do not require the use of human embryos and have the added advantage of producing patient-specific cardiomyocytes, allowing both generic and disease- and patient-specific pharmacological screening, as well as drug development through disease modelling. A critical question is whether sufficient standards have been achieved in the reliable and reproducible generation of ‘adult-like’ cardiomyocytes from human fibroblast tissue to progress from validation to safe use in practice and drug discovery. This review will highlight the need for a new experimental system, assess the validity of human induced pluripotent stem cell-derived cardiomyocytes and explore what the future may hold for their use in pharmacology. LINKED ARTICLES This article is part of a themed section on Regenerative Medicine and Pharmacology: A Look to the Future. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-2 PMID:22845396

  18. Bioengineering of functional human induced pluripotent stem cell-derived intestinal grafts.

    Science.gov (United States)

    Kitano, Kentaro; Schwartz, Dana M; Zhou, Haiyang; Gilpin, Sarah E; Wojtkiewicz, Gregory R; Ren, Xi; Sommer, Cesar A; Capilla, Amalia V; Mathisen, Douglas J; Goldstein, Allan M; Mostoslavsky, Gustavo; Ott, Harald C

    2017-10-10

    Patients with short bowel syndrome lack sufficient functional intestine to sustain themselves with enteral intake alone. Transplantable vascularized bioengineered intestine could restore nutrient absorption. Here we report the engineering of humanized intestinal grafts by repopulating decellularized rat intestinal matrix with human induced pluripotent stem cell-derived intestinal epithelium and human endothelium. After 28 days of in vitro culture, hiPSC-derived progenitor cells differentiate into a monolayer of polarized intestinal epithelium. Human endothelial cells seeded via native vasculature restore perfusability. Ex vivo isolated perfusion testing confirms transfer of glucose and medium-chain fatty acids from lumen to venous effluent. Four weeks after transplantation to RNU rats, grafts show survival and maturation of regenerated epithelium. Systemic venous sampling and positron emission tomography confirm uptake of glucose and fatty acids in vivo. Bioengineering intestine on vascularized native scaffolds could bridge the gap between cell/tissue-scale regeneration and whole organ-scale technology needed to treat intestinal failure patients.There is a need for humanised grafts to treat patients with intestinal failure. Here, the authors generate intestinal grafts by recellularizing native intestinal matrix with human induced pluripotent stem cell-derived epithelium and human endothelium, and show nutrient absorption after transplantation in rats.

  19. In-vitro stem cell derived red blood cells for transfusion: are we there yet?

    Science.gov (United States)

    Kim, Hyun Ok

    2014-03-01

    To date, the use of red blood cells (RBCs) produced from stem cells in vitro has not proved practical for routine transfusion. However, the perpetual and widespread shortage of blood products, problems related to transfusion-transmitted infections, and new emerging pathogens elicit an increasing demand for artificial blood. Worldwide efforts to achieve the goal of RBC production through stem cell research have received vast attention; however, problems with large-scale production and cost effectiveness have yet to prove practical usefulness. Some progress has been made, though, as cord blood stem cells and embryonic stem cells have shown an ability to differentiate and proliferate, and induced pluripotent stem cells have been shown to be an unlimited source for RBC production. However, transfusion of stem cell-derived RBCs still presents a number of challenges to overcome. This paper will summarize an up to date account of research and advances in stem cell-derived RBCs, delineate our laboratory protocol in producing RBCs from cord blood, and introduce the technological developments and limitations to current RBC production practices.

  20. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Ono, Hiromasa [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan); Database Center for Life Science (DBCLS), Research Organization of Information and Systems (ROIS), Faculty of Engineering Bldg.12 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Oki, Yoshinao [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan); Bono, Hidemasa [Database Center for Life Science (DBCLS), Research Organization of Information and Systems (ROIS), Faculty of Engineering Bldg.12 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Kano, Koichiro, E-mail: kkano@brs.nihon-u.ac.jp [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan)

    2011-04-15

    Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

  1. Stem cell-derived exosomes: A promising strategy for fracture healing.

    Science.gov (United States)

    Hao, Zi-Chen; Lu, Jun; Wang, Shan-Zheng; Wu, Hao; Zhang, Yun-Tong; Xu, Shuo-Gui

    2017-10-01

    To describe the biological characteristics of exosomes and to summarize the current status of stem cell-derived exosomes on fracture healing. Meanwhile, future challenges, limitations and perspectives are also discussed. Search and analyze the related articles in pubmed database through the multi-combination of keywords like "stem cells","exosomes","bone regeneration" and "fracture healing". Stem cell-derived exosome therapy for fracture healing has been enjoying popularity and is drawing increasing attention. This strategy helps to promote proliferation and migration of cells, as well as osteogenesis and angiogenesis, in the process of bone formation. Although the exact mechanisms remain elusive, exosomal miRNAs seem to play vital roles. Future studies are required to solve multiple problems before clinical application, including comprehensive and thorough understanding of exosomes, the exact roles of exosomes in regulating bone formation, and the optimal source, dose and frequency of treatment, as well as technical and safety issues. Moreover, studies based on fracture models of large animals are could offer guidance and are in demand. © 2017 John Wiley & Sons Ltd.

  2. Mesenchymal stromal cell derived endothelial progenitor treatment in patients with refractory angina

    DEFF Research Database (Denmark)

    Friis, Tina; Haack-Sørensen, Mandana; Mathiasen, Anders B

    2011-01-01

    Abstract Aims. We evaluated the feasibility, safety and efficacy of intra-myocardial injection of autologous mesenchymal stromal cells derived endothelial progenitor cell (MSC) in patients with stable coronary artery disease (CAD) and refractory angina in this first in man trial. Methods and resu......Abstract Aims. We evaluated the feasibility, safety and efficacy of intra-myocardial injection of autologous mesenchymal stromal cells derived endothelial progenitor cell (MSC) in patients with stable coronary artery disease (CAD) and refractory angina in this first in man trial. Methods...... and results. A total of 31 patients with stable CAD, moderate to severe angina and no further revascularization options, were included. Bone marrow MSC were isolated and culture expanded for 6-8 weeks. It was feasible and safe to establish in-hospital culture expansion of autologous MSC and perform intra......-myocardial injection of MSC. After six months follow-up myocardial perfusion was unaltered, but the patients increased exercise capacity (p Questionnaire (SAQ) evaluations (p

  3. Functional Properties of Human Stem Cell-Derived Neurons in Health and Disease

    Directory of Open Access Journals (Sweden)

    Jason P. Weick

    2016-01-01

    Full Text Available Stem cell-derived neurons from various source materials present unique model systems to examine the fundamental properties of central nervous system (CNS development as well as the molecular underpinnings of disease phenotypes. In order to more accurately assess potential therapies for neurological disorders, multiple strategies have been employed in recent years to produce neuronal populations that accurately represent in vivo regional and transmitter phenotypes. These include new technologies such as direct conversion of somatic cell types into neurons and glia which may accelerate maturation and retain genetic hallmarks of aging. In addition, novel forms of genetic manipulations have brought human stem cells nearly on par with those of rodent with respect to gene targeting. For neurons of the CNS, the ultimate phenotypic characterization lies with their ability to recapitulate functional properties such as passive and active membrane characteristics, synaptic activity, and plasticity. These features critically depend on the coordinated expression and localization of hundreds of ion channels and receptors, as well as scaffolding and signaling molecules. In this review I will highlight the current state of knowledge regarding functional properties of human stem cell-derived neurons, with a primary focus on pluripotent stem cells. While significant advances have been made, critical hurdles must be overcome in order for this technology to support progression toward clinical applications.

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

    Directory of Open Access Journals (Sweden)

    Gang Chang

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

  5. Taxol(®)-induced phosphatidylserine exposure and microvesicle formation in red blood cells is mediated by its vehicle Cremophor(®) EL.

    Science.gov (United States)

    Vader, Pieter; Fens, Marcel Ham; Sachini, Nikoleta; van Oirschot, Brigitte A; Andringa, Grietje; Egberts, Antoine Cg; Gaillard, Carlo Ajm; Rasmussen, Jan T; van Wijk, Richard; van Solinge, Wouter W; Schiffelers, Raymond M

    2013-07-01

    The conventional clinical formulation of paclitaxel (PTX), Taxol®, consists of Cremophor® EL (CrEL) and ethanol. CrEL-formulated PTX is associated with acute hypersensitivity reactions, anemia and cardiovascular events. In this study, the authors investigated the effects of CrEL-PTX on red blood cells (RBCs) and compared these with the effects observed after exposure to the novel nanoparticle albumin-bound PTX, marketed as Abraxane®. The authors demonstrate that CrEL is primarily responsible for RBC lysis and induction of phosphatidylserine exposure. Phosphatidylserine-exposing RBCs showed increased association with endothelial cells in culture. The authors also identified CrEL as being responsible for vesiculation of RBCs. This is the first time that excipients have been shown to be involved in microvesicle formation. Microvesicles were taken up by endothelial cells. These results offer new insights into the side effect profile of Taxol, which is likely to have implications for patients with erythrocyte disorders. Abraxane did not induce any of these effects on RBCs, indicating that the choice of excipients can have a pronounced influence on the efficacy and side effects of drug molecules.

  6. Co-cultures with stem cell-derived human sensory neurons reveal regulators of peripheral myelination.

    Science.gov (United States)

    Clark, Alex J; Kaller, Malte S; Galino, Jorge; Willison, Hugh J; Rinaldi, Simon; Bennett, David L H

    2017-04-01

    See Saporta and Shy (doi:10.1093/awx048) for a scientific commentary on this article.Effective bidirectional signalling between axons and Schwann cells is essential for both the development and maintenance of peripheral nerve function. We have established conditions by which human induced pluripotent stem cell-derived sensory neurons can be cultured with rat Schwann cells, and have produced for the first time long-term and stable myelinating co-cultures with human neurons. These cultures contain the specialized domains formed by axonal interaction with myelinating Schwann cells, such as clustered voltage-gated sodium channels at the node of Ranvier and Shaker-type potassium channel (Kv1.2) at the juxtaparanode. Expression of type III neuregulin-1 (TIIINRG1) in induced pluripotent stem cell-derived sensory neurons strongly enhances myelination, while conversely pharmacological blockade of the NRG1-ErbB pathway prevents myelination, providing direct evidence for the ability of this pathway to promote the myelination of human sensory axons. The β-secretase, BACE1 is a protease needed to generate active NRG1 from the full-length form. Due to the fact that it also cleaves amyloid precursor protein, BACE1 is a therapeutic target in Alzheimer's disease, however, consistent with its role in NRG1 processing we find that BACE1 inhibition significantly impairs myelination in our co-culture system. In order to exploit co-cultures to address other clinically relevant problems, they were exposed to anti-disialosyl ganglioside antibodies, including those derived from a patient with a sensory predominant, inflammatory neuropathy with mixed axonal and demyelinating electrophysiology. The co-cultures reveal that both mouse and human disialosyl antibodies target the nodal axolemma, induce acute axonal degeneration in the presence of complement, and impair myelination. The human, neuropathy-associated IgM antibody is also shown to induce complement-independent demyelination

  7. clickECM: Development of a cell-derived extracellular matrix with azide functionalities.

    Science.gov (United States)

    Ruff, S M; Keller, S; Wieland, D E; Wittmann, V; Tovar, G E M; Bach, M; Kluger, P J

    2017-04-01

    In vitro cultured cells produce a complex extracellular matrix (ECM) that remains intact after decellularization. The biological complexity derived from the variety of distinct ECM molecules makes these matrices ideal candidates for biomaterials. Biomaterials with the ability to guide cell function are a topic of high interest in biomaterial development. However, these matrices lack specific addressable functional groups, which are often required for their use as a biomaterial. Due to the biological complexity of the cell-derived ECM, it is a challenge to incorporate such functional groups without affecting the integrity of the biomolecules within the ECM. The azide-alkyne cycloaddition (click reaction, Huisgen-reaction) is an efficient and specific ligation reaction that is known to be biocompatible when strained alkynes are used to avoid the use of copper (I) as a catalyst. In our work, the ubiquitous modification of a fibroblast cell-derived ECM with azides was achieved through metabolic oligosaccharide engineering by adding the azide-modified monosaccharide Ac4GalNAz (1,3,4,6-tetra-O-acetyl-N-azidoacetylgalactosamine) to the cell culture medium. The resulting azide-modified network remained intact after removing the cells by lysis and the molecular structure of the ECM proteins was unimpaired after a gentle homogenization process. The biological composition was characterized in order to show that the functionalization does not impair the complexity and integrity of the ECM. The azides within this "clickECM" could be accessed by small molecules (such as an alkyne-modified fluorophore) or by surface-bound cyclooctynes to achieve a covalent coating with clickECM. The clickECM was produced by the incorporation of azide-functionalized sugar analogues into the extracellular glycans of fibroblast cell cultures by metabolic oligosaccharide engineering. By introducing these azide groups into the glycan structures, we enabled this cell-derived ECM for bioorthogonal click

  8. Cancer

    Science.gov (United States)

    ... Prostate cancer Lung cancer Colorectal cancer In US women, other than skin cancer the three most common cancers are: Breast cancer Lung cancer Colorectal cancer Some cancers are more common in certain parts of the world. For example, in Japan, there are many cases of stomach cancer . But ...

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

    Science.gov (United States)

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

    2003-11-01

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

  10. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Afford New Opportunities in Inherited Cardiovascular Disease Modeling

    Directory of Open Access Journals (Sweden)

    Daniel R. Bayzigitov

    2016-01-01

    Full Text Available Fundamental studies of molecular and cellular mechanisms of cardiovascular disease pathogenesis are required to create more effective and safer methods of their therapy. The studies can be carried out only when model systems that fully recapitulate pathological phenotype seen in patients are used. Application of laboratory animals for cardiovascular disease modeling is limited because of physiological differences with humans. Since discovery of induced pluripotency generating induced pluripotent stem cells has become a breakthrough technology in human disease modeling. In this review, we discuss a progress that has been made in modeling inherited arrhythmias and cardiomyopathies, studying molecular mechanisms of the diseases, and searching for and testing drug compounds using patient-specific induced pluripotent stem cell-derived cardiomyocytes.

  11. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Afford New Opportunities in Inherited Cardiovascular Disease Modeling

    Science.gov (United States)

    Bayzigitov, Daniel R.; Medvedev, Sergey P.; Dementyeva, Elena V.; Bayramova, Sevda A.; Pokushalov, Evgeny A.; Karaskov, Alexander M.; Zakian, Suren M.

    2016-01-01

    Fundamental studies of molecular and cellular mechanisms of cardiovascular disease pathogenesis are required to create more effective and safer methods of their therapy. The studies can be carried out only when model systems that fully recapitulate pathological phenotype seen in patients are used. Application of laboratory animals for cardiovascular disease modeling is limited because of physiological differences with humans. Since discovery of induced pluripotency generating induced pluripotent stem cells has become a breakthrough technology in human disease modeling. In this review, we discuss a progress that has been made in modeling inherited arrhythmias and cardiomyopathies, studying molecular mechanisms of the diseases, and searching for and testing drug compounds using patient-specific induced pluripotent stem cell-derived cardiomyocytes. PMID:27110425

  12. Retinoid Processing in Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Cultures.

    Science.gov (United States)

    Fields, Mark A; Bowrey, Hannah E; Gong, Jie; Ablonczy, Zsolt; Del Priore, Lucian V

    2015-01-01

    Stem cell therapy for retinal degenerative diseases such as age-related macular degeneration is a promising clinical option for the replacement of photoreceptors and retinal pigment epithelium (RPE). Induced pluripotent stem cell technology has emerged as a viable potential source of cells for transplantation in retinal degenerative disorders. Induced pluripotent stem cells have been used to derive RPE and have been tested for their functional behavior. These cells have the ability to express RPE-specific proteins and morphologically resemble native RPE. Induced pluripotent stem cell-derived RPE are also able to contribute to the visual cycle by their ability to metabolize all-trans retinol, a critical function of RPE in maintaining visual function. Advances in induced pluripotent stem cell technology will contribute to the development of clinical therapies for retinal degenerative diseases as well as provide a tool to understand the pathology of these disorders. © 2015 Elsevier Inc. All rights reserved.

  13. Low immunogenicity of mouse induced pluripotent stem cell-derived neural stem/progenitor cells.

    Science.gov (United States)

    Itakura, Go; Ozaki, Masahiro; Nagoshi, Narihito; Kawabata, Soya; Nishiyama, Yuichiro; Sugai, Keiko; Iida, Tsuyoshi; Kashiwagi, Rei; Ookubo, Toshiki; Yastake, Kaori; Matsubayashi, Kohei; Kohyama, Jun; Iwanami, Akio; Matsumoto, Morio; Nakamura, Masaya; Okano, Hideyuki

    2017-10-11

    Resolving the immunogenicity of cells derived from induced pluripotent stem cells (iPSCs) remains an important challenge for cell transplant strategies that use banked allogeneic cells. Thus, we evaluated the immunogenicity of mouse fetal neural stem/progenitor cells (fetus-NSPCs) and iPSC-derived neural stem/progenitor cells (iPSC-NSPCs) both in vitro and in vivo. Flow cytometry revealed the low expression of immunological surface antigens, and these cells survived in all mice when transplanted syngeneically into subcutaneous tissue and the spinal cord. In contrast, an allogeneic transplantation into subcutaneous tissue was rejected in all mice, and allogeneic cells transplanted into intact and injured spinal cords survived for 3 months in approximately 20% of mice. In addition, cell survival was increased after co-treatment with an immunosuppressive agent. Thus, the immunogenicity and post-transplantation immunological dynamics of iPSC-NSPCs resemble those of fetus-NSPCs.

  14. Electrophysiological properties and calcium handling of embryonic stem cell-derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Jae Boum Youm

    2016-03-01

    Full Text Available Embryonic stem cell-derived cardiomyocytes (ESC-CMs hold great interest in many fields of research including clinical applications such as stem cell and gene therapy for cardiac repair or regeneration. ESC-CMs are also used as a platform tool for pharmacological tests or for investigations of cardiac remodeling. ESC-CMs have many different aspects of morphology, electrophysiology, calcium handling, and bioenergetics compared with adult cardiomyocytes. They are immature in morphology, similar to sinus nodal-like in the electrophysiology, higher contribution of trans-sarcolemmal Ca2+ influx to Ca2+ handling, and higher dependence on anaerobic glycolysis. Here, I review a detailed electrophysiology and Ca2+ handling features of ESC-CMs during differentiation into adult cardiomyocytes to gain insights into how all the developmental changes are related to each other to display cardinal features of developing cardiomyocytes.

  15. NF-kappaB regulates B-cell-derived nerve growth factor expression.

    Science.gov (United States)

    Heese, Klaus; Inoue, Noriko; Sawada, Tohru

    2006-02-01

    In the mammalian brain, four neurotrophins have been identified: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). NGF exerts an important role in the development and functions of the central and peripheral nervous system. However, it has recently been documented that several types of immune cells, such as mast cells, lymphocytes, basophils and eosinophils, produce, store and release NGF. Accumulating preclinical and clinical data indicate that dysfunctions of NGF and the other neurotrophins may contribute to impaired immune responses and concentration of NGF frequently correlates with disease severity. Thus, the aim of this study was to elucidate the potential signaling mechanisms of cytokine- neurotrophins interactions contributing to increased NGF levels. Our data show that the transcription factor NF-kappaB plays a pivotal role in regulating B-cell-derived NGF expression.

  16. Maturation of induced pluripotent stem cell derived hepatocytes by 3D-culture.

    Directory of Open Access Journals (Sweden)

    Richard L Gieseck

    Full Text Available Induced pluripotent stem cell derived hepatocytes (IPSC-Heps have the potential to reduce the demand for a dwindling number of primary cells used in applications ranging from therapeutic cell infusions to in vitro toxicology studies. However, current differentiation protocols and culture methods produce cells with reduced functionality and fetal-like properties compared to adult hepatocytes. We report a culture method for the maturation of IPSC-Heps using 3-Dimensional (3D collagen matrices compatible with high throughput screening. This culture method significantly increases functional maturation of IPSC-Heps towards an adult phenotype when compared to conventional 2D systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves functional longevity to over 75 days. Overall, this research reveals a method to shift the phenotype of existing IPSC-Heps towards primary adult hepatocytes allowing such cells to be a more relevant replacement for the current primary standard.

  17. Maturation of induced pluripotent stem cell derived hepatocytes by 3D-culture.

    Science.gov (United States)

    Gieseck, Richard L; Hannan, Nicholas R F; Bort, Roque; Hanley, Neil A; Drake, Rosemary A L; Cameron, Grant W W; Wynn, Thomas A; Vallier, Ludovic

    2014-01-01

    Induced pluripotent stem cell derived hepatocytes (IPSC-Heps) have the potential to reduce the demand for a dwindling number of primary cells used in applications ranging from therapeutic cell infusions to in vitro toxicology studies. However, current differentiation protocols and culture methods produce cells with reduced functionality and fetal-like properties compared to adult hepatocytes. We report a culture method for the maturation of IPSC-Heps using 3-Dimensional (3D) collagen matrices compatible with high throughput screening. This culture method significantly increases functional maturation of IPSC-Heps towards an adult phenotype when compared to conventional 2D systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves functional longevity to over 75 days. Overall, this research reveals a method to shift the phenotype of existing IPSC-Heps towards primary adult hepatocytes allowing such cells to be a more relevant replacement for the current primary standard.

  18. Mitigating Ischemic Injury of Stem Cell-Derived Insulin-Producing Cells after Transplant

    Directory of Open Access Journals (Sweden)

    Gaetano Faleo

    2017-09-01

    Full Text Available The advent of large-scale in vitro differentiation of human stem cell-derived insulin-producing cells (SCIPC has brought us closer to treating diabetes using stem cell technology. However, decades of experiences from islet transplantation show that ischemia-induced islet cell death after transplant severely limits the efficacy of the therapy. It is unclear to what extent human SCIPC are susceptible to ischemia. In this study, we show that more than half of SCIPC die shortly after transplantation. Nutrient deprivation and hypoxia acted synergistically to kill SCIPC in vitro. Amino acid supplementation rescued SCIPC from nutrient deprivation, likely by providing cellular energy. Generating SCIPC under physiological oxygen tension of 5% conferred hypoxia resistance without affecting their differentiation or function. A two-pronged strategy of physiological oxygen acclimatization during differentiation and amino acid supplementation during transplantation significantly improved SCIPC survival after transplant.

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    Tokunori Ikeda

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

  1. Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.

    Directory of Open Access Journals (Sweden)

    Nicolas Christoforou

    Full Text Available The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS cells, which once differentiated allow for the enrichment of Nkx2-5(+ cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+ cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological

  2. Passaged neural stem cell-derived neuronal networks for a portable biosensor.

    Science.gov (United States)

    O'Shaughnessy, Thomas J; Liu, Jinny L; Ma, Wu

    2009-04-15

    We have previously demonstrated a portable biosensor that utilizes networks of mammalian neurons on microelectrode arrays (MEAs) as the sensing element. These neuronal cultures on MEAs are derived from primary neuronal tissues and are short-lived. In order to extend the shelf life of neuronal networks for use in a fieldable sensor technology, a renewable source of networks is needed. Neural stem and progenitor cells are capable of self-renewal and differentiation into functional neuronal networks. The purpose of this study was to develop a strategy for growing passaged neural stem and progenitor cells on MEAs under controlled conditions to produce differentiated neurons and glia comprising functional neuronal networks. Primary and passaged neuroepithelial stem and progenitor cells dissociated from embryonic day 13 rat cortex were seeded on MEAs and maintained with serum-free medium containing basic fibroblast growth factor (bFGF) combined with brain-derived neurotrophic factor (BDNF). These culture conditions lead to abundant neurons, with astrocytes as supportive cells, forming synaptically linked networks of neurons. Spontaneous action potentials were best recorded from networks derived from primary or passaged progenitor cells 4-5 weeks after initial culture. The passaged progenitor cell-derived networks on MEAs responded to the GABA(A) antagonist bicuculline, the NMDA glutamate inhibitor APV, and the non-NMDA glutamate antagonist CNQX indicating active synapses were present. Passaged neural stem and progenitor cell-derived networks on MEAs have properties similar to networks derived from primary neuronal cultures and can serve as a renewable supply of sensor elements for detection of environmental threats.

  3. Rett syndrome induced pluripotent stem cell-derived neurons reveal novel neurophysiological alterations.

    Science.gov (United States)

    Farra, N; Zhang, W-B; Pasceri, P; Eubanks, J H; Salter, M W; Ellis, J

    2012-12-01

    Rett syndrome (RTT) is a neurodevelopmental autism spectrum disorder caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Here, we describe the first characterization and neuronal differentiation of induced pluripotent stem (iPS) cells derived from Mecp2-deficient mice. Fully reprogrammed wild-type (WT) and heterozygous female iPS cells express endogenous pluripotency markers, reactivate the X-chromosome and differentiate into the three germ layers. We directed iPS cells to produce glutamatergic neurons, which generated action potentials and formed functional excitatory synapses. iPS cell-derived neurons from heterozygous Mecp2(308) mice showed defects in the generation of evoked action potentials and glutamatergic synaptic transmission, as previously reported in brain slices. Further, we examined electrophysiology features not yet studied with the RTT iPS cell system and discovered that MeCP2-deficient neurons fired fewer action potentials, and displayed decreased action potential amplitude, diminished peak inward currents and higher input resistance relative to WT iPS-derived neurons. Deficiencies in action potential firing and inward currents suggest that disturbed Na(+) channel function may contribute to the dysfunctional RTT neuronal network. These phenotypes were additionally confirmed in neurons derived from independent WT and hemizygous mutant iPS cell lines, indicating that these reproducible deficits are attributable to MeCP2 deficiency. Taken together, these results demonstrate that neuronally differentiated MeCP2-deficient iPS cells recapitulate deficits observed previously in primary neurons, and these identified phenotypes further illustrate the requirement of MeCP2 in neuronal development and/or in the maintenance of normal function. By validating the use of iPS cells to delineate mechanisms underlying RTT pathogenesis, we identify deficiencies that can be targeted for in vitro translational screens.

  4. Mesenchymal stem cell derived hematopoietic cells are permissive to HIV-1 infection

    Directory of Open Access Journals (Sweden)

    Mondal Debasis

    2011-01-01

    Full Text Available Abstract Background Tissue resident mesenchymal stem cells (MSCs are multipotent, self-renewing cells known for their differentiation potential into cells of mesenchymal lineage. The ability of single cell clones isolated from adipose tissue resident MSCs (ASCs to differentiate into cells of hematopoietic lineage has been previously demonstrated. In the present study, we investigated if the hematopoietic differentiated (HD cells derived from ASCs could productively be infected with HIV-1. Results HD cells were generated by differentiating clonally expanded cultures of adherent subsets of ASCs (CD90+, CD105+, CD45-, and CD34-. Transcriptome analysis revealed that HD cells acquire a number of elements that increase their susceptibility for HIV-1 infection, including HIV-1 receptor/co-receptor and other key cellular cofactors. HIV-1 infected HD cells (HD-HIV showed elevated p24 protein and gag and tat gene expression, implying a high and productive infection. HD-HIV cells showed decreased CD4, but significant increase in the expression of CCR5, CXCR4, Nef-associated factor HCK, and Vpu-associated factor BTRC. HIV-1 restricting factors like APOBEC3F and TRIM5 also showed up regulation. HIV-1 infection increased apoptosis and cell cycle regulatory genes in HD cells. Although undifferentiated ASCs failed to show productive infection, HIV-1 exposure increased the expression of several hematopoietic lineage associated genes such as c-Kit, MMD2, and IL-10. Conclusions Considering the presence of profuse amounts of ASCs in different tissues, these findings suggest the possible role that could be played by HD cells derived from ASCs in HIV-1 infection. The undifferentiated ASCs were non-permissive to HIV-1 infection; however, HIV-1 exposure increased the expression of some hematopoietic lineage related genes. The findings relate the importance of ASCs in HIV-1 research and facilitate the understanding of the disease process and management strategies.

  5. A Stem Cell-Derived Platform for Studying Single Synaptic Vesicles in Dopaminergic Synapses.

    Science.gov (United States)

    Gu, Haigang; Lazarenko, Roman M; Koktysh, Dmitry; Iacovitti, Lorraine; Zhang, Qi

    2015-08-01

    The exocytotic release of dopamine is one of the most characteristic but also one of the least appreciated processes in dopaminergic neurotransmission. Fluorescence imaging has yielded rich information about the properties of synaptic vesicles and the release of neurotransmitters in excitatory and inhibitory neurons. In contrast, imaging-based studies for in-depth understanding of synaptic vesicle behavior in dopamine neurons are lagging largely because of a lack of suitable preparations. Midbrain culture has been one of the most valuable preparations for the subcellular investigation of dopaminergic transmission; however, the paucity and fragility of cultured dopaminergic neurons limits their use for live cell imaging. Recent developments in stem cell technology have led to the successful production of dopamine neurons from embryonic or induced pluripotent stem cells. Although the dopaminergic identity of these stem cell-derived neurons has been characterized in different ways, vesicle-mediated dopamine release from their axonal terminals has been barely assessed. We report a more efficient procedure to reliably generate dopamine neurons from embryonic stem cells, and it yields more dopamine neurons with more dopaminergic axon projections than midbrain culture does. Using a collection of functional measurements, we show that stem cell-derived dopamine neurons are indistinguishable from those in midbrain culture. Taking advantage of this new preparation, we simultaneously tracked the turnover of hundreds of synaptic vesicles individually using pH-sensitive quantum dots. By doing so, we revealed distinct fusion kinetics of the dopamine-secreting vesicles, which is consistent within both preparations. ©AlphaMed Press.

  6. Different Angiogenic Potentials of Mesenchymal Stem Cells Derived from Umbilical Artery, Umbilical Vein, and Wharton’s Jelly

    Directory of Open Access Journals (Sweden)

    Lu Xu

    2017-01-01

    Full Text Available Human mesenchymal stem cells derived from the umbilical cord (UC are a favorable source for allogeneic cell therapy. Here, we successfully isolated the stem cells derived from three different compartments of the human UC, including perivascular stem cells derived from umbilical arteries (UCA-PSCs, perivascular stem cells derived from umbilical vein (UCV-PSCs, and mesenchymal stem cells derived from Wharton’s jelly (WJ-MSCs. These cells had the similar phenotype and differentiation potential toward adipocytes, osteoblasts, and neuron-like cells. However, UCA-PSCs and UCV-PSCs had more CD146+ cells than WJ-MSCs (P<0.05. Tube formation assay in vitro showed the largest number of tube-like structures and branch points in UCA-PSCs among the three stem cells. Additionally, the total tube length in UCA-PSCs and UCV-PSCs was significantly longer than in WJ-MSCs (P<0.01. Microarray, qRT-PCR, and Western blot analysis showed that UCA-PSCs had the highest expression of the Notch ligand Jagged1 (JAG1, which is crucial for blood vessel maturation. Knockdown of Jagged1 significantly impaired the angiogenesis in UCA-PSCs. In summary, UCA-PSCs are promising cell populations for clinical use in ischemic diseases.

  7. Influence of erythropoietin on microvesicles derived from mesenchymal stem cells protecting renal function of chronic kidney disease.

    Science.gov (United States)

    Wang, Yan; Lu, Xingyan; He, Juan; Zhao, Weihong

    2015-05-22

    Mesenchymal stem cells (MSCs) play a central role in the remediation of cell and tissue damage. Erythropoietin (EPO) may enhance the beneficial influence of MSCs during recovery from tissue and organ injuries. Microvesicles (MVs) released from MSCs contribute to the restoration of kidney damage. We studied the influence of EPO on MVs derived from MSCs, and the protective effects of these factors in subjects with chronic kidney disease (CKD). The MVs derived from untreated MSCs (MSC-MVs) or from MSCs incubated in different concentrations of EPO (1, 10, 100, and 500 IU/ml EPO-MVs) were used to treat renal injury of unilateral ureteral obstruction (UUO) in vivo, and transforming growth factor-β1 (TGF-β1)-induced fibrosis in a human renal proximal tubular epithelial (HK2) cell line in vitro. Western blot and reverse transcription polymerase chain reaction (RT-PCR) analyses were used to evaluate the expression of epithelial and mesenchymal markers in the renal tissue and HK2 cells. Flow cytometry was used to assess apoptosis within the HK2 cells, and microRNA (miRNA) microarray assays were used to determine the expression profiles of miRNA in the MSC-MVs and EPO-MVs. Compared to MSC-MVs (untreated), there was a significant increase in the number of EPO-MVs derived from MSCs treated with 1-100 IU/ml EPO, and these EPO-MVs had a greater benefit in UUO mice on days 7 and 14. Moreover, the EPO-MVs had a better restorative effect following TGF-β1-induced fibrosis in HK2 cells at 24 h and 48 h. The flow cytometry results revealed that both types of MVs, especially EPO-MVs, play an important anti-apoptotic role in HK2 cells treated with TGF-β1. The miRNA profiles of the MVs revealed that EPO-MVs changed 212 miRNAs (fold-change ≥ 1.5), including miR-299, miR-499, miR-302, and miRNA-200, and that 70.28 % of these changes involved upregulation. The changed miRNA in EPO-MVs may have contributed to their enhanced protective effects following renal injury compared to MSC

  8. In vitro Incubation of Platelets with oxLDL Does Not Induce Microvesicle Release When Measured by Sensitive Flow Cytometry.

    Science.gov (United States)

    Nielsen, Tine Bo; Nielsen, Morten Hjuler; Handberg, Aase

    2015-01-01

    Microvesicles (MVs) are submicron vesicles with sizes of 0.1-1.0 μm in diameter, released from various cell types upon activation or apoptosis. Their involvement in a variety of diseases has been intensively investigated. In blood, platelets are potent MV secretors, and oxidized low-density lipoprotein (oxLDL), a platelet ligand, induces platelet activation and thus potentially MV secretion. This interaction occurs through binding of oxLDL with CD36, located on the platelet membrane. In this study, we investigated the effect of in vitro incubation of platelets with oxLDL on MV release. Furthermore, we compared the results obtained when separating MVs larger than 0.5 μm as a measure of results obtained from less sensitive conventional flow cytometers with MVs below the 0.5 μm limit. MV size distribution was analyzed in plasma from 11 healthy volunteers (four females and seven males). MVs were identified as Platelet-rich plasma (PRP) was incubated without and with oxLDL or LDL (as control) to investigate the impact on platelet activation, evident by release of MVs. Size-calibrated fluorescent beads were used to establish the MV gate, and separate small- and large-size vesicles. CD41(+) and CD41(+)CD36(+) MVs increased by six to eightfold in PRP, when left at room temperature, and the presence of cell-specific markers increased. Total MV count was unaffected. Incubations with oxLDL did not increase the MV release or affect the distribution of small- and large-size MVs. We found a large interindividual variation in the fraction of small- and large-size MVs of 73%. In conclusion, we propose that procoagulant activity and activation of platelets induced by interaction of platelet CD36 with oxLDL may not involve release of MVs. Furthermore, our results demonstrate great interindividual variability in size distribution of platelet-derived MVs and thereby stress the importance for generation of standardized protocols for MV quantification by flow cytometry.

  9. miR-200b-containing microvesicles attenuate experimental colitis associated intestinal fibrosis by inhibiting epithelial-mesenchymal transition.

    Science.gov (United States)

    Yang, Jia; Zhou, Cheng-Zhi; Zhu, Rui; Fan, Heng; Liu, Xing-Xing; Duan, Xue-Yun; Tang, Qing; Shou, Zhe-Xing; Zuo, Dong-Mei

    2017-12-01

    Epithelial-mesenchymal transition (EMT), characterized by the decrease of E-cadherin (E-Cad) and increase in vimentin and alpha-smooth muscle actin (α-SMA), was demonstrated to participate in inflammatory bowel disease-related fibrosis. miR-200b plays an anti-fibrosis role in inhibiting EMT by targeting ZEB1 and ZEB2. But the stability of exogenous miR-200b in blood limits its application. Microvesicles (MVs), which can transfer miRNAs among cells and prevent them from degradation, may provide an excellent transport system for the delivery of miR-200b in the treatment of fibrosis. Bone marrow mesenchymal stem cells (BMSCs) were transfected with lentivirus to overexpress miR-200b. The MVs packaged with miRNA-200b were harvested for the anti-fibrotic treatment using in vitro (transforming growth factor beta 1-mediated EMT in intestinal epithelial cells: IEC-6) and in vivo (TNBS-induced intestinal fibrosis in rats) models. The pathological morphology was observed, and the fibrosis related proteins, such as E-Cad, vimentin, α-SMA, ZEB1, and ZEB2, were detected. MiR-200b-MVs would significantly reverse the morphology in TGF-β1-treated IEC-6 cells and improve the TNBS-induced colon fibrosis histologically. The treatment of miR-200b-MVs increased miR-200b levels both in the IEC-6 cells and colon, resulting in a significant prevention EMT and alleviation of fibrosis. The expression of E-Cad was increased, and the expressions of vimentin and α-SMA were decreased. ZBE1 and ZEB2, the targets of miR-200b, were also decreased. miR-200b could be transferred from genetically modified BMSCs to the target cells or tissue by MVs. The mechanisms of miR-200b-MVs in inhibiting colonic fibrosis were related to suppressing the development of EMT by targeting ZEB1and ZEB2. © 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

  10. The effect of oxLDL on microvesicle release from platelets, measured by a sensitive flow cytometry method.

    Directory of Open Access Journals (Sweden)

    Tine Bo Nielsen

    2015-11-01

    Full Text Available Microvesicles (MVs are submicron vesicles with sizes of 0.1-1.0-µm in diameter, released from various cell types upon activation or apoptosis. Their involvement in a variety of diseases has been intensively investigated. In blood, platelets are potent MV secretors, and oxLDL, a platelet ligand, induce platelet activation and thus potentially MV secretion. This interaction occurs through binding of oxLDL with CD36, located on the platelet membrane. In this study we investigated the effect of in vitro incubation of platelets with oxLDL on MV release. Furthermore, we compared the results obtained when separating MVs larger than 0.5-µm as a measure of results obtained from less sensitive conventional flow cytometers with MVs below the 0.5-µm limit. MV size-distribution was analysed in plasma from 11 healthy volunteers (4 females, 7 males. MVs were identified as < 1-μm and positive for lactadherin binding and cell specific markers. Platelet rich plasma (PRP was incubated without and with oxLDL or LDL (as control to investigate the impact on platelet activation, evident by release of MVs. Size-calibrated fluorescent beads were used to establish the MV gate, and separate small- and large-size vesicles. CD41+ and CD41+CD36+ MVs increased by 6-8 fold in PRP, when left at room temperature, and the presence of cell specific markers increased. Total MV count was unaffected. Incubations with oxLDL did not increase the MV release or affect the distribution of small- and large-size MVs. We found a large inter-individual variation in the fraction of small- and large-size MVs of 73%. In conclusion, we propose that pro-coagulant activity and activation of platelets induced by interaction of platelet CD36 with oxLDL may not involve release of MVs. Furthermore, our results demonstrate great inter-individual variability in size-distribution of platelet derived MVs and thereby stresses the importance for generation of standardized protocols for MV quantification

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

    Directory of Open Access Journals (Sweden)

    Rebecca Josowitz

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

  12. Target-cell-derived tRNA-like primers for reverse transcription support retroviral infection at low efficiency

    DEFF Research Database (Denmark)

    Schmitz, Alexander; Lund, Anders H; Hansen, Anette C

    2002-01-01

    Reverse transcription of a retroviral genome takes place in the cytoplasm of an infected cell by a process primed by a producer-cell-derived tRNA annealed to an 18-nucleotide primer-binding site (PBS). By an assay involving primer complementation of PBS-mutated vectors we analyzed whether tRNA...... primers derived from the target cell can sustain reverse transcription during murine leukemia virus (MLV) infection. Transduction efficiencies were 4-5 orders of magnitude below those of comparable producer-cell complementations. However, successful usage of a target-cell-derived tRNA primer was proven...... by cases of correction of single mismatches between Akv-MLV vectors and complementary tRNA primers toward the primer sequence in the integrated vector. Thus, target-cell-derived tRNA-like primers are able to initiate first-strand cDNA synthesis and plus-strand transfer leading to a complete provirus...

  13. Rapid Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded Fluorescent Voltage Sensor

    Directory of Open Access Journals (Sweden)

    Jordan S. Leyton-Mange

    2014-02-01

    Full Text Available In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity.

  14. A Non-invasive Platform for Functional Characterization of Stem-Cell-Derived Cardiomyocytes with Applications in Cardiotoxicity Testing

    Directory of Open Access Journals (Sweden)

    Mahnaz Maddah

    2015-04-01

    Full Text Available We present a non-invasive method to characterize the function of pluripotent stem-cell-derived cardiomyocytes based on video microscopy and image analysis. The platform, called Pulse, generates automated measurements of beating frequency, beat duration, amplitude, and beat-to-beat variation based on motion analysis of phase-contrast images captured at a fast frame rate. Using Pulse, we demonstrate recapitulation of drug effects in stem-cell-derived cardiomyocytes without the use of exogenous labels and show that our platform can be used for high-throughput cardiotoxicity drug screening and studying physiologically relevant phenotypes.

  15. Protein Characterization of Extracellular Microvesicles/Exosomes Released from Cytotoxin-Challenged Rat Cerebrocortical Mixed Culture and Mouse N2a Cells.

    Science.gov (United States)

    Kumar, Dhwani; Manek, Rachna; Raghavan, Vijaya; Wang, Kevin K

    2017-03-10

    A number of neuronal and glial proteins were previously found to be released in free-standing soluble form from cultured brain cells into cell-conditioned media. Here, we sought to examine if similar proteins are also contained in neural and astroglial cell-released extracellular microvesicles/exosomes (MV/E). In this study, MV/E were isolated from cell-conditioned media from control and cytotoxin-challenged rat cerebrocortical mixed culture (CTX) and mouse neuroblastoma N2a cells. Cytotoxin challenges included pro-necrosis calcium ionophore A23187, pro-apoptosis staurosporine (STS), and excitotoxin N-methyl-D-aspartate. Based on established nanoparticle characterization method (dynamic light scattering, NanoTracker, and transmission electron microscopy), we confirmed that these released vesicles are in fact characteristic representation of MV/E by morphology (lipid bilayered vesicles) and by particle size (132-142 nm for CTX and 49-77 nm for N2a cells). We indeed identified neural cell body protein UCH-L1, axonal injury marker αII-spectrin and its breakdown products (SBDPs), astroglial markers GFAP and its breakdown products (GFAP-BDP), dendritic protein BIII-tubulin, synaptic protein synaptophysin, and exosome marker Alix in microvesicles from CTX and/or N2a cells. Furthermore, SBDPs, GFAP-BDP, UCH-L1, and synaptophysin are especially dominant in MV/E isolated from cytotoxin-treated CTX cells. Similarly, SBDPs, βIII-tubulin, and UCH-L1 are more prominently observed in cytotoxin-challenged N2a cells. Lastly, when isolated MV/E from A23187- or STS-challenged N2a cells were introduced to healthy N2a culture, they are capable of evoking cytotoxicity in the latter. Taken together, our study identified that microvesicles/exosomes isolated form healthy and injured brain cells contain certain neural and astroglial proteins, as well as possibly other cytotoxic factors that are capable of propagating cytotoxic effects.

  16. Use of human stem cell derived cardiomyocytes to examine sunitinib mediated cardiotoxicity and electrophysiological alterations

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, J.D., E-mail: jennifer.cohen@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Babiarz, J.E., E-mail: joshua.babiarz@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Abrams, R.M., E-mail: rory.abrams@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Guo, L., E-mail: liang.guo@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Kameoka, S., E-mail: sei.kameoka@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Chiao, E., E-mail: eric.chiao@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Taunton, J., E-mail: taunton@cmp.ucsf.edu [Howard Hughes Medical Institute, Cellular and Molecular Pharmacology, University California San Francisco, San Francisco, CA 94158 (United States); Kolaja, K.L., E-mail: kyle.kolaja@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States)

    2011-11-15

    Sunitinib, an oral tyrosine kinase inhibitor approved to treat advanced renal cell carcinoma and gastrointestinal stroma tumor, is associated with clinical cardiac toxicity. Although the precise mechanism of sunitinib cardiotoxicity is not known, both the key metabolic energy regulator, AMP-activated protein kinase (AMPK), and ribosomal S 6 kinase (RSK) have been hypothesized as causative, albeit based on rodent models. To study the mechanism of sunitinib-mediated cardiotoxicity in a human model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) having electrophysiological and contractile properties of native cardiac tissue were investigated. Sunitinib was cardiotoxic in a dose-dependent manner with an IC{sub 50} in the low micromolar range, observed by a loss of cellular ATP, an increase in oxidized glutathione, and induction of apoptosis in iPSC-CMs. Pretreatment of iPSC-CMs with AMPK activators AICAR or metformin, increased the phosphorylation of pAMPK-T172 and pACC-S79, but only marginally attenuated sunitinib mediated cell death. Furthermore, additional inhibitors of AMPK were not directly cytotoxic to iPSC-CMs up to 250 {mu}M concentrations. Inhibition of RSK with a highly specific, irreversible, small molecule inhibitor (RSK-FMK-MEA) did not induce cytotoxicity in iPSC-CMs below 250 {mu}M. Extensive electrophysiological analysis of sunitinib and RSK-FMK-MEA mediated conduction effects were performed. Taken together, these findings suggest that inhibition of AMPK and RSK are not a major component of sunitinib-induced cardiotoxicity. Although the exact mechanism of cardiotoxicity of sunitinib is not known, it is likely due to inhibition of multiple kinases simultaneously. These data highlight the utility of human iPSC-CMs in investigating the potential molecular mechanisms underlying drug-induced cardiotoxicity. -- Highlights: Black-Right-Pointing-Pointer Cytoxic effect of sunitinib on human stem cell derived cardiomyocytes Black

  17. Mesenchymal Stromal Cell-Derived Extracellular Vesicles Protect the Fetal Brain After Hypoxia-Ischemia.

    Science.gov (United States)

    Ophelders, Daan R M G; Wolfs, Tim G A M; Jellema, Reint K; Zwanenburg, Alex; Andriessen, Peter; Delhaas, Tammo; Ludwig, Anna-Kristin; Radtke, Stefan; Peters, Vera; Janssen, Leon; Giebel, Bernd; Kramer, Boris W

    2016-06-01

    Preterm neonates are susceptible to perinatal hypoxic-ischemic brain injury, for which no treatment is available. In a preclinical animal model of hypoxic-ischemic brain injury in ovine fetuses, we have demonstrated the neuroprotective potential of systemically administered mesenchymal stromal cells (MSCs). The mechanism of MSC treatment is unclear but suggested to be paracrine, through secretion of extracellular vesicles (EVs). Therefore, we investigated in this study the protective effects of mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) in a preclinical model of preterm hypoxic-ischemic brain injury. Ovine fetuses were subjected to global hypoxia-ischemia by transient umbilical cord occlusion, followed by in utero intravenous administration of MSC-EVs. The therapeutic effects of MSC-EV administration were assessed by analysis of electrophysiological parameters and histology of the brain. Systemic administration of MSC-EVs improved brain function by reducing the total number and duration of seizures, and by preserving baroreceptor reflex sensitivity. These functional protections were accompanied by a tendency to prevent hypomyelination. Cerebral inflammation remained unaffected by the MSC-EV treatment. Our data demonstrate that MSC-EV treatment might provide a novel strategy to reduce the neurological sequelae following hypoxic-ischemic injury of the preterm brain. Our study results suggest that a cell-free preparation comprising neuroprotective MSC-EVs could substitute MSCs in the treatment of preterm neonates with hypoxic-ischemic brain injury, thereby circumventing the potential risks of systemic administration of living cells. Bone marrow-derived mesenchymal stromal cells (MSCs) show promise in treating hypoxic-ischemic injury of the preterm brain. Study results suggest administration of extracellular vesicles, rather than intact MSCs, is sufficient to exert therapeutic effects and avoids potential concerns associated with administration

  18. Novel Application of Stem Cell-Derived Neurons to Evaluate the Time-and Dose-Dependent Progression of Excitotoxic Injury

    Science.gov (United States)

    2013-05-14

    3. DATES COVERED (From - To) 4. TITLE AND SUBTITLE Novel Application of Stem Cell -Derived Neurons to Evaluate the Time- and Dose-Dependent 5a...01.10.RC.021). 14. ABSTRACT See reprint. 15. SUBJECT TERMS Mouse stem cell -derived neurons, NMDA receptor, AMPA receptor, Neurotoxicity, Apoptosis...area code) 410-436-8044 Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std. Z39.18 Novel Application of Stem Cell -Derived Neurons to Evaluate the

  19. Neurotrophic requirements of human motor neurons defined using amplified and purified stem cell-derived cultures.

    Directory of Open Access Journals (Sweden)

    Nuno Jorge Lamas

    Full Text Available Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC(50 1-2 pM. The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.

  20. Glial cell-derived neurotrophic factor gene polymorpisms affect severity and functionality of bipolar disorder.

    Science.gov (United States)

    Safari, Roghaiyeh; Tunca, Zeliha; Özerdem, Ayşegül; Ceylan, Deniz; Yalçın, Yaprak; Sakizli, Meral

    2017-01-01

    Glial cell-derived neurotrophic factor and other neurotrophins have important role in the development of mental disorders. Here, we aimed to assess the effects of Single nucleotide polymorphisms at potentially regulated regions of GDNF on severity and functionality of bipolar disorder and GDNF serum levels in bipolar disorder patients and healthy volunteers. Severity and functionality of bipolar disorder were evaluated using the Clinical Global Impression and Global Assessment of Functioning scales in sixty-six bipolar disorder patients. The GDNF serum levels obtained from bipolar disorder patients and healthy volunteers who had been already reported SNPs information by our group. GAF scales were lower and GDNF serum levels were higher in Bipolar disorder patients with T/A genotype at 5:37812784 and 5:37812782 compared to patients with T/T genotype. There were significant difference in severity and functionality scores, but not in GDNF serum levels, between patients with G/G and G/A genotype of rs62360370 G > A SNP.rs2075680 C > A and rs79669773 T > C SNPs had no effect on bipolar disorder severity and functionality scores and GDNF serum levels. The results suggest that some SNPs of GDNF have potential association with severity and functionality of bipolar disorder. In addition, except two SNPs, none of GDNF SNPs had association with GDNF serum levels.

  1. Effects of cardioactive drugs on human induced pluripotent stem cell derived long QT syndrome cardiomyocytes.

    Science.gov (United States)

    Kuusela, Jukka; Kujala, Ville J; Kiviaho, Anna; Ojala, Marisa; Swan, Heikki; Kontula, Kimmo; Aalto-Setälä, Katriina

    2016-01-01

    Human induced pluripotent stem cells (hiPSC) have enabled a major step forward in pathophysiologic studies of inherited diseases and may also prove to be valuable in in vitro drug testing. Long QT syndrome (LQTS), characterized by prolonged cardiac repolarization and risk of sudden death, may be inherited or result from adverse drug effects. Using a microelectrode array platform, we investigated the effects of six different drugs on the electrophysiological characteristics of human embryonic stem cell-derived cardiomyocytes as well as hiPSC-derived cardiomyocytes from control subjects and from patients with type 1 (LQT1) and type 2 (LQT2) of LQTS. At baseline the repolarization time was significantly longer in LQTS cells compared to controls. Isoprenaline increased the beating rate of all cell lines by 10-73 % but did not show any arrhythmic effects in any cell type. Different QT-interval prolonging drugs caused prolongation of cardiac repolarization by 3-13 % (cisapride), 10-20 % (erythromycin), 8-23 % (sotalol), 16-42 % (quinidine) and 12-27 % (E-4031), but we did not find any systematic differences in sensitivity between the control, LQT1 and LQT2 cell lines. Sotalol, quinidine and E-4031 also caused arrhythmic beats and beating arrests in some cases. In summary, the drug effects on these patient-specific cardiomyocytes appear to recapitulate clinical observations and provide further evidence that these cells can be applied for in vitro drug testing to probe their vulnerability to arrhythmia.

  2. Excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Kane, Christopher; Couch, Liam; Terracciano, Cesare M N

    2015-01-01

    Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold enormous potential in many fields of cardiovascular research. Overcoming many of the limitations of their embryonic counterparts, the application of iPSC-CMs ranges from facilitating investigation of familial cardiac disease and pharmacological toxicity screening to personalized medicine and autologous cardiac cell therapies. The main factor preventing the full realization of this potential is the limited maturity of iPSC-CMs, which display a number of substantial differences in comparison to adult cardiomyocytes. Excitation-contraction (EC) coupling, a fundamental property of cardiomyocytes, is often described in iPSC-CMs as being more analogous to neonatal than adult cardiomyocytes. With Ca(2+) handling linked, directly or indirectly, to almost all other properties of cardiomyocytes, a solid understanding of this process will be crucial to fully realizing the potential of this technology. Here, we discuss the implications of differences in EC coupling when considering the potential applications of human iPSC-CMs in a number of areas as well as detailing the current understanding of this fundamental process in these cells.

  3. Excitation–contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes

    Science.gov (United States)

    Kane, Christopher; Couch, Liam; Terracciano, Cesare M. N.

    2015-01-01

    Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold enormous potential in many fields of cardiovascular research. Overcoming many of the limitations of their embryonic counterparts, the application of iPSC-CMs ranges from facilitating investigation of familial cardiac disease and pharmacological toxicity screening to personalized medicine and autologous cardiac cell therapies. The main factor preventing the full realization of this potential is the limited maturity of iPSC-CMs, which display a number of substantial differences in comparison to adult cardiomyocytes. Excitation–contraction (EC) coupling, a fundamental property of cardiomyocytes, is often described in iPSC-CMs as being more analogous to neonatal than adult cardiomyocytes. With Ca2+ handling linked, directly or indirectly, to almost all other properties of cardiomyocytes, a solid understanding of this process will be crucial to fully realizing the potential of this technology. Here, we discuss the implications of differences in EC coupling when considering the potential applications of human iPSC-CMs in a number of areas as well as detailing the current understanding of this fundamental process in these cells. PMID:26484342

  4. Induced pluripotent stem cell derived macrophages as a cellular system to study salmonella and other pathogens.

    Directory of Open Access Journals (Sweden)

    Christine Hale

    Full Text Available A number of pathogens, including several human-restricted organisms, persist and replicate within macrophages (Mφs as a key step in pathogenesis. The mechanisms underpinning such host-restricted intracellular adaptations are poorly understood, in part, due to a lack of appropriate model systems. Here we explore the potential of human induced pluripotent stem cell derived macrophages (iPSDMs to study such pathogen interactions. We show iPSDMs express a panel of established Mφ-specific markers, produce cytokines, and polarise into classical and alternative activation states in response to IFN-γ and IL-4 stimulation, respectively. iPSDMs also efficiently phagocytosed inactivated bacterial particles as well as live Salmonella Typhi and S. Typhimurium and were able to kill these pathogens. We conclude that iPSDMs can support productive Salmonella infection and propose this as a flexible system to study host/pathogen interactions. Furthermore, iPSDMs can provide a flexible and practical cellular platform for assessing host responses in multiple genetic backgrounds.

  5. Human Induced Pluripotent Stem Cell-Derived Endothelial Cells for Three-Dimensional Microphysiological Systems.

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    Kurokawa, Yosuke K; Yin, Rose T; Shang, Michael R; Shirure, Venktesh S; Moya, Monica L; George, Steven C

    2017-08-01

    Microphysiological systems (MPS), or "organ-on-a-chip" platforms, aim to recapitulate in vivo physiology using small-scale in vitro tissue models of human physiology. While significant efforts have been made to create vascularized tissues, most reports utilize primary endothelial cells that hinder reproducibility. In this study, we report the use of human induced pluripotent stem cell-derived endothelial cells (iPS-ECs) in developing three-dimensional (3D) microvascular networks. We established a CDH5-mCherry reporter iPS cell line, which expresses the vascular endothelial (VE)-cadherin fused to mCherry. The iPS-ECs demonstrate physiological functions characteristic of primary endothelial cells in a series of in vitro assays, including permeability, response to shear stress, and the expression of endothelial markers (CD31, von Willibrand factor, and endothelial nitric oxide synthase). The iPS-ECs form stable, perfusable microvessels over the course of 14 days when cultured within 3D microfluidic devices. We also demonstrate that inhibition of TGF-β signaling improves vascular network formation by the iPS-ECs. We conclude that iPS-ECs can be a source of endothelial cells in MPS providing opportunities for human disease modeling and improving the reproducibility of 3D vascular networks.

  6. Functional differentiation of stem cell-derived neurons from different murine backgrounds.

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    Barth, Lydia; Sütterlin, Rosmarie; Nenniger, Markus; Vogt, Kaspar E

    2014-01-01

    Murine stem cell-derived neurons have been used to study a wide variety of neuropsychiatric diseases with a hereditary component, ranging from autism to Alzheimer's. While a significant amount of data on their molecular biology has been generated, there is little data on the physiology of these cultures. Different mouse strains show clear differences in behavioral and other neurobiologically relevant readouts. We have studied the physiology of early differentiation and network formation in neuronal cultures derived from three different mouse embryonic stem cell lines. We have found largely overlapping patterns with some significant differences in the timing of the functional milestones. Neurons from R1 showed the fastest development of intrinsic excitability, while E14Tg2a and J1 were slower. This was also reflected in an earlier appearance of synaptic activity in R1 cultures, while E14Tg2a and J1 were delayed by up to 2 days. In conclusion, stem cells from all backgrounds could be successfully differentiated into functioning neural networks with similar developmental patterns. Differences in the timing of specific milestones, suggest that control cell lines and time-points should be carefully chosen when investigating genetic alterations that lead to subtle deficits in neuronal function.

  7. Engineered Microenvironments for the Maturation and Observation of Human Embryonic Stem Cell Derived Cardiomyocytes

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    Salick, Max R.

    The human heart is a dynamic system that undergoes substantial changes as it develops and adapts to the body's growing needs. To better understand the physiology of the heart, researchers have begun to produce immature heart muscle cells, or cardiomyocytes, from pluripotent stem cell sources with remarkable efficiency. These stem cell-derived cardiomyocytes hold great potential in the understanding and treatment of heart disease; however, even after prolonged culture, these cells continue to exhibit an immature phenotype, as indicated by poor sarcomere organization and calcium handling, among other features. The lack of maturation that is observed in these cardiomyocytes greatly limits their applicability towards drug screening, disease modeling, and cell therapy applications. The mechanical environment surrounding a cell has been repeatedly shown to have a large impact on that cell's behavior. For this reason, we have implemented micropatterning methods to mimic the level of alignment that occurs in the heart in vivo in order to study how this alignment may help the cells to produce a more mature sarcomere phenotype. It was discovered that the level of sarcomere organization of a cardiomyocyte can be strongly influenced by the micropattern lane geometry on which it adheres. Steps were taken to optimize this micropattern platform, and studies of protein organization, gene expression, and myofibrillogenesis were conducted. Additionally, a set of programs was developed to provide quantitative analysis of the level of sarcomere organization, as well as to assist with several other tissue engineering applications.

  8. Transcriptional and functional profiling of human embryonic stem cell-derived cardiomyocytes.

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    Feng Cao

    Full Text Available Human embryonic stem cells (hESCs can serve as a potentially limitless source of cells that may enable regeneration of diseased tissue and organs. Here we investigate the use of human embryonic stem cell-derived cardiomyocytes (hESC-CMs in promoting recovery from cardiac ischemia reperfusion injury in a mouse model. Using microarrays, we have described the hESC-CM transcriptome within the spectrum of changes that occur between undifferentiated hESCs and fetal heart cells. The hESC-CMs expressed cardiomyocyte genes at levels similar to those found in 20-week fetal heart cells, making this population a good source of potential replacement cells in vivo. Echocardiographic studies showed significant improvement in heart function by 8 weeks after transplantation. Finally, we demonstrate long-term engraftment of hESC-CMs by using molecular imaging to track cellular localization, survival, and proliferation in vivo. Taken together, global gene expression profiling of hESC differentiation enables a systems-based analysis of the biological processes, networks, and genes that drive hESC fate decisions, and studies such as this will serve as the foundation for future clinical applications of stem cell therapies.

  9. Differential expression of cell-cycle regulators in human beta-cells derived from insulinoma tissue.

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    Ueberberg, Sandra; Tannapfel, Andrea; Schenker, Peter; Viebahn, Richard; Uhl, Waldemar; Schneider, Stephan; Meier, Juris J

    2016-05-01

    The low frequency of beta-cell replication in the adult human pancreas limits beta-cell regeneration. A better understanding of the regulation of human beta-cell proliferation is crucial to develop therapeutic strategies aiming to enhance beta-cell mass. To identify factors that control beta-cell proliferation, cell-cycle regulation was examined in human insulinomas as a model of increased beta-cell proliferation (n=11) and healthy pancreatic tissue from patients with benign pancreatic tumors (n=9). Tissue sections were co-stained for insulin and cell-cycle proteins. Transcript levels of selected cell-cycle factors in beta-cells were determined by qRT-PCR after performing laser-capture microdissection. The frequency of beta-cell replication was 3.74±0.92% in the insulinomas and 0.11±0.04% in controls (p=0.0016). p21 expression was higher in insulinomas (p=0.0058), and Rb expression was higher by trend (p=0.085), whereas p16 (pcell-cycle factors in beta-cells derived from insulinomas and healthy adults differs markedly. Targeting such differentially regulated cell-cycle proteins may evolve as a future strategy to enhance beta-cell regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. 3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes.

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    Correia, Cláudia; Koshkin, Alexey; Duarte, Patrícia; Hu, Dongjian; Carido, Madalena; Sebastião, Maria J; Gomes-Alves, Patrícia; Elliott, David A; Domian, Ibrahim J; Teixeira, Ana P; Alves, Paula M; Serra, Margarida

    2017-11-27

    Three-dimensional (3D) cultures of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) hold great promise for drug discovery, providing a better approximation to the in vivo physiology over standard two-dimensional (2D) monolayer cultures. However, the transition of CM differentiation protocols from 2D to 3D cultures is not straightforward. In this work, we relied on the aggregation of hPSC-derived cardiac progenitors and their culture under agitated conditions to generate highly pure cardiomyocyte aggregates. Whole-transcriptome analysis and 13 C-metabolic flux analysis allowed to demonstrate at both molecular and fluxome levels that such 3D culture environment enhances metabolic maturation of hiPSC-CMs. When compared to 2D, 3D cultures of hiPSC-CMs displayed down-regulation of genes involved in glycolysis and lipid biosynthesis and increased expression of genes involved in OXPHOS. Accordingly, 3D cultures of hiPSC-CMs had lower fluxes through glycolysis and fatty acid synthesis and increased TCA-cycle activity. Importantly, we demonstrated that the 3D culture environment reproducibly improved both CM purity and metabolic maturation across different hPSC lines, thereby providing a robust strategy to derive enriched hPSC-CMs with metabolic features closer to that of adult CMs. © 2017 Wiley Periodicals, Inc.

  11. Three-dimensional image reconstruction of distribution of Pnmt+ cell-derived cells in murine heart.

    Science.gov (United States)

    Ni, Haibo; Wang, Yange; Crawford, William; Zhang, Shanzhuo; Cheng, Longxian; Zhang, Henggui; Lei, Ming

    2017-09-26

    Elucidating the function of specific cell types in a highly complex multicellular system such as the heart often requires detailed anatomic reconstruction. We recently described a distinctive class of phenylethanolamine n-methyltransferase (Pnmt+) cell-derived cardiomyocytes (PdCMs), a new cardiomyocyte population with a potential endocrine role. In this dataset, a 3D reconstruction was carried out to visualise the distribution of PdCMs throughout the murine heart. Rigid registration (stiff rotation and translation) was applied to properly align the fused heart slice images based on landmarks using TrakEM2, an open source plug-in in Fiji. The registered slices were then analysed and reconstructed using MATLAB (MATLAB®. Version 8.3.0.532). The final reconstructed 3D volume was 561×866×48 pixels (corresponding to spatial resolutions of 5.8, 8.9 and 2.5 mm in the x-, y- and z-direction respectively), and visualised in Paraview. The reconstruction allows for detailed analyses of morphology, projections and cellular features of different cell types, enabling further geometrical and topological analyses. Image data can be accessed and viewed through Figshare.

  12. Stromal cell-derived factor-1α as a novel biomarker for hyperlipidemia.

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    Li, Shou-Lin; Lin, Wei; Zhang, Yan; Zheng, Zhi-Chang; Liu, Li-Jun; Fu, Hao; Liu, Jie; Wang, Guo-Dong; Chen, Si-Yuan; Feng, Li-Hong

    2012-12-01

    Stromal cell-derived factor-1 (SDF-1) is expressed in a wide variety of organs, such as heart, and plays a pivotal role in the mobilization of hematopoietic stem and progenitor cells in bone marrow. SDF-1α, a common subtype of SDF-1, may control hematopoiesis and angiogenesis, but its role in the pathogenesis of hyperlipidemia is unknown. The aim of this study was to determine the role of SDF-1α in the pathogenesis of hyperlipidemia. First, log-transformed SDF-1α serum levels (logSDF-1α) were significantly higher in male patients with borderline high lipid profile (BHLP; n=28; 2.15±0.08 ng/ml) compared to control subjects (n=37; 1.94±0.06 ng/ml; Ppathophysiology in male and female patients with hyperlipidemia. Moreover, flow cytometry analysis showed that expression of the SDF-1α receptor, CXC-chemokine receptor 4, was lower in peripheral blood mononuclear cells of patients with BHLP (n=10) and HLP (n=10), compared to control subjects (n=10; Phyperlipidemia that may be helpful to uncover the pathogenesis of hyperlipidemia.

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

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

    2013-03-01

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

  14. Gene Transfection of Human Turbinate Mesenchymal Stromal Cells Derived from Human Inferior Turbinate Tissues

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    Jin Seon Kwon

    2016-01-01

    Full Text Available Human turbinate mesenchymal stromal cells (hTMSCs are novel stem cells derived from nasal inferior turbinate tissues. They are easy to isolate from the donated tissue after turbinectomy or conchotomy. In this study, we applied hTMSCs to a nonviral gene delivery system using polyethyleneimine (PEI as a gene carrier; furthermore, the cytotoxicity and transfection efficiency of hTMSCs were evaluated to confirm their potential as resources in gene therapy. DNA-PEI nanoparticles (NPs were generated by adding the PEI solution to DNA and were characterized by a gel electrophoresis and by measuring particle size and surface charge of NPs. The hTMSCs were treated with DNA-PEI NPs for 4 h, and toxicity of NPs to hTMSCs and gene transfection efficiency were monitored using MTT assay, fluorescence images, and flow cytometry after 24 h and 48 h. At a high negative-to-positive charge ratio, DNA-PEI NPs treatment led to cytotoxicity of hTMSCs, but the transfection efficiency of DNA was increased due to the electrostatic effect between the NPs and the membranes of hTMSCs. Importantly, the results of this research verified that PEI could deliver DNA into hTMSCs with high efficiency, suggesting that hTMSCs could be considered as untapped resources for applications in gene therapy.

  15. Human pluripotent stem cell-derived neural constructs for predicting neural toxicity.

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    Schwartz, Michael P; Hou, Zhonggang; Propson, Nicholas E; Zhang, Jue; Engstrom, Collin J; Santos Costa, Vitor; Jiang, Peng; Nguyen, Bao Kim; Bolin, Jennifer M; Daly, William; Wang, Yu; Stewart, Ron; Page, C David; Murphy, William L; Thomson, James A

    2015-10-06

    Human pluripotent stem cell-based in vitro models that reflect human physiology have the potential to reduce the number of drug failures in clinical trials and offer a cost-effective approach for assessing chemical safety. Here, human embryonic stem (ES) cell-derived neural progenitor cells, endothelial cells, mesenchymal stem cells, and microglia/macrophage precursors were combined on chemically defined polyethylene glycol hydrogels and cultured in serum-free medium to model cellular interactions within the developing brain. The precursors self-assembled into 3D neural constructs with diverse neuronal and glial populations, interconnected vascular networks, and ramified microglia. Replicate constructs were reproducible by RNA sequencing (RNA-Seq) and expressed neurogenesis, vasculature development, and microglia genes. Linear support vector machines were used to construct a predictive model from RNA-Seq data for 240 neural constructs treated with 34 toxic and 26 nontoxic chemicals. The predictive model was evaluated using two standard hold-out testing methods: a nearly unbiased leave-one-out cross-validation for the 60 training compounds and an unbiased blinded trial using a single hold-out set of 10 additional chemicals. The linear support vector produced an estimate for future data of 0.91 in the cross-validation experiment and correctly classified 9 of 10 chemicals in the blinded trial.

  16. Isolation and Mechanical Measurements of Myofibrils from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

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    Josè Manuel Pioner

    2016-06-01

    Full Text Available Tension production and contractile properties are poorly characterized aspects of excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs. Previous approaches have been limited due to the small size and structural immaturity of early-stage hiPSC-CMs. We developed a substrate nanopatterning approach to produce hiPSC-CMs in culture with adult-like dimensions, T-tubule-like structures, and aligned myofibrils. We then isolated myofibrils from hiPSC-CMs and measured the tension and kinetics of activation and relaxation using a custom-built apparatus with fast solution switching. The contractile properties and ultrastructure of myofibrils more closely resembled human fetal myofibrils of similar gestational age than adult preparations. We also demonstrated the ability to study the development of contractile dysfunction of myofibrils from a patient-derived hiPSC-CM cell line carrying the familial cardiomyopathy MYH7 mutation (E848G. These methods can bring new insights to understanding cardiomyocyte maturation and developmental mechanical dysfunction of hiPSC-CMs with cardiomyopathic mutations.

  17. Evaluation of MDCK cell-derived influenza H7N9 vaccine candidates in ferrets.

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    Min-Yuan Chia

    Full Text Available Avian-origin influenza A (H7N9 viruses emerged as human pathogens in China in early 2013 and have killed >100 persons. Influenza vaccines are mainly manufactured using egg-based technology which could not meet the surging demand during influenza pandemics. In this study, we evaluated cell-based influenza H7N9 vaccines in ferrets. An egg-derived influenza H7N9 reassortant vaccine virus was adapted in MDCK cells. Influenza H7N9 whole virus vaccine antigen was manufactured using a microcarrier-based culture system. Immunogenicity and protection of the vaccine candidates with three different formulations (300 μg aluminum hydroxide, 1.5 μg HA, and 1.5 μg HA plus 300 μg aluminum hydroxide were evaluated in ferrets. In ferrets receiving two doses of vaccination, geometric mean titers of hemagglutination (HA inhibition and neutralizing antibodies were <10 and <40 for the control group (adjuvant only, 17 and 80 for the unadjuvanted (HA only group, and 190 and 640 for the adjuvanted group (HA plus adjuvant, respectively. After challenge with wild-type influenza H7N9 viruses, virus titers in respiratory tracts of the adjuvanted group were significantly lower than that in the control, and unadjuvanted groups. MDCK cell-derived influenza H7N9 whole virus vaccine candidate is immunogenic and protective in ferrets and clinical development is highly warranted.

  18. Cultures of astroglial cells derived from brain of adult cichlid fish.

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    Mack, Andreas F; Tiedemann, Karin

    2013-01-30

    Astroglial cells in teleost fish occur mostly as radial glia. We established a culture system derived from brain tissue of mature cichlid fish Astatotilapia burtoni to study fish astroglial cells in more detail. Cells were passaged several times to expand the cultures, and could be kept in vitro for several months. The cell identity was tested by the presence of glial fibrillary acidic protein (GFAP); in addition, cells expressed the tight junction adaptor protein zonula occludens-1 (ZO-1) known to be present on astroglial cells in fish brain. This is consistent with the radial and epithelial nature of fish astroglial cells derived from neuroepithelium. To characterize the properties of cultured astroglial cells we challenged them in hypo-osmotic conditions. Cells reacted with volume increase, slower but similar to mammalian astrocytes. We also tested whether astroglial cells support growth during axonal elongation. We placed retinal explants on astroglial cultures and found neurites extending readily on these cells, compared to controls which showed no or little growth. Thus, we established a culture system for astroglial cells from the mature fish brain that demonstrates their neuroepithelial properties. This culture system will be useful to study functions in which glial cells are thought to play an important role: e.g. regulation of water homeostasis and supporting axonal regeneration. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Hair Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin Organoids

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    Jiyoon Lee

    2018-01-01

    Full Text Available The mammalian hair follicle arises during embryonic development from coordinated interactions between the epidermis and dermis. It is currently unclear how to recapitulate hair follicle induction in pluripotent stem cell cultures for use in basic research studies or in vitro drug testing. To date, generation of hair follicles in vitro has only been possible using primary cells isolated from embryonic skin, cultured alone or in a co-culture with stem cell-derived cells, combined with in vivo transplantation. Here, we describe the derivation of skin organoids, constituting epidermal and dermal layers, from a homogeneous population of mouse pluripotent stem cells in a 3D culture. We show that skin organoids spontaneously produce de novo hair follicles in a process that mimics normal embryonic hair folliculogenesis. This in vitro model of skin development will be useful for studying mechanisms of hair follicle induction, evaluating hair growth or inhibitory drugs, and modeling skin diseases.

  20. Process Extension from Embryonic Stem Cell-Derived Motor Neurons through Synthetic Extracellular Matrix Mimics

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    McKinnon, Daniel Devaud

    This thesis focuses on studying the extension of motor axons through synthetic poly(ethylene glycol) PEG hydrogels that have been modified with biochemical functionalities to render them more biologically relevant. Specifically, the research strategy is to encapsulate embryonic stem cell-derived motor neurons (ESMNs) in synthetic PEG hydrogels crosslinked through three different chemistries providing three mechanisms for dynamically tuning material properties. First, a covalently crosslinked, enzymatically degradable hydrogel is developed and exploited to study the biophysical dynamics of axon extension and matrix remodeling. It is demonstrated that dispersed motor neurons require a battery of adhesive peptides and growth factors to maintain viability and extend axons while those in contact with supportive neuroglial cells do not. Additionally, cell-degradable crosslinker peptides and a soft modulus mimicking that of the spinal cord are requirements for axon extension. However, because local degradation of the hydrogel results in a cellular environment significantly different than that of the bulk, enzymatically degradable peptide crosslinkers were replaced with reversible covalent hydrazone bonds to study the effect of hydrogel modulus on axon extension. This material is characterized in detail and used to measure forces involved in axon extension. Finally, a hydrogel with photocleavable linkers incorporated into the network structure is exploited to explore motor axon response to physical channels. This system is used to direct the growth of motor axons towards co-cultured myotubes, resulting in the formation of an in vitro neural circuit.

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

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    Lu Wang

    2014-07-01

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

  2. Human embryonic stem cell-derived neuronal cells form spontaneously active neuronal networks in vitro.

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    Heikkilä, Teemu J; Ylä-Outinen, Laura; Tanskanen, Jarno M A; Lappalainen, Riikka S; Skottman, Heli; Suuronen, Riitta; Mikkonen, Jarno E; Hyttinen, Jari A K; Narkilahti, Susanna

    2009-07-01

    The production of functional human embryonic stem cell (hESC)-derived neuronal cells is critical for the application of hESCs in treating neurodegenerative disorders. To study the potential functionality of hESC-derived neurons, we cultured and monitored the development of hESC-derived neuronal networks on microelectrode arrays. Immunocytochemical studies revealed that these networks were positive for the neuronal marker proteins beta-tubulin(III) and microtubule-associated protein 2 (MAP-2). The hESC-derived neuronal networks were spontaneously active and exhibited a multitude of electrical impulse firing patterns. Synchronous bursts of electrical activity similar to those reported for hippocampal neurons and rodent embryonic stem cell-derived neuronal networks were recorded from the differentiated cultures until up to 4 months. The dependence of the observed neuronal network activity on sodium ion channels was examined using tetrodotoxin (TTX). Antagonists for the glutamate receptors NMDA [D(-)-2-amino-5-phosphonopentanoic acid] and AMPA/kainate [6-cyano-7-nitroquinoxaline-2,3-dione], and for GABAA receptors [(-)-bicuculline methiodide] modulated the spontaneous electrical activity, indicating that pharmacologically susceptible neuronal networks with functional synapses had been generated. The findings indicate that hESC-derived neuronal cells can generate spontaneously active networks with synchronous communication in vitro, and are therefore suitable for use in developmental and drug screening studies, as well as for regenerative medicine.

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

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    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.

  4. Potential Therapies by Stem Cell-Derived Exosomes in CNS Diseases: Focusing on the Neurogenic Niche

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    Alejandro Luarte

    2016-01-01

    Full Text Available Neurodegenerative disorders are one of the leading causes of death and disability and one of the biggest burdens on health care systems. Novel approaches using various types of stem cells have been proposed to treat common neurodegenerative disorders such as Alzheimer’s Disease, Parkinson’s Disease, or stroke. Moreover, as the secretome of these cells appears to be of greater benefit compared to the cells themselves, the extracellular components responsible for its therapeutic benefit have been explored. Stem cells, as well as most cells, release extracellular vesicles such as exosomes, which are nanovesicles able to target specific cell types and thus to modify their function by delivering proteins, lipids, and nucleic acids. Exosomes have recently been tested in vivo and in vitro as therapeutic conveyors for the treatment of diseases. As such, they could be engineered to target specific populations of cells within the CNS. Considering the fact that many degenerative brain diseases have an impact on adult neurogenesis, we discuss how the modulation of the adult neurogenic niches may be a therapeutic target of stem cell-derived exosomes. These novel approaches should be examined in cellular and animal models to provide better, more effective, and specific therapeutic tools in the future.

  5. Isolation, Characterization, and Multipotent Differentiation of Mesenchymal Stem Cells Derived from Meniscal Debris

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    Weili Fu

    2016-01-01

    Full Text Available This study aimed to culture and characterize mesenchymal stem cells derived from meniscal debris. Cells in meniscal debris from patients with meniscal injury were isolated by enzymatic digestion, cultured in vitro to the third passage, and analyzed by light microscopy to observe morphology and growth. Third-passage cultures were also analyzed for immunophenotype and ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages. After 4-5 days in culture, cells showed a long fusiform shape and adhered to the plastic walls. After 10–12 days, cell clusters and colonies were observed. Third-passage cells showed uniform morphology and good proliferation. They expressed CD44, CD90, and CD105 but were negative for CD34 and CD45. Cultures induced to differentiate via osteogenesis became positive for Alizarin Red staining as well as alkaline phosphatase activity. Cultures induced to undergo adipogenesis were positive for Oil Red O staining. Cultures induced to undergo chondrogenesis were positive for staining with Toluidine Blue, Alcian Blue, and type II collagen immunohistochemistry, indicating cartilage-specific matrix. These results indicate that the cells we cultured from meniscal debris are mesenchymal stem cells capable of differentiating along three lineages. These stem cells may be valuable source for meniscal regeneration.

  6. Appearance of differentiated cells derived from polar body nuclei in the silkworm, Bombyx mori

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    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. Functional differentiation of stem cell-derived neurons from different murine backgrounds

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    Lydia eBarth

    2014-02-01

    Full Text Available Murine stem cell derived-neurons have been used to study a wide variety of neuropsychiatric diseases with a hereditary component, ranging from autism to Alzheimer’s. While a significant amount of data on their molecular biology has been generated, there is little data on the physiology of these cultures. Different mouse strains show clear differences in behavioural and other neurobiologically relevant readouts. We have studied the physiology of early differentiation and network formation in neuronal cultures derived from three different mouse embryonic stem cell lines. We have found largely overlapping patterns with some significant differences in the timing of the functional milestones. Neurons from R1 showed the fastest development of intrinsic excitability, while E14Tg2a and J1 were slower. This was also reflected in an earlier appearance of synaptic activity in R1 cultures, while E14Tg2a and J1 were delayed by up to two days. In conclusion, stem cells from all backgrounds could be successfully differentiated into functioning neural networks with similar developmental patterns. Differences in the timing of specific milestones, suggest that control cell lines and time-points should be carefully chosen when investigating genetic alterations that lead to subtle deficits in neuronal function.

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

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

    2013-01-01

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

  9. Pathogen sensing pathways in human embryonic stem cell derived-endothelial cells: role of NOD1 receptors.

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    Daniel M Reed

    Full Text Available Human embryonic stem cell-derived endothelial cells (hESC-EC, as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR Toll-like receptor (TLR-4 and nucleotide-binding oligomerisation domain-containing protein (NOD-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC. HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC, and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.

  10. Relative contributions of human types 1 and 2 T-helper cell-derived eosinophilotrophic cytokines to development of eosinophilia

    NARCIS (Netherlands)

    Wierenga, E. A.; Backx, B.; Snoek, M.; Koenderman, L.; Kapsenberg, M. L.

    1993-01-01

    The relative contributions of type 1 and 2 T-helper (Th1 and Th2) cell-derived interleukin (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 were studied in the regulation of sequential events in the development of eosinophilia. Using eosinophils from normal donors and

  11. Endothelial cell-derived matrix promotes the metabolic functional maturation of hepatocyte via integrin-Src signalling.

    Science.gov (United States)

    Guo, Xinyue; Li, Weihong; Ma, Minghui; Lu, Xin; Zhang, Haiyan

    2017-11-01

    The extracellular matrix (ECM) microenvironment is involved in the regulation of hepatocyte phenotype and function. Recently, the cell-derived extracellular matrix has been proposed to represent the bioactive and biocompatible materials of the native ECM. Here, we show that the endothelial cell-derived matrix (EC matrix) promotes the metabolic maturation of human adipose stem cell-derived hepatocyte-like cells (hASC-HLCs) through the activation of the transcription factor forkhead box protein A2 (FOXA2) and the nuclear receptors hepatocyte nuclear factor 4 alpha (HNF4α) and pregnane X receptor (PXR). Reducing the fibronectin content in the EC matrix or silencing the expression of α5 integrin in the hASC-HLCs inhibited the effect of the EC matrix on Src phosphorylation and hepatocyte maturation. The inhibition of Src phosphorylation using the inhibitor PP2 or silencing the expression of Src in hASC-HLCs also attenuated the up-regulation of the metabolic function of hASC-HLCs in a nuclear receptor-dependent manner. These data elucidate integrin-Src signalling linking the extrinsic EC matrix signals and metabolic functional maturation of hepatocyte. This study provides a model for studying the interaction between hepatocytes and non-parenchymal cell-derived matrix. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  12. Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells

    NARCIS (Netherlands)

    Sorkio, A.; Haimi, Suvi; Verdoold, Vincent; Juuti-Uusitalo, K.; Grijpma, Dirk W.; Skottman, H.

    2017-01-01

    Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great

  13. Induced pluripotent stem cell-derived neuron as a human model for testing environmentally induced developmental neurotoxicity

    Science.gov (United States)

    Induced pluripotent stem cell-derived neurons as a human model for testing environmentally induced developmental neurotoxicity Ingrid L. Druwe1, Timothy J. Shafer2, Kathleen Wallace2, Pablo Valdivia3 ,and William R. Mundy2. 1University of North Carolina, Curriculum in Toxicology...

  14. Autographa californica multiple nucleopolyhedrovirus ac75 is required for egress of nucleocapsids from the nucleus and formation of de novo intranuclear membrane microvesicles.

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    Ya-Jun Guo

    Full Text Available In this study, Autographa californica multiple nucleopolyhedrovirus ac75 was functionally characterized. Ac75 has homologs in all sequenced genomes of alphabaculoviruses, betabaculoviruses, and gammabaculoviruses. It was determined to encode a protein that is associated with the nucleocapsid of budded virus and with both envelope and nucleocapsids of occlusion-derived virus. Sf9 cells transfected by an ac75-knockout bacmid resulted in the infection being restricted to single cells. No budded virus were detected although viral DNA replication and late gene expression were unaffected. Electron microscopy revealed that the virogenic stroma, nucleocapsids and occlusion bodies appeared normal in the cells transfected by an ac75-knockout bacmid. However, the nucleocapsids were unenveloped, the occlusion bodies did not contain any virions or nucleocapsids, and no nucleocapsids were found outside the nucleus or spanning the nuclear membrane. In addition, de novo intranuclear membrane microvesicles that are the precursor of occlusion-derived virus envelopes were absent in the nuclei of transfected cells. Confocal microscopy showed that AC75 protein appeared in the cytoplasm as early as 6 hours post infection. It localized to the ring zone at the periphery of the nucleus from 15 to 24 hours post infection and demonstrated light blocky cloud-like distribution in the center of the nucleus. AC75 was found to co-immunoprecipitate with BV and ODV associated envelope protein ODV-E25. The data from this study suggest that ac75 is essential for induction of the intranuclear membrane microvesicles, it appears to be required for the intranuclear envelopment of nucleocapsids, and is also essential for egress of nucleocapsids from the nuclei, in infected cells.

  15. [Analysis of factors related to the number of mesenchymal stem cells derived from synovial fluid of the temporomandibular joint].

    Science.gov (United States)

    Sun, Y P; Zheng, Y H; Zhang, Z G

    2017-06-09

    Objective: To analyze related factors on the number of mesenchymal stem cells in the synovial fluid of the temporomandibular joint (TMJ) and provide an research basis for understanding of the source and biological role of mesenchymal stem cells derived from synovial fluid in TMJ. Methods: One hundred and twenty-two synovial fluid samples from 91 temporomandibular disorders (TMD) patients who visited in Department of TMJ Center, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University from March 2013 to December 2013 were collected in this study, and 6 TMJ synovial fluid samples from 6 normal volunteers who were studying in the North Campus of Sun Yat-sen University were also collected, so did their clinical information. Then the relation between the number of mesenchymal stem cells derived from synovial fluid and the health status of the joints, age of donor, disc perforation, condylar bony destruction, blood containing and visual analogue scale score of pain were investigated using Mann-Whitney U test and Spearman rank correlation test. Results: The number of mesenchymal stem cells derived from synovial fluid had no significant relation with visual analogue scale score of pain (r=0.041, P=0.672), blood containing (P=0.063), condylar bony destruction (P= 0.371). Linear correlation between the number of mesenchymal stem cells derived from synovial fluid and age of donor was very week (r=0.186, P=0.043). The number of mesenchymal stem cells up-regulated when the joint was in a disease state (P=0.001). The disc perforation group had more mesenchymal stem cells in synovial fluid than without disc perforation group (P=0.042). Conclusions: The number of mesenchymal stem cells derived from synovial fluid in TMJ has no correlation with peripheral blood circulation and condylar bony destruction, while has close relation with soft tissue structure damage of the joint.

  16. Nanofibrous scaffolds for the guidance of stem cell-derived neurons for auditory nerve regeneration.

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    Sandra Hackelberg

    Full Text Available Impairment of spiral ganglion neurons (SGNs of the auditory nerve is a major cause for hearing loss occurring independently or in addition to sensory hair cell damage. Unfortunately, mammalian SGNs lack the potential for autonomous regeneration. Stem cell based therapy is a promising approach for auditory nerve regeneration, but proper integration of exogenous cells into the auditory circuit remains a fundamental challenge. Here, we present novel nanofibrous scaffolds designed to guide the integration of human stem cell-derived neurons in the internal auditory meatus (IAM, the foramen allowing passage of the spiral ganglion to the auditory brainstem. Human embryonic stem cells (hESC were differentiated into neural precursor cells (NPCs and seeded onto aligned nanofiber mats. The NPCs terminally differentiated into glutamatergic neurons with high efficiency, and neurite projections aligned with nanofibers in vitro. Scaffolds were assembled by seeding GFP-labeled NPCs on nanofibers integrated in a polymer sheath. Biocompatibility and functionality of the NPC-seeded scaffolds were evaluated in vivo in deafened guinea pigs (Cavia porcellus. To this end, we established an ouabain-based deafening procedure that depleted an average 72% of SGNs from apex to base of the cochleae and caused profound hearing loss. Further, we developed a surgical procedure to implant seeded scaffolds directly into the guinea pig IAM. No evidence of an inflammatory response was observed, but post-surgery tissue repair appeared to be facilitated by infiltrating Schwann cells. While NPC survival was found to be poor, both subjects implanted with NPC-seeded and cell-free control scaffolds showed partial recovery of electrically-evoked auditory brainstem thresholds. Thus, while future studies must address cell survival, nanofibrous scaffolds pose a promising strategy for auditory nerve regeneration.

  17. Evaluation of nefazodone-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes.

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    Lee, Sujeong; Lee, Hyang-Ae; Choi, Sung Woo; Kim, Sung Joon; Kim, Ki-Suk

    2016-04-01

    The recent establishment of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which express the major cardiac ion channels and recapitulate spontaneous mechanical and electrical activities, may provide a possible solution for the lack of in vitro human-based cardiotoxicity testing models. Cardiotoxicity induced by the antidepressant nefazodone was previously revealed to cause an acquired QT prolongation by hERG channel blockade. To elucidate the cellular mechanisms underlying the cardiotoxicity of nefazodone beyond hERG, its effects on cardiac action potentials (APs) and ion channels were investigated using hiPSC-CMs with whole-cell patch clamp techniques. In a proof of principle study, we examined the effects of cardioactive channel blockers on the electrophysiological profile of hiPSC-CMs in advance of the evaluation of nefazodone. Nefazodone dose-dependently prolonged the AP duration at 90% (APD90) and 50% (APD50) repolarization, reduced the maximum upstroke velocity (dV/dtmax) and induced early after depolarizations. Voltage-clamp studies of hiPSC-CMs revealed that nefazodone inhibited various voltage-gated ion channel currents including IKr, IKs, INa, and ICa. Among them, IKr and INa showed relatively higher sensitivity to nefazodone, consistent with the changes in the AP parameters. In summary, hiPSC-CMs enabled an integrated approach to evaluate the complex interactions of nefazodone with cardiac ion channels. These results suggest that hiPSC-CMs can be an effective model for detecting drug-induced arrhythmogenicity beyond the current standard assay of heterologously expressed hERG K(+) channels. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Objective evaluation of the degree of pigmentation in human induced pluripotent stem cell-derived RPE.

    Science.gov (United States)

    Kamao, Hiroyuki; Mandai, Michiko; Wakamiya, Shunji; Ishida, Junko; Goto, Katsutoshi; Ono, Takaaki; Suda, Taiji; Takahashi, Masayo; Kiryu, Junichi

    2014-11-11

    For the transplantation of human induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE), determination of the maturation status of these cells is essential, and the degree of pigmentation (dPG) can serve as a good indicator of this status. The aim of this study was to establish a method of objectively and quantitatively evaluating the dPG of hiPSC-RPE. Two observers determined the dPG subjectively by observing recorded images of hiPSC-RPE as follows: the dPG of a single cell was classified into three different pigmentation stages, and the overall dPG was compared between two cell groups to identify the group with the higher dPG. The κ statistic was applied to assess interobserver reproducibility. Next, the dPG of single cells and cell groups was objectively determined by the lightness of the hue, saturation, and value (HSL) color space, and the correlation between the subjective evaluation and time-dependent change in the objective dPG of hiPSC-RPE was investigated. The κ statistic was 0.88 and 0.81 in the single-cell and cell-group observations, respectively. The objective dPG of single cells and cell groups was highly correlated with the subjective dPG. However, the observers were occasionally unable to subjectively determine the group with the higher dPG. The objective dPG increased in a time-dependent manner. The lightness of the HSL color space can be used to objectively and quantitatively evaluate the dPG of hiPSC-RPE in culture. The objective evaluation was consistent and was able to better identify small differences than subjective evaluation. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  19. 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.

  20. Functional cardiotoxicity assessment of cosmetic compounds using human-induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Chaudhari, Umesh; Nemade, Harshal; Sureshkumar, Poornima; Vinken, Mathieu; Ates, Gamze; Rogiers, Vera; Hescheler, Jürgen; Hengstler, Jan Georg; Sachinidis, Agapios

    2017-09-22

    There is a large demand of a human relevant in vitro test system suitable for assessing the cardiotoxic potential of cosmetic ingredients and other chemicals. Using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we have already established an in vitro cardiotoxicity assay and identified genomic biomarkers of anthracycline-induced cardiotoxicity in our previous work. Here, five cosmetic ingredients were studied by the new hiPSC-CMs test; kojic acid (KJA), triclosan (TS), triclocarban (TCC), 2,7-naphthalenediol (NPT), and basic red 51 (BR51) based on cytotoxicity as well as ATP assays, beating rate, and genomic biomarkers to determine the lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC). The LOEC for beating rate were 400, 10, 3, >400, and 3 µM for KJA, TS, TCC, NPT, and BR51, respectively. The corresponding concentrations for cytotoxicity or ATP depletion were similar, with the exception of TS and TCC, where the cardiomyocyte-beating assay showed positive results at non-cytotoxic concentrations. Functional analysis also showed that the individual compounds caused different effects on hiPSC-CMs. While exposure to KJA, TS, TCC, and BR51 induced significant arrhythmic beating, NPT slightly decreased cell viability, but did not influence beating. Gene expression studies showed that TS and NPT caused down-regulation of cytoskeletal and cardiac ion homeostasis genes. Moreover, TS and NPT deregulated genomic biomarkers known to be affected also by anthracyclines. The present study demonstrates that hiPSC-CMs can be used to determine LOECs and NOECs in vitro, which can be compared to human blood concentrations to determine margins of exposure. Our in vitro assay, which so far has been tested with several anthracyclines and cosmetics, still requires validation by larger numbers of positive and negative controls, before it can be recommended for routine analysis.

  1. Phenotypic Assays for Characterizing Compound Effects on Induced Pluripotent Stem Cell-Derived Cardiac Spheroids.

    Science.gov (United States)

    Sirenko, Oksana; Hancock, Michael K; Crittenden, Carole; Hammer, Matthew; Keating, Sean; Carlson, Coby B; Chandy, Grischa

    Development of more complex, biologically relevant, and predictive cell-based assays for compound screening is a major challenge in drug discovery. The focus of this study was to establish high-throughput compatible three-dimensional (3D) cardiotoxicity assays using human induced pluripotent stem cell-derived cardiomyocytes. Using both high-content imaging and fast kinetic fluorescence imaging, the impact of various compounds on the beating rates and patterns of cardiac spheroids was monitored by changes in intracellular Ca(2+) levels with calcium-sensitive dyes. Advanced image analysis methods were implemented to provide multiparametric characterization of the Ca(2+) oscillation patterns. In addition, we used confocal imaging and 3D analysis methods to characterize compound effects on the morphology of 3D spheroids. This phenotypic assay allows for the characterization of parameters such as beating frequency, amplitude, peak width, rise and decay times, as well as cell viability and morphological characteristics. A set of 22 compounds, including a number of known cardioactive and cardiotoxic drugs, was assayed at different time points, and the calculated EC50 values for compound effects were compared between 3D and two-dimensional (2D) model systems. A significant concordance in the phenotypes was observed for compound effects between the two models, but essential differences in the concentration responses and time dependencies of the compound-induced effects were observed. Together, these results indicate that 3D cardiac spheroids constitute a functionally distinct biological model system from traditional flat 2D cultures. In conclusion, we have demonstrated that phenotypic assays using 3D model systems are enabled for screening and suitable for cardiotoxicity assessment in vitro.

  2. Flow rate calibration to determine cell-derived microparticles and homogeneity of blood components.

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    Noulsri, Egarit; Lerdwana, Surada; Kittisares, Kulvara; Palasuwan, Attakorn; Palasuwan, Duangdao

    2017-08-01

    Cell-derived microparticles (MPs) are currently of great interest to screening transfusion donors and blood components. However, the current approach to counting MPs is not affordable for routine laboratory use due to its high cost. The current study aimed to investigate the potential use of flow-rate calibration for counting MPs in whole blood, packed red blood cells (PRBCs), and platelet concentrates (PCs). The accuracy of flow-rate calibration was investigated by comparing the platelet counts of an automated counter and a flow-rate calibrator. The concentration of MPs and their origins in whole blood (n=100), PRBCs (n=100), and PCs (n=92) were determined using a FACSCalibur. The MPs' fold-changes were calculated to assess the homogeneity of the blood components. Comparing the platelet counts conducted by automated counting and flow-rate calibration showed an r2 of 0.6 (y=0.69x+97,620). The CVs of the within-run and between-run variations of flow-rate calibration were 8.2% and 12.1%, respectively. The Bland-Altman plot showed a mean bias of -31,142platelets/μl. MP enumeration revealed both the difference in MP levels and their origins in whole blood, PRBCs, and PCs. Screening the blood components demonstrated high heterogeneity of the MP levels in PCs when compared to whole blood and PRBCs. The results of the present study suggest the accuracy and precision of flow-rate calibration for enumerating MPs. This flow-rate approach is affordable for assessing the homogeneity of MPs in blood components in routine laboratory practice. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

    Science.gov (United States)

    Kucera, Jan P.; Prudat, Yann; Marcu, Irene C.; Azzarito, Michela; Ullrich, Nina D.

    2015-01-01

    Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs). However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs) and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV) was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands. CV was significantly lower in strands composed purely of SCMs (5.5 ± 1.5 cm/s, n = 11) as compared to PCMs (34.9 ± 2.9 cm/s, n = 21) at similar refractoriness (100% SCMs: 122 ± 25 ms, n = 9; 100% PCMs: 139 ± 67 ms, n = 14). In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV. These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias. PMID:26442264

  4. Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Jan Pavel Kucera

    2015-09-01

    Full Text Available Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs. However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands.CV was significantly lower in strands composed purely of SCMs (5.5±1.5 cm/s, n=11 as compared to PCMs (34.9±2.9 cm/s, n=21 at similar refractoriness (100% SCMs: 122±25 ms, n=9; 100% PCMs: 139±67 ms, n=14. In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV.These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

  5. Inflammation-induced endothelial cell-derived extracellular vesicles modulate the cellular status of pericytes.

    Science.gov (United States)

    Yamamoto, Seiji; Niida, Shumpei; Azuma, Erika; Yanagibashi, Tsutomu; Muramatsu, Masashi; Huang, Ting Ting; Sagara, Hiroshi; Higaki, Sayuri; Ikutani, Masashi; Nagai, Yoshinori; Takatsu, Kiyoshi; Miyazaki, Kenji; Hamashima, Takeru; Mori, Hisashi; Matsuda, Naoyuki; Ishii, Yoko; Sasahara, Masakiyo

    2015-02-17

    Emerging lines of evidence have shown that extracellular vesicles (EVs) mediate cell-to-cell communication by exporting encapsulated materials, such as microRNAs (miRNAs), to target cells. Endothelial cell-derived EVs (E-EVs) are upregulated in circulating blood in different pathological conditions; however, the characteristics and the role of these E-EVs are not yet well understood. In vitro studies were conducted to determine the role of inflammation-induced E-EVs in the cell-to-cell communication between vascular endothelial cells and pericytes/vSMCs. Stimulation with inflammatory cytokines and endotoxin immediately induced release of shedding type E-EVs from the vascular endothelial cells, and flow cytometry showed that the induction was dose dependent. MiRNA array analyses revealed that group of miRNAs were specifically increased in the inflammation-induced E-EVs. E-EVs added to the culture media of cerebrovascular pericytes were incorporated into the cells. The E-EV-supplemented cells showed highly induced mRNA and protein expression of VEGF-B, which was assumed to be a downstream target of the miRNA that was increased within the E-EVs after inflammatory stimulation. The results suggest that E-EVs mediate inflammation-induced endothelial cell-pericyte/vSMC communication, and the miRNAs encapsulated within the E-EVs may play a role in regulating target cell function. E-EVs may be new therapeutic targets for the treatment of inflammatory diseases.

  6. Cell-Derived Nanoparticles are Endogenous Modulators of Sepsis With Therapeutic Potential.

    Science.gov (United States)

    Kunz, Natalia; Xia, Brent T; Kalies, Kai-Uwe; Klinger, Matthias; Gemoll, Timo; Habermann, Jens K; Whitacre, Brynne E; Seitz, Aaron P; Kalies, Kathrin; Caldwell, Charles C

    2017-09-01

    Cell-derived nanoparticles (CDNPs) containing cytosolic proteins and RNAs/DNAs can be isolated from stressed eukaryotic cells. Previously, CDNPs isolated from cultured cells exerted immunomodulatory activities in different infections. Here, we sought to elucidate the role of CDNPs using a murine model of cecal ligation and puncture (CLP). We hypothesized that CDNPs influence the immune response at the site of infection, where severe cellular stress occurs. We observed early CDNP accumulation in the peritoneum after 4 h and continued CDNP presence 24 h after CLP. To determine whether CDNPs influence the host response to sepsis, we isolated CDNPs from a murine fibroblast cell line stressed by nutrient-deprivation, and injected them into septic mice. CDNP-treated mice demonstrated decreased peritoneal interleukin 6 levels and an approximately 2-log lower bacterial load compared with control mice 24 h after CLP. Additionally, a 20% CFU reduction was observed when incubating CDNPs with Pseudomona aeroginosa, indicating that CDNPs are bactericidal. To identify CDNP-responsive cells, CFSE-labeled CDNPs were injected into mice at the time of CLP. We observed that CDNPs were preferentially ingested by F4/80 macrophages, and to a lesser degree, associated with inflammatory monocytes and neutrophils. Strikingly, CDNP-ingesting cells demonstrated elevated CD11b and MHCII expression compared with control cells. Altogether, our data indicate that CDNPs enhance the immune response at the site of infection and promote bacterial clearance, by direct bacterial killing and increasing phagocyte activation. Thus, CDNPs represent a novel, unexplored endogenous sepsis modulator with therapeutic potential.

  7. Bioactive cell-derived matrices combined with polymer mesh scaffold for osteogenesis and bone healing.

    Science.gov (United States)

    Kim, In Gul; Hwang, Mintai P; Du, Ping; Ko, Jaehoon; Ha, Chul-won; Do, Sun Hee; Park, Kwideok

    2015-05-01

    Successful bone tissue engineering generally requires an osteoconductive scaffold that consists of extracellular matrix (ECM) to mimic the natural environment. In this study, we developed a PLGA/PLA-based mesh scaffold coated with cell-derived extracellular matrix (CDM) for the delivery of bone morphogenic protein (BMP-2), and assessed the capacity of this system to provide an osteogenic microenvironment. Decellularized ECM from human lung fibroblasts (hFDM) was coated onto the surface of the polymer mesh scaffolds, upon which heparin was then conjugated onto hFDM via EDC chemistry. BMP-2 was subsequently immobilized onto the mesh scaffolds via heparin, and released at a controlled rate. Human placenta-derived mesenchymal stem cells (hPMSCs) were cultured in such scaffolds and subjected to osteogenic differentiation for 28 days in vitro. The results showed that alkaline phosphatase (ALP) activity, mineralization, and osteogenic marker expression were significantly improved with hPMSCs cultured in the hFDM-coated mesh scaffolds compared to the control and fibronectin-coated ones. In addition, a mouse ectopic and rat calvarial bone defect model was used to examine the feasibility of current platform to induce osteogenesis as well as bone regeneration. All hFDM-coated mesh groups exhibited a significant increase of newly formed bone and in particular, hFDM-coated mesh scaffold loaded with a high dose of BMP-2 exhibited a nearly complete bone defect healing as confirmed via micro-CT and histological observation. This work proposes a great potency of using hFDM (biophysical) coupled with BMP-2 (biochemical) as a promising osteogenic microenvironment for bone tissue engineering applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Knockout of endothelial cell-derived endothelin-1 attenuates skin fibrosis but accelerates cutaneous wound healing.

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    Katsunari Makino

    Full Text Available Endothelin (ET-1 is known for the most potent vasoconstrictive peptide that is released mainly from endothelial cells. Several studies have reported ET-1 signaling is involved in the process of wound healing or fibrosis as well as vasodilation. However, little is known about the role of ET-1 in these processes. To clarify its mechanism, we compared skin fibrogenesis and wound repair between vascular endothelial cell-specific ET-1 knockout mice and their wild-type littermates. Bleomycin-injected fibrotic skin of the knockout mice showed significantly decreased skin thickness and collagen content compared to that of wild-type mice, indicating that bleomycin-induced skin fibrosis is attenuated in the knockout mice. The mRNA levels of transforming growth factor (TGF-β were decreased in the bleomycin-treated skin of ET-1 knockout mice. On the other hand, skin wound healing was accelerated in ET-1 knockout mice, which was indicated by earlier granulation tissue reduction and re-epithelialization in these mice. The mRNA levels of TGF-β, tumor necrosis factor (TNF-α and connective tissue growth factor (CTGF were reduced in the wound of ET-1 knockout mice. In endothelial ET-1 knockout mouse, the expression of TNF-α, CTGF and TGF-β was down-regulated. Bosentan, an antagonist of dual ET receptors, is known to attenuate skin fibrosis and accelerate wound healing in systemic sclerosis, and such contradictory effect may be mediated by above molecules. The endothelial cell-derived ET-1 is the potent therapeutic target in fibrosis or wound healing, and investigations of the overall regulatory mechanisms of these pathological conditions by ET-1 may lead to a new therapeutic approach.

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

    Science.gov (United States)

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

    2002-01-01

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

  10. Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Kucera, Jan P; Prudat, Yann; Marcu, Irene C; Azzarito, Michela; Ullrich, Nina D

    2015-01-01

    Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs). However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs) and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV) was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands. CV was significantly lower in strands composed purely of SCMs (5.5 ± 1.5 cm/s, n = 11) as compared to PCMs (34.9 ± 2.9 cm/s, n = 21) at similar refractoriness (100% SCMs: 122 ± 25 ms, n = 9; 100% PCMs: 139 ± 67 ms, n = 14). In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV. These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

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

    Science.gov (United States)

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

    2012-12-01

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

  12. Degradation of amyloid beta by human induced pluripotent stem cell-derived macrophages expressing Neprilysin-2

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    Koutaro Takamatsu

    2014-11-01

    Full Text Available The purpose of this study was to evaluate the therapeutic potential of human induced pluripotent stem (iPS cell-derived macrophage-like cells for Alzheimer's disease (AD. In previous studies, we established the technology to generate macrophage-like myeloid lineage cells with proliferating capacity from human iPS cells, and we designated the cells iPS-ML. iPS-ML reduced the level of Aβ added into the culture medium, and the culture supernatant of iPS-ML alleviated the neurotoxicity of Aβ. We generated iPS-ML expressing the Fc-receptor-fused form of a single chain antibody specific to Aβ. In addition, we made iPS-ML expressing Neprilysin-2 (NEP2, which is a protease with Aβ-degrading activity. In vitro, expression of NEP2 but not anti-Aβ scFv enhanced the effect to reduce the level of soluble Aβ oligomer in the culture medium and to alleviate the neurotoxicity of Aβ. To analyze the effect of iPS-ML expressing NEP2 (iPS-ML/NEP2 in vivo, we intracerebrally administered the iPS-ML/NEP2 to 5XFAD mice, which is a mouse model of AD. We observed significant reduction in the level of Aβ in the brain interstitial fluid following administration of iPS-ML/NEP2. These results suggested that iPS-ML/NEP2 may be a potential therapeutic agent in the treatment of AD.

  13. Circulating apoptotic endothelial cell-derived microparticles are predicted metabolically unhealthy obesity

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    Alexander E. Berezin

    2017-01-01

    Full Text Available Introduction: Circulating apoptotic endothelial cell-derived micro particles (EMPs are a marker of endothelial dysfunction and cardiovascular (CV risk in type 2 diabetes mellitus patients. There is evidence regarding association between apoptotic EMP number and CV disease in obese individuals. The aim of the study to investigate whether increased number of circulating apoptotic EMPs may predict transformation of Met-HO into Met-UHO. Methods: The study was retrospectively evolved 89 patients with established abdominal obesity (47 patients with Met-UHO determined as MetS and 42 subjects with Met-HO from the large cohort of abdominal obesity patients (n=268. Thirty five healthy volunteers matched for age and sex were involved in the study as a control cohort. Obesity-related biomarker (adiponectin, leptin, vistafin and EMPs were measured at baseline. Flow cytometry was used to determine EMPs with immune phenotype CD31+/annexin V+ and CD144+/annexin V+. Results: There was not found a significant difference between numbers of EMPs labeled CD31+/ Annexin V+ in Met-UHO and Met-HO patients, while Met-UHO patients had a significantly increased level of circulating CD144+/ Annexin V+ compared with Met-HO individuals. Multivariate logistic regression analysis has revealed the HOMA-IR, number of CV risk factors, serum leptin and hs-CRP independently predicted numbers of circulating CD31+/ Annexin V+ and CD144+/ Annexin V+ EMPs in Met-UHO. In Met-HO patients HOMA-IR remained an independent predictor of increased numbers of circulating CD31+/ Annexin V+ and CD144+/ Annexin V+ EMPs. Conclusion: in the investigation we found that the increased number of CD31+/Annexin V+ and CD144+/ Annexin V+ EMPs added to the based predictive model (HOMA-IR may predict transformation of Met-HO into Met-UHO.

  14. Interactions of human embryonic stem cell-derived cardiovascular progenitor cells with immobilized extracellular matrix proteins.

    Science.gov (United States)

    Lu, Jizhen; Kaestle, Katrin; Huang, Jijun; Liu, Qiao; Zhang, Peng; Gao, Ling; Gardiner, James; Thissen, Helmut; Yang, Huang-Tian

    2017-04-01

    Human embryonic stem cell-derived cardiovascular progenitor cells (hESC-CVPCs) hold great promise for cell-based therapies of heart diseases. However, little is known about their niche microenvironment and in particular the required extracellular matrix (ECM) components. Here we screened combinations of surface-immobilized ECM proteins to identify substrates that support the attachment and survival of hESC-CVPCs. Covalent immobilization of ECM proteins laminin (Lm), fibronectin (Fn), collagen I (CI), collagen III (CIII), and collagen IV (CIV) in multiple combinations and concentrations was achieved by reductive amination on transparent acetaldehyde plasma polymer (AAPP) interlayer coatings. We identified that CI, CIII, CIV, and Fn and their combinations were important for hESC-CVPC attachment and survival, while Lm was dispensable. Moreover, for coatings displaying single ECM proteins, CI and CIII performed better than CIV and Fn, while coatings displaying the combined ECM proteins CIII + CIV and Fn + CIII + CIV at 100 µg/mL were comparable to Matrigel in regard to supporting hESC-CVPC attachment and viability. Our results identify ECM proteins required for hESC-CVPCs and demonstrate that coatings displaying multiple immobilized ECM proteins offer a suitable microenvironment for the attachment and survival of hESC-CVPCs. This knowledge contributes to the development of approaches for maintaining hESC-CVPCs and therefore to advances in cardiovascular regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1094-1104, 2017. © 2017 Wiley Periodicals, Inc.

  15. Exogenous stromal cell-derived factor-1 induces modest leukocyte recruitment in vivo.

    Science.gov (United States)

    Kerfoot, Steven M; Andonegui, Graciela; Bonder, Claudine S; Liu, Lixin

    2008-06-01

    Stromal cell-derived factor-1 (SDF-1; CXCL12), a CXC chemokine, has been found to be involved in inflammation models in vivo and in cell adhesion, migration, and chemotaxis in vitro. This study aimed to determine whether exogenous SDF-1 induces leukocyte recruitment in mice. After systemic administration of SDF-1alpha, expression of the adhesion molecules P-selectin and VCAM-1 in mice was measured using a quantitative dual-radiolabeled Ab assay and leukocyte recruitment in various tissues was evaluated using intravital microscopy. The effect of local SDF-1alpha on leukocyte recruitment was also determined in cremaster muscle and compared with the effect of the cytokine TNFalpha and the CXC chemokine keratinocyte-derived chemokine (KC; CXCL1). Systemic administration of SDF-1alpha (10 microg, 4-5 h) induced upregulation of P-selectin, but not VCAM-1, in most tissues in mice. It caused modest leukocyte recruitment responses in microvasculature of cremaster muscle, intestine, and brain, i.e., an increase in flux of rolling leukocytes in cremaster muscle and intestines, leukocyte adhesion in all three tissues, and emigration in cremaster muscle. Local treatment with SDF-1alpha (1 microg, 4-5 h) reduced leukocyte rolling velocity and increased leukocyte adhesion and emigration in cremasteric venules, but the responses were much less profound than those elicited by KC or TNFalpha. SDF-1alpha-induced recruitment was dependent on endothelial P-selectin, but not P-selectin on platelets. We conclude that the exogenous SDF-1alpha enhances leukocyte-endothelial cell interactions and induces modest and endothelial P-selectin-dependent leukocyte recruitment.

  16. Morphological Analysis of Live Undifferentiated Cells Derived from Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Osawa, Yukihiko; Miyamoto, Tomoyuki; Ohno, Setsuyo; Ohno, Eiji

    2017-12-05

    Induced pluripotent stem (iPS) cells possess pluripotency and self-renewal ability. Therefore, iPS cells are expected to be useful in regenerative medicine. However, iPS cells form malignant immature teratomas after transplantation into animals, even after differentiation induction. It has been suggested that undifferentiated cells expressing Nanog that remain after differentiation induction are responsible for teratoma formation. Various methods of removing these undifferentiated cells have therefore been investigated, but few methods involve morphological approaches, which may induce less cell damage. In addition, for cells derived from iPS cells to be applied in regenerative medicine, they must be alive. However, detailed morphological analysis of live undifferentiated cells has not been performed. For the above reasons, we assessed the morphological features of live undifferentiated cells remaining after differentiation induction as a basic investigation into the clinical application of iPS cells. As a result, live undifferentiated cells remaining after differentiation induction exhibited a round or oval cytoplasm about 12 μm in diameter and a nucleus. They exhibited nucleo-cytoplasmic (N/C) ratio of about 60% and eccentric nuclei, and they possessed partially granule-like structures in the cytoplasm and prominent nucleoli. Although they were similar to iPS cells, they were smaller than live iPS cells. Furthermore, very small cells were present among undifferentiated cells after differentiation induction. These results suggest that the removal of undifferentiated cells may be possible using the morphological features of live iPS cells and undifferentiated cells after differentiation induction. In addition, this study supports safe regenerative medicine using iPS cells.

  17. Functional and Transcriptional Characterization of Histone Deacetylase Inhibitor-Mediated Cardiac Adverse Effects in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

    Science.gov (United States)

    Kopljar, Ivan; Gallacher, David J; De Bondt, An; Cougnaud, Laure; Vlaminckx, Eddy; Van den Wyngaert, Ilse; Lu, Hua Rong

    2016-05-01

    Histone deacetylase (HDAC) inhibitors possess therapeutic potential to reverse aberrant epigenetic changes associated with cancers, neurological diseases, and immune disorders. Unfortunately, clinical studies with some HDAC inhibitors displayed delayed cardiac adverse effects, such as atrial fibrillation and ventricular tachycardia. However, the underlying molecular mechanism(s) of HDAC inhibitor-mediated cardiotoxicity remains poorly understood and is difficult to detect in the early stages of preclinical drug development because of a delayed onset of effects. In the present study, we show for the first time in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) that HDAC inhibitors (dacinostat, panobinostat, vorinostat, entinostat, and tubastatin-a) induce delayed dose-related cardiac dysfunction at therapeutic concentrations associated with cardiac adverse effects in humans. HDAC inhibitor-mediated delayed effects on the beating properties of hiPS-CMs developed after 12 hours by decreasing the beat rate, shortening the field potential duration, and inducing arrhythmic behavior under form of sustained contractions and fibrillation-like patterns. Transcriptional changes that are common between the cardiotoxic HDAC inhibitors but different from noncardiotoxic treatments identified cardiac-specific genes and pathways related to structural and functional changes in cardiomyocytes. Combining the functional data with epigenetic changes in hiPS-CMs allowed us to identify molecular targets that might explain HDAC inhibitor-mediated cardiac adverse effects in humans. Therefore, hiPS-CMs represent a valuable translational model to assess HDAC inhibitor-mediated cardiotoxicity and support identification of better HDAC inhibitors with an improved benefit-risk profile. Histone deacetylase (HDAC) inhibitors are a promising class of drugs to treat certain cancers, autoimmune, and neurodegenerative diseases. However, treated patients can experience various

  18. Human endometrial milk fat globule-epidermal growth factor 8 (MFGE8) is up regulated by estradiol at the transcriptional level, and its secretion via microvesicles is stimulated by human chorionic gonadotropin (hCG)

    KAUST Repository

    Sarhan, Abbaa

    2013-10-17

    Objective: We have recently showed that MFGE8, a novel epithelial cell protein in the human endometrium, upregulated during the window of implantation. We hypothesized that MFGE8 may act as a key modulator of endometrial remodeling and trophoblast invasion. The aims of this study were (i) to investigate the in vitro regulation of human endometrial epithelial cells MFGE8 transcription, translation, and secretion by sex steroids and hCG; and (ii) to examine the possibility of MFGE8 secretion via microvesicles. Design: Experimental in vitro study using Ishikawa cells. Setting: University center. Interventions: Treatment with estradiol (E2), progesterone (P4), and human chorionic gonatropin (hCG). Main outcome measures: MFGE8 mRNA and protein expression, and identification of secreted microvesicles by mass spectrometry (MS) and immunoblotting. Results: E2, but not P4 or hCG, significantly upregulated MFGE8 mRNA expression. hCG significantly increased MFGE8 secretion. Microvesicels obtained after ultracentrifugation were visualized with atomic force microscopy ranging from ~100 to 200 nm. In addition to the expected 46 kD protein, the microvesicles contained a second form of secreted MFGE8 measuring ~30 kD which was confirmed by MS. Conclusions: We demonstrated (i) dual effects of E2 and hCG on the regulation of MFGE8, and (ii) MFGE8 protein secretion in association with microvesicles. MFGE8 has the potential to modulate endometrial function and implantation via exocrine and/ or paracrine-autocrine effects. To the best of our knowledge, this is the first demonstration of microvesicular secretion of any regulatory protein by endometrial epithelial cells, providing initial evidence suggestive of microvesicular participation in cellular trafficking information in the non-pregnant and pregnant endometrium.

  19. Signaling by Extracellular Vesicles Advances Cancer Hallmarks.

    Science.gov (United States)

    Kanada, Masamitsu; Bachmann, Michael H; Contag, Christopher H

    2016-02-01

    Mammalian cells secrete various extracellular vesicles (EVs; exosomes, microvesicles, and apoptotic bodies) that differ in biogenesis, composition, and function. Each vesicle type can originate from normal or cancerous cells, transfer molecular cargo to both neighboring and distant cells, and modulate cellular behaviors involved in eubiology and pathology, such as tumor development. Here, we review evidence for the role of EVs in the establishment and maintenance of cancer hallmarks, including sustaining proliferative signaling, evading growth suppression, resisting cell death, reprogramming energy metabolism, acquiring genomic instability, and remodeling the tumor microenvironment. We also discuss how EVs are implicated in the induction of angiogenesis, control of cellular invasion, initiation of premetastatic niches, maintenance of inflammation, and evasion of immune surveillance. The deeper understanding of the biology of EVs and their contribution to the development and progression of tumors is leading to new opportunities in the diagnosis and treatment of cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Role of leukocyte cell-derived chemotaxin 2 as a biomarker in hepatocellular carcinoma.

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    Hirohisa Okabe

    Full Text Available We sought to identify a secreted biomarker for β-catenin activation commonly seen in hepatocellular carcinoma (HCC. By examination of our previously published genearray of hepatocyte-specific β-catenin knockout (KO livers, we identified secreted factors whose expression may be β-catenin-dependent. We verified expression and secretion of the leading factor in HCC cells transfected with mutated (Hep3BS33Y-β-catenin. Serum levels of biomarker were next investigated in a mouse model of HCC with β-catenin gene (Ctnnb1 mutations and eventually in HCC patients. Leukocyte cell-derived chemotaxin-2 (LECT2 expression was decreased in KO livers. Hep3BS33Y expressed and secreted more LECT2 in media as compared to Hep3BWT. Mice developing HCC with Ctnnb1 mutations showed significantly higher serum LECT2 levels. However patients with CTNNB1 mutations showed LECT2 levels of 54.28 ± 22.32 ng/mL (Mean ± SD; n = 8 that were insignificantly different from patients with non-neoplastic chronic liver disease (32.8 ± 21.1 ng/mL; n = 15 or healthy volunteers (33.2 ± 7.2 ng/mL; n = 11. Intriguingly, patients without β-catenin mutations showed significantly higher serum LECT2 levels (54.26 ± 22.25 ng/mL; n = 46. While β-catenin activation was evident in a subset of non-mutant β-catenin HCC group with high LECT2 expression, serum LECT2 was unequivocally similar between β-catenin-active and -normal group. Further analysis showed that LECT2 levels greater than 50 ng/ml diagnosed HCC in patients irrespective of β-catenin mutations with specificity of 96.1% and positive predictive value of 97.0%. Thus, LECT2 is regulated by β-catenin in HCC in both mice and men, but serum LECT2 reflects β-catenin activity only in mice. Serum LECT2 could be a potential biomarker of HCC in patients.

  1. Evaluation of Optogenetic Electrophysiology Tools in Human Stem Cell-Derived Cardiomyocytes

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    Susann Björk

    2017-11-01

    Full Text Available Current cardiac drug safety assessments focus on hERG channel block and QT prolongation for evaluating arrhythmic risks, whereas the optogenetic approach focuses on the action potential (AP waveform generated by a monolayer of human cardiomyocytes beating synchronously, thus assessing the contribution of several ion channels on the overall drug effect. This novel tool provides arrhythmogenic sensitizing by light-induced pacing in combination with non-invasive, all-optical measurements of cardiomyocyte APs and will improve assessment of drug-induced electrophysiological aberrancies. With the help of patch clamp electrophysiology measurements, we aimed to investigate whether the optogenetic modifications alter human cardiomyocytes' electrophysiology and how well the optogenetic analyses perform against this gold standard. Patch clamp electrophysiology measurements of non-transduced stem cell-derived cardiomyocytes compared to cells expressing the commercially available optogenetic constructs Optopatch and CaViar revealed no significant changes in action potential duration (APD parameters. Thus, inserting the optogenetic constructs into cardiomyocytes does not significantly affect the cardiomyocyte's electrophysiological properties. When comparing the two methods against each other (patch clamp vs. optogenetic imaging we found no significant differences in APD parameters for the Optopatch transduced cells, whereas the CaViar transduced cells exhibited modest increases in APD-values measured with optogenetic imaging. Thus, to broaden the screen, we combined optogenetic measurements of membrane potential and calcium transients with contractile motion measured by video motion tracking. Furthermore, to assess how optogenetic measurements can predict changes in membrane potential, or early afterdepolarizations (EADs, cells were exposed to cumulating doses of E-4031, a hERG potassium channel blocker, and drug effects were measured at both spontaneous and

  2. Mesenchymal Stem Cell-Derived Exosomes Promote Fracture Healing in a Mouse Model.

    Science.gov (United States)

    Furuta, Taisuke; Miyaki, Shigeru; Ishitobi, Hiroyuki; Ogura, Toshihiko; Kato, Yoshio; Kamei, Naosuke; Miyado, Kenji; Higashi, Yukihito; Ochi, Mitsuo

    2016-12-01

    : Paracrine signaling by bone-marrow-derived mesenchymal stem cells (MSCs) plays a major role in tissue repair. Although the production of regulatory cytokines by MSC transplantation is a critical modulator of tissue regeneration, we focused on exosomes, which are extracellular vesicles that contain proteins and nucleic acids, as a novel additional modulator of cell-to-cell communication and tissue regeneration. To address this, we used radiologic imaging, histological examination, and immunohistochemical analysis to evaluate the role of exosomes isolated from MSC-conditioned medium (CM) in the healing process in a femur fracture model of CD9(-/-) mice, a strain that is known to produce reduced levels of exosomes. We found that the bone union rate in CD9(-/-) mice was significantly lower than wild-type mice because of the retardation of callus formation. The retardation of fracture healing in CD9(-/-) mice was rescued by the injection of exosomes, but this was not the case after the injection of exosomes-free conditioned medium (CM-Exo). The levels of the bone repair-related cytokines, monocyte chemotactic protein-1 (MCP-1), MCP-3, and stromal cell-derived factor-1 in exosomes were low compared with levels in CM and CM-Exo, suggesting that bone repair may be in part mediated by other exosome components, such as microRNAs. These results suggest that exosomes in CM facilitate the acceleration of fracture healing, and we conclude that exosomes are a novel factor of MSC paracrine signaling with an important role in the tissue repair process. This work focuses on exosomes, which are extracellular vesicles, as a novel additional modulator of cell-to-cell communication. This study evaluated the role of exosomes isolated from mesenchymal stem cell (MSC)-conditioned medium (MSC-CM) in the fracture-healing process of CD9(-/-) mice, a strain that is known to produce reduced levels of exosomes. Retardation of fracture healing in CD9(-/-) mice was rescued by the injection of

  3. Transplantation of Embryonic and Induced Pluripotent Stem Cell-Derived 3D Retinal Sheets into Retinal Degenerative Mice

    Directory of Open Access Journals (Sweden)

    Juthaporn Assawachananont

    2014-05-01

    Full Text Available In this article, we show that mouse embryonic stem cell- or induced pluripotent stem cell-derived 3D retinal tissue developed a structured outer nuclear layer (ONL with complete inner and outer segments even in an advanced retinal degeneration model (rd1 that lacked ONL. We also observed host-graft synaptic connections by immunohistochemistry. This study provides a “proof of concept” for retinal sheet transplantation therapy for advanced retinal degenerative diseases.

  4. The postischemic environment differentially impacts teratoma or tumor formation after transplantation of human embryonic stem cell-derived neural progenitors

    DEFF Research Database (Denmark)

    Seminatore, Christine; Polentes, Jerome; Ellman, Ditte

    2010-01-01

    Risk of tumorigenesis is a major obstacle to human embryonic and induced pluripotent stem cell therapy. Likely linked to the stage of differentiation of the cells at the time of implantation, formation of teratoma/tumors can also be influenced by factors released by the host tissue. We have...... analyzed the relative effects of the stage of differentiation and the postischemic environment on the formation of adverse structures by transplanted human embryonic stem cell-derived neural progenitors....

  5. Autocrine regulation of pulmonary inflammation by effector T-cell derived IL-10 during infection with respiratory syncytial virus.

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    Jie Sun

    2011-08-01

    Full Text Available Respiratory syncytial virus (RSV infection is the leading viral cause of severe lower respiratory tract illness in young infants. Clinical studies have documented that certain polymorphisms in the gene encoding the regulatory cytokine IL-10 are associated with the development of severe bronchiolitis in RSV infected infants. Here, we examined the role of IL-10 in a murine model of primary RSV infection and found that high levels of IL-10 are produced in the respiratory tract by anti-viral effector T cells at the onset of the adaptive immune response. We demonstrated that the function of the effector T cell -derived IL-10 in vivo is to limit the excess pulmonary inflammation and thereby to maintain critical lung function. We further identify a novel mechanism by which effector T cell-derived IL-10 controls excess inflammation by feedback inhibition through engagement of the IL-10 receptor on the antiviral effector T cells. Our findings suggest a potentially critical role of effector T cell-derived IL-10 in controlling disease severity in clinical RSV infection.

  6. Finding the rhythm of sudden cardiac death: new opportunities using induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Sallam, Karim; Li, Yingxin; Sager, Philip T; Houser, Steven R; Wu, Joseph C

    2015-06-05

    Sudden cardiac death is a common cause of death in patients with structural heart disease, genetic mutations, or acquired disorders affecting cardiac ion channels. A wide range of platforms exist to model and study disorders associated with sudden cardiac death. Human clinical studies are cumbersome and are thwarted by the extent of investigation that can be performed on human subjects. Animal models are limited by their degree of homology to human cardiac electrophysiology, including ion channel expression. Most commonly used cellular models are cellular transfection models, which are able to mimic the expression of a single-ion channel offering incomplete insight into changes of the action potential profile. Induced pluripotent stem cell-derived cardiomyocytes resemble, but are not identical, adult human cardiomyocytes and provide a new platform for studying arrhythmic disorders leading to sudden cardiac death. A variety of platforms exist to phenotype cellular models, including conventional and automated patch clamp, multielectrode array, and computational modeling. Induced pluripotent stem cell-derived cardiomyocytes have been used to study long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, hypertrophic cardiomyopathy, and other hereditary cardiac disorders. Although induced pluripotent stem cell-derived cardiomyocytes are distinct from adult cardiomyocytes, they provide a robust platform to advance the science and clinical care of sudden cardiac death. © 2015 American Heart Association, Inc.

  7. Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model.

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    Jan Tønnesen

    Full Text Available Intrastriatal grafts of stem cell-derived dopamine (DA neurons induce behavioral recovery in animal models of Parkinson's disease (PD, but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2 and halorhodopsin (NpHR, respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.

  8. Cell and Microvesicle Urine microRNA Deep Sequencing Profiles from Healthy Individuals: Observations with Potential Impact on Biomarker Studies.

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    Iddo Z Ben-Dov

    Full Text Available Urine is a potential source of biomarkers for diseases of the kidneys and urinary tract. RNA, including microRNA, is present in the urine enclosed in detached cells or in extracellular vesicles (EVs or bound and protected by extracellular proteins. Detection of cell- and disease-specific microRNA in urine may aid early diagnosis of organ-specific pathology. In this study, we applied barcoded deep sequencing to profile microRNAs in urine of healthy volunteers, and characterized the effects of sex, urine fraction (cells vs. EVs and repeated voids by the same individuals.Compared to urine-cell-derived small RNA libraries, urine-EV-derived libraries were relatively enriched with miRNA, and accordingly had lesser content of other small RNA such as rRNA, tRNA and sn/snoRNA. Unsupervised clustering of specimens in relation to miRNA expression levels showed prominent bundling by specimen type (urine cells or EVs and by sex, as well as a tendency of repeated (first and second void samples to neighbor closely. Likewise, miRNA profile correlations between void repeats, as well as fraction counterparts (cells and EVs from the same specimen were distinctly higher than correlations between miRNA profiles overall. Differential miRNA expression by sex was similar in cells and EVs.miRNA profiling of both urine EVs and sediment cells can convey biologically important differences between individuals. However, to be useful as urine biomarkers, careful consideration is needed for biofluid fractionation and sex-specific analysis, while the time of voiding appears to be less important.

  9. Bone Marrow Suppression by c-Kit Blockade Enhances Tumor Growth of Colorectal Metastases through the Action of Stromal Cell-Derived Factor-1

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    Kathrin Rupertus

    2012-01-01

    Full Text Available Background. Mobilization of c-Kit+ hematopoietic cells (HCs contributes to tumor vascularization. Whereas survival and proliferation of HCs are regulated by binding of the stem cell factor to its receptor c-Kit, migration of HCs is directed by stromal cell-derived factor (SDF-1. Therefore, targeting migration of HCs provides a promising new strategy of anti-tumor therapy. Methods. BALB/c mice (=16 were pretreated with an anti-c-Kit antibody followed by implantation of CT26.WT-GFP colorectal cancer cells into dorsal skinfold chambers. Animals (=8 additionally received a neutralizing anti-SDF-1 antibody. Animals (=8 treated with a control antibody served as controls. Investigations were performed using intravital fluorescence microscopy, immunohistochemistry, flow cytometry and western blot analysis. Results. Blockade of c-Kit significantly enhanced tumor cell engraftment compared to controls due to stimulation of tumor cell proliferation and invasion without markedly affecting tumor vascularization. C-Kit blockade significantly increased VEGF and CXCR4 expression within the growing tumors. Neutralization of SDF-1 completely antagonized this anti-c-Kit-associated tumor growth by suppression of tumor neovascularization, inhibition of tumor cell proliferation and reduction of muscular infiltration. Conclusion. Our study indicates that bone marrow suppression via anti-c-Kit pretreatment enhances tumor cell engraftment of colorectal metastases due to interaction with the SDF-1/CXCR4 pathway which is involved in HC-mediated tumor angiogenesis.

  10. The influence of static magnetic fields on canine and equine mesenchymal stem cells derived from adipose tissue.

    Science.gov (United States)

    Marędziak, Monika; Marycz, Krzysztof; Smieszek, Agnieszka; Lewandowski, Daniel; Toker, Nezir Yaşar

    2014-06-01

    The aim of this study was to evaluate the proliferation rate and morphological changes of adipose-derived mesenchymal stem cells of canine and equine origin (Eq- and CaAdMSC). Investigated cells were exposed to a static magnetic field (MF) with the intensity of 0.5 T. Proliferation activity of cells was determined with the Alamar Blue assay. Obtained results, normalized in respect to the control culture, showed that EqAdMSC exposed to MF maintained a high proliferation status, whereas proliferation activity of CaAdMSC cultured in the presence of MF was decreased. Estimations of population doubling time (PDT) also revealed that EqAdMSCs exposed to static MF achieved a twofold increase in the total number of cells in a shorter amount of time than the control culture. The PDT value obtained for investigated CaAdMSCs indicated that MF exposure resulted in the prolongation of population doubling time. Morphology of cells and cellular composition was investigated using a light inverted microscope and a fluorescent microscope. A scanning electron microscope was used for microvesicles (MVs) imaging. Obtained results showed that both cell types maintained fibroblastic morphology and did not reveal signs of apoptosis or necrosis. However, the MF had an influence on the MVs secretion. While EqAdMSCs propagated in the presence of MF were characterized by the abundant MVs presence, CaAdMSCs revealed poor secretory activity. The approach presented provides complex analysis, which enables one to determine changes in equine and canine cytophysiology.

  11. An overview of the role of microparticles/microvesicles in blood components: Are they clinically beneficial or harmful?

    Science.gov (United States)

    Burnouf, Thierry; Chou, Ming-Li; Goubran, Hadi; Cognasse, Fabrice; Garraud, Olivier; Seghatchian, Jerard

    2015-10-01

    Blood cells and tissues generate heterogeneous populations of cell-derived vesicles, ranging from approximately 50 nm to 1 µm in diameter. Under normal physiological conditions and as an essential part of an energy-dependent natural process, microparticles (MPs) are continuously shed into the circulation from membranes of all viable cells such as megakaryocytes, platelets, red blood cells, white blood cells and endothelial cells. MP shedding can also be triggered by pathological activation of inflammatory processes and activation of coagulation or complement systems, or even by shear stress in the circulation. Structurally, MPs have a bilayered phospholipid structure exposing coagulant-active phosphatidylserine and expressing various membrane receptors, and they serve as cell-to-cell shuttles for bioactive molecules such as lipids, growth factors, microRNAs, and mitochondria. It was established that ex vivo processing of blood into its components, involving centrifugation, processing by various apheresis procedures, leucoreduction, pathogen reduction, and finally storage in different media and different types of blood bags, can impact MP generation and content. This is mostly due to exposure of the collected blood to anticoagulant/storage media and due to shear stresses or activation, contact with artificial surfaces, or exposure to various leucocyte-removal filters and pathogen-reduction treatments. Such artificially generated MPs, which are added to the original pool of MPs collected from the donor, may exhibit specific functional characteristics, as MPs are not an inert element of blood components. Not surprisingly, MPs' roles and functionality are therefore increasingly seen to be fully relevant to the field of transfusion medicine, and as a parameter of blood safety that must be considered in haemovigilance programmes. Continual advancements in assessment methods of MPs and storage lesions are gradually leading to a better understanding of the impacts of

  12. Microvesicles released from fat-laden cells promote activation of hepatocellular NLRP3 inflammasome: A pro-inflammatory link between lipotoxicity and non-alcoholic steatohepatitis.

    Science.gov (United States)

    Cannito, Stefania; Morello, Elisabetta; Bocca, Claudia; Foglia, Beatrice; Benetti, Elisa; Novo, Erica; Chiazza, Fausto; Rogazzo, Mara; Fantozzi, Roberto; Povero, Davide; Sutti, Salvatore; Bugianesi, Elisabetta; Feldstein, Ariel E; Albano, Emanuele; Collino, Massimo; Parola, Maurizio

    2017-01-01

    Non-Alcoholic Fatty Liver Disease (NAFLD) is a major form of chronic liver disease in the general population in relation to its high prevalence among overweight/obese individuals and patients with diabetes type II or metabolic syndrome. NAFLD can progress to steatohepatitis (NASH), fibrosis and cirrhosis and end-stage of liver disease but mechanisms involved are still incompletely characterized. Within the mechanisms proposed to mediate the progression of NAFLD, lipotoxicity is believed to play a major role. In the present study we provide data suggesting that microvesicles (MVs) released by fat-laden cells undergoing lipotoxicity can activate NLRP3 inflammasome following internalization by either cells of hepatocellular origin or macrophages. Inflammasome activation involves NF-kB-mediated up-regulation of NLRP3, pro-caspase-1 and pro-Interleukin-1, then inflammasome complex formation and Caspase-1 activation leading finally to an increased release of IL-1β. Since the release of MVs from lipotoxic cells and the activation of NLRP3 inflammasome have been reported to occur in vivo in either clinical or experimental NASH, these data suggest a novel rational link between lipotoxicity and increased inflammatory response.

  13. Microvesicles released from fat-laden cells promote activation of hepatocellular NLRP3 inflammasome: A pro-inflammatory link between lipotoxicity and non-alcoholic steatohepatitis.

    Directory of Open Access Journals (Sweden)

    Stefania Cannito

    Full Text Available Non-Alcoholic Fatty Liver Disease (NAFLD is a major form of chronic liver disease in the general population in relation to its high prevalence among overweight/obese individuals and patients with diabetes type II or metabolic syndrome. NAFLD can progress to steatohepatitis (NASH, fibrosis and cirrhosis and end-stage of liver disease but mechanisms involved are still incompletely characterized. Within the mechanisms proposed to mediate the progression of NAFLD, lipotoxicity is believed to play a major role. In the present study we provide data suggesting that microvesicles (MVs released by fat-laden cells undergoing lipotoxicity can activate NLRP3 inflammasome following internalization by either cells of hepatocellular origin or macrophages. Inflammasome activation involves NF-kB-mediated up-regulation of NLRP3, pro-caspase-1 and pro-Interleukin-1, then inflammasome complex formation and Caspase-1 activation leading finally to an increased release of IL-1β. Since the release of MVs from lipotoxic cells and the activation of NLRP3 inflammasome have been reported to occur in vivo in either clinical or experimental NASH, these data suggest a novel rational link between lipotoxicity and increased inflammatory response.

  14. Endothelial cell-derived exosomes protect SH-SY5Y nerve cells against ischemia/reperfusion injury.

    Science.gov (United States)

    Xiao, Bing; Chai, Yi; Lv, Shigang; Ye, Minhua; Wu, Miaojing; Xie, Liyuan; Fan, Yanghua; Zhu, Xingen; Gao, Ziyun

    2017-10-01

    Cerebral ischemia is a leading cause of death and disability. A previous study indicated that remote ischemic postconditioning (RIP) in the treatment of cerebral ischemia reduces ischemia/reperfusion (I/R) injury. However, the underlying mechanism is not well understood. In the present study, the authors hypothesized that the protective effect of RIP on neurological damage is mediated by exosomes that are released by endothelial cells in femoral arteries. To test this, right middle cerebral artery occlusion/reperfusion with RIP was performed in rats. In addition, an I/R injury cell model was tested that included human umbilical vein endothelial cells (HUVECs) and SH-SY5Y cells. Both the in vivo and in vitro models were examined for injury. Markers of exosomes (CD63, HSP70 and TSG101) were assessed by immunohistochemistry, western blot analysis and flow cytometry. Exosomes were extracted from both animal serum and HUVEC culture medium and identified by electron microscopy. They investigated the role of endothelial cell-derived exosomes in the proliferation, apoptosis, cell cycle, migration and invasion of I/R-injured SH-SY5Y cells. In addition, apoptosis-related molecules caspase-3, Bax and Bcl-2 were detected. RIP was determined to increase the number of exosomes and the expression levels of CD63, HSP70 and TSG101 in plasma, but not in brain hippocampal tissue. The size of exosomes released after I/R in HUVECs was similar to the size of exosomes released in rats subjected to RIP. Endothelial cell-derived exosomes partly suppressed the I/R-induced cell cycle arrest and apoptosis, and inhibited cell proliferation, migration and invasion in SH-SY5Y nerve cells. Endothelial cell-derived exosomes directly protect nerve cells against I/R injury, and are responsible for the protective role of RIP in I/R.

  15. Β-amyloid 1-42 oligomers impair function of human embryonic stem cell-derived forebrain cholinergic neurons.

    Directory of Open Access Journals (Sweden)

    Linn Wicklund

    Full Text Available Cognitive impairment in Alzheimer's disease (AD patients is associated with a decline in the levels of growth factors, impairment of axonal transport and marked degeneration of basal forebrain cholinergic neurons (BFCNs. Neurogenesis persists in the adult human brain, and the stimulation of regenerative processes in the CNS is an attractive prospect for neuroreplacement therapy in neurodegenerative diseases such as AD. Currently, it is still not clear how the pathophysiological environment in the AD brain affects stem cell biology. Previous studies investigating the effects of the β-amyloid (Aβ peptide on neurogenesis have been inconclusive, since both neurogenic and neurotoxic effects on progenitor cell populations have been reported. In this study, we treated pluripotent human embryonic stem (hES cells with nerve growth factor (NGF as well as with fibrillar and oligomeric Aβ1-40 and Aβ1-42 (nM-µM concentrations and thereafter studied the differentiation in vitro during 28-35 days. The process applied real time quantitative PCR, immunocytochemistry as well as functional studies of intracellular calcium signaling. Treatment with NGF promoted the differentiation into functionally mature BFCNs. In comparison to untreated cells, oligomeric Aβ1-40 increased the number of functional neurons, whereas oligomeric Aβ1-42 suppressed the number of functional neurons. Interestingly, oligomeric Aβ exposure did not influence the number of hES cell-derived neurons compared with untreated cells, while in contrast fibrillar Aβ1-40 and Aβ1-42 induced gliogenesis. These findings indicate that Aβ1-42 oligomers may impair the function of stem cell-derived neurons. We propose that it may be possible for future AD therapies to promote the maturation of functional stem cell-derived neurons by altering the brain microenvironment with trophic support and by targeting different aggregation forms of Aβ.

  16. SAFETY OF CELL-DERIVED SUBUNIT ADJUVANTED INFLUENZA VACCINE FOR CHILDREN VACCINATION: DOUBLE-BLIND RANDOMIZED CLINICAL TRIAL

    Directory of Open Access Journals (Sweden)

    S.M. Kharit

    2010-01-01

    Full Text Available This article presents the safety data for cell-derived inactivated subunit adjuvanted influenza vaccine «Grippol Neo» in children 3–17 years old in comparison with reference egg-derived inactivated subunit vaccine «Grippol plus». Good test vaccine tolerability and high efficacy profile is demonstrated. Based on the results obtained vaccine «Grippol Neo» is recommended for mass influenza prophylaxis in pediatry, including National Immunization Schedule.Key words: children, influenza, vaccination, «Grippol Neo».(Voprosy sovremennoi pediatrii — Current Pediatrics. – 2010;9(4:44-49

  17. Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors

    DEFF Research Database (Denmark)

    Avaliani, Natalia; Sørensen, Andreas Toft; Ledri, Marco

    2014-01-01

    properties such as tetrodotoxin-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher...... input resistance, lower resting membrane potential, and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light...

  18. Human-induced pluripotent stem cell-derived cardiomyocytes from cardiac progenitor cells: effects of selective ion channel blockade.

    Science.gov (United States)

    Altomare, Claudia; Pianezzi, Enea; Cervio, Elisabetta; Bolis, Sara; Biemmi, Vanessa; Benzoni, Patrizia; Camici, Giovanni G; Moccetti, Tiziano; Barile, Lucio; Vassalli, Giuseppe

    2016-12-01

    Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are likely to revolutionize electrophysiological approaches to arrhythmias. Recent evidence suggests the somatic cell origin of hiPSCs may influence their differentiation potential. Owing to their cardiomyogenic potential, cardiac-stromal progenitor cells (CPCs) are an interesting cellular source for generation of hiPSC-derived cardiomyocytes. The effect of ionic current blockade in hiPSC-derived cardiomyocytes generated from CPCs has not been characterized yet. Human-induced pluripotent stem cell-derived cardiomyocytes were generated from adult CPCs and skin fibroblasts from the same individuals. The effect of selective ionic current blockade on spontaneously beating hiPSC-derived cardiomyocytes was assessed using multi-electrode arrays. Cardiac-stromal progenitor cells could be reprogrammed into hiPSCs, then differentiated into hiPSC-derived cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin showed higher upregulation of cardiac-specific genes compared with those of fibroblastic origin. Human-induced pluripotent stem cell-derived cardiomyocytes of both somatic cell origins exhibited sensitivity to tetrodotoxin, a blocker of Na(+ )current (INa), nifedipine, a blocker of L-type Ca(2+ )current (ICaL), and E4031, a blocker of the rapid component of delayed rectifier K(+ )current (IKr). Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin exhibited sensitivity to JNJ303, a blocker of the slow component of delayed rectifier K(+ )current (IKs). In hiPSC-derived cardiomyocytes of cardiac origin, INa, ICaL, IKr, and IKs were present as tetrodotoxin-, nifedipine-, E4031-, and JNJ303-sensitive currents, respectively. Although cardiac differentiation efficiency was improved in hiPSCs of cardiac vs. non-cardiac origin, no major functional differences were observed between hiPSC-derived cardiomyocytes of different somatic cell origins

  19. Correction: Magnetic Cell Labeling of Primary and Stem Cell-Derived Pig Hepatocytes for MRI-Based Cell Tracking of Hepatocyte Transplantation

    National Research Council Canada - National Science Library

    Dwayne R Roach; Wesley M Garrett; Glenn Welch; Dorela D Shuboni-Mulligan; Thomas J Caperna; Neil C Talbot; Erik M Shapiro

    2017-01-01

    Roach DR, Garrett WM, Welch G, Caperna TJ, Talbot NC, Shapiro EM (2015) Magnetic Cell Labeling of Primary and Stem Cell-Derived Pig Hepatocytes for MRI-Based Cell Tracking of Hepatocyte Transplantation. (2017) Correction...

  20. Neonatal mouse cortical but not isogenic human astrocyte feeder layers enhance the functional maturation of induced pluripotent stem cell-derived neurons in culture.

    Science.gov (United States)

    Lischka, Fritz W; Efthymiou, Anastasia; Zhou, Qiong; Nieves, Michael D; McCormack, Nikki M; Wilkerson, Matthew D; Sukumar, Gauthaman; Dalgard, Clifton L; Doughty, Martin L

    2018-04-01

    Human induced pluripotent stem (iPS) cell-derived neurons and astrocytes are attractive cellular tools for nervous system disease modeling and drug screening. Optimal utilization of these tools requires differentiation protocols that efficiently generate functional cell phenotypes in vitro. As nervous system function is dependent on networked neuronal activity involving both neuronal and astrocytic synaptic functions, we examined astrocyte effects on the functional maturation of neurons from human iPS cell-derived neural stem cells (NSCs). We first demonstrate human iPS cell-derived NSCs can be rapidly differentiated in culture to either neurons or astrocytes with characteristic cellular, molecular and physiological features. Although differentiated neurons were capable of firing multiple action potentials (APs), few cells developed spontaneous electrical activity in culture. We show spontaneous electrical activity was significantly increased by neuronal differentiation of human NSCs on feeder layers of neonatal mouse cortical astrocytes. In contrast, co-culture on feeder layers of isogenic human iPS cell-derived astrocytes had no positive effect on spontaneous neuronal activity. Spontaneous electrical activity was dependent on glutamate receptor-channel function and occurred without changes in I Na , I K , V m , and AP properties of iPS cell-derived neurons. These data demonstrate co-culture with neonatal mouse cortical astrocytes but not human isogenic iPS cell-derived astrocytes stimulates glutamatergic synaptic transmission between iPS cell-derived neurons in culture. We present RNA-sequencing data for an immature, fetal-like status of our human iPS cell-derived astrocytes as one possible explanation for their failure to enhance synaptic activity in our co-culture system. © 2017 Wiley Periodicals, Inc.

  1. Increased IgG on cell-derived plasma microparticles in systemic lupus erythematosus is associated with autoantibodies and complement activation

    DEFF Research Database (Denmark)

    Nielsen, Christoffer T; Østergaard, Ole; Stener, Line

    2012-01-01

    To quantify immunoglobulin and C1q on circulating cell-derived microparticles (MPs) in patients with systemic lupus erythematosus (SLE) and to determine whether immunoglobulin and C1q levels are correlated with clinical and serologic parameters.......To quantify immunoglobulin and C1q on circulating cell-derived microparticles (MPs) in patients with systemic lupus erythematosus (SLE) and to determine whether immunoglobulin and C1q levels are correlated with clinical and serologic parameters....

  2. Tumour-derived exosomes as a signature of pancreatic cancer - liquid biopsies as indicators of tumour progression.

    Science.gov (United States)

    Nuzhat, Zarin; Kinhal, Vyjayanthi; Sharma, Shayna; Rice, Gregory E; Joshi, Virendra; Salomon, Carlos

    2017-03-07

    Pancreatic cancer is the fourth most common cause of death due to cancer in the world. It is known to have a poor prognosis, mostly because early stages of the disease are generally asymptomatic. Progress in pancreatic cancer research has been slow, leaving several fundamental questions pertaining to diagnosis and treatment unanswered. Recent studies highlight the putative utility of tissue-specific vesicles (i.e. extracellular vesicles) in the diagnosis of disease onset and treatment monitoring in pancreatic cancer. Extracellular vesicles are membrane-limited structures derived from the cell membrane. They contain specific molecules including proteins, mRNA, microRNAs and non-coding RNAs that are secreted in the extracellular space. Extracellular vesicles can be classified according to their size and/or origin into microvesicles (~150-1000 nm) and exosomes (~40-120 nm). Microvesicles are released by budding from the plasmatic membrane, whereas exosomes are released via the endocytic pathway by fusion of multivesicular bodies with the plasmatic membrane. This endosomal origin means that exosomes contain an abundance of cell-specific biomolecules which may act as a 'fingerprint' of the cell of origin. In this review, we discuss our current knowledge in the diagnosis and treatment of pancreatic cancer, particularly the potential role of EVs in these facets of disease management. In particular, we suggest that as exosomes contain cellular protein and RNA molecules in a cell type-specific manner, they may provide extensive information about the signature of the tumour and pancreatic cancer progression.

  3. Teratocarcinomas Arising from Allogeneic Induced Pluripotent Stem Cell-Derived Cardiac Tissue Constructs Provoked Host Immune Rejection in Mice.

    Science.gov (United States)

    Kawamura, Ai; Miyagawa, Shigeru; Fukushima, Satsuki; Kawamura, Takuji; Kashiyama, Noriyuki; Ito, Emiko; Watabe, Tadashi; Masuda, Shigeo; Toda, Koichi; Hatazawa, Jun; Morii, Eiichi; Sawa, Yoshiki

    2016-01-14

    Transplantation of induced pluripotent stem cell-derived cardiac tissue constructs is a promising regenerative treatment for cardiac failure: however, its tumourigenic potential is concerning. We hypothesised that the tumourigenic potential may be eliminated by the host immune response after allogeneic cell transplantation. Scaffold-free iPSC-derived cardaic tissue sheets of C57BL/6 mouse origin were transplanted into the cardiac surface of syngeneic C57BL/6 mice and allogeneic BALB/c mice with or without tacrolimus injection. Syngeneic mice and tacrolimus-injected immunosuppressed allogeneic mice formed teratocarcinomas with identical phenotypes, characteristic, and time courses, as assessed by imaging tools including (18)F-fluorodeoxyglucose-positron emission tomography. In contrast, temporarily immunosuppressed allogeneic mice, following cessation of tacrolimus injection displayed diminished progression of the teratocarcinoma, accompanied by an accumulation of CD4/CD8-positive T cells, and finally achieved complete elimination of the teratocarcinoma. Our results indicated that malignant teratocarcinomas arising from induced pluripotent stem cell-derived cardiac tissue constructs provoked T cell-related host immune rejection to arrest tumour growth in murine allogeneic transplantation models.

  4. Stimulation of GABA-induced Ca2+ influx enhances maturation of human induced pluripotent stem cell-derived neurons.

    Directory of Open Access Journals (Sweden)

    David J Rushton

    Full Text Available Optimal use of patient-derived, induced pluripotent stem cells for modeling neuronal diseases is crucially dependent upon the proper physiological maturation of derived neurons. As a strategy to develop defined differentiation protocols that optimize electrophysiological function, we investigated the role of Ca(2+ channel regulation by astrocyte conditioned medium in neuronal maturation, using whole-cell patch clamp and Ca(2+ imaging. Standard control medium supported basic differentiation of induced pluripotent stem cell-derived neurons, as assayed by the ability to fire simple, single, induced action potentials. In contrast, treatment with astrocyte conditioned medium elicited complex and spontaneous neuronal activity, often with rhythmic and biphasic characteristics. Such augmented spontaneous activity correlated with astrocyte conditioned medium-evoked hyperpolarization and was dependent upon regulated function of L-, N- and R-type Ca(2+ channels. The requirement for astrocyte conditioned medium could be substituted by simply supplementing control differentiation medium with high Ca(2+ or γ-amino butyric acid (GABA. Importantly, even in the absence of GABA signalling, opening Ca(2+ channels directly using Bay K8644 was able to hyperpolarise neurons and enhance excitability, producing fully functional neurons. These data provide mechanistic insight into how secreted astrocyte factors control differentiation and, importantly, suggest that pharmacological modulation of Ca(2+ channel function leads to the development of a defined protocol for improved maturation of induced pluripotent stem cell-derived neurons.

  5. Isolation and characterization of human mesenchymal stem cells derived from shoulder tissues involved in rotator cuff tears.

    Science.gov (United States)

    Utsunomiya, Hajime; Uchida, Soshi; Sekiya, Ichiro; Sakai, Akinori; Moridera, Kuniaki; Nakamura, Toshitaka

    2013-03-01

    Recent studies report a relatively high failure rate for tendon-bone healing after rotator cuff repair. Several studies have investigated biologically augmented rotator cuff repair; however, none has shown the application of synovial mesenchymal stem cells for such repair. To demonstrate whether cells derived from shoulder tissues have mesenchymal stem cell properties and to identify which tissue is the best source of the mesenchymal stem cells. Controlled laboratory study. Forty-two patients with a diagnosed rotator cuff tear preoperatively were enrolled in this study. Human mesenchymal tissues were obtained during arthroscopic surgery for rotator cuff tears from 19 donors who met the inclusion criteria and had investigable amounts of tissue. Colony-forming units, yield obtained, expandability, differentiation potential, epitope profile, and gene expression were compared among the cells from 4 shoulder tissues: synovium of the glenohumeral joint, subacromial bursa, margin of the ruptured supraspinatus tendon, and residual tendon stump on the greater tuberosity (enthesis). The number of live passage 0 cells from whole tissue was significantly higher in cells derived from the subacromial bursa (P source. The findings indicate that the subacromial bursa is a good candidate for the source of mesenchymal stem cells in rotator cuff tears. Synovial cells from the subacromial bursa in patients with rotator cuff tears are a superior cell source in vitro, suggesting that mesenchymal stem cells from this tissue could be good candidates for biological augmentation of rotator cuff repair.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C.; Meer, Berend van; Ward-van Oostwaard, Dorien [Department of Anatomy and Embryology, Leiden University Medical Center, Leiden (Netherlands); Passier, Robert [Department of Anatomy and Embryology, Leiden University Medical Center, Leiden (Netherlands); MIRA, University of Twente (Netherlands); Tertoolen, Leon G.J.; Mummery, Christine L. [Department of Anatomy and Embryology, Leiden University Medical Center, Leiden (Netherlands); Casini, Simona, E-mail: s.casini@amc.uva.nl [Department of Anatomy and Embryology, Leiden University Medical Center, Leiden (Netherlands)

    2015-11-27

    One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation. - Highlights: • Dexamethasone accelerates Ca{sup 2+} transient decay in hESC-CMs. • Dexamethasone enhances SERCA and NCX function in hESC-CMs. • Dexamethasone increases force of contraction and sarcomere length in hESC-CMs. • Dexamethasone does not alter I{sub Ca,L} and action potential characteristics in hESC-CMs.

  7. Defined MicroRNAs Induce Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell-Derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Desy S. Lee

    2015-09-01

    Full Text Available Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs. We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo, to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling.

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

    Science.gov (United States)

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

    2015-10-01

    The human cerebral cortex is peculiar for a six-layered cellular-sheet structure with convolution, which is a consequence of neuronal migration. Dysfunctions of the pathways contributing to this mechanism typically lead to lissencephaly manifesting smooth brain surfaces. To investigate the unknown mechanism underlying neuronal migration disorders, we generated induced pluripotent stem (iPS) cells from two patients with lissencephaly. Whole gene expression study for iPS cells derived from a patient with a LIS1 deletion showed reduced expression of the coiled-coil-helix-coiled-coil-helix domain containing 2 gene (CHCHD2), which was also confirmed in iPS cells derived from a patient with a TUBA1A mutation. CHCHD2 expression was detected in neuronal cells differentiated from normal iPS cells in a time-dependent manner, as well as in the brain of a fetus at 26-28 week gestational age, suggesting development-dependent expression. Migrating neuronal cells showed CHCHD2 expression, suggesting its functional relevance to neuronal migration. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Genome-Edited Human Pluripotent Stem Cell-Derived Macrophages as a Model of Reverse Cholesterol Transport--Brief Report.

    Science.gov (United States)

    Gupta, Rajat M; Meissner, Torsten B; Cowan, Chad A; Musunuru, Kiran

    2016-01-01

    To create isogenic human pluripotent stem cell-derived macrophages with and without ABCA1 expression as a model for reverse cholesterol transport. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) genome-editing system was used to introduce frameshift mutations into the coding sequence of ATP-binding cassette, subfamily A, member 1. Individual human pluripotent stem cell clones with deleterious mutations were identified, expanded, and differentiated into mature macrophages with a cytokine-based, feeder-free differentiation protocol. Wild-type cells demonstrated effective cholesterol efflux to apoAI acceptor, whereas ABCA1(-/-) cells displayed significantly reduced efflux ability and increased expression of proinflammatory cytokines. Human pluripotent stem cell-derived macrophages capable of reverse cholesterol transport can be rapidly generated and genetically edited with CRISPR/Cas9. Introduction of homozygous frameshift mutations results in loss of ABCA1 expression in differentiated macrophages and subsequent reduction of cholesterol efflux capability. This facile genome-editing approach and differentiation protocol pave the way for future studies of the molecular determinants of reverse cholesterol transport and other macrophage properties. © 2015 American Heart Association, Inc.

  10. Airway epithelial cell-derived insulin-like growth factor-1 triggers skewed CD8(+) T cell polarization.

    Science.gov (United States)

    Zou, Jian-Yong; Huang, Shao-hong; Li, Yun; Chen, Hui-guo; Rong, Jian; Ye, Sheng

    2014-10-01

    Skewed CD8(+) T cell responses are important in airway inflammation. This study investigates the role of the airway epithelial cell-derived insulin-like growth factor 1 (IGF1) in contributing to CD8(+) T cell polarization. Expression of IGF1 in the airway epithelial cell line, RPMI2650 cells, was assessed by quantitative real time RT-PCR and Western blotting. The role of IGF1 in regulating CD8(+) T cell activation was observed by coculture of mite allergen-primed RPMI2650 cells and naïve CD8(+) T cells. CD8(+) T cell polarization was assessed by the carboxyfluorescein succinimidyl ester-dilution assay and the determination of cytotoxic cytokine levels in the culture medium. Exposure to mite allergen, Der p1, increased the expression of IGF1 by RPMI2650 cells. The epithelial cell-derived IGF1 prevented the activation-induced cell death by inducing the p53 gene hypermethylation. Mite allergen-primed RPMI2650 cells induced an antigen-specific CD8(+) T cell polarization. We conclude that mite allergens induce airway epithelial cell line, RPMI2650 cells, to produce IGF1; the latter contributes to antigen-specific CD8(+) T cell polarization. © 2014 International Federation for Cell Biology.

  11. From beat rate variability in induced pluripotent stem cell-derived pacemaker cells to heart rate variability in human subjects.

    Science.gov (United States)

    Ben-Ari, Meital; Schick, Revital; Barad, Lili; Novak, Atara; Ben-Ari, Erez; Lorber, Avraham; Itskovitz-Eldor, Joseph; Rosen, Michael R; Weissman, Amir; Binah, Ofer

    2014-10-01

    We previously reported that induced pluripotent stem cell-derived cardiomyocytes manifest beat rate variability (BRV) resembling heart rate variability (HRV) in the human sinoatrial node. We now hypothesized the BRV-HRV continuum originates in pacemaker cells. To investigate whether cellular BRV is a source of HRV dynamics, we hypothesized 3 levels of interaction among different cardiomyocyte entities: (1) single pacemaker cells, (2) networks of electrically coupled pacemaker cells, and (3) the in situ sinoatrial node. We measured BRV/HRV properties in single pacemaker cells, induced pluripotent stem cell-derived contracting embryoid bodies (EBs), and electrocardiograms from the same individual. Pronounced BRV/HRV was present at all 3 levels. The coefficient of variance of interbeat intervals and Poincaré plot indices SD1 and SD2 for single cells were 20 times greater than those for EBs (P heart (the latter two were similar; P > .05). We also compared BRV magnitude among single cells, small EBs (~5-10 cells), and larger EBs (>10 cells): BRV indices progressively increased with the decrease in the cell number (P heart rhythm. The decreased BRV magnitude in transitioning from the single cell to the EB suggests that the HRV of in situ hearts originates from the summation and integration of multiple cell-based oscillators. Hence, complex interactions among multiple pacemaker cells and intracellular Ca(2+) handling determine HRV in humans and cardiomyocyte networks. Copyright © 2014 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  12. Dendritic cell-derived exosomes elicit tumor regression in autochthonous hepatocellular carcinoma mouse models.

    Science.gov (United States)

    Lu, Zhen; Zuo, Bingfeng; Jing, Renwei; Gao, Xianjun; Rao, Quan; Liu, Zhili; Qi, Han; Guo, Hongxing; Yin, HaiFang

    2017-10-01

    Dendritic cell (DC)-derived exosomes (DEXs) form a new class of vaccines for cancer immunotherapy. However, their potency in hepatocellular carcinoma (HCC), a life-threatening malignancy with limited treatment options in the clinic that responds poorly to immunotherapy, remains to be investigated. Exosomes derived from α-fetoprotein (AFP)-expressing DCs (DEXAFP) were investigated in three different HCC mouse models systemically. Tumor growth and microenvironment were monitored. DEXAFP elicited strong antigen-specific immune responses and resulted in significant tumor growth retardation and prolonged survival rates in mice with ectopic, orthotopic and carcinogen-induced HCC tumors that displayed antigenic and pathological heterogeneity. The tumor microenvironment was improved in DEXAFP-treated HCC mice, demonstrated by significantly more γ-interferon (IFN-γ)-expressing CD8+ T lymphocytes, elevated levels of IFN-γ and interleukin-2, and fewer CD25+Foxp3+ regulatory T (Treg) cells and decreased levels of interleukin-10 and transforming growth factor-β in tumor sites. Lack of efficacy in athymic nude mice and CD8+ T cell-depleted mice showed that T cells contribute to DEXAFP-mediated antitumor function. Dynamic examination of the antitumor efficacy and the immune microenvironment in DEXAFP-treated orthotopic HCC mice at different time-points revealed a positive correlation between tumor suppression and immune microenvironment. Our findings provide evidence that AFP-enriched DEXs can trigger potent antigen-specific antitumor immune responses and reshape the tumor microenvironment in HCC mice and thus provide a cell-free vaccine option for HCC immunotherapy. Lay summary: Dendritic cell (DC)-derived exosomes (DEXs) form a new class of vaccines for cancer immunotherapy. However, their potency in hepatocellular carcinoma (HCC) remains unknown. Here, we investigated exosomes from HCC antigen-expressing DCs in three different HCC mouse models and proved their feasibility

  13. Induced Pluripotent Stem Cell-Derived Megakaryocytes and Platelets for Disease Modeling and Clinical Use.

    Science.gov (United States)

    Borst, Sara; Sim, Xiuli; Poncz, Mortimer; French, Deborah L; Gadue, Paul

    2017-10-05

    Platelets, derived from megakaryocytes, are anucleate cytoplasmic discs that circulate in the blood stream and play major roles in hemostasis, inflammation, and vascular biology. Platelet transfusions are used in a variety of medical settings to prevent life-threatening thrombocytopenia because of cancer therapy, other causes of acquired or inherited thrombocytopenia, and trauma. Currently, platelets used for transfusion purposes are donor derived. However, there is a drive to generate nondonor sources of platelets to help supplement donor-derived platelets. Efforts have been made by many laboratories to generate in vitro platelets and optimize their production and quality. In vitro-derived platelets have the potential to be a safer, more uniform product, and genetic manipulation could allow for better treatment of patients who become refractory to donor-derived units. This review focuses on potential clinical applications of in vitro-derived megakaryocytes and platelets, current methods to generate and expand megakaryocytes from pluripotent stem cell sources, and the use of these cells for disease modeling. © 2017 American Heart Association, Inc.

  14. Mesenchymal Stem Cell-Derived Extracellular Vesicles: Roles in Tumor Growth, Progression, and Drug Resistance

    Directory of Open Access Journals (Sweden)

    Xiaoyan Zhang

    2017-01-01

    Full Text Available Mesenchymal stem cells (MSCs are ubiquitously present in many tissues. Due to their unique advantages, MSCs have been widely employed in clinical studies. Emerging evidences indicate that MSCs can also migrate to the tumor surrounding stroma and exert complex effects on tumor growth and progression. However, the effect of MSCs on tumor growth is still a matter of debate. Several studies have shown that MSCs could favor tumor growth. On the contrary, other groups have demonstrated that MSCs suppressed tumor progression. Extracellular vesicles have emerged as a new mechanism of cell-to-cell communication in the development of tumor diseases. MSCs-derived extracellular vesicles (MSC-EVs could mimic the effects of the mesenchymal stem cells from which they originate. Different studies have reported that MSC-EVs may exert various effects on the growth, metastasis, and drug response of different tumor cells by transferring proteins, messenger RNA, and microRNA to recipient cells. In the present review, we summarize the components of MSC-EVs and discuss the roles of MSC-EVs in different malignant diseases, including the related mechanisms that may account for their therapeutic potential. MSC-EVs open up a promising opportunity in the treatment of cancer with increased efficacy.

  15. Large-scale models of signal propagation in human cells derived from discovery phosphoproteomic data.

    Science.gov (United States)

    Terfve, Camille D A; Wilkes, Edmund H; Casado, Pedro; Cutillas, Pedro R; Saez-Rodriguez, Julio

    2015-09-10

    Mass spectrometry is widely used to probe the proteome and its modifications in an untargeted manner, with unrivalled coverage. Applied to phosphoproteomics, it has tremendous potential to interrogate phospho-signalling and its therapeutic implications. However, this task is complicated by issues of undersampling of the phosphoproteome and challenges stemming from its high-content but low-sample-throughput nature. Hence, methods using such data to reconstruct signalling networks have been limited to restricted data sets and insights (for example, groups of kinases likely to be active in a sample). We propose a new method to handle high-content discovery phosphoproteomics data on perturbation by putting it in the context of kinase/phosphatase-substrate knowledge, from which we derive and train logic models. We show, on a data set obtained through perturbations of cancer cells with small-molecule inhibitors, that this method can study the targets and effects of kinase inhibitors, and reconcile insights obtained from multiple data sets, a common issue with these data.

  16. Suppressive effects of tumor cell-derived 5'-deoxy-5'-methylthioadenosine on human T cells.

    Science.gov (United States)

    Henrich, Frederik C; Singer, Katrin; Poller, Kerstin; Bernhardt, Luise; Strobl, Carolin D; Limm, Katharina; Ritter, Axel P; Gottfried, Eva; Völkl, Simon; Jacobs, Benedikt; Peter, Katrin; Mougiakakos, Dimitrios; Dettmer, Katja; Oefner, Peter J; Bosserhoff, Anja-Katrin; Kreutz, Marina P; Aigner, Michael; Mackensen, Andreas

    2016-08-01

    The immunosuppressive tumor microenvironment represents one of the main obstacles for immunotherapy of cancer. The tumor milieu is among others shaped by tumor metabolites such as 5'-deoxy-5'-methylthioadenosine (MTA). Increased intratumoral MTA levels result from a lack of the MTA-catabolizing enzyme methylthioadenosine phosphorylase (MTAP) in tumor cells and are found in various tumor entities. Here, we demonstrate that MTA suppresses proliferation, activation, differentiation, and effector function of antigen-specific T cells without eliciting cell death. Conversely, if MTA is added to highly activated T cells, MTA exerts cytotoxic effects on T cells. We identified the Akt pathway, a critical signal pathway for T cell activation, as a target of MTA, while, for example, p38 remained unaffected. Next, we provide evidence that MTA exerts its immunosuppressive effects by interfering with protein methylation in T cells. To confirm the relevance of the suppressive effects of exogenously added MTA on human T cells, we used an MTAP-deficient tumor cell-line that was stably transfected with the MTAP-coding sequence. We observed that T cells stimulated with MTAP-transfected tumor cells revealed a higher proliferative capacity compared to T cells stimulated with Mock-transfected cells. In conclusion, our findings reveal a novel immune evasion strategy of human tumor cells that could be of interest for therapeutic targeting.

  17. Cancer

    Science.gov (United States)

    Cancer begins in your cells, which are the building blocks of your body. Normally, your body forms ... be benign or malignant. Benign tumors aren't cancer while malignant ones are. Cells from malignant tumors ...

  18. CD20-based Immunotherapy of B-cell Derived Hematologic Malignancies.

    Science.gov (United States)

    Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili

    2017-01-01

    CD20 is a surface antigen, which is expressed at certain stages of B-cell differentiation. Targeting the CD20-positive B-cells with therapeutic monoclonal antibodies (MAbs) has been an effectual strategy in the treatment of hematologic malignancies such as non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Initial success with Rituximab (RTX) has encouraged the creation and development of more effective CD20 based therapeutics. However, treatment with conventional MAbs has not been adequate to overcome the problems such as refractory/ relapsed disease. In this regard, new generations of MAbs with enhanced affinity or improved anti-tumor properties have been developed. CD20 directed therapeutics have heterogeneous features and mechanisms of action. Hence, having sufficient knowledge on the immunological and molecular aspects of CD20 based cancer therapy is necessary for predicting the clinical outcomes and taking the necessary measures. An extensive search was performed in PubMed and similar databases for peer-reviewed articles concerning the biology, function and characteristics of CD20 molecule as well as the mechanisms of action and evolutionary process of CD20 targeting agents. This review provides information about the current situation of CD20 targeting immunotherapeutics including MAbs, bispecific antibodies (which exert multiple functions or involve Tcells in tumor elimination) and CAR T-cells (engineered T-cells armed with chimeric antigen receptors). Moreover, limitations, challenges and available solutions regarding the application of CD20 targeting treatments are addressed. Utilization of CD20-targeted therapeutics, due to their diverse properties, requires special considerations. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. The Role of Extracellular Vesicles: An Epigenetic View of the Cancer Microenvironment.

    Science.gov (United States)

    Qian, Zhongrun; Shen, Qi; Yang, Xi; Qiu, Yongming; Zhang, Wenbin

    2015-01-01

    Exosomes, microvesicles, and other extracellular vesicles are released by many cell types, including cancer cells and cancer-related immune cells. Extracellular vesicles can directly or indirectly facilitate the transfer of bioinformation to recipient cells or to the extracellular environment. In cancer, exosomes have been implicated in tumor initiation, proliferation, and metastasis. Extracellular vesicles can transmit proteins and nucleic acids that participate in DNA methylation, histone modification, and posttranscriptional regulation of RNA. Factors transmitted by extracellular vesicles reflect the donor cell status, and extracellular vesicles derived from tumor cells may be also responsible for altering expression of tumor promoting and tumor suppressing genes in recipient cells. Thus, circulating extracellular vesicles may act as biomarkers of cancer, and detection of these biomarkers may be applied to diagnosis or assessment of prognosis in patients with cancer.

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

    Science.gov (United States)

    Murayama, Asako; Sugiyama, Nao; Wakita, Takaji; Kato, Takanobu

    2016-06-14

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

  1. Induced Pluripotent Stem Cell-Derived Neural Stem Cell Therapy Enhances Recovery in an Ischemic Stroke Pig Model.

    Science.gov (United States)

    Baker, Emily W; Platt, Simon R; Lau, Vivian W; Grace, Harrison E; Holmes, Shannon P; Wang, Liya; Duberstein, Kylee Jo; Howerth, Elizabeth W; Kinder, Holly A; Stice, Steve L; Hess, David C; Mao, Hui; West, Franklin D

    2017-08-30

    Induced pluripotent stem cell-derived neural stem cells (iNSCs) have significant potential as an autologous, multifunctional cell therapy for stroke, which is the primary cause of long term disability in the United States and the second leading cause of death worldwide. Here we show that iNSC transplantation improves recovery through neuroprotective, regenerative, and cell replacement mechanisms in a novel ischemic pig stroke model. Longitudinal multiparametric magnetic resonance imaging (MRI) following iNSC therapy demonstrated reduced changes in white matter integrity, cerebral blood perfusion, and brain metabolism in the infarcted tissue. The observed tissue level recovery strongly correlated with decreased immune response, enhanced neuronal protection, and increased neurogenesis. iNSCs differentiated into neurons and oligodendrocytes with indication of long term integration. The robust recovery response to iNSC therapy in a translational pig stroke model with increased predictive potential strongly supports that iNSCs may be the critically needed therapeutic for human stroke patients.

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

    DEFF Research Database (Denmark)

    Massumi, Mohammad; Pourasgari, Farzaneh; Nalla, Amarnadh

    2016-01-01

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

  3. Neural precursor cells derived from induced pluripotent stem cells exhibit reduced susceptibility to infection with a neurotropic coronavirus.

    Science.gov (United States)

    Mangale, Vrushali; Marro, Brett S; Plaisted, Warren C; Walsh, Craig M; Lane, Thomas E

    2017-11-01

    The present study examines the susceptibility of mouse induced pluripotent stem cell-derived neural precursor cells (iPSC-NPCs) to infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV). Similar to NPCs derived from striatum of day 1 postnatal GFP-transgenic mice (GFP-NPCs), iPSC-derived NPCs (iPSC-NPCs) are able to differentiate into terminal neural cell types and express MHC class I and II in response to IFN-γ treatment. However, in contrast to postnatally-derived NPCs, iPSC-NPCs express low levels of carcinoembryonic antigen-cell adhesion molecule 1a (CEACAM1a), the surface receptor for JHMV, and are less susceptible to infection and virus-induced cytopathic effects. The relevance of this in terms of therapeutic application of NPCs resistant to viral infection is discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Stable engineered vascular networks from human induced pluripotent stem cell-derived endothelial cells cultured in synthetic hydrogels.

    Science.gov (United States)

    Zanotelli, Matthew R; Ardalani, Hamisha; Zhang, Jue; Hou, Zhonggang; Nguyen, Eric H; Swanson, Scott; Nguyen, Bao Kim; Bolin, Jennifer; Elwell, Angela; Bischel, Lauren L; Xie, Angela W; Stewart, Ron; Beebe, David J; Thomson, James A; Schwartz, Michael P; Murphy, William L

    2016-04-15

    Here, we describe an in vitro strategy to model vascular morphogenesis where human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) are encapsulated in peptide-functionalized poly(ethylene glycol) (PEG) hydrogels, either on standard well plates or within a passive pumping polydimethylsiloxane (PDMS) tri-channel microfluidic device. PEG hydrogels permissive towards cellular remodeling were fabricated using thiol-ene photopolymerization to incorporate matrix metalloproteinase (MMP)-degradable crosslinks and CRGDS cell adhesion peptide. Time lapse microscopy, immunofluorescence imaging, and RNA sequencing (RNA-Seq) demonstrated that iPSC-ECs formed vascular networks through mechanisms that were consistent with in vivo vasculogenesis and angiogenesis when cultured in PEG hydrogels. Migrating iPSC-ECs condensed into clusters, elongated into tubules, and formed polygonal networks through sprouting. Genes upregulated for iPSC-ECs cultured in PEG hydrogels relative to control cells on tissue culture polystyrene (TCP) surfaces included adhesion, matrix remodeling, and Notch signaling pathway genes relevant to in vivo vascular development. Vascular networks with lumens were stable for at least 14days when iPSC-ECs were encapsulated in PEG hydrogels that were polymerized within the central channel of the microfluidic device. Therefore, iPSC-ECs cultured in peptide-functionalized PEG hydrogels offer a defined platform for investigating vascular morphogenesis in vitro using both standard and microfluidic formats. Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) cultured in synthetic hydrogels self-assemble into capillary networks through mechanisms consistent with in vivo vascular morphogenesis. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  5. Safety and immunogenicity of an inactivated whole virus Vero cell-derived Ross River virus vaccine: a randomized trial.

    Science.gov (United States)

    Aichinger, Gerald; Ehrlich, Hartmut J; Aaskov, John G; Fritsch, Sandor; Thomasser, Christiane; Draxler, Wolfgang; Wolzt, Michael; Müller, Markus; Pinl, Fritz; Van Damme, Pierre; Hens, Annick; Levy, Jack; Portsmouth, Daniel; Holzer, Georg; Kistner, Otfried; Kreil, Thomas R; Barrett, P Noel

    2011-11-21

    Ross River virus (RRV) is endemic in Australia and several South Pacific Islands. Approximately 5000 cases of RRV disease, which is characterized by debilitating polyarthritis, are recorded each year in Australia. This study describes the first clinical trial of a candidate RRV vaccine. An inactivated whole-virus Vero cell-derived RRV vaccine was tested in 382 healthy, RRV-naïve adults in a phase 1/2 dose-escalation study at ten sites in Austria, Belgium and The Netherlands. Subjects were equally randomized to receive 1.25 μg, 2.5 μg, 5 μg, or 10 μg aluminum hydroxide-adjuvanted or non-adjuvanted RRV vaccine, with a second dose after three weeks and a booster at six months. Vaccine immunogenicity was determined by measurements of serum IgG and neutralizing antibody titers. Vaccine tolerability and safety were monitored over the entire study period. The optimal vaccine formulation was the adjuvanted 2.5 μg dose, as calculated using a repeated mixed model analysis of covariance comparing log-transformed RRV-specific IgG titers between different dose groups. Geometric means of RRV-specific serum antibodies measured 21 days after the third vaccination with the 2.5 μg adjuvanted formulation were 520.9 (90% CI 377.2-719.4) as determined by IgG ELISA and 119.9 (82.6-173.9) as determined by virus neutralization assay, resulting in seropositivity rates of 92.9% (82.6-98.0) and 92.7% (82.2-98.0), respectively. All vaccine formulations and doses were well tolerated after the first, second and third vaccination. The adjuvanted, inactivated whole-virus Vero cell-derived Ross River virus vaccine is highly immunogenic in RRV-naïve adults and well tolerated at all dose levels. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Unique proliferation response in odontoblastic cells derived from human skeletal muscle stem cells by cytokine-induced matrix metalloproteinase-3

    Energy Technology Data Exchange (ETDEWEB)

    Ozeki, Nobuaki; Hase, Naoko; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464-8651, Aichi (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan); Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464-8651, Aichi (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan)

    2015-02-01

    A pro-inflammatory cytokine mixture (CM: interleukin (IL)-1β, tumor necrosis factor-α and interferon-γ) and IL-1β-induced matrix metalloproteinase (MMP)-3 activity have been shown to increase the proliferation of rat dental pulp cells and murine stem cell-derived odontoblast-like cells. This suggests that MMP-3 may regulate wound healing and regeneration in the odontoblast-rich dental pulp. Here, we determined whether these results can be extrapolated to human dental pulp by investigating the effects of CM-induced MMP-3 up-regulation on the proliferation and apoptosis of purified odontoblast-like cells derived from human skeletal muscle stem cells. We used siRNA to specifically reduce MMP-3 expression. We found that CM treatment increased MMP-3 mRNA and protein levels as well as MMP-3 activity. Cell proliferation was also markedly increased, with no changes in apoptosis, upon treatment with CM and following the application of exogenous MMP-3. Endogenous tissue inhibitors of metalloproteinases were constitutively expressed during all experiments and unaffected by MMP-3. Although treatment with MMP-3 siRNA suppressed cell proliferation, it also unexpectedly increased apoptosis. This siRNA-mediated increase in apoptosis could be reversed by exogenous MMP-3. These results demonstrate that cytokine-induced MMP-3 activity regulates cell proliferation and suppresses apoptosis in human odontoblast-like cells. - Highlights: • Pro-inflammatory cytokines induce MMP-3 activity in human odontoblast-like cells. • Increased MMP-3 activity can promote cell proliferation in odontoblasts. • Specific loss of MMP-3 increases apoptosis in odontoblasts. • MMP-3 has potential as a promising new target for pupal repair and regeneration.

  7. Andrographolide - A promising therapeutic agent, negatively regulates glial cell derived neurodegeneration of prefrontal cortex, hippocampus and working memory impairment.

    Science.gov (United States)

    Das, Sudeshna; Mishra, K P; Ganju, Lilly; Singh, S B

    2017-12-15

    Over activation of glial cell derived innate immune factors induces neuro-inflammation that results in neurodegenerative disease, like working memory impairment. In this study, we have investigated the role of andrographolide, a major constituent of Andrographis paniculata plant, in reduction of reactive glial cell derived working memory impairment. Real time PCR, Western bloting, flow cytometric and immunofluorescence studies demonstrated that andrographolide inhibited lipopolysaccharide (LPS)-induced overexpression of HMGB1, TLR4, NFκB, COX-2, iNOS, and release of inflammatory mediators in primary mix glial culture, adult mice prefrontal cortex and hippocampus region. Active microglial and reactive astrocytic makers were also downregulated after andrographolide treatment. Andrographolide suppressed overexpression of microglial MIP-1α, P2X7 receptor and its downstream signaling mediators including-inflammasome NLRP3, caspase1 and mature IL-1β. Furthermore, in vivo maze studies suggested that andrographolide treatment reversed LPS-induced behavioural and working memory disturbances including regulation of expression of protein markers like PKC, p-CREB, amyloid beta, APP, p-tau, synapsin and PSD-95. Andrographolide, by lowering expression of pro apoptotic genes and enhancing the expression of anti-apoptotic gene showed its anti-apoptotic nature that in turn reduces neurodegeneration. Morphology studies using Nissl and FJB staining also showed the neuroprotective effect of andrographolide in the prefrontal cortex region. The above studies indicated that andrographolide prevented neuroinflammation-associated neurodegeneration and improved synaptic plasticity markers in cortical as well as hippocampal region which suggests that andrographolide could be a novel pharmacological countermeasure for the treatment of neuroinflammation and neurological disorders related to memory impairment. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Human embryonic stem cell-derived oligodendrocyte progenitors aid in functional recovery of sensory pathways following contusive spinal cord injury.

    Directory of Open Access Journals (Sweden)

    Angelo H All

    Full Text Available Transplantations of human stem cell derivatives have been widely investigated in rodent models for the potential restoration of function of neural pathways after spinal cord injury (SCI. Studies have already demonstrated cells survival following transplantation in SCI. We sought to evaluate survival and potential therapeutic effects of transplanted human embryonic stem (hES cell-derived oligodendrocyte progenitor cells (OPCs in a contusive injury in rats. Bioluminescence imaging was utilized to verify survivability of cells up to 4 weeks, and somatosensory evoked potential (SSEPs were recorded at the cortex to monitor function of sensory pathways throughout the 6-week recovery period.hES cells were transduced with the firefly luciferase gene and differentiated into OPCs. OPCs were transplanted into the lesion epicenter of rat spinal cords 2 hours after inducing a moderate contusive SCI. The hES-treatment group showed improved SSEPs, including increased amplitude and decreased latencies, compared to the control group. The bioluminescence of transplanted OPCs decreased by 97% in the injured spinal cord compared to only 80% when injected into an uninjured spinal cord. Bioluminescence increased in both experimental groups such that by week 3, no statistical difference was detected, signifying that the cells survived and proliferated independent of injury. Post-mortem histology of the spinal cords showed integration of human cells expressing mature oligodendrocyte markers and myelin basic protein without the expression of markers for astrocytes (GFAP or pluripotent cells (OCT4.hES-derived OPCs transplanted 2 hours after contusive SCI survive and differentiate into OLs that produce MBP. Treated rats demonstrated functional improvements in SSEP amplitudes and latencies compared to controls as early as 1 week post-injury. Finally, the hostile injury microenvironment at 2 hours post-injury initially caused increased cell death but did not affect the long

  9. The in Vitro Assessment of Biochemical Factors in Hepatocyte like Cells Derived from Umbilical Cord Blood Stem Cells

    Directory of Open Access Journals (Sweden)

    A KHoramroodi

    2009-10-01

    Full Text Available Introduction & Objective: Umbilical cord blood (UCB is a source of Hematopoietic Stem Cells (HSC and progenitor cells that can reconstitute the hematopoietic system in patients with malignant and nonmalignant disorders. Mesenchymal stem cell-derived from umbilical cord blood (UCB have been differentiated to some kind of cells, such as osteobblast, adipoblast and chondroblast in Vitro. This study examined the differentiation of Umbilical Cord Blood (UCB derived stem cells to functional hepatocytes. Materials & Methods: The present study was an experimental study which was carried out in the Payam-e-Noor University of Tehran in cooperation with Hamedan University of Medical Sciences in 2008. Umbilical cord blood (UCB was obtained from Fatemieh hospital (Hamadan, Iran. Stem cells were isolated from the cord blood by combining density gradient centrifugation with plastic adherence. When the isolated cells reached 80% confluence, they differentiated to hepatocyte like cells. The medium which was used was consists of DMEM and 10% Fetal Bovine Serum (FBS supplemented with 20 ng/mL Hepatocyte Growth Factor (HGF, 10 ng/mL basic Fibroblast Growth Factor (bFGF and 20 ng/mL Oncostatin M (OSM.The medium was changed every 3 days and stored for Albumin (ALB, Alpha Fetoprotein (AFP, Alkaline Phosphatase (ALP, and urea assay. Finally PAS stain was done to study Glycogen storage in the differentiated cell. Results: Measurement of biochemical factors in different days showed that concentration of albumin (ALB, alpha fetoprotein (AFP, alkaline phosphatase (ALP, and Urea gradually increased. Also, PAS staining showed the storage of glycogen in these cells. Conclusion: Stem cell-derived from human umbilical cord blood (HUCB is a new source of cell types for cell transplantation therapy of hepatic diseases and under certain conditions these cells can differentiate into liver cells.

  10. Polyphosphate induces matrix metalloproteinase-3-mediated proliferation of odontoblast-like cells derived from induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Ozeki, Nobuaki; Hase, Naoko; Yamaguchi, Hideyuki; Hiyama, Taiki; Kawai, Rie [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan); Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan)

    2015-05-01

    Inorganic polyphosphate [Poly(P)] may represent a physiological source of phosphate and has the ability to induce bone differentiation in osteoblasts. We previously reported that cytokine-induced matrix metalloproteinase (MMP)-3 accelerates the proliferation of purified odontoblast-like cells. In this study, MMP-3 small interfering RNA (siRNA) was transfected into odontoblast-like cells derived from induced pluripotent stem cells to investigate whether MMP-3 activity is induced by Poly(P) and/or is associated with cell proliferation and differentiation into odontoblast-like cells. Treatment with Poly(P) led to an increase in both cell proliferation and additional odontoblastic differentiation. Poly(P)-treated cells showed a small but significant increase in dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) mRNA expression, which are markers of mature odontoblasts. The cells also acquired additional odontoblast-specific properties including adoption of an odontoblastic phenotype typified by high alkaline phosphatase (ALP) activity and a calcification capacity. In addition, Poly(P) induced expression of MMP-3 mRNA and protein, and increased MMP-3 activity. MMP-3 siRNA-mediated disruption of the expression of these effectors potently suppressed the expression of odontoblastic biomarkers ALP, DSPP, and DMP-1, and blocked calcification. Interestingly, upon siRNA-mediated silencing of MMP-3, we noted a potent and significant decrease in cell proliferation. Using specific siRNAs, we revealed that a unique signaling cascade, Poly(P)→MMP-3→DSPP and/or DMP-1, was intimately involved in the proliferation of odontoblast-like cells. - Highlights: • Polyphosphate increases proliferation of iPS cell-derived odontoblast-like cells. • Polyphosphate-induced MMP-3 results in an increase of cell proliferation. • Induced cell proliferation involves MMP-3, DSPP, and/or DMP-1 sequentially. • Induced MMP-3 also results in an increase of odontoblastic

  11. Human Stem Cell-Derived Endothelial-Hepatic Platform for Efficacy Testing of Vascular-Protective Metabolites from Nutraceuticals.

    Science.gov (United States)

    Narmada, Balakrishnan Chakrapani; Goh, Yeek Teck; Li, Huan; Sinha, Sanjay; Yu, Hanry; Cheung, Christine

    2017-03-01

    Atherosclerosis underlies many cardiovascular and cerebrovascular diseases. Nutraceuticals are emerging as a therapeutic moiety for restoring vascular health. Unlike small-molecule drugs, the complexity of ingredients in nutraceuticals often confounds evaluation of their efficacy in preclinical evaluation. It is recognized that the liver is a vital organ in processing complex compounds into bioactive metabolites. In this work, we developed a coculture system of human pluripotent stem cell-derived endothelial cells (hPSC-ECs) and human pluripotent stem cell-derived hepatocytes (hPSC-HEPs) for predicting vascular-protective effects of nutraceuticals. To validate our model, two compounds (quercetin and genistein), known to have anti-inflammatory effects on vasculatures, were selected. We found that both quercetin and genistein were ineffective at suppressing inflammatory activation by interleukin-1β owing to limited metabolic activity of hPSC-ECs. Conversely, hPSC-HEPs demonstrated metabolic capacity to break down both nutraceuticals into primary and secondary metabolites. When hPSC-HEPs were cocultured with hPSC-ECs to permit paracrine interactions, the continuous turnover of metabolites mitigated interleukin-1β stimulation on hPSC-ECs. We observed significant reductions in inflammatory gene expressions, nuclear translocation of nuclear factor κB, and interleukin-8 production. Thus, integration of hPSC-HEPs could accurately reproduce systemic effects involved in drug metabolism in vivo to unravel beneficial constituents in nutraceuticals. This physiologically relevant endothelial-hepatic platform would be a great resource in predicting the efficacy of complex nutraceuticals and mechanistic interrogation of vascular-targeting candidate compounds. Stem Cells Translational Medicine 2017;6:851-863. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  12. Tumour cell derived effects on monocyte/macrophage polarization and function and modulatory potential of Viscum album lipophilic extract in vitro.

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    Estko, Myriam; Baumgartner, Stephan; Urech, Konrad; Kunz, Matthias; Regueiro, Ursula; Heusser, Peter; Weissenstein, Ulrike

    2015-04-24

    Macrophages are highly versatile cells that play an important role in tumour microenvironment. Tumour associated macrophages (TAMs) have been linked to both, good or bad prognosis of several cancer types depending on their number, composition and polarization. Viscum album lipophilic extract (VALE) contains several pentacyclic triterpenes known to modulate the activity of monocytes and other immune cells and to exhibit anticancer properties. In our in vitro study, we investigated the effect of tumour cell lines on macrophage polarization and monocyte chemotactic transmigration and examined the modulatory potential of VALE and its predominant triterpene oleanolic acid (OA). Human peripheral blood monocytes were differentiated into monocyte derived macrophages (MDM) using M-CSF and polarized into M1 by IFN-γ and LPS and into M2 macrophages by IL-4 and IL-13 or by co-culture with two different tumour cell lines. Polarized macrophages were subsequently treated with VALE or OA. Phenotypic markers and cytokines were assessed by flow cytometry and immunoanalysis. Migration of human peripheral blood monocytes induced by monocyte chemotactic protein-1 (MCP-1) or supernatants of different tumour cell lines under the influence of VALE or OA was measured in a chemotaxis transmigration assay. In vitro polarized M1 and M2 type macrophages revealed specific phenotypic patterns and tumour cell co-cultured MDM displayed ambiguous phenotypes with M1 as well as M2 associated markers. VALE and OA showed modest influence on cell surface marker profile and cytokine expression of tumour cell co-cultured macrophages. All tumour cell supernatants markedly enhanced the migratory activity of monocytes. VALE and OA significantly inhibited MCP-1 induced monocyte transmigration, whereas monocyte migration initiated by tumour cell derived supernatants was not affected. In our study we reconfirmed that co-culture with different tumour cell lines can result in a mixed macrophage phenotype with M1

  13. Recruitment of RNA molecules by connexin RNA-binding motifs: Implication in RNA and DNA transport through microvesicles and exosomes.

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    Varela-Eirin, Marta; Varela-Vazquez, Adrian; Rodríguez-Candela Mateos, Marina; Vila-Sanjurjo, Anton; Fonseca, Eduardo; Mascareñas, José L; Eugenio Vázquez, M; Mayan, Maria D

    2017-04-01

    Connexins (Cxs) are integral membrane proteins that form high-conductance plasma membrane channels, allowing communication from cell to cell (via gap junctions) and from cells to the extracellular environment (via hemichannels). Initially described for their role in joining excitable cells (nerve and muscle), gap junctions (GJs) are found between virtually all cells in solid tissues and are essential for functional coordination by enabling the direct transfer of small signalling molecules, metabolites, ions, and electrical signals from cell to cell. Several studies have revealed diverse channel-independent functions of Cxs, which include the control of cell growth and tumourigenicity. Connexin43 (Cx43) is the most widespread Cx in the human body. The myriad roles of Cx43 and its implication in the development of disorders such as cancer, inflammation, osteoarthritis and Alzheimer's disease have given rise to many novel questions. Several RNA- and DNA-binding motifs were predicted in the Cx43 and Cx26 sequences using different computational methods. This review provides insights into new, ground-breaking functions of Cxs, highlighting important areas for future work such as transfer of genetic information through extracellular vesicles. We discuss the implication of potential RNA- and DNA-binding domains in the Cx43 and Cx26 sequences in the cellular communication and control of signalling pathways. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  14. The microvesicle component of HIV-1 inocula modulates dendritic cell infection and maturation and enhances adhesion to and activation of T lymphocytes.

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    Sarah K Mercier

    2013-10-01

    Full Text Available HIV-1 is taken up by immature monocyte derived dendritic cells (iMDDCs into tetraspanin rich caves from which the virus can either be transferred to T lymphocytes or enter into endosomes resulting in degradation. HIV-1 binding and fusion with the DC membrane results in low level de novo infection that can also be transferred to T lymphocytes at a later stage. We have previously reported that HIV-1 can induce partial maturation of iMDDCs at both stages of trafficking. Here we show that CD45⁺ microvesicles (MV which contaminate purified HIV-1 inocula due to similar size and density, affect DC maturation, de novo HIV-1 infection and transfer to T lymphocytes. Comparing iMDDCs infected with CD45-depleted HIV-1BaL or matched non-depleted preparations, the presence of CD45⁺ MVs was shown to enhance DC maturation and ICAM-1 (CD54 expression, which is involved in DC∶T lymphocyte interactions, while restricting HIV-1 infection of MDDCs. Furthermore, in the DC culture HIV-1 infected (p24⁺ MDDCs were more mature than bystander cells. Depletion of MVs from the HIV-1 inoculum markedly inhibited DC∶T lymphocyte clustering and the induction of alloproliferation as well as limiting HIV-1 transfer from DCs to T lymphocytes. The effects of MV depletion on these functions were reversed by the re-addition of purified MVs from activated but not non-activated SUPT1.CCR5-CL.30 or primary T cells. Analysis of the protein complement of these MVs and of these HIV-1 inocula before and after MV depletion showed that Heat Shock Proteins (HSPs and nef were the likely DC maturation candidates. Recombinant HSP90α and β and nef all induced DC maturation and ICAM-1 expression, greater when combined. These results suggest that MVs contaminating HIV-1 released from infected T lymphocytes may be biologically important, especially in enhancing T cell activation, during uptake by DCs in vitro and in vivo, particularly as MVs have been detected in the circulation of HIV-1

  15. Murine pluripotent stem cells derived scaffold-free cartilage grafts from a micro-cavitary hydrogel platform.

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    He, Pengfei; Fu, Jiayin; Wang, Dong-An

    2016-04-15

    By means of appropriate cell type and scaffold, tissue-engineering approaches aim to construct grafts for cartilage repair. Pluripotent stem cells especially induced pluripotent stem cells (iPSCs) are of promising cell candidates due to the pluripotent plasticity and abundant cell source. We explored three dimensional (3D) culture and chondrogenesis of murine iPSCs (miPSCs) on an alginate-based micro-cavity hydrogel (MCG) platform in pursuit of fabricating synthetic-scaffold-free cartilage grafts. Murine embryonic stem cells (mESCs) were employed in parallel as the control. Chondrogenesis was fulfilled using a consecutive protocol via mesoderm differentiation followed by chondrogenic differentiation; subsequently, miPSC and mESC-seeded constructs were further respectively cultured in chondrocyte culture (CC) medium. Alginate phase in the constructs was then removed to generate a graft only comprised of induced chondrocytic cells and cartilaginous extracellular matrix (ECMs). We found that from the mESC-seeded constructs, formation of intact grafts could be achieved in greater sizes with relatively fewer chondrocytic cells and abundant ECMs; from miPSC-seeded constructs, relatively smaller sized cartilaginous grafts could be formed by cells with chondrocytic phenotype wrapped by abundant and better assembled collagen type II. This study demonstrated successful creation of pluripotent stem cells-derived cartilage/chondroid graft from a 3D MCG interim platform. By the support of materials and methodologies established from this study, particularly given the autologous availability of iPSCs, engineered autologous cartilage engraftment may be potentially fulfilled without relying on the limited and invasive autologous chondrocytes acquisition. In this study, we explored chondrogenic differentiation of pluripotent stem cells on a 3D micro-cavitary hydrogel interim platform and creation of pluripotent stem cells-derived cartilage/chondroid graft via a consecutive

  16. A preliminary study for constructing a bioartificial liver device with induced pluripotent stem cell-derived hepatocytes

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    Iwamuro Masaya

    2012-12-01

    Full Text Available Abstract Background Bioartificial liver systems, designed to support patients with liver failure, are composed of bioreactors and functional hepatocytes. Immunological rejection of the embedded hepatocytes by the host immune system is a serious concern that crucially degrades the performance of the device. Induced pluripotent stem (iPS cells are considered a desirable source for bioartificial liver systems, because patient-derived iPS cells are free from immunological rejection. The purpose of this paper was to test the feasibility of a bioartificial liver system with iPS cell-derived hepatocyte-like cells. Methods Mouse iPS cells were differentiated into hepatocyte-like cells by a multi-step differentiation protocol via embryoid bodies and definitive endoderm. Differentiation of iPS cells was evaluated by morphology, PCR assay, and functional assays. iPS cell-derived hepatocyte-like cells were cultured in a bioreactor module with a pore size of 0.2 μm for 7 days. The amount of albumin secreted into the circulating medium was analyzed by ELISA. Additionally, after a 7-day culture in a bioreactor module, cells were observed by a scanning electron microscope. Results At the final stage of the differentiation program, iPS cells changed their morphology to a polygonal shape with two nucleoli and enriched cytoplasmic granules. Transmission electron microscope analysis revealed their polygonal shape, glycogen deposition in the cytoplasm, microvilli on their surfaces, and a duct-like arrangement. PCR analysis showed increased expression of albumin mRNA over the course of the differentiation program. Albumin and urea production was also observed. iPS-Heps culture in bioreactor modules showed the accumulation of albumin in the medium for up to 7 days. Scanning electron microscopy revealed the attachment of cell clusters to the hollow fibers of the module. These results indicated that iPS cells were differentiated into hepatocyte-like cells after culture

  17. Identification of Neurexophilin 3 as a Novel Supportive Factor for Survival of Induced Pluripotent Stem Cell-Derived Dopaminergic Progenitors

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    Nishimura, Kaneyasu; Murayama, Shigeo

    2015-01-01

    Successful cell transplantation for Parkinson’s disease (PD) depends on both an optimal host brain environment and ideal donor cells. We report that a secreted peptide, neurexophilin 3 (NXPH3), supports the survival of mouse induced pluripotent stem cell-derived (iPSC-derived) dopaminergic (DA) neurons in vitro and in vivo. We compared the gene expression profiles in the mouse striatum from two different environments: a supportive environment, which we defined as 1 week after acute administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and a nonsupportive environment, defined as 8 weeks after chronic administration of MPTP. NXPH3 expression was higher in the former condition and lower in the latter compared with untreated controls. When we injected mouse iPSC-derived neural cells along with NXPH3 into the mouse striatum, the ratio of tyrosine hydroxylase-positive DA neurons per graft volume was higher at 8 weeks compared with cell injections that excluded NXPH3. In addition, quantitative polymerase chain reaction analyses of the postmortem putamen revealed that the expression level of NXPH3 was lower in PD patients compared with normal controls. These findings will contribute to optimizing the host brain environment and patient recruitment in cell therapy for PD. Significance This study identified neurexophilin 3 (NXPH3), a secreted peptide, through comparison of gene expression profiles in the mouse striatum from various environments generated by different doses of dopaminergic (DA) neuron toxin. When mouse induced pluripotent stem cell-derived neural cells along with NXPH3 were injected into the mouse striatum, the ratio of DA neurons per graft volume was higher at 8 weeks compared with cell injections without NXPH3. In addition, quantitative polymerase chain reaction analyses of the postmortem putamen revealed that the expression level of NXPH3 was lower in patients with Parkinson’s disease (PD) compared with controls without PD. These

  18. Label-free real-time acoustic sensing of microvesicle release from prostate cancer (PC3) cells using a Quartz Crystal Microbalance.

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    Stratton, Dan; Lange, Sigrun; Kholia, Sharad; Jorfi, Samireh; Antwi-Baffour, Samuel; Inal, Jameel

    2014-10-24

    Using a Quartz Crystal Microbalance with dissipation monitoring, QCM-D (label-free system) measuring changes in resonant frequency (Δf) that equate to mass deposited on a sensor, we showed the attachment, over a 60min period, of a monolayer of PC3 cells to the gold electrodes of the quartz crystal sensor, which had been rendered hydrophilic. That MVs were released upon BzATP stimulation of cells was confirmed by NTA analysis (average 250nm diameter), flow cytometry, showing high phosphatidylserine exposition and by fluorescent (Annexin V Alexa Fluor® 488-positive) and electron microscopy. Over a period of 1000s (16.7min) during which early apoptosis increased from 4% plateauing at 10% and late apoptosis rose to 2%, the Δf increased 20Hz, thereupon remaining constant for the last 1000s of the experiment. Using the Sauerbrey equation, the loss in mass, which corresponded to the release of 2.36×10(6)MVs, was calculated to be 23ng. We therefore estimated the mass of an MV to be 0.24pg. With the deposition on the QCM-D of 3.5×10(7)MVs over 200s, the decrease in Δf (Hz) gave an estimate of 0.235pg per MV. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Embryonic Stem Cell-Derived Cardiomyocyte Heterogeneity and the Isolation of Immature and Committed Cells for Cardiac Remodeling and Regeneration

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    Kenneth R. Boheler

    2011-01-01

    Full Text Available Pluripotent stem cells represent one promising source for cell replacement therapy in heart, but differentiating embryonic stem cell-derived cardiomyocytes (ESC-CMs are highly heterogeneous and show a variety of maturation states. In this study, we employed an ESC clonal line that contains a cardiac-restricted ncx1 promoter-driven puromycin resistance cassette together with a mass culture system to isolate ESC-CMs that display traits characteristic of very immature CMs. The cells display properties of proliferation, CM-restricted markers, reduced mitochondrial mass, and hypoxia-resistance. Following transplantation into rodent hearts, bioluminescence imaging revealed that immature cells, but not more mature CMs, survived for at least one month following injection. These data and comparisons with more mature cells lead us to conclude that immature hypoxia resistant ESC-CMs can be isolated in mass in vitro and, following injection into heart, form grafts that may mediate long-term recovery of global and regional myocardial contractile function following infarction.

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

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    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.

  1. A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix.

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    Hellewell, Andrew L; Rosini, Silvia; Adams, Josephine C

    2017-01-04

    The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation. Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

  2. Availability of human induced pluripotent stem cell-derived cardiomyocytes in assessment of drug potential for QT prolongation.

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    Nozaki, Yumiko; Honda, Yayoi; Tsujimoto, Shinji; Watanabe, Hitoshi; Kunimatsu, Takeshi; Funabashi, Hitoshi

    2014-07-01

    Field potential duration (FPD) in human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which can express QT interval in an electrocardiogram, is reported to be a useful tool to predict K(+) channel and Ca(2+) channel blocker effects on QT interval. However, there is no report showing that this technique can be used to predict multichannel blocker potential for QT prolongation. The aim of this study is to show that FPD from MEA (Multielectrode array) of hiPS-CMs can detect QT prolongation induced by multichannel blockers. hiPS-CMs were seeded onto MEA and FPD was measured for 2min every 10min for 30min after drug exposure for the vehicle and each drug concentration. IKr and IKs blockers concentration-dependently prolonged corrected FPD (FPDc), whereas Ca(2+) channel blockers concentration-dependently shortened FPDc. Also, the multichannel blockers Amiodarone, Paroxetine, Terfenadine and Citalopram prolonged FPDc in a concentration dependent manner. Finally, the IKr blockers, Terfenadine and Citalopram, which are reported to cause Torsade de Pointes (TdP) in clinical practice, produced early afterdepolarization (EAD). hiPS-CMs using MEA system and FPDc can predict the effects of drug candidates on QT interval. This study also shows that this assay can help detect EAD for drugs with TdP potential. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy

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    Irène Tatischeff

    2012-11-01

    Full Text Available The joint use of 3 complementary techniques, namely, nanoparticle tracking analysis (NTA, cryo-electron microscopy (Cryo-EM and Raman tweezers microspectroscopy (RTM, is proposed for a rapid characterisation of extracellular vesicles (EVs of various origins. NTA is valuable for studying the size distribution and concentration, Cryo-EM is outstanding for the morphological characterisation, including observation of vesicle heterogeneity, while RTM provides the global chemical composition without using any exogenous label. The capabilities of this approach are evaluated on the example of cell-derived vesicles of Dictyostelium discoideum, a convenient general model for eukaryotic EVs. At least 2 separate species differing in chemical composition (relative amounts of DNA, lipids and proteins, presence of carotenoids were found for each of the 2 physiological states of this non-pathogenic microorganism, that is, cell growth and starvation-induced aggregation. These findings demonstrate the specific potency of RTM. In addition, the first Raman spectra of human urinary exosomes are reported, presumably constituting the primary step towards Raman characterisation of EVs for the purpose of human diseases diagnoses.

  4. Development of correction formula for field potential duration of human induced pluripotent stem cell-derived cardiomyocytes sheets

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    Hiroko Izumi-Nakaseko

    2017-09-01

    Full Text Available Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs have been used in many studies to assess proarrhythmic risks of chemical compounds. In those studies, field potential durations (FPD of hiPSC-CMs have been corrected by clinically used Fridericia's and/or Bazett's formulae, however, the rationale for the use of these formulae has not been well established. In the present study, we developed a correction formula for experiments using hiPSC-CMs. First, we analyzed the effect of beating rate on FPD in the hiPSC-CMs sheets with electrical stimuli and a HCN channel inhibitor zatebradine. Next, we examined the relationship between the electrophysiological properties and the expression levels of ion channel genes in the cell sheets. Zatebradine slowed the beating rate and allowed to analyze FPD changes at various pacing cycle lengths. Rate-dependent change in the repolarization period was smaller in the cell sheets than that reported on the human hearts, which can be partly explained by lower gene expression level of hKCNJ2 and hKCNE1. Thus, non-linear equation for correcting FPD in the cell sheet; FPDc = FPD/RR0.22 with RR given in second was obtained, which may make it feasible to assess net repolarization delay by various chemical compounds with a chronotropic action.

  5. Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Encapsulating Bioactive Hydrogels Improve Rat Heart Function Post Myocardial Infarction.

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    Chow, Andre; Stuckey, Daniel J; Kidher, Emaddin; Rocco, Mark; Jabbour, Richard J; Mansfield, Catherine A; Darzi, Ara; Harding, Sian E; Stevens, Molly M; Athanasiou, Thanos

    2017-10-04

    Tissue engineering offers an exciting possibility for cardiac repair post myocardial infarction. We assessed the effects of combined polyethylene glycol hydrogel (PEG), human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM), and erythropoietin (EPO) therapy in a rat model of myocardial infarction. PEG with/out iPSC-CMs and EPO; iPSC-CMs in saline; or saline alone was injected into infarcted hearts shortly after infarction. Injection of almost any combination of the therapeutics limited acute elevations in chamber volumes. After 10 weeks, attenuation of ventricular remodeling was identified in all groups that received PEG injections, while ejection fractions were significantly increased in the gel-EPO, cell, and gel-cell-EPO groups. In all treatment groups, infarct thickness was increased and regions of muscle were identified within the scar. However, no grafted cells were detected. Hence, iPSC-CM-encapsulating bioactive hydrogel therapy can improve cardiac function post myocardial infarction and increase infarct thickness and muscle content despite a lack of sustained donor-cell engraftment. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Biomaterial-Free Three-Dimensional Bioprinting of Cardiac Tissue using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes.

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    Ong, Chin Siang; Fukunishi, Takuma; Zhang, Huaitao; Huang, Chen Yu; Nashed, Andrew; Blazeski, Adriana; DiSilvestre, Deborah; Vricella, Luca; Conte, John; Tung, Leslie; Tomaselli, Gordon F; Hibino, Narutoshi

    2017-07-04

    We have developed a novel method to deliver stem cells using 3D bioprinted cardiac patches, free of biomaterials. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), fibroblasts (FB) and endothelial cells (EC) were aggregated to create mixed cell spheroids. Cardiac patches were created from spheroids (CM:FB:EC = 70:15:15, 70:0:30, 45:40:15) using a 3D bioprinter. Cardiac patches were analyzed with light and video microscopy, immunohistochemistry, immunofluorescence, cell viability assays and optical electrical mapping. Cardiac tissue patches of all cell ratios beat spontaneously after 3D bioprinting. Patches exhibited ventricular-like action potential waveforms and uniform electrical conduction throughout the patch. Conduction velocities were higher and action potential durations were significantly longer in patches containing a lower percentage of FBs. Immunohistochemistry revealed staining for CM, FB and EC markers, with rudimentary CD31+ blood vessel formation. Immunofluorescence revealed the presence of Cx43, the main cardiac gap junction protein, localized to cell-cell borders. In vivo implantation suggests vascularization of 3D bioprinted cardiac patches with engraftment into native rat myocardium. This constitutes a significant step towards a new generation of stem cell-based treatment for heart failure.

  7. Vildagliptin reduces plasma stromal cell-derived factor-1α in patients with type 2 diabetes compared with glimepiride.

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    Park, Kyeong Seon; Kwak, SooHeon; Cho, Young Min; Park, Kyong Soo; Jang, Hak C; Kim, Seong Yeon; Jung, Hye Seung

    2017-03-01

    Dipeptidyl peptidase-4 inhibitors might have pleiotropic protective effects on cardiovascular disease (CVD), in contrast to sulfonylureas. Therefore, we compared various CVD risk factors between vildagliptin and glimepiride. We carried out a randomized, prospective and crossover trial. A total of 16 patients with type 2 diabetes whose glycated hemoglobin was >7% were randomized to add vildagliptin or glimepiride. After 12-week treatment, each drug was replaced with the other for another 12 weeks. Before and after each treatment, glucose homeostasis and CVD risk factors were assessed, and the continuous glucose monitoring system was applied to calculate glycemic variability. The mean age of the participants was 60 years, 31% were men, body mass index 25.5 kg/m2 and HbA1c 8.41%. Both vildagliptin and glimepiride significantly decreased glycated hemoglobin and glycemic variability indices. Despite the improved glucose homeostasis, favorable change of CVD markers was not prominent in both the arms, along with significant weight gain. Only plasma stromal cell-derived factor (SDF)-1α decreased by 30% in the vildagliptin arm. According to regression analyses, the reduction of SDF-1α was independently associated with vildagliptin usage and serum interleukin-6 changes, but white blood cells were not related with the SDF-1α changes. Compared with glimepiride, vildagliptin arrestingly decreased plasma SDF-1α, and its clinical implications should be further investigated. © 2016 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

  8. A Human Neural Crest Stem Cell-Derived Dopaminergic Neuronal Model Recapitulates Biochemical Abnormalities in GBA1 Mutation Carriers

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    Shi-Yu Yang

    2017-03-01

    Full Text Available Numerically the most important risk factor for the development of Parkinson's disease (PD is the presence of mutations in the glucocerebrosidase GBA1 gene. In vitro and in vivo studies show that GBA1 mutations reduce glucocerebrosidase (GCase activity and are associated with increased α-synuclein levels, reflecting similar changes seen in idiopathic PD brain. We have developed a neural crest stem cell-derived dopaminergic neuronal model that recapitulates biochemical abnormalities in GBA1 mutation-associated PD. Cells showed reduced GCase protein and activity, impaired macroautophagy, and increased α-synuclein levels. Advantages of this approach include easy access to stem cells, no requirement to reprogram, and retention of the intact host genome. Treatment with a GCase chaperone increased GCase protein levels and activity, rescued the autophagic defects, and decreased α-synuclein levels. These results provide the basis for further investigation of GCase chaperones or similar drugs to slow the progression of PD.

  9. Effect of age on pro-inflammatory miRNAs contained in mesenchymal stem cell-derived extracellular vesicles.

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    Fafián-Labora, J; Lesende-Rodriguez, I; Fernández-Pernas, P; Sangiao-Alvarellos, S; Monserrat, L; Arntz, O J; van de Loo, F J; Mateos, J; Arufe, M C

    2017-03-06

    Stem cells possess significant age-dependent differences in their immune-response profile. These differences were analysed by Next-Generation Sequencing of six age groups from bone marrow mesenchymal stem cells. A total of 9,628 genes presenting differential expression between age groups were grouped into metabolic pathways. We focused our research on young, pre-pubertal and adult groups, which presented the highest amount of differentially expressed genes related to inflammation mediated by chemokine and cytokine signalling pathways compared with the newborn group, which was used as a control. Extracellular vesicles extracted from each group were characterized by nanoparticle tracking and flow cytometry analysis, and several micro-RNAs were verified by quantitative real-time polymerase chain reaction because of their relationship with the pathway of interest. Since miR-21-5p showed the highest statistically significant expression in extracellular vesicles from mesenchymal stem cells of the pre-pubertal group, we conducted a functional experiment inhibiting its expression and investigating the modulation of Toll-Like Receptor 4 and their link to damage-associated molecular patterns. Together, these results indicate for the first time that mesenchymal stem cell-derived extracellular vesicles have significant age-dependent differences in their immune profiles.

  10. Elevated Plasma Stromal-Cell-Derived Factor-1 Protein Levels Correlate with Severity in Patients with Community-Acquired Pneumonia

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    Ping-Kun Tsai

    2014-01-01

    Full Text Available Background. The aim of this study was to investigate differential changes in plasma levels of stromal-cell-derived factor-1 (SDF-1 before and after antibiotic treatment in patients with community-acquired pneumonia (CAP and observe the association between the severity of CAP and the plasma SDF-1 level. Methods. We gathered blood specimens from 61 adult CAP patients before and after antibiotic treatment and from 60 healthy controls to measure the plasma concentrations of SDF-1 by using an enzyme-linked immunosorbent assay. Results. The plasma SDF-1 concentration was elevated significantly in patients with CAP before receiving treatment compared with the controls and decreased significantly after the patients received treatment. Leukocyte (WBC and neutrophil counts and C-reactive protein (CRP levels decreased significantly after antibiotic treatment. Moreover, differences in the plasma concentration of SDF-1 were significantly correlated with PSI, CURB-65, and APACHE II scores (r=0.389, P=0.002, and n=61; r=0.449, P<0.001, and n=61; and r=0.363, P=0.004, and n=61, resp.. Conclusions. An elevated plasma SDF-1 concentration can be used as a biological marker for the early diagnosis of CAP and for the early detection of its severity.

  11. The influence of physiological matrix conditions on permanent culture of induced pluripotent stem cell-derived cardiomyocytes.

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    Heras-Bautista, Carlos O; Katsen-Globa, Alisa; Schloerer, Nils E; Dieluweit, Sabine; Abd El Aziz, Osama M; Peinkofer, Gabriel; Attia, Wael A; Khalil, Markus; Brockmeier, Konrad; Hescheler, Jürgen; Pfannkuche, Kurt

    2014-08-01

    Cardiomyocytes (CMs) from induced pluripotent stem (iPS) cells mark an important achievement in the development of in vitro pharmacological, toxicological and developmental assays and in the establishment of protocols for cardiac cell replacement therapy. Using CMs generated from murine embryonic stem cells and iPS cells we found increased cell-matrix interaction and more matured embryoid body (EB) structures in iPS cell-derived EBs. However, neither suspension-culture in form of purified cardiac clusters nor adherence-culture on traditional cell culture plastic allowed for extended culture of CMs. CMs grown for five weeks on polystyrene exhibit signs of massive mechanical stress as indicated by α-smooth muscle actin expression and loss of sarcomere integrity. Hydrogels from polyacrylamide allow adapting of the matrix stiffness to that of cardiac tissue. We were able to eliminate the bottleneck of low cell adhesion using 2,5-Dioxopyrrolidin-1-yl-6-acrylamidohexanoate as a crosslinker to immobilize matrix proteins on the gels surface. Finally we present an easy method to generate polyacrylamide gels with a physiological Young's modulus of 55 kPa and defined surface ligand, facilitating the culture of murine and human iPS-CMs, removing excess mechanical stresses and reducing the risk of tissue culture artifacts exerted by stiff substrates. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Stephanie Friedrichs

    2015-01-01

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

  13. Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip

    Science.gov (United States)

    Musah, Samira; Mammoto, Akiko; Ferrante, Thomas C.; Jeanty, Sauveur S. F.; Hirano-Kobayashi, Mariko; Mammoto, Tadanori; Roberts, Kristen; Chung, Seyoon; Novak, Richard; Ingram, Miles; Fatanat-Didar, Tohid; Koshy, Sandeep; Weaver, James C.; Church, George M.; Ingber, Donald E.

    2017-01-01

    An in vitro model of the human kidney glomerulus — the major site of blood filtration — could facilitate drug discovery and illuminate kidney-disease mechanisms. Microfluidic organ-on-a-chip technology has been used to model the human proximal tubule, yet a kidney-glomerulus-on-a-chip has not been possible because of the lack of functional human podocytes — the cells that regulate selective permeability in the glomerulus. Here, we demonstrate an efficient (> 90%) and chemically defined method for directing the differentiation of human induced pluripotent stem (hiPS) cells into podocytes that express markers of the mature phenotype (nephrin+, WT1+, podocin+, Pax2−) and that exhibit primary and secondary foot processes. We also show that the hiPS-cell-derived podocytes produce glomerular basement-membrane collagen and recapitulate the natural tissue/tissue interface of the glomerulus, as well as the differential clearance of albumin and inulin, when co-cultured with human glomerular endothelial cells in an organ-on-a-chip microfluidic device. The glomerulus-on-a-chip also mimics adriamycin-induced albuminuria and podocyte injury. This in vitro model of human glomerular function with mature human podocytes may facilitate drug development and personalized-medicine applications. PMID:29038743

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

    Directory of Open Access Journals (Sweden)

    Komiyama NH

    2006-06-01

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

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

    Science.gov (United States)

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

    2003-08-01

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

  16. Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes

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    Kyung Hoon Lee

    2016-01-01

    Full Text Available Enrichment of spermatogonial stem cells is important for studying their self-renewal and differentiation. Although germ cell-derived colonies (GDCs have been successfully cultured from neonatal pig testicular cells under 31°C conditions, the short period of in vitro maintenance (<2 months limited their application to further investigations. To develop a culture method that allows for in vitro maintenance of GDCs for long periods, we subcultured the GDCs with freshly prepared somatic cells from neonatal pig testes as feeder cells. The subcultured GDCs were maintained up to passage 13 with the fresh feeder cells (FFCs and then frozen. Eight months later, the frozen GDCs could again form the colonies on FFCs as shown in passages 1 to 13. Immunocytochemistry data revealed that the FFCs expressed GATA-binding protein 4 (GATA4, which is also detected in the cells of neonatal testes and total testicular cells, and that the expression of GATA4 was decreased in used old feeder cells. The subcultured GDCs in each passage had germ and stem cell characteristics, and flow cytometric analyses revealed that ~60% of these cells were GFRα-1 positive. In conclusion, neonatal pig testes-derived GDCs can be maintained for long periods with GATA4-expressing testicular somatic cells.

  17. Magnetically Responsive Bone Marrow Mesenchymal Stem Cell-Derived Smooth Muscle Cells Maintain Their Benefits to Augmenting Elastic Matrix Neoassembly.

    Science.gov (United States)

    Swaminathan, Ganesh; Sivaraman, Balakrishnan; Moore, Lee; Zborowski, Maciej; Ramamurthi, Anand

    2016-04-01

    Abdominal aortic aneurysms (AAA) represent abnormal aortal expansions that result from chronic proteolytic breakdown of elastin and collagen fibers by matrix metalloproteases. Poor elastogenesis by adult vascular smooth muscle cells (SMCs) limits regenerative repair of elastic fibers, critical for AAA growth arrest. Toward overcoming these limitations, we recently demonstrated significant elastogenesis by bone marrow mesenchymal stem cell-derived SMCs (BM-SMCs) and their proelastogenesis and antiproteolytic effects on rat aneurysmal SMCs (EaRASMCs). We currently investigate the effects of super paramagnetic iron oxide nanoparticle (SPION) labeling of BM-SMCs, necessary to magnetically guide them to the AAA wall, on their functional benefits. Our results indicate that SPION-labeling is noncytotoxic and does not adversely impact the phenotype and elastogenesis by BM-SMCs. In addition, SPION-BM-SMCs showed no changes in the ability of the BM-SMCs to stimulate elastin regeneration and attenuate proteolytic activity by EaRASMCs. Together, our results are promising toward the utility of SPIONs for magnetic targeting of BM-SMCs for in situ AAA regenerative repair.

  18. The effect of magnetic nanoparticles on neuronal differentiation of induced pluripotent stem cell-derived neural precursors

    Science.gov (United States)

    Jiráková, Klára; Šeneklová, Monika; Jirák, Daniel; Turnovcová, Karolína; Vosmanská, Magda; Babič, Michal; Horák, Daniel; Veverka, Pavel; Jendelová, Pavla

    2016-01-01

    Introduction Magnetic resonance (MR) imaging is suitable for noninvasive long-term tracking. We labeled human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) with two types of iron-based nanoparticles, silica-coated cobalt zinc ferrite nanoparticles (CZF) and poly-l-lysine-coated iron oxide superparamagnetic nanoparticles (PLL-coated γ-Fe2O3) and studied their effect on proliferation and neuronal differentiation. Materials and methods We investigated the effect of these two contrast agents on neural precursor cell proliferation and differentiation capability. We further defined the intracellular localization and labeling efficiency and analyzed labeled cells by MR. Results Cell proliferation was not affected by PLL-coated γ-Fe2O3 but was slowed down in cells labeled with CZF. Labeling efficiency, iron content and relaxation rates measured by MR were lower in cells labeled with CZF when compared to PLL-coated γ-Fe2O3. Cytoplasmic localization of both types of nanoparticles was confirmed by transmission electron microscopy. Flow cytometry and immunocytochemical analysis of specific markers expressed during neuronal differentiation did not show any significant differences between unlabeled cells or cells labeled with both magnetic nanoparticles. Conclusion Our results show that cells labeled with PLL-coated γ-Fe2O3 are suitable for MR detection, did not affect the differentiation potential of iPSC-NPs and are suitable for in vivo cell therapies in experimental models of central nervous system disorders. PMID:27920532

  19. Productive Infection of Human Embryonic Stem Cell-Derived NKX2.1+ Respiratory Progenitors With Human Rhinovirus

    Science.gov (United States)

    Jenny, Robert A.; Hirst, Claire; Lim, Sue Mei; Goulburn, Adam L.; Micallef, Suzanne J.; Labonne, Tanya; Kicic, Anthony; Ling, Kak-Ming; Stick, Stephen M.; Ng, Elizabeth S.; Trounson, Alan; Giudice, Antonietta; Elefanty, Andrew G.

    2015-01-01

    Airway epithelial cells generated from pluripotent stem cells (PSCs) represent a resource for research into a variety of human respiratory conditions, including those resulting from infection with common human pathogens. Using an NKX2.1-GFP reporter human embryonic stem cell line, we developed a serum-free protocol for the generation of NKX2.1+ endoderm that, when transplanted into immunodeficient mice, matured into respiratory cell types identified by expression of CC10, MUC5AC, and surfactant proteins. Gene profiling experiments indicated that day 10 NKX2.1+ endoderm expressed markers indicative of early foregut but lacked genes associated with later stages of respiratory epithelial cell differentiation. Nevertheless, NKX2.1+ endoderm supported the infection and replication of the common respiratory pathogen human rhinovirus HRV1b. Moreover, NKX2.1+ endoderm upregulated expression of IL-6, IL-8, and IL-1B in response to infection, a characteristic of human airway epithelial cells. Our experiments provide proof of principle for the use of PSC-derived respiratory epithelial cells in the study of cell-virus interactions. Significance This report provides proof-of-principle experiments demonstrating, for the first time, that human respiratory progenitor cells derived from stem cells in the laboratory can be productively infected with human rhinovirus, the predominant cause of the common cold. PMID:25873746

  20. Economic 3D-printing approach for transplantation of human stem cell-derived β-like cells.

    Science.gov (United States)

    Song, Jiwon; Millman, Jeffrey R

    2016-12-01

    Transplantation of human pluripotent stem cells (hPSC) differentiated into insulin-producing β cells is a regenerative medicine approach being investigated for diabetes cell replacement therapy. This report presents a multifaceted transplantation strategy that combines differentiation into stem cell-derived β (SC-β) cells with 3D printing. By modulating the parameters of a low-cost 3D printer, we created a macroporous device composed of polylactic acid (PLA) that houses SC-β cell clusters within a degradable fibrin gel. Using finite element modeling of cellular oxygen diffusion-consumption and an in vitro culture system that allows for culture of devices at physiological oxygen levels, we identified cluster sizes that avoid severe hypoxia within 3D-printed devices and developed a microwell-based technique for resizing clusters within this range. Upon transplantation into mice, SC-β cell-embedded 3D-printed devices function for 12 weeks, are retrievable, and maintain structural integrity. Here, we demonstrate a novel 3D-printing approach that advances the use of differentiated hPSC for regenerative medicine applications and serves as a platform for future transplantation strategies.

  1. Stroma cell-derived factor-1α signaling enhances calcium transients and beating frequency in rat neonatal cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Ielham Hadad

    Full Text Available Stroma cell-derived factor-1α (SDF-1α is a cardioprotective chemokine, acting through its G-protein coupled receptor CXCR4. In experimental acute myocardial infarction, administration of SDF-1α induces an early improvement of systolic function which is difficult to explain solely by an anti-apoptotic and angiogenic effect. We wondered whether SDF-1α signaling might have direct effects on calcium transients and beating frequency.Primary rat neonatal cardiomyocytes were culture-expanded and characterized by immunofluorescence staining. Calcium sparks were studied by fluorescence microscopy after calcium loading with the Fluo-4 acetoxymethyl ester sensor. The cardiomyocyte enriched cellular suspension expressed troponin I and CXCR4 but was vimentin negative. Addition of SDF-1α in the medium increased cytoplasmic calcium release. The calcium response was completely abolished by using a neutralizing anti-CXCR4 antibody and partially suppressed and delayed by preincubation with an inositol triphosphate receptor (IP3R blocker, but not with a ryanodine receptor (RyR antagonist. Calcium fluxes induced by caffeine, a RyR agonist, were decreased by an IP3R blocker. Treatment with forskolin or SDF-1α increased cardiomyocyte beating frequency and their effects were additive. In vivo, treatment with SDF-1α increased left ventricular dP/dtmax.These results suggest that in rat neonatal cardiomyocytes, the SDF-1α/CXCR4 signaling increases calcium transients in an IP3-gated fashion leading to a positive chronotropic and inotropic effect.

  2. Cytotoxicity of Silver Nanoparticles in Human Embryonic Stem Cell-Derived Fibroblasts and an L-929 Cell Line

    Directory of Open Access Journals (Sweden)

    Hui Peng

    2012-01-01

    Full Text Available Consensus about the toxicity of silver nanoparticles (Ag-NPs has not been reached, even though extensive attention has been paid to this issue. This confusion may be due to physicochemical factors of Ag-NPs and the cell model used for biological safety evaluation. In the present study, human embryonic stem cell-derived fibroblasts (EBFs, which have been considered a closer representative of the in vivo response, were used as a novel cell model to assess the cytotoxicity of Ag-NPs (~20 nm and ~100 nm in comparison with L-929 fibroblast cell line. Cell proliferation, cell cycle, apoptosis, p53 expression, and cellular uptake were examined. Results showed that Ag-NPs presented higher cytotoxicity to EBF than to L-929. EBF demonstrated a stronger capacity to ingest Ag-NPs, a higher G2/M arrest, and more upgraduated p53 expression after exposed to Ag-NPs for 48 h when compared with L-929. It could be concluded that EBF exhibited a more sensitive response to Ag-NPs compared with L-929 cells, indicating that EBF may be a valid candidate for cytotoxicity screening assays of nanoparticles.

  3. Human Induced Pluripotent Stem Cell-Derived Macrophages Share Ontogeny with MYB-Independent Tissue-Resident Macrophages.

    Science.gov (United States)

    Buchrieser, Julian; James, William; Moore, Michael D

    2017-02-14

    Tissue-resident macrophages, such as microglia, Kupffer cells, and Langerhans cells, derive from Myb-independent yolk sac (YS) progenitors generated before the emergence of hematopoietic stem cells (HSCs). Myb-independent YS-derived resident macrophages self-renew locally, independently of circulating monocytes and HSCs. In contrast, adult blood monocytes, as well as infiltrating, gut, and dermal macrophages, derive from Myb-dependent HSCs. These findings are derived from the mouse, using gene knockouts and lineage tracing, but their applicability to human development has not been formally demonstrated. Here, we use human induced pluripotent stem cells (iPSCs) as a tool to model human hematopoietic development. By using a CRISPR-Cas9 knockout strategy, we show that human iPSC-derived monocytes/macrophages develop in an MYB-independent, RUNX1-, and SPI1 (PU.1)-dependent fashion. This result makes human iPSC-derived macrophages developmentally related to and a good model for MYB-independent tissue-resident macrophages, such as alveolar and kidney macrophages, microglia, Kupffer cells, and Langerhans cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Characteristics of cells derived from the girdle region of the pre-implantation blastocyst of the donkey.

    Science.gov (United States)

    Whyte, A; Ockleford, C D; Wooding, F B; Hamon, M; Allen, W R; Kellie, S

    1986-01-01

    The establishment of a monolayer culture of cells derived from the girdle region of a 34-day-old donkey conceptus is described. These cells have had over 100 repeated passages in culture. Low levels of pregnant mares' serum gonadotrophin (PMSG, eCG) could be detected in the cells by indirect immunofluorescence using some monoclonal anti-eCG antibodies, but the cells did not secrete eCG as measured by radioimmunoassay or inhibition of haemagglutination. There was marked nuclear polymorphism with binucleate and occasional multinucleate cells. The cells were strongly reactive with wheatgerm agglutinin and concanavalin A suggesting the synthesis of many glycosylated products. Some cells were reactive with antisera to prekeratin, others with antisera to vimentin. The cells also contained actin (showing peculiar intercellular communications), alpha-actinin and tubulin. They were able to metabolize certain steroid precursors, but there was no definitive evidence for the presence of aromatase or delta 5-3 beta-hydroxysteroid dehydrogenase in these cells. This cell line appears to resemble trophectodermal girdle epithelium at a stage of development prior to the onset of eCG production, and may be useful in studies on the control of expression of this substance.

  5. Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1.

    Science.gov (United States)

    Iohara, Koichiro; Imabayashi, Kiyomi; Ishizaka, Ryo; Watanabe, Atsushi; Nabekura, Junichi; Ito, Masataka; Matsushita, Kenji; Nakamura, Hiroshi; Nakashima, Misako

    2011-08-01

    Loss of pulp due to caries and pulpitis leads to loss of teeth and reduced quality of life. Thus, there is an unmet need for regeneration of pulp. A promising approach is stem cell therapy. Autologous pulp stem/progenitor (CD105(+)) cells were transplanted into a root canal with stromal cell-derived factor-1 (SDF-1) after pulpectomy in mature teeth with complete apical closure in dogs. The root canal was successfully filled with regenerated pulp including nerves and vasculature by day 14, followed by new dentin formation along the dentinal wall. The newly regenerated tissue was significantly larger in the transplantation of pulp CD105(+) cells with SDF-1 compared with those of adipose CD105(+) cells with SDF-1 or unfractionated total pulp cells with SDF-1. The pulp CD105(+) cells highly expressed angiogenic/neurotrophic factors compared with other cells and localized in the vicinity of newly formed capillaries after transplantation, demonstrating its potent trophic effects on neovascularization. Two-dimensional electrophoretic analyses and real-time reverse transcription-polymerase chain reaction analyses demonstrated that the qualitative and quantitative protein and mRNA expression patterns of the regenerated pulp were similar to those of normal pulp. Thus, this novel stem cell therapy is the first demonstration of complete pulp regeneration, implying novel treatment to preserve and save teeth.

  6. A basis for comparison: sensitive authentication of stem cell derived RPE using physiological responses of intact RPE monolayers.

    Science.gov (United States)

    Miyagishima, Kiyoharu J; Wan, Qin; Miller, Sheldon S; Bharti, Kapil

    2017-01-01

    The retinal pigment epithelium (RPE) is a monolayer of highly specialized cells that help maintain the chemical composition of its surrounding subretinal and choroidal extracellular spaces. Retinal cells (photoreceptors in particular), RPE, and choroidal endothelial cells together help ensure a homeostatically stable metabolic environment with exquisitely sensitive functional responses to light. Aging and disease of the RPE impairs its supportive functions contributing to the progressive loss of photoreceptors and vision. The prevalence of RPE associated retinal degenerations has prompted researchers to develop new therapies aimed at replacing the affected RPE with induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC) derived RPE. Despite recent attempts to characterize stem cell derived RPE and to truly authenticate RPE for clinical applications, there remains a significant unmet need to explore the heterogeneity resulting from donor to donor variation as well as the variations inherent in the current processes of cell manufacture. Additionally, it remains unknown whether the starting cell type influences the resulting RPE phenotype following reprogramming and differentiation. To address these questions, we performed a comprehensive evaluation (genomic, structural, and functional) of 15 iPSC derived RPE originating from different donors and tissues and compiled a reference data set for the authentication of iPSC-derived RPE and RPE derived from other stem cell sources.

  7. Development of correction formula for field potential duration of human induced pluripotent stem cell-derived cardiomyocytes sheets.

    Science.gov (United States)

    Izumi-Nakaseko, Hiroko; Kanda, Yasunari; Nakamura, Yuji; Hagiwara-Nagasawa, Mihoko; Wada, Takeshi; Ando, Kentaro; Naito, Atsuhiko T; Sekino, Yuko; Sugiyama, Atsushi

    2017-09-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used in many studies to assess proarrhythmic risks of chemical compounds. In those studies, field potential durations (FPD) of hiPSC-CMs have been corrected by clinically used Fridericia's and/or Bazett's formulae, however, the rationale for the use of these formulae has not been well established. In the present study, we developed a correction formula for experiments using hiPSC-CMs. First, we analyzed the effect of beating rate on FPD in the hiPSC-CMs sheets with electrical stimuli and a HCN channel inhibitor zatebradine. Next, we examined the relationship between the electrophysiological properties and the expression levels of ion channel genes in the cell sheets. Zatebradine slowed the beating rate and allowed to analyze FPD changes at various pacing cycle lengths. Rate-dependent change in the repolarization period was smaller in the cell sheets than that reported on the human hearts, which can be partly explained by lower gene expression level of hKCNJ2 and hKCNE1. Thus, non-linear equation for correcting FPD in the cell sheet; FPDc = FPD/RR(0.22) with RR given in second was obtained, which may make it feasible to assess net repolarization delay by various chemical compounds with a chronotropic action. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  8. Three-Dimensional Culture of Human Embryonic Stem Cell Derived Hepatic Endoderm and Its Role in Bioartificial Liver Construction

    Directory of Open Access Journals (Sweden)

    Ruchi Sharma

    2010-01-01

    Full Text Available The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays.

  9. Atherosclerosis amelioration by moderate alcohol consumption is associated with increased circulating levels of stromal cell-derived factor-1.

    Science.gov (United States)

    Gil-Bernabe, Paloma; Boveda-Ruiz, Daniel; D'Alessandro-Gabazza, Corina; Toda, Masaaki; Miyake, Yasushi; Mifuji-Moroka, Rumi; Iwasa, Motoh; Morser, John; Gabazza, Esteban C; Takei, Yoshiyuki

    2011-01-01

    A moderate intake of alcohol is associated with lower cardiovascular mortality, and the role of circulating progenitor cells in the beneficial effect of alcohol on atherosclerosis is unclear. The hypothesis of this study was that alcohol ameliorates atherosclerosis by modulating the circulating levels of stromal cell-derived growth factor (SDF)-1 and vascular progenitor cells. Atherosclerosis was induced by infusion of angiotensin II in apolipoprotein-E deficient mice, which were treated with high and low doses of ethanol for 28 days by intraperitoneal injection. Mice treated with low-dose ethanol had significantly less dilatation and fewer atheromatous lesions than mice receiving the high-dose ethanol. The number of circulating fibrocytes was significantly lower in mice treated with high-dose ethanol compared with mice with atherosclerosis untreated with ethanol. The plasma CXCL12/SDF-1 level was significantly increased in mice treated with low-dose ethanol compared with mice treated with a high dose, and the plasma concentration of transforming growth factor-β1 was significantly increased in mice treated with high-dose ethanol compared with control mice. Ethanol regulated the secretion of SDF-1 and vascular endothelial growth factor from fibroblasts in a dose-dependent and bimodal fashion. The circulating level of CXCL12/SDF-1 may be involved, at least in part, in the differential effects of alcohol consumption on atherosclerosis.

  10. Human natural killer cells promote cross-presentation of tumor cell-derived antigens by dendritic cells.

    Science.gov (United States)

    Deauvieau, Florence; Ollion, Vincent; Doffin, Anne-Claire; Achard, Carole; Fonteneau, Jean-François; Verronese, Estelle; Durand, Isabelle; Ghittoni, Raffaella; Marvel, Jacqueline; Dezutter-Dambuyant, Colette; Walzer, Thierry; Vie, Henri; Perrot, Ivan; Goutagny, Nadège; Caux, Christophe; Valladeau-Guilemond, Jenny

    2015-03-01

    Dendritic cells (DCs) cross-present antigen (Ag) to initiate T-cell immunity against most infections and tumors. Natural killer (NK) cells are innate cytolytic lymphocytes that have emerged as key modulators of multiple DC functions. Here, we show that human NK cells promote cross-presentation of tumor cell-derived Ag by DC leading to Ag-specific CD8(+) T-cell activation. Surprisingly, cytotoxic function of NK cells was not required. Instead, we highlight a critical and nonredundant role for IFN-γ and TNF-α production by NK cells to enhance cross-presentation by DC using two different Ag models. Importantly, we observed that NK cells promote cell-associated Ag cross-presentation selectively by monocytes-derived DC (Mo-DC) and CD34-derived CD11b(neg) CD141(high) DC subsets but not by myeloid CD11b(+) DC. Moreover, we demonstrate that triggering NK cell activation by monoclonal antibodies (mAbs)-coated tumor cells leads to efficient DC cross-presentation, supporting the concept that NK cells can contribute to therapeutic mAbs efficiency by inducing downstream adaptive immunity. Taken together, our findings point toward a novel role of human NK cells bridging innate and adaptive immunity through selective induction of cell-associated Ag cross-presentation by CD141(high) DC, a process that could be exploited to better harness Ag-specific cellular immunity in immunotherapy. © 2014 UICC.

  11. Engraftment and morphological development of vascularized human iPS cell-derived 3D-cardiomyocyte tissue after xenotransplantation.

    Science.gov (United States)

    Narita, Hirokazu; Shima, Fumiaki; Yokoyama, Junya; Miyagawa, Shigeru; Tsukamoto, Yoshinari; Takamura, Yasushi; Hiura, Ayami; Fukumoto, Ken; Chiba, Tomohiro; Watanabe, Seiji; Sawa, Yoshiki; Akashi, Mitsuru; Shimoda, Hiroshi

    2017-10-20

    One of the major challenges in cell-based cardiac regenerative medicine is the in vitro construction of three-dimensional (3D) tissues consisting of induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) and a blood vascular network supplying nutrients and oxygen throughout the tissue after implantation. We have successfully built a vascularized iPSC-CM 3D-tissue using our validated cell manipulation technique. In order to evaluate an availability of the 3D-tissue as a biomaterial, functional morphology of the tissues was examined by light and transmission electron microscopy through their implantation into the rat infarcted heart. Before implantation, the tissues showed distinctive myofibrils within iPSC-CMs and capillary-like endothelial tubes, but their profiles were still like immature. In contrast, engraftment of the tissues to the rat heart led the iPSC-CMs and endothelial tubes into organization of cell organelles and junctional apparatuses and prompt development of capillary network harboring host blood supply, respectively. A number of capillaries in the implanted tissues were derived from host vascular bed, whereas the others were likely to be composed by fusion of host and implanted endothelial cells. Thus, our vascularized iPSC-CM 3D-tissues may be a useful regenerative paradigm which will require additional expanded and long-term studies.

  12. Transplanted Human Stem Cell-Derived Interneuron Precursors Mitigate Mouse Bladder Dysfunction and Central Neuropathic Pain after Spinal Cord Injury.

    Science.gov (United States)

    Fandel, Thomas M; Trivedi, Alpa; Nicholas, Cory R; Zhang, Haoqian; Chen, Jiadong; Martinez, Aida F; Noble-Haeusslein, Linda J; Kriegstein, Arnold R

    2016-10-06

    Neuropathic pain and bladder dysfunction represent significant quality-of-life issues for many spinal cord injury patients. Loss of GABAergic tone in the injured spinal cord may contribute to the emergence of these symptoms. Previous studies have shown that transplantation of rodent inhibitory interneuron precursors from the medial ganglionic eminence (MGE) enhances GABAergic signaling in the brain and spinal cord. Here we look at whether transplanted MGE-like cells derived from human embryonic stem cells (hESC-MGEs) can mitigate the pathological effects of spinal cord injury. We find that 6 months after transplantation into injured mouse spinal cords, hESC-MGEs differentiate into GABAergic neuron subtypes and receive synaptic inputs, suggesting functional integration into host spinal cord. Moreover, the transplanted animals show improved bladder function and mitigation of pain-related symptoms. Our results therefore suggest that this approach may be a valuable strategy for ameliorating the adverse effects of spinal cord injury. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Stromal cell derived factor-1α (SDF-1α) directed chemoattraction of transiently CXCR4 overexpressing mesenchymal stem cells into functionalized three-dimensional biomimetic scaffolds

    DEFF Research Database (Denmark)

    Thieme, S; Ryser, Martin; Gentsch, Marcus

    2009-01-01

    into deeper structures of 3D porous bone substitute scaffolds. Here we show that transient overexpression of CXCR4 in human BMSCs induced by mRNA transfection enhances stromal cell-derived factor-1alpha (SDF-1alpha)-directed chemotactic capacity to invade internal compartments of porous 3D bone substitute...

  14. Expression of vascular endothelial growth factor, stromal cell-derived factor-1, and CXCR4 in human limb muscle with acute and chronic ischemia

    NARCIS (Netherlands)

    Weel, V. van; Seghers, L.; Vries, M.R. de; Kuiper, E.J.; Schlingemann, R.O.; Bajema, I.M.; Lindeman, J.H.N.; Delis-Diemen, P.M. van; Hinsbergh, V.W.M. van; Bockel, J.H. van; Quax, P.H.A.

    2007-01-01

    OBJECTIVE - Vascular endothelial growth factor (VEGF)-induced stromal cell-derived factor-1 (SDF-1) has been implicated in angiogenesis in ischemic tissues by recruitment of CXCR4-positive bone marrow-derived circulating cells with paracrine functions in preclinical models. Here, evidence for this

  15. Incorporation of stromal cell-derived factor-1α in PCL/gelatin electrospun membranes for guided bone regeneration.

    Science.gov (United States)

    Ji, Wei; Yang, Fang; Ma, Jinling; Bouma, Margaretha J; Boerman, Otto C; Chen, Zhi; van den Beucken, Jeroen J J P; Jansen, John A

    2013-01-01

    The goal of this work was to evaluate the effect of membrane functionalization with a chemotactic factor on cell recruitment and bone formation in order to develop a bioactive membrane for guided bone regeneration (GBR) applications. To this end, GBR membranes were prepared by electrospinning using poly(ε-caprolactone) (PCL) blended with type B-gelatin, and functionalized with stromal cell derived factor-1α (SDF-1α) via physical adsorption. Firstly, the obtained membranes were evaluated in vitro for SDF-1α release and chemotactic effect on bone marrow stromal cells (BMSCs). Subsequently, in vivo BMSCs recruitment and bone regeneration in response to SDF-1α loaded PCL/gelatin electrospun membranes were assessed in rat cranial defects. The results showed that PCL/gelatin electrospun membranes provided a diffusion-controlled SDF-1α release profile. Furthermore, the membranes loaded with different amounts of SDF-1α (50-400 ng) significantly induced stimulated chemotactic migration of BMSCs in vitro without dose-dependent effects. Eight weeks after implantation in rat cranial defects, SDF-1α loaded membranes yielded a 6-fold increase in the amount of bone formation compared to the bare membranes, albeit that contribution of in vivo BMSCs recruitment to the bone regeneration could not be ascertained. In conclusion, the results of current study indicate the potential for using SDF-1α loaded PCL/gelatin electrospun membrane as a bioactive membrane, which is beneficial for optimizing clinical application of GBR strategies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. A novel method to generate and culture human mast cells: Peripheral CD34+ stem cell-derived mast cells (PSCMCs).

    Science.gov (United States)

    Schmetzer, Oliver; Valentin, Patricia; Smorodchenko, Anna; Domenis, Rossana; Gri, Giorgia; Siebenhaar, Frank; Metz, Martin; Maurer, Marcus

    2014-11-01

    The identification and characterization of human mast cell (MC) functions are hindered by the shortage of MC populations suitable for investigation. Here, we present a novel technique for generating large numbers of well differentiated and functional human MCs from peripheral stem cells (=peripheral stem cell-derived MCs, PSCMCs). Innovative and key features of this technique include 1) the use of stem cell concentrates, which are routinely discarded by blood banks, as the source of CD34+ stem cells, 2) cell culture in serum-free medium and 3) the addition of LDL as well as selected cytokines. In contrast to established and published protocols that use CD34+ or CD133+ progenitor cells from full blood, we used a pre-enriched cell population obtained from stem cell concentrates, which yielded up to 10(8) differentiated human MCs per batch after only three weeks of culture starting with 10(6) total CD34+ cells. The total purity on MCs (CD117+, FcεR1+) generated by this method varied between 55 and 90%, of which 4-20% were mature MCs that contain tryptase and chymase and show expression of FcεRI and CD117 in immunohistochemistry. PSCMCs showed robust histamine release in response to stimulation with anti-FcεR1 or IgE/anti-IgE, and increased proliferation and differentiation in response to IL-1β or IFN-γ. Taken together, this new protocol of the generation of large numbers of human MCs provides for an innovative and suitable option to investigate the biology of human MCs. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Synaptic inputs from stroke-injured brain to grafted human stem cell-derived neurons activated by sensory stimuli.

    Science.gov (United States)

    Tornero, Daniel; Tsupykov, Oleg; Granmo, Marcus; Rodriguez, Cristina; Grønning-Hansen, Marita; Thelin, Jonas; Smozhanik, Ekaterina; Laterza, Cecilia; Wattananit, Somsak; Ge, Ruimin; Tatarishvili, Jemal; Grealish, Shane; Brüstle, Oliver; Skibo, Galina; Parmar, Malin; Schouenborg, Jens; Lindvall, Olle; Kokaia, Zaal

    2017-03-01

    Transplanted neurons derived from stem cells have been proposed to improve function in animal models of human disease by various mechanisms such as neuronal replacement. However, whether the grafted neurons receive functional synaptic inputs from the recipient's brain and integrate into host neural circuitry is unknown. Here we studied the synaptic inputs from the host brain to grafted cortical neurons derived from human induced pluripotent stem cells after transplantation into stroke-injured rat cerebral cortex. Using the rabies virus-based trans-synaptic tracing method and immunoelectron microscopy, we demonstrate that the grafted neurons receive direct synaptic inputs from neurons in different host brain areas located in a pattern similar to that of neurons projecting to the corresponding endogenous cortical neurons in the intact brain. Electrophysiological in vivo recordings from the cortical implants show that physiological sensory stimuli, i.e. cutaneous stimulation of nose and paw, can activate or inhibit spontaneous activity in grafted neurons, indicating that at least some of the afferent inputs are functional. In agreement, we find using patch-clamp recordings that a portion of grafted neurons respond to photostimulation of virally transfected, channelrhodopsin-2-expressing thalamo-cortical axons in acute brain slices. The present study demonstrates, for the first time, that the host brain regulates the activity of grafted neurons, providing strong evidence that transplanted human induced pluripotent stem cell-derived cortical neurons can become incorporated into injured cortical circuitry. Our findings support the idea that these neurons could contribute to functional recovery in stroke and other conditions causing neuronal loss in cerebral cortex. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. PDGF-BB Carried by Endothelial Cell-derived Extracellular Vesicles Reduces Vascular Smooth Muscle Cell Apoptosis in Diabetes.

    Science.gov (United States)

    Togliatto, Gabriele; Dentelli, Patrizia; Rosso, Arturo; Lombardo, Giusy; Gili, Maddalena; Gallo, Sara; Gai, Chiara; Solini, Anna; Camussi, Giovanni; Brizzi, Maria Felice

    2018-01-31

    Endothelial cell-derived extracellular vesicles (CD31EVs) are a new entity for therapeutic/prognostic purposes. The roles of CD31EVs as mediators of smooth muscle cell (VSMC) dysfunction in type 2 diabetes (T2D) is investigated herein.We demonstrated that, unlike non-diabetic, diabetic serum-derived-EVs (D-CD31EVs) boosted apoptosis resistance of VSMCs cultured in hyperglycaemic condition. Biochemical analysis revealed that this effect relies on changes in the balance between anti-apoptotic/pro-apoptotic signals: increase of bcl-2 and decrease of bak/bax. D-CD31EV cargo analysis demonstrated that D-CD31EVs are enriched in membrane-bound-platelet-derived-growth-factor-BB (mbPDGF-BB). Thus, we postulated that mbPDGF-BB transfer by D-CD31EVs could account for VSMC resistance to apoptosis. By depleting CD31EVs of PDGF-BB or blocking the PDGF-BB-receptorβ on VSMCs, we demonstrated that mbPDGF-BB contributes to D-CD31EV-mediated bak/bax and bcl-2 levels. Moreover, we found that bak expression is under the control of PDGF-BB-mediated miR-296-5p expression. In fact, while PDGF-BB-treatment recapitulated D-CD31EV-mediated anti-apoptotic program and VSMC resistance to apoptosis, PDGF-BB-depleted CD31EVs failed. D-CD31EVs also increased VSMC migration and recruitment to neovessels, by means of PDGF-BB. Finally, we found that VSMCs, from human atherosclerotic arteries of T2D individuals, express low bak/bax and high bcl-2 and miR-296-5p levels.This study identifies the mbPDGF-BB in D-CD31EVs as a relevant mediator of diabetes-associated VSMC resistance to apoptosis. © 2018 by the American Diabetes Association.

  19. Regenerative medicine for Parkinson's disease using differentiated nerve cells derived from human buccal fat pad stem cells.

    Science.gov (United States)

    Takahashi, Haruka; Ishikawa, Hiroshi; Tanaka, Akira

    2017-04-01

    The purpose of this study was to evaluate the utility of human adipose stem cells derived from the buccal fat pad (hBFP-ASCs) for nerve regeneration. Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive death of dopaminergic neurons. PD is a candidate disease for cell replacement therapy because it has no fundamental therapeutic methods. We examined the properties of neural-related cells induced from hBFP-ASCs as a cell source for PD treatment. hBFP-ASCs were cultured in neurogenic differentiation medium for about 2 weeks. After the morphology of hBFP-ASCs changed to neural-like cells, the medium was replaced with neural maintenance medium. Cells differentiated from hBFP-ASCs showed neuron-like structures and expressed neuron markers (β3-tubulin, neurofilament 200, and microtubule-associated protein 2), an astrocyte marker (glial fibrillary acidic protein), or dopaminergic neuron-related marker (tyrosine hydroxylase). Induced neural cells were transplanted into a 6-hydroxydopamine (6-OHDA)-lesioned rat hemi-parkinsonian model. At 4 weeks after transplantation, 6-OHDA-lesioned rats were subjected to apomorphine-induced rotation analysis. The transplanted cells survived in the brain of rats as dopaminergic neural cells. No tumor formation was found after cell transplantation. We demonstrated differentiation of hBFP-ASCs into neural cells, and that transplantation of these neural cells improved the symptoms of model rats. Our results suggest that neurons differentiated from hBFP-ASCs would be applicable to cell replacement therapy of PD.

  20. Mammalian Cell-Derived Respiratory Syncytial Virus-Like Particles Protect the Lower as well as the Upper Respiratory Tract.

    Directory of Open Access Journals (Sweden)

    Pramila Walpita

    Full Text Available Globally, Respiratory Syncytial Virus (RSV is a leading cause of bronchiolitis and pneumonia in children less than one year of age and in USA alone, between 85,000 and 144,000 infants are hospitalized every year. To date, there is no licensed vaccine. We have evaluated vaccine potential of mammalian cell-derived native RSV virus-like particles (RSV VLPs composed of the two surface glycoproteins G and F, and the matrix protein M. Results of in vitro testing showed that the VLPs were functionally assembled and immunoreactive, and that the recombinantly expressed F protein was cleaved intracellularly similarly to the virus-synthesized F protein to produce the F1 and F2 subunits; the presence of the F1 fragment is critical for vaccine development since all the neutralizing epitopes present in the F protein are embedded in this fragment. Additional in vitro testing in human macrophage cell line THP-1 showed that both virus and the VLPs were sensed by TLR-4 and induced a Th1-biased cytokine response. Cotton rats vaccinated with RSV VLPs adjuvanted with alum and monophosphoryl lipid A induced potent neutralizing antibody response, and conferred protection in the lower as well as the upper respiratory tract based on substantial virus clearance from these sites. To the best of our knowledge, this is the first VLP/virosome vaccine study reporting protection of the lower as well as the upper respiratory tract: Prevention from replication in the nose is an important consideration if the target population is infants < 6 months of age. This is because continued virus replication in the nose results in nasal congestion and babies at this age are obligate nose breathers. In conclusion, these results taken together suggest that our VLPs show promise to be a safe and effective vaccine for RSV.

  1. A Systemized Approach to Investigate Ca(2+) Synchronization in Clusters of Human Induced Pluripotent Stem-Cell Derived Cardiomyocytes.

    Science.gov (United States)

    Jones, Aled R; Edwards, David H; Cummins, Michael J; Williams, Alan J; George, Christopher H

    2015-01-01

    Induced pluripotent stem cell-derived cardiomyocytes (IPS-CM) are considered by many to be the cornerstone of future approaches to repair the diseased heart. However, current methods for producing IPS-CM typically yield highly variable populations with low batch-to-batch reproducibility. The underlying reasons for this are not fully understood. Here we report on a systematized approach to investigate the effect of maturation in embryoid bodies (EB) vs. "on plate" culture on spontaneous activity and regional Ca(2+) synchronization in IPS-CM clusters. A detailed analysis of the temporal and spatial organization of Ca(2+) spikes in IPS-CM clusters revealed that the disaggregation of EBs between 0.5 and 2 weeks produced IPS-CM characterized by spontaneous beating and high levels of regional Ca(2+) synchronization. These phenomena were typically absent in IPS-CM obtained from older EBs (>2 weeks). The maintenance of all spontaneously active IPS-CM clusters under "on plate" culture conditions promoted the progressive reduction in regional Ca(2+) synchronization and the loss of spontaneous Ca(2+) spiking. Raising the extracellular [Ca(2+)] surrounding these quiescent IPS-CM clusters from ~0.4 to 1.8 mM unmasked discrete behaviors typified by either (a) long-lasting Ca(2+) elevation that returned to baseline or (b) persistent, large-amplitude Ca(2+) oscillations around an increased cytoplasmic [Ca(2+)]. The different responses of IPS-CM to elevated extracellular [Ca(2+)] could be traced back to their routes of derivation. The data point to the possibility of predictably influencing IPS-CM phenotype and response to external activation via defined interventions at early stages in their maturation.

  2. Stromal Cell-Derived Factor-1 Is Associated with Angiogenesis and Inflammatory Cell Infiltration in Aneurysm Walls

    Science.gov (United States)

    Hoh, Brian L.; Hosaka, Koji; Downes, Daniel P.; Nowicki, Kamil W.; Wilmer, Erin N.; Velat, Gregory J.; Scott, Edward W.

    2013-01-01

    Object A small percentage of cerebral aneurysms rupture, but when they do, the effects are devastating. Current management of unruptured aneurysms consist of surgery, endovascular treatment, or watchful waiting. If the biology of how aneurysms grow and rupture were better known, a novel drug could be developed to prevent unruptured aneurysms from rupturing. Ruptured cerebral aneurysms are characterized by inflammation-mediated wall remodeling. We studied the role of stromal cell-derived factor-1 (SDF-1) in inflammation-mediated wall remodeling in cerebral aneurysms. Methods Human aneurysms; murine carotid aneurysms; and murine intracranial aneurysms were studied by immunohistochemistry. Flow cytometry analysis was performed on blood from mice developing carotid aneurysms or intracranial aneurysms. The effect of SDF-1 on endothelial cells and macrophages was studied by chemotaxis cell migration assay and capillary tube formation assay. Anti-SDF-1 blocking antibody was given to mice and compared to control (vehicle)-administered mice for its effects on the walls of carotid aneurysms and the development of intracranial aneurysms. Results Human aneurysms, murine carotid aneurysms, and murine intracranial aneurysms, all express SDF-1; and mice with developing carotid aneurysms or intracranial aneurysms have increased progenitor cells expressing CXCR4, the receptor for SDF-1 (Paneurysms and murine carotid aneurysms have endothelial cells, macrophages, and capillaries in the walls of the aneurysms; and the presence of capillaries in the walls of human aneurysms is associated with presence of macrophages (P=0.01). SDF-1 promotes endothelial cell and macrophage migration (Paneurysm wall. Mice given anti-SDF-1 blocking antibody develop significantly fewer intracranial aneurysms (33% versus 89% in mice given control IgG)(Paneurysms, and may have a role in the development of intracranial aneurysms. PMID:24160472

  3. Nucleus pulposus cells derived IGF-1 and MCP-1 enhance osteoclastogenesis and vertebrae disruption in lumbar disc herniation

    Science.gov (United States)

    Zhu, Zhongjiao; Huang, Peng; Chong, Yanxue; George, Suraj K; Wen, Bingtao; Han, Na; Liu, Zhiqiang; Kang, Lixin; Lin, Nie

    2014-01-01

    Study design: Chronic strained lumbar disc herniation (LDH) cases were classified into bulging LDH, herniated LDH and prolapse LDH types according to imaging examination, and vertebrae disruptions were evaluated. Cytokines derived from the nucleus pulposus cells were detected, and their effects on osteoclastogenesis, as well as the mechanisms involved, were studied via an in vitro osteoclast differentiation system. Objective: To clarify the mechanisms of lumbar vertebrae resorption induced by lumbar herniation. Summary and background data: Chronic strained lumbar disc herniation induced vertebrae erosion exacerbates quality of patients’ life and clinical outcome. Although nucleus pulposus cells derived cytokines were reported to play an important role in this pathogenesis, the fundamental mechanisms underlying this process are still unclear. Methods: Chronic strained lumbar disc herniation patients were diagnosed with CT scan and T2-weighted magnetic resonance imaging. RNA was extracted from 192 surgical specimens of the herniated lumbar disc and 29 surgical excisions of the lumbar disc from spinal injury patients. The expressions of osteoclastogenesis related cytokines and chemokines were examined using real time PCR. Monocytes were induced into osteoclast with M-CSF and RANKL in vitro, while the IGF-1 and MCP-1 were added into the differentiation procedure in order to evaluate the effects and explore the molecular mechanisms. Results: Vertebrae erosion had a positive relationship with lumbar disc herniation severity types. In all of the osteoclastogenesis related cytokines, the IGF-1 and MCP-1 were the most highly expressed in the nucleus pulposus cells. IGF-1 enhances activation of NF-kB signaling directly, but MCP-1 upregulated the expression of RANK, so that enhanced cellular sensitivity to RANKL resulted in increasing osteoclastogenesis and activity. Conclusion: Lumbar herniation induced overexpression of IGF-1 and MCP-1 in nucleus pulposus cells aggravated

  4. Assessment of acute and chronic toxicity of doxorubicin in human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Louisse, Jochem; Wüst, Rob C I; Pistollato, Francesca; Palosaari, Taina; Barilari, Manuela; Macko, Peter; Bremer, Susanne; Prieto, Pilar

    2017-08-01

    The present study assesses acute and chronic toxicity of doxorubicin in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), with the aim to obtain in vitro biomarkers that can be used as readouts to predict in vivo cardiotoxicity. Possible acute toxicity was investigated by assessing effects on the beating rate and the field potential duration (FPD) of doxorubicin-exposed cardiomyocytes by measuring electrical activity using multi-electrode array (MEA) analyses. No effects on the beating rate and FPD were found at concentrations up to 6μM, whereas at 12μM no electrical activity was recorded, indicating that the cardiomyocytes stopped beating. Acute and chronic effects of doxorubicin on mitochondria, which have been reported to be affected in doxorubicin-induced cardiotoxicity, were assessed using high content imaging techniques. To this end hiPSC-CMs were exposed to 150 or 300nM doxorubicin using both single dosing (3h and 2days) and repetitive dosing (3 times, of 2days each), including washout studies to assess delayed effects (assessment at day 14) and effects on cell number, mitochondrial density, mitochondrial membrane potential, mitochondrial superoxide levels and mitochondrial calcium levels were assessed. No effects of doxorubicin were found on mitochondrial density and mitochondrial superoxide levels, whereas doxorubicin reduced cell survival and slightly altered mitochondrial membrane potential and mitochondrial calcium levels, which was most profound in the washout studies. Altogether, the results of the present study show that concentrations of doxorubicin in the micromolar range were required to affect electrical activity of hiPSC-CMs, whereas nanomolar concentrations already affected cell viability and caused mitochondrial disturbances. Integration of these data with other in vitro data may enable the selection of a series of in vitro biomarkers that can be used as readouts to screen chemicals for possible cardiotoxicity

  5. Connective Tissue Growth Factor Promotes Efficient Generation of Human Induced Pluripotent Stem Cell-Derived Choroidal Endothelium.

    Science.gov (United States)

    Songstad, Allison E; Worthington, Kristan S; Chirco, Kathleen R; Giacalone, Joseph C; Whitmore, S Scott; Anfinson, Kristin R; Ochoa, Dalyz; Cranston, Cathryn M; Riker, Megan J; Neiman, Maurine; Stone, Edwin M; Mullins, Robert F; Tucker, Budd A

    2017-06-01

    Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the Western world. Although, the majority of stem cell research to date has focused on production of retinal pigment epithelial (RPE) and photoreceptor cells for the purpose of evaluating disease pathophysiology and cell replacement, there is strong evidence that the choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first to be lost in this disease. As such, to accurately evaluate disease pathophysiology and develop an effective treatment, production of patient-specific, stem cell-derived CECs will be required. In this study, we report for the first time a stepwise differentiation protocol suitable for generating human iPSC-derived CEC-like cells. RNA-seq analysis of the monkey CEC line, RF/6A, combined with two statistical screens allowed us to develop media comprised of various protein combinations. In both screens, connective tissue growth factor (CTGF) was identified as the key component required for driving CEC development. A second factor tumor necrosis factor (TNF)-related weak inducer of apoptosis receptor was also found to promote iPSC to CEC differentiation by inducing endogenous CTGF secretion. CTGF-driven iPSC-derived CEC-like cells formed capillary tube-like vascular networks, and expressed the EC-specific markers CD31, ICAM1, PLVAP, vWF, and the CEC-restricted marker CA4. In combination with RPE and photoreceptor cells, patient-specific iPSC derived CEC-like cells will enable scientists to accurately evaluate AMD pathophysiology and develop effective cell replacement therapies. Stem Cells Translational Medicine 2017;6:1533-1546. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  6. Sodium channel current loss of function in induced pluripotent stem cell-derived cardiomyocytes from a Brugada syndrome patient.

    Science.gov (United States)

    Selga, Elisabet; Sendfeld, Franziska; Martinez-Moreno, Rebecca; Medine, Claire N; Tura-Ceide, Olga; Wilmut, Sir Ian; Pérez, Guillermo J; Scornik, Fabiana S; Brugada, Ramon; Mills, Nicholas L

    2017-10-09

    Brugada syndrome predisposes to sudden death due to disruption of normal cardiac ion channel function, yet our understanding of the underlying cellular mechanisms is incomplete. Commonly used heterologous expression models lack many characteristics of native cardiomyocytes and, in particular, the individual genetic background of a patient. Patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CM) may uncover cellular phenotypical characteristics not observed in heterologous models. Our objective was to determine the properties of the sodium current in iPS-CM with a mutation in SCN5A associated with Brugada syndrome. Dermal fibroblasts from a Brugada syndrome patient with a mutation in SCN5A (c.1100G>A, leading to Nav1.5_p.R367H) were reprogrammed to iPS cells. Clones were characterized and differentiated to form beating clusters and sheets. Patient and control iPS-CM were structurally indistinguishable. Sodium current properties of patient and control iPS-CM were compared. These results were contrasted with those obtained in tsA201 cells heterologously expressing sodium channels with the same mutation. Patient-derived iPS-CM showed a 33.1-45.5% reduction in INa density, a shift in both activation and inactivation voltage-dependence curves, and faster recovery from inactivation. Co-expression of wild-type and mutant channels in tsA201 cells did not compromise channel trafficking to the membrane, but resulted in a reduction of 49.8% in sodium current density without affecting any other parameters. Cardiomyocytes derived from iPS cells from a Brugada syndrome patient with a mutation in SCN5A recapitulate the loss of function of sodium channel current associated with this syndrome; including pro-arrhythmic changes in channel function not detected using conventional heterologous expression systems. Copyright © 2017. Published by Elsevier Ltd.

  7. Concave Pit-Containing Scaffold Surfaces Improve Stem Cell-Derived Osteoblast Performance and Lead to Significant Bone Tissue Formation

    Science.gov (United States)

    Cusella-De Angelis, Maria Gabriella; Laino, Gregorio; Piattelli, Adriano; Pacifici, Maurizio; De Rosa, Alfredo; Papaccio, Gianpaolo

    2007-01-01

    Background Scaffold surface features are thought to be important regulators of stem cell performance and endurance in tissue engineering applications, but details about these fundamental aspects of stem cell biology remain largely unclear. Methodology and Findings In the present study, smooth clinical-grade lactide-coglyolic acid 85:15 (PLGA) scaffolds were carved as membranes and treated with NMP (N-metil-pyrrolidone) to create controlled subtractive pits or microcavities. Scanning electron and confocal microscopy revealed that the NMP-treated membranes contained: (i) large microcavities of 80–120 µm in diameter and 40–100 µm in depth, which we termed primary; and (ii) smaller microcavities of 10–20 µm in diameter and 3–10 µm in depth located within the primary cavities, which we termed secondary. We asked whether a microcavity-rich scaffold had distinct bone-forming capabilities compared to a smooth one. To do so, mesenchymal stem cells derived from human dental pulp were seeded onto the two types of scaffold and monitored over time for cytoarchitectural characteristics, differentiation status and production of important factors, including bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF). We found that the microcavity-rich scaffold enhanced cell adhesion: the cells created intimate contact with secondary microcavities and were polarized. These cytological responses were not seen with the smooth-surface scaffold. Moreover, cells on the microcavity-rich scaffold released larger amounts of BMP-2 and VEGF into the culture medium and expressed higher alkaline phosphatase activity. When this type of scaffold was transplanted into rats, superior bone formation was elicited compared to cells seeded on the smooth scaffold. Conclusion In conclusion, surface microcavities appear to support a more vigorous osteogenic response of stem cells and should be used in the design of therapeutic substrates to improve bone repair and

  8. Functional Epstein-Barr virus reservoir in plasma cells derived from infected peripheral blood memory B cells.

    Science.gov (United States)

    Al Tabaa, Yassine; Tuaillon, Edouard; Bollore, Karine; Foulongne, Vincent; Petitjean, Gael; Seigneurin, Jean-Marie; Duperray, Christophe; Desgranges, Claude; Vendrell, Jean-Pierre

    2009-01-15

    The Epstein-Barr virus (EBV) causes infectious mononucleosis, establishes latency in resting memory B lymphocytes, and is involved in oncogenesis through poorly understood mechanisms. The EBV lytic cycle is initiated during plasma cell differentiation by mRNAs transcripts encoded by BZLF1, which induce the synthesis of EBV proteins such as the immediate-early antigen ZEBRA and the late membrane antigen gp350. Therefore, we assessed the capacity of circulating EBV-infected B lymphocytes from healthy EBV-seropositive subjects to enter and complete the EBV lytic cycle. Purified B lymphocytes were polyclonally stimulated and BZLF1- or gp350-secreting cells (BZLF1-SCs or gp350-SCs) were enumerated by ELISpot assays. The number of BZLF1-SCs ranged from 50 to 480/107 lymphocytes (median, 80; 25th-75th percentiles, 70-150) and gp350-SCs from 10 to 40/107 lymphocytes (median, 17; 25th-75th percentiles, 10-20). gp350-SCs represented only 7.7% to 28.6% of BZLF1-SCs (median, 15%; 25th-75th percentiles, 10.5%-20%). This EBV functional reservoir was preferentially restricted to plasma cells derived from CD27(+) IgD(-) memory B lymphocytes. In 9 of 13 subjects, EBV DNA quantification in B-cell culture supernatants gave evidence of completion of EBV lytic cycle. These results demonstrate that EBV proteins can be secreted by EBV-infected B lymphocytes from healthy carriers, a majority generating an abortive EBV lytic cycle and a minority completing the cycle.

  9. Circulating Stromal Cell-Derived Factor 1α Levels in Heart Failure: A Matter of Proper Sampling.

    Directory of Open Access Journals (Sweden)

    Lesley Baerts

    Full Text Available The chemokine Stromal cell-derived factor 1α (SDF1α, CXCL12 is currently under investigation as a biomarker for various cardiac diseases. The correct interpretation of SDF1α levels is complicated by the occurrence of truncated forms that possess an altered biological activity.We studied the immunoreactivities of SDF1α forms and evaluated the effect of adding a DPP4 inhibitor in sampling tubes on measured SDF1α levels. Using optimized sampling, we measured DPP4 activity and SDF1α levels in patients with varying degrees of heart failure.The immunoreactivities of SDF1α and its degradation products were determined with three immunoassays. A one hour incubation of SDF1α with DPP4 at 37°C resulted in 2/3 loss of immunoreactivity in each of the assays. Incubation with serum gave a similar result. Using appropriate sampling, SDF1α levels were found to be significantly higher in those heart failure patients with a severe loss of left ventricular function. DPP4 activity in serum was not altered in the heart failure population. However, the DPP4 activity was found to be significantly decreased in patients with high SDF1α levels.We propose that all samples for SDF1α analysis should be collected in the presence of at least a DPP4 inhibitor. In doing so, we found higher SDF1α levels in subgroups of patients with heart failure. Our work supports the need for further research on the clinical relevance of SDF1α levels in cardiac disease.

  10. Platelet expression of stromal cell-derived factor-1 is associated with the degree of valvular aortic stenosis.

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    Thomas Wurster

    Full Text Available BACKGROUND AND PURPOSE: Platelet surface expression of stromal-cell-derived factor-1 (SDF-1 is increased during platelet activation and constitutes an important factor in hematopoetic progenitor cell trafficking at sites of vascular injury and ischemia. Enhanced platelet SDF-1 expression has been reported previously in patients suffering from acute coronary syndrome (ACS. We hypothesized that expression of platelet associated SDF-1 may also be influenced by calcified valvular aortic stenosis (AS. METHODS: We consecutively evaluated 941 patients, who were admitted to the emergency department with dyspnea and chest pain. Platelet surface expression of SDF-1 was determined by flow cytometry, AS was assessed using echocardiography and hemodynamic assessment by heart catheterization. A 1∶1 propensity score matching was implemented to match 218 cases with 109 pairs adjusting for age, sex, cardiovascular risk factors, and medication including ACE inhibitors, angiotensin receptor blockers, beta blockers, statins, aspirin, clopidogrel, GPIIb/IIIa antagonists, and vitamin K antagonists. RESULTS: Patients with valvular AS showed enhanced platelet SDF-1 expression compared to patients without AS (non-valvular disease, NV independent of ACS and stable coronary artery disease (SAP [mean fluorescence intensity (MFI for ACS (AS vs. NV: 75±40.4 vs. 39.5±23.3; P = 0.002; for SAP (AS vs. NV: 54.9±44.6 vs. 24.3±11.2; P = 0.008]. Moreover, the degree of AS significantly correlated with SDF-1 platelet surface expression (r = 0.462; P = 0.002. CONCLUSIONS: Valvular AS is associated with enhanced platelet-SDF-1 expression; moreover the degree of valvular AS correlates with SDF-1 platelet surface expression. These findings may have clinical implications in the future.

  11. Platelet expression of stromal cell-derived factor-1 is associated with the degree of valvular aortic stenosis.

    Science.gov (United States)

    Wurster, Thomas; Tegtmeyer, Roland; Borst, Oliver; Rath, Dominik; Geisler, Tobias; Gawaz, Meinrad; Bigalke, Boris

    2014-01-01

    Platelet surface expression of stromal-cell-derived factor-1 (SDF-1) is increased during platelet activation and constitutes an important factor in hematopoetic progenitor cell trafficking at sites of vascular injury and ischemia. Enhanced platelet SDF-1 expression has been reported previously in patients suffering from acute coronary syndrome (ACS). We hypothesized that expression of platelet associated SDF-1 may also be influenced by calcified valvular aortic stenosis (AS). We consecutively evaluated 941 patients, who were admitted to the emergency department with dyspnea and chest pain. Platelet surface expression of SDF-1 was determined by flow cytometry, AS was assessed using echocardiography and hemodynamic assessment by heart catheterization. A 1∶1 propensity score matching was implemented to match 218 cases with 109 pairs adjusting for age, sex, cardiovascular risk factors, and medication including ACE inhibitors, angiotensin receptor blockers, beta blockers, statins, aspirin, clopidogrel, GPIIb/IIIa antagonists, and vitamin K antagonists. Patients with valvular AS showed enhanced platelet SDF-1 expression compared to patients without AS (non-valvular disease, NV) independent of ACS and stable coronary artery disease (SAP) [mean fluorescence intensity (MFI) for ACS (AS vs. NV): 75±40.4 vs. 39.5±23.3; P = 0.002; for SAP (AS vs. NV): 54.9±44.6 vs. 24.3±11.2; P = 0.008]. Moreover, the degree of AS significantly correlated with SDF-1 platelet surface expression (r = 0.462; P = 0.002). Valvular AS is associated with enhanced platelet-SDF-1 expression; moreover the degree of valvular AS correlates with SDF-1 platelet surface expression. These findings may have clinical implications in the future.

  12. Concentrations of stromal cell-derived factor-1 in serum, plasma, and synovial fluid of horses with osteochondral injury.

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    Dymock, David C; Brown, Murray P; Merritt, Kelly A; Trumble, Troy N

    2014-08-01

    To determine whether stromal cell-derived factor-1 (SDF-1) concentrations in serum, plasma, and synovial fluid differed among untrained, race-trained, and osteochondral-injured Thoroughbred racehorses. 22 racehorses without osteochondral injury and 37 racehorses with osteochondral injury. Horses without osteochondral injury were examined before and after 5 to 6 months of race training. Horses with osteochondral injury were undergoing arthroscopic surgery for removal of osteochondral fragments from carpal or metacarpophalangeal or metatarsophalangeal joints (fetlock joints). Serum, plasma, and fetlock or carpal synovial fluid samples were obtained and analyzed for SDF-1 concentration by use of an ELISA. In horses with fetlock or carpal joint injury, mean synovial fluid SDF-1 concentrations were significantly higher, serum SDF-1 concentrations were significantly lower, and synovial fluid-to-serum SDF-1 ratios were significantly higher than in untrained and trained horses. Synovial fluid SDF-1 concentrations were not significantly different between trained and untrained horses. Plasma SDF-1 concentrations were not different among the 3 groups. Results obtained with serum, compared with synovial fluid and plasma, had better sensitivity for differentiating between osteochondral-injured horses and uninjured horses. In horses with fetlock joint osteochondral injury, serum SDF-1 concentrations were correlated with radiographic and arthroscopic inflammation scores, but not arthroscopic cartilage scores. Results suggested that serum SDF-1 concentrations were more sensitive than plasma and synovial fluid concentrations for detection of osteochondral injury in the fetlock or carpal joint of racehorses. Analysis of serum and synovial SDF-1 concentrations in horses with experimentally induced joint injury may help define the onset and progression of post-traumatic osteoarthritis and aid in the evaluation of anti-inflammatory treatments.

  13. Endothelial cell-derived nitric oxide enhances aerobic glycolysis in astrocytes via HIF-1α-mediated target gene activation.

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    Brix, Britta; Mesters, Jeroen R; Pellerin, Luc; Jöhren, Olaf

    2012-07-11

    Astrocytes exhibit a prominent glycolytic activity, but whether such a metabolic profile is influenced by intercellular communication is unknown. Treatment of primary cultures of mouse cortical astrocytes with the nitric oxide (NO) donor DetaNONOate induced a time-dependent enhancement in the expression of genes encoding various glycolytic enzymes as well as transporters for glucose and lactate. Such an effect was shown to be dependent on the hypoxia-inducible factor HIF-1α, which is stabilized and translocated to the nucleus to exert its transcriptional regulation. NO action was dependent on both the PI3K/Akt/mTOR and MEK signaling pathways and required the activation of COX, but was independent of the soluble guanylate cyclase pathway. Furthermore, as a consequence of NO treatment, an enhanced lactate production and release by astrocytes was evidenced, which was prevented by downregulating HIF-1α. Several brain cell types represent possible sources of NO. It was found that endothelial cells, which express the endothelial NO synthase (eNOS) isoform, constitutively produced the largest amount of NO in culture. When astrocytes were cocultured with primary cultures of brain vascular endothelial cells, stabilization of HIF-1α and an enhancement in glucose transporter-1, hexokinase-2, and monocarboxylate transporter-4 expression as well as increased lactate production was found in astrocytes. This effect was inhibited by the NOS inhibitor l-NAME and was not seen when astrocytes were cocultured with primary cultures of cortical neurons. Our findings suggest that endothelial cell-derived NO participates to the maintenance of a high glycolytic activity in astrocytes mediated by astrocytic HIF-1α activation.

  14. Patch-Clamp Recording from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Improving Action Potential Characteristics through Dynamic Clamp.

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    Verkerk, Arie O; Veerman, Christiaan C; Zegers, Jan G; Mengarelli, Isabella; Bezzina, Connie R; Wilders, Ronald

    2017-08-30

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current (IK1) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in "ventricular-like" and "atrial-like" hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial IK1 (computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic IK1 through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon IK1 injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic IK1. In conclusion, the dynamic clamp-based approach of IK1 injection has broad applicability for detailed AP measurements in hiPSC-CMs.

  15. Patch-Clamp Recording from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Improving Action Potential Characteristics through Dynamic Clamp

    Science.gov (United States)

    Veerman, Christiaan C.; Zegers, Jan G.; Mengarelli, Isabella; Bezzina, Connie R.

    2017-01-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current (IK1) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in “ventricular-like” and “atrial-like” hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial IK1 (computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic IK1 through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon IK1 injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic IK1. In conclusion, the dynamic clamp-based approach of IK1 injection has broad applicability for detailed AP measurements in hiPSC-CMs. PMID:28867785

  16. Evaluation of the cardiotoxicity of mitragynine and its analogues using human induced pluripotent stem cell-derived cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Jun Lu

    Full Text Available Mitragynine is a major bioactive compound of Kratom, which is derived from the leave extracts of Mitragyna speciosa Korth or Mitragyna speciosa (M. speciosa, a medicinal plant from South East Asia used legally in many countries as stimulant with opioid-like effects for the treatment of chronic pain and opioid-withdrawal symptoms. Fatal incidents with Mitragynine have been associated with cardiac arrest. In this study, we determined the cardiotoxicity of Mitragynine and other chemical constituents isolated using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs.The rapid delayed rectifier potassium current (IKr, L-type Ca2+ current (ICa,L and action potential duration (APD were measured by whole cell patch-clamp. The expression of KCNH2 and cytotoxicity was determined by real-time PCR and Caspase activity measurements. After significant IKr suppression by Mitragynine (10 µM was confirmed in hERG-HEK cells, we systematically examined the effects of Mitragynine and other chemical constituents in hiPSC-CMs. Mitragynine, Paynantheine, Speciogynine and Speciociliatine, dosage-dependently (0.1∼100 µM suppressed IKr in hiPSC-CMs by 67%∼84% with IC50 ranged from 0.91 to 2.47 µM. Moreover, Mitragynine (10 µM significantly prolonged APD at 50 and 90% repolarization (APD50 and APD90 (439.0±11.6 vs. 585.2±45.5 ms and 536.0±22.6 vs. 705.9±46.1 ms, respectively and induced arrhythmia, without altering the L-type Ca2+ current. Neither the expression, and intracellular distribution of KCNH2/Kv11.1, nor the Caspase 3 activity were significantly affected by Mitragynine.Our study indicates that Mitragynine and its analogues may potentiate Torsade de Pointes through inhibition of IKr in human cardiomyocytes.

  17. Dendritic cells derived from bone marrow cells fail to acquire and present major histocompatibility complex antigens from other dendritic cells

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    Bedford, Penelope A; Burke, Fiona; Stagg, Andrew J; Knight, Stella C

    2008-01-01

    Dendritic cells stimulate primary T-cell responses and a major activation route is via presentation of antigens pre-processed by other dendritic cells. This presentation of pre-processed antigens most likely proceeds through transfer of functional major histocompatibility complex (MHC) antigens through exosomes, ‘live nibbling’ or apoptotic vesicles. We hypothesized that not all dendritic cell populations may both donate MHC antigen to dendritic cells and present antigens acquired from other dendritic cells. All populations tested, including those derived from bone marrow precursor cells stimulated primary, allogeneic T-cell responses and acted as accessory cells for mitogen stimulation. Populations of responder type, splenic dendritic cells promoted allogeneic responses indirectly but those derived from bone marrow cells blocked rather than promoted T-cell proliferation. To identify mechanisms underlying this difference we studied transfer of I-A antigens between cells. Active, two-way transfer of allogeneic I-A occurred between splenic primary antigen presenting cells including CD8α+ lymphoid dendritic cells, CD8α− myeloid dendritic cells and B220+ cells; all these cell types donated as well as acquired MHC molecules. By contrast, the bone marrow-derived dendritic cells donated I-A antigens but acquired negligible amounts. Thus, dendritic cells derived directly from bone marrow cells may stimulate primary T-cell responses through transferring functional MHC to other dendritic cells but may not be able to acquire and present antigens from other dendritic cells. The evidence suggests that T-cell activation may be blocked by the presence of dendritic cells that have not matured through lymphoid tissues which are unable to acquire and present antigens pre-processed by other dendritic cells. PMID:18266716

  18. Engineered myocardial tissues constructed in vivo using cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells in rats

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    Xing Yujie

    2012-01-01

    Full Text Available Abstract Background To explore the feasibility of constructing engineered myocardial tissues (EMTs in vivo, using polylactic acid -co-glycolic acid (PLGA for scaffold and cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells (BMMSCs for seeded cells. Methods BMMSCs were isolated from femur and tibia of Sprague-Dawley (SD rats by density-gradient centrifugation. The third passage cells were treated with 10 μmol/L 5-azacytidine (5-aza and 0.1 μmol/L angiotensin II (Ang II for 24 h, followed by culturing in complete medium for 3 weeks to differentiated into cardiomyocyte-like cells. The cardiomyocyte-like cells were seeded into PLGA scaffolds to form the grafts. The grafts were cultured in the incubator for three days and then implanted into the peritoneal cavity of SD rats. Four weeks later, routine hematoxylin-eosin (HE staining, immunohistochemical staining for myocardium-specific cardiac troponin I (cTnI, scanning electron microscopy and transmission electron microscopy were used to analyze the morphology and microconstruction of the EMTs in host rats. Results HE staining showed that the cardiomyocyte-like cells distributed equally in the PLGA scaffold, and the nuclei arranged in the spindle shape. Immunohistochemical staining revealed that majority of engrafted cells in the PLGA -Cardiomyocyte-like cells group were positive for cTnI. Scanning electron microscopy showed that the inoculated cells well attached to PLGA and grew in 3 dimensions in construct. Transmission electron microscopy showed that the EMTs contained well arranged myofilaments paralleled to the longitudinal cell axis, the cells were rich in endoplasmic reticulum and mitochondria, while desmosomes, gap junction and Z line-like substances were also can be observed as well within the engrafted cells. Conclusion We have developed an in vivo method to construct engineered myocardial tissue. The in vivo microenvironment helped engrafted cells/tissue survive and

  19. Evaluation of the Cardiotoxicity of Mitragynine and Its Analogues Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

    Science.gov (United States)

    Wu, Jianjun; Jamil, Mohd Fadzly Amar; Tan, Mei Lan; Adenan, Mohd Ilham; Wong, Philip; Shim, Winston

    2014-01-01

    Introduction Mitragynine is a major bioactive compound of Kratom, which is derived from the leave extracts of Mitragyna speciosa Korth or Mitragyna speciosa (M. speciosa), a medicinal plant from South East Asia used legally in many countries as stimulant with opioid-like effects for the treatment of chronic pain and opioid-withdrawal symptoms. Fatal incidents with Mitragynine have been associated with cardiac arrest. In this study, we determined the cardiotoxicity of Mitragynine and other chemical constituents isolated using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods and Results The rapid delayed rectifier potassium current (IKr), L-type Ca2+ current (ICa,L) and action potential duration (APD) were measured by whole cell patch-clamp. The expression of KCNH2 and cytotoxicity was determined by real-time PCR and Caspase activity measurements. After significant IKr suppression by Mitragynine (10 µM) was confirmed in hERG-HEK cells, we systematically examined the effects of Mitragynine and other chemical constituents in hiPSC-CMs. Mitragynine, Paynantheine, Speciogynine and Speciociliatine, dosage-dependently (0.1∼100 µM) suppressed IKr in hiPSC-CMs by 67% ∼84% with IC50 ranged from 0.91 to 2.47 µM. Moreover, Mitragynine (10 µM) significantly prolonged APD at 50 and 90% repolarization (APD50 and APD90) (439.0±11.6 vs. 585.2±45.5 ms and 536.0±22.6 vs. 705.9±46.1 ms, respectively) and induced arrhythmia, without altering the L-type Ca2+ current. Neither the expression,and intracellular distribution of KCNH2/Kv11.1, nor the Caspase 3 activity were significantly affected by Mitragynine. Conclusions Our study indicates that Mitragynine and its analogues may potentiate Torsade de Pointes through inhibition of IKr in human cardiomyocytes. PMID:25535742

  20. [Effect of PC cell-derived growth factor RNA interference on biological behavior of esophageal squamous carcinoma cells].

    Science.gov (United States)

    Zheng, Qing-feng; Liu, Shuo-yan; Wang, Hai-yan; Wang, Feng; Wang, Zhen; Chen, Xiao-feng; Wang, Jian-jian; Ying, Min-gang; Zheng, Xiong-wei; Lin, Xian-dong; Zhou, Zhi-feng; Gong, Fu-sheng; Xie, Yun-qing

    2013-09-01

    To investigate the effect of PC cell-derived growth factor (PCDGF) RNA interference on esophageal squamous carcinoma cells Eca-109 in vitro. The PCDGF-shRNA expression vector was transfected into the Eca-109 cells by liposome. After transfection, the mRNA and protein expressions of PCDGF were detected by RT-PCR and Western-blot respectively. Cell Counting Kit-8 (CCK-8) assay and Boyden chamber method were performed to measure the cell proliferation and invasion ability respectively. The expression levels of PCDGF mRNA and protein were both decreased in Eca-109 cells transfected with PCDGF-shRNA expression vector (transfection group). Twenty-four, 48 and 72 h after transfection, the cells proliferation in the transfection group was inhibited, and the inhibition rate was 20.4%, 21.1% and 20.9% respectively. The cell proliferation activity in the transfection group was significantly lower than that in the non-transfection group, liposome group and negative vector group (all Pgroup,negative vector group, liposome group and transfection group was 118.8±12.0, 100.8±9.0, 114.3±4.7, and 53.5±16.3 respectively. The differences were statistically significant between the transfection group and the other 3 groups (all P<0.05). PCDGF RNA interference can inhibit the proliferation and invasion abilities of esophageal squamous carcinoma cells in vitro. PCDGF gene may be the new target of gene therapy.

  1. Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium.

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    Ryuhei Hayashi

    Full Text Available Induced pluripotent stem (iPS cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF-derived iPS cells (253G1 and human adult corneal limbal epithelial cells (HLEC-derived iPS cells (L1B41. We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA differentiation method, as Pax6(+/K12(+ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.

  2. Cytokeratin expression of engrafted three-dimensional culture tissues using epithelial cells derived from porcine periodontal ligaments.

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    Yamada, Rie; Kitajima, Kayoko; Arai, Kyoko; Igarashi, Masaru

    2014-09-01

    This study investigated the differentiation and proliferation of epithelial cells derived from periodontal ligaments after three-dimensional culture using collagen gel with fibroblasts in vitro and in vivo. Epithelial cells and fibroblasts were derived from porcine periodontal ligaments. Epithelial cells were labeled using a fluorescent red membrane marker (PKH-26GL) and were seeded onto collagen gel with fibroblasts, followed by incubation in an air-liquid interface for 7 days. Three-dimensional cultures were grafted onto the backs of nude mice and removed at 1, 7, and 14 days after surgery (in vivo model). Unfixed sections (5 μm) were used to detect the presence of red fluorescent cells. Paraffin sections were analyzed histologically and immunohistochemically. Specimens were compared with three-dimensional culture tissues at 8, 14 and 21 days (in vitro model). Grafted three-dimensional cultures formed a stratified epithelial structure similar to skin in vivo. Epithelial cells were sequenced in basal-layer-like structures at 14 days in vivo. Immunohistochemical findings showed that the expression of cytokeratin was detected in the epithelial layer in in vitro and in vivo models. Ck8 + 18 + 19 was expressed in the upper epithelial layer in the in vitro model at 14 and 21 days, but not in vivo. Involucrin was expressed in the certified layers in vitro at 14 days, but not in vivo. Laminin was detected at the dermo-epidermal junction in vivo at 7 and 14 days, but not in vitro. These results suggest that differentiation of three-dimensional culture tissues differs in vivo and in vitro. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Notch1-WISP-1 axis determines the regulatory role of mesenchymal stem cell-derived stromal fibroblasts in melanoma metastasis.

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    Shao, Hongwei; Cai, Long; Moller, Mecker; Issac, Biju; Zhang, Leiming; Owyong, Mark; Moscowitz, Anna Elizabeth; Vazquez-Padron, Roberto; Radtke, Freddy; Liu, Zhao-Jun

    2016-11-29

    Mesenchymal stem cells-derived fibroblasts (MSC-DF) constitute a significant portion of stromal fibroblasts in the tumor microenvironment (TME) and are key modulators of tumor progression. However, the molecular mechanisms that determine their tumor-regulatory function are poorly understood. Here, we uncover the Notch1 pathway as a molecular determinant that selectively controls the regulatory role of MSC-DF in melanoma metastasis. We demonstrate that the Notch1 pathway's activity is inversely correlated with the metastasis-regulating function of fibroblasts and can determine the metastasis-promoting or -suppressing phenotype of MSC-DF. When co-grafted with melanoma cells, MSC-DFNotch1-/- selectively promote, while MSC-DFN1IC+/+ preferentially suppress melanoma metastasis, but not growth, in mouse models. Consistently, conditioned media (CM) from MSC-DFNotch1-/- and MSC-DFN1IC+/+ oppositely, yet selectively regulates migration, but not growth of melanoma cells in vitro. Additionally, when co-cultured with metastatic melanoma cells in vitro, MSC-DFNotch1-/- support, while MSC-DFN1IC+/+ inhibit melanoma cells in the formation of spheroids. These findings expand the repertoire of Notch1 signaling as a molecular switch in determining the tumor metastasis-regulating function of MSC-DF. We also identified Wnt-induced secreted protein-1 (WISP-1) as a key downstream secretory mediator of Notch1 signaling to execute the influential role of MSC-DF on melanoma metastasis. These findings reveal the Notch1-WISP-1 axis as a crucial molecular determinant in governing stromal regulation of melanoma metastasis; thus, establishing this axis as a potential therapeutic target for melanoma metastasis.

  4. The effects of human platelet lysate on dental pulp stem cells derived from impacted human third molars.

    Science.gov (United States)

    Chen, Bo; Sun, Hai-Hua; Wang, Han-Guo; Kong, Hui; Chen, Fa-Ming; Yu, Qing

    2012-07-01

    Human platelet lysate (PL) has been suggested as a substitute for fetal bovine serum (FBS) in the large-scale expansion of dental pulp stem cells (DPSCs). However, the biological effects and the optimal concentrations of PL for the proliferation and differentiation of human DPSCs remain unexplored. We isolated and expanded stem cells from the dental pulp of extracted third molars and evaluated the effects of PL on the cells' proliferative capacity and differentiation potential in vitro and in vivo. Before testing, immunocytochemical staining and flow cytometry-based cell sorting showed that the cells derived from human dental pulp contained mesenchymal stem cell populations. Cells were grown on tissue culture plastic or on hydroxyapatite-tricalcium phosphate (HA/TCP) biomaterials and were incubated with either normal or odontogenic/osteogenic media in the presence or absence of various concentrations of human PL for further investigation. The proliferation of DPSCs was significantly increased when the cells were cultured in 5% PL under all testing conditions (P models. We conclude that the appropriate concentration of PL enhances the proliferation and mineralized differentiation of human DPSCs both in vitro and in vivo, which supports the use of PL as an alternative to FBS or a nonzoonotic adjuvant for cell culture in future clinical trials. However, the elucidation of the molecular complexity of PL products and the identification of both the essential growth factors that determine the fate of a specific stem cell and the criteria to establish dosing require further investigation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Could a B-1 cell derived phagocyte "be one" of the peritoneal macrophages during LPS-driven inflammation?

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    Ana Flavia Popi

    Full Text Available The inflammatory response is driven by signals that recruit and elicit immune cells to areas of tissue damage or infection. The concept of a mononuclear phagocyte system postulates that monocytes circulating in the bloodstream are recruited to inflamed tissues where they give rise to macrophages. A recent publication demonstrated that the large increase in the macrophages observed during infection was the result of the multiplication of these cells rather than the recruitment of blood monocytes. We demonstrated previously that B-1 cells undergo differentiation to acquire a mononuclear phagocyte phenotype in vitro (B-1CDP, and we propose that B-1 cells could be an alternative origin for peritoneal macrophages. A number of recent studies that describe the phagocytic and microbicidal activity of B-1 cells in vitro and in vivo support this hypothesis. Based on these findings, we further investigated the differentiation of B-1 cells into phagocytes in vivo in response to LPS-induced inflammation. Therefore, we investigated the role of B-1 cells in the composition of the peritoneal macrophage population after LPS stimulation using osteopetrotic mice, BALB/Xid mice and the depletion of monocytes/macrophages by clodronate treatment. We show that peritoneal macrophages appear in op/op((-/- mice after LPS stimulation and exhibit the same Ig gene rearrangement (VH11 that is often found in B-1 cells. These results strongly suggest that op/op((-/- peritoneal "macrophages" are B-1CDP. Similarly, the LPS-induced increase in the macrophage population was observed even following monocyte/macrophage depletion by clodronate. After monocyte/macrophage depletion by clodronate, LPS-elicited macrophages were observed in BALB/Xid mice only following the transfer of B-1 cells. Based on these data, we confirmed that B-1 cell differentiation into phagocytes also occurs in vivo. In conclusion, the results strongly suggest that B-1 cell derived phagocytes are a component of

  6. Hepatic ischemia and reperfusion injury in the absence of myeloid cell-derived COX-2 in mice.

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    Sergio Duarte

    Full Text Available Cyclooxygenase-2 (COX-2 is a mediator of hepatic ischemia and reperfusion injury (IRI. While both global COX-2 deletion and pharmacologic COX-2 inhibition ameliorate liver IRI, the clinical use of COX-2 inhibitors has been linked to increased risks of heart attack and stroke. Therefore, a better understanding of the role of COX-2 in different cell types may lead to improved therapeutic strategies for hepatic IRI. Macrophages of myeloid origin are currently considered to be important sources of the COX-2 in damaged livers. Here, we used a Cox-2flox conditional knockout mouse (COX-2-M/-M to examine the function of COX-2 expression in myeloid cells during liver IRI. COX-2-M/-M mice and their WT control littermates were subjected to partial liver ischemia followed by reperfusion. COX-2-M/-M macrophages did not express COX-2 upon lipopolysaccharide stimulation and COX-2-M/-M livers showed reduced levels of COX-2 protein post-IRI. Nevertheless, selective deletion of myeloid cell-derived COX-2 failed to ameliorate liver IRI; serum transaminases and histology were comparable in both COX-2-M/-M and WT mice. COX-2-M/-M livers, like WT livers, developed extensive necrosis, vascular congestion, leukocyte infiltration and matrix metalloproteinase-9 (MMP-9 expression post-reperfusion. In addition, myeloid COX-2 deletion led to a transient increase in IL-6 levels after hepatic reperfusion, when compared to controls. Administration of celecoxib, a selective COX-2 inhibitor, resulted in significantly improved liver function and histology in both COX-2-M/-M and WT mice post-reperfusion, providing evidence that COX-2-mediated liver IRI is caused by COX-2 derived from a source(s other than myeloid cells. In conclusion, these results support the view that myeloid COX-2, including myeloid-macrophage COX-2, is not responsible for the hepatic IRI phenotype.

  7. Visceral fat area is a strong predictor of leukocyte cell-derived chemotaxin 2, a potential biomarker of dyslipidemia.

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    Tanisawa, Kumpei; Taniguchi, Hirokazu; Sun, Xiaomin; Ito, Tomoko; Kawakami, Ryoko; Sakamoto, Shiz