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Sample records for cancer cell-derived microvesicles

  1. Microfluidic isolation of cancer-cell-derived microvesicles from hetergeneous extracellular shed vesicle populations

    PubMed Central

    Santana, Steven M.; Antonyak, Marc A.; Cerione, Richard A.

    2015-01-01

    Extracellular shed vesicles, including exosomes and microvesicles, are disseminated throughout the body and represent an important conduit of cell communication. Cancer-cell-derived microvesicles have potential as a cancer biomarker as they help shape the tumor microenvironment to promote the growth of the primary tumor and prime the metastatic niche. It is likely that, in cancer cell cultures, the two constituent extracellular shed vesicle subpopulations, observed in dynamic light scattering, represent an exosome population and a cancer-cell-specific microvesicle population and that extracellular shed vesicle size provides information about provenance and cargo. We have designed and implemented a novel microfluidic technology that separates microvesicles, as a function of diameter, from heterogeneous populations of cancer-cell-derived extracellular shed vesicles. We measured cargo carried by the microvesicle subpopulation processed through this microfluidic platform. Such analyses could enable future investigations to more accurately and reliably determine provenance, functional activity, and mechanisms of transformation in cancer. PMID:25342569

  2. Improved Flow Cytometric Assessment Reveals Distinct Microvesicle (Cell-Derived Microparticle) Signatures in Joint Diseases

    PubMed Central

    György, Bence; Szabó, Tamás G.; Turiák, Lilla; Wright, Matthew; Herczeg, Petra; Lédeczi, Zsigmond; Kittel, Ágnes; Polgár, Anna; Tóth, Kálmán; Dérfalvi, Beáta; Zelenák, Gergő; Böröcz, István; Carr, Bob; Nagy, György; Vékey, Károly; Gay, Steffen; Falus, András; Buzás, Edit I.

    2012-01-01

    Introduction Microvesicles (MVs), earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. Methods In this study, we analyzed synovial fluid (SF) samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM), Nanoparticle Tracking Analysis (NTA) and mass spectrometry (MS). For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. Results EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3+ and CD8+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p = 0.027 and p = 0.009, respectively, after Bonferroni corrections). In JIA, we identified reduced numbers of B cell-derived MVs (p = 0.009, after Bonferroni correction). Conclusions Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures. PMID:23185418

  3. Cranial grafting of stem cell-derived microvesicles improves cognition and reduces neuropathology in the irradiated brain

    PubMed Central

    Baulch, Janet E.; Acharya, Munjal M.; Allen, Barrett D.; Ru, Ning; Chmielewski, Nicole N.; Martirosian, Vahan; Giedzinski, Erich; Syage, Amber; Park, Audrey L.; Benke, Sarah N.; Parihar, Vipan K.; Limoli, Charles L.

    2016-01-01

    Cancer survivors face a variety of challenges as they cope with disease recurrence and a myriad of normal tissue complications brought on by radio- and chemotherapeutic treatment regimens. For patients subjected to cranial irradiation for the control of CNS malignancy, progressive and debilitating cognitive dysfunction remains a pressing unmet medical need. Although this problem has been recognized for decades, few if any satisfactory long-term solutions exist to resolve this serious unintended side effect of radiotherapy. Past work from our laboratory has demonstrated the neurocognitive benefits of human neural stem cell (hNSC) grafting in the irradiated brain, where intrahippocampal transplantation of hNSC ameliorated radiation-induced cognitive deficits. Using a similar strategy, we now provide, to our knowledge, the first evidence that cranial grafting of microvesicles secreted from hNSC affords similar neuroprotective phenotypes after head-only irradiation. Cortical- and hippocampal-based deficits found 1 mo after irradiation were completely resolved in animals cranially grafted with microvesicles. Microvesicle treatment was found to attenuate neuroinflammation and preserve host neuronal morphology in distinct regions of the brain. These data suggest that the neuroprotective properties of microvesicles act through a trophic support mechanism that reduces inflammation and preserves the structural integrity of the irradiated microenvironment. PMID:27044087

  4. Cranial grafting of stem cell-derived microvesicles improves cognition and reduces neuropathology in the irradiated brain.

    PubMed

    Baulch, Janet E; Acharya, Munjal M; Allen, Barrett D; Ru, Ning; Chmielewski, Nicole N; Martirosian, Vahan; Giedzinski, Erich; Syage, Amber; Park, Audrey L; Benke, Sarah N; Parihar, Vipan K; Limoli, Charles L

    2016-04-26

    Cancer survivors face a variety of challenges as they cope with disease recurrence and a myriad of normal tissue complications brought on by radio- and chemotherapeutic treatment regimens. For patients subjected to cranial irradiation for the control of CNS malignancy, progressive and debilitating cognitive dysfunction remains a pressing unmet medical need. Although this problem has been recognized for decades, few if any satisfactory long-term solutions exist to resolve this serious unintended side effect of radiotherapy. Past work from our laboratory has demonstrated the neurocognitive benefits of human neural stem cell (hNSC) grafting in the irradiated brain, where intrahippocampal transplantation of hNSC ameliorated radiation-induced cognitive deficits. Using a similar strategy, we now provide, to our knowledge, the first evidence that cranial grafting of microvesicles secreted from hNSC affords similar neuroprotective phenotypes after head-only irradiation. Cortical- and hippocampal-based deficits found 1 mo after irradiation were completely resolved in animals cranially grafted with microvesicles. Microvesicle treatment was found to attenuate neuroinflammation and preserve host neuronal morphology in distinct regions of the brain. These data suggest that the neuroprotective properties of microvesicles act through a trophic support mechanism that reduces inflammation and preserves the structural integrity of the irradiated microenvironment.

  5. Embryonic Stem Cell-Derived Microvesicles: Could They be Used for Retinal Regeneration?

    PubMed

    Farber, Debora B; Katsman, Diana

    2016-01-01

    Mouse embryonic stem cells (mESCs) release into the medium in which they are cultured heterogeneous populations of microvesicles (mESMVs), important components of cell-cell communication, that transfer their contents not only to other stem cells but also to cells of other origins. The purpose of these studies was to demonstrate that ESMVs could be the signals that lead the retinal progenitor Müller cells to de-differentiate and re-entry the cell cycle, followed by differentiation along retinal lineages. Indeed, we found that ESMVs induce these processes and change Müller cells' microenvironment towards a more permissive state for tissue regeneration.

  6. Microvesicles released from hormone-refractory prostate cancer cells facilitate mouse pre-osteoblast differentiation.

    PubMed

    Itoh, Tomohiro; Ito, Yuko; Ohtsuki, Yoshinori; Ando, Masashi; Tsukamasa, Yasuyuki; Yamada, Nami; Naoe, Tomoki; Akao, Yukihiro

    2012-10-01

    Bone metastasis is often occurs in patients with prostate cancer. There is a vicious cycle for bone metastases involving prostate cancer cells, osteoblasts, and osteoclasts. Acting among those cells during the process of metastasis are several molecules such as bone morphogenetic proteins, platelet-derived growth factor, endothelin-1, matrix metalloproteases, vascular endothelial growth factor, transforming growth factor-β, and insulin-like growth factors. Cell-derived microvesicles are endogenous carriers transporting proteins, mRNAs and miRNAs between cells, which is a candidate for participation in the bone metastasis of these cells. Here, we demonstrated that prostate cancer cells in vitro released microvesicles into the culture medium (PCa-MVs), which was shown by electron microscopic study and nanoparticle tracking analysis. In this study, we found for the first time that these PCa-MVs enhanced osteoblast differentiation mainly through the delivery of PCa cell-derived v-ets erythroblastosis virus E26 oncogene homolog 1, which is an osteoblast differentiation related-transcriptional factor.

  7. [Application Prospect of Stem Cell-derived Microvesicles in Regeneration of Injured Tissues].

    PubMed

    Yin, Huiqun; Jiang, Hong

    2015-06-01

    More and more evidence indicates that microvesicles (MVs) play a key role in cell-to-cell communication. The MVs are circular fragments of membrane released from the endosomal compartment as exosomes or shed from the cell surface membranes of most types. Components of donor cells are incorporated into MVs that contain bioactive lipids, proteins, genetic cargoes. MVs derived from stem cells may reprogram cells that survived in injury tissue and favor tissue regeneration by delivering their bioactive cargoes to influence the behaviors of recipient cells. Compared with mesenchymal stem cells (MSCs), MVs derived from MSCs were found to mimic the beneficial effects of these cells. These proregenerative effects mediated by MVs can be explained by the fact that MVs are enriched in bioactive lipids, anti-apoptotic and pro-stimulatory growth factors or cytokines, and deliver mRNAs, regulatory miRNAs and proteins that improve overall cell function. Therefore, it opens novel perspectives in exploiting these MVs in tissue regeneration and repair. In addition, the use of MVs instead of stem cells could represent a safe and potentially more advantageous alternative to cell-therapy approaches.

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

    PubMed

    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.

  9. Longitudinal effects of menopausal hormone treatments on platelet characteristics and cell-derived microvesicles.

    PubMed

    Miller, Virginia M; Lahr, Brian D; Bailey, Kent R; Heit, John A; Harman, S Mitchell; Jayachandran, Muthuvel

    2016-01-01

    Activated platelets serve as a catalyst for thrombin generation and a source of vasoactive and mitogenic factors affecting vascular remodeling. Oral menopausal hormone treatments (MHT) may carry greater thrombotic risk than transdermal products. This study compared effects of oral and transdermal MHT on platelet characteristics, platelet proteins, and platelet-derived microvesicles (MV) in recently menopausal women. Platelets and MV were prepared from blood of a subset of women (n = 117) enrolled in the Kronos Early Estrogen Prevention Study prior to and after 48 months of treatment with either oral conjugated equine estrogen (0.45 mg/day), transdermal 17β-estradiol (50 µg/day), each with intermittent progesterone (200 mg/day for 12 days a month), or placebo pills and patch. Platelet count and expression of platelet P-selectin and fibrinogen receptors were similar across groups. An aggregate measure of 4-year change in vasoactive and mitogenic factors in platelet lysate, by principle component analysis, indicated significantly lower values in both MHT groups compared to placebo. Increases in numbers of tissue factor positive and platelet-derived MV were significantly greater in the transdermal compared to placebo group. MHT was associated with significantly reduced platelet content of vasoactive and mitogenic factors representing a potential mechanism by which MHT may affect vascular remodeling. Various hormonal compositions and doses of MHT could differentially regulate nuclear transcription in bone marrow megakaryocytes and non-genomic pathways in circulating platelets thus determining numbers and characteristics of circulating MV. Thrombotic risk associated with oral MHT most likely involves liver-derived inflammatory/coagulation proteins rather than circulating platelets per se.

  10. Embryonic Stem Cell-Derived Microvesicles Induce Gene Expression Changes in Müller Cells of the Retina

    PubMed Central

    Katsman, Diana; Stackpole, Emma J.; Domin, Daniel R.; Farber, Debora B.

    2012-01-01

    Cell-derived microvesicles (MVs), recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs) have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC) mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation. PMID:23226281

  11. Embryonic stem cell-derived microvesicles induce gene expression changes in Müller cells of the retina.

    PubMed

    Katsman, Diana; Stackpole, Emma J; Domin, Daniel R; Farber, Debora B

    2012-01-01

    Cell-derived microvesicles (MVs), recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs) have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC) mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation.

  12. Microvesicles as mediators of intercellular communication in cancer.

    PubMed

    Antonyak, Marc A; Cerione, Richard A

    2014-01-01

    The discovery that cancer cells generate large membrane-enclosed packets of epigenetic information, known as microvesicles (MVs), that can be transferred to other cells and influence their behavior (Antonyak et al., Small GTPases 3:219-224, 2012; Cocucci et al., Trends Cell Biol 19:43-51, 2009; Rak, Semin Thromb Hemost 36:888-906, 2010; Skog et al., Nat Cell Biol 10:1470-1476, 2008) has added a unique perspective to the classical paracrine signaling paradigm. This is largely because, in addition to growth factors and cytokines, MVs contain a variety of components that are not usually thought to be released into the extracellular environment by viable cells including plasma membrane-associated proteins, cytosolic- and nuclear-localized proteins, as well as nucleic acids, particularly RNA transcripts and micro-RNAs (Skog et al., Nat Cell Biol 10:1470-1476, 2008; Al-Nedawi et al., Nat Cell Biol 10:619-624, 2008; Antonyak et al., Proc Natl Acad Sci U S A 108:4852-4857, 2011; Balaj et al., Nat Commun 2:180, 2011; Choi et al., J Proteome Res 6:4646-4655, 2007; Del Conde et al., Blood 106:1604-1611, 2005; Gallo et al., PLoS One 7:e30679, 2012; Graner et al., FASEB J 23:1541-1557, 2009; Grange et al., Cancer Res 71:5346-5356, 2011; Hosseini-Beheshti et al., Mol Cell Proteomics 11:863-885, 2012; Martins et al., Curr Opin Oncol 25:66-75, 2013; Noerholm et al., BMC Cancer 12:22, 2012; Zhuang et al., EMBO J 31:3513-3523, 2012). When transferred between cancer cells, MVs have been shown to stimulate signaling events that promote cell growth and survival (Al-Nedawi et al., Nat Cell Biol 10:619-624, 2008). Cancer cell-derived MVs can also be taken up by normal cell types that surround the tumor, an outcome that helps shape the tumor microenvironment, trigger tumor vascularization, and even confer upon normal recipient cells the transformed characteristics of a cancer cell (Antonyak et al., Proc Natl Acad Sci U S A 108:4852-4857, 2011; Martins et al., Curr Opin Oncol 25

  13. The emerging role of exosome and microvesicle- (EMV-) based cancer therapeutics and immunotherapy.

    PubMed

    Moore, Colin; Kosgodage, Uchini; Lange, Sigrun; Inal, Jameel M

    2017-03-01

    There is an urgent need to develop new combination therapies beyond existing surgery, radio- and chemo-therapy, perhaps initially combining chemotherapy with the targeting specificities of immunotherapy. For this, strategies to limit inflammation and immuno-suppression and evasion in the tumour microenvironment are also needed. To devise effective new immunotherapies we must first understand tumour immunology, including the roles of T cells, macrophages, myeloid suppressor cells and of exosomes and microvesicles (EMVs) in promoting angiogenesis, tumour growth, drug resistance and metastasis. One promising cancer immunotherapy discussed uses cationic liposomes carrying tumour RNA (RNA-lipoplexes) to provoke a strong anti-viral-like (cytotoxic CD8(+) ) anti-tumour immune response. Mesenchymal stem cell-derived EMVs, with their capacity to migrate towards inflammatory areas including solid tumours, have also been used. As tumour EMVs clearly exacerbate the tumour microenvironment, another therapy option could involve EMV removal. Affinity-based methods to deplete EMVs, including an immuno-depletion, antibody-based affinity substrate, are therefore considered. Finally EMV and exosome-mimetic nanovesicles (NVs) delivery of siRNA or chemotherapeutic drugs that target tumours using peptide ligands for cognate receptors on the tumour cells are discussed. We also touch upon the reversal of drug efflux in EMVs from cancer cells which can sensitize cells to chemotherapy. The use of immunotherapy in combination with the advent of EMVs provides potent therapies to various cancers. This article is protected by copyright. All rights reserved.

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

    PubMed Central

    Mao, Mao; Zhou, Liang

    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. PMID:28194184

  15. Platelet microvesicles in health and disease.

    PubMed

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

    2017-01-19

    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.

  16. Detection and isolation of circulating exosomes and microvesicles for cancer monitoring and diagnostics using micro-/nano-based devices

    PubMed Central

    Ko, Jina

    2016-01-01

    In the last several years, nanoscale vesicles that originate from tumor cells and which can be found circulating in the blood (i.e. exosomes and microvesicles) have been discovered to contain a wealth of proteomic and genetic information to monitor cancer progression, metastasis, and drug efficacy. However, the use of exosomes and microvesicles as biomarkers to improve patient care has been limited by their small size (30 nm–1 μm) and the extensive sample preparation required for their isolation and measurement. In this Critical Review, we explore the emerging use of micro and nano-technology to isolate and detect exosomes and microvesicles in clinical samples and the application of this technology to the monitoring and diagnosis of cancer. PMID:26378496

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

    SciTech Connect

    Stratton, Dan; Moore, Colin; Antwi-Baffour, Samuel; Lange, Sigrun; Inal, Jameel

    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.

  18. Microvesicle removal of anticancer drugs contributes to drug resistance in human pancreatic cancer cells

    PubMed Central

    Muralidharan-Chari, Vandhana; Kohan, Hamed Gilzad; Asimakopoulos, Alexandros G.; Sudha, Thangirala; Sell, Stewart; Kannan, Kurunthachalam; Boroujerdi, Mehdi; Davis, Paul J.; Mousa, Shaker A.

    2016-01-01

    High mortality in pancreatic cancer patients is partly due to resistance to chemotherapy. We describe that human pancreatic cancer cells acquire drug resistance by a novel mechanism in which they expel and remove chemotherapeutic drugs from the microenvironment via microvesicles (MVs). Using human pancreatic cancer cells that exhibit varied sensitivity to gemcitabine (GEM), we show that GEM exposure triggers the cancer cells to release MVs in an amount that correlates with that cell line's sensitivity to GEM. The importance of MV-release in gaining drug resistance in GEM-resistant pancreatic cancer cells was confirmed when the inhibition of MV-release sensitized the cells to GEM treatment, both in vitro and in vivo. Mechanistically, MVs remove drugs that are internalized into the cells and that are in the microenvironment. The differences between the drug-resistant and drug-sensitive pancreatic cancer cell lines tested here are explained based on the variable content of influx/efflux proteins present on MVs, which directly dictates the ability of MVs either to trap GEM or to allow GEM to flow back to the microenvironment. PMID:27391262

  19. Extracellular vesicles in cancer: exosomes, microvesicles and the emerging role of large oncosomes.

    PubMed

    Minciacchi, Valentina R; Freeman, Michael R; Di Vizio, Dolores

    2015-04-01

    Since their first description, extracellular vesicles (EVs) have been the topic of avid study in a variety of physiologic contexts and are now thought to play an important role in cancer. The state of knowledge on biogenesis, molecular content and horizontal communication of diverse types of cancer EVs has expanded considerably in recent years. As a consequence, a plethora of information about EV composition and molecular function has emerged, along with the notion that cancer cells rely on these particles to invade tissues and propagate oncogenic signals at distance. The number of in vivo studies, designed to achieve a deeper understanding of the extent to which EV biology can be applied to clinically relevant settings, is rapidly growing. This review summarizes recent studies on cancer-derived EV functions, with an overview about biogenesis and molecular cargo of exosomes, microvesicles and large oncosomes. We also discuss current challenges and emerging technologies that might improve EV detection in various biological systems. Further studies on the functional role of EVs in specific steps of cancer formation and progression will expand our understanding of the diversity of paracrine signaling mechanisms in malignant growth.

  20. Microvesicles as mediators of intercellular communication in cancer--the emerging science of cellular 'debris'.

    PubMed

    Lee, Tae Hoon; D'Asti, Esterina; Magnus, Nathalie; Al-Nedawi, Khalid; Meehan, Brian; Rak, Janusz

    2011-09-01

    Cancer cells emit a heterogeneous mixture of vesicular, organelle-like structures (microvesicles, MVs) into their surroundings including blood and body fluids. MVs are generated via diverse biological mechanisms triggered by pathways involved in oncogenic transformation, microenvironmental stimulation, cellular activation, stress, or death. Vesiculation events occur either at the plasma membrane (ectosomes, shed vesicles) or within endosomal structures (exosomes). MVs are increasingly recognized as mediators of intercellular communication due to their capacity to merge with and transfer a repertoire of bioactive molecular content (cargo) to recipient cells. Such processes may occur both locally and systemically, contributing to the formation of microenvironmental fields and niches. The bioactive cargo of MVs may include growth factors and their receptors, proteases, adhesion molecules, signalling molecules, as well as DNA, mRNA, and microRNA (miRs) sequences. Tumour cells emit large quantities of MVs containing procoagulant, growth regulatory and oncogenic cargo (oncosomes), which can be transferred throughout the cancer cell population and to non-transformed stromal cells, endothelial cells and possibly to the inflammatory infiltrates (oncogenic field effect). These events likely impact tumour invasion, angiogenesis, metastasis, drug resistance, and cancer stem cell hierarchy. Ongoing studies explore the molecular mechanisms and mediators of MV-based intercellular communication (cancer vesiculome) with the hope of using this information as a possible source of therapeutic targets and disease biomarkers in cancer.

  1. Tumor Cell-Derived Microvesicles Induced Not Epithelial-Mesenchymal Transition but Apoptosis in Human Proximal Tubular (HK-2) Cells: Implications for Renal Impairment in Multiple Myeloma

    PubMed Central

    Zhao, Aiqi; Kong, Fancong; Liu, Chun-Jie; Yan, Guoxin; Gao, Fei; Guo, Hao; Guo, An-Yuan; Chen, Zhichao; Li, Qiubai

    2017-01-01

    Renal impairment (RI) is one of the hallmarks of multiple myeloma (MM) and carries a poor prognosis. Microvesicles (MVs) are membrane vesicles and play an important role in disease progression. Here, we investigated the role of MVs derived from MM cells (MM-MVs) in RI of MM. We found that MM-MVs significantly inhibited viability and induced apoptosis, but not epithelial-mesenchymal transition in human kidney-2 (HK-2), a human renal tubular epithelial cell line. The protein levels of cleaved caspase-3, 8, and 9, and E-cadherin, were increased, but vementin levels were decreased in the HK-2 cells treated with MM-MVs. Through a comparative sequencing and analysis of RNA content between the MVs from RPMI8226 MM cells (RPMI8226-MVs) and K562 leukemia cells, RPMI8226-MVs were enriched with more renal-pathogenic miRNAs, in which the selective miRNAs may participate in the up-regulation of the levels of cleaved caspase-3. Furthermore, the levels of CD138+ circulating MVs (cirMVs) in the peripheral blood were positively correlated with the severity of RI in newly-diagnosed MM. Our study supports MM-MVs representing a previously undescribed factor and playing a potential role in the development of RI of MM patients, and sheds light on the potential application of CD138+ cirMV counts in precise diagnosis of RI in MM and exploring MM-MVs as a therapeutic target. PMID:28264449

  2. Identification and characterization of proteins isolated from microvesicles derived from human lung cancer pleural effusions.

    PubMed

    Park, Jung Ok; Choi, Do-Young; Choi, Dong-Sic; Kim, Hee Joung; Kang, Jeong Won; Jung, Jae Hun; Lee, Jeong Hwa; Kim, Jayoung; Freeman, Michael R; Lee, Kye Young; Gho, Yong Song; Kim, Kwang Pyo

    2013-07-01

    Microvesicles (MVs, also known as exosomes, ectosomes, microparticles) are released by various cancer cells, including lung, colorectal, and prostate carcinoma cells. MVs released from tumor cells and other sources accumulate in the circulation and in pleural effusion. Although recent studies have shown that MVs play multiple roles in tumor progression, the potential pathological roles of MV in pleural effusion, and their protein composition, are still unknown. In this study, we report the first global proteomic analysis of highly purified MVs derived from human nonsmall cell lung cancer (NSCLC) pleural effusion. Using nano-LC-MS/MS following 1D SDS-PAGE separation, we identified a total of 912 MV proteins with high confidence. Three independent experiments on three patients showed that MV proteins from PE were distinct from MV obtained from other malignancies. Bioinformatics analyses of the MS data identified pathologically relevant proteins and potential diagnostic makers for NSCLC, including lung-enriched surface antigens and proteins related to epidermal growth factor receptor signaling. These findings provide new insight into the diverse functions of MVs in cancer progression and will aid in the development of novel diagnostic tools for NSCLC.

  3. Human Mesenchymal Stem Cell-Derived Microvesicles Prevent the Rupture of Intracranial Aneurysm in Part by Suppression of Mast Cell Activation via a PGE2-Dependent Mechanism

    PubMed Central

    Liu, Jia; Kuwabara, Atsushi; Kamio, Yoshinobu; Hu, Shuling; Park, Jeonghyun; Hashimoto, Tomoki; Lee, Jae-Woo

    2017-01-01

    Background Activation of mast cells participates in the chronic inflammation associated with cerebral arteries in intracranial aneurysm formation and rupture. Several studies have shown that the anti-inflammatory effect of mesenchymal stem cells (MSCs) is beneficial for the treatment of aneurysms. However, some long-term safety concerns exist regarding stem cell-based therapy for clinical use. Objective We investigated the therapeutic potential of microvesicles (MVs) derived from human MSCs, anuclear membrane bound fragments with reparative properties, in preventing the rupture of intracranial aneurysm in mice, particularly in the effect of MVs on mast cell activation. Methods and Results Intracranial aneurysm was induced in C57BL/6 mice by the combination of systemic hypertension and intrathecal elastase injection. Intravenous administration of MSC-derived MVs on day 6 and day 9 after aneurysm induction significantly reduced the aneurysmal rupture rate, which was associated with reduced number of activated mast cells in the brain. A23187-induced activation of both primary cultures of murine mast cells and a human mast cell line, LAD2, was suppressed by MVs treatment, leading to a decrease in cytokine release and tryptase and chymase activities. Up-regulation of prostaglandin E2 (PGE2) production and E-prostanoid 4 (EP4) receptor expression were also observed on mast cells with MVs treatment. Administration of an EP4 antagonist with the MVs eliminated the protective effect of MVs against the aneurysmal rupture in vivo. Conclusions Human MSC-derived MVs prevented the rupture of intracranial aneurysm, in part due to their anti-inflammatory effect on mast cells, which was mediated by PGE2 production and EP4 activation. PMID:27350036

  4. Human Mesenchymal Stem Cell-Derived Microvesicles Prevent the Rupture of Intracranial Aneurysm in Part by Suppression of Mast Cell Activation via a PGE2-Dependent Mechanism.

    PubMed

    Liu, Jia; Kuwabara, Atsushi; Kamio, Yoshinobu; Hu, Shuling; Park, Jeonghyun; Hashimoto, Tomoki; Lee, Jae-Woo

    2016-12-01

    Activation of mast cells participates in the chronic inflammation associated with cerebral arteries in intracranial aneurysm formation and rupture. Several studies have shown that the anti-inflammatory effect of mesenchymal stem cells (MSCs) is beneficial for the treatment of aneurysms. However, some long-term safety concerns exist regarding stem cell-based therapy for clinical use. We investigated the therapeutic potential of microvesicles (MVs) derived from human MSCs, anuclear membrane bound fragments with reparative properties, in preventing the rupture of intracranial aneurysm in mice, particularly in the effect of MVs on mast cell activation. Intracranial aneurysm was induced in C57BL/6 mice by the combination of systemic hypertension and intrathecal elastase injection. Intravenous administration of MSC-derived MVs on day 6 and day 9 after aneurysm induction significantly reduced the aneurysmal rupture rate, which was associated with reduced number of activated mast cells in the brain. A23187-induced activation of both primary cultures of murine mast cells and a human mast cell line, LAD2, was suppressed by MVs treatment, leading to a decrease in cytokine release and tryptase and chymase activities. Upregulation of prostaglandin E2 (PGE2) production and E-prostanoid 4 (EP4) receptor expression were also observed on mast cells with MVs treatment. Administration of an EP4 antagonist with the MVs eliminated the protective effect of MVs against the aneurysmal rupture in vivo. Human MSC-derived MVs prevented the rupture of intracranial aneurysm, in part due to their anti-inflammatory effect on mast cells, which was mediated by PGE2 production and EP4 activation. Stem Cells 2016;34:2943-2955.

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

    PubMed

    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

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

    PubMed Central

    Chen, Xiaomei; Xiong, Wei; Li, Huiyu

    2016-01-01

    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

  7. Ultrasmall Magnetically Engineered Ag2Se Quantum Dots for Instant Efficient Labeling and Whole-Body High-Resolution Multimodal Real-Time Tracking of Cell-Derived Microvesicles.

    PubMed

    Zhao, Jing-Ya; Chen, Gang; Gu, Yi-Ping; Cui, Ran; Zhang, Zhi-Ling; Yu, Zi-Li; Tang, Bo; Zhao, Yi-Fang; Pang, Dai-Wen

    2016-02-17

    Cell-derived microvesicles (MVs) are natural carriers that can transport biological molecules between cells, which are expected to be promising delivery vehicles for therapeutic purposes. Strategies to label MVs are very important for investigation and application of MVs. Herein, ultrasmall Mn-magnetofunctionalized Ag2Se quantum dots (Ag2Se@Mn QDs) integrated with excellent near-infrared (NIR) fluorescence and magnetic resonance (MR) imaging capabilities have been developed for instant efficient labeling of MVs for their in vivo high-resolution dual-mode tracking. The Ag2Se@Mn QDs were fabricated by controlling the reaction of Mn(2+) with the Ag2Se nanocrystals having been pretreated in 80 °C NaOH solution, with an ultrasmall size of ca. 1.8 nm, water dispersibility, high NIR fluorescence quantum yield of 13.2%, and high longitudinal relaxivity of 12.87 mM(-1) s(-1) (almost four times that of the commercial contrast agent Gd-DTPA). The ultrasmall size of the Ag2Se@Mn QDs enables them to be directly and efficiently loaded into MVs by electroporation, instantly and reliably conferring both NIR fluorescence and MR traceability on MVs. Our method for labeling MVs of different origins is universal and free of unfavorable influence on intrinsic behaviors of MVs. The complementary imaging capabilities of the Ag2Se@Mn QDs have made the long-term noninvasive whole-body high-resolution dual-mode tracking of MVs in vivo realized, by which the dynamic biodistribution of MVs has been revealed in a real-time and in situ quantitative manner. This work not only opens a new window for labeling with QDs, but also facilitates greatly the investigation and application of MVs.

  8. Metastatic Mechanisms in Follicular Cell-Derived Thyroid Cancer

    PubMed Central

    Phay, John E.; Ringel, Matthew D.

    2013-01-01

    Thyroid cancer incidence is rising annually largely related to enhanced detection and of early stage well-differentiated primary tumors. The prognosis for patients with early stage thyroid cancer is outstanding with most patients being cured with surgery. In selected cases, I-131 is administered to treat known or suspected residual or metastatic disease. Even patients with loco-regional metastases typically have an outstanding long-term prognosis, albeit with monitoring and occasional intervention for residual or recurrent disease. In contrast, individuals with distant metastases from thyroid cancer, particular older patients with larger metastatic burdens and those with poorly differentiated tumors, have a poor prognosis. Patients with metastatic anaplastic thyroid cancer have a particularly poor prognosis. Published clinical trials indicate that transient disease control and partial remissions can be achieved with kinase inhibitor therapy directed toward angiogenic targets, and that in some cases, I-131 uptake can be enhanced. However, the direct targets of activity in metastatic lesions are incompletely defined and clear evidence that these treatments increase the duration or quality of life of patients is lacking, underscoring the need for improved knowledge regarding the metastatic process to inform the development of new therapies. In this review, we will focus on current data and hypotheses regarding key regulators of metastatic dormancy, metastatic progression, and the role of putative cancer stem cells. PMID:24036131

  9. Tumor-derived exosomes and microvesicles in head and neck cancer: implications for tumor biology and biomarker discovery.

    PubMed

    Principe, Simona; Hui, Angela Bik-Yu; Bruce, Jeff; Sinha, Ankit; Liu, Fei-Fei; Kislinger, Thomas

    2013-05-01

    Exosomes and microvesicles (MVs) are nanometer-sized, membranous vesicles secreted from many cell types into their surrounding extracellular space and into body fluids. These two classes of extracellular vesicles are regarded as a novel mechanism through which cancer cells, including virally infected cancer cells, regulate their micro-environment via the horizontal transfer of bioactive molecules: proteins, lipids, and nucleic acids (DNA, mRNA, micro-RNAs; oncogenic cargo hence often referred to as oncosomes). In head and neck cancer (HNC), exosomes and MVs have been described in Epstein Barr Virus (EBV)-associated nasopharyngeal cancer (NPC), as well as being positively correlated with oral squamous cell carcinoma (OSCC) progression. It has therefore been suggested that HNC-derived vesicles could represent a useful source for biomarker discovery, enriched in tumor antigens and cargo; hence fundamentally important for cancer progression. This current review offers an overall perspective on the roles of exosomes and MVs in HNC biology, focusing on EBV-associated NPC and OSCC. We also highlight the importance of saliva as a proximal and easily accessible bio-fluid for HNC detection, and propose that salivary vesicles might serve as an alternative model in the discovery of novel HNC biomarkers.

  10. iPS-cell derived dendritic cells and macrophages for cancer therapy.

    PubMed

    Senju, Satoru

    2016-08-01

    Antibody-based anti-cancer immunotherapy was recently recognized as one of the truly effective therapies for cancer patients. Antibodies against cell surface cancer antigens, such as CD20, and also those against immune-inhibitory molecules called "immune checkpoint blockers", such as CTLA4 or PD1, have emerged. Large-scale clinical trials have confirmed that, in some cases, antibody-based drugs are superior to conventional chemotherapeutic agents. These antibody-based drugs are now being manufactured employing a mass-production system by pharmaceutical companies. Anti-cancer therapy by immune cells, i.e. cell-based immunotherapy, is expected to be more effective than antibody therapy, because immune cells can recognize, infiltrate, and act in cancer tissues more directly than antibodies. In order to achieve cell-based anti-cancer immunotherapy, it is necessary to develop manufacturing systems for mass-production of immune cells. Our group has been studying immunotherapy with myeloid cells derived from ES cells or iPS cells. These pluripotent stem cells can be readily propagated under constant culture conditions, with expansion into a large quantity. We consider these stem cells to be the most suitable cellular source for mass-production of immune cells. This review introduces our studies on anti-cancer therapy with iPS cell-derived dendritic cells and iPS cell-derived macrophages.

  11. Cancer immunotherapy using dendritic cell-derived exosomes.

    PubMed

    Amigorena, S

    2000-01-01

    Dendritic cells (DCs) are the most potent antigen presenting cells and the only ones capable of inducing primary cytotoxic immune responses. We found that DCs secrete a population of membrane vesicles, called exosomes. Exosomes are 60-80 nm vesicles of endocytic origin. The protein composition of exosomes was subjected to a systematic proteomic analysis. Besides MHC and co-stimulatory molecules, exosomes bear several adhesion proteins, most likely involved in their specific subjected to targeting. We also found that exosomes accumulate several cytosolic factors, probably involved in their endosomal biogenesis. Like DCs, exosomes induced immune responses in vivo. Indeed, a single injection of DC-derived exosomes sensitized with tumor peptides induced potent anti tumor immune responses in mice and the eradication of established tumors. Tumor-specific cytotoxic T lymphocytes were found in the spleen of exosome-treated mice, and the anti tumor effect of exosomes was sensitive to in vivo depletion of CD8+ T cells. These results show that exosomes induce potent anti tumor effects in vivo, and strongly support the implementation of human DC-derived exosomes for cancer immunotherapy.

  12. Breast cancer cell-derived matrix supports vascular morphogenesis.

    PubMed

    Hielscher, Abigail C; Qiu, Connie; Gerecht, Sharon

    2012-04-15

    The extracellular matrix (ECM), important for maintaining tissue homeostasis, is abnormally expressed in mammary tumors and additionally plays a crucial role in angiogenesis. We hypothesize that breast cancer cells (BCCs) deposit ECM that supports unique patterns of vascular morphogenesis of endothelial cells (ECs). Evaluation of ECM expression revealed that a nontumorigenic cell line (MCF10A), a tumorigenic cell line (MCF7), and a metastatic cell line (MDA-MB-231) express collagens I and IV, fibronectin, and laminin, with tenascin-C limited to MCF10A and MCF7. The amount of ECM deposited by BCCs was found to be higher in MCF10A compared with MCF7 and MDA231, with all ECM differing in their gross structure but similar in mean fiber diameter. Nonetheless, deposition of ECM from BCC lines was overall difficult to detect and insufficient to support capillary-like structure (CLS) formation of ECs. Therefore, a coculture approach was undertaken in which individual BCC lines were cocultured with fibroblasts. Variation in abundance of deposited ECM, deposition of ECM proteins, such as absent collagen I deposition from MDA231-fibroblast cocultures, and fibril organization was found. Deposited ECM from fibroblasts and each coculture supported rapid CLS formation of ECs. Evaluation of capillary properties revealed that CLS grown on ECM deposited from MDA231-fibroblast cocultures possessed significantly larger lumen diameters, occupied the greatest percentage of area, expressed the highest levels of von Willebrand factor, and expressed the greatest amount of E-selectin, which was upregulated independent of exposure to TNF-α. To our knowledge, this is the first study to report tumor cell ECM-mediated differences in vascular capillary features, and thus offers the framework for future investigations interrogating the role of the tumor ECM in supporting vascular morphogenesis.

  13. Circulating Microvesicles Are Elevated Acutely following Major Burns Injury and Associated with Clinical Severity

    PubMed Central

    Tirlapur, Nikhil; Katbeh, Umar; Singh, Suveer; Handy, Jonathan M.; Takata, Masao

    2016-01-01

    Microvesicles are cell-derived signaling particles emerging as important mediators and biomarkers of systemic inflammation, but their production in severe burn injury patients has not been described. In this pilot investigation, we measured circulating microvesicle levels following severe burns, with severe sepsis patients as a comparator group. We hypothesized that levels of circulating vascular cell-derived microvesicles are elevated acutely following burns injury, mirroring clinical severity due to the early onset and prevalence of systemic inflammatory response syndrome (SIRS) in these patients. Blood samples were obtained from patients with moderate to severe thermal injury burns, with severe sepsis, and from healthy volunteers. Circulating microvesicles derived from total leukocytes, granulocytes, monocytes, and endothelial cells were quantified in plasma by flow cytometry. All circulating microvesicle subpopulations were elevated in burns patients on day of admission (day 0) compared to healthy volunteers (leukocyte-microvesicles: 3.5-fold, p = 0.005; granulocyte-microvesicles: 12.8-fold, p<0.0001; monocyte-microvesicles: 20.4-fold, p<0.0001; endothelial- microvesicles: 9.6-fold, p = 0.01), but decreased significantly by day 2. Microvesicle levels were increased with severe sepsis, but less consistently between patients. Leukocyte- and granulocyte-derived microvesicles on day 0 correlated with clinical assessment scores and were higher in burns ICU non-survivors compared to survivors (leukocyte MVs 4.6 fold, p = 0.002; granulocyte MVs 4.8 fold, p = 0.003). Mortality prediction analysis of area under receiver operating characteristic curve was 0.92 (p = 0.01) for total leukocyte microvesicles and 0.85 (p = 0.04) for granulocyte microvesicles. These findings demonstrate, for the first time, acute increases in circulating microvesicles following burns injury in patients and point to their potential role in propagation of sterile SIRS-related pathophysiology

  14. Role of stromal cell-derived factor 1α pathway in bone metastatic prostate cancer

    PubMed Central

    Gupta, Nisha; Duda, Dan G.

    2016-01-01

    Abstract Metastatic prostate cancer is one of the leading causes of cancer-related death in men. The primary site of metastasis from prostate cancers is the bone. During the last decade, multiple studies have pointed to the role of the stromal cell-derived factor 1 alpha (SDF1α)/CXCR4 axis in the metastatic spread of the disease, but the mechanisms that underlie this effect are still incompletely understood. In this review, we summarize the current understanding of the role of the SDF1α/CXCR4 pathway in bone metastatic prostate cancer. We also discuss the therapeutic potential of disrupting the interaction between prostate tumor cells and bone environment with focus on the SDF1α pathway. PMID:27533927

  15. Fas signal promotes lung cancer growth by recruiting myeloid-derived suppressor cells via cancer cell-derived PGE2.

    PubMed

    Zhang, Yongliang; Liu, Qiuyan; Zhang, Minggang; Yu, Yizhi; Liu, Xia; Cao, Xuetao

    2009-03-15

    Fas/FasL system has been extensively investigated with respect to its capacity to induce cellular apoptosis. However, accumulated evidences show that Fas signaling also exhibits nonapoptotic functions, such as induction of cell proliferation and differentiation. Lung cancer is one of cancer's refractory to the immunotherapy, however, the underlying mechanisms remain to be fully understood. In this study, we show that Fas overexpression does not affect in vitro growth of 3LL cells, but promotes lung cancer growth in vivo. However, such tumor-promoting effect is not observed in FasL-deficient (gld) mice, and also not observed in the immune competent mice once inoculation with domain-negative Fas-overexpressing 3LL cells, suggesting the critical role of Fas signal in the promotion of lung cancer growth in vivo. More accumulation of myeloid-derived suppressor cells (MDSC) and Foxp3(+) regulatory T cells is found in tumors formed by inoculation with Fas-overexpressing 3LL cells, but not domain-negative Fas-overexpressing 3LL cells. Accordingly, Fas-ligated 3LL lung cancer cells can chemoattract more MDSC but not regulatory T cells in vitro. Furthermore, Fas ligation induces 3LL lung cancer cells to produce proinflammatory factor PGE(2) by activating p38 pathway, and in turn, 3LL cells-derived PGE(2) contribute to the Fas ligation-induced MDSC chemoattraction. Furthermore, in vivo administration of cyclooxygenase-2 inhibitor can significantly reduce MDSC accumulation in the Fas-overexpressing tumor. Therefore, our results demonstrate that Fas signal can promote lung cancer growth by recruiting MDSC via cancer cell-derived PGE(2), thus providing new mechanistic explanation for the role of inflammation in cancer progression and immune escape.

  16. Regulated Delivery of Molecular Cargo to Invasive Tumor-derived Microvesicles

    PubMed Central

    Clancy, James W.; Sedgwick, Alanna; Rosse, Carine; Muralidharan-Chari, Vandhana; Raposo, Graca; Method, Michael; Chavrier, Philippe; D'Souza-Schorey, Crislyn

    2015-01-01

    Cells release multiple, distinct, forms of extracellular vesicles including structures known as microvesicles which are known to alter the extracellular environment. Despite growing understanding of microvesicle biogenesis, function, and contents, mechanisms regulating cargo delivery and enrichment remain largely unknown. Here we demonstrate that in amoeboid-like invasive tumor cell lines, the v-SNARE, VAMP3, regulates delivery of microvesicle cargo such as the membrane-type 1 matrix metalloprotease (MT1-MMP) to shedding microvesicles. MT1-MMP delivery to nascent microvesicles depends on the association of VAMP3 with the tetraspanin CD9 and facilitates the maintenance of amoeboid cell invasion. VAMP3-shRNA expression depletes shed vesicles of MT1-MMP and decreases cell invasiveness when embedded in cross-linked collagen matrices. Finally, we describe functionally similar microvesicles isolated from bodily fluids of ovarian cancer patients. Together these studies demonstrate the importance of microvesicle cargo sorting in matrix degradation and disease progression. PMID:25897521

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

    PubMed Central

    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

    Background 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. Methods 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. Results 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. Conclusions Our findings suggest that cancer cell-derived EVs mediate coagulopathy resulting in ischemic stroke via TF-independent mechanisms. PMID:27427978

  18. Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells

    PubMed Central

    2013-01-01

    Background Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. Methods To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. Results Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. Conclusions Our findings suggest that chronic

  19. Induced Pluripotent Stem Cell-Derived Natural Killer Cells for Treatment of Ovarian Cancer.

    PubMed

    Hermanson, David L; Bendzick, Laura; Pribyl, Lee; McCullar, Valarie; Vogel, Rachel Isaksson; Miller, Jeff S; Geller, Melissa A; Kaufman, Dan S

    2016-01-01

    Natural killer (NK) cells can provide effective immunotherapy for ovarian cancer. Here, we evaluated the ability of NK cells isolated from peripheral blood (PB) and NK cells derived from induced pluripotent stem cell (iPSC) to mediate killing of ovarian cancer cells in a mouse xenograft model. A mouse xenograft model was used to evaluate the intraperitoneal delivery of three different NK cell populations: iPSC-derived NK cells, PB-NK cells that had been activated and expanded in long-term culture, and overnight activated PB-NK cells that were isolated through CD3/CD19 depletion of PB B and T cells. Bioluminescent imaging was used to monitor tumor burden of luciferase expressing tumor lines. Tumors were allowed to establish prior to administering NK cells via intraperitoneal injection. These studies demonstrate a single dose of any of the three NK cell populations significantly reduced tumor burden. When mice were given three doses of either iPSC-NK cells or expanded PB-NK cells, the median survival improved from 73 days in mice untreated to 98 and 97 days for treated mice, respectively. From these studies, we conclude iPSC-derived NK cells mediate antiovarian cancer killing at least as well as PB-NK cells, making these cells a viable resource for immunotherapy for ovarian cancer. Due to their ability to be easily differentiated into NK cells and their long-term expansion potential, iPSCs can be used to produce large numbers of well-defined NK cells that can be banked and used to treat a large number of patients including treatment with multiple doses if necessary.

  20. A Comprehensive Review on Exosomes and Microvesicles as Epigenetic Factors.

    PubMed

    Bakhshandeh, Behnaz; Kamaleddin, Mohammad Amin; Aalishah, Khadijeh

    2017-01-01

    Exosomes and microvesicles, which are released by most of the cells, play important roles in intracellular correspondence by transferring DNA, messenger RNA, micro RNA, and other types of RNA and proteins. Exosomes and microvesicles may contribute to the distribution of cancers and diseases through delivering the pathogenic agents to the non-infected cells; in cancers, they can modify the cells in the tumor niche and lead them to transformation. In addition, these vesicles can affect stem cell activity and their physiological properties. On the other hand, exosomes and microvesicles can be applied in the therapeutic strategies as they are small, non-viral, flexible and able to cross biological barriers. In this review, we focused on some details about the exosomes and microvesicles both functionally and structurally.

  1. Revisiting tumor angiogenesis: vessel co-option, vessel remodeling, and cancer cell-derived vasculature formation.

    PubMed

    Qian, Chao-Nan; Tan, Min-Han; Yang, Jun-Ping; Cao, Yun

    2016-01-08

    Tumor growth and metastasis depend on the establishment of tumor vasculature to provide oxygen, nutrients, and other essential factors. The well-known vascular endothelial growth factor (VEGF) signaling is crucial for sprouting angiogenesis as well as recruitment of circulating progenitor endothelial cells to tumor vasculature, which has become therapeutic targets in clinical practice. However, the survival benefits gained from targeting VEGF signaling have been very limited, with the inevitable development of treatment resistance. In this article, we discuss the most recent findings and understanding on how solid tumors evade VEGF-targeted therapy, with a special focus on vessel co-option, vessel remodeling, and tumor cell-derived vasculature establishment. Vessel co-option may occur in tumors independently of sprouting angiogenesis, and sprouting angiogenesis is not always required for tumor growth. The differences between vessel-like structure and tubule-like structure formed by tumor cells are also introduced. The exploration of the underlying mechanisms of these alternative angiogenic approaches would not only widen our knowledge of tumor angiogenesis but also provide novel therapeutic targets for better controlling cancer growth and metastasis.

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

    PubMed

    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.

  3. Induction of the MCP chemokine cluster cascade in the periphery by cancer cell-derived Ccl3.

    PubMed

    Farmaki, Elena; Kaza, Vimala; Papavassiliou, Athanasios G; Chatzistamou, Ioulia; Kiaris, Hippokratis

    2017-03-28

    The induction of localized pro-inflammatory niches in the periphery is instrumental in metastasis. In order to better understand how tumors engage distal sites and activate a pro-inflammatory response we utilized syngeneic breast cancers as a model and showed that soluble factors from the neoplastic epithelium activate the expression of the monocyte chemoattractive protein (MCP) chemokines of the mouse 11C cluster that include Ccl1, Ccl2, Ccl7, Ccl8, Ccl11 and Ccl12. Tissues such as the lungs and the brain, that are more prone to colonization by breast cancer cells, were more sensitive to MCP cluster chemokine induction than others such as the liver. Subsequent analyses involving chemokine arrays in breast cancer cells and media followed by functional validation assays in in vitro and in vivo identified the cytokine Ccl3 as the principle mediator of the communication between the neoplastic epithelium and the peripheral tissues in terms of MCP cluster chemokine induction. Our results show that MCP chemokines are activated in peripheral tissues of breast cancer-bearing mice, by a mechanism that involves breast cancer cell-derived Ccl3. Interference with the expression of cancer cell-derived Ccl3 may find application in the management of breast cancer metastases.

  4. Cancer cell-derived IL-1α induces CCL22 and the recruitment of regulatory T cells.

    PubMed

    Wiedemann, Gabriela Maria; Knott, Max Martin Ludwig; Vetter, Viola Katharina; Rapp, Moritz; Haubner, Sascha; Fesseler, Julia; Kühnemuth, Benjamin; Layritz, Patrick; Thaler, Raffael; Kruger, Stephan; Ormanns, Steffen; Mayr, Doris; Endres, Stefan; Anz, David

    2016-01-01

    In cancer patients, immunosuppression through regulatory T cells (Treg) is a crucial component of tumor immune evasion and contributes to disease progression. Tumor-infiltrating Treg in particular suppress local effector T cell responses and are associated with poor prognosis in tumors such as human pancreatic cancer or hepatocellular carcinoma (HCC). The chemokine CCL22 is known to recruit Treg into the tumor tissue and many types of human tumors are known to express high levels of CCL22. The mechanisms leading to intratumoral secretion of CCL22 are so far unknown. We demonstrate here that intratumoral CCL22 is induced in tumor-infiltrating immune cells through cancer cell-derived interleukin-1 (IL-1α). In pancreatic cancer and HCC, CCL22 is produced by intratumoral dendritic cells, while the cancer cells themselves do not secrete CCL22 in vitro and in vivo. Incubation of human peripheral blood mononuclear cells (PBMC) or murine splenocytes with tumor cells or tumor cell supernatants strongly induced CCL22 secretion in vitro. Tumor cell supernatants contained IL-1 and CCL22 induction in PBMC could be specifically prevented by the IL-1 receptor antagonist anakinra or by transfection of tumor cell lines with IL-1 siRNA, leading to a suppression of Treg migration. In conclusion, we identify here tumor cell-derived IL-1α as a major inducer of the Treg attracting chemokine CCL22 in human cancer cells. Therapeutic blockade of the IL-1 pathway could represent a promising strategy to inhibit tumor-induced immunosuppression.

  5. Silencing of human papillomavirus (HPV) E6/E7 oncogene expression affects both the contents and the amounts of extracellular microvesicles released from HPV-positive cancer cells.

    PubMed

    Honegger, Anja; Leitz, Jenny; Bulkescher, Julia; Hoppe-Seyler, Karin; Hoppe-Seyler, Felix

    2013-10-01

    The human papillomavirus (HPV) E6/E7 oncogenes play a crucial role in the HPV-induced carcinogenesis. In this study, the authors investigated whether silencing of endogenous HPV E6/E7 expression may influence the contents or amounts of extracellular microvesicles (eMVs) released from HPV-positive cancer cells. It was found that eMVs secreted from HeLa cells are enriched for Survivin protein. RNA interference studies revealed that maintenance of both intracellular and microvesicular Survivin amounts was strongly dependent on continuous E6/E7 expression. This indicates that intracellular HPV activities are translated into visible alterations of protein contents in eMVs. Besides Survivin, eMVs from HeLa cells contain additional members of the inhibitor of apoptosis protein (IAP) family (XIAP, c-IAP1 and Livin). In contrast, no evidence for the presence of the HPV E6 and E7 oncoproteins in eMVs was obtained. Moreover, it was found that silencing of HPV E6/E7 expression led to a significant increase of exosomes-representing eMVs of endocytic origin-released from HeLa cells. This effect was associated with the reinduction of p53, stimulation of the p53 target genes TSAP6 and CHMP4C that can enhance exosome production and induction of senescence. Taken together, these results show that silencing of HPV E6/E7 oncogene expression profoundly affects both the composition and amounts of eMVs secreted by HPV-positive cancer cells. This indicates that HPVs can induce molecular signatures in eMVs that may affect intercellular communication and could be explored for diagnostic purposes.

  6. Therapeutic potential of CAR-T cell-derived exosomes: a cell-free modality for targeted cancer therapy

    PubMed Central

    Zhang, Li; Yang, Zhuo-Shun; Zou, Dan-Dan; Wang, Bin; Warnock, Garth L.; Dai, Long-Jun; Luo, Jie

    2015-01-01

    Chimeric antigen receptor (CAR)-based T-cell adoptive immunotherapy is a distinctively promising therapy for cancer. The engineering of CARs into T cells provides T cells with tumor-targeting capabilities and intensifies their cytotoxic activity through stimulated cell expansion and enhanced cytokine production. As a novel and potent therapeutic modality, there exists some uncontrollable processes which are the potential sources of adverse events. As an extension of this impactful modality, CAR-T cell-derived exosomes may substitute CAR-T cells to act as ultimate attackers, thereby overcoming some limitations. Exosomes retain most characteristics of parent cells and play an essential role in intercellular communications via transmitting their cargo to recipient cells. The application of CAR-T cell-derived exosomes will make this cell-based therapy more clinically controllable as it also provides a cell-free platform to diversify anticancer mediators, which responds effectively to the complexity and volatility of cancer. It is believed that the appropriate application of both cellular and exosomal platforms will make this effective treatment more practicable. PMID:26496034

  7. Therapeutic potential of CAR-T cell-derived exosomes: a cell-free modality for targeted cancer therapy.

    PubMed

    Tang, Xiang-Jun; Sun, Xu-Yong; Huang, Kuan-Ming; Zhang, Li; Yang, Zhuo-Shun; Zou, Dan-Dan; Wang, Bin; Warnock, Garth L; Dai, Long-Jun; Luo, Jie

    2015-12-29

    Chimeric antigen receptor (CAR)-based T-cell adoptive immunotherapy is a distinctively promising therapy for cancer. The engineering of CARs into T cells provides T cells with tumor-targeting capabilities and intensifies their cytotoxic activity through stimulated cell expansion and enhanced cytokine production. As a novel and potent therapeutic modality, there exists some uncontrollable processes which are the potential sources of adverse events. As an extension of this impactful modality, CAR-T cell-derived exosomes may substitute CAR-T cells to act as ultimate attackers, thereby overcoming some limitations. Exosomes retain most characteristics of parent cells and play an essential role in intercellular communications via transmitting their cargo to recipient cells. The application of CAR-T cell-derived exosomes will make this cell-based therapy more clinically controllable as it also provides a cell-free platform to diversify anticancer mediators, which responds effectively to the complexity and volatility of cancer. It is believed that the appropriate application of both cellular and exosomal platforms will make this effective treatment more practicable.

  8. High-Throughput Single-Cell Derived Sphere Formation for Cancer Stem-Like Cell Identification and Analysis

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Chih; Ingram, Patrick N.; Fouladdel, Shamileh; McDermott, Sean P.; Azizi, Ebrahim; Wicha, Max S.; Yoon, Euisik

    2016-06-01

    Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis.

  9. High-Throughput Single-Cell Derived Sphere Formation for Cancer Stem-Like Cell Identification and Analysis

    PubMed Central

    Chen, Yu-Chih; Ingram, Patrick N.; Fouladdel, Shamileh; McDermott, Sean P.; Azizi, Ebrahim; Wicha, Max S.; Yoon, Euisik

    2016-01-01

    Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis. PMID:27292795

  10. Tumor-associated macrophages induce capillary morphogenesis of lymphatic endothelial cells derived from human gastric cancer.

    PubMed

    Tauchi, Yukie; Tanaka, Hiroaki; Kumamoto, Kanako; Tokumoto, Mao; Sakimura, Chie; Sakurai, Katsunobu; Kimura, Kenjiro; Toyokawa, Takahiro; Amano, Ryosuke; Kubo, Naoshi; Muguruma, Kazuya; Yashiro, Masakazu; Maeda, Kiyoshi; Ohira, Masaichi; Hirakawa, Kosei

    2016-08-01

    Tumor lymphangiogenesis is a major prognostic indicator of gastric cancer. Tumor-induced inflammation has been shown to attract tumor-associated macrophages that affect lymphangiogenesis. However, detailed mechanisms of macrophage-induced lymphangiogenesis have not been elucidated. Here, we evaluated the interaction between tumor-associated macrophages and lymphatic endothelial cells (LECs) derived from lymph nodes (LNs) of human gastric cancer. Lymphatic endothelial cells were directly or indirectly cocultured with macrophages from healthy human blood, with or without the supernatant of the gastric cancer cell line, OCUM-12. We analyzed the effect of cancer pretreated macrophages and of macrophages from metastatic LNs of gastric cancer on LECs. We observed morphological changes of LECs in coculture and assessed the gene expression of possible lymphangiogenic molecules of macrophages and LECs after contact coculture, and of cancer pretreated macrophages, by quantitative RT-PCR. Specimens of metastatic LN of gastric cancer were immunofluorescently stained. We found that tubulogenesis of LECs was observed only in the contact coculture model. OCUM-12 cells promoted macrophage-induced tubulogenesis of LECs. Relative gene expression of MMP and adhesion molecules was significantly upregulated in both capillary-forming LECs and cocultured macrophages. Cancer pretreated macrophages upregulated lymphangiogenic factors including inflammatory cytokines, MMPs, adhesion molecules, and vascular endothelial growth factor-C. Blocking of intercellular adhesion molecule-1 and macrophage activation suppressed tubulogenesis of LECs. Immunohistochemistry showed macrophages localized around lymphatic vessels. Our results suggested that interaction between LECs and macrophages may be an important initial step of tumor lymphangiogenesis developing LN metastasis. Understanding of its mechanisms could be useful for future therapeutics of gastric cancer.

  11. Proteomic analysis of microvesicles from plasma of healthy donors reveals high individual variability.

    PubMed

    Bastos-Amador, Patricia; Royo, Felix; Gonzalez, Esperanza; Conde-Vancells, Javier; Palomo-Diez, Laura; Borras, Francesc E; Falcon-Perez, Juan M

    2012-06-27

    Healthy blood plasma is required for several therapeutic procedures. To maximize successful therapeutic outcomes it is critical to control the quality of blood plasma. Clearly initiatives to improve the safety of blood transfusions will have a high economical and social impact. A detailed knowledge of the composition of healthy blood plasma is essential to facilitate such improvements. Apart from free proteins, lipids and metabolites, blood plasma also contains cell-derived microvesicles, including exosomes and microparticles from several different cellular origins. In this study, we have purified microvesicles smaller than 220nm from plasma of healthy donors and performed proteomic, ultra-structural, biochemical and functional analyses. We have detected 161 microvesicle-associated proteins, including many associated with the complement and coagulation signal-transduction cascades. Several proteases and protease inhibitors associated with acute phase responses were present, indicating that these microvesicles may be involved in these processes. There was a remarkably high variability in the protein content of plasma from different donors. In addition, we report that this variability could be relevant for their interaction with cellular systems. This work provides valuable information on plasma microvesicles and a foundation to understand microvesicle biology and clinical implications.

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

    PubMed Central

    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 RASwt 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− RASwt, EGFR+ RASmut, and EGFR+ BRAFmut 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−RASwt (42 ± 8 versus 67 ± 7%), EGFR+ RASmut (20 ± 2 versus 37 ± 6%), and EGFR+ BRAFmut (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+ RASmut 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. PMID:28220124

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

    PubMed

    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.

  14. Chemotherapeutic Vulnerability of Triple-negative Breast Cancer Cell-derived Tumors to Pretreatment with Vernonia amygdalina Aqueous Extracts

    PubMed Central

    Howard, Carolyn B.; Mcdowell, Roderick; Feleke, Kidus; Deer, Evangeline; Stamps, Symone; Thames, Easter; Singh, Vikash; Pervin, Shehla

    2016-01-01

    Background Unresponsive to most clinical therapies, triple-negative breast cancer (TNBC) is the dominant biological cause of population-based racioethnic disparities in breast cancer mortality in the United States. We report the chemotherapeutic vulnerability of TNBC cells and stem cell-derived tumors to Vernonia amygdalina aqueous leaf extracts (VA extracts). VA extracts arrest cell proliferation and induce apoptosis in vitro and inhibit growth of implanted tumors and show chemo-preventive efficacy in vivo. Materials and Methods HRAS cells and MDA-MB-468 cells were subcutaneously implanted into nude mice with or without pretreatment with VA extracts before chemotherapeutic treatment with VA extracts and/or paclitaxel to evaluate their ability to inhibit tumor growth. Results The most significant reduction in tumor volume was observed in the MDA-MB-468 cell-induced tumors following VA extract pre-treatment compared to those from HRAS cell implantation. Conclusion VA extracts induce apoptosis, exhibit additive effects, inhibit tumor growth and display chemo-preventive actions against TNBCs. PMID:27466496

  15. Dendritic cell-derived interleukin-15 is crucial for therapeutic cancer vaccine potency

    PubMed Central

    Zhang, Yi; Tian, Shenghe; Liu, Zuqiang; Zhang, Jiying; Zhang, Meili; Bosenberg, Marcus W; Kedl, Ross M; Waldmann, Thomas A; Storkus, Walter J; Falo, Louis D; You, Zhaoyang

    2014-01-01

    IL-15 supports improved antitumor immunity. How to best incorporate IL-15 into vaccine formulations for superior cancer immunotherapy remains a challenge. DC-derived IL-15 (DCIL-15) notably has the capacity to activate DC, to substitute for CD4+ Th and to potentiate vaccine efficacy making IL-15-based therapies attractive treatment options. We observed in transplantable melanoma, glioma and metastatic breast carcinoma models that DCIL-15-based DNA vaccines in which DC specifically express IL-15 and simultaneously produce tumor Aghsp70 were able to mediate potent therapeutic efficacy that required both host Batf3+ DC and CD8+ T cells. In an inducible BrafV600E/Pten-driven murine melanoma model, DCIL-15 (not rIL-15)-based DNA vaccines elicited durable therapeutic CD8+ T cell-dependent antitumor immunity. DCIL-15 was found to be superior to rIL-15 in “licensing” both mouse and human DC, and for activating CD8+ T cells. Such activation occurred even in the presence of Treg, without a need for CD4+ Th, but was IL-15/IL-15Rα-dependent. A single low-dose of DCIL-15 (not rIL-15)-based DC vaccines induced therapeutic antitumor immunity. CD14+ DC emigrating from human skin explants genetically-immunized by IL-15 and Aghsp70 were more effective than similar DC emigrating from the explants genetically-immunized by Aghsp70 in the presence of rIL-15 in expressing membrane-bound IL-15/IL-15Rα and activating CD8+ T cells. These results support future clinical use of DCIL-15 as a therapeutic agent in battling cancer. PMID:25941586

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

    PubMed

    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.

  17. Hyaluronan production enhances shedding of plasma membrane-derived microvesicles.

    PubMed

    Rilla, Kirsi; Pasonen-Seppänen, Sanna; Deen, Ashik J; Koistinen, Ville V T; Wojciechowski, Sara; Oikari, Sanna; Kärnä, Riikka; Bart, Genevieve; Törrönen, Kari; Tammi, Raija H; Tammi, Markku I

    2013-08-01

    Many cell types secrete plasma membrane-bound microvesicles, suggested to play an important role in tissue morphogenesis, wound healing, and cancer spreading. However, the mechanisms of their formation have remained largely unknown. It was found that the tips of long microvilli induced in cells by overexpression of hyaluronan synthase 3 (HAS3) were detach into the culture medium as microvesicles. Moreover, several cell types with naturally active hyaluronan synthesis released high numbers of plasma membrane-derived vesicles, and inhibition of hyaluronan synthesis reduced their formation. The vesicles contained HAS, and were covered with a thick hyaluronan coat, a part of which was retained even after purification with high-speed centrifugation. HAS3 overexpressing MDCK cells cultured in a 3-D matrix as epithelial cysts released large amounts of HAS- and hyaluronan-positive vesicles from their basal surfaces into the extracellular matrix. As far as we know, hyaluronan synthesis is one of the first molecular mechanisms shown to stimulate the production of microvesicles. The microvesicles have a potential to deliver the hyaluronan synthase machinery and membrane and cytoplasmic materials to other cells, influencing tissue regeneration, inflammation and tumor progression.

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

    NASA Astrophysics Data System (ADS)

    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 (<=1.5 hour) and reliable diagnostic differentiation of bacterial infection from non-infectious inflammation in a double-blind pilot study. Our study demonstrates the potential of microvesicle activities for 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 (<=1.5 hour) and reliable diagnostic differentiation of bacterial infection from non-infectious inflammation in a double-blind pilot study. Our study demonstrates the potential of microvesicle activities for sepsis diagnosis and

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

    PubMed

    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.

  20. Characterization of membrane-shed microvesicles from cytokine-stimulated β-cells using proteomics strategies.

    PubMed

    Palmisano, Giuseppe; Jensen, Søren Skov; Le Bihan, Marie-Catherine; Lainé, Jeanne; McGuire, James N; Pociot, Flemming; Larsen, Martin Røssel

    2012-08-01

    Microparticles and exosomes are two of the most well characterized membrane-derived microvesicles released either directly from the plasma membrane or released through the fusion of intracellular multivesicular 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 β-cell-derived microvesicles generated after cytokine induced apoptosis using stable isotope labeled amino acids in cell culture combined with mass spectrometry. We identified and quantified a large number of β-cell-specific proteins and proteins previously described in microvesicles from other cell types in addition to new proteins located to these vesicles. In addition, we quantified specific sites of protein phosphorylation and N-linked sialylation in proteins associated with microvesicles from β-cells. Using pathway analysis software, we were able to map the most distinctive changes between microvesicles generated during growth and after cytokine stimulation to several cell death and cell signaling molecules including tumor necrosis factor receptor superfamily member 1A, tumor necrosis factor, α-induced protein 3, tumor necrosis factor-interacting kinase receptor-interacting serine-threonine kinase 1, and intercellular adhesion molecule 1.

  1. MicroRNA-containing microvesicles regulating inflammation in association with atherosclerotic disease.

    PubMed

    Hulsmans, Maarten; Holvoet, Paul

    2013-10-01

    In addition to intracellular organelles, eukaryotic cells contain extracellular organelles which are released, or shed, into the microenvironment. In practice, most human studies have examined mixed populations containing both exosomes and shedding microvesicles (also called ectosomes or microparticles); only a few studies have rigorously distinguished between the two. Accordingly, in this review, exosomes and shedding microvesicles are collectively called microvesicles. The first aim of this review was to discuss the role of microvesicles in cell-to-cell communication in general and in specific interactions between cells in chronic inflammation associated with atherosclerotic disease. Hereby, we focused on cell-specific microvesicles derived from platelets, endothelial cells and monocyte and monocyte-derived cells. The latter were also found to be associated with inflammation in obesity and type 2 diabetes prior to atherosclerotic disease, and cancer. Our second aim was to discuss specific changes in microvesicle content in relation with inflammation associated with metabolic and atherosclerotic disease, and cancer. Because many studies supported the putative diagnostic value of microRNAs, we emphasized therein changes in microRNA content rather than protein or lipid content. The most interesting microRNAs in inflammatory microvesicles in association with metabolic and cardiovascular diseases were found to be the let-7 family, miR-17/92 family, miR-21, miR-29, miR-126, miR-133, miR-146, and miR-155. These data warrant further investigation of the potential of microvesicles as putative biomarkers and as novel carriers for the cell-specific transfer of microRNAs and other therapeutic agents.

  2. [Cancer vaccine therapy using genetically modified induced pluripotent stem cell-derived dendritic cells expressing the TAA gene].

    PubMed

    Iwamoto, Hiromitsu; Ojima, Toshiyasu; Nakamori, Mikihito; Nakamura, Masaki; Hayata, Keiji; Katsuda, Masahiro; Iida, Takeshi; Miyazawa, Motoki; Iwahashi, Makoto; Yamaue, Hiroki

    2013-11-01

    It is generally accepted that the difficulty in obtaining a sufficient number of functional dendritic cells (DCs) poses a serious problem in DC-based immunotherapy. Therefore, we used induced pluripotent stem (iPS) cell-derived DCs (iPSDCs) instead. If the therapeutic efficacy of iPSDCs was equivalent to that of bone marrow-derived DCs( BMDCs), then the above-mentioned problems may be solved. In this study, we generated iPSDCs from iPS cells and compared their capacity to mature and migrate to the regional lymph nodes with that of BMDCs. We adenovirally transduced the hgp100 gene, which codes for a natural tumor antigen, into the DCs and immunized the mice with these genetically modified DCs. The cytotoxic activity of CD8( +) cytotoxic T lymphocytes( CTLs) was assayed using a 51Cr-release assay. The therapeutic efficacy of the vaccination was examined in a subcutaneous tumor model. Our results demonstrated that iPSDCs equaled BMDCs in terms of their maturation and migration capacity. Furthermore, hgp100-specific CTLs were generated in mice that were immunized with the genetically modified iPSDCs. These CTLs exhibited a high level of cytotoxicity against B16 cells, which is similar to that exhibited by CTLs generated in BMDCs immunized mice. Moreover, vaccination with genetically modified iPSDCs elicited a high level of therapeutic efficacy equaling that of vaccination with BMDCs. This study clarified experimentally that genetically modified iPSDCs are equivalent to BMDCs in terms of tumor-associated antigen-specific therapeutic antitumor immunity. This vaccination strategy may therefore be useful for future clinical application as a cancer vaccine.

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

    PubMed

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

    2012-05-01

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

  4. Blood/plasma secretome and microvesicles.

    PubMed

    Inal, Jameel M; Kosgodage, Uchini; Azam, Sarah; Stratton, Dan; Antwi-Baffour, Samuel; Lange, Sigrun

    2013-11-01

    A major but hitherto overseen component of the blood/plasma secretome is that of extracellular vesicles (EVs) which are shed from all blood cell types. These EVs are made up of microvesicles (MVs) and exosomes. MVs, 100nm-1μm in diameter, are released from the cell surface, and are a rich source of non-conventionally secreted proteins lacking a conventional signal peptide, and thus not secreted by the classical secretory pathways. Exosomes are smaller vesicles (≤100nm) having an endocytic origin and released upon multivesicular body fusion with the plasma membrane. Both vesicle types play major roles in intercellular cross talk and constitute an important component of the secretome especially in the area of biomarkers for cancer. The release of EVs, which are found in all the bodily fluids, is enhanced in cancer and a major focus of cancer proteomics is therefore targeted at EVs. The blood/plasma secretome is also a source of EVs, potentially diagnostic of infectious disease, whether from EVs released from infected cells or from the pathogens themselves. Despite the great excitement in this field, as is stated here and in other parts of this Special issue entitled: An Updated Secretome, much of the EV research, whether proteomic or functional in nature, urgently needs standardisation both in terms of nomenclature and isolation protocols. This article is part of a Special Issue entitled: An Updated Secretome.

  5. Exosomes and microvesicles: extracellular vesicles for genetic information transfer and gene therapy.

    PubMed

    Lee, Yi; El Andaloussi, Samir; Wood, Matthew J A

    2012-10-15

    Exosomes and microvesicles are extracellular nanovesicles released by most but not all cells. They are specifically equipped to mediate intercellular communication via the transfer of genetic information, including the transfer of both coding and non-coding RNAs, to recipient cells. As a result, both exosomes and microvesicles play a fundamental biological role in the regulation of normal physiological as well as aberrant pathological processes, via altered gene regulatory networks and/or via epigenetic programming. For example, microvesicle-mediated genetic transfer can regulate the maintenance of stem cell plasticity and induce beneficial cell phenotype modulation. Alternatively, such vesicles play a role in tumor pathogenesis and the spread of neurodegenerative diseases via the transfer of specific microRNAs and pathogenic proteins. Given this natural property for genetic information transfer, the possibility of exploiting these vesicles for therapeutic purposes is now being investigated. Stem cell-derived microvesicles appear to be naturally equipped to mediate tissue regeneration under certain conditions, while recent evidence suggests that exosomes might be harnessed for the targeted delivery of human genetic therapies via the introduction of exogenous genetic cargoes such as siRNA. Thus, extracellular vesicles are emerging as potent genetic information transfer agents underpinning a range of biological processes and with therapeutic potential.

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

    SciTech Connect

    Stratton, Dan; Lange, Sigrun; Kholia, Sharad; Jorfi, Samireh; Antwi-Baffour, Samuel; Inal, Jameel

    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.

  7. Exosomes for Immunoregulation and Therapeutic Intervention in Cancer

    PubMed Central

    Zhang, Xuan; Pei, Zenglin; Chen, Jinyun; Ji, Chunxia; Xu, Jianqing; Zhang, Xiaoyan; Wang, Jin

    2016-01-01

    Exosomes, as a subset of extracellular vesicles, function as a mode of intercellular communication and molecular transfer, and facilitate the direct extracellular transfer of proteins, lipids, and miRNAs/mRNAs/DNAs between cells. Cancers have adapted exosomes and related microvesicles as a pathway that can suppress the immune system and establish a fertile local and distant environment to support neoplastic growth, invasion, and metastasis; these tumor-derived exosomes affect immunoregulation mechanisms, including immune activation and immune suppression. Immune cell-derived exosomes can modulate the immune response in cancer, which supports the belief that these membranous vesicles are immunotherapeutic reagents. In this review, we discuss the recent advances in the cancer immunotherapy, roles of exosomes in cancer, immunoregulation of tumor-derived exosomes, and immunomodulation by immune cell-derived exosomes. The topics covered here highlight novel insights into the development of efficient exosome-based cancer vaccines for cancer therapeutic intervention. PMID:27326251

  8. Sphere formation assay is not an effective method for cancer stem cell derivation and characterization from the Caco-2 colorectal cell line.

    PubMed

    Wu, Hui; Zhang, Haihong; Hu, Yue; Xia, Qiu; Liu, Chenlu; Li, Yingnan; Yu, Bin; Gu, Tiejun; Zhang, Xizhen; Yu, Xianghui; Kong, Wei

    2014-03-01

    Although the existence of cancer stem cells (CSCs) has been demonstrated in colorectal cancer, further investigation is hindered by controversies over their surface markers. The sphere formation assay is widely used as in vitro method for derivation and characterization of CSCs based on the intrinsic self-renewal property of these cells. Isolated cancer cells that form tumorspheres are generally recognized as CSCs with self-renewal and tumorigenic capacities. In this study, colon spheres grown from Caco-2 cells in the sphere formation assay were separated from other differentiated cells and characterized. Compared with Caco-2 cells, the derived colon spheres lost several CSC properties. The colon spheres contained decreased levels of specific colorectal CSC surface markers as well as low levels of ATP-binding cassette (ABC) transporters typically overexpressed in CSCs, resulting in the near loss of their chemoresistance ability. Furthermore, cells that developed as colon spheres with strong self-renewal ability in vitro lost their tumorigenic capacity in vivo compared with Caco-2 cells, which could establish tumors in non-obese diabetic/severe-combined immunodeficient (NOD/SCID) mice. The results indicated that the Caco-2 cell derived colon spheres did not consist of colorectal CSCs. Thus, the well-accepted sphere formation assay may not be an effective method for CSC isolation and characterization from the Caco-2 colorectal cancer cell line.

  9. Acoustic purification of extracellular microvesicles.

    PubMed

    Lee, Kyungheon; Shao, Huilin; Weissleder, Ralph; Lee, Hakho

    2015-03-24

    Microvesicles (MVs) are an increasingly important source for biomarker discovery and clinical diagnostics. The small size of MVs and their presence in complex biological environment, however, pose technical challenges in sample preparation, particularly when sample volumes are small. We herein present an acoustic nanofilter system that size-specifically separates MVs in a continuous and contact-free manner. The separation uses ultrasound standing waves to exert differential acoustic force on MVs according to their size and density. By optimizing the design of the ultrasound transducers and underlying electronics, we were able to achieve a high separation yield and resolution. The "filter size-cutoff" can be controlled electronically in situ, which enables versatile MV-size selection. We applied the acoustic nanofilter to isolate nanoscale (<200 nm) vesicles from cell culture media as well as MVs in stored red blood cell products. With the capacity for rapid and contact-free MV isolation, the developed system could become a versatile preparatory tool for MV analyses.

  10. Cultivation and identification of colon cancer stem cell-derived spheres from the Colo205 cell line.

    PubMed

    Li, Ying-fei; Xiao, Bing; Tu, San-fang; Wang, Yuan-yuan; Zhang, Xiao-lang

    2012-03-01

    Our group established a method to culture spheres under serum-free culture condition. However, the biological characteristics and the tumorigenicity of spheres are unknown. Here, we demonstrate that sphere cells expressed high levels of the putative colorectal cancer stem cell markers CD133 and CD44. The CD133-positive rates were 13.27 ± 5.62, 52.71 ± 16.97 and 16.47 ± 2.45% in sphere cells, regular Colo205 cells and differentiated sphere cells, respectively, while the CD44-positive rates were 62.92 ± 8.38, 79.06 ± 12.10 and 47.80 ± 2.5%, respectively, and the CD133/CD44-double-positive rates were 10.77 ± 4.96, 46.89 ± 19.17 and 12.41 ± 2.27%, respectively (P < 0.05). Cancer sphere cells formed crypt-like structures in 3-D culture. Moreover, cells from cancer spheres exhibited more tumorigenicity than regular Colo205 cells in a xenograft assay. The cancer sphere cells displayed much higher oncogenicity than regular Colo205 cells to initiate neoplasms, as assayed by H&E staining, Musashi-1 staining and electron microscopy. Our findings indicated that the sphere cells were enriched with cancer stem cells (CSCs), and exhibited more proliferation capacity, more differentiation potential and especially more tumorigenicity than regular Colo205 cells in vitro and in vivo. Further isolation and characterization of these CSCs may provide new insights for novel therapeutic targets and prognostic markers.

  11. Cancer stem-like cells derived from chemoresistant tumors have a unique capacity to prime tumorigenic myeloid cells.

    PubMed

    Yamashina, Tsunaki; Baghdadi, Muhammad; Yoneda, Akihiro; Kinoshita, Ichiro; Suzu, Shinya; Dosaka-Akita, Hirotoshi; Jinushi, Masahisa

    2014-05-15

    Resistance to anticancer therapeutics greatly affects the phenotypic and functional properties of tumor cells, but how chemoresistance contributes to the tumorigenic activities of cancer stem-like cells remains unclear. In this study, we found that a characteristic of cancer stem-like cells from chemoresistant tumors (CSC-R) is the ability to produce a variety of proinflammatory cytokines and to generate M2-like immunoregulatory myeloid cells from CD14(+) monocytes. Furthermore, we identified the IFN-regulated transcription factor IRF5 as a CSC-R-specific factor critical for promoting M-CSF production and generating tumorigenic myeloid cells. Importantly, myeloid cells primed with IRF5(+) CSC-R facilitate the tumorigenic and stem cell activities of bulk tumors. Importantly, the activation of IRF5/M-CSF pathways in tumor cells were correlated with the number of tumor-associated CSF1 receptor(+) M2 macrophages in patients with non-small lung cancer. Collectively, our findings show how chemoresistance affects the properties of CSCs in their niche microenvironments.

  12. Identifying microRNA-mRNA regulatory network in gemcitabine-resistant cells derived from human pancreatic cancer cells.

    PubMed

    Shen, Yehua; Pan, Yan; Xu, Litao; Chen, Lianyu; Liu, Luming; Chen, Hao; Chen, Zhen; Meng, Zhiqiang

    2015-06-01

    Pancreatic cancer is unresectable in over 80 % of patients owing to difficulty in early diagnosis. Chemotherapy is the most frequently adopted therapy for advanced pancreatic cancer. The development of drug resistance to gemcitabine (GEM), which is always used in standard chemotherapy, often results in therapeutic failure. However, the molecular mechanisms underlying the gemcitabine resistance remain unclear. Therefore, we sought to explore the microRNA-mRNA network that is associated with the development of gemcitabine resistance and to identify molecular targets for overcoming the gemcitabine resistance. By exposing SW1990 pancreatic cancer cells to long-term gemcitabine with increasing concentrations, we established a gemcitabine-resistant cell line (SW1990/GEM) with a high IC50 (the concentration needed for 50 % growth inhibition, 847.23 μM). The mRNA and microRNA expression profiles of SW1990 cells and SW1990/GEM cells were determined using RNA-seq analysis. By comparing the results in control SW1990 cells, 507 upregulated genes and 550 downregulated genes in SW1990/GEM cells were identified as differentially expressed genes correlated with gemcitabine sensitivity. Gene ontology (GO) analysis showed that the differentially expressed genes were related to diverse biological processes. The upregulated genes were mainly associated with drug response and apoptosis, and the downregulated genes were correlated with cell cycle progression and RNA splicing. Concurrently, the differentially expressed microRNAs, which are the important player in drug resistance development, were also examined in SW1990/GEM cells, and 56 differential microRNAs were identified. Additionally, the expression profiles of selected genes and microRNAs were confirmed by using Q-PCR assays. Furthermore, combining the differentially expressed microRNAs and mRNAs as well as the predicted targets for these microRNAs, a core microRNA-mRNA regulatory network was constructed, which included hub micro

  13. Cancer-initiating cells derived from established cervical cell lines exhibit stem-cell markers and increased radioresistance

    PubMed Central

    2012-01-01

    Background Cancer-initiating cells (CICs) are proposed to be responsible for the generation of metastasis and resistance to therapy. Accumulating evidences indicates CICs are found among different human cancers and cell lines derived from them. Few studies address the characteristics of CICs in cervical cancer. We identify biological features of CICs from four of the best-know human cell lines from uterine cervix tumors. (HeLa, SiHa, Ca Ski, C-4 I). Methods Cells were cultured as spheres under stem-cell conditions. Flow cytometry was used to detect expression of CD34, CD49f and CD133 antigens and Hoechst 33342 staining to identify side population (SP). Magnetic and fluorescence-activated cell sorting was applied to enrich and purify populations used to evaluate tumorigenicity in nude mice. cDNA microarray analysis and in vitro radioresistance assay were carried out under standard conditions. Results CICs, enriched as spheroids, were capable to generate reproducible tumor phenotypes in nu-nu mice and serial propagation. Injection of 1 × 103 dissociated spheroid cells induced tumors in the majority of animals, whereas injection of 1 × 105 monolayer cells remained nontumorigenic. Sphere-derived CICs expressed CD49f surface marker. Gene profiling analysis of HeLa and SiHa spheroid cells showed up-regulation of CICs markers characteristic of the female reproductive system. Importantly, epithelial to mesenchymal (EMT) transition-associated markers were found highly expressed in spheroid cells. More importantly, gene expression analysis indicated that genes required for radioresistance were also up-regulated, including components of the double-strand break (DSB) DNA repair machinery and the metabolism of reactive oxygen species (ROS). Dose-dependent radiation assay indicated indeed that CICs-enriched populations exhibit an increased resistance to ionizing radiation (IR). Conclusions We characterized a self-renewing subpopulation of CICs found among four well known human

  14. Stromal cell-derived factor-1{alpha} (SDF-1{alpha}/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation

    SciTech Connect

    Porcile, Carola; Bajetto, Adriana . E-mail: bajetto@cba.unige.it; Barbieri, Federica; Barbero, Simone; Bonavia, Rudy; Biglieri, Marianna; Pirani, Paolo; Florio, Tullio . E-mail: florio@cba.unige.it; Schettini, Gennaro

    2005-08-15

    Ovarian cancer (OC) is the leading cause of death in gynecologic diseases in which there is evidence for a complex chemokine network. Chemokines are a family of proteins that play an important role in tumor progression influencing cell proliferation, angiogenic/angiostatic processes, cell migration and metastasis, and, finally, regulating the immune cells recruitment into the tumor mass. We previously demonstrated that astrocytes and glioblastoma cells express both the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1), and that SDF-1{alpha} treatment induced cell proliferation, supporting the hypothesis that chemokines may play an important role in tumor cells' growth in vitro. In the present study, we report that CXCR4 and SDF-1 are expressed in OC cell lines. We demonstrate that SDF-1{alpha} induces a dose-dependent proliferation in OC cells, by the specific interaction with CXCR4 and a biphasic activation of ERK1/2 and Akt kinases. Our results further indicate that CXCR4 activation induces EGF receptor (EGFR) phosphorylation that in turn was linked to the downstream intracellular kinases activation, ERK1/2 and Akt. In addition, we provide evidence for cytoplasmic tyrosine kinase (c-Src) involvement in the SDF-1/CXCR4-EGFR transactivation. These results suggest a possible important 'cross-talk' between SDF-1/CXCR4 and EGFR intracellular pathways that may link signals of cell proliferation in ovarian cancer.

  15. Silencing stem cell factor attenuates stemness and inhibits migration of cancer stem cells derived from Lewis lung carcinoma cells.

    PubMed

    Wang, Li; Wang, JianTao; Li, Zhixi; Liu, YanYang; Jiang, Ming; Li, Yan; Cao, Dan; Zhao, Maoyuan; Wang, Feng; Luo, Feng

    2016-06-01

    Stem cell factor (SCF) plays an important role in tumor growth and metastasis. However, the function of SCF in regulating stemness and migration of cancer stem cells (CSCs) remains largely undefined. Here, we report that non-adhesive culture system can enrich and expand CSCs derived from Lewis lung carcinoma (LLC) cells and that the expression level of SCF in CSCs was higher than those in LLC cells. Silencing SCF via short hairpin (sh) RNA lentivirus transduction attenuated sphere formation and inhibited expressions of stemness genes, ALDH1, Sox2, and Oct4 of CSCs in vitro and in vivo. Moreover, SCF-silenced CSCs inhibited the migration and epithelial-mesenchymal transition, with decreased expression of N-cadherin, Vimentin, and increased expression of E-cadherin in vitro and in vivo. Finally, SCF-short hairpin RNA (shRNA) lentivirus transduction suppressed tumorigenicity of CSCs. Taken together, our findings unraveled an important role of SCF in CSCs derived from LLC cells. SCF might serve as a novel target for lung cancer therapy.

  16. Hypoxia Affects the Structure of Breast Cancer Cell-Derived Matrix to Support Angiogenic Responses of Endothelial Cells.

    PubMed

    Hielscher, Abigail; Qiu, Connie; Porterfield, Josh; Smith, Quinton; Gerecht, Sharon

    2013-01-01

    Hypoxia, a common feature of the tumor environment and participant in tumor progression, is known to alter gene and protein expression of several Extracellular Matrix (ECM) proteins, many of which have roles in angiogenesis. Previously, we reported that ECM deposited from co-cultures of Neonatal Fibroblasts (NuFF) with breast cancer cells, supported 3-dimensional vascular morphogenesis. Here, we sought to characterize the hypoxic ECM and to identify whether the deposited ECM induce angiogenic responses in Endothelial Cells (ECs). NuFF and MDA-MB-231 breast cancer cells were co-cultured, subjected to alternating cycles of 24 hours of 1% (hypoxia) and 21% (atmospheric) oxygen and de-cellularized for analyses of deposited ECM. We report differences in mRNA expression profiles of matrix proteins and crosslinking enzymes relevant to angiogenesis in hypoxia-exposed co-cultures. Interestingly, overt differences in the expression of ECM proteins were not detected in the de-cellularized ECM; however, up-regulation of the cell-binding fragment of fibronecin was observed in the conditioned media of hypoxic co-cultures. Ultrastructure analyses of the de-cellularized ECM revealed differences in fiber morphology with hypoxic fibers more compact and aligned, occupying a greater percent area and having larger diameter fibers than atmospheric ECM. Examining the effect of hypoxic ECM on angiogenic responses of ECs, morphological differences in Capillary-Like Structures (CLS) formed atop de-cellularized hypoxic and atmospheric ECM were not evident. Interestingly, we found that hypoxic ECM regulated the expression of angiogenic factors and matrix metalloproteinases in CLS. Overall, we report that in vitro, hypoxia does not alter the composition of the ECM deposited by co-cultures of NuFF/MDA-MB-231, but rather alters fiber morphology, and induces vascular expression of angiogenic growth factors and metalloproteinases. Taken together, these results have important implications for

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

    PubMed

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

    2017-01-16

    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.

  18. Extracellular vesicles: Exosomes, microvesicles, and friends

    PubMed Central

    Stoorvogel, Willem

    2013-01-01

    Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and RNA. Deficiencies in our knowledge of the molecular mechanisms for EV formation and lack of methods to interfere with the packaging of cargo or with vesicle release, however, still hamper identification of their physiological relevance in vivo. In this review, we focus on the characterization of EVs and on currently proposed mechanisms for their formation, targeting, and function. PMID:23420871

  19. Tumour-derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes.

    PubMed

    Baj-Krzyworzeka, Monika; Szatanek, Rafał; Weglarczyk, Kazimierz; Baran, Jarosław; Urbanowicz, Barbara; Brański, Piotr; Ratajczak, Mariusz Z; Zembala, Marek

    2006-07-01

    This study was designed to determine the characteristics of tumour cell-derived microvesicles (TMV) and their interactions with human monocytes. TMV were shed spontaneously by three different human cancer cell lines but their release was significantly increased upon activation of the cells with phorbol 12-myristate 13-acetate (PMA). TMV showed the presence of several surface determinants of tumour cells, e.g. HLA class I, CD29, CD44v7/8, CD51, chemokine receptors (CCR6, CX3CR1), extracellular matrix metalloproteinase inducer (EMMPRIN), epithelial cell adhesion molecule (EpCAM), but their level of expression differed from that on cells they originated from. TMV also carried mRNA for growth factors: vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), interleukin-8 (IL-8) and surface determinants (CD44H). TMV were localized at the monocytes surface following their short exposure to TMV, while at later times intracellularly. TMV transferred CCR6 and CD44v7/8 to monocytes, exerted antiapoptotic effect on monocytes and activated AKT kinase (Protein Kinase B). Thus, TMV interact with monocytes, alter their immunophenotype and biological activity. This implicates the novel mechanism by which tumour infiltrating macrophages may be affected by tumour cells not only by a direct cell to cell contact, soluble factors but also by TMV.

  20. C-src Enriched Serum Microvesicles Are Generated in Malignant Plasma Cell Dyscrasia

    PubMed Central

    Zendrini, Andrea; Radeghieri, Annalisa; Caimi, Luigi; Ricotta, Doris

    2013-01-01

    Plasma cell dyscrasias are immunosecretory disorders that can lead to hematological malignancies such as Multiple Myeloma (MM). MM accounts for 15% of all hematologic cancers, and those diagnosed with MM typically become severely ill and have a low life expectancy. Monoclonal immunoglobulin Free Light Chains (FLC) are present in the serum and urine of many patients with plasma cell diseases. The biological differences between monoclonal FLCs, produced under malignant or benign dyscrasias, has not yet been characterized. In the present study, we show that endothelial and heart muscle cell lines internalize kappa and lambda FLCs. After internalization, FLCs are rerouted in the extracellular space via microvesicles and exosomes that can be re-internalized in contiguous cells. Only FLCs secreted from malignant B Lymphocytes were carried in Hsp70, annexin V, and c-src positive vesicles. In both MM and AL Amyloidosis patients we observed an increase in microvesicle and exosome production. Isolated serum vesicles from MM, AL Amyloidosis and monoclonal gammopathy of undetermined significance (MGUS) patients contained FLCs. Furthermore MM and AL amyloidosis vesicles were strongly positive for Hsp70, annexin V, and c-src compared to MGUS and control patients. These are the first data implying that FLCs reroute via microvesicles in the blood stream, and also suggest a potential novel mechanism of c-src activation in plasma cell dyscrasia. PMID:23940647

  1. C-src enriched serum microvesicles are generated in malignant plasma cell dyscrasia.

    PubMed

    Di Noto, Giuseppe; Paolini, Lucia; Zendrini, Andrea; Radeghieri, Annalisa; Caimi, Luigi; Ricotta, Doris

    2013-01-01

    Plasma cell dyscrasias are immunosecretory disorders that can lead to hematological malignancies such as Multiple Myeloma (MM). MM accounts for 15% of all hematologic cancers, and those diagnosed with MM typically become severely ill and have a low life expectancy. Monoclonal immunoglobulin Free Light Chains (FLC) are present in the serum and urine of many patients with plasma cell diseases. The biological differences between monoclonal FLCs, produced under malignant or benign dyscrasias, has not yet been characterized. In the present study, we show that endothelial and heart muscle cell lines internalize kappa and lambda FLCs. After internalization, FLCs are rerouted in the extracellular space via microvesicles and exosomes that can be re-internalized in contiguous cells. Only FLCs secreted from malignant B Lymphocytes were carried in Hsp70, annexin V, and c-src positive vesicles. In both MM and AL Amyloidosis patients we observed an increase in microvesicle and exosome production. Isolated serum vesicles from MM, AL Amyloidosis and monoclonal gammopathy of undetermined significance (MGUS) patients contained FLCs. Furthermore MM and AL amyloidosis vesicles were strongly positive for Hsp70, annexin V, and c-src compared to MGUS and control patients. These are the first data implying that FLCs reroute via microvesicles in the blood stream, and also suggest a potential novel mechanism of c-src activation in plasma cell dyscrasia.

  2. Role of Platelet-Derived Microvesicles As Crosstalk Mediators in Atherothrombosis and Future Pharmacology Targets: A Link between Inflammation, Atherosclerosis, and Thrombosis.

    PubMed

    Badimon, Lina; Suades, Rosa; Fuentes, Eduardo; Palomo, Iván; Padró, Teresa

    2016-01-01

    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.

  3. Role of Platelet-Derived Microvesicles As Crosstalk Mediators in Atherothrombosis and Future Pharmacology Targets: A Link between Inflammation, Atherosclerosis, and Thrombosis

    PubMed Central

    Badimon, Lina; Suades, Rosa; Fuentes, Eduardo; Palomo, Iván; Padró, Teresa

    2016-01-01

    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. PMID:27630570

  4. Colon cancer cell-derived 12(S)-HETE induces the retraction of cancer-associated fibroblast via MLC2, RHO/ROCK and Ca(2+) signalling.

    PubMed

    Stadler, Serena; Nguyen, Chi Huu; Schachner, Helga; Milovanovic, Daniela; Holzner, Silvio; Brenner, Stefan; Eichsteininger, Julia; Stadler, Mira; Senfter, Daniel; Krenn, Liselotte; Schmidt, Wolfgang M; Huttary, Nicole; Krieger, Sigurd; Koperek, Oskar; Bago-Horvath, Zsuzsanna; Brendel, Konstantin Alexander; Marian, Brigitte; de Wever, Oliver; Mader, Robert M; Giessrigl, Benedikt; Jäger, Walter; Dolznig, Helmut; Krupitza, Georg

    2016-12-24

    Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca(2+) levels were measured and pharmacological- or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca(2+), Ca(2+)-calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca(2+) release. Thus, Ca(2+) signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour-stroma interaction.

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

    PubMed

    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.

  6. Microglial Microvesicle Secretion and Intercellular Signaling

    PubMed Central

    Turola, Elena; Furlan, Roberto; Bianco, Fabio; Matteoli, Michela; Verderio, Claudia

    2012-01-01

    Microvesicles (MVs) are released from almost all cell brain types into the microenvironment and are emerging as a novel way of cell-to-cell communication. This review focuses on MVs discharged by microglial cells, the brain resident myeloid cells, which comprise ∼10–12% of brain population. We summarize first evidence indicating that MV shedding is a process activated by the ATP receptor P2X7 and that shed MVs represent a secretory pathway for the inflammatory cytokine IL-β. We then discuss subsequent findings which clarify how IL-1 β can be locally processed and released from MVs into the extracellular environment. In addition, we describe the current understanding about the mechanism of P2X7-dependent MV formation and membrane abscission, which, by involving sphingomyelinase activity and ceramide formation, may share similarities with exosome biogenesis. Finally we report our recent results which show that microglia-derived MVs can stimulate neuronal activity and participate to the propagation of inflammatory signals, and suggest new areas for future investigation. PMID:22661954

  7. Neuroendocrine-like cells -derived CXCL10 and CXCL11 induce the infiltration of tumor-associated macrophage leading to the poor prognosis of colorectal cancer

    PubMed Central

    Liu, Lu; Xu, He-Yang; Wang, Jie; Chu, Zhong-Hua

    2016-01-01

    Our previous study revealed that neuroendocrine differentiation in colorectal cancer is one of the important factors leading to worse prognosis. In this study, we apply immunohistochemical staining, Western-blot, RT-PCR and ELISA to investigate the underlying mechanism that how the neuroendocrine differentiation to affect the prognosis of colorectal cancer. The interaction of colorectal cancer cells, neuroendocrine-like cells and tumor-associated macrophages in colorectal cancer progress is also investigated. By analyzing 82 cases of colorectal cancer patients treated in our institution, we found that colorectal adenocarcinoma with neuroendocrine differentiation had increasing number of tumor-associated macrophages and worse prognosis. Further evaluation of cytology showed that neuroendocrine cells have the ability to recruit tumor-associated macrophages to infiltrate the tumor tissue, and the tumor-associated macrophages enhance the proliferation and invasion abilities of the colon cancer cells. Moreover, we confirmed that CXCL10 and CXCL11 are the key chemokines in neuroendocrine-like cells and they promote the chemotaxis activity of tumor-associated macrophages. The secretion of CXCL10 and CXCL11 by neuroendocrine-like cells can recruit tumor-associated macrophages to infiltrate in tumor tissues. The latter enhances the proliferation and invasion of colorectal cancer cell and lead to poor prognosis. PMID:27034164

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

    PubMed Central

    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

  9. High Expression of Stromal Cell-Derived Factor 1 (SDF-1) and NF-κB Predicts Poor Prognosis in Cervical Cancer

    PubMed Central

    Song, Zhiwang; Zhang, Xia; Ye, Xiaojuan; Feng, Chan; Yang, Guang; Lu, Yonglin; Lin, Yun; Dong, Chunyan

    2017-01-01

    Background SDF-1 and NF-κB are associated with the prognosis of a wide range of cancers, but their value in cervical cancer remains controversial. The aim of this study was to investigate the expression of SDF-1and NF-κB in cervical cancer and their significance in clinical prognosis. Material/Methods The expression of SDF-1and NF-κB in 105 formalin-fixed, paraffin-embedded cervical cancer tissues and the adjacent tissues was examined by immunohistochemistry (IHC). The results were semi-quantitatively scored and analyzed by chi-square test. The overall survival times (OS) were collected by follow-up and analyzed by Kaplan-Meier analysis. Results The expression level of both SDF-1and NF-κB in cervical cancer are higher than that in the adjacent tissues (P<0.05). SDF-1 expression are correlated with tumor size and FIGO histology grade (P<0.05). NF-κB expression are correlated with tumor size and FIGO histology grade, and lymph node metastasis (LNM) status (P<0.05). The patients with a positive expression of SDF-1or NF-κB tended to have much shorter survival time than patients with negative expression. In addition, multivariate Cox regression analysis demonstrated that SDF-1 expression and lymph node metastasis are independent predictors of the OS in cervical cancer patients. Conclusions The expression of SDF-1 is significantly associated with tumor size and FIGO histology grade. The expression of NF-κB is significantly associated with tumor size, FIGO histology grade, and lymph node metastasis. The positive SDF-1or NF-κB expression is significantly correlated with poor prognosis. These may be valuable biomarkers for the prognosis and the potential therapeutic targets of cervical cancer. PMID:28074045

  10. Paclitaxel-resistant cancer cell-derived secretomes elicit ABCB1-associated docetaxel cross-resistance and escape from apoptosis through FOXO3a-driven glycolytic regulation

    PubMed Central

    Aldonza, Mark Borris D; Hong, Ji-Young; Lee, Sang Kook

    2017-01-01

    Chemotherapy-induced cancer cell secretomes promote resistance due, in part, to a predominant glycolytic energy metabolism, which drives aggressive cancer cell proliferation. However, the characterization of these secretomes and the molecular events that associate them with acquired drug resistance remain poorly understood. In this study, we show that secretomes of cancer cells with high-level paclitaxel resistance stimulated cell proliferation and suppressed drug-induced apoptosis of drug-sensitive cells. We also found that drug (docetaxel)-stimulated induction of interferon-α (IFN-α), IFN-λ and tumor necrosis factor-α (TNF-α) release in drug-sensitive cells was lowered by these secretomes. The promotion of cell proliferation by paclitaxel-resistant (PacR) cancer cell secretomes was associated, in part, with an increase in S phase of the cell cycle and downregulation of the cell death pathway that supports escape from apoptosis. In addition, we also found that the regulation of targeted glycolysis in PacR cancer cells alters the effects of the secretomes on cell growth, apoptosis, ATP generation and acquired drug resistance. Further study revealed that the deletion of FOXO3a transcription exacerbates glycolytic shift-induced apoptosis by rescuing TRAIL expression. By generating a docetaxel–cross-resistant PacR cancer cell line (PacR/DCT), we further clarified the role of FOXO3a in glycolysis-associated mediation of P-glycoprotein/ABCB1 hyperactivity that induces docetaxel cross-resistance. These findings suggest that suppression of the cellular energy supply by targeting glycolysis may inhibit the multiplicity of acquired chemotherapy resistance. Therefore, the therapeutic inhibition of FOXO3a might direct glycolysis to induce apoptosis and overcome multidrug resistance in cancer cells. PMID:28104912

  11. High Expression of Stromal Cell-Derived Factor 1 (SDF-1) and NF-κB Predicts Poor Prognosis in Cervical Cancer.

    PubMed

    Song, Zhiwang; Zhang, Xia; Ye, Xiaojuan; Feng, Chan; Yang, Guang; Lu, Yonglin; Lin, Yun; Dong, Chunyan

    2017-01-11

    BACKGROUND SDF-1 and NF-κB are associated with the prognosis of a wide range of cancers, but their value in cervical cancer remains controversial. The aim of this study was to investigate the expression of SDF-1and NF-κB in cervical cancer and their significance in clinical prognosis. MATERIAL AND METHODS The expression of SDF-1and NF-κB in 105 formalin-fixed, paraffin-embedded cervical cancer tissues and the adjacent tissues was examined by immunohistochemistry (IHC). The results were semi-quantitatively scored and analyzed by chi-square test. The overall survival times (OS) were collected by follow-up and analyzed by Kaplan-Meier analysis. RESULTS The expression level of both SDF-1and NF-κB in cervical cancer are higher than that in the adjacent tissues (P<0.05). SDF-1 expression are correlated with tumor size and FIGO histology grade (P<0.05). NF-κB expression are correlated with tumor size and FIGO histology grade, and lymph node metastasis (LNM) status (P<0.05). The patients with a positive expression of SDF-1or NF-κB tended to have much shorter survival time than patients with negative expression. In addition, multivariate Cox regression analysis demonstrated that SDF-1 expression and lymph node metastasis are independent predictors of the OS in cervical cancer patients. CONCLUSIONS The expression of SDF-1 is significantly associated with tumor size and FIGO histology grade. The expression of NF-κB is significantly associated with tumor size, FIGO histology grade, and lymph node metastasis. The positive SDF-1or NF-κB expression is significantly correlated with poor prognosis. These may be valuable biomarkers for the prognosis and the potential therapeutic targets of cervical cancer.

  12. Macrophages are recruited to hypoxic tumor areas and acquire a Pro-Angiogenic M2-Polarized phenotype via hypoxic cancer cell derived cytokines Oncostatin M and Eotaxin

    PubMed Central

    Tripathi, Chakrapani; Tewari, Brij Nath; Kanchan, Ranjana Kumari; Baghel, Khemraj Singh; Nautiyal, Naveen; Shrivastava, Richa; Kaur, Harbeer; Bhatt, Madan Lal Bramha; Bhadauria, Smrati

    2014-01-01

    TAMs, a unique and distinct M2-skewed myeloid population of tumor stroma, exhibiting pro-tumor functions is fast emerging as a potential target for anti-cancer immunotherapy. Macrophage-recruitment and M2-polarization represent key TAMs-related phenomenon that are amenable to therapeutic intervention. However successful translation of these approaches into effective therapeutic regimen requires better characterization of tumor-microenvironment derived signals that regulate macrophage recruitment and their polarization. Owing to hypoxic milieu being a persistent feature of tumor-microenvironment and a major contributor to malignancy and treatment resistance, the current study was planned with an aim to decipher tumor cell responses to hypoxia vis-a-vis macrophage homing and phenotype switching. Here, we show that hypoxia-primed cancer cells chemoattract and polarize macrophages to pro-angiogenic M2-polarized subtype via Eotaxin and Oncostatin M. Concordantly, hypoxic regions of human breast-cancer specimen exhibited elevated Eotaxin and Oncostatin M levels with concurrently elevated M2-macrophage content. Blockade of Eotaxin/Oncostatin M not only prevented hypoxic breast-cancer cells from recruiting and polarizing macrophages towards an M2-polarized phenotype and retarded tumor progression in 4T1/BALB/c-syngenic-mice-model of breast-cancer but also enhanced the efficacy of anti-angiogenic Bevacizumab. The findings established these two cytokines as novel targets for devising effective anticancer therapy particularly for tumors that are refractory or develop resistance to anti-angiogenic therapeutics. PMID:25051364

  13. Evidence for epithelial-mesenchymal transition in cancer stem-like cells derived from carcinoma cell lines of the cervix uteri.

    PubMed

    Lin, Jiaying; Liu, Xishi; Ding, Ding

    2015-01-01

    The cancer stem cell (CSC) paradigm is one possible way to understand the genesis of cancer, and cervical cancer in particular. We quantified and enriched ALDH1(+) cells within cervical cancer cell lines and subsequently characterized their phenotypical and functional properties like invasion capacity and epithelial-mesenchymal transition (EMT). ALDH1 expression in spheroid-derived cells (SDC) and the parental monolayer-derived cell (MDC) line was compared by flow-cytometry. Invasion capability was evaluated by Matrigel assay and expression of EMT-related genes Twist 1, Twist 2, Snail 1, Snail 2, Vimentin and E-cadherin by real-time PCR. ALDH1 expression was significantly higher in SDC. ALDH1(+) cells showed increased colony-formation. SDC expressed lower levels of E-cadherin and elevated levels of Twist 1, Twist 2, Snail 1, Snail 2 and Vimentin compared to MDC. Cervical cancer cell lines harbor potential CSC, characterized by ALDH1 expression as well as properties like invasiveness, colony-forming ability, and EMT. CSC can be enriched by anchorage-independent culture techniques, which may be important for the investigation of their contribution to therapy resistance, tumor recurrence and metastasis.

  14. Circulating Membrane-Derived Microvesicles in Redox Biology

    PubMed Central

    Larson, Michael Craig; Hillery, Cheryl A.; Hogg, Neil

    2015-01-01

    Microparticles or microvesicles (MV) are sub-cellular membrane blebs shed from all cells in response to various stimuli. MVs carry a battery of signaling molecules, many of them related to redox-regulated processes. The role of MVs, either as a cause or result of cellular redox signaling has been increasingly recognized over the past decade. This is in part due to advances in flow cytometry and its detection of MVs. Notably, recent studies have shown circulating MVs from platelets and endothelial cells drive reactive species-dependent angiogenesis; circulating MVs in cancer alter the microenvironment and enhance invasion through horizontal transfer of mutated proteins and nucleic acids, and harbor redox-regulated matrix metalloproteinases and pro-coagulative surface molecules; and circulating MVs from RBCs and other cells modulate cell-cell interactions through scavenging or production of nitric oxide and other free radicals. While our recognition of MVs in redox-related processes is growing, especially in the vascular biology field, much remains unknown regarding the various biologic and pathologic functions of MVs. Like reactive oxygen and nitrogen species, MVs were originally believed to have a solely a pathological role in biology. And like our understanding of reactive species, it is now clear that MVs also play an important role in normal growth, development, and homeostasis. We are just beginning to understand how MVs are involved in various biological processes—developmental, homeostatic and pathological—and the role of MVs in redox signaling is an rich and exciting area of investigation. PMID:24751526

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

    PubMed

    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.

  16. High-efficiency lysis of cervical cancer by allogeneic NK cells derived from umbilical cord progenitors is independent of HLA status.

    PubMed

    Veluchamy, John P; Heeren, A Marijne; Spanholtz, Jan; van Eendenburg, Jaap D H; Heideman, Daniëlle A M; Kenter, Gemma G; Verheul, Henk M; van der Vliet, Hans J; Jordanova, Ekaterina S; de Gruijl, Tanja D

    2017-01-01

    Down-regulation of HLA in tumor cells, low numbers and dysfunctionality of NK cells are commonly observed in patients with end-stage cervical cancer. Adoptive transfer of high numbers of cytotoxic NK cells might be a promising treatment approach in this setting. Here, we explored the cytotoxic efficacy on ten cervical cancer cell lines of activated allogeneic NK cells from two sources, i.e., peripheral blood (PBNK) with and without cetuximab (CET), a tumor-specific monoclonal antibody directed against EGFR, or derived from umbilical cord blood (UCB-NK). Whereas CET monotherapy was ineffective against the panel of cervical cancer cell lines, irrespective of their EGFR expression levels and despite their RAS (wt) status, it significantly enhanced the in vitro cytotoxic efficacy of activated PBNK (P = 0.002). Equally superior cytotoxicity over activated PBNK alone was achieved by UCB-NK (P < 0.001). Both PBNK- and UCB-NK-mediated cytotoxic activity was dependent on the NK-activating receptors natural killer group 2, member D receptor (NKG2D) and DNAX accessory molecule-1 (DNAM-1) (P < 0.05) and unrelated to expression levels of the inhibitory receptors HLA-E and/or HLA-G. Most strikingly, whereas the PBNK's cytotoxic activity was inversely correlated with HLA-ABC levels (P = 0.036), PBNK + CET and UCB-NK cytotoxicity were entirely independent of HLA-ABC expression. In conclusion, this study provides a rationale to initiate a clinical trial for cervical cancer with adoptively transferred allogeneic NK cells, employing either UCB-NK or PBNK + CET for EGFR-expressing tumors. Adoptive transfer of UCB-NK might serve as a generally applicable treatment for cervical cancer, enabled by HLA-, histology- and HPV-independent killing mechanisms.

  17. Breast Cancer Cell-Derived GM-CSF Licenses Regulatory Th2 Induction by Plasmacytoid Predendritic Cells in Aggressive Disease Subtypes.

    PubMed

    Ghirelli, Cristina; Reyal, Fabien; Jeanmougin, Marine; Zollinger, Raphaël; Sirven, Philémon; Michea, Paula; Caux, Christophe; Bendriss-Vermare, Nathalie; Donnadieu, Marie-Hélène; Caly, Martial; Fourchotte, Virginie; Vincent-Salomon, Anne; Sigal-Zafrani, Brigitte; Sastre-Garau, Xavier; Soumelis, Vassili

    2015-07-15

    Reciprocal interactions between tumor cells and their microenvironment vitally impact tumor progression. In this study, we show that GM-CSF produced by primary breast tumor cells induced the activation of plasmacytoid predendritic cells (pDC), a cell type critical to anti-viral immunity. pDC that expressed the GM-CSF receptor were increased in breast tumors compared with noninvolved adjacent breast tissue. Tumor-activated pDC acquired naïve CD4(+) T-cell stimulatory capacity and promoted a regulatory Th2 response. Finally, the concomitant increase of GM-CSF and pDC was significantly associated with relatively more aggressive breast cancer subtypes. Our results characterize the first tumor-derived factor that can activate pDC to promote a regulatory Th2 response, with implications for therapeutic targeting of a tumor-immune axis of growing recognition in its significance to cancer.

  18. [STRUCTURAL CHARACTERIZATION OF PLATELETS AND PLATELET-DERIVED MICROVESICLES].

    PubMed

    Ponomareva, A A; Nevzorova, T A; Mordakhanova, E R; Andrianova, I A; Litvinov, R I

    2016-01-01

    Platelets are the anucleated blood cells, wich together with the fibrin stop bleeding (hemostasis). Cellular microvesicles are membrane-surrounded microparticles released into extracellular space upon activation and/or apoptosis of various cells. Platelet-derived macrovesicles from the major population of circulating blood microparticles that play an important role in hemostasis and thrombosis. Despite numerous studies on the pathophysiology of platelet-derived macrovesicles, mechanisms of their formation and structural details remain poorly understood. Here we investigated the ultrastructure of parental platelets and platelet-derived microvesicles formed in vitro by quiescent cells as well as by cells stimulated with one of the following activators: arachidonic acid, ADP, thrombin, calcium ionophore A23187. Using transmission electron microscopy of human platelets and isolated microvesicles, we analyzed the intracellular origin, steps of formation, structural diversity, and size distributions of the subcellular particles. We have revealed that thrombin, unlike other stimuli, not only induced vesiculation of the plasma membrane but also caused break-up of the cells followed by formation of microparticles that are comparable with microvesicles by size. A fraction of these microparticles contained cellular organelles surrounded by a thin membrane. The size of platelet-derived macrovesicles varied from 30 nm to 500 nm, however, the size distributions depended on the nature of a cell-activating stimulus. The results obtained provide new information about the formation of platelet-derived macrovesicles and their structural diversity, wich is important to understand their multiple functions in normal and disease states.

  19. Tumor-derived mesenchymal stem cells and orthotopic site increase the tumor initiation potential of putative mouse mammary cancer stem cells derived from MMTV-PyMT mice.

    PubMed

    Lanza, Denise Grant; Ma, Jun; Guest, Ian; Uk-Lim, Chang; Glinskii, Anna; Glinsky, Gennadi; Sell, Stewart

    2012-12-01

    The ability to transplant mammary cancer stem cells, identified by the phenotype CD24(+)CD29(+)CD49f(+)Sca-1(low), is dependent on the microenvironment in which the cells are placed. Using the MMTV-PyMT mouse model of mammary cancer, we now report two methods of tumor growth enhancement: contributions of tumor stroma in the form of tumor-derived mesenchymal stem cells and orthotopic vs. heterotopic transplantation sites. To support evidence of stem cell function, tumor-derived mesenchymal stem cells differentiated into adipocyte- and osteocyte-like cells after culture in specific medium. Co-injection of tumor-initiating cells with tumor-derived mesenchymal stem cells significantly increased tumor initiation compared to subcutaneous injection of TICs alone; co-injection also allowed tumor initiation with a single TIC. Interestingly, we observed the formation of sarcomas after co-injections of tumor-derived mesenchymal stem cells or mouse embryonic fibroblasts with TICs; sarcomas are not observed in spontaneous MMTV-PyMT tumors and rarely observed in injections of TICs alone. Tumor initiation was also significantly increased in the orthotopic injection site compared to heterotopic injections. We conclude that tumor stroma and orthotopic sites both enhance tumor initiation by mammary cancer stem cells.

  20. Macrophage microvesicles induce macrophage differentiation and miR-223 transfer

    PubMed Central

    Ismail, Noura; Wang, Yijie; Dakhlallah, Duaa; Moldovan, Leni; Agarwal, Kitty; Batte, Kara; Shah, Prexy; Wisler, Jon; Eubank, Tim D.; Tridandapani, Susheela; Paulaitis, Michael E.

    2013-01-01

    Microvesicles are small membrane-bound particles comprised of exosomes and various-sized extracellular vesicles. These are released by several cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets, while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and explored their role in the differentiation of naive monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including mono cytes, endothelial cells, epithelial cells, and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation. PMID:23144169

  1. Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

    PubMed Central

    Miranda, Kevin C.; Bond, Daniel T.; McKee, Mary; Skog, Johan; Păunescu, Teodor G.; Da Silva, Nicolas; Brown, Dennis; Russo, Leileata M.

    2015-01-01

    Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers for renal disease. We sought to identify whether these microvesicles contain nucleic acids. We isolated microvesicles from human urine in the same density range as that previously described for urinary exosomes and found them to have an RNA integrity profile similar to that of kidney tissue, including 18S and 28S rRNA. This profile was better preserved in urinary microvesicles compared with whole cells isolated from urine, suggesting that microvesicles may protect RNA during urine passage. We were able to detect mRNA in the human urinary microvesicles encoding proteins from all regions of the nephron and the collecting duct. Further, to provide a proof of principle, we found that microvesicles isolated from the urine of the V-ATPase B1 subunit knockout mice lacked mRNA of this subunit while containing a normal amount of the B2 subunit and aquaporin 2. The microvesicles were found to be contaminated with extraneous DNA potentially on their surface; therefore, we developed a rapid and reliable means to isolate nucleic acids from within urine microvesicles devoid of this extraneous contamination. Our study provides an experimental strategy for the routine isolation and use of urinary microvesicles as a novel and non-invasive source of nucleic acids to further renal disease biomarker discovery. PMID:20428099

  2. MET-dependent cancer invasion may be preprogrammed by early alterations of p53-regulated feedforward loop and triggered by stromal cell-derived HGF.

    PubMed

    Hwang, Chang-Il; Choi, Jinhyang; Zhou, Zongxiang; Flesken-Nikitin, Andrea; Tarakhovsky, Alexander; Nikitin, Alexander Yu

    2011-11-15

    MET, a receptor protein tyrosine kinase activated by hepatocyte growth factor (HGF), is a crucial determinant of metastatic progression. Recently, we have identified p53 as an important regulator of MET-dependent cell motility and invasion. This regulation occurs via feedforward loop suppressing MET expression by miR-34-dependent and -independent mechanisms. Here, by using Dicer conditional knockout, we provide further evidence for microRNA-independent MET regulation by p53. Furthermore, we show that while MET levels increase immediately after p53 inactivation, mutant cells do not contain active phosphorylated MET and remain non-invasive for a long latency period at contrary to cell culture observations. Evaluation of mouse models of ovarian and prostate carcinogenesis indicates that formation of desmoplastic stroma, associated production of HGF by stromal cells and coinciding MET phosphorylation precede cancer invasion. Thus, initiation mutation of p53 is sufficient for preprogramming motile and invasive properties of epithelial cells, but the stromal reaction may represent a critical step for their manifestation during cancer progression.

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

    SciTech Connect

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

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer We sorted SP cells from a human gallbladder carcinoma cell lines, SGC-996. Black-Right-Pointing-Pointer SP cells displayed higher proliferation and stronger clonal-generating capability. Black-Right-Pointing-Pointer SP cells showed more migratory and invasive abilities. Black-Right-Pointing-Pointer SP cells were more resistant and tumorigenic than non-SP counterparts. Black-Right-Pointing-Pointer ABCG2 might be a candidate as a marker for SP cells. -- Abstract: 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.

  4. Colorectal cancer cell-derived microRNA200 modulates the resistance of adjacent blood endothelial barriers in vitro.

    PubMed

    Holzner, Silvio; Senfter, Daniel; Stadler, Serena; Staribacher, Anna; Nguyen, Chi Huu; Gaggl, Anna; Geleff, Silvana; Huttary, Nicole; Krieger, Sigurd; Jäger, Walter; Dolznig, Helmut; Mader, Robert M; Krupitza, Georg

    2016-11-01

    Since cancer cells, when grown as spheroids, display drug sensitivity and radiation resistance patterns such as seen in vivo we recently established a three‑dimensional (3D) in vitro model recapitulating colorectal cancer (CRC)-triggered lymphatic endothelial cell (LEC)‑barrier breaching to study mechanisms of intra‑/extravasation. CRC metastasizes not only through lymphatics but also through blood vessels and here we extend the 3D model to the interaction of blood endothelial cells (BECs) with naïve and 5‑fluorouracil (5‑FU)‑resistant CRC CCL227 cells. The 3D model enabled quantifying effects of tumour‑derived microRNA200 (miR200) miR200a, miR200b, miR200c, miR141 and miR429 regarding the induction of so-called 'circular chemorepellent‑induced defects' (CCIDs) within the BEC‑barrier, which resemble gates for tumour transmigration. For this, miR200 precursors were individually transfected and furthermore, the modulation of ZEB family expression was analysed by western blotting. miR200c, miR141 and miR429, which are contained in exosomes from naïve CCL227 cells, downregulated the expression of ZEB2, SNAI and TWIST in BECs. The exosomes of 5‑FU‑resistant CCL227‑RH cells, which are devoid of miR200, accelerated CCID formation in BEC monolayers as compared to exosomes from naïve CCL227 cells. This confirmed the reported role of ZEB2 and SNAI in CRC metastasis and highlighted the active contribution of the stroma in the metastatic process. CCL227 spheroids affected the integrity of BEC and LEC barriers alike, which was in agreement with the observation that CRC metastasizes via blood stream (into the liver) as well as via lymphatics (into lymph nodes and lungs). This further validated the CRC/LEC and CRC/BEC in vitro model to study mechanisms of CRC spreading through vascular systems. Treatment of CCL227‑RH cells with the HDAC inhibitors mocetinostat and sulforaphane reduced CCID formation to the level triggered by naïve CCL227

  5. Microvesicle Production after Trauma and Its Clinical Impact on Venothromboembolism

    DTIC Science & Technology

    2014-12-01

    bleeding and/or a clot in their veins which cause a life-threatening event known as venous thromboembolism (VTE). We began enrollment of patients into...the study on 2 February 2011. As of 1 October 2014, we have successfully enrolled and collected blood samples on 1139 patients and 89 healthy...found both in plasma and microvesicles (MVs) derived from blood cells and injured tissues. The specific Aims of this study are: Aim 1: · • Identify

  6. Regenerating the injured kidney with human umbilical cord mesenchymal stem cell-derived exosomes.

    PubMed

    Dorronsoro, Akaitz; Robbins, Paul D

    2013-04-25

    Transplantation of adult stem cells is being used to facilitate repair or regeneration of damaged or diseased tissues. However, in many cases, the therapeutic effects of the injected stem cells are mediated by factors secreted by stem cells and not by differentiation of the transplanted stem cells. Recent reports have identified a class of microvesicles, termed exosomes, released by stem cells that are able to confer therapeutic effects on injured renal and cardiac tissue. In this issue of Stem Cell Research & Therapy, Zhou and colleagues demonstrate the ability of exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs), but not non-stem cell-derived exosomes, to improve acute kidney injury induced by cisplatin in rats. The authors demonstrate that hucMSC exosomes can reduce cisplatin-mediated renal oxidative stress and apoptosis in vivo and increase renal epithelial cell proliferation in culture. These results suggest that stem cell-derived exosomes, which are easy to isolate and safer to use than the parental stem cells, could have significant clinical utility.

  7. Exosomes as Biomarker Enriched Microvesicles: Characterization of Exosomal Proteins Derived from a Panel of Prostate Cell Lines with Distinct AR Phenotypes

    PubMed Central

    Hosseini-Beheshti, Elham; Pham, Steven; Adomat, Hans; Li, Na; Tomlinson Guns, Emma S.

    2012-01-01

    Prostate cancer is the leading type of cancer diagnosed in men. In 2010, ∼217,730 new cases of prostate cancer were reported in the United States. Prompt diagnosis of the disease can substantially improve its clinical outcome. Improving capability for early detection, as well as developing new therapeutic targets in advanced disease are research priorities that will ultimately lead to better patient survival. Eukaryotic cells secrete proteins via distinct regulated mechanisms which are either ER/Golgi dependent or microvesicle mediated. The release of microvesicles has been shown to provide a novel mechanism for intercellular communication. Exosomes are nanometer sized cup-shaped membrane vesicles which are secreted from normal and cancerous cells. They are present in various biological fluids and are rich in characteristic proteins. Exosomes may thus have potential both in facilitating early diagnosis via less invasive procedures or be candidates for novel therapeutic approaches for castration resistance prostate cancer. Because exosomes have been shown previously to have a role in cell-cell communication in the local tumor microenvironment, conferring activation of numerous survival mechanisms, we characterized constitutive lipids, cholesterol and proteins from exosomes derived from six prostate cell lines and tracked their uptake in both cancerous and benign prostate cell lines respectively. Our comprehensive proteomic and lipidomic analysis of prostate derived exosomes could provide insight for future work on both biomarker and therapeutic targets for the treatment of prostate cancer. PMID:22723089

  8. Urinary Microvesicle-Bound Uromodulin: A Potential Molecular Biomarker in Diabetic Kidney Disease

    PubMed Central

    Lou, Neng-jun; Ni, Yi-hong; Jia, Hong-ying; Deng, Jing-ti; Jiang, Lu; Zheng, Feng-jie

    2017-01-01

    This study was designed to investigate the changes of urinary microvesicle-bound uromodulin and total urinary uromodulin levels in human urine and the correlations with the severity of diabetic kidney disease (DKD). 31 healthy subjects without diabetes and 100 patients with type 2 diabetes mellitus (T2DM) were included in this study. The patients with T2DM were divided into three groups based on the urinary albumin/creatinine ratio (UACR): normoalbuminuria group (DM, n = 46); microalbuminuria group (DN1, n = 32); and macroalbuminuria group (DN2, n = 22). We use a specific monoclonal antibody AD-1 to capture the urinary microvesicles. Urinary microvesicle-bound uromodulin and total urinary uromodulin levels were determined by enzyme-linked immunosorbent assay (ELISA). Our results showed that the levels of urinary microvesicle-bound uromodulin in DN1 and DN2 groups were significantly higher than those in control group and DM group (P < 0.01). Multiple stepwise linear regression analysis showed that UACR was independent determinant for urinary microvesicle-bound uromodulin (P < 0.05) but not for total urinary uromodulin. These findings suggest that the levels of urinary microvesicle-bound uromodulin are associated with the severity of DKD. The uromodulin in urinary microvesicles may be a specific marker of DKD and potentially may be used to predict the onset and/or monitor the progression of DKD. PMID:28182086

  9. Quantitative proteomics of extracellular vesicles derived from human primary and metastatic colorectal cancer cells.

    PubMed

    Choi, Dong-Sic; Choi, Do-Young; Hong, Bok Sil; Jang, Su Chul; Kim, Dae-Kyum; Lee, Jaewook; Kim, Yoon-Keun; Kim, Kwang Pyo; Gho, Yong Song

    2012-01-01

    Cancer cells actively release extracellular vesicles (EVs), including exosomes and microvesicles, into surrounding tissues. These EVs play pleiotropic roles in cancer progression and metastasis, including invasion, angiogenesis, and immune modulation. However, the proteomic differences between primary and metastatic cancer cell-derived EVs remain unclear. Here, we conducted comparative proteomic analysis between EVs derived from human primary colorectal cancer cells (SW480) and their metastatic derivatives (SW620). Using label-free quantitation, we identified 803 and 787 proteins in SW480 EVs and SW620 EVs, respectively. Based on comparison between the estimated abundance of EV proteins, we identified 368 SW480 EV-enriched and 359 SW620 EV-enriched proteins. SW480 EV-enriched proteins played a role in cell adhesion, but SW620 EV-enriched proteins were associated with cancer progression and functioned as diagnostic indicators of metastatic cancer; they were overexpressed in metastatic colorectal cancer and played roles in multidrug resistance. As the first proteomic analysis comparing primary and metastatic cancer-derived EVs, this study increases our understanding of the pathological function of EVs in the metastatic process and provides useful biomarkers for cancer metastasis.

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

    PubMed Central

    Lai, Charles Pin-Kuang; Breakefield, Xandra Owen

    2012-01-01

    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. PMID:22754538

  11. Procoagulant and immunogenic properties of melanoma exosomes, microvesicles and apoptotic vesicles

    PubMed Central

    Muhsin-Sharafaldine, Morad-Rémy; Saunderson, Sarah C.; Dunn, Amy C.; Faed, James M.; Kleffmann, Torsten; McLellan, Alexander D.

    2016-01-01

    Extracellular vesicles (EV) are lipid particles released from eukaryotic cells into the extracellular fluid. Depending on the cell type or mechanism of release, vesicles vary in form and function and exert distinct functions in coagulation and immunity. Tumor cells may constitutively shed vesicles known as exosomes or microvesicles (MV). Alternatively, apoptosis induces the release of apoptotic blebs or vesicles (ApoV) from the plasma membrane. EV have been implicated in thrombotic events (the second highest cause of death in cancer patients) and tumor vesicles contribute to the anti-cancer immune response. In this study, we utilized the well characterized B16 melanoma model to determine the molecular composition and procoagulant and immunogenic potential of exosomes, MV and ApoV. Distinct patterns of surface and cytoplasmic molecules (tetraspanins, integrins, heat shock proteins and histones) were expressed between the vesicle types. Moreover, in vitro coagulation assays revealed that membrane-derived vesicles, namely MV and ApoV, were more procoagulant than exosomes–with tissue factor and phosphatidylserine critical for procoagulant activity. Mice immunized with antigen-pulsed ApoV and challenged with B16 tumors were protected out to 60 days, while lower protection rates were afforded by MV and exosomes. Together the results demonstrate distinct phenotypic and functional differences between vesicle types, with important procoagulant and immunogenic functions emerging for membrane-derived MV and ApoV versus endosome-derived exosomes. This study highlights the potential of EV to contribute to the prothrombotic state, as well as to anti-cancer immunity. PMID:27462921

  12. Exosomes as Novel microRNA-Delivery Vehicles to Modulate Prostate Cancer Progression

    DTIC Science & Technology

    2015-10-01

    specifically packaged in these 40-100 nm microvesicles and secreted from prostate cancer cells are important in the progression to aggressive disease...behavior of aggressive human prostate cancer cell lines in vitro as well as in vivo using mouse xenograft models. 15. SUBJECT TERMS microRNAs, exosomes...microvesicles and secreted from prostate cancer cells are important in the progression to aggressive disease. In this exploratory award, we are

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

    PubMed Central

    Velling Magnussen, Line; Nielsen, Morten Hjuler; Nielsen, Tine Bo; Højlund, Kurt; Andersen, Marianne Skovsager; Handberg, Aase

    2017-01-01

    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 of platelet, monocyte, and endothelial cell origin were identified by cell-specific markers and their expression of CD36 was investigated. Results. Triglycerides correlated positively with all investigated microvesicle phenotypes in this study (p < 0.05), and indicators of hepatic fat accumulation, alanine aminotransferase, and gamma glutamyltransferase correlated with platelet and endothelial microvesicles and CD36-expressing microvesicles from platelets and monocytes (p < 0.05). BMI, waist circumference, and fat percentage correlated with CD36-expressing monocyte microvesicles (p < 0.05), while insulin sensitivity did 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 pathogenesis. Although testosterone therapy improved body composition measures, microvesicle phenotype levels were unaffected. This trial was registered at ClinicalTrials.gov (NCT01560546). PMID:28168203

  14. Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome.

    PubMed

    Rood, Ilse M; Deegens, Jeroen K J; Merchant, Michael L; Tamboer, Wim P M; Wilkey, Daniel W; Wetzels, Jack F M; Klein, Jon B

    2010-10-01

    Urinary microvesicles, such as 40-100 nm exosomes and 100-1000 nm microparticles, contain many proteins that may serve as biomarkers of renal disease. Microvesicles have been isolated by ultracentrifugation or nanomembrane ultrafiltration from normal urine; however, little is known about the efficiency of these methods in isolating microvesicles from patients with nephrotic-range proteinuria. Here we compared three techniques to isolate microvesicles from nephrotic urine: nanomembrane ultrafiltration, ultracentrifugation, and ultracentrifugation followed by size-exclusion chromatography (UC-SEC). Highly abundant urinary proteins were still present in sufficient quantity after ultrafiltration or ultracentrifugation to blunt detection of less abundant microvesicular proteins by MALDI-TOF-TOF mass spectrometry. The microvesicular markers neprilysin, aquaporin-2, and podocalyxin were highly enriched following UC-SEC compared with preparations by ultrafiltration or ultracentrifugation alone. Electron microscopy of the UC-SEC fractions found microvesicles of varying size, compatible with the presence of both exosomes and microparticles. Thus, UC-SEC following ultracentrifugation to further enrich and purify microparticles facilitates the search for prognostic biomarkers that might be used to predict the clinical course of nephrotic syndrome.

  15. Microvesicles and exosomes: new players in metabolic and cardiovascular disease.

    PubMed

    Lawson, Charlotte; Vicencio, Jose M; Yellon, Derek M; Davidson, Sean M

    2016-02-01

    The past decade has witnessed an exponential increase in the number of publications referring to extracellular vesicles (EVs). For many years considered to be extracellular debris, EVs are now seen as novel mediators of endocrine signalling via cell-to-cell communication. With the capability of transferring proteins and nucleic acids from one cell to another, they have become an attractive focus of research for different pathological settings and are now regarded as both mediators and biomarkers of disease including cardio-metabolic disease. They also offer therapeutic potential as signalling agents capable of targeting tissues or cells with specific peptides or miRNAs. In this review, we focus on the role that microvesicles (MVs) and exosomes, the two most studied classes of EV, have in diabetes, cardiovascular disease, endothelial dysfunction, coagulopathies, and polycystic ovary syndrome. We also provide an overview of current developments in MV/exosome isolation techniques from plasma and other fluids, comparing different available commercial and non-commercial methods. We describe different techniques for their optical/biochemical characterization and quantitation. We also review the signalling pathways that exosomes and MVs activate in target cells and provide some insight into their use as biomarkers or potential therapeutic agents. In summary, we give an updated focus on the role that these exciting novel nanoparticles offer for the endocrine community.

  16. Expression of blood group antigens on red cell microvesicles.

    PubMed

    Oreskovic, R T; Dumaswala, U J; Greenwalt, T J

    1992-01-01

    The purpose of this study was to determine whether epitopes of the A, B, D, Fya, M, N, S, s, and K blood group antigens are present on microvesicle membranes shed by red cells during storage. Vesicles were isolated from outdated units of blood having and lacking the specified antigens. Diluted antisera were absorbed with fixed quantities of vesicles from red cells with the test antigen and red cells lacking that antigen (controls). The adsorbed and unadsorbed antisera were titrated and scored by using panel cells from persons known to be heterozygous for all the non-AB antigens. The mean titration scores following adsorption with the vesicles from A, B, D, M+N-, M-N+, S+s-, S-s+, and Fy(a+b-) units were appreciably lower than the control scores (0, 0, 3, 2, 2, 0, 4, and 4 vs. 19, 23, 34, 13, 12, 16, 18, and 29, respectively), which indicated the presence of these epitopes on the membrane of shed vesicles. The results following adsorption with K:1,2 vesicles were equivocal.

  17. Thrombocyte adhesion and release of extracellular microvesicles correlate with surface morphology of adsorbent polymers for lipid apheresis.

    PubMed

    Weiss, René; Spittler, Andreas; Schmitz, Gerd; Fischer, Michael B; Weber, Viktoria

    2014-07-14

    Whole blood lipid apheresis is clinically applied to reduce low density lipoprotein cholesterol in patients with homozygous familial hypercholesterolemia. Here, we studied the correlation between physicochemical parameters, in particular, surface roughness and blood compatibility, of two polyacrylate-based and a dextran sulfate-based polymer for lipid apheresis. The adsorbent surface roughness was assessed by atomic force microscopy. Freshly isolated human thrombocytes were circulated over adsorbent columns downscaled equivalent to clinical use to study thrombocyte adhesion and microvesicle generation. Quantification of thrombocytes and microvesicles in the flow-through of the columns revealed that both thrombocyte adhesion and microvesicle generation increased with increasing adsorbent surface roughness. Activation of thrombocytes with thrombin receptor-activating peptide-6 favored their adhesion to the adsorbents, as demonstrated by preferential depletion of CD62(+) and PAC-1(+) thrombocytes. Taken together, enhanced polymer surface roughness fostered cell adhesion and microvesicle release, underscoring the role of extracellular microvesicles as markers of cellular activation and of blood compatibility.

  18. Human adipose mesenchymal stem cell-derived exosomal-miRNAs are critical factors for inducing anti-proliferation signalling to A2780 and SKOV-3 ovarian cancer cells

    PubMed Central

    Reza, Abu Musa Md Talimur; Choi, Yun-Jung; Yasuda, Hideyo; Kim, Jin-Hoi

    2016-01-01

    An enigmatic question exists concerning the pro- or anti-cancer status of mesenchymal stem cells (MSCs). Despite growing interest, this question remains unanswered, and the debate became intensified with new evidences backing each side. Here, we showed that human adipose MSC (hAMSC)-derived conditioned medium (CM) exhibited inhibitory effects on A2780 human ovarian cancer cells by blocking the cell cycle, and activating mitochondria-mediated apoptosis signalling. Explicitly, we demonstrated that exosomes, an important biological component of hAMSC-CM, could restrain proliferation, wound-repair and colony formation ability of A2780 and SKOV-3 cancer cells. Furthermore, hAMSC-CM-derived exosomes induced apoptosis signalling by upregulating different pro-apoptotic signalling molecules, such as BAX, CASP9, and CASP3, as well as downregulating the anti-apoptotic protein BCL2. More specifically, cancer cells exhibited reduced viability following fresh or protease-digested exosome treatment; however, treatment with RNase-digested exosomes could not inhibit the proliferation of cancer cells. Additionally, sequencing of exosomal RNAs revealed a rich population of microRNAs (miRNAs), which exhibit anti-cancer activities by targeting different molecules associated with cancer survival. Our findings indicated that exosomal miRNAs are important players involved in the inhibitory influence of hAMSC-CM towards ovarian cancer cells. Therefore, we believe that these comprehensive results will provide advances concerning ovarian cancer research and treatment. PMID:27929108

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

    NASA Astrophysics Data System (ADS)

    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

  20. Nonsedimentable Microvesicles from Senescing Bean Cotyledons Contain Gel Phase-Forming Phospholipid Degradation Products 1

    PubMed Central

    Yao, Kening; Paliyath, Gopinadhan; Thompson, John E.

    1991-01-01

    A mixture of liquid-crystalline and gel-phase lipid domains is detectable by wide angle x-ray diffraction in smooth microsomal membranes isolated from senescent 7-day-old cotyledons, whereas corresponding membranes from young 2-day-old cotyledons are exclusively liquid-crystalline. The gel-phase domains in the senescent membranes comprise phospholipid degradation products including diacylglycerols, free fatty acids, long-chain aldehydes, and long-chain hydrocarbons. The same complement of phospholipid degradation products is also present in nonsedimentable microvesicles isolated from senescent 7-day-old cotyledons by filtration of a 250,000g, 12-hour supernatant through a 300,000 dalton cut-off filter. The phospholipid degradation products in the microvesicles form gel-phase lipid domains when reconstituted into phospholipid liposomes. Nonsedimentable microvesicles of a similar size, which are again enriched in the same gel-phase-forming phospholipid degradation products, are also generated in vitro from smooth microsomal membranes isolated from 2-day-old cotyledons when Ca2+ is added to activate membrane-associated lipolytic enzymes. The Ca2+-treated membranes do not contain detectable gel-phase domains, suggesting that the phospholipid degradation products are completely removed by microvesiculation. The observations collectively indicate that these nonsedimentable microvesicles serve as a vehicle for moving phospholipid degradation products out of membrane bilayers into the cytosol. As noted previously (Yao K, Paliyath G, Humphrey RW, Hallett FR, Thompson JE [1991] Proc Natl Acad Sci USA 88: 2269-2273), the term “deteriosome” connotes this putative function and would serve to distinguish these microvesicles from other cytoplasmic microvesicles unrelated to deterioration. ImagesFigure 2Figure 3Figure 4Figure 5Figure 6Figure 7 PMID:16668427

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

    PubMed Central

    Müller, Günter

    2012-01-01

    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 complete individual pathogenetic pathways underlying metabolic diseases. A critical step in further validation of EMVs as biomarkers will rely on

  2. Impact of Biofluid Viscosity on Size and Sedimentation Efficiency of the Isolated Microvesicles

    PubMed Central

    Momen-Heravi, Fatemeh; Balaj, Leonora; Alian, Sara; Trachtenberg, Alexander J.; Hochberg, Fred H.; Skog, Johan; Kuo, Winston Patrick

    2012-01-01

    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, 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 ultracentrifugation when processing any biofluids. PMID:22661955

  3. Silica Microspheres Are Superior to Polystyrene for Microvesicle Analysis by Flow Cytometry

    DTIC Science & Technology

    2015-02-16

    Sustained elevated amounts of circulating procoagulant membrane microparticles and soluble GPV after acute myocardial infarction in diabetes mellitus...cerebral spinal fluid, and alveolar lavage fluid and have been associated with several pathological conditions that include thrombosis, diabetes, myocardial ... infarction , traumatic brain injury, pulmonary hypertension, brain hemor- rhage, and acute respiratory distress syndrome [3–7]. Microvesicles have

  4. Monocyte derived microvesicles deliver a cell death message via encapsulated caspase-1.

    PubMed

    Sarkar, Anasuya; Mitra, Srabani; Mehta, Sonya; Raices, Raquel; Wewers, Mark D

    2009-09-25

    Apoptosis depends upon the activation of intracellular caspases which are classically induced by either an intrinsic (mitochondrial based) or extrinsic (cytokine) pathway. However, in the process of explaining how endotoxin activated monocytes are able to induce apoptosis of vascular smooth muscle cells when co-cultured, we uncovered a transcellular apoptosis inducing pathway that utilizes caspase-1 containing microvesicles. Endotoxin stimulated monocytes induce the cell death of VSMCs but this activity is found in 100,000 g pellets of cell free supernatants of these monocytes. This activity is not a direct effect of endotoxin, and is inhibited by the caspase-1 inhibitor YVADcmk but not by inhibitors of Fas-L, IL-1beta and IL-18. Importantly, the apoptosis inducing activity co-purifies with 100 nm sized microvesicles as determined by TEM of the pellets. These microvesicles contain caspase-1 and caspase-1 encapsulation is required since disruption of microvesicular integrity destroys the apoptotic activity but not the caspase-1 enzymatic activity. Thus, monocytes are capable of delivering a cell death message which depends upon the release of microvesicles containing functional caspase-1. This transcellular apoptosis induction pathway describes a novel pathway for inflammation induced programmed cell death.

  5. Monocyte Derived Microvesicles Deliver a Cell Death Message via Encapsulated Caspase-1

    PubMed Central

    Sarkar, Anasuya; Mitra, Srabani; Mehta, Sonya; Raices, Raquel; Wewers, Mark D.

    2009-01-01

    Apoptosis depends upon the activation of intracellular caspases which are classically induced by either an intrinsic (mitochondrial based) or extrinsic (cytokine) pathway. However, in the process of explaining how endotoxin activated monocytes are able to induce apoptosis of vascular smooth muscle cells when co-cultured, we uncovered a transcellular apoptosis inducing pathway that utilizes caspase-1 containing microvesicles. Endotoxin stimulated monocytes induce the cell death of VSMCs but this activity is found in 100,000 g pellets of cell free supernatants of these monocytes. This activity is not a direct effect of endotoxin, and is inhibited by the caspase-1 inhibitor YVADcmk but not by inhibitors of Fas-L, IL-1β and IL-18. Importantly, the apoptosis inducing activity co-purifies with 100 nm sized microvesicles as determined by TEM of the pellets. These microvesicles contain caspase-1 and caspase-1 encapsulation is required since disruption of microvesicular integrity destroys the apoptotic activity but not the caspase-1 enzymatic activity. Thus, monocytes are capable of delivering a cell death message which depends upon the release of microvesicles containing functional caspase-1. This transcellular apoptosis induction pathway describes a novel pathway for inflammation induced programmed cell death. PMID:19779610

  6. Translocation of Endogenous Danger Signal HMGB1 From Nucleus to Membrane Microvesicles in Macrophages.

    PubMed

    Chen, Yan; Li, Guangping; Liu, Yanxia; Werth, Victoria P; Williams, Kevin Jon; Liu, Ming-Lin

    2016-11-01

    High mobility group box 1 (HMGB1) is a nuclear protein that can be released from activated or dead cells. Extracellular HMGB1 can serve as a "danger signal" and novel cytokine that mediates sterile inflammation. In addition to its soluble form, extracellular HMGB1 can also be carried by membrane microvesicles. However, the cellular mechanisms responsible for nuclear HMGB1 translocation to the plasma membrane and release onto membrane microvesicles have not been investigated. Tobacco smoking is a major cause of sterile inflammation in many diseases. Smoking also increases blood levels of HMGB1. In this study, we found that exposure of macrophages to tobacco smoke extract (TSE) stimulated HMGB1 expression, redistribution, and release into the extracellular milieu both as a soluble molecule and, surprisingly, as a microvesicle-associated form (TSE-MV). Inhibition of chromosome region maintenance-1 (CRM1), a nuclear exporter, attenuated TSE-induced HMGB1 redistribution from the nucleus to the cytoplasm, and then its release on TSE-MVs. Our study demonstrates a novel mechanism for the translocation of nuclear HMGB1 to the plasma membrane, and then its release in a microvesicle-associated form. J. Cell. Physiol. 231: 2319-2326, 2016. © 2016 Wiley Periodicals, Inc.

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

    PubMed

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

    EPA Pesticide Factsheets

    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

  9. Linneg Sca-1high CD49fhigh prostate cancer cells derived from the Hi-Myc mouse model are tumor-initiating cells with basal-epithelial characteristics and differentiation potential in vitro and in vivo.

    PubMed

    Saha, Achinto; Blando, Jorge; Fernandez, Irina; Kiguchi, Kaoru; DiGiovanni, John

    2016-05-03

    A cell line was established from ventral prostate (VP) tumors of one-year-old Hi-Myc mice. These cells, called HMVP2 cells, are LinnegSca-1highCD49fhigh with high CD44 and CD29 expression and express CK14, Sca-1 and CD49f (but not CK8), suggesting basal-epithelial characteristics. Furthermore, HMVP2 cells form spheroids and both the cells and spheroids produce tumors in syngeneic mice. After four days of culture, HMVP2 spheroids underwent a gradual transition from LinnegSca-1highCD49fhigh expression to LinnegSca-1lowCD49flow while a subpopulation of the cells retained the original LinnegSca-1highCD49fhigh expression pattern. Additional cell subpopulations expressing Lin positive markers were also present suggesting further differentiation of HMVP2 spheroids. Two additional highly tumorigenic cell lines (HMVP2A1 and HMVP2A2) were isolated from HMVP2 cells after subsequent tumor formation in FVB/N mice. Concurrently, we also established cell lines from the VP of 6 months old Hi-Myc mice (named as HMVP1) and FVB/N mice (called NMVP) having less aggressive growth properties compared to the other three cell lines. AR expression was reduced in HMVP2 cells compared to NMVP and HMVP1 cells and almost absent in HMVP2A1 and HMVP2A2 cells. These cell lines will provide valuable tools for further mechanistic studies as well as preclinical studies to evaluate preventive and/or therapeutic agents for prostate cancer.

  10. Linneg Sca-1high CD49fhigh prostate cancer cells derived from the Hi-Myc mouse model are tumor-initiating cells with basal-epithelial characteristics and differentiation potential in vitro and in vivo

    PubMed Central

    Fernandez, Irina; Kiguchi, Kaoru; DiGiovanni, John

    2016-01-01

    A cell line was established from ventral prostate (VP) tumors of one-year-old Hi-Myc mice. These cells, called HMVP2 cells, are LinnegSca-1highCD49fhigh with high CD44 and CD29 expression and express CK14, Sca-1 and CD49f (but not CK8), suggesting basal-epithelial characteristics. Furthermore, HMVP2 cells form spheroids and both the cells and spheroids produce tumors in syngeneic mice. After four days of culture, HMVP2 spheroids underwent a gradual transition from LinnegSca-1highCD49fhigh expression to LinnegSca-1lowCD49flow while a subpopulation of the cells retained the original LinnegSca-1highCD49fhigh expression pattern. Additional cell subpopulations expressing Lin positive markers were also present suggesting further differentiation of HMVP2 spheroids. Two additional highly tumorigenic cell lines (HMVP2A1 and HMVP2A2) were isolated from HMVP2 cells after subsequent tumor formation in FVB/N mice. Concurrently, we also established cell lines from the VP of 6 months old Hi-Myc mice (named as HMVP1) and FVB/N mice (called NMVP) having less aggressive growth properties compared to the other three cell lines. AR expression was reduced in HMVP2 cells compared to NMVP and HMVP1 cells and almost absent in HMVP2A1 and HMVP2A2 cells. These cell lines will provide valuable tools for further mechanistic studies as well as preclinical studies to evaluate preventive and/or therapeutic agents for prostate cancer. PMID:26910370

  11. Fabrication of a microvesicles-incorporated fibrous membrane for controlled delivery applications in tissue engineering.

    PubMed

    Nair, Bindu P; Vaikkath, Dhanesh; Mohan, Dhanya S; Nair, Prabha D

    2014-10-13

    A scaffold, which can provide mechanical support for tissue regeneration and simultaneously release functionally active biomolecules are highly desirable for tissue engineering applications. Herein, we report the fabrication of a fibrous mesh of polycaprolactone (PCL) incorporating PCL-pluronic (F127) microvesicles through electrospinning, by exploiting the slow dissolution of PCL in glacial acetic acid (g-AA). Micro-vesicles 1-10 μm in diameter were fabricated through a non-solubility driven spontaneous self-assembly and stabilization of F127 with low molecular weight PCL in tetrahydrofuran-water mixture. Time-dependent stability of the vesicles in g-AA was confirmed prior to the electrospinning. The electrospun membrane was found to be comprised of microvesicles entangled in a fibrous mesh of PCL with a fiber diameter ranging from 50-300 nm. Significant reduction in the release rate of rhodamine-B, an indicator dye from the electrospun membrane, when compared to that from the vesicle alone, evidences the surface coating of the vesicles with high molecular weight PCL during electrospinning. The vesicle incorporated membrane exhibited increased hydrophilicity when compared to the control PCL membrane, possibly due to surface unevenness and the hydrophilic F127. This enhanced surface hydrophilicity led to an increased cell viability of L929 cells on the membrane.

  12. Mechanisms of Infectivity and Evasion Derived from Microvesicles Cargo Produced by Trypanosoma cruzi

    PubMed Central

    Borges, Bruna C.; Uehara, Isadora A.; Dias, Laysa O. S.; Brígido, Paula C.; da Silva, Claudio V.; Silva, Marcelo J. B.

    2016-01-01

    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 twentieth 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. PMID:27921011

  13. Mechanisms of Infectivity and Evasion Derived from Microvesicles Cargo Produced by Trypanosoma cruzi.

    PubMed

    Borges, Bruna C; Uehara, Isadora A; Dias, Laysa O S; Brígido, Paula C; da Silva, Claudio V; Silva, Marcelo J B

    2016-01-01

    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 twentieth 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.

  14. A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis

    PubMed Central

    Feng, Qiyu; Zhang, Chengliang; Lum, David; Druso, Joseph E.; Blank, Bryant; Wilson, Kristin F.; Welm, Alana; Antonyak, Marc A.; Cerione, Richard A.

    2017-01-01

    Non-classical secretory vesicles, collectively referred to as extracellular vesicles (EVs), have been implicated in different aspects of cancer cell survival and metastasis. Here, we describe how a specific class of EVs, called microvesicles (MVs), activates VEGF receptors and tumour angiogenesis through a unique 90 kDa form of VEGF (VEGF90K). We show that VEGF90K is generated by the crosslinking of VEGF165, catalysed by the enzyme tissue transglutaminase, and associates with MVs through its interaction with the chaperone Hsp90. We further demonstrate that MV-associated VEGF90K has a weakened affinity for Bevacizumab, causing Bevacizumab to be ineffective in blocking MV-dependent VEGF receptor activation. However, treatment with an Hsp90 inhibitor releases VEGF90K from MVs, restoring the sensitivity of VEGF90K to Bevacizumab. These findings reveal a novel mechanism by which cancer cell-derived MVs influence the tumour microenvironment and highlight the importance of recognizing their unique properties when considering drug treatment strategies. PMID:28205552

  15. Potential role of corneal epithelial cell-derived exosomes in corneal wound healing and neovascularization

    PubMed Central

    Han, Kyu-Yeon; Tran, Jennifer A.; Chang, Jin-Hong; Azar, Dimitri T.; Zieske, James D.

    2017-01-01

    Specific factors from the corneal epithelium underlying the stimulation of stromal fibrosis and myofibroblast formation in corneal wound healing have not been fully elucidated. Given that exosomes are known to transfer bioactive molecules among cells and play crucial roles in wound healing, angiogenesis, and cancer, we hypothesized that corneal epithelial cell-derived exosomes may gain access to the underlying stromal fibroblasts upon disruption of the epithelial basement membrane and that they induce signaling events essential for corneal wound healing. In the present study, exosome-like vesicles were observed between corneal epithelial cells and the stroma during wound healing after corneal epithelial debridement. These vesicles were also found in the stroma following anterior stromal keratectomy, in which surgical removal of the epithelium, basement membrane, and anterior stroma was performed. Exosomes secreted by mouse corneal epithelial cells were found to fuse to keratocytes in vitro and to induce myofibroblast transformation. In addition, epithelial cell-derived exosomes induced endothelial cell proliferation and ex vivo aortic ring sprouting. Our results indicate that epithelial cell-derived exosomes mediate communication between corneal epithelial cells and corneal keratocytes as well as vascular endothelial cells. These findings demonstrate that epithelial-derived exosomes may be involved in corneal wound healing and neovascularization, and thus, may serve as targets for potential therapeutic interventions. PMID:28165027

  16. Infrared spectroscopic characterization of monocytic microvesicles (microparticles) released upon lipopolysaccharide stimulation.

    PubMed

    Lee, Joonsup; Wen, Beryl; Carter, Elizabeth A; Combes, Valery; Grau, Georges E R; Lay, Peter A

    2017-03-17

    Microvesicles (MVs) are involved in cell-cell interactions, including disease pathogenesis. Nondestructive Fourier-transform infrared (FTIR) spectra from MVs were assessed as a technique to provide new biochemical insights into a LPS-induced monocyte model of septic shock. FTIR spectroscopy provided a quick method to investigate relative differences in biomolecular content of different MV populations that was complementary to traditional semiquantitative omics approaches, with which it is difficult to provide information on relative changes between classes (proteins, lipids, nucleic acids, carbohydrates) or protein conformations. Time-dependent changes were detected in biomolecular contents of MVs and in the monocytes from which they were released. Differences in phosphatidylcholine and phosphatidylserine contents were observed in MVs released under stimulation, and higher relative concentrations of RNA and α-helical structured proteins were present in stimulated MVs compared with MVs from resting cells. FTIR spectra of stimulated monocytes displayed changes that were consistent with those observed in the corresponding MVs they released. LPS-stimulated monocytes had reduced concentrations of nucleic acids, α-helical structured proteins, and phosphatidylcholine compared with resting monocytes but had an increase in total lipids. FTIR spectra of MV biomolecular content will be important in shedding new light on the mechanisms of MVs and the different roles they play in physiology and disease pathogenesis.-Lee, J., Wen, B., Carter, E. A., Combes, V., Grau, G. E. R., Lay. P. A. Infrared spectroscopic characterization of monocytic microvesicles (microparticles) released upon lipopolysaccharide stimulation.

  17. Extracellular microvesicle microRNAs in children with sickle cell anaemia with divergent clinical phenotypes.

    PubMed

    Khalyfa, Abdelnaby; Khalyfa, Ahamed A; Akbarpour, Mahzad; Connes, Phillippe; Romana, Marc; Lapping-Carr, Gabrielle; Zhang, Chunling; Andrade, Jorge; Gozal, David

    2016-09-01

    Sickle cell anaemia (SCA) is the most frequent genetic haemoglobinopathy, which exhibits a highly variable clinical course characterized by hyper-coagulable and pro-inflammatory states, as well as endothelial dysfunction. Extracellular microvesicles are released into biological fluids and play a role in modifying the functional phenotype of target cells. We hypothesized that potential differences in plasma-derived extracellular microvesicles (EV) function and cargo from SCA patients may underlie divergent clinical trajectories. Plasma EV from SCA patients with mild, intermediate and severe clinical disease course were isolated, and primary endothelial cell cultures were exposed. Endothelial cell activation, monocyte adhesion, barrier disruption and exosome cargo (microRNA microarrays) were assessed. EV disrupted the endothelial barrier and induced expression of adhesion molecules and monocyte adhesion in a SCA severity-dependent manner compared to healthy children. Microarray approaches identified a restricted signature of exosomal microRNAs that readily distinguished severe from mild SCA, as well as from healthy children. The microRNA candidates were further validated using quantitative real time polymerase chain reaction assays, and revealed putative gene targets. Circulating exosomal microRNAs may play important roles in predicting the clinical course of SCA, and in delineation of individually tailored, mechanistically-based clinical treatment approaches of SCA patients in the near future.

  18. Microvesicles Produced by Poly(dimethylsiloxane-b-ferrocenylsilane) Block Copolymers in Selective Solvents

    NASA Astrophysics Data System (ADS)

    Frankowski, David; Power-Billard, Nicole; Raez, Jose; Khan, Saad; Manners, Ian; Spontak, Richard

    2004-03-01

    The formation of nanoscopic aggregates via the self-assembly of block copolymers in a selective solvent for one of the blocks is currently a flourishing area of polymer and materials science, and shows tremendous potential for encapsulation applications. In this work, we use transmission electron microscopy (TEM) to investigate the self-organized morphologies of poly(dimethylsiloxaneb-ferrocenylsilane) (PDMS-b-PFS) diblock copolymers in the presence of a selective solvent. In aqueous medium, one copolymer containing a hydrophilic cationic polyferrocene block organizes into microvesicles with a diameter of ˜85 nm in which the organometallic block is located on both the outside and the inside of the aggregates. Cyclic voltammetry confirms that the ferrocene units are subject to sequential oxidation and reduction, in which case these nanoscopic aggregates are considered redox-active. Exposure of a highly asymmetric PDMS-b-PFS copolymer capable of forming nanotubes to high-pressure CO2, a selective solvent for the PDMS block, results in the gradual breakup of the nanotubes and the concurrent formation of micelles. The size of these micelles is sensitive to CO2 pressure and saturation time. At elevated temperatures, microvesicles measuring ˜63 nm in diameter develop.

  19. Structural Phenotyping of Stem Cell-Derived Cardiomyocytes

    PubMed Central

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

    2015-01-01

    Summary Structural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes. PMID:25733020

  20. Application of the PAMONO-Sensor for Quantification of Microvesicles and Determination of Nano-Particle Size Distribution

    PubMed Central

    Shpacovitch, Victoria; Sidorenko, Irina; Lenssen, Jan Eric; Temchura, Vladimir; Weichert, Frank; Müller, Heinrich; Überla, Klaus; Zybin, Alexander; Schramm, Alexander; Hergenröder, Roland

    2017-01-01

    The PAMONO-sensor (plasmon assisted microscopy of nano-objects) demonstrated an ability to detect and quantify individual viruses and virus-like particles. However, another group of biological vesicles—microvesicles (100–1000 nm)—also attracts growing interest as biomarkers of different pathologies and needs development of novel techniques for characterization. This work shows the applicability of a PAMONO-sensor for selective detection of microvesicles in aquatic samples. The sensor permits comparison of relative concentrations of microvesicles between samples. We also study a possibility of repeated use of a sensor chip after elution of the microvesicle capturing layer. Moreover, we improve the detection features of the PAMONO-sensor. The detection process utilizes novel machine learning techniques on the sensor image data to estimate particle size distributions of nano-particles in polydisperse samples. Altogether, our findings expand analytical features and the application field of the PAMONO-sensor. They can also serve for a maturation of diagnostic tools based on the PAMONO-sensor platform. PMID:28134825

  1. Application of the PAMONO-Sensor for Quantification of Microvesicles and Determination of Nano-Particle Size Distribution.

    PubMed

    Shpacovitch, Victoria; Sidorenko, Irina; Lenssen, Jan Eric; Temchura, Vladimir; Weichert, Frank; Müller, Heinrich; Überla, Klaus; Zybin, Alexander; Schramm, Alexander; Hergenröder, Roland

    2017-01-27

    The PAMONO-sensor (plasmon assisted microscopy of nano-objects) demonstrated an ability to detect and quantify individual viruses and virus-like particles. However, another group of biological vesicles-microvesicles (100-1000 nm)-also attracts growing interest as biomarkers of different pathologies and needs development of novel techniques for characterization. This work shows the applicability of a PAMONO-sensor for selective detection of microvesicles in aquatic samples. The sensor permits comparison of relative concentrations of microvesicles between samples. We also study a possibility of repeated use of a sensor chip after elution of the microvesicle capturing layer. Moreover, we improve the detection features of the PAMONO-sensor. The detection process utilizes novel machine learning techniques on the sensor image data to estimate particle size distributions of nano-particles in polydisperse samples. Altogether, our findings expand analytical features and the application field of the PAMONO-sensor. They can also serve for a maturation of diagnostic tools based on the PAMONO-sensor platform.

  2. Initial evidence that blood-borne microvesicles are biomarkers for recurrence and survival in newly diagnosed glioblastoma patients.

    PubMed

    Evans, Sydney M; Putt, Mary; Yang, Xiang-Yang; Lustig, Robert A; Martinez-Lage, Maria; Williams, Dewight; Desai, Arati; Wolf, Ronald; Brem, Steven; Koch, Cameron J

    2016-04-01

    The purpose of this pilot study was to determine whether blood-borne microvesicles from newly diagnosed glioblastoma patients could be used as biomarkers. We collected 2.8 mL blood from 16 post-operative patients at the time that they were being simulated for chemoradiation therapy (radiation with concurrent temozolomide). Two additional samples were collected during chemoradiation therapy and a final sample was collected at the end of chemoradiation therapy. Patients continued with the therapy suggested by their physicians, based on tumor conference consensus and were followed for recurrence and overall survival. Microvesicles were isolated using serial centrifugation and stained for surface markers (Annexin V for phosphotidyl serine, CD41 for platelets, anti-EGFR for tumor cells, and CD235 for red blood cells). Flow cytometry analysis was performed. Our findings provide initial evidence that increases in Annexin V positive microvesicle levels during chemoradiation therapy are associated with earlier recurrence and shorter overall survival in newly diagnosed glioblastoma patients. The effect is dramatic, with over a four-fold increase in the hazard ratio for an individual at the 75th versus the 25th percentile. Moreover the pattern of Annexin V positive microvesicles remain significant after adjustment for confounding clinical variables that have previously been shown to be prognostic for recurrence and survival. Inclusion of neutrophil levels at the start of chemoradiation therapy in the model yielded the largest attenuation of the observed association. Further studies will be needed to verify and further investigate the association between these two entities.

  3. Quality Metrics for Stem Cell-Derived Cardiac Myocytes

    PubMed Central

    Sheehy, Sean P.; Pasqualini, Francesco; Grosberg, Anna; Park, Sung Jin; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

    2014-01-01

    Summary Advances in stem cell manufacturing methods have made it possible to produce stem cell-derived cardiac myocytes at industrial scales for in vitro muscle physiology research purposes. Although FDA-mandated quality assurance metrics address safety issues in the manufacture of stem cell-based products, no standardized guidelines currently exist for the evaluation of stem cell-derived myocyte functionality. As a result, it is unclear whether the various stem cell-derived myocyte cell lines on the market perform similarly, or whether any of them accurately recapitulate the characteristics of native cardiac myocytes. We propose a multiparametric quality assessment rubric in which genetic, structural, electrophysiological, and contractile measurements are coupled with comparison against values for these measurements that are representative of the ventricular myocyte phenotype. We demonstrated this procedure using commercially available, mass-produced murine embryonic stem cell- and induced pluripotent stem cell-derived myocytes compared with a neonatal mouse ventricular myocyte target phenotype in coupled in vitro assays. PMID:24672752

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

    PubMed Central

    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. PMID:27597941

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

    PubMed

    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.

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

    PubMed

    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.

  7. Noncoding RNAs in Cancer Immunology.

    PubMed

    Li, Qian; Liu, Qiang

    2016-01-01

    Cancer immunology is the study of interaction between cancer cells and immune system by the application of immunology principle and theory. With the recent approval of several new drugs targeting immune checkpoints in cancer, cancer immunology has become a very attractive field of research and is thought to be the new hope to conquer cancer. This chapter introduces the aberrant expression and function of noncoding RNAs, mainly microRNAs and long noncoding RNAs, in tumor-infiltrating immune cells, and their significance in tumor immunity. It also illustrates how noncoding RNAs are shuttled between tumor cells and immune cells in tumor microenvironments via exosomes or other microvesicles to modulate tumor immunity.

  8. Application of nano-LC-MALDI-TOF/TOF-MS for proteomic analysis of microvesicles.

    PubMed

    Kasprzyk, Joanna; Stępień, Ewa; Piekoszewski, Wojciech

    2017-03-01

    Activated platelets and platelet derived microvesicles (PMVs) emerged recently to be promising biomarkers. There is no universal procedure to carry out the proteomic analysis on microvesicles. In this study we proposed a nano-liquid chromatography (nano-LC) technique coupled off-line with a spectrometric measurement MALDI-TOF-MS/MS as a throughput and time-saving procedure. In this study we developed a simplified method to evaluate the protein composition of platelet organelles and PMVs. Platelet-rich plasma (PRP) was collected from healthy donors. PMVs were generated from washed and thrombin activated platelets. Activated platelets from every donor were used to compare the PMV proteome. Enzymatic digestion of protein lysate was carried out using Filter Aided Sample Preparation (FASP) method with trypsin as a proteolytic enzyme. Tryptic peptides derived from PMVs and activated platelets were analysed using nano-LC coupled off-line mode with a MALDI-TOF/TOF-MS. PMV and platelet protein identification was performed using the Mascot engine for searching against the Swiss-Prot human database. The precision tolerance was 100ppm for peptide masses and 0.7Da for fragment ion masses. Individual peptide matches with a score above 28 were considered statistically significant. In total, 446 proteins were identified in PMVs and 513 proteins in activated platelets. Among them 190 were specific for activated platelets and 123 were PMV specific. Cellular component analysis of identified proteins revealed that PMVs contained relatively more extracellular proteins than activated platelets (9.6 vs. 6.0 %) and unique synaptic proteins (0.3%). A new simplified bottom-up method for PMV proteome analysis allowed eliminating the drawbacks of the previously used protocols. This approach can be used in PMV proteome identification.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26344757

  10. Scaling and automation of a high-throughput single-cell-derived tumor sphere assay chip.

    PubMed

    Cheng, Yu-Heng; Chen, Yu-Chih; Brien, Riley; Yoon, Euisik

    2016-10-07

    Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.

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

    PubMed

    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.

  12. Electrophysiological Properties of Embryonic Stem Cell-Derived Neurons

    PubMed Central

    Risner-Janiczek, Jessica R.; Ungless, Mark A.; Li, Meng

    2011-01-01

    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. PMID:21887381

  13. Hypoxic tumor-derived microvesicles negatively regulate NK cell function by a mechanism involving TGF-β and miR23a transfer

    PubMed Central

    Berchem, Guy; Noman, Muhammad Zaeem; Bosseler, Manon; Paggetti, Jerome; Baconnais, Sonia; Le cam, Eric; Nanbakhsh, Arash; Moussay, Etienne; Mami-Chouaib, Fathia; Janji, Bassam; Chouaib, Salem

    2016-01-01

    ABSTRACT Tumor-derived microvesicles (TD-MVs) are key mediators which are shed by cancer cells and can sensitize neighboring cells in the tumor microenvironment. TD-MVs are extracellular vesicles composed of exosomes and MVs and promote cancer invasion and metastasis. Intratumoral hypoxia is an integral component of all solid tumors. The relationship between hypoxic tumor-shed MVs and NK-mediated cytotoxicity remains unknown. In this paper, we reported that MVs derived from hypoxic tumor cells qualitatively differ from those derived from normoxic tumor cells. Using multiple tumor models, we showed that hypoxic MVs inhibit more NK cell function as compared to normoxic MVs. Hypoxic TD-MVs package two immunosuppressive factors involved in the impairment of natural killer (NK) cell cytotoxicity against different tumor cells in vitro and in vivo. We showed that following their uptake by NK cells, hypoxic TD-MVs transfer TGF-β1 to NK cells, decreasing the cell surface expression of the activating receptor NKG2D, thereby inhibiting NK cell function. MicroRNA profiling revealed the presence of high levels of miR-210 and miR-23a in hypoxic TD-MVs. We demonstrated that miR-23a in hypoxic TD-MVs operates as an additional immunomosuppressive factor, since it directly targets the expression of CD107a in NK cells. To our knowledge, this is the first study to show that hypoxic tumor cells by secreting MVs can educate NK cells and decrease their antitumor immune response. This study highlights the existence of a novel mechanism of immune suppression mediated by hypoxic TD-MVs and further improves our understanding of the immunosuppressive mechanisms prevailing in the hypoxic tumor microenvironment. PMID:27141372

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

    PubMed

    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.

  15. Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment*

    PubMed Central

    Liang, Yaxuan; Eng, William S.; Colquhoun, David R.; Dinglasan, Rhoel R.; Graham, David R.; Mahal, Lara K.

    2014-01-01

    Exosomes, also known as microvesicles (EMVs), are nano-sized membranous particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell-cell signaling, immune activation, and suppression and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein composition, which can dictate pathogenicity. Although some mechanisms have been proposed for the regulation of EMV protein trafficking, little attention has been paid to N-linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from that of the parent cell membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we further explore the role of glycosylation in EMV protein trafficking. We identify EMV glycoproteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Furthermore, we show that genetic manipulation of the glycosylation levels of a specific EMV glycoprotein, EWI-2, directly impacts its recruitment as a function of N-linked glycan sites. Taken together, our data provide strong evidence that N-linked glycosylation directs glycoprotein sorting into EMVs. PMID:25261472

  16. Complex N-linked glycans serve as a determinant for exosome/microvesicle cargo recruitment.

    PubMed

    Liang, Yaxuan; Eng, William S; Colquhoun, David R; Dinglasan, Rhoel R; Graham, David R; Mahal, Lara K

    2014-11-21

    Exosomes, also known as microvesicles (EMVs), are nano-sized membranous particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell-cell signaling, immune activation, and suppression and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein composition, which can dictate pathogenicity. Although some mechanisms have been proposed for the regulation of EMV protein trafficking, little attention has been paid to N-linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from that of the parent cell membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we further explore the role of glycosylation in EMV protein trafficking. We identify EMV glycoproteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Furthermore, we show that genetic manipulation of the glycosylation levels of a specific EMV glycoprotein, EWI-2, directly impacts its recruitment as a function of N-linked glycan sites. Taken together, our data provide strong evidence that N-linked glycosylation directs glycoprotein sorting into EMVs.

  17. Microvesicles released from Giardia intestinalis disturb host-pathogen response in vitro.

    PubMed

    Evans-Osses, Ingrid; Mojoli, Andres; Monguió-Tortajada, Marta; Marcilla, Antonio; Aran, Veronica; Amorim, Maria; Inal, Jameel; Borràs, Francesc E; Ramirez, Marcel I

    2017-03-01

    Giardia intestinalis (G.I), is an anaerobic protozoan and the aetiological agent of giardiasis, a diarrhoea present worldwide and associated with poverty. G.I has a simple life cycle alternating between cyst and trophozoite. Cysts are transmitted orally to the stomach and transform to trophozoites in the intestine by a multifactorial process. Recently, microvesicles (MVs) have been found to be released from a wide range of eukaryotic cells. We have observed a release of MVs during the life cycle of G.I., identifying MVs from active trophozoites and from trophozoites differentiating to the cyst form. The aim of the current work was to investigate the role of MVs from G.I in the pathogenesis of giardiasis. MVs from log phase were able to increase the attachment of G. intestinalis trophozoites to Caco-2 cells. Moreover, MVs from G. intestinalis could be captured by human immature dendritic cells, resulting in increased activation and allostimulation of human dendritic cells. Lipid rafts participate in the MV biogenesis and in the attachment to Caco-2 cells. Nevertheless, proteomic analysis from two types of MVs has shown slight differences at the protein levels. An understanding of biogenesis and content of MVs derived from trophozoites might have important implications in the pathogenesis of the disease.

  18. Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients.

    PubMed

    Muntión, Sandra; Ramos, Teresa L; Diez-Campelo, María; Rosón, Beatriz; Sánchez-Abarca, Luis Ignacio; Misiewicz-Krzeminska, Irena; Preciado, Silvia; Sarasquete, María-Eugenia; de Las Rivas, Javier; González, Marcos; Sánchez-Guijo, Fermín; Del Cañizo, María-Consuelo

    2016-01-01

    Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34+ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34+ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34+ cells as well as viability and clonogenic assays of CD34+ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34+ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34+ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties.

  19. MicroRNA biomarkers of Parkinson's disease in serum exosome-like microvesicles.

    PubMed

    Cao, Xiang-Yang; Lu, Jing-Min; Zhao, Zhi-Qiang; Li, Ming-Chao; Lu, Ting; An, Xu-Sheng; Xue, Liu-Jun

    2017-02-20

    Parkinson's disease (PD) is a progressive age-related debilitating motor disorder and the second most common neurodegenerative disease after Alzheimer's disease. In this study, we aimed to investigate the expression of 24 candidate miRNAs in PD and to assess their diagnostic value in patients with PD. We collected serum samples from 109 patients with PD and 40 age- and sex-matched healthy volunteers (control group). RNAs encapsulated in exosome-like microvesicles in serum were extracted and reverse transcribed. Serum miRNAs were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the ability of the miRNAs to accurately discriminate PD was analyzed by receiver operating characteristic curves. Based on our analysis, we further validated the downregulation of miR-19b and the upregulation of miR-195 and miR-24 in patients with PD. When compared with the control group, the area under the curve (AUC) values for miR-19b, miR-24, and miR-195 were 0.753, 0.908, and 0.697, respectively. Therefore, analysis of the expression levels of miR-19b, miR-24, and miR-195 in serum may be useful for the diagnosis of PD.

  20. Proteinase 3 Is a Phosphatidylserine-binding Protein That Affects the Production and Function of Microvesicles.

    PubMed

    Martin, Katherine R; Kantari-Mimoun, Chahrazade; Yin, Min; Pederzoli-Ribeil, Magali; Angelot-Delettre, Fanny; Ceroi, Adam; Grauffel, Cédric; Benhamou, Marc; Reuter, Nathalie; Saas, Philippe; Frachet, Philippe; Boulanger, Chantal M; Witko-Sarsat, Véronique

    2016-05-13

    Proteinase 3 (PR3), the autoantigen in granulomatosis with polyangiitis, is expressed at the plasma membrane of resting neutrophils, and this membrane expression increases during both activation and apoptosis. Using surface plasmon resonance and protein-lipid overlay assays, this study demonstrates that PR3 is a phosphatidylserine-binding protein and this interaction is dependent on the hydrophobic patch responsible for membrane anchorage. Molecular simulations suggest that PR3 interacts with phosphatidylserine via a small number of amino acids, which engage in long lasting interactions with the lipid heads. As phosphatidylserine is a major component of microvesicles (MVs), this study also examined the consequences of this interaction on MV production and function. PR3-expressing cells produced significantly fewer MVs during both activation and apoptosis, and this reduction was dependent on the ability of PR3 to associate with the membrane as mutating the hydrophobic patch restored MV production. Functionally, activation-evoked MVs from PR3-expressing cells induced a significantly larger respiratory burst in human neutrophils compared with control MVs. Conversely, MVs generated during apoptosis inhibited the basal respiratory burst in human neutrophils, and those generated from PR3-expressing cells hampered this inhibition. Given that membrane expression of PR3 is increased in patients with granulomatosis with polyangiitis, MVs generated from neutrophils expressing membrane PR3 may potentiate oxidative damage of endothelial cells and promote the systemic inflammation observed in this disease.

  1. Microvesicles and diabetic complications — novel mediators, potential biomarkers and therapeutic targets

    PubMed Central

    Wang, Ying; Chen, Li-ming; Liu, Ming-lin

    2014-01-01

    Microvesicles (MVs), also known as microparticles, are small membrane vesicles released from different cell types under different conditions. MVs have been detected in the circulation and in organs/tissues in various diseases, including diabetes. Patients with different types of diabetes and complications have different cellular MV patterns. Studies have shown that MVs may mediate vascular thrombosis, vascular inflammation, angiogenesis, and other pathological processes of the disease through their procoagulant, pro-inflammatory, pro-angiogenic, proteolytic, and other properties. Therefore, MVs contribute to the development of diabetic macrovascular and microvascular complications. In addition, clinical studies have indicated that changes in MV number and composition may reflect the pathophysiological conditions of disease, and therefore, may serve as potential biomarkers for diagnostic and prognostic use. Understanding MVs' involvement in the pathophysiological conditions may provide insight into disease mechanisms and would also be helpful for the development of novel therapeutic strategies in the future. Here, we review the latest publications from our group and other groups and focus on the involvement of MVs in diabetic complications. PMID:24608676

  2. Microvesicles from Mesenchymal Stromal Cells Are Involved in HPC-Microenvironment Crosstalk in Myelodysplastic Patients

    PubMed Central

    Muntión, Sandra; Ramos, Teresa L.; Diez-Campelo, María; Rosón, Beatriz; Sánchez-Abarca, Luis Ignacio; Misiewicz-Krzeminska, Irena; Preciado, Silvia; Sarasquete, María-Eugenia; de las Rivas, Javier; González, Marcos; Sánchez-Guijo, Fermín; del Cañizo, María-Consuelo

    2016-01-01

    Exosomes/microvesicles (MVs) provide a mechanism of intercellular communication. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodysplastic syndrome (MDS) patients could modify CD34+ cells properties by MVs. They were isolated from MSC from MDS patients and healthy donors (HD). MVs from 30 low-risk MDS patients and 27 HD were purified by ExoQuick-TC™ or ultracentrifugation and identified by transmission electron microscopy, flow cytometry (FC) and western blot for CD63. Incorporation of MVs into CD34+ cells was analyzed by FC, and confocal and fluorescence microscopy. Changes in hematopoietic progenitor cell (HPC) properties were assessed from modifications in microRNAs and gene expression in CD34+ cells as well as viability and clonogenic assays of CD34+ cells after MVs incorporation. Some microRNAs were overexpressed in MVs from patients MSC and two of them, miR-10a and miR-15a, were confirmed by RT-PCR. These microRNAs were transferred to CD34+ cells, modifying the expression of MDM2 and P53 genes, which was evaluated by RT-PCR and western blot. Finally, examining CD34+ cells properties after incorporation, higher cell viability (p = 0.025) and clonogenic capacity (p = 0.037) were observed when MVs from MDS patients were incorporated. In summary, we show that BM-MSC release MVs with a different cargo in MDS patients compared with HD. These structures are incorporated into HPC and modify their properties. PMID:26836120

  3. Characterization of Induced Pluripotent Stem Cell Microvesicle Genesis, Morphology and Pluripotent Content

    PubMed Central

    Zhou, Jing; Ghoroghi, Shima; Benito-Martin, Alberto; Wu, Hao; Unachukwu, Uchenna John; Einbond, Linda Saxe; Guariglia, Sara; Peinado, Hector; Redenti, Stephen

    2016-01-01

    Microvesicles (MVs) are lipid bilayer-covered cell fragments that range in diameter from 30 nm–1uM and are released from all cell types. An increasing number of studies reveal that MVs contain microRNA, mRNA and protein that can be detected in the extracellular space. In this study, we characterized induced pluripotent stem cell (iPSC) MV genesis, content and fusion to retinal progenitor cells (RPCs) in vitro. Nanoparticle tracking revealed that iPSCs released approximately 2200 MVs cell/hour in the first 12 hrs with an average diameter of 122 nm. Electron and light microscopic analysis of iPSCs showed MV release via lipid bilayer budding. The mRNA content of iPSC MVs was characterized and revealed the presence of the transcription factors Oct-3/4, Nanog, Klf4, and C-Myc. The protein content of iPSCs MVs, detected by immunogold electron microscopy, revealed the presence of the Oct-3/4 and Nanog. Isolated iPSC MVs were shown to fuse with RPCs in vitro at multiple points along the plasma membrane. These findings demonstrate that the mRNA and protein cargo in iPSC MVs have established roles in maintenance of pluripotency. Building on this work, iPSC derived MVs may be shown to be involved in maintaining cellular pluripotency and may have application in regenerative strategies for neural tissue. PMID:26797168

  4. Ewing sarcoma cells secrete EWS/Fli-1 fusion mRNA via microvesicles.

    PubMed

    Tsugita, Masanori; Yamada, Nami; Noguchi, Shunsuke; Yamada, Kazunari; Moritake, Hiroshi; Shimizu, Katsuji; Akao, Yukihiro; Ohno, Takatoshi

    2013-01-01

    Tumours defined as Ewing sarcoma (ES) constitute a group of highly malignant neoplasms that most often affect children and young adults in the first 2 decades of life. The EWS/Fli-1 fusion gene, a product of the translocation t(11;22) (q24; 12), is detected in 95% of ES patients. Recently, it was validated that cells emit a heterogeneous mixture of vesicular, organelle-like structures (microvesicles, MVs) into their surroundings including blood and body fluids, and that these MVs contain a selected set of tumor-related proteins and high levels of mRNAs and miRNAs. In this present study, we detected the Ewing sarcoma-specific EWS/Fli-1 mRNA in MVs from the culture medium of ES cell lines carrying t(11;22) (q24; 12). Also, we detected this fusion gene in approximately 40% of the blood samples from mice inoculated with xenografts of TC135 or A673 cells. These findings indicate the EWS/Fli-1 mRNA in MVs might be a new non-invasive diagnostic marker for specific cases of Ewing sarcoma.

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

    SciTech Connect

    Guescini, M.; Leo, G.; Genedani, S.; Carone, C.; Pederzoli, F.; Ciruela, F.; Guidolin, D.; Stocchi, V.; Mantuano, M.; Borroto-Escuela, D.O.; Fuxe, K.; Agnati, L.F.

    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.

  6. Engineering musculoskeletal tissues with human embryonic germ cell derivatives.

    PubMed

    Varghese, Shyni; Hwang, Nathaniel S; Ferran, Angela; Hillel, Alexander; Theprungsirikul, Parnduangjai; Canver, Adam C; Zhang, Zijun; Gearhart, John; Elisseeff, Jennifer

    2010-04-01

    The cells derived from differentiating embryoid bodies of human embryonic germ (hEG) cells express a broad spectrum of gene markers and have been induced toward ecto- and endodermal lineages. We describe here in vitro and in vivo differentiation of hEG-derived cells (LVEC line) toward mesenchymal tissues. The LVEC cells express many surface marker proteins characteristic of mesenchymal stem cells and differentiated into cartilage, bone, and fat. Homogenous hyaline cartilage was generated from cells after 63 population doublings. In vivo results demonstrate cell survival, differentiation, and tissue formation. The high proliferative capacity of hEG-derived cells and their ability to differentiate and form three-dimensional mesenchymal tissues without teratoma formation underscores their significant potential for regenerative medicine. The adopted coculture system also provides new insights into how a microenvironment comprised of extracellular and cellular components may be harnessed to generate hierarchically complex tissues from pluripotent cells.

  7. Functional Characterization of Human Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Kirsch, Authors Glenn E.; Obejero-Paz, Carlos A.; Bruening-Wright, Andrew

    2014-01-01

    Cardiac toxicity is a leading contributor to late-stage attrition in the drug discovery process and to withdrawal of approved from the market. In vitro assays that enable earlier and more accurate testing for cardiac risk provide early stage predictive indicators that aid in mitigating risk. Human cardiomyocytes, the most relevant subjects for early stage testing, are severely limited in supply. But human stem cell-derived cardiomyocytes (SC-hCM) are readily available from commercial sources and are increasingly used in academic research, drug discovery and safety pharmacology. As a result, SC-hCM electrophysiology has become a valuable tool to assess cardiac risk associated with drugs. This unit describes techniques for recording individual currents carried by sodium, calcium and potassium ions, as well as single cell action potentials, and impedance recordings from contracting syncytia of thousands of interconnected cells. PMID:25152802

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

    PubMed

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

    2017-01-13

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

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

    SciTech Connect

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

    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.

  10. Cartilage repair using human embryonic stem cell-derived chondroprogenitors.

    PubMed

    Cheng, Aixin; Kapacee, Zoher; Peng, Jiang; Lu, Shibi; Lucas, Robert J; Hardingham, Timothy E; Kimber, Susan J

    2014-11-01

    In initial work, we developed a 14-day culture protocol under potential GMP, chemically defined conditions to generate chondroprogenitors from human embryonic stem cells (hESCs). The present study was undertaken to investigate the cartilage repair capacity of these cells. The chondrogenic protocol was optimized and validated with gene expression profiling. The protocol was also applied successfully to two lines of induced pluripotent stem cells (iPSCs). Chondrogenic cells derived from hESCs were encapsulated in fibrin gel and implanted in osteochondral defects in the patella groove of nude rats, and cartilage repair was evaluated by histomorphology and immunocytochemistry. Genes associated with chondrogenesis were upregulated during the protocol, and pluripotency-related genes were downregulated. Aggregation of chondrogenic cells was accompanied by high expression of SOX9 and strong staining with Safranin O. Culture with PluriSln1 was lethal for hESCs but was tolerated by hESC chondrogenic cells, and no OCT4-positive cells were detected in hESC chondrogenic cells. iPSCs were also shown to generate chondroprogenitors in this protocol. Repaired tissue in the defect area implanted with hESC-derived chondrogenic cells was stained for collagen II with little collagen I, but negligible collagen II was observed in the fibrin-only controls. Viable human cells were detected in the repair tissue at 12 weeks. The results show that chondrogenic cells derived from hESCs, using a chemically defined culture system, when implanted in focal defects were able to promote cartilage repair. This is a first step in evaluating these cells for clinical application for the treatment of cartilage lesions.

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

    PubMed

    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.

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

    PubMed Central

    Lao, Zheng; Kelly, Catherine J.; Yang, Xiang-Yang; Jenkins, W. Timothy; Toorens, Erik; Ganguly, Tapan; Evans, Sydney M.; Koch, Cameron J.

    2015-01-01

    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. PMID:26208323

  13. Extracellular microvesicles from astrocytes contain functional glutamate transporters: regulation by protein kinase C and cell activation

    PubMed Central

    Gosselin, Romain-Daniel; Meylan, Patrick; Decosterd, Isabelle

    2013-01-01

    Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT)-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes) and because the protein kinase C (PKC) family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA) reorganizes EAAT-1 distribution and reduces functional [3H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [3H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI) is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release. PMID:24368897

  14. Intrinsic repair protects cells from pore-forming toxins by microvesicle shedding.

    PubMed

    Romero, Matthew; Keyel, Michelle; Shi, Guilan; Bhattacharjee, Pushpak; Roth, Robyn; Heuser, John E; Keyel, Peter A

    2017-02-10

    Pore-forming toxins (PFTs) are used by both the immune system and by pathogens to disrupt cell membranes. Cells attempt to repair this disruption in various ways, but the exact mechanism(s) that cells use are not fully understood, nor agreed upon. Current models for membrane repair include (1) patch formation (e.g., fusion of internal vesicles with plasma membrane defects), (2) endocytosis of the pores, and (3) shedding of the pores by blebbing from the cell membrane. In this study, we sought to determine the specific mechanism(s) that cells use to resist three different cholesterol-dependent PFTs: Streptolysin O, Perfringolysin O, and Intermedilysin. We found that all three toxins were shed from cells by blebbing from the cell membrane on extracellular microvesicles (MVs). Unique among the cells studied, we found that macrophages were 10 times more resistant to the toxins, yet they shed significantly smaller vesicles than the other cells. To examine the mechanism of shedding, we tested whether toxins with engineered defects in pore formation or oligomerization were shed. We found that oligomerization was necessary and sufficient for membrane shedding, suggesting that calcium influx and patch formation were not required for shedding. However, pore formation enhanced shedding, suggesting that calcium influx and patch formation enhance repair. In contrast, monomeric toxins were endocytosed. These data indicate that cells use two interrelated mechanisms of membrane repair: lipid-dependent MV shedding, which we term 'intrinsic repair', and patch formation by intracellular organelles. Endocytosis may act after membrane repair is complete by removing inactivated and monomeric toxins from the cell surface.Cell Death and Differentiation advance online publication, 10 February 2017; doi:10.1038/cdd.2017.11.

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

    PubMed Central

    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. PMID:23935504

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

    PubMed Central

    Wang, De-jing; Wang, Chen-meiyi; Wang, Yi-ting; Qiao, Hai; Fang, Liao-qiong; Wang, Zhi-biao

    2016-01-01

    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/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 β-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 (P<0.05). Conclusions Umbilical cord blood MVs contain many lactation-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. PMID:27885249

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

    PubMed Central

    Haraszti, Reka A.; Didiot, Marie-Cecile; Sapp, Ellen; Leszyk, John; Shaffer, Scott A.; Rockwell, Hannah E.; Gao, Fei; Narain, Niven R.; DiFiglia, Marian; Kiebish, Michael A.; Aronin, Neil; Khvorova, Anastasia

    2016-01-01

    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. PMID:27863537

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

    PubMed

    Sullivan, Robert

    2015-01-01

    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.

  19. Extracellular Microvesicle Production by Human Eosinophils Activated by “Inflammatory” Stimuli

    PubMed Central

    Akuthota, Praveen; Carmo, Lívia A. S.; Bonjour, Kennedy; Murphy, Ryann O.; Silva, Thiago P.; Gamalier, Juliana P.; Capron, Kelsey L.; Tigges, John; Toxavidis, Vasilis; Camacho, Virginia; Ghiran, Ionita; Ueki, Shigeharu; Weller, Peter F.; Melo, Rossana C. N.

    2016-01-01

    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 (MVs) outwardly 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 to 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 responses. The

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

    PubMed

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

    2011-11-01

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

  1. Large-scale generation of cell-derived nanovesicles.

    PubMed

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

    2014-10-21

    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. Large-scale generation of cell-derived nanovesicles

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

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

  3. Enriched retinal ganglion cells derived from human embryonic stem cells

    PubMed Central

    Gill, Katherine P.; Hung, Sandy S. C.; Sharov, Alexei; Lo, Camden Y.; Needham, Karina; Lidgerwood, Grace E.; Jackson, Stacey; Crombie, Duncan E.; Nayagam, Bryony A.; Cook, Anthony L.; Hewitt, Alex W.; Pébay, Alice; Wong, Raymond C. B.

    2016-01-01

    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453

  4. Mesenchymal stem cell-derived exosomes from different sources selectively promote neuritic outgrowth.

    PubMed

    Lopez-Verrilli, M A; Caviedes, A; Cabrera, A; Sandoval, S; Wyneken, U; Khoury, M

    2016-04-21

    Mesenchymal stem cells (MSCs) obtained from bone marrow (BM) have been shown to promote neuronal growth and survival. However, the comparative effects of MSCs of different sources, including menstrual MSCs (MenSCs), BM, umbilical cord and chorion stem cells on neurite outgrowth have not yet been explored. Moreover, the modulatory effects of MSCs may be mediated by paracrine mechanisms, i.e. by molecules contained in the MSC secretome that includes soluble factors and extracellular vesicles such as microvesicles and/or exosomes. The biogenesis of microvesicles, characterized by a vesicle diameter of 50 to 1000 nm, involves membrane shedding while exosomes, of 30 to 100 nm in diameter, originate in the multivesicular bodies within cells. Both vesicle types, which can be harvested from the conditioned media of cell cultures by differential centrifugation steps, regulate the function of target cells due to their molecular content of microRNA, mRNA, proteins and lipids. Here, we compared the effect of human menstrual MSCs (MenSCs) mediated by cell-cell contact, by their total secretome or by secretome-derived extracellular vesicles on neuritic outgrowth in primary neuronal cultures. The contact of MenSCs with cortical neurons inhibited neurite outgrowth while their total secretome enhanced it. The extracellular vesicle fractions showed a distinctive effect: while the exosome-enriched fraction enhanced neurite outgrowth, the microvesicle-enriched fraction displayed an inhibitory effect. When we compared exosome fractions of different human MSC sources, MenSC exosomes showed superior effects on the growth of the longest neurite in cortical neurons and had a comparable effect to BM-SC exosomes on neurite outgrowth in dorsal root ganglia neurons. Thus, the growth-stimulating effects of exosomes derived from MenSCs as well as the opposing effects of both extracellular vesicle fractions provide important information regarding the potential use of MenSCs as therapeutic

  5. Islet Endothelial Cells Derived From Mouse Embryonic Stem Cells.

    PubMed

    Jain, Neha; Lee, Eun Jung

    2016-01-01

    The islet endothelium comprises a specialized population of islet endothelial cells (IECs) expressing unique markers such as nephrin and α-1 antitrypsin (AAT) that are not found in endothelial cells in surrounding tissues. However, due to difficulties in isolating and maintaining a pure population of these cells, the information on these islet-specific cells is currently very limited. Interestingly, we have identified a large subpopulation of endothelial cells exhibiting IEC phenotype, while deriving insulin-producing cells from mouse embryonic stem cells (mESCs). These cells were identified by the uptake of low-density lipoprotein (LDL) and were successfully isolated and subsequently expanded in endothelial cell culture medium. Further analysis demonstrated that the mouse embryonic stem cell-derived endothelial cells (mESC-ECs) not only express classical endothelial markers, such as platelet endothelial cell adhesion molecule (PECAM1), thrombomodulin, intercellular adhesion molecule-1 (ICAM-1), and endothelial nitric oxide synthase (eNOS) but also IEC-specific markers such as nephrin and AAT. Moreover, mESC-ECs secrete basement membrane proteins such as collagen type IV, laminin, and fibronectin in culture and form tubular networks on a layer of Matrigel, demonstrating angiogenic activity. Further, mESC-ECs not only express eNOS, but also its eNOS expression is glucose dependent, which is another characteristic phenotype of IECs. With the ability to obtain highly purified IECs derived from pluripotent stem cells, it is possible to closely examine the function of these cells and their interaction with pancreatic β-cells during development and maturation in vitro. Further characterization of tissue-specific endothelial cell properties may enhance our ability to formulate new therapeutic angiogenic approaches for diabetes.

  6. Muse Cells Derived from Dermal Tissues Can Differentiate into Melanocytes.

    PubMed

    Tian, Ting; Zhang, Ru-Zhi; Yang, Yu-Hua; Liu, Qi; Li, Di; Pan, Xiao-Ru

    2017-02-07

    The objective of the authors has been to obtain multilineage-differentiating stress-enduring cells (Muse cells) from primary cultures of dermal fibroblasts, identify their pluripotency, and detect their ability to differentiate into melanocytes. The distribution of SSEA-3-positive cells in human scalp skin was assessed by immunohistochemistry, and the distribution of Oct4, Sox2, Nanog, and SSEA-3-positive cells was determined by immunofluorescence staining. The expression levels of Sox2, Oct4, hKlf4, and Nanog mRNAs and proteins in Muse cells were determined by reverse transcription polymerase chain reaction (RT-PCR) analyses and Western blots, respectively. These Muse cells differentiated into melanocytes in differentiation medium. The SSEA-3-positive cells were scattered in the basement membrane zone and the dermis, with comparatively more in the sebaceous glands, vascular and sweat glands, as well as the outer root sheath of hair follicles, the dermal papillae, and the hair bulbs. Muse cells, which have the ability to self-renew, were obtained from scalp dermal fibroblasts by flow cytometry sorting with an anti-SSEA-3 antibody. The results of RT-PCR, Western blot, and immunofluorescence staining showed that the expression levels of Oct4, Nanog, Sox2, and Klf4 mRNAs and proteins in Muse cells were significantly different from their parental dermal fibroblasts. Muse cells differentiated into melanocytes when cultured in melanocyte differentiation medium, and the Muse cell-derived melanocytes expressed the melanocyte-specific marker HMB45. Muse cells could be obtained by flow cytometry from primary cultures of scalp dermal fibroblasts, which possessed the ability of pluripotency and self-renewal, and could differentiate into melanocytes in vitro.

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

    PubMed Central

    Matijevic, Nena; Wang, Yao-Wei W.; Holcomb, John B.; Kozar, Rosemary; Cardenas, Jessica C.; Wade, Charles E.

    2015-01-01

    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. 1

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

    PubMed Central

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

    2017-01-01

    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. PMID

  9. Mesenchymal stromal cell derived extracellular vesicles rescue radiation damage to murine marrow hematopoietic cells

    PubMed Central

    Wen, Sicheng; Dooner, Mark; Cheng, Yan; Papa, Elaine; Del Tatto, Michael; Pereira, Mandy; Deng, Yanhui; Goldberg, Laura; Aliotta, Jason; Chatterjee, Devasis; Stewart, Connor; Carpanetto, Andrea; Collino, Federica; Bruno, Stefania; Camussi, Giovanni; Quesenberry, Peter

    2016-01-01

    Mesenchymal stromal cells (MSC) have been shown to reverse radiation damage to marrow stem cells. We have evaluated the capacity of MSC-derived extracellular vesicles (MSC-EVs) to mitigate radiation injury to marrow stem cells at 4 hours to 7 days after irradiation. Significant restoration of marrow stem cell engraftment at 4, 24 and 168 hours post-irradiation by exposure to MSC-EVs was observed at 3 weeks to 9 months after transplant and further confirmed by secondary engraftment. Intravenous injection of MSC-EVs to 500cGy exposed mice led to partial recovery of peripheral blood counts and restoration of the engraftment of marrow. The murine hematopoietic cell line, FDC-P1 exposed to 500 cGy, showed reversal of growth inhibition, DNA damage and apoptosis on exposure to murine or human MSC-EVs. Both murine and human MSC-EVs reverse radiation damage to murine marrow cells and stimulate normal murine marrow stem cell/progenitors to proliferate. A preparation with both exosomes and microvesicles was found to be superior to either microvesicles or exosomes alone. Biologic activity was seen in freshly isolated vesicles and in vesicles stored for up to 6 months in 10% DMSO at −80°C. These studies indicate that MSC-EVs can reverse radiation damage to bone marrow stem cells. PMID:27150009

  10. Modeling Doxorubicin-Induced Cardiotoxicity in Human Pluripotent Stem Cell Derived-Cardiomyocytes

    PubMed Central

    Maillet, Agnes; Tan, Kim; Chai, Xiaoran; Sadananda, Singh N.; Mehta, Ashish; Ooi, Jolene; Hayden, Michael R.; Pouladi, Mahmoud A.; Ghosh, Sujoy; Shim, Winston; Brunham, Liam R.

    2016-01-01

    Doxorubicin is a highly efficacious anti-cancer drug but causes cardiotoxicity in many patients. The mechanisms of doxorubicin-induced cardiotoxicity (DIC) remain incompletely understood. We investigated the characteristics and molecular mechanisms of DIC in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). We found that doxorubicin causes dose-dependent increases in apoptotic and necrotic cell death, reactive oxygen species production, mitochondrial dysfunction and increased intracellular calcium concentration. We characterized genome-wide changes in gene expression caused by doxorubicin using RNA-seq, as well as electrophysiological abnormalities caused by doxorubicin with multi-electrode array technology. Finally, we show that CRISPR-Cas9-mediated disruption of TOP2B, a gene implicated in DIC in mouse studies, significantly reduces the sensitivity of hPSC-CMs to doxorubicin-induced double stranded DNA breaks and cell death. These data establish a human cellular model of DIC that recapitulates many of the cardinal features of this adverse drug reaction and could enable screening for protective agents against DIC as well as assessment of genetic variants involved in doxorubicin response. PMID:27142468

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

    PubMed Central

    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

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

    PubMed

    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.

  13. Stromal cell-derived factor-1 (SDF-1) as a target in liver diseases.

    PubMed

    Liepelt, Anke; Tacke, Frank

    2016-08-01

    The chemokine stromal cell-derived factor-1 (SDF-1) or CXCL12 is constitutively expressed in healthy liver. However, its expression increases following acute or chronic liver injury. Liver sinusoidal endothelial cells (LSEC), hepatic stellate cells (HSC), and malignant hepatocytes are important sources of SDF-1/CXCL12 in liver diseases. CXCL12 is able to activate two chemokine receptors with different downstream signaling pathways, CXCR4 and CXCR7. CXCR7 expression is relevant on LSEC, while HSC, mesenchymal stem cells, and tumor cells mainly respond via CXCR4. Here, we summarize recent developments in the field of liver diseases involving this chemokine and its receptors. SDF-1-dependent signaling contributes to modulating acute liver injury and subsequent tissue regeneration. By activating HSC and recruiting mesenchymal cells from bone marrow, CXCL12 can promote liver fibrosis progression, while CXCL12-CXCR7 interactions endorse proregenerative responses in chronic injury. Moreover, the SDF-1 pathway is linked to development of hepatocellular carcinoma (HCC) by promoting tumor growth, angiogenesis, and HCC metastasis. High hepatic CXCR4 expression has been suggested as a biomarker indicating poor prognosis of HCC patients. Tumor-infiltrating myeloid-derived suppressor cells (MDSC) also express CXCR4 and migrate toward CXCL12. Thus CXCL12 inhibition might not only directly block HCC growth but also modulate the tumor microenvironment (angiogenesis, MDSC), thereby sensitizing HCC patients to conventional or emerging novel cancer therapies (e.g., sorafenib, regorafenib, nivolumab, pembrolizumab). We herein summarize the current knowledge on the complex interplay between CXCL12 and CXCR4/CXCR7 in liver diseases and discuss approaches on the therapeutic targeting of these axes in hepatitis, fibrosis, and liver cancer.

  14. Biochemical and Biologic Characterization of Exosomes and Microvesicles as Facilitators of HIV-1 Infection in Macrophages1

    PubMed Central

    Kadiu, Irena; Narayanasamy, Prabagaran; Dash, Prasanta K.; Zhang, Wei; Gendelman, Howard E.

    2013-01-01

    Exosomes and microvesicles are cell membranous sacs originating from multivesicular bodies and plasma membranes that facilitate long-distance intercellular communications. Lipidomic, proteomic and cell biologic approaches uncovered processes by which the human immunodeficiency virus type-1 (HIV-1) can use exosomes and MV to facilitate its dissemination. Macrophage MV and exosomes were isolated by immunoaffinity and sucrose cushion centrifugation and characterized by morphologic, biochemical and molecular assays. HIV-1 was “entrapped” in exosome aggregates. Robust HIV-1 replication followed infection with exosome-enhanced fractions isolated from infected cell supernatants. MV and exosomes facilitated viral infection that was affected by a range of cell surface receptors and adhesion proteins. HIV-1 readily completed its life cycle in human monocyte-derived macrophages but not in CD4 negative cells. The data support a significant role for exosomes as facilitators of viral infection. PMID:22711894

  15. Immunogold detection of L-glutamate and D-serine in small synaptic-like microvesicles in adult hippocampal astrocytes.

    PubMed

    Bergersen, L H; Morland, C; Ormel, L; Rinholm, J E; Larsson, M; Wold, J F H; Røe, A T; Stranna, A; Santello, M; Bouvier, D; Ottersen, O P; Volterra, A; Gundersen, V

    2012-07-01

    Glutamate and the N-methyl-D-aspartate receptor ligand D-serine are putative gliotransmitters. Here, we show by immunogold cytochemistry of the adult hippocampus that glutamate and D-serine accumulate in synaptic-like microvesicles (SLMVs) in the perisynaptic processes of astrocytes. The estimated concentration of fixed glutamate in the astrocytic SLMVs is comparable to that in synaptic vesicles of excitatory nerve terminals (≈ 45 and ≈ 55 mM, respectively), whereas the D-serine level is about 6 mM. The vesicles are organized in small spaced clusters located near the astrocytic plasma membrane. Endoplasmic reticulum is regularly found in close vicinity to SLMVs, suggesting that astrocytes contain functional nanodomains, where a local Ca(2+) increase can trigger release of glutamate and/or D-serine.

  16. An AP-3-dependent mechanism drives synaptic-like microvesicle biogenesis in pancreatic islet beta-cells.

    PubMed

    Suckow, Arthur T; Craige, Branch; Faundez, Victor; Cain, William J; Chessler, Steven D

    2010-07-01

    Pancreatic islet beta-cells contain synaptic-like microvesicles (SLMVs). The origin, trafficking, and role of these SLMVs are poorly understood. In neurons, synaptic vesicle (SV) biogenesis is mediated by two different cytosolic adaptor protein complexes, a ubiquitous AP-2 complex and the neuron-specific AP-3B complex. Mice lacking AP-3B subunits exhibit impaired GABAergic (inhibitory) neurotransmission and reduced neuronal vesicular GABA transporter (VGAT) content. Since beta-cell maturation and exocytotic function seem to parallel that of the inhibitory synapse, we predicted that AP-3B-associated vesicles would be present in beta-cells. Here, we test the hypothesis that AP-3B is expressed in islets and mediates beta-cell SLMV biogenesis. A secondary aim was to test whether the sedimentation properties of INS-1 beta-cell microvesicles are identical to those of bona fide SLMVs isolated from PC12 cells. Our results show that the two neuron-specific AP-3 subunits beta3B and mu3B are expressed in beta-cells, the first time these proteins have been found to be expressed outside the nervous system. We found that beta-cell SLMVs share the same sedimentation properties as PC12 SLMVs and contain SV proteins that sort specifically to AP-3B-associated vesicles in the brain. Brefeldin A, a drug that interferes with AP-3-mediated SV biogenesis, inhibits the delivery of AP-3 cargoes to beta-cell SLMVs. Consistent with a role for AP-3 in the biogenesis of GABAergic SLMV in beta-cells, INS-1 cell VGAT content decreases upon inhibition of AP-3 delta-subunit expression. Our findings suggest that beta-cells and neurons share molecules and mechanisms important for mediating the neuron-specific membrane trafficking pathways that underlie synaptic vesicle formation.

  17. Characterization and prognostic relevance of circulating microvesicles in chronic lymphocytic leukemia.

    PubMed

    De Luca, Luciana; D'Arena, Giovanni; Simeon, Vittorio; Trino, Stefania; Laurenzana, Ilaria; Caivano, Antonella; La Rocca, Francesco; Villani, Oreste; Mansueto, Giovanna; Deaglio, Silvia; Innocenti, Idanna; Laurenti, Luca; Molica, Stefano; Pietrantuono, Giuseppe; De Stradis, Angelo; Del Vecchio, Luigi; Musto, Pellegrino

    2017-06-01

    Microvescicles (MV) are shedding particles released by normal and neoplastic cells, whose levels in biological fluids highlight their potential role as disease biomarkers and therapeutic targets. By analyzing 131 newly diagnosed chronic lymphocytic leukemia (CLL), we found that the absolute number of serum CLL MV was significantly higher than in controls, in particular in advanced stages of disease. In addition, CD19 + and CD37+, B-cell derived MV, significantly correlated with high tumor burden. Absolute MV number cutoff selected by ROC analysis distinguished Rai stage 0 patients with shorter time to treatment (TTT) from those with more stable disease. Likewise, in the entire cohort, two groups of patients with different overall survival (OS) and different TTT were identified. At multivariate analysis, serum MV independently predicted for OS (along with Rai stage) and TTT (along with Rai stage, lymphocytes and CD38). In conclusion, circulating MV represent a new potential prognostic biomarker in CLL.

  18. Preclinical Studies of Induced Pluripotent Stem Cell-Derived Astrocyte Transplantation in ALS

    DTIC Science & Technology

    2012-10-01

    Pluripotent Stem Cell -Derived Astrocyte Transplantation in ALS PRINCIPAL INVESTIGATOR: Nicholas J. Maragakis, M.D...Pluripotent Stem Cell -Derived Astrocyte Transplantation in ALS 5b. GRANT NUMBER W81XWH-10-1-0520 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d...into astrocytes following transplantation. 15. SUBJECT TERMS Stem Cells , iPS cells , astrocytes, familial ALS 16. SECURITY CLASSIFICATION OF

  19. Cell-Derived Extracellular Matrix: Basic Characteristics and Current Applications in Orthopedic Tissue Engineering.

    PubMed

    Zhang, Weixiang; Zhu, Yun; Li, Jia; Guo, Quanyi; Peng, Jiang; Liu, Shichen; Yang, Jianhua; Wang, Yu

    2016-06-01

    The extracellular matrix (ECM) is a dynamic and intricate microenvironment with excellent biophysical, biomechanical, and biochemical properties, which can directly or indirectly regulate cell proliferation, adhesion, migration, and differentiation, as well as plays key roles in homeostasis and regeneration of tissues and organs. The ECM has attracted a great deal of attention with the rapid development of tissue engineering in the field of regenerative medicine. Tissue-derived ECM scaffolds (also referred to as decellularized tissues and whole organs) are considered a promising therapy for the repair of musculoskeletal defects, including those that are widely used in orthopedics, although there are a few shortcomings. Similar to tissue-derived ECM scaffolds, cell-derived ECM scaffolds also have highly advantageous biophysical and biochemical properties, in particular their ability to be produced in vitro from a number of different cell types. Furthermore, cell-derived ECM scaffolds more closely resemble native ECM microenvironments. The products of cell-derived ECM have a wide range of biomedical applications; these include reagents for cell culture substrates and biomaterials for scaffolds, hybrid scaffolds, and living cell sheet coculture systems. Although cell-derived ECM has only just begun to be investigated, it has great potential as a novel approach for cell-based tissue repair in orthopedic tissue engineering. This review summarizes and analyzes the various types of cell-derived ECM products applied in cartilage, bone, and nerve tissue engineering in vitro or in vivo and discusses future directions for investigation of cell-derived ECM.

  20. Radiation response of mesenchymal stem cells derived from bone marrow and human pluripotent stem cells.

    PubMed

    Islam, Mohammad S; Stemig, Melissa E; Takahashi, Yutaka; Hui, Susanta K

    2015-03-01

    Mesenchymal stem cells (MSCs) isolated from human pluripotent stem cells are comparable with bone marrow-derived MSCs in their function and immunophenotype. The purpose of this exploratory study was comparative evaluation of the radiation responses of mesenchymal stem cells derived from bone marrow- (BMMSCs) and from human embryonic stem cells (hESMSCs). BMMSCs and hESMSCs were irradiated at 0 Gy (control) to 16 Gy using a linear accelerator commonly used for cancer treatment. Cells were harvested immediately after irradiation, and at 1 and 5 days after irradiation. Cell cycle analysis, colony forming ability (CFU-F), differentiation ability, and expression of osteogenic-specific runt-related transcription factor 2 (RUNX2), adipogenic peroxisome proliferator-activated receptor gamma (PPARγ), oxidative stress-specific dismutase-1 (SOD1) and Glutathione peroxidase (GPX1) were analyzed. Irradiation arrested cell cycle progression in BMMSCs and hESMSCs. Colony formation ability of irradiated MSCs decreased in a dose-dependent manner. Irradiated hESMSCs showed higher adipogenic differentiation compared with BMMSCs, together with an increase in the adipogenic PPARγ expression. PPARγ expression was upregulated as early as 4 h after irradiation, along with the expression of SOD1. More than 70% downregulation was found in Wnt3A, Wnt4, Wnt 7A, Wnt10A and Wnt11 in BMMSCs, but not in hESMSCs. hESMSCs are highly proliferative but radiosensitive compared with BMMSCs. Increased PPARγ expression relative to RUNX2 and downregulation of Wnt ligands in irradiated MSCs suggest Wnt mediated the fate determination of irradiated MSCs.

  1. The Role of Tumor Cell-Derived Connective Tissue Growth Factor (CTGF/CCN2) in Pancreatic Tumor Growth

    PubMed Central

    Bennewith, Kevin L.; Huang, Xin; Ham, Christine M.; Graves, Edward E.; Erler, Janine T.; Kambham, Neeraja; Feazell, Jonathan; Yang, George P.; Koong, Albert

    2009-01-01

    Pancreatic cancer is highly aggressive and refractory to existing therapies. Connective tissue growth factor (CTGF/CCN2) is a fibrosis-related gene that is thought to play a role in pancreatic tumor progression. However, CCN2 can be expressed in a variety of cell types, and the contribution of CCN2 derived from either tumor cells or stromal cells as it affects the growth of pancreatic tumors is unknown. Using genetic inhibition of CCN2, we have discovered that CCN2 derived from tumor cells is a critical regulator of pancreatic tumor growth. Pancreatic tumor cells derived from CCN2 shRNA-expressing clones showed dramatically reduced growth in soft agar and when implanted subcutaneously. We also observed a role for CCN2 in the growth of pancreatic tumors implanted orthotopically, with tumor volume measurements obtained by PET imaging. Mechanistically, CCN2 protects cells from hypoxia-mediated apoptosis, providing an in vivo selection for tumor cells that express high levels of CCN2. We found that CCN2 expression and secretion was increased in hypoxic pancreatic tumor cells in vitro, and we observed co-localization of CCN2 and hypoxia in pancreatic tumor xenografts and clinical pancreatic adenocarcinomas. Furthermore, we found increased CCN2 staining in clinical pancreatic tumor tissue relative to stromal cells surrounding the tumor, supporting our assertion that tumor cell-derived CCN2 is important for pancreatic tumor growth. Taken together, these data improve our understanding of the mechanisms responsible for pancreatic tumor growth and progression, and also indicate that CCN2 produced by tumor cells represents a viable therapeutic target for the treatment of pancreatic cancer. PMID:19179545

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

    NASA Astrophysics Data System (ADS)

    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.

  3. Functional analysis of carboxylesterase in human induced pluripotent stem cell-derived enterocytes.

    PubMed

    Kabeya, Tomoki; Matsumura, Wakana; Iwao, Takahiro; Hosokawa, Masakiyo; Matsunaga, Tamihide

    2017-04-22

    Human carboxylesterase (CES) is a key esterase involved in the metabolism and biotransformation of drugs. Hydrolysis activity in the human small intestine is predominantly mediated by CES2A1 rather than CES1A. In drug development studies, Caco-2 cells are commonly used as a model to predict drug absorption in the human small intestine. However, the expression patterns of CES2A1 and CES1A in Caco-2 cells differ from those in the human small intestine. There are also species-specific differences in CES expression patterns between human and experimental animals. Furthermore, it is difficult to obtain primary human intestinal epithelial cells. Therefore, there is currently no system that can precisely predict features of drug absorption, such as CES-mediated metabolism, in the human intestine. To develop a novel system to evaluate intestinal pharmacokinetics, we analyzed CES expression and function in human induced pluripotent stem (iPS) cell-derived enterocytes. CES2A1 mRNA and protein levels in human iPS cell-derived enterocytes were comparable to Caco-2 cells, whereas CES1A levels were lower in human iPS cell-derived enterocytes compared with Caco-2 cells. p-nitrophenyl acetate hydrolysis in human iPS cell-derived enterocytes was significantly inhibited by the CES2A1-specific inhibitor telmisartan. Hydrolysis levels of the CES2A1-specific substrate aspirin were similar in human iPS cell-derived enterocytes and Caco-2 cells, whereas hydrolysis of the CES1A-specific substrate monoethylglycylxylidine was observed in Caco-2 cells but not in human iPS cell-derived enterocytes. These findings demonstrated that the expression and activity of CES isozymes in human iPS cell-derived enterocytes are more similar to the human small intestine compared with Caco-2 cells.

  4. Leukocyte Cell-Derived Chemotaxin 2-Associated Amyloidosis: A Recently Recognized Disease with Distinct Clinicopathologic Characteristics.

    PubMed

    Nasr, Samih H; Dogan, Ahmet; Larsen, Christopher P

    2015-11-06

    Amyloidosis derived from leukocyte cell-derived chemotaxin 2 is a recently recognized form of amyloidosis, and it has already been established as a frequent form of systemic amyloidosis in the United States, with predominant involvement of kidney and liver. The disease has a strong ethnic bias, affecting mainly Hispanics (particularly Mexicans). Additional ethnic groups prone to develop amyloidosis derived from leukocyte cell-derived chemotaxin 2 include Punjabis, First Nations people in British Columbia, and Native Americans. Most patients are elderly who present with chronic renal insufficiency and bland urinary sediment. Proteinuria is variable, being absent altogether in about one third of patients. Liver involvement is frequently an incidental finding. Amyloidosis derived from leukocyte cell-derived chemotaxin 2 deposits shows a characteristic distribution: in the kidney, there is consistent involvement of cortical interstitium, whereas in the liver, there is a preferential involvement of periportal and pericentral vein regions. Concurrent renal disease is frequent, with diabetic nephropathy and IgA nephropathy being the most common. Patient survival is excellent, likely because of the rarity of cardiac involvement, whereas renal survival is guarded, with a median renal survival of 62 months in those without concurrent renal disease. There is currently no efficacious therapy for amyloidosis derived from leukocyte cell-derived chemotaxin 2 amyloidosis. Renal transplantation seems to be a reasonable treatment for patients with advanced renal failure, although the disease may recur in the allograft. The pathogenesis of amyloidosis derived from leukocyte cell-derived chemotaxin 2 amyloidosis has not yet been elucidated. It could be a result of leukocyte cell-derived chemotaxin 2 overexpression by hepatocytes either constitutively (controlled by yet-uncharacterized genetic defects) or secondary to hepatocellular damage. It is critical not to misdiagnose amyloidosis

  5. Endometrial exosomes/microvesicles in the uterine microenvironment: a new paradigm for embryo-endometrial cross talk at implantation.

    PubMed

    Ng, York Hunt; Rome, Sophie; Jalabert, Audrey; Forterre, Alexis; Singh, Harmeet; Hincks, Cassandra L; Salamonsen, Lois A

    2013-01-01

    Exosomes are nanoparticles (∼100 nm diameter) released from cells, which can transfer small RNAs and mRNA via the extracellular environment to cells at distant sites. We hypothesised that exosomes or the slightly larger microvesicles (100-300 nm) are released from the endometrial epithelium into the uterine cavity, and that these contain specific micro (mi)RNA that could be transferred to either the trophectodermal cells of the blastocyst or to endometrial epithelial cells, to promote implantation. The aim of this study was to specifically identify and characterise exosomes/microvesicles (mv) released from endometrial epithelial cells and to determine whether exosomes/mv are present in uterine fluid. Immunostaining demonstrated that the tetraspanins, CD9 and CD63 used as cell surface markers of exosomes are present on the apical surfaces of endometrial epithelial cells in tissue sections taken across the menstrual cycle: CD63 showed cyclical regulation. Exosome/mv pellets were prepared from culture medium of endometrial epithelial cell (ECC1 cells) and from uterine fluid and its associated mucus by sequential ultracentifugation. Exosomes/mv were positively identified in all preparations by FACS and immunofluorescence staining following exosome binding to beads. Size particle analysis confirmed the predominance of particles of 50-150 nm in each of these fluids. MiRNA analysis of the ECC1 cells and their exosomes/mv demonstrated sorting of miRNA into exosomes/mv: 13 of the 227 miRNA were specific to exosomes/mv, while a further 5 were not present in these. The most abundant miRNA in exosomes/mv were hsa-miR-200c, hsa-miR-17 and hsa-miR-106a. Bioinformatic analysis showed that the exosome/mv-specific miRNAs have potential targets in biological pathways highly relevant for embryo implantation. Thus exosomes/mv containing specific miRNA are present in the microenvironment in which embryo implantation occurs and may contribute to the endometrial-embryo cross talk

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

    PubMed

    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.

  7. Microvesicles from brain-extract—treated mesenchymal stem cells improve neurological functions in a rat model of ischemic stroke

    PubMed Central

    Lee, Ji Yong; Kim, Eiru; Choi, Seong-Mi; Kim, Dong-Wook; Kim, Kwang Pyo; Lee, Insuk; Kim, Han-Soo

    2016-01-01

    Transplantation of mesenchymal stem cells (MSCs) was reported to improve functional outcomes in a rat model of ischemic stroke, and subsequent studies suggest that MSC-derived microvesicles (MVs) can replace the beneficial effects of MSCs. Here, we evaluated three different MSC-derived MVs, including MVs from untreated MSCs (MSC-MVs), MVs from MSCs treated with normal rat brain extract (NBE-MSC-MVs), and MVs from MSCs treated with stroke-injured rat brain extract (SBE-MSC-MVs), and tested their effects on ischemic brain injury induced by permanent middle cerebral artery occlusion (pMCAO) in rats. NBE-MSC-MVs and SBE-MSC-MVs had significantly greater efficacy than MSC-MVs for ameliorating ischemic brain injury with improved functional recovery. We found similar profiles of key signalling proteins in NBE-MSC-MVs and SBE-MSC-MVs, which account for their similar therapeutic efficacies. Immunohistochemical analyses suggest that brain-extract—treated MSC-MVs reduce inflammation, enhance angiogenesis, and increase endogenous neurogenesis in the rat brain. We performed mass spectrometry proteomic analyses and found that the total proteomes of brain-extract—treated MSC-MVs are highly enriched for known vesicular proteins. Notably, MSC-MV proteins upregulated by brain extracts tend to be modular for tissue repair pathways. We suggest that MSC-MV proteins stimulated by the brain microenvironment are paracrine effectors that enhance MSC therapy for stroke injury. PMID:27609711

  8. Compound K attenuates stromal cell-derived growth factor 1 (SDF-1)-induced migration of C6 glioma cells

    PubMed Central

    Kim, Hyuck; Roh, Hyo Sun; Kim, Jai Eun; Park, Sun Dong; Park, Won Hwan

    2016-01-01

    BACKGROUND/OBJECTIVES Stromal cell-derived growth factor 1 (SDF-1), also known as chemokine ligand 12, and chemokine receptor type 4 are involved in cancer cell migration. Compound K (CK), a metabolite of protopanaxadiol-type ginsenoside by gut microbiota, is reported to have therapeutic potential in cancer therapy. However, the inhibitory effect of CK on SDF-1 pathway-induced migration of glioma has not yet been established. MATERIALS/METHODS Cytotoxicity of CK in C6 glioma cells was determined using an EZ-Cytox cell viability assay kit. Cell migration was tested using the wound healing and Boyden chamber assay. Phosphorylation levels of protein kinase C (PKC)α and extracellular signal-regulated kinase (ERK) were measured by western blot assay, and matrix metallopeptidases (MMP) were measured by gelatin-zymography analysis. RESULTS CK significantly reduced the phosphorylation of PKCα and ERK1/2, expression of MMP9 and MMP2, and inhibited the migration of C6 glioma cells under SDF-1-stimulated conditions. CONCLUSIONS CK is a cell migration inhibitor that inhibits C6 glioma cell migration by regulating its downstream signaling molecules including PKCα, ERK1/2, and MMPs. PMID:27247721

  9. Two dimensional electrophysiological characterization of human pluripotent stem cell-derived cardiomyocyte system

    PubMed Central

    Zhu, Huanqi; Scharnhorst, Kelsey S.; Stieg, Adam Z.; Gimzewski, James K.; Minami, Itsunari; Nakatsuji, Norio; Nakano, Haruko; Nakano, Atsushi

    2017-01-01

    Stem cell-derived cardiomyocytes provide a promising tool for human developmental biology, regenerative therapies, disease modeling, and drug discovery. As human pluripotent stem cell-derived cardiomyocytes remain functionally fetal-type, close monitoring of electrophysiological maturation is critical for their further application to biology and translation. However, to date, electrophysiological analyses of stem cell-derived cardiomyocytes has largely been limited by biologically undefined factors including 3D nature of embryoid body, sera from animals, and the feeder cells isolated from mouse. Large variability in the aforementioned systems leads to uncontrollable and irreproducible results, making conclusive studies difficult. In this report, a chemically-defined differentiation regimen and a monolayer cell culture technique was combined with multielectrode arrays for accurate, real-time, and flexible measurement of electrophysiological parameters in translation-ready human cardiomyocytes. Consistent with their natural counterpart, amplitude and dV/dtmax of field potential progressively increased during the course of maturation. Monolayer culture allowed for the identification of pacemaking cells using the multielectrode array platform and thereby the estimation of conduction velocity, which gradually increased during the differentiation of cardiomyocytes. Thus, the electrophysiological maturation of the human pluripotent stem cell-derived cardiomyocytes in our system recapitulates in vivo development. This system provides a versatile biological tool to analyze human heart development, disease mechanisms, and the efficacy/toxicity of chemicals. PMID:28266620

  10. Maturation of Stem Cell-Derived Beta-cells Guided by the Expression of Urocortin 3

    PubMed Central

    van der Meulen, Talitha; Huising, Mark O.

    2014-01-01

    Type 1 diabetes (T1D) is a devastating disease precipitated by an autoimmune response directed at the insulin-producing beta-cells of the pancreas for which no cure exists. Stem cell-derived beta-cells show great promise for a cure as they have the potential to supply unlimited numbers of cells that could be derived from a patient's own cells, thus eliminating the need for immunosuppression. Current in vitro protocols for the differentiation of stem cell-derived beta-cells can successfully generate pancreatic endoderm cells. In diabetic rodents, such cells can differentiate further along the beta-cell lineage until they are eventually capable of restoring normoglycemia. While these observations demonstrate that stem cell-derived pancreatic endoderm has the potential to differentiate into mature, glucose-responsive beta-cells, the signals that direct differentiation and maturation from pancreatic endoderm onwards remain poorly understood. In this review, we analyze the sequence of events that culminates in the formation of beta-cells during embryonic development. and summarize how current protocols to generate beta-cells have sought to capitalize on this ontogenic template. We place particular emphasis on the current challenges and opportunities which occur in the later stages of beta-cell differentiation and maturation of transplantable stem cell-derived beta-cells. Another focus is on the question how the use of recently identified maturation markers such as urocortin 3 can be instrumental in guiding these efforts. PMID:25148370

  11. Ethical aspects of the use of stem cell derived gametes for reproduction.

    PubMed

    Mertes, Heidi; Pennings, Guido

    2010-09-01

    A lot of interest has been generated by the possibility of deriving gametes from embryonic stem cells and bone marrow stem cells. These stem cell derived gametes may become useful for research and for the treatment of infertility. In this article we consider prospectively the ethical issues that will arise if stem cell derived gametes are used in the clinic, making a distinction between concerns that only apply to embryonic stem cell derived gametes and concerns that are also relevant for gametes derived from adult stem cells. At present, it appears preferable to use non-embryonic stem cells for the derivation of gametes. Adult stem cell derived gametes do not present any problems with regard to the moral status of the human embryo, bypass the safety risks linked to SCNT and do not present any ambiguity or novel problems with regard to informed consent, psychological consequences for the child or genetic parenthood. A remaining ethical concern, however, regards the safety of the procedure in terms of the welfare of the resulting children. This should spark a thorough reflection on how far one must go to accommodate a person's wish to have a genetically related child.

  12. Molecular and in vivo characterization of cancer-propagating cells derived from MYCN-dependent medulloblastoma.

    PubMed

    Ahmad, Zai; Jasnos, Lukasz; Gil, Veronica; Howell, Louise; Hallsworth, Albert; Petrie, Kevin; Sawado, Tomoyuki; Chesler, Louis

    2015-01-01

    Medulloblastoma (MB) is the most common malignant pediatric brain tumor. While the pathways that are deregulated in MB remain to be fully characterized, amplification and/or overexpression of the MYCN gene, which is has a critical role in cerebellar development as a regulator of neural progenitor cell fate, has been identified in several MB subgroups. Phenotypically, aberrant expression of MYCN is associated with the large-cell/anaplastic MB variant, which accounts for 5-15% of cases and is associated with aggressive disease and poor clinical outcome. To better understand the role of MYCN in MB in vitro and in vivo and to aid the development of MYCN-targeted therapeutics we established tumor-derived neurosphere cell lines from the GTML (Glt1-tTA/TRE-MYCN-Luc) genetically engineered mouse model. A fraction of GTML neurospheres were found to be growth factor independent, expressed CD133 (a marker of neural stem cells), failed to differentiate upon MYCN withdrawal and were highly tumorigenic when orthotopically implanted into the cerebellum. Principal component analyzes using single cell RNA assay data suggested that the clinical candidate aurora-A kinase inhibitor MLN8237 converts GTML neurospheres to resemble non-MYCN expressors. Correlating with this, MLN8237 significantly extended the survival of mice bearing GTML MB allografts. In summary, our results demonstrate that MYCN plays a critical role in expansion and survival of aggressive MB-propagating cells, and establish GTML neurospheres as an important resource for the development of novel therapeutic strategies.

  13. Growth suppression of MCF-7 cancer cell-derived xenografts in nude mice by caveolin-1

    SciTech Connect

    Wu Ping; Wang Xiaohui; Li Fei; Qi Baoju; Zhu Hua; Liu Shuang; Cui Yeqing; Chen Jianwen

    2008-11-07

    Caveolin-1 is an essential structural constituent of caveolae membrane domains that has been implicated in mitogenic signaling and oncogenesis. However, the exact functional role of caveolin-1 still remains controversial. In this report, utilizing MCF-7 human breast adenocarcinoma cells stably transfected with caveolin-1 (MCF-7/cav-1 cells), we demonstrate that caveolin-1 expression dramatically inhibits invasion and migration of these cells. Importantly, in vivo experiments employing xenograft tumor models demonstrated that expression of caveolin-1 results in significant growth inhibition of breast tumors. Moreover, a dramatic delay in tumor progression was observed in MCF-7/cav-1 cells as compared with MCF-7 cells. Histological analysis of tumor sections demonstrated a marked decrease in the percentage of proliferating tumor cells (Ki-67 assay) along with an increase in apoptotic tumor cells (TUNEL assay) in MCF-7/cav-1-treated animals. Our current findings provide for the first time in vivo evidence that caveolin-1 can indeed function as a tumor suppressor in human breast adenocarcinoma derived from MCF-7 cells rather than as a tumor promoter.

  14. Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles.

    PubMed

    Théry, C; Boussac, M; Véron, P; Ricciardi-Castagnoli, P; Raposo, G; Garin, J; Amigorena, S

    2001-06-15

    Dendritic cells constitutively secrete a population of small (50-90 nm diameter) Ag-presenting vesicles called exosomes. When sensitized with tumor antigenic peptides, dendritic cells produce exosomes, which stimulate anti-tumor immune responses and the rejection of established tumors in mice. Using a systematic proteomic approach, we establish the first extensive protein map of a particular exosome population; 21 new exosomal proteins were thus identified. Most proteins present in exosomes are related to endocytic compartments. New exosomal residents include cytosolic proteins most likely involved in exosome biogenesis and function, mainly cytoskeleton-related (cofilin, profilin I, and elongation factor 1alpha) and intracellular membrane transport and signaling factors (such as several annexins, rab 7 and 11, rap1B, and syntenin). Importantly, we also identified a novel category of exosomal proteins related to apoptosis: thioredoxin peroxidase II, Alix, 14-3-3, and galectin-3. These findings led us to analyze possible structural relationships between exosomes and microvesicles released by apoptotic cells. We show that although they both represent secreted populations of membrane vesicles relevant to immune responses, exosomes and apoptotic vesicles are biochemically and morphologically distinct. Therefore, in addition to cytokines, dendritic cells produce a specific population of membrane vesicles, exosomes, with unique molecular composition and strong immunostimulating properties.

  15. Microfluidic fabrication of cell-derived nanovesicles as endogenous RNA carriers.

    PubMed

    Jo, Wonju; Jeong, Dayeong; Kim, Junho; Cho, Siwoo; Jang, Su Chul; Han, Chungmin; Kang, Ji Yoon; Gho, Yong Song; Park, Jaesung

    2014-04-07

    Exosomes/microvesicles are known to shuttle biological signals between cells, possibly by transferring biological signal components such as encapsulated RNAs and proteins, plasma membrane proteins, or both. Therefore exosomes are being considered for use as RNA and protein delivery vehicles for various therapeutic applications. However, living cells in nature secrete only a small number of exosomes, and procedures to collect them are complex; these complications impede their use in mass delivery of components to targeted cells. We propose a novel and efficient method that forces cells through hydrophilic microchannels to generate artificial nanovesicles. These mimetic nanovesicles contain mRNAs, intracellular proteins and plasma membrane proteins, and are shaped like cell-secreted exosomes. When recipient cells are exposed to nanovesicles from embryonic stem cells, mRNAs of Oct 3/4 and Nanog are transferred from embryonic stem cells to the target cells. This result suggests that mimetic nanovesicles can be used as vehicles to deliver RNA. This nanovesicle formation method is expected to be used in exosome research and to have applications in drug and RNA-delivery systems.

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

    PubMed Central

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

    2014-01-01

    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 most comprehensive

  17. Transgene Reactivation in Induced Pluripotent Stem Cell Derivatives and Reversion to Pluripotency of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells

    PubMed Central

    Galat, Yekaterina; Perepitchka, Mariana; Jennings, Lawrence J.; Iannaccone, Philip M.; Hendrix, Mary J.C.

    2016-01-01

    Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with nonintegrative constructs. Numerous studies, however, including those describing disease models, are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs, but in mesenchymal and endothelial iPSC derivatives, the transgenes experienced random upregulation of Nanog and c-Myc. Additionally, we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies, which use cellular products derived from iPSCs generated with retro- or lentiviruses, should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work, however, is to communicate the possibility of transgene reactivation in retro- or lenti-iPSC derivatives and the associated loss of cellular fidelity in vitro, which may impact the outcomes of disease modeling and related experimentation. PMID:27193052

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

    PubMed

    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.

  19. Membrane anchoring of the autoantigen GAD65 to microvesicles in pancreatic beta-cells by palmitoylation in the NH2-terminal domain

    PubMed Central

    1992-01-01

    Pancreatic beta-cells and gamma-aminobutyric acid (GABA)-secreting neurons both express the enzyme glutamic acid decarboxylase (GAD) which is a major target of autoantibodies associated with beta-cell destruction and impairment of GABA-ergic neurotransmitter pathways. The predominant form of GAD in pancreatic beta-cells, GAD65, is synthesized as a soluble hydrophilic molecule, which is modified to become firmly membrane anchored. Here we show by immunogold electron microscopy that GAD65 is localized to the membrane of small vesicles which are identical in size to small synaptic-like microvesicles in pancreatic beta-cells. The NH2-terminal domain of GAD65 is the site of a two-step modification, the last of which results in a firm membrane anchoring that involves posttranslational hydroxylamine sensitive palmitoylation. GAD65 can be released from the membrane by an apparent enzyme activity in islets, suggesting that the membrane anchoring step is reversible and potentially regulated. The hydrophobic modifications and consequent membrane anchoring of GAD65 to microvesicles that store its product GABA may be of functional importance and, moreover, significant for its selective role as an autoantigen. PMID:1321158

  20. Therapeutic Potential of Lung Epithelial Progenitor Cells Derived from Embryonic and Induced Pluripotent Stem Cells

    PubMed Central

    Wetsel, Rick A.; Wang, Dachun; Calame, Daniel G.

    2015-01-01

    Embryonic stem (ES) cells derived from preimplantation blastocysts and induced pluripotent stem (iPS) cells generated from somatic cell sources are pluripotent and capable of indefinite expansion in vitro. They provide a possible unlimited source of cells that could be differentiated into lung progenitor cells for potential clinical use in pulmonary regenerative medicine. Because of inherent difficulties in deriving endodermal cells from undifferentiated cell cultures, applications using lung epithelial cells derived from ES and iPS cells have lagged behind similar efforts devoted to other tissues, such as the heart and spinal cord. However, during the past several years, significant advances in culture, differentiation, and purification protocols, as well as in bioengineering methodologies, have fueled enthusiasm for the development of stem cell–based lung therapeutics. This article provides an overview of recent research achievements and discusses future technical challenges that must be met before the promise of stem cell applications for lung disease can be realized. PMID:21226612

  1. Pluripotent stem cell derived hepatocytes: using materials to define cellular differentiation and tissue engineering

    PubMed Central

    Lucendo-Villarin, B.; Rashidi, H.; Cameron, K.

    2016-01-01

    Pluripotent stem cell derived liver cells (hepatocytes) represent a promising alternative to primary tissue for biological and clinical applications. To date, most hepatocyte maintenance and differentiation systems have relied upon the use of animal derived components. This serves as a significant barrier to large scale production and application of stem cell derived hepatocytes. Recently, the use of defined biologics has overcome those limitations in two-dimensional monolayer culture. In order to improve the cell phenotype further, three-dimensional culture systems have been employed to better mimic the in vivo situation, drawing upon materials chemistry, engineering and biology. In this review we discuss efforts in the field, to differentiate pluripotent stem cells towards hepatocytes under defined conditions. PMID:27746914

  2. Pluripotent stem cell derived hepatocytes: using materials to define cellular differentiation and tissue engineering.

    PubMed

    Lucendo-Villarin, B; Rashidi, H; Cameron, K; Hay, D C

    2016-05-28

    Pluripotent stem cell derived liver cells (hepatocytes) represent a promising alternative to primary tissue for biological and clinical applications. To date, most hepatocyte maintenance and differentiation systems have relied upon the use of animal derived components. This serves as a significant barrier to large scale production and application of stem cell derived hepatocytes. Recently, the use of defined biologics has overcome those limitations in two-dimensional monolayer culture. In order to improve the cell phenotype further, three-dimensional culture systems have been employed to better mimic the in vivo situation, drawing upon materials chemistry, engineering and biology. In this review we discuss efforts in the field, to differentiate pluripotent stem cells towards hepatocytes under defined conditions.

  3. Stem cell derived interneuron transplants as a treatment for schizophrenia: preclinical validation in a rodent model

    PubMed Central

    Donegan, Jennifer J.; Tyson, Jennifer A.; Branch, Sarah Y.; Beckstead, Michael J.; Anderson, Stewart A.; Lodge, Daniel J.

    2016-01-01

    An increasing literature suggests that schizophrenia is associated with a reduction in hippocampal interneuron function. Thus, we posit that stem cell-derived interneuron transplants may be an effective therapeutic strategy to reduce hippocampal hyperactivity and attenuate behavioral deficits in schizophrenia. Here we used a dual-reporter embryonic stem cell line to generate enriched populations of parvalbumin (PV)- or somatostatin (SST)-positive interneurons, which were transplanted into the ventral hippocampus of the methylazoxymethanol (MAM) rodent model of schizophrenia. These interneuron transplants integrate within the existing circuitry, reduce hippocampal hyperactivity, and normalize aberrant dopamine neuron activity. Further, interneuron transplants alleviate behaviors that model negative and cognitive symptoms, including deficits in social interaction and cognitive inflexibility. Interestingly, PV- and SST-enriched transplants produced differential effects on behavior, with PV-enriched populations effectively normalizing all the behaviors examined. These data suggest that stem cell-derived interneuron transplants may represent a novel therapeutic strategy for schizophrenia. PMID:27480492

  4. Autonomous beating rate adaptation in human stem cell-derived cardiomyocytes

    PubMed Central

    Eng, George; Lee, Benjamin W.; Protas, Lev; Gagliardi, Mark; Brown, Kristy; Kass, Robert S.; Keller, Gordon; Robinson, Richard B.; Vunjak-Novakovic, Gordana

    2016-01-01

    The therapeutic success of human stem cell-derived cardiomyocytes critically depends on their ability to respond to and integrate with the surrounding electromechanical environment. Currently, the immaturity of human cardiomyocytes derived from stem cells limits their utility for regenerative medicine and biological research. We hypothesize that biomimetic electrical signals regulate the intrinsic beating properties of cardiomyocytes. Here we show that electrical conditioning of human stem cell-derived cardiomyocytes in three-dimensional culture promotes cardiomyocyte maturation, alters their automaticity and enhances connexin expression. Cardiomyocytes adapt their autonomous beating rate to the frequency at which they were stimulated, an effect mediated by the emergence of a rapidly depolarizing cell population, and the expression of hERG. This rate-adaptive behaviour is long lasting and transferable to the surrounding cardiomyocytes. Thus, electrical conditioning may be used to promote cardiomyocyte maturation and establish their automaticity, with implications for cell-based reduction of arrhythmia during heart regeneration. PMID:26785135

  5. Large scale production of a mammalian cell derived quadrivalent hepatitis C virus like particle vaccine.

    PubMed

    Earnest-Silveira, L; Christiansen, D; Herrmann, S; Ralph, S A; Das, S; Gowans, E J; Torresi, J

    2016-10-01

    A method for the large-scale production of a quadrivalent mammalian cell derived hepatitis C virus-like particles (HCV VLPs) is described. The HCV core E1 and E2 coding sequences of genotype 1a, 1b, 2a or 3a were co-expressed in Huh7 cell factories using a recombinant adenoviral expression system. The structural proteins self-assembled into VLPs that were purified from Huh7 cell lysates by iodixanol ultracentrifugation and Stirred cell ultrafiltration. Electron microscopy, revealed VLPs of the different genotypes that are morphologically similar. Our results show that it is possible to produce large quantities of individual HCV genotype VLPs with relative ease thus making this approach an alternative for the manufacture of a quadrivalent mammalian cell derived HCV VLP vaccine.

  6. Glucose responsive insulin production from human embryonic germ (EG) cell derivatives

    SciTech Connect

    Clark, Gregory O.; Yochem, Robert L.; Axelman, Joyce; Sheets, Timothy P.; Kaczorowski, David J.; Shamblott, Michael J. . E-mail: mshambl1@jhmi.edu

    2007-05-11

    Type 1 diabetes mellitus subjects millions to a daily burden of disease management, life threatening hypoglycemia and long-term complications such as retinopathy, nephropathy, heart disease, and stroke. Cell transplantation therapies providing a glucose-regulated supply of insulin have been implemented clinically, but are limited by safety, efficacy and supply considerations. Stem cells promise a plentiful and flexible source of cells for transplantation therapies. Here, we show that cells derived from human embryonic germ (EG) cells express markers of definitive endoderm, pancreatic and {beta}-cell development, glucose sensing, and production of mature insulin. These cells integrate functions necessary for glucose responsive regulation of preproinsulin mRNA and expression of insulin C-peptide in vitro. Following transplantation into mice, cells become insulin and C-peptide immunoreactive and produce plasma C-peptide in response to glucose. These findings suggest that EG cell derivatives may eventually serve as a source of insulin producing cells for the treatment of diabetes.

  7. Xenotransplantation of embryonic stem cell-derived motor neurons into the developing chick spinal cord.

    PubMed

    Wichterle, Hynek; Peljto, Mirza; Nedelec, Stephane

    2009-01-01

    A growing number of specific cell types have been successfully derived from embryonic stem cells (ES cells), including a variety of neural cells. In vitro generated cells need to be extensively characterized to establish functional equivalency with their in vivo counterparts. The ultimate test for the ability of ES cell-derived neurons to functionally integrate into neural networks is transplantation into the developing central nervous system, a challenging technique limited by the poor accessibility of mammalian embryos. Here we describe xenotransplantation of mouse embryonic stem cell-derived motor neurons into the developing chick neural tube as an alternative for testing the ability of in vitro generated neurons to survive, integrate, extend axons, and form appropriate synaptic contacts with functionally relevant targets in vivo. Similar methods can be adapted to study functionality of other mammalian cells, including derivatives of human ES cells.

  8. PIEZO2 is required for mechanotransduction in human stem cell-derived touch receptors.

    PubMed

    Schrenk-Siemens, Katrin; Wende, Hagen; Prato, Vincenzo; Song, Kun; Rostock, Charlotte; Loewer, Alexander; Utikal, Jochen; Lewin, Gary R; Lechner, Stefan G; Siemens, Jan

    2015-01-01

    Human sensory neurons are inaccessible for functional examination, and thus little is known about the mechanisms mediating touch sensation in humans. Here we demonstrate that the mechanosensitivity of human embryonic stem (hES) cell-derived touch receptors depends on PIEZO2. To recapitulate sensory neuron development in vitro, we established a multistep differentiation protocol and generated sensory neurons via the intermediate production of neural crest cells derived from hES cells or human induced pluripotent stem (hiPS) cells. The generated neurons express a distinct set of touch receptor-specific genes and convert mechanical stimuli into electrical signals, their most salient characteristic in vivo. Strikingly, mechanosensitivity is lost after CRISPR/Cas9-mediated PIEZO2 gene deletion. Our work establishes a model system that resembles human touch receptors, which may facilitate mechanistic analysis of other sensory subtypes and provide insight into developmental programs underlying sensory neuron diversity.

  9. Preclinical Studies of Induced Pluripotent Stem Cell-Derived Astrocyte Transplantation in ALS

    DTIC Science & Technology

    2014-12-01

    transporter GLT1, connexin 43, aquaporin 4, the cell surface marker CD44 (a marker of astrocyte precursor identity), and the intermediate filament ... print ] 2. iPSC-Derived GRPs from FUS H517Q ALS do not significantly differ from control iPSC-derived GRP lines One question is whether...Cell-Derived Astrocyte Progenitors Following Spinal Cord Engraftment. Stem Cells Transl Med. 2014 Mar 6. [Epub ahead of print ] (see Appendix) Amanda

  10. Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells.

    PubMed

    Araki, Ryoko; Uda, Masahiro; Hoki, Yuko; Sunayama, Misato; Nakamura, Miki; Ando, Shunsuke; Sugiura, Mayumi; Ideno, Hisashi; Shimada, Akemi; Nifuji, Akira; Abe, Masumi

    2013-02-07

    The advantages of using induced pluripotent stem cells (iPSCs) instead of embryonic stem (ES) cells in regenerative medicine centre around circumventing concerns about the ethics of using ES cells and the likelihood of immune rejection of ES-cell-derived tissues. However, partial reprogramming and genetic instabilities in iPSCs could elicit immune responses in transplant recipients even when iPSC-derived differentiated cells are transplanted. iPSCs are first differentiated into specific types of cells in vitro for subsequent transplantation. Although model transplantation experiments have been conducted using various iPSC-derived differentiated tissues and immune rejections have not been observed, careful investigation of the immunogenicity of iPSC-derived tissue is becoming increasingly critical, especially as this has not been the focus of most studies done so far. A recent study reported immunogenicity of iPSC- but not ES-cell-derived teratomas and implicated several causative genes. Nevertheless, some controversy has arisen regarding these findings. Here we examine the immunogenicity of differentiated skin and bone marrow tissues derived from mouse iPSCs. To ensure optimal comparison of iPSCs and ES cells, we established ten integration-free iPSC and seven ES-cell lines using an inbred mouse strain, C57BL/6. We observed no differences in the rate of success of transplantation when skin and bone marrow cells derived from iPSCs were compared with ES-cell-derived tissues. Moreover, we observed limited or no immune responses, including T-cell infiltration, for tissues derived from either iPSCs or ES cells, and no increase in the expression of the immunogenicity-causing Zg16 and Hormad1 genes in regressing skin and teratoma tissues. Our findings suggest limited immunogenicity of transplanted cells differentiated from iPSCs and ES cells.

  11. Learning-induced synaptic potentiation in implanted neural precursor cell-derived neurons

    PubMed Central

    Park, Kyungjoon; Heo, Hwon; Han, Ma Eum; Choi, Kyuhyun; Yi, Jee Hyun; Kang, Shin Jung; Kwon, Yunhee Kim; Shin, Ki Soon

    2015-01-01

    Neuronal loss caused by neurodegenerative diseases, traumatic brain injury and stroke results in cognitive dysfunctioning. Implantation of neural stem/precursor cells (NPCs) can improve the brain function by replacing lost neurons. Proper synaptic integration following neuronal differentiation of implanted cells is believed to be a prerequisite for the functional recovery. In the present study, we characterized the functional properties of immortalized neural progenitor HiB5 cells implanted into the rat hippocampus with chemically induced lesion. The implanted HiB5 cells migrated toward CA1 pyramidal layer and differentiated into vGluT1-positive glutamatergic neurons with morphological and electrophysiological properties of endogenous CA1 pyramidal cells. Functional synaptic integration of HiB5 cell-derived neurons was also evidenced by immunohistochemical and electrophysiological data. Lesion-caused memory deficit was significantly recovered after the implantation when assessed by inhibitory avoidance (IA) learning. Remarkably, IA learning preferentially produced long-term potentiation (LTP) at the synapses onto HiB5 cell-derived neurons, which occluded paring protocol-induced LTP ex vivo. We conclude that the implanted HiB5 cell-derived neurons actively participate in learning process through LTP formation, thereby counteracting lesion-mediated memory impairment. PMID:26634434

  12. Methods for Assessing the Electromechanical Integration of Human Pluripotent Stem Cell-Derived Cardiomyocyte Grafts

    PubMed Central

    Zhu, Wei-Zhong; Filice, Dominic; Palpant, Nathan J.; Laflamme, Michael A.

    2014-01-01

    Cardiomyocytes derived from human pluripotent stem cells show tremendous promise for the replacement of myocardium and contractile function lost to infarction. However, until recently, no methods were available to directly determine whether these stem cell-derived grafts actually couple with host myocardium and fire synchronously following transplantation in either intact or injured hearts. To resolve this uncertainty, our group has developed techniques for the intravital imaging of hearts engrafted with stem cell-derived cardiomyocytes that have been modified to express the genetically encoded protein calcium sensor, GCaMP. When combined with the simultaneously recorded electrocardiogram, this protocol allows one to make quantitative assessments as to the presence and extent of host–graft electrical coupling as well as the timing and pattern of graft activation. As described here, this system has been employed to investigate the electromechanical integration of human embryonic stem cell-derived cardiomyocytes in a guinea pig model of cardiac injury, but analogous approaches should be applicable to other human graft cell types and animal models. PMID:25070341

  13. Methods for assessing the electromechanical integration of human pluripotent stem cell-derived cardiomyocyte grafts.

    PubMed

    Zhu, Wei-Zhong; Filice, Dominic; Palpant, Nathan J; Laflamme, Michael A

    2014-01-01

    Cardiomyocytes derived from human pluripotent stem cells show tremendous promise for the replacement of myocardium and contractile function lost to infarction. However, until recently, no methods were available to directly determine whether these stem cell-derived grafts actually couple with host myocardium and fire synchronously following transplantation in either intact or injured hearts. To resolve this uncertainty, our group has developed techniques for the intravital imaging of hearts engrafted with stem cell-derived cardiomyocytes that have been modified to express the genetically encoded protein calcium sensor, GCaMP. When combined with the simultaneously recorded electrocardiogram, this protocol allows one to make quantitative assessments as to the presence and extent of host-graft electrical coupling as well as the timing and pattern of graft activation. As described here, this system has been employed to investigate the electromechanical integration of human embryonic stem cell-derived cardiomyocytes in a guinea pig model of cardiac injury, but analogous approaches should be applicable to other human graft cell types and animal models.

  14. Angiogenic activity mediates bone repair from human pluripotent stem cell-derived osteogenic cells

    PubMed Central

    Zou, Li; Chen, Qingshan; Quanbeck, Zachary; Bechtold, Joan E.; Kaufman, Dan S.

    2016-01-01

    Human pluripotent stem cells provide a standardized resource for bone repair. However, criteria to determine which exogenous cells best heal orthopedic injuries remain poorly defined. We evaluated osteogenic progenitor cells derived from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Phenotypic and genotypic analyses demonstrated that these hESCs/hiPSCs are similar in their osteogenic differentiation efficiency and they generate osteogenic cells comparable to osteogenic cells derived from mesenchymal stromal cells (BM-MSCs). However, expression of angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor in these osteogenic progenitor cells are markedly different, suggesting distinct pro-angiogenic potential of these stem cell derivatives. Studies to repair a femur non-union fracture demonstrate only osteogenic progenitor cells with higher pro-angiogenic potential significantly enhance bone repair in vivo. Together, these studies highlight a key role of pro-angiogenic potential of transplanted osteogenic cells for effective cell-mediated bone repair. PMID:26980556

  15. Mechanical Injury Induces Brain Endothelial-Derived Microvesicle Release: Implications for Cerebral Vascular Injury during Traumatic Brain Injury

    PubMed Central

    Andrews, Allison M.; Lutton, Evan M.; Merkel, Steven F.; Razmpour, Roshanak; Ramirez, Servio H.

    2016-01-01

    It is well established that the endothelium responds to mechanical forces induced by changes in shear stress and strain. However, our understanding of vascular remodeling following traumatic brain injury (TBI) remains incomplete. Recently published studies have revealed that lung and umbilical endothelial cells produce extracellular microvesicles (eMVs), such as microparticles, in response to changes in mechanical forces (blood flow and mechanical injury). Yet, to date, no studies have shown whether brain endothelial cells produce eMVs following TBI. The brain endothelium is highly specialized and forms the blood-brain barrier (BBB), which regulates diffusion and transport of solutes into the brain. This specialization is largely due to the presence of tight junction proteins (TJPs) between neighboring endothelial cells. Following TBI, a breakdown in tight junction complexes at the BBB leads to increased permeability, which greatly contributes to the secondary phase of injury. We have therefore tested the hypothesis that brain endothelium responds to mechanical injury, by producing eMVs that contain brain endothelial proteins, specifically TJPs. In our study, primary human adult brain microvascular endothelial cells (BMVEC) were subjected to rapid mechanical injury to simulate the abrupt endothelial disruption that can occur in the primary injury phase of TBI. eMVs were isolated from the media following injury at 2, 6, 24, and 48 h. Western blot analysis of eMVs demonstrated a time-dependent increase in TJP occludin, PECAM-1 and ICAM-1 following mechanical injury. In addition, activation of ARF6, a small GTPase linked to extracellular vesicle production, was increased after injury. To confirm these results in vivo, mice were subjected to sham surgery or TBI and blood plasma was collected 24 h post-injury. Isolation and analysis of eMVs from blood plasma using cryo-EM and flow cytometry revealed elevated levels of vesicles containing occludin following brain trauma

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

    PubMed

    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.

  17. Marrow Hematopoietic Stem Cells Revisited: They Exist in a Continuum and are Not Defined by Standard Purification Approaches; Then There are the Microvesicles

    PubMed Central

    Quesenberry, Peter J.; Goldberg, Laura; Aliotta, Jason; Dooner, Mark

    2013-01-01

    with the purification. This system, where the marrow stem cell continuously and reversibly changes with obligate cell cycle transit, is further complicated by the consideration of the impact of tissue microvesicles on the cell phenotypes. Tissue microvesicles have been found to alter the phenotype of marrow cells, possibly explaining the observations of “stem cell plasticity.” These alterations, short-term, are due to transfer of originator cell mRNA and as yet undefined transcription factors. Long-term phenotype change is due to transcriptional modulation; a stable epigenetic change. Thus, the stem cell system is characterized by continuous cycle and microvesicle-related change. The challenge of the future is to define the stem cell population. PMID:24772390

  18. The effect of PVDF-TrFE scaffolds on stem cell derived cardiovascular cells.

    PubMed

    Hitscherich, Pamela; Wu, Siliang; Gordan, Richard; Xie, Lai-Hua; Arinzeh, Treena; Lee, Eun Jung

    2016-07-01

    Recently, electrospun polyvinylidene fluoride (PVDF) and polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) scaffolds have been developed for tissue engineering applications. These materials have piezoelectric activity, wherein they can generate electric charge with minute mechanical deformations. Since the myocardium is an electroactive tissue, the unique feature of a piezoelectric scaffold is attractive for cardiovascular tissue engineering applications. In this study, we examined the cytocompatibility and function of pluripotent stem cell derived cardiovascular cells including mouse embryonic stem cell-derived cardiomyocytes (mES-CM) and endothelial cells (mES-EC) on PVDF-TrFE scaffolds. MES-CM and mES-EC adhered well to PVDF-TrFE and became highly aligned along the fibers. When cultured on scaffolds, mES-CM spontaneously contracted, exhibited well-registered sarcomeres and expressed classic cardiac specific markers such as myosin heavy chain, cardiac troponin T, and connexin43. Moreover, mES-CM cultured on PVDF-TrFE scaffolds responded to exogenous electrical pacing and exhibited intracellular calcium handling behavior similar to that of mES-CM cultured in 2D. Similar to cardiomyocytes, mES-EC also demonstrated high viability and maintained a mature phenotype through uptake of low-density lipoprotein and expression of classic endothelial cell markers including platelet endothelial cell adhesion molecule, endothelial nitric oxide synthase, and the arterial specific marker, Notch-1. This study demonstrates the feasibility of PVDF-TrFE scaffold as a candidate material for developing engineered cardiovascular tissues utilizing stem cell-derived cells. Biotechnol. Bioeng. 2016;113: 1577-1585. © 2015 Wiley Periodicals, Inc.

  19. Magnetic Resonance Imaging of Iron Oxide-Labeled Human Embryonic Stem Cell-Derived Cardiac Progenitors

    PubMed Central

    Skelton, Rhys J.P.; Khoja, Suhail; Almeida, Shone; Rapacchi, Stanislas; Han, Fei; Engel, James; Zhao, Peng; Hu, Peng; Stanley, Edouard G.; Elefanty, Andrew G.; Kwon, Murray

    2016-01-01

    Given the limited regenerative capacity of the heart, cellular therapy with stem cell-derived cardiac cells could be a potential treatment for patients with heart disease. However, reliable imaging techniques to longitudinally assess engraftment of the transplanted cells are scant. To address this issue, we used ferumoxytol as a labeling agent of human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) to facilitate tracking by magnetic resonance imaging (MRI) in a large animal model. Differentiating hESCs were exposed to ferumoxytol at different time points and varying concentrations. We determined that treatment with ferumoxytol at 300 μg/ml on day 0 of cardiac differentiation offered adequate cell viability and signal intensity for MRI detection without compromising further differentiation into definitive cardiac lineages. Labeled hESC-CPCs were transplanted by open surgical methods into the left ventricular free wall of uninjured pig hearts and imaged both ex vivo and in vivo. Comprehensive T2*-weighted images were obtained immediately after transplantation and 40 days later before termination. The localization and dispersion of labeled cells could be effectively imaged and tracked at days 0 and 40 by MRI. Thus, under the described conditions, ferumoxytol can be used as a long-term, differentiation-neutral cell-labeling agent to track transplanted hESC-CPCs in vivo using MRI. Significance The development of a safe and reproducible in vivo imaging technique to track the fate of transplanted human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) is a necessary step to clinical translation. An iron oxide nanoparticle (ferumoxytol)-based approach was used for cell labeling and subsequent in vivo magnetic resonance imaging monitoring of hESC-CPCs transplanted into uninjured pig hearts. The present results demonstrate the use of ferumoxytol labeling and imaging techniques in tracking the location and dispersion of cell grafts

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

    NASA Astrophysics Data System (ADS)

    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

  1. Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids.

    PubMed

    Tauro, Bow J; Greening, David W; Mathias, Rommel A; Mathivanan, Suresh; Ji, Hong; Simpson, Richard J

    2013-03-01

    Exosomes are naturally occurring biological nanomembranous vesicles (∼40 to 100 nm) of endocytic origin that are released from diverse cell types into the extracellular space. They have pleiotropic functions such as antigen presentation and intercellular transfer of protein cargo, mRNA, microRNA, lipids, and oncogenic potential. Here we describe the isolation, via sequential immunocapture using anti-A33- and anti-EpCAM-coupled magnetic beads, of two distinct populations of exosomes released from organoids derived from human colon carcinoma cell line LIM1863. The exosome populations (A33-Exos and EpCAM-Exos) could not be distinguished via electron microscopy and contained stereotypical exosome markers such as TSG101, Alix, and HSP70. The salient finding of this study, revealed via gel-based LC-MS/MS, was the exclusive identification in EpCAM-Exos of the classical apical trafficking molecules CD63 (LAMP3), mucin 13 and the apical intestinal enzyme sucrase isomaltase and increased expression of dipeptidyl peptidase IV and the apically restricted pentaspan membrane glycoprotein prominin 1. In contrast, the A33-Exos preparation was enriched with basolateral trafficking molecules such as early endosome antigen 1, the Golgi membrane protein ADP-ribosylation factor, and clathrin. Our observations are consistent with EpCAM- and A33-Exos being released from the apical and basolateral surfaces, respectively, and the EpCAM-Exos proteome profile with widely published stereotypical exosomes. A proteome analysis of LIM1863-derived shed microvesicles (sMVs) was also performed in order to clearly distinguish A33- and EpCAM-Exos from sMVs. Intriguingly, several members of the MHC class I family of antigen presentation molecules were exclusively observed in A33-Exos, whereas neither MHC class I nor MHC class II molecules were observed via MS in EpCAM-Exos. Additionally, we report for the first time in any extracellular vesicle study the colocalization of EpCAM, claudin-7, and CD44

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

    PubMed

    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.

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

    PubMed Central

    Boylan, Brendan T.; Moreira, Fernando R.; Carlson, Tim W.

    2017-01-01

    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. PMID:28187142

  4. Dendritic Cell-Derived Exosomes Express a Streptococcus pneumoniae Capsular Polysaccharide Type 14 Cross-Reactive Antigen That Induces Protective Immunoglobulin Responses against Pneumococcal Infection in Mice

    DTIC Science & Technology

    2007-01-01

    exosome biogenesis (9). Thus, dendritic cell (DC)-derived exo- somes are enriched in major histocompatibility complex (MHC), T-cell costimulatory and...biotinylated by activation with cyanogen bromide and coupling to biotin-LC- Hydrazide (Pierce, Rockford, IL) essentially as described by Lucas et al. (25...in the enzyme-linked immunosorbent assays (ELISAs). Detection and quantitation of tetraspan-containing microvesicles. The pres- ence of complexed

  5. Melanoma cell-derived exosomes alter macrophage and dendritic cell functions in vitro.

    PubMed

    Marton, Annamaria; Vizler, Csaba; Kusz, Erzsebet; Temesfoi, Viktoria; Szathmary, Zsuzsa; Nagy, Krisztina; Szegletes, Zsolt; Varo, Gyorgy; Siklos, Laszlo; Katona, Robert L; Tubak, Vilmos; Howard, O M Zack; Duda, Erno; Minarovits, Janos; Nagy, Katalin; Buzas, Krisztina

    2012-01-01

    To clarify controversies in the literature of the field, we have purified and characterized B16F1 melanoma cell derived exosomes (mcd-exosomes) then we attempted to dissect their immunological activities. We tested how mcd-exosomes influence CD4+ T cell proliferation induced by bone marrow derived dendritic cells; we quantified NF-κB activation in mature macrophages stimulated with mcd-exosomes, and we compared the cytokine profile of LPS-stimulated, IL-4 induced, and mcd-exosome treated macrophages. We observed that mcd-exosomes helped the maturation of dendritic cells, enhancing T cell proliferation induced by the treated dendritic cells. The exosomes also activated macrophages, as measured by NF-κB activation. The cytokine and chemokine profile of macrophages treated with tumor cell derived exosomes showed marked differences from those induced by either LPS or IL-4, and it suggested that exosomes may play a role in the tumor progression and metastasis formation through supporting tumor immune escape mechanisms.

  6. Human Cytomegalovirus Carries a Cell-Derived Phospholipase A2 Required for Infectivity

    PubMed Central

    Allal, Cuider; Buisson-Brenac, Claire; Marion, Vincent; Claudel-Renard, Clotilde; Faraut, Thomas; Dal Monte, Paola; Streblow, Daniel; Record, Michel

    2004-01-01

    Human cytomegalovirus (HCMV) is known to carry host cell-derived proteins and mRNAs whose role in cell infection is not understood. We have identified a phospholipase A2 (PLA2) activity borne by HCMV by using an assay based on the hydrolysis of fluorescent phosphatidylcholine. This activity was found in all virus strains analyzed and in purified strains. It was calcium dependent and was sensitive to inhibitors of cytosolic PLA2 (cPLA2) but not to inhibitors of soluble PLA2 or calcium-independent PLA2. No other phospholipase activity was detected in the virus. Purified virus was found to contain human cellular cPLA2α, as detected by monoclonal antibody. No homology with PLA2 was found in the genome of HCMV, indicating that HCMV does not code for a PLA2. Decreased de novo expression of immediate-early proteins 1 and 2 (IE1 and IE2), tegument phosphoprotein pp65, and virus production was observed when HCMV was treated with inhibitors of cPLA2. Cell entry of HCMV was not altered by those inhibitors, suggesting the action of cPLA2 was postentry. Together, our results indicate a selective sorting of a cell-derived cPLA2 during HCMV maturation, which is further required for infectivity. PMID:15220446

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

    SciTech Connect

    Ono, Hiromasa; Oki, Yoshinao; Bono, Hidemasa; Kano, Koichiro

    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.

  8. Decellularized Tissue and Cell-Derived Extracellular Matrices as Scaffolds for Orthopaedic Tissue Engineering

    PubMed Central

    Cheng, Christina W.; Solorio, Loran D.; Alsberg, Eben

    2014-01-01

    The reconstruction of musculoskeletal defects is a constant challenge for orthopaedic surgeons. Musculoskeletal injuries such as fractures, chondral lesions, infections and tumor debulking can often lead to large tissue voids requiring reconstruction with tissue grafts. Autografts are currently the gold standard in orthopaedic tissue reconstruction; however, there is a limit to the amount of tissue that can be harvested before compromising the donor site. Tissue engineering strategies using allogeneic or xenogeneic decellularized bone, cartilage, skeletal muscle, tendon and ligament have emerged as promising potential alternative treatment. The extracellular matrix provides a natural scaffold for cell attachment, proliferation and differentiation. Decellularization of in vitro cell-derived matrices can also enable the generation of autologous constructs from tissue specific cells or progenitor cells. Although decellularized bone tissue is widely used clinically in orthopaedic applications, the exciting potential of decellularized cartilage, skeletal muscle, tendon and ligament cell-derived matrices has only recently begun to be explored for ultimate translation to the orthopaedic clinic. PMID:24417915

  9. Partial Reprogramming of Pluripotent Stem Cell-Derived Cardiomyocytes into Neurons.

    PubMed

    Chuang, Wenpo; Sharma, Arun; Shukla, Praveen; Li, Guang; Mall, Moritz; Rajarajan, Kuppusamy; Abilez, Oscar J; Hamaguchi, Ryoko; Wu, Joseph C; Wernig, Marius; Wu, Sean M

    2017-03-22

    Direct reprogramming of somatic cells has been demonstrated, however, it is unknown whether electrophysiologically-active somatic cells derived from separate germ layers can be interconverted. We demonstrate that partial direct reprogramming of mesoderm-derived cardiomyocytes into neurons is feasible, generating cells exhibiting structural and electrophysiological properties of both cardiomyocytes and neurons. Human and mouse pluripotent stem cell-derived CMs (PSC-CMs) were transduced with the neurogenic transcription factors Brn2, Ascl1, Myt1l and NeuroD. We found that CMs adopted neuronal morphologies as early as day 3 post-transduction while still retaining a CM gene expression profile. At week 1 post-transduction, we found that reprogrammed CMs expressed neuronal markers such as Tuj1, Map2, and NCAM. At week 3 post-transduction, mature neuronal markers such as vGlut and synapsin were observed. With single-cell qPCR, we temporally examined CM gene expression and observed increased expression of neuronal markers Dcx, Map2, and Tubb3. Patch-clamp analysis confirmed the neuron-like electrophysiological profile of reprogrammed CMs. This study demonstrates that PSC-CMs are amenable to partial neuronal conversion, yielding a population of cells exhibiting features of both neurons and CMs.

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

    PubMed

    Ono, Hiromasa; Oki, Yoshinao; Bono, Hidemasa; Kano, Koichiro

    2011-04-15

    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.

  11. Partial Reprogramming of Pluripotent Stem Cell-Derived Cardiomyocytes into Neurons

    PubMed Central

    Chuang, Wenpo; Sharma, Arun; Shukla, Praveen; Li, Guang; Mall, Moritz; Rajarajan, Kuppusamy; Abilez, Oscar J.; Hamaguchi, Ryoko; Wu, Joseph C.; Wernig, Marius; Wu, Sean M.

    2017-01-01

    Direct reprogramming of somatic cells has been demonstrated, however, it is unknown whether electrophysiologically-active somatic cells derived from separate germ layers can be interconverted. We demonstrate that partial direct reprogramming of mesoderm-derived cardiomyocytes into neurons is feasible, generating cells exhibiting structural and electrophysiological properties of both cardiomyocytes and neurons. Human and mouse pluripotent stem cell-derived CMs (PSC-CMs) were transduced with the neurogenic transcription factors Brn2, Ascl1, Myt1l and NeuroD. We found that CMs adopted neuronal morphologies as early as day 3 post-transduction while still retaining a CM gene expression profile. At week 1 post-transduction, we found that reprogrammed CMs expressed neuronal markers such as Tuj1, Map2, and NCAM. At week 3 post-transduction, mature neuronal markers such as vGlut and synapsin were observed. With single-cell qPCR, we temporally examined CM gene expression and observed increased expression of neuronal markers Dcx, Map2, and Tubb3. Patch-clamp analysis confirmed the neuron-like electrophysiological profile of reprogrammed CMs. This study demonstrates that PSC-CMs are amenable to partial neuronal conversion, yielding a population of cells exhibiting features of both neurons and CMs. PMID:28327614

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

    PubMed Central

    Weick, Jason P.

    2016-01-01

    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. PMID:27274733

  13. Microcircuit formation following transplantation of mouse embryonic stem cell-derived neurons in peripheral nerve.

    PubMed

    Magown, Philippe; Rafuse, Victor F; Brownstone, Robert M

    2017-04-01

    Motoneurons derived from embryonic stem cells can be transplanted in the tibial nerve, where they extend axons to functionally innervate target muscle. Here, we studied spontaneous muscle contractions in these grafts 3 mo following transplantation. One-half of the transplanted grafts generated rhythmic muscle contractions of variable patterns, either spontaneously or in response to brief electrical stimulation. Activity generated by transplanted embryonic stem cell-derived neurons was driven by glutamate and was modulated by muscarinic and GABAergic/glycinergic transmission. Furthermore, rhythmicity was promoted by the same transmitter combination that evokes rhythmic locomotor activity in spinal cord circuits. These results demonstrate that there is a degree of self-assembly of microcircuits in these peripheral grafts involving embryonic stem cell-derived motoneurons and interneurons. Such spontaneous activity is reminiscent of embryonic circuit development in which spontaneous activity is essential for proper connectivity and function and may be necessary for the grafts to form functional connections with muscle.NEW & NOTEWORTHY This manuscript demonstrates that, following peripheral transplantation of neurons derived from embryonic stem cells, the grafts are spontaneously active. The activity is produced and modulated by a number of transmitter systems, indicating that there is a degree of self-assembly of circuits in the grafts.

  14. Expansion and Characterization of Human Embryonic Stem Cell-Derived Osteoblast-Like Cells

    PubMed Central

    Arpornmaeklong, Premjit; Wang, Zhuo; Pressler, Michael J.; Brown, Shelley E.

    2010-01-01

    Abstract Human embryonic stem cells (hESCs) have the potential to serve as a repository of cells for the replacement of damaged or diseased tissues and organs. However, to use hESCs in clinically relevant scenarios, a large number of cells are likely to be required. The aim of this study was to demonstrate an alternative cell culture method to increase the quantity of osteoblast-like cells directly derived from hESCs (hESCs-OS). Undifferentiated hESCs were directly cultivated and serially passaged in osteogenic medium (hESC-OS), and exhibited similar expression patterns of osteoblast-related genes to osteoblast-like cells derived from mesenchymal stem cells derived from hESCs (hESCs-MSCs-OS) and human bone marrow stromal cells (hBMSCs-OS). In comparison to hESCs-MSCs-OS, the hESCs-OS required a shorter expansion time to generate a homogenous population of osteoblast-like cells that did not contain contaminating undifferentiated hESCs. Identification of human specific nuclear antigen (HuNu) in the newly formed bone in calvarial defects verified the role of the transplanted hESCs-OS as active bone forming cells in vivo. Taken together, this study suggests that osteoblast-like cells directly derived from hESCs have the potential to serve as an alternative source of osteoprogenitors for bone tissue engineering strategies. PMID:20698777

  15. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

    PubMed

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza

    2016-06-01

    Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments.

  16. Functional astrocyte-neuron lactate shuttle in a human stem cell-derived neuronal network.

    PubMed

    Tarczyluk, Marta A; Nagel, David A; O'Neil, John D; Parri, H Rheinallt; Tse, Erin H Y; Coleman, Michael D; Hill, Eric J

    2013-09-01

    The NT2.D1 cell line is one of the most well-documented embryocarcinoma cell lines, and can be differentiated into neurons and astrocytes. Great focus has also been placed on defining the electrophysiological properties of the neuronal cells, and more recently we have investigated the functional properties of their associated astrocytes. We now show for the first time that human stem cell-derived astrocytes produce glycogen and that co-cultures of these cells demonstrate a functional astrocyte-neuron lactate shuttle (ANLS). The ANLS hypothesis proposes that during neuronal activity, glutamate released into the synaptic cleft is taken up by astrocytes and triggers glucose uptake, which is converted into lactate and released via monocarboxylate transporters for neuronal use. Using mixed cultures of NT2-derived neurons and astrocytes, we have shown that these cells modulate their glucose uptake in response to glutamate. Additionally, we demonstrate that in response to increased neuronal activity and under hypoglycaemic conditions, co-cultures modulate glycogen turnover and increase lactate production. Similar results were also shown after treatment with glutamate, potassium, isoproterenol, and dbcAMP. Together, these results demonstrate for the first time a functional ANLS in a human stem cell-derived co-culture.

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

    PubMed

    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.

  18. Ape1/Ref-1 Induces Glial Cell-Derived Neurotropic Factor (GDNF) Responsiveness by Upregulating GDNF Receptor α1 Expression ▿ ‡

    PubMed Central

    Kim, Mi-Hwa; Kim, Hong-Beum; Acharya, Samudra; Sohn, Hong-Moon; Jun, Jae Yeoul; Chang, In-Youb; You, Ho Jin

    2009-01-01

    Apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) dysregulation has been identified in several human tumors and in patients with a variety of neurodegenerative diseases. However, the function of Ape1/Ref-1 is unclear. We show here that Ape1/Ref-1 increases the expression of glial cell-derived neurotropic factor (GDNF) receptor α1 (GFRα1), a key receptor for GDNF. Expression of Ape1/Ref-1 led to an increase in the GDNF responsiveness in human fibroblast. Ape1/Ref-1 induced GFRα1 transcription through enhanced binding of NF-κB complexes to the GFRα1 promoter. GFRα1 levels correlate proportionally with Ape1/Ref-1 in cancer cells. The knockdown of endogenous Ape1/Ref-1 in pancreatic cancer cells markedly suppressed GFRα1 expression and invasion in response to GNDF, while overexpression of GFRα1 restored invasion. In neuronal cells, the Ape1/Ref-1-mediated increase in GDNF responsiveness not only stimulated neurite outgrowth but also protected the cells from β-amyloid peptide and oxidative stress. Our results show that Ape1/Ref-1 is a novel physiological regulator of GDNF responsiveness, and they also suggest that Ape1/Ref-1-induced GFRα1 expression may play important roles in pancreatic cancer progression and neuronal cell survival. PMID:19188437

  19. [The micro-particles of blood plasma, micro-vesicles, exosomes, apoptotic bodies and Kupffer macrophage in liver: late in phylogenesis system of realization of biological function of endoecology].

    PubMed

    Titov, V N

    2014-07-01

    Probably, at early stages of phylogenesis and on the stage of first contacts of single cells in environment the mode of intercommunication was developed via formation of micro-vesicles. It is quite possible that this so complicated way was used during billions of years to develop the very early cenosises of functionally different cells. Later on, diffusion of humoral mediators within the framework of group of cells but without vesicles resulted in formation of very early regulated cenosises of various cells. In billions years, these paracrin regulated cenosises became structural and functional units of every organ. It is difficult to imagine that mode of early in phylogenesis humoral intercommunication of cells could preserve its significance in present conditions. At the same time, according to methodological approach of biological continuity in becoming of biological functions and biological reactions, micro-vesicles continue to function but with somehow different purposes. It is surmised that microparticles of blood plasma consist a heterogeneous population of micro-vesicles initially formed by cells, exosomes and apoptotic bodies. This population is a foundation of spontaneous, physical chemical formation of complexes in blood plasma on principles of absorption, hydrophobicity and ionic interaction of structural cells'components insoluble in water medium. It is assumed that presently formation of micro-particles in blood is functionally a kind of realization of phylogenetically late variant of biological function of endoecology, in inter-cellular medium, in local pool of intravascular blood plasma. This variant includes: a) microparticles, micro-vesicles, exosomes and apoptotic bodies as elements of biological reaction of support of "purity" of inter-cellular medium; b) in many respects highly specialized variant of phagocytosis of micro-particles by Kupffer macrophages in liver In the aggregate, this is a phylogenetically late medium of realization of

  20. Optical control of muscle function by transplantation of stem cell-derived motor neurons in mice.

    PubMed

    Bryson, J Barney; Machado, Carolina Barcellos; Crossley, Martin; Stevenson, Danielle; Bros-Facer, Virginie; Burrone, Juan; Greensmith, Linda; Lieberam, Ivo

    2014-04-04

    Damage to the central nervous system caused by traumatic injury or neurological disorders can lead to permanent loss of voluntary motor function and muscle paralysis. Here, we describe an approach that circumvents central motor circuit pathology to restore specific skeletal muscle function. We generated murine embryonic stem cell-derived motor neurons that express the light-sensitive ion channel channelrhodopsin-2, which we then engrafted into partially denervated branches of the sciatic nerve of adult mice. These engrafted motor neurons not only reinnervated lower hind-limb muscles but also enabled their function to be restored in a controllable manner using optogenetic stimulation. This synthesis of regenerative medicine and optogenetics may be a successful strategy to restore muscle function after traumatic injury or disease.

  1. Chondrogenic differentiation of human embryonic germ cell derived cells in hydrogels.

    PubMed

    Varghese, Shyni; Theprungsirikul, Paranduangji; Ferran, Angela; Hwang, Nathaniel; Canver, Adam; Elisseeff, Jennifer

    2006-01-01

    Human embryonic germ (hEG) cells have the potential to self-renew over long periods of time and differentiate into various lineages. Cells derived from embryoid bodies of hEG cells express a broad spectrum of gene markers and have been induced towards cells of ecto-dermal and recently endo-dermal and mesenchymal lineages. LVEC cells express a number of surface marker proteins characteristic of mesenchymal stem cells (MSCs), indicating the potential of these cells to differentiate into mesenchymal tissues. Here we demonstrate the homogenous differentiation of LVEC cells into hyaline cartilage. Three dimensional tissue formation is achieved by encapsulating cells in synthetic hydrogels followed by incubation in chondrocyte-conditioned culture medium. Homogenous hyaline cartilage was produced, even after 63 population doublings (13 passages). The high proliferative capacity of these cells without teratoma formation, homogenous differentiation, and three-dimensional cartilage tissue formation suggests the significant potential of LVEC cells for cartilage tissue engineering applications.

  2. Induced pluripotent stem cell-derived cardiomyocytes: boutique science or valuable arrhythmia model?

    PubMed

    Knollmann, Björn C

    2013-03-15

    This article reviews the strengths and limitations of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) as models of cardiac arrhythmias. Specifically, the article attempts to answer the following questions: Which clinical arrhythmias can be modeled by iPSC-CM? How well can iPSC-CM model adult ventricular myocytes? What are the strengths and limitations of published iPSC-CM arrhythmia models? What new mechanistic insight has been gained? What is the evidence that would support using iPSC-CM to personalize antiarrhythmic drug therapy? The review also discusses the pros and cons of using the iPSC-CM technology for modeling specific genetic arrhythmia disorders, such as long QT syndrome, Brugada Syndrome, or Catecholaminergic Polymorphic Ventricular Tachycardia.

  3. Maturation of Induced Pluripotent Stem Cell Derived Hepatocytes by 3D-Culture

    PubMed Central

    Gieseck III, 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. PMID:24466060

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

    PubMed Central

    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

  5. Myeloid and T Cell-Derived TNF Protects against Central Nervous System Tuberculosis

    PubMed Central

    Hsu, Nai-Jen; Francisco, Ngiambudulu M.; Keeton, Roanne; Allie, Nasiema; Quesniaux, Valérie F. J.; Ryffel, Bernhard; Jacobs, Muazzam

    2017-01-01

    Tuberculosis of the central nervous system (CNS-TB) is a devastating complication of tuberculosis, and tumor necrosis factor (TNF) is crucial for innate immunity and controlling the infection. TNF is produced by many cell types upon activation, in particularly the myeloid and T cells during neuroinflammation. Here we used mice with TNF ablation targeted to myeloid and T cell (MT-TNF−/−) to assess the contribution of myeloid and T cell-derived TNF in immune responses during CNS-TB. These mice exhibited impaired innate immunity and high susceptibility to cerebral Mycobacterium tuberculosis infection, a similar phenotype to complete TNF-deficient mice. Further, MT-TNF−/− mice were not able to control T cell responses and cytokine/chemokine production. Thus, our data suggested that collective TNF production by both myeloid and T cells are required to provide overall protective immunity against CNS-TB infection. PMID:28280495

  6. Bioengineering Approaches to Mature Human Pluripotent Stem Cell-Derived Cardiomyocytes.

    PubMed

    Sun, Xuetao; Nunes, Sara S

    2017-01-01

    Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) represent a potential unlimited cell supply for cardiac tissue engineering and possibly regenerative medicine applications. However, hPSC-CMs produced by current protocols are not representative of native adult human cardiomyocytes as they display immature gene expression profile, structure and function. In order to improve hPSC-CM maturity and function, various approaches have been developed, including genetic manipulations to induce gene expression, delivery of biochemical factors, such as triiodothyronine and alpha-adrenergic agonist phenylephrine, induction of cell alignment in 3D tissues, mechanical stress as a mimic of cardiac load and electrical stimulation/pacing or a combination of these. In this mini review, we discuss biomimetic strategies for the maturation for hPSC-CMs with a particular focus on electromechanical conditioning methods.

  7. The phenotype and tissue-specific nature of multipotent cells derived from human mature adipocytes.

    PubMed

    Kou, Liang; Lu, Xiao-Wen; Wu, Min-Ke; Wang, Hang; Zhang, Yu-Jiao; Sato, Soh; Shen, Jie-Fei

    2014-02-21

    Dedifferentiated fat (DFAT) cells derived from mature adipocytes have been considered to be a homogeneous group of multipotent cells, which present to be an alternative source of adult stem cells for regenerative medicine. However, many aspects of the cellular nature about DFAT cells remained unclarified. This study aimed to elucidate the basic characteristics of DFAT cells underlying their functions and differentiation potentials. By modified ceiling culture technique, DFAT cells were converted from human mature adipocytes from the human buccal fat pads. Flow cytometry analysis revealed that those derived cells were a homogeneous population of CD13(+) CD29(+) CD105(+) CD44(+) CD31(-) CD34(-) CD309(-) α-SMA(-) cells. DFAT cells in this study demonstrated tissue-specific differentiation properties with strong adipogenic but much weaker osteogenic capacity. Neither did they express endothelial markers under angiogenic induction.

  8. Bioengineering Approaches to Mature Human Pluripotent Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Sun, Xuetao; Nunes, Sara S.

    2017-01-01

    Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) represent a potential unlimited cell supply for cardiac tissue engineering and possibly regenerative medicine applications. However, hPSC-CMs produced by current protocols are not representative of native adult human cardiomyocytes as they display immature gene expression profile, structure and function. In order to improve hPSC-CM maturity and function, various approaches have been developed, including genetic manipulations to induce gene expression, delivery of biochemical factors, such as triiodothyronine and alpha-adrenergic agonist phenylephrine, induction of cell alignment in 3D tissues, mechanical stress as a mimic of cardiac load and electrical stimulation/pacing or a combination of these. In this mini review, we discuss biomimetic strategies for the maturation for hPSC-CMs with a particular focus on electromechanical conditioning methods. PMID:28337437

  9. Myeloid and T Cell-Derived TNF Protects against Central Nervous System Tuberculosis.

    PubMed

    Hsu, Nai-Jen; Francisco, Ngiambudulu M; Keeton, Roanne; Allie, Nasiema; Quesniaux, Valérie F J; Ryffel, Bernhard; Jacobs, Muazzam

    2017-01-01

    Tuberculosis of the central nervous system (CNS-TB) is a devastating complication of tuberculosis, and tumor necrosis factor (TNF) is crucial for innate immunity and controlling the infection. TNF is produced by many cell types upon activation, in particularly the myeloid and T cells during neuroinflammation. Here we used mice with TNF ablation targeted to myeloid and T cell (MT-TNF(-/-)) to assess the contribution of myeloid and T cell-derived TNF in immune responses during CNS-TB. These mice exhibited impaired innate immunity and high susceptibility to cerebral Mycobacterium tuberculosis infection, a similar phenotype to complete TNF-deficient mice. Further, MT-TNF(-/-) mice were not able to control T cell responses and cytokine/chemokine production. Thus, our data suggested that collective TNF production by both myeloid and T cells are required to provide overall protective immunity against CNS-TB infection.

  10. Replication of Human Noroviruses in Stem Cell-Derived Human Enteroids

    PubMed Central

    Ettayebi, Khalil; Crawford, Sue E.; Murakami, Kosuke; Broughman, James R.; Karandikar, Umesh; Tenge, Victoria R.; Neill, Frederick H.; Blutt, Sarah E.; Zeng, Xi-Lei; Qu, Lin; Kou, Baijun; Opekun, Antone R.; Burrin, Douglas; Graham, David Y.; Ramani, Sasirekha; Atmar, Robert L.

    2016-01-01

    The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the leading cause of gastroenteritis worldwide. We report successful cultivation of multiple HuNoV strains in enterocytes in stem cell-derived, nontransformed human intestinal enteroid monolayer cultures. Bile, a critical factor of the intestinal milieu, is required for strain-dependent HuNoV replication. Lack of appropriate histoblood group antigen expression in intestinal cells restricts virus replication, and infectivity is abrogated by inactivation (e.g., irradiation, heating) and serum neutralization. This culture system recapitulates the human intestinal epithelium, permits human host-pathogen studies of previously noncultivatable pathogens, and allows the assessment of methods to prevent and treat HuNoV infections. PMID:27562956

  11. Application potential of mesenchymal stem cells derived from Wharton's jelly in liver tissue engineering.

    PubMed

    Zhang, Lei; Zhao, Yong-Hen; Guan, Zheng; Ye, Jun-Song; de Isla, Natalia; Stoltz, Jean-François

    2015-01-01

    The shortage of organ resource has been limiting the application of liver transplantation. Bioartificial liver construction is increasingly focused as a replacement treatment. To product a bioartificial liver, three elements must be considered: seeding cells, scaffold and bioreactor. Recent studies have shown that several methods can successfully differentiate MSC (mesenchymal stem cells) derived from Wharton's jelly into hepatocyte, such as stimulating MSC by cytokines and growth factors, direct and indirect co-culture MSC with hepatocytes, or promote MSC differentiation by 3-dimensional matrix. In some cases, differentiation of MSC into hepatocytes can also be an alternative approach for whole organ transplantation in treatment of acute and chronic liver diseases. In this review, the characterization of MSC from Wharton's jelly, their potential of application in liver tissue engineering on base of decellularized scaffold, their status of banking and their preclinical work performed will be discussed.

  12. A Method for Sectioning and Immunohistochemical Analysis of Stem Cell-Derived 3-D Organoids.

    PubMed

    Wiley, Luke A; Beebe, David C; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

    2016-05-12

    This unit describes a protocol for embedding, sectioning, and immunocytochemical analysis of pluripotent stem cell-derived 3-D organoids. Specifically, we describe a method to embed iPSC-derived retinal cups in low-melt agarose, acquire thick sections using a vibratome tissue slicer, and perform immunohistochemical analysis. This method includes an approach for antibody labeling that minimizes the amount of antibody needed for individual experiments and that utilizes large-volume washing to increase the signal-to-noise ratio, allowing for clean, high-resolution imaging of developing cell types. The universal methods described can be employed regardless of the type of pluripotent stem cell used and 3-D organoid generated. © 2016 by John Wiley & Sons, Inc.

  13. Cryopreservation of Human Pluripotent Stem Cell-derived Cardiomyocytes: Strategies, Challenges, and Future Directions

    PubMed Central

    Preininger, Marcela K.; Singh, Monalisa; Xu, Chunhui

    2017-01-01

    In recent years, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as a vital cell source for in vitro modeling of genetic cardiovascular disorders, drug screening, and in vivo cardiac regeneration research. Looking forward, the ability to efficiently cryopreserve hPSC-CMs without compromising their normal biochemical and physiologic functions will dramatically facilitate their various biomedical applications. Although working protocols for freezing, storing, and thawing hPSC-CMs have been established, the question remains as to whether they are optimal. In this chapter, we discuss our current understanding of cryopreservation appertaining to hPSC-CMs, and proffer key questions regarding the mechanical, contractile, and regenerative properties of cryopreserved hPSC-CMs. PMID:27837559

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

    PubMed

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

    2016-12-10

    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.

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

    PubMed Central

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

    2016-01-01

    Background Human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electrical impulse propagation velocity and immature action potential profiles. Methods and Results Here we have identified an optimal extracellular matrix (ECM) for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal ECM combination have impulse propagation velocities ~2X faster than previously reported (43.6±7.0 cm·s−1 n=9) and have mature cardiomyocyte action potential profiles including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s, N=5 monolayers). In addition the optimal ECM promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1 and Connexin43) and myofilament markers (cTroponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of β1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase (FAK) activation prevented structural maturation. Conclusions Maturation of human stem cell derived cardiomyocyte monolayers is achieved in a one week period by plating cardiomyocytes on PDMS coverslips rather than on conventional 2D cell culture formats such as glass coverslips or plastic dishes. Activation of integrin signaling and FAK are essential for significant maturation of human cardiac monolayers. PMID:27069088

  16. Stem Cells Derived from Tooth Periodontal Ligament Enhance Functional Angiogenesis by Endothelial Cells

    PubMed Central

    Yeasmin, Shamima; Ceccarelli, Jacob; Vigen, Marina; Carrion, Bita; Putnam, Andrew J.; Tarle, Susan A.

    2014-01-01

    In regenerative medicine approaches involving cell therapy, selection of the appropriate cell type is important in that the cells must directly (differentiation) or indirectly (trophic effects) participate in the regenerative response. Regardless of the mode of action of the cells, angiogenesis underlies the success of these approaches. Stem cells derived from tooth tissues, specifically the periodontal ligament of teeth (periodontal ligament stem cells [PDLSCs]), have recently been identified as a good source of multipotent cells for cell therapies. PDLSCs have demonstrated properties similar to mesenchymal stem cells (MSCs), yet, unlike MSCs, their vascular potential has not been previously demonstrated. Thus, the aim of this study was to determine if PDLSCs could modulate angiogenesis. In comparison to MSCs and stem cells derived from tooth pulp tissues (SHEDs), we first determined if PDLSCs released soluble proangiogenic factors with the capacity to induce vessel formation by endothelial cells (ECs). Next, the ability of PDLSCs to modulate angiogenesis was examined through their cotransplantation with ECs in subcutaneous sites of immunocompromised mice. Finally, the stability of the PDLSC-mediated vasculature was determined through evaluation of the maturity and functionality of the vessels formed following PDLSC transplantation. It was determined that PDLSCs produced appreciable levels of vascular endothelial growth factor and basic fibroblast growth factor-2, and additionally, were able to initiate in vitro angiogenesis of ECs comparable to MSC- and SHED-mediated angiogenesis. In vivo cotransplantation of ECs with PDLSCs significantly (>50% increase) enhanced the number of blood vessels formed relative to transplantation of ECs alone. Finally, vessels formed following PDLSC cotransplantation were more mature and less permeable than those formed after transplantation of EC alone. These data demonstrate for the first time that PDLSCs have vascular potential

  17. Stem cells derived from tooth periodontal ligament enhance functional angiogenesis by endothelial cells.

    PubMed

    Yeasmin, Shamima; Ceccarelli, Jacob; Vigen, Marina; Carrion, Bita; Putnam, Andrew J; Tarle, Susan A; Kaigler, Darnell

    2014-04-01

    In regenerative medicine approaches involving cell therapy, selection of the appropriate cell type is important in that the cells must directly (differentiation) or indirectly (trophic effects) participate in the regenerative response. Regardless of the mode of action of the cells, angiogenesis underlies the success of these approaches. Stem cells derived from tooth tissues, specifically the periodontal ligament of teeth (periodontal ligament stem cells [PDLSCs]), have recently been identified as a good source of multipotent cells for cell therapies. PDLSCs have demonstrated properties similar to mesenchymal stem cells (MSCs), yet, unlike MSCs, their vascular potential has not been previously demonstrated. Thus, the aim of this study was to determine if PDLSCs could modulate angiogenesis. In comparison to MSCs and stem cells derived from tooth pulp tissues (SHEDs), we first determined if PDLSCs released soluble proangiogenic factors with the capacity to induce vessel formation by endothelial cells (ECs). Next, the ability of PDLSCs to modulate angiogenesis was examined through their cotransplantation with ECs in subcutaneous sites of immunocompromised mice. Finally, the stability of the PDLSC-mediated vasculature was determined through evaluation of the maturity and functionality of the vessels formed following PDLSC transplantation. It was determined that PDLSCs produced appreciable levels of vascular endothelial growth factor and basic fibroblast growth factor-2, and additionally, were able to initiate in vitro angiogenesis of ECs comparable to MSC- and SHED-mediated angiogenesis. In vivo cotransplantation of ECs with PDLSCs significantly (>50% increase) enhanced the number of blood vessels formed relative to transplantation of ECs alone. Finally, vessels formed following PDLSC cotransplantation were more mature and less permeable than those formed after transplantation of EC alone. These data demonstrate for the first time that PDLSCs have vascular potential

  18. A cloned toy poodle produced from somatic cells derived from an aged female dog.

    PubMed

    Jang, G; Hong, S G; Oh, H J; Kim, M K; Park, J E; Kim, H J; Kim, D Y; Lee, B C

    2008-03-15

    To date, dogs have been cloned with somatic cell nuclear transfer (SCNT), using donor cells derived from large-breed dogs 2 months to 3 years of age. The objective of the present study was to use SCNT to produce a small-breed dog from ear fibroblasts of an aged poodle, using large-breed oocyte donors and surrogate females, and to determine the origin of its mitochondrial DNA (mtDNA) and the length of its telomeres. Oocytes were derived from large-breed donors, matured in vivo, collected by flushing oviducts, and reconstructed with somatic cells derived from an aged (14-year-old) female toy poodle. Oocytes and donor cells were fused by electric stimuli, activated chemically, and transferred into the oviducts of large-breed recipient females. Overall, 358 activated couplets were surgically transferred into the oviducts of 20 recipient dogs. Two recipients became pregnant; only one maintained pregnancy to term, and a live puppy (weighing 190 g) was delivered by Caesarean section. The cloned poodle was phenotypically and genetically identical to the nuclear donor dog; however, its mtDNA was from the oocyte donor, and its mean telomere length was not significantly different from that of the nuclear donor. In summary, we demonstrated that a small-breed dog could be cloned by transferring activated couplets produced by fusion of somatic cells from a small-breed, aged donor female with enucleated in-vivo-matured oocytes of large-breed females, and transferred into the oviduct of large-breed recipient female dogs.

  19. Effects of tacrolimus on morphology, proliferation and differentiation of mesenchymal stem cells derived from gingiva tissue

    PubMed Central

    HA, DONG-HO; YONG, CHUL SOON; KIM, JONG OH; JEONG, JEE-HEON; PARK, JUN-BEOM

    2016-01-01

    Tacrolimus is a 23-membered macrolide lactone with potent immunosuppressive activity that is effective in the prophylaxis of organ rejection following kidney, heart and liver transplantation. Tacrolimus also exerts a variety of actions on bone metabolism. The aim of the present study was to evaluate the effects of different concentrations of tacrolimus on the morphology and viability of human stem cells derived from the gingiva. Gingival-derived stem cells were grown in the presence of tacrolimus at final concentrations ranging from 0.001 to 100 µg/ml. The morphology of the cells was viewed under an inverted microscope and the cell viability was analyzed using Cell Counting kit-8 (CCK-8) on days 1, 3, 5 and 7. Alizarin Red S staining was used to assess mineralization of treated cells. The control group showed spindle-shaped, fibroblast-like morphology and the shapes of the cells in 0.001, 0.01, 0.1, 1 and 10 µg/ml tacrolimus were similar to those of the control group. All groups except the 100 µg/ml group showed increased cell proliferation over time. Cultures grown in the presence of tacrolimus at 0.001, 0.01, 0.1, 1 and 10 µg/ml were not identified to be significantly different compared with the control at days 1, 3 and 5 using the CCK-8 assays. Increased mineralized deposits were noted with increased incubation time. Treatment with tacrolimus from 0.001 to 1 µg/ml led to an increase in mineralization compared with the control group. Within the limits of this study, tacrolimus at the tested concentrations (ranging from 0.001 to 10 µg/ml) did not result in differences in the viability of stem cells derived from gingiva; however it did enhance osteogenic differentiation of the stem cells. PMID:27177273

  20. Reversible Commitment to Differentiation by Human Multipotent Stromal Cells (MSCs) in Single-Cell Derived Colonies

    PubMed Central

    Ylöstalo, Joni; Bazhanov, Nikolay; Prockop, Darwin J

    2008-01-01

    Objective Human multipotent stromal cells (MSCs) readily form single-cell derived colonies when plated at clonal densities. However, the colonies are heterogeneous since the cells from a colony form new colonies that vary in size and differentiation potential when re-plated at clonal densities. The experiments here tested the hypothesis that the cells in the inner regions of colonies are partially differentiated but the differentiation is reversible. Materials and Methods Cells were separately isolated from the dense inner regions (IN) and less dense outer regions (OUT) of single-cell derived colonies. The cells were then compared by assays of their transcriptomes and proteins, and for clonogenicity and differentiation. Results The IN cells expressed fewer cell-cycle genes and higher levels of genes for extracellular matrix than the OUT cells. When transferred to differentiation medium, differentiation of the colonies occurred primarily in the IN regions. However, the IN cells were indistinguishable from OUT cells when re-plated at clonal densities and assayed for rates of propagation and clonogenicity. Also, the colonies formed by IN cells were similar to colonies formed by OUT cells in that they had distinct IN and OUT regions. Cultures of IN and OUT cells remained indistinguishable through multiple passages (30-75 population doublings), and both cells formed colonies that were looser and less dense as they were expanded. Conclusions The results demonstrated that the cells in the inner region of single-derived colonies are partially differentiated but the differentiation can be reversed by re-plating the cells at clonal densities. PMID:18619725

  1. Immunogenicity of single-dose Vero cell-derived Japanese encephalitis vaccine in Japanese adults.

    PubMed

    Takeshita, Nozomi; Lim, Chang-Kweng; Mizuno, Yasutaka; Shimbo, Takuro; Kotaki, Akira; Ujiie, Mugen; Hayakawa, Kayoko; Kato, Yasuyuki; Kanagawa, Shuzo; Kaku, Mitsuo; Takasaki, Tomohiko

    2014-04-01

    In Japan, intensive immunization against Japanese encephalitis (JE) was performed from 1967 to 1976, and regular JE immunization was performed thereafter. However, for Japanese adults facing JE risk, dates of vaccination with new inactivated Vero cell-derived JE vaccine are unavailable. This study investigated how a single dose of Vero cell-derived JE vaccine affects Japanese adults. Neutralizing antibodies were measured pre- and post-JE vaccination in 79 participants (age 40.7 ± 9.4 years), enrolled between October 2009 and March 2011, whose JE-vaccination data were gathered from vaccination records and history taking. Before vaccination, the participants' seroprotection rate (SPR) was 51.9%, whereas SPR after vaccination was 93.7%. The seroconversion rate (SCR), which measures seronegative cases that turn seropositive after vaccination, was 86.8%. The geometric mean titer (GMT) was 14.7 before vaccination and 70.1 after vaccination. Age was a significant difference between seroprotected (42.8 years) and non-seroprotected (38.7 years) groups before vaccination. Then the difference of age, SCR, pre-vaccination GMT, post-vaccination GMT and sex ratio were also significant in participants aged 25-39 years and ≥40 years, who represent generations born when Japan's JE-vaccination policy changed. SCR was 100% in participants aged 25-39 years with a vaccination recorded 55.6% in participants aged 25-39 without a vaccination record, and 96.0% in participants aged ≥40 years. Thus, more participants aged 25-39 years were seroprotected before vaccination, but SCR was higher in those aged ≥40 years. Most Japanese adults can be protected after one-dose vaccination, but this may be insufficient for people aged 25-39 years without recorded JE vaccination.

  2. Neuronal-like cell differentiation of non-adherent bone marrow cell-derived mesenchymal stem cells.

    PubMed

    Wu, Yuxin; Zhang, Jinghan; Ben, Xiaoming

    2013-08-05

    Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were separated and cultured using the "pour-off" method. Non-adherent bone marrow cell-derived mesenchymal stem cells developed colony-forming unit-fibroblasts, and could be expanded by supplementation with epidermal growth factor. Immunocytochemistry showed that the non-adherent bone marrow cell-derived mesenchymal stem cells exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers, neurofilament-200 and NeuN, in vitro. Non-adherent bone marrow cell-derived mesenchymal stem cells from β-galactosidase transgenic mice were also transplanted into focal ischemic brain (right corpus striatum) of C57BL/6J mice. At 8 weeks, cells positive for LacZ and β-galactosidase staining were observed in the ischemic tissues, and cells co-labeled with both β-galactosidase and NeuN were seen by double immunohistochemical staining. These findings suggest that the non-adherent bone marrow cell-derived mesenchymal stem cells could differentiate into neuronal-like cells in vitro and in vivo.

  3. Cancer

    MedlinePlus

    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 ...

  4. Effect of lentivirus-mediated survivin transfection on the morphology and apoptosis of nucleus pulposus cells derived from degenerative human disc in vitro

    PubMed Central

    MA, XUEXIAO; LIN, YAZHOU; YANG, KUN; YUE, BIN; XIANG, HONGFEI; CHEN, BOHUA

    2015-01-01

    Lower back pain is a common concern, and 40% of all cases involve the degeneration of the intervertebral disc (IVD). However, the excessive apoptosis of disc cells plays an important role in IVD degeneration, particularly in the nucleus pulposus (NP). Thus, anti-apoptotic gene therapy to attenuate or reverse the degenerative process within the NP is being developed. Survivin is a unique inhibitor of apoptosis (IAP) and has been extensively investigated in cancer cells. However, little is known of the effects of survivin transfection on NP cells derived from degenerative human disc. In this study, we aimed to investigate the effects of lentivirus (LV)-mediated survivin transfection on the morphology and apoptosis of NP cells derived from degenerative human disc in vitro. NP cells were transfected with LV-mediated survivin. Subsequently, cell morphology was observed and the survivin mRNA expression levels were measured by RT-qPCR. Apoptosis was analyzed by flow cytometry and by measuring caspase-3 activity. The results revealed that the morphology of the NP cells derived from degenerative human disc transfected with LV-mediated survivin was significantly altered as evidenced by cytomorphosis, the reduction of the cytoplasm and cell shrinkage. Following transfection, survivin gene expression significantly increased in the transfected cells and subsequent generation cells; however, no significant differences in the cell apoptotic rate and caspase-3 activity were observed. We found that transfection of the survivin gene into NP cells led to the stable expression of survivin and induced marked changes in cell morphology. Furthermore, no significant anti-apoptotic effects were observed following LV-mediated survivin transfection. Overall, our findings demonstrate that LV carrying surviving may be used to successfully enforce the expression of survivin in NP cells. However, cell morphology was evidently altered, whereas the apoptotic rate did not decrease. Comprehensive

  5. Direct comparison of progenitor cells derived from adipose, muscle, and bone marrow from wild-type or craniosynostotic rabbits

    PubMed Central

    GM, Cooper; EL, Lensie; JJ, Cray; MR, Bykowski; GE, DeCesare; MA, Smalley; MP, Mooney; PG, Campbell; JE, Losee

    2010-01-01

    Background Reports have identified cells capable of osteogenic differentiation in bone marrow, muscle, and adipose tissues, but there are few direct comparisons of these different cell-types. Also, few have investigated the potential connection between a tissue-specific pathology and cells derived from seemingly unrelated tissues. Here, we compare cells isolated from wild-type rabbits or rabbits with nonsyndromic craniosynostosis, defined as the premature fusion of one or more of the cranial sutures. Methods Cells were derived from bone marrow, adipose, and muscle of 10 day-old wild-type rabbits (WT; n=17) or from age-matched rabbits with familial nonsyndromic craniosynostosis (CS; n=18). Cells were stimulated with bone morphogenetic protein 4 (BMP4) and alkaline phosphatase expression and cell proliferation were assessed. Results In WT rabbits, cells derived from muscle had more alkaline phosphatase activity than cells derived from either adipose or bone marrow. The cells derived from CS rabbit bone marrow and muscle were significantly more osteogenic than WT. Adipose-derived cells demonstrated no significant differences. While muscle-derived cells were most osteogenic in WT rabbits, bone marrow-derived cells were most osteogenic in CS rabbits. Conclusions Results suggest that cells from different tissues have different potentials for differentiation. Furthermore, cells derived from rabbits with craniosynostosis were different from wild-type derived cells. Interestingly, cells derived from the craniosynostotic rabbits were not uniformly more responsive compared with wild-type cells, suggesting that specific tissue-derived cells may react differently in individuals with craniosynostosis. PMID:20871482

  6. Label-free imaging of metabolism and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes

    PubMed Central

    Datta, Rupsa; Heylman, Christopher; George, Steven C.; Gratton, Enrico

    2016-01-01

    In this work we demonstrate a label-free optical imaging technique to assess metabolic status and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes by two-photon fluorescence lifetime imaging of endogenous fluorophores. Our results show the sensitivity of this method to detect shifts in metabolism and oxidative stress in the cardiomyocytes upon pathological stimuli of hypoxia and cardiotoxic drugs. This non-invasive imaging technique could prove beneficial for drug development and screening, especially for in vitro cardiac models created from stem cell-derived cardiomyocytes and to study the pathogenesis of cardiac diseases and therapy. PMID:27231614

  7. Label-free imaging of metabolism and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes.

    PubMed

    Datta, Rupsa; Heylman, Christopher; George, Steven C; Gratton, Enrico

    2016-05-01

    In this work we demonstrate a label-free optical imaging technique to assess metabolic status and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes by two-photon fluorescence lifetime imaging of endogenous fluorophores. Our results show the sensitivity of this method to detect shifts in metabolism and oxidative stress in the cardiomyocytes upon pathological stimuli of hypoxia and cardiotoxic drugs. This non-invasive imaging technique could prove beneficial for drug development and screening, especially for in vitro cardiac models created from stem cell-derived cardiomyocytes and to study the pathogenesis of cardiac diseases and therapy.

  8. Tumour-derived exosomes as a signature of pancreatic cancer - liquid biopsies as indicators of tumour progression.

    PubMed

    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.

  9. Proinflammatory and prothrombotic effects on human vascular endothelial cells of Immune-cell-derived LIGHT

    PubMed Central

    2009-01-01

    Objective LIGHT (TNFSF 14) belongs to the tumor necrosis factor superfamily and is expressed by activated T cells as well as various types of antigen presenting cells. LIGHT binds to its cellular receptors TR2 and LTßR and has a co-stimulatory role in T cell activation. Here, we compared the relative expression of LIGHT in different immune cells and the biological activity of immune cell-derived LIGHT on endothelial cells. Methods and Results Surface expression of LIGHT and mRNA production by PBMC and isolated T cells (CD4+ or CD8+) significantly increased after stimulation with PMA (Phorbolester-12-Myristat-13-Acetat) + ionomycin. No LIGHT expression on PMA stimulated monocytes or monocytic-like THP-1 cells could be detected; differentiation of monocytes and THP-1 cells into macrophages, however, resulted in up-regulation of LIGHT. Supernatants of stimulated T cells contained higher concentrations of soluble LIGHT than macrophage supernatants normalized to cell numbers; release of soluble LIGHT was found to be dependent on metalloproteinase activity. Size determination of released soluble LIGHT by size exclusion chromatography revealed a molecular mass of ~60 kDa, suggesting a trimeric form. Released soluble LIGHT induced expression of proinflammatory antigens ICAM-1, tissue factor and IL-8 in human endothelial cells and caused apoptosis of IFN-γ pretreated endothelial cells. Soluble LIGHT was detected at low levels in sera of healthy controls and was significantly enhanced in sera of patients with chronic hepatitis C and rheumatoid arthritis (24.93 ± 9.41 vs.129.53 ± 49.14 and 172.13 ± 77.64; p < 0.0005). Conclusion These findings suggest that among immune cells activated T lymphocytes are the main source of soluble LIGHT with released amounts of soluble LIGHT markedly higher compared to platelets. Immune cell-derived membrane-bound and soluble trimeric LIGHT is biologically active, inducing proinflammatory changes in endothelial cells. Enhanced plasma

  10. Stem Cell-Derived Immature Human Dorsal Root Ganglia Neurons to Identify Peripheral Neurotoxicants

    PubMed Central

    Klima, Stefanie; Karreman, Christiaan; Grinberg, Marianna; Meisig, Johannes; Henry, Margit; Rotshteyn, Tamara; Rahnenführer, Jörg; Blüthgen, Nils; Sachinidis, Agapios; Waldmann, Tanja; Leist, Marcel

    2016-01-01

    Safety sciences and the identification of chemical hazards have been seen as one of the most immediate practical applications of human pluripotent stem cell technology. Protocols for the generation of many desirable human cell types have been developed, but optimization of neuronal models for toxicological use has been astonishingly slow, and the wide, clinically important field of peripheral neurotoxicity is still largely unexplored. A two-step protocol to generate large lots of identical peripheral human neuronal precursors was characterized and adapted to the measurement of peripheral neurotoxicity. High content imaging allowed an unbiased assessment of cell morphology and viability. The computational quantification of neurite growth as a functional parameter highly sensitive to disturbances by toxicants was used as an endpoint reflecting specific neurotoxicity. The differentiation of cells toward dorsal root ganglia neurons was tracked in relation to a large background data set based on gene expression microarrays. On this basis, a peripheral neurotoxicity (PeriTox) test was developed as a first toxicological assay that harnesses the potential of human pluripotent stem cells to generate cell types/tissues that are not otherwise available for the prediction of human systemic organ toxicity. Testing of more than 30 chemicals showed that human neurotoxicants and neurite growth enhancers were correctly identified. Various classes of chemotherapeutic agents causing human peripheral neuropathies were identified, and they were missed when tested on human central neurons. The PeriTox test we established shows the potential of human stem cells for clinically relevant safety testing of drugs in use and of new emerging candidates. Significance The generation of human cells from pluripotent stem cells has aroused great hopes in biomedical research and safety sciences. Neurotoxicity testing is a particularly important application for stem cell-derived somatic cells, as

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

    SciTech Connect

    Cohen, J.D.; Babiarz, J.E.; Abrams, R.M.; Guo, L.; Kameoka, S.; Chiao, E.; Taunton, J.; Kolaja, K.L.

    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

  12. Resveratrol-Induced Apoptosis and Increased Radiosensitivity in CD133-Positive Cells Derived From Atypical Teratoid/Rhabdoid Tumor

    SciTech Connect

    Kao, C.-L.; Huang, P.-I; Tsai, P.-H.; Tsai, M.-L.; Lo, J.-F.; Lee, Y.-Y.; Chen, Y.-J.; Chen, Y.-W.; Chiou, S.-H.

    2009-05-01

    Purpose: CD133 has recently been proposed as a marker for cancer stem-like cells (CSC) in brain tumors. The aim of the present study was to investigate the possible role of resveratrol (RV) in radiosensitivity of CD133-positive/-negative cells derived from atypical teratoid/rhabdoid tumors (AT/RT-CD133{sup +/-}). Materials and Methods: AT/RT-CD133{sup +/-} were isolated and characterized by flow cytometry and quantitative real-time reverse transcription-polymerase chain reaction, and then treated with RV at different doses. Migratory ability, colony formation, apoptotic activity, and xenotransplantation were assessed for RV alone, ionizing radiation (IR) alone, and IR with RV conditions. Results: AT/RT-CD133{sup +} displayed enhanced self-renewal and highly coexpressed 'stem cell' genes and drug-resistant genes, in addition to showing significant resistance to chemotherapeutic agents and radiotherapy as compared with CD133{sup -} cells. After treatment with 200 {mu}M RV, the in vitro proliferation rates and in vivo tumor restoration abilities of ATRT-CD133{sup +} were dramatically inhibited. Importantly, treatment with 150 {mu}M RV can effectively inhibit the expression of drug-resistant genes in AT/RT-CD133{sup +}, and further facilitate to the differentiation of CD133{sup +} into CD133{sup -}. In addition, treatment with 150 {mu}M RV could significantly enhance the radiosensitivity and IR-mediated apoptosis in RV-treated ATRT-CD133{sup +/-}. Kaplan-Meier survival analysis indicated that the mean survival rate of mice with ATRT-CD133{sup +} that were treated with IR could be significantly improved when IR was combined with 150 {mu}M RV treatment. Conclusions: AT/RT-CD133{sup +} exhibit CSC properties and are refractory to IR treatment. Our results suggest that RV treatment plays crucial roles in antiproliferative, proapoptotic, and radiosensitizing effects on treated-CD133{sup +/-}; RV may therefore improve the clinical treatment of AT/RT.

  13. Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism

    PubMed Central

    James, S. Jill; Rose, Shannon; Melnyk, Stepan; Jernigan, Stefanie; Blossom, Sarah; Pavliv, Oleksandra; Gaylor, David W.

    2009-01-01

    Research into the metabolic phenotype of autism has been relatively unexplored despite the fact that metabolic abnormalities have been implicated in the pathophysiology of several other neurobehavioral disorders. Plasma biomarkers of oxidative stress have been reported in autistic children; however, intracellular redox status has not yet been evaluated. Lymphoblastoid cells (LCLs) derived from autistic children and unaffected controls were used to assess relative concentrations of reduced glutathione (GSH) and oxidized disulfide glutathione (GSSG) in cell extracts and isolated mitochondria as a measure of intracellular redox capacity. The results indicated that the GSH/GSSG redox ratio was decreased and percentage oxidized glutathione increased in both cytosol and mitochondria in the autism LCLs. Exposure to oxidative stress via the sulfhydryl reagent thimerosal resulted in a greater decrease in the GSH/GSSG ratio and increase in free radical generation in autism compared to control cells. Acute exposure to physiological levels of nitric oxide decreased mitochondrial membrane potential to a greater extent in the autism LCLs, although GSH/GSSG and ATP concentrations were similarly decreased in both cell lines. These results suggest that the autism LCLs exhibit a reduced glutathione reserve capacity in both cytosol and mitochondria that may compromise antioxidant defense and detoxification capacity under prooxidant conditions.—James, S. J., Rose, S., Melnyk, S., Jernigan, S., Blossom, S., Pavliv, O., Gaylor, D. W. Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism. PMID:19307255

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

    PubMed

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

    2016-01-01

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

  15. [Effects of radiation on capillary endothelial cells derived from Mongolian gerbil brain].

    PubMed

    Mori, S; Tanaka, R; Minakawa, T; Onda, K

    1990-09-01

    Confluent monolayers of capillary endothelial cells derived from Mongolian gerbil brain were irradiated with a single exposure of x-rays, and their radiosensitivity and sequential changes in morphology, staining intensity for factor VIII-related antigen (F VIII RAg), and capacity to produce prostacyclin (PGI2) were examined. The radiobiologic parameters that characterized the dose-response survival curve for these cells were found to be n = 1.9, Dq = 140 rad, and D0 = 190 rad. Morphologically, nuclear and cytoplasmic swelling, vacuolation of cytoplasm, and giant cell formation occurred in a dose dependent manner after 24 hours from irradiation. Decreased staining intensity for F VIII RAg was observed in morphologically affected cells. The capacity to synthesize PGI2 was significantly enhanced at 24 hours, but less significant at 72 hours after irradiation. The present data suggest that the radiosensitivity of brain capillary endothelial cells may be somewhat lower than that of endothelial cells originated from larger vessels, and that radiation induced morphological and functional changes in the brain capillary endothelial cells may be quantitatively similar to the changes in endothelial cells of larger vessels.

  16. Stromal cell-derived factor-1 potentiates bone morphogenetic protein-2 induced bone formation.

    PubMed

    Higashino, Kosaku; Viggeswarapu, Manjula; Bargouti, Maggie; Liu, Hui; Titus, Louisa; Boden, Scott D

    2011-02-01

    The mechanisms driving bone marrow stem cell mobilization are poorly understood. A recent murine study found that circulating bone marrow-derived osteoprogenitor cells (MOPCs) were recruited to the site of recombinant human bone morphogenetic protein-2 (BMP-2)-induced bone formation. Stromal cell-derived factor-1α (SDF-1α) and its cellular receptor CXCR4 have been shown to mediate the homing of stem cells to injured tissues. We hypothesized that chemokines, such as SDF-1, are also involved with mobilization of bone marrow cells. The CD45(-) fraction is a major source of MOPCs. In this report we determined that the addition of BMP-2 or SDF-1 to collagen implants increased the number of MOPCs in the peripheral blood. BMP-2-induced mobilization was blocked by CXCR4 antibody, confirming the role of SDF-1 in mobilization. We determined for the first time that addition of SDF-1 to implants containing BMP-2 enhances mobilization, homing of MOPCs to the implant, and ectopic bone formation induced by suboptimal BMP-2 doses. These results suggest that SDF-1 increases the number of osteoprogenitor cells that are mobilized from the bone marrow and then home to the implant. Thus, addition of SDF-1 to BMP-2 may improve the efficiency of BMPs in vivo, making their routine use for orthopaedic applications more affordable and available to more patients.

  17. Electric impedance of human embryonic stem cell-derived retinal pigment epithelium.

    PubMed

    Onnela, Niina; Savolainen, Virpi; Juuti-Uusitalo, Kati; Vaajasaari, Hanna; Skottman, Heli; Hyttinen, Jari

    2012-02-01

    The barrier properties of epithelium are conventionally defined by transepithelial resistance (TER). TER provides information about the tightness of the epithelium. Electrical impedance spectroscopy (EIS) provides additional information regarding cell membrane properties, such as changes in electric capacitance and possible parallel or serial pathways that may correlate with the morphology of the cell layer. This study presents EIS of retinal pigment epithelial (RPE) cell model of the putative RPE differentiated from human embryonic stem cells (hESC-RPE). The generally utilized RPE cell model, ARPE-19, was used as immature control. The measured EIS was analyzed by fitting an equivalent electrical circuit model describing the resistive and capacitive properties of the RPE. Our results indicated that TER of hESC-RPE cells was close to the values of human RPE presented in the literature. This provides evidence that the stem cell-derived RPE in vitro can reach high-barrier function. Furthermore, hESC-RPE cells produced impedance spectra that can be modeled by the equivalent circuit of one time constant. ARPE-19 cells produced low-barrier properties, that is, an impedance spectra that suggested poor maturation of ARPE-19 cells. To conclude, EIS could give us means for non-invasively estimating the functionality and maturation of differentiated-RPE cells.

  18. Importance of being Nernst: Synaptic activity and functional relevance in stem cell-derived neurons

    PubMed Central

    Bradford, Aaron B; McNutt, Patrick M

    2015-01-01

    Functional synaptogenesis and network emergence are signature endpoints of neurogenesis. These behaviors provide higher-order confirmation that biochemical and cellular processes necessary for neurotransmitter release, post-synaptic detection and network propagation of neuronal activity have been properly expressed and coordinated among cells. The development of synaptic neurotransmission can therefore be considered a defining property of neurons. Although dissociated primary neuron cultures readily form functioning synapses and network behaviors in vitro, continuously cultured neurogenic cell lines have historically failed to meet these criteria. Therefore, in vitro-derived neuron models that develop synaptic transmission are critically needed for a wide array of studies, including molecular neuroscience, developmental neurogenesis, disease research and neurotoxicology. Over the last decade, neurons derived from various stem cell lines have shown varying ability to develop into functionally mature neurons. In this review, we will discuss the neurogenic potential of various stem cells populations, addressing strengths and weaknesses of each, with particular attention to the emergence of functional behaviors. We will propose methods to functionally characterize new stem cell-derived neuron (SCN) platforms to improve their reliability as physiological relevant models. Finally, we will review how synaptically active SCNs can be applied to accelerate research in a variety of areas. Ultimately, emphasizing the critical importance of synaptic activity and network responses as a marker of neuronal maturation is anticipated to result in in vitro findings that better translate to efficacious clinical treatments. PMID:26240679

  19. Human Embryonic Stem Cell-Derived Cardiomyocytes Regenerate Non-Human Primate Hearts

    PubMed Central

    Chong, James J.H.; Yang, Xiulan; Don, Creighton W.; Minami, Elina; Liu, Yen-Wen; Weyers, Jill J; Mahoney, William M.; Van Biber, Benjamin; Cook, Savannah M.; Palpant, Nathan J; Gantz, Jay; Fugate, James A.; Muskheli, Veronica; Gough, G. Michael; Vogel, Keith W.; Astley, Cliff A.; Hotchkiss, Charlotte E.; Baldessari, Audrey; Pabon, Lil; Reinecke, Hans; Gill, Edward A.; Nelson, Veronica; Kiem, Hans-Peter; Laflamme, Michael A.; Murry, Charles E.

    2014-01-01

    Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure 1 by providing human cardiomyocytes to support heart regeneration 2. Studies of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in small animal models have shown favorable effects of this treatment 3–7. It remains unknown, however, whether clinical scale hESC-CMs transplantation is feasible, safe or can provide large-scale myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (>1 billion cells/batch) and cryopreserved with good viability. Using a non-human primate (NHP) model of myocardial ischemia-reperfusion, we show that that cryopreservation and intra-myocardial delivery of 1 billion hESC-CMs generates significant remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a three-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small animal models 7, non-fatal ventricular arrhythmias were observed in hESC-CM engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome. PMID:24776797

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

    PubMed Central

    Fu, Weili; Xie, Xing; Li, Qi; Chen, Gang; Zhang, Chenghao; Tang, Xin

    2016-01-01

    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. PMID:28044083

  1. Upscaling of hiPS Cell-Derived Neurons for High-Throughput Screening.

    PubMed

    Traub, Stefanie; Stahl, Heiko; Rosenbrock, Holger; Simon, Eric; Heilker, Ralf

    2017-03-01

    The advent of human-induced pluripotent stem (hiPS) cell-derived neurons promised to provide better model cells for drug discovery in the context of the central nervous system. This work demonstrates both the upscaling of cellular expansion and the acceleration of neuronal differentiation to accommodate the immense material needs of a high-throughput screening (HTS) approach. Using GRowth factor-driven expansion and INhibition of NotCH (GRINCH) during maturation, the derived cells are here referred to as GRINCH neurons. GRINCH cells displayed neuronal markers, and their functional activity could be demonstrated by electrophysiological recordings. In an application of GRINCH neurons, the brain-derived neurotrophic factor (BDNF)-mediated activation of tropomyosin receptor kinase (TrkB) was investigated as a promising drug target to treat synaptic dysfunctions. To assess the phosphorylation of endogenous TrkB in the GRINCH cells, the highly sensitive amplified luminescent proximity homogeneous assay LISA (AlphaLISA) format was established as a primary screen. A high-throughput reverse transcription (RT)-PCR format was employed as a secondary assay to analyze TrkB-mediated downstream target gene expression. In summary, an optimized differentiation protocol, highly efficient cell upscaling, and advanced assay miniaturization, combined with increased detection sensitivity, pave the way for a new generation of predictive cell-based drug discovery.

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

    PubMed Central

    Luarte, Alejandro; Bátiz, Luis Federico; Wyneken, Ursula; Lafourcade, Carlos

    2016-01-01

    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. PMID:27195011

  3. Induced Pluripotent Stem Cell Derived Macrophages as a Cellular System to Study Salmonella and Other Pathogens

    PubMed Central

    Hale, Christine; Yeung, Amy; Goulding, David; Pickard, Derek; Alasoo, Kaur; Powrie, Fiona; Dougan, Gordon; Mukhopadhyay, Subhankar

    2015-01-01

    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. PMID:25946027

  4. Multielectrode Array (MEA) Assay for Profiling Electrophysiological Drug Effects in Human Stem Cell-Derived Cardiomyocytes.

    PubMed

    Clements, Mike

    2016-05-04

    More relevant and reliable preclinical cardiotoxicity tests are required to improve drug safety and reduce the cost of drug development. Human stem cell-derived cardiomyocytes (hSC-CMs) provide a potential model for the development of superior assays for preclinical drug safety screening. One such hSC-CM assay that has shown significant potential for enabling more predictive drug cardiac risk assessment is the MEA assay. The Multi-electrode Array (MEA) assay is an electrophysiology-based technique that uses microelectrodes embedded in the culture surface of each well to measure fluctuations in extracellular field potential (FP) generated from spontaneously beating hSC-CMs. Perturbations to the recorded FP waveform can be used as an unbiased method of predicting the identity of ion channel(s) impacted on drug exposure. Here, a higher throughput MEA assay using hSC-CMs in 48-well MEA plates is described for profiling compound-induced effects on cardiomyocyte electrophysiology. Techniques for preparing hSC-CM monolayers in MEA plates and methods to contextualize MEA assay experimental results are also covered. © 2016 by John Wiley & Sons, Inc.

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

    PubMed Central

    McCabe, Kathryn L.; Kunzevitzky, Noelia J.; Chiswell, Brian P.; Xia, Xin; Goldberg, Jeffrey L.; Lanza, Robert

    2015-01-01

    Aim To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies. Materials and Methods Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression. Results hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1) and Na+/K+ATPaseα1 (ATPA1) on the apical surface in monolayer culture, and produced the key proteins of Descemet’s membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2). Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis. Conclusion hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium. PMID:26689688

  6. Increasing doublecortin expression promotes migration of human embryonic stem cell-derived neurons.

    PubMed

    Filipovic, Radmila; Santhosh Kumar, Saranya; Fiondella, Chris; Loturco, Joseph

    2012-09-01

    Human embryonic stem cell-derived neuronal progenitors (hNPs) provide a potential source for cellular replacement following neurodegenerative diseases. One of the greatest challenges for future neuron replacement therapies will be to control extensive cell proliferation and stimulate cell migration of transplanted cells. The doublecortin (DCX) gene encodes the protein DCX, a microtubule-associated protein essential for the migration of neurons in the human brain. In this study, we tested whether increasing the expression of DCX in hNPs would favorably alter their proliferation and migration. Migration and proliferation of hNPs was compared between hNPs expressing a bicistronic DCX/IRES-GFP transgene and those expressing a green fluorescent protein (GFP) transgene introduced by piggyBac-mediated transposition. The DCX-transfected hNPs showed a significant decrease in their proliferation and migrated significantly further on two different substrates, Matrigel and brain slices. Additionally, a dense network of nestin-positive (+) and vimentin+ fibers were found to extend from neurospheres transplanted onto brain slices, and this fiber growth was increased from neurospheres containing DCX-transfected hNPs. In summary, our results show that increased DCX expression inhibits proliferation and promotes migration of hNPs.

  7. Long Term Liver Engraftment of Functional Hepatocytes Obtained from Germline Cell-Derived Pluripotent Stem Cells

    PubMed Central

    Fagoonee, Sharmila; Famulari, Elvira Smeralda; Silengo, Lorenzo; Tolosano, Emanuela; Altruda, Fiorella

    2015-01-01

    One of the major hurdles in liver gene and cell therapy is availability of ex vivo-expanded hepatocytes. Pluripotent stem cells are an attractive alternative. Here, we show that hepatocyte precursors can be isolated from male germline cell-derived pluripotent stem cells (GPSCs) using the hepatoblast marker, Liv2, and induced to differentiate into hepatocytes in vitro. These cells expressed hepatic-specific genes and were functional as demonstrated by their ability to secrete albumin and produce urea. When transplanted in the liver parenchyma of partially hepatectomised mice, Liv2-sorted cells showed regional and heterogeneous engraftment in the injected lobe. Moreover, approximately 50% of Y chromosome-positive, GPSC-derived cells were found in the female livers, in the region of engraftment, even one month after cell injection. This is the first study showing that Liv2-sorted GPSCs-derived hepatocytes can undergo long lasting engraftment in the mouse liver. Thus, GPSCs might offer promise for regenerative medicine. PMID:26323094

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

    PubMed

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

    2014-10-01

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

  9. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.

    PubMed

    Rezania, Alireza; Bruin, Jennifer E; Arora, Payal; Rubin, Allison; Batushansky, Irina; Asadi, Ali; O'Dwyer, Shannon; Quiskamp, Nina; Mojibian, Majid; Albrecht, Tobias; Yang, Yu Hsuan Carol; Johnson, James D; Kieffer, Timothy J

    2014-11-01

    Transplantation of pancreatic progenitors or insulin-secreting cells derived from human embryonic stem cells (hESCs) has been proposed as a therapy for diabetes. We describe a seven-stage protocol that efficiently converts hESCs into insulin-producing cells. Stage (S) 7 cells expressed key markers of mature pancreatic beta cells, including MAFA, and displayed glucose-stimulated insulin secretion similar to that of human islets during static incubations in vitro. Additional characterization using single-cell imaging and dynamic glucose stimulation assays revealed similarities but also notable differences between S7 insulin-secreting cells and primary human beta cells. Nevertheless, S7 cells rapidly reversed diabetes in mice within 40 days, roughly four times faster than pancreatic progenitors. Therefore, although S7 cells are not fully equivalent to mature beta cells, their capacity for glucose-responsive insulin secretion and rapid reversal of diabetes in vivo makes them a promising alternative to pancreatic progenitor cells or cadaveric islets for the treatment of diabetes.

  10. Tomosyn Negatively Regulates Arginine Vasopressin Secretion in Embryonic Stem Cell-Derived Neurons

    PubMed Central

    Takeuchi, Seiji; Iwama, Shintaro; Takagi, Hiroshi; Kiyota, Atsushi; Nakashima, Kohtaro; Izumida, Hisakazu; Fujisawa, Haruki; Iwata, Naoko; Suga, Hidetaka; Watanabe, Takashi; Kaibuchi, Kozo; Oiso, Yutaka; Arima, Hiroshi; Sugimura, Yoshihisa

    2016-01-01

    Arginine vasopressin (AVP) is secreted via exocytosis; however, the precise molecular mechanism underlying the exocytosis of AVP remains to be elucidated. To better understand the mechanisms of AVP secretion, in our study we have identified proteins that bind with a 25 kDa synaptosomal-associated protein (SNAP25). SNAP25 plays a crucial role in exocytosis, in the posterior pituitary. Embryonic stem (ES) cell-derived AVP neurons were established to investigate the functions of the identified proteins. Using glutathione S-transferase (GST)-pulldown assays and proteomic analyses, we identified tomosyn-1 (syntaxin-binding protein 5) as a SNAP25-binding protein in the posterior pituitary. Coimmunoprecipitation assays indicated that tomosyn formed N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with SNAP25 and syntaxin1. Immunohistochemistry showed that tomosyn localized to the posterior pituitary. Mouse ES cells self-differentiated into AVP neurons (mES-AVP) that expressed tomosyn and two transmembrane SNARE proteins, including SNAP25 and syntaxin1. KCl increased AVP secretion in mES-AVP, and overexpression of tomosyn-1 reduced KCl-stimulated AVP secretion. Downregulation of tomosyn-1 with siRNA increased KCl-stimulated AVP secretion. These results suggested that tomosyn-1 negatively regulated AVP secretion in mES-AVP and further suggest the possibility of using mES-AVP culture systems to evaluate the role of synaptic proteins from AVP neurons. PMID:27732637

  11. Mitochondrial gene replacement in human pluripotent stem cell-derived neural progenitors.

    PubMed

    Iyer, S; Xiao, E; Alsayegh, K; Eroshenko, N; Riggs, M J; Bennett, J P; Rao, R R

    2012-05-01

    Human pluripotent stem cell-derived neural progenitor (hNP) cells are an excellent resource for understanding early neural development and neurodegenerative disorders. Given that many neurodegenerative disorders can be correlated with defects in the mitochondrial genome, optimal utilization of hNP cells requires an ability to manipulate and monitor changes in the mitochondria. Here, we describe a novel approach that uses recombinant human mitochondrial transcription factor A (rhTFAM) protein to transfect and express a pathogenic mitochondrial genome (mtDNA) carrying the G11778A mutation associated with Leber's hereditary optic neuropathy (LHON) disease, into dideoxycytidine (ddC)-treated hNPs. Treatment with ddC reduced endogenous mtDNA and gene expression, without loss of hNP phenotypic markers. Entry of G11778A mtDNA complexed with the rhTFAM was observed in mitochondria of ddC-hNPs. Expression of the pathogenic RNA was confirmed by restriction enzyme analysis of the SfaN1-digested cDNA. On the basis of the expression of neuron-specific class III beta-tubulin, neuronal differentiation occurred. Our results show for the first time that pathogenic mtDNA can be introduced and expressed into hNPs without loss of phenotype or neuronal differentiation potential. This mitochondrial gene replacement technology allows for creation of in vitro stem cell-based models useful for understanding neuronal development and treatment of neurodegenerative disorders.

  12. Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.

    PubMed

    Chong, James J H; Yang, Xiulan; Don, Creighton W; Minami, Elina; Liu, Yen-Wen; Weyers, Jill J; Mahoney, William M; Van Biber, Benjamin; Cook, Savannah M; Palpant, Nathan J; Gantz, Jay A; Fugate, James A; Muskheli, Veronica; Gough, G Michael; Vogel, Keith W; Astley, Cliff A; Hotchkiss, Charlotte E; Baldessari, Audrey; Pabon, Lil; Reinecke, Hans; Gill, Edward A; Nelson, Veronica; Kiem, Hans-Peter; Laflamme, Michael A; Murry, Charles E

    2014-06-12

    Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

  13. Evaluation of MDCK Cell-Derived Influenza H7N9 Vaccine Candidates in Ferrets

    PubMed Central

    Tseng, Yu-Fen; Weng, Tsai-Chuan; Lai, Chia-Chun; Lin, Jun-Yang; Chen, Po-Ling; Wang, Ya-Fang; Chao, Sin-Ru; Chang, Jui-Yuan; Hwang, Yi-Shiuh; Yeh, Chia-Tsui; Yu, Cheng-Ping; Chen, Yee-Chun; Su, Ih-Jen; Lee, Min-Shi

    2015-01-01

    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 (300μg aluminum hydroxide, 1.5μg HA, and 1.5μg HA plus 300μg 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. PMID:25799397

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Mitochondrial Toxicity of Perfluorooctane Sulfonate in Mouse Embryonic Stem Cell-Derived Cardiomyocytes.

    PubMed

    Tang, Lei-Lei; Wang, Jia-Dan; Xu, Ting-Ting; Zhao, Zhe; Zheng, Jia-Jie; Ge, Ren-Shan; Zhu, Dan-Yan

    2017-03-10

    Perfluorooctane sulfonate (PFOS) is a persistent organic contaminant that may cause cardiotoxicity in animals and humans. However, little is known about the underlying mechanism by which it affects the organelle toxicity in cardiomyocytes during the cardiogenesis. Our previous proteomic study showed that differences of protein expression mainly existed in mitochondria of cardiomyocytes differentiated from embryonic stem (ES) cells after exposure to PFOS. Here, we focused on mitochondrial toxicity of PFOS in ES cell-derived cardiomyocytes. The cardiomyogenesis from ES cells in vitro was inhibited, and the expression of L-type Ca(2+) channel (LTCC) was decreased to interrupt [Ca(2+)]c transient amplitude in cardiomyocytes after PFOS treatment. Transmission electron microscope revealed that swollen mitochondrion with vacuole in PFOS-treated cells. Meanwhile, mitochondrial transmembrane potential (ΔYm) was declined and ATP production was lowered. These changes were related to the increased EGFR phosphorylation, activated Rictor signaling, then mediated HK2 binding to mitochondrial membrane. Furthermore, PFOS reduced the interaction of IP3R-Grp75-VDAC and accumulated intracellular fatty acids by activating Rictor, thereby attenuating PGC-1a and Mfn2 expressions, then destroying mitochondria-associated endoplasmic reticulum membrane (MAM), which resulted in the decrease of [Ca(2+)]mito transient amplitude triggered by ATP. In conclusion, mitochondrial structure damages and abnormal Ca(2+) shuttle were the important aspects in PFOS-induced cardiomyocytes toxicity from ES cells by activating Rictor signaling pathway.

  17. Endothelial differentiation in multipotent cells derived from mouse and human white mature adipocytes.

    PubMed

    Jumabay, Medet; Abdmaulen, Raushan; Urs, Sumithra; Heydarkhan-Hagvall, Sepideh; Chazenbalk, Gregorio D; Jordan, Maria C; Roos, Kenneth P; Yao, Yucheng; Boström, Kristina I

    2012-12-01

    White mature adipocytes give rise to multipotent cells, so-called de-differentiated fat (DFAT) cells, when losing their fat in culture. The objective of this study was to examine the ability of DFAT cells to give rise to endothelial cells (ECs) in vitro and vivo. We demonstrate that mouse and human DFAT cells, derived from adipose tissue and lipospirate, respectively, initially lack expression of CD34, CD31, CD146, CD45 and pericyte markers, distinguishing them from progenitor cells previously identified in adipose stroma. The DFAT cells spontaneously differentiate into vascular ECs in vitro, as determined by real-time PCR, fluorescence activated cell sorting, immunostaining, and formation of tube structures. Treatment with bone morphogenetic protein (BMP)4 and BMP9, important in regulating angiogenesis, significantly enhances the EC differentiation. Furthermore, adipocyte-derived cells from Green Fluorescent Protein-transgenic mice were detected in the vasculature of infarcted myocardium up to 6 weeks after ligation of the left anterior descending artery in mice. We conclude that adipocyte-derived multipotent cells are able to spontaneously give rise to ECs, a process that is promoted by BMPs and may be important in cardiovascular regeneration and in physiological and pathological changes in fat and other tissues.

  18. Dedifferentiated follicular granulosa cells derived from pig ovary can transdifferentiate into osteoblasts

    PubMed Central

    Oki, Yoshinao; Ono, Hiromasa; Motohashi, Takeharu; Sugiura, Nobuki; Nobusue, Hiroyuki; Kano, Koichiro

    2012-01-01

    Transdifferentiation is the conversion of cells from one differentiated cell type into another. How functionally differentiated cells already committed to a specific cell lineage can transdifferentiate into other cell types is a key question in cell biology and regenerative medicine. In the present study we show that porcine ovarian follicular GCs (granulosa cells) can transdifferentiate into osteoblasts in vitro and in vivo. Pure GCs isolated and cultured in Dulbecco's modified Eagle's medium supplemented with 20% FBS (fetal bovine serum) proliferated and dedifferentiated into fibroblast-like cells. We referred to these cells as DFOG (dedifferentiated follicular granulosa) cells. Microarray analysis showed that DFOG cells lost expression of GC-specific marker genes, but gained the expression of osteogenic marker genes during dedifferentiation. After osteogenic induction, DFOG cells underwent terminal osteoblast differentiation and matrix mineralization in vitro. Furthermore, when DFOG cells were transplanted subcutaneously into SCID mice, these cells formed ectopic osteoid tissue. These results indicate that DFOG cells derived from GCs can differentiate into osteoblasts in vitro and in vivo. We suggest that GCs provide a useful model for studying the mechanisms of transdifferentiation into other cell lineages in functionally differentiated cells. PMID:22839299

  19. Endothelial Differentiation in Multipotent Cells Derived from Mouse and Human White Mature Adipocytes

    PubMed Central

    Jumabay, Medet; Abdmaulen, Raushan; Urs, Sumithra; Heydarkhan-Hagvall, Sepideh; Chazenbalk, Gregorio D.; Jordan, Maria C.; Roos, Kenneth P.; Yao, Yucheng; Boström, Kristina I.

    2012-01-01

    White mature adipocytes give rise to multipotent cells, so-called de-differentiated fat (DFAT) cells, when losing their fat in culture. The objective of this study was to examine the ability of DFAT cells to give rise to endothelial cells (ECs) in vitro and vivo. We demonstrate that mouse and human DFAT cells, derived from adipose tissue and lipospirate, respectively, initially lack expression of CD34, CD31, CD146, CD45 and pericyte markers, distinguishing them from progenitor cells previously identified in adipose stroma. The DFAT cells spontaneously differentiate into vascular ECs in vitro, as determined by real-time PCR, fluorescence activated cell sorting, immunostaining, and formation of tube structures. Treatment with bone morphogenetic protein (BMP)4 and BMP9, important in regulating angiogenesis, significantly enhance the EC differentiation. Furthermore, adipocyte-derived cells from Green Fluorescent Protein-transgenic mice were detected in the vasculature of infarcted myocardium up to 6 weeks after ligation of the left anterior descending artery in mice. We conclude that adipocyte-derived multipotent cells are able to spontaneously give rise to ECs, a process that is promoted by BMPs and may be important in cardiovascular regeneration and in physiological and pathological changes in fat and other tissues. PMID:22999861

  20. In vitro patterning of pluripotent stem cell-derived intestine recapitulates in vivo human development

    PubMed Central

    Tsai, Yu-Hwai; Nattiv, Roy; Dedhia, Priya H.; Nagy, Melinda S.; Chin, Alana M.; Thomson, Matthew; Klein, Ophir D.

    2017-01-01

    ABSTRACT The intestine plays a central role in digestion, nutrient absorption and metabolism, with individual regions of the intestine having distinct functional roles. Many examples of region-specific gene expression in the adult intestine are known, but how intestinal regional identity is established during development is a largely unresolved issue. Here, we have identified several genes that are expressed in a region-specific manner in the developing human intestine. Using human embryonic stem cell-derived intestinal organoids, we demonstrate that the duration of exposure to active FGF and WNT signaling controls regional identity. Short-term exposure to FGF4 and CHIR99021 (a GSK3β inhibitor that stabilizes β-catenin) resulted in organoids with gene expression patterns similar to developing human duodenum, whereas longer exposure resulted in organoids similar to ileum. When region-specific organoids were transplanted into immunocompromised mice, duodenum-like organoids and ileum-like organoids retained their regional identity, demonstrating that regional identity of organoids is stable after initial patterning occurs. This work provides insights into the mechanisms that control regional specification of the developing human intestine and provides new tools for basic and translational research. PMID:27927684

  1. Stromal Cell-Derived Factor-1 Alpha is Cardioprotective After Myocardial Infarction

    PubMed Central

    Saxena, Ankur; Fish, Jason E.; White, Michael D.; Yu, Sangho; Smyth, James WP; Shaw, Robin M.; DiMaio, J. Michael; Srivastava, Deepak

    2009-01-01

    Background Heart disease is a leading cause of mortality throughout the world. Tissue damage from vascular occlusive events results in the replacement of contractile myocardium by nonfunctional scar tissue. The potential of new technologies to regenerate damaged myocardium is significant, although cell-based therapies must overcome several technical barriers. One possible cell-independent alternative is the direct administration of small proteins to damaged myocardium. Methods and Results Here we show that the secreted signaling protein stromal cell-derived factor-1 alpha (SDF-1α), which activates the cell-survival factor protein kinase B (PKB/Akt) via the G-protein-coupled receptor CXCR4, protected tissue after an acute ischemic event in mice and activated Akt within endothelial cells and myocytes of the heart. Significantly better cardiac function than in control mice was evident as early as 24 hours post-infarction as well as at 3, 14 and 28 days post-infarction. Prolonged survival of hypoxic myocardium was followed by an increase in levels of vascular endothelial growth factor (VEGF) protein and neo-angiogenesis. Consistent with improved cardiac function, mice exposed to SDF-1α demonstrated significantly decreased scar formation than control mice. Conclusions These findings suggest that SDF-1α may serve a tissue-protective and regenerative role for solid organs suffering a hypoxic insult. PMID:18427137

  2. Automated Electrophysiological and Pharmacological Evaluation of Human Pluripotent Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Rajamohan, Divya; Kalra, Spandan; Duc Hoang, Minh; George, Vinoj; Staniforth, Andrew; Russell, Hugh; Yang, Xuebin

    2016-01-01

    Automated planar patch clamp systems are widely used in drug evaluation studies because of their ability to provide accurate, reliable, and reproducible data in a high-throughput manner. Typically, CHO and HEK tumorigenic cell lines overexpressing single ion channels are used since they can be harvested as high-density, homogenous, single-cell suspensions. While human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are physiologically more relevant, these cells are fragile, have complex culture requirements, are inherently heterogeneous, and are expensive to produce, which has restricted their use on automated patch clamp (APC) devices. Here, we used high efficiency differentiation protocols to produce cardiomyocytes from six different hPSC lines for analysis on the Patchliner (Nanion Technologies GmbH) APC platform. We developed a two-step cell preparation protocol that yielded cell catch rates and whole-cell breakthroughs of ∼80%, with ∼40% of these cells allowing electrical activity to be recorded. The protocol permitted formation of long-lasting (>15 min), high quality seals (>2 GΩ) in both voltage- and current-clamp modes. This enabled density of sodium, calcium, and potassium currents to be evaluated, along with dose–response curves to their respective channel inhibitors, tetrodotoxin, nifedipine, and E-4031. Thus, we show the feasibility of using the Patchliner platform for automated evaluation of the electrophysiology and pharmacology of hPSC-CMs, which will enable considerable increase in throughput for reliable and efficient drug evaluation. PMID:26906236

  3. Embryonic stem cell-derived M2-like macrophages delay cutaneous wound healing.

    PubMed

    Dreymueller, Daniela; Denecke, Bernd; Ludwig, Andreas; Jahnen-Dechent, Willi

    2013-01-01

    In adults, repair of deeply injured skin wounds results in the formation of scar tissue, whereas in embryos wounds heal almost scar-free. Macrophages are important mediators of wound healing and secrete cytokines and tissue remodeling enzymes. In contrast to host defense mediated by inflammatory M1 macrophages, wound healing and tissue repair involve regulatory M2/M2-like macrophages. Embryonic/fetal macrophages are M2-like, and this may promote scar-free wound healing. In the present study, we asked whether atopical application of ex vivo generated, embryonic stem cell-derived macrophages (ESDM) improve wound healing in mice. ESDM were tested side by side with bone marrow-derived macrophages (BMDM). Compared to BMDM, ESDM resembled a less inflammatory and more M2-like macrophage subtype as indicated by their reduced responsiveness to lipopolysaccharide, reduced expression of Toll-like receptors, and reduced bacterial phagocytosis. Despite this anti-inflammatory phenotype in cell culture, ESDM prolonged the healing of deep skin wounds even more than BMDM. Healed wounds had more scar formation compared to wounds receiving BMDM or cell-free treatment. Our data indicate that atopical application of ex vivo generated macrophages is not a suitable cell therapy of dermal wounds.

  4. Functional Neurons Generated from T Cell-Derived Induced Pluripotent Stem Cells for Neurological Disease Modeling

    PubMed Central

    Matsumoto, Takuya; Fujimori, Koki; Andoh-Noda, Tomoko; Ando, Takayuki; Kuzumaki, Naoko; Toyoshima, Manabu; Tada, Hirobumi; Imaizumi, Kent; Ishikawa, Mitsuru; Yamaguchi, Ryo; Isoda, Miho; Zhou, Zhi; Sato, Shigeto; Kobayashi, Tetsuro; Ohtaka, Manami; Nishimura, Ken; Kurosawa, Hiroshi; Yoshikawa, Takeo; Takahashi, Takuya; Nakanishi, Mahito; Ohyama, Manabu; Hattori, Nobutaka; Akamatsu, Wado; Okano, Hideyuki

    2016-01-01

    Summary Modeling of neurological diseases using induced pluripotent stem cells (iPSCs) derived from the somatic cells of patients has provided a means of elucidating pathogenic mechanisms and performing drug screening. T cells are an ideal source of patient-specific iPSCs because they can be easily obtained from samples. Recent studies indicated that iPSCs retain an epigenetic memory relating to their cell of origin that restricts their differentiation potential. The classical method of differentiation via embryoid body formation was not suitable for T cell-derived iPSCs (TiPSCs). We developed a neurosphere-based robust differentiation protocol, which enabled TiPSCs to differentiate into functional neurons, despite differences in global gene expression between TiPSCs and adult human dermal fibroblast-derived iPSCs. Furthermore, neurons derived from TiPSCs generated from a juvenile patient with Parkinson's disease exhibited several Parkinson's disease phenotypes. Therefore, we conclude that TiPSCs are a useful tool for modeling neurological diseases. PMID:26905201

  5. Nitric oxide regulates cell behavior on an interactive cell-derived extracellular matrix scaffold.

    PubMed

    Xing, Qi; Zhang, Lijun; Redman, Travis; Qi, Shaohai; Zhao, Feng

    2015-12-01

    During tissue injury and wound healing process, there are dynamic reciprocal interactions among cells, extracellular matrix (ECM), and mediating molecules which are crucial for functional tissue repair. Nitric oxide (NO) is one of the key mediating molecules that can positively regulate various biological activities involved in wound healing. Various ECM components serve as binding sites for cells and mediating molecules, and the interactions further stimulate cellular activities. Human mesenchymal stem cells (hMSCs) can migrate to the wound site and contribute to tissue regeneration through differentiation and paracrine signaling. The objective of this work was to investigate the regulatory effect of NO on hMSCs in an interactive ECM-rich microenvironment. In order to mimic the in vivo stromal environment in wound site, a cell-derived ECM scaffold that was able to release NO within the range of in vivo wound fluid NO level was fabricated. Results showed that the micro-molar level of NO released from the ECM scaffold had an inhibitory effect on cellular activities of hMSCs. The NO impaired cell growth, altered cell morphology, disrupted the F-actin organization, also decreased the expression of focal adhesion related molecules integrin α5 and paxillin. These results may contribute to the elucidation of how NO acts on hMSCs in wound healing process.

  6. Engraftment of embryonic stem cell-derived myogenic progenitors in a dominant model of muscular dystrophy.

    PubMed

    Darabi, Radbod; Baik, June; Clee, Mark; Kyba, Michael; Tupler, Rossella; Perlingeiro, Rita C R

    2009-11-01

    Muscular dystrophies (MDs) consist of a genetically heterogeneous group of disorders, recessive or dominant, characterized by progressive skeletal muscle weakening. To date, no effective treatment is available. Experimental strategies pursuing muscle regeneration through the transplantation of stem cell preparations have brought hope to patients affected by this disorder. Efficacy has been demonstrated in recessive MD models through contribution of wild-type nuclei to the muscle fiber heterokaryon; however, to date, there has been no study investigating the efficacy of a cell therapy in a dominant model of MD. We have recently demonstrated that Pax3-induced embryonic stem (ES) cell-derived myogenic progenitors are able to engraft and improve muscle function in mdx mice, a recessive mouse model for Duchenne MD. To assess whether this therapeutic effect can be extended to a dominant type of muscle disorder, here we transplanted these cells into FRG1 transgenic mice, a dominant model that has been associated with facioscapulohumeral muscular dystrophy. Our results show that Pax3-induced ES-derived myogenic progenitors are capable of significant engraftment after intramuscular or systemic transplantation into Frg1 mice. Analyses of contractile parameters revealed functional improvement in treated muscles of male mice, but not females, which are less severely affected. This study is the first to use Frg1 transgenic mice to assess muscle regeneration as well as to support the use of a cell-based therapy for autosomal dominant types of MD.

  7. Stem cell-derived exosomes as a therapeutic tool for cardiovascular disease

    PubMed Central

    Suzuki, Etsu; Fujita, Daishi; Takahashi, Masao; Oba, Shigeyoshi; Nishimatsu, Hiroaki

    2016-01-01

    Mesenchymal stem cells (MSCs) have been used to treat patients suffering from acute myocardial infarction (AMI) and subsequent heart failure. Although it was originally assumed that MSCs differentiated into heart cells such as cardiomyocytes, recent evidence suggests that the differentiation capacity of MSCs is minimal and that injected MSCs restore cardiac function via the secretion of paracrine factors. MSCs secrete paracrine factors in not only naked forms but also membrane vesicles including exosomes containing bioactive substances such as proteins, messenger RNAs, and microRNAs. Although the details remain unclear, these bioactive molecules are selectively sorted in exosomes that are then released from donor cells in a regulated manner. Furthermore, exosomes are specifically internalized by recipient cells via ligand-receptor interactions. Thus, exosomes are promising natural vehicles that stably and specifically transport bioactive molecules to recipient cells. Indeed, stem cell-derived exosomes have been successfully used to treat cardiovascular disease (CVD), such as AMI, stroke, and pulmonary hypertension, in animal models, and their efficacy has been demonstrated. Therefore, exosome administration may be a promising strategy for the treatment of CVD. Furthermore, modifications of exosomal contents may enhance their therapeutic effects. Future clinical studies are required to confirm the efficacy of exosome treatment for CVD. PMID:27679686

  8. FUS affects circular RNA expression in murine embryonic stem cell-derived motor neurons

    PubMed Central

    Errichelli, Lorenzo; Dini Modigliani, Stefano; Laneve, Pietro; Colantoni, Alessio; Legnini, Ivano; Capauto, Davide; Rosa, Alessandro; De Santis, Riccardo; Scarfò, Rebecca; Peruzzi, Giovanna; Lu, Lei; Caffarelli, Elisa; Shneider, Neil A.; Morlando, Mariangela; Bozzoni, Irene

    2017-01-01

    The RNA-binding protein FUS participates in several RNA biosynthetic processes and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Here we report that FUS controls back-splicing reactions leading to circular RNA (circRNA) production. We identified circRNAs expressed in in vitro-derived mouse motor neurons (MNs) and determined that the production of a considerable number of these circRNAs is regulated by FUS. Using RNAi and overexpression of wild-type and ALS-associated FUS mutants, we directly correlate the modulation of circRNA biogenesis with alteration of FUS nuclear levels and with putative toxic gain of function activities. We also demonstrate that FUS regulates circRNA biogenesis by binding the introns flanking the back-splicing junctions and that this control can be reproduced with artificial constructs. Most circRNAs are conserved in humans and specific ones are deregulated in human-induced pluripotent stem cell-derived MNs carrying the FUSP525L mutation associated with ALS. PMID:28358055

  9. Engraftment of embryonic stem cell-derived myogenic progenitors in a dominant model of muscular dystrophy

    PubMed Central

    Darabi, Radbod; Baik, June; Clee, Mark; Kyba, Michael; Tupler, Rossella; Perlingeiro, Rita C.R.

    2009-01-01

    Muscular dystrophies (MD) consist of a genetically heterogeneous group of disorders, recessive or dominant, characterized by progressive skeletal muscle weakening. To date, no effective treatment is available. Experimental strategies pursuing muscle regeneration through the transplantation of stem cell preparations have brought hope to patients affected by this disorder. Efficacy has been demonstrated in recessive MD models through contribution of wild-type nuclei to the muscle fiber heterokaryon, however to date, there has been no study investigating the efficacy of a cell therapy in a dominant model of MD. We have recently demonstrated that Pax3-induced embryonic stem (ES) cell- derived myogenic progenitors are able to engraft and improve muscle function in mdx mice, a recessive mouse model for Duchenne MD. To assess whether this therapeutic effect can be extended to a dominant type of muscle disorder, here we transplanted these cells into FRG1 transgenic mice, a dominant model that has been associated with Facioscapulohumeral muscular dystrophy. Our results show that Pax3-induced ES-derived myogenic progenitors are capable of significant engraftment after intramuscular or systemic transplantation into Frg1 mice. Analyses of contractile parameters revealed functional improvement in treated muscles of male mice, but not females, which are less severely affected. This study is the first to use Frg1 transgenic mice to assess muscle regeneration as well as to support the use of a cell-based therapy for autosomal dominant types of MD. PMID:19682990

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

    SciTech Connect

    Robinson, Claire; Kolb, Andreas F.

    2009-02-01

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

  11. In vitro patterning of pluripotent stem cell-derived intestine recapitulates in vivo human development.

    PubMed

    Tsai, Yu-Hwai; Nattiv, Roy; Dedhia, Priya H; Nagy, Melinda S; Chin, Alana M; Thomson, Matthew; Klein, Ophir D; Spence, Jason R

    2017-03-15

    The intestine plays a central role in digestion, nutrient absorption and metabolism, with individual regions of the intestine having distinct functional roles. Many examples of region-specific gene expression in the adult intestine are known, but how intestinal regional identity is established during development is a largely unresolved issue. Here, we have identified several genes that are expressed in a region-specific manner in the developing human intestine. Using human embryonic stem cell-derived intestinal organoids, we demonstrate that the duration of exposure to active FGF and WNT signaling controls regional identity. Short-term exposure to FGF4 and CHIR99021 (a GSK3β inhibitor that stabilizes β-catenin) resulted in organoids with gene expression patterns similar to developing human duodenum, whereas longer exposure resulted in organoids similar to ileum. When region-specific organoids were transplanted into immunocompromised mice, duodenum-like organoids and ileum-like organoids retained their regional identity, demonstrating that regional identity of organoids is stable after initial patterning occurs. This work provides insights into the mechanisms that control regional specification of the developing human intestine and provides new tools for basic and translational research.

  12. Stromal cell derived factor-1 (SDF-1) targeting reperfusion reduces myocardial infarction in isolated rat hearts.

    PubMed

    Jang, Young-Ho; Kim, June-Hong; Ban, Changill; Ahn, Kyohan; Cheong, Jae-Hun; Kim, Hyung-Hoi; Kim, Jung-Soo; Park, Yong-Hyun; Kim, Jun; Chun, Kook-Jin; Lee, Gyeong-Ho; Kim, Miju; Kim, Cheolmin; Xu, Zhelong

    2012-10-01

    Recent studies have shown that stromal cell derived factor-1 (SDF-1), first known as a cytokine involved in recruiting stem cells into injured organs, confers myocardial protection in myocardial infarction, which is not dependent on stem cell recruitment but related with modulation of ischemia-reperfusion (I/R) injury. However, the effect of SDF has been studied only in a preischemic exposure model, which is not clinically relevant if SDF is to be used as a therapeutic agent. Our study was aimed at evaluating whether or not SDF-1 confers cardioprotection during the reperfusion period. Hearts from SD rats were isolated and perfused with the Langendorff system. Proximal left coronary artery ligation, reperfusion, and SDF perfusion in KH buffer was done according to study protocol. Area of necrosis (AN) relative to area at risk (AR) was the primary endpoint of the study. Significant reduction of AN/AR by SDF in an almost dose-dependent manner was noted during both the preischemic exposure and reperfusion periods. In particular, infusion of a high concentration of SDF (25 nM/L) resulted in a dramatic reduction of infarct size, which was greater than that achieved with ischemic pre- or postconditioning. SDF perfusion during reperfusion was associated with a similar significant reduction of infarct size as preischemic SDF exposure. Further studies are warranted to assess the potential of SDF as a therapeutic agent for reducing I/R injury in clinical practice.

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

    PubMed

    Sakai, Hiroki; Yokoyama, Takeshi; Abe, Hiroaki; Fujii, Tsuguru; Suzuki, Masataka G

    2013-01-01

    In Bombyx mori, polar body nuclei are observed until 9 h 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 ~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.

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

    PubMed

    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.

  15. FUS affects circular RNA expression in murine embryonic stem cell-derived motor neurons.

    PubMed

    Errichelli, Lorenzo; Dini Modigliani, Stefano; Laneve, Pietro; Colantoni, Alessio; Legnini, Ivano; Capauto, Davide; Rosa, Alessandro; De Santis, Riccardo; Scarfò, Rebecca; Peruzzi, Giovanna; Lu, Lei; Caffarelli, Elisa; Shneider, Neil A; Morlando, Mariangela; Bozzoni, Irene

    2017-03-30

    The RNA-binding protein FUS participates in several RNA biosynthetic processes and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Here we report that FUS controls back-splicing reactions leading to circular RNA (circRNA) production. We identified circRNAs expressed in in vitro-derived mouse motor neurons (MNs) and determined that the production of a considerable number of these circRNAs is regulated by FUS. Using RNAi and overexpression of wild-type and ALS-associated FUS mutants, we directly correlate the modulation of circRNA biogenesis with alteration of FUS nuclear levels and with putative toxic gain of function activities. We also demonstrate that FUS regulates circRNA biogenesis by binding the introns flanking the back-splicing junctions and that this control can be reproduced with artificial constructs. Most circRNAs are conserved in humans and specific ones are deregulated in human-induced pluripotent stem cell-derived MNs carrying the FUS(P525L) mutation associated with ALS.

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

    PubMed Central

    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

  17. Accurate prediction of drug-induced liver injury using stem cell-derived populations.

    PubMed

    Szkolnicka, Dagmara; Farnworth, Sarah L; Lucendo-Villarin, Baltasar; Storck, Christopher; Zhou, Wenli; Iredale, John P; Flint, Oliver; Hay, David C

    2014-02-01

    Despite major progress in the knowledge and management of human liver injury, there are millions of people suffering from chronic liver disease. Currently, the only cure for end-stage liver disease is orthotopic liver transplantation; however, this approach is severely limited by organ donation. Alternative approaches to restoring liver function have therefore been pursued, including the use of somatic and stem cell populations. Although such approaches are essential in developing scalable treatments, there is also an imperative to develop predictive human systems that more effectively study and/or prevent the onset of liver disease and decompensated organ function. We used a renewable human stem cell resource, from defined genetic backgrounds, and drove them through developmental intermediates to yield highly active, drug-inducible, and predictive human hepatocyte populations. Most importantly, stem cell-derived hepatocytes displayed equivalence to primary adult hepatocytes, following incubation with known hepatotoxins. In summary, we have developed a serum-free, scalable, and shippable cell-based model that faithfully predicts the potential for human liver injury. Such a resource has direct application in human modeling and, in the future, could play an important role in developing renewable cell-based therapies.

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

    PubMed

    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.

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

    PubMed Central

    Sakai, Hiroki; Yokoyama, Takeshi; Abe, Hiroaki; Fujii, Tsuguru; Suzuki, Masataka G.

    2013-01-01

    In Bombyx mori, polar body nuclei are observed until 9 h 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 ~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. PMID:24027530

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

    EPA Science Inventory

    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...

  1. Identification of Autographa californica nucleopolyhedrovirus ac93 as a core gene and its requirement for intranuclear microvesicle formation and nuclear egress of nucleocapsids.

    PubMed

    Yuan, Meijin; Huang, Zhenqiu; Wei, Denghui; Hu, Zhaoyang; Yang, Kai; Pang, Yi

    2011-11-01

    Autographa californica nucleopolyhedrovirus (AcMNPV) orf93 (ac93) is a highly conserved uncharacterized gene that is found in all of the sequenced baculovirus genomes except for Culex nigripalpus NPV. In this report, using bioinformatics analyses, ac93 and odv-e25 (ac94) were identified as baculovirus core genes and thus p33-ac93-odv-e25 represent a cluster of core genes. To investigate the role of ac93 in the baculovirus life cycle, an ac93 knockout AcMNPV bacmid was constructed via homologous recombination in Escherichia coli. Fluorescence and light microscopy showed that the AcMNPV ac93 knockout did not spread by infection, and titration assays confirmed a defect in budded virus (BV) production. However, deletion of ac93 did not affect viral DNA replication. Electron microscopy indicated that ac93 was required for the egress of nucleocapsids from the nucleus and the formation of intranuclear microvesicles, which are precursor structures of occlusion-derived virus (ODV) envelopes. Immunofluorescence analyses showed that Ac93 was concentrated toward the cytoplasmic membrane in the cytoplasm and in the nuclear ring zone in the nucleus. Western blot analyses showed that Ac93 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV. Our results suggest that ac93, although not previously recognized as a core gene, is one that plays an essential role in the formation of the ODV envelope and the egress of nucleocapsids from the nucleus.

  2. Microvesicles released from fat-laden cells promote activation of hepatocellular NLRP3 inflammasome: A pro-inflammatory link between lipotoxicity and non-alcoholic steatohepatitis

    PubMed Central

    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. PMID:28249038

  3. An overview of the role of microparticles/microvesicles in blood components: Are they clinically beneficial or harmful?

    PubMed

    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

  4. Cancer

    MedlinePlus

    ... Two kinds of lymphocytes can attack and kill cancer cells: T-cells and B-cells. Immunotherapy aims to boost the ability of the T-cell and B-cell lymphocytes to kill cancer. This kind of therapy can also be used ...

  5. Pancreatic Satellite Cells Derived Galectin-1 Increase the Progression and Less Survival of Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Gao, Jun; Wang, Sen; Ye, Nianyuan; Li, Ping; Gao, Sujun; Miao, Yi; Wang, Daorong; Jiang, Kuirong

    2014-01-01

    Background Galectin-1, a member of carbohydrate-binding proteins with a polyvalent function on tumor progression, was found strongly expressed in pancreatic satellite cells (PSCs), which partner in crime with cancer cells and promote the development of pancreatic ductal adenocarcinoma (PDAC). We evaluated the effects of PSCs derived Galectin-1 on the progression of PDAC, as well as the tumor establishment and development in mouse xenografts. Methods The relationship between immunohistochemistry staining intensity of Galectin-1 and clinicopathologic variables were assessed in 66 PDAC tissues, 18 chronic pancreatitis tissues and 10 normal controls. The roles of PSCs isolated from PDAC and normal pancreas on the proliferative activity, MMP2 and MMP9 expression, and the invasion of CFPAC-1 in the co-cultured system, as well as on the tumor establishment and development in mouse xenografts by mixed implanting with CFPAC-1 subcutaneously were evaluated. Results Galectin-1 expression was gradually increased from normal pancreas (negative), chronic pancreatitis (weak) to PDAC (strong), in which Galectin-1 expression was also increased from well, moderately to poorly differentiated PDAC. Galectin-1 staining intensity of pancreatic cancer tissue was associated with increase in tumor size, lymph node metastasis, perineural invasion and differentiation and UICC stage, and served as the independent prognostic indicator of poor survival of pancreatic cancer. In vitro and in vivo experiments indicated that TGF-β1 upregulated Galectin-1 expression in PSCs, which could further promotes the proliferative activity, MMP2 and MMP9 expression, and invasion of pancreatic cancer cells, as well as the tumor establishment and growth. Conclusion Galectin-1 expression in stromal cells of pancreatic cancer suggests that this protein plays a role in the promotion of cancer cells invasion and metastasis and provides a therapeutic target for the treatment of pancreatic cancer. PMID:24595374

  6. Tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras.

    PubMed

    Karagenç, Levent; Sandikci, Mustafa

    2010-01-01

    The objective of the current study was to determine the tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras. Quail-chick chimeras were constructed by transferring dissociated cells from the area opaca of the stage X-XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. The distribution of quail cells in embryonic as well as extra-embryonic tissues of the recipient embryo were examined using the QCPN monoclonal antibody after 6 days of incubation in serial sections taken at 100-mum intervals. Data gathered in the present study demonstrated that, when introduced into the subgerminal cavity of a recipient embryo, cells of the area opaca are able to populate not only extra-embryonic structures such as the amnion and the yolk sac, but also various embryonic tissues derived from the ectoderm and less frequently the mesoderm. Ectodermal chimerism was confined mainly to the head region and was observed in tissues derived from the neural ectoderm and the surface ectoderm, including the optic cup, diencephalon and lens. Although the possibility of random incorporation of transplanted cells into these embryonic structures cannot be excluded, these results would suggest that area opaca, a peripheral ring of cells in the avian embryo destined to form the extra-embryonic ectoderm and endoderm of the yolk sac, might harbor cells that have the potential to give rise to various cell types in the recipient chick embryo, including those derived from the surface ectoderm and neural ectoderm.

  7. Analysis of Signaling Endosome Composition and Dynamics Using SILAC in Embryonic Stem Cell-Derived Neurons*

    PubMed Central

    Debaisieux, Solène; Encheva, Vesela; Chakravarty, Probir; Snijders, Ambrosius P.; Schiavo, Giampietro

    2016-01-01

    Neurons require efficient transport mechanisms such as fast axonal transport to ensure neuronal homeostasis and survival. Neurotrophins and their receptors are conveyed via fast axonal retrograde transport of signaling endosomes to the soma, where they elicit transcriptional responses. Despite the essential roles of signaling endosomes in neuronal differentiation and survival, little is known about their molecular identity, dynamics, and regulation. Gaining a better mechanistic understanding of these organelles and their kinetics is crucial, given the growing evidence linking vesicular trafficking deficits to neurodegeneration. Here, we exploited an affinity purification strategy using the binding fragment of tetanus neurotoxin (HCT) conjugated to monocrystalline iron oxide nanoparticles (MIONs), which in motor neurons, is transported in the same carriers as neurotrophins and their receptors. To quantitatively assess the molecular composition of HCT-containing signaling endosomes, we have developed a protocol for triple Stable Isotope Labeling with Amino acids in Cell culture (SILAC) in embryonic stem cell-derived motor neurons. After HCT internalization, retrograde carriers were magnetically isolated at different time points and subjected to mass-spectrometry and Gene Ontology analyses. This purification strategy is highly specific, as confirmed by the presence of essential regulators of fast axonal transport in the make-up of these organelles. Our results indicate that signaling endosomes undergo a rapid maturation with the acquisition of late endosome markers following a specific time-dependent kinetics. Strikingly, signaling endosomes are specifically enriched in proteins known to be involved in neurodegenerative diseases and neuroinfection. Moreover, we highlighted the presence of novel components, whose precise temporal recruitment on signaling endosomes might be essential for proper sorting and/or transport of these organelles. This study provides the first

  8. Insulin - producing cells derived from stem cells: recent progress and future directions

    PubMed Central

    Santana, A; Enseñat - Waser, R; Arribas, Maria Isabel; Reig, J A; Roche, E

    2006-01-01

    Type 1 diabetes is characterized by the selective destruction of pancreatic β-cells caused by an autoimmune attack. Type 2 diabetes is a more complex pathology which, in addition to β-cell loss caused by apoptotic programs, includes β-cell dedifferentiation and peripheric insulin resistance. β-Cells are responsible for insulin production, storage and secretion in accordance to the demanding concentrations of glucose and fatty acids. The absence of insulin results in death and therefore diabetic patients require daily injections of the hormone for survival. However, they cannot avoid the appearance of secondary complications affecting the peripheral nerves as well as the eyes, kidneys and cardiovascular system. These afflictions are caused by the fact that external insulin injection does not mimic the tight control that pancreaticderived insulin secretion exerts on the body’s glycemia. Restoration of damaged β-cells by transplantation from exogenous sources or by endocrine pancreas regeneration would be ideal therapeutic options. In this context, stem cells of both embryonic and adult origin (including β-cell/islet progenitors) offer some interesting alternatives, taking into account the recent data indicating that these cells could be the building blocks from which insulin secreting cells could be generated in vitro under appropriate culture conditions. Although in many cases insulin-producing cells derived from stem cells have been shown to reverse experimentally induced diabetes in animal models, several concerns need to be solved before finding a definite medical application. These refer mainly to the obtainment of a cell population as similar as possible to pancreatic β-cells, and to the problems related with the immune compatibility and tumor formation. This review will summarize the different approaches that have been used to obtain insulin-producing cells from embryonic and adult stem cells, and the main problems that hamper the clinical

  9. Ceruloplasmin copper induces oxidant damage by a redox process utilizing cell-derived superoxide as reductant

    NASA Technical Reports Server (NTRS)

    Mukhopadhyay, C. K.; Fox, P. L.

    1998-01-01

    Oxidative damage by transition metals bound to proteins may be an important pathogenic mechanism. Ceruloplasmin (Cp) is a Cu-containing plasma protein thought to be involved in oxidative modification of lipoproteins. We have previously shown that Cp increased cell-mediated low-density lipoprotein (LDL) oxidation by a process requiring cell-derived superoxide, but the underlying chemical mechanism(s) is (are) unknown. We now show that superoxide reduction of Cp Cu is a critical reaction in cellular LDL oxidation. By bathocuproine disulfonate (BCS) binding and by superoxide utilization, we showed that exogenous superoxide reduces a single Cp Cu atom, the same Cu required for LDL oxidation. The Cu atom remained bound to Cp during the redox cycle. Three avenues of evidence showed that vascular cells reduce Cp Cu by a superoxide-dependent process. The 2-fold higher rate of Cp Cu reduction by smooth muscle cells (SMC) compared to endothelial cells (EC) was consistent with their relative rates of superoxide release. Furthermore, Cp Cu reduction by cells was blocked by Cu,Zn superoxide dismutase (SOD1). Finally, the level of superoxide produced by EC and SMC was sufficient to cause the amount of Cu reduction observed. An important role of Cp Cu reduction in LDL oxidation was suggested by results showing that SOD1 inhibited Cp Cu reduction and LDL oxidation by SMC with equal potency, while tumor necrosis factor-alpha stimulated both processes. In summary, these results show that superoxide is a critical cellular reductant of divalent transition metals involved in oxidation, and that protein-bound Cu is a substrate for this reaction. The role of these mechanisms in oxidative processes in vivo has yet to be defined.

  10. Fetal stromal niches enhance human embryonic stem cell-derived hematopoietic differentiation and globin switch.

    PubMed

    Lee, King Yiu; Fong, Benny Shu Pan; Tsang, Kam Sze; Lau, Tze Kin; Ng, Pak Cheung; Lam, Audrey Carmen; Chan, Kathy Yuen Yee; Wang, Chi Chiu; Kung, Hsiang Fu; Li, Chi Kong; Li, Karen

    2011-01-01

    Hematopoiesis during mammalian embryonic development has been perceived as a migratory phenomenon, from the yolk sac blood island to the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), and subsequently, the fetal bone marrow. In this study, we investigated the effects of primary stromal cells from fetal hematopoietic niches and their conditioned media (CM), applied singly or in sequential orders, on induction of human embryonic stem cells, H1, H9, and H14 lines, to hematopoietic cells. Our results demonstrated that stromal support of FL, AGM + FL, and AGM + FL + fetal bone marrow significantly increased the proliferation of embryoid bodies (EB) at day 18 of hematopoietic induction in the presence of thrombopoietin, stem cell factor, and Flt-3 ligand. AGM + FL also increased hematopoietic colony-forming unit (CFU) formation. CM did not enhance EB proliferation but CM of FL and AGM + FL significantly increased the density of total CFU and early erythroid (burst-forming unit) progenitors. Increased commitment to the hematopoietic lineage was demonstrated by enhanced expressions of CD45, alpha-, beta-, and gamma-globins in CFU at day 32, compared with EB at day 18. CM of FL significantly increased these globin expressions, indicating enhanced switches from embryonic to fetal and adult erythropoiesis. Over 50% and 10% of cells derived from CFU expressed CD45 and beta-globin proteins, respectively. Expressions of hematopoietic regulatory genes (Bmi-1, β-Catenin, Hox B4, GATA-1) were increased in EB or CFU cultures supported by FL or sequential CM. Our study has provided a strategy for derivation of hematopoietic cells from embryonic stem cells under the influence of primary hematopoietic niches and CM, particularly the FL.

  11. Knockout of Endothelial Cell-Derived Endothelin-1 Attenuates Skin Fibrosis but Accelerates Cutaneous Wound Healing

    PubMed Central

    Makino, Katsunari; Jinnin, Masatoshi; Aoi, Jun; Kajihara, Ikko; Makino, Takamitsu; Fukushima, Satoshi; Sakai, Keisuke; Nakayama, Kazuhiko; Emoto, Noriaki; Yanagisawa, Masashi; Ihn, Hironobu

    2014-01-01

    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. PMID:24853267

  12. Profiling ethanol-targeted transcription factors in human carcinoma cell-derived embryoid bodies.

    PubMed

    Mandal, Chanchal; Halder, Debasish; Chai, Jin Choul; Lee, Young Seek; Jung, Kyoung Hwa; Chai, Young Gyu

    2016-01-15

    Fetal alcohol spectrum disorder is a collective term that represents fetal abnormalities associated with maternal alcohol consumption. Prenatal alcohol exposure and related anomalies are well characterized, but the molecular mechanism behind this phenomenon is not yet understood. Few insights have been gained from genetic and epigenetic studies of fetal alcohol spectrum disorder. Our aim was to profile the important molecular regulators of ethanol-related alterations of the genome. For this purpose, we have analyzed the gene expression pattern of human carcinoma cell-derived embryoid bodies in the absence or presence of ethanol. A cDNA microarray analysis was used to profile mRNA expression in embryoid bodies at day 7 with or without ethanol treatment. A total of 493 differentially expressed genes were identified in response to 50 mM ethanol exposure. Of these, 111 genes were up-regulated, and 382 were down-regulated. Gene ontology term enrichment analysis revealed that these genes are involved in important biological processes: neurological system processes, cognition, behavior, sensory perception of smell, taste and chemical stimuli and synaptic transmission. Similarly, the enrichment of disease-related genes included relevant categories such as neurological diseases, developmental disorders, skeletal and muscular disorders, and connective tissue disorders. Furthermore, we have identified a group of 26 genes that encode transcription factors. We validated the relative gene expression of several transcription factors using quantitative real time PCR. We hope that our study substantially contributes to the understanding of the molecular mechanisms underlying the pathology of alcohol-mediated anomalies and facilitates further research.

  13. A novel broadband impedance method for detection of cell-derived microparticles

    PubMed Central

    Lvovich, Vadim; Srikanthan, Sowmya; Silverstein, Roy L.

    2010-01-01

    A novel label-free method is presented to detect and quantify cell-derived microparticles (MPs) by the electrochemical potential-modulated electrochemical impedance spectroscopy (EIS). MPs are present in elevated concentrations during pathological conditions and play a major role in the establishment and pathogenesis of many diseases. Considering this, accurate detection and quantification of MPs is very important in clinical diagnostics and therapeutics. A combination of bulk solution electrokinetic sorting and interfacial impedance responses allows achieving detection limits as low as several MPs per µL. By fitting resulting EIS spectra with an equivalent electrical circuit, the bulk solution electrokinetic and interfacial impedance responses were characterized. In the bulk solution two major relaxations were prominent - β-relaxation in low MHz region due to the MP capacitive membrane bridging, and α-relaxation at ∼ 10 kHz due to counter ions diffusion. At low frequencies (10-0.1 Hz) at electrochemical potentials exceeding −100 mV, a facile interfacial Faradaic process of oxidation in MPs coupled with diffusion and non Faradaic double layer charging dominate, probably due to oxidation of phospholipids and/or proteins on the MP surface and MP lysis. Buffer influence on the MP detection demonstrated that that a relatively low conductivity Tyrode’s buffer background solution is preferential for the MP electrokinetic separation and characterization. This study also demonstrated that standard laboratory methods such as flow cytometry underestimate MP concentrations, especially those with smaller average sizes, by as much as a factor of 2 to 40. PMID:20729061

  14. Cell-Derived Nanoparticles are Endogenous Modulators of Sepsis with Therapeutic Potential.

    PubMed

    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

    2017-02-22

    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.

  15. Calcium activated potassium channel expression during human iPS cell-derived neurogenesis.

    PubMed

    Linta, Leonhard; Boeckers, Tobias M; Kleger, Alexander; Liebau, Stefan

    2013-07-01

    The family of calcium activated potassium channels of low and intermediate conductance, known as SK channels, consists of four members (SK1-4). These channels are widely expressed throughout the organism and involved in various cellular processes, such as the afterhyperpolarization in excitable cells but also in differentiation processes of various tissues. To date, the role of SK channels in developmental processes has been merely a marginal focus of investigation, although it is well accepted that cell differentiation and maturation affect the expression patterns of certain ion channels. Recently, several studies from our laboratory delineated the influence of SK channel expression and their respective activity on cytoskeletal reorganization in neural and pluripotent stem cells and regulation of cell fate determination toward the cardiac lineage in human and mouse pluripotent stem cells. Herein, we have now analyzed SK channel expression patterns and distribution at various stages of human induced pluripotent stem cell-derived neurogenesis particularly focusing on undifferentiated iPS cells, neural progenitors and mature neurons. All family members could be detected starting at the iPS cell level and were differentially expressed during the subsequent maturation process. Intriguingly, we found obvious discrepancies between mRNA and protein expression pointing toward a complex regulatory mechanism. Inhibition of SK channels with either apamin or clotrimazol did not have any significant effects on the speed or amount of neurogenesis in vitro. The abundance and specific regulation of SK channel expression during iPS cell differentiation indicates distinct roles of these ion channels not only for the cardiac but also for neuronal cell differentiation and in vitro neurogenesis.

  16. Enhanced Human-Induced Pluripotent Stem Cell Derived Cardiomyocyte Maturation Using a Dual Microgradient Substrate

    PubMed Central

    2016-01-01

    Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) raise many possibilities for cardiac research but they exhibit an immature phenotype, which influences experimental outcomes. The aim of our research is to investigate the effects of a topographical gradient substrate on the morphology and function of commercially available hiPSC-CM. The lateral dimensions the microgrooves on the substrate varied from 8 to 100 μm space between the 8 μm grooves on one axis and from ∼5 nm to ∼1 μm in depth on the other axis. Cells were seeded homogeneously across the substrate and according to the manufacturers protocols. At days 4 and 10, measures of eccentricity, elongation, orientation, sarcomere length (SL), and contractility of the hiPSC-CM were taken. Only the deepest and widest region (8–30 μm wide and 0.85–1 μm deep) showed a significantly higher percentage of hiPSC-CM with an increased eccentricity (31.3 ± 6.4%), elongation (10.4 ± 4.3%), and orientation (<10°) (32.1 ± 2.7%) when compared with the control (flat substrate) (15.8 ± 5.0%, 3.4 ± 2.7%, and 10.6 ± 1.1%, respectively). Additionally, during stimulus-induced contraction, the relaxation phase of the twitch was prolonged (400 ms) compared to nonelongated cells (200 ms). These findings support the potential use of dual microgradient substrates to investigate substrate topographies that stimulate migration and/or maturation of hiPSC-CM. PMID:27990488

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

    PubMed

    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.

  18. ROCK Inhibition Extends Passage of Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium.

    PubMed

    Croze, Roxanne H; Buchholz, David E; Radeke, Monte J; Thi, William J; Hu, Qirui; Coffey, Peter J; Clegg, Dennis O

    2014-09-01

    Human embryonic stem cells (hESCs) offer a potentially unlimited supply of cells for emerging cell-based therapies. Unfortunately, the process of deriving distinct cell types can be time consuming and expensive. In the developed world, age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with more than 7.2 million people afflicted in the U.S. alone. Both hESC-derived retinal pigmented epithelium (hESC-RPE) and induced pluripotent stem cell-derived RPE (iPSC-RPE) are being developed for AMD therapies by multiple groups, but their potential for expansion in culture is limited. To attempt to overcome this passage limitation, we examined the involvement of Rho-associated, coiled-coil protein kinase (ROCK) in hESC-RPE and iPSC-RPE culture. We report that inhibiting ROCK1/2 with Y-27632 allows extended passage of hESC-RPE and iPSC-RPE. Microarray analysis suggests that ROCK inhibition could be suppressing an epithelial-to-mesenchymal transition through various pathways. These include inhibition of key ligands of the transforming growth factor-β pathway (TGFB1 and GDF6) and Wnt signaling. Two important processes are affected, allowing for an increase in hESC-RPE expansion. First, ROCK inhibition promotes proliferation by inducing multiple components that are involved in cell cycle progression. Second, ROCK inhibition affects many pathways that could be converging to suppress RPE-to-mesenchymal transition. This allows hESC-RPE to remain functional for an extended but finite period in culture.

  19. Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

    SciTech Connect

    Yang, Yingbin; Cai, Shaoxi; Yang, Li; Yu, Shuhui; Jiang, Jiahuan; Yan, Xiaoqing; Zhang, Haoxing; Liu, Lan; Liu, Qun; Du, Jun; Cai, Shaohui; Sung, K.L. Paul

    2010-12-10

    Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

  20. Structure and chromosomal localization of the human stromal cell-derived factor 1 (SDF1) gene

    SciTech Connect

    Shirozu, Michio; Takano, Toru; Tada, Hideaki; Honjo, Tasuku

    1995-08-10

    Stromal cell-derived factors 1{alpha} and 1{beta} are small cytokines belonging to the intercrine CXC subfamily and originally isolated from a murine bone-marrow stroma cell line by the signal sequence trap method. cDNA and genomic clones of human SDF1{alpha} and SDF1{beta} (SDF1A and SDF1B) were isolated and characterized. cDNAs of SDF1{alpha} and SDF1{beta} encode proteins of 89 and 93 amino acids, respectively. SDF1{alpha} and SDF1{beta} sequences are more than 92% identical to those of the human counterparts. The genomic structure of the SDF1 gene revealed that human SDF1{alpha} and SDF1{beta} are encoded by a single gene and arise by alternative splicing. SDF1{alpha} and SDF1{beta} are encoded by 3 and 4 exons, respectively. Ubiquitous expression of the SDF1 gene, except in blood cells, was consistent with the presence of the GC-rich sequence in the 5{prime}-flanking region of the SDF1 gene, as is often the case in the {open_quotes}housekeeping{close_quotes} genes. Although genes encoding other members of the intercrine family are localized on chromosome 4q or 17q, the human SDF1 gene was mapped to chromosome 10q by fluorescence in situ hybridization. Strong evolutionary conservation and unique chromosomal localization of the SDF1 gene suggest that SDF1{alpha} and SDF1{beta} may have important functions distinct from those of other members of the intercrine family. 37 refs., 5 figs.

  1. Continuous delivery of stromal cell-derived factor-1 from alginate scaffolds accelerates wound healing.

    PubMed

    Rabbany, Sina Y; Pastore, Joseph; Yamamoto, Masaya; Miller, Tim; Rafii, Shahin; Aras, Rahul; Penn, Marc

    2010-01-01

    Proper wound diagnosis and management is an increasingly important clinical challenge and is a large and growing unmet need. Pressure ulcers, hard-to-heal wounds, and problematic surgical incisions are emerging at increasing frequencies. At present, the wound-healing industry is experiencing a paradigm shift towards innovative treatments that exploit nanotechnology, biomaterials, and biologics. Our study utilized an alginate hydrogel patch to deliver stromal cell-derived factor-1 (SDF-1), a naturally occurring chemokine that is rapidly overexpressed in response to tissue injury, to assess the potential effects SDF-1 therapy on wound closure rates and scar formation. Alginate patches were loaded with either purified recombinant human SDF-1 protein or plasmid expressing SDF-1 and the kinetics of SDF-1 release were measured both in vitro and in vivo in mice. Our studies demonstrate that although SDF-1 plasmid- and protein-loaded patches were able to release therapeutic product over hours to days, SDF-1 protein was released faster (in vivo K(d) 0.55 days) than SDF-1 plasmid (in vivo K(d) 3.67 days). We hypothesized that chronic SDF-1 delivery would be more effective in accelerating the rate of dermal wound closure in Yorkshire pigs with acute surgical wounds, a model that closely mimics human wound healing. Wounds treated with SDF-1 protein (n = 10) and plasmid (n = 6) loaded patches healed faster than sham (n = 4) or control (n = 4). At day 9, SDF-1-treated wounds significantly accelerated wound closure (55.0 +/- 14.3% healed) compared to nontreated controls (8.2 +/- 6.0%, p < 0.05). Furthermore, 38% of SDF-1-treated wounds were fully healed at day 9 (vs. none in controls) with very little evidence of scarring. These data suggest that patch-mediated SDF-1 delivery may ultimately provide a novel therapy for accelerating healing and reducing scarring in clinical wounds.

  2. The nature of activatory and tolerogenic dendritic cell-derived signal II

    PubMed Central

    Bakdash, Ghaith; Sittig, Simone P.; van Dijk, Tjeerd; Figdor, Carl G.; de Vries, I. Jolanda M.

    2013-01-01

    Dendritic cells (DCs) are central in maintaining the intricate balance between immunity and tolerance by orchestrating adaptive immune responses. Being the most potent antigen presenting cells, DCs are capable of educating naïve T cells into a wide variety of effector cells ranging from immunogenic CD4+ T helper cells and cytotoxic CD8+ T cells to tolerogenic regulatory T cells. This education is based on three fundamental signals. Signal I, which is mediated by antigen/major histocompatibility complexes binding to antigen-specific T cell receptors, guarantees antigen specificity. The co-stimulatory signal II, mediated by B7 family molecules, is crucial for the expansion of the antigen-specific T cells. The final step is T cell polarization by signal III, which is conveyed by DC-derived cytokines and determines the effector functions of the emerging T cell. Although co-stimulation is widely recognized to result from the engagement of T cell-derived CD28 with DC-expressed B7 molecules (CD80/CD86), other co-stimulatory pathways have been identified. These pathways can be divided into two groups based on their impact on primed T cells. Whereas pathways delivering activatory signals to T cells are termed co-stimulatory pathways, pathways delivering tolerogenic signals to T cells are termed co-inhibitory pathways. In this review, we discuss how the nature of DC-derived signal II determines the quality of ensuing T cell responses and eventually promoting either immunity or tolerance. A thorough understanding of this process is instrumental in determining the underlying mechanism of disorders demonstrating distorted immunity/tolerance balance, and would help innovating new therapeutic approaches for such disorders. PMID:23450201

  3. Cancer

    MedlinePlus

    ... weaken. Talk with family, friends, or a support group about your feelings. Work with your health care providers throughout your treatment. Helping yourself can make you feel more in control. Support Groups The diagnosis and treatment of cancer often causes ...

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

    DTIC Science & Technology

    2013-09-01

    formation, are associated with gastrointestinal problems when taken orally and may cause osteonecrosis in cancer patients and bone pain in other...expression of osteoblastic phenotypic markers on iPS derived MSC cultured on nanotopographic biofilms . While we have not yet examined the effect

  5. G-protein Coupled Receptor Signaling in Pluripotent Stem Cell-derived Cardiovascular Cells: Implications for Disease Modeling

    PubMed Central

    Dolatshad, Nazanin F.; Hellen, Nicola; Jabbour, Richard J.; Harding, Sian E.; Földes, Gabor

    2015-01-01

    Human pluripotent stem cell derivatives show promise as an in vitro platform to study a range of human cardiovascular diseases. A better understanding of the biology of stem cells and their cardiovascular derivatives will help to understand the strengths and limitations of this new model system. G-protein coupled receptors (GPCRs) are key regulators of stem cell maintenance and differentiation and have an important role in cardiovascular cell signaling. In this review, we will therefore describe the state of knowledge concerning the regulatory role of GPCRs in both the generation and function of pluripotent stem cell derived-cardiomyocytes, -endothelial, and -vascular smooth muscle cells. We will consider how far the in vitro disease models recapitulate authentic GPCR signaling and provide a useful basis for discovery of disease mechanisms or design of therapeutic strategies. PMID:26697426

  6. Sensitive detection and estimation of cell-derived peroxynitrite fluxes using fluorescein-boronate.

    PubMed

    Rios, Natalia; Piacenza, Lucía; Trujillo, Madia; Martínez, Alejandra; Demicheli, Verónica; Prolo, Carolina; Álvarez, María Noel; López, Gloria V; Radi, Rafael

    2016-12-01

    more sensitive than the coumarin boronate due to a higher molar absorption coefficient and quantum yield. Overall, our results show that Fl-B is a kinetically selective and highly sensitive probe for the direct detection of cell-derived peroxynitrite.

  7. Producing fully ES cell-derived mice from eight-cell stage embryo injections.

    PubMed

    DeChiara, Thomas M; Poueymirou, William T; Auerbach, Wojtek; Frendewey, David; Yancopoulos, George D; Valenzuela, David M

    2010-01-01

    In conventional methods for the generation of genetically modified mice, gene-targeted embryonic stem (ES) cells are injected into blastocyst-stage embryos or are aggregated with morula-stage embryos, which are then transferred to the uterus of a surrogate mother. F0 generation mice born from the embryos are chimeras composed of genetic contributions from both the modified ES cells and the recipient embryos. Obtaining a mouse strain that carries the gene-targeted mutation requires breeding the chimeras to transmit the ES cell genetic component through the germ line to the next (F1) generation (germ line transmission, GLT). To skip the chimera stage, we developed the VelociMouse method, in which injection of genetically modified ES cells into eight-cell embryos followed by maturation to the blastocyst stage and transfer to a surrogate mother produces F0 generation mice that are fully derived from the injected ES cells and exhibit a 100% GLT efficiency. The method is simple and flexible. Both male and female ES cells can be introduced into the eight-cell embryo by any method of injection or aggregation and using all ES cell and host embryo combinations from inbred, hybrid, and outbred genetic backgrounds. The VelociMouse method provides several unique opportunities for shortening project timelines and reducing mouse husbandry costs. First, as VelociMice exhibit 100% GLT, there is no need to test cross chimeras to establish GLT. Second, because the VelociMouse method permits efficient production of ES cell-derived mice from female ES cells, XO ES cell subclones, identified by screening for spontaneous loss of the Y chromosome, can be used to generate F0 females that can be bred with isogenic F0 males derived from the original targeted ES cell clone to obtain homozygous mutant mice in the F1 generation. Third, as VelociMice are genetically identical to the ES cells from which they were derived, the VelociMouse method opens up myriad possibilities for creating mice with

  8. Comparison of mesenchymal stem cells derived from gingival tissue and periodontal ligament in different incubation conditions.

    PubMed

    Yang, Hao; Gao, Li-Na; An, Ying; Hu, Cheng-Hu; Jin, Fang; Zhou, Jun; Jin, Yan; Chen, Fa-Ming

    2013-09-01

    Gingival tissue-derived mesenchymal stem cells (MSCs) were recently identified and characterized as having multipotential differentiation and immunomodulatory properties in vitro and in vivo, and they represent new postnatal stem cell types for cytotherapy and regenerative medicine. However, the utility of gingival MSCs (GMSCs) as alternatives to periodontal ligament stem cells (PDLSCs), which have been demonstrated to be effective but with limited cell availability and reduced clinical feasibility, for periodontal regeneration in a previously diseased/inflamed environment remains obscure. In this study, patient-matched human GMSCs and PDLSCs were evaluated in terms of their colony-forming ability, proliferative capacity, cell surface epitopes, multi-lineage differentiation potentials, and related gene expression when incubated in different designed culture conditions, with or without the presence of inflammatory cytokines. An in vivo ectopic transplantation model using transplants from inflammatory cytokine-treated or untreated cells was applied to assess bone formation. We found that cells derived from both tissues expressed MSC markers, including CD146, CD105, CD90, CD29, and STRO-1. Both cells successfully differentiated under osteogenic, adipogenic, and chondrogenic microenvironments; PDLSCs displayed a more effective differentiation potential in all of the incubation conditions compared to GMSCs (P < 0.01). Although inflammatory cytokine-treated GMSCs and PDLSCs are inferior to normally cultured, patient and tissue-matched cells in terms of their osteogenic capacity and regenerative potential (P < 0.05), they retain the capacity for osteoblastic and adipose differentiation, as well as ectopic bone formation, similar to what has been demonstrated for other MSCs. Interestingly, GMSCs exhibited fewer inflammation-related changes in terms of osteogenic potential in vitro and bone formation in vivo compared to PDLSCs (P < 0.01). These results suggest

  9. Muscle cell derived angiopoietin-1 contributes to both myogenesis and angiogenesis in the ischemic environment

    PubMed Central

    McClung, Joseph M.; Reinardy, Jessica L.; Mueller, Sarah B.; McCord, Timothy J.; Kontos, Christopher D.; Brown, David A.; Hussain, Sabah N. A.; Schmidt, Cameron A.; Ryan, Terence E.; Green, Tom D.

    2015-01-01

    Recent strategies to treat peripheral arterial disease (PAD) have focused on stem cell based therapies, which are believed to result in local secretion of vascular growth factors. Little is known, however, about the role of ischemic endogenous cells in this context. We hypothesized that ischemic muscle cells (MC) are capable of secreting growth factors that act as potent effectors of the local cellular regenerative environment. Both muscle and endothelial cells (ECs) were subjected to experimental ischemia, and conditioned medium (CM) from each was collected and analyzed to assess myogenic and/or angiogenic potential. In muscle progenitors, mRNA expression of VEGF and its cognate receptors (Nrp1, Flt, Flk) was present and decreased during myotube formation in vitro, and EC CM or VEGF increased myoblast proliferation. Angiopoietin-1 (Ang-1), Tie1, and Tie2 mRNA increased during MC differentiation in vitro. Exogenous Ang-1 enhanced myogenic (MyoD and Myogenin) mRNA in differentiating myoblasts and increased myosin heavy chain protein. Myotube formation was enhanced by MC CM and inhibited by EC CM. Ang-1 protein was present in CM from MCs isolated from both the genetically ischemia-susceptible BALB/c and ischemia-resistant C57BL/6 mouse strains, and chimeric Tie2 receptor trapping in situ ablated Ang-1's myogenic effects in vitro. Ang-1 or MC CM enhanced myotube formation in a mixed isolate of muscle progenitors as well as a myoblast co-culture with pluripotent mesenchymal cells (10T1/2) and this effect was abrogated by viral expression of the extracellular domain of Tie2 (AdsTie2). Furthermore, mesh/tube formation by HUVECs was enhanced by Ang-1 or MC CM and abrogated by Tie2 chimeric receptor trapping. Our results demonstrate the ability of muscle and endothelial cell-derived vascular growth factors, particularly Ang-1, to serve as multi-functional stimuli regulating crosstalk between blood vessels and muscle cells during regeneration from ischemic myopathy. PMID

  10. Vascular morphogenesis of human umbilical vein endothelial cells on cell-derived macromolecular matrix microenvironment.

    PubMed

    Du, Ping; Subbiah, Ramesh; Park, Jung-Hwan; Park, Kwideok

    2014-09-01

    Extracellular matrix (ECM) is a highly organized network of proteins and other macromolecules that plays a critical role in cell adhesion, migration, and differentiation. In this study, we hypothesize that ECM derived from in-vitro-cultured cells possesses unique surface texture, topography, and mechanical property, and consequently carries some distinct cues for vascular morphogenesis of human umbilical vein endothelial cells (ECs). Cell-derived matrix (CDM) was obtained by culturing fibroblasts, preosteoblasts, and chondrocytes, respectively, on coverslips and then by decellularizing them using detergents and enzymes. These matrices were named fibroblast-derived matrix (FDM), preosteoblast-derived matrix (PDM), and chondrocyte-derived matrix (CHDM). Immunofluorescence of each CDM shows that some of the matrix components are fibronectin (FN), type I collagen, and laminin. Atomic force microscopy analysis presented that average fiber diameter ranged from 2 to 7 μm and FDM holds much larger fibers. The matrix elasticity measurements revealed that average Young's modulus of CHDM (17.7 ± 4.2 kPa) was much greater than that of PDM (10.5 ± 1.1 kPa) or FDM (5.7 ± 0.5 kPa). During 5-day culture, EC morphologies were dramatically changed on PDM and FDM, but those on CHDM and gelatin were rather stable, regardless of time lapse. Cell migration assay discovered quicker repopulation of the scratched areas on PDM and FDM than on gelatin and CHDM. A capillary-like structure (CLS) assembly was also notable only in the PDM and FDM, as compared with CHDM, gelatin, or FN that were very poor in CLS formation. Quantitative analysis of mean CLS branch points and branch lengths demonstrated much better angiogenic activity of ECs on PDM and FDM. Interestingly, CLS formation was closely associated with matrix remodeling by ECs and the matrix clearance on PDM with time was sharply contrasted with that on CHDM that majority of the matrix FN was reserved. It was notable that membrane

  11. Peripheral antinociceptive effects of exogenous and immune cell-derived endomorphins in prolonged inflammatory pain.

    PubMed

    Labuz, Dominika; Berger, Stephan; Mousa, Shaaban A; Zöllner, Christian; Rittner, Heike L; Shaqura, Mohammed A; Segovia-Silvestre, Toni; Przewlocka, Barbara; Stein, Christoph; Machelska, Halina

    2006-04-19

    Endomorphins (EMs) are endogenous selective mu-opioid receptor agonists. Their role in inflammatory pain has not been fully elucidated. Here we examine peripheral antinociception elicited by exogenously applied EM-1 and EM-2 and the contribution of EM-containing leukocytes to stress- and corticotropin-releasing factor (CRF)-induced antinociception. To this end, we applied behavioral (paw pressure) testing, radioligand binding, immunohistochemistry, and flow cytometry in rats with unilateral hindpaw inflammation induced with Freund's adjuvant. EMs injected directly into both hindpaws produced antinociception exclusively in inflamed paws. This was blocked by locally applied mu-receptor-selective (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) but not kappa-receptor-selective (nor-binaltorphimine) antagonists. Delta-receptor antagonists (naltrindole and N,N-diallyl-Tyr-Aib-Aib-Phe-Leu) did not influence EM-1-induced but dose-dependently decreased EM-2-induced antinociception. Antibodies against beta-endorphin, methionine-enkephalin, or leucine-enkephalin did not significantly change EM-2-induced antinociception. Both EMs displaced binding of [3H]-[D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin to mu-receptors in dorsal root ganglia (DRG). Using [3H]-naltrindole or [(125)I]-[D-Pen2,5]-enkephalin, no detectable delta-binding was found in DRG of inflamed hindlimbs. Numerous beta-endorphin-containing and fewer EM-1- and EM-2-containing leukocytes were detected in subcutaneous tissue of inflamed paws. Leukocyte-depleting serum decreased the number of immigrating opioid-containing immune cells and attenuated swim stress- and CRF-induced antinociception in inflamed paws. Both forms of antinociception were strongly attenuated by anti-beta-endorphin and to a lesser degree by anti-EM-1 and anti-EM-2 antibodies injected into inflamed paws. Together, exogenously applied and immune cell-derived EMs alleviate prolonged inflammatory pain through selective activation of peripheral opioid receptors

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

    PubMed Central

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

    2015-01-01

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

  13. Long-term culture of mouse embryonic stem cell-derived adherent neurospheres and functional neurons.

    PubMed

    Hayashi, Mirian A F; Guerreiro, Juliano R; Cassola, Antonio C; Lizier, Nelson F; Kerkis, Alexandre; Camargo, Antonio C M; Kerkis, Irina

    2010-12-01

    Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of

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

    PubMed Central

    Assawachananont, Juthaporn; Mandai, Michiko; Okamoto, Satoshi; Yamada, Chikako; Eiraku, Mototsugu; Yonemura, Shigenobu; Sasai, Yoshiki; Takahashi, Masayo

    2014-01-01

    Summary 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. PMID:24936453

  15. Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

    DTIC Science & Technology

    2015-02-05

    derived 5a. CONTRACT NUMBER central nervous system neurons 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Hubbard, K, Beske, PH...numbers CBM.THRTOX.01.10.RC.023 and CBM.THRTOX.01.RC.014). 14. ABSTRACT See reprint. 15. SUBJECT TERMS embryonic stem cells, stem cell-derived neurons ...botulinum neurotoxin detection, electrophysiology, synapse, neuronal networks, glutamatergic synapse, GABAergic synapse 16. SECURITY CLASSIFICATION

  16. Internalization of Tissue Factor-Rich Microvesicles by Platelets Occurs Independently of GPIIb-IIIa, and Involves CD36 Receptor, Serotonin Transporter and Cytoskeletal Assembly.

    PubMed

    Lopez-Vilchez, Irene; Diaz-Ricart, Maribel; Galan, Ana M; Roque, Merce; Caballo, Carolina; Molina, Patricia; White, James G; Escolar, Gines

    2016-02-01

    Platelets are important in hemostasis, but also detect particles and pathogens in the circulation. Phagocytic and endocytic activities of platelets are widely recognized; however, receptors and mechanisms involved remain poorly understood. We previously demonstrated that platelets internalize and store phospholipid microvesicles enriched in human tissue factor (TF+MVs) and that platelet-associated TF enhances thrombus formation at sites of vascular damage. Here, we investigate the mechanisms implied in the interactions of TF+MVs with platelets and the effects of specific inhibitory strategies. Aggregometry and electron microscopy were used to assess platelet activation and TF+MVs uptake. Cytoskeletal assembly and activation of phosphoinositide 3-kinase (PI3K) and RhoA were analyzed by western blot and ELISA. Exposure of platelets to TF+MVs caused reversible platelet aggregation, actin polymerization and association of contractile proteins to the cytoskeleton being maximal at 1 min. The same kinetics were observed for activation of PI3K and translocation of RhoA to the cytoskeleton. Inhibitory strategies to block glycoprotein IIb-IIIa (GPIIb-IIIa), scavenger receptor CD36, serotonin transporter (SERT) and PI3K, fully prevented platelet aggregation by TF+MVs. Ultrastructural techniques revealed that uptake of TF+MVs was efficiently prevented by anti-CD36 and SERT inhibitor, but only moderately interfered by GPIIb-IIIa blockade. We conclude that internalization of TF+MVs by platelets occurs independently of receptors related to their main hemostatic function (GPIIb-IIIa), involves the scavenger receptor CD36, SERT and engages PI3-Kinase activation and cytoskeletal assembly. CD36 and SERT appear as potential therapeutic targets to interfere with the association of TF+MVs with platelets and possibly downregulate their prothrombotic phenotype.

  17. Growth and turning properties of adult glial cell-derived neurotrophic factor coreceptor α1 nonpeptidergic sensory neurons.

    PubMed

    Guo, GuiFang; Singh, Vandana; Zochodne, Douglas W

    2014-09-01

    An overlapping population of adult primary sensory neurons that innervate the skin express the glial cell-derived neurotrophic factor coreceptor α1 (GFRα1), the lectin IB4, and the "regenerative brake" phosphatase and tensin homolog deleted on chromosome 10. Using an adapted turning and growth assay, we analyzed the growth cone behavior of adult immunoselected GFRα1 sensory neurons. These neurons had less robust baseline growth and reluctant responsiveness to individual growth factors but responded to synergistic types of input from glial cell-derived neurotrophic factor, hepatocyte growth factor, a phosphatase and tensin homolog deleted on chromosome 10 inhibitor, or a downstream Rho kinase inhibitor. Hepatocyte growth factor and the phosphatase and tensin homolog deleted on chromosome 10 inhibitor were associated with growth cone turning. A gradient of protein extracted from skin samples, a primary target of GFRα1 axons, replicated the impact of synergistic support. Within the skin, glial cell-derived neurotrophic factor was expressed within epidermal axons, indicating an autocrine role accompanying local hepatocyte growth factor synthesis. Taken together, our findings identify unique growth properties and plasticity of a distinct population of epidermal axons that are relevant to neurologic repair and skin reinnervation.

  18. Expression of epithelial cell-derived cytokine genes in the duodenal and colonic mucosae of dogs with chronic enteropathy

    PubMed Central

    OSADA, Hironari; OGAWA, Misato; HASEGAWA, Ayana; NAGAI, Makoto; SHIRAI, Junsuke; SASAKI, Kazuaki; SHIMODA, Minoru; ITOH, Hiroshi; KONDO, Hirotaka; OHMORI, Keitaro

    2016-01-01

    It remains unclear whether epithelial cell-derived cytokines, including interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP), contribute to development of canine chronic enteropathy (CE), which includes antibiotic-responsive enteropathy (ARE), food-responsive enteropathy (FRE) and inflammatory bowel disease (IBD). In the present study, we examined mRNA expression of il-25, il-33 and tslp in the duodenal and colonic mucosae of dogs with ARE, FRE and IBD. Real-time PCR analysis revealed that mRNA expression of il-33 was significantly lower in the duodenum in dogs with FRE than in healthy dogs. The results suggest that epithelial cell-derived cytokines may not be an inducer of Th2-type immunity in the gut of dogs with CE, and decreased expression of IL-33 may be involved in induction of FRE. Further studies are required to clarify roles of epithelial cell-derived cytokines, especially IL-33, in the pathogenesis of canine CE. PMID:28049868

  19. Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles

    PubMed Central

    Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

    2016-01-01

    Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs. PMID:27022410

  20. Expression of epithelial cell-derived cytokine genes in the duodenal and colonic mucosae of dogs with chronic enteropathy.

    PubMed

    Osada, Hironari; Ogawa, Misato; Hasegawa, Ayana; Nagai, Makoto; Shirai, Junsuke; Sasaki, Kazuaki; Shimoda, Minoru; Itoh, Hiroshi; Kondo, Hirotaka; Ohmori, Keitaro

    2017-02-28

    It remains unclear whether epithelial cell-derived cytokines, including interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP), contribute to development of canine chronic enteropathy (CE), which includes antibiotic-responsive enteropathy (ARE), food-responsive enteropathy (FRE) and inflammatory bowel disease (IBD). In the present study, we examined mRNA expression of il-25, il-33 and tslp in the duodenal and colonic mucosae of dogs with ARE, FRE and IBD. Real-time PCR analysis revealed that mRNA expression of il-33 was significantly lower in the duodenum in dogs with FRE than in healthy dogs. The results suggest that epithelial cell-derived cytokines may not be an inducer of Th2-type immunity in the gut of dogs with CE, and decreased expression of IL-33 may be involved in induction of FRE. Further studies are required to clarify roles of epithelial cell-derived cytokines, especially IL-33, in the pathogenesis of canine CE.

  1. The influence of static magnetic fields on canine and equine mesenchymal stem cells derived from adipose tissue.

    PubMed

    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.

  2. Podoplanin is a component of extracellular vesicles that reprograms cell-derived exosomal proteins and modulates lymphatic vessel formation

    PubMed Central

    Andrés, Germán; Gopal, Shashi K.; Martín-Villar, Ester; Renart, Jaime; Simpson, Richard J.; Quintanilla, Miguel

    2016-01-01

    Podoplanin (PDPN) is a transmembrane glycoprotein that plays crucial roles in embryonic development, the immune response, and malignant progression. Here, we report that cells ectopically or endogenously expressing PDPN release extracellular vesicles (EVs) that contain PDPN mRNA and protein. PDPN incorporates into membrane shed microvesicles (MVs) and endosomal-derived exosomes (EXOs), where it was found to colocalize with the canonical EV marker CD63 by immunoelectron microscopy. We have previously found that expression of PDPN in MDCK cells induces an epithelial-mesenchymal transition (EMT). Proteomic profiling of MDCK-PDPN cells compared to control cells shows that PDPN-induced EMT is associated with upregulation of oncogenic proteins and diminished expression of tumor suppressors. Proteomic analysis of exosomes reveals that MDCK-PDPN EXOs were enriched in protein cargos involved in cell adhesion, cytoskeletal remodeling, signal transduction and, importantly, intracellular trafficking and EV biogenesis. Indeed, expression of PDPN in MDCK cells stimulated both EXO and MV production, while knockdown of endogenous PDPN in human HN5 squamous carcinoma cells reduced EXO production and inhibited tumorigenesis. EXOs released from MDCK-PDPN and control cells both stimulated in vitro angiogenesis, but only EXOs containing PDPN were shown to promote lymphatic vessel formation. This effect was mediated by PDPN on the surface of EXOs, as demonstrated by a neutralizing specific monoclonal antibody. These results contribute to our understanding of PDPN-induced EMT in association to tumor progression, and suggest an important role for PDPN in EV biogenesis and/or release and for PDPN-EXOs in modulating lymphangiogenesis. PMID:26893367

  3. Exosome-based tumor antigens-adjuvant co-delivery utilizing genetically engineered tumor cell-derived exosomes with immunostimulatory CpG DNA.

    PubMed

    Morishita, Masaki; Takahashi, Yuki; Matsumoto, Akihiro; Nishikawa, Makiya; Takakura, Yoshinobu

    2016-12-01

    For cancer immunotherapy via tumor antigen vaccination in combination with an adjuvant, major challenges include the identification of a particular tumor antigen and efficient delivery of the antigen as well as adjuvant to antigen-presenting cells. In this study, we proposed an efficient exosome-based tumor antigens-adjuvant co-delivery system using genetically engineered tumor cell-derived exosomes containing endogenous tumor antigens and immunostimulatory CpG DNA. Murine melanoma B16BL6 cells were transfected with a plasmid vector encoding a fusion streptavidin (SAV; a protein that binds to biotin with high affinity)-lactadherin (LA; an exosome-tropic protein) protein, yielding genetically engineered SAV-LA-expressing exosomes (SAV-exo). SAV-exo were combined with biotinylated CpG DNA to prepare CpG DNA-modified exosomes (CpG-SAV-exo). Fluorescent microscopic observation revealed the successful modification of exosomes with CpG DNA by SAV-biotin interaction. CpG-SAV-exo showed efficient and simultaneous delivery of exosomes with CpG DNA to murine dendritic DC2.4 cells in culture. Treatment with CpG-SAV-exo effectively activated DC2.4 cells and enhanced tumor antigen presentation capacity. Immunization with CpG-SAV-exo exhibited stronger in vivo antitumor effects in B16BL6 tumor-bearing mice than simple co-administration of exosomes and CpG DNA. Thus, genetically engineered CpG-SAV-exo is an effective exosome-based tumor antigens-adjuvant co-delivery system that will be useful for cancer immunotherapy.

  4. A Computational Model for Investigating Tumor Apoptosis Induced by Mesenchymal Stem Cell-Derived Secretome

    PubMed Central

    Sudiono, Janti

    2016-01-01

    Apoptosis is a programmed cell death that occurs naturally in physiological and pathological conditions. Defective apoptosis can trigger the development and progression of cancer. Experiments suggest the ability of secretome derived from mesenchymal stem cells (MSC) to induce apoptosis in cancer cells. We develop a hybrid discrete-continuous multiscale model to further investigate the effect of MSC-derived secretome in tumor growth. The model encompasses three biological scales. At the molecular scale, a system of ordinary differential equations regulate the expression of proteins involved in apoptosis signaling pathways. At the cellular scale, discrete equations control cellular migration, phenotypic switching, and proliferation. At the extracellular scale, a system of partial differential equations are employed to describe the dynamics of microenvironmental chemicals concentrations. The simulation is able to produce both avascular tumor growth rate and phenotypic patterns as observed in the experiments. In addition, we obtain good quantitative agreements with the experimental data on the apoptosis of HeLa cancer cells treated with MSC-derived secretome. We use this model to predict the growth of avascular tumor under various secretome concentrations over time. PMID:27956936

  5. Stromal Cell-Derived Factor 1 Polymorphism in Retinal Vein Occlusion

    PubMed Central

    Szigeti, Andrea; Ecsedy, Mónika; Schneider, Miklós; Lénárt, Lilla; Lesch, Balázs; Nagy, Zoltán Zsolt

    2016-01-01

    Background Stromal cell-derived factor 1 (SDF1) has crucial role in the regulation of angiogenesis and ocular neovascularisation (NV). The purpose of this study was to evaluate the association between SDF1-3’G(801)A polymorphism and NV complications of retinal vein occlusion (RVO). Methods 130 patients with RVO (median age: 69.0, range 35–93 years; male/female– 58/72; 55 patients had central RVO, 75 patients had branch RVO) were enrolled in this study. In the RVO group, 40 (30.8%) patients were diagnosed with NV complications of RVO and 90 (69.2%) patients without NVs. The median follow up period was 40.3 months (range: 18–57 months). The SDF1-3’G(801)A polymorphism was detected by PCR-RFLP. Allelic prevalence was related to reference values obtained in the control group consisted of 125 randomly selected, age and gender matched, unrelated volunteers (median age: 68.0, range 36–95 years; male/female– 53/72). Statistical analysis of the allele and genotype differences between groups (RVO patients vs controls; RVO patients with NV vs RVO patients without NV) was determined by chi-squared test. P value of <0.05 was considered statistically significant. Results Hardy-Weinberg criteria was fulfilled in all groups. The SDF1-3’G(801)A allele and genotype frequencies of RVO patients were similar to controls (SDF1-3’A allele: 22.3% vs 20.8%; SDF1-3’(801)AA: 5.4% vs 4.8%, SDF1-3’(801)GG: 60.8% vs 63.2%). The frequency of SDF1-3’(801)AA and SDF1-3’(801)GA genotypes, as well as the SDF1-3’(801)A allele frequency were higher in RVO patients with NV versus in patients without NV complication (SDF1-3’(801)AA+AG genotypes: 57.5% vs 31.1%, p = 0.008; SDF1-3’(801)A allele: 35.0% vs 16.7%, p = 0.002) or versus controls (SDF1-3’(801)AA+AG genotypes 57.5% vs 36.8%, p = 0.021; SDF1-3’(801)A allele: 35.0% vs 20.8% p = 0.01). Carrying of SDF1-3’(801)A allele increased the risk of neovascularisation complications of RVO by 2.69 (OR, 95% CI = 1.47–4

  6. Paracrine Engineering of Human Explant-Derived Cardiac Stem Cells to Over-Express Stromal-Cell Derived Factor 1α Enhances Myocardial Repair.

    PubMed

    Tilokee, Everad L; Latham, Nicholas; Jackson, Robyn; Mayfield, Audrey E; Ye, Bin; Mount, Seth; Lam, Buu-Khanh; Suuronen, Erik J; Ruel, Marc; Stewart, Duncan J; Davis, Darryl R

    2016-07-01

    First generation cardiac stem cell products provide indirect cardiac repair but variably produce key cardioprotective cytokines, such as stromal-cell derived factor 1α, which opens the prospect of maximizing up-front paracrine-mediated repair. The mesenchymal subpopulation within explant derived human cardiac stem cells underwent lentiviral mediated gene transfer of stromal-cell derived factor 1α. Unlike previous unsuccessful attempts to increase efficacy by boosting the paracrine signature of cardiac stem cells, cytokine profiling revealed that stromal-cell derived factor 1α over-expression prevented lv-mediated "loss of cytokines" through autocrine stimulation of CXCR4+ cardiac stem cells. Stromal-cell derived factor 1α enhanced angiogenesis and stem cell recruitment while priming cardiac stem cells to readily adopt a cardiac identity. As compared to injection with unmodified cardiac stem cells, transplant of stromal-cell derived factor 1α enhanced cells into immunodeficient mice improved myocardial function and angiogenesis while reducing scarring. Increases in myocardial stromal-cell derived factor 1α content paralleled reductions in myocyte apoptosis but did not influence long-term engraftment or the fate of transplanted cells. Transplantation of stromal-cell derived factor 1α transduced cardiac stem cells increased the generation of new myocytes, recruitment of bone marrow cells, new myocyte/vessel formation and the salvage of reversibly damaged myocardium to enhance cardiac repair after experimental infarction. Stem Cells 2016;34:1826-1835.

  7. N-terminal sequences from Autographa californica nuclear polyhedrosis virus envelope proteins ODV-E66 and ODV-E25 are sufficient to direct reporter proteins to the nuclear envelope, intranuclear microvesicles and the envelope of occlusion derived virus.

    PubMed

    Hong, T; Summers, M D; Braunagel, S C

    1997-04-15

    Baculovirus occlusion-derived virus (ODV) derives its envelope from an intranuclear membrane source. N-terminal amino acid sequences of the Autographa californica nuclear polyhedrosis virus (AcMNPV) envelope proteins, ODV-E66 and ODV-E25 (23 and 24 amino acids, respectively) are highly hydrophobic. Recombinant viruses that express the two N-terminal amino acid sequences fused to green fluorescent protein (23GFP or 24GFP) provided visual markers to follow protein transport and localization within the nucleus during infection. Autoflourescence was first detected along the cytoplasmic periphery of the nucleus and subsequently localized as foci to discrete locations within the nucleus. Immunoelectron microscopy confirmed that these foci predominantly contained intranuclear microvesicles and the reporter fusion proteins were also detected in cytoplasmic membranes near the nucleus, and the outer and inner nuclear membrane. Therefore, these defined hydrophobic domains are sufficient to direct native and fusion proteins to induced membrane microvesicles within a baculovirus-infected cell nucleus and the viral envelope. In addition, these data suggest that movement of these proteins into the nuclear envelope may initiate through cytoplasmic membranes, such as endoplasmic reticulum, and that transport into the nucleus may be mediated through the outer and inner nuclear membrane.

  8. Recruitment of RNA molecules by connexin RNA-binding motifs: Implication in RNA and DNA transport through microvesicles and exosomes.

    PubMed

    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.

  9. Immune Suppression and Inflammation in the Progression of Breast Cancer

    DTIC Science & Technology

    2006-03-01

    420:860. 5. Kusmartsev , S., and D. I. Gabrilovich. 2002. Immature myeloid cells and cancer- associated immune suppression. Cancer Immunol Immunother 51...166:5398. 20. Kusmartsev , S. A., Y. Li, and S. H. Chen. 2000. Gr-1+ myeloid cells derived from tumor-bearing mice inhibit primary T cell activation...Chronic inflammation and cancer. On- cology 16: 217–226. 4. Coussens, L. M., and Z. Werb. 2002. Inflammation and cancer. Nature 420: 860–867. 5. Kusmartsev

  10. Tumour resistance in induced pluripotent stem cells derived from naked mole-rats.

    PubMed

    Miyawaki, Shingo; Kawamura, Yoshimi; Oiwa, Yuki; Shimizu, Atsushi; Hachiya, Tsuyoshi; Bono, Hidemasa; Koya, Ikuko; Okada, Yohei; Kimura, Tokuhiro; Tsuchiya, Yoshihiro; Suzuki, Sadafumi; Onishi, Nobuyuki; Kuzumaki, Naoko; Matsuzaki, Yumi; Narita, Minoru; Ikeda, Eiji; Okanoya, Kazuo; Seino, Ken-Ichiro; Saya, Hideyuki; Okano, Hideyuki; Miura, Kyoko

    2016-05-10

    The naked mole-rat (NMR, Heterocephalus glaber), which is the longest-lived rodent species, exhibits extraordinary resistance to cancer. Here we report that NMR somatic cells exhibit a unique tumour-suppressor response to reprogramming induction. In this study, we generate NMR-induced pluripotent stem cells (NMR-iPSCs) and find that NMR-iPSCs do not exhibit teratoma-forming tumorigenicity due to the species-specific activation of tumour-suppressor alternative reading frame (ARF) and a disruption mutation of the oncogene ES cell-expressed Ras (ERAS). The forced expression of Arf in mouse iPSCs markedly reduces tumorigenicity. Furthermore, we identify an NMR-specific tumour-suppression phenotype-ARF suppression-induced senescence (ASIS)-that may protect iPSCs and somatic cells from ARF suppression and, as a consequence, tumorigenicity. Thus, NMR-specific ARF regulation and the disruption of ERAS regulate tumour resistance in NMR-iPSCs. Our findings obtained from studies of NMR-iPSCs provide new insight into the mechanisms of tumorigenicity in iPSCs and cancer resistance in the NMR.

  11. Heparanase regulates secretion, composition, and function of tumor cell-derived exosomes.

    PubMed

    Thompson, Camilla A; Purushothaman, Anurag; Ramani, Vishnu C; Vlodavsky, Israel; Sanderson, Ralph D

    2013-04-05

    Emerging evidence indicates that exosomes play a key role in tumor-host cross-talk and that exosome secretion, composition, and functional capacity are altered as tumors progress to an aggressive phenotype. However, little is known regarding the mechanisms that regulate these changes. Heparanase is an enzyme whose expression is up-regulated as tumors become more aggressive and is associated with enhanced tumor growth, angiogenesis, and metastasis. We have discovered that in human cancer cells (myeloma, lymphoblastoid, and breast cancer), when expression of heparanase is enhanced or when tumor cells are exposed to exogenous heparanase, exosome secretion is dramatically increased. Heparanase enzyme activity is required for robust enhancement of exosome secretion because enzymatically inactive forms of heparanase, even when present in high amounts, do not dramatically increase exosome secretion. Heparanase also impacts exosome protein cargo as reflected by higher levels of syndecan-1, VEGF, and hepatocyte growth factor in exosomes secreted by heparanase-high expressing cells as compared with heparanase-low expressing cells. In functional assays, exosomes from heparanase-high cells stimulated spreading of tumor cells on fibronectin and invasion of endothelial cells through extracellular matrix better than did exosomes secreted by heparanase-low cells. These studies reveal that heparanase helps drive exosome secretion, alters exosome composition, and facilitates production of exosomes that impact both tumor and host cell behavior, thereby promoting tumor progression.

  12. Completely ES cell-derived mice produced by tetraploid complementation using inner cell mass (ICM) deficient blastocysts.

    PubMed

    Wen, Duancheng; Saiz, Nestor; Rosenwaks, Zev; Hadjantonakis, Anna-Katerina; Rafii, Shahin

    2014-01-01

    Tetraploid complementation is often used to produce mice from embryonic stem cells (ESCs) by injection of diploid (2n) ESCs into tetraploid (4n) blastocysts (ESC-derived mice). This method has also been adapted to mouse cloning and the derivation of mice from induced pluripotent stem (iPS) cells. However, the underlying mechanism(s) of the tetraploid complementation remains largely unclear. Whether this approach can give rise to completely ES cell-derived mice is an open question, and has not yet been unambiguously proven. Here, we show that mouse tetraploid blastocysts can be classified into two groups, according to the presence or absence of an inner cell mass (ICM). We designate these as type a (presence of ICM at blastocyst stage) or type b (absence of ICM). ESC lines were readily derived from type a blastocysts, suggesting that these embryos retain a pluripotent epiblast compartment; whereas the type b blastocysts possessed very low potential to give rise to ESC lines, suggesting that they had lost the pluripotent epiblast. When the type a blastocysts were used for tetraploid complementation, some of the resulting mice were found to be 2n/4n chimeric; whereas when type b blastocysts were used as hosts, the resulting mice are all completely ES cell-derived, with the newborn pups displaying a high frequency of abdominal hernias. Our results demonstrate that completely ES cell-derived mice can be produced using ICM-deficient 4n blastocysts, and provide evidence that the exclusion of tetraploid cells from the fetus in 2n/4n chimeras can largely be attributed to the formation of ICM-deficient blastocysts.

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

    PubMed

    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.

  14. Resolving the "egg supply problem" in human embryonic stem cell derivation through technical means--a legal and ethical analysis.

    PubMed

    Hammond, Natasha; Holm, Søren

    2008-03-01

    This paper seeks to briefly discuss the legal and ethical problems connected to scientific developments in the field of human embryonic stem cell derivation aimed at solving the "egg supply problem" in stem cell research. The legal situation is discussed in respect of the UK's current regulatory regime, proposed reform and the Oviedo Convention. The scientific developments which are examined are chimeric embryos, in vitro maturation of oocytes, derivation of stem cell lines in connection with pre-implantation genetic diagnosis and the derivation of oocytes from existing stem cell lines.

  15. Disease Modeling in Stem Cell-Derived 3D Organoid Systems.

    PubMed

    Dutta, Devanjali; Heo, Inha; Clevers, Hans

    2017-03-21

    Organoids are 3D in vitro culture systems derived from self-organizing stem cells. They can recapitulate the in vivo architecture, functionality, and genetic signature of original tissues. Thus, organoid technology has been rapidly applied to understanding stem cell biology, organogenesis, and various human pathologies. The recent development of human patient-derived organoids has enabled disease modeling with precision, highlighting their great potential in biomedical applications, translational medicine, and personalized therapy. In light of recent breakthroughs using organoids, it is only apt that we appreciate the advantages and shortcomings of this technology to exploit its full potential. We discuss recent advances in the application of organoids in studying cancer and hereditary diseases, as well as in the examination of host cell-microorganism interactions.

  16. Metastatic MHC class I-negative mouse cells derived by transformation with human papillomavirus type 16

    PubMed Central

    Šmahel, M; Sobotková, E; Bubeník, J; Šímová, J; Žák, R; Ludvíková, V; Hájková, R; Kovařík, J; Jelínek, F; Povýšil, C; Marinov, J; Vonka, V

    2001-01-01

    In the endeavour to develop a model for studying gene therapy of cancers associated with human papillomaviruses (HPVs), mouse cells were transformed with the HPV type 16 (HPV16) and activated H-ras oncogenes. This was done by contransfection of plasmid p16HHMo, carrying the HPV16 E6/E7 oncogenes, and plasmid pEJ6.6, carrying the gene coding for human H-ras oncoprotein activated by G12V mutation, into secondary C57BL/6 mouse kidney cells. An oncogenic cell line, designated MK16/1/IIIABC, was derived. The epithelial origin of the cells was confirmed by their expression of cytokeratins. No MHC class I and class II molecules were detected on the surface of MK16/1/IIIABC cells. Spontaneous metastases were observed in lymphatic nodes and lungs after prolonged growth of MK16/1/IIIABC-induced subcutaneous tumours. Lethally irradiated MK16/1/IIIABC cells induced protection against challenge with 105homologous cells, but not against a higher cell dose (5 × 105). Plasmids p16HHMo and pEJ6.6 were also used for preventive immunization of mice. In comparison with a control group injected with pBR322, they exhibited moderate protection, in terms of prolonged survival, against MK16/1/IIIABC challenge (P< 0.03). These data suggest that MK16/1/IIIABC cells may serve as a model for studying immune reactions against HPV16-associated human tumours. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11225590

  17. Application of a Persistent Heparin Treatment Inhibits the Malignant Potential of Oral Squamous Carcinoma Cells Induced by Tumor Cell-Derived Exosomes.

    PubMed

    Sento, Shinya; Sasabe, Eri; Yamamoto, Tetsuya

    2016-01-01

    Exosomes are 30-100 nm-sized membranous vesicles, secreted from a variety of cell types into their surrounding extracellular space. Various exosome components including lipids, proteins, and nucleic acids are transferred to recipient cells and affect their function and activity. Numerous studies have showed that tumor cell-derived exosomes play important roles in tumor growth and progression. However, the effect of exosomes released from oral squamous cell carcinoma (OSCC) into the tumor microenvironment remains unclear. In the present study, we isolated exosomes from OSCC cells and investigated the influence of OSCC cell-derived exosomes on the tumor cell behavior associated with tumor development. We demonstrated that OSCC cell-derived exosomes were taken up by OSCC cells themselves and significantly promoted proliferation, migration, and invasion through the activation of the PI3K/Akt, MAPK/ERK, and JNK-1/2 pathways in vitro. These effects of OSCC cell-derived exosomes were obviously attenuated by treatment with PI3K, ERK-1/2, and JNK-1/2 pharmacological inhibitors. Furthermore, the growth rate of tumor xenografts implanted into nude mice was promoted by treatment with OSCC cell-derived exosomes. The uptake of exosomes by OSCC cells and subsequent tumor progression was abrogated in the presence of heparin. Taken together, these data suggest that OSCC cell-derived exosomes might be a novel therapeutic target and the use of heparin to inhibit the uptake of OSCC-derived exosomes by OSCC cells may be useful for treatment.

  18. Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues

    PubMed Central

    Ravenscroft, Stephanie M.; Pointon, Amy; Williams, Awel W.; Cross, Michael J.; Sidaway, James E.

    2016-01-01

    The immature phenotype of stem cell derived cardiomyocytes is a significant barrier to their use in translational medicine and pre-clinical in vitro drug toxicity and pharmacological analysis. Here we have assessed the contribution of non-myocyte cells on the contractile function of co-cultured human embryonic stem cell derived cardiomyocytes (hESC-CMs) in spheroid microtissue format. Microtissues were formed using a scaffold free 96-well cell suspension method from hESC-CM cultured alone (CM microtissues) or in combination with human primary cardiac microvascular endothelial cells and cardiac fibroblasts (CMEF microtissues). Contractility was characterized with fluorescence and video-based edge detection. CMEF microtissues displayed greater Ca2+ transient amplitudes, enhanced spontaneous contraction rate and remarkably enhanced contractile function in response to both positive and negative inotropic drugs, suggesting a more mature contractile phenotype than CM microtissues. In addition, for several drugs the enhanced contractile response was not apparent when endothelial cell or fibroblasts from a non-cardiac tissue were used as the ancillary cells. Further evidence of maturity for CMEF microtissues was shown with increased expression of genes that encode proteins critical in cardiac Ca2+ handling (S100A1), sarcomere assembly (telethonin/TCAP) and β-adrenergic receptor signalling. Our data shows that compared with single cell-type cardiomyocyte in vitro models, CMEF microtissues are superior at predicting the inotropic effects of drugs, demonstrating the critical contribution of cardiac non-myocyte cells in mediating functional cardiotoxicity. PMID:27125969

  19. Comparative gene expression profiling in human-induced pluripotent stem cell--derived cardiocytes and human and cynomolgus heart tissue.

    PubMed

    Puppala, Dinesh; Collis, Leon P; Sun, Sunny Z; Bonato, Vinicius; Chen, Xian; Anson, Blake; Pletcher, Mathew; Fermini, Bernard; Engle, Sandra J

    2013-01-01

    Cardiotoxicity is one of the leading causes of drug attrition. Current in vitro models insufficiently predict cardiotoxicity, and there is a need for alternative physiologically relevant models. Here we describe the gene expression profile of human-induced pluripotent stem cell-derived cardiocytes (iCC) postthaw over a period of 42 days in culture and compare this profile to human fetal and adult as well as adult cynomolgus nonhuman primate (NHP, Macaca fascicularis) heart tissue. Our results indicate that iCC express relevant cardiac markers such as ion channels (SCN5A, KCNJ2, CACNA1C, KCNQ1, and KCNH2), tissue-specific structural markers (MYH6, MYLPF, MYBPC3, DES, TNNT2, and TNNI3), and transcription factors (NKX2.5, GATA4, and GATA6) and lack the expression of stem cell markers (FOXD3, GBX2, NANOG, POU5F1, SOX2, and ZFP42). Furthermore, we performed a functional evaluation of contractility of the iCC and showed functional and pharmacological correlations with myocytes isolated from adult NHP hearts. These results suggest that stem cell-derived cardiocytes may represent a novel in vitro model to study human cardiac toxicity with potential ex vivo and in vivo translation.

  20. Tumor cell-derived exosome-targeted dendritic cells stimulate stronger CD8+ CTL responses and antitumor immunities.

    PubMed

    Yao, Ye; Chen, Linjun; Wei, Wei; Deng, Xiaohui; Ma, Liyuan; Hao, Siguo

    2013-06-21

    Tumor cell-derived exosomes (TEX) have been widely used to induce antitumor immune responses in animal models and clinical trials. However, the efficiency of the antitumor immunity that is induced by TEX is still relatively weak. In this study, we compared the antitumor immunities between EG7 tumor cell-derived exosomes (EXO(EG7)) and EXO(EG7)-targeted dendritic cells (DC(EXO)). We found that EXO(EG7) harbored OVA and peptide major histocompatibility complex I (pMHC-I), which were expressed on its parental EG7 tmor cells, and they could transfer OVA and pMHC-I to dendritic cells (DCs) in vitro. DC(EXO) could more efficiently induce antitumor immunity than EXO(EG7). In addition, we showed that the immune stimulatory effects of EXO(EG7) were dependent on the host DCs and, whereas those of DC(EXO) were not, indicating the important role of the host DCs in TEX vaccines. Taken together, TEX-targeted DCs may be more effective for EXO-based vaccines for the induction of antitumor immunity.

  1. Analysis of stromal cell secretomes reveals a critical role for stromal cell-derived HGF and fibronectin in angiogenesis

    PubMed Central

    Newman, Andrew C.; Chou, Wayne; Welch-Reardon, Katrina M.; Fong, Ashley H.; Popson, Stephanie A.; Phan, Duc Thien; Sandoval, Daniel R.; Nguyen, Dananh P.; Gershon, Paul D.; Hughes, Christopher C. W.

    2013-01-01

    Objective Angiogenesis requires tightly coordinated cross-talk between endothelial cells and stromal cells such as fibroblasts and smooth muscle cells. The specific molecular mechanisms moderating this process are still poorly understood. Method and Results Stromal cell-derived factors are essential for endothelial cell sprouting and lumen formation. We therefore compared the abilities of two primary fibroblast isolates and a primary smooth muscle cell isolate to promote in vitro angiogenesis and analyzed their secretomes using a combination of nanoLC-MS/MS, qPCR and ELISA. Each isolate exhibited a different level of angiogenic ability. Using quantitative MS, we then compared the secretomes of a fibroblast isolate exhibiting low angiogenic activity, a fibroblast isolate exhibiting high angiogenic activity and human umbilical vein endothelial cells. High angiogenic fibroblast supernatants exhibited an over-abundance of proteins associated with extracellular matrix constituents compared to low angiogenic fibroblasts or endothelial cells. Finally, siRNA technology and purified protein were used to confirm a role for stromal cell-derived hepatocyte growth factor and fibronectin in inducing endothelial cell sprouting. Conclusion Differences in stromal cell ability to induce angiogenesis are due to differences in the secreted proteomes of both extracellular matrix proteins and pro-angiogenic growth factors. PMID:23288153

  2. Stem cell-derived interneuron transplants as a treatment for schizophrenia: preclinical validation in a rodent model.

    PubMed

    Donegan, J J; Tyson, J A; Branch, S Y; Beckstead, M J; Anderson, S A; Lodge, D J

    2016-08-02

    An increasing literature suggests that schizophrenia is associated with a reduction in hippocampal interneuron function. Thus, we posit that stem cell-derived interneuron transplants may be an effective therapeutic strategy to reduce hippocampal hyperactivity and attenuate behavioral deficits in schizophrenia. Here we used a dual-reporter embryonic stem cell line to generate enriched populations of parvalbumin (PV)- or somatostatin (SST)-positive interneurons, which were transplanted into the ventral hippocampus of the methylazoxymethanol rodent model of schizophrenia. These interneuron transplants integrate within the existing circuitry, reduce hippocampal hyperactivity and normalize aberrant dopamine neuron activity. Further, interneuron transplants alleviate behaviors that model negative and cognitive symptoms, including deficits in social interaction and cognitive inflexibility. Interestingly, PV- and SST-enriched transplants produced differential effects on behavior, with PV-enriched populations effectively normalizing all the behaviors examined. These data suggest that the stem cell-derived interneuron transplants may represent a novel therapeutic strategy for schizophrenia.Molecular Psychiatry advance online publication, 2 August 2016; doi:10.1038/mp.2016.121.

  3. Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.

    PubMed

    Shah, Sandeep N; Gelderman, Monique P; Lewis, Emily M A; Farrel, John; Wood, Francine; Strader, Michael Brad; Alayash, Abdu I; Vostal, Jaroslav G

    2016-01-01

    Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as 'the storage lesion'. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.

  4. Pulmonary Epithelial Cell-Derived Cytokine TGF-β1 Is a Critical Cofactor for Enhanced Innate Lymphoid Cell Function

    PubMed Central

    Denney, Laura; Byrne, Adam J.; Shea, Thomas J.; Buckley, James S.; Pease, James E.; Herledan, Gaelle M.F.; Walker, Simone A.; Gregory, Lisa G.; Lloyd, Clare M.

    2015-01-01

    Summary Epithelial cells orchestrate pulmonary homeostasis and pathogen defense and play a crucial role in the initiation of allergic immune responses. Maintaining the balance between homeostasis and inappropriate immune activation and associated pathology is particularly complex at mucosal sites that are exposed to billions of potentially antigenic particles daily. We demonstrated that epithelial cell-derived cytokine TGF-β had a central role in the generation of the pulmonary immune response. Mice that specifically lacked epithelial cell-derived TGF-β1 displayed a reduction in type 2 innate lymphoid cells (ILCs), resulting in suppression of interleukin-13 and hallmark features of the allergic response including airway hyperreactivity. ILCs in the airway lumen were primed to respond to TGF-β by expressing the receptor TGF-βRII and ILC chemoactivity was enhanced by TGF-β. These data demonstrate that resident epithelial cells instruct immune cells, highlighting the central role of the local environmental niche in defining the nature and magnitude of immune reactions. PMID:26588780

  5. Prominent role for T cell-derived Tumour Necrosis Factor for sustained control of Mycobacterium tuberculosis infection

    PubMed Central

    Allie, Nasiema; Grivennikov, Sergei I.; Keeton, Roanne; Hsu, Nai-Jen; Bourigault, Marie-Laure; Court, Nathalie; Fremond, Cecile; Yeremeev, Vladimir; Shebzukhov, Yuriy; Ryffel, Bernhard; Nedospasov, Sergei A.; Quesniaux, Valerie F. J.; Jacobs, Muazzam

    2013-01-01

    Tumour Necrosis Factor (TNF) is critical for host control of M. tuberculosis, but the relative contribution of TNF from innate and adaptive immune responses during tuberculosis infection is unclear. Myeloid versus T-cell-derived TNF function in tuberculosis was investigated using cell type-specific TNF deletion. Mice deficient for TNF expression in macrophages/neutrophils displayed early, transient susceptibility to M. tuberculosis but recruited activated, TNF-producing CD4+ and CD8+ T-cells and controlled chronic infection. Strikingly, deficient TNF expression in T-cells resulted in early control but susceptibility and eventual mortality during chronic infection with increased pulmonary pathology. TNF inactivation in both myeloid and T-cells rendered mice critically susceptible to infection with a phenotype resembling complete TNF deficient mice, indicating that myeloid and T-cells are the primary TNF sources collaborating for host control of tuberculosis. Thus, while TNF from myeloid cells mediates early immune function, T-cell derived TNF is essential to sustain protection during chronic tuberculosis infection. PMID:23657146

  6. Optimization of surface-immobilized extracellular matrices for the proliferation of neural progenitor cells derived from induced pluripotent stem cells.

    PubMed

    Komura, Takashi; Kato, Koichi; Konagaya, Shuhei; Nakaji-Hirabayashi, Tadashi; Iwata, Hiroo

    2015-11-01

    Neural progenitor cells derived from induced pluripotent stem cells have been considered as a potential source for cell-transplantation therapy of central nervous disorders. However, efficient methods to expand neural progenitor cells are further required for their clinical applications. In this study, a protein array was fabricated with nine extracellular matrices and used to screen substrates suitable for the expansion of neural progenitor cells derived from mouse induced pluripotent stem cells. The results showed that neural progenitor cells efficiently proliferated on substrates with immobilized laminin-1, laminin-5, or Matrigel. Based on this result, further attempts were made to develop clinically compliant substrates with immobilized polypeptides that mimic laminin-1, one of the most effective extracellular matrices as identified in the array-based screening. We used here recombinant DNA technology to prepare polypeptide containing the globular domain 3 of laminin-1 and immobilized it onto glass-based substrates. Our results showed that neural progenitor cells selectively proliferated on substrate with the immobilized polypeptide while maintaining their differentiated state.

  7. Teratocarcinomas Arising from Allogeneic Induced Pluripotent Stem Cell-Derived Cardiac Tissue Constructs Provoked Host Immune Rejection in Mice

    PubMed Central

    Kawamura, Ai; Miyagawa, Shigeru; Fukushima, Satsuki; Kawamura, Takuji; Kashiyama, Noriyuki; Ito, Emiko; Watabe, Tadashi; Masuda, Shigeo; Toda, Koichi; Hatazawa, Jun; Morii, Eiichi; Sawa, Yoshiki

    2016-01-01

    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 18F-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. PMID:26763872

  8. Region-Specific Integration of Embryonic Stem Cell-Derived Neuronal Precursors into a Pre-Existing Neuronal Circuit

    PubMed Central

    Neuser, Franziska; Polack, Martin; Annaheim, Christine; Tucker, Kerry L.; Korte, Martin

    2013-01-01

    Enduring reorganization is accepted as a fundamental process of adult neural plasticity. The most dramatic example of this reorganization is the birth and continuously occurring incorporation of new neurons into the pre-existing network of the adult mammalian hippocampus. Based on this phenomenon we transplanted murine embryonic stem (ES)-cell derived neuronal precursors (ESNPs) into murine organotypic hippocampal slice cultures (OHC) and examined their integration. Using a precise quantitative morphological analysis combined with a detailed electrophysiology, we show a region-specific morphological integration of transplanted ESNPs into different subfields of the hippocampal tissue, resulting in pyramidal neuron-like embryonic stem cell-derived neurons (ESNs) in the Cornu Ammonis (CA1 and CA3) and granule neuron-like ESNs in the dentate gyrus (DG), respectively. Subregion specific structural maturation was accompanied by the development of dendritic spines and the generation of excitatory postsynaptic currents (EPSCs). This cell type specific development does not depend upon NMDA-receptor-dependent synaptic transmission. The presented integration approach was further used to determine the cell-autonomous function of the pan-neurotrophin receptor p75 (P75NTR), as a possible negative regulator of ESN integration. By this means we used p75NTR-deficient ESNPs to study their integration into a WT organotypic environment. We show here that p75NTR is not necessary for integration per se but plays a suppressing role in dendritic development. PMID:23840491

  9. Opposite polarity of virus budding and of viral envelope glycoprotein distribution in epithelial cells derived from different tissues

    PubMed Central

    1992-01-01

    We compared the surface envelope glycoprotein distribution and the budding polarity of four RNA viruses in Fischer rat thyroid (FRT) cells and in CaCo-2 cells derived from a human colon carcinoma. Whereas both FRT and CaCo-2 cells sort similarly influenza hemagglutinin and vesicular stomatitis virus (VSV) G protein, respectively, to apical and basolateral membrane domains, they differ in their handling of two togaviruses, Sindbis and Semliki Forest virus (SFV). By conventional EM Sindbis virus and SFV were shown to bud apically in FRT cells and basolaterally in CaCo-2 cells. Consistent with this finding, the distribution of the p62/E2 envelope glycoprotein of SFV, assayed by immunoelectronmicroscopy and by domain-selective surface biotinylation was predominantly apical on FRT cells and basolateral on CaCo-2 cells. We conclude that a given virus and its envelope glycoprotein can be delivered to opposite membrane domains in epithelial cells derived from different tissues. The tissue specificity in the polarity of virus budding and viral envelope glycoprotein distribution indicate that the sorting machinery varies considerably between different epithelial cell types. PMID:1572895

  10. Dendritic cell-derived exosomes as cell-free peptide-based vaccines.

    PubMed

    Taïeb, Julien; Chaput, Nathalie; Zitvogel, Laurence

    2005-01-01

    Dendritic cells (DC) are professional antigen-presenting cells and the only ones capable of inducing primary cytotoxic immune responses both in vivo and in vitro. DCs secrete a 60-100 nm membrane vesicle population of endocytic origin, called exosomes. The lipid and protein composition of DC-derived exosomes (DEX) is now well characterized. Besides MHC and costimulatory molecules, DEX bear several adhesion proteins, which are probably involved in their specific targeting. DEX also accumulate several cytosolic factors, most likely involved in exosome's biogenesis in late endosomes. In 1998, we reported that DEX are immunogenic in mice and lead to tumor rejection. These findings have renewed the interest in DEX. The current challenge consists of understanding the mechanisms and the physiological relevance of DEX, which could contribute to the design of the optimal DEX-based vaccination. In this review, we focus on the biological features of DEX and their immunostimulatory functions in mice and humans, and we discuss their potential clinical implementation in the immunotherapy of cancer.

  11. Mesenchymal Stem Cell-Derived Extracellular Vesicles: Roles in Tumor Growth, Progression, and Drug Resistance

    PubMed Central

    Tu, Huaijun; Yang, Yazhi; Wu, Qiong

    2017-01-01

    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. PMID:28377788

  12. Modeling human development and disease in pluripotent stem cell-derived gastric organoids

    PubMed Central

    McCracken, Kyle W.; Catá, Emily M.; Crawford, Calyn M.; Sinagoga, Katie L.; Schumacher, Michael; Rockich, Briana E.; Tsai, Yu-Hwai; Mayhew, Christopher N.; Spence, Jason R.; Zavros, Yana; Wells, James M.

    2014-01-01

    Gastric diseases, including peptic ulcer disease and gastric cancer, affect 10% of the world’s population and are largely due to chronic H. pylori infection1–3. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis4, and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells (hPSCs). We identified that temporal manipulation of the FGF, WNT, BMP, retinoic acid and EGF signaling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains, proliferative zones containing LGR5-expressing cells, surface and antral mucous cells, and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signaling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease, we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor, activation of signaling and induction of epithelial proliferation. Together, these studies describe a novel and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease. PMID:25363776

  13. Effects of Asiasari radix on the morphology and viability of mesenchymal stem cells derived from the gingiva.

    PubMed

    Jeong, Su-Hyeon; Lee, Ji-Eun; Jin, Seong-Ho; Ko, Youngkyung; Park, Jun-Beom

    2014-12-01

    Medicinal herbs used in traditional Oriental medicine, which have been in use clinically for thousands of years, are attractive sources of novel therapeutics or preventatives. Asiasari radix (A. radix) has been suggested for use in the treatment of dental diseases, including toothache and aphthous stomatitis. The aim of this study was to evaluate the effects of A. radix extracts on the morphology and viability of human stem cells derived from the gingiva. An Asiasarum heterotropoides extract was centrifuged and freeze-dried in a lyophilizer. Stem cells derived from the gingiva were grown in the presence of A. radix at concentrations ranging between 0.1 µg/ml and 1 mg/ml (0, 0.1, 1, 10, 100 and 1,000 µg/ml). Cell morphology was evaluated with an optical microscope and the viability of the cells was quantitatively analyzed with a cell counting kit-8 (CCK-8) assay for up to seven days. The untreated control group exhibited normal fibroblast morphology. The shapes of the cells following 0.1, 1, 10 and 100 µg/ml A. radix treatments were similar to those of the control group. However, a significant change was noted in the 1,000 µg/ml group on day 1, when compared with the untreated group. Furthermore, on day 7, the shapes of the cells following 100 and 1,000 µg/ml A. radix treatments were rounder and fewer cells were present, when compared with those of the control group. The cultures that grew in the presence of A. radix did not exhibit any changes in the CCK‑8 assay on day 2; however, significant reductions in cell viability were noticed following 100 and 1,000 µg/ml A. radix treatment on days 5 and 7. Within the limits of this study, A. radix influenced the viability of the stem cells derived from the gingiva. Thus, the direct application of A. radix to oral tissues may produce adverse effects at high doses. Therefore, the concentration and application time of A. radix requires meticulous control to obtain optimal results. These effects require consideration

  14. Stem cell-derived erythrocytes as upcoming players in blood transfusion

    PubMed Central

    Migliaccio, A. R.

    2015-01-01

    Background Blood transfusion is current standard-of-care for genetic forms of anemia that would be otherwise lethal and allows implementation of aggressive cytotoxic/surgical therapies developed for numerous types of cancer. In developed countries the blood supply is adequate and sporadically even in excess. However, difficulties exist in finding blood with rare phenotypes to treat alloimmunized patients and the progressive ageing of the human population predicts that blood will become scarce by 2050. These considerations establish the need for the development of techniques to generate cultured red blood cell (cRBCs) as transfusion products. Materials and Methods Recent progress in cell culture techniques is revolutionizing organ replacement therapies. Two new disciplines, cell therapy and tissue engineering, have been developed to generate in vitro therapeutic products for a variety of applications ranging from skin grafts to organ-function repairs. It is currently believed that these advances will eventually allow ex-vivo production of various cell types in numbers so great that, in the case of red cells, would be clinically adequate for transfusion. Results Proof-of-principle in animal models indicate that cRBCs generated from murine embryonic stem cells protect mice from lethal anemia. Conditions to generate small amounts of clinical grade cRBCs have been established and the first-in-man administration of autologous cRBCs perfomed. The results of this trial indicate that cRBCs survive in vivo at least as long as their natural counterpart. Discussion These ground-breaking reports have raised great excitement for clinical evaluation of cRBCs for transfusion. However, skepticism still persist that production of cRBCs in numbers sufficient for transfusion will ever be possible. This paper will discuss diagnostic and clinical goals pursuable with numbers of cRBCs that may be generated with current technology. Conclusion We are confident that development of relevant

  15. Shedding of Endogenous Interleukin-6 Receptor (IL-6R) Is Governed by A Disintegrin and Metalloproteinase (ADAM) Proteases while a Full-length IL-6R Isoform Localizes to Circulating Microvesicles*

    PubMed Central

    Schumacher, Neele; Meyer, Dörte; Mauermann, Andre; von der Heyde, Jan; Wolf, Janina; Schwarz, Jeanette; Knittler, Katharina; Murphy, Gillian; Michalek, Matthias; Garbers, Christoph; Bartsch, Jörg W.; Guo, Songbo; Schacher, Beate; Eickholz, Peter; Chalaris, Athena; Rose-John, Stefan; Rabe, Björn

    2015-01-01

    Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation. PMID:26359498

  16. Integrated Analysis of Contractile Kinetics, Force Generation, and Electrical Activity in Single Human Stem Cell-Derived Cardiomyocytes.

    PubMed

    Kijlstra, Jan David; Hu, Dongjian; Mittal, Nikhil; Kausel, Eduardo; van der Meer, Peter; Garakani, Arman; Domian, Ibrahim J

    2015-12-08

    The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) through simultaneous quantitative analysis of contraction kinetics, force generation, and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness, we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly, our approach has the potential for broad application in the study of cardiac disease, drug discovery, and cardiotoxicity screening.

  17. Monitoring the Differentiation and Migration Patterns of Neural Cells Derived from Human Embryonic Stem Cells Using a Microfluidic Culture System

    PubMed Central

    Lee, Nayeon; Park, Jae Woo; Kim, Hyung Joon; Yeon, Ju Hun; Kwon, Jihye; Ko, Jung Jae; Oh, Seung-Hun; Kim, Hyun Sook; Kim, Aeri; Han, Baek Soo; Lee, Sang Chul; Jeon, Noo Li; Song, Jihwan

    2014-01-01

    Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells. PMID:24938227

  18. Galactosylated collagen matrix enhanced in vitro maturation of human embryonic stem cell-derived hepatocyte-like cells.

    PubMed

    Ghodsizadeh, Arefeh; Hosseinkhani, Hossein; Piryaei, Abbas; Pournasr, Behshad; Najarasl, Mostafa; Hiraoka, Yosuke; Baharvand, Hossein

    2014-05-01

    Due to their important biomedical applications, functional human embryonic stem cell-derived hepatocyte-like cells (hESC-HLCs) are an attractive topic in the field of stem cell differentiation. Here, we have initially differentiated hESCs into functional hepatic endoderm (HE) and continued the differentiation by replating them onto galactosylated collagen (GC) and collagen matrices. The differentiation of hESC-HE cells into HLCs on GC substrate showed significant up-regulation of hepatic-specific genes such as ALB, HNF4α, CYP3A4, G6P, and ASGR1. There was more albumin secretion and urea synthesis, as well as more cytochrome p450 activity, in differentiated HLCs on GC compared to the collagen-coated substrate. These results suggested that GC substrate has the potential to be used for in vitro maturation of hESC-HLCs.

  19. Monitoring the differentiation and migration patterns of neural cells derived from human embryonic stem cells using a microfluidic culture system.

    PubMed

    Lee, Nayeon; Park, Jae Woo; Kim, Hyung Joon; Yeon, Ju Hun; Kwon, Jihye; Ko, Jung Jae; Oh, Seung-Hun; Kim, Hyun Sook; Kim, Aeri; Han, Baek Soo; Lee, Sang Chul; Jeon, Noo Li; Song, Jihwan

    2014-06-01

    Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.

  20. Stromal cell-derived factor-1 promotes migration of cells from the upper rhombic lip in cerebellar development.

    PubMed

    Yu, Tao; Huang, Hai; Li, Hui-Fang

    2010-10-01

    During cerebellar development, the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha) has been shown to play an important role in recruiting cells from the upper rhombic lip (URL) and external granule cell layer (EGL). However, its function in cerebellar development is still poorly understood. Our results have demonstrated that SDF-1 is necessary for EGL development, and URL cells stream to the SDF-1 source in vitro. Results of embryonic URL explant assays and transwell assays indicated that SDF-1 induces neural cell migration from the URL region in chemotactic and chemokinetic responses. The time-lapse results showed that the migration speed of granule cell progenitors out of the URL was accelerated by the addition of recombinant SDF-1 alpha. Collectively, our study shows that SDF-1 increases the motility of URL cells in the absence of a gradient and promotes the migration of granule cell progenitors during cerebellar development.

  1. Integrated Analysis of Contractile Kinetics, Force Generation, and Electrical Activity in Single Human Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Kijlstra, Jan David; Hu, Dongjian; Mittal, Nikhil; Kausel, Eduardo; van der Meer, Peter; Garakani, Arman; Domian, Ibrahim J.

    2015-01-01

    Summary The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) through simultaneous quantitative analysis of contraction kinetics, force generation, and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness, we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly, our approach has the potential for broad application in the study of cardiac disease, drug discovery, and cardiotoxicity screening. PMID:26626178

  2. Establishment of an ES cell-derived murine megakaryocytic cell line, MKD1, with features of primary megakaryocyte progenitors.

    PubMed

    Chagraoui, Hedia; Porcher, Catherine

    2012-01-01

    Because of the scarcity of megakaryocytes in hematopoietic tissues, studying megakaryopoiesis heavily relies on the availability of appropriate cellular models. Here, we report the establishment of a new mouse embryonic stem (ES) cell-derived megakaryocytic cell line, MKD1. The cells are factor-dependent, their cell surface immunophenotype and gene expression profile closely resemble that of primary megakaryocyte progenitors (MkPs) and they further differentiate along the megakaryocyte lineage upon valproic acid treatment. At a functional level, we show that ablation of SCL expression, a transcription factor critical for MkP maturation, leads to gene expression alterations similar to that observed in primary, Scl-excised MkPs. Moreover, the cell line is amenable to biochemical and transcriptional analyses, as we report for GpVI, a direct target of SCL. Thus, the MKD1 cell line offers a pertinent experimental model to study the cellular and molecular mechanisms underlying MkP biology and more broadly megakaryopoiesis.

  3. Mouse embryonic stem cell-derived cells reveal niches that support neuronal differentiation in the adult rat brain.

    PubMed

    Maya-Espinosa, Guadalupe; Collazo-Navarrete, Omar; Millán-Aldaco, Diana; Palomero-Rivero, Marcela; Guerrero-Flores, Gilda; Drucker-Colín, René; Covarrubias, Luis; Guerra-Crespo, Magdalena

    2015-02-01

    A neurogenic niche can be identified by the proliferation and differentiation of its naturally residing neural stem cells. However, it remains unclear whether "silent" neurogenic niches or regions suitable for neural differentiation, other than the areas of active neurogenesis, exist in the adult brain. Embryoid body (EB) cells derived from embryonic stem cells (ESCs) are endowed with a high potential to respond to specification and neuralization signals of the embryo. Hence, to identify microenvironments in the postnatal and adult rat brain with the capacity to support neuronal differentiation, we transplanted dissociated EB cells to conventional neurogenic and non-neurogenic regions. Our results show a neuronal differentiation pattern of EB cells that was dependent on the host region. Efficient neuronal differentiation of EB cells occurred within an adjacent region to the rostral migratory stream. EB cell differentiation was initially patchy and progressed toward an even distribution along the graft by 15-21 days post-transplantation, giving rise mostly to GABAergic neurons. EB cells in the striatum displayed a lower level of neuronal differentiation and derived into a significant number of astrocytes. Remarkably, when EB cells were transplanted to the striatum of adult rats after a local ischemic stroke, increased number of neuroblasts and neurons were observed. Unexpectedly, we determined that the adult substantia nigra pars compacta, considered a non-neurogenic area, harbors a robust neurogenic environment. Therefore, neurally uncommitted cells derived from ESCs can detect regions that support neuronal differentiation within the adult brain, a fundamental step for the development of stem cell-based replacement therapies.

  4. Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model

    PubMed Central

    Lewis, Emily M. A.; Farrel, John; Wood, Francine; Strader, Michael Brad; Alayash, Abdu I.; Vostal, Jaroslav G.

    2016-01-01

    Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as ‘the storage lesion’. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product. PMID:27959920

  5. Human Stem Cell-Derived Endothelial-Hepatic Platform for Efficacy Testing of Vascular-Protective Metabolites from Nutraceuticals.

    PubMed

    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.

  6. Spatiotemporal recapitulation of central nervous system development by murine embryonic stem cell-derived neural stem/progenitor cells.

    PubMed

    Okada, Yohei; Matsumoto, Arifumi; Shimazaki, Takuya; Enoki, Ryosuke; Koizumi, Amane; Ishii, Seiji; Itoyama, Yasuto; Sobue, Gen; Okano, Hideyuki

    2008-12-01

    Neural stem/progenitor cells (NS/PCs) can generate a wide variety of neural cells. However, their fates are generally restricted, depending on the time and location of NS/PC origin. Here we demonstrate that we can recapitulate the spatiotemporal regulation of central nervous system (CNS) development in vitro by using a neurosphere-based culture system of embryonic stem (ES) cell-derived NS/PCs. This ES cell-derived neurosphere system enables the efficient derivation of highly neurogenic fibroblast growth factor-responsive NS/PCs with early temporal identities and high cell-fate plasticity. Over repeated passages, these NS/PCs exhibit temporal progression, becoming epidermal growth factor-responsive gliogenic NS/PCs with late temporal identities; this change is accompanied by an alteration in the epigenetic status of the glial fibrillary acidic protein promoter, similar to that observed in the developing brain. Moreover, the rostrocaudal and dorsoventral spatial identities of the NS/PCs can be successfully regulated by sequential administration of several morphogens. These NS/PCs can differentiate into early-born projection neurons, including cholinergic, catecholaminergic, serotonergic, and motor neurons, that exhibit action potentials in vitro. Finally, these NS/PCs differentiate into neurons that form synaptic contacts with host neurons after their transplantation into wild-type and disease model animals. Thus, this culture system can be used to obtain specific neurons from ES cells, is a simple and powerful tool for investigating the underlying mechanisms of CNS development, and is applicable to regenerative treatment for neurological disorders.

  7. Phenotypic and functional characterization of cytokine-induced killer cells derived from preterm and term infant cord blood.

    PubMed

    Zhang, Qian; Wang, Lili; Luo, Chenghan; Shi, Zanyang; Cheng, Xinru; Zhang, Zhen; Yang, Yi; Zhang, Yi

    2014-11-01

    Cord blood has gradually become an important source for hematopoietic stem cell transplantation (HSCT) in the human, particularly in pediatric patients. Adoptive cellular immunotherapy of patients with hematologic malignancies after umbilical cord blood transplant is crucial. Cytokine‑induced killer (CIK) cells derived from cord blood are a new type of antitumor immune effector cells in tumor prevention and treatment and have increasingly attracted the attention of researchers. On the other hand, it has been suggested that preterm infant cord blood retains an early differentiation phenotype suitable for immunotherapy. Therefore, we determined the phenotypic and functional characterization of CIK cells derived from preterm infant cord blood (PCB-CIK) compared with CIK cells from term infant cord blood (TCB-CIK). Twenty cord blood samples were collected and classified into two groups based on gestational age. Cord blood mononuclear cells (CBMCs) were isolated, cultured and induced to CIK cells in vitro. We used flow cytometry to detect cell surface markers, FlowJo software to analyze the proliferation profile and intracellular staining to test the secretion of cytokines. Finally, we evaluated the antitumor activity of CIK cells against K562 in vitro. Compared with TCB-CIK, PCB-CIK cells demonstrated faster proliferation and higher expression of activated cell surface markers. The secretion of IL-10 was lower in PCB-CIK cells while the expression of perforin and CD107a had no significant difference between the two cell groups. PCB-CIK cells exhibited a high proliferation rate while the cytotoxic activity had no difference between the PCB-CIK and TCB-CIK cells. Hence preterm infant cord blood may be a potential source for immunotherapy.

  8. Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitors Aid in Functional Recovery of Sensory Pathways following Contusive Spinal Cord Injury

    PubMed Central

    All, Angelo H.; Bazley, Faith A.; Gupta, Siddharth; Pashai, Nikta; Hu, Charles; Pourmorteza, Amir; Kerr, Candace

    2012-01-01

    Background 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. Principal Findings 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). Conclusions 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

  9. Generation of Neural Crest-Like Cells From Human Periodontal Ligament Cell-Derived Induced Pluripotent Stem Cells.

    PubMed

    Tomokiyo, Atsushi; Hynes, Kim; Ng, Jia; Menicanin, Danijela; Camp, Esther; Arthur, Agnes; Gronthos, Stan; Mark Bartold, Peter

    2017-02-01

    Neural crest cells (NCC) hold great promise for tissue engineering, however the inability to easily obtain large numbers of NCC is a major factor limiting their use in studies of regenerative medicine. Induced pluripotent stem cells (iPSC) are emerging as a novel candidate that could provide an unlimited source of NCC. In the present study, we examined the potential of neural crest tissue-derived periodontal ligament (PDL) iPSC to differentiate into neural crest-like cells (NCLC) relative to iPSC generated from a non-neural crest derived tissue, foreskin fibroblasts (FF). We detected high HNK1 expression during the differentiation of PDL and FF iPSC into NCLC as a marker for enriching for a population of cells with NCC characteristics. We isolated PDL iPSC- and FF iPSC-derived NCLC, which highly expressed HNK1. A high proportion of the HNK1-positive cell populations generated, expressed the MSC markers, whilst very few cells expressed the pluripotency markers or the hematopoietic markers. The PDL and FF HNK1-positive populations gave rise to smooth muscle, neural, glial, osteoblastic and adipocytic like cells and exhibited higher expression of smooth muscle, neural, and glial cell-associated markers than the PDL and FF HNK1-negative populations. Interestingly, the HNK1-positive cells derived from the PDL-iPSC exhibited a greater ability to differentiate into smooth muscle, neural, glial cells and adipocytes, than the HNK1-positive cells derived from the FF-iPSC. Our work suggests that HNK1-enriched NCLC from neural crest tissue-derived iPSC more closely resemble the phenotypic and functional hallmarks of NCC compared to the HNK1-low population and non-neural crest iPSC-derived NCLC. J. Cell. Physiol. 232: 402-416, 2017. © 2016 Wiley Periodicals, Inc.

  10. Polyphosphate induces matrix metalloproteinase-3-mediated proliferation of odontoblast-like cells derived from induced pluripotent stem cells

    SciTech Connect

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

    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. Isolation and Assessment of Mesenchymal Stem Cells Derived From Bone Marrow: Histologic and Histomorphometric Study in a Canine Periodontal Defect.

    PubMed

    Paknejad, Mojgan; Eslaminejad, Mohamadreza Baghaban; Ghaedi, Baharak; Rokn, Amir-Reza; Khorsand, Afshin; Etemad-Moghadam, Shahroo; Alaeddini, Mojgan; Dehghan, Mohammad Mehdi; Moslemi, Neda; Nowzari, Hessam

    2015-06-01

    The aim of the present study was to investigate an isolation procedure to culture mesenchymal stem cells derived from bone marrow and evaluate their potential in periodontal regeneration. Potential stem cells from bone marrow, aspirated from the iliac crest of nine mongrel canines 1 to 2 years of age, were cultivated. After the examination of surface epitopes of the isolated cells, the total RNA from osteogenic, adipogenic, and chondrogenic cell cultures were analyzed by reverse transcription polymerase chain reaction (RT-PCR) to confirm stem cell gene expressions. 2 × 10(7) mL of the stem cells were loaded on 0.2 mL of anorganic bovine bone mineral (ABBM) granules. In each animal, bilateral acute/chronic intrabony periodontal defects were created surgically and by placement of ligatures around the cervical aspect of the teeth. At week 5, after flap debridement, the bilateral defects were randomly assigned to 2 treatment groups: the control group received ABBM, and the test group received BMSCs-loaded ABBM. Eight weeks after transplantation, regenerative parameters were analyzed histologically and histometrically. The RNA expressions confirmed the cultivation of mesenchymal stem cell. More new cementum and periodontal ligament (PDL) were measured in the test group (cementum: 3.33 ± 0.94 vs 2.03 ± 1.30, P = 0.027; PDL: 2.69 ± 0.73 vs 1.53 ± 1.21, P = 0.026). New bone formation was similar in both groups (2.70 ± 0.86 vs 1.99 ± 1.31; P = 0.193). Mesenchymal stem cells derived from bone marrow should be considered a promising technique for use in patients with periodontal attachment loss and merits further investigations.

  12. Early maturation and distinct tau pathology in induced pluripotent stem cell-derived neurons from patients with MAPT mutations.

    PubMed

    Iovino, Mariangela; Agathou, Sylvia; González-Rueda, Ana; Del Castillo Velasco-Herrera, Martin; Borroni, Barbara; Alberici, Antonella; Lynch, Timothy; O'Dowd, Sean; Geti, Imbisaat; Gaffney, Daniel; Vallier, Ludovic; Paulsen, Ole; Káradóttir, Ragnhildur Thóra; Spillantini, Maria Grazia

    2015-11-01

    Tauopathies, such as Alzheimer's disease, some cases of frontotemporal dementia, corticobasal degeneration and progressive supranuclear palsy, are characterized by aggregates of the microtubule-associated protein tau, which are linked to neuronal death and disease development and can be caused by mutations in the MAPT gene. Six tau isoforms are present in the adult human brain and they differ by the presence of 3(3R) or 4(4R) C-terminal repeats. Only the shortest 3R isoform is present in foetal brain. MAPT mutations found in human disease affect tau binding to microtubules or the 3R:4R isoform ratio by altering exon 10 splicing. We have differentiated neurons from induced pluripotent stem cells derived from fibroblasts of controls and patients with N279K and P301L MAPT mutations. Induced pluripotent stem cell-derived neurons recapitulate developmental tau expression, showing the adult brain tau isoforms after several months in culture. Both N279K and P301L neurons exhibit earlier electrophysiological maturation and altered mitochondrial transport compared to controls. Specifically, the N279K neurons show abnormally premature developmental 4R tau expression, including changes in the 3R:4R isoform ratio and AT100-hyperphosphorylated tau aggregates, while P301L neurons are characterized by contorted processes with varicosity-like structures, some containing both alpha-synuclein and 4R tau. The previously unreported faster maturation of MAPT mutant human neurons, the developmental expression of 4R tau and the morphological alterations may contribute to disease development.

  13. Unique proliferation response in odontoblastic cells derived from human skeletal muscle stem cells by cytokine-induced matrix metalloproteinase-3

    SciTech Connect

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

    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.

  14. Identification and Characterization of 293T Cell-Derived Exosomes by Profiling the Protein, mRNA and MicroRNA Components

    PubMed Central

    Li, Dameng; Wang, Jifeng; Hou, Dongxia; Jiang, Xiaohong; Zhang, Junfeng; Wang, Jin; Zen, Ke; Yang, Fuquan; Zhang, Chen-Yu

    2016-01-01

    Cell-derived exosomes are leading candidates for in vivo drug delivery carriers. In particular, exosomes derived from 293T cells are used most frequently, although exosome dosing has varied greatly among studies. Considering their biological origin, it is crucial to characterize the molecular composition of exosomes if large doses are to be administered in clinical settings. In this study, we present the first comprehensive analysis of the protein, messenger RNA and microRNA profiles of 293T cell-derived exosomes; then, we characterized these data using Gene Ontology annotation and Kyoto Encyclopedia for Genes and Genomes pathway analysis. Our study will provide the basis for the selection of 293T cell-derived exosome drug delivery systems. Profiling the exosomal signatures of 293T cells will lead to a better understanding of 293T exosome biology and will aid in the identification of any harmful factors in exosomes that could cause adverse clinical effects. PMID:27649079

  15. The polyphosphate/factor XII pathway in cancer-associated thrombosis: novel perspectives for safe anticoagulation in patients with malignancies.

    PubMed

    Nickel, Katrin F; Labberton, Linda; Long, Andrew T; Langer, Florian; Fuchs, Tobias A; Stavrou, Evi X; Butler, Lynn M; Renné, Thomas

    2016-05-01

    Cancer is an established risk factor for venous thromboembolism (VTE) and VTE is the second leading cause of death in patients with cancer. The incidence of cancer-related thrombosis is rising and is associated with worse outcomes. Despite our growing understanding on tumor-driven procoagulant mechanisms including cancer-released procoagulant proteases, expression of tissue factor on cancer cells and derived microvesicles, as well as alterations in the extracellular matrix of the cancer cell milieu, anticoagulation therapy in cancer patients has remained challenging. This review comments on a newly discovered cancer-associated procoagulant pathway. Experimental VTE models in mice and studies on patient cancer material revealed that prostate cancer cells and associated exosomes display the inorganic polymer polyphosphate on their plasma membrane. Polyphosphate activates blood coagulation factor XII and initiates thrombus formation via the intrinsic pathway of coagulation. Pharmacologic inhibition of factor XII activity protects mice from VTE and reduces thrombin coagulant activity in plasma of prostate cancer patients. Factor XII inhibitors provide thrombo-protection without impairing hemostatic mechanisms and thus, unlike currently used anticoagulants, do not increase bleeding risk. Interference with the polyphosphate/factor XII pathway may provide the novel opportunity for safe anticoagulation therapy in patients with malignancies.

  16. Upregulation of CD4+T-Cell Derived MiR-223 in The Relapsing Phase of Multiple Sclerosis Patients

    PubMed Central

    Hosseini, Aref; Ghaedi, Kamran; Tanhaei, Somayeh; Ganjalikhani-Hakemi, Mazdak; Teimuri, Shohreh; Etemadifar, Masoud; Nasr Esfahani, Mohammad Hossein

    2016-01-01

    Objective MicroRNAs (miRNA) are a class of non-coding RNAs which play key roles in post-transcriptional gene regulation. Previous studies indicate that miRNAs are dysregulated in patients with multiple sclerosis (MS). Th17 and regulatory T (Treg) cells are two subsets of CD4+T-cells which have critical functions in the onset and progression of MS. The current study seeks to distinguish fluctuations in expression of CD4+T-cell derived miR-223 during the relapsing-remitting (RR) phase of MS (RR-MS), as well as the expressions of Th17 and Treg cell markers. Materials and Methods This experimental study used real-time quantitative polymerase chain reaction (qRT-PCR) to evaluate CD4+ T cell derived miR-223 expression patterns in patients that experienced either of the RR-MS phases (n=40) compared to healthy controls (n=12), along with RNA markers for Th17 and Treg cells. We conducted flow cytometry analyses of forkhead box P3 (FOXP3) and RAR-related orphan receptor γt (RORγt) in CD4+T-cells. Putative and validated targets of miR-223 were investigated in the miRWalk and miRTarBase databases, respectively. Results miR-223 significantly upregulated in CD4+T-cells during the relapsing phase of RR-MS compared to the remitting phase (P=0.000) and healthy individuals (P=0.036). Expression of RORγt, a master transcription factor of Th17, upregulated in the relapsing phase, whereas FOXP3 upregulated in the remitting phase. Additionally, potential targets of miR-223, STAT1, FORKHEAD BOX O (FOXO1) and FOXO3 were predicted by in silico studies. Conclusion miR-223 may have a potential role in MS progression. Therefore, suppression of miR-223 can be proposed as an appropriate approach to control progression of the relapsing phase of MS. PMID:27602319

  17. Venous Thrombosis and Cancer: from Mouse Models to Clinical Trials

    PubMed Central

    Hisada, Y.; Geddings, J. E.; Ay, C.; Mackman, N.

    2015-01-01

    Cancer patients have a ~4 fold increased risk of venous thromboembolism (VTE) compared with the general population and this is associated with significant morbidity and mortality. This review summarizes our current knowledge of VTE and cancer from mouse models to clinical studies. Notably, risk of VTE varies depending on the type and stage of cancer. For instance, pancreatic and brain cancer patients have a higher risk of VTE than breast and prostate cancer patients. Moreover, patients with metastatic disease have a higher risk than those with localized tumors. Tumor-derived procoagulant factors and growth factors may directly and indirectly enhance VTE. For example, increased levels of circulating tumor-derived, tissue factor-positive microvesicles may trigger VTE. In a mouse model of ovarian cancer, tumor-derived IL-6 and hepatic thrombopoietin has been linked to increased platelet production and thrombosis. In addition, mouse models of mammary and lung cancer showed that tumor-derived granulocyte colony-stimulating factor causes neutrophilia and activation of neutrophils. Activated neutrophils can release neutrophil extracellular traps (NETs) that enhance thrombosis. Cell-free DNA in the blood derived from cancer cells, NETs and treatment with cytotoxic drugs can activate the clotting cascade. These studies suggest that there are multiple mechanisms for VTE in patients with different types of cancer. Preventing and treating VTE in cancer patients is challenging; the current recommendations are to use low molecular weight heparin. Understanding the underlying mechanisms may allow the development of new therapies to safely prevent VTE in cancer patients. PMID:25988873

  18. Patient-specific cardiovascular progenitor cells derived from integration-free induced pluripotent stem cells for vascular tissue regeneration

    PubMed Central

    Hu, Jiang; Wang, Yongyu; Jiao, Jiao; Liu, Zhongning; Zhao, Chao; Zhou, Zhou; Zhang, Zhanpeng; Forde, Kaitlynn; Wang, Lunchang; Wang, Jiangang; Baylink, David J.; Zhang, Xiao-Bing; Gao, Shaorong; Yang, Bo; Chen, Y. Eugene; Ma, Peter X.

    2015-01-01

    Tissue-engineered blood vessels (TEBVs) are promising in regenerating a live vascular replacement. However, the vascular cell source is limited, and it is crucial to develop a scaffold that accommodates new type of vascular progenitor cells and facilitates in vivo lineage specification of the cells into functional vascular smooth muscle cells (VSMCs) to regenerate vascular tissue. In the present study, integration-free human induced pluripotent stem cells (hiPSCs) were established from patient peripheral blood mononuclear cells through episomal vector nucleofection of reprogramming factors. The established hiPSCs were then induced into mesoderm-originated cardiovascular progenitor cells (CVPCs) with a highly efficient directed lineage specification method. The derived CVPCs were demonstrated to be able to differentiate into functional VSMCs. Subcutaneous implantation of CVPCs seeded on macroporous nanofibrous poly(l-lactide) scaffolds led to in vivo VSMC lineage specification and matrix deposition inside the scaffolds. In summary, we established integration-free patient-specific hiPSCs from peripheral blood mononuclear cells, derived CVPCs through directed lineage specification, and developed an advanced scaffold for these progenitor cells to further differentiate in vivo into VSMCs and regenerate vascular tissue in a subcutaneous implantation model. This study has established an efficient patient-specific approach towards in vivo regeneration of vascular tissue. PMID:26398309

  19. The effect of magnetic nanoparticles on neuronal differentiation of induced pluripotent stem cell-derived neural precursors

    PubMed Central

    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

  20. A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

    PubMed Central

    Hellewell, Andrew L.; Rosini, Silvia; Adams, Josephine C.

    2017-01-01

    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. PMID:28117783

  1. Recommended Ethical Safeguards on Fertilization of Human Germ Cells Derived from Pluripotent Stem Cells Solely for Research Purposes.

    PubMed

    Mizuno, Hiroshi

    2016-08-01

    Production of human fertilized embryos by using germ cells derived from pluripotent stem cells (PSCs) entails ethical issues that differ fundamentally depending on the aim. If the aim is solely to conduct research, then embryo generation, utilization and destruction must respect for the human embryo as having the innate potential to develop into a human being. If the aim is human reproduction, this technology must never be used to manipulate human life, confuse social order, or negatively affect future generations. Researchers should distinguish the aims and then accordingly establish a consensus on the safeguards needed to proceed with scientifically significant and socially accepted research, or otherwise set a moratorium. Currently, in Japan, germ cell production from human PSCs is permitted, whereas fertilization of these germ cells is not. The Japanese Expert Panel on Bioethics in the Cabinet Office has proposed that all of the following conditions must be met to approve fertilization for research purposes: (1) the research is significant for the life sciences and medicine; (2) the benefits or anticipated benefits are socially accepted; (3) human safety is assured; and (4) safeguards are put in place. If fertilization is ethically approved, I recommend the following safeguards: limitation of the purpose to improving conventional ART as an initial step; permitted culture of human embryos until the appearance of the primitive streak; restriction of the number of embryos produced to the minimum necessary; prohibition of transplantation into a human or animal uterus; and provision of human-derived ova that are not required for ART treatment.

  2. Conditional-ready mouse embryonic stem cell derived macrophages enable the study of essential genes in macrophage function

    PubMed Central

    Yeung, A. T. Y.; Hale, C.; Xia, J.; Tate, P. H.; Goulding, D.; Keane, J. A.; Mukhopadhyay, S.; Forrester, L.; Billker, O.; Skarnes, W. C.; Hancock, R. E. W.; Dougan, G.

    2015-01-01

    The ability to differentiate genetically modified mouse embryonic stem (ES) cells into functional macrophages provides a potentially attractive resource to study host-pathogen interactions without the need for animal experimentation. This is particularly useful in instances where the gene of interest is essential and a knockout mouse is not available. Here we differentiated mouse ES cells into macrophages in vitro and showed, through a combination of flow cytometry, microscopic imaging, and RNA-Seq, that ES cell-derived macrophages responded to S. Typhimurium, in a comparable manner to mouse bone marrow derived macrophages. We constructed a homozygous mutant mouse ES cell line in the Traf2 gene that is known to play a role in tumour necrosis factor-α signalling but has not been studied for its role in infections or response to Toll-like receptor agonists. Interestingly, traf2-deficient macrophages produced reduced levels of inflammatory cytokines in response to lipopolysaccharide (LPS) or flagellin stimulation and exhibited increased susceptibility to S. Typhimurium infection. PMID:25752829