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Sample records for affects cell expansion

  1. Culture materials affect ex vivo expansion of hematopoietic progenitor cells.

    PubMed

    LaIuppa, J A; McAdams, T A; Papoutsakis, E T; Miller, W M

    1997-09-05

    Ex vivo expansion of hematopoietic cells is important for applications such as cancer treatment, gene therapy, and transfusion medicine. While cell culture systems are widely used to evaluate the biocompatibility of materials for implantation, the ability of materials to support proliferation of primary human cells in cultures for reinfusion into patients has not been addressed. We screened a variety of commercially available polymer (15 types), metal (four types), and glass substrates for their ability to support expansion of hematopoietic cells when cultured under conditions that would be encountered in a clinical setting. Cultures of peripheral blood (PB) CD34+ cells and mononuclear cells (MNC) were evaluated for expansion of total cells and colony-forming unit-granulocyte monocyte (CFU-GM; progenitors committed to the granulocyte and/or monocyte lineage). Human hematopoietic cultures in serum-free medium were found to be extremely sensitive to the substrate material. The only materials tested that supported expansion at or near the levels of polystyrene were tissue culture polystyrene, Teflon perfluoroalkoxy, Teflon fluorinated ethylene propylene, cellulose acetate, titanium, new polycarbonate, and new polymethylpentene. MNC were less sensitive to the substrate materials than the primitive CD34+ progenitors, although similar trends were seen for expansion of the two cell populations on the substrates tested. CFU-GM expansion was more sensitive to substrate materials than was total cell expansion. The detrimental effects of a number of the materials on hematopoietic cultures appear to be caused by protein adsorption and/or leaching of toxins. Factors such as cleaning, sterilization, and reuse significantly affected the performance of some materials as culture substrates. We also used PB CD34+ cell cultures to examine the biocompatibility of gas-permeable cell culture and blood storage bags and several types of tubing commonly used with biomedical equipment

  2. The novel herbicide oxaziclomefone inhibits cell expansion in maize cell cultures without affecting turgor pressure or wall acidification.

    PubMed

    O'Looney, Nichola; Fry, Stephen C

    2005-11-01

    Oxaziclomefone [OAC; IUPAC name 3-(1-(3,5-dichlorophenyl)-1-methylethyl)-3,4-dihydro-6-methyl-5-phenyl-2H-1,3-oxazin-4-one] is a new herbicide that inhibits cell expansion in grass roots. Its effects on cell cultures and mode of action were unknown. In principle, cell expansion could be inhibited by a decrease in either turgor pressure or wall extensibility. Cell expansion was estimated as settled cell volume; cell division was estimated by cell counting. Membrane permeability to water was measured by a novel method involving simultaneous assay of the efflux of (3)H(2)O and [(14)C]mannitol from a 'bed' of cultured cells. Osmotic potential was measured by depression of freezing point. OAC inhibited cell expansion in cultures of maize (Zea mays), spinach (Spinacia oleracea) and rose (Rosa sp.), with an ID(50) of 5, 30 and 250 nm, respectively. In maize cultures, OAC did not affect cell division for the first 40 h. It did not affect the osmotic potential of cell sap or culture medium, nor did it impede water transport across cell membranes. It did not affect cells' ability to acidify the apoplast (medium), which may be necessary for 'acid growth'. As OAC did not diminish turgor pressure, its ability to inhibit cell expansion must depend on changes in wall extensibility. It could be a valuable tool for studies on cell expansion.

  3. Expansion of adipose mesenchymal stromal cells is affected by human platelet lysate and plating density.

    PubMed

    Cholewa, Dominik; Stiehl, Thomas; Schellenberg, Anne; Bokermann, Gudrun; Joussen, Sylvia; Koch, Carmen; Walenda, Thomas; Pallua, Norbert; Marciniak-Czochra, Anna; Suschek, Christoph V; Wagner, Wolfgang

    2011-01-01

    The composition of mesenchymal stromal cells (MSCs) changes in the course of in vitro culture expansion. Little is known how these cell preparations are influenced by culture media, plating density, or passaging. In this study, we have isolated MSCs from human adipose tissue in culture medium supplemented with either fetal calf serum (FCS) or human platelet lysate (HPL). In addition, culture expansion was simultaneously performed at plating densities of 10 or 10,000 cells/cm(2). The use of FCS resulted in larger cells, whereas HPL significantly enhanced proliferation. Notably, HPL also facilitated expansion for more population doublings than FCS (43 ± 3 vs. 22 ± 4 population doubling; p < 0.001), while plating density did not have a significant effect on long-term growth curves. To gain further insight into population dynamics, we conceived a cellular automaton model to simulate expansion of MSCS. It is based on the assumptions that the number of cell divisions is limited and that due to contact inhibition proliferation occurs only at the rim of colonies. The model predicts that low plating densities result in more heterogeneity with regard to cell division history, and favor subpopulations of higher migratory activity. In summary, HPL is a suitable serum supplement for isolation of MSC from adipose tissue and facilitates more population doublings than FCS. Cellular automaton computer simulations provided additional insights into how complex population dynamics during long-term expansion are affected by plating density and migration.

  4. The conflict between cell proliferation and expansion primarily affects stem organogenesis in Arabidopsis.

    PubMed

    Maeda, Saori; Gunji, Shizuka; Hanai, Kenya; Hirano, Tomonari; Kazama, Yusuke; Ohbayashi, Iwai; Abe, Tomoko; Sawa, Shinichiro; Tsukaya, Hirokazu; Ferjani, Ali

    2014-11-01

    Plant shoot organs such as stems, leaves and flowers are derived from specialized groups of stem cells organized at the shoot apical meristem (SAM). Organogenesis involves two major processes, namely cell proliferation and differentiation, whereby the former contributes to increasing the cell number and the latter involves substantial increases in cell volume through cell expansion. Co-ordination between the above processes in time and space is essential for proper organogenesis. To identify regulatory factors involved in proper organogenesis, heavy-ion beam-irradiated de-etiolated (det) 3-1 seeds have been used to identify striking phenotypes in the A#26-2; det3-1 mutant. In addition to the stunted plant stature mimicking det3-1, the A#26-2; det3-1 mutant exhibited stem thickening, increased floral organ number and a fruit shape reminiscent of clavata (clv) mutants. DNA sequencing analysis demonstrated that A#26-2; det3-1 harbors a mutation in the CLV3 gene. Importantly, A#26-2; det3-1 displayed cracks that randomly occurred on the main stem with a frequency of approximately 50%. Furthermore, the double mutants clv3-8 det3-1, clv1-4 det3-1 and clv2-1 det3-1 consistently showed stem cracks with frequencies of approximately 97, 38 and 35%, respectively. Cross-sections of stems further revealed an increase in vascular bundle number, cell number and size in the pith of clv3-8 det3-1 compared with det3-1. These findings suggest that the stem inner volume increase due to clv mutations exerts an outward mechanical stress; that in a det3-1 background (defective in cell expansion) resulted in cracking of the outermost layer of epidermal cells.

  5. Functional and differential proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesenchymal stromal cells

    PubMed Central

    2013-01-01

    Background Multilineage differentiation, immunomodulation and secretion of trophic factors render mesenchymal stromal cells (MSC) highly attractive for clinical application. Human platelet derivatives such as pooled human platelet lysate (pHPL) and thrombin-activated platelet releasate in plasma (tPRP) have been introduced as alternatives to fetal bovine serum (FBS) to achieve GMP-compliance. However, whereas both pHPL and tPRP support similar proliferation kinetics of lipoaspirate-derived MSC (LA-MSC), only pHPL significantly accelerates bone marrow-derived MSC (BM-MSC) expansion. To identify functionally bioactive factors affecting ex vivo MSC expansion, a differential proteomic approach was performed and identified candidate proteins were evaluated within a bioassay. Results Two dimensional difference gel electrophoresis (2D-DIGE), MALDI-TOF analyses and complementary Western blotting revealed 20 differential protein species. 14 candidate proteins occured at higher concentrations in pHPL compared to tPRP and 6 at higher concentrations in tPRP. The candidate proteins fibrinogen and apolipoprotein A1 differentially affected LA- and BM-MSC proliferation. In a second set of experiments, reference cytokines known to foster proliferation in FBS were tested for their effects in the human supplements. Interestingly although these cytokines promoted proliferation in FBS, they failed to do so when added to the humanized system. Conclusions The differential proteomic approach identified novel platelet derived factors differentially acting on human MSC proliferation. Complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to FBS. The data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, GMP-compliant conditions for MSC expansion. PMID:24168020

  6. 182 MATURATION OF BOVINE CUMULUS-OOCYTE COMPLEXES WITH FOLLICLE FLUID VARYING IN ESTRADIOL CONTENT AFFECTS CUMULUS CELL EXPANSION WITHOUT AFFECTING SUBSEQUENT EMBRYO DEVELOPMENT IN VITRO.

    PubMed

    Harl, A W; Larimore, E L; Al Naib, A; Wooldridge, L K; Ealy, A D; Perry, G A; Rhoads, M L

    2016-01-01

    The objective of this work was to determine how characteristics of bovine follicle fluid (FF; especially oestradiol content) affect cumulus cell expansion and oocyte competence. In the first study, FF was collected from abattoir-derived ovaries and pooled separately for large follicles (≥10mm) and small follicles (≤3mm). A portion of the FF from each category was charcoal stripped. These 4 types of FF were then used as the primary ingredient (75% vol/vol) in oocyte maturation media. A separate control group lacking FF but containing BSA was included to monitor potential impacts of protein on outcomes (control; without FF). Some of the cumulus-oocyte complexes (COC; n=250) were matured in individual drops for analysis of cumulus expansion (photographed and measured at 0 and 21h of maturation). Other COC (n=770) were matured in groups of 12 to 25 in the previously described media, and then subjected to IVF procedures. Cleavage rates were recorded on Day 3, and blastocyst rates were recorded on Day 8 post-fertilization. Cumulus cell expansion was greatest when COC were matured in medium containing FF from large follicles, wherein it even exceeded the controls (P<0.02). Maturation in FF from small follicles resulted in cumulus expansion that was intermediate between large and control. Maturation in charcoal-stripped FF severely restricted cumulus cell expansion (P<0.05) compared with those matured in untreated FF. Despite the observed improvement in cumulus cell expansion, COC that had been matured in media containing FF were less likely to cleave (P<0.05) and also less likely to develop to the blastocyst stage (P<0.01) than those matured in control medium. Cleavage and blastocyst rates did not differ among any of the maturation media containing FF. In the second study, oestrous cycles of 9 crossbred cows were synchronized and FF samples were collected 36 to 42h after prostaglandin F2α injection. Samples from individual cows were categorized as having high

  7. Feline foamy virus adversely affects feline mesenchymal stem cell culture and expansion: implications for animal model development.

    PubMed

    Arzi, Boaz; Kol, Amir; Murphy, Brian; Walker, Naomi J; Wood, Joshua A; Clark, Kaitlin; Verstraete, Frank J M; Borjesson, Dori L

    2015-04-01

    Mesenchymal stem cells (MSCs) are a promising therapeutic option for various immune-mediated and inflammatory disorders due to their potent immunomodulatory and trophic properties. Naturally occurring diseases in large animal species may serve as surrogate animal models of human disease, as they may better reflect the complex genetic, environmental, and physiologic variation present in outbred populations. We work with naturally occurring diseases in large animal species to better understand how MSCs work and to facilitate optimal translation of MSC-based therapies. We are investigating the use of MSC therapy for a chronic oral inflammatory disease in cats. During our efforts to expand fat-derived feline MSCs (fMSCs), we observed that∼50% of the cell lines developed giant foamy multinucleated cells in later passages. These morphologic alterations were associated with proliferation arrest. We hypothesized that the cytopathic effects were caused by infection with a retrovirus, feline foamy virus (FFV). Using transmission electron microscopy, polymerase chain reaction, and in vitro assays, we determined that syncytial cell formation and proliferation arrest in fMSCs were caused by FFV strains that were highly homologous to previously reported FFV strains. We determined that the antiretroviral drug, tenofovir, may be used to support ex vivo expansion and salvage of FFV-infected fMSC lines. MSC lines derived from specific pathogen-free cats do not appear to be infected with FFV and may be a source of allogeneic fMSCs for clinical application. FFV infection of fMSC lines may hinder large-scale expansion of autologous MSC for therapeutic use in feline patients.

  8. DMSO-free cryopreservation of adipose-derived mesenchymal stromal cells: expansion medium affects post-thaw survival.

    PubMed

    Rogulska, Olena; Petrenko, Yuri; Petrenko, Alexander

    2017-04-01

    Off-the-shelf availability of human adipose-derived mesenchymal stromal cells (ASCs) for regenerative medicine application requires the development of nontoxic, safe, and efficient protocols for cryopreservation. Favorably, such cell processing protocols should not contain xenogeneic or toxic components, such as fetal bovine serum (FS) and dimethyl sulfoxide (DMSO). The objective of the study was to assess the sensitivity of ASCs to DMSO-free cryopreservation protocol depending on their expansion conditions: conventional, based on the application of FS or xeno-free, using PL as a medium supplement. ASCs expansion was carried out in α-MEM supplemented either with FS or PL. For DMSO- and xeno-free cryopreservation ASCs were pretreated with different concentrations of sucrose during 24 h of culture. Pretreated ASCs were cryopreserved in α-MEM containing 100-300 mM of sucrose with the cooling rate of 1 degree/min. ASCs were tested for survival (Trypan Blue test), viability (MTT test), recovery (Alamar Blue test), proliferation and ability to multilineage differentiation. The optimal concentrations of sucrose for ASCs pretreatment and as an additive in cryoprotective solution, which provided highest cell survival, comprised 100 and 200 mM, correspondingly. Survival and recovery rates of platelet lysate (PL)-expanded ASCs after DMSO-free cryopreservation comprised 59 and 51%, and were higher than in FS-cultured cells. After DMSO-free cryopreservation PL-processed ASCs had a shorter population doubling time and higher capacity for osteogenic differentiation than FS-processed cultures. The described DMSO- and xeno-free processing may form the basis for the development of safe and efficient protocols for manufacturing and banking of ASCs, providing their off-the-shelf availability for regenerative medicine applications.

  9. Feline Foamy Virus Adversely Affects Feline Mesenchymal Stem Cell Culture and Expansion: Implications for Animal Model Development

    PubMed Central

    Kol, Amir; Murphy, Brian; Walker, Naomi J.; Wood, Joshua A.; Clark, Kaitlin; Verstraete, Frank J.M.; Borjesson, Dori L.

    2015-01-01

    Abstract Mesenchymal stem cells (MSCs) are a promising therapeutic option for various immune-mediated and inflammatory disorders due to their potent immunomodulatory and trophic properties. Naturally occurring diseases in large animal species may serve as surrogate animal models of human disease, as they may better reflect the complex genetic, environmental, and physiologic variation present in outbred populations. We work with naturally occurring diseases in large animal species to better understand how MSCs work and to facilitate optimal translation of MSC-based therapies. We are investigating the use of MSC therapy for a chronic oral inflammatory disease in cats. During our efforts to expand fat-derived feline MSCs (fMSCs), we observed that∼50% of the cell lines developed giant foamy multinucleated cells in later passages. These morphologic alterations were associated with proliferation arrest. We hypothesized that the cytopathic effects were caused by infection with a retrovirus, feline foamy virus (FFV). Using transmission electron microscopy, polymerase chain reaction, and in vitro assays, we determined that syncytial cell formation and proliferation arrest in fMSCs were caused by FFV strains that were highly homologous to previously reported FFV strains. We determined that the antiretroviral drug, tenofovir, may be used to support ex vivo expansion and salvage of FFV-infected fMSC lines. MSC lines derived from specific pathogen-free cats do not appear to be infected with FFV and may be a source of allogeneic fMSCs for clinical application. FFV infection of fMSC lines may hinder large-scale expansion of autologous MSC for therapeutic use in feline patients. PMID:25404388

  10. Anisotropic Cell Expansion Is Affected through the Bidirectional Mobility of Cellulose Synthase Complexes and Phosphorylation at Two Critical Residues on CESA31[OPEN

    PubMed Central

    Liu, Yanmei; Bauer, Stefan

    2016-01-01

    Here we report that phosphorylation status of S211 and T212 of the CESA3 component of Arabidopsis (Arabidopsis thaliana) cellulose synthase impacts the regulation of anisotropic cell expansion as well as cellulose synthesis and deposition and microtubule-dependent bidirectional mobility of CESA complexes. Mutation of S211 to Ala caused a significant decrease in the length of etiolated hypocotyls and primary roots, while root hairs were not significantly affected. By contrast, the S211E mutation stunted the growth of root hairs, but primary roots were not significantly affected. Similarly, T212E caused a decrease in the length of root hairs but not root length. However, T212E stunted the growth of etiolated hypocotyls. Live-cell imaging of fluorescently labeled CESA showed that the rate of movement of CESA particles was directionally asymmetric in etiolated hypocotyls of S211A and T212E mutants, while similar bidirectional velocities were observed with the wild-type control and S211E and T212A mutant lines. Analysis of cell wall composition and the innermost layer of cell wall suggests a role for phosphorylation of CESA3 S211 and T212 in cellulose aggregation into fibrillar bundles. These results suggest that microtubule-guided bidirectional mobility of CESA complexes is fine-tuned by phosphorylation of CESA3 S211 and T212, which may, in turn, modulate cellulose synthesis and organization, resulting in or contributing to the observed defects of anisotropic cell expansion. PMID:26969722

  11. How will oil palm expansion affect biodiversity?

    PubMed

    Fitzherbert, Emily B; Struebig, Matthew J; Morel, Alexandra; Danielsen, Finn; Brühl, Carsten A; Donald, Paul F; Phalan, Ben

    2008-10-01

    Oil palm is one of the world's most rapidly increasing crops. We assess its contribution to tropical deforestation and review its biodiversity value. Oil palm has replaced large areas of forest in Southeast Asia, but land-cover change statistics alone do not allow an assessment of where it has driven forest clearance and where it has simply followed it. Oil palm plantations support much fewer species than do forests and often also fewer than other tree crops. Further negative impacts include habitat fragmentation and pollution, including greenhouse gas emissions. With rising demand for vegetable oils and biofuels, and strong overlap between areas suitable for oil palm and those of most importance for biodiversity, substantial biodiversity losses will only be averted if future oil palm expansion is managed to avoid deforestation.

  12. Angiogenin is upregulated during the alloreactive immune response and has no effect on the T-cell expansion phase, whereas it affects the contraction phase by inhibiting CD4+ T-cell apoptosis

    PubMed Central

    Eleftheriadis, Theodoros; Pissas, Georgios; Sounidaki, Maria; Antoniadis, Nikolaos; Antoniadi, Georgia; Liakopoulos, Vassilios; Stefanidis, Ioannis

    2016-01-01

    Under growth conditions, angiogenin is translocated into the nucleus, where it enhances ribosomal RNA transcription, facilitating increased protein synthesis and cellular proliferation. During stress conditions, angiogenin is sequestered in the cytoplasm, where it cleaves transfer RNA (tRNA) to produce tRNA-derived, stress-induced small RNAs (tiRNAs) that inhibit global protein synthesis, but increase the translation of anti-apoptotic factors. In the present study, the role of angiogenin in the human alloreactive immune response was evaluated using mixed lymphocyte reactions (MLRs) and neamine, an inhibitor of angiogenin nuclear translocation. In MLRs, angiogenin production was significantly (P<0.001) increased compared with resting peripheral blood mononuclear cells. The addition of neamine had no effect on cell proliferation, but did significantly (P<0.001) increase expression of Bcl-2-associated X protein and protein levels of activated caspase-3 in CD4+ T-cells isolated from the MLRs, indicating that angiogenin reduces apoptosis. In conclusion, angiogenin is upregulated during the alloreactive immune response, in which it does not affect the T-cell expansion phase, but inhibits the T-cell contraction phase by protecting against CD4+ T-cell apoptosis. PMID:27882181

  13. Coupling of solute transport and cell expansion in pea stems

    NASA Technical Reports Server (NTRS)

    Schmalstig, J. G.; Cosgrove, D. J.

    1990-01-01

    As cells expand and are displaced through the elongation zone of the epicotyl of etiolated pea (Pisum sativum L. var Alaska) seedlings, there is little net dilution of the cell sap, implying a coordination between cell expansion and solute uptake from the phloem. Using [14C] sucrose as a phloem tracer (applied to the hypogeous cotyledons), the pattern of label accumulation along the stem closely matched the growth rate pattern: high accumulation in the growing zone, little accumulation in nongrowing regions. Several results suggest that a major portion of phloem contents enters elongating cells through the symplast. We propose that the coordination between phloem transport and cell expansion is accomplished via regulatory pathways affecting both plasmodesmata conductivity and cell expansion.

  14. Hematopoietic Stem Cell Expansion and Gene Therapy

    PubMed Central

    Watts, Korashon Lynn; Adair, Jennifer; Kiem, Hans-Peter

    2012-01-01

    Hematopoietic stem cell (HSC) gene therapy remains a highly attractive treatment option for many disorders including hematologic conditions, immunodeficiencies including HIV/AIDS, and other genetic disorders like lysosomal storage diseases, among others. In this review, we discuss the successes, side effects, and limitations of current gene therapy protocols. In addition, we describe the opportunities presented by implementing ex vivo expansion of gene-modified HSCs, as well as summarize the most promising ex vivo expansion techniques currently available. We conclude by discussing how some of the current limitations of HSC gene therapy could be overcome by combining novel HSC expansion strategies with gene therapy. PMID:21999373

  15. Ex vivo expansion of hematopoietic stem cells.

    PubMed

    Xie, JingJing; Zhang, ChengCheng

    2015-09-01

    Ex vivo expansion of hematopoietic stem cells (HSCs) would benefit clinical applications in several aspects, to improve patient survival, utilize cord blood stem cells for adult applications, and selectively propagate stem cell populations after genetic manipulation. In this review we summarize and discuss recent advances in the culture systems of mouse and human HSCs, which include stroma/HSC co-culture, continuous perfusion and fed-batch cultures, and those supplemented with extrinsic ligands, membrane transportable transcription factors, complement components, protein modification enzymes, metabolites, or small molecule chemicals. Some of the expansion systems have been tested in clinical trials. The optimal condition for ex vivo expansion of the primitive and functional human HSCs is still under development. An improved understanding of the mechanisms for HSC cell fate determination and the HSC culture characteristics will guide development of new strategies to overcome difficulties. In the future, development of a combination treatment regimen with agents that enhance self-renewal, block differentiation, and improve homing will be critical. Methods to enhance yields and lower cost during collection and processing should be employed. The employment of an efficient system for ex vivo expansion of HSCs will facilitate the further development of novel strategies for cell and gene therapies including genome editing.

  16. Lumenal protein within secretory granules affects fusion pore expansion.

    PubMed

    Weiss, Annita Ngatchou; Anantharam, Arun; Bittner, Mary A; Axelrod, Daniel; Holz, Ronald W

    2014-07-01

    It is often assumed that upon fusion of the secretory granule membrane with the plasma membrane, lumenal contents are rapidly discharged and dispersed into the extracellular medium. Although this is the case for low-molecular-weight neurotransmitters and some proteins, there are numerous examples of the dispersal of a protein being delayed for many seconds after fusion. We have investigated the role of fusion-pore expansion in determining the contrasting discharge rates of fluorescent-tagged neuropeptide-Y (NPY) (within 200 ms) and tissue plasminogen activator (tPA) (over many seconds) in adrenal chromaffin cells. The endogenous proteins are expressed in separate chromaffin cell subpopulations. Fusion pore expansion was measured by two independent methods, orientation of a fluorescent probe within the plasma membrane using polarized total internal reflection fluorescence microscopy and amperometry of released catecholamine. Together, they probe the continuum of the fusion-pore duration, from milliseconds to many seconds after fusion. Polarized total internal reflection fluorescence microscopy revealed that 71% of the fusion events of tPA-cer-containing granules maintained curvature for >10 s, with approximately half of the structures likely connected to the plasma membrane by a short narrow neck. Such events were not commonly observed upon fusion of NPY-cer-containing granules. Amperometry revealed that the expression of tPA-green fluorescent protein (GFP) prolonged the duration of the prespike foot ∼2.5-fold compared to NPY-GFP-expressing cells and nontransfected cells, indicating that expansion of the initial fusion pore in tPA granules was delayed. The t1/2 of the main catecholamine spike was also increased, consistent with a prolonged delay of fusion-pore expansion. tPA added extracellularly bound to the lumenal surface of fused granules. We propose that tPA within the granule lumen controls its own discharge. Its intrinsic biochemistry determines not only

  17. Cell wall, cytoskeleton, and cell expansion in higher plants.

    PubMed

    Bashline, Logan; Lei, Lei; Li, Shundai; Gu, Ying

    2014-04-01

    To accommodate two seemingly contradictory biological roles in plant physiology, providing both the rigid structural support of plant cells and the adjustable elasticity needed for cell expansion, the composition of the plant cell wall has evolved to become an intricate network of cellulosic, hemicellulosic, and pectic polysaccharides and protein. Due to its complexity, many aspects of the cell wall influence plant cell expansion, and many new and insightful observations and technologies are forthcoming. The biosynthesis of cell wall polymers and the roles of the variety of proteins involved in polysaccharide synthesis continue to be characterized. The interactions within the cell wall polymer network and the modification of these interactions provide insight into how the plant cell wall provides its dual function. The complex cell wall architecture is controlled and organized in part by the dynamic intracellular cytoskeleton and by diverse trafficking pathways of the cell wall polymers and cell wall-related machinery. Meanwhile, the cell wall is continually influenced by hormonal and integrity sensing stimuli that are perceived by the cell. These many processes cooperate to construct, maintain, and manipulate the intricate plant cell wall--an essential structure for the sustaining of the plant stature, growth, and life.

  18. [Feedback control mechanisms of plant cell expansion

    SciTech Connect

    Cosgrove, D.J.

    1992-01-01

    We have generated considerable evidence for the significance of wall stress relaxation in the control of plant growth and found that several agents (gibberellin, light, genetic loci for dwarf stature) influence growth rate via alteration of wall relaxation. We have refined our methods for measuring wall relaxation and, moreover, have found that wall relaxation properties bear only a distance relationship to wall mechanical properties. We have garnered novel insights into the nature of cell expansion mechanisms by analyzing spontaneous fluctuations of plant growth rate in seedlings. These experiments involved the application of mathematical techniques for analyzing growth rate fluctuations and the development of new instrumentation for measuring and forcing plant growth in a controlled fashion. These studies conclude that growth rate fluctuations generated by the plant as consequence of a feedback control system. This conclusion has important implications for the nature of wall loosening processes and demands a different framework for thinking about growth control. It also implies the existence of a growth rate sensor.

  19. LECT2 drives haematopoietic stem cell expansion and mobilization via regulating the macrophages and osteolineage cells

    PubMed Central

    Lu, Xin-Jiang; Chen, Qiang; Rong, Ye-Jing; Yang, Guan-Jun; Li, Chang-Hong; Xu, Ning-Yi; Yu, Chao-Hui; Wang, Hui-Ying; Zhang, Shun; Shi, Yu-Hong; Chen, Jiong

    2016-01-01

    Haematopoietic stem cells (HSCs) can differentiate into cells of all lineages in the blood. However, the mechanisms by which cytokines in the blood affect HSC homeostasis remain largely unknown. Here we show that leukocyte cell-derived chemotaxin 2 (LECT2), a multifunctional cytokine, induces HSC expansion and mobilization. Recombinant LECT2 administration results in HSC expansion in the bone marrow and mobilization to the blood via CD209a. The effect of LECT2 on HSCs is reduced after specific depletion of macrophages or reduction of osteolineage cells. LECT2 treatment reduces the tumour necrosis factor (TNF) expression in macrophages and osteolineage cells. In TNF knockout mice, the effect of LECT2 on HSCs is reduced. Moreover, LECT2 induces HSC mobilization in irradiated mice, while granulocyte colony-stimulating factor does not. Our results illustrate that LECT2 is an extramedullar cytokine that contributes to HSC homeostasis and may be useful to induce HSC mobilization. PMID:27596364

  20. Biomaterial Strategies for Stem Cell Maintenance During In Vitro Expansion

    PubMed Central

    Yan, Xiang-Zhen; van den Beucken, Jeroen J.J.P.; Both, Sanne K.; Yang, Pi-Shan; Jansen, John A.

    2014-01-01

    Stem cells, having the potential for self-renewal and multilineage differentiation, are the building blocks for tissue/organ regeneration. Stem cells can be isolated from various sources but are, in general, available in too small numbers to be used directly for clinical purpose without intermediate expansion procedures in vitro. Although this in vitro expansion of undifferentiated stem cells is necessary, stem cells typically diminish their ability to self-renew and proliferate during passaging. Consequently, maintaining the stemness of stem cells has been recognized as a major challenge in stem cell-based research. This review focuses on the latest developments in maintaining the self-renewal ability of stem cells during in vitro expansion by biomaterial strategies. Further, this review highlights what should be the focus for future studies using stem cells for regenerative applications. PMID:24168361

  1. Coupling of Solute Transport and Cell Expansion in Pea Stems 1

    PubMed Central

    Schmalstig, Judy Gougler; Cosgrove, Daniel J.

    1990-01-01

    As cells expand and are displaced through the elongation zone of the epicotyl of etiolated pea (Pisum sativum L. var Alaska) seedlings, there is little net dilution of the cell sap, implying a coordination between cell expansion and solute uptake from the phloem. Using [14C]sucrose as a phloem tracer (applied to the hypogeous cotyledons), the pattern of label accumulation along the stem closely matched the growth rate pattern: high accumulation in the growing zone, little accumulation in nongrowing regions. Several results suggest that a major portion of phloem contents enters elongating cells through the symplast. We propose that the coordination between phloem transport and cell expansion is accomplished via regulatory pathways affecting both plasmodesmata conductivity and cell expansion. PMID:11537472

  2. Novel chemical attempts at ex vivo hematopoietic stem cell expansion.

    PubMed

    Zhang, Yu; Gao, Yingdai

    2016-05-01

    Hematopoietic stem cells (HSCs) are the most extensively studied stem cell type in adults, and the only stem cell type with proof of clinical utility. However, the greatest challenge for the broader use of HSCs remains the true expansion of the stem cells ex vivo. The development of researches on small-molecule compounds that support the safe and efficient ex vivo expansion of HSCs would help to promote the clinical application of HSCs. In recent years, several novel small-molecule compounds have been reported to improve ex vivo HSC expansion by promoting self-renewal, delaying differentiation, increasing homing, and inhibiting apoptosis. Here, we review recent chemical developments in stem cell research and the mechanisms underlying these compounds' effects.

  3. Nanofiber Expansion of Umbilical Cord Blood Hematopoietic Stem Cells

    PubMed Central

    Eskandari, F; Allahverdi, A; Nasiri, H; Azad, M; Kalantari, N; Soleimani, M; Zare-Zardini, H

    2015-01-01

    Background The aim of this study was the ex vivo expansion of Umbilical Cord Blood hematopoietic stem cells on biocompatible nanofiber scaffolds. Materials and Methods CD133+ hematopoietic stem cells were separated from umbilical cord blood using MidiMacs (positive selection) system by means of monocolonal antibody CD133 (microbeads); subsequently, flowcytometry method was done to assess the purity of separated cells. Isolated cells were cultured on plate (2 Dimensional) and fibronectin conjugated polyethersulfon nanofiber scaffold, simultaneously (3 Dimensional). Colony assay test was performed to show colonization ability of expanded cells. Results Cell count analysis revealed that expansion of hematopoietic stem cells in 2dimensional (2D) environment was greater than 3dimensional (3D) condition (p= 0.01). Assessment of stem cell- phenotype after expansions was performed by flowcytometric analysis which is showed that the maintenance of CD133 marker in expanded cells in 3 dimensional condition were higher than expanded cells in 2 dimensional condition (p=0.01). Moreover, colony assay test was performed before and after of expansion to show colonization ability of expanded cells both in 3D and 2D culture and results revealed more ability of 3D culture compared with 2D culture (p= 0.03). Conclusion The results of current study confirmed that umbilical cord blood CD133+ haematopoietic stem cells are able to expand on fibronectin conjugated polyethersulfon scaffold. These findings indicated that 3D is a proper and valuable cell culture system for hematopoietic stem cells expansion, compared to 2D in invitro situation. PMID:26985349

  4. (Rapid regulatory control of plant cell expansion and wall relaxation)

    SciTech Connect

    Cosgrove, D.J.

    1990-01-01

    This section presents a brief overview of accomplishments related to this project in the past 3-year period. Our work has focused on the basic mechanisms of plant cell expansion, particularly on the interrelations of water and solute transport with cell wall relaxation and expansion. To study these processes, we have developed new methods and used these methods to analyze the dynamic behavior of growth processes and to examine how various agents (GA, drought, light, genetic lesions) alter the growth machinery of the cell.

  5. T Cell Maturation Stage Prior to and During GMP Processing Informs on CAR T Cell Expansion in Patients.

    PubMed

    Klaver, Yarne; van Steenbergen, Sabine C L; Sleijfer, Stefan; Debets, Reno; Lamers, Cor H J

    2016-01-01

    Autologous T cells were genetically modified to express a chimeric antigen receptor (CAR) directed toward carboxy-anhydrase-IX (CAIX) and used to treat patients with CAIX-positive metastatic renal cell carcinoma. In this study, we questioned whether the T cell maturation stage in the pre-infusion product affected CAIX CAR expression and function in vitro as well as in vivo CAR T cell numbers and expansion. During the 14 days expansion of CAR T cells prior to administration, we observed shifts from a predominant CD4 to a CD8 T cell phenotype and from a significant fraction of naïve to central effector T cells. Surface expression of the CAR was equally distributed among different T cell subsets and T cell maturation stages. During T cell culture days 14-18 (which covered patient treatment days 1-5), T cells demonstrated a decline in CAR expression level per cell irrespective of T cell maturation stage, although the proportion of CAR-positive T cells and CAR-mediated T cell effector functions remained similar for both CD4 and CD8 T cell populations. Notably, patients with a higher fraction of naïve CD8 T cells at baseline (prior to genetic modification) or central effector CD8 T cells at 2 weeks of CAR T cell culture demonstrated a higher fold expansion and absolute numbers of circulating CAR T cells at 1 month after start of therapy. We conclude that the T cell maturation stage prior to and during CAR T cell expansion culture is related to in vivo CAR T cell expansion.

  6. Effects of androgen receptor polyglutamine tract expansion on proliferation of NG108-15 cells.

    PubMed

    Nakajima, H; Kimura, F; Nakagawa, T; Ikemoto, T; Furutama, D; Shinoda, K; Kato, S; Shimizu, A; Ohsawa, N

    1997-01-31

    Expansion of the polyglutamine tracts in the androgen receptor (AR) has been recognized as a cause of X-linked spinal and bulbar muscular atrophy (SBMA). In the present study, NG108-15 cells were stably transfected with expression vectors coding for either the wild type (WT) AR gene (CAG repeat number = 22) or a mutated (MT) AR gene (CAG repeat number = 52). Cells proliferation and cell cycle parameters were evaluated for NG108-15-WT and NG108-15-MT cells in the presence or absence of androgen. NG108-15-WT cells demonstrated an androgen-dependent increase in cell number, while NG108-15-MT cells did not. Our results demonstrate that expansion of polyglutamine tracts in the AR may affect the proliferation and differentiation of nerve cells.

  7. The cell biology of fat expansion

    PubMed Central

    Rutkowski, Joseph M.; Stern, Jennifer H.

    2015-01-01

    Adipose tissue is a complex, multicellular organ that profoundly influences the function of nearly all other organ systems through its diverse metabolite and adipokine secretome. Adipocytes are the primary cell type of adipose tissue and play a key role in maintaining energy homeostasis. The efficiency with which adipose tissue responds to whole-body energetic demands reflects the ability of adipocytes to adapt to an altered nutrient environment, and has profound systemic implications. Deciphering adipocyte cell biology is an important component of understanding how the aberrant physiology of expanding adipose tissue contributes to the metabolic dysregulation associated with obesity. PMID:25733711

  8. Methodology for Optimal In Vitro Cell Expansion in Tissue Engineering

    NASA Astrophysics Data System (ADS)

    Melero-Martin, J. M.; Santhalingam, S.; Al-Rubeai, M.

    Expansion of the cell population in vitro has become an essential step in the process of tissue engineering and also the systematic optimization of culture conditions is now a fundamental problem that needs to be addressed. Herein, we provide a rational methodology for searching culture conditions that optimize the acquisition of large quantities of cells following a sequential expansion process. In particular, the analysis of both seeding density and passage length was consid ered crucial, and their correct selection should be taken as a requisite to establish culture conditions for monolayer systems. This methodology also introduces addi tional considerations concerning the running cost of the expansion process. The selection of culture conditions will be a compromise between optimal cell expan sion and acceptable running cost. This compromise will normally translate into an increase of passage length further away from the optimal value dictated by the growth kinetic of the cells. Finally, the importance of incorporating functional assays to validate the phenotypical and functional characteristics of the expanded cells has been highlighted. The optimization approach presented will contribute to the development of feasible large scale expansion of cells required by the tissue engineering industry.

  9. Ex Vivo Expansion of Dendritic Cells for Breast Cancer Treatment

    DTIC Science & Technology

    2002-06-01

    healthy consented peripheral blood mononuclear cell (PBMC) volunteers. Adherent cells or CD14 + immunoselected ( Miltenyi , CA) monocytes were collected from...from patients/donors, we began the evaluation of our culture conditions for expansion of DCs using CD14 ’ selected or adherent cells from normal...formulated for macrophages and lymphocytes is currently being used in several protocols so was selected as a standard for DC production. Purity of CD14

  10. Wall relaxation and the driving forces for cell expansive growth

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1987-01-01

    When water uptake by growing cells is prevented, the turgor pressure and the tensile stress in the cell wall are reduced by continued wall loosening. This process, termed in vivo stress relaxation, provides a new way to study the dynamics of wall loosening and to measure the wall yield threshold and the physiological wall extensibility. Stress relaxation experiments indicate that wall stress supplies the mechanical driving force for wall yielding. Cell expansion also requires water absorption. The driving force for water uptake during growth is created by wall relaxation, which lowers the water potential of the expanding cells. New techniques for measuring this driving force show that it is smaller than believed previously; in elongating stems it is only 0.3 to 0.5 bar. This means that the hydraulic resistance of the water transport pathway is small and that rate of cell expansion is controlled primarily by wall loosening and yielding.

  11. Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells.

    PubMed

    Himburg, Heather A; Muramoto, Garrett G; Daher, Pamela; Meadows, Sarah K; Russell, J Lauren; Doan, Phuong; Chi, Jen-Tsan; Salter, Alice B; Lento, William E; Reya, Tannishtha; Chao, Nelson J; Chute, John P

    2010-04-01

    Hematopoietic stem cell (HSC) self-renewal is regulated by both intrinsic and extrinsic signals. Although some of the pathways that regulate HSC self-renewal have been uncovered, it remains largely unknown whether these pathways can be triggered by deliverable growth factors to induce HSC growth or regeneration. Here we show that pleiotrophin, a neurite outgrowth factor with no known function in hematopoiesis, efficiently promotes HSC expansion in vitro and HSC regeneration in vivo. Treatment of mouse bone marrow HSCs with pleiotrophin caused a marked increase in long-term repopulating HSC numbers in culture, as measured in competitive repopulating assays. Treatment of human cord blood CD34(+)CDCD38(-)Lin(-) cells with pleiotrophin also substantially increased severe combined immunodeficient (SCID)-repopulating cell counts in culture, compared to input and cytokine-treated cultures. Systemic administration of pleiotrophin to irradiated mice caused a pronounced expansion of bone marrow stem and progenitor cells in vivo, indicating that pleiotrophin is a regenerative growth factor for HSCs. Mechanistically, pleiotrophin activated phosphoinositide 3-kinase (PI3K) signaling in HSCs; antagonism of PI3K or Notch signaling inhibited pleiotrophin-mediated expansion of HSCs in culture. We identify the secreted growth factor pleiotrophin as a new regulator of both HSC expansion and regeneration.

  12. Ex vivo expansion of human hematopoietic stem and progenitor cells

    PubMed Central

    Dahlberg, Ann; Delaney, Colleen

    2011-01-01

    Despite progress in our understanding of the growth factors that support the progressive maturation of the various cell lineages of the hematopoietic system, less is known about factors that govern the self-renewal of hematopoietic stem and progenitor cells (HSPCs), and our ability to expand human HSPC numbers ex vivo remains limited. Interest in stem cell expansion has been heightened by the increasing importance of HSCs in the treatment of both malignant and nonmalignant diseases, as well as their use in gene therapy. To date, most attempts to ex vivo expand HSPCs have used hematopoietic growth factors but have not achieved clinically relevant effects. More recent approaches, including our studies in which activation of the Notch signaling pathway has enabled a clinically relevant ex vivo expansion of HSPCs, have led to renewed interest in this arena. Here we briefly review early attempts at ex vivo expansion by cytokine stimulation followed by an examination of our studies investigating the role of Notch signaling in HSPC self-renewal. We will also review other recently developed approaches for ex vivo expansion, primarily focused on the more extensively studied cord blood–derived stem cell. Finally, we discuss some of the challenges still facing this field. PMID:21436068

  13. Expansion of human cord blood hematopoietic stem cells for transplantation.

    PubMed

    Chou, Song; Chu, Pat; Hwang, William; Lodish, Harvey

    2010-10-08

    A recent Science paper reported a purine derivative that expands human cord blood hematopoietic stem cells in culture (Boitano et al., 2010) by antagonizing the aryl hydrocarbon receptor. Major problems need to be overcome before ex vivo HSC expansion can be used clinically.

  14. UV radiation reduces epidermal cell expansion in leaves of Arabidopsis thaliana.

    PubMed

    Hectors, Kathleen; Jacques, Eveline; Prinsen, Els; Guisez, Yves; Verbelen, Jean-Pierre; Jansen, Marcel A K; Vissenberg, Kris

    2010-10-01

    Plants have evolved a broad spectrum of mechanisms to ensure survival under changing and suboptimal environmental conditions. Alterations of plant architecture are commonly observed following exposure to abiotic stressors. The mechanisms behind these environmentally controlled morphogenic traits are, however, poorly understood. In this report, the effects of a low dose of chronic ultraviolet (UV) radiation on leaf development are detailed. Arabidopsis rosette leaves exposed for 7, 12, or 19 d to supplemental UV radiation expanded less compared with non-UV controls. The UV-mediated decrease in leaf expansion is associated with a decrease in adaxial pavement cell expansion. Elevated UV does not affect the number and shape of adaxial pavement cells, nor the stomatal index. Cell expansion in young Arabidopsis leaves is asynchronous along a top-to-base gradient whereas, later in development, cells localized at both the proximal and distal half expand synchronously. The prominent, UV-mediated inhibition of cell expansion in young leaves comprises effects on the early asynchronous growing stage. Subsequent cell expansion during the synchronous phase cannot nullify the UV impact established during the asynchronous phase. The developmental stage of the leaf at the onset of UV treatment determines whether UV alters cell expansion during the synchronous and/or asynchronous stage. The effect of UV radiation on adaxial epidermal cell size appears permanent, whereas leaf shape is transiently altered with a reduced length/width ratio in young leaves. The data show that UV-altered morphogenesis is a temporal- and spatial-dependent process, implying that common single time point or single leaf zone analyses are inadequate.

  15. Integrating human stem cell expansion and neuronal differentiation in bioreactors

    PubMed Central

    Serra, Margarida; Brito, Catarina; Costa, Eunice M; Sousa, Marcos FQ; Alves, Paula M

    2009-01-01

    Background Human stem cells are cellular resources with outstanding potential for cell therapy. However, for the fulfillment of this application, major challenges remain to be met. Of paramount importance is the development of robust systems for in vitro stem cell expansion and differentiation. In this work, we successfully developed an efficient scalable bioprocess for the fast production of human neurons. Results The expansion of undifferentiated human embryonal carcinoma stem cells (NTera2/cl.D1 cell line) as 3D-aggregates was firstly optimized in spinner vessel. The media exchange operation mode with an inoculum concentration of 4 × 105 cell/mL was the most efficient strategy tested, with a 4.6-fold increase in cell concentration achieved in 5 days. These results were validated in a bioreactor where similar profile and metabolic performance were obtained. Furthermore, characterization of the expanded population by immunofluorescence microscopy and flow cytometry showed that NT2 cells maintained their stem cell characteristics along the bioreactor culture time. Finally, the neuronal differentiation step was integrated in the bioreactor process, by addition of retinoic acid when cells were in the middle of the exponential phase. Neurosphere composition was monitored and neuronal differentiation efficiency evaluated along the culture time. The results show that, for bioreactor cultures, we were able to increase significantly the neuronal differentiation efficiency by 10-fold while reducing drastically, by 30%, the time required for the differentiation process. Conclusion The culture systems developed herein are robust and represent one-step-forward towards the development of integrated bioprocesses, bridging stem cell expansion and differentiation in fully controlled bioreactors. PMID:19772662

  16. Expansion of Multipotent Stem Cells from the Adult Human Brain

    PubMed Central

    Murrell, Wayne; Palmero, Emily; Bianco, John; Stangeland, Biljana; Joel, Mrinal; Paulson, Linda; Thiede, Bernd; Grieg, Zanina; Ramsnes, Ingunn; Skjellegrind, Håvard K.; Nygård, Ståle; Brandal, Petter; Sandberg, Cecilie; Vik-Mo, Einar; Palmero, Sheryl; Langmoen, Iver A.

    2013-01-01

    The discovery of stem cells in the adult human brain has revealed new possible scenarios for treatment of the sick or injured brain. Both clinical use of and preclinical research on human adult neural stem cells have, however, been seriously hampered by the fact that it has been impossible to passage these cells more than a very few times and with little expansion of cell numbers. Having explored a number of alternative culturing conditions we here present an efficient method for the establishment and propagation of human brain stem cells from whatever brain tissue samples we have tried. We describe virtually unlimited expansion of an authentic stem cell phenotype. Pluripotency proteins Sox2 and Oct4 are expressed without artificial induction. For the first time multipotency of adult human brain-derived stem cells is demonstrated beyond tissue boundaries. We characterize these cells in detail in vitro including microarray and proteomic approaches. Whilst clarification of these cells’ behavior is ongoing, results so far portend well for the future repair of tissues by transplantation of an adult patient’s own-derived stem cells. PMID:23967194

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

    SciTech Connect

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

    2011-04-15

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

  18. Differential response of cell-cycle and cell-expansion regulators to heat stress in apple (Malus domestica) fruitlets.

    PubMed

    Flaishman, Moshe A; Peles, Yuval; Dahan, Yardena; Milo-Cochavi, Shira; Frieman, Aviad; Naor, Amos

    2015-04-01

    Temperature is one of the most significant factors affecting physiological and biochemical aspects of fruit development. Current and progressing global warming is expected to change climate in the traditional deciduous fruit tree cultivation regions. In this study, 'Golden Delicious' trees, grown in a controlled environment or commercial orchard, were exposed to different periods of heat treatment. Early fruitlet development was documented by evaluating cell number, cell size and fruit diameter for 5-70 days after full bloom. Normal activities of molecular developmental and growth processes in apple fruitlets were disrupted under daytime air temperatures of 29°C and higher as a result of significant temporary declines in cell-production and cell-expansion rates, respectively. Expression screening of selected cell cycle and cell expansion genes revealed the influence of high temperature on genetic regulation of apple fruitlet development. Several core cell-cycle and cell-expansion genes were differentially expressed under high temperatures. While expression levels of B-type cyclin-dependent kinases and A- and B-type cyclins declined moderately in response to elevated temperatures, expression of several cell-cycle inhibitors, such as Mdwee1, Mdrbr and Mdkrps was sharply enhanced as the temperature rose, blocking the cell-cycle cascade at the G1/S and G2/M transition points. Moreover, expression of several expansin genes was associated with high temperatures, making them potentially useful as molecular platforms to enhance cell-expansion processes under high-temperature regimes. Understanding the molecular mechanisms of heat tolerance associated with genes controlling cell cycle and cell expansion may lead to the development of novel strategies for improving apple fruit productivity under global warming.

  19. Arabidopsis cell expansion is controlled by a photothermal switch

    PubMed Central

    Johansson, Henrik; Jones, Harriet J.; Foreman, Julia; Hemsted, Joseph R.; Stewart, Kelly; Grima, Ramon; Halliday, Karen J.

    2014-01-01

    In Arabidopsis, the seedling hypocotyl has emerged as an exemplar model system to study light and temperature control of cell expansion. Light sensitivity of this organ is epitomized in the fluence rate response where suppression of hypocotyl elongation increases incrementally with light intensity. This finely calibrated response is controlled by the photoreceptor, phytochrome B, through the deactivation and proteolytic destruction of phytochrome-interacting factors (PIFs). Here we show that this classical light response is strictly temperature dependent: a shift in temperature induces a dramatic reversal of response from inhibition to promotion of hypocotyl elongation by light. Applying an integrated experimental and mathematical modelling approach, we show how light and temperature coaction in the circuitry drives a molecular switch in PIF activity and control of cell expansion. This work provides a paradigm to understand the importance of signal convergence in evoking different or non-intuitive alterations in molecular signalling. PMID:25258215

  20. Selection and expansion of natural killer cells for NK cell-based immunotherapy.

    PubMed

    Becker, Petra S A; Suck, Garnet; Nowakowska, Paulina; Ullrich, Evelyn; Seifried, Erhard; Bader, Peter; Tonn, Torsten; Seidl, Christian

    2016-04-01

    Natural killer (NK) cells have been used in several clinical trials as adaptive immunotherapy. The low numbers of these cells in peripheral blood mononuclear cells (PBMC) have resulted in various approaches to preferentially expand primary NK cells from PBMC. While some clinical trials have used the addition of interleukin 2 (IL-2) to co-stimulate the expansion of purified NK cells from allogeneic donors, recent studies have shown promising results in achieving in vitro expansion of NK cells to large numbers for adoptive immunotherapy. NK cell expansion requires multiple cell signals for survival, proliferation and activation. Thus, expansion strategies have been focused either to substitute these factors using autologous feeder cells or to use genetically modified allogeneic feeder cells. Recent developments in the clinical use of genetically modified NK cell lines with chimeric antigen receptors, the development of expansion protocols for the clinical use of NK cell from human embryonic stem cells and induced pluripotent stem cells are challenging improvements for NK cell-based immunotherapy. Transfer of several of these protocols to clinical-grade production of NK cells necessitates adaptation of good manufacturing practice conditions, and the development of freezing conditions to establish NK cell stocks will require some effort and, however, should enhance the therapeutic options of NK cells in clinical medicine.

  1. Epigenetic reprogramming induces the expansion of cord blood stem cells

    PubMed Central

    Chaurasia, Pratima; Gajzer, David C.; Schaniel, Christoph; D’Souza, Sunita; Hoffman, Ronald

    2014-01-01

    Cord blood (CB) cells that express CD34 have extensive hematopoietic capacity and rapidly divide ex vivo in the presence of cytokine combinations; however, many of these CB CD34+ cells lose their marrow-repopulating potential. To overcome this decline in function, we treated dividing CB CD34+ cells ex vivo with several histone deacetylase inhibitors (HDACIs). Treatment of CB CD34+ cells with the most active HDACI, valproic acid (VPA), following an initial 16-hour cytokine priming, increased the number of multipotent cells (CD34+CD90+) generated; however, the degree of expansion was substantially greater in the presence of both VPA and cytokines for a full 7 days. Treated CD34+ cells were characterized based on the upregulation of pluripotency genes, increased aldehyde dehydrogenase activity, and enhanced expression of CD90, c-Kit (CD117), integrin α6 (CD49f), and CXCR4 (CD184). Furthermore, siRNA-mediated inhibition of pluripotency gene expression reduced the generation of CD34+CD90+ cells by 89%. Compared with CB CD34+ cells, VPA-treated CD34+ cells produced a greater number of SCID-repopulating cells and established multilineage hematopoiesis in primary and secondary immune–deficient recipient mice. These data indicate that dividing CB CD34+ cells can be epigenetically reprogrammed by treatment with VPA so as to generate greater numbers of functional CB stem cells for use as transplantation grafts. PMID:24762436

  2. Mesenchymal Stem Cells in ex vivo Cord Blood Expansion

    PubMed Central

    Robinson, Simon N.; Simmons, Paul J.; Yang, Hong; Alousi, Amin M; de Lima, Marcos J.

    2013-01-01

    Umbilical cord blood (CB) is becoming an important source of haematopoietic support for transplant patients lacking human leukocyte antigen matched donors. The ethnic diversity, relative ease of collection, ready availability as cryopreserved units from CB banks, reduced incidence and severity of graft versus host disease and tolerance of higher degrees of HLA disparity between donor and recipient, are positive attributes when compared to bone marrow or cytokine-mobilized peripheral blood. However, CB transplantation is associated with significantly delayed neutrophil and platelet engraftment and an elevated risk of graft failure. These hurdles are thought to be due, at least in part, to low total nucleated cell and CD34+ cell doses transplanted. Here, current strategies directed at improving TNC and CD34+ cell doses at transplant are discussed, with particular attention paid to the use of a mesenchymal stem cell (MSC)/CB mononuclear cell ex vivo co-culture expansion system. PMID:21396596

  3. CD101 inhibits the expansion of colitogenic T cells

    PubMed Central

    Schey, Regina; Dornhoff, Heike; Baier, Julia L.C.; Purtak, Martin; Opoka, Robert; Koller, Anna Katharina; Atreya, Raya; Rau, Tilman T.; Daniel, Christoph; Amann, Kerstin; Bogdan, Christian; Mattner, Jochen

    2015-01-01

    CD101 exerts negative-costimulatory effects in vitro, but its function in vivo remains poorly defined. CD101 is abundantly expressed on lymphoid and myeloid cells in intestinal tissues, but absent from naïve splenic T cells. Here, we assessed the impact of CD101 on the course of inflammatory bowel disease (IBD). Using a T cell transfer model of chronic colitis, we found that in recipients of naïve T cells from CD101+/+ donors up to 30% of the recovered lymphocytes expressed CD101, correlating with an increased IL-2-mediated FoxP3-expression. Transfer of CD101−/− T cells caused more severe colitis and was associated with an expansion of IL-17-producing T cells and an enhanced expression of IL-2Rα/β independently of FoxP3. The co-transfer of naïve and regulatory T cells (Treg) protected most effectively from colitis, when both donor and recipient mice expressed CD101. While the expression of CD101 on T cells was sufficient for Treg-function and the inhibition of T cell proliferation, sustained IL-10-production required additional CD101-expression by myeloid cells. Finally, in patients with IBD a reduced CD101-expression on peripheral and intestinal monocytes and CD4+ T cells correlated with enhanced IL-17-production and disease activity. Thus, CD101-deficiency is a novel marker for progressive colitis and potential target for therapeutic intervention. PMID:26813346

  4. Characterization of Human AB Serum for Mesenchymal Stromal Cell Expansion

    PubMed Central

    dos Santos, Vanessa Tieko Marques; Mizukami, Amanda; Orellana, Maristela Delgado; Caruso, Samia Rigotto; da Silva, Fernanda Borges; Traina, Fabiola; de Lima Prata, Karen; Covas, Dimas Tadeu; Swiech, Kamilla

    2017-01-01

    Background So far, using human blood-derived components appears to be the most efficient and safest approach available for mesenchymal stromal cell (MSC) expansion. In this paper, we report on the characterization of human AB serum (AB HS) produced by using different plasma sources, and its use as an alternative supplement to MSC expansion. Methods Two plasma sources were used for AB HS production: plasma removed from whole blood after 24 h of collection (PC > 24 h) and plasma, cryoprecipitate reduced (PCryoR). The biochemical profile and quality of the produced AB HS batches were analyzed and their ability to support MSC cell growth after different storage times (0, 3, 6, 9 and 12 months) was evaluated. Results The two plasma sources used showed similar characteristics regarding biochemical constituents and quality parameters and were effective in promoting MSC growth. MSCs cultured in medium supplemented with 10% AB HS presented similar doubling times and cumulative population doublings when compared to the 10% fetal bovine serum(FBS)-supplemented culture while maintaining immunophenotype, functional features, and cytogenetic profile. Conclusion Overall, the results indicate that AB HS is an efficient FBS substitute and can be used for at least 12 months after production without impairing cell proliferation and quality. PMID:28275329

  5. Rapid regulatory control of plant cell expansion and wall relaxation

    SciTech Connect

    Cosgrove, D.J.

    1991-08-14

    The aim of this project is to elucidate the biophysical and cellular mechanisms that control plant cell expansion. At present we are attempting to characterize the kinetics of the system(s) responsible for regulatory and compensatory behavior of growing cells and tissues. This work is significantly because it indicates that biochemical loosening and biophysical stress relaxation of the wall are part of a feedback loop controlling growth. This report briefly summarizes the efforts and results of the past 12 months. In large part, we have been trying to analyze the nature of growth rate noise,'' i.e. spontaneous and often erratic variations in growth rate. We are obtaining evidence that such noise'' is not random, but rather reveals an underlying growth mechanism with complex dynamics.

  6. Immunoproteasomes are essential for survival and expansion of T cells in virus-infected mice.

    PubMed

    Moebius, Jacqueline; van den Broek, Maries; Groettrup, Marcus; Basler, Michael

    2010-12-01

    Immunoproteasomes containing the IFN-inducible subunits β1i (LMP2), β2i (MECL-1) and β5i (LMP7) alter proteasomal cleavage preference and optimize the generation of peptide ligands of MHC class I molecules. Here, we report on an unexpected new function of immunoproteasome subunits for the survival and expansion of CD4(+) and CD8(+) T cells during viral infection of mice. The effect of immunoproteasome subunit deficiency on T-cell survival upon adoptive transfer was most prominent for the lack of LMP7 followed by MECL-1 and LMP2. The survival of T cells in uninfected mice or the homeostatic expansion after transfer into RAG-2(-/-) mice was not affected by the lack of the immunosubunits. Lymphocytic choriomeningitis virus (LCMV)-specific CD8(+) T cells lacking LMP7 or MECL-1 started to divide after transfer into LCMV-infected mice but experienced a considerable cell loss within 2 days after transfer. We provide strong evidence that the loss of immunoproteasome-deficient T cells after transfer is not a consequence of graft rejection by the host, but instead is based on the requirement for immunoproteasomes for the survival of T cells in LCMV-infected mice. Therefore, the immunoproteasome may qualify as a potential new target for the suppression of undesired proinflammatory T-cell responses.

  7. IGFBP2 promotes glioma tumor stem cell expansion and survival

    SciTech Connect

    Hsieh, David; Hsieh, Antony; Stea, Baldassarre; Ellsworth, Ron

    2010-06-25

    IGFBP2 is overexpressed in the most common brain tumor, glioblastoma (GBM), and its expression is inversely correlated to GBM patient survival. Previous reports have demonstrated a role for IGFBP2 in glioma cell invasion and astrocytoma development. However, the function of IGFBP2 in the restricted, self-renewing, and tumorigenic GBM cell population comprised of tumor-initiating stem cells has yet to be determined. Herein we demonstrate that IGFBP2 is overexpressed within the stem cell compartment of GBMs and is integral for the clonal expansion and proliferative properties of glioma stem cells (GSCs). In addition, IGFBP2 inhibition reduced Akt-dependent GSC genotoxic and drug resistance. These results suggest that IGFBP2 is a selective malignant factor that may contribute significantly to GBM pathogenesis by enriching for GSCs and mediating their survival. Given the current dearth of selective molecular targets against GSCs, we anticipate our results to be of high therapeutic relevance in combating the rapid and lethal course of GBM.

  8. Antagonistic relationship between AtRALF1 and brassinosteroid regulates cell expansion-related genes.

    PubMed

    Bergonci, Tábata; Silva-Filho, Marcio C; Moura, Daniel S

    2014-01-01

    Rapid alkalinization factor (RALF) is a peptide signal that plays a role in plant cell expansion. We have recently proposed that AtRALF1 negatively regulates root cell elongation and lateral root formation by opposing the effects of brassinosteroid (BR). We reported 6 AtRALF1-inducible cell wall-related genes and 2 P450 monooxygenase -encoding genes involved in the BR biosynthetic pathway. The AtRALF1-inducible genes implicated in cell wall remodeling were not downregulated by brassinolide (BL) treatment alone; their induction was only compromised following simultaneous treatment with AtRALF1 and BL. We further examined the cell wall-remodeling gene EXPANSIN A5 (AtEXPA5), which is upregulated by BL and has been shown to positively affect root cell elongation. Herein, we report that AtEXPA5 expression is downregulated by AtRALF1 in a dose-dependent manner in the roots and hypocotyls of Arabidopsis plants. AtEXPA5 is also downregulated in plants that overexpress AtRALF1, and it is upregulated in plants in which the AtRALF1 gene is partially silenced. The AtRALF1 peptide is also able to repress AtEXPA5 induction following a pre-treatment with BL. A schematic diagram showing the gene regulatory network connecting the recently reported genes with the regulation of cell expansion by AtEXPA5 is presented.

  9. Stepwise B-cell-dependent expansion of T helper clonotypes diversifies the T-cell response

    PubMed Central

    Merkenschlager, Julia; Ploquin, Mickaël J.; Eksmond, Urszula; Andargachew, Rakieb; Thorborn, Georgina; Filby, Andrew; Pepper, Marion; Evavold, Brian; Kassiotis, George

    2016-01-01

    Antigen receptor diversity underpins adaptive immunity by providing the ground for clonal selection of lymphocytes with the appropriate antigen reactivity. Current models attribute T cell clonal selection during the immune response to T-cell receptor (TCR) affinity for either foreign or self peptides. Here, we report that clonal selection of CD4+ T cells is also extrinsically regulated by B cells. In response to viral infection, the antigen-specific TCR repertoire is progressively diversified by staggered clonotypic expansion, according to functional avidity, which correlates with self-reactivity. Clonal expansion of lower-avidity T-cell clonotypes depends on availability of MHC II-expressing B cells, in turn influenced by B-cell activation. B cells clonotypically diversify the CD4+ T-cell response also to vaccination or tumour challenge, revealing a common effect. PMID:26728651

  10. Phloem unloading and cell expansion in pea stems

    SciTech Connect

    Schmalstig, J.G.; Cosgrove, D.J. )

    1989-04-01

    Phloem unloading into elongating stems of dark-grown pea seedlings was greater in regions with higher relative growth rates. Phloem transport was monitored over 1 h by measuring accumulation of radiolabel from {sup 14}C-sucrose added between the cotyledons. The apical hook and plumule and 8 mm of the growing region of an intact plant were sealed in a pressure chamber and the pressure was raised to stop elongation. Phloem unloading was inhibited in the pressurized zone of elongation and accelerated in the apical hook and plumule, with the result that the magnitude of phloem transport into the stem was unchanged. The results demonstrate a coupling between cell expansion and phloem unloading.

  11. Three-dimensional patterns of cell division and expansion throughout the development of Arabidopsis thaliana leaves.

    PubMed

    Kalve, Shweta; Fotschki, Joanna; Beeckman, Tom; Vissenberg, Kris; Beemster, Gerrit T S

    2014-12-01

    Variations in size and shape of multicellular organs depend on spatio-temporal regulation of cell division and expansion. Here, cell division and expansion rates were quantified relative to the three spatial axes in the first leaf pair of Arabidopsis thaliana. The results show striking differences in expansion rates: the expansion rate in the petiole is higher than in the leaf blade; expansion rates in the lateral direction are higher than longitudinal rates between 5 and 10 days after stratification, but become equal at later stages of leaf blade development; and anticlinal expansion co-occurs with, but is an order of magnitude slower than periclinal expansion. Anticlinal expansion rates also differed greatly between tissues: the highest rates occurred in the spongy mesophyll and the lowest in the epidermis. Cell division rates were higher and continued for longer in the epidermis compared with the palisade mesophyll, causing a larger increase of palisade than epidermal cell area over the course of leaf development. The cellular dynamics underlying the effect of shading on petiole length and leaf thickness were then investigated. Low light reduced leaf expansion rates, which was partly compensated by increased duration of the growth phase. Inversely, shading enhanced expansion rates in the petiole, so that the blade to petiole ratio was reduced by 50%. Low light reduced leaf thickness by inhibiting anticlinal cell expansion rates. This effect on cell expansion was preceded by an effect on cell division, leading to one less layer of palisade cells. The two effects could be uncoupled by shifting plants to contrasting light conditions immediately after germination. This extended kinematic analysis maps the spatial and temporal heterogeneity of cell division and expansion, providing a framework for further research to understand the molecular regulatory mechanisms involved.

  12. Fetal hepatic progenitors support long-term expansion of hematopoietic stem cells.

    PubMed

    Chou, Song; Flygare, Johan; Lodish, Harvey F

    2013-05-01

    We have developed a coculture system that establishes DLK(+) fetal hepatic progenitors as the authentic supportive cells for expansion of hematopoietic stem (HSCs) and progenitor cells. In 1-week cultures supplemented with serum and supportive cytokines, both cocultured DLK(+) fetal hepatic progenitors and their conditioned medium supported rapid expansion of hematopoietic progenitors and a small increase in HSC numbers. In 2- and 3-week cultures DLK(+) cells, but not their conditioned medium, continuously and significantly (>20-fold) expanded both hematopoietic stem and progenitor cells. Physical contact between HSCs and DLK(+) cells was crucial to maintaining this long-term expansion. Similar HSC expansion (approximately sevenfold) was achieved in cocultures using a serum-free, low cytokine- containing medium. In contrast, DLK(-) cells are incapable of expanding hematopoietic cells, demonstrating that hepatic progenitors are the principle supportive cells for HSC expansion in the fetal liver.

  13. Sex-dependent mechanisms for expansions and contractions of the CAG repeat on affected Huntington disease chromosomes

    SciTech Connect

    Kremer, B.; Theilmann, J.; Spence, N.

    1995-08-01

    A total of 254 affected parent-child pairs with Huntington disease (HD) and 440 parent-child pairs with CAG size in the normal range were assessed to determine the nature and frequency of intergenerational CAG changes in the HD gene. Intergenerational CAG changes are extremely rare (3/440 [0.68%]) on normal chromosomes. In contrast, on HD chromosomes, changes in CAG size occur in {approximately}70% of meioses on HD chromosomes, with expansions accounting for 73% of these changes. These intergenerational CAG changes make a significant but minor contribution to changes in age at onset (r{sup 2}=.19). The size of the CAG repeat influenced larger intergenerational expansions (>7 CAG repeats), but the likelihood of smaller expansions or contractions was not influenced by CAG size. Large expansions (>7 CAG repeats) occur almost exclusively through paternal transmission (0.96%; P<10{sub -7}), while offspring of affected mothers are more likely to show no change (P=.01) or contractions in CAG size (P=.002). This study demonstrates that sex of the transmitting parent is the major determinant for CAG intergenerational changes in the HD gene. Similar paternal sex effects are seen in the evolution of new mutations for HD from intermediate alleles and for large expansions on affected chromosomes. Affected mothers almost never transmit a significantly expanded CAG repeat, despite the fact that many have similar large-sized alleles, compared with affected fathers. The sex-dependent effects of major expansion and contractions of the CAG repeat in the HD gene implicate different effects of gametogenesis, in males versus females, on intergenerational CAG repeat stability. 22 refs., 4 figs., 3 tabs.

  14. Multiple mechanisms support oligoclonal T cell expansion in rheumatoid synovitis.

    PubMed Central

    Rittner, H. L.; Zettl, A.; Jendro, M. C.; Bartz-Bazzanella, P.; Goronzy, J. J.; Weyand, C. M.

    1997-01-01

    BACKGROUND: The synovial T cell infiltrate in rheumatoid arthritis (RA) is diverse but contains clonally expanded CD4+ populations. Recent reports have emphasized that RA patients have a tendency to develop CD4+ T cell oligoclonality which also manifests in the peripheral blood. Clonal dominance in the tissue may thus result from antigen specific stimulation in the synovial membrane or may reflect the infiltration of expanded clonotypes present throughout the lymphoid system. We have explored to what extent clonal populations amongst tissue CD4+ T cells display joint specificity as defined by their restriction to the joint, their persistence over time, and their expression of markers indicative for local activation. MATERIALS AND METHODS: Matched samples of peripheral blood and synovial fluid or synovial tissue were collected from 14 patients with active RA and CD4+ IL-2R+ and CD4+ IL-2R- T cells from both compartments were purified. Clonal populations of CD4+ T cells were detected by RT-PCR amplification of T cell receptor (TCR) transcripts with BV and BJ specific primers followed by size fractionation and direct sequencing of dominant size classes of TCR transcripts. RESULTS: Clonal CD4+ T cells were detected in the synovial fluid and synovial tissue of all patients. All patients carried synovial clonotypes that were undetectable in the blood but were present in independent joints or at several non-adjacent areas of the same joint. These joint restricted CD4+ clonotypes were generally small in size, were preferentially found in the IL-2R+ subpopulation, and persisted over time. A second type of clonogenic T cells in the synovial infiltrate had an unrestricted tissue distribution and was present at similar frequencies amongst activated and nonactivated T cells in the blood and affected joints. Ubiquitous clonotypes isolated from two different patients expressed sequence homologies of the TCR beta chain. CONCLUSIONS: Two types of expanded CD4+ clonotypes contribute

  15. The endoplasmic reticulum exerts control over organelle streaming during cell expansion.

    PubMed

    Stefano, Giovanni; Renna, Luciana; Brandizzi, Federica

    2014-03-01

    Cytoplasmic streaming is crucial for cell homeostasis and expansion but the precise driving forces are largely unknown. In plants, partial loss of cytoplasmic streaming due to chemical and genetic ablation of myosins supports the existence of yet-unknown motors for organelle movement. Here we tested a role of the endoplasmic reticulum (ER) as propelling force for cytoplasmic streaming during cell expansion. Through quantitative live-cell analyses in wild-type Arabidopsis thaliana cells and mutants with compromised ER structure and streaming, we demonstrate that cytoplasmic streaming undergoes profound changes during cell expansion and that it depends on motor forces co-exerted by the ER and the cytoskeleton.

  16. Influence of stress restraint on the expansive behaviour of concrete affected by alkali-silica reaction

    SciTech Connect

    Berra, M.; Faggiani, G.; Mangialardi, T.; Paolini, A.E.

    2010-09-15

    The primary objective of this study was to ascertain whether the Threshold Alkali Level (TAL) of the concrete aggregates may be taken as a suitable reactivity parameter for the selection of aggregates susceptible of alkali-silica reaction (ASR), even when ASR expansion in concrete develops under restrained conditions. Concrete mixes made with different alkali contents and two natural siliceous aggregates with very different TALs were tested for their expansivity at 38 {sup o}C and 100% RH under unrestrained and restrained conditions. Four compressive stress levels over the range from 0.17 to 3.50 N/mm{sup 2} were applied by using a new appositely designed experimental equipment. The lowest stress (0.17 N/mm{sup 2}) was selected in order to estimate the expansive pressure developed by the ASR gel under 'free' expansion conditions. It was found that, even under restrained conditions, the threshold alkali level proves to be a suitable reactivity parameter for designing concrete mixes that are not susceptible of deleterious ASR expansion. An empirical relationship between expansive pressure, concrete alkali content and aggregate TAL was developed in view of its possible use for ASR diagnosis and/or safety evaluation of concrete structures.

  17. Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse behaviour

    PubMed Central

    Hernández-Hernández, Oscar; Guiraud-Dogan, Céline; Sicot, Géraldine; Huguet, Aline; Luilier, Sabrina; Steidl, Esther; Saenger, Stefanie; Marciniak, Elodie; Obriot, Hélène; Chevarin, Caroline; Nicole, Annie; Revillod, Lucile; Charizanis, Konstantinos; Lee, Kuang-Yung; Suzuki, Yasuhiro; Kimura, Takashi; Matsuura, Tohru; Cisneros, Bulmaro; Swanson, Maurice S.; Trovero, Fabrice; Buisson, Bruno; Bizot, Jean-Charles; Hamon, Michel; Humez, Sandrine; Bassez, Guillaume; Metzger, Friedrich; Buée, Luc; Munnich, Arnold; Sergeant, Nicolas; Gourdon, Geneviève

    2013-01-01

    Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology. PMID:23404338

  18. Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse behaviour.

    PubMed

    Hernández-Hernández, Oscar; Guiraud-Dogan, Céline; Sicot, Géraldine; Huguet, Aline; Luilier, Sabrina; Steidl, Esther; Saenger, Stefanie; Marciniak, Elodie; Obriot, Hélène; Chevarin, Caroline; Nicole, Annie; Revillod, Lucile; Charizanis, Konstantinos; Lee, Kuang-Yung; Suzuki, Yasuhiro; Kimura, Takashi; Matsuura, Tohru; Cisneros, Bulmaro; Swanson, Maurice S; Trovero, Fabrice; Buisson, Bruno; Bizot, Jean-Charles; Hamon, Michel; Humez, Sandrine; Bassez, Guillaume; Metzger, Friedrich; Buée, Luc; Munnich, Arnold; Sergeant, Nicolas; Gourdon, Geneviève; Gomes-Pereira, Mário

    2013-03-01

    Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology.

  19. Cryopreservation method affects DNA fragmentation in trophectoderm and the speed of re-expansion in bovine blastocysts.

    PubMed

    Inaba, Yasushi; Miyashita, Satoshi; Somfai, Tamás; Geshi, Masaya; Matoba, Satoko; Dochi, Osamu; Nagai, Takashi

    2016-04-01

    This study investigated re-expansion dynamics during culture of bovine blastocysts cryopreserved either by slow-freezing or vitrification. Also, the extent and localization of membrane damage and DNA fragmentation in re-expanded embryos were studied. Frozen-thawed embryos showed a significantly lower re-expansion rate during 24 h of post-thawing culture compared to vitrified embryos. Vitrified embryos reached the maximum level of re-expansion rate by 12 h of culture whereas frozen embryos showed a gradual increase in re-expansion rate by 24 h of culture. When assayed by Hoechst/propidium iodide staining there was no difference in the numbers and ratio of membrane damaged cells between re-expanded frozen and vitrified embryos; however, the extent of membrane damage in blastomeres was significantly higher in both groups compared with non-cryopreserved embryos (control). TUNEL assay combined with differential ICM and TE staining revealed a significantly higher number and ratio of TE cells showing DNA-fragmentation in frozen-thawed re-expanded blastocysts compared to vitrified ones; however, vitrification also resulted in an increased extent of DNA fragmentation in TE cells compared with control blastocysts. In frozen-thawed blastocysts increased extent of DNA fragmentation was associated with reduced numbers and proportion of TE cells compared with vitrified and control embryos. The number and ratio of ICM cells and the extent of DNA fragmentation in ICM did not differ among control, frozen and vitrified groups. In conclusion, compared with vitrified embryos, blastocysts preserved by slow-freezing showed a delayed timing of re-expansion which was associated with an increased frequency of DNA fragmentation in TE cells.

  20. Type I IFN promotes NK cell expansion during viral infection by protecting NK cells against fratricide.

    PubMed

    Madera, Sharline; Rapp, Moritz; Firth, Matthew A; Beilke, Joshua N; Lanier, Lewis L; Sun, Joseph C

    2016-02-08

    Type I interferon (IFN) is crucial in host antiviral defense. Previous studies have described the pleiotropic role of type I IFNs on innate and adaptive immune cells during viral infection. Here, we demonstrate that natural killer (NK) cells from mice lacking the type I IFN-α receptor (Ifnar(-/-)) or STAT1 (which signals downstream of IFNAR) are defective in expansion and memory cell formation after mouse cytomegalovirus (MCMV) infection. Despite comparable proliferation, Ifnar(-/-) NK cells showed diminished protection against MCMV infection and exhibited more apoptosis compared with wild-type NK cells. Furthermore, we show that Ifnar(-/-) NK cells express increased levels of NK group 2 member D (NKG2D) ligands during viral infection and are susceptible to NK cell-mediated fratricide in a perforin- and NKG2D-dependent manner. Adoptive transfer of Ifnar(-/-) NK cells into NK cell-deficient mice reverses the defect in survival and expansion. Our study reveals a novel type I IFN-dependent mechanism by which NK cells evade mechanisms of cell death after viral infection.

  1. LPS-TLR4 Pathway Mediates Ductular Cell Expansion in Alcoholic Hepatitis

    PubMed Central

    Odena, Gemma; Chen, Jiegen; Lozano, Juan Jose; Altamirano, Jose; Rodrigo-Torres, Daniel; Affo, Silvia; Morales-Ibanez, Oriol; Matsushita, Hiroshi; Zou, Jian; Dumitru, Raluca; Caballeria, Juan; Gines, Pere; Arroyo, Vicente; You, Min; Rautou, Pierre-Emmanuel; Valla, Dominique; Crews, Fulton; Seki, Ekihiro; Sancho-Bru, Pau; Bataller, Ramon

    2016-01-01

    Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease for which there are no effective therapies. Patients with AH show impaired hepatocyte proliferation, expansion of inefficient ductular cells and high lipopolysaccharide (LPS) levels. It is unknown whether LPS mediates ductular cell expansion. We performed transcriptome studies and identified keratin 23 (KRT23) as a new ductular cell marker. KRT23 expression correlated with mortality and LPS serum levels. LPS-TLR4 pathway role in ductular cell expansion was assessed in human and mouse progenitor cells, liver slices and liver injured TLR4 KO mice. In AH patients, ductular cell expansion correlated with portal hypertension and collagen expression. Functional studies in ductular cells showed that KRT23 regulates collagen expression. These results support a role for LPS-TLR4 pathway in promoting ductular reaction in AH. Maneuvers aimed at decreasing LPS serum levels in AH patients could have beneficial effects by preventing ductular reaction development. PMID:27752144

  2. Microcarrier Culture for Efficient Expansion and Osteogenic Differentiation of Human Fetal Mesenchymal Stem Cells

    PubMed Central

    Goh, Tony Kwang-Poh; Zhang, Zhi-Yong; Chen, Allen Kuan-Liang; Reuveny, Shaul; Choolani, Mahesh

    2013-01-01

    Abstract Stirred microcarrier (MC) culture has been suggested as the method of choice for supplying large volumes of mesenchymal stem cells (MSCs) for bone tissue engineering. In this study, we show that in addition to the improvement in cell expansion capacity, MSCs propagated and harvested from MC culture also demonstrate higher osteogenic potency when differentiated in vivo or in vitro in three-dimensional (3D) scaffold cultures as compared with traditional monolayer (MNL) cultures. Cytodex 3 microcarrier-expanded human fetal MSC (hfMSC) cultures (MC-hfMSCs) achieved 12- to 16-fold expansion efficiency (6×105–8×105 cells/mL) compared to 4- to 6-fold (1.2×105–1.8×105 cells/mL) achieved by traditional MNL-expanded hfMSC culture (MNL-hfMSCs; p<0.05). Both MC-hfMSCs and MNL-hfMSCs maintained similar colony-forming capacity, doubling times, and immunophenotype postexpansion. However, when differentiated under in vitro two-dimensional (2D) osteogenic conditions, MC-hfMSCs exhibited a 45-fold reduction in alkaline phosphatase level and a 37.5% decrease in calcium deposition compared with MNL-hfMSCs (p<0.05). Surprisingly, when MC-hfMSCs and MNL-hfMSCs were seeded on 3D macroporous scaffold culture or subcutaneously implanted into nonobese diabetic/severe combined immunodeficient mice, MC-hfMSCs deposited 63.5% (p<0.05) more calcium and formed 47.2% (p<0.05) more bone volume, respectively. These results suggest that the mode of hfMSC growth in the expansion phase affects the osteogenic potential of hfMSCs differently in various differentiation platforms. In conclusion, MC cultures are advantageous over MNL cultures in bone tissue engineering because MC-hfMSCs have improved cell expansion capacity and exhibit higher osteogenic potential than MNL-hfMSCs when seeded in vitro into 3D scaffolds or implanted in vivo. PMID:23593561

  3. Balanced cell proliferation and expansion is essential for flowering stem growth control.

    PubMed

    Ferjani, Ali; Hanai, Kenya; Gunji, Shizuka; Maeda, Saori; Sawa, Shinichiro; Tsukaya, Hirokazu

    2015-01-01

    The postembryonic development of aboveground plant organs relies on a continuous supply of cells from the shoot apical meristem. Previous studies of developmental regulation in leaves and flowers have revealed the crucial role of coordinated cell proliferation and differentiation during organogenesis. However, the importance of this coordination has not been examined in flowering stems. Very recently, we attempted to identify regulatory factors that maintain flowering stem integrity. We found that the increased cell number in clavata (clv) mutants and the decreased cell size in de-etiolated (det)3-1 resulted in flowering stems that were thicker and thinner, respectively, than in wild-type (WT) plants. Interestingly, in the cell proliferation- and cell expansion-defective double mutant clv det3-1, the flowering stems often exhibited severe cracking, resulting in exposure of their inner tissues. In this study, further quantification of the cellular phenotypes in the cotyledons and leaves revealed no differences between det3-1 and clv3 det3-1. Together, the above findings suggest that the clv3 mutation in a det3-1 background primarily affects flowering stems, while its effect on other organs is likely negligible. We propose that the coordination between cell proliferation and differentiation is not only important during leaf development, but also plays a role in the growth control of Arabidopsis flowering stems.

  4. Balanced cell proliferation and expansion is essential for flowering stem growth control

    PubMed Central

    Ferjani, Ali; Hanai, Kenya; Gunji, Shizuka; Maeda, Saori; Sawa, Shinichiro; Tsukaya, Hirokazu

    2015-01-01

    The postembryonic development of aboveground plant organs relies on a continuous supply of cells from the shoot apical meristem. Previous studies of developmental regulation in leaves and flowers have revealed the crucial role of coordinated cell proliferation and differentiation during organogenesis. However, the importance of this coordination has not been examined in flowering stems. Very recently, we attempted to identify regulatory factors that maintain flowering stem integrity. We found that the increased cell number in clavata (clv) mutants and the decreased cell size in de-etiolated (det)3-1 resulted in flowering stems that were thicker and thinner, respectively, than in wild-type (WT) plants. Interestingly, in the cell proliferation- and cell expansion-defective double mutant clv det3-1, the flowering stems often exhibited severe cracking, resulting in exposure of their inner tissues. In this study, further quantification of the cellular phenotypes in the cotyledons and leaves revealed no differences between det3-1 and clv3 det3-1. Together, the above findings suggest that the clv3 mutation in a det3-1 background primarily affects flowering stems, while its effect on other organs is likely negligible. We propose that the coordination between cell proliferation and differentiation is not only important during leaf development, but also plays a role in the growth control of Arabidopsis flowering stems. PMID:25831425

  5. Does Schooling Affect Health Behavior? Evidence from the Educational Expansion in Western Germany

    ERIC Educational Resources Information Center

    Jurges, Hendrik; Reinhold, Steffen; Salm, Martin

    2011-01-01

    During the postwar period German states pursued policies to increase the share of young Germans obtaining a university entrance diploma (Abitur) by building more academic track schools, but the timing of educational expansion differed between states. This creates exogenous variation in the availability of upper secondary schooling, which allows…

  6. CD133+ cell selection is an alternative to CD34+ cell selection for ex vivo expansion of hematopoietic stem cells.

    PubMed

    Kobari, L; Giarratana, M C; Pflumio, F; Izac, B; Coulombel, L; Douay, L

    2001-04-01

    CD133 is a new stem cell antigen that may provide an alternative to CD34 for the selection and expansion of hematopoietic cells for transplantation. This study compared the expansion capacities of CD133(+) and CD34(+) cells isolated from the same cord blood (CB) samples. After 14 days culture in stroma-free, serum-free medium in the presence of stem cell factor (SCF), Flt3-1, megakaryocyte growth and development factor (MGDF), and granulocyte colony-stimulating factor (G-CSF), the CD133(+) and CD34(+) fractions displayed comparable expansion of the myeloid compartment (CFC, LTC-IC, and E-LTC-IC). The expansion of CD133(+) CB cells was up to 1262-fold for total cells, 99-fold for CD34(+) cells, 109-fold for CD34(+) CD133(+) cells, 133-fold for CFU-GM, 14.5-fold for LTC-IC, and 7.5-fold for E-LTC-IC. Moreover, the expanded population was able to generate lymphoid B (CD19(+)), NK (CD56(+)), and T (CD4(+) CD8(+)) cells in liquid or fetal thymic organ cultures, while expression of the homing antigen CXCR4 was similar on expanded and nonexpanded CD133(+) or CD34(+) cells. Thus, the CD133(+) subset could be expanded in the same manner as the CD34(+) subset and conserved its multilineage capacity, which would support the relevance of CD133 for clinical hematopoietic selection.

  7. Cell Expansion-Dependent Inflammatory and Metabolic Profile of Human Bone Marrow Mesenchymal Stem Cells

    PubMed Central

    Prieto, Patricia; Fernández-Velasco, María; Fernández-Santos, María E.; Sánchez, Pedro L.; Terrón, Verónica; Martín-Sanz, Paloma; Fernández-Avilés, Francisco; Boscá, Lisardo

    2016-01-01

    Stem cell therapy has emerged as a promising new area in regenerative medicine allowing the recovery of viable tissues. Among the many sources of adult stem cells, bone marrow-derived are easy to expand in culture via plastic adherence and their multipotentiality for differentiation make them ideal for clinical applications. Interestingly, several studies have indicated that MSCs expansion in vitro may be limited mainly due to “cell aging” related to the number of cell divisions in culture. We have determined that MSCs exhibit a progressive decline across successive passages in the expression of stem cell markers, in plasticity and in the inflammatory response, presenting low immunogenicity. We have exposed human MSCs after several passages to TLRs ligands and analyzed their inflammatory response. These cells responded to pro-inflammatory stimuli (i.e., NOS-2 expression) and to anti-inflammatory cytokines (i.e., HO1 and Arg1) until two expansions, rapidly declining upon subculture. Moreover, in the first passages, MSCs were capable to release IL1β, IL6, and IL8, as well as to produce active MMPs allowing them to migrate. Interestingly enough, after two passages, anaerobic glycolysis was enhanced releasing high levels of lactate to the extracellular medium. All these results may have important implications for the safety and efficacy of MSCs-based cell therapies. PMID:27899899

  8. Chemical expansion affected oxygen vacancy stability in different oxide structures from first principles calculations

    SciTech Connect

    Aidhy, Dilpuneet S.; Liu, Bin; Zhang, Yanwen; Weber, William J.

    2015-01-21

    We study the chemical expansion for neutral and charged oxygen vacancies in fluorite, rocksalt, perovskite and pyrochlores materials using first principles calculations. We show that the neutral oxygen vacancy leads to lattice expansion whereas the charged vacancy leads to lattice contraction. In addition, we show that there is a window of strain within which an oxygen vacancy is stable; beyond that range, the vacancy can become unstable. Using CeO2|ZrO2 interface structure as an example, we show that the concentration of oxygen vacancies can be manipulated via strain, and the vacancies can be preferentially stabilized. Furthermore, these results could serve as guiding principles in predicting oxygen vacancy stability in strained systems and in the design of vacancy stabilized materials.

  9. Chemical expansion affected oxygen vacancy stability in different oxide structures from first principles calculations

    SciTech Connect

    Aidhy, Dilpuneet S.; Liu, Bin; Zhang, Yanwen; Weber, William J.

    2015-03-01

    We study the chemical expansion for neutral and charged oxygen vacancies in fluorite, rocksalt, perovskite and pyrochlores materials using first principles calculations. We show that the neutral oxygen vacancy leads to lattice expansion whereas the charged vacancy leads to lattice contraction. In addition, we show that there is a window of strain within which an oxygen vacancy is stable; beyond that range, the vacancy can become unstable. Using CeO2|ZrO2 interface structure as an example, we show that the concentration of oxygen vacancies can be manipulated via strain, and the vacancies can be preferentially stabilized. These results could serve as guiding principles in predicting oxygen vacancy stability in strained systems and in the design of vacancy stabilized materials.

  10. Chemical expansion affected oxygen vacancy stability in different oxide structures from first principles calculations

    DOE PAGES

    Aidhy, Dilpuneet S.; Liu, Bin; Zhang, Yanwen; ...

    2015-01-21

    We study the chemical expansion for neutral and charged oxygen vacancies in fluorite, rocksalt, perovskite and pyrochlores materials using first principles calculations. We show that the neutral oxygen vacancy leads to lattice expansion whereas the charged vacancy leads to lattice contraction. In addition, we show that there is a window of strain within which an oxygen vacancy is stable; beyond that range, the vacancy can become unstable. Using CeO2|ZrO2 interface structure as an example, we show that the concentration of oxygen vacancies can be manipulated via strain, and the vacancies can be preferentially stabilized. Furthermore, these results could serve asmore » guiding principles in predicting oxygen vacancy stability in strained systems and in the design of vacancy stabilized materials.« less

  11. Induced p53 loss in mouse luminal cells causes clonal expansion and development of mammary tumours

    PubMed Central

    Tao, Luwei; Xiang, Dongxi; Xie, Ying; Bronson, Roderick T.; Li, Zhe

    2017-01-01

    Most breast cancers may have a luminal origin. TP53 is one of the most frequently mutated genes in breast cancers. However, how p53 deficiency contributes to breast tumorigenesis from luminal cells remains elusive. Here we report that induced p53 loss in Krt8+ mammary luminal cells leads to their clonal expansion without directly affecting their luminal identity. All induced mice develop mammary tumours with 9qA1 (Yap1) and/or 6qA2 (Met) amplification(s). These tumours exhibit a mammary stem cell (MaSC)-like expression signature and most closely resemble claudin-low breast cancer. Thus, although p53 does not directly control the luminal fate, its loss facilitates acquisition of MaSC-like properties by luminal cells and predisposes them to development of mammary tumours with loss of luminal identity. Our data also suggest that claudin-low breast cancer can develop from luminal cells, possibly via a basal-like intermediate state, although further study using a different luminal promoter is needed to fully support this conclusion. PMID:28194015

  12. Paracrine WNT5A Signaling Inhibits Expansion of Tumor-Initiating Cells.

    PubMed

    Borcherding, Nicholas; Kusner, David; Kolb, Ryan; Xie, Qing; Li, Wei; Yuan, Fang; Velez, Gabriel; Askeland, Ryan; Weigel, Ronald J; Zhang, Weizhou

    2015-05-15

    It is not well understood how paracrine communication between basal and luminal cell populations in the mammary gland affects tumorigenesis. During ErbB2-induced mammary tumorigenesis, enriched mammary stem cells that represent a subpopulation of basal cells exhibit enhanced tumorigenic capacity compared with the corresponding luminal progenitors. Transcript profiling of tumors derived from basal and luminal tumor-initiating cells (TIC) revealed preferential loss of the noncanonical Wnt ligand WNT5A in basal TIC-derived tumors. Heterozygous loss of WNT5A was correlated with shorter survival of breast cancer patients. In a mouse model of ErbB2-induced breast cancer, Wnt5a heterozygosity promoted tumor multiplicity and pulmonary metastasis. As a TGFβ substrate, luminal cell-produced WNT5A induced a feed-forward loop to activate SMAD2 in a RYK and TGFβR1-dependent manner to limit the expansion of basal TIC in a paracrine fashion, a potential explanation for the suppressive effect of WNT5A in mammary tumorigenesis. Our results identify the WNT5A/RYK module as a spatial regulator of the TGFβ-SMAD signaling pathway in the context of mammary gland development and carcinogenesis, offering a new perspective on tumor suppression provided by basal-luminal cross-talk in normal mammary tissue.

  13. Paracrine WNT5A signaling inhibits expansion of tumor-initiating cells

    PubMed Central

    Borcherding, Nicholas; Kusner, David; Kolb, Ryan; Xie, Qing; Li, Wei; Yuan, Fang; Velez, Gabriel; Askeland, Ryan; Weigel, Ronald J.; Zhang, Weizhou

    2015-01-01

    It is not well understood how paracrine communication between basal and luminal cell populations in the mammary gland affects tumorigenesis. During ErbB2-induced mammary tumorigenesis, enriched mammary stem cells that represent a subpopulation of basal cells exhibit enhanced tumorigenic capacity compared to the corresponding luminal progenitors. Transcript profiling of tumors derived from basal and luminal tumor-initiating cells (TIC) revealed preferential loss of the noncanonical Wnt ligand WNT5A in basal TIC-derived tumors. Heterozygous loss of WNT5A was correlated with shorter survival of breast cancer patients. In a mouse model of ErbB2-induced breast cancer, Wnt5a heterozygosity promoted tumor multiplicity and pulmonary metastasis. As a TGFβ substrate, luminal cell-produced WNT5A induced a feed-forward loop to activate SMAD2 in a RYK and TGFβR1-dependent manner to limit the expansion of basal TIC in a paracrine fashion, a potential explanation for the suppressive effect of WNT5A in mammary tumorigenesis. Our results identify the WNT5A/RYK module as a spatial regulator of TGFβ/SMAD signaling pathway in the context of mammary gland development and carcinogenesis, offering a new perspective on tumor suppression provided by basal-luminal crosstalk in normal mammary tissue. PMID:25769722

  14. Cytokines regulate postnatal hematopoietic stem cell expansion: opposing roles of thrombopoietin and LNK

    PubMed Central

    Buza-Vidas, Natalija; Antonchuk, Jennifer; Qian, Hong; Månsson, Robert; Luc, Sidinh; Zandi, Sasan; Anderson, Kristina; Takaki, Satoshi; Nygren, Jens M.; Jensen, Christina T.; Jacobsen, Sten Eirik W.

    2006-01-01

    The role of cytokines as regulators of hematopoietic stem cell (HSC) expansion remains elusive. Herein, we identify thrombopoietin (THPO) and the cytokine signaling inhibitor LNK, as opposing physiological regulators of HSC expansion. Lnk−/− HSCs continue to expand postnatally, up to 24-fold above normal by 6 mo of age. Within the stem cell compartment, this expansion is highly selective for self-renewing long-term HSCs (LT-HSCs), which show enhanced THPO responsiveness. Lnk−/− HSC expansion is dependent on THPO, and 12-wk-old Lnk−/−Thpo−/− mice have 65-fold fewer LT-HSCs than Lnk−/− mice. Expansions of multiple myeloid, but not lymphoid, progenitors in Lnk−/− mice also proved THPO-dependent. PMID:16882979

  15. Effector T cells boost regulatory T cell expansion by IL-2, TNF, OX40, and plasmacytoid dendritic cells depending on the immune context.

    PubMed

    Baeyens, Audrey; Saadoun, David; Billiard, Fabienne; Rouers, Angéline; Grégoire, Sylvie; Zaragoza, Bruno; Grinberg-Bleyer, Yenkel; Marodon, Gilles; Piaggio, Eliane; Salomon, Benoît L

    2015-02-01

    CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells play a major role in peripheral tolerance. Multiple environmental factors and cell types affect their biology. Among them, activated effector CD4(+) T cells can boost Treg cell expansion through TNF or IL-2. In this study, we further characterized this effector T (Teff) cell-dependent Treg cell boost in vivo in mice. This phenomenon was observed when both Treg and Teff cells were activated by their cognate Ag, with the latter being the same or different. Also, when Treg cells highly proliferated on their own, there was no additional Treg cell boost by Teff cells. In a condition of low inflammation, the Teff cell-mediated Treg cell boost involved TNF, OX40L, and plasmacytoid dendritic cells, whereas in a condition of high inflammation, it involved TNF and IL-2. Thus, this feedback mechanism in which Treg cells are highly activated by their Teff cell counterparts depends on the immune context for its effectiveness and mechanism. This Teff cell-dependent Treg cell boost may be crucial to limit inflammatory and autoimmune responses.

  16. Allogeneic Cell Therapy Bioprocess Economics and Optimization: Single-Use Cell Expansion Technologies

    PubMed Central

    Simaria, Ana S; Hassan, Sally; Varadaraju, Hemanthram; Rowley, Jon; Warren, Kim; Vanek, Philip; Farid, Suzanne S

    2014-01-01

    For allogeneic cell therapies to reach their therapeutic potential, challenges related to achieving scalable and robust manufacturing processes will need to be addressed. A particular challenge is producing lot-sizes capable of meeting commercial demands of up to 109 cells/dose for large patient numbers due to the current limitations of expansion technologies. This article describes the application of a decisional tool to identify the most cost-effective expansion technologies for different scales of production as well as current gaps in the technology capabilities for allogeneic cell therapy manufacture. The tool integrates bioprocess economics with optimization to assess the economic competitiveness of planar and microcarrier-based cell expansion technologies. Visualization methods were used to identify the production scales where planar technologies will cease to be cost-effective and where microcarrier-based bioreactors become the only option. The tool outputs also predict that for the industry to be sustainable for high demand scenarios, significant increases will likely be needed in the performance capabilities of microcarrier-based systems. These data are presented using a technology S-curve as well as windows of operation to identify the combination of cell productivities and scale of single-use bioreactors required to meet future lot sizes. The modeling insights can be used to identify where future R&D investment should be focused to improve the performance of the most promising technologies so that they become a robust and scalable option that enables the cell therapy industry reach commercially relevant lot sizes. The tool outputs can facilitate decision-making very early on in development and be used to predict, and better manage, the risk of process changes needed as products proceed through the development pathway. Biotechnol. Bioeng. 2014;111: 69–83. © 2013 Wiley Periodicals, Inc. PMID:23893544

  17. A Peptide Hormone and Its Receptor Protein Kinase Regulate Plant Cell Expansion

    PubMed Central

    Haruta, Miyoshi; Sabat, Grzegorz; Stecker, Kelly; Minkoff, Benjamin B.; Sussman, Michael R.

    2015-01-01

    Plant cells are immobile; thus, plant growth and development depend on cell expansion rather than cell migration. The molecular mechanism by which the plasma membrane initiates changes in the cell expansion rate remains elusive. We found that a secreted peptide, RALF (rapid alkalinization factor), suppresses cell elongation of the primary root by activating the cell surface receptor FERONIA in Arabidopsis thaliana. A direct peptide-receptor interaction is supported by specific binding of RALF to FERONIA and reduced binding and insensitivity to RALF-induced growth inhibition in feronia mutants. Phosphoproteome measurements demonstrate that the RALF-FERONIA interaction causes phosphorylation of plasma membrane H+–adenosine triphosphatase 2 at Ser899, mediating the inhibition of proton transport. The results reveal a molecular mechanism for RALF-induced extracellular alkalinization and a signaling pathway that regulates cell expansion. PMID:24458638

  18. Discovery of a Novel Polymer for Human Pluripotent Stem Cell Expansion and Multilineage Differentiation.

    PubMed

    Celiz, Adam D; Smith, James G W; Patel, Asha K; Hook, Andrew L; Rajamohan, Divya; George, Vinoj T; Flatt, Luke; Patel, Minal J; Epa, Vidana C; Singh, Taranjit; Langer, Robert; Anderson, Daniel G; Allen, Nicholas D; Hay, David C; Winkler, David A; Barrett, David A; Davies, Martyn C; Young, Lorraine E; Denning, Chris; Alexander, Morgan R

    2015-07-15

    A scalable and cost-effective synthetic polymer substrate that supports robust expansion and subsequent multilineage differentiation of human pluripotent stem cells (hPSCs) with defined commercial media is presented. This substrate can be applied to common cultureware and used off-the-shelf after long-term storage. Expansion and differentiation of hPSCs are performed entirely on the polymeric surface, enabling the clinical potential of hPSC-derived cells to be realized.

  19. 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

  20. Substrates for Expansion of Corneal Endothelial Cells towards Bioengineering of Human Corneal Endothelium

    PubMed Central

    Navaratnam, Jesintha; Utheim, Tor P.; Rajasekhar, Vinagolu K.; Shahdadfar, Aboulghassem

    2015-01-01

    Corneal endothelium is a single layer of specialized cells that lines the posterior surface of cornea and maintains corneal hydration and corneal transparency essential for vision. Currently, transplantation is the only therapeutic option for diseases affecting the corneal endothelium. Transplantation of corneal endothelium, called endothelial keratoplasty, is widely used for corneal endothelial diseases. However, corneal transplantation is limited by global donor shortage. Therefore, there is a need to overcome the deficiency of sufficient donor corneal tissue. New approaches are being explored to engineer corneal tissues such that sufficient amount of corneal endothelium becomes available to offset the present shortage of functional cornea. Although human corneal endothelial cells have limited proliferative capacity in vivo, several laboratories have been successful in in vitro expansion of human corneal endothelial cells. Here we provide a comprehensive analysis of different substrates employed for in vitro cultivation of human corneal endothelial cells. Advances and emerging challenges with ex vivo cultured corneal endothelial layer for the ultimate goal of therapeutic replacement of dysfunctional corneal endothelium in humans with functional corneal endothelium are also presented. PMID:26378588

  1. Human Head and Neck Squamous Cell Carcinoma-Associated Semaphorin 4D Induces Expansion of Myeloid-Derived Suppressor Cells.

    PubMed

    Younis, Rania H; Han, Kyu Lee; Webb, Tonya J

    2016-02-01

    One of the mechanisms by which malignancies can induce immune suppression is through the production of cytokines that affect the maturation and differentiation of inflammatory cells in the tumor microenvironment. Semaphorin 4D (Sema4D) is a proangiogenic cytokine produced by several malignancies, which has been described in the regulation of the immune system. In the present study, we examined the role of human head and neck squamous cell carcinoma (HNSCC)-secreted Sema4D on myeloid cell differentiation. CD33(+) cells cultured in HNSCC cell line-derived conditioned medium differentiated into myeloid derived suppressor cells (MDSC) (CD33(+)CD11b(+)HLA-DR(-/low)). The addition of anti-Sema4D Ab to HNSCC conditioned medium significantly reduced the expansion of the MDSC population. Similarly, knockdown of Sema4D in an HNSCC cell line resulted in a loss of MDSC function as shown by a decrease in the production of the immune-suppressive cytokines arginase-1, TGF-β, and IL-10 by MDSC, concomitant with recovery of T cell proliferation and IFN-γ production following stimulation of CD3/CD28. Importantly, CD33(+) myeloid and T cells cultured in conditioned medium of HNSCC cells in which Sema4D was knocked down promoted antitumor inflammatory profile, through recovery of the effector T cells (CD4(+)T-bet(+) and CD8(+)T-bet(+)), as well as a decrease in regulatory T cells (CD4(+)CD25(+)FOXP3(+)). We also showed that Sema4D was comparable to GM-CSF in its induction of MDSC. Collectively, this study describes a novel immunosuppressive role for Sema4D in HNSCC through induction of MDSC, and it highlights Sema4D as a therapeutic target for future studies to enhance the antitumorigenic inflammatory response in HNSCC and other epithelial malignancies.

  2. Activated CD8+ T cells induce expansion of Vβ5+ regulatory T cells via TNFR2 signaling

    PubMed Central

    Joedicke, Jara J; Myers, Lara; Carmody, Aaron B; Messer, Ronald J; Wajant, Harald; Lang, Karl S; Lang, Philipp A; Mak, Tak W; Hasenkrug, Kim J; Dittmer, Ulf

    2014-01-01

    Vβ5+ regulatory T cells (Tregs), which are specific for a mouse endogenous retroviral superantigen, become activated and proliferate in response to Friend retrovirus (FV) infection. We previously reported that FV-induced expansion of this Treg subset was dependent on CD8+ T cells and TNFα, but independent of IL-2. We now show that the inflammatory milieu associated with FV infection is not necessary for induction of Vβ5+ Treg expansion. Rather, it is the presence of activated CD8+ T cells that is critical for their expansion. The data indicate that the mechanism involves signaling between the membrane-bound form of TNFα (memTNFα) on activated CD8+ T cells and TNF receptor 2 (TNFR2) on Tregs. CD8+ T cells expressing memTNFα but no soluble TNFα (solTNFα) remained competent to induce strong Vβ5+ Treg expansion in vivo. In addition, Vβ5+ Tregs expressing only TNFR2 but no TNFR1 were still responsive to expansion. Finally, treatment of naïve mice with solTNFα did not induce Vβ5+ Treg expansion, but treatment with a TNFR2-specific agonist did. These results reveal a new mechanism of intercellular communication between activated CD8+ T cell effectors and Tregs that results in the activation and expansion of a Treg subset that subsequently suppresses CD8+ T cell functions. PMID:25098294

  3. Numerical Investigation of Blade Lean and Sweep affecting Secondary Flows in an Axial Expansion Turbine

    NASA Astrophysics Data System (ADS)

    Neipp, A.; Riedelbauch, S.

    2016-11-01

    Based on an axial expansion turbine used for energy recovery from working fluids an automated CFD-flow optimization was performed to increase the turbine efficiency without narrowing the operating range. The optimization results showed that even small changes in the optimization target had a significant influence on the lean and sweep of the new blade designs. The resulting blade shapes were extraordinary. The lean and sweep resembled more that of thermal turbomachinery than hydraulic machinery. It became clear that the special blade shapes were a result of the very low aspect ratio of the turbine and the resulting large influence of secondary flows. The blade lean and sweep induce secondary flow structures which are of decisive importance for the turbine performance. The simulation results showed that positive compound lean increases the efficiency and positive compound sweep improves the cavitation behavior of the investigated axial expansion turbine. However the complexity of the occurring secondary flows make an universally valid statement on the effect of blade lean and sweep on the flow behavior of an axial turbine impossible.

  4. Enhanced ex vivo expansion of adult mesenchymal stem cells by fetal mesenchymal stem cell ECM.

    PubMed

    Ng, Chee Ping; Sharif, Abdul Rahim Mohamed; Heath, Daniel E; Chow, John W; Zhang, Claire B Y; Chan-Park, Mary B; Hammond, Paula T; Chan, Jerry K Y; Griffith, Linda G

    2014-04-01

    Large-scale expansion of highly functional adult human mesenchymal stem cells (aMSCs) remains technologically challenging as aMSCs lose self renewal capacity and multipotency during traditional long-term culture and their quality/quantity declines with donor age and disease. Identification of culture conditions enabling prolonged expansion and rejuvenation would have dramatic impact in regenerative medicine. aMSC-derived decellularized extracellular matrix (ECM) has been shown to provide such microenvironment which promotes MSC self renewal and "stemness". Since previous studies have demonstrated superior proliferation and osteogenic potential of human fetal MSCs (fMSCs), we hypothesize that their ECM may promote expansion of clinically relevant aMSCs. We demonstrated that aMSCs were more proliferative (∼ 1.6 ×) on fMSC-derived ECM than aMSC-derived ECMs and traditional tissue culture wares (TCPS). These aMSCs were smaller and more uniform in size (median ± interquartile range: 15.5 ± 4.1 μm versus 17.2 ± 5.0 μm and 15.5 ± 4.1 μm for aMSC ECM and TCPS respectively), exhibited the necessary biomarker signatures, and stained positive for osteogenic, adipogenic and chondrogenic expressions; indications that they maintained multipotency during culture. Furthermore, fMSC ECM improved the proliferation (∼ 2.2 ×), size (19.6 ± 11.9 μm vs 30.2 ± 14.5 μm) and differentiation potential in late-passaged aMSCs compared to TCPS. In conclusion, we have established fMSC ECM as a promising cell culture platform for ex vivo expansion of aMSCs.

  5. Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells.

    PubMed

    Zhang, Cheng Cheng; Kaba, Megan; Ge, Guangtao; Xie, Kathleen; Tong, Wei; Hug, Christopher; Lodish, Harvey F

    2006-02-01

    Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of crucial questions of stem cell biology. Here we show, using microarray studies, that the HSC-supportive mouse fetal liver CD3(+) cells specifically express the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). We observed a 24- or 30-fold net expansion of long-term HSCs by reconstitution analysis when we cultured highly enriched HSCs for 10 days in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors. The coiled-coil domain of Angptl2 was capable of stimulating expansion of HSCs. Furthermore, angiopoietin-like 5, angiopoietin-like 7 and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.

  6. Pregnancy persistently affects memory T cell populations.

    PubMed

    Kieffer, Tom E C; Faas, Marijke M; Scherjon, Sicco A; Prins, Jelmer R

    2017-02-01

    Pregnancy is an immune challenge to the maternal immune system. The effects of pregnancy on maternal immunity and particularly on memory T cells during and after pregnancy are not fully known. This observational study aims to show the short term and the long term effects of pregnancy on the constitution, size and activation status of peripheral human memory T-lymphocyte populations. Effector memory (EM) and central memory (CM) T-lymphocytes were analyzed using flow cytometry of peripheral blood from 14 nulligravid, 12 primigravid and 15 parous women that were on average 18 months postpartum. The short term effects were shown by the significantly higher CD4+ EM cell and activated CD4+ memory cell proportions in primigravid women compared to nulligravid women. The persistent effects found in this study were the significantly higher proportions of CD4+ EM, CD4+ CM and activated memory T cells in parous women compared to nulligravid women. In contrast to CD4+ cells, activation status of CD8+ memory cells did not differ between the groups. This study shows that pregnancy persistently affects the pre-pregnancy CD4+ memory cell pool in human peripheral blood. During pregnancy, CD4+ T-lymphocytes might differentiate into EM cells followed by persistent higher proportions of CD4+ CM and EM cells postpartum. The persistent effects of pregnancy on memory T cells found in this study support the hypothesis that memory T cells are generated during pregnancy and that these cells could be involved in the lower complication risks in multiparous pregnancies in humans.

  7. Expansion of undifferentiated murine embryonic stem cells as aggregates in suspension culture bioreactors.

    PubMed

    Cormier, Jaymi T; zur Nieden, Nicole I; Rancourt, Derrick E; Kallos, Michael S

    2006-11-01

    Pluripotent embryonic stem cells (ESCs) have recently been considered as a primary material for regenerating tissues lost to injuries and degenerative diseases. For clinical implementation of this technology, a quality controlled, reproducible culture system is necessary for the expansion and differentiation of the cells. Used in many bioprocess applications, suspension bioreactors have gained considerable attention for the regulated large-scale expansion of cells. The current study presents a bioreactor process for the large-scale expansion of undifferentiated murine ESCs as aggregates. In this system, the level of ESC aggregation and differentiation was effectively controlled by adjusting shear forces and inoculation density, achieving a 31-fold expansion in 5 days. Pluripotency markers Oct-4, Nanog, SSEA-1, ALP, and rex-1 were assessed using flow cytometry analysis and gene expression profiles and showed that the undifferentiated nature of the cells within the ESC aggregates was maintained. Colony-forming efficiencies and embryoid body formation tests of the expanded cultures demonstrated that characteristic functional attributes of undifferentiated cells were not lost. Overcoming a major impediment in the area of ESC expansion, this study describes a successful process for the controlled and reproducible largescale expansion of ESCs using suspension culture bioreactors.

  8. Engineered artificial antigen presenting cells facilitate direct and efficient expansion of tumor infiltrating lymphocytes

    PubMed Central

    2011-01-01

    Background Development of a standardized platform for the rapid expansion of tumor-infiltrating lymphocytes (TILs) with anti-tumor function from patients with limited TIL numbers or tumor tissues challenges their clinical application. Methods To facilitate adoptive immunotherapy, we applied genetically-engineered K562 cell-based artificial antigen presenting cells (aAPCs) for the direct and rapid expansion of TILs isolated from primary cancer specimens. Results TILs outgrown in IL-2 undergo rapid, CD28-independent expansion in response to aAPC stimulation that requires provision of exogenous IL-2 cytokine support. aAPCs induce numerical expansion of TILs that is statistically similar to an established rapid expansion method at a 100-fold lower feeder cell to TIL ratio, and greater than those achievable using anti-CD3/CD28 activation beads or extended IL-2 culture. aAPC-expanded TILs undergo numerical expansion of tumor antigen-specific cells, remain amenable to secondary aAPC-based expansion, and have low CD4/CD8 ratios and FOXP3+ CD4+ cell frequencies. TILs can also be expanded directly from fresh enzyme-digested tumor specimens when pulsed with aAPCs. These "young" TILs are tumor-reactive, positively skewed in CD8+ lymphocyte composition, CD28 and CD27 expression, and contain fewer FOXP3+ T cells compared to parallel IL-2 cultures. Conclusion Genetically-enhanced aAPCs represent a standardized, "off-the-shelf" platform for the direct ex vivo expansion of TILs of suitable number, phenotype and function for use in adoptive immunotherapy. PMID:21827675

  9. Peptide specific expansion of CD8(+) T cells by recombinant plate bound MHC/peptide complexes.

    PubMed

    Schmidt, Esben G W; Buus, Søren; Thorn, Mette; Stryhn, Anette; Leisner, Christian; Claesson, Mogens H

    2009-01-01

    Development of methods for efficient in vitro stimulation and expansion of peptide specific CD8(+) T cells is compelling not only with respect to adoptive T cell therapy but also regarding analysis of T cell responses and search for new immunogenic peptides. In the present study, a new approach to in vitro T cell stimulation was investigated. By use of an antigenic peptide derived from the cytomegalovirus (CMVp) we tested the stimulatory efficacy of recombinant plate bound MHC molecules (PB-MHC), being immobilized in culture plates. A single stimulation of non-adherent peripheral blood mononuclear cells (NA-PBMCs) with PB-MHC/CMVp resulted in significant expansion of CMVp specific CD8(+) T cells, which was comparable to that achieved by CMVp pulsed mature dendritic cells (DCs). By repeated exposure of NA-PBMCs to PB-MHC/CMVp more than 60% CMVp specific CD8(+) T cells, representing a 240-fold expansion, were reached after only two stimulations. Although stimulation with PB-MHC/CMVp clearly demonstrated efficient peptide specific expansion of CD8(+) T cells, there was a tendency to proliferative exhaustion of the cells after 3-4 stimulations. Thus, it will be of interest to examine the effect of new stimulatory cocktails, e.g. cytokines and co-stimulatory molecules, by use of the present rapid and easy-to-use method of expanding peptide specific T cells.

  10. Cryptococcal Cell Morphology Affects Host Cell Interactions and Pathogenicity

    PubMed Central

    Nielsen, Judith N.; Charlier, Caroline; Baltes, Nicholas J.; Chrétien, Fabrice; Heitman, Joseph; Dromer, Françoise; Nielsen, Kirsten

    2010-01-01

    Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 µm. Cell enlargement was observed in vivo, producing cells up to 100 µm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3aΔ pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection. PMID:20585559

  11. Stroma-conditioned media improve expansion of human primitive hematopoietic stem cells and progenitor cells.

    PubMed

    Breems, D A; Blokland, E A; Ploemacher, R E

    1997-01-01

    It has been reported that stroma-dependent cultures support proliferation of hematopoietic stem cells (HSC). In order to investigate the effect of soluble stromal factors, we developed short-term serum-low liquid cultures in which the effect of stroma-conditioned media (SCM) from the murine FBMD-1, and human L87/4 and L88/5 cell lines was studied on the maintenance and expansion of various human HSC subsets in CD34-positive selected mobilized peripheral blood stem cells (PBSC) from autologous transplants of lymphoma and multiple myeloma patients. The human cobblestone area forming cell (CAFC) assay was employed to determine the frequencies of both the CAFC weeks 2 to 4 as tentative indicators of progenitor and transiently repopulating HSC, and the more primitive CAFC weeks 6 to 8 as indicators of long-term repopulating HSC. In 7-day liquid cultures containing interleukin-3 (IL-3), stem cell factor (SCF) and IL-6, we recovered 3.0-fold more colony-forming cells (CFC) and 1.7- to 1.9-fold more CAFC weeks 2 and 4. The absolute number of primitive CAFC weeks 6 and 8 were only maintained (1.1- to 1.4-fold) in these liquid cultures. This modest expansion was significantly improved by the addition of SCM from the FBMD-1, L87/4 or L88/5 cell lines. Output CFC numbers were 6.8-, 5.8- and 9.9-fold higher, respectively, than the input values, while absolute CAFC week 2 to 4 numbers were 4.5-, 10.2- and 10.2-fold expanded, respectively. The addition of SCM also improved expansion of the more primitive CAFC week 6 to 8 stem cell subsets by 2.2-, 4.5- and 4.9-fold, respectively. The addition of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), IL-1beta, IL-11 or macrophage inflammatory protein-1alpha to cultures containing IL-3, SCF and IL-6 could not explain the SCM effect and in all these combinations SCM addition further increased the recovery of HSC subsets. Similarly, addition of anti-cytokine antibodies (ie alpha-G-CSF, alpha-GM-CSF, alpha

  12. Mechanism of rapid suppression of cell expansion in cucumber hypocotyls after blue-light irradiation

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1988-01-01

    Rapid suppression of hypocotyl elongation by blue light in cucumber (Cucumis sativus L.) was studied to examine possible hydraulic and wall changes responsible for diminished growth. Cell-sap osmotic pressure, measured by vapor-pressure osmometry, was not decreased by blue light; turgor pressure, measured by the pressure-probe technique, remained constant during the growth inhibition; and stem hydraulic conductance, measured by dynamic and static methods, was likewise unaffected by blue light. Wall yielding properties were assessed by the pressure-block technique for in-vivo stress relaxation. Blue light reduced the initial rate of relaxation by 77%, but had little effect on the final amount of relaxation. The results demonstrate that blue irradiation acts to decrease the wall yielding coefficient, but not the yield threshold. Stress-strain (Instron) analysis showed that irradiation of the seedlings had little effect on the mechanical extensibilities of the isolated wall. The results indicate that blue light can reduce cell-wall loosening without affecting bulk viscoelastic properties, and indicate a chemorheological mechanism of cell-wall expansion.

  13. SH2B1 in β-cells regulates glucose metabolism by promoting β-cell survival and islet expansion.

    PubMed

    Chen, Zheng; Morris, David L; Jiang, Lin; Liu, Yong; Rui, Liangyou

    2014-02-01

    IGF-1 and insulin promote β-cell expansion by inhibiting β-cell death and stimulating β-cell proliferation, and the phosphatidylinositol (PI) 3-kinase/Akt pathway mediates insulin and IGF-1 action. Impaired β-cell expansion is a risk factor for type 2 diabetes. Here, we identified SH2B1, which is highly expressed in β-cells, as a novel regulator of β-cell expansion. Silencing of SH2B1 in INS-1 832/13 β-cells attenuated insulin- and IGF-1-stimulated activation of the PI 3-kinase/Akt pathway and increased streptozotocin (STZ)-induced apoptosis; conversely, overexpression of SH2B1 had the opposite effects. Activation of the PI 3-kinase/Akt pathway in β-cells was impaired in pancreas-specific SH2B1 knockout (PKO) mice fed a high-fat diet (HFD). HFD-fed PKO mice also had increased β-cell apoptosis, decreased β-cell proliferation, decreased β-cell mass, decreased pancreatic insulin content, impaired insulin secretion, and exacerbated glucose intolerance. Furthermore, PKO mice were more susceptible to STZ-induced β-cell destruction, insulin deficiency, and hyperglycemia. These data indicate that SH2B1 in β-cells is an important prosurvival and proproliferative protein and promotes compensatory β-cell expansion in the insulin-resistant state and in response to β-cell stress.

  14. FORMOSA controls cell division and expansion during floral development in Antirrhinum majus.

    PubMed

    Delgado-Benarroch, Luciana; Causier, Barry; Weiss, Julia; Egea-Cortines, Marcos

    2009-05-01

    Control of organ size is the product of coordinated cell division and expansion. In plants where one of these pathways is perturbed, organ size is often unaffected as compensation mechanisms are brought into play. The number of founder cells in organ primordia, dividing cells, and the period of cell proliferation determine cell number in lateral organs. We have identified the Antirrhinum FORMOSA (FO) gene as a specific regulator of floral size. Analysis of cell size and number in the fo mutant, which has increased flower size, indicates that FO is an organ-specific inhibitor of cell division and activator of cell expansion. Increased cell number in fo floral organs correlated with upregulation of genes involved in the cell cycle. In Arabidopsis the AINTEGUMENTA (ANT) gene promotes cell division. In the fo mutant increased cell number also correlates with upregulation of an Antirrhinum ANT-like gene (Am-ANT) in inflorescences that is very closely related to ANT and shares a similar expression pattern, suggesting that they may be functional equivalents. Increased cell proliferation is thought to be compensated for by reduced cell expansion to maintain organ size. In Arabidopsis petal cell expansion is inhibited by the BIGPETAL (BPE) gene, and in the fo mutant reduced cell size corresponded to upregulation of an Antirrhinum BPE-like gene (Am-BPE). Our data suggest that FO inhibits cell proliferation by negatively regulating Am-ANT, and acts upstream of Am-BPE to coordinate floral organ size. This demonstrates that organ size is modulated by the organ-specific control of both general and local gene networks.

  15. microRNAs as potential regulators of myeloid-derived suppressor cell expansion.

    PubMed

    El Gazzar, Mohamed

    2014-04-01

    Proper development and activation of cells of the myeloid lineage are critical for supporting innate immunity. This myelopoiesis is orchestrated by interdependent interactions between cytokine receptors, transcription factors and, as recently described, microRNAs (miRNAs). miRNAs contribute to normal and dysregulated myelopoiesis. Alterations in myelopoiesis underlie myeloid-derived suppressor cell (MDSC) expansion, a poorly understood heterogeneous population of immature and suppressive myeloid cells that expand in nearly all diseases where inflammation exists. MDSCs associated with inflammation often have immunosuppressive properties, but molecular mechanisms responsible for MDSC expansion are unclear. Emerging data implicate miRNAs in MDSC expansion. This review focuses on miRNAs that contribute to myeloid lineage differentiation and maturation under physiological conditions, and introduces the concept that altered miRNA expression my underlie expansion and accumulation of MDSCs. We divide our miRNAs into those with potential to promote MDSC expansion and two with known direct links to MDSC expansion, miR-223 and miR-494.

  16. Expansion of CD8+ cells in autoimmune hemolytic anemia.

    PubMed

    Smirnova, S Ju; Sidorova, Ju V; Tsvetaeva, N V; Nikulina, O F; Biderman, B V; Nikulina, E E; Kulikov, S M; Sudarikov, A B

    2016-01-01

    Autoimmune hemolytic anemia (AIHA) is a rare blood disease associated with the production of auto-antibodies and autoimmune hemolysis. A critical role of B-cells in the development of AIHA has been demonstrated before. Here, we present the analysis of the clonal T-cell populations in patients with AIHA. Thirty-three patients with AIHA were included in this study. Thirteen patients with other anemias, 14 patients with other autoimmune conditions (SLE - 6, RA - 8) and 20 healthy donors were included in the study as a control group. The clonality of T-cell was evaluated by the assessment of the T-cell receptor gamma and beta chain gene rearrangements (TCRG and TCRB). The incidence of T-cell monoclonality detected in patients with AIHA was significantly higher compared to the control group. The persistence of T-cell clones did not correlate with the level of hemoglobin and other signs of remission or relapse and did not disappear after the therapy and clinical improvement (observation period was between 1 and 10 years). There was no correlation between the T-cell clonality and the gender, age, splenectomy, duration or severity of the disease. Fractionation of T-lymphocytes (CD4+, CD8+, CD4+25+) revealed that the monoclonal T-cells belonged to the CD8+ sub-population. We assume that besides a possible causative role of the T-cell clones in AIHA to autoimmune process, these clones do not directly participate in the development and maintenance of hemolysis. Most of the AIHA patients (48.5%) demonstrated a T-cell monoclonality, which requires monitoring and should be distinguished from T-cell tumors.

  17. HOT CELL BUILDING, TRA632. FLOOR PLAN OF EXPANSION SHOWS LOCATION ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    HOT CELL BUILDING, TRA-632. FLOOR PLAN OF EXPANSION SHOWS LOCATION OF NEW CELLS, "HEAVY" CELL AT WEST END, "LIGHT" CELLS AT EAST. MOCK-UP AND STORAGE AREAS IN SOUTH HALF OF FLOOR. H.K. FERGUSON 895-MTR-ETR-632-A1, 12/1958. INL INDEX NO. 531-0632-00-279-101924, REV. 4. - Idaho National Engineering Laboratory, Test Reactor Area, Materials & Engineering Test Reactors, Scoville, Butte County, ID

  18. Augmented lymphocyte expansion from solid tumors with engineered cells for costimulatory enhancement

    PubMed Central

    Friedman, Kevin M; DeVillier, Laura E; Feldman, Steven A; Rosenberg, Steven A; Dudley, Mark E

    2011-01-01

    Treatment of patients with adoptive T cell therapy requires expansion of unique tumor-infiltrating lymphocyte (TIL) cultures from single cell suspensions processed from melanoma biopsies. Strategies which increase the expansion and reliability of TIL generation from tumor digests are necessary to improve access to TIL therapy. Prior work evaluated artificial antigen presenting cells (aAPCs) for their antigen-specific and costimulatory properties. We investigated engineered cells for co-stimulatory enhancement (ECCE) consisting of K562 cells which express 4-1BBL in the absence of artificial antigen stimulation. ECCE accelerated TIL expansion and significantly improved TIL numbers (p=0.001) from single cell melanoma suspensions. TIL generated with ECCE contain significantly more CD8+CD62L+ and CD8+CD27+ T cells then comparable IL-2-expanded TIL and maintained anti-tumor reactivity. Moreover, ECCE improved TIL expansion from non-melanoma cell suspensions similar to that seen with melanoma tumors. These data demonstrate that ECCE addition to TIL production will enable treatment of patients ineligible using current methods. PMID:21989413

  19. Augmented lymphocyte expansion from solid tumors with engineered cells for costimulatory enhancement.

    PubMed

    Friedman, Kevin M; Devillier, Laura E; Feldman, Steven A; Rosenberg, Steven A; Dudley, Mark E

    2011-01-01

    Treatment of patients with adoptive T-cell therapy requires expansion of unique tumor-infiltrating lymphocyte (TIL) cultures from single-cell suspensions processed from melanoma biopsies. Strategies which increase the expansion and reliability of TIL generation from tumor digests are necessary to improve access to TIL therapy. Previous studies have evaluated artificial antigen presenting cells for their antigen-specific and costimulatory properties. We investigated engineered cells for costimulatory enhancement (ECCE) consisting of K562 cells that express 4-1BBL in the absence of artificial antigen stimulation. ECCE accelerated TIL expansion and significantly improved TIL numbers (P=0.001) from single-cell melanoma suspensions. TIL generated with ECCE contain significantly more CD8CD62L and CD8CD27 T cells then comparable interleukin-2-expanded TIL and maintained antitumor reactivity. Moreover, ECCE improved TIL expansion from nonmelanoma-cell suspensions similar to that seen with melanoma tumors. These data demonstrate that the addition of ECCE to TIL production will enable the treatment of patients that are ineligible using current methods.

  20. Suitability of human mesenchymal stem cells for gene therapy depends on the expansion medium

    SciTech Connect

    Apel, Anja; Groth, Ariane; Schlesinger, Sabine; Bruns, Helge; Schemmer, Peter; Buechler, Markus W.; Herr, Ingrid

    2009-02-01

    Great hope is set in the use of mesenchymal stem cells for gene therapy and regenerative medicine. Since the frequency of this subpopulation of stem cells in bone marrow is low, mesenchymal stem cells are expanded ex vivo and manipulated prior to experimental or clinical use. Different methods for isolation and expansion are available, but the particular effect on the stem cell character is unclear. While the isolation of mesenchymal stem cells by density centrifugation followed by selection of the plastic adherent fraction is frequently used, the composition of expansion media differs. Thus, in the present study we cultured mesenchymal stem cells isolated from five healthy young volunteers in three widely used expansion media and performed a detailed analysis of the effect on morphology, proliferation, clonogenicity, passaging, differentiation and senescence. By this way we clearly show that the type of expansion medium used determines the stem cell character and time of senescence which is critical for future gene therapeutic and regenerative approaches using mesenchymal stem cells.

  1. The Arabidopsis ARGOS-LIKE gene regulates cell expansion during organ growth.

    PubMed

    Hu, Yuxin; Poh, Huay Mei; Chua, Nam-Hai

    2006-07-01

    Cell expansion, and its coordination with cell division, plays a critical role in the growth and development of plant organs. However, the genes controlling cell expansion during organogenesis are largely unknown. Here, we demonstrate that a novel Arabidopsis gene, ARGOS-LIKE (ARL), which has some sequence homology to the ARGOS gene, is involved in this process. Reduced expression or overexpression of ARL in Arabidopsis results in smaller or larger cotyledons and leaves as well as other lateral organs, respectively. Anatomical examination of cotyledons and leaves in ARL transgenic plants demonstrates that the alteration in size can be attributed to changes in cell size rather than cell number, indicating that ARL plays a role in cell expansion-dependent organ growth. ARL is upregulated by brassinosteroid (BR) and this induction is impaired in the BR-insensitive mutant bri1, but not in the BR-deficient mutant det2. Ectopic expression of ARL in bri1-119 partially restores cell growth in cotyledons and leaves. Our results suggest that ARL acts downstream of BRI1 and partially mediates BR-related cell expansion signals during organ growth.

  2. In vitro expansion of the mammary stem/progenitor cell population by xanthosinetreatment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Mammary stem cells are critical for growth and maintenance of the mammary gland and therefore of considerable interest for improving productivity and efficiency of dairy animals. Xanthosine (Xs) treatment has been demonstrated to promote expansion of putative mammary stem cells in vivo ...

  3. α-Tocopherol induces hematopoietic stem/progenitor cell expansion and ERK1/2-mediated differentiation.

    PubMed

    Nogueira-Pedro, Amanda; Barbosa, Christiano M V; Segreto, Helena Regina Comodo; Lungato, Lisandro; D'Almeida, Vania; Moraes, Andrea Aparecida F S; Miranda, Antonio; Paredes-Gamero, Edgar Julian; Ferreira, Alice Teixeira

    2011-12-01

    Tocopherols promote or inhibit growth in different cell types. In the hematopoietic system, the radioprotective property of tocopherols is thought to act through the expansion of primitive hematopoietic cells. However, the mechanisms activated by tocopherols and which HPs are affected remain poorly understood. To better address these questions, mice were treated with α-tocopherol, and its effects were investigated in the BM microenvironment. α-Tocopherol induced increased proliferation in HSC/HP cells, leading to BM hyperplasia. In addition, differentiation to the granulocytic/monocytic lineage was enhanced by α-tocopherol treatment. α-Tocopherol treatment resulted in decreased basal phosphorylation of ERK1/2, PKC, and STAT-5 in HSC/HP cells. In contrast, α-tocopherol enhanced ERK1/2 activation in response to IL-3 stimulation in HSC/HP cells without altering the expression of IL-3Rs. Moreover, α-tocopherol-induced differentiation and ERK1/2 activation were abolished in mice pretreated with a MEK inhibitor (PD98059); however, pretreatment with PD98059 did not reduce the α-tocopherol-mediated increase in HSC/HP cells but instead, further enhanced their proliferation. Therefore, α-tocopherol induces expansion of HSC/HP cells by a nonidentified intracellular pathway and granulocytic/monocytic differentiation through ERK1/2 activation.

  4. Production Process for Stem Cell Based Therapeutic Implants: Expansion of the Production Cell Line and Cultivation of Encapsulated Cells

    NASA Astrophysics Data System (ADS)

    Weber, C.; Pohl, S.; Poertner, R.; Pino-Grace, Pablo; Freimark, D.; Wallrapp, C.; Geigle, P.; Czermak, P.

    Cell based therapy promises the treatment of many diseases like diabetes mellitus, Parkinson disease or stroke. Microencapsulation of the cells protects them against host-vs-graft reactions and thus enables the usage of allogenic cell lines for the manufacturing of cell therapeutic implants. The production process of such implants consists mainly of the three steps expansion of the cells, encapsulation of the cells, and cultivation of the encapsulated cells in order to increase their vitality and thus quality. This chapter deals with the development of fixed-bed bioreactor-based cultivation procedures used in the first and third step of production. The bioreactor system for the expansion of the stem cell line (hMSC-TERT) is based on non-porous glass spheres, which support cell growth and harvesting with high yield and vitality. The cultivation process for the spherical cell based implants leads to an increase of vitality and additionally enables the application of a medium-based differentiation protocol.

  5. Multiple effects of IL-21 on human NK cells in ex vivo expansion.

    PubMed

    Li, Qi; Ye, Lin-Jie; Ren, Hai-Long; Huyan, Ting; Li, Jing; Shi, Jun-Ling; Huang, Qing-Sheng

    2015-07-01

    Natural killer (NK) cells (CD56(+)CD3(-)) are large, granular immunocytes that play a very pivotal role in the anti-inflammatory response and tumor surveillance. As an ideal cytotoxic lymphocyte (CTL), NK cells have attracted much attention in clinical trials. However, an insufficient number and their limited life span are bottlenecks that limit the application of NK cells in adoptive immunotherapy. Interleukins such as IL-2, IL-15 and IL-18 are recognized as factors that stimulate NK cells and have been used in NK cells ex vivo expansion. Similar to IL-2 and IL-15, IL-21 is a common γ-chain cytokine that is important in NK cell activation, maturation and proliferation. The present study aims to assess the effects of membrane-bound and soluble IL-21 on primary human NK cells during ex vivo expansion. IL-21 was found to have multiple effects on NK cells, increasing their cytotoxicity in a concentration-dependent manner by up-regulating IFN-γ and Granzyme-B expression. Nevertheless, at a high concentration (50 ng/mL), IL-21 curtailed the life span of NK cells by significantly inducing apoptosis. Moreover, when treated with IL-21, the number of NKT (CD56(+)CD3(+)) cells increased among peripheral blood mononuclear cells (PBMCs) during ex vivo expansion in a concentration-dependent manner. IL-21 also promoted expanded cells to enter into S phase of the cell cycle during the first to second weeks of culture. All these results suggest that IL-21 has multiple effects on NK cell development and functions. More attention should be given to the dosage and multiple effects of IL-21 when it was applied to NK cells in ex vivo expansion.

  6. Allogenic human serum, a clinical grade serum supplement for promoting human periodontal ligament stem cell expansion.

    PubMed

    Arpornmaeklong, Premjit; Sutthitrairong, Chotika; Jantaramanant, Piyathida; Pripatnanont, Prisana

    2016-12-13

    Exposing human periodontal ligament stem cells (hPDLSCs) to animal proteins during cell expansion would compromise quality and safety of the hPDLSCs for clinical applications. The current study aimed to evaluate the replacement of animal based serum by human serum for the expansion of hPDLSCs. Human PDLSCs were cultured in culture media supplemented with 4 types of serums, Group A: fetal bovine serum (FBS), Group B: allogeneic human male AB serum (HS) and Group C in-house autologous (Auto-HS) and Group D: in-house allogeneic human serums (Allo-HS). Exhibitions of mesenchymal stem cell (MSC) characteristics of hPDLSCs were examined. Then growth and osteogenic differentiation potential of hPDLSCs in FBS and HS at passages 5 and 15 were compared to investigate effects of serum supplements on growth and expansion stability of the expanded hPDLSCs. After that, growth and osteogenic differentiation of hPDLSCs in Auto- and Allo-HS were investigated. Flow cytometrical analyses, functional differentiations, cell growth kinetic, cytogenetic analysis, alkaline phosphatase (ALP) and calcium content assays and oil red O and von Kossa staining were performed. Results showed that at passage 5, HS promoted growth and osteogenic differentiation of hPDLSCs and extensive cell expansion, decreased growth and differentiation potential of the expanded hPDLSCs, particularly in HS. Growth and osteogenic differentiation of hPDLSCs in Auto-HS and Allo-HS were not different. In summary, allogeneic human serum could be a replacement to FBS for hPDLSC expansion. In vitro cell expansion of hPDLSCs should be minimal to ensure optimal cell quality. This article is protected by copyright. All rights reserved.

  7. Enrichment and Expansion with Nanoscale Artificial Antigen Presenting Cells for Adoptive Immunotherapy.

    PubMed

    Perica, Karlo; Bieler, Joan Glick; Schütz, Christian; Varela, Juan Carlos; Douglass, Jacqueline; Skora, Andrew; Chiu, Yen Ling; Oelke, Mathias; Kinzler, Kenneth; Zhou, Shibin; Vogelstein, Bert; Schneck, Jonathan P

    2015-07-28

    Adoptive immunotherapy (AIT) can mediate durable regression of cancer, but widespread adoption of AIT is limited by the cost and complexity of generating tumor-specific T cells. Here we develop an Enrichment + Expansion strategy using paramagnetic, nanoscale artificial antigen presenting cells (aAPC) to rapidly expand tumor-specific T cells from rare naïve precursors and predicted neo-epitope responses. Nano-aAPC are capable of enriching rare tumor-specific T cells in a magnetic column and subsequently activating them to induce proliferation. Enrichment + Expansion resulted in greater than 1000-fold expansion of both mouse and human tumor-specific T cells in 1 week, with nano-aAPC based enrichment conferring a proliferation advantage during both in vitro culture and after adoptive transfer in vivo. Robust T cell responses were seen not only for shared tumor antigens, but also for computationally predicted neo-epitopes. Streamlining the rapid generation of large numbers of tumor-specific T cells in a cost-effective fashion through Enrichment + Expansion can be a powerful tool for immunotherapy.

  8. Blockade of TNF-α signaling benefits cancer therapy by suppressing effector regulatory T cell expansion.

    PubMed

    Chang, Li-Yuan; Lin, Yung-Chang; Chiang, Jy-Ming; Mahalingam, Jayashri; Su, Shih-Huan; Huang, Ching-Tai; Chen, Wei-Ting; Huang, Chien-Hao; Jeng, Wen-Juei; Chen, Yi-Cheng; Lin, Shi-Ming; Sheen, I-Shyan; Lin, Chun-Yen

    2015-10-01

    Effector but not naive regulatory T cells (Treg cells) can accumulate in the peripheral blood as well as the tumor microenvironment, expand during tumor progression and be one of the main suppressors for antitumor immunity. However, the underlying mechanisms for effector Treg cell expansion in tumor are still unknown. We demonstrate that effector Treg cell-mediated suppression of antitumor CD8(+) T cells is tumor-nonspecific. Furthermore, TNFR2 expression is increased in these Treg cells by Affymetrix chip analysis which was confirmed by monoclonal antibody staining in both hepatocellular carcinoma (HCC) and colorectal cancer (CRC) patients and murine models. Correspondingly, increased levels of TNF-α in both tissue and serum were also demonstrated. Interestingly, TNF-α could not only expand effector Treg cells through TNFR2 signaling, but also enhanced their suppressive activity against antitumor immunity of CD8(+) T cells. Furthermore, targeting TNFR2 signaling with a TNF-α inhibitor could selectively reduce rapid resurgence of effector Treg cells after cyclophosphamide-induced lymphodepletion and markedly inhibit the growth of established tumors. Herein, we propose a novel mechanism in which TNF-α could promote tumor-associated effector Treg cell expansion and suggest a new cancer immunotherapy strategy using TNF-α inhibitors to reduce effector Treg cells expansion after cyclophosphamide-induced lymphodepletion.

  9. A microcarrier-based cultivation system for expansion of primary mesenchymal stem cells.

    PubMed

    Frauenschuh, Simone; Reichmann, Elisabeth; Ibold, Yvonne; Goetz, Peter M; Sittinger, Michael; Ringe, Jochen

    2007-01-01

    Microcarrier cultures have been shown to allow extensive cell expansion of tissue engineering relevant cells, such as chondrocytes, while maintaining their phenotype. Our aim was to investigate the in vitro three-dimensional expansion of porcine bone-marrow-derived primary mesenchymal stem cells (MSC) using commercially available Cytodex type 1, type 2, and type 3 microcarriers. In comparison, the Cytodex type 1 microcarriers showed the best results for adherence with over 80% adherent cells after 3 h of incubation, analyzed by the Poisson distribution. Different start cell densities ranging from 1 to 3 x 106 cells per 100 cm2 had only a minor influence on adhesion. The proliferation was examined on Cytodex type 1 microcarriers over a cultivation time of 28 days, which could reveal cell growth and proof of cells recolonizing freshly added microcarriers. Scanning electron microscopy displayed appropriate cell morphology and confirmed cell proliferation. After enzymatic harvest from microcarriers, the osteogenic and chondrogenic differentiation of these cells was induced and shown by relevant histochemistry, such as von Kossa and Alcian blue staining. Totaling the results, we have shown that the three-dimensional expansion of MSC on microcarriers represents a beneficial alternative to the conventional two-dimensional monolayer cultivation method.

  10. Affective agnosia: Expansion of the alexithymia construct and a new opportunity to integrate and extend Freud's legacy.

    PubMed

    Lane, Richard D; Weihs, Karen L; Herring, Anne; Hishaw, Alex; Smith, Ryan

    2015-08-01

    We describe a new type of agnosia consisting of an impairment in the ability to mentally represent or know what one is feeling. Freud the neurologist coined the term "agnosia" in 1891 before creating psychoanalysis in 1895 but the term has not been previously applied to the domain of affective processing. We propose that the concept of "affective agnosia" advances the theory, measurement and treatment of what is now called "alexithymia," meaning "lack of words for emotion." We trace the origin of the alexithymia construct and discuss the strengths and limitations of extant research. We review evidence that the ability to represent and put emotions into words is a developmental achievement that strongly influences one's ability to experience, recognize, understand and use one's own emotional responses. We describe the neural substrates of emotional awareness and affective agnosia and compare and contrast these with related conditions. We then describe how this expansion of the conceptualization and measurement of affective processing deficits has important implications for basic emotion research and clinical practice.

  11. An NAC transcription factor controls ethylene-regulated cell expansion in flower petals.

    PubMed

    Pei, Haixia; Ma, Nan; Tian, Ji; Luo, Jing; Chen, Jiwei; Li, Jing; Zheng, Yi; Chen, Xiang; Fei, Zhangjun; Gao, Junping

    2013-10-01

    Cell expansion is crucial for plant growth. It is well known that the phytohormone ethylene functions in plant development as a key modulator of cell expansion. However, the role of ethylene in the regulation of this process remains unclear. In this study, 2,189 ethylene-responsive transcripts were identified in rose (Rosa hybrida) petals using transcriptome sequencing and microarray analysis. Among these transcripts, an NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])-domain transcription factor gene, RhNAC100, was rapidly and dramatically induced by ethylene in the petals. Interestingly, accumulation of the RhNAC100 transcript was modulated by ethylene via microRNA164-dependent posttranscriptional regulation. Overexpression of RhNAC100 in Arabidopsis (Arabidopsis thaliana) substantially reduced the petal size by repressing petal cell expansion. By contrast, silencing of RhNAC100 in rose petals using virus-induced gene silencing significantly increased petal size and promoted cell expansion in the petal abaxial subepidermis (P < 0.05). Expression analysis showed that 22 out of the 29 cell expansion-related genes tested exhibited changes in expression in RhNAC100-silenced rose petals. Moreover, of those genes, one cellulose synthase and two aquaporin genes (Rosa hybrida Cellulose Synthase2 and R. hybrida Plasma Membrane Intrinsic Protein1;1/2;1) were identified as targets of RhNAC100. Our results suggest that ethylene regulates cell expansion by fine-tuning the microRNA164/RhNAC100 module and also provide new insights into the function of NAC transcription factors.

  12. Expansion induced microRNA changes in bone marrow mesenchymal stromal cells reveals interplay between immune regulation and cell cycle

    PubMed Central

    Kilpinen, Lotta; Parmar, Amarjit; Greco, Dario; Korhonen, Matti; Lehenkari, Petri; Saavalainen, Päivi; Laitinen, Saara

    2016-01-01

    Mesenchymal stromal cells (MSC) are currently used in many cell based therapies. Prior to use in therapy, extensive expansion is required. We used microarray profiling to investigate expansion induced miRNA and mRNA expression changes of bone marrow MSCs (BM-MSCs) derived from old and young donors. The expression levels of 36 miRNAs were altered in cells derived from the old and respectively 39 miRNAs were altered in cells derived from young donors. Of these, only 12 were differentially expressed in both young and old donor BM-MSCs, and their predicted target mRNAs, were mainly linked to cell proliferation and senescence. Further qPCR verification showed that the expression of miR-1915-3p, miR-1207, miR-3665, and miR-762 correlated with the expansion time at passage 8. Previously described BM-MSC-specific miRNA fingerprints were also detected but these remained unchanged during expansion. Interestingly, members of well-studied miR-17/92 cluster, involved in cell cycle regulation, aging and also development of immune system, were down-regulated specifically in cells from old donors. The role of this cluster in MSC functionality is worth future studies since it links expansion, aging and immune system together. PMID:27852979

  13. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer.

    PubMed

    Miller, Jeffrey S; Soignier, Yvette; Panoskaltsis-Mortari, Angela; McNearney, Sarah A; Yun, Gong H; Fautsch, Susan K; McKenna, David; Le, Chap; Defor, Todd E; Burns, Linda J; Orchard, Paul J; Blazar, Bruce R; Wagner, John E; Slungaard, Arne; Weisdorf, Daniel J; Okazaki, Ian J; McGlave, Philip B

    2005-04-15

    We previously demonstrated that autologous natural killer (NK)-cell therapy after hematopoietic cell transplantation (HCT) is safe but does not provide an antitumor effect. We hypothesize that this is due to a lack of NK-cell inhibitory receptor mismatching with autologous tumor cells, which may be overcome by allogeneic NK-cell infusions. Here, we test haploidentical, related-donor NK-cell infusions in a nontransplantation setting to determine safety and in vivo NK-cell expansion. Two lower intensity outpatient immune suppressive regimens were tested: (1) low-dose cyclophosphamide and methylprednisolone and (2) fludarabine. A higher intensity inpatient regimen of high-dose cyclophosphamide and fludarabine (Hi-Cy/Flu) was tested in patients with poor-prognosis acute myeloid leukemia (AML). All patients received subcutaneous interleukin 2 (IL-2) after infusions. Patients who received lower intensity regimens showed transient persistence but no in vivo expansion of donor cells. In contrast, infusions after the more intense Hi-Cy/Flu resulted in a marked rise in endogenous IL-15, expansion of donor NK cells, and induction of complete hematologic remission in 5 of 19 poor-prognosis patients with AML. These findings suggest that haploidentical NK cells can persist and expand in vivo and may have a role in the treatment of selected malignancies used alone or as an adjunct to HCT.

  14. Safety and efficient ex vivo expansion of stem cells using platelet-rich plasma technology.

    PubMed

    Anitua, Eduardo; Prado, Roberto; Orive, Gorka

    2013-09-01

    The goal of this Review is to provide an overview of the cell culture media supplements used in the ex vivo expansion of stem cells intended for cell therapy. Currently, the gold standard is the culture supplemented with fetal bovine serum, however, their use in cell therapy raises many concerns. The alternatives to its use are presented, ranging from the use of human serum to platelet-rich plasma (PRP), to serum-free media or extracellular matrix components. Finally, various growth factors present in PRP are described, which make it a safe and effective stem cell expansion supplement. These growth factors could be responsible for their efficiency, as they increase both stem cell proliferation and survival. The different PRP formulations are also discussed, as well as the need for protocol standardization.

  15. Ex Vivo Expansion of Human Mobilized Peripheral Blood Stem Cells Using Epigenetic Modifiers

    PubMed Central

    Saraf, Santosh; Araki, Hiroto; Petro, Benjamin; Park, Youngmin; Taioli, Simona; Yoshinaga, Kazumi G; Koca, Emre; Rondelli, Damiano; Mahmud, Nadim

    2014-01-01

    Background Epigenetic modifications likely control fate of hematopoietic stem cells (HSC). The chromatin modifying agents (CMA), 5-aza-2’-deoxyctidine (5azaD) and trichostatin A (TSA) have previously been shown to expand HSC from cord blood and bone marrow. Here we assessed whether CMA can also expand HSCs present in growth factor mobilized human peripheral blood (MPB). Study Design & Methods 5azaD and TSA were sequentially added to CD34+ MPB cells in the presence of cytokines and the cells were cultured for nine days. Results Following culture, a 3.6 ± 0.5 fold expansion of CD34+CD90+ cells, a 10.1 ± 0.5 fold expansion of primitive colony forming unit (CFU)-mix, and a 2.2 ± 0.5 fold expansion of long-term cobble stone-area forming cells (CAFC) was observed in 5azaD/TSA expanded cells. By contrast, cells cultured in cytokines without 5azaD/TSA displayed no expansion; rather a reduction in CD34+CD90+ cells (0.7 ± 0.1 fold) and CAFCs (0.3 ± 0.1) from their initial numbers was observed. Global hypomethylation corresponding with increased transcript levels of several genes implicated in HSC self-renewal, including HOXB4, GATA2, and EZH2, was observed in 5azaD/TSA expanded MPB cells in contrast to controls. 5azaD/TSA expanded MPB cells retained in vivo hematopoietic engraftment capacity. Conclusion MPB CD34+ cells from donors can be expanded using 5azaD/TSA and these expanded cells retain in vivo hematopoietic reconstitution capacity. This strategy may prove to be potentially useful to augment HSCs numbers for patients who fail to mobilize. PMID:25363624

  16. A TNFR2-Agonist Facilitates High Purity Expansion of Human Low Purity Treg Cells.

    PubMed

    He, Xuehui; Landman, Sija; Bauland, Stijn C G; van den Dolder, Juliette; Koenen, Hans J P M; Joosten, Irma

    2016-01-01

    Regulatory T cells (Treg) are important for immune homeostasis and are considered of great interest for immunotherapy. The paucity of Treg numbers requires the need for ex vivo expansion. Although therapeutic Treg flow-sorting is feasible, most centers aiming at Treg-based therapy focus on magnetic bead isolation of CD4+CD25+ Treg using a good manufacturing practice compliant closed system that achieves lower levels of cell purity. Polyclonal Treg expansion protocols commonly use anti-CD3 plus anti-CD28 monoclonal antibody (mAb) stimulation in the presence of rhIL-2, with or without rapamycin. However, the resultant Treg population is often heterogeneous and pro-inflammatory cytokines like IFNγ and IL-17A can be produced. Hence, it is crucial to search for expansion protocols that not only maximize ex vivo Treg proliferative rates, but also maintain Treg stability and preserve their suppressive function. Here, we show that ex vivo expansion of low purity magnetic bead isolated Treg in the presence of a TNFR2 agonist mAb (TNFR2-agonist) together with rapamycin, results in a homogenous stable suppressive Treg population that expresses FOXP3 and Helios, shows low expression of CD127 and hypo-methylation of the FOXP3 gene. These cells reveal a low IL-17A and IFNγ producing potential and hardly express the chemokine receptors CCR6, CCR7 and CXCR3. Restimulation of cells in a pro-inflammatory environment did not break the stability of this Treg population. In a preclinical humanized mouse model, the TNFR2-agonist plus rapamycin expanded Treg suppressed inflammation in vivo. Importantly, this Treg expansion protocol enables the use of less pure, but more easily obtainable cell fractions, as similar outcomes were observed using either FACS-sorted or MACS-isolated Treg. Therefore, this protocol is of great interest for the ex vivo expansion of Treg for clinical immunotherapy.

  17. Targeted erythropoietin selectively stimulates red blood cell expansion in vivo

    PubMed Central

    Burrill, Devin R.; Vernet, Andyna; Collins, James J.; Silver, Pamela A.; Way, Jeffrey C.

    2016-01-01

    The design of cell-targeted protein therapeutics can be informed by natural protein–protein interactions that use cooperative physical contacts to achieve cell type specificity. Here we applied this approach in vivo to the anemia drug erythropoietin (EPO), to direct its activity to EPO receptors (EPO-Rs) on red blood cell (RBC) precursors and prevent interaction with EPO-Rs on nonerythroid cells, such as platelets. Our engineered EPO molecule was mutated to weaken its affinity for EPO-R, but its avidity for RBC precursors was rescued via tethering to an antibody fragment that specifically binds the human RBC marker glycophorin A (huGYPA). We systematically tested the impact of these engineering steps on in vivo markers of efficacy, side effects, and pharmacokinetics. huGYPA transgenic mice dosed with targeted EPO exhibited elevated RBC levels, with only minimal platelet effects. This in vivo selectivity depended on the weakening EPO mutation, fusion to the RBC-specific antibody, and expression of huGYPA. The terminal plasma half-life of targeted EPO was ∼28.3 h in transgenic mice vs. ∼15.5 h in nontransgenic mice, indicating that huGYPA on mature RBCs acted as a significant drug sink but did not inhibit efficacy. In a therapeutic context, our targeting approach may allow higher restorative doses of EPO without platelet-mediated side effects, and also may improve drug pharmacokinetics. These results demonstrate how rational drug design can improve in vivo specificity, with potential application to diverse protein therapeutics. PMID:27114509

  18. B cell repertoire expansion occurs in meningeal ectopic lymphoid tissue

    PubMed Central

    Lehmann-Horn, Klaus; Wang, Sheng-zhi; Sagan, Sharon A.; Zamvil, Scott S.

    2016-01-01

    Ectopic lymphoid tissues (ELT) can be found in multiple sclerosis (MS) and other organ-specific inflammatory conditions. Whether ELT in the meninges of central nervous system (CNS) autoimmune disease exhibit local germinal center (GC) activity remains unknown. In an experimental autoimmune encephalomyelitis model of CNS autoimmunity, we found activation-induced cytidine deaminase, a GC-defining enzyme, in meningeal ELT (mELT) densely populated by B and T cells. To determine GC activity in mELT, we excised meningeal lymphoid aggregates using laser capture microscopy and evaluated B cell repertoires in mELT and secondary lymphoid organs by next-generation immune repertoire sequencing. We found immunoglobulin heavy chain variable region sequences that were unique to mELT and had accumulated functionally relevant somatic mutations, together indicating localized antigen-driven affinity maturation. Our results suggest that B cells in mELT actively participate in CNS autoimmunity, which may be relevant to mELT in MS and ELT in other chronic inflammatory conditions. PMID:27942581

  19. Further clonal expansion of T cells upon rechallenge of superantigen staphylococcal enterotoxin A.

    PubMed

    Aoki, Y; Yoshikai, Y

    1997-01-01

    Superantigens are known to induce clonal anergy and/or deletion in reactive T cells peripherally. This study was undertaken to investigate the T-cell status early after exposure to staphylococcal enterotoxin A (SEA) in vivo and in vitro. At the peak of clonal expansion following the administration of 5 microg SEA (i.e., 2 days after the injection), C57BL/6 mice were rechallenged with the same dose of SEA in vivo. The secondary stimulation augmented clonal expansion of the T cells bearing Vbeta3 and Vbeta11 in both CD4+ and CD8+ populations. In vitro restimulation of the spleen cells taken from the SEA-primed mice also induced further expansion of the Vbeta3+ T cells during 2 days of culturing, whereas without restimulation, a marked reduction of Vbeta3+ T cells occurred. The spleen cells from the SEA-primed mice were hyper-reactive to in vitro restimulation with SEA as measured by 3H-TdR uptake on day 1 of culturing, but augmented proliferation leveled off thereafter. By day 3, the values of 3H-TdR uptake were less than 20% of those of the controls in which spleen cells from native mice were stimulated with SEA in vitro. These results suggest that T cells exposed to SEA in vivo are still capable of proliferating upon SEA rechallenge, but subsequently, the proliferation starts to wane.

  20. Human platelet lysate gel provides a novel three dimensional-matrix for enhanced culture expansion of mesenchymal stromal cells.

    PubMed

    Walenda, Gudrun; Hemeda, Hatim; Schneider, Rebekka K; Merkel, Rudolf; Hoffmann, Bernd; Wagner, Wolfgang

    2012-12-01

    Cell culture in regenerative medicine needs to facilitate efficient expansion according to good manufacturing practice requirements. Human platelet lysate (HPL) can be used as a substitute for fetal calf serum without the risk of xenogeneic immune reactions or transmission of bovine pathogens. Heparin needs to be added as anticoagulant before addition of HPL to culture medium; otherwise, HPL-medium forms a gel within 1 h. Here, we demonstrated that such HPL-gels provide a suitable 3D-matrix for cell culture that-apart from heparin-consists of the same components as the over-layered culture medium. Mesenchymal stromal cells (MSCs) grew in several layers at the interface between HPL-gel and HPL-medium without contact with any artificial biomaterials. Notably, proliferation of MSCs was much higher on HPL-gel compared with tissue culture plastic. Further, the frequency of initial fibroblastoid colony forming units (CFU-f) increased on HPL-gel. The viscous consistency of HPL-gel enabled passaging with a convenient harvesting and reseeding procedure by pipetting cells together with their HPL-matrix-this method does not require washing steps and can easily be automated. The immunophenotype and in vitro differentiation potential toward adipogenic, osteogenic, and chondrogenic lineage were not affected by culture-isolation on HPL-gel. Taken together, HPL-gel has many advantages over conventional plastic surfaces: it facilitates enhanced CFU-f outgrowth, increased proliferation rates, higher cell densities, and nonenzymatic passaging procedures for culture expansion of MSCs.

  1. Cyclin D3 is selectively required for proliferative expansion of germinal center B cells.

    PubMed

    Cato, Matthew H; Chintalapati, Suresh K; Yau, Irene W; Omori, Sidne A; Rickert, Robert C

    2011-01-01

    The generation of robust T-cell-dependent humoral immune responses requires the formation and expansion of germinal center structures within the follicular regions of the secondary lymphoid tissues. B-cell proliferation in the germinal center drives ongoing antigen-dependent selection and the generation of high-affinity class-switched plasma and memory B cells. However, the mechanisms regulating B-cell proliferation within this microenvironment are largely unknown. Here, we report that cyclin D3 is uniquely required for germinal center progression. Ccnd3(-/-) mice exhibit a B-cell-intrinsic defect in germinal center maturation and fail to generate an affinity-matured IgG response. We determined that the defect resulted from failed proliferative expansion of GL7(+) IgD(-) PNA(+) B cells. Mechanistically, sustained expression of cyclin D3 was found to be regulated at the level of protein stability and controlled by glycogen synthase kinase 3 in a cyclic AMP-protein kinase A-dependent manner. The specific defect in proliferative expansion of GL7(+) IgD(-) PNA(+) B cells in Ccnd3(-/-) mice defines an underappreciated step in germinal center progression and solidifies a role for cyclin D3 in the immune response, and as a potential therapeutic target for germinal center-derived B-cell malignancies.

  2. Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein.

    PubMed

    Amsellem, Sophie; Pflumio, Françoise; Bardinet, Dominique; Izac, Brigitte; Charneau, Pierre; Romeo, Paul-Henri; Dubart-Kupperschmitt, Anne; Fichelson, Serge

    2003-11-01

    Expansion of human hematopoietic stem cells (HSCs) is a major challenge in cellular therapy, and currently relies on the use of recombinant cytokines or on gene transfer of transcription factors. Of these, the HOXB4 homeoprotein protein is of particular interests as it promotes the expansion of mouse HSCs without inducing the development of leukemia. To eliminate any deleterious effects that might be associated with stable HOXB4 gene transfer into human cells, we took advantage of the ability of HOX proteins to passively translocate through cell membranes. Here we show that when cultured on stromal cells genetically engineered to secrete HOXB4, human long-term culture-initiating cells (LTC-ICs) and nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mouse repopulating cells (SRCs) were expanded by more than 20- and 2.5-fold, respectively, over their input numbers. This expansion was associated with enhanced stem cell repopulating capacity in vivo and maintenance of pluripotentiality. This method provides a basis for developing cell therapy strategies using expanded HSCs that are not genetically modified.

  3. A thermoresponsive and magnetic colloid for 3D cell expansion and reconfiguration.

    PubMed

    Saeed, Aram; Francini, Nora; White, Lisa; Dixon, James; Gould, Toby; Rashidi, Hassan; Al Ghanami, Racha Cheikh; Hruschka, Veronika; Redl, Heinz; Saunders, Brian R; Alexander, Cameron; Shakesheff, Kevin M

    2015-01-27

    A dual thermoresponsive and magnetic colloidal gel matrix is described for enhanced stem-cell culture. The combined properties of the material allow enzyme-free passaging and expansion of mesenchymal stem cells, as well as isolation of cells postculture by the simple process of lowering the temperature and applying an external magnetic field. The colloidal gel can be reconfigured with thermal and magnetic stimuli to allow patterning of cells in discrete zones and to control movement of cells within the porous matrix during culture.

  4. Contribution of endothelial cells to human bone-derived cells expansion in coculture.

    PubMed

    Leszczynska, Joanna; Zyzynska-Granica, Barbara; Koziak, Katarzyna; Ruminski, Slawomir; Lewandowska-Szumiel, Malgorzata

    2013-02-01

    Creating a functional vascularized bone tissue remains one of the main goals of bone tissue engineering. Recently, a growing interest in the crosstalk between endothelial cells (EC) and osteoblasts (OB), the two main players in a new bone formation, has been observed. However, only a few reports have addressed a mutual influence of OB and EC on cell proliferation. Our study focuses on this issue by investigating cocultures of human bone-derived cells (HBDC) and human umbilical vein endothelial cells (HUVEC). Three various proportions of cells have been used that is, HBDC:HUVEC 1:1, 1:4, and 4:1 and the cocultures were investigated on day 1, 4, and 7, while HUVEC and HBDC monocultures served as reference. We have detected enhanced alkaline phosphatase (ALP) activity in a direct HBDC-HUVEC coculture. This effect was not observed when cells were separated by an insert, which is consistent with other reports on various OB-EC lineages. The appearance of gap-junctions in coculture was confirmed by a positive staining for connexin 43. The number of cells of both phenotypes has been determined by flow cytometry: CD-31-positive cells have been considered EC, while CD-31-negative have been counted as OB. We have observed an over 14-fold increase in OB number after a week in the 1:4 HBDC:HUVEC coculture as compared with less than fourfold in monoculture. The increase in HBDC number in 1:1 coculture has been less pronounced and has reached the value of about sevenfold. These results correspond well with the cell proliferation rate, which has been measured by 5-bromo-2'-deoxyuridine incorporation. Moreover, at day 7 EC have been still present in the coculture, which is inconsistent with some other reports. Real-time polymerase chain reaction analysis has revealed the upregulation of ALP and collagen type I genes, but not osteocalcin gene, in all the cocultures grown without pro-osteogenic additives. Our study indicates that HUVEC significantly promote HBDC expansion and

  5. Satellite cell activity, without expansion, after nonhypertrophic stimuli

    PubMed Central

    Joanisse, Sophie; McKay, Bryon R.; Nederveen, Joshua P.; Scribbans, Trisha D.; Gurd, Brendon J.; Gillen, Jenna B.; Gibala, Martin J.; Tarnopolsky, Mark

    2015-01-01

    The purpose of the present studies was to determine the effect of various nonhypertrophic exercise stimuli on satellite cell (SC) pool activity in human skeletal muscle. Previously untrained men and women (men: 29 ± 9 yr and women: 29 ± 2 yr, n = 7 each) completed 6 wk of very low-volume high-intensity sprint interval training. In a separate study, recreationally active men (n = 16) and women (n = 3) completed 6 wk of either traditional moderate-intensity continuous exercise (n = 9, 21 ± 4 yr) or low-volume sprint interval training (n = 10, 21 ± 2 yr). Muscle biopsies were obtained from the vastus lateralis before and after training. The fiber type-specific SC response to training was determined, as was the activity of the SC pool using immunofluorescent microscopy of muscle cross sections. Training did not induce hypertrophy, as assessed by muscle cross-sectional area, nor did the SC pool expand in any group. However, there was an increase in the number of active SCs after each intervention. Specifically, the number of activated (Pax7+/MyoD+, P ≤ 0.05) and differentiating (Pax7−/MyoD+, P ≤ 0.05) SCs increased after each training intervention. Here, we report evidence of activated and cycling SCs that may or may not contribute to exercise-induced adaptations while the SC pool remains constant after three nonhypertrophic exercise training protocols. PMID:26333785

  6. Tc1 clonal T cell expansion during chronic graft-versus-host disease-associated hypereosinophilia.

    PubMed

    Clave, Emmanuel; Xhaard, Aliénor; Douay, Corrine; Adès, Lionel; Cayuela, Jean Michel; Peffault de Latour, Régis; Robin, Marie; Toubert, Antoine; Socié, Gérard

    2014-05-01

    Although hypereosinophilia (HE) associated with chronic graft-versus-host disease (GVHD) has long been recognized, biological data on this phenomenon are scarce. Here we compare patients with chronic GVHD with HE together with a clonal T cell expansion and control patients with acute or chronic GVHD but without HE. These clonal expansions share a CD8(+) TC1 phenotype rather than a CD4(+) Th2 profile. In contrast to the drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome, these allogeneic CD8(+) clones do not recognize the epitopes of herpesviruses. Furthermore, these TC1 clones do not produce IL-17 as described in the DRESS syndrome.

  7. Exogenous TNFR2 activation protects from acute GvHD via host T reg cell expansion

    PubMed Central

    Chopra, Martin; Brandl, Andreas; Amich, Jorge; Mottok, Anja; Jordán-Garrote, Ana-Laura; Bäuerlein, Carina A.; Brede, Christian; Ribechini, Eliana; Fick, Andrea; Polz, Johannes; Nishikii, Hidekazu; Mattenheimer, Katharina; Schwinn, Stefanie; Winter, Thorsten; Krappmann, Sven; Einsele, Hermann; Reddehase, Matthias J.; Lutz, Manfred B.

    2016-01-01

    Donor CD4+Foxp3+ regulatory T cells (T reg cells) suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HCT [allo-HCT]). Current clinical study protocols rely on the ex vivo expansion of donor T reg cells and their infusion in high numbers. In this study, we present a novel strategy for inhibiting GvHD that is based on the in vivo expansion of recipient T reg cells before allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in T reg cell biology. Expanding radiation-resistant host T reg cells in recipient mice using a mouse TNFR2-selective agonist before allo-HCT significantly prolonged survival and reduced GvHD severity in a TNFR2- and T reg cell–dependent manner. The beneficial effects of transplanted T cells against leukemia cells and infectious pathogens remained unaffected. A corresponding human TNFR2-specific agonist expanded human T reg cells in vitro. These observations indicate the potential of our strategy to protect allo-HCT patients from acute GvHD by expanding T reg cells via selective TNFR2 activation in vivo. PMID:27526711

  8. Expansion of activated lymphocytes obtained from renal cell carcinoma in an automated hollow fiber bioreactor.

    PubMed

    Hillman, G G; Wolf, M L; Montecillo, E; Younes, E; Ali, E; Pontes, J E; Haas, G P

    1994-01-01

    Immunotherapy using IL-2 alone or combined with activated lymphocytes has been promising for metastatic renal cell carcinoma. Cytotoxic lymphocytes can be isolated from tumors, expanded in vitro with IL-2, and adoptively transferred back into the tumor-bearing host. These cells can also be transduced with the genes coding for cytokines for local delivery to tumor sites. A major drawback in adoptive immunotherapy is the cumbersome and expensive culture technology associated with the growth of large numbers of cells required for their therapeutic effect. To reduce the cost, resources, and manpower, we have developed the methodology for lymphocyte activation and expansion in the automated hollow fiber bioreactor IMMUNO*STAR Cell Expander (ACT BIOMEDICAL, INC). Tumor Infiltrating Lymphocytes (TIL) isolated from human renal cell carcinoma tumor specimens were inoculated at a number of 10(8) cells in a small bioreactor of 30 ml extracapillary space volume. We have determined the medium flow rates and culture conditions to obtain a significant and repeated expansion of TIL at weekly intervals. The lymphocytes cultured in the bioreactor demonstrated the same phenotype and cytotoxic activity as those expanded in parallel in tissue culture plates. Lymphocyte expansion in the hollow fiber bioreactor required lower volumes of medium, human serum, IL-2 and minimal labor. This technology may facilitate the use of adoptive immunotherapy for the treatment of refractory malignancies.

  9. Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system.

    PubMed

    Mizukami, Amanda; Orellana, Maristela D; Caruso, Sâmia R; de Lima Prata, Karen; Covas, Dimas T; Swiech, Kamilla

    2013-01-01

    The need for efficient and reliable technologies for clinical-scale expansion of mesenchymal stromal cells (MSC) has led to the use of disposable bioreactors and culture systems. Here, we evaluate the expansion of cord blood-derived MSC in a disposable fixed bed culture system. Starting from an initial cell density of 6.0 × 10(7) cells, after 7 days of culture, it was possible to produce of 4.2(±0.8) × 10(8) cells, which represents a fold increase of 7.0 (±1.4). After enzymatic retrieval from Fibra-Cell disks, the cells were able to maintain their potential for differentiation into adipocytes and osteocytes and were positive for many markers common to MSC (CD73, CD90, and CD105). The results obtained in this study demonstrate that MSC can be efficiently expanded in the culture system. This novel approach presents several advantages over the current expansion systems, based on culture flasks or microcarrier-based spinner flasks and represents a key element for MSC cellular therapy according to GMP compliant clinical-scale production system.

  10. The grape berry-specific basic helix–loop–helix transcription factor VvCEB1 affects cell size

    PubMed Central

    Lecourieux, Fatma

    2013-01-01

    The development of fleshy fruits involves complex physiological and biochemical changes. After fertilization, fruit growth usually begins with cell division, continues with both cell division and expansion, allowing fruit set to occur, and ends with cell expansion only. In spite of the economical importance of grapevine, the molecular mechanisms controlling berry growth are not fully understood. The present work identified and characterized Vitis vinifera cell elongation bHLH protein (VvCEB1), a basic helix–loop–helix (bHLH) transcription factor controlling cell expansion in grape. VvCEB1 was expressed specifically in berry-expanding tissues with a maximum around veraison. The study of VvCEB1 promoter activity in tomato confirmed its specific fruit expression during the expansion phase. Overexpression of VvCEB1 in grape embryos showed that this protein stimulates cell expansion and affects the expression of genes involved in cell expansion, including genes of auxin metabolism and signalling. Taken together, these data show that VvCEB1 is a fruit-specific bHLH transcription factor involved in grape berry development. PMID:23314819

  11. The grape berry-specific basic helix-loop-helix transcription factor VvCEB1 affects cell size.

    PubMed

    Nicolas, Philippe; Lecourieux, David; Gomès, Eric; Delrot, Serge; Lecourieux, Fatma

    2013-02-01

    The development of fleshy fruits involves complex physiological and biochemical changes. After fertilization, fruit growth usually begins with cell division, continues with both cell division and expansion, allowing fruit set to occur, and ends with cell expansion only. In spite of the economical importance of grapevine, the molecular mechanisms controlling berry growth are not fully understood. The present work identified and characterized Vitis vinifera cell elongation bHLH protein (VvCEB1), a basic helix-loop-helix (bHLH) transcription factor controlling cell expansion in grape. VvCEB1 was expressed specifically in berry-expanding tissues with a maximum around veraison. The study of VvCEB1 promoter activity in tomato confirmed its specific fruit expression during the expansion phase. Overexpression of VvCEB1 in grape embryos showed that this protein stimulates cell expansion and affects the expression of genes involved in cell expansion, including genes of auxin metabolism and signalling. Taken together, these data show that VvCEB1 is a fruit-specific bHLH transcription factor involved in grape berry development.

  12. Expansion of CD8+CD57+ T Cells in an Immunocompetent Patient with Acute Toxoplasmosis

    PubMed Central

    García-Muñoz, R.; Rodríguez-Otero, P.; Galar, A.; Merino, J.; Beunza, J. J.; Páramo, J. A.; Lecumberri, R.

    2009-01-01

    CD57+ T cells increase in several viral infections like cytomegalovirus, herpesvirus, parvovirus, HIV and hepatitis C virus and are associated with several clinical conditions related to immune dysfunction and ageing. We report for the first time an expansion of CD8+ CD57+ T cells in a young patient with an acute infection with Toxoplasma gondii. Our report supports the concept that CD8+ CD57+ T cells could be important in the control of chronic phase of intracellular microorganisms and that the high numbers of these cells may reflect the continuing survey of the immune system, searching for parasite proliferation in the tissues. PMID:19946421

  13. Expansion of CD8+CD57+ T Cells in an Immunocompetent Patient with Acute Toxoplasmosis.

    PubMed

    García-Muñoz, R; Rodríguez-Otero, P; Galar, A; Merino, J; Beunza, J J; Páramo, J A; Lecumberri, R

    2009-01-01

    CD57+ T cells increase in several viral infections like cytomegalovirus, herpesvirus, parvovirus, HIV and hepatitis C virus and are associated with several clinical conditions related to immune dysfunction and ageing. We report for the first time an expansion of CD8+ CD57+ T cells in a young patient with an acute infection with Toxoplasma gondii. Our report supports the concept that CD8+ CD57+ T cells could be important in the control of chronic phase of intracellular microorganisms and that the high numbers of these cells may reflect the continuing survey of the immune system, searching for parasite proliferation in the tissues.

  14. Cytokine combinations on the potential for ex vivo expansion of murine hematopoietic stem cells.

    PubMed

    Lui, Wing Chi; Chan, Yuen Fan; Chan, Li Chong; Ng, Ray Kit

    2014-08-01

    Hematopoietic stem cell (HSC) is a rare cell population, which is capable of self-renewal and differentiation to all blood lineages. The clinical potential of HSCs for treating hematological disorders has led to the use of cytokine stimulation for ex vivo expansion. However, little is known about the molecular features of the HSC populations expanded under different cytokine combinations. We studied the expansion of murine HSCs cultured with six different cytokine combinations under serum-containing or serum-free conditions for 14days. We found that all the cytokine combinations promoted expansion of murine HSCs. Although SCF/IL-3/IL-6 induced the highest expansion of the immunophenotypic Lineage(-)Sca-1(+)c-Kit(+) (LSK) cells at day 14, over 90% of them were FcεRIα(+) mast cells. In contrast, the serum-free medium with SCF/Flt3-L/IL-11 effectively promoted the expansion of LSK/FcεRIα(-) HSCs by over 50-fold. HSCs expanded by SCF/Flt3-L/IL-11 combination formed compact hematopoietic colonies and demonstrated a higher degree of multipotency compared to the HSCs cultured with other cytokine combinations. Surprisingly, despite the same LSK/FcεRIα(-) immunophenotype, HSCs cultured with different cytokine combinations demonstrated differential patterns of hematopoietic gene expression. HSCs cultured with SCF/Flt3-L/IL-11 maintained a transcription profile resembling that of freshly isolated HSCs. We propose that serum-free medium supplemented with SCF/Flt3-L/IL-11 is the optimal culture condition to maintain the stemness of ex vivo expanded HSCs. This study used molecular characterization of cytokine-expanded murine HSCs to facilitate the selection of cytokine combinations that could induce fully competent HSC for clinical applications.

  15. IL-15 promotes activation and expansion of CD8+ T cells in HIV-1 infection

    PubMed Central

    Younes, Souheil-Antoine; Freeman, Michael L.; Mudd, Joseph C.; Shive, Carey L.; Reynaldi, Arnold; Estes, Jacob D.; Deleage, Claire; Lucero, Carissa; Anderson, Jodi; Schacker, Timothy W.; Davenport, Miles P.; McCune, Joseph M.; Hunt, Peter W.; Lee, Sulggi A.; Debernardo, Robert L.; Jacobson, Jeffrey M.; Canaday, David H.; Sekaly, Rafick-Pierre; Sieg, Scott F.; Lederman, Michael M.

    2016-01-01

    In HIV-1–infected patients, increased numbers of circulating CD8+ T cells are linked to increased risk of morbidity and mortality. Here, we identified a bystander mechanism that promotes CD8 T cell activation and expansion in untreated HIV-1–infected patients. Compared with healthy controls, untreated HIV-1–infected patients have an increased population of proliferating, granzyme B+, CD8+ T cells in circulation. Vβ expression and deep sequencing of CDR3 revealed that in untreated HIV-1 infection, cycling memory CD8 T cells possess a broad T cell repertoire that reflects the repertoire of the resting population. This suggests that cycling is driven by bystander activation, rather than specific antigen exposure. Treatment of peripheral blood mononuclear cells with IL-15 induced a cycling, granzyme B+ phenotype in CD8+ T cells. Moreover, elevated IL-15 expression in the lymph nodes of untreated HIV-1–infected patients correlated with circulating CD8+ T cell counts and was normalized in these patients following antiretroviral therapy. Together, these results suggest that IL-15 drives bystander activation of CD8+ T cells, which predicts disease progression in untreated HIV-1–infected patients and suggests that elevated IL-15 may also drive CD8+ T cell expansion that is linked to increased morbidity and mortality in treated patients. PMID:27322062

  16. Ex vivo expansion of circulating CD34+ cells enhances the regenerative effect on rat liver cirrhosis

    PubMed Central

    Nakamura, Toru; Koga, Hironori; Iwamoto, Hideki; Tsutsumi, Victor; Imamura, Yasuko; Naitou, Masako; Masuda, Atsutaka; Ikezono, Yu; Abe, Mitsuhiko; Wada, Fumitaka; Sakaue, Takahiko; Ueno, Takato; Ii, Masaaki; Alev, Cantas; Kawamoto, Atsuhiko; Asahara, Takayuki; Torimura, Takuji

    2016-01-01

    Ex vivo expansion of autologous cells is indispensable for cell transplantation therapy of patients with liver cirrhosis. The aim of this study was to investigate the efficacy of human ex vivo-expanded CD34+ cells for treatment of cirrhotic rat liver. Recipient rats were intraperitoneally injected with CCl4 twice weekly for 3 weeks before administration of CD34+ cells. CCl4 was then re-administered twice weekly for 3 more weeks, and the rats were sacrificed. Saline, nonexpanded or expanded CD34+ cells were injected via the spleen. After 7 days, CD34+ cells were effectively expanded in a serum-free culture medium. Expanded CD34+ cells were also increasingly positive for cell surface markers of VE-cadherin, VEGF receptor-2, and Tie-2. The expression of proangiogenic growth factors and adhesion molecules in expanded CD34+ cells increased compared with nonexpanded CD34+ cells. Expanded CD34+ cell transplantation reduced liver fibrosis, with a decrease of αSMA+ cells. Assessments of hepatocyte and sinusoidal endothelial cell proliferative activity indicated the superior potency of expanded CD34+ cells over non-expanded CD34+ cells. The inhibition of integrin αvβ3 and αvβ5 disturbed the engraftment of transplanted CD34+ cells and aggravated liver fibrosis. These findings suggest that expanded CD34+ cells enhanced the preventive efficacy of cell transplantation in a cirrhotic model. PMID:27162932

  17. Expansion and homing of umbilical cord blood hematopoietic stem and progenitor cells for clinical transplantation.

    PubMed

    Bari, Sudipto; Seah, Kevin Kwee Hong; Poon, Zhiyong; Cheung, Alice Man Sze; Fan, Xiubo; Ong, Shin-Yeu; Li, Shang; Koh, Liang Piu; Hwang, William Ying Khee

    2015-06-01

    The successful expansion of hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood (UCB) for transplantation could revolutionize clinical practice by improving transplantation-related outcomes and making available UCB units that have suboptimal cell doses for transplantation. New cytokine combinations appear able to promote HSPC growth with minimal differentiation into mature precursors and new agents, such as insulin-like growth factor-binding protein 2, are being used in clinical trials. Molecules that simulate the HSPC niche, such as Notch ligand, have also shown promise. Further improvements have been made with the use of mesenchymal stromal cells, which have made possible UCB expansion without a potentially deleterious prior CD34/CD133 cell selection step. Chemical molecules, such as copper chelators, nicotinamide, and aryl hydrocarbon antagonists, have shown excellent outcomes in clinical studies. The use of bioreactors could further add to HSPC studies in future. Drugs that could improve HSPC homing also appear to have potential in improving engraftment times in UCB transplantation. Technologies to expand HSPC from UCB and to enhance the homing of these cells appear to have attained the goal of accelerating hematopoietic recovery. Further discoveries and clinical studies are likely to make the goal of true HSPC expansion a reality for many applications in future.

  18. Fallopian tube secretory cell expansion: a sensitive biomarker for ovarian serous carcinogenesis.

    PubMed

    Wang, Yiying; Li, Li; Wang, Yue; Tang, Sarah Ngocvi; Zheng, Wenxin

    2015-01-01

    Recent advances suggest that precancerous lesions of pelvic serous carcinoma originate from tubal secretory cells. The purpose of our study was to determine if an increased number of secretory cells varies with age or location in the fallopian tube and to examine its association with serous neoplasia. Three groups (benign control, high-risk, and pelvic serous carcinoma) of age-matched patients were studied. The age data were stratified into 10-year intervals ranging from 20-29 to older than 80. The number of secretory and ciliated cells from both tubal fimbria and ampulla segments was counted by microscopy and immunohistochemical staining methods. The data were analyzed by standard contingency table and Poisson distribution methods after age justification. We found that the absolute number of tubal secretory cells increased significantly with age in all three groups. But a more dramatic increase of secretory cells was observed in high-risk and pelvic serous carcinoma patients. Secretory cell expansion is more prevalent than secretory cell outgrowth in both fimbria and ampulla tubal segments and is significantly associated with serous neoplasia (P < 0.001). Furthermore, age remained a significant risk factor for serous neoplasia after age adjustment. These findings suggest that secretory cell expansion could serve as a potential sensitive biomarker for early serous carcinogenesis within the fallopian tube. The study also supports a relationship between serous neoplasia and increased secretory to ciliated cell ratios, and the relationship between frequency of secretory cell expansion within the fallopian tube and increasing age and-more significantly-presence of high-risk factors or co-existing serous cancers.

  19. Fallopian tube secretory cell expansion: a sensitive biomarker for ovarian serous carcinogenesis.

    PubMed

    Wang, Yiying; Li, Li; Wang, Yue; Tang, Sarah Ngocvi; Zheng, Wenxin

    2016-01-01

    Recent advances suggest that precancerous lesions of pelvic serous carcinoma originate from tubal secretory cells. The purpose of our study was to determine if an increased number of secretory cells vary with age or location in the fallopian tube and to examine its association with serous neoplasia. Three groups (benign control, high-risk, and pelvic serous carcinoma) of age-matched patients were studied. The age data were stratified into 10-year intervals ranging from 20-29 to older than 80. The number of secretory and ciliated cells from both tubal fimbria and ampulla segments was counted by microscopy and immunohistochemical staining methods. The data were analyzed by standard contingency table and Poisson distribution methods after age justification. We found that the absolute number of tubal secretory cells increased significantly with age in all three groups. But a more dramatic increase of secretory cells was observed in high-risk and pelvic serous carcinoma patients. Secretory cell expansion is more prevalent than secretory cell outgrowth in both fimbria and ampulla tubal segments and is significantly associated with serous neoplasia (p < 0.001). Furthermore, age remained a significant risk factor for serous neoplasia after age adjustment. These findings suggest that secretory cell expansion could serve as a potential sensitive biomarker for early serous carcinogenesis within the fallopian tube. The study also supports a relationship between serous neoplasia and increased secretory to ciliated cell ratios, and the relationship between frequency of secretory cell expansion within the fallopian tube and increasing age and-more significantly-presence of high-risk factors or co-existing serous cancers.

  20. Ex vivo expansion of normal and chronic myeloid leukemic stem cells without functional alteration using a NUP98HOXA10homeodomain fusion gene

    PubMed Central

    Sloma, I; Imren, S; Beer, P A; Zhao, Y; Lecault, V; Leung, D; Raghuram, K; Brimacombe, C; Lambie, K; Piret, J; Hansen, C; Humphries, R K; Eaves, C J

    2013-01-01

    HOX genes have been implicated as regulators of normal and leukemic stem cell functionality, but the extent to which these activities are linked is poorly understood. Previous studies revealed that transduction of primitive mouse hematopoietic cells with a NUP98HOXA10homeodomain (NA10HD) fusion gene enables a subsequent rapid and marked expansion in vitro of hematopoietic stem cell numbers without causing their transformation or deregulated expansion in vivo. To determine whether forced expression of NA10HD in primitive human cells would have a similar effect, we compared the number of long-term culture-initiating cells (LTC-ICs) present in cultures of lenti-NA10HD versus control virus-transduced CD34+ cells originally isolated from human cord blood and chronic phase (CP) chronic myeloid leukemia (CML) patients. We found that NA10HD greatly increases outputs of both normal and Ph+/BCR-ABL+ LTC-ICs, and this effect is particularly pronounced in cultures containing growth factor-producing feeders. Interestingly, NA10HD did not affect the initial cell cycle kinetics of the transduced cells nor their subsequent differentiation. Moreover, immunodeficient mice repopulated with NA10HD-transduced CP-CML cells for more than 8 months showed no evidence of altered behavior. Thus, NA10HD provides a novel tool to enhance both normal and CP-CML stem cell expansion in vitro, without apparently altering other properties. PMID:22868969

  1. PILAR is a novel modulator of human T-cell expansion

    PubMed Central

    Huarte, Eduardo; Cubillos-Ruiz, Juan R.; Nesbeth, Yolanda C.; Scarlett, Uciane K.; Martinez, Diana G.; Engle, Xavier A.; Rigby, William F.; Pioli, Patricia A.; Guyre, Paul M.

    2008-01-01

    Robust T-cell responses without autoimmunity are only possible through a fine balance between activating and inhibitory signals. We have identified a novel modulator of T-cell expansion named proliferation-induced lymphocyte-associated receptor (PILAR). Surface PILAR is markedly up-regulated on CD4 and, to a lesser extent, on CD8 T cells on T-cell receptor engagement. In absence of CD28 costimulation, PILAR signaling through CD161 supports CD3 antibody-dependent and antigen-specificT-cell proliferation by increasing the expression of antiapoptotic Bcl-xL and induces secretion of T helper type 1 cytokines. These effects are abrogated by PILAR blockade with specific antibodies, which decrease surface levels of CD28. In contrast, PILAR induces apoptotic death on naive and early activated T cells if CD161 engagement is blocked. PILAR is expressed by approximately 7% to 10% of CD4 T cells in 2 samples of inflammatory synovial fluid, suggesting a potential role in the pathogenesis of joint inflammation. In addition, in the ovarian cancer microenvironment, effector T cells express PILAR, but not CD161, although expression of both can be augmented ex vivo. Our results indicate that PILAR plays a central role in modulating the extent of T-cell expansion. Manipulation of PILAR signaling may be important for treatment of autoimmune diseases and cancer. PMID:18550855

  2. Phenotypic analysis of cell surface markers and gene expression of human mesenchymal stem cells and chondrocytes during monolayer expansion.

    PubMed

    Cournil-Henrionnet, Christel; Huselstein, Céline; Wang, Yun; Galois, Laurent; Mainard, Didier; Decot, Véronique; Netter, Patrick; Stoltz, Jean-François; Muller, Sylvaine; Gillet, Pierre; Watrin-Pinzano, Astrid

    2008-01-01

    Both chondrocytes and mensenchymal stem cells (MSCs) are the most used cell sources for cartilage tissue engineering. However, monolayer expansion to obtain sufficient cells leads to a rapid chondrocyte dedifferentiation and a subsequent ancillary reduced ability of MSCs to differentiate into chondrocytes, thus limiting their application in cartilage repair. The aim of this study was to investigate the influence of the monolayer expansion on the immunophenotype and the gene expression profile of both cell types, and to find the appropriate compromise between monolayer expansion and the remaining chondrogenic characteristics. To this end, human chondrocytes, isolated enzymatically from femoral head slice, and human MSCs, derived from bone marrow, were maintained in monolayer culture up to passage 5. The respective expressions of cell surface markers (CD34, CD45, CD73, CD90, CD105, CD166) and several chondrogenic-related genes for each passage (P0-P5) of those cells were then analyzed using flow cytometry and quantitative real-time PCR, respectively. Flow cytometry analyses showed that, during the monolayer expansion, some qualitative and quantitative regulations occur for the expression of cell surface markers. A rapid increase in mRNA expression of type 1 collagen occurs whereas a significant decrease of type 2 collagen and Sox 9 was observed in chondrocytes through the successive passages. On the other hand, the expansion did not induced obvious change in MSCs gene expression. In conclusion, our results suggest that passage 1 might be the up-limit for chondrocytes in order to achieve their subsequent redifferentiation in 3D scaffold. Nevertheless, MSCs could be expanded in monolayer until passage 5 without loosing their undifferentiated phenotypes.

  3. Artificial Polymeric Scaffolds as Extracellular Matrix Substitutes for Autologous Conjunctival Goblet Cell Expansion

    PubMed Central

    He, Min; Storr-Paulsen, Thomas; Wang, Annie L.; Ghezzi, Chiara E.; Wang, Siran; Fullana, Matthew; Karamichos, Dimitrios; Utheim, Tor P.; Islam, Rakibul; Griffith, May; Islam, M. Mirazul; Hodges, Robin R.; Wnek, Gary E.; Kaplan, David L.; Dartt, Darlene A.

    2016-01-01

    Purpose We fabricated and investigated polymeric scaffolds that can substitute for the conjunctival extracellular matrix to provide a substrate for autologous expansion of human conjunctival goblet cells in culture. Methods We fabricated two hydrogels and two silk films: (1) recombinant human collagen (RHC) hydrogel, (2) recombinant human collagen 2-methacryloylxyethyl phosphorylcholine (RHC-MPC) hydrogel, (3) arginine-glycine-aspartic acid (RGD) modified silk, and (4) poly-D-lysine (PDL) coated silk, and four electrospun scaffolds: (1) collagen, (2) poly(acrylic acid) (PAA), (3) poly(caprolactone) (PCL), and (4) poly(vinyl alcohol) (PVA). Coverslips and polyethylene terephthalate (PET) were used for comparison. Human conjunctival explants were cultured on scaffolds for 9 to 15 days. Cell viability, outgrowth area, and the percentage of cells expressing markers for stratified squamous epithelial cells (cytokeratin 4) and goblet cells (cytokeratin 7) were determined. Results Most of cells grown on all scaffolds were viable except for PCL in which only 3.6 ± 2.2% of the cells were viable. No cells attached to PVA scaffold. The outgrowth was greatest on PDL-silk and PET. Outgrowth was smallest on PCL. All cells were CK7-positive on RHC-MPC while 84.7 ± 6.9% of cells expressed CK7 on PDL-silk. For PCL, 87.10 ± 3.17% of cells were CK7-positive compared to PET where 67.10 ± 12.08% of cells were CK7-positive cells. Conclusions Biopolymer substrates in the form of hydrogels and silk films provided for better adherence, proliferation, and differentiation than the electrospun scaffolds and could be used for conjunctival goblet cell expansion for eventual transplantation once undifferentiated and stratified squamous cells are included. Useful polymer scaffold design characteristics have emerged from this study. PMID:27832279

  4. Selective Expansion of Skeletal Muscle Stem Cells From Bulk Muscle Cells in Soft Three-Dimensional Fibrin Gel.

    PubMed

    Zhu, Pei; Zhou, Yalu; Wu, Furen; Hong, Yuanfan; Wang, Xin; Shekhawat, Gajendra; Mosenson, Jeffrey; Wu, Wen-Shu

    2017-02-28

    Muscle stem cells (MuSCs) exhibit robust myogenic potential in vivo, thus providing a promising curative treatment for muscle disorders. Ex vivo expansion of adult MuSCs is highly desired to achieve a therapeutic cell dose because of their scarcity in limited muscle biopsies. Sorting of pure MuSCs is generally required for all the current culture systems. Here we developed a soft three-dimensional (3D) salmon fibrin gel culture system that can selectively expand mouse MuSCs from bulk skeletal muscle preparations without cell sorting and faithfully maintain their regenerative capacity in culture. Our study established a novel platform for convenient ex vivo expansion of MuSCs, thus greatly advancing stem cell-based therapies for various muscle disorders. © Stem Cells Translational Medicine 2017.

  5. Clonal Expansion of Lgr5-Positive Cells from Mammalian Cochlea and High-Purity Generation of Sensory Hair Cells.

    PubMed

    McLean, Will J; Yin, Xiaolei; Lu, Lin; Lenz, Danielle R; McLean, Dalton; Langer, Robert; Karp, Jeffrey M; Edge, Albert S B

    2017-02-21

    Death of cochlear hair cells, which do not regenerate, is a cause of hearing loss in a high percentage of the population. Currently, no approach exists to obtain large numbers of cochlear hair cells. Here, using a small-molecule approach, we show significant expansion (>2,000-fold) of cochlear supporting cells expressing and maintaining Lgr5, an epithelial stem cell marker, in response to stimulation of Wnt signaling by a GSK3β inhibitor and transcriptional activation by a histone deacetylase inhibitor. The Lgr5-expressing cells differentiate into hair cells in high yield. From a single mouse cochlea, we obtained over 11,500 hair cells, compared to less than 200 in the absence of induction. The newly generated hair cells have bundles and molecular machinery for transduction, synapse formation, and specialized hair cell activity. Targeting supporting cells capable of proliferation and cochlear hair cell replacement could lead to the discovery of hearing loss treatments.

  6. Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

    PubMed Central

    Fluri, David A.; Tonge, Peter D.; Song, Hannah; Baptista, Ricardo P.; Shakiba, Nika; Shukla, Shreya; Clarke, Geoffrey; Nagy, Andras; Zandstra, Peter W.

    2016-01-01

    We demonstrate derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension (S) reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor expressing cells based on their differential survival and proliferation in suspension. Seamless integration of SiPSC reprogramming and directed differentiation enabled the scalable production of functionally and phenotypically defined cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step towards the development of a robust PSC generation, expansion and differentiation technology. PMID:22447133

  7. Selective expansion of T cells expressing V beta 2 in toxic shock syndrome

    PubMed Central

    1990-01-01

    Infection with Staphylococcus aureus and the production of toxic shock syndrome toxin-1 (TSST-1) have been implicated in the pathogenesis of toxic shock syndrome. Previous in vitro studies have demonstrated that TSST-1 is a powerful but selective stimulator of human T cells, and that the majority of activated cells express the TCR V beta 2 gene segment. We therefore studied patients with toxic shock syndrome using a modification of the PCR to determine if expansion of V beta 2+ T cells is a marker of the in vivo disease process. Five of eight patients studied demonstrated markedly elevated levels of circulating V beta 2+ T cells, whereas none showed significantly elevated levels of T cells expressing other V beta gene segments. The results suggest that toxin-mediated T cell activation, which involves a large fraction of the human T cell repertoire, may be critical in the pathogenesis of this disease. PMID:2117641

  8. Prevention of B220+ T cell expansion and prolongation of lifespan induced by Lactobacillus casei in MRL/lpr mice

    PubMed Central

    Mike, A; Nagaoka, N; Tagami, Y; Miyashita, M; Shimada, S; Uchida, K; Nanno, M; Ohwaki, M

    1999-01-01

    We examined the therapeutic effect of heat-killed Lactobacillus casei (LC) on MRL/lpr mice. Ingestion of a diet containing 0.05% (w/w) LC from the weaning period prolonged the lifespan and tended to reduce the proportion of B220+ T cells in the spleen and mesenteric lymph nodes (MLN) of MRL/lpr mice. When LC was intraperitoneally injected once a week after the age of 8 weeks, I-A− macrophages accumulated in the spleen as well as the peritoneum and macrophage progenitors increased in the bone marrow. Moreover, the amount of IL-6 mRNA in peritoneal macrophages was reduced by LC injection. Splenocytes from LC-injected MRL/lpr mice exhibited lower proliferative responses to mitogens than those from control MRL/lpr mice and the increase in number of B220+ T cells in the spleen and MLN was prevented by LC injection. However, LC injection affected neither expression of interferon-gamma (IFN-γ) and IL-4 mRNAs nor proliferative capacities of splenic T cells. Our findings demonstrate that LC injection accelerates macrophage recruitment and prevents the expansion of B220+ T cells without affecting the functions of T cells in MRL/lpr mice. These immunological modulations induced by LC may lead to prolongation of the lifespan of MRL/lpr mice. PMID:10444272

  9. Modeling diseases of noncoding unstable repeat expansions using mutant pluripotent stem cells

    PubMed Central

    Yanovsky-Dagan, Shira; Mor-Shaked, Hagar; Eiges, Rachel

    2015-01-01

    Pathogenic mutations involving DNA repeat expansions are responsible for over 20 different neuronal and neuromuscular diseases. All result from expanded tracts of repetitive DNA sequences (mostly microsatellites) that become unstable beyond a critical length when transmitted across generations. Nearly all are inherited as autosomal dominant conditions and are typically associated with anticipation. Pathologic unstable repeat expansions can be classified according to their length, repeat sequence, gene location and underlying pathologic mechanisms. This review summarizes the current contribution of mutant pluripotent stem cells (diseased human embryonic stem cells and patient-derived induced pluripotent stem cells) to the research of unstable repeat pathologies by focusing on particularly large unstable noncoding expansions. Among this class of disorders are Fragile X syndrome and Fragile X-associated tremor/ataxia syndrome, myotonic dystrophy type 1 and myotonic dystrophy type 2, Friedreich ataxia and C9 related amyotrophic lateral sclerosis and/or frontotemporal dementia, Facioscapulohumeral Muscular Dystrophy and potentially more. Common features that are typical to this subclass of conditions are RNA toxic gain-of-function, epigenetic loss-of-function, toxic repeat-associated non-ATG translation and somatic instability. For each mechanism we summarize the currently available stem cell based models, highlight how they contributed to better understanding of the related mechanism, and discuss how they may be utilized in future investigations. PMID:26131313

  10. Oxidative stress causes DNA triplet expansion in Huntington's disease mouse embryonic stem cells.

    PubMed

    Jonson, Ida; Ougland, Rune; Klungland, Arne; Larsen, Elisabeth

    2013-11-01

    Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded trinucleotide CAG repeat in the Huntingtin (Htt) gene. The molecular basis for the development and progression of HD is currently poorly understood. However, different DNA repair pathways have been implicated in both somatic expansion and disease progression. Embryonic stem cells provide a remarkable in vitro system to study HD and could have implications for understanding disease development and for therapeutic treatment. Here, we derive pluripotent stem cells from the mouse R6/1 HD model and demonstrate that repeated exposure to genotoxic agents inducing oxidative DNA damage gave a significant and dose dependent increase in somatic triplet expansion. Further investigation into specific steps of DNA repair revealed impaired double stranded break repair in exposed R6/1 cells, accompanied by the induction of apoptosis. We also found that differentiation status, and consequently DNA repair efficiency influenced somatic expansion. Our data underscore the importance of DNA damage and repair for the stability of the HD triplet in pluripotent stem cells.

  11. Differential Clonal Expansion in an Invading Cell Population: Clonal Advantage or Dumb Luck?

    PubMed

    Newgreen, Donald F; Zhang, Dongcheng; Cheeseman, Bevan L; Binder, Benjamin J; Landman, Kerry A

    2017-01-01

    In neoplastic cell growth, clones and subclones are variable both in size and mutational spectrum. The largest of these clones are believed to represent those cells with mutations that make them the most "fit," in a Darwinian sense, for expansion in their microenvironment. Thus, the degree of quantitative clonal expansion is regarded as being determined by innate qualitative differences between the cells that originate each clone. Here, using a combination of mathematical modelling and clonal labelling experiments applied to the developmental model system of the forming enteric nervous system, we describe how cells which are qualitatively identical may consistently produce clones of dramatically different sizes: most clones are very small while a few clones we term "superstars" contribute most of the cells to the final population. The basis of this is minor stochastic variations ("luck") in the timing and direction of movement and proliferation of individual cells, which builds a local advantage for daughter cells that is cumulative. This has potentially important consequences. In cancers, especially before strongly selective cytotoxic therapy, the assumption that the largest clones must be the cells with deterministic proliferative ability may not always hold true. In development, the gradual loss of clonal diversity as "superstars" take over the population may erode the resilience of the system to somatic mutations, which may have occurred early in clonal growth.

  12. Expansion of mesenchymal stem cells using a microcarrier-based cultivation system: growth and metabolism.

    PubMed

    Schop, D; Janssen, F W; Borgart, E; de Bruijn, J D; van Dijkhuizen-Radersma, R

    2008-01-01

    For the continuous and fast expansion of mesenchymal stem cells (MSCs), microcarriers have gained increasing interest. The aim of this study was to evaluate the growth and metabolism profiles of MSCs, expanded in a microcarrier-based cultivation system. We investigated various cultivation conditions to expand goat mesenchymal stem cells on Cytodex 1 microcarriers. These conditions differed in feeding regime, i.e. the addition of fresh proliferation medium, with or without new microcarriers. For all conditions, cell attachment, cell proliferation, energy source consumption, metabolite production, and cell distribution on the microcarriers were studied. Attachment efficiencies of 40% were obtained followed by successful expansion up to 15 cultivation days. Depending on the feeding regime, an exponential growth, stationary growth, and decline growth phase could be distinguished. Addition of 30% fresh medium containing microcarriers every three days showed the longest continuous proliferation of goat MSCs on microcarriers. This feeding regime has the advantage that metabolites, such as ammonia, are diluted and that new energy sources, such as glucose and glutamine, and additional surface area are provided to the cells. In addition, by adding extra microcarriers a more homogenous cell distribution on the microcarriers is obtained as a result of bead-to-bead transfer. A correlation between nutrient consumption, metabolite production and cell growth was observed. The decreasing yield of lactate from glucose over time indicated a possible shift in cellular metabolism.

  13. An intermittent rocking platform for integrated expansion and differentiation of human pluripotent stem cells to cardiomyocytes in suspended microcarrier cultures.

    PubMed

    Ting, Sherwin; Chen, Allen; Reuveny, Shaul; Oh, Steve

    2014-09-01

    The development of novel platforms for large scale production of human embryonic stem cells (hESC) derived cardiomyocytes (CM) becomes more crucial as the demand for CMs in preclinical trials, high throughput cardio toxicity assays and future regenerative therapeutics rises. To this end, we have designed a microcarrier (MC) suspension agitated platform that integrates pluripotent hESC expansion followed by CM differentiation in a continuous, homogenous process. Hydrodynamic shear stresses applied during the hESC expansion and CM differentiation steps drastically reduced the capability of the cells to differentiate into CMs. Applying vigorous stirring during pluripotent hESC expansion on Cytodex 1 MC in spinner cultures resulted in low CM yields in the following differentiation step (cardiac troponin-T (cTnT): 22.83±2.56%; myosin heavy chain (MHC): 19.30±5.31%). Whereas the lower shear experienced in side to side rocker (wave type) platform resulted in higher CM yields (cTNT: 47.50±7.35%; MHC: 42.85±2.64%). The efficiency of CM differentiation is also affected by the hydrodynamic shear stress applied during the first 3days of the differentiation stage. Even low shear applied continuously by side to side rocker agitation resulted in very low CM differentiation efficiency (cTnT<5%; MHC<2%). Simply by applying intermittent agitation during these 3days followed by continuous agitation for the subsequent 9days, CM differentiation efficiency can be substantially increased (cTNT: 65.73±10.73%; MHC: 59.73±9.17%). These yields are 38.3% and 39.3% higher (for cTnT and MHC respectively) than static culture control. During the hESC expansion phase, cells grew on continuously agitated rocker platform as pluripotent cell/MC aggregates (166±88×10(5)μm(2)) achieving a cell concentration of 3.74±0.55×10(6)cells/mL (18.89±2.82 fold expansion) in 7days. These aggregates were further differentiated into CMs using a WNT modulation differentiation protocol for the subsequent

  14. Expansion of Lithium Ion Pouch Cell Batteries: Observations from Neutron Imaging

    DTIC Science & Technology

    2012-12-21

    low C-rates the measured battery thickness was a function of State of Charge (SOC) and independent of a small external clamping force. A 0.3...Lee et al. in [4] measured the dimensional changes in lithium cobalt oxide pouch cells during cycling using a specialized dilatometer setup. They...and rates on electrode expansion was investigated. At low C-rates the measured battery thickness was a function of State of Charge (SOC) and independent

  15. The SAUR19 subfamily of SMALL AUXIN UP RNA genes promote cell expansion

    PubMed Central

    Spartz, Angela K.; Lee, Sang H.; Wenger, Jonathan P.; Gonzalez, Nathalie; Itoh, Hironori; Inzé, Dirk; Peer, Wendy A.; Murphy, Angus S.; Overvoorde, Paul J.; Gray, William M.

    2012-01-01

    SUMMARY The plant hormone auxin controls numerous aspects of plant growth and development by regulating the expression of hundreds of genes. SMALL AUXIN UP RNA (SAUR) genes comprise the largest family of auxin-responsive genes, but their function is unknown. Although prior studies have correlated the expression of some SAUR genes with auxin-mediated cell expansion, genetic evidence implicating SAURs in cell expansion has not been reported. The Arabidopsis SAUR19, SAUR20, SAUR21, SAUR22, SAUR23, and SAUR24 (SAUR19– 24) genes encode a subgroup of closely related SAUR proteins. We demonstrate that these SAUR proteins are highly unstable in Arabidopsis. However, the addition of an N-terminal GFP or epitope tag dramatically increases the stability of SAUR proteins. Expression of these stabilized SAUR fusion proteins in Arabidopsis confers numerous auxin-related phenotypes indicative of increased and/or unregulated cell expansion, including increased hypocotyl and leaf size, defective apical hook maintenance, and altered tropic responses. Furthermore, seedlings expressing an artificial microRNA targeting multiple members of the SAUR19–24 subfamily exhibit short hypocotyls and reduced leaf size. Together, these findings demonstrate that SAUR19– 24 function as positive effectors of cell expansion. This regulation may be achieved through the modulation of auxin transport, as SAUR gain-of-function and loss-of-function seedlings exhibit increased and reduced basipetal indole-3-acetic acid transport, respectively. Consistent with this possibility, SAUR19–24 proteins predominantly localize to the plasma membrane. PMID:22348445

  16. Potential role of 20S proteasome in maintaining stem cell integrity of human bone marrow stromal cells in prolonged culture expansion

    SciTech Connect

    Lu, Li; Song, Hui-Fang; Zhang, Wei-Guo; Liu, Xue-Qin; Zhu, Qian; Cheng, Xiao-Long; Yang, Gui-Jiao; Li, Ang; Xiao, Zhi-Cheng

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer Prolonged culture expansion retards proliferation and induces senescence of hBMSCs. Black-Right-Pointing-Pointer Reduced 20S proteasomal activity and expression potentially contribute to cell aging. Black-Right-Pointing-Pointer MG132-mediated 20S proteasomal inhibition induces senescence-like phenotype. Black-Right-Pointing-Pointer 18{alpha}-GA stimulates proteasomal activity and restores replicative senescence. Black-Right-Pointing-Pointer 18{alpha}-GA retains differentiation without affecting stem cell characterizations. -- Abstract: Human bone marrow stromal cells (hBMSCs) could be used in clinics as precursors of multiple cell lineages following proper induction. Such application is impeded by their characteristically short lifespan, together with the increasing loss of proliferation capability and progressive reduction of differentiation potential after the prolonged culture expansion. In the current study, we addressed the possible role of 20S proteasomes in this process. Consistent with prior reports, long-term in vitro expansion of hBMSCs decreased cell proliferation and increased replicative senescence, accompanied by reduced activity and expression of the catalytic subunits PSMB5 and PSMB1, and the 20S proteasome overall. Application of the proteasome inhibitor MG132 produced a senescence-like phenotype in early passages, whereas treating late-passage cells with 18{alpha}-glycyrrhetinic acid (18{alpha}-GA), an agonist of 20S proteasomes, delayed the senescence progress, enhancing the proliferation and recovering the capability of differentiation. The data demonstrate that activation of 20S proteasomes assists in counteracting replicative senescence of hBMSCs expanded in vitro.

  17. Recombinant cells in the lung increase with age via de novo recombination events and clonal expansion.

    PubMed

    Kimoto, Takafumi; Kay, Jennifer E; Li, Na; Engelward, Bevin P

    2017-04-01

    Homologous recombination (HR) is a critical DNA repair pathway, which is usually error-free, but can sometimes lead to cancer-promoting mutations. Despite the importance of HR as a driver of mutations, the spontaneous frequency of such mutations has proven difficult to study. To gain insight to location, cell type, and subsequent proliferation of mutated cells, we used the Rosa26 Direct Repeat (RaDR) mice for in situ detection and quantification of recombinant cells in the lung. We developed a method for automated enumeration of recombinant cells in lung tissue using the Metafer 4 slide-scanning platform. The mean spontaneous HR frequencies of the lung tissue in young and aged mice were 2 × 10(-6) and 30 × 10(-6) , respectively, which is consistent with our previous reports that mutated cells accumulate with age. In addition, by using the capability of Metafer 4 to mark the position of fluorescent cells, we found that recombinant cells from the aged mice formed clusters in the lung tissue, likely due to clonal expansion of a single mutant cell. The recombinant cells primarily consisted of alveolar epithelial type II or club (previously known as Clara) cells, both of which have the potential to give rise to cancer. This approach to tissue image analysis reveals the location and cell types that have undergone HR. Being able to quantify mutant cells in situ within lung tissue opens doors to studies of exposure-induced mutations and clonal expansion, giving rise to new opportunities for understanding how genetic and environmental factors cause tumorigenic mutations. Environ. Mol. Mutagen. 58:135-145, 2017. © 2017 Wiley Periodicals, Inc.

  18. A conserved bacterial protein induces pancreatic beta cell expansion during zebrafish development

    PubMed Central

    Hill, Jennifer Hampton; Franzosa, Eric A; Huttenhower, Curtis; Guillemin, Karen

    2016-01-01

    Resident microbes play important roles in the development of the gastrointestinal tract, but their influence on other digestive organs is less well explored. Using the gnotobiotic zebrafish, we discovered that the normal expansion of the pancreatic β cell population during early larval development requires the intestinal microbiota and that specific bacterial members can restore normal β cell numbers. These bacteria share a gene that encodes a previously undescribed protein, named herein BefA (β Cell Expansion Factor A), which is sufficient to induce β cell proliferation in developing zebrafish larvae. Homologs of BefA are present in several human-associated bacterial species, and we show that they have conserved capacity to stimulate β cell proliferation in larval zebrafish. Our findings highlight a role for the microbiota in early pancreatic β cell development and suggest a possible basis for the association between low diversity childhood fecal microbiota and increased diabetes risk. DOI: http://dx.doi.org/10.7554/eLife.20145.001 PMID:27960075

  19. Development of a vascular niche platform for expansion of repopulating human cord blood stem and progenitor cells.

    PubMed

    Butler, Jason M; Gars, Eric J; James, Daylon J; Nolan, Daniel J; Scandura, Joseph M; Rafii, Shahin

    2012-08-09

    Transplantation of ex vivo expanded human umbilical cord blood cells (hCB) only partially enhances the hematopoietic recovery after myelosuppressive therapy. Incubation of hCB with optimal combinations of cytokines and niche cells, such as endothelial cells (ECs), could augment the efficiency of hCB expansion. We have devised an approach to cultivate primary human ECs (hECs) in serum-free culture conditions. We demonstrate that coculture of CD34(+) hCB in direct cellular contact with hECs and minimal concentrations of thrombopoietin/Kit-ligand/Flt3-ligand resulted in a 400-fold expansion of total hematopoietic cells, 150-fold expansion of CD45(+)CD34(+) progenitor cells, and 23-fold expansion of CD45(+) Lin(-)CD34(hi+)CD45RA(-)CD49f(+) stem and progenitor cells over a 12-day period. Compared with cytokines alone, coculture of hCB with hECs permitted greater expansion of cells capable of multilineage engraftment and serial transplantation, hallmarks of long-term repopulating hematopoietic stem cells. Therefore, hECs establish a cellular platform for expansion of hematopoietic stem and progenitor cells and treatment of hematologic disorders.

  20. ANGPTL4 deficiency in haematopoietic cells promotes monocyte expansion and atherosclerosis progression

    PubMed Central

    Aryal, Binod; Rotllan, Noemi; Araldi, Elisa; Ramírez, Cristina M.; He, Shun; Chousterman, Benjamin G.; Fenn, Ashley M.; Wanschel, Amarylis; Madrigal-Matute, Julio; Warrier, Nikhil; Martín-Ventura, Jose L.; Swirski, Filip K.; Suárez, Yajaira; Fernández-Hernando, Carlos

    2016-01-01

    Lipid accumulation in macrophages has profound effects on macrophage gene expression and contributes to the development of atherosclerosis. Here, we report that angiopoietin-like protein 4 (ANGPTL4) is the most highly upregulated gene in foamy macrophages and it's absence in haematopoietic cells results in larger atherosclerotic plaques, characterized by bigger necrotic core areas and increased macrophage apoptosis. Furthermore, hyperlipidemic mice deficient in haematopoietic ANGPTL4 have higher blood leukocyte counts, which is associated with an increase in the common myeloid progenitor (CMP) population. ANGPTL4-deficient CMPs have higher lipid raft content, are more proliferative and less apoptotic compared with the wild-type (WT) CMPs. Finally, we observe that ANGPTL4 deficiency in macrophages promotes foam cell formation by enhancing CD36 expression and reducing ABCA1 localization in the cell surface. Altogether, these findings demonstrate that haematopoietic ANGPTL4 deficiency increases atherogenesis through regulating myeloid progenitor cell expansion and differentiation, foam cell formation and vascular inflammation. PMID:27460411

  1. ANGPTL4 deficiency in haematopoietic cells promotes monocyte expansion and atherosclerosis progression

    NASA Astrophysics Data System (ADS)

    Aryal, Binod; Rotllan, Noemi; Araldi, Elisa; Ramírez, Cristina M.; He, Shun; Chousterman, Benjamin G.; Fenn, Ashley M.; Wanschel, Amarylis; Madrigal-Matute, Julio; Warrier, Nikhil; Martín-Ventura, Jose L.; Swirski, Filip K.; Suárez, Yajaira; Fernández-Hernando, Carlos

    2016-07-01

    Lipid accumulation in macrophages has profound effects on macrophage gene expression and contributes to the development of atherosclerosis. Here, we report that angiopoietin-like protein 4 (ANGPTL4) is the most highly upregulated gene in foamy macrophages and it's absence in haematopoietic cells results in larger atherosclerotic plaques, characterized by bigger necrotic core areas and increased macrophage apoptosis. Furthermore, hyperlipidemic mice deficient in haematopoietic ANGPTL4 have higher blood leukocyte counts, which is associated with an increase in the common myeloid progenitor (CMP) population. ANGPTL4-deficient CMPs have higher lipid raft content, are more proliferative and less apoptotic compared with the wild-type (WT) CMPs. Finally, we observe that ANGPTL4 deficiency in macrophages promotes foam cell formation by enhancing CD36 expression and reducing ABCA1 localization in the cell surface. Altogether, these findings demonstrate that haematopoietic ANGPTL4 deficiency increases atherogenesis through regulating myeloid progenitor cell expansion and differentiation, foam cell formation and vascular inflammation.

  2. Expansion of CD133+ Umbilical Cord Blood Derived Hematopoietic Stem Cells on a Biocompatible Microwells

    PubMed Central

    Soufizomorrod, Mina; Soleimani, Masoud; Hajifathali, Abbas; Mohammadi, Majid Mossahebi; Abroun, Saeed

    2013-01-01

    Umbilical cord Blood (UCB) as a source of Hematopoietic Stem/Progenitor cells (HSPCs) used for Umbilical cord blood transplantation (UCBT). The main obstacle in application of this source as an appropriate source of HSPCs is low volume of this product. So ex vivo expansion of these cells in a microenvironment which mimic body condition is important. In current study we designed biocompatible microwells in which collagene type I is coated by softlitography method. Our findings designated that in 3-Dimensional (3D) microenvironment CD133+ UCB derived HSC expanded significantly compared to 2-Dimensional (2D) microenvironment. PMID:24505514

  3. Ex vivo expansion of hematopoietic stem cell by fusion protein TAT-Zfx

    SciTech Connect

    Xu Chong; Zhang Yanbing; Jiang Hua

    2009-02-13

    The relative inability of hemopoietic stem cells (HSCs) to reproduce themselves (self-renew) ex vivo imposes substantial limitations on the current use of HSC transplantation. Recently, the transcription factor Zfx has been demonstrated that played an important in controlling the self-renewal of hematopoietic stem cells. Here, we reported that Zfx could enable high-level expansion of HSCs in vitro, by combination of protein transduction domain, TAT. Furthermore, we also demonstrated that expanded HSCs population retains their normal in vivo potential of pluripotency. It is thus that TAT-Zfx has the potential to expand HSCs significantly in vitro, and will have enormous clinical potentials.

  4. Single CD28 stimulation induces stable and polyclonal expansion of human regulatory T cells

    PubMed Central

    He, Xuehui; Smeets, Ruben L.; van Rijssen, Esther; Boots, Annemieke M. H.; Joosten, Irma; Koenen, Hans J. P. M.

    2017-01-01

    CD4+FOXP3+ Treg are essential for immune tolerance. Phase-1 clinical trials of Treg-therapy to treat graft-versus-host-disease reported safety and potential therapeutic efficacy. Treg-based trials have started in organ-transplant patients. However, efficient ex vivo expansion of a stable Treg population remains a challenge and exploring novel ways for Treg expansion is a pre-requisite for successful immunotherapy. Based on the recent finding that CD28-signaling is crucial for survival and proliferation of mouse Treg, we studied single-CD28 stimulation of human Treg, without T cell receptor stimulation. Single-CD28 stimulation of human Treg in the presence of recombinant human IL-2(rhIL-2), as compared to CD3/CD28/rhIL-2 stimulation, led to higher expression levels of FOXP3. Although the single-CD28 expanded Treg population was equally suppressive to CD3/CD28 expanded Treg, pro-inflammatory cytokine (IL-17A/IFNγ) production was strongly inhibited, indicating that single-CD28 stimulation promotes Treg stability. As single-CD28 stimulation led to limited expansion rates, we examined a CD28-superagonist antibody and demonstrate a significant increased Treg expansion that was more efficient than standard anti-CD3/CD28-bead stimulation. CD28-superagonist stimulation drove both naïve and memory Treg proliferation. CD28-superagonist induction of stable Treg appeared both PI3K and mTOR dependent. Regarding efficient and stable expansion of Treg for adoptive Treg-based immunotherapy, application of CD28-superagonist stimulation is of interest. PMID:28223693

  5. Single CD28 stimulation induces stable and polyclonal expansion of human regulatory T cells.

    PubMed

    He, Xuehui; Smeets, Ruben L; van Rijssen, Esther; Boots, Annemieke M H; Joosten, Irma; Koenen, Hans J P M

    2017-02-22

    CD4+FOXP3+ Treg are essential for immune tolerance. Phase-1 clinical trials of Treg-therapy to treat graft-versus-host-disease reported safety and potential therapeutic efficacy. Treg-based trials have started in organ-transplant patients. However, efficient ex vivo expansion of a stable Treg population remains a challenge and exploring novel ways for Treg expansion is a pre-requisite for successful immunotherapy. Based on the recent finding that CD28-signaling is crucial for survival and proliferation of mouse Treg, we studied single-CD28 stimulation of human Treg, without T cell receptor stimulation. Single-CD28 stimulation of human Treg in the presence of recombinant human IL-2(rhIL-2), as compared to CD3/CD28/rhIL-2 stimulation, led to higher expression levels of FOXP3. Although the single-CD28 expanded Treg population was equally suppressive to CD3/CD28 expanded Treg, pro-inflammatory cytokine (IL-17A/IFNγ) production was strongly inhibited, indicating that single-CD28 stimulation promotes Treg stability. As single-CD28 stimulation led to limited expansion rates, we examined a CD28-superagonist antibody and demonstrate a significant increased Treg expansion that was more efficient than standard anti-CD3/CD28-bead stimulation. CD28-superagonist stimulation drove both naïve and memory Treg proliferation. CD28-superagonist induction of stable Treg appeared both PI3K and mTOR dependent. Regarding efficient and stable expansion of Treg for adoptive Treg-based immunotherapy, application of CD28-superagonist stimulation is of interest.

  6. Donor bone marrow cells are essential for iNKT cell-mediated Foxp3+ Treg cell expansion in a murine model of transplantation tolerance.

    PubMed

    Miyairi, Satoshi; Hirai, Toshihito; Ishii, Rumi; Okumi, Masayoshi; Nunoda, Shinichi; Yamazaki, Kenji; Ishii, Yasuyuki; Tanabe, Kazunari

    2017-01-26

    Mixed chimerism induction is the most reliable method for establishing transplantation tolerance. We previously described a novel treatment using a suboptimal dose of anti-CD40 ligand (anti-CD40L) and liposomal formulation of a ligand for invariant natural killer T cells administered to sub-lethally irradiated recipient mice after donor bone marrow cell (BMC) transfer. Recipient mice treated with this regimen showed expansion of a Foxp3-positive regulatory T(Treg) cell phenotype, and formation of mixed chimera. However, the mechanism of expansion and bioactivity of Treg cells remains unclear. Here, we examine the role of donor BMCs in the expansion of bioactive Treg cells. The mouse model was transplanted with a heart allograft the day after treatment. The results showed that transfer of spleen cells in place of BMCs failed to deplete host interferon (IFN)-γ-producing CD8(+) T cells, expand host Ki67(+) CD4(+) CD25(+) Foxp3(+) Treg cells, and prolong graft survival. Severe combined immunodeficiency mice who received Treg cells obtained from BMC-recipients accepted skin grafts in an allo-specific manner. Myeloid-derived suppressor cells, which were a copious cell subset in BMCs, enhanced the Ki67 expression of Treg cells. This suggests that donor BMCs are indispensable for the expansion of host bioactive Treg cells in our novel treatment for transplant tolerance induction.

  7. Embryonic stem cells remain highly pluripotent following long term expansion as aggregates in suspension bioreactors.

    PubMed

    zur Nieden, Nicole I; Cormier, Jaymi T; Rancourt, Derrick E; Kallos, Michael S

    2007-05-01

    Increasing attention has been drawn towards pluripotent embryonic stem cells (ESCs) and their potential use as the primary material in various tissue engineering applications. Successful clinical implementation of this technology would require a quality controlled reproducible culture system for the expansion of the cells to be used in the generation of functional tissues. Recently, we showed that suspension bioreactors could be used in the regulated large-scale expansion of highly pluripotent murine ESCs. The current study illustrates that these bioreactor protocols can be adapted for long term culture and that murine ESC cultures remain highly undifferentiated, when serially passaged in suspension bioreactors for extended periods. Flow cytometry analysis and gene expression profiles of several pluripotency markers, in addition to colony and embryoid body (EB) formation tests were conducted at the start and end of the experiment and all showed that the ESC cultures remained highly undifferentiated over extended culture time in suspension. In vivo teratoma formation and in vitro differentiation into neural, cardiomyocyte, osteoblast and chondrocyte lineages, performed at the end of the long term culture, further supported the presence of functional and undifferentiated ESCs in the expanded population. Overall, this system enables the controlled expansion of highly pluripotent murine ESC populations.

  8. The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division.

    PubMed

    Ambrose, J Christian; Shoji, Tsubasa; Kotzer, Amanda M; Pighin, Jamie A; Wasteneys, Geoffrey O

    2007-09-01

    Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein-CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.

  9. Methods of ex vivo expansion of human cord blood cells: challenges, successes and clinical implications.

    PubMed

    Baron, Frédéric; Ruggeri, Annalisa; Nagler, Arnon

    2016-03-01

    More than 40,000 unrelated cord blood transplantations (UCBT) have been performed worldwide as treatment for patients with malignant or non-malignant life threatening hematologic disorders. However, low absolute numbers of hematopoietic stem and progenitor cells (HSPCs) within a single cord blood unit has remained a limiting factor for this transplantation modality, particularly in adult recipients. Further, because UCB contains low numbers of mostly naïve T cells, immune recovery after UCBT is slow, predisposing patients to severe infections. Other causes of UCBT failure has included graft-versus-host disease (GVHD) and relapse of the underlying disease. In this article, we first review the current landscape of cord blood engineering aimed at improving engraftment. This includes approaches of UCB-HSPCs expansion and methods aimed at improving UCB-HSCPs homing. We then discuss recent approaches of cord blood engineering developed to prevent infection [generation of multivirus-specific cytotoxic T cells (VSTs) from UCB], relapse [transduction of UCB-T cells with tumor-specific chimeric receptor antigens (CARs)] and GVHD (expansion of regulatory T cells from UCB). Although many of these techniques of UCB engineering remain currently technically challenging and expensive, they are likely to revolutionize the field of UCBT in the next decades.

  10. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies

    PubMed Central

    Tillberg, Paul W.; Chen, Fei; Piatkevich, Kiryl D.; Zhao, Yongxin; Yu, Chih-Chieh (Jay); English, Brian P.; Gao, Linyi; Martorell, Anthony; Suk, Ho-Jun; Yoshida, Fumiaki; DeGennaro, Ellen M.; Roossien, Douglas H.; Gong, Guanyu; Seneviratne, Uthpala; Tannenbaum, Steven R.; Desimone, Robert; Cai, Dawen; Boyden, Edward S.

    2016-01-01

    Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first expansion microscopy method was unable to retain native proteins in the gel and used custom made reagents not widely available. Here, we describe protein retention ExM (proExM), a variant of ExM that anchors proteins to the swellable gel allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validate and demonstrate utility of proExM for multi-color super-resolution (~70 nm) imaging of cells and mammalian tissues on conventional microscopes. PMID:27376584

  11. In vitro expansion of antigen-specific CD8(+) T cells distorts the T-cell repertoire.

    PubMed

    Koning, Dan; Costa, Ana I; Hasrat, Raiza; Grady, Bart P X; Spijkers, Sanne; Nanlohy, Nening; Keşmir, Can; van Baarle, Debbie

    2014-03-01

    Short-term in vitro expansion of antigen-specific T cells is an appreciated assay for the analysis of small memory T-cell populations. However, how well short-term expanded T cells represent the direct ex vivo situation remains to be elucidated. In this study we compared the clonality of Epstein-Barr virus (EBV) and cytomegalovirus (CMV)-specific CD8(+) T cells directly ex vivo and after in vitro stimulation with antigen. Our data show that the antigen-specific T cell repertoire significantly alters after in vitro culture. Clear shifts in clonotype hierarchy were observed, with the most dominant ex vivo clonotype decreasing after stimulation at the expense of several previously subdominant clonotypes. Notably, these alterations were more pronounced in polyclonal T-cell populations compared to mono- or oligoclonal repertoires. Furthermore, TCR diversity significantly increased after culture with antigen. These results suggest that the T-cell repertoire is highly subjective to variation after in vitro stimulation with antigen. Hence, although short-term expansion of T cells provides a simple and efficient tool to examine antigen-specific immune responses, caution is required if T-cell populations are expanded prior to detailed, clonotypic analyses or other repertoire-based investigations.

  12. In vitro expansion of Lin+ and Lin- mononuclear cells from human peripheral blood

    NASA Astrophysics Data System (ADS)

    Norhaiza, H. Siti; Rohaya, M. A. W.; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul

    2013-11-01

    Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin-) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin+) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin- cell population. The ability of Lin+ and Lin- to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin+ and Lin- were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin+ mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin- stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation medium support Lin

  13. DJ-1 deficiency attenuates expansion of liver progenitor cells through modulating the inflammatory and fibrogenic niches

    PubMed Central

    Chen, L; Luo, M; Sun, X; Qin, J; Yu, C; Wen, Y; Zhang, Q; Gu, J; Xia, Q; Kong, X

    2016-01-01

    Our previous study suggested that DJ-1 has a critical role in initiating an inflammatory response, but its role in the liver progenitor cell (LPC) expansion, a process highly dependent on the inflammatory niche, remains elusive. The objective of this study is to determine the role of DJ-1 in LPC expansion. The correlation of DJ-1 expression with LPC markers was examined in the liver of patients with hepatitis B or hepatitis C virus (HBV and HCV, respectively) infection, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), nonalcoholic fatty liver disease (NAFLD), cirrhosis or hepatocellular carcinoma (HCC), respectively. The role of DJ-1 in LPC expansion and the formation of LPC-associated fibrosis and inflammation was examined in a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced liver injury murine model. We also determined the ability of hepatic stellate cells (HSCs) in recruiting macrophages in DJ-1 knockout (KO) mice. The expression levels of DJ-1 were upregulated in the liver of HBV, HCV, PBC and PSC patients and DDC-fed mice. Additionally, DJ-1 expression was positively correlated with LPC proliferation in patients with liver injury and mice with DDC exposure. DJ-1 has no direct effect on LPC proliferation. Reduced activation of HSCs and collagen deposition were observed in DJ-1 KO mice. Furthermore, infiltrated CD11b+Gr-1low macrophages and pro-inflammatory factors (IL-6, TNF-α) were attenuated in DJ-1 KO mice. Mechanistically, we found that HSCs isolated from DJ-1 KO mice had decreased secretion of macrophage-mobilizing chemokines, such as CCL2 and CX3CL1, resulting in impaired macrophage infiltration. DJ-1 positively correlates with LPC expansion during liver injury. DJ-1 deficiency negatively regulates LPC proliferation by impairing the formation of LPC-associated fibrosis and inflammatory niches. PMID:27277679

  14. [Advances in ex vivo expansion and immunotherapy application of regulatory T cells].

    PubMed

    Yan, Li; Shao, Zong-Hong

    2015-04-01

    CD4+ CD25+ regulatory T cells (Treg) play a fundamental role in the establishment and maintenance of immune tolerance. In a some of experimental models, it was found that Tregs can quench autoimmune diseases, maintain allogeneic transplants, and prevent allergic diseases. A major obstacle to their clinical application is related to their definitive phenotype and very limited number of these cells in peripheral circulation, no more than 5%-10% of total CD4+ T cells. Recent progress of technologies for Treg sorting with multicolor flow cytometry and immuno-absorbing columns has overcome these obstacles, and opened the doors to the clinical application of Treg. This review highlight the characteristics of Treg, describe the current information of cell sorting and ex vivo expansion techniques, and outline the adoptive transfer experiments and clinical trials of immunotherapy that have been developed in recent years. It is foreseeable that Treg adoptive transfusion will be a promising immunosuppressive therapy.

  15. Immune complex formation and in situ B-cell clonal expansion in human cerebral cavernous malformations.

    PubMed

    Shi, Changbin; Shenkar, Robert; Kinloch, Andrew; Henderson, Scott G; Shaaya, Mark; Chong, Anita S; Clark, Marcus R; Awad, Issam A

    2014-07-15

    Cerebral cavernous malformations (CCMs) represent clusters of dilated vascular channels, predisposing to hemorrhagic stroke and seizures. They are associated with defective blood brain barrier, hemorrhages of different ages and a robust inflammatory cell infiltrate. We report for the first time evidence of co-localized IgG and complement membrane attack complexes in CCM lesions. CD4(+) and CD8(+) T-cells are aggregated with CD20(+) B-cells. And IgG repertoire analyses demonstrate in situ B-cell clonal expansion and antigen-driven affinity maturation in CCMs. These results suggest an organ-intrinsic adaptive immune response in CCMs that should be further characterized as a potential therapeutic target.

  16. Local Oxidative Stress Expansion through Endothelial Cells – A Key Role for Gap Junction Intercellular Communication

    PubMed Central

    Feine, Ilan; Pinkas, Iddo; Salomon, Yoram; Scherz, Avigdor

    2012-01-01

    Background Major circulation pathologies are initiated by oxidative insult expansion from a few injured endothelial cells to distal sites; this possibly involves mechanisms that are important to understanding circulation physiology and designing therapeutic management of myocardial pathologies. We tested the hypothesis that a localized oxidative insult of endothelial cells (ECs) propagates through gap junction inter-cellular communication (GJIC). Methodology/Principal Findings Cultures comprising the bEnd.3 cell line, that have been established and recognized as suitable for examining communication among ECs, were used to study the propagation of a localized oxidative insult to remote cells. Spatially confined near infrared illumination of parental or genetically modified bEnd.3 cultures, pretreated with the photosensitizer WST11, generated O2•− and •OH radicals in the illuminated cells. Time-lapse fluorescence microscopy, utilizing various markers, and other methods, were used to monitor the response of non-illuminated bystander and remote cells. Functional GJIC among ECs was shown to be mandatory for oxidative insult propagation, comprising de-novo generation of reactive oxygen and nitrogen species (ROS and RNS, respectively), activation and nuclear translocation of c-Jun N-terminal kinase, followed by massive apoptosis in all bystander cells adjacent to the primarily injured ECs. The oxidative insult propagated through GJIC for many hours, over hundreds of microns from the primary photogeneration site. This wave is shown to be limited by intracellular ROS scavenging, chemical GJIC inhibition or genetic manipulation of connexin 43 (a key component of GJIC). Conclusion/Significance Localized oxidative insults propagate through GJIC between ECs, while stimulating de-novo generation of ROS and RNS in bystander cells, thereby driving the insult's expansion. PMID:22911831

  17. Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells from Umbilical Cord Blood

    PubMed Central

    Sotnezova, E.V.; Andreeva, E.R.; Grigoriev, A.I.; Buravkova, L.B.

    2016-01-01

    Transplantation of umbilical cord blood cells is currently widely used in modern cell therapy. However, the limited number of hematopoietic stem and progenitor cells (HSPCs) and prolonged time of recovery after the transplantation are significant limitations in the use of cord blood. Ex vivo expansion with various cytokine combinations is one of the most common approaches for increasing the number of HSPCs from one cord blood unit. In addition, there are protocols that enable ex vivo amplification of cord blood cells based on native hematopoietic microenvironmental cues, including stromal components and the tissue-relevant oxygen level. The newest techniques for ex vivo expansion of HSPCs are based on data from the elucidation of the molecular mechanisms governing the hematopoietic niche function. Application of these methods has provided an improvement of several important clinical outcomes. Alternative methods of cord blood transplantation enhancement based on optimization of HPSC homing and engraftment in patient tissues have also been successful. The goal of the present review is to analyze recent methodological approaches to cord blood HSPC ex vivo amplification. PMID:27795840

  18. Evaluating Clonal Expansion of HIV-Infected Cells: Optimization of PCR Strategies to Predict Clonality

    PubMed Central

    Laskey, Sarah B.; Pohlmeyer, Christopher W.; Bruner, Katherine M.; Siliciano, Robert F.

    2016-01-01

    In HIV-infected individuals receiving suppressive antiretroviral therapy, the virus persists indefinitely in a reservoir of latently infected cells. The proliferation of these cells may contribute to the stability of the reservoir and thus to the lifelong persistence of HIV-1 in infected individuals. Because the HIV-1 replication process is highly error-prone, the detection of identical viral genomes in distinct host cells provides evidence for the clonal expansion of infected cells. We evaluated alignments of unique, near-full-length HIV-1 sequences to determine the relationship between clonality in a short region and clonality in the full genome. Although it is common to amplify and sequence short, subgenomic regions of the viral genome for phylogenetic analysis, we show that sequence identity of these amplicons does not guarantee clonality across the full viral genome. We show that although longer amplicons capture more diversity, no subgenomic region can recapitulate the diversity of full viral genomes. Consequently, some identical subgenomic amplicons should be expected even from the analysis of completely unique viral genomes, and the presence of identical amplicons alone is not proof of clonally expanded HIV-1. We present a method for evaluating evidence of clonal expansion in the context of these findings. PMID:27494508

  19. Nutrient Regulation by Continuous Feeding Removes Limitations on Cell Yield in the Large-Scale Expansion of Mammalian Cell Spheroids

    PubMed Central

    Weegman, Bradley P.; Nash, Peter; Carlson, Alexandra L.; Voltzke, Kristin J.; Geng, Zhaohui; Jahani, Marjan; Becker, Benjamin B.; Papas, Klearchos K.; Firpo, Meri T.

    2013-01-01

    Cellular therapies are emerging as a standard approach for the treatment of several diseases. However, realizing the promise of cellular therapies across the full range of treatable disorders will require large-scale, controlled, reproducible culture methods. Bioreactor systems offer the scale-up and monitoring needed, but standard stirred bioreactor cultures do not allow for the real-time regulation of key nutrients in the medium. In this study, β-TC6 insulinoma cells were aggregated and cultured for 3 weeks as a model of manufacturing a mammalian cell product. Cell expansion rates and medium nutrient levels were compared in static, stirred suspension bioreactors (SSB), and continuously fed (CF) SSB. While SSB cultures facilitated increased culture volumes, no increase in cell yields were observed, partly due to limitations in key nutrients, which were consumed by the cultures between feedings, such as glucose. Even when glucose levels were increased to prevent depletion between feedings, dramatic fluctuations in glucose levels were observed. Continuous feeding eliminated fluctuations and improved cell expansion when compared with both static and SSB culture methods. Further improvements in growth rates were observed after adjusting the feed rate based on calculated nutrient depletion, which maintained physiological glucose levels for the duration of the expansion. Adjusting the feed rate in a continuous medium replacement system can maintain the consistent nutrient levels required for the large-scale application of many cell products. Continuously fed bioreactor systems combined with nutrient regulation can be used to improve the yield and reproducibility of mammalian cells for biological products and cellular therapies and will facilitate the translation of cell culture from the research lab to clinical applications. PMID:24204645

  20. Human saphenous vein endothelial cell adhesion and expansion on micropatterned polytetrafluoroethylene.

    PubMed

    Boivin, Marie-Claude; Chevallier, Pascale; Hoesli, Corinne A; Lagueux, Jean; Bareille, Reine; Rémy, Murielle; Bordenave, Laurence; Durrieu, Marie-Christine; Laroche, Gaétan

    2013-03-01

    Intimal hyperplasia and thrombosis are responsible for the poor patency rates of small-diameter vascular grafts. These complications could be avoided by a rapid and strong adhesion of endothelial cells to the prosthetic surfaces, which typically consist of expanded polytetrafluoroethylene (PTFE) for small-diameter vessels. We have previously described two peptide micropatterning strategies that increase the endothelialization rates of PTFE. The micropatterns were generated either by inkjet printing 300 μm squares or by spraying 10.1 ± 0.1 μm diameter droplets of the CGRGDS cell adhesion peptide, while the remaining surface was functionalized using the CWQPPRARI cell migration peptide. We now directly compare these two micropatterning strategies and examine the effect of hydrodynamic stress on human saphenous vein endothelial cells grown on the patterned surfaces. No significant differences in cell adhesion were observed between the two micropatterning methods. When compared to unpatterned surfaces treated with a uniform mixture of the two peptides, the cell expansion was significantly higher on sprayed or printed surfaces after 9 days of static cell culture. In addition, after 6 h of exposure to hydrodynamic stress, the cell retention and cell cytoskeleton reorganization on the patterned surfaces was improved when compared to untreated or random treated surfaces. These results indicate that micropatterned surfaces lead to improved rates of PTFE endothelialization with higher resistance to hydrodynamic stress.

  1. Gαi/o-coupled receptor signaling restricts pancreatic β-cell expansion.

    PubMed

    Berger, Miles; Scheel, David W; Macias, Hector; Miyatsuka, Takeshi; Kim, Hail; Hoang, Phuong; Ku, Greg M; Honig, Gerard; Liou, Angela; Tang, Yunshuo; Regard, Jean B; Sharifnia, Panid; Yu, Lisa; Wang, Juehu; Coughlin, Shaun R; Conklin, Bruce R; Deneris, Evan S; Tecott, Laurence H; German, Michael S

    2015-03-03

    Gi-GPCRs, G protein-coupled receptors that signal via Gα proteins of the i/o class (Gαi/o), acutely regulate cellular behaviors widely in mammalian tissues, but their impact on the development and growth of these tissues is less clear. For example, Gi-GPCRs acutely regulate insulin release from pancreatic β cells, and variants in genes encoding several Gi-GPCRs--including the α-2a adrenergic receptor, ADRA2A--increase the risk of type 2 diabetes mellitus. However, type 2 diabetes also is associated with reduced total β-cell mass, and the role of Gi-GPCRs in establishing β-cell mass is unknown. Therefore, we asked whether Gi-GPCR signaling regulates β-cell mass. Here we show that Gi-GPCRs limit the proliferation of the insulin-producing pancreatic β cells and especially their expansion during the critical perinatal period. Increased Gi-GPCR activity in perinatal β cells decreased β-cell proliferation, reduced adult β-cell mass, and impaired glucose homeostasis. In contrast, Gi-GPCR inhibition enhanced perinatal β-cell proliferation, increased adult β-cell mass, and improved glucose homeostasis. Transcriptome analysis detected the expression of multiple Gi-GPCRs in developing and adult β cells, and gene-deletion experiments identified ADRA2A as a key Gi-GPCR regulator of β-cell replication. These studies link Gi-GPCR signaling to β-cell mass and diabetes risk and identify it as a potential target for therapies to protect and increase β-cell mass in patients with diabetes.

  2. Thymic epithelial cell expansion through matricellular protein CYR61 boosts progenitor homing and T-cell output

    NASA Astrophysics Data System (ADS)

    Emre, Yalin; Irla, Magali; Dunand-Sauthier, Isabelle; Ballet, Romain; Meguenani, Mehdi; Jemelin, Stephane; Vesin, Christian; Reith, Walter; Imhof, Beat A.

    2013-11-01

    Thymic epithelial cells (TEC) are heterogeneous stromal cells that generate microenvironments required for the formation of T cells within the thymus. Defects in TEC lead to immunodeficiency or autoimmunity. Here we identify TEC as the major source of cysteine-rich protein 61 (CYR61), a matricellular protein implicated in cell proliferation and migration. Binding of CYR61 to LFA-1, ICAM-1 and integrin α6 supports the adhesion of TEC and thymocytes as well as their interaction. Treatment of thymic lobes with recombinant CYR61 expands the stromal compartment by inducing the proliferation of TEC and activates Akt signalling. Engraftment of CYR61-overexpressing thymic lobes into athymic nude mice drastically boosts the yield of thymic output via expansion of TEC. This increases the space for the recruitment of circulating hematopoietic progenitors and the development of T cells. Our discovery paves the way for therapeutic interventions designed to restore thymus stroma and T-cell generation.

  3. Expansion of CD56Bright natural killer cells in the peripheral blood of multiple sclerosis patients treated with interferon-beta.

    PubMed

    Saraste, M; Irjala, H; Airas, L

    2007-06-01

    We studied how interferon-beta (IFN-beta) treatment of relapsing-remitting multiple sclerosis (MS) affects subgroups of natural killer cells (NK cells). Following IFN-beta treatment, there was an expansion of CD56(Bright) NK-cells in the peripheral blood of MS patients, while at the same time the proportion of CD56(Dim) cells was diminished. In a control group, the proportion of CD56(Bright) NK-cells was significantly higher in secondary lymphoid tissues compared to the peripheral blood of the same individual. Our findings confirm that CD56(Bright) NK-cells preferably locate within the secondary lymphoid tissues, where they may interact with T cells and thereby contribute to the control of the disease activity in MS.

  4. Klebsiella pneumoniae alleviates influenza-induced acute lung injury via limiting NK cell expansion.

    PubMed

    Wang, Jian; Li, Fengqi; Sun, Rui; Gao, Xiang; Wei, Haiming; Tian, Zhigang

    2014-08-01

    A protective effect induced by bacterial preinfection upon a subsequent lethal influenza virus infection has been observed, but the underlying immune mechanisms have not yet been fully elucidated. In this study, we used a mouse model of Klebsiella pneumoniae preinfection to gain insight into how bacterial preinfection influences the subsequent lethal influenza virus infection. We found that K. pneumoniae preinfection significantly attenuated lung immune injury and decreased mortality during influenza virus infection, but K. pneumoniae-specific immunity was not involved in this cross-protection against influenza virus. K. pneumoniae preinfection limited NK cell expansion, which was involved in influenza-induced immune injury and death. Furthermore, K. pneumoniae preinfection could not control NK cell expansion and death during influenza virus infection in Rag1(-/-) mice, but adoptive transfer of T cells from wild-type mice was able to restore this protective effect. Our data suggest that the adaptive immune response activated by bacterial infection limits the excessive innate immune response induced by a subsequent influenza infection, ultimately protecting mice from death.

  5. CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins

    PubMed Central

    Zurawel, Ashley A.; Kabeche, Ruth; DiGregorio, Sonja E.; Deng, Lin; Menon, Kartikeya M.; Opalko, Hannah

    2016-01-01

    ABSTRACT Proteins containing polyglutamine (polyQ) regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt) with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington’s disease (HD). To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt) in Schizosaccharomyces pombe. In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms. PMID:27677791

  6. Compositional dependence of hematopoietic stem cells expansion on bioceramic composite scaffolds for bone tissue engineering.

    PubMed

    Mishra, Sarika; Rajyalakshmi, A; Balasubramanian, K

    2012-09-01

    Bioceramics are although well known for their osteoinductive and osseointegrative properties in bone tissue regeneration, yet, they are inappropriate for load bearing applications due to inadequate mechanical strength. In this article, the authors report the expansion of hematopoietic stem cells (HSCs) on as-synthesized composite scaffolds from hydroxyapatite and β-tricalcium phosphate for bone tissue engineering, in an adequate load-bearing application. The physical, structural, and mechanical properties of the composite scaffolds have been examined and correlated with the in vitro adhesion pattern of HSCs. The results indicated that the response of HSCs varies with change in the stoichiometry of composite scaffolds. The H2T2 scaffolds have exhibited the highest expansion of CD34+ cells and long-term culture initiating cells when compared with other stoichiometries. The results suggest that H2T2 composite can be a potential strategic bone-graft substitute in contrast with monolithic bioceramics, serving a dual role of bioresorbability and enhanced load-bearing capacity.

  7. Expansion of T Cells with Interleukin-21 for Adoptive Immunotherapy of Murine Mammary Carcinoma

    PubMed Central

    Zoon, Christine K.; Wan, Wen; Graham, Laura; Bear, Harry D.

    2017-01-01

    We previously demonstrated that culturing antigen-sensitized draining lymph node (DLN) lymphocytes from BALB/c mice in interleukin (IL)-7/15 after activation with bryostatin/ionomycin (B/I) is superior to culture in IL-2 for expansion, differentiation to cluster of differentiation (CD)8+ cells and anti-tumor activity. We sought to determine whether the substitution or addition of IL-21 to culture had a similar effect. DLN lymphocytes were antigen-sensitized with 4T1 mammary carcinoma 10 days prior to harvest, activated with B/I, and expanded in culture for 7 days with either IL-2, IL-21, IL-2/21, IL-7/15, or IL-7/15/21. Cellular expansion, phenotype, interferon (IFN)-γ responses, and in vivo anti-tumor activity were compared. We found that T cells grown in IL7/15/21 demonstrated significantly greater lymphocyte expansion than IL-2, IL-21, IL-2/21, and IL-7/15 (38.4-fold vs. 5.5, 6.6, 9.5, and 23.9-fold, respectively). Of these expanded cells, IL-7/15/21 significantly expanded the greatest percentage of CD8+ cells (67.1% vs. 22.2%, 47.2%, 47.4%, and 55.3%, respectively), and the greatest number of T central memory cells (TCM) compared to IL-2, IL-21 and IL-2/21 (45.8% vs. 11.1%, 7.7%, and 12.1%, respectively). IL-21 and IL-2/21-expanded T cells preferentially differentiated into T naïve cells (TN) vs. those expanded in IL-2, IL-7/15 and IL-7/15/21 (27.6% and 23.2% vs. 1.7%, 4.5%, and 10.4%, respectively), and demonstrated the highest IFN-γ levels in vitro. In vivo adoptive immunotherapy (AIT) experiments demonstrated anti-tumor efficacy was equally effective using IL-2, IL-21, IL-2/21, IL-7/15 and IL-7/15/21-cultured lymphocytes vs. control or cyclophosphamide alone, even at lower doses or with greater initial size of tumor prior to treatment. PMID:28146052

  8. Clonal expansion of antitumor T cells in breast cancer correlates with response to neoadjuvant chemotherapy

    PubMed Central

    Park, Jae-Hyun; Jang, Miran; Tarhan, Yunus Emre; Katagiri, Toyomasa; Sasa, Mitsunori; Miyoshi, Yasuo; Kalari, Krishna R.; Suman, Vera J.; Weinshilboum, Richard; Wang, Liewei; Boughey, Judy C.; Goetz, Matthew P.; Nakamura, Yusuke

    2016-01-01

    The immune microenvironment of tumor plays a critical role in therapeutic responses to chemotherapy. Cancer tissues are composed of a complex network between anti-tumor and pro-tumor immune cells and molecules; therefore a comprehensive analysis of the tumor immune condition is imperative for better understanding of the roles of the immune microenvironment in anticancer treatment response. In this study, we performed T cell receptor (TCR) repertoire analysis of tumor infiltrating T cells (TILs) in cancer tissues of pre- and post-neoadjuvant chemotherapy (NAC) from 19 breast cancer patients; five cases showed CR (complete response), ten showed PR (partial response), and four showed SD/PD (stable disease/progressive disease) to the treatment. From the TCR sequencing results, we calculated the diversity index of the TCRβ chain and found that clonal expansion of TILs could be detected in patients who showed CR or PR to NAC. Noteworthy, the diversity of TCR was further reduced in the post-NAC tumors of CR patients. Our quantitative RT-PCR also showed that expression ratio of CD8/Foxp3 was significantly elevated in the post-NAC tumors of CR cases (p=0.0032), indicating that antitumor T cells were activated and enriched in these tumors. Collectively, our findings suggest that the clonal expansion of antitumor T cells may be a critical factor associated with response to chemotherapy and that their TCR sequences might be applicable for the development of TCR-engineered T cells treatment for individual breast cancer patients when their tumors relapse. PMID:27278091

  9. IL-10 conditioning of human skin affects the distribution of migratory dendritic cell subsets and functional T cell differentiation.

    PubMed

    Lindenberg, Jelle J; Oosterhoff, Dinja; Sombroek, Claudia C; Lougheed, Sinéad M; Hooijberg, Erik; Stam, Anita G M; Santegoets, Saskia J A M; Tijssen, Henk J; Buter, Jan; Pinedo, Herbert M; van den Eertwegh, Alfons J M; Scheper, Rik J; Koenen, Hans J P M; van de Ven, Rieneke; de Gruijl, Tanja D

    2013-01-01

    In cancer patients pervasive systemic suppression of Dendritic Cell (DC) differentiation and maturation can hinder vaccination efficacy. In this study we have extensively characterized migratory DC subsets from human skin and studied how their migration and T cell-stimulatory abilities were affected by conditioning of the dermal microenvironment through cancer-related suppressive cytokines. To assess effects in the context of a complex tissue structure, we made use of a near-physiological skin explant model. By 4-color flow cytometry, we identified migrated Langerhans Cells (LC) and five dermis-derived DC populations in differential states of maturation. From a panel of known tumor-associated suppressive cytokines, IL-10 showed a unique ability to induce predominant migration of an immature CD14(+)CD141(+)DC-SIGN(+) DC subset with low levels of co-stimulatory molecules, up-regulated expression of the co-inhibitory molecule PD-L1 and the M2-associated macrophage marker CD163. A similarly immature subset composition was observed for DC migrating from explants taken from skin overlying breast tumors. Whereas predominant migration of mature CD1a(+) subsets was associated with release of IL-12p70, efficient Th cell expansion with a Th1 profile, and expansion of functional MART-1-specific CD8(+) T cells, migration of immature CD14(+) DDC was accompanied by increased release of IL-10, poor expansion of CD4(+) and CD8(+) T cells, and skewing of Th responses to favor coordinated FoxP3 and IL-10 expression and regulatory T cell differentiation and outgrowth. Thus, high levels of IL-10 impact the composition of skin-emigrated DC subsets and appear to favor migration of M2-like immature DC with functional qualities conducive to T cell tolerance.

  10. Narrow-Band Ultraviolet B Phototherapy Ameliorates Acute Graft-Versus-Host Disease of the Intestine by Expansion of Regulatory T Cells.

    PubMed

    Hashimoto, Akari; Sato, Tsutomu; Iyama, Satoshi; Yoshida, Masahiro; Ibata, Soushi; Tatekoshi, Ayumi; Kamihara, Yusuke; Horiguchi, Hiroto; Murase, Kazuyuki; Kawano, Yutaka; Takada, Kohichi; Miyanishi, Koji; Kobune, Masayoshi; Ichimiya, Shingo; Kato, Junji

    2016-01-01

    Narrowband ultraviolet B (NB-UVB) has been widely used in dermatological phototherapy. As for the application of NB-UVB phototherapy to graft-versus-host disease (GVHD), we previously reported that it was highly efficacious for cutaneous lesions of acute GVHD (aGVHD) and that expansion of regulatory T (Treg) cells induced by NB-UVB might be one of the mechanisms. In order to examine whether NB-UVB irradiation through expansion of Treg cells is effective for the treatment of not only cutaneous aGVHD but also aGVHD of inner organs such as the intestine or liver, we conducted experiments in which a murine lethal aGVHD model, characterized by severe involvement of the intestine, was irradiated with NB-UVB. We found that NB-UVB irradiation improved the clinical score and survival rate. The pathological score of aGVHD was improved in all affected organs: intestine, liver, and skin. In the serum of mice irradiated with NB-UVB, the levels of Treg cells-associated cytokines such as transforming growth factor beta (TGFβ) and interleukin-10 (IL-10) were elevated. The numbers of infiltrating Treg cells in inflamed tissue of the intestine and those in spleen were increased in mice treated with NB-UVB. This is the first report demonstrating that NB-UVB phototherapy has the ability to ameliorate intestinal aGVHD through the expansion of Treg cells.

  11. Essential Role for Survivin in the Proliferative Expansion of Progenitor and Mature B Cells.

    PubMed

    Miletic, Ana V; Jellusova, Julia; Cato, Matthew H; Lee, Charlotte R; Baracho, Gisele V; Conway, Edward M; Rickert, Robert C

    2016-03-01

    Survivin is a member of the inhibitor of apoptosis family of proteins and a biomarker of poor prognosis in aggressive B cell non-Hodgkin's lymphoma. In addition to its role in inhibition of apoptosis, survivin also regulates mitosis. In this article, we show that deletion of survivin during early B cell development results in a complete block at the cycling pre-B stage. In the periphery, B cell homeostasis is not affected, but survivin-deficient B cells are unable to mount humoral responses. Correspondingly, we show that survivin is required for cell division in response to mitogenic stimulation. Thus, survivin is essential for proliferation of B cell progenitors and activated mature B cells, but is dispensable for B cell survival. Moreover, a small-molecule inhibitor of survivin strongly impaired the growth of representative B lymphoma lines in vitro, supporting the validity of survivin as an attractive therapeutic target for high-grade B cell non-Hodgkin's lymphoma.

  12. Aging affects initiation and continuation of T cell proliferation.

    PubMed

    Jiang, Jiu; Gross, Diara; Elbaum, Philip; Murasko, Donna M

    2007-04-01

    Aging is associated with a decline in immune responses, particularly within the T cell compartment. While the expansion of specific T cells in response to virus infections is consistently decreased in aged mice, the differences in T cell activation between young and aged mice as demonstrated in each round of proliferation remain poorly defined. In the present study, we utilized the T cell mitogen, ConA, to explore if fewer T cells of aged mice initiate proliferation upon mitogen stimulation or if similar numbers of T cells of aged mice begin proliferation but undergo fewer rounds of division. We also examined whether these age-associated changes in proliferation are reflected by differences in T cell activation by comparing activation markers (CD25, CD69, CD44, and CD62L) on T cells of young and aged mice at each round of proliferation. Not only was the kinetics of the expression of these markers greatly different between young and aged mice on the entire CD8 T cell population, but also at each round of proliferation. Our results demonstrate that a larger percentage of CD8 T cells of aged mice do not proliferate at all upon stimulation. Of the CD8 T cells of aged mice that do proliferate, a larger percentage start later and stop sooner. These results suggest that multiple levels of alteration may need to be considered when trying to maximize the immune response of aged individuals.

  13. TNFα facilitates clonal expansion of JAK2V617F positive cells in myeloproliferative neoplasms

    PubMed Central

    Aichberger, Karl J.; Luty, Samuel B.; Bumm, Thomas G.; Petersen, Curtis L.; Doratotaj, Shirin; Vasudevan, Kavin B.; LaTocha, Dorian H.; Yang, Fei; Press, Richard D.; Loriaux, Marc M.; Pahl, Heike L.; Silver, Richard T.; Agarwal, Anupriya; O'Hare, Thomas; Druker, Brian J.; Bagby, Grover C.

    2011-01-01

    Proinflammatory cytokines such as TNFα are elevated in patients with myeloproliferative neoplasms (MPN), but their contribution to disease pathogenesis is unknown. Here we reveal a central role for TNFα in promoting clonal dominance of JAK2V617F expressing cells in MPN. We show that JAK2V617F kinase regulates TNFα expression in cell lines and primary MPN cells and TNFα expression is correlated with JAK2V617F allele burden. In clonogenic assays, normal controls show reduced colony formation in the presence of TNFα while colony formation by JAK2V617F-positive progenitor cells is resistant or stimulated by exposure to TNFα. Ectopic JAK2V617F expression confers TNFα resistance to normal murine progenitor cells and overcomes inherent TNFα hypersensitivity of Fanconi anemia complementation group C deficient progenitors. Lastly, absence of TNFα limits clonal expansion and attenuates disease in a murine model of JAK2V617F-positive MPN. Altogether our data are consistent with a model where JAK2V617F promotes clonal selection by conferring TNFα resistance to a preneoplastic TNFα sensitive cell, while simultaneously generating a TNFα-rich environment. Mutations that confer resistance to environmental stem cell stressors are a recognized mechanism of clonal selection and leukemogenesis in bone marrow failure syndromes and our data suggest that this mechanism is also critical to clonal selection in MPN. PMID:21860020

  14. Proteome analysis of human Wharton's jelly cells during in vitro expansion

    PubMed Central

    2010-01-01

    Background The human umbilical cord contains mucoid connective tissue and fibroblast-like cells. These cells named Wharton's jelly cells, (WJCs) display properties similar to mesenchymal stem cells therefore representing a rich source of primitive cells to be potentially used in regenerative medicine. Results To better understand their self-renewal and potential in vitro expansion capacity, a reference 2D map was constructed as a proteomic data set. 158 unique proteins were identified. More than 30% of these proteins belong to cytoskeleton compartment. We also found that several proteins including Shootin1, Adenylate kinase 5 isoenzyme and Plasminogen activator-inhibitor 2 are no longer expressed after the 2nd passage of in vitro replication. This indicates that the proliferative potency of these cells is reduced after the initial stage of in vitro growing. At the end of cellular culturing, new synthesized proteins, including, ERO1-like protein alpha, Aspartyl-tRNA synthetase and Prolyl-4-hydroxylase were identified. It is suggested that these new synthesized proteins are involved in the impairment of cellular surviving during replication and differentiation time. Conclusions Our work represents an essential step towards gaining knowledge of the molecular properties of WJCs so as to better understand their possible use in the field of cell therapy and regenerative medicine. PMID:20346146

  15. Cumulus cell-expressed type I interferons induce cumulus expansion in mice.

    PubMed

    Jang, You-Jee; Park, Jae-Il; Moon, Won-Jin; Dam, Phuong T M; Cho, Moon-Kyoung; Chun, Sang-Young

    2015-01-01

    Ovulation resembles the inflammatory response. The purpose of the present study was to examine the expression and role of type I interferons (IFNs) Ifnalpha and Ifnbeta in mouse ovaries during the process of ovulation. An in vivo injection of equine chorionic gonadotropin (CG)-human CG (hCG) stimulated Ifnalpha and Ifnbeta mRNA in cumulus-oocyte complexes (COCs) within 6 h. Type I IFN receptor (Ifnar1 and Ifnar2) genes were also expressed in preovulatory follicles without a change by hCG. Immunofluorescent study revealed the expression of protein signals of Ifnalpha, Ifnbeta, and Ifnar1 in cumulus cells. Treatment of COCs with Ifnalpha or Ifnbeta in vitro induced cumulus expansion that was comparable to that mediated by epiregulin. In cultured COCs, the levels of Ifnalpha and Ifnbeta mRNA increased by epiregulin and follicle-stimulating hormone, but not by prostaglandin E2. Ifnalpha and Ifnbeta activated multiple signaling events (signal transducer and activator of transcription-1/3, Akt, and mitogen-activated protein kinase 1/2) and stimulated the expression of genes known to impact COC expansion (Has2, Ptx3, Tnfaip6, and Ptgs2). Interestingly, treatment of COCs with Toll-like receptor (TLR) 2 and TLR4 ligands (lipopolysaccharides, Pam3Cys, and hyaluronan fragments) increased Ifnalpha and Ifnbeta mRNA, while coculture with anti-TLR2/4 neutralizing antibody abolished these effects. Taken together, these results demonstrate that the type I IFN system is operating in mouse cumulus cells and plays a role in the induction of cumulus expansion during the ovulatory process in mice.

  16. Brain and muscle Arnt-like 1 promotes skeletal muscle regeneration through satellite cell expansion

    SciTech Connect

    Chatterjee, Somik; Yin, Hongshan; Nam, Deokhwa; Li, Yong; Ma, Ke

    2015-02-01

    Circadian clock is an evolutionarily conserved timing mechanism governing diverse biological processes and the skeletal muscle possesses intrinsic functional clocks. Interestingly, although the essential clock transcription activator, Brain and muscle Arnt-like 1 (Bmal1), participates in maintenance of muscle mass, little is known regarding its role in muscle growth and repair. In this report, we investigate the in vivo function of Bmal1 in skeletal muscle regeneration using two muscle injury models. Bmal1 is highly up-regulated by cardiotoxin injury, and its genetic ablation significantly impairs regeneration with markedly suppressed new myofiber formation and attenuated myogenic induction. A similarly defective regenerative response is observed in Bmal1-null mice as compared to wild-type controls upon freeze injury. Lack of satellite cell expansion accounts for the regeneration defect, as Bmal1{sup −/−} mice display significantly lower satellite cell number with nearly abolished induction of the satellite cell marker, Pax7. Furthermore, satellite cell-derived primary myoblasts devoid of Bmal1 display reduced growth and proliferation ex vivo. Collectively, our results demonstrate, for the first time, that Bmal1 is an integral component of the pro-myogenic response that is required for muscle repair. This mechanism may underlie its role in preserving adult muscle mass and could be targeted therapeutically to prevent muscle-wasting diseases. - Highlights: • Bmal1 is highly inducible by muscle injury and myogenic stimuli. • Genetic ablation of Bmal1 significantly impairs muscle regeneration. • Bmal1 promotes satellite cell expansion during muscle regeneration. • Bmal1-deficient primary myoblasts display attenuated growth and proliferation.

  17. Expansion of monocytic myeloid-derived suppressor cells dampens T cell function in HIV-1-seropositive individuals.

    PubMed

    Qin, Aiping; Cai, Weiping; Pan, Ting; Wu, Kang; Yang, Qiong; Wang, Nina; Liu, Yufeng; Yan, Dehong; Hu, Fengyu; Guo, Pengle; Chen, Xiaoping; Chen, Ling; Zhang, Hui; Tang, Xiaoping; Zhou, Jie

    2013-02-01

    T lymphocyte dysfunction contributes to human immunodeficiency virus type 1 (HIV-1) disease progression by impairing antivirus cellular immunity. However, the mechanisms of HIV-1 infection-mediated T cell dysfunction are not completely understood. Here, we provide evidence that expansion of monocytic myeloid-derived suppressor cells (M-MDSCs) suppressed T cell function in HIV-1-infected individuals. We observed a dramatic elevation of M-MDSCs (HLA-DR(-/low) CD11b(+) CD33(+/high) CD14(+) CD15(-) cells) in the peripheral blood of HIV-1-seropositive subjects (n = 61) compared with healthy controls (n = 51), despite efficacious antiretroviral therapy for nearly 2 years. The elevated M-MDSC frequency in HIV-1(+) subjects correlated with prognostic HIV-1 disease markers, including the HIV-1 load (r = 0.5957; P < 0.0001), CD4(+) T cell loss (r = -0.5312; P < 0.0001), and activated T cells (r = 0.4421; P = 0.0004). Functional studies showed that M-MDSCs from HIV-1(+) subjects suppressed T cell responses in both HIV-1-specific and antigen-nonspecific manners; this effect was dependent on the induction of arginase 1 and required direct cell-cell contact. Further investigations revealed that direct HIV-1 infection or culture with HIV-1-derived Tat protein significantly enhanced human MDSC generation in vitro, and MDSCs from healthy donors could be directly infected by HIV-1 to facilitate HIV-1 replication and transmission, indicating that a positive-feedback loop between HIV-1 infection and MDSC expansion existed. In summary, our studies revealed a novel mechanism of T cell dysfunction in HIV-1-infected individuals and suggested that targeting MDSCs may be a promising strategy for HIV-1 immunotherapy.

  18. Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

    PubMed Central

    Gowers, Kate H. C.; Lee, Dani Do Hyang; Brown, James M.; Crowley, Claire; Teixeira, Vitor H.; Smith, Claire M.; Urbani, Luca; Hamilton, Nicholas J.; Thakrar, Ricky M.; Booth, Helen L.; Birchall, Martin A.; De Coppi, Paolo; Giangreco, Adam; O’Callaghan, Christopher

    2016-01-01

    Rationale: Stem cell–based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell–seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. Objectives: To define a scalable cell culture system to deliver airway epithelium to clinical grafts. Methods: Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air–liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air–liquid interface cultures. Measurements and Main Results: 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. Conclusions: Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical

  19. Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications.

    PubMed

    Doucet, Christelle; Ernou, Isabelle; Zhang, Yizhou; Llense, Jean-Roch; Begot, Laurent; Holy, Xavier; Lataillade, Jean-Jacques

    2005-11-01

    Mesenchymal stem cells (MSCs) are considered as emergent "universal" cells and various tissue repair programs using MSCs are in development. In vitro expansion of MSCs is conventionally achieved in medium containing fetal calf serum (FCS) and is increased by addition of growth factors. However, for widespread clinical applications, contact of MSCs with FCS must be minimized since it is a putative source of prion or virus transmission. Therefore, because platelets are a natural source of growth factors, we sought to investigate in vitro MSC expansion in response to platelet lysates (PL) obtained from platelet-rich plasma. Human MSCs were expanded in FCS (+/-bFGF)- or PL-supplemented medium through a process of subculture. We demonstrated that PL-containing medium is enriched by growth factors (platelet-derived growth factors (PDGFs), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-beta), insulin-like growth factor-1 (IGF-1) ...) and showed that PL is able to promote MSC expansion, to decrease the time required to reach confluence, and to increase CFU-F size, as compared to the FCS medium. Furthermore, we demonstrated that MSCs cultured in the presence of PL maintain their osteogenic, chondrogenic, and adipogenic differentiation properties and retain their immunosuppressive activity. Therefore, we propose that PL may be a powerful and safe substitute for FCS in development of tissue- and cellular-engineered products in clinical settings using MSCs.

  20. Making the Switch: Alternatives to Fetal Bovine Serum for Adipose-Derived Stromal Cell Expansion

    PubMed Central

    Dessels, Carla; Potgieter, Marnie; Pepper, Michael S.

    2016-01-01

    Adipose-derived stromal cells (ASCs) are being used extensively in clinical trials. These trials require that ASCs are prepared using good manufacturing practices (GMPs) and are safe for use in humans. The majority of clinical trials in which ASCs are expanded make use of fetal bovine serum (FBS). While FBS is used traditionally in the research setting for in vitro expansion, it does carry the risk of xenoimmunization and zoonotic transmission when used for expanding cells destined for therapeutic purposes. In order to ensure a GMP quality product for cellular therapy, in vitro expansion of ASCs has been undertaken using xeno-free (XF), chemically-defined, and human blood-derived alternatives. These investigations usually include the criteria proposed by the International Society of Cellular Therapy (ISCT) and International Fat Applied Technology Society (IFATS). The majority of studies use these criteria to compare plastic-adherence, morphology, the immunophenotype and the trilineage differentiation of ASCs under the different medium supplemented conditions. Based on these studies, all of the alternatives to FBS seem to be suitable replacements; however, each has its own advantages and drawbacks. Very few studies have investigated the effects of the supplements on the immunomodulation of ASCs; the transcriptome, proteome and secretome; and the ultimate effects in appropriate animal models. The selection of medium supplementation will depend on the downstream application of the ASCs and their efficacy and safety in preclinical studies. PMID:27800478

  1. Progenitor cell expansion and impaired hepatocyte regeneration in explanted livers from alcoholic hepatitis

    PubMed Central

    Dubuquoy, Laurent; Louvet, Alexandre; Lassailly, Guillaume; Truant, Stéphanie; Boleslawski, Emmanuel; Artru, Florent; Maggiotto, François; Gantier, Emilie; Buob, David; Leteurtre, Emmanuelle; Cannesson, Amélie; Dharancy, Sébastien; Moreno, Christophe; Pruvot, François-René; Bataller, Ramon; Mathurin, Philippe

    2015-01-01

    Objective In alcoholic hepatitis (AH), development of targeted therapies is crucial and requires improved knowledge of cellular and molecular drivers in liver dysfunction. The unique opportunity of using explanted livers from patients with AH having undergone salvage liver transplantation allowed to perform more in-depth molecular translational studies. Design We studied liver explants from patients with AH submitted to salvage transplantation (n=16), from patients with alcoholic cirrhosis without AH (n=12) and fragments of normal livers (n=16). Hepatic cytokine content was quantified. Hepatocyte function and proliferation and the presence of hepatic progenitor cells (HPCs) were evaluated by immunohistochemistry, western blot or quantitative PCR. Mitochondrial morphology was evaluated by electron microscopy. Results Livers from patients with AH showed decreased cytokine levels involved in liver regeneration (tumour necrosis factor α and interleukin-6), as well as a virtual absence of markers of hepatocyte proliferation compared with alcoholic cirrhosis and normal livers. Electron microscopy revealed obvious mitochondrial abnormalities in AH hepatocytes. Importantly, livers from patients with AH showed substantial accumulation of HPCs that, unexpectedly, differentiate only into biliary cells. AH livers predominantly express laminin (extracellular matrix protein favouring cholangiocyte differentiation); consequently, HPC expansion is inefficient at yielding mature hepatocytes. Conclusions AH not responding to medical therapy is associated with lack of expression of cytokines involved in liver regeneration and profound mitochondrial damage along with lack of proliferative hepatocytes. Expansion of HPCs is inefficient to yield mature hepatocytes. Manoeuvres aimed at promoting differentiation of HPCs into mature hepatocytes should be tested in AH. PMID:25731872

  2. Suppression of IL-7-dependent Effector T-cell Expansion by Multipotent Adult Progenitor Cells and PGE2

    PubMed Central

    Reading, James L; Vaes, Bart; Hull, Caroline; Sabbah, Shereen; Hayday, Thomas; Wang, Nancy S; DiPiero, Anthony; Lehman, Nicholas A; Taggart, Jen M; Carty, Fiona; English, Karen; Pinxteren, Jef; Deans, Robert; Ting, Anthony E; Tree, Timothy I M

    2015-01-01

    T-cell depletion therapy is used to prevent acute allograft rejection, treat autoimmunity and create space for bone marrow or hematopoietic cell transplantation. The evolved response to T-cell loss is a transient increase in IL-7 that drives compensatory homeostatic proliferation (HP) of mature T cells. Paradoxically, the exaggerated form of this process that occurs following lymphodepletion expands effector T-cells, often causing loss of immunological tolerance that results in rapid graft rejection, autoimmunity, and exacerbated graft-versus-host disease (GVHD). While standard immune suppression is unable to treat these pathologies, growing evidence suggests that manipulating the incipient process of HP increases allograft survival, prevents autoimmunity, and markedly reduces GVHD. Multipotent adult progenitor cells (MAPC) are a clinical grade immunomodulatory cell therapy known to alter γ-chain cytokine responses in T-cells. Herein, we demonstrate that MAPC regulate HP of human T-cells, prevent the expansion of Th1, Th17, and Th22 effectors, and block the development of pathogenic allograft responses. This occurs via IL-1β-primed secretion of PGE2 and activates T-cell intrinsic regulatory mechanisms (SOCS2, GADD45A). These data provide proof-of-principle that HP of human T-cells can be targeted by cellular and molecular therapies and lays a basis for the development of novel strategies to prevent immunopathology in lymphodepleted patients. PMID:26216515

  3. Endothelin-1 supports clonal derivation and expansion of cardiovascular progenitors derived from human embryonic stem cells.

    PubMed

    Soh, Boon-Seng; Ng, Shi-Yan; Wu, Hao; Buac, Kristina; Park, Joo-Hye C; Lian, Xiaojun; Xu, Jiejia; Foo, Kylie S; Felldin, Ulrika; He, Xiaobing; Nichane, Massimo; Yang, Henry; Bu, Lei; Li, Ronald A; Lim, Bing; Chien, Kenneth R

    2016-03-08

    Coronary arteriogenesis is a central step in cardiogenesis, requiring coordinated generation and integration of endothelial cell and vascular smooth muscle cells. At present, it is unclear whether the cell fate programme of cardiac progenitors to generate complex muscular or vascular structures is entirely cell autonomous. Here we demonstrate the intrinsic ability of vascular progenitors to develop and self-organize into cardiac tissues by clonally isolating and expanding second heart field cardiovascular progenitors using WNT3A and endothelin-1 (EDN1) human recombinant proteins. Progenitor clones undergo long-term expansion and differentiate primarily into endothelial and smooth muscle cell lineages in vitro, and contribute extensively to coronary-like vessels in vivo, forming a functional human-mouse chimeric circulatory system. Our study identifies EDN1 as a key factor towards the generation and clonal derivation of ISL1(+) vascular intermediates, and demonstrates the intrinsic cell-autonomous nature of these progenitors to differentiate and self-organize into functional vasculatures in vivo.

  4. Strategies to direct the enrichment, expansion, and recruitment of regulatory cells for the treatment of disease

    PubMed Central

    Glowacki, Andrew J.; Gottardi, Riccardo; Yoshizawa, Sayuri; Cavalla, Franco; Garlet, Gustavo P.; Sfeir, Charles; Little, Steven R.

    2014-01-01

    Disease and injury perturb the balance of processes associated with inflammation and tissue remodeling, resulting in positive feedback loops, exacerbation of disease and compromised tissue repair. Conversely, under homeostatic healthy conditions, these processes are tightly regulated through the expansion and/or recruitment of specific cell populations, promoting a balanced steady-state. Better understanding of these regulatory processes and recent advances in biomaterials and biotechnology have prompted strategies to utilize cells for the treatment and prevention of disease through regulation of inflammation and promotion of tissue repair. Herein, we describe how cells that regulate these processes can be increased in prevalence at a site of disease or injury. We review several relevant cell therapy approaches as well as new strategies for directing endogenous regulatory cells capable of promoting environmental homeostasis and even the establishment of a pro-regenerative micro-environment. Collectively, these examples may provide a blueprint for next-generation “medicine” that spurs the body’s own cells to action and replaces conventional drugs. PMID:25245220

  5. Ex vivo nanofiber expansion and genetic modification of human cord blood-derived progenitor/stem cells enhances vasculogenesis.

    PubMed

    Das, Hiranmoy; Abdulhameed, Nasreen; Joseph, Matthew; Sakthivel, Ramasamy; Mao, Hai-Quan; Pompili, Vincent J

    2009-01-01

    The stem cell therapy for treating ischemic diseases is promising; however, the limited availability and compromised quality of progenitor cells in aged and diseased patients limit its therapeutic use. Here we report a nanofiber-based ex vivo stem cell expansion technology and proangiogenic growth factors overexpression of human umbilical cord blood (UCB)-derived progenitor cells to enhance angiogenic potential of therapeutic stem cells. The progenitor cells were expanded approximately 225-fold on nanofiber-based serum-free ex vivo expansion culture technique without inducing differentiation. The expanded cells express high levels of stem cell homing receptor, CXCR4, and adhesion molecule, LFA-1. The nanofiber-expanded stem cells uptake AcLDL effectively, and migrate efficiently in an in vitro transmigration assay. These expanded cells can also differentiate into endothelial and smooth muscle cells in vitro. In a NOD/SCID mouse hind limb vascular injury model, nanofiber-expanded cells were more effective in blood flow restoration and this effect was further augmented by VEGF(164) and PDGF-BB, growth factor overexpression. The data indicate that nanofiber-based ex vivo expansion technology can provide an essential number of therapeutic stem cells. Additionally, proangiogenic growth factors overexpression in progenitor cells can potentially improve autologous or allogeneic stem cell therapy for ischemic diseases.

  6. Th17 cells are refractory to senescence and retain robust antitumor activity after long-term ex vivo expansion

    PubMed Central

    Bowers, Jacob S.; Nelson, Michelle H.; Majchrzak, Kinga; Bailey, Stefanie R.; Rohrer, Baerbel; Kaiser, Andrew D.M.; Atkinson, Carl; Paulos, Chrystal M.

    2017-01-01

    Adoptive immunotherapy for solid tumors relies on infusing large numbers of T cells to mediate successful antitumor responses in patients. While long-term rapid-expansion protocols (REPs) produce sufficient numbers of CD8+ T cells for treatment, they also cause decline in the cell’s therapeutic fitness. In contrast, we discovered that IL-17–producing CD4+ T cells (Th17 cells) do not require REPs to expand 5,000-fold over 3 weeks. Also, unlike Th1 cells, Th17 cells do not exhibit hallmarks of senescence or apoptosis, retaining robust antitumor efficacy in vivo. Three-week-expanded Th17 cells eliminated melanoma as effectively as Th17 cells expanded for 1 week when infused in equal numbers into mice. However, treating mice with large recalcitrant tumors required the infusion of all cells generated after 2 or 3 weeks of expansion, while the cell yield obtained after 1-week expansion was insufficient. Long-term-expanded Th17 cells also protected mice from tumor rechallenge including lung metastasis. Importantly, 2-week-expanded human chimeric antigen receptor–positive (CAR+) Th17 cells also retained their ability to regress human mesothelioma, while CAR+ Th1 cells did not. Our results indicate that tumor-reactive Th17 cells are an effective cell therapy for cancer, remaining uncompromised when expanded for a long duration owing to their resistance to senescence. PMID:28289713

  7. Rapid regulatory control of plant cell expansion and wall relaxation. Annual technical progress report

    SciTech Connect

    Cosgrove, D.J.

    1991-08-14

    The aim of this project is to elucidate the biophysical and cellular mechanisms that control plant cell expansion. At present we are attempting to characterize the kinetics of the system(s) responsible for regulatory and compensatory behavior of growing cells and tissues. This work is significantly because it indicates that biochemical loosening and biophysical stress relaxation of the wall are part of a feedback loop controlling growth. This report briefly summarizes the efforts and results of the past 12 months. In large part, we have been trying to analyze the nature of growth rate ``noise,`` i.e. spontaneous and often erratic variations in growth rate. We are obtaining evidence that such ``noise`` is not random, but rather reveals an underlying growth mechanism with complex dynamics.

  8. Persistent expansion of CD4+ effector memory T cells in Wegener's granulomatosis.

    PubMed

    Abdulahad, W H; van der Geld, Y M; Stegeman, C A; Kallenberg, C G M

    2006-09-01

    In order to test the hypothesis that Wegener's granulomatosis (WG) is associated with an ongoing immune effector response, even in remission, we examined the distribution of peripheral naive and memory T-lymphocytes in this disease, and analyzed the function-related phenotypes of the memory T-cell population. Peripheral blood mononuclear cells (PBMCs) were freshly isolated from WG-patients in remission (R-WG, n=40), active WG-patients (A-WG, n=17), and age-matched healthy controls (HCs, n=21). Expression of CD4, CD8, CD45RO, CCR7, interleukin (IL)-18Ralpha, ST2L, and FoxP3 were determined by four-color flow cytometric analysis. CD45RO and CCR7 were used for distinction between naive and memory T cells, IL-18Ralpha, ST2L, and FoxP3 for the assessment of Type1, Type2, and regulatory T-cells, respectively. In R-WG, the CD4+CD45RO+CCR7- effector memory T-cell subpopulation (TEM) was relatively increased, whereas the CD4+CD45RO-CCR7+ naive T-cell population (TNaive) was decreased as compared to HC. The distribution of naive and memory CD8+T cells did not differ between R-WG, A-WG, and HC, nor did CD4+CD45RO+CCR7+ central memory T cells (TCM). In contrast to HC, the percentage of CD4+TNaive cells in R-WG correlated negatively with age, whereas CD4+TEM cells showed a positive correlation. In R-WG, a skewing towards Type2 T cells was observed in CD4+TEM cells. No differences were detected in FoxP3+CD4+TEM cells between R-WG and A-WG, whereas the FoxP3-CD4+TEM cells were increased in R-WG and decreased in A-WG as compared to HC. Collectively, peripheral blood homeostasis of CD4+T cells is disturbed in R-WG with the persistent expansion of non-regulatory CD4+TEM cells. These cells might be involved in relapse and may constitute a target for therapy.

  9. Epicutaneous sensitization results in IgE-dependent intestinal mast cell expansion and food anaphylaxis

    PubMed Central

    Bartnikas, Lisa M.; Gurish, Michael F.; Burton, Oliver T.; Leisten, Sabine; Janssen, Erin; Oettgen, Hans C.; Beaupré, Jacqueline; Lewis, Christopher N.; Austen, K. Frank; Schulte, Stephanie; Hornick, Jason L.; Geha, Raif S.; Oyoshi, Michiko K.

    2013-01-01

    Background Sensitization to food antigen may occur through cutaneous exposure. Objective Test the hypothesis that epicutaneous (EC) sensitization with food antigen predisposes to IgE-mediated anaphylaxis upon oral allergen challenge. Methods BALB/c mice were EC sensitized by repeated application of ovalbumin (OVA) to tape-stripped skin over 7 weeks, or orally immunized with OVA and cholera toxin (CT) weekly for 8 weeks, then orally challenged with OVA. Body temperature was monitored and serum mouse mast cell protease 1 (mMCP-1) level was determined following challenge. Tissue mast cells (MCs) were examined by chloroacetate esterase (CAE) staining. Serum OVA-specific IgE and IgG1 antibodies, and cytokines in supernatants of OVA-stimulated splenocytes, were measured by ELISA. Serum interleukin-4 (IL-4) levels were measured using an in vivo cytokine capture assay (IVCCA). Results EC sensitized mice exhibited expansion of connective tissue MC in the jejunum, increased serum IL-4 levels, and systemic anaphylaxis following oral challenge, as evidenced by decreased body temperature and increased serum mMCP-1 level. Intestinal MC expansion and anaphylaxis were IgE-dependent, as they did not occur in EC sensitized IgE−/− mice. Mice orally immunized with OVA+CT failed to increase serum IL-4 levels, expand their intestinal MCs, or develop anaphylaxis following oral challenge, despite OVA-specific IgE levels and splenocyte cytokine production in response to OVA stimulation, which were comparable to those of EC sensitized mice. Conclusion EC sensitized mice, but not mice orally immunized with antigen+CT, develop expansion of intestinal MCs and IgE-mediated anaphylaxis following single oral antigen challenge. IgE is necessary but not sufficient for food anaphylaxis, and MC expansion in the gut may play an important role in the development of anaphylaxis. Clinical Implications The skin may be an important route of sensitization to food antigens. Avoidance of cutaneous

  10. WVD2 and WDL1 modulate helical organ growth and anisotropic cell expansion in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Yuen, Christen Y L.; Pearlman, Rebecca S.; Silo-Suh, Laura; Hilson, Pierre; Carroll, Kathleen L.; Masson, Patrick H.

    2003-01-01

    Wild-type Arabidopsis roots develop a wavy pattern of growth on tilted agar surfaces. For many Arabidopsis ecotypes, roots also grow askew on such surfaces, typically slanting to the right of the gravity vector. We identified a mutant, wvd2-1, that displays suppressed root waving and leftward root slanting under these conditions. These phenotypes arise from transcriptional activation of the novel WAVE-DAMPENED2 (WVD2) gene by the cauliflower mosaic virus 35S promoter in mutant plants. Seedlings overexpressing WVD2 exhibit constitutive right-handed helical growth in both roots and etiolated hypocotyls, whereas the petioles of WVD2-overexpressing rosette leaves exhibit left-handed twisting. Moreover, the anisotropic expansion of cells is impaired, resulting in the formation of shorter and stockier organs. In roots, the phenotype is accompanied by a change in the arrangement of cortical microtubules within peripheral cap cells and cells at the basal end of the elongation zone. WVD2 transcripts are detectable by reverse transcriptase-polymerase chain reaction in multiple organs of wild-type plants. Its predicted gene product contains a conserved region named "KLEEK," which is found only in plant proteins. The Arabidopsis genome possesses seven other genes predicted to encode KLEEK-containing products. Overexpression of one of these genes, WVD2-LIKE 1, which encodes a protein with regions of similarity to WVD2 extending beyond the KLEEK domain, results in phenotypes that are highly similar to wvd2-1. Silencing of WVD2 and its paralogs results in enhanced root skewing in the wild-type direction. Our observations suggest that at least two members of this gene family may modulate both rotational polarity and anisotropic cell expansion during organ growth.

  11. Stem cell expansion during carcinogenesis in stem cell-depleted conditional telomeric repeat factor 2 null mutant mice.

    PubMed

    Bojovic, B; Ho, H-Y; Wu, J; Crowe, D L

    2013-10-24

    To examine the role of telomeric repeat-binding factor 2 (TRF2) in epithelial tumorigenesis, we characterized conditional loss of TRF2 expression in the basal layer of mouse epidermis. These mice exhibit some characteristics of dyskeratosis congenita, a human stem cell depletion syndrome caused by telomere dysfunction. The epidermis in conditional TRF2 null mice exhibited DNA damage response and apoptosis, which correlated with stem cell depletion. The stem cell population in conditional TRF2 null epidermis exhibited shorter telomeres than those in control mice. Squamous cell carcinomas induced in conditional TRF2 null mice developed with increased latency and slower growth due to reduced numbers of proliferating cells as the result of increased apoptosis. TRF2 null epidermal stem cells were found in both primary and metastatic tumors. Despite the low-grade phenotype of the conditional TRF2 null primary tumors, the number of metastatic lesions was similar to control cancers. Basal cells from TRF2 null tumors demonstrated extreme telomere shortening and dramatically increased numbers of telomeric signals by fluorescence in situ hybridization due to increased genomic instability and aneuploidy in these cancers. DNA damage response signals were detected at telomeres in TRF2 null tumor cells from these mice. The increased genomic instability in these tumors correlated with eightfold expansion of the transformed stem cell population compared with that in control cancers. We concluded that genomic instability resulting from loss of TRF2 expression provides biological advantages to the cancer stem cell population.

  12. Purification and long-term expansion of multipotent endothelial-like cells with potential cardiovascular regeneration.

    PubMed

    Marchal, Juan A; Picón, Manuel; Perán, Macarena; Bueno, Clara; Jiménez-Navarro, Manuel; Carrillo, Esmeralda; Boulaiz, Houria; Rodríguez, Noela; Álvarez, Pablo; Menendez, Pablo; de Teresa, Eduardo; Aránega, Antonia

    2012-03-01

    Endothelial progenitor cells (EPC) represent a relatively rare cell population, and expansion of sufficient cell numbers remains a challenge. Nevertheless, human adipose-derived stem cells (hASC) can be easily isolated and possess the ability to differentiate into endothelial cells. Here, we propose the isolation and characterization of multipotent endothelial-like cells (ME-LC) with the capacity to maintain their vascular progenitor properties for long periods. hASC were isolated from lipoaspirates and cultured through distinct consecutive culture stages for 2 months to enrich ME-LC: first in Dulbecco's modified Eagle's medium-fetal bovine serum (stage I), followed by a stage of culture in absent of fetal bovine serum (stage II), a culture in SFO3 medium (stage III), and, finally, the culture of ME-LC into collagen IV-coated flasks in endothelial growth medium (EGM-2) (stage IV). ME-LC display increased expression levels of endothelial and hematopoietic lineage markers (CD45, KDR, and CXCR4) and EPC markers (CD34 and CD133), whereas the expression of CD31 was barely detectable. Reverse transcription (RT)-polymerase chain reaction assays showed expression of genes involved in early stages of EPC differentiation and decreased expression of genes associated to differentiated EPC (TIE-2, DLL4, and FLT-1). ME-LC formed capillary-like structures when grown on Matrigel, secreted increased levels of stromal cell-derived factor-1 (SDF-1), and showed the ability to migrate attracted by SDF-1, vascular endothelial growth factor, and hematopoietic growth factor cytokines. Importantly, ME-LC retained the capacity to differentiate into cardiomyocyte-like cells. We present a simplified and efficient method to generate large numbers of autologous ME-LC from lipoaspirates-derived hASC, opening up potential cell-based therapies for cardiovascular regenerative medicine.

  13. Expansion of CD11b(+)Ly6G (+)Ly6C (int) cells driven by medroxyprogesterone acetate in mice bearing breast tumors restrains NK cell effector functions.

    PubMed

    Spallanzani, Raúl Germán; Dalotto-Moreno, Tomás; Raffo Iraolagoitia, Ximena Lucía; Ziblat, Andrea; Domaica, Carolina Inés; Avila, Damián Ezequiel; Rossi, Lucas Ezequiel; Fuertes, Mercedes Beatriz; Battistone, María Agustina; Rabinovich, Gabriel Adrián; Salatino, Mariana; Zwirner, Norberto Walter

    2013-12-01

    The progesterone analog medroxyprogesterone acetate (MPA) is widely used as a hormone replacement therapy in postmenopausal women and as contraceptive. However, prolonged administration of MPA is associated with increased incidence of breast cancer through ill-defined mechanisms. Here, we explored whether exposure to MPA during mammary tumor growth affects myeloid-derived suppressor cells (MDSCs; CD11b(+)Gr-1(+), mostly CD11b(+)Ly6G(+)Ly6C(int) and CD11b(+)Ly6G(-)Ly6C(high) cells) and natural killer (NK) cells, potentially restraining tumor immunosurveillance. We used the highly metastatic 4T1 breast tumor (which does not express the classical progesterone receptor and expands MDSCs) to challenge BALB/c mice in the absence or in the presence of MPA. We observed that MPA promoted the accumulation of NK cells in spleens of tumor-bearing mice, but with reduced degranulation ability and in vivo cytotoxic activity. Simultaneously, MPA induced a preferential expansion of CD11b(+)Ly6G(+)Ly6C(int) cells in spleen and bone marrow of 4T1 tumor-bearing mice. In vitro, MPA promoted nuclear mobilization of the glucocorticoid receptor (GR) in 4T1 cells and endowed these cells with the ability to promote a preferential differentiation of bone marrow cells into CD11b(+)Ly6G(+)Ly6C(int) cells that displayed suppressive activity on NK cell degranulation. Sorted CD11b(+)Gr-1(+) cells from MPA-treated tumor-bearing mice exhibited higher suppressive activity on NK cell degranulation than CD11b(+)Gr-1(+) cells from vehicle-treated tumor-bearing mice. Thus, MPA, acting through the GR, endows tumor cells with an enhanced capacity to expand CD11b(+)Ly6G(+)Ly6C(int) cells that subsequently display a stronger suppression of NK cell-mediated anti-tumor immunity. Our results describe an alternative mechanism by which MPA may affect immunosurveillance and have potential implication in breast cancer incidence.

  14. In vivo expansion and activation of γδ T cells as immunotherapy for refractory neuroblastoma

    PubMed Central

    Pressey, Joseph G.; Adams, Julia; Harkins, Lualhati; Kelly, David; You, Zhiying; Lamb, Lawrence S.

    2016-01-01

    expansion of Natural Killer cells. Progressive increases in circulating CD4+ T cells with a regulatory phenotype cells may offset beneficial effects of this therapy. PMID:27684826

  15. Factors Affecting Polymer Electrolyte Fuel Cells Performance and Reproducibility

    SciTech Connect

    Moller-Holst S.

    1998-11-01

    Development of fuel cells is often based on small-scale laboratory studies. Due to limited time and budgets, a minimum number of cells are usually prepared and tested, thus, conclusions about improved performance are often drawn from studies of a few cells. Generally, statistics showing the significance of an effect are seldom reported. In this work a simple PEM fuel cell electrode optimization experiment is used as an example to illustrate the importance of statistical evaluation of factors affecting cell performance. The use of fractional factorial design of experiments to reduce the number of cells that have to be studied is also addressed.

  16. Adventitial lymphatic capillary expansion impacts on plaque T cell accumulation in atherosclerosis

    PubMed Central

    Rademakers, Timo; van der Vorst, Emiel P. C.; Daissormont, Isabelle T. M. N.; Otten, Jeroen J. T.; Theodorou, Kosta; Theelen, Thomas L.; Gijbels, Marion; Anisimov, Andrey; Nurmi, Harri; Lindeman, Jan H. N.; Schober, Andreas; Heeneman, Sylvia; Alitalo, Kari; Biessen, Erik A. L.

    2017-01-01

    During plaque progression, inflammatory cells progressively accumulate in the adventitia, paralleled by an increased presence of leaky vasa vasorum. We here show that next to vasa vasorum, also the adventitial lymphatic capillary bed is expanding during plaque development in humans and mouse models of atherosclerosis. Furthermore, we investigated the role of lymphatics in atherosclerosis progression. Dissection of plaque draining lymph node and lymphatic vessel in atherosclerotic ApoE−/− mice aggravated plaque formation, which was accompanied by increased intimal and adventitial CD3+ T cell numbers. Likewise, inhibition of VEGF-C/D dependent lymphangiogenesis by AAV aided gene transfer of hVEGFR3-Ig fusion protein resulted in CD3+ T cell enrichment in plaque intima and adventitia. hVEGFR3-Ig gene transfer did not compromise adventitial lymphatic density, pointing to VEGF-C/D independent lymphangiogenesis. We were able to identify the CXCL12/CXCR4 axis, which has previously been shown to indirectly activate VEGFR3, as a likely pathway, in that its focal silencing attenuated lymphangiogenesis and augmented T cell presence. Taken together, our study not only shows profound, partly CXCL12/CXCR4 mediated, expansion of lymph capillaries in the adventitia of atherosclerotic plaque in humans and mice, but also is the first to attribute an important role of lymphatics in plaque T cell accumulation and development. PMID:28349940

  17. A Novel Ex Vivo Isolation and Expansion Procedure for Chimeric Antigen Receptor Engrafted Human T Cells

    PubMed Central

    Cartellieri, Marc; Koristka, Stefanie; Arndt, Claudia; Feldmann, Anja; Stamova, Slava; von Bonin, Malte; Töpfer, Katrin; Krüger, Thomas; Geib, Mathias; Michalk, Irene; Temme, Achim; Bornhäuser, Martin; Lindemann, Dirk; Ehninger, Gerhard; Bachmann, Michael P.

    2014-01-01

    Genetically engineered T lymphocytes are a promising option for cancer therapy. Prior to adoptive transfer they have to be expanded in vitro to reach therapeutically sufficient numbers. So far, no universal method exists for selective in vitro expansion of engineered T lymphocytes. In order to overcome this problem and for proof of concept we incorporated a novel unique peptide sequence of ten amino acids as epitope (E-Tag) into the binding domains of two novel chimeric antigen receptors (ECARs) directed against either prostate stem cell antigen (PSCA) for the treatment of prostate cancer (PCa) or CD33 for the treatment of acute myeloide leukemia (AML). The epitope tag then was utilized for expanding ECAR engrafted T cells by triggering the modified T cells via a monoclonal antibody directed against the E-Tag (Emab). Moreover, the E-Tag served as an efficient selection epitope for immunomagnetic isolation of modified T cells to high purity. ECAR engrafted T cells were fully functional and mediated profound anti-tumor effects in the respective models of PCa or AML both in vitro and in vivo. The method can be integrated straightforward into clinical protocols to improve therapeutic efficiency of tumor treatment with CAR modified T lymphocytes. PMID:24699869

  18. An Integrated Miniature Bioprocessing for Personalized Human Induced Pluripotent Stem Cell Expansion and Differentiation into Neural Stem Cells

    PubMed Central

    Lin, Haishuang; Li, Qiang; Lei, Yuguo

    2017-01-01

    Human induced pluripotent stem cells (iPSCs) are ideal cell sources for personalized cell therapies since they can be expanded to generate large numbers of cells and differentiated into presumably all the cell types of the human body in vitro. In addition, patient specific iPSC-derived cells induce minimal or no immune response in vivo. However, with current cell culture technologies and bioprocessing, the cost for biomanufacturing clinical-grade patient specific iPSCs and their derivatives are very high and not affordable for majority of patients. In this paper, we explored the use of closed and miniature cell culture device for biomanufacturing patient specific neural stem cells (NSCs) from iPSCs. We demonstrated that, with the assist of a thermoreversible hydrogel scaffold, the bioprocessing including iPSC expansion, iPSC differentiation into NSCs, the subsequent depletion of undifferentiated iPSCs from the NSCs, and concentrating and transporting the purified NSCs to the surgery room, could be integrated and completed within two closed 15 ml conical tubes. PMID:28057917

  19. Ex Vivo Expansion of Human Hematopoietic Stem Cells by Garcinol, a Potent Inhibitor of Histone Acetyltransferase

    PubMed Central

    Nishino, Taito; Wang, Changshan; Mochizuki-Kashio, Makiko; Osawa, Mitsujiro; Nakauchi, Hiromitsu; Iwama, Atsushi

    2011-01-01

    Background Human cord blood (hCB) is the main source of hematopoietic stem and progenitor cells (HSCs/PCs) for transplantation. Efforts to overcome relative shortages of HSCs/PCs have led to technologies to expand HSCs/PCs ex vivo. However, methods suitable for clinical practice have yet to be fully established. Methodology/Principal Findings In this study, we screened biologically active natural products for activity to promote expansion of hCB HSCs/PCs ex vivo, and identified Garcinol, a plant-derived histone acetyltransferase (HAT) inhibitor, as a novel stimulator of hCB HSC/PC expansion. During a 7-day culture of CD34+CD38– HSCs supplemented with stem cell factor and thrombopoietin, Garcinol increased numbers of CD34+CD38– HSCs/PCs more than 4.5-fold and Isogarcinol, a derivative of Garcinol, 7.4-fold. Furthermore, during a 7-day culture of CD34+ HSCs/PCs, Garcinol expanded the number of SCID-repopulating cells (SRCs) 2.5-fold. We also demonstrated that the capacity of Garcinol and its derivatives to expand HSCs/PCs was closely correlated with their inhibitory effect on HAT. The Garcinol derivatives which expanded HSCs/PCs inhibited the HAT activity and acetylation of histones, while inactive derivatives did not. Conclusions/Significance Our findings identify Garcinol as the first natural product acting on HSCs/PCs and suggest the inhibition of HAT to be an alternative approach for manipulating HSCs/PCs. PMID:21931675

  20. Influence of IL-3 functional fragment on cord blood stem cell ex vivo expansion and differentiation

    PubMed Central

    Ren, Zhihua; Zhang, Yu; Zhang, Yanxi; Jiang, Wenhong; Dai, Wei; Ding, Xinxin

    2016-01-01

    Background Recombinant human interleukin-3 (rhIL-3) is a multiple hematopoietic growth factor, which enhances stem cell expansion and hematopoiesis regeneration in vitro and in vivo, when administrated in combination with other cytokines. However, the structure-function study of rhIL-3 remains rarely studied, so far. The purpose of this study was to recognize the short peptide with similar function as rhIL-3, and assess the hematopoietic efficacy in umbilical cord blood (UCB) stem cell culture as well. Methods Two novel monoclonal antibodies (mAb) (C1 and E1) were generated against rhIL-3 using hybridoma technique. Eleven short peptides were depicted and synthesized to overlap covering the full length sequence of rhIL-3. ELISA was employed to distinguish the antibody-binding peptide from the negative peptides. In addition, the multi-potential hematopoiesis capabilities of the positive peptides were evaluated by adding 25 ng/mL of each peptide to the culture medium of hematopoietic stem cells (HSCs) derived from UCB. Total nucleated cell number and the CD34+ cell number from each individual treatment group were calculated on day 7. Correlated antibodies at 0.5 or 2 molar fold to each peptide were also tested in the stem cell expansion experiment, to further confirm the bioactivity of the peptides. Results Two peptides were recognized by the novel generated antibodies, using ELISA. Peptide 3 and 8 exhibited comparable hematopoiesis potentials, with 25.01±0.14 fold, and 19.89±0.12 fold increase of total nucleated cell number on day 7, respectively, compared with the basal medium control (4.93±0.55 fold). These biological effects were neutralized by adding the corresponding mAb at a dose dependent manner. Conclusions Our results identified two specific regions of rhIL-3 responsible for HSC proliferation and differentiation, which were located from 28 to 49 amino acids (P3), and 107 to 127 amino acids (P8), respectively. The short peptide 3 and 8 might act

  1. Low- and high-LET radiation drives clonal expansion of lung progenitor cells in vivo.

    PubMed

    Farin, Alicia M; Manzo, Nicholas D; Kirsch, David G; Stripp, Barry R

    2015-01-01

    Abundant populations of epithelial progenitor cells maintain the epithelium along the proximal-to-distal axis of the airway. Exposure of lung tissue to ionizing radiation leads to tissue remodeling and potential cancer initiation or progression. However, little is known about the effects of ionizing radiation on airway epithelial progenitor cells. We hypothesized that ionizing radiation exposure will alter the behavior of airway epithelial progenitor cells in a radiation dose- and quality-dependent manner. To address this hypothesis, we cultured primary airway epithelial cells isolated from mice exposed to various doses of 320 kVp X ray or 600 MeV/nucleon (56)Fe ions in a 3D epithelial-fibroblast co-culture system. Colony-forming efficiency of the airway epithelial progenitor cells was assessed at culture day 14. In vivo clonogenic and proliferative potentials of airway epithelial progenitor cells were measured after exposure to ionizing radiation by lineage tracing and IdU incorporation. Exposure to both X rays and (56)Fe resulted in a dose-dependent decrease in the ability of epithelial progenitors to form colonies in vitro. In vivo evidence for increased clonogenic expansion of epithelial progenitors was observed after exposure to both X rays and (56)Fe. Interestingly, we found no significant increase in the epithelial proliferative index, indicating that ionizing radiation does not promote increased turnover of the airway epithelium. Therefore, we propose a model in which radiation induces a dose-dependent decrease in the pool of available progenitor cells, leaving fewer progenitors able to maintain the airway long-term. This work provides novel insights into the effects of ionizing radiation exposure on airway epithelial progenitor cell behavior.

  2. Planning for the impacts of Hadley cell expansion on water supply in Victoria, Australia

    NASA Astrophysics Data System (ADS)

    Post, David; Timbal, Bertrand; Chiew, Francis; Hendon, Harry; Moran, Rae

    2013-04-01

    South-eastern Australia has recently experienced the worst drought of the instrumental record; the so-called Millennium drought (1997-2009). This drought was primarily a cool season phenomenon, with large reductions in rainfall observed during the Austral winter (April to October). This reduction in winter rainfall, combined with an absence of wet years and very wet months led to a reduction in streamflow across the region far greater than would be anticipated based on the 12 percent reduction in mean annual rainfall alone. These conditions have proven particularly challenging for water management and planning processes. Our research has linked the extent, duration and severity of this drought to the ongoing observed expansion of the Southern Hemisphere Hadley cell of 0.5 degrees per decade. This expansion has intensified the sub-tropical ridge over southern Australia, pushing cool season mid-latitude storm tracks further south, leading to a reduction in winter rainfall over the region. Modelling has shown that the expansion of the Hadley cell can only be reproduced if anthropogenic influences (greenhouse gases, aerosols and stratospheric ozone) are included in the models, thus providing evidence that the Millennium drought was at least partly attributable to climate change. Climate model projections also indicate that the expansion of the Hadley Cell is likely to continue. This means that the traditional winter "filling season" for water supply systems in Victoria may be less reliable in the future. However, in the light of the expansion of tropical influences, the cool season rainfall deficit may be offset to some extent by increased rainfall in the warmer months of the year. Given that the Millennium drought has been shown to be at least partly attributable to climate change and the considerable uncertainties associated with projected future changes, water planning and management processes in Victoria have been modified to be more robust, adaptive and flexible

  3. Clinical-scale expansion of CD34(+) cord blood cells amplifies committed progenitors and rapid scid repopulation cells.

    PubMed

    Casamayor-Genescà, Alba; Pla, Arnau; Oliver-Vila, Irene; Pujals-Fonts, Noèlia; Marín-Gallén, Sílvia; Caminal, Marta; Pujol-Autonell, Irma; Carrascal, Jorge; Vives-Pi, Marta; Garcia, Joan; Vives, Joaquim

    2017-03-25

    Umbilical cord blood (UCB) transplantation is associated with long periods of aplastic anaemia. This undesirable situation is due to the low cell dose available per unit of UCB and the immaturity of its progenitors. To overcome this, we present a cell culture strategy aimed at the expansion of the CD34(+) population and the generation of granulocyte lineage-committed progenitors. Two culture products were produced after either 6 or 14days of in vitro expansion, and their characteristics compared to non-expanded UCB CD34(+) controls in terms of phenotype, colony-forming activity and multilineage repopulation potential in NOD-scid IL2Rγ(null) mice. Both expanded cell products maintained rapid SCID repopulation activity similar to the non-expanded control, but 14-day cultured cells showed impaired long term SCID repopulation activity. The process was successfully scaled up to clinically relevant doses of 89×10(6) CD34(+) cells committed to the granulocytic lineage and 3.9×10(9) neutrophil precursors in different maturation stages. Cell yields and biological properties presented by the cell product obtained after 14days in culture were superior and therefore this is proposed as the preferred production setup in a new type of dual transplant strategy to reduce aplastic periods, producing a transient repopulation before the definitive engraftment of the non-cultured UCB unit. Importantly, human telomerase reverse transcriptase activity was undetectable, c-myc expression levels were low and no genetic abnormalities were found, as determined by G-banding karyotype, further confirming the safety of the expanded product.

  4. Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells.

    PubMed

    Osiecki, Michael J; Michl, Thomas D; Kul Babur, Betul; Kabiri, Mahboubeh; Atkinson, Kerry; Lott, William B; Griesser, Hans J; Doran, Michael R

    2015-01-01

    Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm2) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm2 format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.

  5. Proliferating cell nuclear antigen prevents trinucleotide repeat expansions by promoting repeat deletion and hairpin removal

    PubMed Central

    Beaver, Jill M.; Lai, Yanhao; Rolle, Shantell J.; Liu, Yuan

    2017-01-01

    DNA base lesions and base excision repair (BER) within trinucleotide repeat (TNR) tracts modulate repeat instability through the coordination among the key BER enzymes DNA polymerase β, flap endonuclease 1 (FEN1) and DNA ligase I (LIG I). However, it remains unknown whether BER cofactors can also alter TNR stability. In this study, we discovered that proliferating cell nuclear antigen (PCNA), a cofactor of BER, promoted CAG repeat deletion and removal of a CAG repeat hairpin during BER in a duplex CAG repeat tract and CAG hairpin loop, respectively. We showed that PCNA stimulated LIG I activity on a nick across a small template loop during BER in a duplex (CAG)20 repeat tract promoting small repeat deletions. Surprisingly, we found that during BER in a hairpin loop, PCNA promoted reannealing of the upstream flap of a double-flap intermediate, thereby facilitating the formation of a downstream flap and stimulating FEN1 cleavage activity and hairpin removal. Our results indicate that PCNA plays a critical role in preventing CAG repeat expansions by modulating the structures of dynamic DNA via cooperation with BER enzymes. We provide the first evidence that PCNA prevents CAG repeat expansions during BER by promoting CAG repeat deletion and removal of a TNR hairpin. PMID:27793507

  6. Angiotensin II type-1 receptor (AT1R) regulates expansion, differentiation, and functional capacity of antigen-specific CD8+ T cells

    PubMed Central

    Silva-Filho, João Luiz; Caruso-Neves, Celso; Pinheiro, Ana Acacia Sá

    2016-01-01

    Angiotensin II (Ang II) and its receptor AT1 (AT1R), an important effector axis of renin-angiotensin system (RAS), have been demonstrated to regulate T-cell responses. However, these studies characterized Ang II and AT1R effects using pharmacological tools, which do not target only Ang II/AT1R axis. The specific role of AT1R expressed by antigen-specific CD8+ T cells is unknown. Then we immunized transgenic mice expressing a T-cell receptor specific for SIINFEKL epitope (OT-I mice) with sporozoites of the rodent malaria parasite Plasmodium berghei expressing the cytotoxic epitope SIINFEKL. Early priming events after immunization were not affected but the expansion and contraction of AT1R-deficient (AT1R−/−) OT-I cells was decreased. Moreover, they seemed more activated, express higher levels of CTLA-4, PD-1, LAG-3, and have decreased functional capacity during the effector phase. Memory AT1R−/− OT-I cells exhibited higher IL-7Rα expression, activation, and exhaustion phenotypes but less cytotoxic capacity. Importantly, AT1R−/− OT-I cells show better control of blood parasitemia burden and ameliorate mice survival during lethal disease induced by blood-stage malaria. Our study reveals that AT1R in antigen-specific CD8+ T cells regulates expansion, differentiation, and function during effector and memory phases of the response against Plasmodium, which could apply to different infectious agents. PMID:27782175

  7. Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches.

    PubMed

    Abecasis, Bernardo; Aguiar, Tiago; Arnault, Émilie; Costa, Rita; Gomes-Alves, Patricia; Aspegren, Anders; Serra, Margarida; Alves, Paula M

    2017-03-20

    Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. We demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4%) and perfusion at 1.3day(-1) dilution rate to improve hiPSC growth as aggregates in a xeno-free medium. This strategy allowed for increased cell specific growth rate, maximum volumetric concentrations (4.7×10(6)cell/mL) and expansion factors (approximately 19 in total cells), resulting in a 2.6-fold overall improvement in cell yields. Extensive cell characterization, including whole proteomic analysis, was performed to confirm that cells' pluripotent phenotype was maintained during culture. A scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using mechanical dissociation for aggregate disruption and cell passaging. A total expansion factor of 1100 in viable cells was obtained in 11days of culture, while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability after 3 sequential passages in bioreactors.

  8. C9ORF72 intermediate repeat expansion in patients affected by atypical parkinsonian syndromes or Parkinson's disease complicated by psychosis or dementia in a Sardinian population.

    PubMed

    Cannas, Antonino; Solla, Paolo; Borghero, Giuseppe; Floris, Gian Luca; Chio, Adriano; Mascia, Marcello Mario; Modugno, Nicola; Muroni, Antonella; Orofino, Gianni; Di Stefano, Francesca; Calvo, Andrea; Moglia, Cristina; Restagno, Gabriella; Meloni, Mario; Farris, Rita; Ciaccio, Daniela; Puddu, Roberta; Vacca, Melisa Iris; Melis, Rosanna; Murru, Maria Rita; Tranquilli, Stefania; Corongiu, Daniela; Rolesu, Marcella; Cuccu, Stefania; Marrosu, Maria Giovanna; Marrosu, Francesco

    2015-11-01

    The hexanucleotide repeat expansion GGGGCC in the C9ORF72 gene larger than 30 repeats has been identified as a major genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent papers investigated the possible pathogenic role and associated clinical phenotypes of intermediate C9ORF72 repeat expansion ranging between 20 and 30 repeats. Some studies suggested its pathogenicity for typical Parkinson's disease (PD), atypical parkinsonian syndromes, FTD with/without parkinsonism, and ALS with/without parkinsonism or with/without dementia. In our study, we aimed to screen patients affected by atypical parkinsonian syndromes or PD complicated by psychosis or dementia for the presence of C9ORF72 repeat expansions, and in unrelated age- and sex-matched healthy controls. Consecutive unrelated patients with atypical parkinsonian syndromes and patients with PD complicated by psychosis or dementia were included in this study. Atypical parkinsonian syndromes were further divided into two groups: one with patients who met the criteria for the classic forms of atypical parkinsonism [multiple system atrophy (MSA), Lewy body disease (LBD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD)] ;and patients who did not meet the above criteria, named non-classical atypical parkinsonism with or without dementia. Ninety-two unrelated patients (48 men, 44 women) were enrolled. None of the patients was found to be carriers of C9ORF72 repeat expansions with more than 30 repeats. Intermediate 20-30 repeat expansions were detected in four female patients (4.3 %). Three of them presented clinical features of atypical parkinsonian syndromes, two with non-classical atypical parkinsonism and dementia FTD-like, and one with non-classical atypical parkinsonism without dementia. The other patient presented clinical features of typical PD complicated by psychosis. Among 121 control subjects, none presented long or short expansion for the C9ORF

  9. Mesenchymal stem cells derived from adipose tissue are not affected by renal disease.

    PubMed

    Roemeling-van Rhijn, Marieke; Reinders, Marlies E J; de Klein, Annelies; Douben, Hannie; Korevaar, Sander S; Mensah, Fane K F; Dor, Frank J M F; IJzermans, Jan N M; Betjes, Michiel G H; Baan, Carla C; Weimar, Willem; Hoogduijn, Martin J

    2012-10-01

    Mesenchymal stem cells are a potential therapeutic agent in renal disease and kidney transplantation. Autologous cell use in kidney transplantation is preferred to avoid anti-HLA reactivity; however, the influence of renal disease on mesenchymal stem cells is unknown. To investigate the feasibility of autologous cell therapy in patients with renal disease, we isolated these cells from subcutaneous adipose tissue of healthy controls and patients with renal disease and compared them phenotypically and functionally. The mesenchymal stem cells from both groups showed similar morphology and differentiation capacity, and were both over 90% positive for CD73, CD105, and CD166, and negative for CD31 and CD45. They demonstrated comparable population doubling times, rates of apoptosis, and were both capable of inhibiting allo-antigen- and anti-CD3/CD28-activated peripheral blood mononuclear cell proliferation. In response to immune activation they both increased the expression of pro-inflammatory and anti-inflammatory factors. These mesenchymal stem cells were genetically stable after extensive expansion and, importantly, were not affected by uremic serum. Thus, mesenchymal stem cells of patients with renal disease have similar characteristics and functionality as those from healthy controls. Hence, our results indicate the feasibility of their use in autologous cell therapy in patients with renal disease.

  10. Density of founder cells affects spatial pattern formation and cooperation in Bacillus subtilis biofilms.

    PubMed

    van Gestel, Jordi; Weissing, Franz J; Kuipers, Oscar P; Kovács, Akos T

    2014-10-01

    In nature, most bacteria live in surface-attached sedentary communities known as biofilms. Biofilms are often studied with respect to bacterial interactions. Many cells inhabiting biofilms are assumed to express 'cooperative traits', like the secretion of extracellular polysaccharides (EPS). These traits can enhance biofilm-related properties, such as stress resilience or colony expansion, while being costly to the cells that express them. In well-mixed populations cooperation is difficult to achieve, because non-cooperative individuals can reap the benefits of cooperation without having to pay the costs. The physical process of biofilm growth can, however, result in the spatial segregation of cooperative from non-cooperative individuals. This segregation can prevent non-cooperative cells from exploiting cooperative neighbors. Here we examine the interaction between spatial pattern formation and cooperation in Bacillus subtilis biofilms. We show, experimentally and by mathematical modeling, that the density of cells at the onset of biofilm growth affects pattern formation during biofilm growth. At low initial cell densities, co-cultured strains strongly segregate in space, whereas spatial segregation does not occur at high initial cell densities. As a consequence, EPS-producing cells have a competitive advantage over non-cooperative mutants when biofilms are initiated at a low density of founder cells, whereas EPS-deficient cells have an advantage at high cell densities. These results underline the importance of spatial pattern formation for competition among bacterial strains and the evolution of microbial cooperation.

  11. Hematopoietic stem cell expansion and generation: the ways to make a breakthrough

    PubMed Central

    Park, Bokyung

    2015-01-01

    Hematopoietic stem cell transplantation (HSCT) is the first field where human stem cell therapy was successful. Flooding interest on human stem cell therapy to cure previously incurable diseases is largely indebted to HSCT success. Allogeneic HSCT has been an important modality to cure various diseases including hematologic malignancies, various non-malignant hematologic diseases, primary immunodeficiency diseases, and inborn errors of metabolism, while autologous HSCT is generally performed to rescue bone marrow aplasia following high-dose chemotherapy for solid tumors or multiple myeloma. Recently, HSCs are also spotlighted in the field of regenerative medicine for the amelioration of symptoms caused by neurodegenerative diseases, heart diseases, and others. Although the demand for HSCs has been growing, their supply often fails to meet the demand of the patients needing transplant due to a lack of histocompatible donors or a limited cell number. This review focuses on the generation and large-scale expansion of HSCs, which might overcome current limitations in the application of HSCs for clinical use. Furthermore, current proof of concept to replenish hematological homeostasis from non-hematological origin will be covered. PMID:26770947

  12. Xpg limits the expansion of haematopoietic stem and progenitor cells after ionising radiation

    PubMed Central

    Avila, Alush I.; Illing, Anett; Becker, Friedrich; Maerz, Lars D.; Morita, Yohei; Philipp, Melanie; Burkhalter, Martin D.

    2016-01-01

    Reduced capacity of genome maintenance represents a problem for any organism, potentially causing premature death, carcinogenesis, or accelerated ageing. Strikingly though, loss of certain genome stability factors can be beneficial, especially for the maintenance of tissue stem cells of the intestine and the haematopoietic system. We therefore screened for genome stability factors negatively impacting maintenance of haematopoietic stem cells (HSC) in the context of ionising radiation (IR). We found that in vivo knock down of Xeroderma pigmentosum, complementation group G (Xpg) causes elevation of HSC numbers after IR treatment, while numbers of haematopoietic progenitors are elevated to a lesser extent. IR rapidly induces Xpg both on mRNA and on protein level. Prevention of this induction does not influence activation of the checkpoint cascade, yet attenuates late checkpoint steps such as induction of p21 and Noxa. This causes a leaky cell cycle arrest and lower levels of apoptosis, both contributing to increased colony formation and transformation rates. Xpg thus helps to adequately induce DNA damage responses after IR, thereby keeping the expansion of damaged cells under control. This represents a new function of Xpg in the response to IR, in addition to its well-characterized role in nucleotide excision repair. PMID:27137888

  13. A New Ex Vivo Method for Effective Expansion and Activation of Human Natural Killer Cells for Anti-Tumor Immunotherapy.

    PubMed

    Yang, Hui; Tang, Ruihua; Li, Jing; Liu, Yaxiong; Ye, Linjie; Shao, Dongyan; Jin, Mingliang; Huang, Qingsheng; Shi, Junling

    2015-12-01

    Preserving the activities of natural killer (NK) cells in human peripheral blood mononuclear cells (PBMCs) after ex vivo expansion and activation is critical for NK cell-based therapy. Collected from human PBMCs, the NK cells were expanded and activated. The expressions of surface receptors, cytotoxicity against tumor cells, and antibody-dependent cell-mediated cytotoxicity (ADCC) of the NK cells before and after expansion and activation were, respectively, compared. After expansion, the ADCC activity of healthy human NK cells was improved by 32 %, and the cytotoxicity against four types of tumor cells was increased by 19, 29, 26, and 28 %, respectively. The positive expression rates for the activating receptors NKG2D, CD94, NKp46, NKp30, and NKp44 of healthy human NK cells expanded ex vivo were increased by 60, 40, 20, 40, and 63 %, respectively, whereas those for the inhibitory receptors CD158b, NKB1, and NKAT showed no significant changes. The addition of an immunologically active peptide, "TKD," during cell expansion further increased NK cytotoxicity by approximately 10 %. The expanded and activated NK cells from cancer patients achieved average purity which was greater than 90 %, and the cytotoxicity against K562 cells was increased by more than 17 %. Compared with resting NK cells, NK cells both from healthy volunteers and cancer patients expanded and activated ex vivo using our method were significantly more active and demonstrated significantly increased anti-tumor activity. This method could be therefore used as a new and effective approach to meet requirements for anti-tumor immunotherapy.

  14. FMR1 epigenetic silencing commonly occurs in undifferentiated fragile X-affected embryonic stem cells.

    PubMed

    Avitzour, Michal; Mor-Shaked, Hagar; Yanovsky-Dagan, Shira; Aharoni, Shira; Altarescu, Gheona; Renbaum, Paul; Eldar-Geva, Talia; Schonberger, Oshrat; Levy-Lahad, Ephrat; Epsztejn-Litman, Silvina; Eiges, Rachel

    2014-11-11

    Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from epigenetic silencing of the X-linked FMR1 gene by a CGG expansion in its 5'-untranslated region. Taking advantage of a large set of FXS-affected human embryonic stem cell (HESC) lines and isogenic subclones derived from them, we show that FMR1 hypermethylation commonly occurs in the undifferentiated state (six of nine lines, ranging from 24% to 65%). In addition, we demonstrate that hypermethylation is tightly linked with FMR1 transcriptional inactivation in undifferentiated cells, coincides with loss of H3K4me2 and gain of H3K9me3, and is unrelated to CTCF binding. Taken together, these results demonstrate that FMR1 epigenetic gene silencing takes place in FXS HESCs and clearly highlights the importance of examining multiple cell lines when investigating FXS and most likely other epigenetically regulated diseases.

  15. A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

    NASA Astrophysics Data System (ADS)

    Lei, Yuguo; Schaffer, David V.

    2013-12-01

    Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising for numerous biomedical applications, such as cell replacement therapies, tissue and whole-organ engineering, and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however, the scalable expansion and differentiation of hPSCs, especially for clinical utilization, remains a challenge. We report a simple, defined, efficient, scalable, and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions, free of any human- or animal-derived factors, and entailing only recombinant protein factors. Under an optimized protocol, the 3D system enables long-term, serial expansion of multiple hPSCs lines with a high expansion rate (∼20-fold per 5-d passage, for a 1072-fold expansion over 280 d), yield (∼2.0 × 107 cells per mL of hydrogel), and purity (∼95% Oct4+), even with single-cell inoculation, all of which offer considerable advantages relative to current approaches. Moreover, the system enabled 3D directed differentiation of hPSCs into multiple lineages, including dopaminergic neuron progenitors with a yield of ∼8 × 107 dopaminergic progenitors per mL of hydrogel and ∼80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales, from basic biological investigation to clinical development.

  16. Role of lymphocyte-specific protein tyrosine kinase (LCK) in the expansion of glioma-initiating cells by fractionated radiation

    SciTech Connect

    Kim, Rae-Kwon; Yoon, Chang-Hwan; Hyun, Kyung-Hwan; Lee, Hyejin; An, Sungkwan; Park, Myung-Jin; Kim, Min-Jung; Lee, Su-Jae

    2010-11-26

    Research highlights: {yields} Activation of Lymphocyte-specific protein tyrosine kinase (LCK) is involved in the fractionated radiation-induced expansion of glioma stem-like cells. {yields} Inhibition of LCK prevents acquisition of fractionated radiation-induced resistance to chemotherapeutic treatment. {yields} LCK activity is critical for the maintenance of self-renewal in glioma stem-like cells. -- Abstract: Brain cancers frequently recur or progress as focal masses after treatment with ionizing radiation. Radiation used to target gliomas may expand the cancer stem cell population and enhance the aggressiveness of tumors; however, the mechanisms underlying the expansion of cancer stem cell population after radiation have remained unclear. In this study, we show that LCK (lymphocyte-specific protein tyrosine kinase) is involved in the fractionated radiation-induced expansion of the glioma-initiating cell population and acquisition of resistance to anticancer treatments. Fractionated radiation caused a selective increase in the activity of LCK, a Src family non-receptor tyrosine kinase. The activities of other Src family kinases Src, Fyn, and Lyn were not significantly increased. Moreover, knockdown of LCK expression with a specific small interfering RNA (siRNA) effectively blocked fractionated radiation-induced expansion of the CD133{sup +} cell population. siRNA targeting of LCK also suppressed fractionated radiation-induced expression of the glioma stem cell marker proteins CD133, Nestin, and Musashi. Expression of the known self-renewal-related proteins Notch2 and Sox2 in glioma cells treated with fractionated radiation was also downregulated by LCK inhibition. Moreover, siRNA-mediated knockdown of LCK effectively restored the sensitivity of glioma cells to cisplatin and etoposide. These results indicate that the non-receptor tyrosine kinase LCK is critically involved in fractionated radiation-induced expansion of the glioma-initiating cell population and

  17. A Robust Single Primate Neuroepithelial Cell Clonal Expansion System for Neural Tube Development and Disease Studies

    PubMed Central

    Zhu, Xiaoqing; Li, Bo; Ai, Zongyong; Xiang, Zheng; Zhang, Kunshang; Qiu, Xiaoyan; Chen, Yongchang; Li, Yuemin; Rizak, Joshua D.; Niu, Yuyu; Hu, Xintian; Sun, Yi Eve; Ji, Weizhi; Li, Tianqing

    2015-01-01

    Summary Developing a model of primate neural tube (NT) development is important to promote many NT disorder studies in model organisms. Here, we report a robust and stable system to allow for clonal expansion of single monkey neuroepithelial stem cells (NESCs) to develop into miniature NT-like structures. Single NESCs can produce functional neurons in vitro, survive, and extensively regenerate neuron axons in monkey brain. NT formation and NESC maintenance depend on high metabolism activity and Wnt signaling. NESCs are regionally restricted to a telencephalic fate. Moreover, single NESCs can turn into radial glial progenitors (RGPCs). The transition is accurately regulated by Wnt signaling through regulation of Notch signaling and adhesion molecules. Finally, using the “NESC-TO-NTs” system, we model the functions of folic acid (FA) on NT closure and demonstrate that FA can regulate multiple mechanisms to prevent NT defects. Our system is ideal for studying NT development and diseases. PMID:26584544

  18. The C. elegans engrailed homolog ceh-16 regulates the self-renewal expansion division of stem cell-like seam cells.

    PubMed

    Huang, Xinxin; Tian, E; Xu, Yanhua; Zhang, Hong

    2009-09-15

    Stem cells undergo symmetric and asymmetric division to maintain the dynamic equilibrium of the stem cell pool and also to generate a variety of differentiated cells. The homeostatic mechanism controlling the choice between self-renewal and differentiation of stem cells is poorly understood. We show here that ceh-16, encoding the C. elegans ortholog of the transcription factor Engrailed, controls symmetric and asymmetric division of stem cell-like seam cells. Loss of function of ceh-16 causes certain seam cells, which normally undergo symmetric self-renewal expansion division with both daughters adopting the seam cell fate, to divide asymmetrically with only one daughter retaining the seam cell fate. The human engrailed homolog En2 functionally substitutes the role of ceh-16 in promoting self-renewal expansion division of seam cells. Loss of function of apr-1, encoding the C. elegans homolog of the Wnt signaling component APC, results in transformation of self-renewal maintenance seam cell division to self-renewal expansion division, leading to seam cell hyperplasia. The apr-1 mutation suppresses the seam cell division defect in ceh-16 mutants. Our study reveals that ceh-16 interacts with the Wnt signaling pathway to control the choice between self-renewal expansion and maintenance division and also demonstrates an evolutionarily conserved function of engrailed in promoting cell proliferation.

  19. Soluble factors secreted by glioblastoma cell lines facilitate recruitment, survival, and expansion of regulatory T cells: implications for immunotherapy

    PubMed Central

    Crane, Courtney A.; Ahn, Brian J.; Han, Seunggu J.; Parsa, Andrew T.

    2012-01-01

    In patients with glioma, the tumor microenvironment can significantly impact pro-inflammatory immune cell functions. However, the mechanisms by which this occurs are poorly defined. Because immunosuppressive regulatory T cells (Treg) are over represented in the tumor microenvironment compared with peripheral blood, we hypothesized that the tumor may have an effect on Treg survival, migration, expansion, and/or induction of a regulatory phenotype from non-Treg conventional CD4+ T cells. We defined the impact of soluble factors produced by tumor cells on Treg from healthy patients in vitro to determine mechanisms by which gliomas influence T cell populations. We found that tumor-derived soluble factors allowed for preferential proliferation and increased chemotaxis of Treg, compared with conventional T cells, indicating that these mechanisms may contribute to the increased Treg in the tumor microenvironment. Conventional T cells also exhibited a significantly increased expression of pro-apoptotic transcripts in the presence of tumor-derived factors, indicating that survival of Treg in the tumor site is driven by exposure to soluble factors produced by the tumor. Together, these data suggest that tumor burden may induce increased Treg infiltration, proliferation, and survival, negating productive anti-tumor immune responses in patients treated with immunotherapies. Collectively, our data indicate that several mechanisms of Treg recruitment and retention in the tumor microenvironment exist and may need to be addressed to improve the specificity of immunotherapies seeking to eliminate Treg in patients with glioma. PMID:22406925

  20. N-isopropylacrylamide-based thermoresponsive polyelectrolyte multilayer films for human mesenchymal stem cell expansion.

    PubMed

    Liao, Tianqing; Moussallem, Maroun D; Kim, Junho; Schlenoff, Joseph B; Ma, Teng

    2010-01-01

    Human mesenchymal stem cells (hMSCs) are colony-forming unit fibroblasts (CFU-F) derived from adult bone marrow and have significant potential for many cell-based tissue-engineering applications. Their therapeutic potential, however, is restricted by their diminishing plasticity as they are expanded in culture. In this study, we used N-isopropylacrylamide (NIPAM)-based thermoresponsive polyelectrolyte multilayer (N-PEMU) films as culture substrates to support hMSC expansion and evaluated their effects on cell properties. The N-PEMU films were made via layer-by-layer adsorption of thermoresponsive monomers copolymerized with charged monomers, positively charged allylamine hydrochloride (PAH), or negatively charged styrene sulfonic acid (PSS) and compared to fetal bovine serum (FBS) coated surfaces. Surface charges were shown to alter the extracellular matrix (ECM) structure and subsequently regulate hMSC responses including adhesion, proliferation, integrin expression, detachment, and colony forming ability. The positively charged thermal responsive surfaces improved cell adhesion and growth in a range comparable to control surfaces while maintaining significantly higher CFU-F forming ability. Immunostaining and Western blot results indicate that the improved cell adhesion and growth on the positively charged surfaces resulted from the elevated adhesion of ECM proteins such as fibronectin on the positively charge surfaces. These results demonstrate that the layer-by-layer approach is an efficient way to form PNIPAM-based thermal responsive surfaces for hMSC growth and removal without enzymatic treatment. The results also show that surface charge regulates ECM adhesion, which in turn influences not only cell adhesion but also CFU-forming ability and their multi-lineage differentiation potential.

  1. Highly efficient IL-21 and feeder cell-driven ex vivo expansion of human NK cells with therapeutic activity in a xenograft mouse model of melanoma.

    PubMed

    Granzin, Markus; Stojanovic, Ana; Miller, Matthias; Childs, Richard; Huppert, Volker; Cerwenka, Adelheid

    2016-01-01

    Natural killer (NK) cells are promising antitumor effector cells, but the generation of sufficient NK cell numbers for adoptive immunotherapy remains challenging. Therefore, we developed a method for highly efficient ex vivo expansion of human NK cells. Ex vivo expansion of NK cells in medium containing IL-2 and irradiated clinical-grade feeder cells (EBV-LCL) induced a 22-fold NK cell expansion after one week that was significantly increased to 53-fold by IL-21. Repeated stimulation with irradiated EBV-LCL and IL-2 and addition of IL-21 at the initiation of the culture allowed sustained NK cell proliferation with 10(11)-fold NK cell expansion after 6 weeks. Compared to naive NK cells, expanded NK cells upregulated TRAIL, NKG2D, and DNAM-1, had superior cytotoxicity against tumor cell lines in vitro and produced more IFNγ and TNF-α upon PMA/Iono stimulation. Most importantly, adoptive transfer of NK cells expanded using feeder cells, IL-2 and IL-21 led to significant inhibition of tumor growth in a melanoma xenograft mouse model, which was greater than with NK cells activated with IL-2 alone. Intriguingly, adoptively transferred NK cells maintained their enhanced production of IFNγ and TNF-α upon ex vivo restimulation, although they rapidly lost their capacity to degranulate and mediate tumor cytotoxicity after the in vivo transfer. In conclusion, we developed a protocol for ex vivo NK cell expansion that results in outstanding cell yields. The expanded NK cells possess potent antitumor activity in vitro and in vivo and could be utilized at high numbers for adoptive immunotherapy in the clinic.

  2. A Rapid Cell Expansion Process for Production of Engineered Autologous CAR-T Cell Therapies.

    PubMed

    Lu, Tangying Lily; Pugach, Omar; Somerville, Robert; Rosenberg, Steven A; Kochendefer, James N; Better, Marc; Feldman, Steven A

    2016-12-01

    The treatment of B-cell malignancies by adoptive cell transfer (ACT) of anti-CD19 chimeric antigen receptor T cells (CD19 CAR-T) has proven to be a highly successful therapeutic modality in several clinical trials.(1-6) The anti-CD19 CAR-T cell production method used to support initial trials relied on numerous manual, open process steps, human serum, and 10 days of cell culture to achieve a clinical dose.(7) This approach limited the ability to support large multicenter clinical trials, as well as scale up for commercial cell production. Therefore, studies were completed to streamline and optimize the original National Cancer Institute production process by removing human serum from the process in order to minimize the risk of viral contamination, moving process steps from an open system to functionally closed system operations in order to minimize the risk of microbial contamination, and standardizing additional process steps in order to maximize process consistency. This study reports a procedure for generating CD19 CAR-T cells in 6 days, using a functionally closed manufacturing process and defined, serum-free medium. This method is able to produce CD19 CAR-T cells that are phenotypically and functionally indistinguishable from cells produced for clinical trials by the previously described production process.

  3. Planar cell polarity-mediated induction of neural stem cell expansion during axolotl spinal cord regeneration

    PubMed Central

    Rost, Fabian; Nowoshilow, Sergej; Chara, Osvaldo; Tanaka, Elly M

    2015-01-01

    Axolotls are uniquely able to mobilize neural stem cells to regenerate all missing regions of the spinal cord. How a neural stem cell under homeostasis converts after injury to a highly regenerative cell remains unknown. Here, we show that during regeneration, axolotl neural stem cells repress neurogenic genes and reactivate a transcriptional program similar to embryonic neuroepithelial cells. This dedifferentiation includes the acquisition of rapid cell cycles, the switch from neurogenic to proliferative divisions, and the re-expression of planar cell polarity (PCP) pathway components. We show that PCP induction is essential to reorient mitotic spindles along the anterior-posterior axis of elongation, and orthogonal to the cell apical-basal axis. Disruption of this property results in premature neurogenesis and halts regeneration. Our findings reveal a key role for PCP in coordinating the morphogenesis of spinal cord outgrowth with the switch from a homeostatic to a regenerative stem cell that restores missing tissue. DOI: http://dx.doi.org/10.7554/eLife.10230.001 PMID:26568310

  4. Water sorption and expansion of an ionomer membrane constrained by fuel cell electrodes

    NASA Astrophysics Data System (ADS)

    Goulet, Marc-Antoni; Arbour, Spencer; Lauritzen, Michael; Kjeang, Erik

    2015-01-01

    This article reveals that catalyst coated membranes (CCM) and membrane electrode assemblies (MEA) expand and contract differently than pure ionomer membranes during hydration and dehydration. Pure membranes are shown to generate twice as much longitudinal peak and residual stress during dehydration than CCMs, reflecting the higher modulus of the pure ionomer material. Moreover, the stronger confinement imposed by the lamination of relatively stiff gas diffusion layers to the CCM prevents the ionomer membrane from expanding in the in-plane direction. This is shown to lead to a significant increase in the through-plane stress and strain during hydration of MEAs versus CCMs and pure ionomer membranes. Supplementary measurements indicate that the water sorption properties of the ionomer (at equilibrium) are not altered by the lamination of catalyst layers and gas diffusion layers; hence, the changes in expansion behavior in the MEA are attributed to the mechanical confinement provided by the other layers. These features should be captured by finite element modeling of fuel cell stacks for accurate cell design and may have important implications for fuel cell durability.

  5. Single-Cell Analysis of the Plasmablast Response to Vibrio cholerae Demonstrates Expansion of Cross-Reactive Memory B Cells

    PubMed Central

    Kauffman, Robert C.; Bhuiyan, Taufiqur R.; Nakajima, Rie; Mayo-Smith, Leslie M.; Rashu, Rasheduzzaman; Hoq, Mohammad Rubel; Chowdhury, Fahima; Khan, Ashraful Islam; Rahman, Atiqur; Bhaumik, Siddhartha K.; Harris, Levelle; O'Neal, Justin T.; Trost, Jessica F.; Alam, Nur Haq; Jasinskas, Algis; Dotsey, Emmanuel; Kelly, Meagan; Charles, Richelle C.; Xu, Peng; Kováč, Pavol; Calderwood, Stephen B.; Ryan, Edward T.; Felgner, Phillip L.; Qadri, Firdausi

    2016-01-01

    ABSTRACT We characterized the acute B cell response in adults with cholera by analyzing the repertoire, specificity, and functional characteristics of 138 monoclonal antibodies (MAbs) generated from single-cell-sorted plasmablasts. We found that the cholera-induced responses were characterized by high levels of somatic hypermutation and large clonal expansions. A majority of the expansions targeted cholera toxin (CT) or lipopolysaccharide (LPS). Using a novel proteomics approach, we were able to identify sialidase as another major antigen targeted by the antibody response to Vibrio cholerae infection. Antitoxin MAbs targeted both the A and B subunits, and most were also potent neutralizers of enterotoxigenic Escherichia coli heat-labile toxin. LPS-specific MAbs uniformly targeted the O-specific polysaccharide, with no detectable responses to either the core or the lipid moiety of LPS. Interestingly, the LPS-specific antibodies varied widely in serotype specificity and functional characteristics. One participant infected with the Ogawa serotype produced highly mutated LPS-specific antibodies that preferentially bound the previously circulating Inaba serotype. This demonstrates durable memory against a polysaccharide antigen presented at the mucosal surface and provides a mechanism for the long-term, partial heterotypic immunity seen following cholera. PMID:27999163

  6. Genistein affects proliferation and migration of bovine oviductal epithelial cells.

    PubMed

    García, Daniela C; Valdecantos, Pablo A; Miceli, Dora C; Roldán-Olarte, Mariela

    2017-03-08

    Genistein is one of the most abundant isoflavones in soybean. This molecule induces cell cycle arrest and apoptosis in different normal and cancer cells. Genistein has been of considerable interest due to its adverse effects on bovine reproduction, altering estrous cycle, implantation and fetal development and producing subfertility or infertility. The objective of this work was to study the effects of genistein on the expression of selected genes involved in the regulation of cell cycle and apoptosis. Primary cultures of bovine oviductal epithelial cells (BOEC) were treated with different genistein concentrations (0.2, 2 and 10μM) to analyze CYCLIN B1, BCL-2 and BAX gene expression by Real-time RT-PCR. Results showed that genistein down-regulated CYCLIN B1 expression, affecting cell cycle progression, and caused a decrease in the BCL-2/BAX ratio starting at 2μM of genistein. In addition, in order to determine if genistein affects BOEC migration, in vitro wound healing assays were performed. A significant reduction in cell migration after 12h of culture was observed at both 0.2 and 10μM genistein concentrations. Also, in the presence of genistein the percentage of mitotic cells decreased, although apoptotic cells percentages were not affected. These findings indicate that genistein has an inhibitory effect on BOEC proliferation and migration, suggesting that it could influence the normal physiology of the oviductal epithelium.

  7. Angiopoietin 2 stimulates TIE2-expressing monocytes to suppress T cell activation and to promote regulatory T cell expansion.

    PubMed

    Coffelt, Seth B; Chen, Yung-Yi; Muthana, Munitta; Welford, Abigail F; Tal, Andrea O; Scholz, Alexander; Plate, Karl H; Reiss, Yvonne; Murdoch, Craig; De Palma, Michele; Lewis, Claire E

    2011-04-01

    Angiopoietin 2 (ANGPT2) is a proangiogenic cytokine whose expression is often upregulated by endothelial cells in tumors. Expression of its receptor, TIE2, defines a highly proangiogenic subpopulation of myeloid cells in circulation and tumors called TIE2-expressing monocytes/macrophages (TEMs). Genetic depletion of TEMs markedly reduces tumor angiogenesis in various tumor models, emphasizing their essential role in driving tumor progression. Previously, we demonstrated that ANGPT2 augments the expression of various proangiogenic genes, the potent immunosuppressive cytokine, IL-10, and a chemokine for regulatory T cells (Tregs), CCL17 by TEMs in vitro. We now show that TEMs also express higher levels of IL-10 than TIE2(-) macrophages in tumors and that ANGPT2-stimulated release of IL-10 by TEMs suppresses T cell proliferation, increases the ratio of CD4(+) T cells to CD8(+) T cells, and promotes the expansion of CD4(+)CD25(high)FOXP3(+) Tregs. Furthermore, syngeneic murine tumors expressing high levels of ANGPT2 contained not only high numbers of TEMs but also increased numbers of Tregs, whereas genetic depletion of tumor TEMs resulted in a marked reduction in the frequency of Tregs in tumors. Taken together, our data suggest that ANGPT2-stimulated TEMs represent a novel, potent immunosuppressive force in tumors.

  8. Expansion of inflammatory innate lymphoid cells in patients with common variable immune deficiency

    PubMed Central

    Cols, Montserrat; Rahman, Adeeb; Maglione, Paul J.; Garcia-Carmona, Yolanda; Simchoni, Noa; Ko, Huai-Bin M.; Radigan, Lin; Cerutti, Andrea; Blankenship, Derek; Pascual, Virginia; Cunningham-Rundles, Charlotte

    2016-01-01

    Background Common variable immunodeficiency (CVID) is an antibody deficiency treated with immunoglobulin; however, patients can have noninfectious inflammatory conditions that lead to heightened morbidity and mortality. Objectives Modular analyses of RNA transcripts in whole blood previously identified an upregulation of many interferon-responsive genes. In this study we sought the cell populations leading to this signature. Methods Lymphoid cells were measured in peripheral blood of 55 patients with CVID (31 with and 24 without inflammatory/autoimmune complications) by using mass cytometry and flow cytometry. Surface markers, cytokines, and transcriptional characteristics of sorted innate lymphoid cells (ILCs) were defined by using quantitative PCR. Gastrointestinal and lung biopsy specimens of subjects with inflammatory disease were stained to seek ILCs in tissues. Results The linage-negative, CD127+, CD161+ lymphoid population containing T-box transcription factor, retinoic acid–related orphan receptor (ROR) γt, IFN-γ, IL-17A, and IL-22, all hallmarks of type 3 innate lymphoid cells, were expanded in the blood of patients with CVID with inflammatory conditions (mean, 3.7% of PBMCs). ILCs contained detectable amounts of the transcription factors inhibitor of DNA binding 2, T-box transcription factor, and RORγt and increased mRNA transcripts for IL-23 receptor (IL-23R) and IL-26, demonstrating inflammatory potential. In gastrointestinal and lung biopsy tissues of patients with CVID, numerous IFN-γ+RORγt+CD3− cells were identified, suggesting a role in these mucosal inflammatory states. Conclusions An expansion of this highly inflammatory ILC population is a characteristic of patients with CVID with inflammatory disease; ILCs and the interferon signature are markers for the uncontrolled inflammatory state in these patients. PMID:26542033

  9. Co-operative signalling mechanisms required for erythroid precursor expansion in response to erythropoietin and stem cell factor.

    PubMed

    Arcasoy, Murat O; Jiang, Xiaohong

    2005-07-01

    The regeneration of circulating red blood cells in response to anaemia associated with blood loss or haemolysis involves an increased rate of erythropoiesis and expansion of proerythroblasts, the bone marrow precursor cells that terminally differentiate into mature erythrocytes. This study investigated the mechanisms by which erythropoietin (Epo) and stem cell factor (Scf) modulate the expansion of proerythroblasts. Homogenous populations of primary human proerythroblasts were generated in liquid cultures of CD34(+) cells. In serum-free cultures, proerythroblasts failed to survive in the presence of Epo or Scf alone, but exhibited synergistic proliferation in response to combined Epo and Scf treatment, exhibiting one-log expansion in 5 d. Intracellular signal transduction in response to Epo and Scf revealed that tyrosine phosphorylation of signal transducers and activators of transcription (Stat) 5, a downstream target for the non-receptor tyrosine kinase, Janus kinase 2 (Jak2), was mediated by Epo but not Scf. The mitogen-activated protein kinases (MAPKs) extracellular regulated kinase (Erk) 1-2 were phosphorylated in response to either Epo or Scf. Phosphorylation of Akt, a signalling molecule downstream of phosphatidylinositol 3-kinase (PI3K), was observed following Scf but not Epo treatment. To determine the contribution of specific signalling pathways to synergistic expansion of proerythroblasts in response to co-operative effects of Epo and Scf, cells were treated with kinase inhibitors targeting Jak2, PI3K and MAPK kinase. There was a significant, dose-dependent inhibition of proerythroblast expansion in response to all three kinase inhibitors. In conclusion, Epo- and Scf-mediated co-operative, synergistic expansion of primary erythroid precursors requires selective activation of multiple signalling pathways, including the Jak-Stat, PI3K and MAPK pathways.

  10. Preparation, quality criteria, and properties of human blood platelet lysate supplements for ex vivo stem cell expansion.

    PubMed

    Shih, Daniel Tzu-Bi; Burnouf, Thierry

    2015-01-25

    Most clinical applications of human multipotent mesenchymal stromal cells (MSCs) for cell therapy, tissue engineering, regenerative medicine, and treatment of immune and inflammatory diseases require a phase of isolation and ex vivo expansion allowing a clinically meaningful cell number to be reached. Conditions used for cell isolation and expansion should meet strict quality and safety requirements. This is particularly true for the growth medium used for MSC isolation and expansion. Basal growth media used for MSC expansion are supplemented with multiple nutrients and growth factors. Fetal bovine serum (FBS) has long been the gold standard medium supplement for laboratory-scale MSC culture. However, FBS has a poorly characterized composition and poses risk factors, as it may be a source of xenogenic antigens and zoonotic infections. FBS has therefore become undesirable as a growth medium supplement for isolating and expanding MSCs for human therapy protocols. In recent years, human blood materials, and most particularly lysates and releasates of platelet concentrates have emerged as efficient medium supplements for isolating and expanding MSCs from various origins. This review analyzes the advantages and limits of using human platelet materials as medium supplements for MSC isolation and expansion. We present the modes of production of allogeneic and autologous platelet concentrates, measures taken to ensure optimal pathogen safety profiles, and methods of preparing PLs for MSC expansion. We also discuss the supply of such blood preparations. Produced under optimal conditions of standardization and safety, human platelet materials can become the future 'gold standard' supplement for ex vivo production of MSCs for translational medicine and cell therapy applications.

  11. Todralazine Protects Zebrafish from Lethal Effects of Ionizing Radiation: Role of Hematopoietic Cell Expansion

    PubMed Central

    Dimri, Manali; Joshi, Jayadev; Chakrabarti, Rina; Sehgal, Neeta; Sureshbabu, Angara

    2015-01-01

    Abstract The Johns Hopkins Clinical Compound Library (JHCCL), a collection of Food and Drug Administration (FDA)-approved small molecules (1400), was screened in silico for identification of novel β2AR blockers and tested for hematopoietic stem cell (HSC) expansion and radioprotection in zebrafish embryos. Docking studies, followed by the capacity to hasten erythropoiesis, identified todralazine (Binding energy, −8.4 kcal/mol) as a potential HSC-modulating agent. Todralazine (5 μM) significantly increased erythropoiesis in caudal hematopoietic tissue (CHT) in wild-type and anemic zebrafish embryos (2.33- and 1.44-folds, respectively) when compared with untreated and anemic control groups. Todralazine (5 μM) treatment also led to an increased number of erythroid progenitors, as revealed from the increased expression of erythroid progenitor-specific genes in the CHT region. Consistent with these effects, zebrafish embryos, Tg(cmyb:gfp), treated with 5 μM todralazine from 24 to 36 hours post fertilization (hpf) showed increased (approximately two-folds) number of HSCs at the aorta-gonad-mesonephros region (AGM). Similarly, expression of HSC marker genes, runx1 (3.3-folds), and cMyb (1.41-folds) also increased in case of todralazine-treated embryos, further supporting its HSC expansion potential. Metoprolol, a known beta blocker, also induced HSC expansion (1.36- and 1.48-fold increase in runx1 and cMyb, respectively). Todralazine (5 μM) when added 30 min before 20 Gy gamma radiation, protected zebrafish from radiation-induced organ toxicity, apoptosis, and improved survival (80% survival advantage over 6 days). The 2-deoxyribose degradation test further suggested hydroxyl (OH) radical scavenging potential of todralazine, and the same is recapitulated in vivo. These results suggest that todralazine is a potential HSC expanding agent, which might be acting along with important functions, such as antioxidant and free radical scavenging, in

  12. Low oxygen tension favored expansion and hematopoietic reconstitution of CD34(+) CD38(-) cells expanded from human cord blood-derived CD34(+) Cells.

    PubMed

    Wang, Ziyan; Du, Zheng; Cai, Haibo; Ye, Zhaoyang; Fan, Jinli; Tan, Wen-Song

    2016-07-01

    Oxygen tension is an important factor that regulates hematopoietic stem cells (HSCs) in both in vivo hematopoietic microenvironment and ex vivo culture system. Although the effect of oxygen tension on ex vivo expansion of HSCs was extensively studied, there were no clear descriptions on physiological function and gene expression analysis of HSCs under different oxygen tensions. In this study, the effects of oxygen tension on ex vivo expansion characteristics of human umbilical cord blood (UCB)-derived CD34(+) cells are evaluated. Moreover, the physiological function of expanded CD34(+) cells was assessed by secondary expansion ability ex vivo and hematopoietic reconstitution ability in vivo. Also, genetic profiling was applied to analyze the expression of genes related to cell function. It was found that low oxygen tension favored expansion of CD34(+) CD38(-) cells. Additionally, CD34(+) cells expanded under low oxygen tension showed better secondary expansion ability and reconstitution ability than those under atmospheric oxygen concentration. Finally, the genetic profiling of CD34(+) CD38(-) cells cultured under low oxygen tension was more akin to freshly isolated cells. These results collectively demonstrate that low oxygen tension was able to better maintain both self-renewal and hematopoietic reconstitution potential and may lay an experimental basis for clinical transplantation of HSCs.

  13. Cell flexibility affects the alignment of model myxobacteria.

    PubMed

    Janulevicius, Albertas; van Loosdrecht, Mark C M; Simone, Angelo; Picioreanu, Cristian

    2010-11-17

    Myxobacteria are social bacteria that exhibit a complex life cycle culminating in the development of multicellular fruiting bodies. The alignment of rod-shaped myxobacteria cells within populations is crucial for development to proceed. It has been suggested that myxobacteria align due to mechanical interactions between gliding cells and that cell flexibility facilitates reorientation of cells upon mechanical contact. However, these suggestions have not been based on experimental or theoretical evidence. Here we created a computational mass-spring model of a flexible rod-shaped cell that glides on a substratum periodically reversing direction. The model was formulated in terms of experimentally measurable mechanical parameters, such as engine force, bending stiffness, and drag coefficient. We investigated how cell flexibility and motility engine type affected the pattern of cell gliding and the alignment of a population of 500 mechanically interacting cells. It was found that a flexible cell powered by engine force at the rear of the cell, as suggested by the slime extrusion hypothesis for myxobacteria motility engine, would not be able to glide in the direction of its long axis. A population of rigid reversing cells could indeed align due to mechanical interactions between cells, but cell flexibility impaired the alignment.

  14. Coordination of cell proliferation and cell expansion mediated by ribosome-related processes in the leaves of Arabidopsis thaliana.

    PubMed

    Fujikura, Ushio; Horiguchi, Gorou; Ponce, María Rosa; Micol, José Luis; Tsukaya, Hirokazu

    2009-08-01

    Co-ordination of cell proliferation and cell expansion is a key regulatory process in leaf-size determination, but its molecular details are unknown. In Arabidopsis thaliana, mutations in a positive regulator of cell proliferation often trigger excessive cell enlargement post-mitotically in leaves. This phenomenon, called compensation syndrome, is seen in the mutant angustifolia3 (an3), which is defective in a transcription co-activator. Such compensation, however, does not occur in response to a decrease in cell number in oligocellula (oli). oli2, oli5 and oli7 did not exhibit compensation and the reduction in cell number in these mutants was moderate. However, when an oli mutation was combined with a different oli mutation to create a double mutant, cell number was further reduced and compensation was induced. Similarly, weak suppression of AN3 expression reduced cell number moderately but did not induce compensation compared with an an3 null mutant. Furthermore, double mutants of either oli2, oli5 or oli7 and an3 showed markedly enhanced compensation. These results suggest that compensation is triggered when cell proliferation regulated by OLI2/OLI5/OLI7 and AN3 is compromised in a threshold-dependent manner. OLI2 encodes a Nop2 homolog in Saccharomyces cerevisiae that is involved in ribosome biogenesis, whereas OLI5 and OLI7 encode ribosome proteins RPL5A and RPL5B, respectively. This suggests that a factor involved in the induction of compensation may be under the dual control of AN3 and a ribosome-related process.

  15. Generation of autologous tumor-specific T cells for adoptive transfer based on vaccination, in vitro restimulation and CD3/CD28 dynabead-induced T cell expansion.

    PubMed

    Brimnes, Marie Klinge; Gang, Anne Ortved; Donia, Marco; Thor Straten, Per; Svane, Inge Marie; Hadrup, Sine Reker

    2012-08-01

    Adoptive cell transfer (ACT) of in vitro expanded autologous tumor-infiltrating lymphocytes (TIL) has been shown to exert therapeutic efficacy in melanoma patients. We aimed to develop an ACT protocol based on tumor-specific T cells isolated from peripheral blood and in vitro expanded by Dynabeads® ClinExVivo™CD3/CD28. We show here that the addition of an in vitro restimulation step with relevant peptides prior to bead expansion dramatically increased the proportion of tumor-specific T cells in PBMC-cultures. Importantly, peptide-pulsed dendritic cells (DCs) as well as allogeneic tumor lysate-pulsed DCs from the DC vaccine preparation could be used with comparable efficiency to peptides for in vitro restimulation, to increase the tumor-specific T-cell response. Furthermore, we tested the use of different ratios and different types of Dynabeads® CD3/CD28 and CD3/CD28/CD137 T-cell expander, for optimized expansion of tumor-specific T cells. A ratio of 1:3 of Dynabeads® CD3/CD28 T-cell expander to T cells resulted in the maximum number of tumor-specific T cells. The addition of CD137 did not improve functionality or fold expansion. Both T-cell expansion systems could generate tumor-specific T cells that were both cytotoxic and effective cytokine producers upon antigen recognition. Dynabeads®-expanded T-cell cultures shows phenotypical characteristics of memory T cells with potential to migrate and expand in vivo. In addition, they possess longer telomeres compared to TIL cultures. Taken together, we demonstrate that in vitro restimulation of tumor-specific T cells prior to bead expansion is necessary to achieve high numbers of tumor-specific T cells. This is effective and easily applicable in combination with DC vaccination, by use of vaccine-generated DCs, either pulsed with peptide or tumor-lysate.

  16. Comparative Analysis of Media and Supplements on Initiation and Expansion of Adipose-Derived Stem Cells

    PubMed Central

    Riis, Simone; Nielsen, Frederik Mølgaard; Pennisi, Cristian Pablo; Zachar, Vladimir

    2016-01-01

    Adipose-derived stem cells (ASCs) are being tested in clinical trials related to cell-based regenerative therapies. Although most of the current expansion protocols for ASCs use fetal calf serum (FCS), xenogeneic-free medium supplements are greatly desired. This study aims to compare the effect of FCS, human platelet lysate (hPL), and a fully defined medium on the initiation and maintenance of ASC cultures. ASCs obtained from five donors were cultured in five different media: StemPro, Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% hPL, or α-minimum essential medium (A-MEM) supplemented with 5% hPL, 10% hPL, or 10% FCS. The effect of media on proliferation, colony-forming units (CFUs), attachment, and morphology was assessed along with cell size, granularity, and immunophenotype. StemPro greatly compromised the initiation of ASC cultures, which could not survive more than a few passages. Cells cultured in A-MEM proliferated at a faster rate than in DMEM, and hPL significantly enhanced cell size, granularity, and proliferation compared with FCS. All media except StemPro supported CFUs equally well. Analysis of surface markers revealed higher levels of CD73 and CD105 in FCS-cultured ASCs, whereas increased levels of CD146 were found in hPL-cultured cells. Multiparametric flow cytometric analysis performed after seven passages revealed the existence of four distinct ASC subpopulations, all positive for CD73, CD90, and CD105, which mainly differed by their expression of CD146 and CD271. Analysis of the different subpopulations might represent an important biological measure when assessing different medium formulations for a particular clinical application. Significance In most clinical trials using adipose-derived stem cells (ASCs), the cells have been expanded in culture media supplemented with fetal calf serum. However, there is much interest in replacing fetal calf serum with human platelet lysate or using completely serum- and xenogeneic

  17. Short term ex-vivo expansion of circulating head and neck tumour cells

    PubMed Central

    Kulasinghe, Arutha; Perry, Chris; Warkiani, Majid E.; Blick, Tony; Davies, Anthony; O'Byrne, Ken; Thompson, Erik W.; Nelson, Colleen C.; Vela, Ian; Punyadeera, Chamindie

    2016-01-01

    Minimally invasive techniques are required for the identification of head and neck cancer (HNC) patients who are at an increased risk of metastasis, or are not responding to therapy. An approach utilised in other solid cancers is the identification and enumeration of circulating tumour cells (CTCs) in the peripheral blood of patients. Low numbers of CTCs has been a limiting factor in the HNC field to date. Here we present a methodology to expand HNC patient derived CTCs ex-vivo. As a proof of principle study, 25 advanced stage HNC patient bloods were enriched for circulating tumour cells through negative selection and cultured in 2D and 3D culture environments under hypoxic conditions (2% O2, 5% CO2). CTCs were detected in 14/25 (56%) of patients (ranging from 1–15 CTCs/5 mL blood). Short term CTC cultures were successfully generated in 7/25 advanced stage HNC patients (5/7 of these cultures were from HPV+ patients). Blood samples from which CTC culture was successful had higher CTC counts (p = 0.0002), and were predominantly from HPV+ patients (p = 0.007). This is, to our knowledge, the first pilot study to culture HNC CTCs ex-vivo. Further studies are warranted to determine the use of short term expansion in HNC and the role of HPV in promoting culture success. PMID:27517751

  18. Physiological Integration Affects Expansion of an Amphibious Clonal Plant from Terrestrial to Cu-Polluted Aquatic Environments.

    PubMed

    Xu, Liang; Zhou, Zhen-Feng

    2017-03-08

    The effects of physiological integration on clonal plants growing in aquatic and terrestrial habitats have been extensively studied, but little is known about the role in the extension of amphibious clonal plants in the heterogeneous aquatic-terrestrial ecotones, especially when the water environments are polluted by heavy metals. Ramets of the amphibious clonal herb Alternanthera philoxeroides were rooted in unpolluted soil and polluted water at three concentrations of Cu. The extension of populations from unpolluted terrestrial to polluted aqueous environments mainly relied on stem elongation rather than production of new ramets. The absorbed Cu in the ramets growing in polluted water could be spread horizontally to other ramets in unpolluted soil via physiological integration and redistributed in different organs. The performances of ramets in both terrestrial and aquatic habitats were negatively correlated with Cu intensities in different organs of plants. It is concluded that physiological integration might lessen the fitness of connected ramets in heterogeneously polluted environments. The mechanical strength of the stems decreased with increasing Cu levels, especially in polluted water. We suggest that, except for direct toxicity to growth and expansion, heavy metal pollution might also increase the mechanical risk in breaking failure of plants.

  19. Physiological Integration Affects Expansion of an Amphibious Clonal Plant from Terrestrial to Cu-Polluted Aquatic Environments

    PubMed Central

    Xu, Liang; Zhou, Zhen-Feng

    2017-01-01

    The effects of physiological integration on clonal plants growing in aquatic and terrestrial habitats have been extensively studied, but little is known about the role in the extension of amphibious clonal plants in the heterogeneous aquatic-terrestrial ecotones, especially when the water environments are polluted by heavy metals. Ramets of the amphibious clonal herb Alternanthera philoxeroides were rooted in unpolluted soil and polluted water at three concentrations of Cu. The extension of populations from unpolluted terrestrial to polluted aqueous environments mainly relied on stem elongation rather than production of new ramets. The absorbed Cu in the ramets growing in polluted water could be spread horizontally to other ramets in unpolluted soil via physiological integration and redistributed in different organs. The performances of ramets in both terrestrial and aquatic habitats were negatively correlated with Cu intensities in different organs of plants. It is concluded that physiological integration might lessen the fitness of connected ramets in heterogeneously polluted environments. The mechanical strength of the stems decreased with increasing Cu levels, especially in polluted water. We suggest that, except for direct toxicity to growth and expansion, heavy metal pollution might also increase the mechanical risk in breaking failure of plants. PMID:28272515

  20. Physiological Integration Affects Expansion of an Amphibious Clonal Plant from Terrestrial to Cu-Polluted Aquatic Environments

    NASA Astrophysics Data System (ADS)

    Xu, Liang; Zhou, Zhen-Feng

    2017-03-01

    The effects of physiological integration on clonal plants growing in aquatic and terrestrial habitats have been extensively studied, but little is known about the role in the extension of amphibious clonal plants in the heterogeneous aquatic-terrestrial ecotones, especially when the water environments are polluted by heavy metals. Ramets of the amphibious clonal herb Alternanthera philoxeroides were rooted in unpolluted soil and polluted water at three concentrations of Cu. The extension of populations from unpolluted terrestrial to polluted aqueous environments mainly relied on stem elongation rather than production of new ramets. The absorbed Cu in the ramets growing in polluted water could be spread horizontally to other ramets in unpolluted soil via physiological integration and redistributed in different organs. The performances of ramets in both terrestrial and aquatic habitats were negatively correlated with Cu intensities in different organs of plants. It is concluded that physiological integration might lessen the fitness of connected ramets in heterogeneously polluted environments. The mechanical strength of the stems decreased with increasing Cu levels, especially in polluted water. We suggest that, except for direct toxicity to growth and expansion, heavy metal pollution might also increase the mechanical risk in breaking failure of plants.

  1. Pharmacological preconditioning for short-term ex vivo expansion of human umbilical cord blood hematopoietic stem cells by filgrastim

    PubMed Central

    Grigoriadis, Nikolaos G; Grigoriadis, Ioannis G; Markoula, Sofia; Paschopoulos, Minas; Zikopoulos, Konstantinos; Apostolakopoulos, Panagiotis Gr; Vizirianakis, Ioannis S; Georgiou, Ioannis

    2016-01-01

    Although umbilical cord blood (UCB) hematopoietic stem cell transplantation (UCBT) has emerged as a promising haematological reconstitution therapy for leukemias and other related disorders, the insufficient UCB stem cell dosage still hinders better clinical outcomes. Previous research efforts, by focusing on ex vivo UCB expansion capabilities have sought to benefit from well-known mechanisms of self-renewal characteristics of UCB stem cells. However, the long-term (> 21 days) in vitro culture period and the low neutrophil recovery significantly reduce the transplantability of such ex vivo expanded UCB stem cells. To overcome the latter hurdles in this study, a post-thaw, short-term ex vivo expansion methodology of UCB mononuclear (UCB-MN) and CD34+ cells has been established. Notably, such effort was achieved through pharmacological preconditioned of UCB cultures by filgrastim agent already used in the clinical setting. In crucial cell populations implicated in the promotion of functional engraftment, the progression of free survival rates (PFS), a marked increase of 6.65 to 9.34 fold for UCB-MN and 35 to 49 fold for CD34+ cells has been noticed. Overall, these results indicate that transplantation of pharmacologically-preconditioned ex vivo expansion of UCB stem and progenitor cells keep high promise upon transplantation to enhance therapeutic potential in everyday clinical practice. PMID:27335700

  2. Role of PTGS2-generated PGE2 during gonadotrophin-induced bovine oocyte maturation and cumulus cell expansion.

    PubMed

    Marei, Waleed F; Abayasekara, D Robert E; Wathes, D Claire; Fouladi-Nashta, Ali A

    2014-03-01

    Prostaglandin E2 (PGE2) is an autocrine/paracrine factor which mediates gonadotrophin (Gn) stimulation of cumulus expansion and oocyte maturation in rodents. Its role in bovine oocyte maturation is less characterized. This study detected PTGS2 (COX2) and PGE synthases (PTGES1, PTGES2 and PTGES3) in bovine cumulus-oocyte complexes (COC). Only PTGS2 and PTGES1 expression changed during maturation. In Gn-free media, no cumulus expansion and ∼45% nuclear maturation was achieved, while Gn-induced maturation showed full cumulus expansion (score 3) and ∼87% maturation. PGE2 supplementation without Gn induced mild cumulus expansion (score 0.5-1) but increased nuclear maturation to levels similar to those obtained with Gn alone. In the presence of Gn, exogenous PGE2 did not affect expansion or nuclear maturation and subsequent embryo development. Treatment with PTGS2 selective inhibitor (NS398), PTGS2-specific siRNA or PTGER2-receptor antagonist (AH6809) resulted in ∼20-25% reduction in nuclear maturation. NS398 and AH6809 did not affect cumulus expansion. Most oocytes not reaching metaphase of second meiosis (MII) following NS398, AH6809 and PTGS2-specific siRNA treatments were at MI. After longer maturation, NS398-treated oocytes had normal MII rate and uncompromised embryo development. PGE2 has a limited role in cumulus expansion in bovine COC but is important for the timing of Gn-induced nuclear maturation. We confirmed that genes involved in the synthesis of prostaglandin E2 (PGE2) are expressed by cumulus-oocyte complexes (or eggs) of cows and that PGE2 is synthesized during oocyte maturation in the presence of gonadotrophin hormones. When we inhibited synthesis of PGE2 or blocked its receptors, oocyte maturation, but not cumulus expansion, was compromised. Further investigation showed that oocyte maturation is delayed but not arrested when PGE2 synthesis is inhibited. On the other hand, addition of exogenous PGE2 induced a high maturation rate and mild cumulus

  3. Three-dimensional carbon nanotube scaffolds for long-term maintenance and expansion of human mesenchymal stem cells.

    PubMed

    Lalwani, Gaurav; D'agati, Michael; Gopalan, Anu; Patel, Sunny C; Talukdar, Yahfi; Sitharaman, Balaji

    2017-03-10

    Expansion of mesenchymal stem cells (MSCs) and maintenance of their self-renewal capacity in vitro requires specialized robust cell culture systems. Conventional approaches using animal-derived or artificial matrices and a cocktail of growth factors have limitations such as consistency, scalability, pathogenicity, and loss of MSC phenotype. Herein, we report the use of all-carbon 3-D single- and multiwalled carbon nanotube scaffolds (SWCNTs and MWCNTs) as artificial matrices for long-term maintenance and expansion of human MSCs. Three-dimensional SWCNT and MWCNT scaffolds were fabricated using a novel radical initiated thermal cross-linking method that covalently cross-links CNTs to form 3-D macroporous all-carbon architectures. Adipose-derived human MSCs showed good cell viability, attachment, proliferation, and infiltration in MWCNT and SWCNT scaffolds comparable to poly(lactic-co-glycolic) acid (PLGA) scaffolds (baseline control). ADSCs retained stem cell phenotype after 30 days and satisfied the International Society for Cellular Therapy's (ISCT) minimal criteria for MSCs. Post expansion, (1) ADSCs showed in vitro adherence to tissue culture polystyrene (TCPS); (2) MSC surface antigen expression [CD14(-), CD19(-), CD34(-), CD45(-), CD73(+), CD90(+), CD105(+)]; and (3) trilineage differentiation into osteoblasts, adipocytes, and chondrocytes. Results show that cross-linked 3-D MWCNTs and SWCNTs scaffolds are suitable for ex vivo expansion and maintenance of MSCs for therapeutic applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2017.

  4. Glyphosate-based pesticides affect cell cycle regulation.

    PubMed

    Marc, Julie; Mulner-Lorillon, Odile; Bellé, Robert

    2004-04-01

    Cell-cycle dysregulation is a hallmark of tumor cells and human cancers. Failure in the cell-cycle checkpoints leads to genomic instability and subsequent development of cancers from the initial affected cell. A worldwide used product Roundup 3plus, based on glyphosate as the active herbicide, was suggested to be of human health concern since it induced cell cycle dysfunction as judged from analysis of the first cell division of sea urchin embryos, a recognized model for cell cycle studies. Several glyphosate-based pesticides from different manufacturers were assayed in comparison with Roundup 3plus for their ability to interfere with the cell cycle regulation. All the tested products, Amega, Cargly, Cosmic, and Roundup Biovert induced cell cycle dysfunction. The threshold concentration for induction of cell cycle dysfunction was evaluated for each product and suggests high risk by inhalation for people in the vicinity of the pesticide handling sprayed at 500 to 4000 times higher dose than the cell-cycle adverse concentration.

  5. A cGMP-applicable expansion method for aggregates of human neural stem and progenitor cells derived from pluripotent stem cells or fetal brain tissue.

    PubMed

    Shelley, Brandon C; Gowing, Geneviève; Svendsen, Clive N

    2014-06-15

    A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as "chopping" that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

  6. Co-Expansion of Cytokine-Induced Killer Cells and Vγ9Vδ2 T Cells for CAR T-Cell Therapy.

    PubMed

    Du, Shou-Hui; Li, Zhendong; Chen, Can; Tan, Wee-Kiat; Chi, Zhixia; Kwang, Timothy Weixin; Xu, Xue-Hu; Wang, Shu

    2016-01-01

    Gamma delta (γδ) T cells and cytokine-induced killer (CIK) cells, which are a heterogeneous population of T lymphocytes and natural killer T (NKT) cells, have been separately expanded ex vivo and shown to be capable of targeting and mediating cytotoxicity against various tumor cells in a major histocompatibility complex-unrestricted manner. However, the co-expansion and co-administration of these immune cells have not been explored. In this study we describe an efficient method to expand simultaneously both CIK and Vγ9Vδ2 T cells, termed as CIKZ cells, from human peripheral blood mononuclear cells (PBMCs) using Zometa, interferon-gamma (IFN-γ), interleukin 2 (IL-2), anti-CD3 antibody and engineered K562 feeder cells expressing CD64, CD137L and CD86. A 21-day culture of PBMCs with this method yielded nearly 20,000-fold expansion of CIKZ cells with γδ T cells making up over 20% of the expanded population. The expanded CIKZ cells exhibited antitumor cytotoxicity and could be modified to express anti-CD19 chimeric antigen receptor (CAR), anti-CEA CAR, and anti-HER2 CAR to enhance their specificity and cytotoxicity against CD19-, CEA-, or HER2-positive tumor cells. The tumor inhibitory activity of anti-CD19 CAR-modified CIKZ cells was further demonstrated in vivo in a Raji tumor mouse model. The findings herein substantiate the feasibility of co-expanding CIK and γδ cells for adoptive cellular immunotherapy applications such as CAR T-cell therapy against cancer.

  7. Co-Expansion of Cytokine-Induced Killer Cells and Vγ9Vδ2 T Cells for CAR T-Cell Therapy

    PubMed Central

    Chen, Can; Tan, Wee-Kiat; Chi, Zhixia; Xu, Xue-Hu; Wang, Shu

    2016-01-01

    Gamma delta (γδ) T cells and cytokine-induced killer (CIK) cells, which are a heterogeneous population of T lymphocytes and natural killer T (NKT) cells, have been separately expanded ex vivo and shown to be capable of targeting and mediating cytotoxicity against various tumor cells in a major histocompatibility complex-unrestricted manner. However, the co-expansion and co-administration of these immune cells have not been explored. In this study we describe an efficient method to expand simultaneously both CIK and Vγ9Vδ2 T cells, termed as CIKZ cells, from human peripheral blood mononuclear cells (PBMCs) using Zometa, interferon-gamma (IFN-γ), interleukin 2 (IL-2), anti-CD3 antibody and engineered K562 feeder cells expressing CD64, CD137L and CD86. A 21-day culture of PBMCs with this method yielded nearly 20,000-fold expansion of CIKZ cells with γδ T cells making up over 20% of the expanded population. The expanded CIKZ cells exhibited antitumor cytotoxicity and could be modified to express anti-CD19 chimeric antigen receptor (CAR), anti-CEA CAR, and anti-HER2 CAR to enhance their specificity and cytotoxicity against CD19-, CEA-, or HER2-positive tumor cells. The tumor inhibitory activity of anti-CD19 CAR-modified CIKZ cells was further demonstrated in vivo in a Raji tumor mouse model. The findings herein substantiate the feasibility of co-expanding CIK and γδ cells for adoptive cellular immunotherapy applications such as CAR T-cell therapy against cancer. PMID:27598655

  8. OX40 controls effector CD4+ T-cell expansion, not follicular T helper cell generation in acute Listeria infection.

    PubMed

    Marriott, Clare L; Mackley, Emma C; Ferreira, Cristina; Veldhoen, Marc; Yagita, Hideo; Withers, David R

    2014-08-01

    To investigate the importance of OX40 signals for physiological CD4(+) T-cell responses, an endogenous antigen-specific population of CD4(+) T cells that recognise the 2W1S peptide was assessed and temporal control of OX40 signals was achieved using blocking or agonistic antibodies (Abs) in vivo. Following infection with Listeria monocytogenes expressing 2W1S peptide, OX40 was briefly expressed by the responding 2W1S-specific CD4(+) T cells, but only on a subset that co-expressed effector cell markers. This population was specifically expanded by Ab-ligation of OX40 during priming, which also caused skewing of the memory response towards effector memory cells. Strikingly, this greatly enhanced effector response was accompanied by the loss of T follicular helper (TFH) cells and germinal centres. Mice deficient in OX40 and CD30 showed normal generation of TFH cells but impaired numbers of 2W1S-specific effector cells. OX40 was not expressed by 2W1S-specific memory cells, although it was rapidly up-regulated upon challenge whereupon Ab-ligation of OX40 specifically affected the effector subset. In summary, these data indicate that for CD4(+) T cells, OX40 signals are important for generation of effector T cells rather than TFH cells in this response to acute bacterial infection.

  9. Early peroxisome proliferator-activated receptor gamma regulated genes involved in expansion of pancreatic beta cell mass

    PubMed Central

    2011-01-01

    Background The progression towards type 2 diabetes depends on the allostatic response of pancreatic beta cells to synthesise and secrete enough insulin to compensate for insulin resistance. The endocrine pancreas is a plastic tissue able to expand or regress in response to the requirements imposed by physiological and pathophysiological states associated to insulin resistance such as pregnancy, obesity or ageing, but the mechanisms mediating beta cell mass expansion in these scenarios are not well defined. We have recently shown that ob/ob mice with genetic ablation of PPARγ2, a mouse model known as the POKO mouse failed to expand its beta cell mass. This phenotype contrasted with the appropriate expansion of the beta cell mass observed in their obese littermate ob/ob mice. Thus, comparison of these models islets particularly at early ages could provide some new insights on early PPARγ dependent transcriptional responses involved in the process of beta cell mass expansion Results Here we have investigated PPARγ dependent transcriptional responses occurring during the early stages of beta cell adaptation to insulin resistance in wild type, ob/ob, PPARγ2 KO and POKO mice. We have identified genes known to regulate both the rate of proliferation and the survival signals of beta cells. Moreover we have also identified new pathways induced in ob/ob islets that remained unchanged in POKO islets, suggesting an important role for PPARγ in maintenance/activation of mechanisms essential for the continued function of the beta cell. Conclusions Our data suggest that the expansion of beta cell mass observed in ob/ob islets is associated with the activation of an immune response that fails to occur in POKO islets. We have also indentified other PPARγ dependent differentially regulated pathways including cholesterol biosynthesis, apoptosis through TGF-β signaling and decreased oxidative phosphorylation. PMID:22208362

  10. Real-Time Imaging of Cellulose Reorientation during Cell Wall Expansion in Arabidopsis Roots1[W][OA

    PubMed Central

    Anderson, Charles T.; Carroll, Andrew; Akhmetova, Laila; Somerville, Chris

    2010-01-01

    Cellulose forms the major load-bearing network of the plant cell wall, which simultaneously protects the cell and directs its growth. Although the process of cellulose synthesis has been observed, little is known about the behavior of cellulose in the wall after synthesis. Using Pontamine Fast Scarlet 4B, a dye that fluoresces preferentially in the presence of cellulose and has excitation and emission wavelengths suitable for confocal microscopy, we imaged the architecture and dynamics of cellulose in the cell walls of expanding root cells. We found that cellulose exists in Arabidopsis (Arabidopsis thaliana) cell walls in large fibrillar bundles that vary in orientation. During anisotropic wall expansion in wild-type plants, we observed that these cellulose bundles rotate in a transverse to longitudinal direction. We also found that cellulose organization is significantly altered in mutants lacking either a cellulose synthase subunit or two xyloglucan xylosyltransferase isoforms. Our results support a model in which cellulose is deposited transversely to accommodate longitudinal cell expansion and reoriented during expansion to generate a cell wall that is fortified against strain from any direction. PMID:19965966

  11. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

    PubMed

    Peremyslov, Valera V; Cole, Rex A; Fowler, John E; Dolja, Valerian V

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.

  12. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development

    PubMed Central

    Peremyslov, Valera V.; Cole, Rex A.; Fowler, John E.; Dolja, Valerian V.

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6–1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development. PMID:26426395

  13. Current state and perspectives in modeling and control of human pluripotent stem cell expansion processes in stirred-tank bioreactors.

    PubMed

    Galvanauskas, Vytautas; Grincas, Vykantas; Simutis, Rimvydas; Kagawa, Yuki; Kino-Oka, Masahiro

    2016-12-26

    Implementation of model-based practices for process development, control, automation, standardization, and validation are important factors for therapeutic and industrial applications of human pluripotent stem cells. As robust cultivation strategies for pluripotent stem cell expansion and differentiation have yet to be determined, process development could be enhanced by application of mathematical models and advanced control systems to optimize growth conditions. Therefore, it is important to understand both the potential of possible applications and the apparent limitations of existing mathematical models to improve pluripotent stem cell cultivation technologies. In the present review, the authors focus on these issues as they apply to stem cell expansion processes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017.

  14. In Vitro Expansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage

    PubMed Central

    Hare, Ian; Gencheva, Marieta; Evans, Rebecca; Fortney, James; Piktel, Debbie; Vos, Jeffrey A.; Howell, David; Gibson, Laura F.

    2016-01-01

    Mesenchymal stem cells (MSCs) are of interest for use in diverse cellular therapies. Ex vivo expansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs with in vitro passage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs), the most severe of DNA lesions. To investigate the response to DSB stress with passage in vitro, primary human MSCs were exposed to etoposide (VP16) at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ) transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR) transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passaged in vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate that ex vivo expansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation. PMID:26880992

  15. In Vitro Expansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage.

    PubMed

    Hare, Ian; Gencheva, Marieta; Evans, Rebecca; Fortney, James; Piktel, Debbie; Vos, Jeffrey A; Howell, David; Gibson, Laura F

    2016-01-01

    Mesenchymal stem cells (MSCs) are of interest for use in diverse cellular therapies. Ex vivo expansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs with in vitro passage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs), the most severe of DNA lesions. To investigate the response to DSB stress with passage in vitro, primary human MSCs were exposed to etoposide (VP16) at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ) transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR) transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passaged in vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate that ex vivo expansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation.

  16. Clonal expansion of CD8 T cells in the systemic circulation precedes development of ipilimumab-induced toxicities

    PubMed Central

    Subudhi, Sumit K.; Aparicio, Ana; Gao, Jianjun; Zurita, Amado J.; Araujo, John C.; Logothetis, Christopher J.; Tahir, Salahaldin A.; Korivi, Brinda R.; Slack, Rebecca S.; Vence, Luis; Emerson, Ryan O.; Yusko, Erik; Vignali, Marissa; Robins, Harlan S.; Sun, Jingjing; Allison, James P.; Sharma, Padmanee

    2016-01-01

    Immune checkpoint therapies, such as ipilimumab, induce dramatic antitumor responses in a subset of patients with advanced malignancies, but they may also induce inflammatory responses and toxicities termed immune-related adverse events (irAEs). These irAEs are often low grade and manageable, but severe irAEs may lead to prolonged hospitalizations or fatalities. Early intervention is necessary to minimize morbidities that occur with severe irAEs. However, correlative biomarkers are currently lacking. In a phase II clinical trial that treated 27 patients with metastatic prostate cancer, we aimed to test the safety and efficacy of androgen deprivation therapy plus ipilimumab. In this study, we observed grade 3 toxicities in >40% of treated patients, which led to early closure of the study. Because ipilimumab enhances T-cell responses, we hypothesized that increased clonal T-cell responses in the systemic circulation may contribute to irAEs. Sequencing of the T-cell receptor β-chains in purified T cells revealed clonal expansion of CD8 T cells, which occurred in blood samples collected before the onset of grade 2–3 irAEs. These initial results suggested that expansion of ≥55 CD8 T-cell clones preceded the development of severe irAEs. We further evaluated available blood samples from a second trial and determined that patients who experienced grade 2–3 irAEs also had expansion of ≥55 CD8 T-cell clones in blood samples collected before the onset of irAEs. We propose that CD8 T-cell clonal expansion may be a correlative biomarker to enable close monitoring and early intervention for patients receiving ipilimumab. PMID:27698113

  17. Large-scale expansion of pre-isolated bone marrow mesenchymal stromal cells in serum-free conditions.

    PubMed

    Gottipamula, Sanjay; Muttigi, Manjunatha S; Chaansa, S; Ashwin, K M; Priya, Nancy; Kolkundkar, Udaykumar; SundarRaj, Swathi; Majumdar, Anish Sen; Seetharam, Raviraja N

    2016-02-01

    The regenerative potential of mesenchymal stromal or stem cells (MSCs) has generated tremendous interest for treating various degenerative diseases. Regulatory preference is to use a culture medium that is devoid of bovine components for stem cell expansion intended for therapeutic applications. However, a clear choice an alternative to fetal bovine serum (FBS) has not yet emerged. We have screened five different commercially available serum-free media (SFM) for their ability to support the growth and expansion of pre-isolated undifferentiated bone marrow-derived MSCs (BM-MSCs) and compared the results with cells grown in standard FBS-containing medium as control. In addition, based on initial screening results, BD Mosaic™ Mesenchymal Stem Cell Serum-free (BD-SFM) medium was evaluated in large-scale cultures for the performance and culture characteristics of BM-MSCs. Of the five different serum-free media, BD-SFM enhanced BM-MSCs growth and expansion in Cell STACK (CS), but the cell yield per CS-10 was less when compared to the control medium. The characteristics of MSCs were measured in terms of population doubling time (PDT), cell yield and expression of MSC-specific markers. Significant differences were observed between BD-SFM and control medium in terms of population doublings (PDs), cell yield, CFU-F and morphological features, whereas surface phenotype and differentiation potentials were comparable. The BD-SFM-cultured MSCs were also found to retain the differentiation potential, immune-privileged status and immunosuppressive properties inherent to MSCs. Our results suggest that BD-SFM supports large-scale expansion of BM-MSCs for therapeutic use.

  18. Clonal expansion of CD8 T cells in the systemic circulation precedes development of ipilimumab-induced toxicities.

    PubMed

    Subudhi, Sumit K; Aparicio, Ana; Gao, Jianjun; Zurita, Amado J; Araujo, John C; Logothetis, Christopher J; Tahir, Salahaldin A; Korivi, Brinda R; Slack, Rebecca S; Vence, Luis; Emerson, Ryan O; Yusko, Erik; Vignali, Marissa; Robins, Harlan S; Sun, Jingjing; Allison, James P; Sharma, Padmanee

    2016-10-18

    Immune checkpoint therapies, such as ipilimumab, induce dramatic antitumor responses in a subset of patients with advanced malignancies, but they may also induce inflammatory responses and toxicities termed immune-related adverse events (irAEs). These irAEs are often low grade and manageable, but severe irAEs may lead to prolonged hospitalizations or fatalities. Early intervention is necessary to minimize morbidities that occur with severe irAEs. However, correlative biomarkers are currently lacking. In a phase II clinical trial that treated 27 patients with metastatic prostate cancer, we aimed to test the safety and efficacy of androgen deprivation therapy plus ipilimumab. In this study, we observed grade 3 toxicities in >40% of treated patients, which led to early closure of the study. Because ipilimumab enhances T-cell responses, we hypothesized that increased clonal T-cell responses in the systemic circulation may contribute to irAEs. Sequencing of the T-cell receptor β-chains in purified T cells revealed clonal expansion of CD8 T cells, which occurred in blood samples collected before the onset of grade 2-3 irAEs. These initial results suggested that expansion of ≥55 CD8 T-cell clones preceded the development of severe irAEs. We further evaluated available blood samples from a second trial and determined that patients who experienced grade 2-3 irAEs also had expansion of ≥55 CD8 T-cell clones in blood samples collected before the onset of irAEs. We propose that CD8 T-cell clonal expansion may be a correlative biomarker to enable close monitoring and early intervention for patients receiving ipilimumab.

  19. Retention of stemness and vasculogenic potential of human umbilical cord blood stem cells after repeated expansions on PES-nanofiber matrices

    PubMed Central

    Joseph, Matthew; Das, Manjusri; Kanji, Suman; Lu, Jingwei; Aggarwal, Reeva; Chakraborty, Debanjan; Sarkar, Chandrani; Yu, Hongmei; Mao, Hai-Quan; Basu, Sujit; Pompili, Vincent J.; Das, Hiranmoy

    2014-01-01

    Despite recent advances in cardiovascular medicine, ischemic diseases remain a major cause of morbidity and mortality. Although stem cell-based therapies for the treatment of ischemic diseases show great promise, limited availability of biologically functional stem cells mired the application of stem cell-based therapies. Previously, we reported a PES-nanofiber based ex vivo stem cell expansion technology, which supports expansion of human umbilical cord blood (UCB)-derived CD133+/CD34+ progenitor cells ~225 fold. Herein, we show that using similar technology and subsequent re-expansion methods, we can achieve ~5 million-fold yields within 24 days of the initial seeding. Interestingly, stem cell phenotype was preserved during the course of the multiple expansions. The high level of the stem cell homing receptor, CXCR4 was expressed in the primary expansion cells, and was maintained throughout the course of re-expansions. In addition, re-expanded cells preserved their multi-potential differential capabilities in vitro such as, endothelial and smooth muscle lineages. Moreover, biological functionality of the re-expanded cells was preserved and was confirmed by a murine hind limb ischemia model for revascularization. These cells could also be genetically modified for enhanced vasculogenesis. Immunohistochemical evidences support enhanced expression of angiogenic factors responsible for this enhanced neovascularization. These data further confirms that nanofiber-based ex-vivo expansion technology can generate sufficient numbers of biologically functional stem cells for potential clinical applications. PMID:25002260

  20. Antigen-specific CD4{sup +} effector T cells: Analysis of factors regulating clonal expansion and cytokine production

    SciTech Connect

    Ohnuki, Kazunobu; Watanabe, Yuri; Takahashi, Yusuke; Kobayashi, Sakiko; Watanabe, Shiho; Ogawa, Shuhei; Kotani, Motoko; Kozono, Haruo; Tanabe, Kazunari; Abe, Ryo

    2009-03-20

    In order to fully understand T cell-mediated immunity, the mechanisms that regulate clonal expansion and cytokine production by CD4{sup +} antigen-specific effector T cells in response to a wide range of antigenic stimulation needs clarification. For this purpose, panels of antigen-specific CD4{sup +} T cell clones with different thresholds for antigen-induced proliferation were generated by repeated stimulation with high- or low-dose antigen. Differences in antigen sensitivities did not correlate with expression of TCR, CD4, adhesion or costimulatory molecules. There was no significant difference in antigen-dependent cytokine production by TG40 cells transfected with TCR obtained from either high- or low-dose-responding T cell clones, suggesting that the affinity of TCRs for their ligands is not primary determinant of T cell antigen reactivity. The proliferative responses of all T cell clones to both peptide stimulation and to TCR{beta} crosslinking revealed parallel dose-response curves. These results suggest that the TCR signal strength of effector T cells and threshold of antigen reactivity is determined by an intrinsic property, such as the TCR signalosome and/or intracellular signaling machinery. Finally, the antigen responses of high- and low-peptide-responding T cell clones reveal that clonal expansion and cytokine production of effector T cells occur independently of antigen concentration. Based on these results, the mechanisms underlying selection of high 'avidity' effector and memory T cells in response to pathogen are discussed.

  1. Thermal expansion coefficient prediction of fuel-cell seal materials from silica sand

    NASA Astrophysics Data System (ADS)

    Hidayat, Nurul; Triwikantoro, Baqiya, Malik A.; Pratapa, Suminar

    2013-09-01

    This study is focused on the prediction of coefficient of thermal expansion (CTE) of silica-sand-based fuel-cell seal materials (FcSMs) which in principle require a CTE value in the range of 9.5-12 ppm/°C. A semi-quantitative theoretical method to predict the CTE value is proposed by applying the analyzed phase compositions from XRD data and characterized density-porosity behavior. A typical silica sand was milled at 150 rpm for 1 hour followed by heating at 1000 °C for another hour. The sand and heated samples were characterized by means of XRD to perceive the phase composition correlation between them. Rietveld refinement was executed to investigate the weight fraction of the phase contained in the samples, and then converted to volume fraction for composite CTE calculations. The result was applied to predict their potential physical properties for FcSM. Porosity was taken into account in the calculation after which it was directly measured by the Archimedes method.

  2. Action of obestatin in skeletal muscle repair: stem cell expansion, muscle growth, and microenvironment remodeling.

    PubMed

    Gurriarán-Rodríguez, Uxía; Santos-Zas, Icía; González-Sánchez, Jessica; Beiroa, Daniel; Moresi, Viviana; Mosteiro, Carlos S; Lin, Wei; Viñuela, Juan E; Señarís, José; García-Caballero, Tomás; Casanueva, Felipe F; Nogueiras, Rubén; Gallego, Rosalía; Renaud, Jean-Marc; Adamo, Sergio; Pazos, Yolanda; Camiña, Jesús P

    2015-06-01

    The development of therapeutic strategies for skeletal muscle diseases, such as physical injuries and myopathies, depends on the knowledge of regulatory signals that control the myogenic process. The obestatin/GPR39 system operates as an autocrine signal in the regulation of skeletal myogenesis. Using a mouse model of skeletal muscle regeneration after injury and several cellular strategies, we explored the potential use of obestatin as a therapeutic agent for the treatment of trauma-induced muscle injuries. Our results evidenced that the overexpression of the preproghrelin, and thus obestatin, and GPR39 in skeletal muscle increased regeneration after muscle injury. More importantly, the intramuscular injection of obestatin significantly enhanced muscle regeneration by simulating satellite stem cell expansion as well as myofiber hypertrophy through a kinase hierarchy. Added to the myogenic action, the obestatin administration resulted in an increased expression of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) and the consequent microvascularization, with no effect on collagen deposition in skeletal muscle. Furthermore, the potential inhibition of myostatin during obestatin treatment might contribute to its myogenic action improving muscle growth and regeneration. Overall, our data demonstrate successful improvement of muscle regeneration, indicating obestatin is a potential therapeutic agent for skeletal muscle injury and would benefit other myopathies related to muscle regeneration.

  3. Action of Obestatin in Skeletal Muscle Repair: Stem Cell Expansion, Muscle Growth, and Microenvironment Remodeling

    PubMed Central

    Gurriarán-Rodríguez, Uxía; Santos-Zas, Icía; González-Sánchez, Jessica; Beiroa, Daniel; Moresi, Viviana; Mosteiro, Carlos S; Lin, Wei; Viñuela, Juan E; Señarís, José; García-Caballero, Tomás; Casanueva, Felipe F; Nogueiras, Rubén; Gallego, Rosalía; Renaud, Jean-Marc; Adamo, Sergio; Pazos, Yolanda; Camiña, Jesús P

    2015-01-01

    The development of therapeutic strategies for skeletal muscle diseases, such as physical injuries and myopathies, depends on the knowledge of regulatory signals that control the myogenic process. The obestatin/GPR39 system operates as an autocrine signal in the regulation of skeletal myogenesis. Using a mouse model of skeletal muscle regeneration after injury and several cellular strategies, we explored the potential use of obestatin as a therapeutic agent for the treatment of trauma-induced muscle injuries. Our results evidenced that the overexpression of the preproghrelin, and thus obestatin, and GPR39 in skeletal muscle increased regeneration after muscle injury. More importantly, the intramuscular injection of obestatin significantly enhanced muscle regeneration by simulating satellite stem cell expansion as well as myofiber hypertrophy through a kinase hierarchy. Added to the myogenic action, the obestatin administration resulted in an increased expression of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) and the consequent microvascularization, with no effect on collagen deposition in skeletal muscle. Furthermore, the potential inhibition of myostatin during obestatin treatment might contribute to its myogenic action improving muscle growth and regeneration. Overall, our data demonstrate successful improvement of muscle regeneration, indicating obestatin is a potential therapeutic agent for skeletal muscle injury and would benefit other myopathies related to muscle regeneration. PMID:25762009

  4. [Feedback control mechanisms of plant cell expansion]. Progress report, [June 1989--June 1992

    SciTech Connect

    Cosgrove, D.J.

    1992-12-31

    We have generated considerable evidence for the significance of wall stress relaxation in the control of plant growth and found that several agents (gibberellin, light, genetic loci for dwarf stature) influence growth rate via alteration of wall relaxation. We have refined our methods for measuring wall relaxation and, moreover, have found that wall relaxation properties bear only a distance relationship to wall mechanical properties. We have garnered novel insights into the nature of cell expansion mechanisms by analyzing spontaneous fluctuations of plant growth rate in seedlings. These experiments involved the application of mathematical techniques for analyzing growth rate fluctuations and the development of new instrumentation for measuring and forcing plant growth in a controlled fashion. These studies conclude that growth rate fluctuations generated by the plant as consequence of a feedback control system. This conclusion has important implications for the nature of wall loosening processes and demands a different framework for thinking about growth control. It also implies the existence of a growth rate sensor.

  5. Osteoprogenitor cells from bone marrow and cortical bone: understanding how the environment affects their fate.

    PubMed

    Corradetti, Bruna; Taraballi, Francesca; Powell, Sebastian; Sung, David; Minardi, Silvia; Ferrari, Mauro; Weiner, Bradley K; Tasciotti, Ennio

    2015-05-01

    Bone is a dynamic organ where skeletal progenitors and hematopoietic cells share and compete for space. Presumptive mesenchymal stem cells (MSC) have been identified and harvested from the bone marrow (BM-MSC) and cortical bone fragments (CBF-MSC). In this study, we demonstrate that despite the cells sharing a common ancestor, the differences in the structural properties of the resident tissues affect cell behavior and prime them to react differently to stimuli. Similarly to the bone marrow, the cortical portion of the bone contains a unique subset of cells that stains positively for the common MSC-associated markers. These cells display different multipotent differentiation capability, clonogenic expansion, and immunosuppressive potential. In particular, when compared with BM-MSC, CBF-MSC are bigger in size, show a lower proliferation rate at early passages, have a greater commitment toward the osteogenic lineage, constitutively produce nitric oxide as a mediator for bone remodeling, and more readily respond to proinflammatory cytokines. Our data suggest that the effect of the tissue's microenvironment makes the CBF-MSC a superior candidate in the development of new strategies for bone repair.

  6. Genetic background affects the expansion of macrophage subsets in the lungs of Mycobacterium tuberculosis-infected hosts.

    PubMed

    Bertolini, Thais Barboza; de Souza, Alexandre Ignacio; Gembre, Ana Flávia; Piñeros, Annie Rocio; Prado, Rafael de Queiroz; Silva, João Santana; Ramalho, Leandra Naira Zambelli; Bonato, Vânia Luiza Deperon

    2016-05-01

    M1 macrophages are more effective in the induction of the inflammatory response and clearance of Mycobacterium tuberculosis than M2 macrophages. Infected C57BL/6 mice generate a stronger cellular immune response compared with BALB/c mice. We hypothesized that infected C57BL/6 mice would exhibit a higher frequency and function of M1 macrophages than infected BALB/c mice. Our findings show a higher ratio of macrophages to M2 macrophages in the lungs of chronically infected C57BL/6 mice compared with BALB/c mice. However, there was no difference in the functional ability of M1 and M2 macrophages for the two strains in vitro. In vivo, a deleterious role for M2 macrophages was confirmed by M2 cell transfer, which rendered the infected C57BL/6, but not the BALB/c mice, more susceptible and resulted in mild lung inflammation compared with C57BL/6 mice that did not undergo cell transfer. M1 cell transfer induced a higher inflammatory response, although not protective, in infected BALB/c mice compared with their counterparts that did not undergo cell transfer. These findings demonstrate that an inflammation mediated by M1 macrophages may not induce bacterial tolerance because protection depends on the host genetic background, which drives the magnitude of the inflammatory response against M. tuberculosis in the pulmonary microenvironment. The contribution of our findings is that although M1 macrophage is an effector leucocyte with microbicidal machinery, its dominant role depends on the balance of M1 and M2 subsets, which is driven by the host genetic background.

  7. Angiopoietin-Like Protein 3 Promotes Preservation of Stemness during Ex Vivo Expansion of Murine Hematopoietic Stem Cells

    PubMed Central

    Farahbakhshian, Elnaz; Verstegen, Monique M.; Visser, Trudi P.; Kheradmandkia, Sima; Geerts, Dirk; Arshad, Shazia; Riaz, Noveen; Grosveld, Frank; van Til, Niek P.; Meijerink, Jules P. P.

    2014-01-01

    Allogeneic hematopoietic stem cell (HSC) transplantations from umbilical cord blood or autologous HSCs for gene therapy purposes are hampered by limited number of stem cells. To test the ability to expand HSCs in vitro prior to transplantation, two growth factor cocktails containing stem cell factor, thrombopoietin, fms-related tyrosine kinase-3 ligand (STF) or stem cell factor, thrombopoietin, insulin-like growth factor-2, fibroblast growth factor-1 (STIF) either with or without the addition of angiopoietin-like protein-3 (Angptl3) were used. Culturing HSCs in STF and STIF media for 7 days expanded long-term repopulating stem cells content in vivo by ∼6-fold and ∼10-fold compared to freshly isolated stem cells. Addition of Angptl3 resulted in increased expansion of these populations by ∼17-fold and ∼32-fold, respectively, and was further supported by enforced expression of Angptl3 in HSCs through lentiviral transduction that also promoted HSC expansion. As expansion of highly purified lineage-negative, Sca-1+, c-Kit+ HSCs was less efficient than less pure lineage-negative HSCs, Angptl3 may have a direct effect on HCS but also an indirect effect on accessory cells that support HSC expansion. No evidence for leukemia or toxicity was found during long-term follow up of mice transplanted with ex vivo expanded HSCs or manipulated HSC populations that expressed Angptl3. We conclude that the cytokine combinations used in this study to expand HSCs ex vivo enhances the engraftment in vivo. This has important implications for allogeneic umbilical cord-blood derived HSC transplantations and autologous HSC applications including gene therapy. PMID:25170927

  8. Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells.

    PubMed

    Lohmann, Michael; Walenda, Gudrun; Hemeda, Hatim; Joussen, Sylvia; Drescher, Wolf; Jockenhoevel, Stefan; Hutschenreuter, Gabriele; Zenke, Martin; Wagner, Wolfgang

    2012-01-01

    The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (<35 years) as compared to older donors (>45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.

  9. Length-dependent CTG·CAG triplet-repeat expansion in myotonic dystrophy patient-derived induced pluripotent stem cells

    PubMed Central

    Du, Jintang; Campau, Erica; Soragni, Elisabetta; Jespersen, Christine; Gottesfeld, Joel M.

    2013-01-01

    Myotonic dystrophy type 1 (DM1) is an inherited dominant muscular dystrophy caused by expanded CTG·CAG triplet repeats in the 3′ untranslated region of the DMPK1 gene, which produces a toxic gain-of-function CUG RNA. It has been shown that the severity of disease symptoms, age of onset and progression are related to the length of the triplet repeats. However, the mechanism(s) of CTG·CAG triplet-repeat instability is not fully understood. Herein, induced pluripotent stem cells (iPSCs) were generated from DM1 and Huntington's disease patient fibroblasts. We isolated 41 iPSC clones from DM1 fibroblasts, all showing different CTG·CAG repeat lengths, thus demonstrating somatic instability within the initial fibroblast population. During propagation of the iPSCs, the repeats expanded in a manner analogous to the expansion seen in somatic cells from DM1 patients. The correlation between repeat length and expansion rate identified the interval between 57 and 126 repeats as being an important length threshold where expansion rates dramatically increased. Moreover, longer repeats showed faster triplet-repeat expansion. However, the overall tendency of triplet repeats to expand ceased on differentiation into differentiated embryoid body or neurospheres. The mismatch repair components MSH2, MSH3 and MSH6 were highly expressed in iPSCs compared with fibroblasts, and only occupied the DMPK1 gene harboring longer CTG·CAG triplet repeats. In addition, shRNA silencing of MSH2 impeded CTG·CAG triplet-repeat expansion. The information gained from these studies provides new insight into a general mechanism of triplet-repeat expansion in iPSCs. PMID:23933738

  10. Direct visualization of endogenous Salmonella-specific B cells reveals a marked delay in clonal expansion and germinal center development.

    PubMed

    Nanton, Minelva R; Lee, Seung-Joo; Atif, Shaikh M; Nuccio, Sean-Paul; Taylor, Justin J; Bäumler, Andreas J; Way, Sing Sing; McSorley, Stephen J

    2015-02-01

    CD4(+) T cells and B cells are both essential for acquired immunity to Salmonella infection. It is well established that Salmonella inhibit host CD4(+) T-cell responses, but a corresponding inhibitory effect on B cells is less well defined. Here, we utilize an Ag tetramer and pull-down enrichment strategy to directly visualize OVA-specific B cells in mice, as they respond to infection with Salmonella-OVA. Surprisingly, OVA-specific B-cell expansion and germinal center formation was not detected until bacteria were cleared from the host. Furthermore, Salmonella infection also actively inhibited both B- and T-cell responses to the same coinjected Ag but this did not require the presence of iNOS. The Salmonella Pathogenicity Island 2 (SPI2) locus has been shown to be responsible for inhibition of Salmonella-specific CD4(+) T-cell responses, and an examination of SPI2-deficient bacteria demonstrated a recovery in B-cell expansion in infected mice. Together, these data suggest that Salmonella can simultaneously inhibit host B- and T-cell responses using SPI2-dependent mechanisms.

  11. Glucocorticoid-induced leucine zipper enhanced expression in dendritic cells is sufficient to drive regulatory T cells expansion in vivo.

    PubMed

    Calmette, Joseph; Ellouze, Mehdi; Tran, Thi; Karaki, Soumaya; Ronin, Emilie; Capel, Francis; Pallardy, Marc; Bachelerie, Françoise; Krzysiek, Roman; Emilie, Dominique; Schlecht-Louf, Géraldine; Godot, Véronique

    2014-12-15

    Tolerance induction by dendritic cells (DCs) is, in part, mediated by the activation of regulatory T cells (Tregs). We have previously shown in vitro that human DCs treated with glucocorticoids (GCs), IL-10, or TGF-β upregulate the GC-Induced Leucine Zipper protein (GILZ). GILZ overexpression promotes DC differentiation into regulatory cells that generate IL-10-producing Ag-specific Tregs. To investigate whether these observations extend in vivo, we have generated CD11c-GILZ(hi) transgenic mice. DCs from these mice constitutively overexpress GILZ to levels observed in GC-treated wild-type DCs. In this article, we establish that GILZ(hi) DCs display an accumulation of Foxp3(+) Tregs in the spleens of young CD11c-GILZ(hi) mice. In addition, we show that GILZ(hi) DCs strongly increase the Treg pool in central and peripheral lymphoid organs of aged animals. Upon adoptive transfer to wild-type recipient mice, OVA-loaded GILZ(hi) bone marrow-derived DCs induce a reduced activation and proliferation of OVA-specific T cells as compared with control bone marrow-derived DCs, associated with an expansion of thymus-derived CD25(+)Foxp3(+) CD4 T cells. Transferred OVA-loaded GILZ(hi) DCs produce significantly higher levels of IL-10 and express reduced levels of MHC class II molecules as compared with OVA-loaded control DCs, emphasizing the regulatory phenotype of GILZ(hi) DCs in vivo. Thus, our work demonstrates in vivo that the GILZ overexpression alone is sufficient to promote a tolerogenic mode of function in DCs.

  12. Extensive Ex Vivo Expansion of Functional Human Erythroid Precursors Established From Umbilical Cord Blood Cells by Defined Factors

    PubMed Central

    Huang, Xiaosong; Shah, Siddharth; Wang, Jing; Ye, Zhaohui; Dowey, Sarah N; Tsang, Kit Man; Mendelsohn, Laurel G; Kato, Gregory J; Kickler, Thomas S; Cheng, Linzhao

    2014-01-01

    There is a constant shortage of red blood cells (RBCs) from sufficiently matched donors for patients who need chronic transfusion. Ex vivo expansion and maturation of human erythroid precursors (erythroblasts) from the patients or optimally matched donors could represent a potential solution. Proliferating erythroblasts can be expanded from umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 106–107-fold (in ~50 days) before proliferation arrest and reaching sufficient number for broad application. Here, we report that ectopic expression of three genetic factors (Sox2, c-Myc, and an shRNA against TP53 gene) associated with iPSC derivation enables CB-derived erythroblasts to undergo extended expansion (~1068-fold in ~12 months) in a serum-free culture condition without change of cell identity or function. These expanding erythroblasts maintain immature erythroblast phenotypes and morphology, a normal diploid karyotype and dependence on a specific combination of growth factors for proliferation throughout expansion period. When being switched to a terminal differentiation condition, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes that can bind and release oxygen. Our result may ultimately lead to an alternative approach to generate unlimited numbers of RBCs for personalized transfusion medicine. PMID:24002691

  13. Rosmarinic Acid and Melissa officinalis Extracts Differently Affect Glioblastoma Cells.

    PubMed

    Ramanauskiene, Kristina; Raudonis, Raimondas; Majiene, Daiva

    Lemon balm (Melissa officinalis L.) has many biological effects but especially important is its neuroprotective activity. The aim of the study is to produce different extracts of Melissa officinalis and analyse their chemical composition and biological properties on rat glioblastoma C6 cells. Results revealed that rosmarinic acid (RA) is the predominant compound of lemon balm extracts. RA has cytotoxic effect on glioblastoma cells (LC50 290.5 μM after the incubation of 24 h and LC50 171.3 μM after 48 h). RA at concentration 80-130 μM suppresses the cell proliferation and has an antioxidant effect. 200 μM and higher concentrations of RA have a prooxidant effect and initiate cell death through necrosis. The aqueous extract of lemon balm is also enriched in phenolic compounds: protocatechuic, caftaric, caffeic, ferulic, and cichoric acids and flavonoid luteolin-7-glucoside. This extract at concentrations 50 μM-200 μM RA has cytotoxic activity and initiates cell death through apoptosis. Extracts prepared with 70% ethanol contain the biggest amount of active compounds. These extracts have the highest cytotoxic activity on glioblastoma cells. They initiate generation of intracellular ROS and cell death through apoptosis and necrosis. Our data suggest that differently prepared lemon balm extracts differently affect glioblastoma cells and can be used as neuroprotective agents in several therapeutic strategies.

  14. Rosmarinic Acid and Melissa officinalis Extracts Differently Affect Glioblastoma Cells

    PubMed Central

    Ramanauskiene, Kristina; Raudonis, Raimondas

    2016-01-01

    Lemon balm (Melissa officinalis L.) has many biological effects but especially important is its neuroprotective activity. The aim of the study is to produce different extracts of Melissa officinalis and analyse their chemical composition and biological properties on rat glioblastoma C6 cells. Results revealed that rosmarinic acid (RA) is the predominant compound of lemon balm extracts. RA has cytotoxic effect on glioblastoma cells (LC50 290.5 μM after the incubation of 24 h and LC50 171.3 μM after 48 h). RA at concentration 80–130 μM suppresses the cell proliferation and has an antioxidant effect. 200 μM and higher concentrations of RA have a prooxidant effect and initiate cell death through necrosis. The aqueous extract of lemon balm is also enriched in phenolic compounds: protocatechuic, caftaric, caffeic, ferulic, and cichoric acids and flavonoid luteolin-7-glucoside. This extract at concentrations 50 μM–200 μM RA has cytotoxic activity and initiates cell death through apoptosis. Extracts prepared with 70% ethanol contain the biggest amount of active compounds. These extracts have the highest cytotoxic activity on glioblastoma cells. They initiate generation of intracellular ROS and cell death through apoptosis and necrosis. Our data suggest that differently prepared lemon balm extracts differently affect glioblastoma cells and can be used as neuroprotective agents in several therapeutic strategies. PMID:27688825

  15. Sunitinib pretreatment improves tumor-infiltrating lymphocyte expansion by reduction in intratumoral content of myeloid-derived suppressor cells in human renal cell carcinoma.

    PubMed

    Guislain, Aurelie; Gadiot, Jules; Kaiser, Andrew; Jordanova, Ekaterina S; Broeks, Annegien; Sanders, Joyce; van Boven, Hester; de Gruijl, Tanja D; Haanen, John B A G; Bex, Axel; Blank, Christian U

    2015-10-01

    Targeted therapy with sunitinib, pazopanib or everolimus has improved treatment outcome for patients with metastatic renal cell carcinoma patients (RCC). However, despite considerable efforts in sequential or combined modalities, durable remissions are rare. Immunotherapy like cytokine therapy with interleukin-2, T cell checkpoint blockade or adoptive T cell therapies can achieve long-term benefit and even cure. This raises the question of whether combining targeted therapy with immunotherapy could also be an effective treatment option for RCC patients. Sunitinib, one of the most frequently administered therapeutics in RCC patients has been implicated in impairing T cell activation and proliferation in vitro. In this work, we addressed whether this notion holds true for expansion of tumor-infiltrating lymphocytes (TILs) in sunitinib-treated patients. We compared resected primary RCC tumor material of patients pretreated with sunitinib with resection specimen from sunitinib-naïve patients. We found improved TIL expansion from sunitinib-pretreated tumor digests. These TIL products contained more PD-1 expressing TIL, while the regulatory T cell infiltration was not altered. The improved TIL expansion was associated with reduced intratumoral myeloid-derived suppressor cell (MDSC) content. Depletion of MDSCs from sunitinib-naïve RCC tissue-digest improved TIL expansion, proving the functional relevance of the MDSC alteration by sunitinib. Our in vivo results do not support previous in vitro observations of sunitinib inhibiting T cell function, but do provide a possible rationale for the combination of sunitinib with immunotherapy.

  16. Promoting Effects of Heparin on ex vivo Expansion of Megakaryocytopoiesis from Human Cord Blood CD34+ Cells

    PubMed Central

    Maurer, Anne-Marie; Gezer, Altay

    2013-01-01

    Summary Introduction Transfusion of ex vivo expanded megakaryocytes (MKs) has been proposed to sustain platelet recovery after cord blood (CB) hematopoietic stem cell transplantation. In this study, we investigated the effects of heparin on ex vivo colony forming unit-megakaryocytes (CFU-MKs) and MKs expansion from CB CD34+ cells. Methods CB CD34+ cells were stimulated by a combination of thrombopoietin (TPO), stem cell factor (SCF), Flt3-Ligand (FL), IL-6, and IL-11 supplemented with autologous serum and heparin during 14 days. Expanded cells were analyzed by flow cytometry and cultured in a CFU-MK assay. Results Compared to control cultures, the 5-factor combination with heparin induced significantly (p ≤ 0.05) higher numbers of: CFU-MKs and CD41+ cells on days 7 and 14; CD41+ cells displaying hyperploidy levels (≥8N) on day 14; platelets on day 14. The culture-derived platelets were activated upon collagen stimulation. Conclusion Heparin can significantly enhance the stimulating effects of a combination of TPO, SCF, FL, IL-6, and IL-11 supplemented with autologous serum on CFU-MK, MK, and platelet production from CB CD34+ cells. This expansion system could represent a promising method to generate CFU-MKs and MKs cells for transfusion to sustain platelet reconstitution following CB transplantation. PMID:24273488

  17. High antigen levels induce an exhausted phenotype in a chronic infection without impairing T cell expansion and survival

    PubMed Central

    Alfei, Francesca; Roelli, Patrick; Delorenzi, Mauro; Pinschewer, Daniel D.

    2016-01-01

    Chronic infections induce T cells showing impaired cytokine secretion and up-regulated expression of inhibitory receptors such as PD-1. What determines the acquisition of this chronic phenotype and how it impacts T cell function remain vaguely understood. Using newly generated recombinant antigen variant-expressing chronic lymphocytic choriomeningitis virus (LCMV) strains, we uncovered that T cell differentiation and acquisition of a chronic or exhausted phenotype depend critically on the frequency of T cell receptor (TCR) engagement and less significantly on the strength of TCR stimulation. In fact, we noted that low-level antigen exposure promotes the formation of T cells with an acute phenotype in chronic infections. Unexpectedly, we found that T cell populations with an acute or chronic phenotype are maintained equally well in chronic infections and undergo comparable primary and secondary expansion. Thus, our observations contrast with the view that T cells with a typical chronic infection phenotype are severely functionally impaired and rapidly transition into a terminal stage of differentiation. Instead, our data unravel that T cells primarily undergo a form of phenotypic and functional differentiation in the early phase of a chronic LCMV infection without inheriting a net survival or expansion deficit, and we demonstrate that the acquired chronic phenotype transitions into the memory T cell compartment. PMID:27455951

  18. [Multi-scenario simulation and prediction of ecosystem services as affected by urban expansion: A case study in coastal area of Tianjin, North China].

    PubMed

    Huang, Huan-Chun; Yun, Ying-Xia; Miao, Zhan-Tang; Hao, Cui; Li, Hong-yuan

    2013-03-01

    Based on the modified Logistic-CA model, and taking the coastal area of Tianjin as a case, this paper simulated the spatial evolution patterns of ecosystem services as affected by the urban expansion in 2011-2020 under the scenarios of historical extrapolation, endogenous development, and exogenous development. Overall, the total ecosystem services of the study area under the three scenarios were generally the same, and the functional region with the lowest level ecosystem services had the identical spatial pattern. However, the spatial evolution patterns of the ecosystem services of the study area under the three scenarios had a great difference. The functional regions with lower-level ecosystem services grew in a cross-shaped pattern, with the Tanggu downtown as a center, and finally formed a full connectivity area along the Haihe River and coastal zone.

  19. AtDOF5.4/OBP4, a DOF Transcription Factor Gene that Negatively Regulates Cell Cycle Progression and Cell Expansion in Arabidopsis thaliana.

    PubMed

    Xu, Peipei; Chen, Haiying; Ying, Lu; Cai, Weiming

    2016-06-14

    In contrast to animals, plant development involves continuous organ formation, which requires strict regulation of cell proliferation. The core cell cycle machinery is conserved across plants and animals, but plants have developed new mechanisms that precisely regulate cell proliferation in response to internal and external stimuli. Here, we report that the DOF transcription factor OBP4 negatively regulates cell proliferation and expansion. OBP4 is a nuclear protein. Constitutive and inducible overexpression of OBP4 reduced the cell size and number, resulting in dwarf plants. Inducible overexpression of OBP4 in Arabidopsis also promoted early endocycle onset and inhibited cell expansion, while inducible overexpression of OBP4 fused to the VP16 activation domain in Arabidopsis delayed endocycle onset and promoted plant growth. Furthermore, gene expression analysis showed that cell cycle regulators and cell wall expansion factors were largely down-regulated in the OBP4 overexpression lines. Short-term inducible analysis coupled with in vivo ChIP assays indicated that OBP4 targets the CyclinB1;1, CDKB1;1 and XTH genes. These results strongly suggest that OBP4 is a negative regulator of cell cycle progression and cell growth. These findings increase our understanding of the transcriptional regulation of the cell cycle in plants.

  20. Large scale expansion of human umbilical cord cells in a rotating bed system bioreactor for cardiovascular tissue engineering applications.

    PubMed

    Reichardt, Anne; Polchow, Bianca; Shakibaei, Mehdi; Henrich, Wolfgang; Hetzer, Roland; Lueders, Cora

    2013-01-01

    Widespread use of human umbilical cord cells for cardiovascular tissue engineering requires production of large numbers of well-characterized cells under controlled conditions. In current research projects, the expansion of cells to be used to create a tissue construct is usually performed in static cell culture systems which are, however, often not satisfactory due to limitations in nutrient and oxygen supply. To overcome these limitations dynamic cell expansion in bioreactor systems under controllable conditions could be an important tool providing continuous perfusion for the generation of large numbers of viable pre-conditioned cells in a short time period. For this purpose cells derived from human umbilical cord arteries were expanded in a rotating bed system bioreactor for up to 9 days. For a comparative study, cells were cultivated under static conditions in standard culture devices. Our results demonstrated that the microenvironment in the perfusion bioreactor was more favorable than that of the standard cell culture flasks. Data suggested that cells in the bioreactor expanded 39 fold (38.7 ± 6.1 fold) in comparison to statically cultured cells (31.8 ± 3.0 fold). Large-scale production of cells in the bioreactor resulted in more than 3 x 10(8) cells from a single umbilical cord fragment within 9 days. Furthermore cell doubling time was lower in the bioreactor system and production of extracellular matrix components was higher. With this study, we present an appropriate method to expand human umbilical cord artery derived cells with high cellular proliferation rates in a well-defined bioreactor system under GMP conditions.

  1. Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves. II. Quantity and quality of light required

    NASA Technical Reports Server (NTRS)

    Van Volkenburgh, E.; Cleland, R. E.; Watanabe, M.

    1990-01-01

    The quantity and quality of light required for light-stimulated cell expansion in leaves of Phaseolus vulgaris L. have been determined. Seedlings were grown in dim red light (RL; 4 micromoles photons m-2 s-1) until cell division in the primary leaves was completed, then excised discs were incubated in 10 mM sucrose plus 10 mM KCl in a variety of light treatments. The growth response of discs exposed to continuous white light (WL) for 16 h was saturated at 100 micromoles m-2 s-1, and did not show reciprocity. Extensive, but not continuous, illumination was needed for maximal growth. The wavelength dependence of disc expansion was determined from fluence-response curves obtained from 380 to 730 nm provided by the Okazaki Large Spectrograph. Blue (BL; 460 nm) and red light (RL; 660 nm) were most effective in promoting leaf cell growth, both in photosynthetically active and inhibited leaf discs. Far-red light (FR; 730 nm) reduced the effectiveness of RL, but not BL, indicating that phytochrome and a separate blue-light receptor mediate expansion of leaf cells.

  2. In vitro TNF blockade enhances ex vivo expansion of regulatory T cells in patients with immune thrombocytopenia

    PubMed Central

    Zhong, Hui; Bussel, James; Yazdanbakhsh, Karina

    2017-01-01

    Summary Tumour necrosis factor-α (TNF) is an inflammatory cytokine that is elevated in a number of autoimmune diseases including immune thrombocytopenia (ITP), a bleeding disorder characterized by low platelet counts. In vitro TNF blockade increases expansion of the regulatory T cell (Treg) IKZF2 (also termed Helios) subset in T cell-monocyte cocultures from healthy donors, but its role on proliferative responses of Tregs in ITP patients, who have altered immunoregulatory compartment, remains unclear. TNF in CD4+ T cells from patients with chronic ITP were elevated and negatively correlated with peripheral Treg frequencies, suggesting a possible inhibitory effect of TNF on ITP Tregs. In vitro antibody neutralization with anti-TNF in T cell-monocyte cocultures resulted in a robust expansion of pre-existing ITP Tregs, higher than in healthy controls. Similar to the effects of anti-TNF antibodies, TNF blockade with antibodies against TNFRSF1B (anti-TNFRSF1B, previously termed anti-TNFRII) almost doubled ITP Treg expansion whereas neutralization with anti-TNFRSF1A (anti-TNFRI) antibodies had no effect on proliferative responses of Tregs. In addition, TNFRSF1B levels on ITP Tregs were significantly elevated, which may explain the increased susceptibility of patient Tregs to the actions of TNF blockade. Altogether, these data raise the possibility that TNF blockers, through their ability to increase Treg proliferation, may be efficacious in ITP patients. PMID:25252160

  3. Meis1 Is Required for Adult Mouse Erythropoiesis, Megakaryopoiesis and Hematopoietic Stem Cell Expansion

    PubMed Central

    Miller, Michelle Erin; Rosten, Patty; Lemieux, Madeleine E.; Lai, Courteney; Humphries, R. Keith

    2016-01-01

    Meis1 is recognized as an important transcriptional regulator in hematopoietic development and is strongly implicated in the pathogenesis of leukemia, both as a Hox transcription factor co-factor and independently. Despite the emerging recognition of Meis1’s importance in the context of both normal and leukemic hematopoiesis, there is not yet a full understanding of Meis1’s functions and the relevant pathways and genes mediating its functions. Recently, several conditional mouse models for Meis1 have been established. These models highlight a critical role for Meis1 in adult mouse hematopoietic stem cells (HSCs) and implicate reactive oxygen species (ROS) as a mediator of Meis1 function in this compartment. There are, however, several reported differences between these studies in terms of downstream progenitor populations impacted and effectors of function. In this study, we describe further characterization of a conditional knockout model based on mice carrying a loxP-flanked exon 8 of Meis1 which we crossed onto the inducible Cre localization/expression strains, B6;129-Gt(ROSA)26Sortm1(Cre/ERT)Nat/J or B6.Cg-Tg(Mx1-Cre)1Cgn/J. Findings obtained from these two inducible Meis1 knockout models confirm and extend previous reports of the essential role of Meis1 in adult HSC maintenance and expansion and provide new evidence that highlights key roles of Meis1 in both megakaryopoiesis and erythropoiesis. Gene expression analyses point to a number of candidate genes involved in Meis1’s role in hematopoiesis. Our data additionally support recent evidence of a role of Meis1 in ROS regulation. PMID:26986211

  4. Competency in mismatch repair prohibits clonal expansion of cancer cells treated with N-methyl-N'-nitro-N-nitrosoguanidine.

    PubMed Central

    Carethers, J M; Hawn, M T; Chauhan, D P; Luce, M C; Marra, G; Koi, M; Boland, C R

    1996-01-01

    The phenomenon of alkylation tolerance has been observed in cells that are deficient in some component of the DNA mismatch repair (MMR) system. An alkylation-induced cell cycle arrest had been reported previously in one MMR-proficient cell line, whereas a MMR-defective clone derived from this line escapes from this arrest. We examined human cancer cell lines to determine if the cell cycle arrest were dependent upon the MMR system. Growth characteristics and cell cycle analysis after MNNG treatment were ascertained in seven MMR-deficient and proficient cell lines, with and without confirmed mutations in hMLH1 or hMSH2 by an in vitro transcription/translation assay. MMR-proficient cells underwent growth arrest in the G2 phase of the cell cycle after the first S phase, whereas MMR-deficient cells escaped an initial G2 delay and resumed a normal growth pattern. In the HCT116 line corrected for defective MMR by chromosome 3 transfer, the G2 phase arrest lasted more than five days. In another MMR-proficient colon cancer cell line, SW480, cell death occurred five days after MNNG treatment. A competent MMR system appears to be necessary for G2 arrest or cell death after alkylation damage, and this cell cycle checkpoint may allow the cell to repair damaged DNA, or prevent the replication of mutated DNA by prohibiting clonal expansion. PMID:8690794

  5. Evaluation of TCR Vbeta subfamily T cell expansion in NOD/SCID mice transplanted with human cord blood hematopoietic stem cells.

    PubMed

    Lin, Chen; Chen, Shaohua; Yang, Lijian; Tan, Yubo; Bai, Xue; Li, Yangqiu

    2007-08-01

    Examination of the T cell receptor (TCR) gene repertoire is important in the analysis of the immune status of models, because clonal expansion of T cells permits the identification of specific antigen responses of T cells. Little is known about T-cell immunity in the humanized NOD/SCID mouse model. TCR Vbeta repertoire usage and clonality were analyzed to investigate the distribution and clonal expansion of TCR Vbeta subfamily T cells in NOD/SCID mice transplanted with human cord blood (CB) hematopoietic stem cells. The NOD/SCID mice were sublethally irradiated ((60)Co, 300cGy) to eliminate residual innate immunity in the host. The experimental mice were transplanted intravenously with CB CD34(+) cells sorted by MACS. After 6 weeks, RNA was obtained from peripheral blood, bone marrow and thymus of the study animals. The gene expression and clonality of the TCR Vbeta repertoire were determined by RT-PCR and GeneScan techniques. A restricted range of TCR Vbeta usage was exhibited in the bone marrow of mice, which included TCR Vbeta 1, 2, 9, 13 and 19. Further, oligoclonal expression of some TCR Vbeta subfamilies (Vbeta9, 13, 19) was identified by GeneScan technique. To investigate the reason for oligoclonal expansion of the TCR Vbeta subfamily T cells from CB in mouse models, the T-cell culture with tissue-antigen of NOD/SCID mouse was performed in vitro. The cells from peripheral blood mononuclear cells and bone marrow, spleen, thymus in NOD/SCID mice were frozen and thawed, and used as tissue-antigen. CB mononuclear cells were separately cultured with the component from those murine cells for 15-20 days. Oligoclonal expression or oligoclonal trend of some TCR Vbeta subfamilies (Vbeta10, 11 and Vbeta2, 15, 16, 19) was detected in T cells after stimulation with tissue-antigen of NOD/SCID mouse. Interestingly, a similar clonal expansion of the TCR Vbeta11 subfamily was found in T cells cultured with peripheral blood, bone marrow and spleen respectively. The TCR Vbeta

  6. Dynamic expansion of gastric mucosal doublecortin-like kinase 1-expressing cells in response to parietal cell loss is regulated by gastrin.

    PubMed

    Choi, Eunyoung; Petersen, Christine P; Lapierre, Lynne A; Williams, Janice A; Weis, Victoria G; Goldenring, James R; Nam, Ki Taek

    2015-08-01

    Doublecortin-like kinase 1 (Dclk1) is considered a reliable marker for tuft cells in the gastrointestinal tract. We investigated the dynamic changes of tuft cells associated with mouse models of oxyntic atrophy and metaplasia in the stomach. Increases in the numbers of Dclk1-positive tuft cells were observed in several models of parietal cell loss. However, the expanded population of Dclk1-expressing cells showed a morphologically distinct structure in apical microvilli and acetylated microtubules, which was not seen in the tuft cells present in the normal gastric mucosa. These microvillar sensory cells (MVSCs) showed no evidence of proliferation. The expansion of the MVSCs induced by oxyntic atrophy was reversible after the return of parietal cells. More important, expansion of MVSCs after induced parietal cell loss was not observed in Gast(-/-) mice. Although the Dclk1-expressing cells in the normal gastric mucosa were in part derived from Lrig1-expressing stem cells, the Lrig1-lineaged cells did not produce the expanded Dclk1-expressing cells associated with oxyntic atrophy. These studies indicate that loss of parietal cells leads to the reversible emergence of a novel Dclk1-expressing sensory cell population in the gastric mucosa.

  7. Dynamic Expansion of Gastric Mucosal Doublecortin-Like Kinase 1–Expressing Cells in Response to Parietal Cell Loss Is Regulated by Gastrin

    PubMed Central

    Choi, Eunyoung; Petersen, Christine P.; Lapierre, Lynne A.; Williams, Janice A.; Weis, Victoria G.; Goldenring, James R.; Nam, Ki Taek

    2016-01-01

    Doublecortin-like kinase 1 (Dclk1) is considered a reliable marker for tuft cells in the gastrointestinal tract. We investigated the dynamic changes of tuft cells associated with mouse models of oxyntic atrophy and metaplasia in the stomach. Increases in the numbers of Dclk1-positive tuft cells were observed in several models of parietal cell loss. However, the expanded population of Dclk1-expressing cells showed a morphologically distinct structure in apical microvilli and acetylated microtubules, which was not seen in the tuft cells present in the normal gastric mucosa. These microvillar sensory cells (MVSCs) showed no evidence of proliferation. The expansion of the MVSCs induced by oxyntic atrophy was reversible after the return of parietal cells. More important, expansion of MVSCs after induced parietal cell loss was not observed in Gast–/– mice. Although the Dclk1-expressing cells in the normal gastric mucosa were in part derived from Lrig1-expressing stem cells, the Lrig1-lineaged cells did not produce the expanded Dclk1-expressing cells associated with oxyntic atrophy. These studies indicate that loss of parietal cells leads to the reversible emergence of a novel Dclk1-expressing sensory cell population in the gastric mucosa. PMID:26073039

  8. Thermal Expansion of Three Closed Cell Polymeric Foams at Cryogenic Temperatures

    NASA Technical Reports Server (NTRS)

    Stokes, Eric

    2006-01-01

    The Space Shuttle External Tank (ET) contains the liquid H2 fuel and liquid oxygen oxidizer and supplies them under pressure to the three space shuttle main engines (SSME) in the orbiter during lift-off and ascent. The ET thermal protection system consists of sprayed-on foam insulation and pre-molded ablator materials. The closed-cell foams are the external coating on the ET and are responsible for minimizing the amount of moisture that condenses out and freezes on the tank from the humid air in Florida while it is on the pad with cryogenic propellant awaiting launch. This effort was part of the overall drive to understand the behavior of these materials under use-conditions. There are four specially-engineered closed-cell foams used on the tank. The thermal expansion (contraction) of three of the polyurethane and polyisocyanurate foams were measured from -423 F (the temperature of liquid hydrogen) to 125 F under atmospheric conditions and under vacuum. One of them, NCFI 24-124, is a mechanically-applied material and covers the main acreage of the tank, accounting for 77 percent of the total foam used. Another, BX-265, is also a mechanically-applied and hand-sprayed material used on the tank's "closeout" areas. PDL 1034 is a hand-poured foam used for filling odd-shaped cavities in the tank, Measurements were made in triplicate in the three primary material directions in the case of the first two materials and the two primary material directions in the case of the last. Task 1 was developing the techniques for getting a uniform heating rate and minimizing axial and radial thermal gradients in the specimens. Temperature measurements were made at four locations in the specimens during this initial development phase of testing. Major challenges that were overcome include developing techniques for transferring the coolant, liquid helium (-452 F), from its storage container to the test facility with a minimal transfer of heat to the coolant and control of the heating

  9. Melanopsin, Photosensitive Ganglion Cells, and Seasonal Affective Disorder

    PubMed Central

    Roecklein, Kathryn A.; Wong, Patricia M.; Miller, Megan A.; Donofry, Shannon D.; Kamarck, Marissa L.; Brainard, George C.

    2013-01-01

    ROECKLEIN, K.A., WONG, P.M., MILLER, M.A., DONOFRY, S.D., KAMARCK, M.L., BRAINARD, G.C. Melanopsin, Photosensitive Ganglion Cells, and Seasonal Affective Disorder…NEUROSCI BIOBEHAV REV x(x) XXX-XXX, 2012. In two recent reports, melanopsin gene variations were associated with seasonal affective disorder (SAD), and in changes in the timing of sleep and activity in healthy individuals. New studies have deepened our understanding of the retinohypothalamic tract, which translates environmental light received by the retina into neural signals sent to a set of nonvisual nuclei in the brain that are responsible for functions other than sight including circadian, neuroendocrine and neurobehavioral regulation. Because this pathway mediates seasonal changes in physiology, behavior, and mood, individual variations in the pathway may explain why approximately 1–2% of the North American population develops mood disorders with a seasonal pattern (i.e., Major Depressive and Bipolar Disorders with a seasonal pattern, also known as seasonal affective disorder/SAD). Components of depression including mood changes, sleep patterns, appetite, and cognitive performance can be affected by the biological and behavioral responses to light. Specifically, variations in the gene sequence for the retinal photopigment, melanopsin, may be responsible for significant increased risk for mood disorders with a seasonal pattern, and may do so by leading to changes in activity and sleep timing in winter. The retinal sensitivity of SAD is hypothesized to be decreased compared to controls, and that further decrements in winter light levels may combine to trigger depression in winter. Here we outline steps for new research to address the possible role of melanopsin in seasonal affective disorder including chromatic pupillometry designed to measure the sensitivity of melanopsin containing retinal ganglion cells. PMID:23286902

  10. Human progenitor cells isolated from the developing cortex undergo decreased neurogenesis and eventual senescence following expansion in vitro

    SciTech Connect

    Wright, Lynda S.; Prowse, Karen R.; Wallace, Kyle; Linskens, Maarten H.K.; Svendsen, Clive N. . E-mail: svendsen@waisman.wisc.edu

    2006-07-01

    Isolation of a true self-renewing stem cell from the human brain would be of great interest as a reliable source of neural tissue. Here, we report that human fetal cortical cells grown in epidermal growth factor expressed low levels of telomerase and telomeres in these cultures shortened over time leading to growth arrest after 30 weeks. Following leukemia inhibitory factor (LIF) supplementation, growth rates and telomerase expression increased. This was best demonstrated following cell cycle synchronization and staining for telomerase using immunocytochemistry. This increase in activity resulted in the maintenance of telomeres at approximately 7 kb for more than 60 weeks in vitro. However, all cultures displayed a lack of oligodendrotye production, decreases in neurogenesis over time and underwent replicative senescence associated with increased expression of p21 before 70 weeks in vitro. Thus, under our culture conditions, these cells are not stable, multipotent, telomerase expressing self-renewing stem cells. They may be more accurately described as human neural progenitor cells (hNPC) with limited lifespan and bi-potent potential (neurons/astrocytes). Interestingly, hNPC follow a course of proliferation, neuronal production and growth arrest similar to that seen during expansion and development of the human cortex, thus providing a possible model neural system. Furthermore, due to their high expansion potential and lack of tumorogenicity, these cells remain a unique and safe source of tissue for clinical transplantation.

  11. The immune receptor Trem1 cooperates with diminished DNA damage response to induce preleukemic stem cell expansion.

    PubMed

    Du, W; Amarachintha, S; Wilson, A; Pang, Q

    2017-02-01

    Fanconi anemia (FA) is an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Here we investigate the relationship between DNA damage response (DDR) and leukemogenesis using the Fanca knockout mouse model. We found that chronic exposure of the Fanca(-/-) hematopoietic stem cells to DNA crosslinking agent mitomycin C in vivo leads to diminished DDR, and the emergence/expansion of pre-leukemia stem cells (pre-LSCs). Surprisingly, although genetic correction of Fanca deficiency in the pre-LSCs restores DDR and reduces genomic instability, but fails to prevent pre-LSC expansion or delay leukemia development in irradiated recipients. Furthermore, we identified transcription program underlying dysregulated DDR and cell migration, myeloid proliferation, and immune response in the Fanca(-/-) pre-LSCs. Forced expression of the downregulated DNA repair genes, Rad51c or Trp53i13, in the Fanca(-/-) pre-LSCs partially rescues DDR but has no effect on leukemia, whereas shRNA knockdown of the upregulated immune receptor genes Trem1 or Pilrb improves leukemia-related survival, but not DDR or genomic instability. Furthermore, Trem1 cooperates with diminished DDR in vivo to promote Fanca(-/-) pre-LSC expansion and leukemia development. Our study implicates diminishing DDR as a root cause of FA leukemogenesis, which subsequently collaborates with other signaling pathways for leukemogenic transformation.

  12. STAT5-regulated microRNA-193b controls haematopoietic stem and progenitor cell expansion by modulating cytokine receptor signalling

    PubMed Central

    Haetscher, Nadine; Feuermann, Yonatan; Wingert, Susanne; Rehage, Maike; Thalheimer, Frederic B.; Weiser, Christian; Bohnenberger, Hanibal; Jung, Klaus; Schroeder, Timm; Serve, Hubert; Oellerich, Thomas; Hennighausen, Lothar; Rieger, Michael A.

    2015-01-01

    Haematopoietic stem cells (HSCs) require the right composition of microRNAs (miR) for proper life-long balanced blood regeneration. Here we show a regulatory circuit that prevents excessive HSC self-renewal by upregulation of miR-193b upon self-renewal promoting thrombopoietin (TPO)-MPL-STAT5 signalling. In turn, miR-193b restricts cytokine signalling, by targeting the receptor tyrosine kinase c-KIT. We generated a miR-193b knockout mouse model to unravel the physiological function of miR-193b in haematopoiesis. MiR-193b−/− mice show a selective gradual enrichment of functional HSCs, which are fully competent in multilineage blood reconstitution upon transplantation. The absence of miR-193b causes an accelerated expansion of HSCs, without altering cell cycle or survival, but by decelerating differentiation. Conversely, ectopic miR-193b expression restricts long-term repopulating HSC expansion and blood reconstitution. MiR-193b-deficient haematopoietic stem and progenitor cells exhibit increased basal and cytokine-induced STAT5 and AKT signalling. This STAT5-induced microRNA provides a negative feedback for excessive signalling to restrict uncontrolled HSC expansion. PMID:26603207

  13. Melanopsin, photosensitive ganglion cells, and seasonal affective disorder.

    PubMed

    Roecklein, Kathryn A; Wong, Patricia M; Miller, Megan A; Donofry, Shannon D; Kamarck, Marissa L; Brainard, George C

    2013-03-01

    In two recent reports, melanopsin gene variations were associated with seasonal affective disorder (SAD), and in changes in the timing of sleep and activity in healthy individuals. New studies have deepened our understanding of the retinohypothalamic tract, which translates environmental light received by the retina into neural signals sent to a set of nonvisual nuclei in the brain that are responsible for functions other than sight including circadian, neuroendocrine and neurobehavioral regulation. Because this pathway mediates seasonal changes in physiology, behavior, and mood, individual variations in the pathway may explain why approximately 1-2% of the North American population develops mood disorders with a seasonal pattern (i.e., Major Depressive and Bipolar Disorders with a seasonal pattern, also known as seasonal affective disorder/SAD). Components of depression including mood changes, sleep patterns, appetite, and cognitive performance can be affected by the biological and behavioral responses to light. Specifically, variations in the gene sequence for the retinal photopigment, melanopsin, may be responsible for significant increased risk for mood disorders with a seasonal pattern, and may do so by leading to changes in activity and sleep timing in winter. The retinal sensitivity of SAD is hypothesized to be decreased compared to controls, and that further decrements in winter light levels may combine to trigger depression in winter. Here we outline steps for new research to address the possible role of melanopsin in seasonal affective disorder including chromatic pupillometry designed to measure the sensitivity of melanopsin containing retinal ganglion cells.

  14. Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells

    SciTech Connect

    Baconnais, S.; Delavoie, F. |; Zahm, J.M.; Milliot, M.; Castillon, N.; Terryn, C.; Banchet, V.; Michel, J.; Danos, O.; Merten, M.; Chinet, T.; Zierold, K.; Bonnet, N.; Puchelle, E. , E-Mail: edith.puchelle@univ-reims.fr; Balossier, G.

    2005-10-01

    The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na{sup +} absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na{sup +}, Mg{sup 2+}, P, S and Cl{sup -}) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR{sub inh}-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.

  15. T Helper Cell Activation and Expansion Is Sensitive to Glutaminase Inhibition under Both Hypoxic and Normoxic Conditions

    PubMed Central

    Sener, Zeynep; Cederkvist, Fritjof H.; Volchenkov, Roman; Holen, Halvor L.; Skålhegg, Bjørn S.

    2016-01-01

    Immune responses often take place where nutrients and O2 availability are limited. This has an impact on T cell metabolism and influences activation and effector functions. T cell proliferation and expansion are associated with increased consumption of glutamine which is needed in a number of metabolic pathways and regulate various physiological processes. The first step in endogenous glutamine metabolism is reversible and is regulated by glutaminase (GLS1 and GLS2) and glutamine synthase (GLUL). There are two isoforms of GLS1, Kidney type glutaminase (KGA) and Glutaminase C (GAC). The aim of this study is to investigate the expression, localization and role of GLS1 and GLUL in naïve and activated human CD4+ T cells stimulated through the CD3 and CD28 receptors under normoxia and hypoxia. In proliferating cells, GAC was upregulated and KGA was downregulated, and both enzymes were located to the mitochondria irrespective of O2 levels. By contrast GLUL is localized to the cytoplasm and was upregulated under hypoxia. Proliferation was dependent on glutamine consumption, as glutamine deprivation and GLS1 inhibition decreased proliferation and expression of CD25 and CD226, regardless of O2 availability. Again irrespective of O2, GLS1 inhibition decreased the proportion of CCR6 and CXCR3 expressing CD4+ T cells as well as cytokine production. We propose that systemic Th cell activation and expansion might be dependent on glutamine but not O2 availability. PMID:27467144

  16. Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions

    PubMed Central

    Beers, Jeanette; Gulbranson, Daniel R.; George, Nicole; Siniscalchi, Lauren I.; Jones, Jeffrey; Thomson, James A.; Chen, Guokai

    2013-01-01

    This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (iPSCs). In this protocol, passaging one six-well or 10 cm plate of cells takes about 6–7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization, centrifugation, or drug treatment. It also allows for higher throughput, requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation, colony expansion, cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion, and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages, this procedure provides a consistent and universal approach to passaging human pluripotent stem cells in E8 medium. PMID:23099485

  17. Cross-reactivity and expansion of dengue-specific T cells during acute primary and secondary infections in humans.

    PubMed

    Friberg, Heather; Bashyam, Hema; Toyosaki-Maeda, Tomoko; Potts, James A; Greenough, Thomas; Kalayanarooj, Siripen; Gibbons, Robert V; Nisalak, Ananda; Srikiatkhachorn, Anon; Green, Sharone; Stephens, Henry A F; Rothman, Alan L; Mathew, Anuja

    2011-01-01

    Serotype-cross-reactive memory T cells responding to secondary dengue virus (DENV) infection are thought to contribute to disease. However, epitope-specific T cell responses have not been thoroughly compared between subjects with primary versus secondary DENV infection. We studied CD8(+) T cells specific for the HLA-A*1101-restricted NS3(133) epitope in a cohort of A11(+) DENV-infected patients throughout acute illness and convalescence. We compared the expansion, serotype-cross-reactivity, and activation of these cells in PBMC from patients experiencing primary or secondary infection and mild or severe disease by flow cytometry. Our results show expansion and activation of DENV-specific CD8(+) T cells during acute infection, which are predominantly serotype-cross-reactive regardless of DENV infection history. These data confirm marked T cell activation and serotype-cross-reactivity during the febrile phase of dengue; however, A11-NS3(133)-specific responses did not correlate with prior antigenic exposure or current disease severity.

  18. Surgical retrieval, isolation and in vitro expansion of human anterior cruciate ligament-derived cells for tissue engineering applications.

    PubMed

    Gupta, Ashim; Sharif, Kevin; Walters, Megan; Woods, Mia D; Potty, Anish; Main, Benjamin J; El-Amin, Saadiq F

    2014-04-30

    Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities. The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 ºC or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is

  19. Tumor-associated mesenchymal stem cells inhibit naïve T cell expansion by blocking cysteine export from dendritic cells.

    PubMed

    Ghosh, Tithi; Barik, Subhasis; Bhuniya, Avishek; Dhar, Jesmita; Dasgupta, Shayani; Ghosh, Sarbari; Sarkar, Madhurima; Guha, Ipsita; Sarkar, Koustav; Chakrabarti, Pinak; Saha, Bhaskar; Storkus, Walter J; Baral, Rathindranath; Bose, Anamika

    2016-11-01

    Mesenchymal stem cells (MSCs) represent an important cellular constituent of the tumor microenvironment, which along with tumor cells themselves, serve to regulate protective immune responses in support of progressive disease. We report that tumor MSCs prevent the ability of dendritic cells (DC) to promote naïve CD4(+) and CD8(+) T cell expansion, interferon gamma secretion and cytotoxicity against tumor cells, which are critical to immune-mediated tumor eradication. Notably, tumor MSCs fail to prevent DC-mediated early T cell activation events or the ability of responder T cells to produce IL-2. The immunoregulatory activity of tumor MSCs is IL-10- and STAT3-dependent, with STAT3 repressing DC expression of cystathionase, a critical enzyme that converts methionine-to-cysteine. Under cysteine-deficient priming conditions, naïve T cells exhibit defective cellular metabolism and proliferation. Bioinformatics analyses as well as in vitro observations suggest that STAT3 may directly bind to a GAS-like motif within the cystathionase promoter (-269 to -261) leading to IL-10-STAT3 mediated repression of cystathionase gene transcription. Our collective results provide evidence for a novel mechanism of tumor MSC-mediated T cell inhibition within tumor microenvironment.

  20. Contribution of Intronic miR-338-3p and Its Hosting Gene AATK to Compensatory β-Cell Mass Expansion.

    PubMed

    Jacovetti, Cécile; Jimenez, Veronica; Ayuso, Eduard; Laybutt, Ross; Peyot, Marie-Line; Prentki, Marc; Bosch, Fatima; Regazzi, Romano

    2015-05-01

    The elucidation of the mechanisms directing β-cell mass regeneration and maintenance is of interest, because the deficit of β-cell mass contributes to diabetes onset and progression. We previously found that the level of the microRNA (miRNA) miR-338-3p is decreased in pancreatic islets from rodent models displaying insulin resistance and compensatory β-cell mass expansion, including pregnant rats, diet-induced obese mice, and db/db mice. Transfection of rat islet cells with oligonucleotides that specifically block miR-338-3p activity increased the fraction of proliferating β-cells in vitro and promoted survival under proapoptotic conditions without affecting the capacity of β-cells to release insulin in response to glucose. Here, we evaluated the role of miR-338-3p in vivo by injecting mice with an adeno-associated viral vector permitting specific sequestration of this miRNA in β-cells. We found that the adeno-associated viral construct increased the fraction of proliferating β-cells confirming the data obtained in vitro. miR-338-3p is generated from an intron of the gene coding for apoptosis-associated tyrosine kinase (AATK). Similarly to miR-338-3p, we found that AATK is down-regulated in rat and human islets and INS832/13 β-cells in the presence of the cAMP-raising agents exendin-4, estradiol, and a G-protein-coupled Receptor 30 agonist. Moreover, AATK expression is reduced in islets of insulin resistant animal models and selective silencing of AATK in INS832/13 cells by RNA interference promoted β-cell proliferation. The results point to a coordinated reduction of miR-338-3p and AATK under insulin resistance conditions and provide evidence for a cooperative action of the miRNA and its hosting gene in compensatory β-cell mass expansion.

  1. Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry

    SciTech Connect

    Pham, Son; Tabarin, Thibault; Garvey, Megan; Pade, Corinna; Rossy, Jérémie; Monaghan, Paul; Hyatt, Alex; Böcking, Till; Leis, Andrew; Gaus, Katharina; Mak, Johnson

    2015-12-15

    Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the ‘lynchpin’ for size expansion is highly specific. We demonstrate that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection. - Highlights: • Cell free viruses are able to receive external trigger that leads to apparent size expansion. • Virus envelope and CD4 receptor engagement is the lynchpin of virus size expansion. • Internal capsid organisation can influence receptor mediated virus size expansion. • Pre-existing virus-associated lipid membrane in cell free virus can accommodate the receptor mediated virus size expansion.

  2. Isolation and expansion of human pluripotent stem cell-derived hepatic progenitor cells by growth factor defined serum-free culture conditions.

    PubMed

    Fukuda, Takayuki; Takayama, Kazuo; Hirata, Mitsuhi; Liu, Yu-Jung; Yanagihara, Kana; Suga, Mika; Mizuguchi, Hiroyuki; Furue, Miho K

    2017-03-15

    Limited growth potential, narrow ranges of sources, and difference in variability and functions from batch to batch of primary hepatocytes cause a problem for predicting drug-induced hepatotoxicity during drug development. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells in vitro are expected as a tool for predicting drug-induced hepatotoxicity. Several studies have already reported efficient methods for differentiating hPSCs into hepatocyte-like cells, however its differentiation process is time-consuming, labor-intensive, cost-intensive, and unstable. In order to solve this problem, expansion culture for hPSC-derived hepatic progenitor cells, including hepatic stem cells and hepatoblasts which can self-renewal and differentiate into hepatocytes should be valuable as a source of hepatocytes. However, the mechanisms of the expansion of hPSC-derived hepatic progenitor cells are not yet fully understood. In this study, to isolate hPSC-derived hepatic progenitor cells, we tried to develop serum-free growth factor defined culture conditions using defined components. Our culture conditions were able to isolate and grow hPSC-derived hepatic progenitor cells which could differentiate into hepatocyte-like cells through hepatoblast-like cells. We have confirmed that the hepatocyte-like cells prepared by our methods were able to increase gene expression of cytochrome P450 enzymes upon encountering rifampicin, phenobarbital, or omeprazole. The isolation and expansion of hPSC-derived hepatic progenitor cells in defined culture conditions should have advantages in terms of detecting accurate effects of exogenous factors on hepatic lineage differentiation, understanding mechanisms underlying self-renewal ability of hepatic progenitor cells, and stably supplying functional hepatic cells.

  3. Spatial and Temporal Effects of Free-Air CO2 Enrichment (POPFACE) on Leaf Growth, Cell Expansion, and Cell Production in a Closed Canopy of Poplar1

    PubMed Central

    Taylor, Gail; Tricker, Penny J.; Zhang, Fang Z.; Alston, Victoria J.; Miglietta, Franco; Kuzminsky, Elena

    2003-01-01

    Leaf expansion in the fast-growing tree, Populus × euramericana was stimulated by elevated [CO2] in a closed-canopy forest plantation, exposed using a free air CO2 enrichment technique enabling long-term experimentation in field conditions. The effects of elevated [CO2] over time were characterized and related to the leaf plastochron index (LPI), and showed that leaf expansion was stimulated at very early (LPI, 0–3) and late (LPI, 6–8) stages in development. Early and late effects of elevated [CO2] were largely the result of increased cell expansion and increased cell production, respectively. Spatial effects of elevated [CO2] were also marked and increased final leaf size resulted from an effect on leaf area, but not leaf length, demonstrating changed leaf shape in response to [CO2]. Leaves exhibited a basipetal gradient of leaf development, investigated by defining seven interveinal areas, with growth ceasing first at the leaf tip. Interestingly, and in contrast to other reports, no spatial differences in epidermal cell size were apparent across the lamina, whereas a clear basipetal gradient in cell production rate was found. These data suggest that the rate and timing of cell production was more important in determining leaf shape, given the constant cell size across the leaf lamina. The effect of elevated [CO2] imposed on this developmental gradient suggested that leaf cell production continued longer in elevated [CO2] and that basal increases in cell production rate were also more important than altered cell expansion for increased final leaf size and altered leaf shape in elevated [CO2]. PMID:12529526

  4. Toll-like receptor 2 controls expansion and function of regulatory T cells

    PubMed Central

    Sutmuller, Roger P.M.; den Brok, Martijn H.M.G.M.; Kramer, Matthijs; Bennink, Erik J.; Toonen, Liza W.J.; Kullberg, Bart-Jan; Joosten, Leo A.; Akira, Shizuo; Netea, Mihai G.; Adema, Gosse J.

    2006-01-01

    Tregs play a central role in the suppression of immune reactions and prevention of autoimmune responses harmful to the host. During acute infection, however, Tregs might hinder effector T cell activity directed toward the elimination of the pathogenic challenge. Pathogen recognition receptors from the TLR family expressed by innate immune cells are crucial for the generation of effective immunity. We have recently shown the CD4+CD25+ Treg subset in TLR2–/– mice to be significantly reduced in number compared with WT littermate control mice, indicating a link between Tregs and TLR2. Here, we report that the TLR2 ligand Pam3Cys, but not LPS (TLR4) or CpG (TLR9), directly acts on purified Tregs in a MyD88-dependent fashion. Moreover, when combined with TCR stimulation, TLR2 triggering augmented Treg proliferation in vitro and in vivo and resulted in a temporal loss of the suppressive Treg phenotype in vitro by directly affecting Tregs. Importantly, WT Tregs adoptively transferred into TLR2–/– mice were neutralized by systemic administration of TLR2 ligand during the acute phase of a Candida albicans infection, resulting in a 100-fold reduced C. albicans outgrowth. This demonstrates that in vivo TLR2 also controls the function of Tregs and establishes a direct link between TLRs and the control of immune responses through Tregs. PMID:16424940

  5. Genome rearrangement affects RNA virus adaptability on prostate cancer cells.

    PubMed

    Pesko, Kendra; Voigt, Emily A; Swick, Adam; Morley, Valerie J; Timm, Collin; Yin, John; Turner, Paul E

    2015-01-01

    Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wild type gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense non-segmented RNA viruses (order Mononegavirales) have specific genome architecture: 3' UTR - core protein genes - envelope protein genes - RNA-dependent RNA-polymerase gene - 5' UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV) variants with the nucleocapsid (N) gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N gene translocation toward the 5' end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC-3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function), especially on PC-3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture) adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective environments for a given gene

  6. Injury-induced GR-1+ macrophage expansion and activation occurs independently of CD4 T-cell influence.

    PubMed

    O'Leary, Fionnuala M; Tajima, Goro; Delisle, Adam J; Ikeda, Kimiko; Dolan, Sinead M; Hanschen, Marc; Mannick, John A; Lederer, James A

    2011-08-01

    Burn injury initiates an enhanced inflammatory condition referred to as the systemic inflammatory response syndrome or the two-hit response phenotype. Prior reports indicated that macrophages respond to injury and demonstrate a heightened reactivity to Toll-like receptor stimulation. Since we and others observed a significant increase in splenic GR-1 F4/80 CD11b macrophages in burn-injured mice, we wished to test if these macrophages might be the primary macrophage subset that shows heightened LPS reactivity. We report here that burn injury promoted higher level TNF-α expression in GR-1, but not GR-1 macrophages, after LPS activation both in vivo and ex vivo. We next tested whether CD4 T cells, which are known to suppress injury-induced inflammatory responses, might control the activation and expansion of GR-1 macrophages. Interestingly, we found that GR-1 macrophage expansion and LPS-induced TNF-α expression were not significantly different between wild-type and CD4 T cell-deficient CD4(-/-) mice. However, further investigations showed that LPS-induced TNF-α production was significantly influenced by CD4 T cells. Taken together, these data indicate that GR-1 F4/80 CD11b macrophages represent the primary macrophage subset that expands in response to burn injury and that CD4 T cells do not influence the GR-1 macrophage expansion process, but do suppress LPS-induced TNF-α production. These data suggest that modulating GR-1 macrophage activation as well as CD4 T cell responses after severe injury may help control the development of systemic inflammatory response syndrome and the two-hit response phenotype.

  7. Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion

    PubMed Central

    Kusuma, Gina D.; Brennecke, Shaun P.; O’Connor, Andrea J.; Kalionis, Bill

    2017-01-01

    Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies. PMID:28152107

  8. Human periosteal-derived cell expansion in a perfusion bioreactor system: proliferation, differentiation and extracellular matrix formation.

    PubMed

    Sonnaert, M; Papantoniou, I; Bloemen, V; Kerckhofs, G; Luyten, F P; Schrooten, J

    2017-02-01

    Perfusion bioreactor systems have shown to be a valuable tool for the in vitro development of three-dimensional (3D) cell-carrier constructs. Their use for cell expansion, however, has been much less explored. Since maintenance of the initial cell phenotype is essential in this process, it is imperative to obtain insight into the bioreactor-related variables determining cell fate. Therefore, this study investigated the influence of fluid flow-induced shear stress on the proliferation, differentiation and matrix deposition of human periosteal-derived cells in the absence of additional differentiation-inducing stimuli; 120 000 cells were seeded on additive manufactured 3D Ti6Al4V scaffolds and cultured for up to 28 days at different flow rates in the range 0.04-6 ml/min. DNA measurements showed, on average, a three-fold increase in cell content for all perfused conditions in comparison to static controls, whereas the magnitude of the flow rate did not have an influence. Contrast-enhanced nanofocus X-ray computed tomography showed substantial formation of an engineered neotissue in all perfused conditions, resulting in a filling (up to 70%) of the total internal void volume, and no flow rate-dependent differences were observed. The expression of key osteogenic markers, such as RunX2, OCN, OPN and Col1, did not show any significant changes in comparison to static controls after 28 days of culture, with the exception of OSX at high flow rates. We therefore concluded that, in the absence of additional osteogenic stimuli, the investigated perfusion conditions increased cell proliferation but did not significantly enhance osteogenic differentiation, thus allowing for this process to be used for cell expansion. Copyright © 2014 John Wiley & Sons, Ltd.

  9. A novel method using blinatumomab for efficient, clinical-grade expansion of polyclonal T cells for adoptive immunotherapy.

    PubMed

    Golay, Josée; D'Amico, Anna; Borleri, Gianmaria; Bonzi, Michela; Valgardsdottir, Rut; Alzani, Rachele; Cribioli, Sabrina; Albanese, Clara; Pesenti, Enrico; Finazzi, Maria Chiara; Quaresmini, Giulia; Nagorsen, Dirk; Introna, Martino; Rambaldi, Alessandro

    2014-11-01

    Current treatment of chronic lymphocytic leukemia (CLL) patients often results in life-threatening immunosuppression. Furthermore, CLL is still an incurable disease due to the persistence of residual leukemic cells. These patients may therefore benefit from immunotherapy approaches aimed at immunoreconstitution and/or the elimination of residual disease following chemotherapy. For these purposes, we designed a simple GMP-compliant protocol for ex vivo expansion of normal T cells from CLL patients' peripheral blood for adoptive therapy, using bispecific Ab blinatumomab (CD3 × CD19), acting both as T cell stimulator and CLL depletion agent, and human rIL-2. Starting from only 10 ml CLL peripheral blood, a mean 515 × 10(6) CD3(+) T cells were expanded in 3 wk. The resulting blinatumomab-expanded T cells (BET) were polyclonal CD4(+) and CD8(+) and mostly effector and central memory cells. The Th1 subset was slightly prevalent over Th2, whereas Th17 and T regulatory cells were <1%. CMV-specific clones were detected in equivalent proportion before and after expansion. Interestingly, BET cells had normalized expression of the synapse inhibitors CD272 and CD279 compared with starting T cells and were cytotoxic against CD19(+) targets in presence of blinatumomab in vitro. In support of their functional capacity, we observed that BET, in combination with blinatumomab, had significant therapeutic activity in a systemic human diffuse large B lymphoma model in NOD-SCID mice. We propose BET as a therapeutic tool for immunoreconstitution of heavily immunosuppressed CLL patients and, in combination with bispecific Ab, as antitumor immunotherapy.

  10. In vitro expansion of Lin{sup +} and Lin{sup −} mononuclear cells from human peripheral blood

    SciTech Connect

    Norhaiza, H. Siti; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul; Rohaya, M. A. W.

    2013-11-27

    Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin{sup −}) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin{sup +}) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin{sup −} cell population. The ability of Lin{sup +} and Lin{sup −} to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin{sup +} and Lin{sup −} were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin{sup +} mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin{sup −} stem cells were not able to survive in proliferation medium however

  11. Cell-Cell Contact Area Affects Notch Signaling and Notch-Dependent Patterning.

    PubMed

    Shaya, Oren; Binshtok, Udi; Hersch, Micha; Rivkin, Dmitri; Weinreb, Sheila; Amir-Zilberstein, Liat; Khamaisi, Bassma; Oppenheim, Olya; Desai, Ravi A; Goodyear, Richard J; Richardson, Guy P; Chen, Christopher S; Sprinzak, David

    2017-03-13

    During development, cells undergo dramatic changes in their morphology. By affecting contact geometry, these morphological changes could influence cellular communication. However, it has remained unclear whether and how signaling depends on contact geometry. This question is particularly relevant for Notch signaling, which coordinates neighboring cell fates through direct cell-cell signaling. Using micropatterning with a receptor trans-endocytosis assay, we show that signaling between pairs of cells correlates with their contact area. This relationship extends across contact diameters ranging from micrometers to tens of micrometers. Mathematical modeling predicts that dependence of signaling on contact area can bias cellular differentiation in Notch-mediated lateral inhibition processes, such that smaller cells are more likely to differentiate into signal-producing cells. Consistent with this prediction, analysis of developing chick inner ear revealed that ligand-producing hair cell precursors have smaller apical footprints than non-hair cells. Together, these results highlight the influence of cell morphology on fate determination processes.

  12. Expansion Microscopy

    PubMed Central

    Chen, Fei; Tillberg, Paul W.; Boyden, Edward S.

    2014-01-01

    In optical microscopy, fine structural details are resolved by using refraction to magnify images of a specimen. Here we report the discovery that, by synthesizing a swellable polymer network within a specimen, it can be physically expanded, resulting in physical magnification. By covalently anchoring specific labels located within the specimen directly to the polymer network, labels spaced closer than the optical diffraction limit can be isotropically separated and optically resolved, a process we call expansion microscopy (ExM). Thus, this process can be used to perform scalable super-resolution microscopy with diffraction-limited microscopes. We demonstrate ExM with effective ~70 nm lateral resolution in both cultured cells and brain tissue, performing three-color super-resolution imaging of ~107 μm3 of the mouse hippocampus with a conventional confocal microscope. PMID:25592419

  13. The role of children's bone marrow mesenchymal stromal cells in the ex vivo expansion of autologous and allogeneic hematopoietic stem cells.

    PubMed

    Pelagiadis, Iordanis; Stiakaki, Eftichia; Choulaki, Christianna; Kalmanti, Maria; Dimitriou, Helen

    2015-10-01

    The recognition of the role of Mesenchymal Stromal Cells (MSC) in hematopoiesis, as part of the bone marrow microenvironment, renewed the interest for cord blood (CB) ex vivo expansion as a source of HSC for transplantation. MSC from children are recognized to have different biological properties compared to the ones from adults. The current study focuses on the evaluation of the effects of children's bone marrow MSC on the ex vivo expansion capacity of both allogeneic cord blood and autologous bone marrow (BM) CD34(+) hematopoietic stem cells (HSCs) when used as a cell feeder layer with or without recombinant cytokines. Our results showed that children's bone marrow-derived MSC expand more primitive populations in co culture with CD34 and that the expansion is further enhanced when the culture is supplemented with growth factors. No additive effect was seen either with the early- or late-acting growth factors' cocktails used. Biological features of CB hematopoietic progenitors seem to make them more suitable than their BM counterparts for ex vivo expansion. Clinical implementation will be facilitated by methodological standardization and guidelines' establishment.

  14. Infection-responsive expansion of the hematopoietic stem and progenitor cell compartment in zebrafish is dependent upon inducible nitric oxide.

    PubMed

    Hall, Christopher J; Flores, Maria Vega; Oehlers, Stefan H; Sanderson, Leslie E; Lam, Enid Y; Crosier, Kathryn E; Crosier, Philip S

    2012-02-03

    Hematopoietic stem cells (HSCs) are rare multipotent cells that contribute to all blood lineages. During inflammatory stress, hematopoietic stem and progenitor cells (HSPCs) can be stimulated to proliferate and differentiate into the required immune cell lineages. Manipulating signaling pathways that alter HSPC capacity holds great promise in the treatment of hematological malignancies. To date, signaling pathways that influence HSPC capacity, in response to hematopoietic stress, remain largely unknown. Using a zebrafish model of demand-driven granulopoiesis to explore the HSPC response to infection, we present data supporting a model where the zebrafish ortholog of the cytokine-inducible form of nitric oxide synthase (iNOS/NOS2) Nos2a acts downstream of the transcription factor C/ebpβ to control expansion of HSPCs following infection. These results provide new insights into the reactive capacity of HSPCs and how the blood system is "fine-tuned" in response to inflammatory stress.

  15. Granulocytic Myeloid Derived Suppressor Cells Expansion during Active Pulmonary Tuberculosis Is Associated with High Nitric Oxide Plasma Level

    PubMed Central

    El Daker, Sary; Sacchi, Alessandra; Tempestilli, Massimo; Carducci, Claudia; Goletti, Delia; Vanini, Valentina; Colizzi, Vittorio; Lauria, Francesco Nicola; Martini, Federico; Martino, Angelo

    2015-01-01

    Tuberculosis (TB) is still the principal cause of death caused by a single infectious agent, and the balance between the bacillus and host defense mechanisms reflects the different manifestations of the pathology. The aim of this work was to study the role of myeloid-derived suppressor cells (MDSCs) during active pulmonary tuberculosis at the site of infection. We observed an expansion of MDSCs in the lung and blood of patients with active TB, which are correlated with an enhanced amount of nitric oxide in the plasma. We also found that these cells have the remarkable ability to suppress T-cell response, suggesting an important role in the modulation of the immune response against TB. Interestingly, a trend in the diminution of MDSCs was found after an efficacious anti-TB therapy, suggesting that these cells may be used as a potential biomarker for monitoring anti-TB therapy efficacy. PMID:25879532

  16. T cell expansion is the limiting factor of virus control in mice with attenuated TCR signaling: implications for human immunodeficiency.

    PubMed

    Hillen, Kristina M; Gather, Ruth; Enders, Anselm; Pircher, Hanspeter; Aichele, Peter; Fisch, Paul; Blumenthal, Britta; Schamel, Wolfgang W; Straub, Tobias; Goodnow, Christopher C; Ehl, Stephan

    2015-03-15

    Defining the minimal thresholds for effective antiviral T cell immunity is important for clinical decisions in immunodeficient patients. TCR signaling is critical for T cell development, activation, and effector functions. In this article, we analyzed which of these TCR-mediated processes is limiting for antiviral immunity in a mouse strain with reduced expression of SLP-76 (twp mice). Despite severe T cell activation defects in vitro, twp mice generated a normal proportion of antiviral effector T cells postinfection with lymphocytic choriomeningitis virus (LCMV). Twp CD8(+) T cells showed impaired polyfunctional cytokine production, whereas cytotoxicity as the crucial antiviral effector function for LCMV control was normal. The main limiting factor in the antiviral response of twp mice was impaired T cell proliferation and survival, leading to a 5- to 10-fold reduction of antiviral T cells at the peak of the immune response. This was still sufficient to control infection with the LCMV Armstrong strain, but the more rapidly replicating LCMV-WE induced T cell exhaustion and viral persistence. Thus, under conditions of impaired TCR signaling, reduced T cell expansion was the limiting factor in antiviral immunity. These findings have implications for understanding antiviral immunity in patients with T cell deficiencies.

  17. CD27 costimulation contributes substantially to the expansion of functional memory CD8(+) T cells after peptide immunization.

    PubMed

    Taraban, Vadim Y; Rowley, Tania F; Kerr, Jonathan P; Willoughby, Jane E; Johnson, Peter M W; Al-Shamkhani, Aymen; Buchan, Sarah L

    2013-12-01

    Naive T cells require signals from multiple costimulatory receptors to acquire full effector function and differentiate to long-lived memory cells. The costimulatory receptor, CD27, is essential for optimal T-cell priming and memory differentiation in a variety of settings, although whether CD27 is similarly required during memory CD8(+) T-cell reactivation remains controversial. We have used OVA and anti-CD40 to establish a memory CD8(+) T-cell population and report here that their secondary expansion, driven by peptide and anti-CD40, polyI:C, or LPS, requires CD27. Furthermore, antigenic peptide and a soluble form of the CD27 ligand, CD70 (soluble recombinant CD70 (sCD70)), is sufficient for secondary memory CD8(+) T-cell accumulation at multiple anatomical sites, dependent on CD80/86. Prior to boost, resting effector- and central-memory CD8(+) T cells both expressed CD27 with greater expression on central memory cells. Nonetheless, both populations upregulated CD27 after TCR engagement and accumulated in proportion after boosting with Ag and sCD70. Mechanistically, sCD70 increased the frequency of divided and cytolytic memory T cells, conferred resistance to apoptosis and enabled retardation of tumor growth in vivo. These data demonstrate the central role played by CD27/70 during secondary CD8(+) T-cell activation to a peptide Ag, and identify sCD70 as an immunotherapeutic adjuvant for antitumor immunity.

  18. Simulation of Magnetic Field Guided Plasma Expansion

    NASA Astrophysics Data System (ADS)

    Ebersohn, Frans; Sheehan, J. P.; Gallimore, Alec; Shebalin, John

    2015-09-01

    Magnetic field guided expansion of a radio-frequency plasma was simulated with a quasi-one-dimensional particle-in-cell code. Two-dimensional effects were included in a one-dimensional particle-in-cell code by varying the cross-sectional area of the one dimensional domain and including forces due to the magnetic field. Acceleration of electrons by the magnetic field forces leads to the formation of potential structures which then accelerate the ions into a beam. Density changes due to the plasma expansion only weakly affect the ion acceleration. Rapidly diverging magnetic fields lead to more rapid acceleration and the electrons cool as they expand.

  19. Expansion of CD3+CD4-CD8- T cell population expressing high levels of IL-5 in Omenn's syndrome.

    PubMed Central

    Melamed, I; Cohen, A; Roifman, C M

    1994-01-01

    Omenn's syndrome is a fatal, autosomal-recessive combined immune deficiency characterized by several erythematous exfoliative eruptions, lymphadenopathy, hepatosplenomegaly, and elevated eosinophil count. In some of these patients an expansion of CD3+CD4-CD8- double negative (DN) T cell population was observed. We show here that the DN population represents a clonal expansion of T cells which preferentially use V beta 14 in their T cell receptor complex. Using polymerase chain reaction, we show that patient's DN cells express spontaneously high levels of IL-5, thus possibly explaining the abundance of eosinophils in this disorder. The increase of IgE observed in patients with Omenn's syndrome is unlikely to be related to IL-4 production, as IL-4 levels in patient samples were low. However, patient's low expression of interferon-gamma (IFN-gamma), which has been reported to inhibit IgE production, may explain the elevated levels of IgE in this patient. The results thus highlight the importance of the inhibitory effect of IFN-gamma on regulation of IgE production. Images Fig. 4 Fig. 5 Fig. 6 PMID:8287598

  20. The tarani mutation alters surface curvature in Arabidopsis leaves by perturbing the patterns of surface expansion and cell division

    PubMed Central

    Karidas, Premananda; Challa, Krishna Reddy; Nath, Utpal

    2015-01-01

    The leaf surface usually stays flat, maintained by coordinated growth. Growth perturbation can introduce overall surface curvature, which can be negative, giving a saddle-shaped leaf, or positive, giving a cup-like leaf. Little is known about the molecular mechanisms that underlie leaf flatness, primarily because only a few mutants with altered surface curvature have been isolated and studied. Characterization of mutants of the CINCINNATA-like TCP genes in Antirrhinum and Arabidopsis have revealed that their products help maintain flatness by balancing the pattern of cell proliferation and surface expansion between the margin and the central zone during leaf morphogenesis. On the other hand, deletion of two homologous PEAPOD genes causes cup-shaped leaves in Arabidopsis due to excess division of dispersed meristemoid cells. Here, we report the isolation and characterization of an Arabidopsis mutant, tarani (tni), with enlarged, cup-shaped leaves. Morphometric analyses showed that the positive curvature of the tni leaf is linked to excess growth at the centre compared to the margin. By monitoring the dynamic pattern of CYCLIN D3;2 expression, we show that the shape of the primary arrest front is strongly convex in growing tni leaves, leading to excess mitotic expansion synchronized with excess cell proliferation at the centre. Reduction of cell proliferation and of endogenous gibberellic acid levels rescued the tni phenotype. Genetic interactions demonstrated that TNI maintains leaf flatness independent of TCPs and PEAPODs. PMID:25711708

  1. The tarani mutation alters surface curvature in Arabidopsis leaves by perturbing the patterns of surface expansion and cell division.

    PubMed

    Karidas, Premananda; Challa, Krishna Reddy; Nath, Utpal

    2015-04-01

    The leaf surface usually stays flat, maintained by coordinated growth. Growth perturbation can introduce overall surface curvature, which can be negative, giving a saddle-shaped leaf, or positive, giving a cup-like leaf. Little is known about the molecular mechanisms that underlie leaf flatness, primarily because only a few mutants with altered surface curvature have been isolated and studied. Characterization of mutants of the CINCINNATA-like TCP genes in Antirrhinum and Arabidopsis have revealed that their products help maintain flatness by balancing the pattern of cell proliferation and surface expansion between the margin and the central zone during leaf morphogenesis. On the other hand, deletion of two homologous PEAPOD genes causes cup-shaped leaves in Arabidopsis due to excess division of dispersed meristemoid cells. Here, we report the isolation and characterization of an Arabidopsis mutant, tarani (tni), with enlarged, cup-shaped leaves. Morphometric analyses showed that the positive curvature of the tni leaf is linked to excess growth at the centre compared to the margin. By monitoring the dynamic pattern of CYCLIN D3;2 expression, we show that the shape of the primary arrest front is strongly convex in growing tni leaves, leading to excess mitotic expansion synchronized with excess cell proliferation at the centre. Reduction of cell proliferation and of endogenous gibberellic acid levels rescued the tni phenotype. Genetic interactions demonstrated that TNI maintains leaf flatness independent of TCPs and PEAPODs.

  2. T-cell defect in diffuse large B-cell lymphomas involves expansion of myeloid-derived suppressor cells.

    PubMed

    Azzaoui, Imane; Uhel, Fabrice; Rossille, Delphine; Pangault, Celine; Dulong, Joelle; Le Priol, Jerome; Lamy, Thierry; Houot, Roch; Le Gouill, Steven; Cartron, Guillaume; Godmer, Pascal; Bouabdallah, Krimo; Milpied, Noel; Damaj, Gandhi; Tarte, Karin; Fest, Thierry; Roussel, Mikael

    2016-08-25

    In diffuse large B-cell lymphoma (DLBCL), the number of circulating monocytes and neutrophils represents an independent prognostic factor. These cell subsets include monocytic and granulocytic myeloid-derived suppressor cells (M- and G-MDSCs) defined by their ability to suppress T-cell responses. MDSCs are a heterogeneous population described in inflammatory and infectious diseases and in numerous tumors including multiple myeloma, chronic lymphocytic leukemia, and DLBCL. However, their mechanisms of action remain unclear. We broadly assessed the presence and mechanisms of suppression of MDSC subsets in DLBCL. First, a myeloid suppressive signature was identified by gene expression profiling in DLBCL peripheral blood. Accordingly, we identified, in a cohort of 66 DLBCL patients, an increase in circulating G-MDSC (Lin(neg)HLA-DR(neg)CD33(pos)CD11b(pos)) and M-MDSC (CD14(pos)HLA-DR(low)) counts. Interestingly, only M-MDSC number was correlated with the International Prognostic Index, event-free survival, and number of circulating Tregs. Furthermore, T-cell proliferation was restored after monocyte depletion. Myeloid-dependent T-cell suppression was attributed to a release of interleukin-10 and S100A12 and increased PD-L1 expression. In summary, we identified expanded MDSC subsets in DLBCL, as well as new mechanisms of immunosuppression in DLBCL.

  3. Closely related T-memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15.

    PubMed

    Xu, Yang; Zhang, Ming; Ramos, Carlos A; Durett, April; Liu, Enli; Dakhova, Olga; Liu, Hao; Creighton, Chad J; Gee, Adrian P; Heslop, Helen E; Rooney, Cliona M; Savoldo, Barbara; Dotti, Gianpietro

    2014-06-12

    Adoptive transfer of T lymphocytes expressing a CD19-specific chimeric antigen receptor (CAR.CD19) induces complete tumor regression in patients with lymphoid malignancies. Although in vivo persistence of CAR-T cells correlates with clinical responses, it remains unknown whether specific cell subsets within the CAR-T-cell product correlate with their subsequent in vivo expansion and persistence. We analyzed 14 patients with B-cell malignancies infused with autologous CAR.CD19-redirected T cells expanded ex vivo using IL-2, and found that their in vivo expansion only correlated with the frequency within the infused product of a CD8(+)CD45RA(+)CCR7(+) subset, whose phenotype is closest to "T-memory stem cells." Preclinical models showed that increasing the frequency of CD8(+)CD45RA(+)CCR7(+) CAR-T cells in the infused line by culturing the cells with IL-7 and IL-15 produced greater antitumor activity of CAR-T cells mediated by increased resistance to cell death, following repetitive encounters with the antigen, while preserving their migration to secondary lymphoid organs. This trial was registered at www.clinicaltrials.gov as #NCT00586391 and #NCT00709033.

  4. MicroRNAs affect dendritic cell function and phenotype

    PubMed Central

    Smyth, Lesley A; Boardman, Dominic A; Tung, Sim L; Lechler, Robert; Lombardi, Giovanna

    2015-01-01

    MicroRNA (miRNA) are small, non-coding RNA molecules that have been linked with immunity through regulating/modulating gene expression. A role for these molecules in T-cell and B-cell development and function has been well established. An increasing body of literature now highlights the importance of specific miRNA in dendritic cell (DC) development as well as their maturation process, antigen presentation capacity and cytokine release. Given the unique role of DC within the immune system, linking the innate and adaptive immune responses, understanding how specific miRNA affect DC function is of importance for understanding disease. In this review we summarize recent developments in miRNA and DC research, highlighting the requirement of miRNA in DC lineage commitment from bone marrow progenitors and for the development of subsets such as plasmacytoid DC and conventional DC. In addition, we discuss how infections and tumours modulate miRNA expression and consequently DC function. PMID:25244106

  5. Wnt3a Protein Reduces Growth Factor-Driven Expansion of Human Hematopoietic Stem and Progenitor Cells in Serum-Free Cultures

    PubMed Central

    Duinhouwer, Lucia E.; Tüysüz, Nesrin; Rombouts, Elwin W. J. C.; ter Borg, Mariette N. D.; Mastrobattista, Enrico; Spanholtz, Jan; Cornelissen, Jan J.; ten Berge, Derk; Braakman, Eric

    2015-01-01

    Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in robust proliferation, it is accompanied with extensive differentiation and loss of self-renewal capacity. Wnt signaling has been implicated in regulating HSPC fate decisions in vivo and in promoting HSPC self-renewal by inhibition of differentiation, but the effects of Wnt on the ex vivo expansion of HSPC are controversial. Here, we demonstrate that exogenous Wnt3a protein suppresses rather than promotes the expansion of UCB-derived CD34+ cells in serum free expansion cultures. The reduced expansion was also observed in cultures initiated with Lin-CD34+CD38lowCD45RA-CD90+ cells which are highly enriched in HSC and was also observed in response to activation of beta-catenin signaling by GSK3 inhibition. The presence of Wnt3a protein during the culture reduced the frequency of multilineage CFU-GEMM and the long-term repopulation ability of the expanded HSPC. These data suggest that Wnt signaling reduces expansion of human HSPC in growth factor-driven expansion cultures by promoting differentiation of HSPC. PMID:25807521

  6. Cell proliferation in type C gastritis affecting the intact stomach

    PubMed Central

    Mac, D; Willis, P; Prescott, R; Lamonby, S; Lynch, D

    2000-01-01

    Aims—Type C gastritis caused by bile reflux has a characteristic appearance, similar to that seen in other forms of chemical gastritis, such as those associated with NSAIDs or alcohol. An increase in mucosal cell proliferation increases the likelihood of a neoplastic clone of epithelial cells emerging, particularly where there is chronic epithelial injury associated with bile reflux. It has been shown previously that type C gastritis is associated with increased cell proliferation in the postsurgical stomach. The aim of this study was to determine cell proliferation in type C gastritis caused by bile reflux affecting the intact stomach. Methods—Specimens from 15 patients with a histological diagnosis of type C gastritis on antral biopsy were obtained from the pathology archives between 1994 and 1997. A control group of nine normal antral biopsies was also selected and all underwent MIB-1 immunostaining. The gastric glands were divided into three zones (zone 1, gastric pit; zone 2, isthmus; and zone 3, gland base) and the numbers of positively staining nuclei for 500 epithelial cell nuclei were counted in each zone to determine the percentage labelling index (LI%). Results—Cell proliferation was significantly higher in all three zones of the gastric glands with type C gastritis compared with controls as follows: zone 1, median LI% in type C gastritis 64.7 (range, 7.8–99.2), controls 4.7 (range, 2.0–11.3); zone 2, median LI% in type C gastritis 94.7 (range, 28.8–98.7), controls 40.2 (range, 23.1–70.3); and zone 3, median LI% in type C gastritis 20.0 (range, 1.3–96.0), controls 2.6 (range, 0.9–8.7). Conclusions—Bile reflux is thought to act as a promoter of gastric carcinogenesis in the postsurgical stomach. The same may be true in the intact stomach. Key Words: cell proliferation • epithelial kinetics • chemical gastritis PMID:11064674

  7. Identification of natural killer cell receptor clusters in the platypus genome reveals an expansion of C-type lectin genes.

    PubMed

    Wong, Emily S W; Sanderson, Claire E; Deakin, Janine E; Whittington, Camilla M; Papenfuss, Anthony T; Belov, Katherine

    2009-08-01

    Natural killer (NK) cell receptors belong to two unrelated, but functionally analogous gene families: the immunoglobulin superfamily, situated in the leukocyte receptor complex (LRC) and the C-type lectin superfamily, located in the natural killer complex (NKC). Here, we describe the largest NK receptor gene expansion seen to date. We identified 213 putative C-type lectin NK receptor homologs in the genome of the platypus. Many have arisen as the result of a lineage-specific expansion. Orthologs of OLR1, CD69, KLRE, CLEC12B, and CLEC16p genes were also identified. The NKC is split into at least two regions of the genome: 34 genes map to chromosome 7, two map to a small autosome, and the remainder are unanchored in the current genome assembly. No NK receptor genes from the LRC were identified. The massive C-type lectin expansion and lack of Ig-domain-containing NK receptors represents the most extreme polarization of NK receptors found to date. We have used this new data from platypus to trace the possible evolutionary history of the NK receptor clusters.

  8. The mechanism of expansion of late erythroid progenitors during erythroid regeneration: target cells and effects of erythropoietin and interleukin-3.

    PubMed

    Umemura, T; Papayannopoulou, T; Stamatoyannopoulos, G

    1989-05-15

    Through immunologic means we have been able to separate primate bone marrow cells into populations containing late erythroid progenitors (colony forming units [CFUe] and e-clusters) but depleted of early erythroid progenitors (burst-forming units [BFUe]) or populations enriched in BFUe in relation to late progenitors. We used these fractionated populations in a two stage liquid/semisolid culture system and have assessed the effect of erythropoietin (Epo) and interleukin-3 (IL-3) on the proliferation and differentiation of erythroid progenitors in the presence or absence of early progenitors. We found that populations that contained CFUe but were depleted of BFUe failed to show any amplification of CFUe or e-clusters in the presence of Epo (or Epo plus IL-3). In contrast, populations containing BFUe yielded a striking (sixfold for CFUe; 23-fold for e-clusters) expansion of late progenitors in the presence of Epo. Maximum amplification (15-fold for CFUe; 32-fold for e-clusters) was achieved when both IL-3 and Epo were present in culture. Our results imply that CFUe and e-clusters lack the capacity to amplify their numbers and suggests that the expansion of late erythroid progenitors during rapid erythroid regeneration is accomplished by influx of BFUe rather than amplification of CFUe. These data are of relevance to models of acute marrow expansion and to the mechanism of activation of fetal hemoglobin production during rapid erythroid regeneration.

  9. JAK2V617F-mutant megakaryocytes contribute to hematopoietic stem/progenitor cell expansion in a model of murine myeloproliferation

    PubMed Central

    Zhan, H; Ma, Y; Lin, CHS; Kaushansky, K

    2016-01-01

    The myeloproliferative neoplasms (MPNs) are characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of mature blood cells. The JAK2V617F mutation is present in hematopoietic cells in a majority of patients with MPNs, but the mechanism(s) responsible for MPN stem cell expansion remain incomplete. One hallmark feature of the marrow in patients with MPNs is megakaryocyte (MK) hyperplasia. We report here that mice bearing a human JAK2V617F gene restricted exclusively to the MK lineage develop many of the features of a MPN. Specifically, these mice exhibit thrombocytosis, splenomegaly, increased numbers of marrow and splenic hematopoietic progenitors and a substantial expansion of HSPCs. In addition, wild-type mice transplanted with cells from JAK2V617F-bearing MK marrow develop a myeloproliferative syndrome with thrombocytosis and erythrocytosis as well as pan-hematopoietic progenitor and stem cell expansion. As marrow histology in this murine model of myeloproliferation reveals a preferentially perivascular localization of JAK2V617F-mutant MKs and an increased marrow sinusoid vascular density, it adds to accumulating data that MKs are an important component of the marrow HSPC niche, and that MK expansion might indirectly contribute to the critical role of the thrombopoietin/c-Mpl signaling pathway in HSPC maintenance and expansion. PMID:27133820

  10. Embryonic Poly(A)-Binding Protein (EPAB) Is Required for Granulosa Cell EGF Signaling and Cumulus Expansion in Female Mice.

    PubMed

    Yang, Cai-Rong; Lowther, Katie M; Lalioti, Maria D; Seli, Emre

    2016-01-01

    Embryonic poly(A)-binding protein (EPAB) is the predominant poly(A)-binding protein in Xenopus, mouse, and human oocytes and early embryos before zygotic genome activation. EPAB is required for translational activation of maternally stored mRNAs in the oocyte and Epab(-/-) female mice are infertile due to impaired oocyte maturation, cumulus expansion, and ovulation. The aim of this study was to characterize the mechanism of follicular somatic cell dysfunction in Epab(-/-) mice. Using a coculture system of oocytectomized cumulus oophorus complexes (OOXs) with denuded oocytes, we found that when wild-type OOXs were cocultured with Epab(-/-) oocytes, or when Epab(-/-) OOXs were cocultured with WT oocytes, cumulus expansion failed to occur in response to epidermal growth factor (EGF). This finding suggests that oocytes and cumulus cells (CCs) from Epab(-/-) mice fail to send and receive the necessary signals required for cumulus expansion. The abnormalities in Epab(-/-) CCs are not due to lower expression of the oocyte-derived factors growth differentiation factor 9 or bone morphogenetic protein 15, because Epab(-/-) oocytes express these proteins at comparable levels with WT. Epab(-/-) granulosa cells (GCs) exhibit decreased levels of phosphorylated MEK1/2, ERK1/2, and p90 ribosomal S6 kinase in response to lutenizing hormone and EGF treatment, as well as decreased phosphorylation of the EGF receptor. In conclusion, EPAB, which is oocyte specific, is required for the ability of CCs and GCs to become responsive to LH and EGF signaling. These results emphasize the importance of oocyte-somatic communication for GC and CC function.

  11. Phosphatidylinositol 3-Kinase Mediates Bronchioalveolar Stem Cell Expansion in Mouse Models of Oncogenic K-ras-Induced Lung Cancer

    PubMed Central

    Yang, Yanan; Iwanaga, Kentaro; Raso, Maria Gabriela; Wislez, Marie; Hanna, Amy E.; Wieder, Eric D.; Molldrem, Jeffrey J.; Wistuba, Ignacio I.; Powis, Garth; Demayo, Francesco J.; Kim, Carla F.; Kurie, Jonathan M.

    2008-01-01

    Background Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in Western countries. Developing more effective NSCLC therapeutics will require the elucidation of the genetic and biochemical bases for this disease. Bronchioalveolar stem cells (BASCs) are a putative cancer stem cell population in mouse models of oncogenic K-ras-induced lung adenocarcinoma, an histologic subtype of NSCLC. The signals activated by oncogenic K-ras that mediate BASC expansion have not been fully defined. Methodology/Principal Findings We used genetic and pharmacologic approaches to modulate the activity of phosphatidylinositol 3-kinase (PI3K), a key mediator of oncogenic K-ras, in two genetic mouse models of lung adenocarcinoma. Oncogenic K-ras-induced BASC accumulation and tumor growth were blocked by treatment with a small molecule PI3K inhibitor and enhanced by inactivation of phosphatase and tensin homologue deleted from chromosome 10, a negative regulator of PI3K. Conclusions/Significance We conclude that PI3K is a critical regulator of BASC expansion, supporting treatment strategies to target PI3K in NSCLC patients. PMID:18493606

  12. Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells

    PubMed Central

    Haller, Thomas; Dietl, Paul; Pfaller, Kristian; Frick, Manfred; Mair, Norbert; Paulmichl, Markus; Hess, Michael W.; Fürst, Johannes; Maly, Karl

    2001-01-01

    In alveolar type II cells, the release of surfactant is considerably delayed after the formation of exocytotic fusion pores, suggesting that content dispersal may be limited by fusion pore diameter and subject to regulation at a postfusion level. To address this issue, we used confocal FRAP and N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl) pyridinium dibromide (FM 1-43), a dye yielding intense localized fluorescence of surfactant when entering the vesicle lumen through the fusion pore (Haller, T., J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. 1998. Proc. Natl. Acad. Sci. USA. 95:1579–1584). Thus, we have been able to monitor the dynamics of individual fusion pores up to hours in intact cells, and to calculate pore diameters using a diffusion model derived from Fick's law. After formation, fusion pores were arrested in a state impeding the release of vesicle contents, and expanded at irregular times thereafter. The expansion rate of initial pores and the probability of late expansions were increased by elevation of the cytoplasmic Ca2+ concentration. Consistently, content release correlated with the occurrence of Ca2+ oscillations in ATP-treated cells, and expanded fusion pores were detectable by EM. This study supports a new concept in exocytosis, implicating fusion pores in the regulation of content release for extended periods after initial formation. PMID:11604423

  13. Platelet lysate favours in vitro expansion of human bone marrow stromal cells for bone and cartilage engineering.

    PubMed

    Zaky, S H; Ottonello, A; Strada, P; Cancedda, R; Mastrogiacomo, M

    2008-12-01

    The heterogeneous population of non-haematopoietic cells residing in the bone marrow (bone marrow stromal cells, BMSCs) and the different fractions and components obtained from platelet-rich plasma provide an invaluable source of autologous cells and growth factors for bone and other connective tissue reconstruction. In this study, we investigated the effect of an allogenic platelet lysate on human BMSCs proliferation and differentiation. Cell proliferation and number of performed cell doublings were enhanced in cultures supplemented with the platelet-derived growth factors (platelet lysate, PL), either with or without the concomitant addition of fetal bovine serum (FBS), compared to cultures performed in the presence of FBS and FGF2. Both in vitro and in vivo osteogenic differentiation were unaltered in cells maintained in medium supplemented with PL and not FBS (Only PL) and in cells maintained in medium containing FBS and FGF2. Interestingly, the in vitro cartilage formation was more effective in the pellet of BMSCs expanded in the Only PL medium. In particular, a chondrogenic differentiation was observed in pellets of some in vitro-expanded BMSCs in the Only PL medium, whereas pellets from parallel cell cultures in medium containing FBS did not respond to the chondrogenic induction. We conclude that the platelet lysate from human source is an effective and even more beneficial substitute for fetal bovine serum to support the in vitro expansion of human BMSCs for subsequent tissue-engineering applications.

  14. Differentiation state affects morphine induced cell regulation in neuroblastoma cultured cells.

    PubMed

    Fiore, Giovina; Ghelardini, Carla; Bruni, Giancarlo; Guarna, Massimo; Bianchi, Enrica

    2013-10-25

    Neuroblastoma (NB) is the most common extracranial solid cancer in childhood and the most common cancer in infancy. Our purpose was to investigate in vitro how cancer cell survival occurs in presence of morphine in undifferentiated and differentiated SHSY-5Y human neuroblastoma cultured cell line. Exposure of differentiated cells to morphine dose-dependently induced apoptosis in these cells through c-Jun N-terminal kinase (JNK)/caspase pathway. Otherwise, morphine induced activation for mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway, caused positive regulation of cell survival in undifferentiated cells. Therefore, cell differentiation state bimodally affects the cellular regulation activity triggered by morphine in isolated cultured neuroblastoma cells raising concerns about the application of morphine to this type of cancer patients.

  15. Clonal expansion of T cells in abdominal aortic aneurysm: a role for doxycycline as drug of choice?

    PubMed

    Kroon, Albert M; Taanman, Jan-Willem

    2015-05-18

    Most reported studies with animal models of abdominal aortic aneurysm (AAA) and several studies with patients have suggested that doxycycline favourably modifies AAA; however, a recent large long-term clinical trial found that doxycycline did not limit aneurysm growth. Thus, there is currently no convincing evidence that doxycycline reduces AAA expansion. Here, we critically review the available experimental and clinical information about the effects of doxycycline when used as a pharmacological treatment for AAA. The view that AAA can be considered an autoimmune disease and the observation that AAA tissue shows clonal expansion of T cells is placed in the light of the well-known inhibition of mitochondrial protein synthesis by doxycycline. In T cell leukaemia animal models, this inhibitory effect of the antibiotic has been shown to impede T cell proliferation, resulting in complete tumour eradication. We suggest that the available evidence of doxycycline action on AAA is erroneously ascribed to its inhibition of matrix metalloproteinases (MMPs) by competitive binding of the zinc ion co-factor. Although competitive binding may explain the inhibition of proteolytic activity, it does not explain the observed decreases of MMP mRNA levels. We propose that the observed effects of doxycycline are secondary to inhibition of mitochondrial protein synthesis. Provided that serum doxycycline levels are kept at adequate levels, the inhibition will result in a proliferation arrest, especially of clonally expanding T cells. This, in turn, leads to the decrease of proinflammatory cytokines that are normally generated by these cells. The drastic change in cell type composition may explain the changes in MMP mRNA and protein levels in the tissue samples.

  16. Targeting STAT3 in adoptively transferred T cells promotes their in vivo expansion and antitumor effects

    PubMed Central

    Kujawski, Maciej; Zhang, Chunyan; Herrmann, Andreas; Reckamp, Karen; Scuto, Anna; Jensen, Michael; Deng, Jiehui; Forman, Stephen; Figlin, Robert; Yu, Hua

    2010-01-01

    Adoptive cell therapy with engineered T cells to improve natural immune response and antitumor functions has shown promise for treating cancer. However, the requirement for extensive ex vivo manipulation of T cells and the immunosuppressive effects of the tumor microenvironment limit this therapeutic modality. In the present study, we investigated the possibility to circumvent these limitations by engineering Stat3-deficient CD8+ T cells or by targeting Stat3 in the tumor microenvironment. We show that ablating Stat3 in CD8+ T cells prior to their transfer allows their efficient tumor infiltration and robust proliferation, resulting in increased tumor antigen-specific T cell activity and tumor growth inhibition. For potential clinical translation, we combined adoptive T cell therapy with an FDA-approved tyrosine kinase inhibitor, sunitinib, in renal cell carcinoma and melanoma tumor models. Sunitinib inhibited Stat3 in dendritic cells and T cells, reduced conversion of transferred Foxp3− T cells to tumor-associated T regulatory cells while increasing transferred CD8+ T cell infiltration and activation at the tumor site, leading to inhibition of primary tumor growth. These data demonstrate that adoptively transferred T cells can be expanded and activated in vivo either by engineering Stat3 silenced T cells or by targeting Stat3 systemically with small-molecule inhibitors. PMID:21118964

  17. Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves. I. Growth can occur without photosynthesis

    NASA Technical Reports Server (NTRS)

    Van Volkenburgh, E.; Cleland, R. E.

    1990-01-01

    Cell expansion in dicotyledonous leaves is strongly stimulated by bright white light (WL), at least in part as a result of light-induced acidification of the cell walls. It has been proposed that photosynthetic reactions are required for light-stimulated transport processes across plasma membranes of leaf cells, including proton excretion. The involvement of photosynthesis in growth and wall acidification of primary leaves of bean has been tested by inhibiting photosynthesis in two ways: by reducing chlorophyll content of intact plants with tentoxin (TX) and by treating leaf discs with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Exposure to bright WL stimulated growth of intact leaves of TX-treated plants. Discs excised from green as well as from TX-or DCMU-treated leaves also responded by growing faster in WL, as long as exogenous sucrose was supplied to the photosynthetically inhibited tissues. The WL caused acidification of the epidermal surface of intact TX-leaves, but acidification of the incubation medium by mesophyll cells only occurred when photosynthesis was not inhibited. It is concluded that light-stimulated cell enlargement of bean leaves, and the necessary acidification of epidermal cell walls, are mediated by a pigment other than chlorophyll. Light-induced proton excretion by mesophyll cells, on the other hand, may require both a photosynthetic product (or exogenous sugars) and a non-photosynthetic light effect.

  18. Prolonged expansion of human nucleus pulposus cells expressing human telomerase reverse transcriptase mediated by lentiviral vector.

    PubMed

    Wu, Jianhong; Wang, Deli; Ruan, Dike; He, Qing; Zhang, Yan; Wang, Chaofeng; Xin, Hongkui; Xu, Cheng; Liu, Yue

    2014-01-01

    Human degenerative disc disease (DDD) is characterized by progressive loss of human nucleus pulposus (HNP) cells and extracellular matrix, in which the massive deposition are secreted by HNP cells. Cell therapy to supplement HNP cells to degenerated discs has been thought to be a promising strategy to treat DDD. However, obtaining a large quality of fully functional HNP cells has been severely hampered by limited proliferation capacity of HNP cells in vitro. Previous studies have used lipofectamine or recombinant adeno-associated viral (rAAV) vectors to deliver human telomerase reverse transcriptase (hTERT) into ovine or HNP cells to prolong the activity of nucleus pulposus cells with limited success. Here we developed a lentiviral vector bearing both hTERT and a gene encoding green fluorescence protein (L-hTERT/EGFP). This vector efficiently mediated both hTERT and EGFP into freshly isolated HNP cells. The expressions of both transgenes in L-hTERT/EGFP transduced HNP cells were detected up to day 210 post viral infection, which was twice as long as rAAV vector did. Furthermore, we observed restored telomerase activity, maintained telomere length, delayed cell senescence, and increased cell proliferation rate in those L-hTERT/EGFP transduced HNP cells. Our study suggests that lentiviral vector might be a useful gene delivery vehicle for HNP cell therapy to treat DDD.

  19. Effects of agitation speed on the ex vivo expansion of cord blood hematopoietic stem/progenitor cells in stirred suspension culture.

    PubMed

    Jing, Qiang; Cai, Haibo; Du, Zheng; Ye, Zhaoyang; Tan, Wen-song

    2013-04-01

    The mononuclear cells were cultivated in stirred flasks at different agitation speeds of 30 rpm, 45 rpm, 60 rpm and 80 rpm. At the agitation speed of 30 rpm, total cells achieved higher expansion folds and the CFC density increased. When at higher agitation speed of 60 rpm or 80 rpm, the number of cells dropped rapidly and characteristics of hematopoietic stem/progenitor cells (HSPCs) were not maintained. Moreover, the culture duration of 6-9 days was better for HSPCs ex vivo expansion. These data indicated that HSPCs should be cultured at relatively low agitation speed and for a short-term period when cultured in stirred suspension system.

  20. Concise Review: Ex Vivo Expansion of Cord Blood-Derived Hematopoietic Stem and Progenitor Cells: Basic Principles, Experimental Approaches, and Impact in Regenerative Medicine

    PubMed Central

    Flores-Guzmán, Patricia; Fernández-Sánchez, Verónica

    2013-01-01

    Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) play key roles in the production of mature blood cells and in the biology and clinical outcomes of hematopoietic transplants. The numbers of these cells, however, are extremely low, particularly in umbilical cord blood (UCB); thus, ex vivo expansion of human UCB-derived HSCs and HPCs has become a priority in the biomedical field. Expansion of progenitor cells can be achieved by culturing such cells in the presence of different combinations of recombinant stimulatory cytokines; in contrast, expansion of actual HSCs has proved to be more difficult because, in addition to needing recombinant cytokines, HSCs seem to deeply depend on the presence of stromal cells and/or elements that promote the activation of particular self-renewal signaling pathways. Hence, there is still controversy regarding the optimal culture conditions that should be used to achieve this. To date, UCB transplants using ex vivo-expanded cells have already been performed for the treatment of different hematological disorders, and although results are still far from being optimal, the advances are encouraging. Recent studies suggest that HSCs may also give rise to nonhematopoietic cells, such as neural, cardiac, mesenchymal, and muscle cells. Such plasticity and the possibility of producing nonhematopoietic cells at the clinical scale could bring new alternatives for the treatment of neural, metabolic, orthopedic, cardiac, and neoplastic disorders. Once standardized, ex vivo expansion of human HSCs/HPCs will surely have a positive impact in regenerative medicine. PMID:24101670

  1. Concise review: ex vivo expansion of cord blood-derived hematopoietic stem and progenitor cells: basic principles, experimental approaches, and impact in regenerative medicine.

    PubMed

    Flores-Guzmán, Patricia; Fernández-Sánchez, Verónica; Mayani, Hector

    2013-11-01

    Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) play key roles in the production of mature blood cells and in the biology and clinical outcomes of hematopoietic transplants. The numbers of these cells, however, are extremely low, particularly in umbilical cord blood (UCB); thus, ex vivo expansion of human UCB-derived HSCs and HPCs has become a priority in the biomedical field. Expansion of progenitor cells can be achieved by culturing such cells in the presence of different combinations of recombinant stimulatory cytokines; in contrast, expansion of actual HSCs has proved to be more difficult because, in addition to needing recombinant cytokines, HSCs seem to deeply depend on the presence of stromal cells and/or elements that promote the activation of particular self-renewal signaling pathways. Hence, there is still controversy regarding the optimal culture conditions that should be used to achieve this. To date, UCB transplants using ex vivo-expanded cells have already been performed for the treatment of different hematological disorders, and although results are still far from being optimal, the advances are encouraging. Recent studies suggest that HSCs may also give rise to nonhematopoietic cells, such as neural, cardiac, mesenchymal, and muscle cells. Such plasticity and the possibility of producing nonhematopoietic cells at the clinical scale could bring new alternatives for the treatment of neural, metabolic, orthopedic, cardiac, and neoplastic disorders. Once standardized, ex vivo expansion of human HSCs/HPCs will surely have a positive impact in regenerative medicine.

  2. Matrix-mediated retention of osteogenic differentiation potential by human adult bone marrow stromal cells during ex vivo expansion.

    PubMed

    Mauney, Joshua R; Kaplan, David L; Volloch, Vladimir

    2004-07-01

    During prolonged cultivation ex vivo, adult bone marrow stromal stem cells (BMSCs) undergo two probably interdependent processes, replicative aging and a decline in differentiation potential. Recently, our results with primary human fibroblasts indicated that growth on denatured collagen (DC) matrix results in the reduction of the rate of cellular aging. The present study has been undertaken to test whether the growth of human BMSCs under the same conditions would translate into preservation of cellular aging-attenuated functions, such as the ability to express HSP70 in response to stress as well as of osteogenic differentiation potential. We report here that growth of BMSCs on a DC matrix versus tissue culture polystyrene significantly reduced one of the main manifestations of cellular aging, the attenuation of the ability to express a major protective stress response component, HSP70, increased the proliferation capacity of ex vivo expanded BMSCs, reduced the rate of morphological changes, and resulted in a dramatic increase in the retention of the potential to express osteogenic-specific functions and markers upon treatment with osteogenic stimulants. BMSCs are a promising and increasingly important cell source for tissue engineering as well as cell and gene therapeutic strategies. For use of BMSCs in these applications, ex vivo expansion is necessary to obtain a sufficient, therapeutically useful, number of cells; however, this results in the loss of differentiation potential. This problem is especially acute in older patients where more extensive in vitro expansion of smaller number of stem/progenitor cells is needed. The finding that growth on certain biomaterials preserves aging-attenuated functions, enhances proliferation capacity, and maintains differentiation potential of BMSCs indicates a promising approach to address this problem.

  3. Oriented cell division affects the global stress and cell packing geometry of a monolayer under stretch.

    PubMed

    Xu, Guang-Kui; Liu, Yang; Zheng, Zhaoliang

    2016-02-08

    Cell division plays a vital role in tissue morphogenesis and homeostasis, and the division plane is crucial for cell fate. For isolated cells, extensive studies show that the orientation of divisions is sensitive to cell shape and the direction of extrinsic mechanical forces. However, it is poorly understood that how the cell divides within a cell monolayer and how the local stress change, due to the division, affects the global stress of epithelial monolayers. Here, we use the vertex dynamics models to investigate the effects of division orientation on the configurations and mechanics of a cell monolayer under stretch. We examine three scenarios of the divisions: dividing along the stretch axis, dividing along the geometric long axis of cells, and dividing at a random angle. It is found that the division along the long cell axis can induce the minimal energy difference, and the global stress of the monolayer after stretch releases more rapidly in this case. Moreover, the long-axis division can result in more random cell orientations and more isotropic cell shapes within the monolayer, comparing with other two cases. This study helps understand the division orientation of cells within a monolayer under mechanical stimuli, and may shed light on linking individual cell's behaviors to the global mechanics and patterns of tissues.

  4. The WNT-controlled transcriptional regulator LBH is required for mammary stem cell expansion and maintenance of the basal lineage.

    PubMed

    Lindley, Linsey E; Curtis, Kevin M; Sanchez-Mejias, Avencia; Rieger, Megan E; Robbins, David J; Briegel, Karoline J

    2015-03-01

    The identification of multipotent mammary stem cells (MaSCs) has provided an explanation for the unique regenerative capacity of the mammary gland throughout adult life. However, it remains unclear what genes maintain MaSCs and control their specification into the two epithelial lineages: luminal and basal. LBH is a novel transcription co-factor in the WNT pathway with hitherto unknown physiological function. LBH is expressed during mammary gland development and aberrantly overexpressed in aggressive 'basal' subtype breast cancers. Here, we have explored the in vivo role of LBH in mammopoiesis. We show that in postnatal mammary epithelia, LBH is predominantly expressed in the Lin(-)CD29(high)CD24(+) basal MaSC population. Upon conditional inactivation of LBH, mice exhibit pronounced delays in mammary tissue expansion during puberty and pregnancy, accompanied by increased luminal differentiation at the expense of basal lineage specification. These defects could be traced to a severe reduction in the frequency and self-renewal/differentiation potential of basal MaSCs. Mechanistically, LBH induces expression of key epithelial stem cell transcription factor ΔNp63 to promote a basal MaSC state and repress luminal differentiation genes, mainly that encoding estrogen receptor α (Esr1/ERα). Collectively, these studies identify LBH as an essential regulator of basal MaSC expansion/maintenance, raising important implications for its potential role in breast cancer pathogenesis.

  5. Anabolic androgens affect the competitive interactions in cell migration and adhesion between normal mouse urothelial cells and urothelial carcinoma cells.

    PubMed

    Huang, Chi-Ping; Hsieh, Teng-Fu; Chen, Chi-Cheng; Hung, Xiao-Fan; Yu, Ai-Lin; Chang, Chawnshang; Shyr, Chih-Rong

    2014-09-26

    The urothelium is constantly rebuilt by normal urothelial cells to regenerate damaged tissues caused by stimuli in urine. However, the urothelial carcinoma cells expand the territory by aberrant growth of tumor cells, which migrate and occupy the damaged tissues to spread outside and disrupt the normal cells and organized tissues and form a tumor. Therefore, the interaction between normal urothelial cells and urothelial carcinoma cells affect the initiation and progression of urothelial tumors if normal urothelial cells fail to migrate and adhere to the damages sites to regenerate the tissues. Here, comparing normal murine urothelial cells with murine urothelial carcinoma cells (MBT-2), we found that normal cells had less migration ability than carcinoma cells. And in our co-culture system we found that carcinoma cells had propensity migrating toward normal urothelial cells and carcinoma cells had more advantages to adhere than normal cells. To reverse this condition, we used anabolic androgen, dihyrotestosterone (DHT) to treat normal cells and found that DHT treatment increased the migration ability of normal urothelial cells toward carcinoma cells and the adhesion capacity in competition with carcinoma cells. This study provides the base of a novel therapeutic approach by using anabolic hormone-enforced normal urothelial cells to regenerate the damage urothelium and defend against the occupancy of carcinoma cells to thwart cancer development and recurrence.

  6. Homeostatic T Cell Expansion to Induce Anti-Tumor Autoimmunity in Breast Cancer

    DTIC Science & Technology

    2007-04-01

    Hengartner , H., and Zinkernagel, R.M. 2001. Roles of tumour localization, second signals and cross priming in cytotoxic T-cell induction. Nature 411...irradiation may enhance metastasis by altering the tissue microenviroment, e.g. through induction of cell apoptosis , activation of certain tissue-specific...a more efficient stimulation may be achieved using apoptotic or necrotic tumor cells in themselves. Indeed, cell apoptosis and necrosis occur in a

  7. A model of cell wall expansion based on thermodynamics of polymer networks

    NASA Technical Reports Server (NTRS)

    Veytsman, B. A.; Cosgrove, D. J.

    1998-01-01

    A theory of cell wall extension is proposed. It is shown that macroscopic properties of cell walls can be explained through the microscopic properties of interpenetrating networks of cellulose and hemicellulose. The qualitative conclusions of the theory agree with the existing experimental data. The dependence of the cell wall yield threshold on the secretion of the wall components is discussed.

  8. Expansion and function of Foxp3-expressing T regulatory cells during tuberculosis.

    PubMed

    Scott-Browne, James P; Shafiani, Shahin; Tucker-Heard, Glady's; Ishida-Tsubota, Kumiko; Fontenot, Jason D; Rudensky, Alexander Y; Bevan, Michael J; Urdahl, Kevin B

    2007-09-03

    Mycobacterium tuberculosis (Mtb) frequently establishes persistent infections that may be facilitated by mechanisms that dampen immunity. T regulatory (T reg) cells, a subset of CD4(+) T cells that are essential for preventing autoimmunity, can also suppress antimicrobial immune responses. We use Foxp3-GFP mice to track the activity of T reg cells after aerosol infection with Mtb. We report that during tuberculosis, T reg cells proliferate in the pulmonary lymph nodes (pLNs), change their cell surface phenotype, and accumulate in the pLNs and lung at a rate parallel to the accumulation of effector T cells. In the Mtb-infected lung, T reg cells accumulate in high numbers in all sites where CD4(+) T cells are found, including perivascular/peribronchiolar regions and within lymphoid aggregates of granulomas. To determine the role of T reg cells in the immune response to tuberculosis, we generated mixed bone marrow chimeric mice in which all cells capable of expressing Foxp3 expressed Thy1.1. When T reg cells were depleted by administration of anti-Thy1.1 before aerosol infection with Mtb, we observed approximately 1 log less of colony-forming units of Mtb in the lungs. Thus, after aerosol infection, T reg cells proliferate and accumulate at sites of infection, and have the capacity to suppress immune responses that contribute to the control of Mtb.

  9. The auxin Sl-IAA17 transcriptional repressor controls fruit size via the regulation of endoreduplication-related cell expansion.

    PubMed

    Su, Liyan; Bassa, Carole; Audran, Corinne; Mila, Isabelle; Cheniclet, Catherine; Chevalier, Christian; Bouzayen, Mondher; Roustan, Jean-Paul; Chervin, Christian

    2014-11-01

    Auxin is known to regulate cell division and cell elongation, thus controlling plant growth and development. Part of the auxin signaling pathway depends on the fine-tuned degradation of the auxin/indole acetic acid (Aux/IAA) transcriptional repressors. Recent evidence indicates that Aux/IAA proteins play a role in fruit development in tomato (Solanum lycopersicum Mill.), a model species for fleshy fruit development. We report here on the functional characterization of Sl-IAA17 during tomato fruit development. Silencing of Sl-IAA17 by an RNA interference (RNAi) strategy resulted in the production of larger fruit than the wild type. Histological analyses of the fruit organ and tissues demonstrated that this phenotype was associated with a thicker pericarp, rather than larger locules and/or a larger number of seeds. Microscopic analysis demonstrated that the higher pericarp thickness in Sl-IAA17 RNAi fruits was not due to a larger number of cells, but to the increase in cell size. Finally, we observed that the cell expansion in the transgenic fruits is tightly coupled with higher ploidy levels than in the wild type, suggesting a stimulation of the endoreduplication process. In conclusion, this work provides new insights into the function of the Aux/IAA pathway in fleshy fruit development, especially fruit size and cell size determination in tomato.

  10. VENTX induces expansion of primitive erythroid cells and contributes to the development of acute myeloid leukemia in mice

    PubMed Central

    Gentner, Eva; Vegi, Naidu M.; Mulaw, Medhanie A.; Mandal, Tamoghna; Bamezai, Shiva; Claus, Rainer; Tasdogan, Alpaslan; Quintanilla-Martinez, Leticia; Grunenberg, Alexander; Döhner, Konstanze; Döhner, Hartmut; Bullinger, Lars; Haferlach, Torsten; Buske, Christian

    2016-01-01

    Homeobox genes are key regulators in normal and malignant hematopoiesis. The human Vent-like homeobox gene VENTX, a putative homolog of the Xenopus laevis Xvent-2 gene, was shown to be highly expressed in normal myeloid cells and in patients with acute myeloid leukemia. We now demonstrate that constitutive expression of VENTX suppresses expression of genes responsible for terminal erythroid differentiation in normal CD34+ stem and progenitor cells. Transplantation of bone marrow progenitor cells retrovirally engineered to express VENTX caused massive expansion of primitive erythroid cells and partly acute erythroleukemia in transplanted mice. The leukemogenic potential of VENTX was confirmed in the AML1-ETO transplantation model, as in contrast to AML1-ETO alone co-expression of AML1-ETO and VENTX induced acute myeloid leukemia, partly expressing erythroid markers, in all transplanted mice. VENTX was highly expressed in patients with primary human erythroleukemias and knockdown of VENTX in the erythroleukemic HEL cell line significantly blocked cell growth. In summary, these data indicate that VENTX is able to perturb erythroid differentiation and to contribute to myeloid leukemogenesis when co-expressed with appropriate AML oncogenes and point to its potential significance as a novel therapeutic target in AML. PMID:27888632

  11. Interleukin-2/Anti-Interleukin-2 Immune Complex Attenuates Cardiac Remodeling after Myocardial Infarction through Expansion of Regulatory T Cells.

    PubMed

    Zeng, Zhipeng; Yu, Kunwu; Chen, Long; Li, Weihua; Xiao, Hong; Huang, Zhengrong

    2016-01-01

    CD4+CD25+Foxp3+ regulatory T cells (Treg cells) have protective effects in wound healing and adverse ventricular remodeling after myocardial infarction (MI). We hypothesize that the interleukin- (IL-) 2 complex comprising the recombinant mouse IL-2/anti-IL-2 mAb (JES6-1) attenuates cardiac remodeling after MI through the expansion of Treg. Mice were subjected to surgical left anterior descending coronary artery ligation and treated with either PBS or IL-2 complex. The IL-2 complex significantly attenuates ventricular remodeling, as demonstrated by reduced infarct size, improved left ventricular (LV) function, and attenuated cardiomyocyte apoptosis. The IL-2 complex increased the percentage of CD4+CD25+Foxp3+ Treg cells, which may be recruited to the infarcted heart, and decreased the frequencies of IFN-γ- and IL-17-producing CD4+ T helper (Th) cells among the CD4+Foxp3- T cells in the spleen. Furthermore, the IL-2 complex inhibited the gene expression of proinflammatory cytokines as well as macrophage infiltrates in the infarcted myocardium and induced the differentiation of macrophages from M1 to M2 phenotype in border zone of infarcted myocardium. Our studies indicate that the IL-2 complex may serve as a promising therapeutic approach to attenuate adverse remodeling after MI through expanding Treg cells specifically.

  12. Ectopic expression of a polyalanine expansion mutant of poly(A)-binding protein N1 in muscle cells in culture inhibits myogenesis

    SciTech Connect

    Wang Qishan; Bag, Jnanankur . E-mail: jbag@uoguelph.ca

    2006-02-17

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset dominant genetic disease caused by the expansion of a GCG trinucleotide repeat that encodes the polyalanine tract at the N-terminus of the nuclear poly(A)-binding protein (PABPN1). Presence of intranuclear inclusions (INIs) containing PABPN1 aggregates in the skeletal muscles is the hallmark of OPMD. Here, we show that ectopic expression of the mutant PABPN1 produced INIs in a muscle cell culture model and reduced expression of several muscle-specific proteins including {alpha}-actin, slow troponin C, muscle creatine kinase, and two myogenic transcription factors, myogenin and MyoD. However, the levels of two upstream regulators of the MyoD gene, the Myf-5 and Pax3/7, were not affected, but both proteins co-localized with the PABPN1 aggregates in the mutant PABPN1 overexpressing cells. In these cells, although myogenin and MyoD levels were reduced, these two transcription factors did not co-localize with the mutant PABPN1 aggregates. Therefore, sequestration of Myf5 and Pax3/7 by the mutant PABPN1 aggregates was a specific effect on these factors. Our results suggest that trapping of these two important myogenic determinants may interfere with an early step in myogenesis.

  13. Universal Expansion.

    ERIC Educational Resources Information Center

    McArdle, Heather K.

    1997-01-01

    Describes a week-long activity for general to honors-level students that addresses Hubble's law and the universal expansion theory. Uses a discrepant event-type activity to lead up to the abstract principles of the universal expansion theory. (JRH)

  14. Factors affecting the cryosurvival of mouse two-cell embryos.

    PubMed

    Critser, J K; Arneson, B W; Aaker, D V; Huse-Benda, A R; Ball, G D

    1988-01-01

    A series of 4 experiments was conducted to examine factors affecting the survival of frozen-thawed 2-cell mouse embryos. Rapid addition of 1.5 M-DMSO (20 min equilibration at 25 degrees C) and immediate, rapid removal using 0.5 M-sucrose did not alter the frequency (mean +/- s.e.m.) of blastocyst development in vitro when compared to untreated controls (90.5 +/- 2.7% vs 95.3 +/- 2.8%). There was an interaction between the temperature at which slow cooling was terminated and thawing rate. Termination of slow cooling (-0.3 degrees C/min) at -40 degrees C with subsequent rapid thawing (approximately 1500 degrees C/min) resulted in a lower frequency of blastocyst development than did termination of slow cooling at -80 degrees C with subsequent slow thawing (+8 degrees C/min) (36.8 +/- 5.6% vs 63.9 +/- 5.7%). When slow cooling was terminated between -40 and -60 degrees C, higher survival rates were achieved with rapid thawing. When slow cooling was terminated below -60 degrees C, higher survival rates were obtained with slow thawing rates. In these comparisons absolute survival rates were highest among embryos cooled below -60 degrees C and thawed slowly. However, when slow cooling was terminated at -32 degrees C, with subsequent rapid warming, survival rates were not different from those obtained when embryos were cooled to -80 degrees C and thawed slowly (52.4 +/- 9.5%, 59.5 +/- 8.6%). These results suggest that optimal cryosurvival rates may be obtained from 2-cell mouse embryos by a rapid or slow thawing procedure, as has been found for mouse preimplantation embryos at later stages.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Interleukin-5 Supports the Expansion of Fas Ligand-Expressing Killer B Cells that Induce Antigen-Specific Apoptosis of CD4+ T Cells and Secrete Interleukin-10

    PubMed Central

    Klinker, Matthew W.; Reed, Tamra J.; Fox, David A.; Lundy, Steven K.

    2013-01-01

    Beyond their critical role in humoral immunity, B lymphocytes can employ a variety of immunomodulatory mechanisms including expression of the apoptosis-inducing molecule Fas ligand (FasL; CD178). Here, we extensively characterized the surface phenotype of FasL+ killer B cells, showing they are enriched in the IgMhighCD5+CD1dhigh B cell subset previously reported to contain a higher frequency of B cells producing interleukin-10 (IL-10). A rare population of B cells expressing IL-10 was present among FasL+ B cells, but most FasL+ B cells did not produce IL-10. We also identify interleukin-5 (IL-5) as a novel inducer of killer B cell function. Constitutively FasL+ B cells expressed higher levels of the IL-5 receptor, and treating B cells with IL-5 and CD40L resulted in the expansion of a B cell population enriched for FasL+ cells. B cells stimulated with IL-5 and CD40L were potent inducers of apoptosis in activated primary CD4+ T cells, and this killing function was antigen-specific and dependent upon FasL. IL-5 also enhanced IL-10 secretion in B cells stimulated with CD40L. Taken together these findings elucidate the relationship of FasL+ B cells and IL-10-producing B cells and demonstrate that IL-5 can induce or enhance both killer B cell activity and IL-10 secretion in B cells. Finally, we found that the killer B cell activity induced by IL-5 was completely blocked by IL-4, suggesting the existence of a previously unknown antagonistic relationship between these type-2 cytokines in modulating the activity of killer B cells. Targeting this IL-5/IL-4 signaling axis may therefore represent a novel area of drug discovery in inflammatory disorders. PMID:23940537

  16. Self-renewal of single mouse hematopoietic stem cells is reduced by JAK2V617F without compromising progenitor cell expansion.

    PubMed

    Kent, David G; Li, Juan; Tanna, Hinal; Fink, Juergen; Kirschner, Kristina; Pask, Dean C; Silber, Yvonne; Hamilton, Tina L; Sneade, Rachel; Simons, Benjamin D; Green, Anthony R

    2013-01-01

    Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F

  17. Conditional Repression of AUXIN BINDING PROTEIN1 Reveals That It Coordinates Cell Division and Cell Expansion during Postembryonic Shoot Development in Arabidopsis and Tobacco[W

    PubMed Central

    Braun, Nils; Wyrzykowska, Joanna; Muller, Philippe; David, Karine; Couch, Daniel; Perrot-Rechenmann, Catherine; Fleming, Andrew J.

    2008-01-01

    AUXIN BINDING PROTEIN1 (ABP1) has long been characterized as a potentially important mediator of auxin action in plants. Analysis of the functional requirement for ABP1 during development was hampered because of embryo lethality of the null mutant in Arabidopsis thaliana. Here, we used conditional repression of ABP1 to investigate its function during vegetative shoot development. Using an inducible cellular immunization approach and an inducible antisense construct, we showed that decreased ABP1 activity leads to a severe retardation of leaf growth involving an alteration in cell division frequency, an altered pattern of endocycle induction, a decrease in cell expansion, and a change in expression of early auxin responsive genes. In addition, local repression of ABP1 activity in the shoot apical meristem revealed an additional role for ABP1 in cell plate formation and cell shape. Moreover, cells at the site of presumptive leaf initiation were more sensitive to ABP1 repression than other regions of the meristem. This spatial context-dependent response of the meristem to ABP1 inactivation and the other data presented here are consistent with a model in which ABP1 acts as a coordinator of cell division and expansion, with local auxin levels influencing ABP1 effectiveness. PMID:18952781

  18. Expansion of quiescent lung adenocarcinoma CD8+ T cells by MUC1-8-mer peptide-T2 cell-β2 microglobulin complexes

    PubMed Central

    ATZIN-MÉNDEZ, J.A.; LÓPEZ-GONZÁLEZ, J.S.; BÁEZ, R.; ARENAS-DEL ANGEL, M.C.; MONTAÑO, L.F.; SILVA-ADAYA, D.; LASCURAIN, R.; GOROCICA, P.

    2016-01-01

    Adoptive immunotherapy requires the isolation of CD8+ T cells specific for tumor-associated antigens, their expansion in vitro and their transfusion to the patient to mediate a therapeutic effect. MUC1 is an important adenocarcinoma antigen immunogenic for T cells. The MUC1-derived SAPDTRPA (MUC1-8-mer) peptide is a potent epitope recognized by CD8+ T cells in murine models. Likewise, the T2 cell line has been used as an antigen-presenting cell to activate CD8+ T cells, but so far MUC1 has not been assessed in this context. We evaluated whether the MUC1-8-mer peptide can be presented by T2 cells to expand CD25+CD8+ T cells isolated from HLA-A2+ lung adenocarcinoma patients with stage III or IV tumors. The results showed that MUC1-8-mer peptide-loaded T2 cells activated CD8+ T cells from cancer HLA-A2+ patients when anti-CD2, anti-CD28 antibodies and IL-2 were added. The percentage of CD25+CD8+ T cells was 3-fold higher than those in the non-stimulated cells (P=0.018). HLA-A2+ patient cells showed a significant difference (2.3-fold higher) in activation status than HLA-A2+ healthy control cells (P=0.04). Moreover, 77.6% of MUC1-8-mer peptide-specific CD8+ T cells proliferated following a second stimulation with MUC1-8-mer peptide-loaded T2 cells after 10 days of cell culture. There were significant differences in the percentage of basal CD25+CD8+ T cells in relation to the cancer stage; this difference disappeared after MUC1-8-mer peptide stimulation. In conclusion, expansion of CD25+CD8+ T cells by MUC1-8 peptide-loaded T2 cells plus costimulatory signals via CD2, CD28 and IL-2 can be useful in adoptive immunotherapy. PMID:26498650

  19. An in vitro expansion score for tissue-engineering applications with human bone marrow-derived mesenchymal stem cells.

    PubMed

    Bertolo, Alessandro; Mehr, Marco; Janner-Jametti, Tiziana; Graumann, Ursula; Aebli, Niklaus; Baur, Martin; Ferguson, Stephen J; Stoyanov, Jivko V

    2016-02-01

    Human bone marrow-derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue-engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony-forming unit (CFU) assay, population doubling time (PDT), senescence-associated β-galactosidase (SA-β-Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA-β-Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late-passage cells than in early-passage cells. Early-passage cells underwent efficient osteogenic differentiation, with mid-passage cells performing best in chondrogenic differentiation. Late-passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number.

  20. p53 deficiency induces cancer stem cell pool expansion in a mouse model of triple-negative breast tumors.

    PubMed

    Chiche, A; Moumen, M; Romagnoli, M; Petit, V; Lasla, H; Jézéquel, P; de la Grange, P; Jonkers, J; Deugnier, M-A; Glukhova, M A; Faraldo, M M

    2016-10-24

    Triple-negative breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 overexpression. Triple-negative tumors often display activated Wnt/β-catenin signaling and most have impaired p53 function. We studied the interplay between p53 loss and Wnt/β-catenin signaling in stem cell function and tumorigenesis, by deleting p53 from the mammary epithelium of K5ΔNβcat mice displaying a constitutive activation of Wnt/β-catenin signaling in basal cells. K5ΔNβcat transgenic mice present amplification of the basal stem cell pool and develop triple-negative mammary carcinomas. The loss of p53 in K5ΔNβcat mice led to an early expansion of mammary stem/progenitor cells and accelerated the formation of triple-negative tumors. In particular, p53-deficient tumors expressed high levels of integrins and extracellular matrix components and were enriched in cancer stem cells. They also overexpressed the tyrosine kinase receptor Met, a feature characteristic of human triple-negative breast tumors. The inhibition of Met kinase activity impaired tumorsphere formation, demonstrating the requirement of Met signaling for cancer stem cell growth in this model. Human basal-like breast cancers with predicted mutated p53 status had higher levels of MET expression than tumors with wild-type p53. These results connect p53 loss and β-catenin activation to stem cell regulation and tumorigenesis in triple-negative cancer and highlight the role of Met signaling in maintaining cancer stem cell properties, revealing new cues for targeted therapies.Oncogene advance online publication, 24 October 2016; doi:10.1038/onc.2016.396.

  1. γδ T cells affect IL-4 production and B-cell tolerance

    PubMed Central

    Huang, Yafei; Heiser, Ryan A.; Detanico, Thiago O.; Getahun, Andrew; Kirchenbaum, Greg A.; Casper, Tamara L.; Aydintug, M. Kemal; Carding, Simon R.; Ikuta, Koichi; Huang, Hua; Cambier, John C.; Wysocki, Lawrence J.; O’Brien, Rebecca L.; Born, Willi K.

    2015-01-01

    γδ T cells can influence specific antibody responses. Here, we report that mice deficient in individual γδ T-cell subsets have altered levels of serum antibodies, including all major subclasses, sometimes regardless of the presence of αβ T cells. One strain with a partial γδ deficiency that increases IgE antibodies also displayed increases in IL-4–producing T cells (both residual γδ T cells and αβ T cells) and in systemic IL-4 levels. Its B cells expressed IL-4–regulated inhibitory receptors (CD5, CD22, and CD32) at diminished levels, whereas IL-4–inducible IL-4 receptor α and MHCII were increased. They also showed signs of activation and spontaneously formed germinal centers. These mice displayed IgE-dependent features found in hyper-IgE syndrome and developed antichromatin, antinuclear, and anticytoplasmic autoantibodies. In contrast, mice deficient in all γδ T cells had nearly unchanged Ig levels and did not develop autoantibodies. Removing IL-4 abrogated the increases in IgE, antichromatin antibodies, and autoantibodies in the partially γδ-deficient mice. Our data suggest that γδ T cells, controlled by their own cross-talk, affect IL-4 production, B-cell activation, and B-cell tolerance. PMID:25535377

  2. Dopamine favors expansion of glucocorticoid-resistant IL-17-producing T cells in multiple sclerosis.

    PubMed

    Ferreira, Thais B; Barros, Priscila O; Teixeira, Bruna; Cassano, Tatiane; Centurião, Newton; Kasahara, Taissa M; Hygino, Joana; Vasconcelos, Claudia Cristina F; Filho, Helcio Alvarenga; Alvarenga, Regina; Wing, Ana Cristina; Andrade, Regis M; Andrade, Arnaldo F; Bento, Cleonice A M

    2014-10-01

    Dopamine (DA) is a neurotransmitter produced mainly in the central nervous system (CNS) that has immunomodulatory actions on T cells. As the multiple sclerosis (MS) has long been regarded as an autoimmune disease of CNS mediated by T cells, the objective of this study was to evaluate the impact of DA on in vitro functional status of T cells from relapsing-remitting (RR)-MS patients. Peripheral T-cells from RR-MS patients were activated by mitogens and cell proliferation and cytokine production were assayed by [(3)H]-thymidine uptake and ELISA, respectively. Our results demonstrated that DA enhanced in vitro T cell proliferation and Th17-related cytokines in MS-derived cell cultures. In addition, this catecholamine reduced Treg-related cytokines (IL-10 and TGF-β) release by activated CD4(+) T cells. These DA-induced effects on T cells were mainly dependent on IL-6 production by both polyclonally-activated CD4(+) T cells and LPS-stimulated monocytes. Furthermore, the production of IL-17 and IL-6 by MS-derived T cells was directly related with neurological disability (EDSS score), and the release of these cytokines was less sensitive to glucocorticoid inhibition in MS patients than in control group, mainly after DA addition. In conclusion, our data suggest that DA amplifies glucocorticoid-resistant Th17 phenotype in MS patients, and this phenomenon could be, at least in part, due to its ability to induce IL-6 production by monocytes and CD4(+) T cells.

  3. Expansion of cytotoxic CD8+ CD28- T cells in healthy ageing people, including centenarians.

    PubMed Central

    Fagnoni, F F; Vescovini, R; Mazzola, M; Bologna, G; Nigro, E; Lavagetto, G; Franceschi, C; Passeri, M; Sansoni, P

    1996-01-01

    Ageing is associated with complex remodelling in the phenotypic and functional profiles of T lymphocytes. We investigated whether expression of CD28 antigen on T cells is conserved throughout adulthood and ageing in humans. For this purpose we analysed T cells obtained from peripheral blood of 102 healthy people of ages ranging from 20 to 105 years. We found an age-related increase of CD28- T cells in percentage and absolute number, predominantly among CD8+ T cells. CD28- T cells from aged donors analysed by flow cytometry appeared as resting cells (not expressing CD25, CD38, CD69, CD71, DR), bearing markers of cytotoxic activity (CD 11b and CD 57) and with a phenotype compatible with 'memory' cells (up-regulated CD2 and CD11a; CD62L absent). At the functional level, freshly isolated purified CD28- CD8+ T cells showed high anti-CD3 redirected cytotoxic activity against Fc-bearing P815 cells. The same activity tested on freshly isolated bulk T lymphocytes was significantly augmented with age. We found a positive correlation between age, number of CD8+ CD28- T cells and anti-CD3 redirected cytotoxicity by freshly isolated T cells. These data suggest that an activation of unknown nature within the cytotoxic arm of the immune system occurs with age. We speculate that these cytotoxic T lymphocytes (CTL) in vivo may constitute armed effector cells for immediate killing of targets bearing peptides from pathogens of intracellular origin. PMID:8881749

  4. Organ-specific scaffolds for in vitro expansion, differentiation, and organization of primary lung cells.

    PubMed

    Shamis, Yulia; Hasson, Eilat; Soroker, Avigail; Bassat, Elad; Shimoni, Yael; Ziv, Tamar; Sionov, Ronit Vogt; Mitrani, Eduardo

    2011-08-01

    In light of the increasing need for differentiated primary cells for cell therapy and the rapid dedifferentiation occurring during standard in vitro cultivation techniques, there is an urgent need for developing three-dimensional in vitro systems in which expanded cells display in vivo-like differentiated phenotypes. It is becoming clear that the natural microenvironment provides the optimal conditions for achieving this aim. To this end, we prepared natural decellularized scaffolds of microscopic dimensions that would allow appropriate diffusion of gases and nutrients to all seeded cells. Scaffolds from either the lung or the liver were analyzed for their ability to support growth and differentiation of progenitor alveolar cells and hepatocytes. We observed that progenitor alveolar cells that have been expanded on plastic culture and thus dedifferentiated grew within the lung-derived scaffolds into highly organized structures and regained differentiation markers classical for type I and type II alveolar cells. The cells generated proper alveolar structures, and only 15%-30% of them secreted surfactant proteins in a localized manner for extended periods. Vice versa, liver-derived scaffolds supported the differentiation state of primary hepatocytes. We further demonstrate that the natural scaffolds are organ specific, that is, only cells derived from the same organ become properly differentiated. A proteomic analysis shows significant different composition of lung and liver scaffolds, for example, decorin, thrombospondin 1, vimentin, and various laminin isoforms are especially enriched in the lung. Altogether, our data demonstrate that complex interactions between the seeded cells and a highly organized, organ-specific stroma are required for proper localized cell differentiation. Thus, our novel in vitro culture system can be used for ex vivo differentiation and organization of expanded primary cells.

  5. Functional niche of inflamed synovium for Th17-cell expansion and activation in rheumatoid arthritis: implication to clinical therapeutics.

    PubMed

    Dong, Weijia; Zhu, Ping

    2012-10-01

    Th17 cells selectively produce the signature cytokines such as IL-17, IL-21 and IL-22, and play a critical role for the chronic inflammatory response and subsequent tissue damage in synovial joints of patients with rheumatoid arthritis (RA). The preliminary clinical study indicates that IL-17 neutralizing therapy can ameliorate inflammatory cascades within peripheral synovial joints in the major population of patients with active RA. Multiple cellular and molecular modulations for the Th17-cell-polarized responses could exist, however, in the inflamed synovium, possibly resulting in a functional niche for the generation and activation of pathogenic Th17 cells. This might establish a vicious cycle culminating in the striking marginal erosions of cartilage and bone in the RA joints, and at least partially abrogate the potential therapeutic benefits related to IL-17 antagonizing or Th17-cell depleting therapy. This article is aimed to discuss the cellular and molecular pathways critically involved in the expansion and activation of pathogenic Th17 cells in RA synovium, with emphasis on the potential therapeutic implications for targeting these pathways to the present and future RA clinics.

  6. Fuel cell integral bundle assembly including ceramic open end seal and vertical and horizontal thermal expansion control

    DOEpatents

    Zafred, Paolo R [Murrysville, PA; Gillett, James E [Greensburg, PA

    2012-04-24

    A plurality of integral bundle assemblies contain a top portion with an inlet fuel plenum and a bottom portion containing a base support, the base supports a dense, ceramic air exhaust manifold having four supporting legs, the manifold is below and connects to air feed tubes located in a recuperator zone, the air feed tubes passing into the center of inverted, tubular, elongated, hollow electrically connected solid oxide fuel cells having an open end above a combustion zone into which the air feed tubes pass and a closed end near the inlet fuel plenum, where the open end of the fuel cells rest upon and within a separate combination ceramic seal and bundle support contained in a ceramic support casting, where at least one flexible cushion ceramic band seal located between the recuperator and fuel cells protects and controls horizontal thermal expansion, and where the fuel cells operate in the fuel cell mode and where the base support and bottom ceramic air exhaust manifolds carry from 85% to all of the weight of the generator.

  7. Fast isolation and expansion of multipotent cells from adipose tissue based on chitosan-selected primary culture.

    PubMed

    Huang, Guo-Shiang; Tseng, Ting-Chen; Dai, Niann-Tzyy; Fu, Keng-Yen; Dai, Lien-Guo; Hsu, Shan-Hui

    2015-10-01

    Adipose-derived adult stem cells (ASCs) have gained much attention because of their multipotency and easy access. Here we describe a novel chitosan-based selection (CS) system instead of the conventional plastic adherence (PA) to obtain the primary ASCs. The minimal amount of adipose tissue for consistent isolation of ASCs is reduced from 10 mL to 5 mL. The selection is based on the specific interaction between cells and chitosan materials, which separate ASCs by forming spheroids during primary culture. The primary culture period was reduced from 4 days to one day and more ASCs (ten-fold expansion) were achieved in a week. The average duration for obtaining 1 × 10(7) cells takes about seven days from 5 mL of adipose tissue, compared to 14 days using the conventional PA method from 10 mL of adipose tissue. The replicative senescence of CS-ASCs is not evident until the fifteenth passage (vs. eighth for the PA-ASCs). The obtained ASCs (CS-ASCs) have less doubling time for the same passage of cells and show greater stemness than those obtained from the conventional PA method (PA-ASCs). Moreover, CS-ASCs undergo trilineage differentiation more effectively than PA-ASCs. The greater differentiation potential of CS-ASCs may be associated with the enrichment and maintenance of CD271 positive cells by chitosan selection of primary culture.

  8. CD4+ CD25+ regulatory T cells prevent type 1 diabetes preceded by dendritic cell-dominant invasive insulitis by affecting chemotaxis and local invasiveness of dendritic cells.

    PubMed

    Lee, Mi-Heon; Lee, Wen-Hui; Todorov, Ivan; Liu, Chih-Pin

    2010-08-15

    Development of type 1 diabetes (T1D) is preceded by invasive insulitis. Although CD4(+)CD25(+) regulatory T cells (nTregs) induce tolerance that inhibits insulitis and T1D, the in vivo cellular mechanisms underlying this process remain largely unclear. Using an adoptive transfer model and noninvasive imaging-guided longitudinal analyses, we found nTreg depletion did not affect systemic trafficking and tissue localization of diabetogenic CD4(+) BDC2.5 T (BDC) cells in recipient mice prior to development of T1D. In addition, neither the initial expansion/activation of BDC cells nor the number of CD11c(+) or NK cells in islets and pancreatic lymph nodes were altered. Unexpectedly, our results showed nTreg depletion led to accelerated invasive insulitis dominated by CD11c(+) dendritic cells (ISL-DCs), not BDC cells, which stayed in the islet periphery. Compared with control mice, the phenotype of ISL-DCs and their ability to stimulate BDC cells did not change during invasive insulitis development. However, ISL-DCs from nTreg-deficient recipient mice showed increased in vitro migration toward CCL19 and CCL21. These results demonstrated invasive insulitis dominated by DCs, not CD4(+) T cells, preceded T1D onset in the absence of nTregs, and suggested a novel in vivo function of nTregs in T1D prevention by regulating local invasiveness of DCs into islets, at least partly, through regulation of DC chemotaxis toward CCL19/CCL21 produced by the islets.

  9. A novel feeder-free culture system for expansion of mouse spermatogonial stem cells.

    PubMed

    Choi, Na Young; Park, Yo Seph; Ryu, Jae-Sung; Lee, Hye Jeong; Araúzo-Bravo, Marcos J; Ko, Kisung; Han, Dong Wook; Schöler, Hans R; Ko, Kinarm

    2014-06-01

    Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost- effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.

  10. Autoimmune memory T helper 17 cell function and expansion are dependent on interleukin-23.

    PubMed

    Haines, Christopher J; Chen, Yi; Blumenschein, Wendy M; Jain, Renu; Chang, Charlie; Joyce-Shaikh, Barbara; Porth, Katherine; Boniface, Katia; Mattson, Jeanine; Basham, Beth; Anderton, Stephen M; McClanahan, Terrill K; Sadekova, Svetlana; Cua, Daniel J; McGeachy, Mandy J

    2013-05-30

    Interleukin-23 (IL-23) is essential for the differentiation of pathogenic effector T helper 17 (Th17) cells, but its role in memory Th17 cell responses is unclear. Using the experimental autoimmune encephalomyelitis (EAE) model, we report that memory Th17 cells rapidly expanded in response to rechallenge and migrated to the CNS in high numbers, resulting in earlier onset and increased severity of clinical disease. Memory Th17 cells were generated from IL-17+ and RORγt+ precursors, and the stability of the Th17 cell phenotype depended on the amount of time allowed for the primary response. IL-23 was required for this enhanced recall response. IL-23 receptor blockade did not directly impact IL-17 production, but did impair the subsequent proliferation and generation of effectors coexpressing the Th1 cell-specific transcription factor T-bet. In addition, many genes required for cell-cycle progression were downregulated in Th17 cells that lacked IL-23 signaling, showing that a major mechanism for IL-23 in primary and memory Th17 cell responses operates via regulation of proliferation-associated pathways.

  11. Cell cycle-coupled expansion of AR activity promotes cancer progression.

    PubMed

    McNair, C; Urbanucci, A; Comstock, C E S; Augello, M A; Goodwin, J F; Launchbury, R; Zhao, S G; Schiewer, M J; Ertel, A; Karnes, J; Davicioni, E; Wang, L; Wang, Q; Mills, I G; Feng, F Y; Li, W; Carroll, J S; Knudsen, K E

    2017-03-23

    The androgen receptor (AR) is required for prostate cancer (PCa) survival and progression, and ablation of AR activity is the first line of therapeutic intervention for disseminated disease. While initially effective, recurrent tumors ultimately arise for which there is no durable cure. Despite the dependence of PCa on AR activity throughout the course of disease, delineation of the AR-dependent transcriptional network that governs disease progression remains elusive, and the function of AR in mitotically active cells is not well understood. Analyzing AR activity as a function of cell cycle revealed an unexpected and highly expanded repertoire of AR-regulated gene networks in actively cycling cells. New AR functions segregated into two major clusters: those that are specific to cycling cells and retained throughout the mitotic cell cycle ('Cell Cycle Common'), versus those that were specifically enriched in a subset of cell cycle phases ('Phase Restricted'). Further analyses identified previously unrecognized AR functions in major pathways associated with clinical PCa progression. Illustrating the impact of these unmasked AR-driven pathways, dihydroceramide desaturase 1 was identified as an AR-regulated gene in mitotically active cells that promoted pro-metastatic phenotypes, and in advanced PCa proved to be highly associated with development of metastases, recurrence after therapeutic intervention and reduced overall survival. Taken together, these findings delineate AR function in mitotically active tumor cells, thus providing critical insight into the molecular basis by which AR promotes development of lethal PCa and nominate new avenues for therapeutic intervention.

  12. Aggregate formation affects ultrasonic disruption of microalgal cells.

    PubMed

    Wang, Wei; Lee, Duu-Jong; Lai, Juin-Yih

    2015-12-01

    Ultrasonication is a cell disruption process of low energy efficiency. This study dosed K(+), Ca(2+) and Al(3+) to Chlorella vulgaris cultured in Bold's Basal Medium at 25°C and measured the degree of cell disruption under ultrasonication. Adding these metal ions yielded less negatively charged surfaces of cells, while with the latter two ions large and compact cell aggregates were formed. The degree of cell disruption followed: control=K(+)>Ca(2+)>Al(3+) samples. Surface charges of cells and microbubbles have minimal effects on the microbubble number in the proximity of the microalgal cells. Conversely, cell aggregates with large size and compact interior resist cell disruption under ultrasonication. Staining tests revealed high diffusional resistance of stains over the aggregate interior. Microbubbles may not be effective generated and collapsed inside the compact aggregates, hence leading to low cell disruption efficiencies. Effective coagulation/flocculation in cell harvesting may lead to adverse effect on subsequent cell disruption efficiency.

  13. Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells.

    PubMed

    Grigoryev, Yevgeniy A; Kurian, Sunil M; Avnur, Zafi; Borie, Dominic; Deng, Jun; Campbell, Daniel; Sung, Joanna; Nikolcheva, Tania; Quinn, Anthony; Schulman, Howard; Peng, Stanford L; Schaffer, Randolph; Fisher, Jonathan; Mondala, Tony; Head, Steven; Flechner, Stuart M; Kantor, Aaron B; Marsh, Christopher; Salomon, Daniel R

    2010-10-14

    A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+)CD62L(-) effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.

  14. Expansion of revertant fibers in dystrophic mdx muscles reflects activity of muscle precursor cells and serves as an index of muscle regeneration.

    PubMed

    Yokota, Toshifumi; Lu, Qi-Long; Morgan, Jennifer E; Davies, Kay E; Fisher, Rosie; Takeda, Shin'ichi; Partridge, Terence A

    2006-07-01

    Duchenne muscular dystrophy and the mdx mouse myopathies reflect a lack of dystrophin in muscles. However, both contain sporadic clusters of revertant fibers (RFs) that express dystrophin. RF clusters expand in size with age in mdx mice. To test the hypothesis that the expansion of clusters is achieved through the process of muscle degeneration and regeneration, we analyzed muscles of mdx mice in which degeneration and regeneration were inhibited by the expression of micro-dystrophins or utrophin transgenes. Postnatal RF expansion was diminished in direct correlation to the protective effect of the transgene expression. Similarly, expansion of RFs was inhibited when muscle regeneration was blocked by irradiation. However, in irradiated muscles, irradiation-tolerant quiescent muscle precursor cells reactivated by notexin effectively restored RF expansion. Our observations demonstrate that revertant events occur initially within a subset of muscle precursor cells. The proliferation of these cells, as part of the regeneration process, leads to the expansion of RF clusters within degenerating muscles. This expansion of revertant clusters depicts the cumulative history of regeneration, thus providing a useful index for functional evaluation of therapies that counteract muscle degeneration.

  15. IRF2BP2 transcriptional repressor restrains naive CD4 T cell activation and clonal expansion induced by TCR triggering.

    PubMed

    Sécca, Cristiane; Faget, Douglas V; Hanschke, Steffi C; Carneiro, Mayra S; Bonamino, Martin H; de-Araujo-Souza, Patricia S; Viola, João P B

    2016-11-01

    CD4 T cell activation and differentiation mechanisms constitute a complex and intricate signaling network involving several regulatory proteins. IRF2BP2 is a transcriptional repressor that is involved in gene-expression regulation in very diverse biologic contexts. Information regarding the IRF2BP2 regulatory function in CD4 T lymphocytes is very limited and suggests a role for this protein in repressing the expression of different cytokine genes. Here, we showed that Irf2bp2 gene expression was decreased in CD4 T cells upon activation. To investigate the possible regulatory roles for IRF2BP2 in CD4 T cell functions, this protein was ectopically expressed in murine primary-activated CD4 T lymphocytes through retroviral transduction. Interestingly, ectopic expression of IRF2BP2 led to a reduction in CD25 expression and STAT5 phosphorylation, along with an impaired proliferative capacity. The CD69 expression was also diminished in IRF2BP2-overexpressing cells, whereas CD44 and CD62L levels were not altered. In vivo, transferred, IRF2BP2-overexpressing, transduced cells displayed an impaired expansion capacity compared with controls. Furthermore, overexpression of IRF2BP2 in differentiated Th cells resulted in slightly reduced IL-4 and pro-TGF-β production in Th2 and iTregs but had no effect on IFN-γ or IL-17 expression in Th1 and Th17 cells, respectively. Taken together, our data suggest a role for IRF2BP2 in regulating CD4 T cell activation by repressing proliferation and the expression of CD25 and CD69 induced by TCR stimuli.

  16. Diversity, cellular origin and autoreactivity of antibody-secreting cell expansions in acute Systemic Lupus Erythematosus

    PubMed Central

    Tipton, Christopher M; Fucile, Christopher F; Darce, Jaime; Chida, Asiya; Ichikawa, Travis; Gregoretti, Ivan; Schieferl, Sandra; Hom, Jennifer; Jenks, Scott; Feldman, Ron J; Mehr, Ramit; Wei, Chungwen; Lee, F. Eun-Hyung; Cheung, Wan Cheung; Rosenberg, Alexander F; Sanz, Iñaki

    2015-01-01

    Acute SLE courses with antibody-secreting cells (ASC) surges whose origin, diversity, and contribution to serum autoantibodies remain unknown. Deep sequencing, autoantibody proteome and single-cell analysis demonstrated highly diversified ASC punctuated by VH4-34 clones that produce dominant serum autoantibodies. A fraction of ASC clones contained unmutated autoantibodies, a finding consistent with differentiation outside the germinal centers. A substantial ASC segment derived from a distinct subset of newly activated naïve cells of significant clonality that persist in the circulation for several months. Thus, selection of SLE autoreactivities occurred during polyclonal activation with prolonged recruitment of recently activated naïve B cells. These findings shed light into SLE pathogenesis, help explain the benefit of anti-B cell agents and facilitate the design of future therapies. PMID:26006014

  17. Control of immune ligands by members of a cytomegalovirus gene expansion suppresses natural killer cell activation

    PubMed Central

    Fielding, Ceri A; Weekes, Michael P; Nobre, Luis V; Ruckova, Eva; Wilkie, Gavin S; Paulo, Joao A; Chang, Chiwen; Suárez, Nicolás M; Davies, James A; Antrobus, Robin; Stanton, Richard J; Aicheler, Rebecca J; Nichols, Hester; Vojtesek, Borek; Trowsdale, John; Davison, Andrew J; Gygi, Steven P

    2017-01-01

    The human cytomegalovirus (HCMV) US12 family consists of ten sequentially arranged genes (US12-21) with poorly characterized function. We now identify novel natural killer (NK) cell evasion functions for four members: US12, US14, US18 and US20. Using a systematic multiplexed proteomics approach to quantify ~1300 cell surface and ~7200 whole cell proteins, we demonstrate that the US12 family selectively targets plasma membrane proteins and plays key roles in regulating NK ligands, adhesion molecules and cytokine receptors. US18 and US20 work in concert to suppress cell surface expression of the critical NKp30 ligand B7-H6 thus inhibiting NK cell activation. The US12 family is therefore identified as a major new hub of immune regulation. DOI: http://dx.doi.org/10.7554/eLife.22206.001 PMID:28186488

  18. Rupture of the cell envelope by induced intracellular gas phase expansion in gas vacuolate bacteria.

    PubMed Central

    Hemmingsen, B B; Hemmingsen, E A

    1980-01-01

    Using a new approach, we estimated the physical strength of the cell envelopes of three species of gram-negative, gas vacuolate bacteria (Microcyclus aquaticus, Prosthecomicrobium pneumaticum, and Meniscus glaucopis). Populations of cells were slowly (0.5 to 2.9 h) saturated with argon, nitrogen, or helium to final pressures up to 100 atm (10, 132 kPa). The gas phases of the vesicles remained intact and, upon rapid (1 to 2 s) decompression to atmospheric pressure, expanded and ruptured the cells; loss of colony-forming units was used as an index of rupture. Because the cell envelope is the cellular component most likely to resist the expanding intracellular gas phase, its strength can be estimated from the minimum gas pressures that produce rupture. The viable counts indicated that these minimum pressures were between 25 and 50 atm; the majority of the cell envelopes were ruptured at pressures between 50 and 100 atm. Cells in which the gas vesicles were collapsed and the gas phases were effectively dissolved by rapid compression tolerated decompression from much higher gas saturations. Cells that do not normally possess gas vesicles (Escherichia coli) or that had been prevented from forming them by addition of L-lysine to the medium (M. aquaticus) were not harmed by decompression from gas saturation pressures up to 300 atm. PMID:7204336

  19. Low Night Temperature Affects the Phloem Ultrastructure of Lateral Branches and Raffinose Family Oligosaccharide (RFO) Accumulation in RFO-Transporting Plant Melon (Cucumismelo L.) during Fruit Expansion

    PubMed Central

    Hao, Jinghong; Gu, Fengying; Zhu, Jie; Lu, Shaowei; Liu, Yifei; Li, Yunfei; Chen, Weizhi; Wang, Liping; Fan, Shuangxi; Xian, Cory J.

    2016-01-01

    Due to the importance and complexity of photo assimilate transport in raffinose family oligosaccharide (RFO)-transporting plants such as melon, it is important to study the features of the transport structure (phloem) particularly of the lateral branches connecting the source leaves and the sink fruits, and its responses to environmental challenges. Currently, it is unclear to what extents the cold environmental temperature stress would alter the phloem ultrastructure and RFO accumulation in RFO-transporting plants. In this study, we firstly utilized electron microscopy to investigate the changes in the phloem ultrastructure of lateral branches and RFO accumulation in melons after being subjected to low night temperatures (12°C and 9°C). The results demonstrated that exposure to 9°C and 12°C altered the ultrastructure of the phloem, with the effect of 9°C being more obvious. The most obvious change was the appearance of plasma membrane invaginations in 99% companion cells and intermediary cells. In addition, phloem parenchyma cells contained chloroplasts with increased amounts of starch grains, sparse cytoplasm and reduced numbers of mitochondria. In the intermediary cells, the volume of cytoplasm was reduced by 50%, and the central vacuole was present. Moreover, the treatment at 9°C during the night led to RFO accumulation in the vascular bundles of the lateral branches and fruit carpopodiums. These ultrastructural changes of the transport structure (phloem) following the treatment at 9°C represented adaptive responses of melons to low temperature stresses. Future studies are required to examine whether these responses may affect phloem transport. PMID:27501301

  20. Cell-cycle coupled expansion of AR activity promotes cancer progression

    PubMed Central

    McNair, Christopher; Urbanucci, Alfonso; Comstock, Clay E.S.; Augello, Michael A.; Goodwin, Jonathan F.; Launchbury, Rosalind; Zhao, Shuang; Schiewer, Mathew J.; Ertel, Adam; Karnes, Jeffrey; Davicioni, Elai; Wang, Liguo; Wang, Qianben; Mills, Ian G.; Feng, Felix Y.; Li, Wei; Carroll, Jason S.; Knudsen, Karen E.

    2016-01-01

    The androgen receptor (AR) is required for prostate cancer (PCa) survival and progression, and ablation of AR activity is the first line of therapeutic intervention for disseminated disease. While initially effective, recurrent tumors ultimately arise for which there is no durable cure. Despite the dependence of PCa on AR activity throughout the course of disease, delineation of the AR-dependent transcriptional network that governs disease progression remains elusive, and the function of AR in mitotically active cells is not well understood. Analyzing AR activity as a function of cell cycle revealed an unexpected and highly expanded repertoire of AR-regulated gene networks in actively cycling cells. New AR functions segregated into two major clusters: those that are specific to cycling cells and retained throughout the mitotic cell cycle (“Cell Cycle Common”), versus those that were specifically enriched in a subset of cell cycle phases (“Phase Restricted”). Further analyses identified previously unrecognized AR functions in major pathways associated with clinical PCa progression. Illustrating the impact of these unmasked AR-driven pathways, dihydroceramide-desaturase 1 (DEGS1) was identified as an AR regulated gene in mitotically active cells that promoted pro-metastatic phenotypes, and in advanced PCa proved to be highly associated with development of metastases, recurrence after therapeutic intervention, and reduced overall survival. Taken together, these findings delineate AR function in mitotically active tumor cells, thus providing critical insight into the molecular basis by which AR promotes development of lethal PCa and nominate new avenues for therapeutic intervention. PMID:27669432

  1. Expansion of CCR8+ inflammatory myeloid cells in cancer patients with urothelial and renal carcinomas

    PubMed Central

    Eruslanov, Evgeniy; Stoffs, Taryn; Kim, Wan-Ju; Daurkin, Irina; Gilbert, Scott M.; Su, Li-Ming; Vieweg, Johannes; Daaka, Yehia; Kusmartsev, Sergei

    2013-01-01

    Purpose Chemokines are involved in cancer-related inflammation and malignant progression. In this study we evaluated expression of CCR8 and its natural cognate ligand CCL1 in patients with urothelial carcinomas of bladder and renal cell carcinomas. Experimental Design We examined CCR8 expression in peripheral blood and tumor tissues from patients with bladder and renal carcinomas. CCR8-positive myeloid cells were isolated from cancer tissues with magnetic beads and tested in vitro for cytokine production and ability to modulate T cell function. Results We demonstrate that monocytic and granulocytic myeloid cell subsets in peripheral blood of cancer patients with urothelial and renal carcinomas display increased expression of chemokine receptor CCR8. Up-regulated expression of CCR8 is also detected within human cancer tissues and primarily limited to tumor-associated macrophages (TAMs). When isolated, CD11b+CCR8+ cell subset produces the highest levels of pro-inflammatory and pro-angiogenic factors among intratumoral CD11b myeloid cells. Tumor-infiltrating CD11b+CCR8+ cells selectively display activated Stat3 and are capable of inducing FoxP3 expression in autologous T lymphocytes. Primary human tumors produce substantial amounts of the natural CCR8 ligand CCL1. Conclusions This study provides the first evidence that CCR8+ myeloid cell subset is expanded in cancer patients. Elevated secretion of CCL1 by tumors, increased presence of CCR8+ myeloid cells in peripheral blood and cancer tissues indicate that CCL1/CCR8 axis is a component of cancer-related inflammation and may contribute to immune evasion. Obtained results also implicate that blockade of CCR8 signals may provide an attractive strategy for therapeutic intervention in human urothelial and renal cancers. PMID:23363815

  2. Proteasome-independent major histocompatibility complex class I cross-presentation mediated by papaya mosaic virus-like particles leads to expansion of specific human T cells.

    PubMed

    Leclerc, Denis; Beauseigle, Diane; Denis, Jérome; Morin, Hélène; Paré, Christine; Lamarre, Alain; Lapointe, Réjean

    2007-02-01

    The development of versatile vaccine platforms is a priority that is recognized by health authorities worldwide; such platforms should induce both arms of the immune system, the humoral and cytotoxic-T-lymphocyte responses. In this study, we have established that a vaccine platform based on the coat protein of papaya mosaic virus (PapMV CP), previously shown to induce a humoral response, can induce major histocompatibility complex (MHC) class I cross-presentation of HLA-A*0201 epitopes from gp100, a melanoma antigen, and from influenza virus M1 matrix protein. PapMV proteins were able to assemble into stable virus-like particles (VLPs) in a crystalline and repetitive structure. When we pulsed HLA-A*0201+ antigen-presenting cells (APCs) with the recombinant PapMV FLU or gp100, we noted that antigen-specific CD8+ T cells were highly reactive to these APCs, demonstrating that the epitope from the VLPs were processed and loaded on the MHC class I complex. APCs were preincubated with two different proteasome inhibitors, which did not affect the efficiency of peptide presentation on MHC class I. Classical presentation from an endogenous antigen was abolished in the same conditions. Clearly, antigen presentation mediated by the PapMV system was proteasome independent. Finally, PapMV-pulsed APCs had the capacity to expand highly avid antigen-specific T cells against the influenza virus M1 HLA-A*0201 epitope when cocultured with autologous peripheral blood mononuclear cells. This study demonstrates the potential of PapMV for MHC class I cross-presentation and for the expansion of human antigen-specific T cells. It makes VLPs from PapMV CP a very attractive platform to trigger cellular responses for vaccine development against chronic infectious diseases and cancers.

  3. In vitro release and expansion of mesenchymal stem cells by a hyaluronic acid scaffold used in combination with bone marrow.

    PubMed

    Spoliti, Marco; Iudicone, Paola; Leone, Rossella; De Rosa, Alessandro; Rossetti, Francesca Romana; Pierelli, Luca

    2012-10-01

    Articular cartilage injuries of the knee are difficult to treat due to the poor healing ability of cartilage and conventional treatment methods often give unsatisfactory results. Mesenchymal Stem Cells (MSCs) have generated interest as an alternative source of cells for cartilage tissue engineering due to their chondrogenic potential and their easy isolation from bone marrow. It has been reported that the use of scaffold in cartilage engineering acts as a support for cell adhesion, keeping the cells in the cartilage defects and therefore facilitating tissue formation, and that Hyaluronic acid (HA) is a molecule of particular interest for producing scaffold for tissue engineering. In this study we evaluated the in vitro selection and expansion of Bone Marrow MSCs (BM-MSCs) and by residual BM+HA membrane (BM-HA-MSCs) used as scaffold. Sixty mL of BM have been aspirated by the posterior iliac crest and HA membrane (Hyalograft-C, Fidia Advanced Biopolimers) was used as scaffold. BM-MSCs were cultured with D-MEM supplemented with Desamethasone, Ascorbic Acid, β-Transforming Growth Factor and Insulin. When cultured in chondrogenic selective medium MSCs from both BM and HA membrane were able to differentiate into chondrogenesis, but BM-HA-MSCs showed a higher staining intensity than BM-MSCs when they were stained with Toluidine blue. The interaction of MSCs with the HA-scaffold seems to promote by itself chondrogenesis.

  4. Culture medium refinement by dialysis for the expansion of human induced pluripotent stem cells in suspension culture.

    PubMed

    Nath, Suman Chandra; Nagamori, Eiji; Horie, Masanobu; Kino-Oka, Masahiro

    2017-01-01

    Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study, after determining the minimum inhibitory level of lactic acid for hiPSCs, a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically, about 87 % of the basic fibroblast growth factor and 80 % of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture, a final cell density of (1.1 ± 0.1) × 10(6) cells mL(-1) was obtained, with 99.5 ± 0.2 % OCT 3/4 and 78.3 ± 1.1 % TRA-1-60 expression, on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method, culture medium refinement by dialysis was established to remove toxic metabolites, recycle autocrine factors as well as other growth factors, and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture.

  5. Human Adipose-Derived Stem Cells Suppress Elastase-Induced Murine Abdominal Aortic Inflammation and Aneurysm Expansion Through Paracrine Factors.

    PubMed

    Xie, Jie; Jones, Thomas J; Feng, Dongni; Cook, Todd G; Jester, Andrea A; Yi, Ru; Jawed, Yameena T; Babbey, Clifford; March, Keith L; Murphy, Michael P

    2017-02-16

    Abdominal aortic aneurysm (AAA) is a potentially lethal disease associated with immune activation-induced aortic degradation. We hypothesized that xenotransplantation of human adipose-derived stem cells (hADSCs) would reduce aortic inflammation and attenuate expansion in a murine AAA model. Modulatory effects of ADSCs on immune cell subtypes associated with AAA progression were investigated using human peripheral blood mononuclear cells (hPBMNCs) cocultured with ADSCs. Murine AAA was induced through elastase application to the abdominal aorta in C57BL/6 mice. ADSCs were administered intravenously, and aortic changes were determined by ultrasonography and videomicrometry. Circulating monocytes, aortic neutrophils, CD28- T cells, FoxP3+ regulatory T cells (Tregs), and CD206+ M2 macrophages were assessed at multiple terminal time points. In vitro, ADSCs induced M2 macrophage and Treg phenotypes while inhibiting neutrophil transmigration and lymphocyte activation without cellular contact. Intravenous ADSC delivery reduced aneurysmal expansion starting from day 4 [from baseline: 54.8% (saline) vs. 16.9% (ADSCs), n = 10 at baseline, n = 4 at day 4, p < 0.001], and the therapeutic effect persists through day 14 (from baseline: 64.1% saline vs. 24.6% ADSCs, n = 4, p < 0.01). ADSC administration increased aortic Tregs by 20-fold (n = 5, p < 0.01), while decreasing CD4+CD28- (-28%), CD8+CD28- T cells (-61%), and Ly6G/C+ neutrophils (-43%, n = 5, p < 0.05). Circulating CD115+CXCR1-LY6C+-activated monocytes decreased in the ADSC-treated group by day 7 (-60%, n = 10, p < 0.05), paralleled by an increase in aortic CD206+ M2 macrophages by 2.4-fold (n = 5, p < 0.05). Intravenously injected ADSCs transiently engrafted in the lung on day 1 without aortic engraftment at any time point. In conclusion, ADSCs exhibit pleiotropic immunomodulatory effects in vitro as well as in vivo during the development of AAA. The temporal evolution

  6. St. John's wort and its component hyperforin alleviate experimental autoimmune encephalomyelitis through expansion of regulatory T-cells.

    PubMed

    Nosratabadi, Reza; Rastin, Maryam; Sankian, Mojtaba; Haghmorad, Dariush; Tabasi, Nafiseh; Zamani, Shahrzad; Aghaee, Azita; Salehipour, Zohre; Mahmoudi, Mahmoud

    2016-05-01

    Multiple sclerosis (MS) is a central nervous system disorder mainly characterized by inflammation, demyelination and axonal injury. Anti-inflammatory agents can be used to ameliorate the disease process. Hypericum perforatum L or St. John's wort is widely used as an anti-depressant and anti-inflammatory remedy in traditional and herbal medicine. Based on St. John's wort properties, the therapeutic potentials of an H. perforatum extract (HPE) and a single component, hyperforin were evaluated for effectiveness against MOG35-55-induced experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis. Female C57BL/6 mice were immunized with specific antigen MOG35-55 and then administered different doses of hyperforin or HPE post-immunization. Clinical symptoms/other relevant parameters were assessed daily. Histological analysis of the spinal cord was performed. T-cell proliferative activity was also evaluated using a BrdU assay. The effect of hyperforin on regulatory T-cells (Treg cells) was assessed using flow cytometry. The results indicate hyperforin and HPE reduced the incidence and severity of EAE, an outcome that closely correlated with an inhibition of pathological features (leukocyte infiltration and demyelination) and antigen-specific T-cell proliferation. The study also showed that hyperforin caused increased Treg cell levels in the spleen. These results indicated that hyperforin and HPE could attenuate EAE autoimmune responses by inhibiting immune cell infiltration and expansion of Treg cell and could eventually be considered as a potential candidate for use in the treatment of MS.

  7. Isolation, expansion and characterization of bone marrow-derived mesenchymal stromal cells in serum-free conditions.

    PubMed

    Gottipamula, Sanjay; Ashwin, K M; Muttigi, Manjunatha S; Kannan, Suresh; Kolkundkar, Udaykumar; Seetharam, Raviraja N

    2014-04-01

    Bone marrow-derived mesenchymal stromal cells (BM-MSCs) heralded a new beginning for regenerative medicine and generated tremendous interest as the most promising source for therapeutic application. Most cell therapies require stringent regulatory compliance and prefer the use of serum-free media (SFM) or xeno-free media (XFM) for the MSC production process, starting from the isolation onwards. Here, we report on serum-free isolation and expansion of MSCs and compare them with cells grown in conventional fetal bovine serum (FBS)-containing media as a control. The isolation, proliferation and morphology analysis demonstrated significant differences between MSCs cultured in various SFM/XFM in addition to their difference with FBS controls. BD Mosaic™ Mesenchymal Stem Cell Serum-Free media (BD-SFM) and Mesencult-XF (MSX) supported the isolation, sequential passaging, tri-lineage differentiation potential and acceptable surface marker expression profile of BM-MSCs. Further, MSCs cultured in SFM showed higher immune suppression and hypo-immunogenicity properties, making them an ideal candidate for allogeneic cell therapy. Although cells cultured in control media have a significantly higher proliferation rate, BM-MSCs cultured in BD-SFM or MSX media are the preferred choice to meet regulatory requirements as they do not contain bovine serum. While BM-MSCs cultured in BD-SFM and MSX media adhered to all MSC characteristics, in the case of few parameters, the performance of cells cultured in BD-SFM was superior to that of MSX media. Pre-clinical safety and efficiency studies are required before qualifying SFM or XFM media-derived MSCs for therapeutic applications.

  8. In vitro generated Th17 cells support the expansion and phenotypic stability of CD4+ Foxp3+ Regulatory T cells in vivo

    PubMed Central

    Zhou, Qiong; Hu, Ya; Howard, O.M. Zack; Oppenheim, Joost J.; Chen, Xin

    2013-01-01

    CD4+ T cells stimulate immune responses through distinct patterns of cytokine produced by Th1, Th2 or Th17 cells, or inhibit immune responses through Foxp3-expressing regulatory T cells (Tregs). Paradoxically, effector T cells were recently shown to activate Tregs, however, it remains unclear which Th subset is responsible for this effect. In this study, we found that Th17 cells expressed the highest levels of TNF among in vitro generated Th subsets, and most potently promoted expansion and stabilized Foxp3 expression by Tregs when co-transferred into Rag1−/− mice. Both TNF and IL-2 produced by Th17 cells contributed to this effect. The stimulatory effect of Th17 cells on Tregs was largely abolished when co-transferred with TNFR2-deficient Tregs. Furthermore, Tregs deficient in TNFR2 also supported a much lower production of IL-17A and TNF expression by co-transferred Th17 cells. Thus, our data indicate that the TNF-TNFR2 pathway plays a crucial role in the reciprocal stimulatory effect of Th17 cells and Tregs. This bidirectional interaction should be taken into account when designing therapy targeting Th17 cells, Tregs, TNF and TNFR2. PMID:24080164

  9. In vitro generated Th17 cells support the expansion and phenotypic stability of CD4(+)Foxp3(+) regulatory T cells in vivo.

    PubMed

    Zhou, Qiong; Hu, Ya; Howard, O M Zack; Oppenheim, Joost J; Chen, Xin

    2014-01-01

    CD4(+) T cells stimulate immune responses through distinct patterns of cytokine produced by Th1, Th2 or Th17 cells, or inhibit immune responses through Foxp3-expressing regulatory T cells (Tregs). Paradoxically, effector T cells were recently shown to activate Tregs, however, it remains unclear which Th subset is responsible for this effect. In this study, we found that Th17 cells expressed the highest levels of TNF among in vitro generated Th subsets, and most potently promoted expansion and stabilized Foxp3 expression by Tregs when co-transferred into Rag1(-/-) mice. Both TNF and IL-2 produced by Th17 cells contributed to this effect. The stimulatory effect of Th17 cells on Tregs was largely abolished when co-transferred with TNFR2-deficient Tregs. Furthermore, Tregs deficient in TNFR2 also supported a much lower production of IL-17A and TNF expression by co-transferred Th17 cells. Thus, our data indicate that the TNF-TNFR2 pathway plays a crucial role in the reciprocal stimulatory effect of Th17 cells and Tregs. This bidirectional interaction should be taken into account when designing therapy targeting Th17 cells, Tregs, TNF and TNFR2.

  10. Maintenance and expansion of hematopoietic stem/progenitor cells in biomimetic osteoblast niche.

    PubMed

    Tan, Jing; Liu, Ting; Hou, Li; Meng, Wentong; Wang, Yuchun; Zhi, Wei; Deng, Li

    2010-10-01

    In this study, we employed bio-derived bone scaffold and composited with the marrow mesenchymal stem cell induced into osteoblast to replicate a "biomimetic niche." The CD34(+) cells or mononuclear cells (MNC) from umbilical cord blood were cultured for 2-5 weeks in the biomimetic niche (3D system) was compared with conventional two dimensional cultures (2D system) without adding cytokine supplement. After 2 weeks in culture, the CD34(+) cells from umbilical cord blood in the 3D system increased 3.3-4.8 folds when compared with the initial CD34(+) cells. CD34(+)/CD38(-) cells accounted for 82-90% of CD34(+) cells. After 5 weeks, CD34(+)/CD38(-) cells in the 3D system increased when compared with initial (1.3 ± 0.3 × 10(3) vs. 1.0 ± 0.5 × 10(4), p < 0.05), but were decreased in the 2D system (1.3 ± 0.3 × 10(3) vs. 2.5 ± 0.7 × 10(2), p < 0.05). The CFU progenitors were produced more in the 3D system than in the 2D system (4.6-9.3 folds vs. 1.0-1.5 folds) after 2 weeks in culture, and the colony distribution in the 3D system manifested higher percentage of BFU-E and CFU-GEMM, but in the 2D system was mainly CFU-GM. The LTC-ICs in the 3D system showed 5.2-7.2 folds increase over input at 2 weeks in culture, and maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5 weeks. In conclusion, this new 3D hematopoietic progenitor cell culture system is the first to utilize natural cancellous bone as scaffold with osteoblasts as supporting cells; it is mimicry of natural bone marrow HSC niche. Our primary work has demonstrated it could maintain and expand HSC/HPC in vitro.

  11. Deoxygenation affects tyrosine phosphoproteome of red cell membrane from patients with sickle cell disease.

    PubMed

    Siciliano, Angela; Turrini, Franco; Bertoldi, Mariarita; Matte, Alessandro; Pantaleo, Antonella; Olivieri, Oliviero; De Franceschi, Lucia

    2010-04-15

    Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder related to the production of a defective form of hemoglobin, hemoglobin S (HbS). One of the hallmarks of SCD is the presence of dense, dehydrate highly adhesive sickle red blood cells (RBCs) that result from persistent membrane damage associated with HbS polymerization, abnormal activation of membrane cation transports and generation of distorted and rigid red cells with membrane perturbation and cytoskeleton dysfunction. Although modulation of phosphorylation state of the proteins from membrane and cytoskeleton networks has been proposed to participate in red cell homeostasis, much still remains to be investigated in normal and diseased red cells. Here, we report that tyrosine (Tyr-) phosphoproteome of sickle red cells was different from normal controls and was affected by deoxygenation. We found proteins, p55 and band 4.1, from the junctional complex, differently Tyr-phosphorylated in SCD RBCs compared to normal RBCs under normoxia and modulated by deoxygenation, while band 4.2 was similarly Tyr-phosphorylated in both conditions. In SCD RBCs we identified the phosphopeptides for protein 4.1R located in the protein FERM domain (Tyr-13) and for alpha-spectrin located near or in a linker region (Tyr-422 and Tyr-1498) involving protein areas crucial for their functions in the context of red cell membrane properties, suggesting that Tyr-phosphorylation may be part of the events involved in maintaining membrane mechanical stability in SCD red cells.

  12. Homeostatic T Cell Expansion to Induce Anti-Tumor Antoimmunity in Breast Cancer

    DTIC Science & Technology

    2005-04-01

    years in defining the nature of tumor antigens and devising innovative approaches for the immunotherapy of cancer. Nevertheless, clinical success has been...Odermatt, M. Pericin, U. Karrer, J. Hermans, S. Hemmi, H. Hengartner , and R.M. Zinkernagel. 2001. Roles of tumour localization, second signals and...cross priming in cytotoxic T-cell induction. Nature 411:1058-1064. 4. Fields, R.C., K. Shimizu, and J.J. Mule. 1998. Murine dendritic cells pulsed with

  13. Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture

    PubMed Central

    Schmal, Olga; Seifert, Jan; Schäffer, Tilman E.; Walter, Christina B.; Aicher, Wilhelm K.; Klein, Gerd

    2016-01-01

    Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard. PMID:26839560

  14. Intra-abdominal desmoplastic small round-cell tumor: expansion of the pathologic profile.

    PubMed

    Dorsey, B V; Benjamin, L E; Rauscher, F; Klencke, B; Venook, A P; Warren, R S; Weidner, N

    1996-06-01

    This report describes an intra-abdominal desmoplastic small round-cell tumor in a 29-year-old man that significantly differed from the classically described appearances of this unique tumor. It showed extensive papillary areas, no necrosis, and very little desmoplasia. The latter was limited, paucicellular, and present in areas separate from the papillary structures. Also, areas of back-to-back, single-cell infiltration, which mimicked lobular breast carcinoma, were present. These epithelial features suggested the diagnosis of adenocarcinoma or peculiar mesothelioma. But, the immunohistochemical features (tumor cells positive for keratin, desmin, and vimentin) were more consistent with an intra-abdominal desmoplastic small round-cell tumor. The diagnosis became clear after application of reverse transcriptase-polymerase chain reaction techniques to formalin-fixed, paraffin-embedded tissue, which showed the presence of a 100-base pair product containing the fusion junction of Ewing's sarcoma-1 exon 7 to Wilms' tumor-1 exon 8. This feature is considered unique to intra-abdominal desmoplastic small round-cell tumors. This case illustrates the less common histologic findings that can be found in intra-abdominal desmoplastic small round-cell tumor. This deviation from the classic histologic findings may be an expression of an uncommon morphologic variant and/or partially produced by the effects of prior chemotherapy. In either event, only by illustrating the various histologic appearances of intra-abdominal desmoplastic small round-cell tumor are the chances increased for the accurate diagnosis of this aggressive neoplasm with a poor prognosis.

  15. The hollow fiber bioreactor as a stroma-supported, serum-free ex vivo expansion platform for human umbilical cord blood cells.

    PubMed

    Xue, Cao; Kwek, Kenneth Y C; Chan, Jerry K Y; Chen, Qingfeng; Lim, Mayasari

    2014-07-01

    The bone marrow microenvironment plays an integral role in the regulation of hematopoiesis. Residing stromal cells and the extracellular matrix in the bone marrow microenvironment provide biological signals that control hematopoietic stem cell (HSC) function. In this study, we developed a bio-mimetic co-culture platform using the hollow fiber bioreactor (HFBR) for ex vivo expansion of HSCs. We evaluated the efficacy of such a platform in comparison to standard cultures performed on tissue culture polystyrene (TCP), using a human stromal cell line (HS-5) as stromal support, co-cultured with lineage-depleted human cord blood cells in serum-free medium supplemented with a cytokine cocktail. Our results showed that the performance of the HFBR in supporting total cell and CD34(+) progenitor cell expansion was comparable to that of cultures on TCP. Cells harvested from the HFBR had a higher clonogenic ability. The performance of ex vivo-expanded cells from the HFBR in hematopoietic reconstitution in humanized mice was comparable to that of the TCP control. Scanning electron microscopy revealed that stroma cell growth inside the HFBR created a three-dimensional cell matrix architecture. These findings demonstrate the feasibility of utilizing the HFBR for creating a complex cell matrix architecture, which may provide good in vitro mimicry of the bone marrow, supporting large-scale expansion of HSCs.

  16. Pen Branch delta expansion

    SciTech Connect

    Nelson, E.A.; Christensen, E.J.; Mackey, H.E.; Sharitz, R.R.; Jensen, J.R.; Hodgson, M.E.

    1984-02-01

    Since 1954, cooling water discharges from K Reactor ({anti X} = 370 cfs {at} 59 C) to Pen Branch have altered vegetation and deposited sediment in the Savannah River Swamp forming the Pen Branch delta. Currently, the delta covers over 300 acres and continues to expand at a rate of about 16 acres/yr. Examination of delta expansion can provide important information on environmental impacts to wetlands exposed to elevated temperature and flow conditions. To assess the current status and predict future expansion of the Pen Branch delta, historic aerial photographs were analyzed using both basic photo interpretation and computer techniques to provide the following information: (1) past and current expansion rates; (2) location and changes of impacted areas; (3) total acreage presently affected. Delta acreage changes were then compared to historic reactor discharge temperature and flow data to see if expansion rate variations could be related to reactor operations.

  17. Expansion of CMV-specific CD8+CD45RA+CD27- T cells in B-cell chronic lymphocytic leukemia.

    PubMed

    Mackus, Wendelina J M; Frakking, Florine N J; Grummels, Annette; Gamadia, Laila E; De Bree, Godelieve J; Hamann, Dorte; Van Lier, Rene A W; Van Oers, Marinus H J

    2003-08-01

    In patients with B-cell chronic lymphocytic leukemia (B-CLL), the absolute number of T cells is increased. Although it h