WorldWideScience

Sample records for plant cell expansion

  1. Area Expansivity Moduli of Regenerating Plant Protoplast Cell Walls Exposed to Shear Flows

    Science.gov (United States)

    Fujimura, Yuu; Iino, Masaaki; Watanabe, Ugai

    2005-05-01

    To control the elasticity of the plant cell wall, protoplasts isolated from cultured Catharanthus roseus cells were regenerated in shear flows of 115 s-1 (high shear) and 19.2 s-1 (low shear, as a control). The surface area expansivity modulus and the surface breaking strength of these regenerating protoplasts were measured by a micropipette aspiration technique. Cell wall synthesis was also measured using a cell wall-specific fluorescent dye. High shear exposure for 3 h doubled both the surface area modulus and breaking strength observed under low shear, significantly decreased cell wall synthesis, and roughly quadrupled the moduli of the cell wall. Based on the cell wall synthesis data, we estimated the three-dimensional modulus of the cell wall to be 4.1± 1.2 GPa for the high shear, and 0.35± 0.2 GPa for the low shear condition, using the surface area expansivity modulus divided by the cell wall thickness, which is identical with the Young’s modulus divided by 2(1-σ), where σ is Poisson's ratio. We concluded that high shear exposure considerably strengthens the newly synthesized cell wall.

  2. Inhibition of cell proliferation, cell expansion and differentiation by the Arabidopsis SUPERMAN gene in transgenic tobacco plants.

    Science.gov (United States)

    Bereterbide, A; Hernould, M; Castera, S; Mouras, A

    2001-11-01

    Plant development depends upon the control of growth, organization and differentiation of cells derived from shoot and root meristems. Among the genes involved in flower organ determination, the cadastral gene SUPERMAN controls the boundary between whorls 3 and 4 and the growth of the adaxial outer ovule integument by down-regulating cell divisions. To determine the precise function of this gene we overexpressed ectopically the Arabidopsis thaliana (L.) Heynh. SUPERMAN gene in tobacco (Nicotiana tabacum L.). The transgenic plants exhibited a dwarf phenotype. Histologically and cytologically detailed analyses showed that dwarfism is correlated with a reduction in cell number, which is in agreement with the SUPERMAN function in Arabidopsis. Furthermore, a reduction in cell expansion and an impairment of cell differentiation were observed in tobacco organs. These traits were observed in differentiated vegetative and floral organs but not in meristem structures. A potential effect of the SUPERMAN transcription factor in the control of gibberellin biosynthesis is discussed.

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

    Directory of Open Access Journals (Sweden)

    Valera V Peremyslov

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  5. Light mediated regulation of cell division, endoreduplication and cell expansion

    NARCIS (Netherlands)

    Okello, R.C.; Visser, de P.H.B.; Heuvelink, E.; Marcelis, L.F.M.; Struik, P.C.

    2016-01-01

    Cell division, endoreduplication and cell expansion are key processes for plant growth and development. Light is the main source of energy for plants and as such has a strong effect on plant growth and development. Insight into the role of light in cellular processes is important for our

  6. A role for arabinogalactan-proteins in plant cell expansion: evidence from studies on the interaction of ß-glucosyl Yariv reagent with seedlings of Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Willats, William George Tycho; Knox, J.P.

    1996-01-01

    Seedlings of Arabidopsis thaliana were germinated and grown in medium containing ß-glucosyl Yariv reagent (ßGlcY), a synthetic phenyl glycoside that interacts specifically with arabinogalactan-proteins (AGPs), a class of plant cell surface proteoglycans. The effect of ßGlcY on the seedlings...... of elongation at the root apex and this was associated with extensive radial expansion of root epidermal cells. ßGlcY penetrated roots as far as the endodermis and it is suggested that the interaction of ßGlcY with AGPs in the load-bearing cell layers inhibited root elongation. When ßGlcY was added to carrot...

  7. Over-expression of Arabidopsis CAP causes decreased cell expansion leading to organ size reduction in transgenic tobacco plants.

    Science.gov (United States)

    Barrero, Roberto A; Umeda, Masaaki; Yamamura, Saburo; Uchimiya, Hirofumi

    2003-04-01

    Cyclase-associated proteins (CAP) are multifunctional proteins involved in Ras-cAMP signalling and regulation of the actin cytoskeleton. It has recently been demonstrated that over-expression of AtCAP1 in transgenic arabidopsis plants causes severe morphological defects owing to loss of actin filaments. To test the generality of the function of AtCAP1 in plants, transgenic tobacco plants over-expressing an arabidopsis CAP (AtCAP1) under the regulation of a glucocorticoid-inducible promoter were produced. Over-expression of AtCAP1 in transgenic tobacco plants led to growth abnormalities, in particular a reduction in the size of leaves. Morphological alterations in leaves were the result of reduced elongation of epidermal and mesophyll cells.

  8. 植物激素在植物细胞壁扩展中的作用%The role of phytohormones in plant cell wall expansion

    Institute of Scientific and Technical Information of China (English)

    陈光辉; 高艳; 陈秀娟; 谢丽琼

    2012-01-01

    细胞壁不仅是植物细胞结构的重要组成部分,而且控制着细胞的大小、形状和生长.细胞经有丝分裂后,原生质体吸水膨胀,细胞壁重塑,新生壁物质合成,纤维素定向沉积等引发细胞壁生长.在这些过程中,乙烯(ethylene,ET)、生长素(auxin)、赤霉素(gibberellin,GA)、油菜素甾醇(brassinosteroids,BR)等植物激素调控细胞壁生长相关酶类如纤维素合酶复合体(cellulose synthase A,CESA)、扩展素(expansin,EXP)、木葡聚糖内糖基转移酶/水解酶(xyloglucan endotran glucosylase/hydrolase,XET/XTH)的表达活性,进而调控细胞壁扩展,促使细胞壁的生长.%The cell wall not only provides basic skeleton to plant cell, but also controls the cell size, shape and growth. After mitosis, protoplast enlarges its size through absorbing water. In this phase, the cell wall is remodeled, with cellulose and other new wall materials synthesized and integrated. It finally causes cell growth. Phytohormones, such as IAA, GA, ET and BR, play vital roles in the process of cell expansion, which alters the expression and activity of cell wall-related enzymes such as cellulose synthase A (CESA), expansin (EXP) and xyloglucan endotran glucosylase /hydrolase (XET/XTH), and these factors regulate the cell wall expansion and finally promote cell growth.

  9. An Arabidopsis kinase cascade influences auxin-responsive cell expansion.

    Science.gov (United States)

    Enders, Tara A; Frick, Elizabeth M; Strader, Lucia C

    2017-10-01

    Mitogen-activated protein kinase (MPK) cascades are conserved mechanisms of signal transduction across eukaryotes. Despite the importance of MPK proteins in signaling events, specific roles for many Arabidopsis MPK proteins remain unknown. Multiple studies have suggested roles for MPK signaling in a variety of auxin-related processes. To identify MPK proteins with roles in auxin response, we screened mpk insertional alleles and identified mpk1-1 as a mutant that displays hypersensitivity in auxin-responsive cell expansion assays. Further, mutants defective in the upstream MAP kinase kinase MKK3 also display hypersensitivity in auxin-responsive cell expansion assays, suggesting that this MPK cascade affects auxin-influenced cell expansion. We found that MPK1 interacts with and phosphorylates ROP BINDING PROTEIN KINASE 1 (RBK1), a protein kinase that interacts with members of the Rho-like GTPases from Plants (ROP) small GTPase family. Similar to mpk1-1 and mkk3-1 mutants, rbk1 insertional mutants display auxin hypersensitivity, consistent with a possible role for RBK1 downstream of MPK1 in influencing auxin-responsive cell expansion. We found that RBK1 directly phosphorylates ROP4 and ROP6, supporting the possibility that RBK1 effects on auxin-responsive cell expansion are mediated through phosphorylation-dependent modulation of ROP activity. Our data suggest a MKK3 • MPK1 • RBK1 phosphorylation cascade that may provide a dynamic module for altering cell expansion. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  10. Can lemmings control the expansion of woody plants on tundra?

    Science.gov (United States)

    Oksanen, Lauri; Oksanen, Tarja; Olofsson, Johan; Virtanen, Risto; Hoset, Katrine; Tuomi, Maria; Kyrö, Kukka

    2013-04-01

    The ongoing expansion of woody vegetation in the arctic, due to global warming, creates a positive feed back loop. Increasing abundance of woody plants reduces surface albedo both directly and via speeding up snow melt. Thus a successively greater fraction of incoming solar radiation is absorbed and converted to heat. Browsing mammals - both big and small - can prevent this by consuming woody plants. However, the grazer/browser community of many tundra areas is dominated by brown/Norwegian lemmings (Lemmus spp.) which eat graminoids and mosses and cannot use woody plants as forage. It would seem a priori likely that in such areas, mammalian herbivores speed up the expansion of woody plants by improving the chances of their seedlings to get established. We studied the impact of lemmings on woody plants by constructing lemming proof exclosures within piece high-altitude tundra at Joatkanjávri, northernmost Norway. The exclosures were constructed in 1998, during a period of low lemming densities, in snow-beds, where Norwegian lemmings (L. lemmus) were the only ecologically significant herbivorous mammals. (Reindeer migrate through the area in May, when snow-beds are inaccessible for them; during the fall migration, the area represents a dead end and is therefore avoided.) We chose pairs of maximally similar vegetation patches of 0.5 by 0.5 m and randomly assigned one of each pair to become an exclosure while the other plot was left open. The initial state of the vegetation was documented by the point frequency method. In 2008, after the 2007 lemming outbreak, the same documentation was repeated; thereafter the plots were harvested, the vegetation was sorted to species, oven dried and weighed. Exclusion of lemmings resulted to pronounced increase in community level plant biomass. Evergreen woody plants were especially favored by the exclusion of lemming: their above-ground biomass in exclosures was 14 times as great as their biomass on open reference plots. The

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

    Science.gov (United States)

    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.

  12. Control of the actin cytoskeleton in plant cell growth

    NARCIS (Netherlands)

    Hussey, P.J.; Ketelaar, M.J.; Deeks, M.J.

    2006-01-01

    Plant cells grow through increases in volume and cell wall surface area. The mature morphology of a plant cell is a product of the differential rates of expansion between neighboring zones of the cell wall during this process. Filamentous actin arrays are associated with plant cell growth, and the a

  13. Distinctive expansion of gene families associated with plant cell wall degradation, secondary metabolism, and nutrient uptake in the genomes of grapevine trunk pathogens.

    Science.gov (United States)

    Morales-Cruz, Abraham; Amrine, Katherine C H; Blanco-Ulate, Barbara; Lawrence, Daniel P; Travadon, Renaud; Rolshausen, Philippe E; Baumgartner, Kendra; Cantu, Dario

    2015-06-19

    Trunk diseases threaten the longevity and productivity of grapevines in all viticulture production systems. They are caused by distantly-related fungi that form chronic wood infections. Variation in wood-decay abilities and production of phytotoxic compounds are thought to contribute to their unique disease symptoms. We recently released the draft sequences of Eutypa lata, Neofusicoccum parvum and Togninia minima, causal agents of Eutypa dieback, Botryosphaeria dieback and Esca, respectively. In this work, we first expanded genomic resources to three important trunk pathogens, Diaporthe ampelina, Diplodia seriata, and Phaeomoniella chlamydospora, causal agents of Phomopsis dieback, Botryosphaeria dieback, and Esca, respectively. Then we integrated all currently-available information into a genome-wide comparative study to identify gene families potentially associated with host colonization and disease development. The integration of RNA-seq, comparative and ab initio approaches improved the protein-coding gene prediction in T. minima, whereas shotgun sequencing yielded nearly complete genome drafts of Dia. ampelina, Dip. seriata, and P. chlamydospora. The predicted proteomes of all sequenced trunk pathogens were annotated with a focus on functions likely associated with pathogenesis and virulence, namely (i) wood degradation, (ii) nutrient uptake, and (iii) toxin production. Specific patterns of gene family expansion were described using Computational Analysis of gene Family Evolution, which revealed lineage-specific evolution of distinct mechanisms of virulence, such as specific cell wall oxidative functions and secondary metabolic pathways in N. parvum, Dia. ampelina, and E. lata. Phylogenetically-informed principal component analysis revealed more similar repertoires of expanded functions among species that cause similar symptoms, which in some cases did not reflect phylogenetic relationships, thereby suggesting patterns of convergent evolution. This study

  14. Catalysts of plant cell wall loosening

    OpenAIRE

    Cosgrove, Daniel J.

    2016-01-01

    The growing cell wall in plants has conflicting requirements to be strong enough to withstand the high tensile forces generated by cell turgor pressure while selectively yielding to those forces to induce wall stress relaxation, leading to water uptake and polymer movements underlying cell wall expansion. In this article, I review emerging concepts of plant primary cell wall structure, the nature of wall extensibility and the action of expansins, family-9 and -12 endoglucanases, family-16 xyl...

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

    Science.gov (United States)

    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.

  16. Plant small monomeric G-proteins (RAC/ROPs) of barley are common elements of susceptibility to fungal leaf pathogens, cell expansion and stomata development.

    Science.gov (United States)

    Pathuri, Indira Priyadarshini; Eichmann, Ruth; Hückelhoven, Ralph

    2009-02-01

    Small monomeric RAC/ROP GTPases act as molecular switches in signal transduction processes of plant development and stress responses. They emerged as crucial players in plant-pathogen interactions either by supporting susceptibility or resistance. In a recent publication, we showed that constitutively activated (CA) mutants of different barley (Hordeum vulgare) RAC/ROPs regulate susceptibility to barley fungal leaf pathogens of different life style in a contrasting way. This illustrates the distinctive signalling roles of RAC/ROPs for different plant-pathogen combinations. We also reported the involvement of RAC/ROPs in plant epidermis development in a monocotyledonous plant. Here we further discuss a failure of CA HvRAC/ROP-expressing barley to normally develop stomata.

  17. Flowering plant physiology triggered the expansion of tropical rainforest

    Science.gov (United States)

    Lee, J.; Boyce, C. K.

    2009-12-01

    Transpiration has long been known to feed precipitation, but unique hydraulic characteristics of flowering plants recently have been recognized to impart transpiration capacities dramatically higher than any other plants, living or extinct. Here we show through climate modeling that the replacement of angiosperm with non-angiosperm vegetation would result in a hotter, drier, and more seasonal Amazon basin—dry season length increases by 80 days over the eastern Amazon and overall area of everwet conditions decreases by a factor of five. Evolution of angiosperm physiology has uniquely facilitated spread of warm everwet forests and their enormous biodiversity, perhaps including their early Cenozoic expansion to extra-tropical latitudes. Divergent responses may be expected to general climate parameters and discrete environmental perturbations before and after evolution of angiosperm dominated ecosystems.

  18. Plant stem cell niches.

    Science.gov (United States)

    Aichinger, Ernst; Kornet, Noortje; Friedrich, Thomas; Laux, Thomas

    2012-01-01

    Multicellular organisms possess pluripotent stem cells to form new organs, replenish the daily loss of cells, or regenerate organs after injury. Stem cells are maintained in specific environments, the stem cell niches, that provide signals to block differentiation. In plants, stem cell niches are situated in the shoot, root, and vascular meristems-self-perpetuating units of organ formation. Plants' lifelong activity-which, as in the case of trees, can extend over more than a thousand years-requires that a robust regulatory network keep the balance between pluripotent stem cells and differentiating descendants. In this review, we focus on current models in plant stem cell research elaborated during the past two decades, mainly in the model plant Arabidopsis thaliana. We address the roles of mobile signals on transcriptional modules involved in balancing cell fates. In addition, we discuss shared features of and differences between the distinct stem cell niches of Arabidopsis.

  19. Atkinesin-13A modulates cell-wall synthesis and cell expansion in Arabidopsis thaliana via the THESEUS1 pathway.

    Directory of Open Access Journals (Sweden)

    Ushio Fujikura

    2014-09-01

    Full Text Available Growth of plant organs relies on cell proliferation and expansion. While an increasingly detailed picture about the control of cell proliferation is emerging, our knowledge about the control of cell expansion remains more limited. We demonstrate here that the internal-motor kinesin AtKINESIN-13A (AtKIN13A limits cell expansion and cell size in Arabidopsis thaliana, with loss-of-function atkin13a mutants forming larger petals with larger cells. The homolog, AtKINESIN-13B, also affects cell expansion and double mutants display growth, gametophytic and early embryonic defects, indicating a redundant role of the two genes. AtKIN13A is known to depolymerize microtubules and influence Golgi motility and distribution. Consistent with this function, AtKIN13A interacts genetically with ANGUSTIFOLIA, encoding a regulator of Golgi dynamics. Reduced AtKIN13A activity alters cell wall structure as assessed by Fourier-transformed infrared-spectroscopy and triggers signalling via the THESEUS1-dependent cell-wall integrity pathway, which in turn promotes the excess cell expansion in the atkin13a mutant. Thus, our results indicate that the intracellular activity of AtKIN13A regulates cell expansion and wall architecture via THESEUS1, providing a compelling case of interplay between cell wall integrity sensing and expansion.

  20. Fetal liver stromal cells promote hematopoietic cell expansion

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Kun; Hu, Caihong [Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030 (China); Zhou, Zhigang [Shanghai 1st People Hospital, Shanghai Jiao Tong University, Shanghai 201620 (China); Huang, Lifang; Liu, Wenli [Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030 (China); Sun, Hanying, E-mail: shanhum@163.com [Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030 (China)

    2009-09-25

    Future application of hematopoietic stem and progenitor cells (HSPCs) in clinical therapies largely depends on their successful expansion in vitro. Fetal liver (FL) is a unique hematopoietic organ in which hematopoietic cells markedly expand in number, but the mechanisms involved remain unclear. Stromal cells (StroCs) have been suggested to provide a suitable cellular environment for in vitro expansion of HSPCs. In this study, murine StroCs derived from FL at E14.5, with a high level of Sonic hedgehog (Shh) and Wnt expression, were found to have an increased ability to support the proliferation of HSPCs. This effect was inhibited by blocking Shh signaling. Supplementation with soluble Shh-N promoted the proliferation of hematopoietic cells by activating Wnt signaling. Our findings suggest that FL-derived StroCs support proliferation of HSPCs via Shh inducing an autocrine Wnt signaling loop. The use of FL-derived StroCs and regulation of the Shh pathway might further enhance HPSC expansion.

  1. Trinucleotide repeat expansions catalyzed by human cell-free extracts

    Institute of Scientific and Technical Information of China (English)

    Jennifer R Stevens; Elaine E Lahue; Guo-Min Li; Robert S Lahue

    2013-01-01

    Trinucleotide repeat expansions cause 17 heritable human neurological disorders.In some diseases,somatic expansions occur in non-proliferating tissues such as brain where DNA replication is limited.This finding stimulated significant interest in replication-independent expansion mechanisms.Aberrant DNA repair is a likely source,based in part on mouse studies showing that somatic expansions are provoked by the DNA repair protein MutSβ (Msh2-Msh3complex).Biochemical studies to date used cell-free extracts or purified DNA repair proteins to yield partial reactions at triplet repeats.The findings included expansions on one strand but not the other,or processing of DNA hairpin structures thought to be important intermediates in the expansion process.However,it has been difficult to recapitulate complete expansions in vitro,and the biochemical role of MutSβ remains controversial.Here,we use a novel in vitro assay to show that human cell-free extracts catalyze expansions and contractions of trinucleotide repeats without the requirement for DNA replication.The extract promotes a size range of expansions that is similar to certain diseases,and triplet repeat length and sequence govern expansions in vitro as in vivo.MutSβ stimulates expansions in the extract,consistent with aberrant repair of endogenous DNA damage as a source of expansions.Overall,this biochemical system retains the key characteristics of somatic expansions in humans and mice,suggesting that this important mutagenic process can be restored in the test tube.

  2. Contemporary evolution of host plant range expansion in an introduced herbivorous beetle Ophraella communa.

    Science.gov (United States)

    Fukano, Y; Doi, H; Thomas, C E; Takata, M; Koyama, S; Satoh, T

    2016-04-01

    Host range expansion of herbivorous insects is a key event in ecological speciation and insect pest management. However, the mechanistic processes are relatively unknown because it is difficult to observe the ongoing host range expansion in natural population. In this study, we focused on the ongoing host range expansion in introduced populations of the ragweed leaf beetle, Ophraella communa, to estimate the evolutionary process of host plant range expansion of a herbivorous insect. In the native range of North America, O. communa does not utilize Ambrosia trifida, as a host plant, but this plant is extensively utilized in the beetle's introduced range. Larval performance and adult preference experiments demonstrated that native O. communa beetles show better survival on host plant individuals from introduced plant populations than those from native plant populations and they also oviposit on the introduced plant, but not on the native plant. Introduced O. communa beetles showed significantly higher performance on and preference for both introduced and native A. trifida plants, when compared with native O. communa. These results indicate the contemporary evolution of host plant range expansion of introduced O. communa and suggest that the evolutionary change of both the host plant and the herbivorous insect involved in the host range expansion.

  3. The Plant Cell Surface

    Institute of Scientific and Technical Information of China (English)

    Anne-Mie C.Emons; Kurt V.Fagerstedt

    2010-01-01

    @@ Multicellular organization and tissue construction has evolved along essentially different lines in plants and animals. Since plants do not run away, but are anchored in the soil, their tissues are more or less firm and stiff. This strength stems from the cell walls, which encase the fragile cytoplasm, and protect it.

  4. Plant Stem Cells

    National Research Council Canada - National Science Library

    Greb, Thomas; Lohmann, Jan U

    2016-01-01

    .... While the promise of organ regeneration and the end of cancer have captured our imagination, it has gone almost unnoticed that plant stem cells represent the ultimate origin of much of the food we...

  5. The extracellular EXO protein mediates cell expansion in Arabidopsis leaves.

    Science.gov (United States)

    Schröder, Florian; Lisso, Janina; Lange, Peggy; Müssig, Carsten

    2009-02-13

    The EXO (EXORDIUM) gene was identified as a potential mediator of brassinosteroid (BR)-promoted growth. It is part of a gene family with eight members in Arabidopsis. EXO gene expression is under control of BR, and EXO overexpression promotes shoot and root growth. In this study, the consequences of loss of EXO function are described. The exo loss of function mutant showed diminished leaf and root growth and reduced biomass production. Light and scanning electron microscopy analyses revealed that impaired leaf growth is due to reduced cell expansion. Epidermis, palisade, and spongy parenchyma cells were smaller in comparison to the wild-type. The exo mutant showed reduced brassinolide-induced cotyledon and hypocotyl growth. In contrast, exo roots were significantly more sensitive to the inhibitory effect of synthetic brassinolide. Apart from reduced growth, exo did not show severe morphological abnormalities. Gene expression analyses of leaf material identified genes that showed robust EXO-dependent expression. Growth-related genes such as WAK1, EXP5, and KCS1, and genes involved in primary and secondary metabolism showed weaker expression in exo than in wild-type plants. However, the vast majority of BR-regulated genes were normally expressed in exo. HA- and GFP-tagged EXO proteins were targeted to the apoplast. The EXO gene is essential for cell expansion in leaves. Gene expression patterns and growth assays suggest that EXO mediates BR-induced leaf growth. However, EXO does not control BR-levels or BR-sensitivity in the shoot. EXO presumably is involved in a signalling process which coordinates BR-responses with environmental or developmental signals. The hypersensitivity of exo roots to BR suggests that EXO plays a diverse role in the control of BR responses in the root.

  6. Spatial organisation of cell expansion by the cytoskeleton

    NARCIS (Netherlands)

    Ketelaar, T.

    2002-01-01

    The shape of plants is determined by the sum of cell division and cell growth. The cytoskeleton plays an important role in both processes. This thesis presents research that pinpoints how the cytoskeleton controls plant cell growth. Root hairs of the model plant Arabidopsis have been used as a model

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

    Science.gov (United States)

    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 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.

  8. Ex vivo Expansion of Hematopoietic Stem Cells

    NARCIS (Netherlands)

    E. Farahbakhshian (Elnaz)

    2013-01-01

    textabstractHematopoiesis is a complex cellular differentiation process resulting in the formation of all blood cell types. In this process, hematopoietic stem cells (HSCs) reside at the top of the hematopoiesis hierarchy and have the capacity to differentiate into all blood cell lineages (multipote

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

    Science.gov (United States)

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

    1997-09-01

    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

  10. Elements of the niche for adult stem cell expansion.

    Science.gov (United States)

    Redondo, Patricia A; Pavlou, Marina; Loizidou, Marilena; Cheema, Umber

    2017-01-01

    Adult stem cells are crucial for tissue homeostasis. These cells reside within exclusive locations in tissues, termed niches, which protect adult stem cell fidelity and regulate their many functions through biophysical-, biochemical- and cellular-mediated mechanisms. There is a growing understanding of how these mechanisms and their components contribute towards maintaining stem cell quiescence, self-renewal, expansion and differentiation patterns. In vitro expansion of adult stem cells is a powerful tool for understanding stem cell biology, and for tissue engineering and regenerative medicine applications. However, it is technically challenging, since adult stem cell removal from their native microenvironment has negative repercussions on their sustainability. In this review, we overview specific elements of the biomimetic niche and how recreating such elements can help in vitro propagation of adult stem cells.

  11. Parathyroid hormone mediates hematopoietic cell expansion through interleukin-6.

    Directory of Open Access Journals (Sweden)

    Flavia Q Pirih

    Full Text Available Parathyroid hormone (PTH stimulates hematopoietic cells through mechanisms of action that remain elusive. Interleukin-6 (IL-6 is upregulated by PTH and stimulates hematopoiesis. The purpose of this investigation was to identify actions of PTH and IL-6 in hematopoietic cell expansion. Bone marrow cultures from C57B6 mice were treated with fms-like tyrosine kinase-3 ligand (Flt-3L, PTH, Flt-3L plus PTH, or vehicle control. Flt-3L alone increased adherent and non-adherent cells. PTH did not directly impact hematopoietic or osteoclastic cells but acted in concert with Flt-3L to further increase cell numbers. Flt-3L alone stimulated proliferation, while PTH combined with Flt-3L decreased apoptosis. Flt-3L increased blasts early in culture, and later increased CD45(+ and CD11b(+ cells. In parallel experiments, IL-6 acted additively with Flt-3L to increase cell numbers and IL-6-deficient bone marrow cultures (compared to wildtype controls but failed to amplify in response to Flt-3L and PTH, suggesting that IL-6 mediated the PTH effect. In vivo, PTH increased Lin(- Sca-1(+c-Kit(+ (LSK hematopoietic progenitor cells after PTH treatment in wildtype mice, but failed to increase LSKs in IL-6-deficient mice. In conclusion, PTH acts with Flt-3L to maintain hematopoietic cells by limiting apoptosis. IL-6 is a critical mediator of bone marrow cell expansion and is responsible for PTH actions in hematopoietic cell expansion.

  12. Rhizobacterial volatile emissions regulate auxin homeostasis and cell expansion in Arabidopsis.

    Science.gov (United States)

    Zhang, Huiming; Kim, Mi-Seong; Krishnamachari, Venkat; Payton, Paxton; Sun, Yan; Grimson, Mark; Farag, Mohamed A; Ryu, Choong-Min; Allen, Randy; Melo, Itamar S; Paré, Paul W

    2007-09-01

    Certain plant growth-promoting rhizobacteria (PGPR), in the absence of physical contact with a plant stimulate growth via volatile organic compound (VOC) emissions, through largely unknown mechanisms. To probe how PGPR VOCs trigger growth in plants, RNA transcript levels of Arabidopsis seedlings exposed to Bacillus subtilus (strain GB03) were examined using oligonucleotide microarrays. In screening over 26,000 protein-coded transcripts, a group of approximately 600 differentially expressed genes related to cell wall modifications, primary and secondary metabolism, stress responses, hormone regulation and other expressed proteins were identified. Transcriptional and histochemical data indicate that VOCs from the PGPR strain GB03 trigger growth promotion in Arabidopsis by regulating auxin homeostasis. Specifically, gene expression for auxin synthesis was up regulated in aerial regions of GB03-exposed plants; auxin accumulation decreased in leaves and increased in roots with GB03 exposure as revealed in a transgenic DR5::GUS Arabidopsis line, suggesting activation of basipetal auxin transport. Application of the auxin transport inhibitor 1-naphthylphthalamic acid (NPA) restricted auxin accumulation to sites of synthesis thereby preventing GB03-mediated decreases in shoot auxin levels as well as thwarting GB03-mediated growth promotion. In addition, microarray data revealed coordinated regulation of cell wall loosening enzymes that implicated cell expansion with GB03 exposure, which was confirmed by comparative cytological measurements. The discovery that bacterial VOCs, devoid of auxin or other known plant hormones regulate auxin homeostasis and cell expansion provides a new paradigm as to how rhizobacteria promote plant growth.

  13. The cell biology of fat expansion.

    Science.gov (United States)

    Rutkowski, Joseph M; Stern, Jennifer H; Scherer, Philipp E

    2015-03-02

    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. © 2015 Rutkowski et al.

  14. The cell biology of fat expansion

    Science.gov (United States)

    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

  15. Stirred bioreactors for the expansion of adult pancreatic stem cells.

    Science.gov (United States)

    Serra, Margarida; Brito, Catarina; Leite, Sofia B; Gorjup, Erwin; von Briesen, Hagen; Carrondo, Manuel J T; Alves, Paula M

    2009-01-01

    Adult pluripotent stem cells are a cellular resource representing unprecedented potential for cell therapy and tissue engineering. Complementary to this promise, there is a need for efficient bioprocesses for their large scale expansion and/or differentiation. With this goal in mind, our work focused on the development of three-dimensional (3-D) culture systems for controlled expansion of adult pancreatic stem cells (PSCs). For this purpose, two different culturing strategies were evaluated, using spinner vessels: cell aggregated cultures versus microcarrier technology. The use of microcarrier supports (Cytodex 1 and Cytodex 3) rendered expanded cell populations which retained their self-renewal ability, cell marker, and the potential to differentiate into adipocytes. This strategy surmounted the drawbacks of aggregates in culture which were demonstrably unfeasible as cells clumped together did not proliferate and lost PSC marker expression. Furthermore, the results obtained showed that although both microcarriers tested here were suitable for sustaining cell expansion, Cytodex 3 provided a better substrate for the promotion of cell adherence and growth. For the latter approach, the potential of bioreactor technology was combined with the efficient Cytodex 3 strategy under controlled environmental conditions (pH-7.2, pO2-30% and temperature-37 degrees C); cell growth was more efficient, as shown by faster doubling time, higher growth rate and higher fold increase in cell concentration, when compared to spinner cultures. This study describes a robust bioprocess for the controlled expansion of adult PSC, representing an efficient starting point for the development of novel technologies for cell therapy.

  16. The cell biology of fat expansion

    OpenAIRE

    Rutkowski, Joseph M.; Stern, Jennifer H.; Scherer, Philipp E.

    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. Deciph...

  17. Molecular evolution and species-specific expansion of the NAP members in plants

    Institute of Scientific and Technical Information of China (English)

    Kai Fan,; Hao Shen,; Noreen Bibi,; Feng Li,; Shuna Yuan,; Ming Wang; Xuede Wang

    2015-01-01

    The NAP (NAC-Like, Activated by AP3/PI) subfamily is one of the important plant-specific transcription factors, and controls many vital biological processes in plants. In the current study, 197 NAP proteins were identified from 31 vascular plants, but no NAP members were found in eight non-vascular plants. Al NAP proteins were phylogenetical y classified into two groups (NAP I and NAP II), and the origin time of the NAP I group might be relatively later than that of the NAP II group. Furthermore, species-specific gene duplications, caused by segmental duplication events, resulted in the expansion of the NAP subfamily after species-divergence. Different groups have different expansion rates, and the NAP group preference was found during the expansion in plants. Moreover, the expansion of NAP proteins may be related to the gain and loss of introns. Besides, functional divergence was limited after the gene duplication. Abscisic acid (ABA) might play an important role in leaf senescence, which is regulated by NAP subfamily. These results could lay an important foundation for expansion and evolutionary analysis of NAP subfamily in plants.

  18. Molecular evolution and species-specific expansion of the NAP members in plants.

    Science.gov (United States)

    Fan, Kai; Shen, Hao; Bibi, Noreen; Li, Feng; Yuan, Shuna; Wang, Ming; Wang, Xuede

    2015-08-01

    The NAP (NAC-Like, Activated by AP3 /PI) subfamily is one of the important plant-specific transcription factors, and controls many vital biological processes in plants. In the current study, 197 NAP proteins were identified from 31 vascular plants, but no NAP members were found in eight non-vascular plants. All NAP proteins were phylogenetically classified into two groups (NAP I and NAP II), and the origin time of the NAP I group might be relatively later than that of the NAP II group. Furthermore, species-specific gene duplications, caused by segmental duplication events, resulted in the expansion of the NAP subfamily after species-divergence. Different groups have different expansion rates, and the NAP group preference was found during the expansion in plants. Moreover, the expansion of NAP proteins may be related to the gain and loss of introns. Besides, functional divergence was limited after the gene duplication. Abscisic acid (ABA) might play an important role in leaf senescence, which is regulated by NAP subfamily. These results could lay an important foundation for expansion and evolutionary analysis of NAP subfamily in plants. © 2015 Institute of Botany, Chinese Academy of Sciences.

  19. Expansion of hematopoietic stem cells for transplantation: current perspectives

    Directory of Open Access Journals (Sweden)

    Schuster Jessica A

    2012-05-01

    Full Text Available Abstract Hematopoietic stem cells (HSCs are rare cells that have the unique ability to self-renew and differentiate into cells of all hematopoietic lineages. The expansion of HSCs has remained an important goal to develop advanced cell therapies for bone marrow transplantation and many blood disorders. Over the last several decades, there have been numerous attempts to expand HSCs in vitro using purified growth factors that are known to regulate HSCs. However, these attempts have been met with limited success for clinical applications. New developments in the HSC expansion field coupled with gene therapy and stem cell transplant should encourage progression in attractive treatment options for many disorders including hematologic conditions, immunodeficiencies, and genetic disorders.

  20. Evolution and diversity of green plant cell walls.

    Science.gov (United States)

    Popper, Zoë A

    2008-06-01

    Plant cells are surrounded by a dynamic cell wall that performs many essential biological roles, including regulation of cell expansion, the control of tissue cohesion, ion-exchange and defence against microbes. Recent evidence shows that the suite of polysaccharides and wall proteins from which the plant cell wall is composed shows variation between monophyletic plant taxa. This is likely to have been generated during the evolution of plant groups in response to environmental stress. Understanding the natural variation and diversity that exists between cell walls from different taxa is key to facilitating their future exploitation and manipulation, for example by increasing lignocellulosic content or reducing its recalcitrance for use in biofuel generation.

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

    Science.gov (United States)

    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.

  2. Nuclear plants in the expansion of the Mexican electrical system;Plantas nucleares en la expansion del sistema electrico mexicano

    Energy Technology Data Exchange (ETDEWEB)

    Estrada S, G. J.; Martin del Campo M, C., E-mail: gestradas@yahoo.co [UNAM, Facultad de Ingenieria, Departamento de Sistemas Energeticos, Ciudad Universitaria, 04510 Mexico D. F. (Mexico)

    2009-10-15

    In this work the results of four studies appear that were realized to analyze plans of long term expansion of Mexican electrical system of generation for the study period 2005-2025. The objective is to identify between the two third generation reactors with greater maturity at present which is it is that it can be integrated better in the expansion of the Mexican electrical system of generation. It was analyzed which of the four cases represents the best expansion plan in terms of two only parameters that are: 1) total cost of generation and, 2) the diversity of generated energy in all the period. In all studies candidates three different units of combined cycle were considered (802, 583 and 291 MW), a turbo gas unit of 267 MW, units of 700 MW with coal base and integrated de sulphur, geo thermo electrical units of 26.95 MW and two different types of nuclear units. In both first studies the Advanced Boiling Water Reactor (A BWR) for the nuclear units is considered, considering that is technology with more maturity of all the third generation reactors. In the following two studies were considered the European Pressurized Reactor (EPR), also of third generation, that uses in essence technology more spread to world-wide level. For this task was used the uni nodal planning model WASP-IV, developed by the IAEA to find the expansion configuration with less generation cost for each study. Considering the present situation of the generation system, the capacity additions begin starting from the year 2012 for the four studies. It is not considered the installation of nuclear plants before 2016 considering that its planning period takes 3 years, and the construction period requires at least of 5 years. In order to evaluate the diversity of each study it was used the Stirling Index or of Shannon-Weiner. In order to classify the studies in cost terms and diversity it was used like decision tool the Savage criterion, called also of minimal repentance. With this data, taking

  3. Portsmouth Gaseous Diffusion Plant expansion: final environmental statement. Volume 1

    Energy Technology Data Exchange (ETDEWEB)

    1977-09-01

    Volume 1 is comprised of chapters on: background and description; environmental impacts of add-on gaseous diffusion plant; unavoidable adverse environmental effects; alternatives; relationship between short-term uses and long-term productivity; relationship of program to land-use plans, policies, and controls; irreversible and irretrievable commitments of resources; cost-benefit analysis; and response to comment letters. (LK)

  4. System curves for 100-K water plant expansion pump analysis

    Energy Technology Data Exchange (ETDEWEB)

    Rudock, E.R.

    1958-06-05

    Modifications to the 100-K water plant will be made, under Project CG-775, to increase total process water flow rates to 175,000 gpm or greater. Included in the modifications will be the installation of new pump impellers for the primary and secondary process water pumps located in the 190-K Buildings.

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

    Directory of Open Access Journals (Sweden)

    Costa Eunice M

    2009-09-01

    Full Text Available Abstract 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.

  6. Cell Expansion-Mediated Organ Growth Is Affected by Mutations in Three EXIGUA Genes

    Science.gov (United States)

    González-Bayón, Rebeca; Muñoz-Viana, Rafael; Borrega, Nero; Mouille, Gregory; Hernández-Romero, Diana; Robles, Pedro; Höfte, Herman; Ponce, María Rosa; Micol, José Luis

    2012-01-01

    Organ growth depends on two distinct, yet integrated, processes: cell proliferation and post-mitotic cell expansion. Although the regulatory networks of plant cell proliferation during organ growth have begun to be unveiled, the mechanisms regulating post-mitotic cell growth remain mostly unknown. Here, we report the characterization of three EXIGUA (EXI) genes that encode different subunits of the cellulose synthase complex specifically required for secondary cell wall formation. Despite this highly specific role of EXI genes, all the cells within the leaf, even those that do not have secondary walls, display small sizes in the exi mutants. In addition, we found a positive correlation between cell size and the DNA ploidy levels in exi mutant leaves, suggesting that both processes share some regulatory components. Our results are consistent with the hypothesis that the collapsed xylem vessels of the exi mutants hamper water transport throughout the plant, which, in turn, limits the turgor pressure levels required for normal post-mitotic cell expansion during leaf growth. PMID:22586475

  7. Cell expansion-mediated organ growth is affected by mutations in three EXIGUA genes.

    Directory of Open Access Journals (Sweden)

    Silvia Rubio-Díaz

    Full Text Available Organ growth depends on two distinct, yet integrated, processes: cell proliferation and post-mitotic cell expansion. Although the regulatory networks of plant cell proliferation during organ growth have begun to be unveiled, the mechanisms regulating post-mitotic cell growth remain mostly unknown. Here, we report the characterization of three EXIGUA (EXI genes that encode different subunits of the cellulose synthase complex specifically required for secondary cell wall formation. Despite this highly specific role of EXI genes, all the cells within the leaf, even those that do not have secondary walls, display small sizes in the exi mutants. In addition, we found a positive correlation between cell size and the DNA ploidy levels in exi mutant leaves, suggesting that both processes share some regulatory components. Our results are consistent with the hypothesis that the collapsed xylem vessels of the exi mutants hamper water transport throughout the plant, which, in turn, limits the turgor pressure levels required for normal post-mitotic cell expansion during leaf growth.

  8. Calcium in plant cells

    Directory of Open Access Journals (Sweden)

    V. V. Schwartau

    2014-04-01

    Full Text Available The paper gives the review on the role of calcium in many physiological processes of plant organisms, including growth and development, protection from pathogenic influences, response to changing environmental factors, and many other aspects of plant physiology. Initial intake of calcium ions is carried out by Ca2+-channels of plasma membrane and they are further transported by the xylem owing to auxins’ attractive ability. The level of intake and selectivity of calcium transport to ove-ground parts of the plant is controlled by a symplast. Ca2+enters to the cytoplasm of endoderm cells through calcium channels on the cortical side of Kaspary bands, and is redistributed inside the stele by the symplast, with the use of Ca2+-АТPases and Ca2+/Н+-antiports. Owing to regulated expression and activity of these calcium transporters, calclum can be selectively delivered to the xylem. Important role in supporting calcium homeostasis is given to the vacuole which is the largest depo of calcium. Regulated quantity of calcium movement through the tonoplast is provided by a number of potential-, ligand-gated active transporters and channels, like Ca2+-ATPase and Ca2+/H+ exchanger. They are actively involved in the inactivation of the calcium signal by pumping Ca2+ to the depo of cells. Calcium ATPases are high affinity pumps that efficiently transfer calcium ions against the concentration gradient in their presence in the solution in nanomolar concentrations. Calcium exchangers are low affinity, high capacity Ca2+ transporters that are effectively transporting calcium after raising its concentration in the cell cytosol through the use of protons gradients. Maintaining constant concentration and participation in the response to stimuli of different types also involves EPR, plastids, mitochondria, and cell wall. Calcium binding proteins contain several conserved sequences that provide sensitivity to changes in the concentration of Ca2+ and when you

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

    Science.gov (United States)

    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.

  10. Plant single-cell and single-cell-type metabolomics.

    Science.gov (United States)

    Misra, Biswapriya B; Assmann, Sarah M; Chen, Sixue

    2014-10-01

    In conjunction with genomics, transcriptomics, and proteomics, plant metabolomics is providing large data sets that are paving the way towards a comprehensive and holistic understanding of plant growth, development, defense, and productivity. However, dilution effects from organ- and tissue-based sampling of metabolomes have limited our understanding of the intricate regulation of metabolic pathways and networks at the cellular level. Recent advances in metabolomics methodologies, along with the post-genomic expansion of bioinformatics knowledge and functional genomics tools, have allowed the gathering of enriched information on individual cells and single cell types. Here we review progress, current status, opportunities, and challenges presented by single cell-based metabolomics research in plants. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. News Release - Ceremonies held at Ashland's Kunshan and Changzhou facilities to celebrate plant expansions

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    @@ KUNSHAN & CHANGZHOU, (China) - Ashland Composite Polymers and Ashland Casting Solutions, business groups of Ashland Specialty Chemical, a division of Ashland Inc. (NYSE:ASH), held a two-day ribbon-cutting ceremony for their plant expansions in Kunshan and Changzhou in June 25-26, 2005.This marks the second public ceremonial event since the establishment of both plants in the late 1990s.

  12. Cell wall integrity signaling and innate immunity in plants.

    Science.gov (United States)

    Nühse, Thomas S

    2012-01-01

    All plant pathogens and parasites have had to develop strategies to overcome cell walls in order to access the host's cytoplasm. As a mechanically strong, multi-layered composite exoskeleton, the cell wall not only enables plants to grow tall but also protects them from such attacks. Many plant pathogens employ an arsenal of cell wall degrading enzymes, and it has long been thought that the detection of breaches in wall integrity contributes to the induction of defense. Cell wall fragments are danger-associated molecular patterns or DAMPs that can trigger defense signaling pathways comparable to microbial signals, but the picture is likely to be more complicated. A wide range of defects in cell wall biosynthesis leads to enhanced pathogen resistance. We are beginning to understand the essential role of cell wall integrity surveillance for plant growth, and the connection of processes like cell expansion, plasma membrane-cell wall contact and secondary wall biosynthesis with plant immunity is emerging.

  13. C4 plant expansion since the late Miocene and the evolution of Asian summer monsoon

    Science.gov (United States)

    Wu, H.; Guo, Z.; Guiot, J.; Yu, Y.; Ge, J.; Zhang, Y.; Peng, S.

    2015-12-01

    A dramatic expansion of C4 plant distribution occurred in the South Asia during the late Miocene and in the East Asia during the Pliocene period, with broad spatial and temporal variations. Although the event is well documented, whether subsequent expansions were caused by a decreased atmospheric CO2 concentration or climate change is a contentious issue. In this study, we used an improved inverse vegetation modeling approach that accounts for the physiological responses of C3 and C4 plants to quantitatively reconstruct the paleoclimate in the Siwalik of South Asia and in the Loess Plateau of East Asia, based on pollen and carbon isotope data. We also studied the sensitivity of the C3 and C4 plants to changes in the climate and the atmospheric CO2 concentration. We suggest that the expansion of the C4 plant distribution since the late Miocene was unlikely caused by reduced CO2 levels alone. The expansion may have been primarily triggered by regional aridification, and seasonal analysis revealed that this climate shift mainly attributed to the summer rainfall decrease. Our findings suggest that this abrupt ecological shift mainly resulted from the weakening of the Asian summer monsoon, which may related to the decrease of SST in the tropic since the late Miocene.

  14. Cell Expansion During Directed Differentiation of Stem Cells Toward the Hepatic Lineage.

    Science.gov (United States)

    Raju, Ravali; Chau, David; Cho, Dong Seong; Park, Yonsil; Verfaillie, Catherine M; Hu, Wei-Shou

    2017-02-15

    The differentiation of human pluripotent stem cells toward the hepatocyte lineage can potentially provide an unlimited source of functional hepatocytes for transplantation and extracorporeal bioartificial liver applications. It is anticipated that the quantities of cells needed for these applications will be in the order of 10(9)-10(10) cells, because of the size of the liver. An ideal differentiation protocol would be to enable directed differentiation to the hepatocyte lineage with simultaneous cell expansion. We introduced a cell expansion stage after the commitment of human embryonic stem cells to the endodermal lineage, to allow for at least an eightfold increase in cell number, with continuation of cell maturation toward the hepatocyte lineage. The progressive changes in the transcriptome were measured by expression array, and the expression dynamics of certain lineage markers was measured by mass cytometry during the differentiation and expansion process. The findings revealed that while cells were expanding they were also capable of progressing in their differentiation toward the hepatocyte lineage. In addition, our transcriptome, protein and functional studies, including albumin secretion, drug-induced CYP450 expression and urea production, all indicated that the hepatocyte-like cells obtained with or without cell expansion are very similar. This method of simultaneous cell expansion and hepatocyte differentiation should facilitate obtaining large quantities of cells for liver cell applications.

  15. Peyer's patch innate lymphoid cells regulate commensal bacteria expansion.

    Science.gov (United States)

    Hashiguchi, Masaaki; Kashiwakura, Yuji; Kojima, Hidefumi; Kobayashi, Ayano; Kanno, Yumiko; Kobata, Tetsuji

    2015-05-01

    Anatomical containment of commensal bacteria in the intestinal mucosa is promoted by innate lymphoid cells (ILCs). However, the mechanism by which ILCs regulate bacterial localization to specific regions remains unknown. Here we show that Peyer's patch (PP) ILCs robustly produce IL-22 and IFN-γ in the absence of exogenous stimuli. Antibiotic treatment of mice decreased both IL-22+ and IFN-γ+ cells in PPs. Blockade of both IL-2 and IL-23 signaling in vitro lowered IL-22 and IFN-γ production. PP ILCs induced mRNA expression of the antibacterial proteins RegIIIβ and RegIIIγ in intestinal epithelial cells. Furthermore, in vivo depletion of ILCs rather than T cells altered bacterial composition and allowed bacterial proliferation in PPs. Collectively, our results show that ILCs regulate the expansion of commensal bacteria in PPs.

  16. Experimental Investigation on the Effect of Phase Change Materials on Compressed Air Expansion in CAES Plants

    Directory of Open Access Journals (Sweden)

    Beatrice Castellani

    2015-07-01

    Full Text Available The integration of renewable energy in the electrical grid is challenging due to the intermittent and non-programmable generated electric power and to the transmission of peak power levels. Several energy storage technologies have been studied to find a solution to these issues. In particular, compressed air energy storage (CAES plants work by pumping and storing air into a vessel or in an underground cavern; then when energy is needed, the pressurized air is expanded in an expansion turbine. Several CAES configurations have been proposed: diabatic, adiabatic and isothermal. The isothermal process seems to be the most promising to improve the overall efficiency. It differs from conventional CAES approaches as it employs near-isothermal compression and expansion. Currently, there are no commercial isothermal CAES implementations worldwide, but several methods are under investigation. In this paper, the use of phase change materials (PCM for isothermal air expansion is discussed. Air expansion tests in presence of PCM were carried out in a high-pressure vessel in order to analyze the effect of PCM on the process. Results show that in presence of PCM near isothermal expansion conditions occur and therefore they affect positively the value of the obtainable expansion work.

  17. Sensor for measuring hydrogen partial pressure in parabolic trough power plant expansion tanks

    Science.gov (United States)

    Glatzmaier, Greg C.; Cooney, Daniel A.

    2017-06-01

    The National Renewable Energy Laboratory and Acciona Energy North America are working together to design and implement a process system that provides a permanent solution to the issue of hydrogen buildup at parabolic trough power plants. We are pursuing a method that selectively removes hydrogen from the expansion tanks that serve as reservoirs for the heat transfer fluid (HTF) that circulates in the collector field and power block components. Our modeling shows that removing hydrogen from the expansion tanks at a design rate reduces and maintains dissolved hydrogen in the circulating HTF to a selected target level. Our collaborative work consists of several tasks that are needed to advance this process concept to a development stage, where it is ready for implementation at a commercial power plant. Our main effort is to design and evaluate likely process-unit operations that remove hydrogen from the expansion tanks at a specified rate. Additionally, we designed and demonstrated a method and instrumentation to measure hydrogen partial pressure and concentration in the expansion-tank headspace gas. We measured hydrogen partial pressure in the headspace gas mixture using a palladium-alloy membrane, which is permeable exclusively to hydrogen. The membrane establishes a pure hydrogen gas phase that is in equilibrium with the hydrogen in the gas mixture. We designed and fabricated instrumentation, and demonstrated its effectiveness in measuring hydrogen partial pressures over a range of three orders of magnitude. Our goal is to install this instrument at the Nevada Solar One power plant and to demonstrate its effectiveness in measuring hydrogen levels in the expansion tanks under normal plant operating conditions.

  18. Sensor for Measuring Hydrogen Partial Pressure in Parabolic Trough Power Plant Expansion Tanks

    Energy Technology Data Exchange (ETDEWEB)

    Glatzmaier, Greg C.; Cooney, Daniel A.

    2017-06-27

    The National Renewable Energy Laboratory and Acciona Energy North America are working together to design and implement a process system that provides a permanent solution to the issue of hydrogen buildup at parabolic trough power plants. We are pursuing a method that selectively removes hydrogen from the expansion tanks that serve as reservoirs for the heat transfer fluid (HTF) that circulates in the collector field and power block components. Our modeling shows that removing hydrogen from the expansion tanks at a design rate reduces and maintains dissolved hydrogen in the circulating HTF to a selected target level. Our collaborative work consists of several tasks that are needed to advance this process concept to a development stage, where it is ready for implementation at a commercial power plant. Our main effort is to design and evaluate likely process-unit operations that remove hydrogen from the expansion tanks at a specified rate. Additionally, we designed and demonstrated a method and instrumentation to measure hydrogen partial pressure and concentration in the expansion-tank headspace gas. We measured hydrogen partial pressure in the headspace gas mixture using a palladium-alloy membrane, which is permeable exclusively to hydrogen. The membrane establishes a pure hydrogen gas phase that is in equilibrium with the hydrogen in the gas mixture. We designed and fabricated instrumentation, and demonstrated its effectiveness in measuring hydrogen partial pressures over a range of three orders of magnitude. Our goal is to install this instrument at the Nevada Solar One power plant and to demonstrate its effectiveness in measuring hydrogen levels in the expansion tanks under normal plant operating conditions.

  19. Organelle Extensions in Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Jaideep Mathur; Alena Mammone; Kiah A.Barton

    2012-01-01

    Cell walls lock each cell in a specific position within the supraorganization of a plant.Despite its fixed location,each cell must be able to sense alterations in its immediate environment and respond rapidly to ensure the optimal functioning,continued growth and development,and eventual long-term survival of the plant.The ultra-structural detail that underlies our present understanding of the plant cell has largely been acquired from fixed and processed material that does not allow an appreciation of the dynamic nature of sub-cellular events in the cell.In recent years,fluorescent proteinaided imaging of living plant cells has added to our understanding of the dynamic nature of the plant cell.One of the major outcomes of live imaging of plant cells is the growing appreciation that organelle shapes are not fixed,and many organelles extend their surface transiently in rapid response to environmental stimuli.In many cases,the extensions appear as tubules extending from the main organelle.Specific terms such as stromules from plastids,matrixules from mitochondria,and peroxules from peroxisomes have been coined to describe the extensions.Here,we review our present understanding of organelle extensions and discuss how they may play potential roles in maintaining cellular homeostasis in plant cells.

  20. Celebrating Plant Cells: A Special Issue on Plant Cell Biology

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ A special issue on plant cell biology is long overdue for JIPB! In the last two decades or so, the plant biology community has been thrilled by explosive discoveries regarding the molecular and genetic basis of plant growth, development, and responses to the environment, largely owing to recent maturation of model systems like Arabidopsis thaliana and the rice Oryza sativa, as well as the rapid development of high throughput technologies associated with genomics and proteomics.

  1. Ex vivo expansion protocol for human tumor specific T cells for adoptive T cell therapy.

    Science.gov (United States)

    Rasmussen, Anne-Marie; Borelli, Gabriel; Hoel, Hanna Julie; Lislerud, Kari; Gaudernack, Gustav; Kvalheim, Gunnar; Aarvak, Tanja

    2010-04-15

    Adoptive T cell therapy is a promising treatment strategy for patients with different types of cancer. The methods used for generation of high numbers of tumor specific T cells usually require long-term ex vivo culture, which frequently lead to generation of terminally differentiated effector cells, demonstrating low persistence in vivo. Therefore, optimization of protocols for generation of T cells for adoptive cell therapy is warranted. The aim of this work was to develop a protocol for expansion of antigen-specific T cells using Dynabeads CD3/CD28 to obtain T cells expressing markers important for in vivo persistence and survival. To achieve high numbers of antigen-specific T cells following expansion, we have tested the effect of depleting regulatory T cells using Dynabeads CD25 and including a pre-stimulation step with peptide prior to the non-specific expansion with Dynabeads. Our data demonstrate that virus- and tumor specific T cells can be expanded to high numbers using Dynabeads CD3/CD28 following optimization of the culture conditions. The expansion protocol presented here results in enrichment of antigen-specific CD8(+) T cells with an early/intermediate memory phenotype. This is observed even when the antigen-specific CD8(+) T cells demonstrated a terminal effector phenotype prior to expansion. This protocol thus results in expanded T cells with a phenotypic profile which may increase the chance of retaining long-term persistence following adoptive transfer. Based on these data we have developed a cGMP protocol for expansion of tumor specific T cells for adoptive T cell therapy.

  2. Expansion of intestinal epithelial stem cells during murine development.

    Directory of Open Access Journals (Sweden)

    Jeffrey J Dehmer

    Full Text Available Murine small intestinal crypt development is initiated during the first postnatal week. Soon after formation, overall increases in the number of crypts occurs through a bifurcating process called crypt fission, which is believed to be driven by developmental increases in the number of intestinal stem cells (ISCs. Recent evidence suggests that a heterogeneous population of ISCs exists within the adult intestine. Actively cycling ISCs are labeled by Lgr5, Ascl2 and Olfm4; whereas slowly cycling or quiescent ISC are marked by Bmi1 and mTert. The goal of this study was to correlate the expression of these markers with indirect measures of ISC expansion during development, including quantification of crypt fission and side population (SP sorting. Significant changes were observed in the percent of crypt fission and SP cells consistent with ISC expansion between postnatal day 14 and 21. Quantitative real-time polymerase chain reaction (RT-PCR for the various ISC marker mRNAs demonstrated divergent patterns of expression. mTert surged earliest, during the first week of life as crypts are initially being formed, whereas Lgr5 and Bmi1 peaked on day 14. Olfm4 and Ascl2 had variable expression patterns. To assess the number and location of Lgr5-expressing cells during this period, histologic sections from intestines of Lgr5-EGFP mice were subjected to quantitative analysis. There was attenuated Lgr5-EGFP expression at birth and through the first week of life. Once crypts were formed, the overall number and percent of Lgr5-EGFP positive cells per crypt remain stable throughout development and into adulthood. These data were supported by Lgr5 in situ hybridization in wild-type mice. We conclude that heterogeneous populations of ISCs are expanding as measured by SP sorting and mRNA expression at distinct developmental time points.

  3. Clonal Expansion and Cytotoxicity of TCRVβ Subfamily T Cells Induced by CML and K562 Cells

    Institute of Scientific and Technical Information of China (English)

    YupingZHang; YangqiuLi; ShaohuaChen; LijianYang; GengxinLuo; XueliZhang

    2004-01-01

    OBJECTIVE To investigate the anti-leukemia effect, the distribution and clonal expansion of TCRVβ subfamily T cells in T cells from cord blood and adult peripheral blood induced by CML cells and K562 cells in vitro. METHODS Peripheral blood T cells from one adult donor and 3 cases of cord blood were stimulated with CML cells and K562 cells and further amplified by a suspended T cell-bulk culture,in order to induce CML specific cytotoxic T lymphocytes. The induced T cells were further analyzed for the specific cytotoxicity in CML by LDH assay, the phenotype identification by indirect immunofiuorescence technique and the distribution and clonal expansion of TCRVβ subfamily by using reverse transcriptase-polymerase chain reaction (RT-PCR) and genescan analysis, respectively. RESULTS Oligoclonal and oligoclonal tendency T cells with higher specific cytotoxicity from cord blood and adult peripheral blood could be induced by stimulation with CML cells and K562 cells. CONCLUSIONS Specific cytotoxic T cells for an anti-CML effect could be induced by CML cells and K562 cells .The induced T cells which have the characteristic of specific cytotoxicity against CML cells may come from the clonal expansion of TCRVβ subfamily T cells.

  4. 2003 Plant Cell Walls Gordon Conference

    Energy Technology Data Exchange (ETDEWEB)

    Daniel J. Cosgrove

    2004-09-21

    This conference will address recent progress in many aspects of cell wall biology. Molecular, genetic, and genomic approaches are yielding major advances in our understanding of the composition, synthesis, and architecture of plant cell walls and their dynamics during growth, and are identifying the genes that encode the machinery needed to make their biogenesis possible. This meeting will bring together international scientists from academia, industry and government labs to share the latest breakthroughs and perspectives on polysaccharide biosynthesis, wood formation, wall modification, expansion and interaction with other organisms, and genomic & evolutionary analyses of wall-related genes, as well as to discuss recent ''nanotechnological'' advances that take wall analysis to the level of a single cell.

  5. Animal and plant stem cells concepts, propagation and engineering

    CERN Document Server

    Pavlović, Mirjana

    2017-01-01

    This book provides a multifaceted look into the world of stem cells and explains the similarities and differences between plant and human stem cells. It explores the intersection between animals and plants and explains their cooperative role in bioengineering studies. The book treats both theoretical and practical aspects of stem cell research. It covers the advantages and limitations of many common applications related to stem cells: their sources, categories, engineering of these cells, reprogramming of their functions, and their role as novel cellular therapeutic approach. Written by experts in the field, the book focuses on aspects of stem cells ranging from expansion-propagation to metabolic reprogramming. It introduces the emergence of cancer stem cells and different modalities in targeted cancer stem cell therapies. It is a valuable source of fresh information for academics and researchers, examining molecular mechanisms of animal and plant stem cell regulation and their usage for therapeutic applicati...

  6. Hematopoietic Stem Cells Expansion in Rotating Wall Vessel

    Institute of Scientific and Technical Information of China (English)

    Yang LIU; Tian-Qing LIU; Xiu-Bo FAN; Dan GE; Zhan-Feng CUI; Xue-Hu MA

    2005-01-01

    @@ 1 Introduction Clinical trials have demonstrated that ex vivo expanded hematopoietic stem cells (HSCs) and progenitors offer great promise in reconstituting in vivo hematopoiesis in patients who have undergone intensive chemotherapy.It is therefore necessary to develop a clinical-scale culture system to provide the expanded HSCs and progenitors.Static culture systems such as T-flasks and gas-permeable blood bags are the most widely used culture devices for expanding hematopoietic cells. But they reveal several inherent limitations: ineffective mixing, lack of control options for dissolved oxygen and pH and difficulty in continuous feeding, which restricts the usefulness of static systems. Several advanced bioreactors have been used in the field of HSCs expansion. But hematopoietic cells are extremely sensitive to shear, so cells in bioreactors such as stirred and perfusion culture systems may suffer physical damage. This problem will be improved by applying the rotating wall vessel (RWV) bioreactor in clinic because of its low shear and unique structure. In this research, cord blood (CB) HSCs were expanded by means of a cell-dilution feeding protocol in RWV.

  7. Identification of invasive and expansive plant species based on airborne hyperspectral and ALS data

    Science.gov (United States)

    Szporak-Wasilewska, Sylwia; Kuc, Gabriela; Jóźwiak, Jacek; Demarchi, Luca; Chormański, Jarosław; Marcinkowska-Ochtyra, Adriana; Ochtyra, Adrian; Jarocińska, Anna; Sabat, Anita; Zagajewski, Bogdan; Tokarska-Guzik, Barbara; Bzdęga, Katarzyna; Pasierbiński, Andrzej; Fojcik, Barbara; Jędrzejczyk-Korycińska, Monika; Kopeć, Dominik; Wylazłowska, Justyna; Woziwoda, Beata; Michalska-Hejduk, Dorota; Halladin-Dąbrowska, Anna

    2017-04-01

    The aim of Natura 2000 network is to ensure the long term survival of most valuable and threatened species and habitats in Europe. The encroachment of invasive alien and expansive native plant species is among the most essential threat that can cause significant damage to protected habitats and their biodiversity. The phenomenon requires comprehensive and efficient repeatable solutions that can be applied to various areas in order to assess the impact on habitats. The aim of this study is to investigate of the issue of invasive and expansive plant species as they affect protected areas at a larger scale of Natura 2000 network in Poland. In order to determine the scale of the problem we have been developing methods of identification of invasive and expansive species and then detecting their occurrence and mapping their distribution in selected protected areas within Natura 2000 network using airborne hyperspectral and airborne laser scanning data. The aerial platform used consists of hyperspectral HySpex scanner (451 bands in VNIR and SWIR), Airborne Laser Scanner (FWF) Riegl Lite Mapper and RGB camera. It allowed to obtain simultaneous 1 meter resolution hyperspectral image, 0.1 m resolution orthophotomaps and point cloud data acquired with 7 points/m2. Airborne images were acquired three times per year during growing season to account for plant seasonal change (in May/June, July/August and September/October 2016). The hyperspectral images were radiometrically, geometrically and atmospherically corrected. Atmospheric correction was performed and validated using ASD FieldSpec 4 measurements. ALS point cloud data were used to generate several different topographic, vegetation and intensity products with 1 m spatial resolution. Acquired data (both hyperspectral and ALS) were used to test different classification methods including Mixture Tuned Matched Filtering (MTMF), Spectral Angle Mapper (SAM), Random Forest (RF), Support Vector Machines (SVM), among others

  8. Embryonic stem cells conditioned medium enhances Wharton’s jelly-derived mesenchymal stem cells expansion under hypoxic condition

    OpenAIRE

    Prasajak, Patcharee; Rattananinsruang, Piyaporn; Chotinantakul, Kamonnaree; Dechsukhum, Chavaboon; Leeanansaksiri, Wilairat

    2014-01-01

    Mesenchymal stem cells (MSCs) are accepted as a promising tool for therapeutic purposes. However, low proliferation and early senescence are still main obstacles of MSCs expansion for using as cell-based therapy. Thus, clinical scale of cell expansion is needed to obtain a large number of cells serving for further applications. In this study, we investigated the value of embryonic stem cells conditioned medium (ESCM) for in vitro expansion of Wharton’s jelly-derived mesenchymal stem cells (WJ...

  9. Expansion of CD25+ Innate Lymphoid Cells Reduces Atherosclerosis

    Science.gov (United States)

    Engelbertsen, Daniel; Foks, Amanda C.; Alberts-Grill, Noah; Kuperwaser, Felicia; Chen, Tao; Lederer, James A.; Jarolim, Petr; Grabie, Nir; Lichtman, Andrew H.

    2015-01-01

    Objective Innate lymphoid cells (ILCs) are a newly discovered subset of immune cells that promote tissue homeostasis and protect against pathogens. ILCs produce cytokines also produced by T lymphocytes that have been shown to affect atherosclerosis, but the influence of ILCs on atherosclerosis has not been explored. Approach and Results We demonstrate that CD25+ ILCs that produce type 2 cytokines (ILC2s) are present in the aorta of atherosclerotic immunodeficient ldlr−/−rag1−/− mice. To investigate the role of ILCs in atherosclerosis, ldlr−/−rag1−/− mice were concurrently fed an atherogenic diet and treated with either ILC-depleting anti-CD90.2 antibodies or with IL-2/anti-IL-2 complexes that expand CD25+ ILCs. Lesion development was not affected by anti-CD90.2 treatment, but was reduced in IL-2/anti-IL-2 -treated mice. These IL-2 treated mice had reduced VLDL cholesterol and increased triglycerides compared to controls and reduced apolipoprotein B100 gene expression in the liver. IL-2/anti-IL-2 treatment caused expansion of ILC2s in aorta and other tissues, elevated levels of IL-5, systemic eosinophila and hepatic eosinophilic inflammation. Blockade of IL-5 reversed the IL-2-complex-induced eosinophilia but did not change lesion size. Conclusions This study demonstrates that expansion of CD25-expressing ILCs by IL-2/anti-IL-2 complexes leads to a reduction in VLDL cholesterol and atherosclerosis. Global depletion of ILCs by anti-CD90.2 did not significantly affect lesion size indicating that different ILC subsets may have divergent effects on atherosclerosis. PMID:26494229

  10. A novel monoclonal antibody of human stem cell factor inhibits umbilical cord blood stem cell ex vivo expansion

    Directory of Open Access Journals (Sweden)

    Fan Jie

    2012-12-01

    Full Text Available Abstract Stem cell factor (SCF activates hematopoietic stem cell (HSC self-renewal and is being used to stimulate the ex vivo expansion of HSCs. The mechanism by which SCF supports expansion of HSCs remains poorly understood. In cord blood ex vivo expansion assays, a newly produced anti-SCF monoclonal antibody (clone 23C8 was found to significantly inhibit the expansion of CD34+ cells. This antibody appears to bind directly to a part of SCF that is critical for biological activity toward expansion of CD34+ cells, which is located in the first 104 amino acids from the NH2-terminus.

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

    Science.gov (United States)

    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.

  12. Microstructure of a Planting Material Consisting of Nutrition-Expansive Perlitic-Cement Composites

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ji-ru; LIU Zu-de

    2003-01-01

    An ecotypic revetment material consisting of nutrition-expansive perlitic-cement composites is introduced. This planting material can combine vegetation recovery with slope protection. The XRD, SEM and image analysis techniques were used to study its composition and microstructure. Its strength was measured by an electro-hydraulic servo-controlled testing machine. The results show the unconfined compressive strength is about 393.6 kPa, and the average elastic modulus is about 47.0 MPa. The quartz, felspar, chlorite and calcite are the main non-clay minerals in the planting material. Its particles are mainly spherical,and the range of the equivalent diameter is 1.83 to 15.96 μm. The results also show the planting material contains a large amount of micro non-capillary and capillary pores, and has a microstructure characteristic of honeycomb and coralline. CSH gel produced by hydration of cement increases the strength and water stability of the particles. The anisotropy and slight orientation of the particles increase the void cross-section area,providing an explanation of the high permeability for the planting material.The better porosity of the planting material is apt tokeep moisture and nutriment, provides oxygen for plant root breathing, and aids to exhaust the carbon dioxide by means of exchanging with atmosphere, hence it can facilitate vegetation.

  13. Range expansion induces variation in a behavioural trait in an ant-plant mutualism

    Science.gov (United States)

    Vittecoq, Marion; Djiéto-Lordon, Champlain; McKey, Doyle; Blatrix, Rumsaïs

    2012-01-01

    Climate oscillations produce dramatic changes in species distribution, even in the tropics. The ant-plant Leonardoxa africana africana hosts and feeds the ant Petalomyrmex phylax, which provides protection against herbivores in return. Both partners of this symbiosis present a recent southward range expansion. To test whether the higher investment in sexuals (and thus lower investment in protective workers) previously documented on the colonization front is compensated by a more effective protective behaviour, we compared ant behavioural investment in plant defence between two populations, one in the core of the range and one on the colonization front. We induced ant patrolling activity by artificially damaging leaflets and measured this activity by counting patrolling ants and calculating the increase relative to constitutive patrolling activity measured on control (undamaged) leaflets. Contrary to our expectation, ant behavioural investment in plant defence was lower on the colonization front. Thus, production of fewer workers is not compensated by more protective behaviour of each. Instead, both traits contribute to a phenotype that is less mutualistic as a whole. By favouring increased allocation to dispersal, range expansion can shape ant behavioural traits and potentially the outcome of mutualism.

  14. Expansion of Endothelial Progenitor Cells in High Density Dot Culture of Rat Bone Marrow Cells

    Science.gov (United States)

    Wang, Ling; Kretlow, James D.; Zhou, Guangdong; Cao, Yilin; Liu, Wei; Zhang, Wen Jie

    2014-01-01

    In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2×105 cells/cm2) by seeding total 9×105 cells into six high density dots or cultured in regular density (1.6×104 cells/cm2) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells. PMID:25254487

  15. Plant-soil interactions in the expansion and native range of a poleward shifting plant species

    NARCIS (Netherlands)

    Grunsven, van R.H.A.; Putten, van der W.H.; Bezemer, T.M.; Berendse, F.; Veenendaal, E.M.

    2010-01-01

    Climate warming causes range shifts of many species toward higher latitudes and altitudes. However, range shifts of host species do not necessarily proceed at the same rates as those of their enemies and symbionts. Here, we examined how a range shifting plant species performs in soil from its

  16. Fatal asbestosis 50 years after brief high intensity exposure in a vermiculite expansion plant.

    Science.gov (United States)

    Wright, Robert S; Abraham, Jerrold L; Harber, Philip; Burnett, Bryan R; Morris, Peter; West, Phil

    2002-04-15

    The authors report the case of a 65-year-old accountant whose only asbestos exposure was during a summer job 50 years earlier in a California vermiculite expansion plant. Vermiculite is a silicate material that is useful in building and agriculture as a filler and insulating agent. He developed extensive fibrocalcific pleural plaques and end-stage pulmonary fibrosis, with rapidly progressive respiratory failure. Careful occupational and environmental history revealed no other source of asbestos exposure, and the initial clinical diagnosis was idiopathic pulmonary fibrosis; open lung biopsy shortly before his death confirmed asbestosis. Electron microscopic lung fiber burden analysis revealed over 8,000,000 asbestos fibers per gram dry lung, 68% of which were tremolite asbestos. Additional asbestiform fibers of composition not matching any of the standard asbestos varieties were also present at over 5,000,000 fibers per gram dry lung. Comparison analysis of a sample of Libby, Montana, vermiculite showed a similar mix of asbestiform fibers including tremolite asbestos. This case analysis raises several concerns: risks of vermiculite induced disease among former workers of the more than 200 expansion plants throughout the United States; health effects of brief but very high-intensity exposures to asbestos; and possible health effects in end-users of consumer products containing vermiculite.

  17. Biased competition between Lgr5 intestinal stem cells driven by oncogenic mutation induces clonal expansion

    NARCIS (Netherlands)

    Snippert, Hugo J; Schepers, Arnout G; van Es, Johan H; Simons, Benjamin D; Clevers, Hans

    2014-01-01

    The concept of 'field cancerization' describes the clonal expansion of genetically altered, but morphologically normal cells that predisposes a tissue to cancer development. Here, we demonstrate that biased stem cell competition in the mouse small intestine can initiate the expansion of such clones.

  18. The Untapped Potential Of Plant Thin Cell Layers

    Directory of Open Access Journals (Sweden)

    Teixeira da Silva Jaime

    2015-12-01

    Full Text Available Thin cell layers (TCLs, which contain a small number of cells or tissues, are explants excised from different organs (stems, leaves, roots, inflorescences, flowers, cotyledons, hypocotyls/epicotyls, and embryos. After almost 45 years of research, this culture system has been used for several monocotyledonous and dicotyledonous plants of commercial importance, and for model plants. The limited amount of cells in a TCL is of paramount importance because marker molecules/genes of differentiation can be easily localized in situ in the target/responsive cells. Thus, the use of TCLs has allowed, and continues to allow, for the expansion of knowledge in plant research in a practical and applied manner into the fields of tissue culture and micropropagation, cell and organ genetics, molecular biology, biochemistry, and development. Starting from a brief historical background, the actual and potential uses of the TCL system are briefly reviewed.

  19. Culture expansion of adipose derived stromal cells. A closed automated Quantum Cell Expansion System compared with manual flask-based culture

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Follin, Bjarke; Juhl, Morten

    2016-01-01

    ) over two passages in the automated and functionally closed Quantum Cell Expansion System (Quantum system) is compared with traditional manual cultivation. Methods: Stromal vascular fraction was isolated from abdominal fat, suspended in α-MEM supplemented with 10% Fetal Bovine Serum and seeded......Background: Adipose derived stromal cells (ASCs) are a rich and convenient source of cells for clinical regenerative therapeutic approaches. However, applications of ASCs often require cell expansion to reach the needed dose. In this study, cultivation of ASCs from stromal vascular fraction (SVF...... enhances ASC expansion rate and yield significantly relative to manual processing in T-flasks, while maintaining the purity and quality essential to safe and robust cell production. Notably, the use of the Quantum system entails significantly reduced working hours and thereby costs....

  20. Fibroblastic reticular cells from lymph nodes attenuate T cell expansion by producing nitric oxide.

    Directory of Open Access Journals (Sweden)

    Stefanie Siegert

    Full Text Available Adaptive immune responses are initiated when T cells encounter antigen on dendritic cells (DC in T zones of secondary lymphoid organs. T zones contain a 3-dimensional scaffold of fibroblastic reticular cells (FRC but currently it is unclear how FRC influence T cell activation. Here we report that FRC lines and ex vivo FRC inhibit T cell proliferation but not differentiation. FRC share this feature with fibroblasts from non-lymphoid tissues as well as mesenchymal stromal cells. We identified FRC as strong source of nitric oxide (NO thereby directly dampening T cell expansion as well as reducing the T cell priming capacity of DC. The expression of inducible nitric oxide synthase (iNOS was up-regulated in a subset of FRC by both DC-signals as well as interferon-γ produced by primed CD8+ T cells. Importantly, iNOS expression was induced during viral infection in vivo in both LN FRC and DC. As a consequence, the primary T cell response was found to be exaggerated in Inos(-/- mice. Our findings highlight that in addition to their established positive roles in T cell responses FRC and DC cooperate in a negative feedback loop to attenuate T cell expansion during acute inflammation.

  1. Nuclear lamina in plant cells

    Institute of Scientific and Technical Information of China (English)

    汪健; 杨澄; 翟中和

    1996-01-01

    By using selective extraction and diethylene glycol distearate (DGD) embedment and embedment-free electron microscopy, the nuclear lamina was demonstrated in carrot and Ginkgo male generative cells. Western blotting revealed that the nuclear lamina was composed of A-type and B-type lamins which contained at least 66-ku and 84-ku or 66-ku and 86-ku polypeptides, respectively. These lamin proteins were localized at the nudear periphery as shown by immunogold-labelling. In situ hybridization for light microscope and electron microscope showed that plant cells have the homologous sequences of animal lamin cDNA. The sorting site of lamin mRNA is mainly distributed in the cytoplasm near the nudear envelope. The data have verified that there indeed exists nudear lamina in plant cells.

  2. Bimodal ex vivo expansion of T cells from patients with head and neck squamous cell carcinoma

    DEFF Research Database (Denmark)

    Junker, Niels; Andersen, Mads Hald; Wenandy, Lynn

    2011-01-01

    Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has proven effective in metastatic melanoma and should therefore be explored in other types of cancer. The aim of this study was to examine the feasibility of potentially expanding clinically relevant quantities of tumor-specific T-cell...... cultures from TIL from patients with head and neck squamous cell carcinoma (HNSCC) using a more rapid expansion procedure compared with previous HNSCC studies....

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

    Science.gov (United States)

    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

  4. The mitochondrial genome of Malus domestica and the import-driven hypothesis of mitochondrial genome expansion in seed plants.

    Science.gov (United States)

    Goremykin, Vadim V; Lockhart, Peter J; Viola, Roberto; Velasco, Riccardo

    2012-08-01

    Mitochondrial genomes of spermatophytes are the largest of all organellar genomes. Their large size has been attributed to various factors; however, the relative contribution of these factors to mitochondrial DNA (mtDNA) expansion remains undetermined. We estimated their relative contribution in Malus domestica (apple). The mitochondrial genome of apple has a size of 396 947 bp and a one to nine ratio of coding to non-coding DNA, close to the corresponding average values for angiosperms. We determined that 71.5% of the apple mtDNA sequence was highly similar to sequences of its nuclear DNA. Using nuclear gene exons, nuclear transposable elements and chloroplast DNA as markers of promiscuous DNA content in mtDNA, we estimated that approximately 20% of the apple mtDNA consisted of DNA sequences imported from other cell compartments, mostly from the nucleus. Similar marker-based estimates of promiscuous DNA content in the mitochondrial genomes of other species ranged between 21.2 and 25.3% of the total mtDNA length for grape, between 23.1 and 38.6% for rice, and between 47.1 and 78.4% for maize. All these estimates are conservative, because they underestimate the import of non-functional DNA. We propose that the import of promiscuous DNA is a core mechanism for mtDNA size expansion in seed plants. In apple, maize and grape this mechanism contributed far more to genome expansion than did homologous recombination. In rice the estimated contribution of both mechanisms was found to be similar.

  5. Plant Cell Wall Matrix Polysaccharide Biosynthesis

    Institute of Scientific and Technical Information of China (English)

    Ajay Pal S. Sandhu; Gursharn S. Randhawa; Kanwarpal S. Dhugga

    2009-01-01

    The wall of an expanding plant cell consists primarily of cellulose microfibrils embedded in a matrix of hemi-cellulosic and pectic polysaccharides along with small amounts of structural and enzymatic proteins. Matrix polysacchar-ides are synthesized in the Golgi and exported to the cell wall by exocytosis, where they intercalate among cellulose microfibrUs, which are made at the plasma membrane and directly deposited into the cell wall. Involvement of Golgi glucan synthesis in auxin-induced cell expansion has long been recognized; however, only recently have the genes corresponding to glucan synthases been identified. Biochemical purification was unsuccessful because of the labile nature and very low abundance of these enzymes. Mutational genetics also proved fruitless. Expression of candidate genes identified through gene expression profiling or comparative genomics in heterologous systems followed by functional characterization has been relatively successful. Several genes from the cellulose synthase-like (Cs/) family have been found to be involved in the synthesis of various hemicellulosic glycans. The usefulness of this approach, however, is limited to those enzymes that probably do not form complexes consisting of unrelated proteins. Nonconventional approaches will continue to incre-mentally unravel the mechanisms of Golgi polysaccharide biosynthesis.

  6. Ferns are less dependent on passive dilution by cell expansion to coordinate leaf vein and stomatal spacing than angiosperms.

    Science.gov (United States)

    Carins Murphy, Madeline R; Jordan, Gregory J; Brodribb, Timothy J

    2017-01-01

    Producing leaves with closely spaced veins is a key innovation linked to high rates of photosynthesis in angiosperms. A close geometric link between veins and stomata in angiosperms ensures that investment in enhanced venous water transport provides the strongest net carbon return to the plant. This link is underpinned by "passive dilution" via expansion of surrounding cells. However, it is not known whether this 'passive dilution' mechanism is present in plant lineages other than angiosperms and is another key feature of the angiosperms' evolutionary success. Consequently, we sought to determine whether the 'passive dilution' mechanism is; (i) exclusive to the angiosperms, (ii) a conserved mechanism that evolved in the common ancestor of ferns and angiosperms, or (iii) has evolved continuously over time. To do this we first we assessed the plasticity of vein and stomatal density and epidermal cell size in ferns in response to light environment. We then compared the relationships between these traits found among ferns with modelled relationships that assume vein and stomatal density respond passively to epidermal cell expansion, and with those previously observed in angiosperms. Vein density, stomatal density and epidermal cell size were linked in ferns with remarkably similar relationships to those observed in angiosperms, except that fern leaves had fewer veins per stomata. However, plasticity was limited in ferns and stomatal spacing was dependent on active stomatal differentiation as well as passive cell expansion. Thus, ferns (like angiosperms) appear to coordinate vein and stomatal density with epidermal cell expansion to some extent to maintain a constant ratio between veins and stomata in the leaf. The different general relationships between vein density and stomatal density in ferns and angiosperms suggests the groups have different optimum balances between the production of vein tissue dedicated to water supply and stomatal tissue for gas exchange.

  7. Optimization of SCF feeding regimen for ex vivo expansion of cord blood hematopoietic stem cells.

    Science.gov (United States)

    Du, Zheng; Cai, Haibo; Ye, Zhaoyang; Tan, Wen-Song

    2012-12-15

    Stem cell factor (SCF) plays important roles in ex vivo expansion of hematopoietic stem cells (HSCs). In this study, the effects of dose and feeding time of SCF on ex vivo expansion of CD34(+) cells were investigated in serum-free medium supplemented with a cytokine cocktail composed of SCF, thrombopoietin (TPO) and flt3-ligand (FL). Among the four tested doses (0, 5, 50 and 500ng/mL), a SCF dose of 50ng/mL was demonstrated to be most favorable for ex vivo expansion of CD34(+) cells, which resulted in 34.22±10.80 and 8.89±1.25 folds of expansion regarding total cells and CD34(+) cells, respectively. Meanwhile, the specific growth rate of cells, the consumption rate of SCF and the percentage of CD34(+)c-kit(+) cells during the 21-day culture process were analyzed. The results indicated that initial 4-day period was a critical stage for SCF functioning on CD34(+) cells during ex vivo expansion. Based on this, a modified SCF feeding regimen was proposed, in which SCF (50ng/mL) was only supplemented on day 0 in the cytokine cocktail and cells were then fed with TPO and FL till the end of culture. It was found that this SCF feeding regimen could expand CD34(+) cells efficiently, thus providing a cost-effect expansion protocol for HSCs.

  8. Diversity begets diversity: host expansions and the diversification of plant-feeding insects

    Directory of Open Access Journals (Sweden)

    Nylin Sören

    2006-01-01

    Full Text Available Abstract Background Plant-feeding insects make up a large part of earth's total biodiversity. While it has been shown that herbivory has repeatedly led to increased diversification rates in insects, there has been no compelling explanation for how plant-feeding has promoted speciation rates. There is a growing awareness that ecological factors can lead to rapid diversification and, as one of the most prominent features of most insect-plant interactions, specialization onto a diverse resource has often been assumed to be the main process behind this diversification. However, specialization is mainly a pruning process, and is not able to actually generate diversity by itself. Here we investigate the role of host colonizations in generating insect diversity, by testing if insect speciation rate is correlated with resource diversity. Results By applying a variant of independent contrast analysis, specially tailored for use on questions of species richness (MacroCAIC, we show that species richness is strongly correlated with diversity of host use in the butterfly family Nymphalidae. Furthermore, by comparing the results from reciprocal sister group selection, where sister groups were selected either on the basis of diversity of host use or species richness, we find that it is likely that diversity of host use is driving species richness, rather than vice versa. Conclusion We conclude that resource diversity is correlated with species richness in the Nymphalidae and suggest a scenario based on recurring oscillations between host expansions – the incorporation of new plants into the repertoire – and specialization, as an important driving force behind the diversification of plant-feeding insects.

  9. Expansion of plants with Crassulacean Acid Metabolism under global environment change

    Science.gov (United States)

    Yu, K.; D'Odorico, P.; Collins, S. L.; Carr, D.

    2016-12-01

    contrast to the case of the strong competitive effect from B. mollis. Overall, these research improves understanding of mechanisms underlying the expansion of CAM plants with important implications on shifts in dryland vegetation composition, bioenergy production, food security, and adaptation to global environment change.

  10. Mesenchymal Stromal Cell Phenotype is not Influenced by Confluence during Culture Expansion

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Hansen, Susanne Kofoed; Hansen, Louise

    2013-01-01

    BACKGROUND: Accumulating preclinical and clinical evidence indicates that human mesenchymal stromal cells (MSCs) are good candidates for cell therapy. For clinical applications of MSCs extensive in vitro expansion is required to obtain an adequate number of cells. It is evident that the pursuit...... for cell quantity must not affect quality, but it is also a fact that in vitro culture conditions affect MSC phenotype. One possible variable is the degree of cell confluence during expansion. METHODS: We investigate the influence of cell density on homogeneity and differentiation during culture expansion...... of un-stimulated MSCs isolated from the bone marrow in DMEM and fetal bovine serum (FBS). MSC morphology, phenotype and differentiation were investigated weekly during 5 weeks culture expansion using electron microscopy, flow cytometry, immunocytochemistry, qualitative RT-PCR and quantitative Q...

  11. Coupling cell proliferation and development in plants.

    Science.gov (United States)

    Gutierrez, Crisanto

    2005-06-01

    Plant genome projects have revealed that both the cell-cycle components and the overall cell-cycle architecture are highly evolutionarily conserved. In addition to the temporal and spatial regulation of cell-cycle progression in individual cells, multicellularity has imposed extra layers of complexity that impinge on the balance of cell proliferation and growth, differentiation and organogenesis. In contrast to animals, organogenesis in plants is a postembryonic and continuous process. Differentiated plant cells can revert to a pluripotent state, proliferate and transdifferentiate. This unique potential is strikingly illustrated by the ability of certain cells to produce a mass of undifferentiated cells or a fully totipotent embryo, which can regenerate mature plants. Conversely, plant cells are highly resistant to oncogenic transformation. This review discusses the role that cell-cycle regulators may have at the interface between cell division and differentiation, and in the context of the high plasticity of plant cells.

  12. Thermal expansion of slag and fly ash from coal gasification in IGCC power plant

    Energy Technology Data Exchange (ETDEWEB)

    M. Aineto; A. Acosta; J.M.A. Rincon; M. Romero [University of Castilla La Mancha, Ciudad Real (Spain). Laboratory of Applied Mineralogy

    2006-11-15

    Integrated gasification in combined cycle (IGCC) is an electrical power generation system which is characterized to be a clean coal technology different than conventional process in combustible treatment. IGCC process gives rise to inorganic solid wastes in the form of vitreous slag and fly ashes with singular thermal properties. The gasification of the fuel takes place at high temperature and pressure in reducing atmosphere. Under that conditions, gases such as H{sub 2}, N{sub 2} or CO, which are the main components of the gas mixture in the gasifier, show a high solubility in the melt and during the cooling remain enclosed in the vitreous slag. When these wastes are afterward thermal treated in oxidizing conditions, two phenomena occur. The development of a crystalline phase by devitrification of the glassy matrix and the releasing of the enclosed gas, which starts at temperatures nearly to the softening point. At higher temperatures the bubbles with increasing kinetic energy tend to ascend with difficulty through the viscous liquid phase and promotes an expansive reaction, giving rise to a foam glass-ceramic product. This paper has been focused on the study of thermal expansion in slag and fly ash samples from the ELCOGAS IGCC power plant located in Puertollano (Spain). 18 refs., 11 figs., 1 tab.

  13. Stem cells: a plant biology perspective

    NARCIS (Netherlands)

    Scheres, B.J.G.|info:eu-repo/dai/nl/07493662X

    2005-01-01

    A recent meeting at the Juan March Foundation in Madrid, Spain brought together plant biologists to discuss the characteristics of plant stem cells that are unique and those that are shared by stem cells from the animal kingdom

  14. Stem cells: a plant biology perspective

    NARCIS (Netherlands)

    Scheres, B.J.G.

    2005-01-01

    A recent meeting at the Juan March Foundation in Madrid, Spain brought together plant biologists to discuss the characteristics of plant stem cells that are unique and those that are shared by stem cells from the animal kingdom

  15. Episodic Neoglacial Cryosphere Expansion Reconstructed from 14C Ages of Ice-Entombed Plants on Svalbard

    Science.gov (United States)

    Miller, G. H.; Lehman, S.; Landvik, J. Y.

    2015-12-01

    The Northern Hemisphere cryosphere's response to the monotonic decline in summer insolation through the Holocene has been irregular expansion, interrupted by periods of retreat, culminating in the Little Ice Age, when most glaciers attained their maximum late Holocene dimensions. This non-linear response to near-linear forcing implies other factors modulate the radiative effects of the regular insolation decline. Understanding how the Earth system accomplishes this modulation is relevant to the development of reliable models for future climate change. Accurately dating the onset of persistent summer coolings across the Arctic is a first step toward developing this understanding. Here we report 52 precise radiocarbon dates on rooted plants emerging from beneath receding glaciers on Svalbard that define times when colder summers led to snowline depression and an expanded cryosphere. The earliest persistent depression of snowline documented by our dates occurred between 4.0 and 3.4 ka, but with little additional summer cooling until early in the first millennium AD. Episodes of subsequent summer cooling were centered on 240-340, 410-540 and 670-770 AD, followed by additional cooling between 1000 and 1230 AD, and between 1300 and 1470 AD. Cooling that occurred after 1470 AD, includes of the Little Ice Age when the Svalbard cryosphere reached its maximum Neoglacial dimensions. We suggest that in addition to insolation forcing, irregular reductions in the strength of the North Atlantic Current and expansions of Arctic Ocean sea ice were dominant factors that led to episodic snowline depression over Svalbard, but the extent to which these changes are linked to radiative forcing from volcanism or solar irradiance as opposed to unforced variability remains unclear. The widespread exposure of entombed plants dating from the first millennium AD suggests that Svalbard's average summer temperatures of the past century now exceed those of any century since at least 700 AD

  16. Expansion and photosynthetic rate of leaves of soybean plants during onset of and recovery from nitrogen stress

    Science.gov (United States)

    Tolley-Henry, L.; Raper, C. D. Jr; Raper CD, J. r. (Principal Investigator)

    1986-01-01

    This study reports on the effects of nitrogen stress and restoration of nitrogen availability after a period of stress on expansion and photosynthetic rate of soybean leaves of differing maturity. We hypothesized that nitrogen resupply would lead to additional accumulation of reduced nitrogen in the leaves and, ultimately, resumption of leaf initiation and expansion and photosynthetic activity. In continuously nitrogen-stressed plants, expansion of middle trifoliolates of main-stem trifoliates and leaf area at full expansion were severely restricted. Leaves showing the greatest effects were initiated after removal of nitrogen. When the reduced nitrogen concentration in mature leaves of continuously stressed plants fell below 9 mg dm-2, the photosynthetic rate per unit leaf decreased rapidly, reaching a minimum of ca. 6-8 mg dm-2 h-1. The older mature leaves tended to abscise at this point, while the youngest leaves remained on the plant and continued to photosynthesize slowly. When nitrogen was resupplied, leaf expansion and final leaf area increased. Leaf initiation was also stimulated as reduced nitrogen levels rose in the leaves. Photosynthetic rates of the oldest and youngest pair of mature leaves returned to values comparable to those of similar-aged leaves of nonstressed soybean plants.

  17. Regio- and stereoselectivities in plant cell biotransformation

    Energy Technology Data Exchange (ETDEWEB)

    Hamada, H. [Okayama Univ. of Science (Japan)

    1995-12-01

    The ability of plant cultured cells to convert foreign substrates into more useful substances is of considerable interest. Therefore I have studied biotransformation of foreign substrate by plant cell suspension cultures. In this presentation, I report regio- and stereoselectivities in biotransformation of steroids and indole alkaloids and taxol by plant (tobacco, periwinkle, moss, orchid) cell suspension cultures.

  18. Ex vivo expansions and transplantations of mouse bone marrow-derived hematopoietic stem/progenitor cells

    Institute of Scientific and Technical Information of China (English)

    WANG Jin-fu(王金福); WU Yi-fan(吴亦凡); HARRINTONG Jenny; McNIECE Ian K.

    2004-01-01

    To examine the effects of co-culture with bone marrow mesenchymal stem cells on expansion of hematopoietic stem/progenitor cells and the capacities of rapid neutrophil engraftment and hematopoietic reconstitution of the expanded cells, we expanded mononuclear cells (MNCs) and CD34+/c-kit+ cells from mouse bone marrow and transplanted the expanded cells into the irradiated mice. MNCs were isolated from mouse bone marrow and CD34+/c-kit+ cells were selected from MNCs by using MoFlo Cell Sorter. MNCs and CD34+/c-kit+ cells were co-cultured with mouse bone marrow-derived mesenchymal stem cells (MSCs) under a two-step expansion. The expanded cells were then transplanted into sublethally irradiated BDF1 mice. Results showed that the co-culture with MSCs resulted in expansions of median total nucleated cells,CD34+ cells, GM-CFC and HPP-CFC respectively by 10.8-, 4.8-, 65.9- and 38.8-fold for the mononuclear cell culture, and respectively by 76.1-, 2.9-, 71.7- and 51.8-fold for the CD34+/c-kit+ cell culture. The expanded cells could rapidly engraft in the sublethally irradiated mice and reconstitute their hematopoiesis. Co-cultures with MSCs in conjunction with two-step expansion increased expansions of total nucleated cells, GM-CFC and HPP-CFC, which led us to conclude MSCs may create favorable environment for expansions of hematopoietic stem/progenitor cells. The availability of increased numbers of expanded cells by the co-culture with MSCs may result in more rapid engraftment ofneutrophils following infusion to transplant recipients.

  19. Regulation of Water in Plant Cells

    Science.gov (United States)

    Kowles, Richard V.

    2010-01-01

    Cell water relationships are important topics to be included in cell biology courses. Differences exist in the control of water relationships in plant cells relative to control in animal cells. One important reason for these differences is that turgor pressure is a consideration in plant cells. Diffusion and osmosis are the underlying factors…

  20. CELL EXPANSION-DEPENDENT INFLAMMATORY AND METABOLIC PROFILE OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS

    Directory of Open Access Journals (Sweden)

    PATRICIA PRIETO

    2016-11-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

  2. Ex vivo expansions and transplantations of mouse bone marrow-derived hematopoietic stem/progenitor cells

    Institute of Scientific and Technical Information of China (English)

    王金福; 吴亦凡; HARRINTONGJenny; McNIECEIanK.

    2004-01-01

    To examine the effects of co-culture with bone marrow mesenchymal stem cells on expansion of hematopoietic tem/progenitor cells and the capacities of rapid neutrophil engraftment and hematopoietic reconstitution of the expanded ells, we expanded mononuclear cells (MNCs) and CD34+/c-kit+ cells from mouse bone marrow and transplanted the expanded cells into the irradiated mice. MNCs were isolated from mouse bone marrow and CD34+/c-kit+ cells were selected from MNCs by using MoFlo Cell Sorter. MNCs and CD34+/c-kit+ cells were co-cultured with mouse bone marrow-derived mesenchymal stem cells (MSCs) under a two-step expansion. The expanded cells were then transplanted into sublethally irradiated BDF 1 mice. Results showed that the co-culture with MSCs resulted in expansions of median total nucleated cells, CD34+ cells, GM-CFC and HPP-CFC respectively by 10.8-, 4.8-, 65.9- and 38.8-fold for the mononuclear cell culture, and respectively by 76.1-, 2.9-, 71.7- and 51.8-fold for the CD34+/c-kit+ cell culture. The expanded cells could rapidly engraft in the sublethally irradiated mice and reconstitute their hematopoiesis. Co-cultures with MSCs in conjunction with two-step expansion increased expansions of total nucleated cells, GM-CFC and HPP-CFC, which led us to conclude MSCs may create favorable environment for expansions of hematopoietic stem/progenitor cells. The availability of increased numbers of expanded ceils by the co-culture with MSCs may result in more rapid engraftment ofneutrophils following infusion to transplant recipients.

  3. Expansion and activation of natural killer cells from PBMC for immunotherapy of hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Bao-Gang Peng; Li-Jian Liang; Qiang He; Jie-Fu Huang; Ming-De Lu

    2004-01-01

    AIM: To induce efficient expansion of natural killer (NK) cells from peripheral blood mononuclear cells (PBMCs) using a culture of anchorage-dependent Wilms tumor cell lines, and to provide a reliable supply for adoptive immunotherapy of hepatocellular carcinoma.METHODS: Culture expansion of NK cells was achieved using PBMCs cultured with Wilms tumor cells. Cytotoxicity was measured using a standard 51Cr release assay and crystal violet staining technique. The proportions of CD3+, CD4+, CD8+,CD16+, and CD56+ cells were determined by flow cytometry.RESULTS: After PBMCs from healthy donors and hepatocellular carcinoma (HCC) were cultured with irradiated HFWT cells for 10-21 d, CD56+ CD16+ cells shared more than 50% of the cell population, and more than 80% of fresh HFWT cells were killed at an effector/target ratio of 2 over 24 h. NK-enriched lymphocyte population from HCC patients killed HCC-1 and 2 cells with sensitivities comparable to fresh TKB-17RGB cells. HCC cells proliferated 196-fold with the irradiated HFWT cells at 18 d. Stimulation by HFWT cells required intimate cell-cell interaction with PBMC. However, neither the soluble factors released from HFWT cells nor the fixed HFWT cells were effective for NK expansion. The lymphocytes expanded with IL-2 killed fresh HFWT target cells more effectively than the lymphocytes expanded with the 4-cytokine cocktail (IL-1 β, IL-2, IL-4 and IL-6). IL-2 was the sole cytokine required for NK expansion.CONCLUSION: Wilms tumor is sensitive to human NK cells and is highly efficient for selective expansion of NK cells from PBMCs.

  4. Plant and algal cell walls: diversity and functionality.

    Science.gov (United States)

    Popper, Zoë A; Ralet, Marie-Christine; Domozych, David S

    2014-10-01

    Although plants and many algae (e.g. the Phaeophyceae, brown, and Rhodophyceae, red) are only very distantly related they are united in their possession of carbohydrate-rich cell walls, which are of integral importance being involved in many physiological processes. Furthermore,wall components have applications within food, fuel, pharmaceuticals, fibres (e.g. for textiles and paper) and building materials and have long been an active topic of research. As shown in the 27 papers in this Special Issue, as the major deposit of photosynthetically fixed carbon, and therefore energy investment, cell walls are of undisputed importance to the organisms that possess them, the photosynthetic eukaryotes ( plants and algae). The complexities of cell wall components along with their interactions with the biotic and abiotic environment are becoming increasingly revealed. The importance of plant and algal cell walls and their individual components to the function and survival of the organism, and for a number of industrial applications, are illustrated by the breadth of topics covered in this issue, which includes papers concentrating on various plants and algae, developmental stages, organs, cell wall components, and techniques. Although we acknowledge that there are many alternative ways in which the papers could be categorized (and many would fit within several topics), we have organized them as follows: (1) cell wall biosynthesis and remodelling, (2) cell wall diversity, and (3) application of new technologies to cell walls. Finally, we will consider future directions within plant cell wall research. Expansion of the industrial uses of cell walls and potentially novel uses of cell wall components are both avenues likely to direct future research activities. Fundamentally, it is the continued progression from characterization (structure, metabolism, properties and localization) of individual cell wall components through to defining their roles in almost every aspect of plant

  5. Human disturbance and upward expansion of plants in a warming climate

    Science.gov (United States)

    Dainese, Matteo; Aikio, Sami; Hulme, Philip E.; Bertolli, Alessio; Prosser, Filippo; Marini, Lorenzo

    2017-08-01

    Climate change is expected to trigger an upward expansion of plants in mountain regions and, although there is strong evidence that many native species have already shifted their distributions to higher elevations, little is known regarding how fast non-native species might respond to climate change. By analysing 131,394 occurrence records of 1,334 plant species collected over 20 years in the European Alps, we found that non-natives are spreading upwards approximately twice as fast as natives. Whereas the spread of natives was enhanced by traits favouring longer dispersal distances, this was not the case for non-natives. This was due to the non-native species pool already being strongly biased towards species that had traits facilitating spread. A large proportion of native and non-native species seemed to be able to spread upwards faster than the current velocity of climate change. In particular, long-distance dispersal events and proximity to roads proved to be key drivers for the observed rapid spread. Our findings highlight that invasions by non-native species into native alpine communities are a potentially significant additional pressure on these vulnerable ecosystems that are already likely to suffer dramatic vegetation changes with ongoing warming and increasing human activity in mountain regions.

  6. Expansive effects of aorta-gonad-mesonephros-derived stromal cells on hematopoietic stem cells from embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    FU Jin-rong; LIU Wen-li; ZHOU Yu-feng; ZHOU Jian-feng; SUN Han-ying; LUO Li; ZHANG Heng; XU Hui-zhen

    2005-01-01

    Background Hematopoietic stem cells (HSCs) give rise to all blood and immune cells and are used in clinical transplantation protocols to treat a wide variety of refractory diseases, but the amplification of HSCs has been difficult to achieve in vitro. In the present study, the expansive effects of aorta-gonad-mesonephros (AGM) region derived stromal cells on HSCs were explored, attempting to improve the efficiency of HSC transplantation in clinical practice.Methods The murine stromal cells were isolated from the AGM region of 12 days postcoitum (dpc) murine embryos and bone marrow(BM)of 6 weeks old mice, respectively. After identification with flow cytometry and immunocytochemistry, the stromal cells were co-cultured with ESCs-derived, cytokines-induced HSCs. The maintenance and expansion of ESCs-derived HSCs were evaluated by detecting the population of CD34+ and CD34+Sca-1+cells with flow cytometry and the blast colony-forming cells (BL-CFCs), high proliferative potential colony-forming cells (HPP-CFCs) by using semi-solid medium colonial culture. Finally, the homing and hematopoietic reconstruction abilities of HSCs were evaluated using a murine model of HSC transplantation in vivo.Results AGM and BM-derived stromal cells were morphologically and phenotypically similar, and had the features of stromal cells. When co-cultured with AGM or BM stromal cells, more primitive progenitor cells (HPP-CFCs ) could be detected in ESCs derived hematopoietic precursor cells, but BL-CFC's expansion could be detected only when co-cultured with AGM-derived stromal cells. The population of CD34+ hematopoietic stem/progenitor cells were expanded 3 times,but no significant expansion in the population of CD34+Sca-1+ cells was noted when co-cultured with BM stromal cells. While both CD34+ hematopoietic stem/progenitor cells and CD34+Sca-1+ cells were expanded 4 to 5 times respectively when co-cultured with AGM stromal cells. AGM region-derived stromal cells, like BM-derived stromal

  7. Culture expansion of adipose derived stromal cells. A closed automated Quantum Cell Expansion System compared with manual flask-based culture

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Follin, Bjarke; Juhl, Morten

    2016-01-01

    BACKGROUND: Adipose derived stromal cells (ASCs) are a rich and convenient source of cells for clinical regenerative therapeutic approaches. However, applications of ASCs often require cell expansion to reach the needed dose. In this study, cultivation of ASCs from stromal vascular fraction (SVF......) over two passages in the automated and functionally closed Quantum Cell Expansion System (Quantum system) is compared with traditional manual cultivation. METHODS: Stromal vascular fraction was isolated from abdominal fat, suspended in α-MEM supplemented with 10% Fetal Bovine Serum and seeded......, and endotoxins, in addition to the assessment of cell counts, viability, immunophenotype, and differentiation potential. RESULTS: The viability of ASCs passage 0 (P0) and P1 was above 96%, regardless of cultivation in flasks or Quantum system. Expression of surface markers and differentiation potential...

  8. Plant caspase-like proteases in plant programmed cell death

    OpenAIRE

    Xu, Qixian; Zhang, Lingrui

    2009-01-01

    Programmed cell death (PCD) is a genetically-controlled disassembly of the cell. In animal systems, the central core execution switch for apoptotic PCD is the activation of caspases (Cysteine-containing Aspartate-specific proteases). Accumulating evidence in recent years suggests the existence of caspase-like activity in plants and its functional involvement in various types of plant PCD, although no functional homologs of animal caspases were identified in plant genome. In this mini-review, ...

  9. Colony Expansion of Socially Motile Myxococcus xanthus Cells Is Driven by Growth, Motility, and Exopolysaccharide Production.

    Science.gov (United States)

    Patra, Pintu; Kissoon, Kimberley; Cornejo, Isabel; Kaplan, Heidi B; Igoshin, Oleg A

    2016-06-01

    Myxococcus xanthus, a model organism for studies of multicellular behavior in bacteria, moves exclusively on solid surfaces using two distinct but coordinated motility mechanisms. One of these, social (S) motility is powered by the extension and retraction of type IV pili and requires the presence of exopolysaccharides (EPS) produced by neighboring cells. As a result, S motility requires close cell-to-cell proximity and isolated cells do not translocate. Previous studies measuring S motility by observing the colony expansion of cells deposited on agar have shown that the expansion rate increases with initial cell density, but the biophysical mechanisms involved remain largely unknown. To understand the dynamics of S motility-driven colony expansion, we developed a reaction-diffusion model describing the effects of cell density, EPS deposition and nutrient exposure on the expansion rate. Our results show that at steady state the population expands as a traveling wave with a speed determined by the interplay of cell motility and growth, a well-known characteristic of Fisher's equation. The model explains the density-dependence of the colony expansion by demonstrating the presence of a lag phase-a transient period of very slow expansion with a duration dependent on the initial cell density. We propose that at a low initial density, more time is required for the cells to accumulate enough EPS to activate S-motility resulting in a longer lag period. Furthermore, our model makes the novel prediction that following the lag phase the population expands at a constant rate independent of the cell density. These predictions were confirmed by S motility experiments capturing long-term expansion dynamics.

  10. Feasibility Analysis of a Seabed Filtration Intake System for the Shoaiba III Expansion Reverse Osmosis Plant

    KAUST Repository

    Rodríguez, Luis Raúl

    2012-06-01

    The ability to economically desalinate seawater in arid regions of the world has become a vital advancement to overcome the problem on freshwater availability, quality, and reliability. In contrast with the major capital and operational costs for desalination plants represented by conventional open ocean intakes, subsurface intakes allow the extraction of high quality feed water at minimum costs and reduced environmental impact. A seabed filter is a subsurface intake that consists of a submerged slow sand filter, with benefits of organic matter removal and pathogens, and low operational cost. A site investigation was carried out through the southern coast of the Red Sea in Saudi Arabia, from King Abdullah University of Science and Technology down to 370 kilometers south of Jeddah. A site adjacent to the Shoaiba desalination plant was selected to assess the viability of constructing a seabed filter. Grain sieve size analysis, porosity and hydraulic conductivity permeameter measurements were performed on the collected sediment samples. Based on these results, it was concluded that the characteristics at the Shoaiba site allow for the construction of a seabed filtration system. A seabed filter design is proposed for the 150,000 m3/d Shoaiba III expansion project, a large-scale Reverse Osmosis desalination plant. A filter design with a filtration rate of 7 m/d through an area of 6,000 m2 is proposed to meet the demand of one of the ten desalination trains operating at the plant. The filter would be located 90 meters offshore where hydraulic conductivity of the sediment is high, and mud percentage is minimal. The thin native marine sediment layer is insufficient to provide enough water filtration, and consequently the proposed solution involves excavating the limestone rock and filling it with different layers of non-native sand and gravel of increasing grain size. An initial assessment of the area around Shoaiba showed similar sedimentological conditions that could

  11. Arabidopsis thaliana homeobox 12 (ATHB12), a homeodomain-leucine zipper protein, regulates leaf growth by promoting cell expansion and endoreduplication.

    Science.gov (United States)

    Hur, Yoon-Sun; Um, Ji-Hyun; Kim, Sunghan; Kim, Kyunga; Park, Hee-Jung; Lim, Jong-Seok; Kim, Woo-Young; Jun, Sang Eun; Yoon, Eun Kyung; Lim, Jun; Ohme-Takagi, Masaru; Kim, Donggiun; Park, Jongbum; Kim, Gyung-Tae; Cheon, Choong-Ill

    2015-01-01

    Arabidopsis thaliana homeobox 12 (ATHB12), a homeodomain-leucine zipper class I (HD-Zip I) gene, is highly expressed in leaves and stems, and induced by abiotic stresses, but its role in development remains obscure. To understand its function during plant development, we studied the effects of loss and gain of function. Expression of ATHB12 fused to the EAR-motif repression domain (SRDX) - P35 S ::ATHB12SRDX (A12SRDX) and PATHB 12 ::ATHB12SRDX - slowed both leaf and root growth, while the growth of ATHB12-overexpressing seedlings (A12OX) was accelerated. Microscopic examination revealed changes in the size and number of leaf cells. Ploidy was reduced in A12SRDX plants, accompanied by decreased cell expansion and increased cell numbers. By contrast, cell size was increased in A12OX plants, along with increased ploidy and elevated expression of cell cycle switch 52s (CCS52s), which are positive regulators of endoreduplication, indicating that ATHB12 promotes leaf cell expansion and endoreduplication. Overexpression of ATHB12 led to decreased phosphorylation of Arabidopsis thaliana ribosomal protein S6 (AtRPS6), a regulator of cell growth. In addition, induction of ATHB12 in the presence of cycloheximide increased the expression of several genes related to cell expansion, such as EXPANSIN A10 (EXPA10) and DWARF4 (DWF4). Our findings strongly suggest that ATHB12 acts as a positive regulator of endoreduplication and cell growth during leaf development.

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

    Science.gov (United States)

    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.

  13. Polyclonal Expansion of NKG2C+ NK Cells in TAP-deficient Patients

    Directory of Open Access Journals (Sweden)

    vivien eBeziat

    2015-10-01

    Full Text Available Adaptive natural killer (NK cell responses to human cytomegalovirus (CMV infection are characterized by the expansion of NKG2C+ NK cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs. Here, we set out to study the HLA class I-dependency of such NKG2C+ NK cell expansions. We demonstrate expansion of NKG2C+ NK cells in patients with transporter associated with antigen presentation (TAP-deficiency, whom express less than 10% of normal HLA class I levels. In contrast to normal individuals, expanded NKG2C+ NK cell populations in TAP-deficient patients display a polyclonal KIR-profile and remain hyporesponsive to HLA class I-negative target cells. Nonetheless, agonistic stimulation of NKG2C on NK cells from TAP-deficient patients yielded significant responses in terms of degranulation and cytokine production. Thus, while interactions with self-HLA class I molecules likely shape the KIR-repertoire of expanding NKG2C+ NK cells during adaptive NK cell responses in normal individuals, they are not a prerequisite for NKG2C+ NK cell expansions to occur. Thus, the emergence of NKG2C-responsive adaptive NK cells in TAP-deficient patients may contribute to anti-viral immunity and potentially explain these patients’ low incidence of severe viral infections.

  14. Polyclonal Expansion of NKG2C+ NK Cells in TAP-Deficient Patients

    Science.gov (United States)

    Béziat, Vivien; Sleiman, Marwan; Goodridge, Jodie P.; Kaarbø, Mari; Liu, Lisa L.; Rollag, Halvor; Ljunggren, Hans-Gustaf; Zimmer, Jacques; Malmberg, Karl-Johan

    2015-01-01

    Adaptive natural killer (NK) cell responses to human cytomegalovirus infection are characterized by the expansion of NKG2C+ NK cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs). Here, we set out to study the HLA class I dependency of such NKG2C+ NK cell expansions. We demonstrate the expansion of NKG2C+ NK cells in patients with transporter associated with antigen presentation (TAP) deficiency, who express less than 10% of normal HLA class I levels. In contrast to normal individuals, expanded NKG2C+ NK cell populations in TAP-deficient patients display a polyclonal KIR profile and remain hyporesponsive to HLA class I-negative target cells. Nonetheless, agonistic stimulation of NKG2C on NK cells from TAP-deficient patients yielded significant responses in terms of degranulation and cytokine production. Thus, while interactions with self-HLA class I molecules likely shape the KIR repertoire of expanding NKG2C+ NK cells during adaptive NK cell responses in normal individuals, they are not a prerequisite for NKG2C+ NK cell expansions to occur. The emergence of NKG2C-responsive adaptive NK cells in TAP-deficient patients may contribute to antiviral immunity and potentially explain these patients’ low incidence of severe viral infections. PMID:26500647

  15. Microfluidic platforms for plant cells studies.

    Science.gov (United States)

    Sanati Nezhad, A

    2014-09-07

    Conventional methods of plant cell analysis rely on growing plant cells in soil pots or agarose plates, followed by screening the plant phenotypes in traditional greenhouses and growth chambers. These methods are usually costly, need a large number of experiments, suffer from low spatial resolution and disorderly growth behavior of plant cells, with lack of ability to locally and accurately manipulate the plant cells. Microfluidic platforms take advantage of miniaturization for handling small volume of liquids and providing a closed environment, with the purpose of in vitro single cell analysis and characterizing cell response to external cues. These platforms have shown their ability for high-throughput cellular analysis with increased accuracy of experiments, reduced cost and experimental times, versatility in design, ability for large-scale and combinatorial screening, and integration with other miniaturized sensors. Despite extensive research on animal cells within microfluidic environments for high-throughput sorting, manipulation and phenotyping studies, the application of microfluidics for plant cells studies has not been accomplished yet. Novel devices such as RootChip, RootArray, TipChip, and PlantChip developed for plant cells analysis, with high spatial resolution on a micrometer scale mimicking the internal microenvironment of plant cells, offering preliminary results on the capability of microfluidics to conquer the constraints of conventional methods. These devices have been used to study different aspects of plant cell biology such as gene expression, cell biomechanics, cellular mechanism of growth, cell division, and cells fusion. This review emphasizes the advantages of current microfluidic systems for plant science studies, and discusses future prospects of microfluidic platforms for characterizing plant cells response to diverse external cues.

  16. Clonal expansion of renal cell carcinoma-infiltrating T lymphocytes

    DEFF Research Database (Denmark)

    Sittig, Simone; Køllgaard, Tania; Grønbæk, Kirsten

    2013-01-01

    T lymphocytes can mediate the destruction of cancer cells by virtue of their ability to recognize tumor-derived antigenic peptides that are presented on the cell surface in complex with HLA molecules and expand. Thus, the presence of clonally expanded T cells within neoplastic lesions...... is an indication of ongoing HLA-restricted T cell-mediated immune responses. Multiple tumors, including renal cell carcinomas (RCCs), are often infiltrated by significant amounts of T cells, the so-called tumor-infiltrating lymphocytes (TILs). In the present study, we analyzed RCC lesions (n = 13) for the presence...... of expanded T-cell clonotypes using T-cell receptor clonotype mapping. Surprisingly, we found that RCCs comprise relatively low numbers of distinct expanded T-cell clonotypes as compared with melanoma lesions. The numbers of different T-cell clonotypes detected among RCC-infiltrating lymphocytes were...

  17. Penium margaritaceum as a model organism for cell wall analysis of expanding plant cells

    DEFF Research Database (Denmark)

    Rydahl, Maja Gro; Fangel, Jonatan Ulrik; Mikkelsen, Maria Dalgaard

    2015-01-01

    have focused primarily upon late divergent multicellular land plants and specialized cell types (e.g., pollen tubes, root hairs). Here, we describe a unicellular green alga, Penium margaritaceum (Penium), which can serve as a valuable model organism for understanding cell expansion and the underlying...

  18. Common molecular pathways involved in human CD133+/CD34+ progenitor cell expansion and cancer

    Directory of Open Access Journals (Sweden)

    Vêncio Ricardo Z

    2007-06-01

    Full Text Available Abstract Background Uncovering the molecular mechanism underlying expansion of hematopoietic stem and progenitor cells is critical to extend current therapeutic applications and to understand how its deregulation relates to leukemia. The characterization of genes commonly relevant to stem/progenitor cell expansion and tumor development should facilitate the identification of novel therapeutic targets in cancer. Methods CD34+/CD133+ progenitor cells were purified from human umbilical cord blood and expanded in vitro. Correlated molecular changes were analyzed by gene expression profiling using microarrays covering up to 55,000 transcripts. Genes regulated during progenitor cell expansion were identified and functionally classified. Aberrant expression of such genes in cancer was indicated by in silico SAGE. Differential expression of selected genes was assessed by real-time PCR in hematopoietic cells from chronic myeloid leukemia patients and healthy individuals. Results Several genes and signaling pathways not previously associated with ex vivo expansion of CD133+/CD34+ cells were identified, most of which associated with cancer. Regulation of MEK/ERK and Hedgehog signaling genes in addition to numerous proto-oncogenes was detected during conditions of enhanced progenitor cell expansion. Quantitative real-time PCR analysis confirmed down-regulation of several newly described cancer-associated genes in CD133+/CD34+ cells, including DOCK4 and SPARCL1 tumor suppressors, and parallel results were verified when comparing their expression in cells from chronic myeloid leukemia patients Conclusion Our findings reveal potential molecular targets for oncogenic transformation in CD133+/CD34+ cells and strengthen the link between deregulation of stem/progenitor cell expansion and the malignant process.

  19. Selective in vitro expansion and efficient retroviral transduction of human CD34(+) CD38(-) haematopoietic stem cells

    NARCIS (Netherlands)

    Ng, YY; Bloem, AC; van Kessel, B; Lokhorst, H; Logtenberg, T; Staal, FJT

    2002-01-01

    Ex vivo expansion of primitive human haematopoietic stem cells (HSC) is clinically relevant for stem cell transplantation and gene therapy. Here, we demonstrate the selective expansion of CD34(+) CD38(-) cells from purified CD34(+) cells upon stimulation with Flt3-ligand, stem cell factor and thromb

  20. Cumulus Cell Expansion, Its Role in Oocyte Biology and Perspectives of Measurement: A Review

    Directory of Open Access Journals (Sweden)

    Nevoral J.

    2015-01-01

    Full Text Available Cumulus expansion of the cumulus-oocyte complex is necessary for meiotic maturation and acquiring developmental competence. Cumulus expansion is based on extracellular matrix synthesis by cumulus cells. Hyaluronic acid is the most abundant component of this extracellular matrix. Cumulus expansion takes place during meiotic oocyte maturation under in vivo and in vitro conditions. Quantification and measurement of cumulus expansion intensity is one possible method of determining oocyte quality and optimizing conditions for in vitro cultivation. Currently, subjective methods of expanded area and more exact cumulus expansion measurement by hyaluronic acid assessment are available. Among the methods of hyaluronic acid measurement is the use of radioactively labelled synthesis precursors. Alternatively, immunological and analytical methods, including enzyme-linked immunosorbent assay (ELISA, spectrophotometry, and high-performance liquid chromatography (HPLC in UV light, could be utilized. The high sensitivity of these methods could provide a precise analysis of cumulus expansion without the use of radioisotopes. Therefore, the aim of this review is to summarize and compare available approaches of cumulus expansion measurement, respecting special biological features of expanded cumuli, and to suggest possible solutions for exact cumulus expansion analysis.

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

    Science.gov (United States)

    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

  2. Cell cycle activation by plant parasitic nematodes

    NARCIS (Netherlands)

    Goverse, A.; Almeida Engler, de J.; Verhees, J.; Krol, van der S.; Helder, J.; Gheysen, G.

    2000-01-01

    Sedentary nematodes are important pests of crop plants. They are biotrophic parasites that can induce the (re)differentiation of either differentiated or undifferentiated plant cells into specialized feeding cells. This (re)differentiation includes the reactivation of the cell cycle in specific

  3. Cell cycle activation by plant parasitic nematodes

    NARCIS (Netherlands)

    Goverse, A.; Almeida Engler, de J.; Verhees, J.; Krol, van der S.; Helder, J.; Gheysen, G.

    2000-01-01

    Sedentary nematodes are important pests of crop plants. They are biotrophic parasites that can induce the (re)differentiation of either differentiated or undifferentiated plant cells into specialized feeding cells. This (re)differentiation includes the reactivation of the cell cycle in specific plan

  4. Physiological functions of plant cell coverings.

    Science.gov (United States)

    Hoson, Takayuki

    2002-08-01

    The cell coverings of plants have two important functions in plant life. Plant cell coverings are deeply involved in the regulation of the life cycle of plants: each stage of the life cycle, such as germination, vegetative growth, reproductive growth, and senescence, is strongly influenced by the nature of the cell coverings. Also, the apoplast, which consists of the cell coverings, is the field where plant cells first encounter the outer environment, and so becomes the major site of plant responses to the environment. In the regulation of each stage of the life cycle and the response to each environmental signal, some specific constituents of the cell coverings, such as xyloglucans in dicotyledons and 1,3,1,4-beta-glucans in Gramineae, act as the key component. The physiological functions of plant cell coverings are sustained by the metabolic turnover of these components. The components of the cell coverings are supplied from the symplast, but then they are modified or degraded in the apoplast. Thus, the metabolism of the cell coverings is regulated through the cross-talk between the symplast and the apoplast. The understanding of physiological functions of plant cell coverings will be greatly advanced by the use of genomic approaches. At the same time, we need to introduce nanobiological techniques for clarifying the minute changes in the cell coverings that occur in a small part within each cell.

  5. Simulation of thermal and sodium expansion stress in aluminum reduction cells

    Institute of Scientific and Technical Information of China (English)

    LI Jie; WU Yu-yun; LAI Yan-qing; LIU Wei; WANG Zhi-gang; LIU Jie; LIU Ye-xiang

    2008-01-01

    Two finite element(FE) models were built up for analysis of stress field in the lining of aluminum electrolysis cells. Distribution of sodium concentration in cathode carbon blocks was calculated by one FE model of a cathode block. Thermal stress field was calculated by the other slice model of the cell at the end of the heating-up. Then stresses coupling thermal and sodium expansion were considered after 30 d start-up. The results indicate that sodium penetrates to the bottom of the cathode block after 30 d start-up. The semi-graphitic carbon block has the largest stress at the thermal stage. After 30 d start-up the anthracitic carbon has the greatest sodium expansion stress and the graphitized carbon has the lowest sodium expansion stress. Sodium penetration can cause larger deformation and stress in the cathode carbon block than thermal expansion.

  6. Multi-scale visualization and characterization of lignocellulosic plant cell wall deconstruction during thermochemical pretreatment

    Science.gov (United States)

    Shishir P. S. Chundawat; Bryon S. Donohoe; Leonardo da Costa Sousa; Thomas Elder; Umesh P. Agarwal; Fachuang Lu; John Ralph; Michael E. Himmel; Venkatesh Balan; Bruce E. Dale

    2011-01-01

    Deconstruction of lignocellulosic plant cell walls to fermentable sugars by thermochemical and/or biological means is impeded by several poorly understood ultrastructural and chemical barriers. A promising thermochemical pretreatment called ammonia fiber expansion (AFEX) overcomes the native recalcitrance of cell walls through subtle morphological and physicochemical...

  7. Arabidopsis Kinesins HINKEL and TETRASPORE Act Redundantly to Control Cell Plate Expansion during Cytokinesis in the Male Garnetophyte

    Institute of Scientific and Technical Information of China (English)

    Sung-Aeong Oh; Valérie Bourdon; Madhumita Das'Pal; Hugh Dickinson; David Twell

    2008-01-01

    Asymmetric cell division at pollen mitosis I(PMI)is required to specify the differentiaI fate of the daughter vegetative and generative cells.Cytokinesis at PMI displays specialized features,and it has been suggested that there might be distinct molecular pathways underpinning different modes of cytokinesis in plants.Activation of the NACKPQR MAP kinase signaling pathway,which is essentiaI for somatic cell cytokinesis in tobacco,depends upon the NACK1and NACK2 kinesin-related proteins.Their Arabidopsis orthologs.HINKEL(HIK)and TETRAsPORE(TES).were reported to be essential for cytokinesis in somatic cells and in microsporoctes.respectively.More recently,HIK and TES were shown to have a functionally redundant role in female gametophytic cvtokinesis.We report here that HIK and TES are co-expressed in microspores and developing pollen,and,through analysis of microspore and pollen development in double heterozygote mutants.the occurrence of cell plate expansion defects during cytokinesis at PMI.The data demonstrate a functionally redundant role for HIK and TES in cell plate expansion during male gametophytic cytokinesis.extending the concept that different modes of cytokinesis are executed by a common signaling pathway,but reinforcing the individuality of gametophytic cytokinesis in its requirement for either TES or HIK.

  8. Obesity promotes colonic stem cell expansion during cancer initiation.

    Science.gov (United States)

    DeClercq, V; McMurray, D N; Chapkin, R S

    2015-12-28

    There is an urgent need to elucidate the mechanistic links between obesity and colon cancer. Convincing evidence for the role of Lgr5(+) stem cells in colon tumorigenesis has been established; however, the influence of obesity on stem cell maintenance is unknown. We assessed the effects of high fat (HF) feeding on colonic stem cell maintenance during cancer initiation (AOM induced) and the responsiveness of stem cells to adipokine signaling pathways. The number of colonic GFP(+) stem cells was significantly higher in the AOM-injected HF group compared to the LF group. The Lgr5(+) stem cells of the HF fed mice exhibited statistically significant increases in cell proliferation and decreases in apoptosis in response to AOM injection compared to the LF group. Colonic organoid cultures from lean mice treated with an adiponectin receptor agonist exhibited a reduction in Lgr5-GPF(+) stem cell number and an increase in apoptosis; however, this response was diminished in the organoid cultures from obese mice. These results suggest that the responsiveness of colonic stem cells to adiponectin in diet-induced obesity is impaired and may contribute to the stem cell accumulation observed in obesity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. Cell-penetrating peptides: From mammalian to plant cells

    OpenAIRE

    Eudes, François; Chugh, Archana

    2008-01-01

    Internalization of cell-penetrating peptides, well described in mammalian cell system, has recently been reported in a range of plant cells by three independent groups. Despite fundamental differences between animal cell and plant cell composition, the CPP uptake pattern between the mammalian system and the plant system is very similar. Tat, Tat-2 pVEC and transportan internalisation is concentration dependent and non saturable, enhanced at low temperature (4°C), and receptor independent. The...

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

    OpenAIRE

    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

    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...

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

    Science.gov (United States)

    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.

  12. Plant stem cells as innovation in cosmetics.

    Science.gov (United States)

    Moruś, Martyna; Baran, Monika; Rost-Roszkowska, Magdalena; Skotnicka-Graca, Urszula

    2014-01-01

    The stem cells thanks to their ability of unlimited division number or transformation into different cell types creating organs, are responsible for regeneration processes. Depending on the organism in which the stem cells exists, they divide to the plant or animal ones. The later group includes the stem cells existing in both embryo's and adult human's organs. It includes, among others, epidermal stem cells, located in the hair follicle relieves and also in its basal layers, and responsible for permanent regeneration of the epidermis. Temporary science looks for method suitable for stimulation of the epidermis stem cells, amongst the other by delivery of e.g., growth factors for proliferation that decrease with the age. One of the methods is the use of the plant cell culture technology, including a number of methods that should ensure growth of plant cells, issues or organs in the environment with the microorganism-free medium. It uses abilities of the different plant cells to dedifferentiation into stem cells and coming back to the pluripotent status. The extracts obtained this way from the plant stem cells are currently used for production of both common or professional care cosmetics. This work describes exactly impact of the plant stem cell extract, coming from one type of the common apple tree (Uttwiler Spätlauber) to human skin as one of the first plant sorts, which are used in cosmetology and esthetic dermatology.

  13. Epitope-Specific Vaccination Limits Clonal Expansion of Heterologous Naive T Cells during Viral Challenge

    Directory of Open Access Journals (Sweden)

    Lexus R. Johnson

    2016-10-01

    Full Text Available Despite robust secondary T cell expansion primed by vaccination, the impact on primary immune responses to heterotypic antigens remains undefined. Here we show that secondary expansion of epitope-specific memory CD8+ T cells primed by prior infection with recombinant pathogens limits the primary expansion of naive CD8+ T cells with specificity to new heterologous antigens, dampening protective immunity against subsequent pathogen challenge. The degree of naive T cell repression directly paralleled the magnitude of the recall response. Suppressed primary T cell priming reflects competition for antigen accessibility, since clonal expansion was not inhibited if the primary and secondary epitopes were expressed on different dendritic cells. Interestingly, robust recall responses did not impact antigen-specific NK cells, suggesting that adaptive and innate lymphocyte responses possess different activation requirements or occur in distinct anatomical locations. These findings have important implications in pathogen vaccination strategies that depend on the targeting of multiple T cell epitopes.

  14. Wnt3a nanodisks promote ex vivo expansion of hematopoietic stem and progenitor cells

    DEFF Research Database (Denmark)

    Lalefar, Nahal R.; Witkowski, Andrzej; Simonsen, Jens Bæk;

    2016-01-01

    -elutes with ND. In signaling assays, Wnt3a ND induced β-catenin stabilization in mouse fibroblasts as well as hematopoietic stem and progenitor cells (HSPC). Prolonged exposure of HSPC to Wnt3a ND stimulated proliferation and expansion of Lin- Sca-1+ c-Kit+ cells. Surprisingly, ND lacking Wnt3a contributed...

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

    Science.gov (United States)

    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.

  16. Hematopoietic stem cells: ex-vivo expansion and therapeutic potential for myocardial ischemia

    Directory of Open Access Journals (Sweden)

    Jingwei Lu

    2010-03-01

    Full Text Available Jingwei Lu, Vincent J Pompili, Hiranmoy DasCardiovascular Stem Cell Research Laboratory, The Dorothy M Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, OH 43210, USAAbstract: Despite recent advances in cardiovascular medicine, ischemic heart disease remains the major cause of death in the United States and abroad. Cell-based therapy for degenerative diseases like myocardial ischemia using stem cells is currently under serious investigation. Various types of stem cells are being considered to be candidates for cell transplantation in cell-based therapy. Hematopoietic stem cells are one of the most promising cell types as several studies demonstrated their ability to improve ischemic cardiac functions by enhancing neovascularization and by reducing the total size of scar tissue. However, in order to procure sufficient numbers of functional stem cells, ex-vivo expansion technology became critically important. In this review, we focus on the state-of-the-art ex-vivo technology for the expansion of hematopoietic stem cells, and the underlying mechanisms regulating stem cell self-renewal as well as differentiation.Keywords: ischemic heart disease, ex-vivo expansion, hematopoietic stem cells, cytokines, nanofibers

  17. [On plant stem cells and animal stem cells].

    Science.gov (United States)

    You, Yun; Jiang, Chao; Huang, Lu-Qi

    2014-01-01

    A comparison of plant and animal stem cells can highlight core aspects of stem-cell biology. In both kingdoms, stem cells are defined by their clonogenic properties and are maintained by intercellular signals. The signaling molecules are different in plants and animals stem cell niches, but the roles of argonaute and polycomb group proteins suggest that there are some molecular similarities.

  18. Suppression of cell expansion by ectopic expression of the Arabidopsis SUPERMAN gene in transgenic petunia and tobacco.

    Science.gov (United States)

    Kater, M M; Franken, J; van Aelst, A; Angenent, G C

    2000-08-01

    Molecular and genetic analyses have shown that the Arabidopsis thaliana gene SUPERMAN (SUP) has at least two functions in Arabidopsis flower development. SUP is necessary to control the correct distribution of cells with either a stamen or carpel fate, and is essential for proper outgrowth of the ovule outer integument. Both these functions indicate a role for SUP in cell proliferation. To study the function of the Arabidopsis SUP gene in more detail, we over-expressed the SUP gene in petunia and tobacco in a tissue-specific manner. The petunia FLORAL BINDING PROTEIN 1 (FBP1) gene promoter was used to restrict the expression of SUP to petals and stamens. The development of petals and stamens was severely affected in both petunia and tobacco plants over-expressing SUP. Petals remained small and did not unfold, resulting in closed flowers. Stamen filaments were thin and very short. Detailed analysis of these floral organs from the petunia transformants showed that cell expansion was dramatically reduced without affecting cell division. These results reveal a novel activity for SUP as a regulator of cell expansion.

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

    Science.gov (United States)

    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.

  20. Plants on the move: The role of seed dispersal and initial population establishment for climate-driven range expansions

    Science.gov (United States)

    Hampe, Arndt

    2011-11-01

    Recent climate change will presumably allow many plant species to expand their geographical range up to several hundred kilometres towards the poles within a few decades. Much uncertainty exists however to which extent species will actually be able to keep pace with a rapidly changing climate. A suite of direct and indirect research approaches have explored the phenomenon of range expansions, and the existing evidence is scattered across the literature of diverse research subdisciplines. Here I attempt to synthesise the available information within a population ecological framework in order to evaluate implications of patterns of seed dispersal and initial population establishment for range expansions. After introducing different study approaches and their respective contributions, I review the empirical evidence for the role of long-distance seed dispersal in past and ongoing expansions. Then I examine how some major ecological determinants of seed dispersal and colonisation processes - population fecundity, dispersal pathways, arrival site conditions, and biotic interactions during recruitment - could be altered by a rapidly changing climate. While there is broad consensus that long-distance dispersal is likely to be critical for rapid range expansions, it remains challenging to relate dispersal processes and pathways with the establishment of pioneer populations ahead of the continuous species range. Further transdisciplinary efforts are clearly needed to address this link, key for understanding how plant populations 'move' across changing landscapes.

  1. Morphological classification of plant cell deaths

    DEFF Research Database (Denmark)

    van Doorn, W.G.; Beers, E.P.; Dangl, J.L.

    2011-01-01

    Programmed cell death (PCD) is an integral part of plant development and of responses to abiotic stress or pathogens. Although the morphology of plant PCD is, in some cases, well characterised and molecular mechanisms controlling plant PCD are beginning to emerge, there is still confusion about...... the classification of PCD in plants. Here we suggest a classification based on morphological criteria. According to this classification, the use of the term 'apoptosis' is not justified in plants, but at least two classes of PCD can be distinguished: vacuolar cell death and necrosis. During vacuolar cell death......, the cell contents are removed by a combination of autophagy-like process and release of hydrolases from collapsed lytic vacuoles. Necrosis is characterised by early rupture of the plasma membrane, shrinkage of the protoplast and absence of vacuolar cell death features. Vacuolar cell death is common during...

  2. Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves. I. Growth can occur without photosynthesis

    Science.gov (United States)

    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.

  3. [Genetic regulation of plant shoot stem cells].

    Science.gov (United States)

    Al'bert, E V; Ezhova, T A

    2013-02-01

    This article describes the main features of plant stem cells and summarizes the results of studies of the genetic control of stem cell maintenance in the apical meristem of the shoot. It is demonstrated that the WUS-CLV gene system plays a key role in the maintenance of shoot apical stem cells and the formation of adventitious buds and somatic embryos. Unconventional concepts of plant stem cells are considered.

  4. Stem cell function during plant vascular development.

    Science.gov (United States)

    Miyashima, Shunsuke; Sebastian, Jose; Lee, Ji-Young; Helariutta, Yka

    2013-01-23

    The plant vascular system, composed of xylem and phloem, evolved to connect plant organs and transport various molecules between them. During the post-embryonic growth, these conductive tissues constitutively form from cells that are derived from a lateral meristem, commonly called procambium and cambium. Procambium/cambium contains pluripotent stem cells and provides a microenvironment that maintains the stem cell population. Because vascular plants continue to form new tissues and organs throughout their life cycle, the formation and maintenance of stem cells are crucial for plant growth and development. In this decade, there has been considerable progress in understanding the molecular control of the organization and maintenance of stem cells in vascular plants. Noticeable advance has been made in elucidating the role of transcription factors and major plant hormones in stem cell maintenance and vascular tissue differentiation. These studies suggest the shared regulatory mechanisms among various types of plant stem cell pools. In this review, we focus on two aspects of stem cell function in the vascular cambium, cell proliferation and cell differentiation.

  5. Pathological modifications of plant stem cell destiny

    Science.gov (United States)

    In higher plants, the shoot apex contains undifferentiated stem cells that give rise to various tissues and organs. The fate of these stem cells determines the pattern of plant growth as well as reproduction; and such fate is genetically preprogrammed. We found that a bacterial infection can derai...

  6. Plant cells: immobilization and oxygen transfer.

    NARCIS (Netherlands)

    Hulst, A.C.

    1987-01-01

    The study described in this thesis is part of the integrated project 'Biotechnological production of non-persistent bioinsecticides by means of plant cells invitro ' and was done in close cooperation with the research Institute Ital within the framework of NOVAPLANT. The plant cells us

  7. Wall extensibility: its nature, measurement and relationship to plant cell growth

    Science.gov (United States)

    Cosgrove, D. J.

    1993-01-01

    Expansive growth of plant cells is controlled principally by processes that loosen the wall and enable it to expand irreversibly. The central role of wall relaxation for cell expansion is reviewed. The most common methods for assessing the extension properties of plant cell walls ( wall extensibility') are described, categorized and assessed critically. What emerges are three fundamentally different approaches which test growing cells for their ability (a) to enlarge at different values of turgor, (b) to induce wall relaxation, and (c) to deform elastically or plastically in response to an applied tensile force. Analogous methods with isolated walls are similarly reviewed. The results of these different assays are related to the nature of plant cell growth and pertinent biophysical theory. I argue that the extensibilities' measured by these assays are fundamentally different from one another and that some are more pertinent to growth than others.

  8. Cell fusion and nuclear fusion in plants.

    Science.gov (United States)

    Maruyama, Daisuke; Ohtsu, Mina; Higashiyama, Tetsuya

    2016-12-01

    Eukaryotic cells are surrounded by a plasma membrane and have a large nucleus containing the genomic DNA, which is enclosed by a nuclear envelope consisting of the outer and inner nuclear membranes. Although these membranes maintain the identity of cells, they sometimes fuse to each other, such as to produce a zygote during sexual reproduction or to give rise to other characteristically polyploid tissues. Recent studies have demonstrated that the mechanisms of plasma membrane or nuclear membrane fusion in plants are shared to some extent with those of yeasts and animals, despite the unique features of plant cells including thick cell walls and intercellular connections. Here, we summarize the key factors in the fusion of these membranes during plant reproduction, and also focus on "non-gametic cell fusion," which was thought to be rare in plant tissue, in which each cell is separated by a cell wall.

  9. Microencapsulation technology: a powerful tool for integrating expansion and cryopreservation of human embryonic stem cells.

    Science.gov (United States)

    Serra, Margarida; Correia, Cláudia; Malpique, Rita; Brito, Catarina; Jensen, Janne; Bjorquist, Petter; Carrondo, Manuel J T; Alves, Paula M

    2011-01-01

    The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors.The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics.Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks.This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications.

  10. Human peripheral blood-born hematosphere as a niche for hematopoietic stem cell expansion

    Institute of Scientific and Technical Information of China (English)

    Jin Hur; Eun Ju Lee; Hyun-Jai Cho; Hyun-Jae Kang; Byung-Hee Oh; Young-Bae Park; Hyo-Soo Kim; Jonghanne Park; Sang Eun Lee; Chang-Hwan Yoon; Jae Hee Jang; Ji Min Yang; Tae-Kyu Lee; Jae-Il Choi; Han-Mo Yang

    2011-01-01

    @@ Dear Editor, Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) derived from the adult peripheral blood has been widely used in the treatment of various hematological diseases [1].However,the small number of circulating HSPC is the major limitation and necessitates additional interventions such as G-CSF mobilization and leukapheresis.There have been several attempts to overcome the limitation with ex vivo expansion of HSPC.These strategies are largely based on supplementation of one or more "stem cell niche components"such as supporting-cells,growth factors,extracellular matrix (ECM) or physicochemical microenvironment in the bone marrow [2].Spheroid culture methods of stem ceils from different tissues have been successfully used for expansion of cardiac and neural stem cells.These spheres sensitize target stem cells to growth factors and provide sufficient cell-to-cell and cell-to-matrix contacts,mimicking the in vivo stem cell niche [3,4].Here we asked whether spheroid culture of blood mononuclear cells (MNCs) would potentiate the expansion of circulating blood HSPC.

  11. Microencapsulation technology: a powerful tool for integrating expansion and cryopreservation of human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Margarida Serra

    Full Text Available The successful implementation of human embryonic stem cells (hESCs-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i single cells, ii aggregates and iii immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors.The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration and high cell recovery yields (>70% after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics.Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks.This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications.

  12. Analysis of the Clonal Expansion of TCR VβT Cells in Patients

    Institute of Scientific and Technical Information of China (English)

    LIYangqiu; YANGLijian; 等

    2002-01-01

    Objective To investigate the clonal expansion of T cell receptor(TCR)Vβ subfamily T cells which were considered as GVL effective cells after donor lymphocytes infusion(DLI)in patients with relapse chronic myelogenous leukemia(CML)after allogeneic bone marrow transplantation(allo-BMT).Methods The CDR3 of TCR Vβ24 subfamily genes were amplified in samples of peripheral blood mononuclear cells at different time points before and after DLI,which were drawn from 2 cases of relapse CML treated by allo-BMT,to observe the usage of TCR Vβrepertoire.The PCR products were further labeled with fluorescent and analyzed by genescan technique for identification of the CDR3 size,to evaluate the clonality of the detectable TCR VβT cells.Results Only 4-11 VβT subfamily T cells could be identified in CML cases before DLI,and 12-21 Vβ subfamily T cells could be deected in samples from CML which display remission after DLI.Genescan analysis showed that new clonal expansion TCR Vβ subfamily T cells could be found in samples after DLI.Conclusion The skew distribution of TCR Vβ subfamily T cells could be found on patients with relapse CML after allo-BMT,and this skewing pattern may stage to stage to normal pattern during the complete remission.The GVL effect may exert through some clonal expansion TCR Vβ subfamily T cells during the treatment of DLI in relapse CML.

  13. Clinical scale rapid expansion of lymphocytes for adoptive cell transfer therapy in the WAVE® bioreactor

    Science.gov (United States)

    2012-01-01

    Background To simplify clinical scale lymphocyte expansions, we investigated the use of the WAVE®, a closed system bioreactor that utilizes active perfusion to generate high cell numbers in minimal volumes. Methods We have developed an optimized rapid expansion protocol for the WAVE bioreactor that produces clinically relevant numbers of cells for our adoptive cell transfer clinical protocols. Results TIL and genetically modified PBL were rapidly expanded to clinically relevant scales in both static bags and the WAVE bioreactor. Both bioreactors produced comparable numbers of cells; however the cultures generated in the WAVE bioreactor had a higher percentage of CD4+ cells and had a less activated phenotype. Conclusions The WAVE bioreactor simplifies the process of rapidly expanding tumor reactive lymphocytes under GMP conditions, and provides an alternate approach to cell generation for ACT protocols. PMID:22475724

  14. Clinical scale rapid expansion of lymphocytes for adoptive cell transfer therapy in the WAVE® bioreactor

    Directory of Open Access Journals (Sweden)

    Somerville Robert PT

    2012-04-01

    Full Text Available Abstract Background To simplify clinical scale lymphocyte expansions, we investigated the use of the WAVE®, a closed system bioreactor that utilizes active perfusion to generate high cell numbers in minimal volumes. Methods We have developed an optimized rapid expansion protocol for the WAVE bioreactor that produces clinically relevant numbers of cells for our adoptive cell transfer clinical protocols. Results TIL and genetically modified PBL were rapidly expanded to clinically relevant scales in both static bags and the WAVE bioreactor. Both bioreactors produced comparable numbers of cells; however the cultures generated in the WAVE bioreactor had a higher percentage of CD4+ cells and had a less activated phenotype. Conclusions The WAVE bioreactor simplifies the process of rapidly expanding tumor reactive lymphocytes under GMP conditions, and provides an alternate approach to cell generation for ACT protocols.

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

    Science.gov (United States)

    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.

  16. Transport vesicle formation in plant cells.

    Science.gov (United States)

    Hwang, Inhwan; Robinson, David G

    2009-12-01

    In protein trafficking, transport vesicles bud from donor compartments and carry cargo proteins to target compartments with which they fuse. Thus, vesicle formation is an essential step in protein trafficking. As for mammals, plant cells contain the three major types of vesicles: COPI, COPII, and CCV and the major molecular players in vesicle-mediated protein transport are also present. However, plant cells generally contain more isoforms of the coat proteins, ARF GTPases and their regulatory proteins, as well as SNAREs. In addition, plants have established some unique subfamilies, which may reflect plant cell-specific conditions such as the absence of an ER-Golgi intermediate compartment and the combined activities of the TGN and early endosome. Thus, even though we are still at an early stage in understanding the physiological function of these proteins, it is already clear that vesicle-mediated protein transport in plant cells displays both similarities as well as differences in animal cells.

  17. Measuring the Mechanical Properties of Plant Cell Walls

    Directory of Open Access Journals (Sweden)

    Hannes Vogler

    2015-03-01

    Full Text Available The size, shape and stability of a plant depend on the flexibility and integrity of its cell walls, which, at the same time, need to allow cell expansion for growth, while maintaining mechanical stability. Biomechanical studies largely vanished from the focus of plant science with the rapid progress of genetics and molecular biology since the mid-twentieth century. However, the development of more sensitive measurement tools renewed the interest in plant biomechanics in recent years, not only to understand the fundamental concepts of growth and morphogenesis, but also with regard to economically important areas in agriculture, forestry and the paper industry. Recent advances have clearly demonstrated that mechanical forces play a crucial role in cell and organ morphogenesis, which ultimately define plant morphology. In this article, we will briefly review the available methods to determine the mechanical properties of cell walls, such as atomic force microscopy (AFM and microindentation assays, and discuss their advantages and disadvantages. But we will focus on a novel methodological approach, called cellular force microscopy (CFM, and its automated successor, real-time CFM (RT-CFM.

  18. A new isotropic cell for studying the thermo-mechanical behavior of unsaturated expansive clays

    CERN Document Server

    Tang, Anh-Minh; Barnel, Nathalie

    2007-01-01

    This paper presents a new suction-temperature controlled isotropic cell that can be used to study the thermo-mechanical behavior of unsaturated expansive clays. The vapor equilibrium technique is used to control the soil suction; the temperature of the cell is controlled using a thermostat bath. The isotropic pressure is applied using a volume/pressure controller that is also used to monitor the volume change of soil specimen. Preliminary experimental results showed good performance of the cell.

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

    Directory of Open Access Journals (Sweden)

    Xuehui He

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  1. Microtubule networks for plant cell division

    NARCIS (Netherlands)

    Keijzer, de Jeroen; Mulder, B.M.; Janson, M.E.

    2014-01-01

    During cytokinesis the cytoplasm of a cell is divided to form two daughter cells. In animal cells, the existing plasma membrane is first constricted and then abscised to generate two individual plasma membranes. Plant cells on the other hand divide by forming an interior dividing wall, the so-called

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

    OpenAIRE

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

    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 mEL...

  3. Quantitative analysis of changes in actin microfilament contribution to cell plate development in plant cytokinesis

    Directory of Open Access Journals (Sweden)

    Sano Toshio

    2008-07-01

    Full Text Available Abstract Background Plant cells divide by the formation of new cross walls, known as cell plates, from the center to periphery of each dividing cell. Formation of the cell plate occurs in the phragmoplast, a complex structure composed of membranes, microtubules (MTs and actin microfilaments (MFs. Disruption of phragmoplast MTs was previously found to completely inhibit cell plate formation and expansion, indicative of their crucial role in the transport of cell plate membranes and materials. In contrast, disruption of MFs only delays cell plate expansion but does not completely inhibit cell plate formation. Despite such findings, the significance and molecular mechanisms of MTs and MFs remain largely unknown. Results Time-sequential changes in MF-distribution were monitored by live imaging of tobacco BY-2 cells stably expressing the GFP-actin binding domain 2 (GFP-ABD2 fusion protein, which vitally co-stained with the endocytic tracer, FM4-64, that labels the cell plate. During cytokinesis, MFs accumulated near the newly-separated daughter nuclei towards the emerging cell plate, and subsequently approached the expanding cell plate edges. Treatment with an actin polymerization inhibitor caused a decrease in the cell plate expansion rate, which was quantified using time-lapse imaging and regression analysis. Our results demonstrated time-sequential changes in the contribution of MFs to cell plate expansion; MF-disruption caused about a 10% decrease in the cell plate expansion rate at the early phase of cytokinesis, but about 25% at the late phase. MF-disruption also caused malformation of the emerging cell plate at the early phase, indicative of MF involvement in early cell plate formation and expansion. The dynamic movement of endosomes around the cell plate was also inhibited by treatment with an actin polymerization inhibitor and a myosin ATPase inhibitor, respectively. Furthermore, time-lapse imaging of the endoplasmic reticulum (ER revealed

  4. Enzymatic Modification of Plant Cell Wall Polysaccharides

    DEFF Research Database (Denmark)

    Øbro, Jens; Hayashi, Takahisa; Mikkelsen, Jørn Dalgaard

    2011-01-01

    for sustainable processes that replace chemical treatments with white biotechnology. Plants can contribute significantly to this sustainable process by producing plant or microbialenzymes in planta that are necessary for plant cell wall modification or total degradation. This will give rise to superior food......Plant cell walls are intricate structures with remarkable properties, widely used in almost every aspect of our life. Cell walls consist largely of complex polysaccharides and there is often a need for chemical and biochemical processing before industrial use. There is an increasing demand...... fibres, hydrocolloids, paper,textile, animal feeds or biofuels. Classical microbial-based fermentation systems could in the future face serious competition from plant-based expression systems for enzyme production. Plant expressed enzymes can either be targeted to specific cellular compartments...

  5. Enzymatic Modification of Plant Cell Wall Polysaccharides

    DEFF Research Database (Denmark)

    Øbro, Jens; Hayashi, Takahisa; Mikkelsen, Jørn Dalgaard

    2011-01-01

    fibres, hydrocolloids, paper,textile, animal feeds or biofuels. Classical microbial-based fermentation systems could in the future face serious competition from plant-based expression systems for enzyme production. Plant expressed enzymes can either be targeted to specific cellular compartments......Plant cell walls are intricate structures with remarkable properties, widely used in almost every aspect of our life. Cell walls consist largely of complex polysaccharides and there is often a need for chemical and biochemical processing before industrial use. There is an increasing demand...... for sustainable processes that replace chemical treatments with white biotechnology. Plants can contribute significantly to this sustainable process by producing plant or microbialenzymes in planta that are necessary for plant cell wall modification or total degradation. This will give rise to superior food...

  6. Dendritic cells control fibroblastic reticular network tension and lymph node expansion.

    Science.gov (United States)

    Acton, Sophie E; Farrugia, Aaron J; Astarita, Jillian L; Mourão-Sá, Diego; Jenkins, Robert P; Nye, Emma; Hooper, Steven; van Blijswijk, Janneke; Rogers, Neil C; Snelgrove, Kathryn J; Rosewell, Ian; Moita, Luis F; Stamp, Gordon; Turley, Shannon J; Sahai, Erik; Reis e Sousa, Caetano

    2014-10-23

    After immunogenic challenge, infiltrating and dividing lymphocytes markedly increase lymph node cellularity, leading to organ expansion. Here we report that the physical elasticity of lymph nodes is maintained in part by podoplanin (PDPN) signalling in stromal fibroblastic reticular cells (FRCs) and its modulation by CLEC-2 expressed on dendritic cells. We show in mouse cells that PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-associated protein kinase (ROCK). Engagement by CLEC-2 causes PDPN clustering and rapidly uncouples PDPN from RhoA/C activation, relaxing the actomyosin cytoskeleton and permitting FRC stretching. Notably, administration of CLEC-2 protein to immunized mice augments lymph node expansion. In contrast, lymph node expansion is significantly constrained in mice selectively lacking CLEC-2 expression in dendritic cells. Thus, the same dendritic cells that initiate immunity by presenting antigens to T lymphocytes also initiate remodelling of lymph nodes by delivering CLEC-2 to FRCs. CLEC-2 modulation of PDPN signalling permits FRC network stretching and allows for the rapid lymph node expansion--driven by lymphocyte influx and proliferation--that is the critical hallmark of adaptive immunity.

  7. Single-Pass, Closed-System Rapid Expansion of Lymphocyte Cultures for Adoptive Cell Therapy

    Science.gov (United States)

    Klapper, Jacob A.; Thomasian, Armen A.; Smith, Douglas M.; Gorgas, Gayle C.; Wunderlich, John R.; Smith, Franz O.; Hampson, Brian S.; Rosenberg, Steven A.; Dudley, Mark E.

    2009-01-01

    Adoptive cell therapy (ACT) for metastatic melanoma involves the ex vivo expansion and re-infusion of tumor infiltrating lymphocytes (TIL) obtained from resected specimens. With an overall objective response rate of fifty-six percent, this T-cell immunotherapy provides an appealing alternative to other therapies, including conventional therapies with lower response rates. However, there are significant regulatory and logistical concerns associated with the ex vivo activation and large scale expansion of these cells. The best current practice uses a rapid expansion protocol (REP) consisting of an ex vivo process that occurs in tissue culture flasks (T-flasks) and gas-permeable bags, utilizes OKT3 (anti-CD3 monoclonal antibody), recombinant human interleukin-2, and irradiated peripheral blood mononuclear cells to initiate rapid lymphocyte growth. A major limitation to the widespread delivery of therapy to large numbers of melanoma patients is the open system in which a REP is initiated. To address this problem, we have investigated the initiation, expansion and harvest at clinical scale of TIL in a closed-system continuous perfusion bioreactor. Each cell product met all safety criteria for patient treatment and by head-to-head comparison had a similar potency and phenotype as cells grown in control T-flasks and gas-permeable bags. However, the currently available bioreactor cassettes were limited in the total cell numbers that could be generated. This bioreactor may simplify the process of the rapid expansion of TIL under stringent regulatory conditions thereby enabling other institutions to pursue this form of ACT. PMID:19389403

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

    Science.gov (United States)

    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.

  9. In Vitro Efficient Expansion of Tumor Cells Deriving from Different Types of Human Tumor Samples

    Directory of Open Access Journals (Sweden)

    Ilaria Turin

    2014-03-01

    Full Text Available Obtaining human tumor cell lines from fresh tumors is essential to advance our understanding of antitumor immune surveillance mechanisms and to develop new ex vivo strategies to generate an efficient anti-tumor response. The present study delineates a simple and rapid method for efficiently establishing primary cultures starting from tumor samples of different types, while maintaining the immuno-histochemical characteristics of the original tumor. We compared two different strategies to disaggregate tumor specimens. After short or long term in vitro expansion, cells analyzed for the presence of malignant cells demonstrated their neoplastic origin. Considering that tumor cells may be isolated in a closed system with high efficiency, we propose this methodology for the ex vivo expansion of tumor cells to be used to evaluate suitable new drugs or to generate tumor-specific cytotoxic T lymphocytes or vaccines.

  10. Induced pluripotent stem cells from patients with Huntington's disease show CAG-repeat-expansion-associated phenotypes.

    Science.gov (United States)

    2012-08-03

    Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded stretch of CAG trinucleotide repeats that results in neuronal dysfunction and death. Here, The HD Consortium reports the generation and characterization of 14 induced pluripotent stem cell (iPSC) lines from HD patients and controls. Microarray profiling revealed CAG-repeat-expansion-associated gene expression patterns that distinguish patient lines from controls, and early onset versus late onset HD. Differentiated HD neural cells showed disease-associated changes in electrophysiology, metabolism, cell adhesion, and ultimately cell death for lines with both medium and longer CAG repeat expansions. The longer repeat lines were however the most vulnerable to cellular stressors and BDNF withdrawal, as assessed using a range of assays across consortium laboratories. The HD iPSC collection represents a unique and well-characterized resource to elucidate disease mechanisms in HD and provides a human stem cell platform for screening new candidate therapeutics.

  11. Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector

    Science.gov (United States)

    2013-01-01

    Introduction The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment. Methods In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry. Results Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene. Conclusions This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine. PMID:24406242

  12. discs large regulates somatic cyst cell survival and expansion in Drosophila testis

    Institute of Scientific and Technical Information of China (English)

    Fani Papagiannouli; Bernard M Mechler

    2009-01-01

    Gonad development requires a coordinated snma-germline interaction that ensures renewal and differentiation of germline and somatic stem cells to ultimately produce mature gametes. The Drosophila tumour suppressor gene discs sion, and formation of neuromuscular junctions. Here, we report the role of dig in testis development and its critical function in somatic cyst cells (SCCs). In these cells dig is primarily required for their survival and expansion, and contributes to spermatocyte cyst differentiation. Cell death primarily occurred in SCCs at the end of spermatogo-nial amplification at a time when Dig becomes restricted in wild-type (wt) testes to the distal somatic cells capping the growing spermatocyte cysts. RNAi depletion of dig transcripts in early SCCs fully prevented testis development, whereas depletion in late SCCs resulted in a breakdown of spermatocyte cyst structure and germ cell individualiza-tion. Specific dig expression in SCCs resulted in developmental rescue of dig mutant testes, whereas its expression in germ cells exerted no such effect, dig overexpression in wt testes led to spermatocyte cyst expansion at the expense of spermatogonial cysts. Our data demonstrate that dig is essentially required in SCCs for their survival, expansion, and differentiation, and for the encapsulation of the germline cells.

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

    Science.gov (United States)

    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.

  14. Regulation of cell division in higher plants. Final technical report

    Energy Technology Data Exchange (ETDEWEB)

    Jacobs, Thomas W.

    2000-02-29

    Research in the latter part of the grant period was divided into two parts: (1) expansion of the macromolecular tool kit for studying plant cell division; (2) experiments in which the roles played by plant cell cycle regulators were to be cast in the light of the emerging yeast and animal cell paradigm for molecular control of the mitotic cycle. The first objectives were accomplished to a very satisfactory degree. With regard to the second part of the project, we were driven to change our objectives for two reasons. First, the families of cell cycle control genes that we cloned encoded such closely related members that the prospects for success at raising distinguishing antisera against each were sufficiently dubious as to be impractical. Epitope tagging is not feasible in Pisum sativum, our experimental system, as this species is not realistically transformable. Therefore, differentiating the roles of diverse cyclins and cyclin-dependent kinases was problematic. Secondly, our procedure for generating mitotically synchronized pea root meristems for biochemical studies was far too labor intensive for the proposed experiments. We therefore shifted our objectives to identifying connections between the conserved proteins of the cell cycle engine and factors that interface it with plant physiology and development. In this, we have obtained some very exciting results.

  15. Clay hydration and crystal growth in expansive anhydritic claystone. The Ascó Power Plant case

    Science.gov (United States)

    Alonso, Eduardo; Ramon, Anna

    2015-04-01

    A large power plant directly founded on a hard claystone experienced, soon after the construction of the foundation slabs, a continuous heave developing at decreasing rate, which has been active for the last 35 years. When undisturbed (i.e., at some depth, in the range of several meters) Ascó claystone exhibits high unconfined compressive strengths (30-40 MPa). In high quality cores the rock has a massive aspect and discontinuities are difficult to observe. The rock has a Tertiary origin and horizontal layers at spacing of 1-4 m could be identified. Whitish seams of gypsum, bassanite or anhydrite are also observed within the reddish rock matrix. Minerals identified in deep cores are quartz (10%), calcite and dolomite (50-70%), clay minerals (10-20%) and gypsum and anhydrite (2-20%). Among the clay minerals, illite dominates (10%). Smectite or smectite-interbedded minerals do not amount in general to more than 5%. The undisturbed rock has a low porosity (6-11%) and low water content (2-5%). Because of the presence of hydrated sulphates, water content and degree of saturation (Sr=0.8-0.9 was found) are somewhat uncertain. However, high suctions were found in recovered cores. This rock changes into a weathered material at shallow depths. Mineralogy is not much affected but porosity increases to 22-29% and water content increases to 10-19%. Strength drops to small values (soil like) and a lower "in situ" suction has been measured (0.4-7.1 MPa). The added pore volume of the weathered material, if compared with the deep rock, is filled with water. The heave of the station was attributed to the hydration of undisturbed rock under the building slabs of the power plant. In fact, large excavations preceded the layout of foundations. As a result, atmospheric water had an easy access to the intact rock. The installation of a compacted soil fill around the buildings allowed the presence of a permanent water table which could infiltrate into the rock. Piezometric data provided

  16. Plant cell proliferation inside an inorganic host.

    Science.gov (United States)

    Perullini, Mercedes; Rivero, María Mercedes; Jobbágy, Matías; Mentaberry, Alejandro; Bilmes, Sara A

    2007-01-10

    In recent years, much attention has been paid to plant cell culture as a tool for the production of secondary metabolites and the expression of recombinant proteins. Plant cell immobilization offers many advantages for biotechnological processes. However, the most extended matrices employed, such as calcium-alginate, cannot fully protect entrapped cells. Sol-gel chemistry of silicates has emerged as an outstanding strategy to obtain biomaterials in which living cells are truly protected. This field of research is rapidly developing and a large number of bacteria and yeast-entrapping ceramics have already been designed for different applications. But even mild thermal and chemical conditions employed in sol-gel synthesis may result harmful to cells of higher organisms. Here we present a method for the immobilization of plant cells that allows cell growth at cavities created inside a silica matrix. Plant cell proliferation was monitored for a 6-month period, at the end of which plant calli of more than 1 mm in diameter were observed inside the inorganic host. The resulting hybrid device had good mechanical stability and proved to be an effective barrier against biological contamination, suggesting that it could be employed for long-term plant cell entrapment applications.

  17. Snf2 family gene distribution in higher plant genomes reveals DRD1 expansion and diversification in the tomato genome.

    Directory of Open Access Journals (Sweden)

    Joachim W Bargsten

    Full Text Available As part of large protein complexes, Snf2 family ATPases are responsible for energy supply during chromatin remodeling, but the precise mechanism of action of many of these proteins is largely unknown. They influence many processes in plants, such as the response to environmental stress. This analysis is the first comprehensive study of Snf2 family ATPases in plants. We here present a comparative analysis of 1159 candidate plant Snf2 genes in 33 complete and annotated plant genomes, including two green algae. The number of Snf2 ATPases shows considerable variation across plant genomes (17-63 genes. The DRD1, Rad5/16 and Snf2 subfamily members occur most often. Detailed analysis of the plant-specific DRD1 subfamily in related plant genomes shows the occurrence of a complex series of evolutionary events. Notably tomato carries unexpected gene expansions of DRD1 gene members. Most of these genes are expressed in tomato, although at low levels and with distinct tissue or organ specificity. In contrast, the Snf2 subfamily genes tend to be expressed constitutively in tomato. The results underpin and extend the Snf2 subfamily classification, which could help to determine the various functional roles of Snf2 ATPases and to target environmental stress tolerance and yield in future breeding.

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

    NARCIS (Netherlands)

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

  19. Plume expansion of a laser-induced plasma studied with the particle-in-cell method

    DEFF Research Database (Denmark)

    Ellegaard, Ole; Nedela, T; Urbassek, H;

    2002-01-01

     The initial stage of laser-induced plasma plume expansion from a solid in vacuum and the effect of the Coulomb field have been studied. We have performed a one-dimensional numerical calculation by mapping the charge on a computational grid according to the particle-in-cell (PIC) method of Birdsall...

  20. Plume expansion of a laser-induced plasma studied with the particle-in-cell method

    DEFF Research Database (Denmark)

    Ellegaard, O.; Nedelea, T.; Schou, Jørgen;

    2002-01-01

    The initial stage of laser-induced plasma plume expansion from a solid in vacuum and the effect of the Coulomb field have been studied. We have performed a one-dimensional numerical calculation by mapping the charge on a computational grid according to the particle-in-cell (PIC) method of Birdsall...

  1. An in vitro model of intra-epithelial expansion of transformed urothelial cells

    NARCIS (Netherlands)

    Rebel, J.M.J.; Boer, de W.I.; Thijssen, C.D.; Vermey, M.; Zwarthoff, E.C.; Kwast, van der T.H.

    1993-01-01

    Replacement of normal urothelium by pre-cancerous epithelium may explain the high recurrence rate of human bladder cancer. An in vitro model was designed in order to study the mechanisms of expansion of transformed urothelial cells at the expense of normal urothelium. For this purpose, mouse bladder

  2. Cell cycle phase expansion in nitrogen-limited cultures of Saccharomyces cerevisiae

    OpenAIRE

    1980-01-01

    The time and coordination of cell cycle events were examined in the budding yeast Saccharomyces cerevisiae. Whole-cell autoradiographic techniques and time-lapse photography were used to measure the duration of the S, G1, and G2 phases, and the cell cycle positions of "start" and bud emergence, in cells whose growth rates were determined by the source of nitrogen. It was observed that the G1, S, and G2 phases underwent a proportional expansion with increasing cell cycle length, with the S pha...

  3. Special plant species determines diet breadth of phytophagous insects: a study on host plant expansion of the host-specialized Aphis gossypii Glover.

    Directory of Open Access Journals (Sweden)

    Wei Wu

    Full Text Available Host specialization is a ubiquitous character of phytophagous insects. The polyphagous population is usually composed of some subpopulations that can use only a few closely related plants. Cotton-melon aphids, Aphis gossypii Glover exhibited strong host specialization, and the cotton- and cucurbits-specialized biotypes had been clearly identified. However, the experimental work that addressed the roles of plant species in determining diet breadth of phytophagous insects is rare. In the present study, we took the artificial host transfer method to assess the role of two special plants, zucchini Cucurbita zucchini L. and cowpea Vigna unguiculata (Linn. Walp, in regulating diet breadth of cotton- and cucurbits-specialized A. gossypii collected from cotton and cucumber fields and reared separately on the native host plant for ten years. The results showed that the cotton-specialized aphids did not directly use cucumber whereas the cucurbits-specialized did not use cotton regardless of the coexistence or separation of cotton and cucumber plants. Neither of the cotton- and cucurbits-specialized aphids could use capsicum Capsicum annuum, eggplant Solanum melongenahttp://en.wikipedia.org/wiki/Carolus_Linnaeus, tomato Solanum lycopersicum, maize Zea mayshttp://en.wikipedia.org/wiki/Carl_Linnaeus, and radish Raphanus sativus, however, both of them could use zucchini and cowpea. Moreover, the feeding experience on zucchini led the cotton-specialized aphids to use cucumber well and finally to be transformed into the cucurbits-specialized biotype. The short-term feeding experience on cowpea resulted in the diet breadth expansion of the cucurbits-specialized aphids to use cotton. On the other hand, the diet breadth expansion of the cucurbits- and cotton-specialized aphids was only realized by different species of plant. It concluded that the special host plant did induce the conversion of feeding habits in the cotton- and cucurbits-specialized aphids, and

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

    Science.gov (United States)

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

    2015-11-01

    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.

  5. Cell Production and Expansion in the Primary Root of Maize in Response to Low-Nitrogen Stress

    Institute of Scientific and Technical Information of China (English)

    GAO Kun; CHEN Fan-jun; YUAN Li-xing; MI Guo-hua

    2014-01-01

    Maize plants respond to low-nitrogen stress by enhancing root elongation. The underlying physiological mechanism remains unknown. Seedlings of maize (Zea mays L., cv. Zhengdan 958) were grown in hydroponics with the control (4 mmol L-1) or low-nitrogen (40 µmol L-1) for 12 d, supplied as nitrate. Low nitrogen enhanced root elongation rate by 4.1-fold, accompanied by increases in cell production rate by 2.2-fold, maximal elemental elongation rate (by 2.5-fold), the length of elongation zone (by 1.5-fold), and ifnal cell length by 1.8-fold. On low nitrogen, the higher cell production rate resulted from a higher cell division rate and in fact the number of dividing cells was reduced. Consequently, the residence time of a cell in the division zone tended to be shorter under low nitrogen. In addition, low nitrogen increased root diameter, an increase that occurred speciifcally in the cortex and was accompanied by an increase in cell number. It is concluded that roots elongates in response to low-nitrogen stress by accelerating cell production and expansion.

  6. PvRACK1 loss-of-function impairs cell expansion and morphogenesis in Phaseolus vulgaris L. root nodules.

    Science.gov (United States)

    Islas-Flores, Tania; Guillén, Gabriel; Alvarado-Affantranger, Xóchitl; Lara-Flores, Miguel; Sánchez, Federico; Villanueva, Marco A

    2011-07-01

    Receptor for activated C kinase (RACK1) is a highly conserved, eukaryotic protein of the WD-40 repeat family. Its peculiar β-propeller structure allows its interaction with multiple proteins in various plant signal-transduction pathways, including those arising from hormone responses, development, and environmental stress. During Phaseolus vulgaris root development, RACK1 (PvRACK1) mRNA expression was induced by auxins, abscissic acid, cytokinin, and gibberellic acid. In addition, during P. vulgaris nodule development, PvRACK1 mRNA was highly accumulated at 12 to 15 days postinoculation, suggesting an important role after nodule meristem initiation and Rhizobium nodule infection. PvRACK1 transcript accumulation was downregulated by a specific RNA interference construct which was expressed in transgenic roots of composite plants of P. vulgaris inoculated with Rhizobium tropici. PvRACK1 downregulated transcript levels were monitored by quantitative reverse-transcription polymerase chain reaction analysis in individual transgenic roots and nodules. We observed a clear phenotype in PvRACK1-knockdown nodules, in which nodule number and nodule cell expansion were impaired, resulting in altered nodule size. Microscopic analysis indicated that, in PvRACK1-knockdown nodules, infected and uninfected cells were considerably smaller (80 and 60%, respectively) than in control nodules. In addition, noninfected cells and symbiosomes in silenced nodules showed significant defects in membrane structure under electron microscopy analysis. These findings indicate that PvRACK1 has a pivotal role in cell expansion and in symbiosome and bacteroid integrity during nodule development.

  7. Arx polyalanine expansion in mice leads to reduced pancreatic α-cell specification and increased α-cell death.

    Directory of Open Access Journals (Sweden)

    Crystal L Wilcox

    Full Text Available ARX/Arx is a homeodomain-containing transcription factor necessary for the specification and early maintenance of pancreatic endocrine α-cells. Many transcription factors important to pancreas development, including ARX/Arx, are also crucial for proper brain development. Although null mutations of ARX in human patients result in the severe neurologic syndrome XLAG (X-linked lissencephaly associated with abnormal genitalia, the most common mutation is the expansion of the first polyalanine tract of ARX, which results primarily in the clinical syndrome ISSX (infantile spasms. Mouse models of XLAG, ISSX and other human ARX mutations demonstrate a direct genotype-phenotype correlation in ARX-related neurologic disorders. Furthermore, mouse models utilizing a polyalanine tract expansion mutation have illustrated critical developmental differences between null mutations and expansion mutations in the brain, revealing context-specific defects. Although Arx is known to be required for the specification and early maintenance of pancreatic glucagon-producing α-cells, the consequences of the Arx polyalanine expansion on pancreas development remain unknown. Here we report that mice with an expansion mutation in the first polyalanine tract of Arx exhibit impaired α-cell specification and maintenance, with gradual α-cell loss due to apoptosis. This is in contrast to the re-specification of α-cells into β- and δ-cells that occurs in mice null for Arx. Overall, our analysis of an Arx polyalanine expansion mutation on pancreatic development suggests that impaired α-cell function might also occur in ISSX patients.

  8. Electron Tomography in Plant Cell Biology

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    This review focuses on the contribution of electron tomography-based techniques to our understanding of cellular processes in plant cells. Electron microscopy techniques have evolved to provide better three-dimensional resolution and improved preservation of the subcellular components. In particular, the combination of cryofixation/freeze substitution and electron tomography have allowed plant cell biologists to image organelles and macromolecular complexes in their native cellular context with unprecedented three-dimensional resolution (4-7 nm). Until now, electron tomography has been applied in plant cell biology for the study of cytokinesis, Golgi structure and trafficking, formation of plant endosome/prevacuolar compartments, and organization of photosynthetic membranes. We discuss in this review the new insights that these tomographic studies have brought to the plant biology field.

  9. Conserved roles of CrRLK1L receptor-like kinases in cell expansion and reproduction from algae to angiosperms

    Directory of Open Access Journals (Sweden)

    Sergio Galindo Trigo

    2016-08-01

    Full Text Available Receptor-like kinases (RLKs are regulators of plant development through allowing cells to sense their extracellular environment. They facilitate detection of local endogenous signals, in addition to external biotic and abiotic stimuli. The Catharanthus roseus RLK1-like (CrRLK1L protein kinase subfamily, which contains FERONIA, plays a central role in regulating fertilization and in cell expansion mechanisms such as cell elongation and tip growth, as well as having indirect links to plant-pathogen interactions. Several components of CrRLK1L signaling pathways have been identified, including an extracellular ligand, coreceptors and downstream signaling elements. The presence and abundance of the CrRLK1L proteins in the plant kingdom suggest an origin within the Streptophyta lineage, with a notable increase in prevalence in the seeded land plants. Given the function of the sole CrRLK1L protein in a charophycean alga, the possibility of a conserved role in detection and/or regulation of cell wall integrity throughout the Strephtophytes is discussed. Orthologs of signaling pathway components are also present in extant representatives of non-vascular land plants and early vascular land plants including the liverwort Marchantia polymorpha, the moss Physcomitrella patens and the lycophyte Selaginella moellendorffii. Deciphering the roles in development of the CrRLK1L protein kinases in early diverging land plants will provide insights into their ancestral function, furthering our understanding of this diversified subfamily of receptors in higher plants.

  10. Portsmouth Gaseous Diffusion Plant expansion: final environmental statement. Volume 2. Appendices. [Appendices only

    Energy Technology Data Exchange (ETDEWEB)

    Liverman, James L.

    1977-09-01

    Volume 2 is comprised of appendices: Portsmouth Gaseous Diffusion Plant Existing Facilities; Ecology; Civic Involvement; Social Analysis; Population Projections; Toxicity of Air Pollutants to Biota at Portsmouth Gaseous Diffusion Plant; and Assessment of Noise Effects of an Add-On to the Portsmouth Gaseous Diffusion Plant. (LK)

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  13. DIRECT FUEL/CELL/TURBINE POWER PLANT

    Energy Technology Data Exchange (ETDEWEB)

    Hossein Ghezel-Ayagh

    2004-05-01

    This report includes the progress in development of Direct FuelCell/Turbine{reg_sign} (DFC/T{reg_sign}) power plants for generation of clean power at very high efficiencies. The DFC/T power system is based on an indirectly heated gas turbine to supplement fuel cell generated power. The DFC/T power generation concept extends the high efficiency of the fuel cell by utilizing the fuel cell's byproduct heat in a Brayton cycle. Features of the DFC/T system include: electrical efficiencies of up to 75% on natural gas, 60% on coal gas, minimal emissions, simplicity in design, direct reforming internal to the fuel cell, reduced carbon dioxide release to the environment, and potential cost competitiveness with existing combined cycle power plants. FCE successfully completed testing of the pre-alpha DFC/T hybrid power plant. This power plant was constructed by integration of a 250kW fuel cell stack and a microturbine. The tests of the cascaded fuel cell concept for achieving high fuel utilizations were completed. The tests demonstrated that the concept results in higher power plant efficiency. Also, the preliminary design of a 40 MW power plant including the key equipment layout and the site plan was completed.

  14. Quantification of fluorescent reporters in plant cells.

    Science.gov (United States)

    Pound, Michael; French, Andrew P; Wells, Darren M

    2015-01-01

    Fluorescent reporters are powerful tools for plant research. Many studies require accurate determination of fluorescence intensity and localization. Here, we describe protocols for the quantification of fluorescence intensity in plant cells from confocal laser scanning microscope images using semiautomated software and image analysis techniques.

  15. The spectrum of chronic CD8+ T-cell expansions: clinical features in 14 patients.

    Directory of Open Access Journals (Sweden)

    Etienne Ghrenassia

    Full Text Available Chronic CD8(+ T-cell expansions can result in parotid gland swelling and other organ infiltration in HIV-infected patients, or in persistent cytopenias. We report 14 patients with a CD8+ T-cell expansion to better characterize the clinical spectrum of this ill-defined entity. Patients (9 women/5 men were 65 year-old (range, 25-74. Six patients had ≥ 1 symptomatic organ infiltration, and 9 had ≥ 1 cytopenia with a CD8(+ (>50% of total lymphocyte count and/or a CD8(+/CD57(+ (>30% of total lymphocyte count T-cell expansion for at least 3 months. One patient had both manifestations. A STAT3 mutation, consistent with the diagnosis of large granular lymphocyte leukemia, was found in 2 patients with cytopenia. Organ infiltration involved lymph nodes, the liver, the colon, the kidneys, the skin and the central nervous system. Three patients had a HIV infection for 8 years (range, 0.5-20 years. Two non-HIV patients with hypogammaglobulinemia had been treated with a B-cell depleting monoclonal antibody (rituximab for a lymphoma. One patient had a myelodysplastic syndrome with colon infiltration and agranulocytosis. The outcome was favorable with efficient antiretroviral therapy and steroids in HIV-infected patients and intravenous immunoglobulins in 2/3 non-HIV patients. Six patients had an agranulocytosis of favorable outcome with granulocyte-colony stimulating factor only (3 cases, cyclophosphamide, methotrexate and cyclosporine A, or no treatment (1 case each. Three patients had a pure red cell aplasia, of favorable outcome in 2 cases with methotrexate and cyclosporine A; one patient was unresponsive. Chronic CD8(+ T-cell expansions with organ infiltration in immunocompromised patients may involve other organs than parotid glands; they are non clonal and of favorable outcome after correction of the immune deficiency and/or steroids. In patients with bone marrow infiltration and unexplained cytopenia, CD8(+ T-cell expansions can be clonal or not

  16. Cytomegalovirus-Driven Adaptive-Like Natural Killer Cell Expansions Are Unaffected by Concurrent Chronic Hepatitis Virus Infections

    Directory of Open Access Journals (Sweden)

    David F. G. Malone

    2017-05-01

    Full Text Available Adaptive-like expansions of natural killer (NK cell subsets are known to occur in response to human cytomegalovirus (CMV infection. These expansions are typically made up of NKG2C+ NK cells with particular killer-cell immunoglobulin-like receptor (KIR expression patterns. Such NK cell expansion patterns are also seen in patients with viral hepatitis infection. Yet, it is not known if the viral hepatitis infection promotes the appearance of such expansions or if effects are solely attributed to underlying CMV infection. In sizeable cohorts of CMV seropositive hepatitis B virus (HBV, hepatitis C virus (HCV, and hepatitis delta virus (HDV infected patients, we analyzed NK cells for expression of NKG2A, NKG2C, CD57, and inhibitory KIRs to assess the appearance of NK cell expansions characteristic of what has been seen in CMV seropositive healthy individuals. Adaptive-like NK cell expansions observed in viral hepatitis patients were strongly associated with CMV seropositivity. The number of subjects with these expansions did not differ between CMV seropositive viral hepatitis patients and corresponding healthy controls. Hence, we conclude that adaptive-like NK cell expansions observed in HBV, HCV, and/or HDV infected individuals are not caused by the chronic hepatitis infections per se, but rather are a consequence of underlying CMV infection.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    R. García-Muñoz

    2009-01-01

    Full Text Available 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.

  19. Roles of host plants in boll weevil range expansion beyond tropical Mesoamerica

    Science.gov (United States)

    New findings on boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), biology and ecology have had repercussions on the current level of understanding about short- and long-range boll weevil dispersal, and range expansion from its original tropical Mesoamerican habitat. The w...

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

    Science.gov (United States)

    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.

  1. Disposable Bioreactors for Plant Micropropagation and Mass Plant Cell Culture

    Science.gov (United States)

    Ducos, Jean-Paul; Terrier, Bénédicte; Courtois, Didier

    Different types of bioreactors are used at Nestlé R&D Centre - Tours for mass propagation of selected plant varieties by somatic embryogenesis and for large scale culture of plants cells to produce metabolites or recombinant proteins. Recent studies have been directed to cut down the production costs of these two processes by developing disposable cell culture systems. Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has recently been set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-L glass bioreactors. An improved process has been developed using a 10-L disposable bioreactor consisting of a bag containing a rigid plastic box ('Box-in-Bag' bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design. For large scale cell culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 L working volumes, validated with several plant species ('Wave and Undertow' and 'Slug Bubble' bioreactors). The advantages and the limits of these new types of bioreactor are discussed, based mainly on our own experience on coffee somatic embryogenesis and mass cell culture of soya and tobacco.

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

    Science.gov (United States)

    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

  3. Control of Leaf Expansion by Nitrogen Nutrition in Sunflower Plants : ROLE OF HYDRAULIC CONDUCTIVITY AND TURGOR.

    Science.gov (United States)

    Radin, J W; Boyer, J S

    1982-04-01

    Nitrogen nutrition strongly affected the growth rate of young sunflower (Helianthus annuus L.) leaves. When plants were grown from seed on either of two levels of N availability, a 33% decrease in tissue N of expanding leaves was associated with a 75% overall inhibition of leaf growth. Almost all of the growth inhibition resulted from a depression of the daytime growth rate. Measurements of pressure-induced water flux through roots showed that N deficiency decreased root hydraulic conductivity by about half. Thus, N deficiency lowered the steady-state water potential of expanding leaves during the daytime when transpiration was occurring. As a result, N-deficient leaves were unable to maintain adequate turgor for growth in the daytime. N deficiency also decreased the hydraulic conductivity for water movement into expanding leaf cells in the absence of transpiration, but growth inhibition at night was much less than in the daytime. N nutrition had no detectable effects on plastic extensibility or the threshold turgor for growth.

  4. Plant cells in vitro under altered gravity.

    Science.gov (United States)

    Klymchuk, D O

    1998-07-01

    Establishing the role of gravity in plant requires information about how gravity regulates the metabolism of individual cells. Plant cells and tissues in vitro are valuable models for such purpose. Disrupted intercellular relations in such models have allowed to elucidate both the gravity role in non-specialised to gravity plant cells and the correlative relation role of an intact plant organism. The data obtained from non-numerous space and clinostat experiments with plant cells in vitro have demonstrated that their metabolism is sensitive to g-environment. The most experiments have shown a decrease in the biomass production and cell proliferation of spaceflight samples compared with ground controls, although there is study reporting of increased biomass production in an anise suspension culture and D. carota crown gall tissue culture. At the same time, results of experiments with single carrot cells and tomato callus culture demonstrated similarities in differentiation process in microgravity and in ground controls. Noted ultrastructural arrangement in cells, especially mitochondria and plastids, have been related to altered energy load and functions of organelles in microgravity, as well as changes in the lipid peroxidation and the content of malonic dyaldehyde in a haplopappus tissue culture under altered gravity supposed with modification of membrane structural-functional state. This article focuses on growth aspects of the cultured cells in microgravity and under clinostat conditions and considers those aspects that require further analysis.

  5. Addition of Interleukin-21 for Expansion of T-Cells for Adoptive Immunotherapy of Murine Melanoma

    Directory of Open Access Journals (Sweden)

    Christine Kathryn Zoon

    2015-04-01

    Full Text Available We previously demonstrated that interleukin (IL-7/15 was superior to IL-2 for expansion of T cells in vitro for adoptive immunotherapy. We sought to ascertain whether IL-21 would further improve yield and therapeutic efficacy of T cells in culture. Naïve T cell receptor (TcR transgenic splenocytes or antigen-sensitized lymph node cells were harvested from PMEL-1 mice and exposed to bryostatin-1 and ionomycin (B/I for 18 h. Cells were then cultured in IL-2, IL-21, IL-7/15 or IL-7/15/21 for six days. Harvested cells were analyzed by flow cytometry and used to treat C57Bl/6 mice injected intravenously with B16 melanoma. Lungs were harvested and metastases counted 14 days after treatment. Culturing lymphocytes in IL-7/15/21 increased expansion compared to IL-2 or IL-7/15. IL-21 and IL-7/15/21 increased CD8+ cells compared to IL-2 or IL-7/15. IL-21 preferentially expanded a CD8+CD44−CD62L+ T “naïve” population, whereas IL-7/15/21 increased CD8+CD44+CD62Lhigh central-memory T cells. T cells grown in IL-7/15/21 were more effective at reducing metastases than IL-2. The addition of IL-21 to IL-7/15 induced greater expansion of lymphocytes in culture and increased the yield of CD8+ T central-memory cells vs. IL-7/15 alone. This may have significant impact on future clinical trials of adoptive immunotherapy, particularly for generating adequate numbers of lymphocytes for treatment.

  6. Isolation of plant cell wall proteins

    OpenAIRE

    Jamet, Elisabeth; Boudart, Georges; Borderies, Gisèle; Charmont, Stéphane; Lafitte, Claude; Rossignol, Michel; Canut, Hervé; Pont-Lezica, Rafael F

    2007-01-01

    The quality of a proteomic analysis of a cell compartment strongly depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific drawbacks: (i) the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP) during the isolation procedure; (ii) polysaccharide networks of cellulose, hemicelluloses and pectins form potential traps for contaminants such as intracellular proteins; (iii) the presence of proteins ...

  7. HOXA4 induces expansion of hematopoietic stem cells in vitro and confers enhancement of pro-B-cells in vivo.

    Science.gov (United States)

    Fournier, Marilaine; Lebert-Ghali, Charles-Étienne; Krosl, Gorazd; Bijl, Janet J

    2012-01-01

    Members of the homeobox (Hox) gene family are known to mediate expansion of hematopoietic stem cells (HSCs) and progenitors. The absence of oncogenic properties promoted HOXB4 as prime candidate in the quest to expand HSCs for clinical purposes. Despite its potential to expand HSCs, studies with mutant mice showed that Hoxb4 is not essential for HSC generation and function under physiological conditions. Expression studies and the existence of functional redundancy in particular between paralog Hox genes suggest that HOXA4 might have potent properties to expand HSCs. Here we measured the ability of HOXA4 to promote ex vivo expansion of HSCs and progenitors using retrovirus-mediated overexpression. Our results provide evidence that HOXA4-transduced HSCs and primitive progenitors expand in culture conditions and demonstrate that the potential of expanded HOXA4 HSCs to give rise to mature myeloid and lymphoid progeny in normal proportions remained intact. Interestingly, constitutive overexpression of HOXA4 resulted in an unbalanced expansion of lymphoid/myeloid progenitors in bone marrow chimeras favorable to B-cell progenitors responsive to interleukin-7. This expansion was specific for these progenitors and not for the more primitive Whitlock-Witte-initiating cells. These data indicate that early stages of B-cell development associated with proliferation are in particular sensitive to HOXA4. Thus, this study supports the potential use of HOXA4 to expand both HSCs and B-cell progenitor populations for therapeutic strategies.

  8. Conserved Roles of CrRLK1L Receptor-Like Kinases in Cell Expansion and Reproduction from Algae to Angiosperms

    Science.gov (United States)

    Galindo-Trigo, Sergio; Gray, Julie E.; Smith, Lisa M.

    2016-01-01

    Receptor-like kinases (RLKs) are regulators of plant development through allowing cells to sense their extracellular environment. They facilitate detection of local endogenous signals, in addition to external biotic and abiotic stimuli. The Catharanthus roseus RLK1-like (CrRLK1L) protein kinase subfamily, which contains FERONIA, plays a central role in regulating fertilization and in cell expansion mechanisms such as cell elongation and tip growth, as well as having indirect links to plant–pathogen interactions. Several components of CrRLK1L signaling pathways have been identified, including an extracellular ligand, coreceptors, and downstream signaling elements. The presence and abundance of the CrRLK1L proteins in the plant kingdom suggest an origin within the Streptophyta lineage, with a notable increase in prevalence in the seeded land plants. Given the function of the sole CrRLK1L protein in a charophycean alga, the possibility of a conserved role in detection and/or regulation of cell wall integrity throughout the Strephtophytes is discussed. Orthologs of signaling pathway components are also present in extant representatives of non-vascular land plants and early vascular land plants including the liverwort Marchantia polymorpha, the moss Physcomitrella patens and the lycophyte Selaginella moellendorffii. Deciphering the roles in development of the CrRLK1L protein kinases in early diverging land plants will provide insights into their ancestral function, furthering our understanding of this diversified subfamily of receptors in higher plants. PMID:27621737

  9. Ex vivo Expansion and Differentiation of Mesenchymal Stem Cells from Goat Bone Marrow

    Directory of Open Access Journals (Sweden)

    Mohamadreza Baghaban Eslaminejad

    2009-06-01

    Full Text Available Objective(sMesenchymal stem cells (MSCs from large animals as goat which is genetically more closely related tohuman have rarely been gained attentions. The present study tried to isolate and characterize MSCs fromgoat bone marrow.Materials and MethodsFibroblastic cells appeared in goat marrow cell culture were expanded through several subcultures.Passaged-3 cells were then differentiated among the osteogenic, adipogenic and chondrogenic cell lineagesto determine their MSC nature. Differentiations were determined by RT-PCR analysis of related geneexpression. To identify the best culture conditions for propagation, passage-3 cells were plated either atvarying cell densities or different fetal bovine serum (FBS concentrations for a week, at the end of whichthe cultures were statistically compared with respect to the cell proliferation. In this study, we alsodetermined goat MSC population doubling time (PDT as the index of their in vitro expansion rate.ResultsPassage-3 fibroblastic cells tended to differentiate into skeletal cell lineages. This was evident in bothspecific staining as well as the specific gene expression profile. Moreover, there appeared to be moreexpansion when the cultures were initiated at 100 cells/cm2 in a medium supplemented with 15% FBS. Arelatively short PDT (24.94±2.67 hr was a reflection of the goat MSC rapid rate of expansion.ConclusionTaken together, fibroblastic cells developed at goat marrow cell culture are able to differentiate into skeletalcell lineages. They undergo extensive proliferation when being plated at low cell density in 15% FBSconcentration.Keywords: Adipogenesis, Bovine serum, Cell seeding density, Chondrogenesis, Goat mesenchymal stemcells, Osteogenesis

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

    Science.gov (United States)

    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

  11. Expansion of gd T cells in patients infected with cutaneous leishmaniasis with and without glucantime therapy

    Directory of Open Access Journals (Sweden)

    Haideh Darabi

    2002-10-01

    Full Text Available The expansion of gd T cells in patients with active cutaneous leishmaniasis, with or without glucantime therapy, was investigated. Twenty patients with local cutaneous leishmaniasis including glucantime-treated (n=10 and untreated (n=10 patients were selected. The controls were healthy individuals (n=10 living in endemic areas. Whole blood was obtained and the T cell subpopulations were analyzed by flow cytometry. Significantly more gd CD3+ T cells were observed in untreated patients (15.9% ± 5.9, when compared with glucantime-treated patients (4.6% ± 1.4 and controls (5.3% ± 2.3. On the other hand, when the percentages of ab CD3+ T-cells were analyzed different results were obtained. A significant increase in ab T cells was seen in glucantime-treated patients (62.4% ± 7.6, when compared to the untreated patients (55.7% ± 5.5 and controls (55.1% ± 9.6. The percentage of total CD3+ T cells was statistically greater in both glucantime-treated (68.8% ± 7.4 and untreated patients (73.4% ± 5.9 when compared to the controls (61% ± 10.3. These results are consistent with previous results on the expansion of gdT cells during the course of cutaneous leishmaniasis. They also indicate that glucantime therapy can reverse the expansion of gdT cells and as a result increase the percentages of ab CD3+ T cells.

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

    Directory of Open Access Journals (Sweden)

    Will J. McLean

    2017-02-01

    Full Text Available 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.

  13. Effects of mutations in the Arabidopsis Cold Shock Domain Protein 3 (AtCSP3) gene on leaf cell expansion.

    Science.gov (United States)

    Yang, Yongil; Karlson, Dale

    2012-08-01

    The cold shock domain is among the most evolutionarily conserved nucleic acid binding domains from prokaryotes to higher eukaryotes, including plants. Although eukaryotic cold shock domain proteins have been extensively studied as transcriptional and post-transcriptional regulators during various developmental processes, their functional roles in plants remains poorly understood. In this study, AtCSP3 (At2g17870), which is one of four Arabidopsis thaliana c old s hock domain proteins (AtCSPs), was functionally characterized. Quantitative RT-PCR analysis confirmed high expression of AtCSP3 in reproductive and meristematic tissues. A homozygous atcsp3 loss-of-function mutant exhibits an overall reduced seedling size, stunted and orbicular rosette leaves, reduced petiole length, and curled leaf blades. Palisade mesophyll cells are smaller and more circular in atcsp3 leaves. Cell size analysis indicated that the reduced size of the circular mesophyll cells appears to be generated by a reduction of cell length along the leaf-length axis, resulting in an orbicular leaf shape. It was also determined that leaf cell expansion is impaired for lateral leaf development in the atcsp3 loss-of-function mutant, but leaf cell proliferation is not affected. AtCSP3 loss-of-function resulted in a dramatic reduction of LNG1 transcript, a gene that is involved in two-dimensional leaf polarity regulation. Transient subcellular localization of AtCSP3 in onion epidermal cells confirmed a nucleocytoplasmic localization pattern. Collectively, these data suggest that AtCSP3 is functionally linked to the regulation of leaf length by affecting LNG1 transcript accumulation during leaf development. A putative function of AtCSP3 as an RNA binding protein is also discussed in relation to leaf development.

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

    Science.gov (United States)

    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.

  15. Pax3-induced expansion enables the genetic correction of dystrophic satellite cells.

    Science.gov (United States)

    Filareto, Antonio; Rinaldi, Fabrizio; Arpke, Robert W; Darabi, Radbod; Belanto, Joseph J; Toso, Erik A; Miller, Auston Z; Ervasti, James M; McIvor, R Scott; Kyba, Michael; Perlingeiro, Rita Cr

    2015-01-01

    Satellite cells (SCs) are indispensable for muscle regeneration and repair; however, due to low frequency in primary muscle and loss of engraftment potential after ex vivo expansion, their use in cell therapy is currently unfeasible. To date, an alternative to this limitation has been the transplantation of SC-derived myogenic progenitor cells (MPCs), although these do not hold the same attractive properties of stem cells, such as self-renewal and long-term regenerative potential. We develop a method to expand wild-type and dystrophic fresh isolated satellite cells using transient expression of Pax3. This approach can be combined with genetic correction of dystrophic satellite cells and utilized to promote muscle regeneration when transplanted into dystrophic mice. Here, we show that SCs from wild-type and dystrophic mice can be expanded in culture through transient expression of Pax3, and these expanded activated SCs can regenerate the muscle. We test this approach in a gene therapy model by correcting dystrophic SCs from a mouse lacking dystrophin using a Sleeping Beauty transposon carrying the human μDYSTROPHIN gene. Transplantation of these expanded corrected cells into immune-deficient, dystrophin-deficient mice generated large numbers of dystrophin-expressing myofibers and improved contractile strength. Importantly, in vitro expanded SCs engrafted the SC compartment and could regenerate muscle after secondary injury. These results demonstrate that Pax3 is able to promote the ex vivo expansion of SCs while maintaining their stem cell regenerative properties.

  16. Quantification of plant cell coupling with live-cell microscopy

    DEFF Research Database (Denmark)

    Liesche, Johannes; Schulz, Alexander

    2015-01-01

    cell wall interface. Transport through plasmodesmata, the cell wall channels that directly connect plant cells, is regulated not only by a fixed size exclusion limit, but also by physiological and pathological adaptation. The noninvasive approach described here offers the possibility of precisely......Movement of nutrients and signaling compounds from cell to cell is an essential process for plant growth and development. To understand processes such as carbon allocation, cell communication, and reaction to pathogen attack it is important to know a specific molecule’s capacity to pass a specific...

  17. Embryonic stem cells conditioned medium enhances Wharton's jelly-derived mesenchymal stem cells expansion under hypoxic condition.

    Science.gov (United States)

    Prasajak, Patcharee; Rattananinsruang, Piyaporn; Chotinantakul, Kamonnaree; Dechsukhum, Chavaboon; Leeanansaksiri, Wilairat

    2015-05-01

    Mesenchymal stem cells (MSCs) are accepted as a promising tool for therapeutic purposes. However, low proliferation and early senescence are still main obstacles of MSCs expansion for using as cell-based therapy. Thus, clinical scale of cell expansion is needed to obtain a large number of cells serving for further applications. In this study, we investigated the value of embryonic stem cells conditioned medium (ESCM) for in vitro expansion of Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) as compared to typical culture medium for MSCs, Dulbecco's modified Eagle's medium with 1.0 g/l glucose (DMEM-LG) supplemented with 10 % FBS, under hypoxic condition. The expanded cells from ESCM (ESCM-MSCs) and DMEM-LG (DMEM-MSCs) were characterized for both phenotype and biological activities including proliferation rate, population doubling time, cell cycle distribution and MSCs characteristics. ESCM and DMEM-LG could enhance WJ-MSCs proliferation as 204.66 ± 10.39 and 113.77 ± 7.89 fold increase at day 12, respectively. ESCM-MSCs could express pluripotency genes including Oct-4, Oct-3/4, Nanog, Klf-4, C-Myc and Sox-2 both in early and late passages whereas the downregulations of Oct-4 and Nanog were detected in late passage cells of DMEM-MSCs. The 2 cell populations also showed common MSCs characteristics including normal cell cycle, fibroblastic morphology, cell surface markers expressions (CD29(+), CD44(+), CD90(+), CD34(-), CD45(-)) and differentiation capacities into adipogenic, chondrogenic and osteogenic lineages. Moreover, our results revealed that ESCM exhibited as a rich source of several factors which are required for supportive WJ-MSCs proliferation. In conclusion, ESCM under hypoxic condition could accelerate WJ-MSCs expansion while maintaining their pluripotency properties. Our knowledge provide short term and cost-saving in WJ-MSCs expansion which has benefit to overcome insufficient cell numbers for clinical applications by reusing the

  18. Quantitative Aspects of Cyclosis in Plant Cells.

    Science.gov (United States)

    Howells, K. F.; Fell, D. A.

    1979-01-01

    Describes an exercise which is currently used in a course in cell physiology at Oxford Polytechnic in England. This exercise can give students some idea of the molecular events involved in bringing about movement of chloroplasts (and other organelles) in plant cells. (HM)

  19. ABA inhibits embryo cell expansion and early cell division events during coffee (Coffea arabica 'Rubi') seed germination.

    Science.gov (United States)

    Da Silva, E A Amaral; Toorop, Peter E; Van Lammeren, André A M; Hilhorst, Henk W M

    2008-09-01

    Coffee seed germination represents an interplay between the embryo and the surrounding endosperm. A sequence of events in both parts of the seed determines whether germination will be successful or not. Following previous studies, the aim here was to further characterize the morphology of endosperm degradation and embryo growth with respect to morphology and cell cycle, and the influence of abscisic acid on these processes. Growth of cells in a fixed region of the axis was quantified from light micrographs. Cell cycle events were measured by flow cytometry and by immunocytochemistry, using antibodies against beta-tubulin. Aspects of the endosperm were visualized by light and scanning electron microscopy. The embryonic axis cells grew initially by isodiametric expansion. This event coincided with reorientation and increase in abundance of microtubules and with accumulation of beta-tubulin. Radicle protrusion was characterized by a shift from isodiametric expansion to elongation of radicle cells and further accumulation of beta-tubulin. Early cell division events started prior to radicle protrusion. Abscisic acid decreased the abundance of microtubules and inhibited the growth of the embryo cells, the reorganization of the microtubules, DNA replication in the embryonic axis, the formation of a protuberance and the completion of germination. The endosperm cap cells had smaller and thinner cell walls than the rest of the endosperm. Cells in the endosperm cap displayed compression followed by loss of cell integrity and the appearance of a protuberance prior to radicle protrusion. Coffee seed germination is the result of isodiametric growth of the embryo followed by elongation, at the expense of integrity of endosperm cap cells. The cell cycle, including cell division, is initiated prior to radicle protrusion. ABA inhibits expansion of the embryo, and hence subsequent events, including germination.

  20. Identification of Two New Mechanisms That Regulate Fruit Growth by Cell Expansion in Tomato

    Directory of Open Access Journals (Sweden)

    Constance Musseau

    2017-06-01

    Full Text Available Key mechanisms controlling fruit weight and shape at the levels of meristem, ovary or very young fruit have already been identified using natural tomato diversity. We reasoned that new developmental modules prominent at later stages of fruit growth could be discovered by using new genetic and phenotypic diversity generated by saturated mutagenesis. Twelve fruit weight and tissue morphology mutants likely affected in late fruit growth were selected among thousands of fruit size and shape EMS mutants available in our tomato EMS mutant collection. Their thorough characterization at organ, tissue and cellular levels revealed two major clusters controlling fruit growth and tissue morphogenesis either through (i the growth of all fruit tissues through isotropic cell expansion or (ii only the growth of the pericarp through anisotropic cell expansion. These likely correspond to new cell expansion modules controlling fruit growth and tissue morphogenesis in tomato. Our study therefore opens the way for the identification of new gene regulatory networks controlling tomato fruit growth and morphology.

  1. Ricin Trafficking in Plant and Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Robert A. Spooner

    2011-06-01

    Full Text Available Ricin is a heterodimeric plant protein that is potently toxic to mammalian and many other eukaryotic cells. It is synthesized and stored in the endosperm cells of maturing Ricinus communis seeds (castor beans. The ricin family has two major members, both, lectins, collectively known as Ricinus communis agglutinin ll (ricin and Ricinus communis agglutinin l (RCA. These proteins are stored in vacuoles within the endosperm cells of mature Ricinus seeds and they are rapidly broken down by hydrolysis during the early stages of post-germinative growth. Both ricin and RCA traffic within the plant cell from their site of synthesis to the storage vacuoles, and when they intoxicate mammalian cells they traffic from outside the cell to their site of action. In this review we will consider both of these trafficking routes.

  2. Novel role for tumor-induced expansion of myeloid-derived cells in cancer cachexia.

    Science.gov (United States)

    Cuenca, Alex G; Cuenca, Angela L; Winfield, Robert D; Joiner, Dallas N; Gentile, Lori; Delano, Matthew J; Kelly-Scumpia, Kindra M; Scumpia, Philip O; Matheny, Michael K; Scarpace, Philip J; Vila, Lizette; Efron, Philip A; LaFace, Drake M; Moldawer, Lyle L

    2014-06-15

    Cancer progression is associated with inflammation, increased metabolic demand, infection, cachexia, and eventually death. Myeloid-derived suppressor cells (MDSCs) commonly expand during cancer and are associated with adaptive immune suppression and inflammatory metabolite production. We propose that cancer-induced cachexia is driven at least in part by the expansion of MDSCs. MDSC expansion in 4T1 mammary carcinoma-bearing hosts is associated with induction of a hepatic acute-phase protein response and altered host energy and fat metabolism, and eventually reduced survival to polymicrobial sepsis and endotoxemia. Similar results are also seen in mice bearing a Lewis lung carcinoma and a C26 colon adenocarcinoma. However, a similar cachexia response is not seen with equivalent growth of the 66C4 subclone of 4T1, in which MDSC expansion does not occur. Importantly, reducing MDSC numbers in 4T1-bearing animals can ameliorate some of these late responses and reduce susceptibility to inflammation-induced organ injury and death. In addition, administering MDSCs from both tumor- and nontumor-bearing mice can produce an acute-phase response. Thus, we propose a previously undescribed mechanism for the development of cancer cachexia, whereby progressive MDSC expansion contributes to changes in host protein and energy metabolism and reduced resistance to infection.

  3. Unravelling the role of host plant expansion in the diversification of a Neotropical butterfly genus.

    Science.gov (United States)

    McClure, Melanie; Elias, Marianne

    2016-06-16

    Understanding the processes underlying diversification is a central question in evolutionary biology. For butterflies, access to new host plants provides opportunities for adaptive speciation. On the one hand, locally abundant host species can generate ecologically significant selection pressure. But a diversity of host plant species within the geographic range of each population and/or species might also eliminate any advantage conferred by specialization. This paper focuses on four Melinaea species, which are oligophagous on the family Solanaceae: M. menophilus, M. satevis, M. marsaeus, and finally, M. mothone. We examined both female preference and larval performance on two host plant species that commonly occur in this butterfly's native range, Juanulloa parasitica and Trianaea speciosa, to determine whether the different Melinaea species show evidence of local adaptation. In choice experiments, M. mothone females used both host plants for oviposition, whereas all other species used J. parasitica almost exclusively. In no choice experiment, M. mothone was the only species that readily accepted T. speciosa as a larval host plant. Larval survival was highest on J. parasitica (82.0 % vs. 60.9 %) and development took longer on T. speciosa (14.12 days vs. 13.35 days), except for M. mothone, which did equally well on both host plants. For all species, average pupal weight was highest on J. parasitica (450.66 mg vs. 420.01 mg), although this difference was least apparent in M. mothone. We did not find that coexisting species of Melinaea partition host plant resources as expected if speciation is primarily driven by host plant divergence. Although M. mothone shows evidence of local adaptation to a novel host plant, T. speciosa, which co-occurs, it does not preferentially lay more eggs on or perform better on this host plant than on host plants used by other Melinaea species and not present in its distributional range. It is likely that diversification in this

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  6. Episodic Neoglacial snowline descent and glacier expansion on Svalbard reconstructed from the 14C ages of ice-entombed plants

    Science.gov (United States)

    Miller, Gifford H.; Landvik, Jon Y.; Lehman, Scott J.; Southon, John R.

    2017-01-01

    The response of the Northern Hemisphere cryosphere to the monotonic decline in summer insolation and variable radiative forcing during the Holocene has been one of irregular expansion culminating in the Little Ice Age, when most glaciers attained their maximum late Holocene dimensions. Although periods of intervening still-stand or ice-retreat can be reconstructed by direct dating of ice-recessional features, defining times of Neoglacial ice growth has been limited to indirect proxies preserved in distal archives. Here we report 45 precise radiocarbon dates on in situ plants emerging from beneath receding glaciers on Svalbard that directly date the onset of snowline descent and glacier expansion, entombing the plants. Persistent snowline lowering occurred between 4.0 and 3.4 ka, but with little additional persistent lowering until early in the first millennium AD. Populations of individual 14C calendar age results and their aggregate calendar age probabilities define discrete episodes of vegetation kill and snowline lowering 240-340 AD, 410-540 AD and 670-750 AD, each with a lower snowline than the preceding episode, followed by additional snowline lowering between 1000 and 1220 AD, and between 1300 and 1450 AD. Snowline changes after 1450 AD, including the maximum ice extent of the Little Ice Age are not resolved by our collections, although snowlines remained lower than their 1450 AD level until the onset of modern warming. A time-distance diagram derived from a 250-m-long transect of dated ice-killed plants documents ice-margin advances ∼750, ∼1100 and after ∼1500 AD, concordant with distributed vegetation kill ages seen in the aggregate data set, supporting our central thesis that vegetation kill ages provide direct evidence of snowline lowering and cryospheric expansion. The mid- to late-Holocene history of snowline lowering on Svalbard is similar to ELA reconstructions of Norwegian and Svalbard cirque glaciers, and consistent with a cryospheric response

  7. Laser-mediated perforation of plant cells

    Science.gov (United States)

    Wehner, Martin; Jacobs, Philipp; Esser, Dominik; Schinkel, Helga; Schillberg, Stefan

    2007-07-01

    The functional analysis of plant cells at the cellular and subcellular levels requires novel technologies for the directed manipulation of individual cells. Lasers are increasingly exploited for the manipulation of plant cells, enabling the study of biological processes on a subcellular scale including transformation to generate genetically modified plants. In our setup either a picosecond laser operating at 1064 nm wavelength or a continuous wave laser diode emitting at 405 nm are coupled into an inverse microscope. The beams are focused to a spot size of about 1.5 μm and the tobacco cell protoplasts are irradiated. Optoporation is achieved when targeting the laser focal spot at the outermost edge of the plasma membrane. In case of the picosecond laser a single pulse with energy of about 0.4 μJ was sufficient to perforate the plasma membrane enabling the uptake of dye or DNA from the surrounding medium into the cytosol. When the ultraviolet laser diode at a power level of 17 mW is employed an irradiation time of 200 - 500 milliseconds is necessary to enable the uptake of macromolecules. In the presence of an EYFP encoding plasmid with a C-terminal peroxisomal signal sequence in the surrounding medium transient transformation of tobacco protoplasts could be achieved in up to 2% of the optoporated cells. Single cell perforation using this novel optoporation method shows that isolated plant cells can be permeabilized without direct manipulation. This is a valuable procedure for cell-specific applications, particularly where the import of specific molecules into plant cells is required for functional analysis.

  8. Isolation, expansion and differentiation of mesenchymal stromal cells from rabbits' bone marrow

    Directory of Open Access Journals (Sweden)

    Renato B. Eleotério

    2016-05-01

    Full Text Available Abstract: Tissue engineering has been a fundamental technique in the regenerative medicine field, once it permits to build tri-dimensional tissue constructs associating undifferentiated mesenchymal cells (or mesenchymal stromal cells - MSCs and scaffolds in vitro. Therefore, many studies have been carried out using these cells from different animal species, and rabbits are often used as animal model for in vivo tissue repair studies. However, most of the information available about MSCs harvesting and characterization is about human and murine cells, which brings some doubts to researchers who desire to work with a rabbit model in tissue repair studies based on MSCs. In this context, this study aimed to add and improve the information available in the scientific literature providing a complete technique for isolation, expansion and differentiation of MSCs from rabbits. Bone marrow mononuclear cells (BMMCs from humerus and femur of rabbits were obtained and to evaluate their proliferation rate, three different culture media were tested, here referred as DMEM-P, DMEM´S and α-MEM. The BMMCs were also cultured in osteogenic, chondrogenic and adipogenic induction media to prove their multipotentiality. It was concluded that the techniques suggested in this study can provide a guideline to harvest and isolate MSCs from bone marrow of rabbits in enough amount to allow their expansion and, based on the laboratory experience where the study was developed, it is also suggested a culture media formulation to provide a better cell proliferation rate with multipotentiality preservation.

  9. UV-Induced cell death in plants.

    Science.gov (United States)

    Nawkar, Ganesh M; Maibam, Punyakishore; Park, Jung Hoon; Sahi, Vaidurya Pratap; Lee, Sang Yeol; Kang, Chang Ho

    2013-01-14

    Plants are photosynthetic organisms that depend on sunlight for energy. Plants respond to light through different photoreceptors and show photomorphogenic development. Apart from Photosynthetically Active Radiation (PAR; 400-700 nm), plants are exposed to UV light, which is comprised of UV-C (below 280 nm), UV-B (280-320 nm) and UV-A (320-390 nm). The atmospheric ozone layer protects UV-C radiation from reaching earth while the UVR8 protein acts as a receptor for UV-B radiation. Low levels of UV-B exposure initiate signaling through UVR8 and induce secondary metabolite genes involved in protection against UV while higher dosages are very detrimental to plants. It has also been reported that genes involved in MAPK cascade help the plant in providing tolerance against UV radiation. The important targets of UV radiation in plant cells are DNA, lipids and proteins and also vital processes such as photosynthesis. Recent studies showed that, in response to UV radiation, mitochondria and chloroplasts produce a reactive oxygen species (ROS). Arabidopsis metacaspase-8 (AtMC8) is induced in response to oxidative stress caused by ROS, which acts downstream of the radical induced cell death (AtRCD1) gene making plants vulnerable to cell death. The studies on salicylic and jasmonic acid signaling mutants revealed that SA and JA regulate the ROS level and antagonize ROS mediated cell death. Recently, molecular studies have revealed genes involved in response to UV exposure, with respect to programmed cell death (PCD).

  10. UV-Induced Cell Death in Plants

    Directory of Open Access Journals (Sweden)

    Chang Ho Kang

    2013-01-01

    Full Text Available Plants are photosynthetic organisms that depend on sunlight for energy. Plants respond to light through different photoreceptors and show photomorphogenic development. Apart from Photosynthetically Active Radiation (PAR; 400–700 nm, plants are exposed to UV light, which is comprised of UV-C (below 280 nm, UV-B (280–320 nm and UV-A (320–390 nm. The atmospheric ozone layer protects UV-C radiation from reaching earth while the UVR8 protein acts as a receptor for UV-B radiation. Low levels of UV-B exposure initiate signaling through UVR8 and induce secondary metabolite genes involved in protection against UV while higher dosages are very detrimental to plants. It has also been reported that genes involved in MAPK cascade help the plant in providing tolerance against UV radiation. The important targets of UV radiation in plant cells are DNA, lipids and proteins and also vital processes such as photosynthesis. Recent studies showed that, in response to UV radiation, mitochondria and chloroplasts produce a reactive oxygen species (ROS. Arabidopsis metacaspase-8 (AtMC8 is induced in response to oxidative stress caused by ROS, which acts downstream of the radical induced cell death (AtRCD1 gene making plants vulnerable to cell death. The studies on salicylic and jasmonic acid signaling mutants revealed that SA and JA regulate the ROS level and antagonize ROS mediated cell death. Recently, molecular studies have revealed genes involved in response to UV exposure, with respect to programmed cell death (PCD.

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

    Science.gov (United States)

    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.

  12. Temporal expression of bacterial proteins instructs host CD4 T cell expansion and Th17 development.

    Directory of Open Access Journals (Sweden)

    Seung-Joo Lee

    2012-01-01

    Full Text Available Pathogens can substantially alter gene expression within an infected host depending on metabolic or virulence requirements in different tissues, however, the effect of these alterations on host immunity are unclear. Here we visualized multiple CD4 T cell responses to temporally expressed proteins in Salmonella-infected mice. Flagellin-specific CD4 T cells expanded and contracted early, differentiated into Th1 and Th17 lineages, and were enriched in mucosal tissues after oral infection. In contrast, CD4 T cells responding to Salmonella Type-III Secretion System (TTSS effectors steadily accumulated until bacterial clearance was achieved, primarily differentiated into Th1 cells, and were predominantly detected in systemic tissues. Thus, pathogen regulation of antigen expression plays a major role in orchestrating the expansion, differentiation, and location of antigen-specific CD4 T cells in vivo.

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

    Science.gov (United States)

    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.

  14. Progenitor cells in liver regeneration: molecular responses controlling their activation and expansion

    DEFF Research Database (Denmark)

    Santoni-Rugiu, Eric; Jelnes, Peter; Thorgeirsson, Snorri S

    2005-01-01

    Although normally quiescent, the adult mammalian liver possesses a great capacity to regenerate after different types of injuries in order to restore the lost liver mass and ensure maintenance of the multiple liver functions. Major players in the regeneration process are mature residual cells...... cells, and recruited inflammatory cells as well as the variety of growth-modulating molecules produced and/or harboured by these elements. The cellular and molecular responses to different regenerative stimuli seem to depend on the injury inflicted and consequently on the molecular microenvironment...... created in the liver by a certain insult. This review will focus on molecular responses controlling activation and expansion of the hepatic progenitor cell niche, emphasizing similarities and differences in the microenvironments orchestrating regeneration by recruitment of progenitor cell populations...

  15. A Single Amino-Acid Substitution at Lysine 40 of an Arabidopsis thaliana α-tubulin Causes Extensive Cell Proliferation and Expansion Defects

    Institute of Scientific and Technical Information of China (English)

    Xue Xiong; Deyang Xu; Zhongnan Yang; HaiHuang; Xiaofeng Cui

    2013-01-01

    Microtubules are highly dynamic cytoskeletal polymers of α/β-tubulin heterodimers that undergo multiple post-translational modifications essential for various cellular functions in eukaryotes.The lysine 40 (K40) is largely conserved in α-tubulins in many eukaryote species,and the post-translational modification by acetylation at K40 is critical for neuronal development in vertebrates.However,the biological function of K40 of α-tubulins in plants remains unexplored.In this study,we show in Arabidopsis thaliana that constitutive expression of mutated forms of α-tubulin6 (TUA6) at K40 (TUA6κ40A or TUA6κ40Q),in which K40 is replaced by alanine or glutamine,result in severely reduced plant size.Phenotypic characterization of the 35S:TUA6κ40A transgenic plants revealed that both cell proliferation and cell expansion were affected.Cytological and biochemical analyses showed that the accumulation ofα-and β-tubulin proteins was significantly reduced in the transgenic plants,and the cortical microtubule arrays were severely disrupted,indicating that K40 of the plant α-tubulin is critical in maintaining microtubule stability.We also constructed 35S:TUA6κ40R transgenic plants in which K40 of the engineered TUA6 protein is replaced by an arginine,and found that the 35S:TUA6K40R plants were phenotypically indistinguishable from the wild-type.Since lysine and arginine are similar in biochemical nature but arginine cannot be acetylated,these results suggest a structural importance for K40 of α-tubulins in cell division and expansion.

  16. VirtualLeaf: an open source framework for cell-based modeling of plant tissue growth and development

    NARCIS (Netherlands)

    R.M.H. Merks (Roeland); M.A. Guravage (Michael); D. Inze; G.T.S. Beemster

    2011-01-01

    htmlabstractPlant organs, including leaves and roots, develop by means of a multi-level crosstalk between gene regulation, patterned cell division and cell expansion, and tissue mechanics. The multi-level regulatory mechanisms complicate classic molecular genetics or functional genomics approaches t

  17. VirtualLeaf: an open source framework for cell-based modeling of plant tissue growth and development

    NARCIS (Netherlands)

    Merks, R.M.H.; Guravage, M.A.; Inze, D.; Beemster, G.T.S.

    2011-01-01

    Plant organs, including leaves and roots, develop by means of a multi-level crosstalk between gene regulation, patterned cell division and cell expansion, and tissue mechanics. The multi-level regulatory mechanisms complicate classic molecular genetics or functional genomics approaches to biological

  18. Quantification of plant cell coupling with live-cell microscopy

    DEFF Research Database (Denmark)

    Liesche, Johannes; Schulz, Alexander

    2015-01-01

    cell wall interface. Transport through plasmodesmata, the cell wall channels that directly connect plant cells, is regulated not only by a fixed size exclusion limit, but also by physiological and pathological adaptation. The noninvasive approach described here offers the possibility of precisely......Movement of nutrients and signaling compounds from cell to cell is an essential process for plant growth and development. To understand processes such as carbon allocation, cell communication, and reaction to pathogen attack it is important to know a specific molecule’s capacity to pass a specific...... determining the plasmodesmata-mediated cell wall permeability for small molecules in living cells. The method is based on photoactivation of the fluorescent tracer caged fluorescein. Non-fluorescent caged fluorescein is applied to a target tissue, where it is taken up passively into all cells. Imaged...

  19. Expansion of CD4+CD25+ helper T cells without regulatory function in smoking and COPD

    Directory of Open Access Journals (Sweden)

    Bucht Anders

    2011-06-01

    Full Text Available Abstract Background Regulatory T cells have been implicated in the pathogenesis of COPD by the increased expression of CD25 on helper T cells along with enhanced intracellular expression of FoxP3 and low/absent CD127 expression on the cell surface. Method Regulatory T cells were investigated in BALF from nine COPD subjects and compared to fourteen smokers with normal lung function and nine never-smokers. Results In smokers with normal lung function, the expression of CD25+CD4+ was increased, whereas the proportions of FoxP3+ and CD127+ were unchanged compared to never-smokers. Among CD4+ cells expressing high levels of CD25, the proportion of FoxP3+ cells was decreased and the percentage of CD127+ was increased in smokers with normal lung function. CD4+CD25+ cells with low/absent CD127 expression were increased in smokers with normal lung function, but not in COPD, when compared to never smokers. Conclusion The reduction of FoxP3 expression in BALF from smokers with normal lung function indicates that the increase in CD25 expression is not associated with the expansion of regulatory T cells. Instead, the high CD127 and low FoxP3 expressions implicate a predominantly non-regulatory CD25+ helper T-cell population in smokers and stable COPD. Therefore, we suggest a smoking-induced expansion of predominantly activated airway helper T cells that seem to persist after COPD development.

  20. The High Yield Expansion and Megakaryocytic Differentiation of Human Umbilical Cord Blood CD133+ Cells

    Directory of Open Access Journals (Sweden)

    Mahin Nikougoftar Zarif

    2011-01-01

    Full Text Available Objective: Despite of many benefits, umbilical cord blood (UCB hematopoietic stem cell(HSC transplantation is associated with low number of stem cells and slow engraftment;in particular of platelets. So, expanded HSCs and co-transfusion of megakaryocyte (MKprogenitor cells can shorten this period. In this study, we evaluated the cytokine conditionsfor maximum expansion and MK differentiation of CD133+ HSCs.Materials and Methods: In this experimental study, The CD133+ cells were separatedfrom three cord blood samples by magnetic activated cell sorting (MACS method, expandedin different cytokine combinations for a week and differentiated in thrombopoietin(TPO for the second week. Differentiation was followed by the flow cytometry detectionof CD41 and CD61 surface markers. Colony forming unit (CFU assay and DNA analysiswere done for colonogenic capacity and ploidy assay.Results: CD133+ cells showed maximum expansion in the stem span medium with stemcell factor (SCF + FMS-like tyrosine kinase 3-ligand (Flt3-L + TPO but the maximum differentiationwas seen when CD133+ cells were expanded in stem span medium with SCF+ Interleukin 3 (IL-3 + TPO for the first and in TPO for the second week. Colony FormingUnit-MK (CFU-MK was formed in three sizes of colonies in the mega-cult medium. In theDNA analysis; 25.2 ± 6.7% of the cells had more than 2n DNA mass.Conclusion: Distinct differences in the MK progenitor cell count were observed when thecells were cultured in stem span medium with TPO, SCF, IL-3 and then the TPO in thesecond week. Such strategy could be applied for optimization of CD133+ cells expansionfollowed by MK differentiation.

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

    Science.gov (United States)

    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.

  2. Osmosis in Poisoned Plant Cells.

    Science.gov (United States)

    Tatina, Robert

    1998-01-01

    Describes two simple laboratory exercises that allow students to test hypotheses concerning the requirement of cell energy for osmosis. The first exercise involves osmotically-caused changes in the length of potato tubers and requires detailed quantitative observations. The second exercise involves osmotically-caused changes in turgor of Elodea…

  3. Portsmouth Gaseous Diffusion Plant expansion, Piketon, Ohio. Volume 2. Draft environmental statement

    Energy Technology Data Exchange (ETDEWEB)

    Pennington, W. H.

    1976-06-01

    The need for additional uranium enrichment facilities and the environmental impacts of the add-on gaseous diffusion plant proposed for the Portsmouth Gaseous Diffusion Plant are discussed. A detailed description of the proposed facilities is included and unavoidable adverse environmental effects, possible alternatives, and anticipated benefits from the proposed facilities are considered. The flora and fauna of the area are tabulated and possible effects of air and water pollution on aquatic and terrestrial ecosystems are postulated. The extent of anticipated noise impact on the vicinity and the anticipated extent of civic envolvement are discussed. (CH)

  4. Portsmouth Gaseous Diffusion Plant expansion, Piketon, Ohio. Volume 1. Draft environmental statement

    Energy Technology Data Exchange (ETDEWEB)

    1976-06-01

    Subject to authorizing legislation and funding, ERDA will proceed with steps for additional uranium enrichment capacity at the Portsmouth Gaseous Diffusion Plant near Piketon, Ohio. This environmental statement was prepared by ERDA to cover this action. The statement was prepared in accordance with the National Environmental Policy Act of 1969, and ERDA's implementing regulations, 10 CFR Chapter III, Part 711. The statement describes the reasonably foreseeable environmental, social, economic and technological costs and benefits of the construction and operation of the expanded enrichment plant and its reasonably available alternatives and their anticipated effects.

  5. In vitro expansion of the mammary stem/progenitor cell population by xanthosine treatment

    Directory of Open Access Journals (Sweden)

    Choudhary Ratan K

    2012-06-01

    Full Text Available Abstract Background Mammary stem cells are critical for growth and maintenance of the mammary gland and therefore are of considerable interest for improving productivity and efficiency of dairy animals. Xanthosine treatment has been demonstrated to promote expansion of putative mammary stem cells in vivo, and hepatic and hair follicle stem cells in vitro. In the latter, xanthosine promoted the symmetrical division of hepatic and hair follicle stem cells. The objective of this study was to determine if treating primary cultures of bovine mammary epithelial cells (MEC with xanthosine increases the stem/progenitor cell population by promoting symmetrical division of mammary stem cells. Results In vitro treatment with xanthosine increased the population of MEC during the exponential phase of cell growth, reducing the doubling time from 86 h in control cultures to 60 h in xanthosine-treated cultures. The bromodeoxyuridine (BrdU labeling index and the proportion of MEC in S-phase both were increased by xanthosine treatment, indicating that increased cell accretion was due to increased cell proliferation. Analysis of daughter-pairs indicated that xanthosine promoted a shift from asymmetric to symmetric cell division. Moreover, the 30 % increase in symmetric cell division was concomitant with an increase in the proportion of MEC that were positive for a putative stem cell marker (FNDC3B and a trend toward increased telomerase activity. These results suggest that xanthosine treatment in vitro can increase cell proliferation, promote symmetric cell division and enhance stem/progenitor cell activity. Conclusions Xanthosine treatment increased the proliferation rate of bovine MEC in vitro. This was likely to be mediated by an increase in the proportion of stem/progenitor cells in the MEC population due to promotion of symmetrical stem cell division by xanthosine.

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

    Science.gov (United States)

    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. A plant cell division algorithm based on cell biomechanics and ellipse-fitting

    Science.gov (United States)

    Abera, Metadel K.; Verboven, Pieter; Defraeye, Thijs; Fanta, Solomon Workneh; Hertog, Maarten L. A. T. M.; Carmeliet, Jan; Nicolai, Bart M.

    2014-01-01

    Background and Aims The importance of cell division models in cellular pattern studies has been acknowledged since the 19th century. Most of the available models developed to date are limited to symmetric cell division with isotropic growth. Often, the actual growth of the cell wall is either not considered or is updated intermittently on a separate time scale to the mechanics. This study presents a generic algorithm that accounts for both symmetrically and asymmetrically dividing cells with isotropic and anisotropic growth. Actual growth of the cell wall is simulated simultaneously with the mechanics. Methods The cell is considered as a closed, thin-walled structure, maintained in tension by turgor pressure. The cell walls are represented as linear elastic elements that obey Hooke's law. Cell expansion is induced by turgor pressure acting on the yielding cell-wall material. A system of differential equations for the positions and velocities of the cell vertices as well as for the actual growth of the cell wall is established. Readiness to divide is determined based on cell size. An ellipse-fitting algorithm is used to determine the position and orientation of the dividing wall. The cell vertices, walls and cell connectivity are then updated and cell expansion resumes. Comparisons are made with experimental data from the literature. Key Results The generic plant cell division algorithm has been implemented successfully. It can handle both symmetrically and asymmetrically dividing cells coupled with isotropic and anisotropic growth modes. Development of the algorithm highlighted the importance of ellipse-fitting to produce randomness (biological variability) even in symmetrically dividing cells. Unlike previous models, a differential equation is formulated for the resting length of the cell wall to simulate actual biological growth and is solved simultaneously with the position and velocity of the vertices. Conclusions The algorithm presented can produce different

  8. Soluble Jagged1 attenuates lateral inhibition, allowing for the clonal expansion of neural crest stem cells.

    Science.gov (United States)

    Nikopoulos, George N; Duarte, Maria; Kubu, Chris J; Bellum, Stephen; Friesel, Robert; Maciag, Thomas; Prudovsky, Igor; Verdi, Joseph M

    2007-12-01

    The activation of Notch signaling in neural crest stem cells (NCSCs) results in the rapid loss of neurogenic potential and differentiation into glia. We now show that the attenuation of endogenous Notch signaling within expanding NCSC clones by the Notch ligand soluble Jagged1 (sJ1), maintains NCSCs in a clonal self-renewing state in vitro without affecting their sensitivity to instructive differentiation signals observed previously during NCSC self-renewal. sJ1 functions as a competitive inhibitor of Notch signaling to modulate endogenous cell-cell communication to levels sufficient to inhibit neural differentiation but insufficient to instruct gliogenic differentiation. Attenuated Notch signaling promotes the induction and nonclassic release of fibroblast growth factor 1 (FGF1). The functions of sJ1 and FGF1 signaling are complementary, as abrogation of FGF signaling diminishes the ability of sJ1 to promote NCSC expansion, yet the secondary NCSCs maintain the dosage sensitivity of the founder. These results validate and build upon previous studies on the role of Notch signaling in stem cell self-renewal and suggest that the differentiation bias or self-renewal potential of NCSCs is intrinsically linked to the level of endogenous Notch signaling. This should provide a unique opportunity for the expansion of NCSCs ex vivo without altering their differentiation bias for clinical cell replacement or transplant strategies in tissue repair. Disclosure of potential conflicts of interest is found at the end of this article.

  9. Plant–soil interactions in the expansion and native range of a poleward shifting plant species

    NARCIS (Netherlands)

    Van Grunsven, R.H.A.; Van der Putten, W.H.; Bezemer, T.M.; Berendse, F.; Veenendaal, E.M.

    2010-01-01

    Climate warming causes range shifts of many species toward higher latitudes and altitudes. However, range shifts of host species do not necessarily proceed at the same rates as those of their enemies and symbionts. Here, we examined how a range shifting plant species performs in soil from its

  10. Climate suitability and human influences combined explain the range expansion of an invasive horticultural plant

    Science.gov (United States)

    Carolyn M. Beans; Francis F. Kilkenny; Laura F. Galloway

    2012-01-01

    Ecological niche models are commonly used to identify regions at risk of species invasions. Relying on climate alone may limit a model's success when additional variables contribute to invasion. While a climate-based model may predict the future spread of an invasive plant, we hypothesized that a model that combined climate with human influences would most...

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

    Science.gov (United States)

    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. GMP-compliant isolation and expansion of bone marrow-derived MSCs in the closed, automated device quantum cell expansion system.

    Science.gov (United States)

    Rojewski, Markus T; Fekete, Natalie; Baila, Stefano; Nguyen, Kim; Fürst, Daniel; Antwiler, Delbert; Dausend, Julia; Kreja, Ludwika; Ignatius, Anita; Sensebé, Luc; Schrezenmeier, Hubert

    2013-01-01

    The estimated frequency of MSCs in BM is about 0.001-0.01% of total nucleated cells. Most commonly, one applied therapeutic cell dose is about 1-5 million MSCs/kg body weight, necessitating a reliable, fast, and safe expansion system. The limited availability of MSCs demands for an extensive ex vivo amplification step to accumulate sufficient cell numbers. Human platelet lysate (PL) has proven to be a safe and feasible alternative to animal-derived serum as supplement for MSC cultivation. We have investigated the functionally closed automated cell culture hollow fiber bioreactor Quantum cell expansion system as an alternative novel tool to conventional tissue flasks for efficient clinical-scale MSC isolation and expansion from bone marrow using PL. Cells expanded in the Quantum system fulfilled MSC criteria as shown by flow cytometry and adipogenic, chondrogenic, and osteogenic differentiation capacity. Cell surface expression of a variety of chemokine receptors, adhesion molecules, and additional MSC markers was monitored for several passages by flow cytometry. The levels of critical media components like glucose and lactate were analyzed. PDGF-AA, PDGF-AB/BB, bFGF, TGF-β1, sICAM-1, sVCAM-1, RANTES, GRO, VEGF, sCD40L, and IL-6 were assessed using a LUMINEX platform. Originally optimized for the use of fetal calf serum (FCS) as supplement and fibronectin as coating reagent, we succeeded to obtain an average of more than 100×10(6) of MSCs from as little as 18.8-28.6 ml of BM aspirate using PL. We obtained similar yields of MSCs/µl BM in the FCS-containing and the xenogen-free expansion system. The Quantum system reliably produces a cellular therapeutic dose in a functionally closed system that requires minimal manipulation. Both isolation and expansion are possible using FCS or PL as supplement. Coating of the hollow fibers of the bioreactor is mandatory when loading MSCs. Fibronectin, PL, and human plasma may serve as coating reagents.

  13. The polyphenol oxidase gene family in land plants: Lineage-specific duplication and expansion

    Directory of Open Access Journals (Sweden)

    Tran Lan T

    2012-08-01

    Full Text Available Abstract Background Plant polyphenol oxidases (PPOs are enzymes that typically use molecular oxygen to oxidize ortho-diphenols to ortho-quinones. These commonly cause browning reactions following tissue damage, and may be important in plant defense. Some PPOs function as hydroxylases or in cross-linking reactions, but in most plants their physiological roles are not known. To better understand the importance of PPOs in the plant kingdom, we surveyed PPO gene families in 25 sequenced genomes from chlorophytes, bryophytes, lycophytes, and flowering plants. The PPO genes were then analyzed in silico for gene structure, phylogenetic relationships, and targeting signals. Results Many previously uncharacterized PPO genes were uncovered. The moss, Physcomitrella patens, contained 13 PPO genes and Selaginella moellendorffii (spike moss and Glycine max (soybean each had 11 genes. Populus trichocarpa (poplar contained a highly diversified gene family with 11 PPO genes, but several flowering plants had only a single PPO gene. By contrast, no PPO-like sequences were identified in several chlorophyte (green algae genomes or Arabidopsis (A. lyrata and A. thaliana. We found that many PPOs contained one or two introns often near the 3’ terminus. Furthermore, N-terminal amino acid sequence analysis using ChloroP and TargetP 1.1 predicted that several putative PPOs are synthesized via the secretory pathway, a unique finding as most PPOs are predicted to be chloroplast proteins. Phylogenetic reconstruction of these sequences revealed that large PPO gene repertoires in some species are mostly a consequence of independent bursts of gene duplication, while the lineage leading to Arabidopsis must have lost all PPO genes. Conclusion Our survey identified PPOs in gene families of varying sizes in all land plants except in the genus Arabidopsis. While we found variation in intron numbers and positions, overall PPO gene structure is congruent with the phylogenetic

  14. Recent advances in plant cell wall proteomics.

    Science.gov (United States)

    Jamet, Elisabeth; Albenne, Cécile; Boudart, Georges; Irshad, Muhammad; Canut, Hervé; Pont-Lezica, Rafael

    2008-02-01

    The plant extracellular matrix contains typical polysaccharides such as cellulose, hemicelluloses, and pectins that interact to form dense interwoven networks. Plant cell walls play crucial roles during development and constitute the first barrier of defense against invading pathogens. Cell wall proteomics has greatly contributed to the description of the protein content of a compartment specific to plants. Around 400 cell wall proteins (CWPs) of Arabidopsis, representing about one fourth of its estimated cell wall proteome, have been described. The main points to note are that: (i) the diversity of enzymes acting on polysaccharides suggests a great plasticity of cell walls; (ii) CWPs such as proteases, polysaccharide hydrolytic enzymes, and lipases may contribute to the generation of signals; (iii) proteins of unknown functions were identified, suggesting new roles for cell walls. Recently, the characterization of PTMs such as N- and O-glycosylations improved our knowledge of CWP structure. The presence of many glycoside hydrolases and proteases suggests a complex regulation of CWPs involving various types of post-translational events. The first 3-D structures to be resolved gave clues about the interactions between CWPs, or between CWPs and polysaccharides. Future work should include: extracting and identifying CWPs still recalcitrant to proteomics, describing the cell wall interactome, improving quantification, and unraveling the roles of each of the CWPs.

  15. Spectro-Microscopy of Living Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Klaus Harter; Alfred J. Meixner; Frank Schleifenbaum

    2012-01-01

    Spectro-microscopy,a combination of fluorescence microscopy with spatially resolved spectroscopic techniques,provides new and exciting tools for functional cell biology in living organisms.This review focuses on recent developments in spectro-microscopic applications for the investigation of living plant cells in their native tissue context.The application of spectro-microscopic methods led to the recent discovery of a fast signal response pathway for the brassinosteroide receptor BRI1 in the plasma membrane of living plant cells.Moreover,the competence of different plant cell types to respond to environmental or endogenous stimuli was determined in vivo by correlation analysis of different optical and spectroscopic readouts such as fluorescence lifetime (FLT).Furthermore,a new spectro-microscopic technique,fluorescence intensity decay shape analysis microscopy (FIDSAM),has been developed.FIDSAM is capable of imaging lowexpressed fluorophore-tagged proteins at high spatial resolution and precludes the misinterpretation of autofluorescence artifacts.In addition,FIDSAM provides a very effective and sensitive tool on the basis of F(o)rster resonance energy transfer (FRET) for the qualitative and quantitative determination of protein-protein interaction.Finally,we report on the quantitative analysis of the photosystem Ⅰ and Ⅱ (PSⅠ/PSⅡ) ratio in the chloroplasts of living Arabidopsis plants at room temperature,using high-resolution,spatially resolved fluorescence spectroscopy.With this technique,it was not only possible to measure PSⅠ/PSⅡ ratios,but also to demonstrate the differential competence of wild-type and carbohydrate-deficient plants to adapt the PSⅠ/PSⅡ ratio to different light conditions.In summary,the information content of standard microscopic images is extended by several dimensions by the use of spectro-microscopic approaches.Therefore,novel cell physiological and molecular topics can be addressed and valuable insights into molecular and

  16. Intrinsic Plasma Cell Differentiation Defects in B Cell Expansion with NF-κB and T Cell Anergy Patient B Cells

    Directory of Open Access Journals (Sweden)

    Swadhinya Arjunaraja

    2017-08-01

    Full Text Available B cell Expansion with NF-κB and T cell Anergy (BENTA disease is a novel B cell lymphoproliferative disorder caused by germline, gain-of-function mutations in the lymphocyte scaffolding protein CARD11, which drives constitutive NF-κB signaling. Despite dramatic polyclonal expansion of naive and immature B cells, BENTA patients also present with signs of primary immunodeficiency, including markedly reduced percentages of class-switched/memory B cells and poor humoral responses to certain vaccines. Using purified naive B cells from our BENTA patient cohort, here we show that BENTA B cells exhibit intrinsic defects in B cell differentiation. Despite a profound in vitro survival advantage relative to normal donor B cells, BENTA patient B cells were severely impaired in their ability to differentiate into short-lived IgDloCD38hi plasmablasts or CD138+ long-lived plasma cells in response to various stimuli. These defects corresponded with diminished IgG antibody production and correlated with poor induction of specific genes required for plasma cell commitment. These findings provide important mechanistic clues that help explain both B cell lymphocytosis and humoral immunodeficiency in BENTA disease.

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

    Science.gov (United States)

    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.

  18. Phytosulfokine-α controls hypocotyl length and cell expansion in Arabidopsis thaliana through phytosulfokine receptor 1.

    Directory of Open Access Journals (Sweden)

    Nils Stührwohldt

    Full Text Available The disulfated peptide growth factor phytosulfokine-α (PSK-α is perceived by LRR receptor kinases. In this study, a role for PSK signaling through PSK receptor PSKR1 in Arabidopsis thaliana hypocotyl cell elongation is established. Hypocotyls of etiolated pskr1-2 and pskr1-3 seedlings, but not of pskr2-1 seedlings were shorter than wt due to reduced cell elongation. Treatment with PSK-α did not promote hypocotyl growth indicating that PSK levels were saturating. Tyrosylprotein sulfotransferase (TPST is responsible for sulfation and hence activation of the PSK precursor. The tpst-1 mutant displayed shorter hypocotyls with shorter cells than wt. Treatment of tpst-1 seedlings with PSK-α partially restored elongation growth in a dose-dependent manner. Hypocotyl elongation was significantly enhanced in tpst-1 seedlings at nanomolar PSK-α concentrations. Cell expansion was studied in hypocotyl protoplasts. WT and pskr2-1 protoplasts expanded in the presence of PSK-α in a dose-dependent manner. By contrast, pskr1-2 and pskr1-3 protoplasts were unresponsive to PSK-α. Protoplast swelling in response to PSK-α was unaffected by ortho-vanadate, which inhibits the plasma membrane H(+-ATPase. In maize (Zea mays L., coleoptile protoplast expansion was similarly induced by PSK-α in a dose-dependent manner and was dependent on the presence of K(+ in the media. In conclusion, PSK-α signaling of hypocotyl elongation and protoplast expansion occurs through PSKR1 and likely involves K(+ uptake, but does not require extracellular acidification by the plasma membrane H(+-ATPase.

  19. Skin cell isolation and expansion for cell transplantation is limited in patients using tobacco, alcohol, or are exhibiting diabetes mellitus.

    Science.gov (United States)

    Johnen, Christa; Hartmann, Bernd; Steffen, Ingo; Bräutigam, Kirsten; Witascheck, Tom; Toman, Nidal; Küntscher, Markus V; Gerlach, Jörg C

    2006-03-01

    The aim of this exploratory study was to investigate the isolation and expansion of keratinocytes and fibroblasts from donors with certain medical histories. Biopsies were taken from donors (N=32) falling into one or more of the following categories: a history of heavy smoking and/or alcohol abuse, drug abuse, diabetes mellitus or steroid treatment. Cells from donors who did not fall into any of the above-mentioned categories were used as controls. Proliferation and growth behaviour of cells were analyzed by measurement of passage duration, absorbance (MTT-assay) and light microscopy. Donors with a specific medical history required larger biopsy areas than the control group for isolating a sufficient number of fibroblasts and keratinocytes. Times to confluence were significantly prolonged and absorbances (MTT) were significantly reduced in several donor groups when compared to control cultures. Biopsies from donors with steroid treatment, drug abuse and combined nicotine and alcohol abuse could not be established beyond passage 0 degrees or 1 degree, respectively. We conclude that isolation and expansion of skin cells from donors with certain medical histories may require larger biopsies, prolonged expansion times or may even result in failure. These findings may therefore be of clinical importance in the field of autologous skin cell transplantation.

  20. Direct FuelCell/Turbine Power Plant

    Energy Technology Data Exchange (ETDEWEB)

    Hossein Ghezel-Ayagh

    2008-09-30

    This report summarizes the progress made in development of Direct FuelCell/Turbine (DFC/T{reg_sign}) power plants for generation of clean power at very high efficiencies. The DFC/T system employs an indirectly heated Turbine Generator to supplement fuel cell generated power. The concept extends the high efficiency of the fuel cell by utilizing the fuel cell's byproduct heat in a Brayton cycle. Features of the DFC/T system include: electrical efficiencies of up to 75% on natural gas, minimal emissions, reduced carbon dioxide release to the environment, simplicity in design, direct reforming internal to the fuel cell, and potential cost competitiveness with existing combined cycle power plants. Proof-of-concept tests using a sub-MW-class DFC/T power plant at FuelCell Energy's (FCE) Danbury facility were conducted to validate the feasibility of the concept and to measure its potential for electric power production. A 400 kW-class power plant test facility was designed and retrofitted to conduct the tests. The initial series of tests involved integration of a full-size (250 kW) Direct FuelCell stack with a 30 kW Capstone microturbine. The operational aspects of the hybrid system in relation to the integration of the microturbine with the fuel cell, process flow and thermal balances, and control strategies for power cycling of the system, were investigated. A subsequent series of tests included operation of the sub-MW Direct FuelCell/Turbine power plant with a Capstone C60 microturbine. The C60 microturbine extended the range of operation of the hybrid power plant to higher current densities (higher power) than achieved in initial tests using the 30kW microturbine. The proof-of-concept test results confirmed the stability and controllability of operating a fullsize (250 kW) fuel cell stack in combination with a microturbine. Thermal management of the system was confirmed and power plant operation, using the microturbine as the only source of fresh air supply

  1. Cytokine-based log-scale expansion of functional murine dendritic cells.

    Directory of Open Access Journals (Sweden)

    Yui Harada

    Full Text Available BACKGROUND: Limitations of the clinical efficacy of dendritic cell (DC-based immunotherapy, as well as difficulties in their industrial production, are largely related to the limited number of autologous DCs from each patient. We here established a possible breakthrough, a simple and cytokine-based culture method to realize a log-scale order of functional murine DCs (>1,000-fold, which cells were used as a model before moving to human studies. METHODOLOGY/PRINCIPAL FINDINGS: Floating cultivation of lineage-negative hematopoietic progenitors from bone marrow in an optimized cytokine cocktail (FLT3-L, IL-3, IL-6, and SCF led to a stable log-scale proliferation of these cells, and a subsequent differentiation study using IL-4/GM-CSF revealed that 3-weeks of expansion was optimal to produce CD11b+/CD11c+ DC-like cells. The expanded DCs had typical features of conventional myeloid DCs in vitro and in vivo, including identical efficacy as tumor vaccines. CONCLUSIONS/SIGNIFICANCE: The concept of DC expansion should make a significant contribution to the progress of DC-based immunotherapy.

  2. Perapion connexum (Schilsky, 1902) (Coleoptera, Apionidae) in Central Europe, a case of plant expansion chase

    OpenAIRE

    Marek Wanat; Attila Podlussány; Karel Schön

    2012-01-01

    Perapion connexum (Schilsky) is recorded for the first time from Hungary and Kyrgyzstan, and new distribution data from Ukraine and Russia are provided. Preliminary placements of this weevil in faunal checklists for Poland and Slovakia are here documented with detailed data. Its occurrence in Austria based on older evidence, is discussed. The neophytic and invasive in Central Europe sorrel Rumex confertus Willd. is confirmed to be its unique host plant in Poland. Morphology of the newcoming w...

  3. Ecological consequences of the expansion of N2-fixing plants in cold biomes

    Science.gov (United States)

    Hiltbrunner, Erika; Aerts, Rien; Bühlmann, Tobias; Huss-Danell, Kerstin; Magnusson, Borgthor; Myrold, David D.; Reed, Sasha C.; Sigurdsson, Bjarni D.; Körner, Christian

    2014-01-01

    Research in warm-climate biomes has shown that invasion by symbiotic dinitrogen (N2)-fixing plants can transform ecosystems in ways analogous to the transformations observed as a consequence of anthropogenic, atmospheric nitrogen (N) deposition: declines in biodiversity, soil acidification, and alterations to carbon and nutrient cycling, including increased N losses through nitrate leaching and emissions of the powerful greenhouse gas nitrous oxide (N2O). Here, we used literature review and case study approaches to assess the evidence for similar transformations in cold-climate ecosystems of the boreal, subarctic and upper montane-temperate life zones. Our assessment focuses on the plant genera Lupinus and Alnus, which have become invasive largely as a consequence of deliberate introductions and/or reduced land management. These cold biomes are commonly located in remote areas with low anthropogenic N inputs, and the environmental impacts of N2-fixer invasion appear to be as severe as those from anthropogenic N deposition in highly N polluted areas. Hence, inputs of N from N2 fixation can affect ecosystems as dramatically or even more strongly than N inputs from atmospheric deposition, and biomes in cold climates represent no exception with regard to the risk of being invaded by N2-fixing species. In particular, the cold biomes studied here show both a strong potential to be transformed by N2-fixing plants and a rapid subsequent saturation in the ecosystem’s capacity to retain N. Therefore, analogous to increases in N deposition, N2-fixing plant invasions must be deemed significant threats to biodiversity and to environmental quality.

  4. Ecological consequences of the expansion of N₂-fixing plants in cold biomes.

    Science.gov (United States)

    Hiltbrunner, Erika; Aerts, Rien; Bühlmann, Tobias; Huss-Danell, Kerstin; Magnusson, Borgthor; Myrold, David D; Reed, Sasha C; Sigurdsson, Bjarni D; Körner, Christian

    2014-09-01

    Research in warm-climate biomes has shown that invasion by symbiotic dinitrogen (N2)-fixing plants can transform ecosystems in ways analogous to the transformations observed as a consequence of anthropogenic, atmospheric nitrogen (N) deposition: declines in biodiversity, soil acidification, and alterations to carbon and nutrient cycling, including increased N losses through nitrate leaching and emissions of the powerful greenhouse gas nitrous oxide (N2O). Here, we used literature review and case study approaches to assess the evidence for similar transformations in cold-climate ecosystems of the boreal, subarctic and upper montane-temperate life zones. Our assessment focuses on the plant genera Lupinus and Alnus, which have become invasive largely as a consequence of deliberate introductions and/or reduced land management. These cold biomes are commonly located in remote areas with low anthropogenic N inputs, and the environmental impacts of N2-fixer invasion appear to be as severe as those from anthropogenic N deposition in highly N polluted areas. Hence, inputs of N from N2 fixation can affect ecosystems as dramatically or even more strongly than N inputs from atmospheric deposition, and biomes in cold climates represent no exception with regard to the risk of being invaded by N2-fixing species. In particular, the cold biomes studied here show both a strong potential to be transformed by N2-fixing plants and a rapid subsequent saturation in the ecosystem's capacity to retain N. Therefore, analogous to increases in N deposition, N2-fixing plant invasions must be deemed significant threats to biodiversity and to environmental quality.

  5. A parametric study of assembly pressure, thermal expansion, and membrane swelling in PEM fuel cells

    Directory of Open Access Journals (Sweden)

    Maher A.R. Sadiq Al-Baghdadi

    2016-01-01

    Full Text Available Proton Exchange membrane (PEM fuel cells are still undergoing intense development, and the combination of new and optimized materials, improved product development, novel architectures, more efficient transport processes, and design optimization and integration are expected to lead to major gains in performance, efficiency, durability, reliability, manufacturability and cost-effectiveness. PEM fuel cell assembly pressure is known to cause large strains in the cell components. All components compression occurs during the assembly process of the cell, but also during fuel cell operation due to membrane swelling when absorbs water and cell materials expansion due to heat generating in catalyst layers. Additionally, the repetitive channel-rib pattern of the bipolar plates results in a highly inhomogeneous compressive load, so that while large strains are produced under the rib, the region under the channels remains approximately at its initial uncompressed state. This leads to significant spatial variations in GDL thickness and porosity distributions, as well as in electrical and thermal bulk conductivities and contact resistances (both at the ribe-GDL and membrane-GDL interfaces. These changes affect the rates of mass, charge, and heat transport through the GDL, thus impacting fuel cell performance and lifetime. In this paper, computational fluid dynamics (CFD model of a PEM fuel cell has been developed to simulate the pressure distribution inside the cell, which are occurring during fuel cell assembly (bolt assembling, and membrane swelling and cell materials expansion during fuel cell running due to the changes of temperature and relative humidity. The PEM fuel cell model simulated includes the following components; two bi-polar plates, two GDLs, and, an MEA (membrane plus two CLs. This model is used to study and analyses the effect of assembling and operating parameters on the mechanical behaviour of PEM. The analysis helped identifying critical

  6. Plant microbial fuel cell applied in wetlands

    NARCIS (Netherlands)

    Wetser, Koen; Liu, Jia; Buisman, Cees; Strik, David

    2015-01-01

    The plant microbial fuel cell (PMFC) has to be applied in wetlands to be able to generate electricity on a large scale. The objective of this PMFC application research is to clarify the differences in electricity generation between a Spartina anglica salt marsh and Phragmites australis peat soil

  7. Plant cells : immobilization and oxygen transfer

    NARCIS (Netherlands)

    Hulst, A.C.

    1987-01-01

    The study described in this thesis is part of the integrated project 'Biotechnological production of non-persistent bioinsecticides by means of plant cells invitro ' and was done in close cooperation with the research Institute Ital within the framework

  8. Inducible cell death in plant immunity

    DEFF Research Database (Denmark)

    Hofius, Daniel; Tsitsigiannis, Dimitrios I; Jones, Jonathan D G

    2006-01-01

    Programmed cell death (PCD) occurs during vegetative and reproductive plant growth, as typified by autumnal leaf senescence and the terminal differentiation of the endosperm of cereals which provide our major source of food. PCD also occurs in response to environmental stress and pathogen attack,...

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

    Science.gov (United States)

    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

  10. Nutrient regulation by continuous feeding removes limitations on cell yield in the large-scale expansion of Mammalian cell spheroids.

    Directory of Open Access Journals (Sweden)

    Bradley P Weegman

    Full Text Available 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.

  11. Nutrient regulation by continuous feeding removes limitations on cell yield in the large-scale expansion of Mammalian cell spheroids.

    Science.gov (United States)

    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.

  12. Identification, isolation and expansion of myoendothelial cells involved in leech muscle regeneration.

    Directory of Open Access Journals (Sweden)

    Annalisa Grimaldi

    Full Text Available Adult skeletal muscle in vertebrates contains myoendothelial cells that express both myogenic and endothelial markers, and which are able to differentiate into myogenic cells to contribute to muscle regeneration. In spite of intensive research efforts, numerous questions remain regarding the role of cytokine signalling on myoendothelial cell differentiation and muscle regeneration. Here we used Hirudo medicinalis (Annelid, leech as an emerging new model to study myoendothelial cells and muscle regeneration. Although the leech has relative anatomical simplicity, it shows a striking similarity with vertebrate responses and is a reliable model for studying a variety of basic events, such as tissue repair. Double immunohistochemical analysis were used to characterize myoendothelial cells in leeches and, by injecting in vivo the matrigel biopolymer supplemented with the cytokine Vascular Endothelial Growth Factor (VEGF, we were able to isolate this specific cell population expressing myogenic and endothelial markers. We then evaluated the effect of VEGF on these cells in vitro. Our data indicate that, similar to that proposed for vertebrates, myoendothelial cells of the leech directly participate in myogenesis both in vivo and in vitro, and that VEGF secretion is involved in the recruitment and expansion of these muscle progenitor cells.

  13. Non-climatic constraints on upper elevational plant range expansion under climate change.

    Science.gov (United States)

    Brown, Carissa D; Vellend, Mark

    2014-11-01

    We are limited in our ability to predict climate-change-induced range shifts by our inadequate understanding of how non-climatic factors contribute to determining range limits along putatively climatic gradients. Here, we present a unique combination of observations and experiments demonstrating that seed predation and soil properties strongly limit regeneration beyond the upper elevational range limit of sugar maple, a tree species of major economic importance. Most strikingly, regeneration beyond the range limit occurred almost exclusively when seeds were experimentally protected from predators. Regeneration from seed was depressed on soil from beyond the range edge when this soil was transplanted to sites within the range, with indirect evidence suggesting that fungal pathogens play a role. Non-climatic factors are clearly in need of careful attention when attempting to predict the biotic consequences of climate change. At minimum, we can expect non-climatic factors to create substantial time lags between the creation of more favourable climatic conditions and range expansion.

  14. Matrix Expansion and Syncytial Aggregation of Syndecan-1+ Cells Underpin Villous Atrophy in Coeliac Disease

    Science.gov (United States)

    Salvestrini, Camilla; Lucas, Mark; Lionetti, Paolo; Torrente, Franco; James, Sean; Phillips, Alan D.; Murch, Simon H.

    2014-01-01

    Background We studied the expression of sulphated glycosaminoglycans (GAGs) in coeliac disease (CD) mucosa, as they are critical determinants of tissue volume, which increases in active disease. We also examined mucosal expression of IL-6, which stimulates excess GAG synthesis in disorders such as Grave's ophthalmopathy. Methods We stained archival jejunal biopsies from 5 children with CD at diagnosis, on gluten-free diet and challenge for sulphated GAGs. We then examined duodenal biopsies from 9 children with CD compared to 9 histological normal controls, staining for sulphated GAGs, heparan sulphate proteoglycans (HSPG), short-chain HSPG (Δ-HSPG) and the proteoglycan syndecan-1 (CD138), which is expressed on epithelium and plasma cells. We confirmed findings with a second monoclonal in another 12 coeliac children. We determined mucosal IL-6 expression by immunohistochemistry and PCR in 9 further cases and controls, and used quantitative real time PCR for other Th17 pathway cytokines in an additional 10 cases and controls. Results In CD, HSPG expression was lost in the epithelial compartment but contrastingly maintained within an expanded lamina propria. Within the upper lamina propria, clusters of syndecan-1+ plasma cells formed extensive syncytial sheets, comprising adherent plasma cells, lysed cells with punctate cytoplasmic staining and shed syndecan ectodomains. A dense infiltrate of IL-6+ mononuclear cells was detected in active coeliac disease, also localised to the upper lamina propria, with significantly increased mRNA expression of IL-6 and IL-17A but not IL-23 p19. Conclusions Matrix expansion, through syndecan-1+ cell recruitment and lamina propria GAG increase, underpins villous atrophy in coeliac disease. The syndecan-1+ cell syncytia and excess GAG production recapitulate elements of the invertebrate encapsulation reaction, itself dependent on insect transglutaminase and glutaminated early response proteins. As in other matrix expansion disorders

  15. Delta-like 4 differentially regulates murine CD4 T cell expansion via BMI1.

    Directory of Open Access Journals (Sweden)

    Matthew A Schaller

    Full Text Available BACKGROUND: Studies have shown that Notch is essential for the maintenance of a T cell Th2 phenotype in vivo. It has also been shown that Notch ligands have diverse functions during T cell activation. We chose to investigate the role of Notch ligands during the Th2 response. PRINCIPAL FINDINGS: We studied the relationship of two Notch ligands, delta-like 4 and jagged-1, to T cell proliferation in C57 Bl/6 mice. Our findings indicate that jagged-1 does not affect the rate of T cell proliferation in any subset examined. However, delta-like 4 causes an increase in the expansion of Th2 memory cells and a decrease in effector cell proliferation. Our in vivo studies indicate that the Notch system is dynamically regulated, and that blocking one Notch ligand increases the effective concentration of other Notch ligands, thus altering the response. Examination of genes related to the Notch pathway revealed that the Notch receptors were increased in memory T cells. Expression of BMI1, a gene involved in T cell proliferation, was also higher in memory T cells. Further experiments demonstrated that Notch directly regulates the expression of the BMI1 gene in T cells and may govern T cell proliferation through this pathway. CONCLUSIONS: From these experiments we can make several novel conclusions about the role of Notch ligands in T cell biology. The first is that delta-like 4 suppresses effector cell proliferation and enhances Th2 memory cell proliferation. The second is that blocking one Notch ligand in vivo effectively increases the concentration of other Notch ligands, which can then alter the response.

  16. Bioreactor systems for tissue engineering II. Strategies for the expansion and directed differentiation of stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Kasper, Cornelia [Hannover Univ. (Germany). Inst. fuer Technische Chemie; Griensven, Martijn van [Ludwig Boltzmann Institut fuer Klinische und Experimentelle Traumatologie, Wien (Austria); Poertner, Ralf (eds.) [Technische Univ. Hamburg-Harburg (Germany). Inst. Biotechnologie und Verfahrenstechnik

    2010-07-01

    Alternative Sources of Adult Stem Cells: Human Amniotic Membrane, by S. Wolbank, M. van Griensven, R. Grillari-Voglauer, and A. Peterbauer-Scherb; - Mesenchymal Stromal Cells Derived from Human Umbilical Cord Tissues: Primitive Cells with Potential for Clinical and Tissue Engineering Applications, by P. Moretti, T. Hatlapatka, D. Marten, A. Lavrentieva, I. Majore, R. Hass and C. Kasper; - Isolation, Characterization, Differentiation, and Application of Adipose-Derived Stem Cells, by J. W. Kuhbier, B. Weyand, C. Radtke, P. M. Vogt, C. Kasper and K. Reimers; - Induced Pluripotent Stem Cells: Characteristics and Perspectives, by T. Cantz and U. Martin; - Induced Pluripotent Stem Cell Technology in Regenerative Medicine and Biology, by D. Pei, J. Xu, Q. Zhuang, H.-F. Tse and M. A. Esteban; - Production Process for Stem Cell Based Therapeutic Implants: Expansion of the Production Cell Line and Cultivation of Encapsulated Cells, by C. Weber, S. Pohl, R. Poertner, P. Pino-Grace, D. Freimark, C. Wallrapp, P. Geigle and P. Czermak; - Cartilage Engineering from Mesenchymal Stem Cells, by C. Goepfert, A. Slobodianski, A.F. Schilling, P. Adamietz and R. Poertner; - Outgrowth Endothelial Cells: Sources, Characteristics and Potential Applications in Tissue Engineering and Regenerative Medicine, by S. Fuchs, E. Dohle, M. Kolbe, C. J. Kirkpatrick; - Basic Science and Clinical Application of Stem Cells in Veterinary Medicine, by I. Ribitsch, J. Burk, U. Delling, C. Geissler, C. Gittel, H. Juelke, W. Brehm; - Bone Marrow Stem Cells in Clinical Application: Harnessing Paracrine Roles and Niche Mechanisms, by R. M. El Backly, R. Cancedda; - Clinical Application of Stem Cells in the Cardiovascular System, C. Stamm, K. Klose, Y.-H. Choi. (orig.)

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

    Science.gov (United States)

    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.

  18. Perapion connexum (Schilsky, 1902) (Coleoptera, Apionidae) in Central Europe, a case of plant expansion chase

    Science.gov (United States)

    Wanat, Marek; Podlussány, Attila; Schön, Karel

    2012-01-01

    Abstract Perapion connexum (Schilsky) is recorded for the first time from Hungary and Kyrgyzstan, and new distribution data from Ukraine and Russia are provided. Preliminary placements of this weevil in faunal checklists for Poland and Slovakia are here documented with detailed data. Its occurrence in Austria based on older evidence, is discussed. The neophytic and invasive in Central Europe sorrel Rumex confertus Willd. is confirmed to be its unique host plant in Poland. Morphology of the newcoming weevil is described and illustrated, and the key to all Central European species of Perapion is presented. PMID:22451784

  19. Perapion connexum (Schilsky, 1902 (Coleoptera, Apionidae in Central Europe, a case of plant expansion chase

    Directory of Open Access Journals (Sweden)

    Marek Wanat

    2012-03-01

    Full Text Available Perapion connexum (Schilsky is recorded for the first time from Hungary and Kyrgyzstan, and new distribution data from Ukraine and Russia are provided. Preliminary placements of this weevil in faunal checklists for Poland and Slovakia are here documented with detailed data. Its occurrence in Austria based on older evidence, is discussed. The neophytic and invasive in Central Europe sorrel Rumex confertus Willd. is confirmed to be its unique host plant in Poland. Morphology of the newcoming weevil is described and illustrated, and the key to all Central European species of Perapion is presented.

  20. Perapion connexum (Schilsky, 1902) (Coleoptera, Apionidae) in Central Europe, a case of plant expansion chase.

    Science.gov (United States)

    Wanat, Marek; Podlussány, Attila; Schön, Karel

    2012-01-01

    Perapion connexum (Schilsky) is recorded for the first time from Hungary and Kyrgyzstan, and new distribution data from Ukraine and Russia are provided. Preliminary placements of this weevil in faunal checklists for Poland and Slovakia are here documented with detailed data. Its occurrence in Austria based on older evidence, is discussed. The neophytic and invasive in Central Europe sorrel Rumex confertus Willd. is confirmed to be its unique host plant in Poland. Morphology of the newcoming weevil is described and illustrated, and the key to all Central European species of Perapion is presented.

  1. Reactivation or expansion of Hotel Baker Hydro plant. Feasibility assessment report

    Energy Technology Data Exchange (ETDEWEB)

    1979-03-01

    The Hotel Baker Hydroelectric Power plant, built in 1928 at St. Charles, IL., has two generating units which until 1970 supplied the hotel with 100 kW output at 6 ft. head. The feasibility of reactivating, upgrading, or replacing these units to again supply power for the hotel was evaluated considering installation and operating costs, financing requirements, environmental impacts, and regulatory requirements. It was concluded that the reactivation project is marginally attractive only with full use of the power output by an expanded hotel and if electric utility rates increase at 8% per year. (LCL)

  2. Current Injection Provokes Rapid Expansion of the Guard Cell Cytosolic Volume and Triggers Ca(2+) Signals.

    Science.gov (United States)

    Voss, Lena J; Hedrich, Rainer; Roelfsema, M Rob G

    2016-03-07

    High-resolution microscopy opens the door for detailed single-cell studies with fluorescent reporter dyes and proteins. We used a confocal spinning disc microscope to monitor fluorescent dyes and the fluorescent protein Venus in tobacco and Arabidopsis guard cells. Multi-barreled microelectrodes were used to inject dyes and apply voltage pulses, which provoke transient rises in the cytosolic Ca(2+) level. Voltage pulses also caused changes in the distribution of Lucifer Yellow and Venus, which pointed to a reversible increase of guard cell cytosolic volume. The dynamic cytosolic volume changes turned out to be provoked by current injection of ions. A reduction of the clamp current, by blocking K(+) uptake channels with Cs(+), strongly suppressed the cytosolic volume changes. Cs(+) not only inhibited the expansion of the cytosol, but also inhibited hyperpolarization-induced elevations of the cytosolic Ca(2+) concentration. A complete loss of voltage-induced Ca(2+) signals occurred when Ca(2+)-permeable plasma membrane channels were simultaneously blocked with La(3+). This shows that two mechanisms cause hyperpolarization-induced elevation of the cytosolic Ca(2+)-concentration: (i) activation of voltage-dependent Ca(2+)-permeable channels, (ii) osmotically induced expansion of the cytosol, which leads to a release of Ca(2+) from intracellular stores.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Ashley A. Zurawel

    2016-09-01

    Full Text Available 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.

  5. Classifying the expansion kinetics and critical surface dynamics of growing cell populations

    CERN Document Server

    Block, M; Drasdo, D

    2006-01-01

    Based on a cellular automaton model the growth kinetics and the critical surface dynamics of cell monolayers is systematically studied by variation of the cell migration activity, the size of the proliferation zone and the cell cycle time distribution over wide ranges. The model design avoids lattice artifacts and ensures high performance. The monolayer expansion velocity derived from our simulations can be interpreted as a generalization of the velocity relationship for a traveling front in the Fisher-Kolmogorov-Petrovskii-Piskounov (FKPP) equation that is frequently used to model tumor growth phenomena by continuum models. The critical surface dynamics corresponds to the Kardar-Parisi-Zhang (KPZ) universality class for all parameters and model variations studied. While the velocity agrees quantitatively with experimental observations by Bru et al, the critical surface dynamics is in contrast to their interpretation as generic molecular-beam-epitaxy-like growth.

  6. Neocortex expansion in development and evolution - from cell biology to single genes.

    Science.gov (United States)

    Wilsch-Bräuninger, Michaela; Florio, Marta; Huttner, Wieland B

    2016-08-01

    Neocortex expansion in development and evolution reflects an increased and prolonged activity of neural progenitor cells. Insight into key aspects of the underlying cell biology has recently been obtained. First, the restriction of apical progenitors to undergo mitosis at the ventricular surface is overcome by generation of basal progenitors, which are free to undergo mitosis at abventricular location, typically the subventricular zone. This process involves basolateral ciliogenesis, delamination from the apical adherens junction belt, and loss of apical cell polarity. Second, proliferative capacity of basal progenitors is supported by self-produced extracellular matrix constituents, which in turn promote growth factor signalling. Humans amplify these processes by characteristic alterations in expression of key regulatory genes (PAX6), and via human-specific genes (ARHGAP11B).

  7. Evaluation of the expansion of umbilical cord blood derived from CD133+ cells on biocompatible microwells

    Directory of Open Access Journals (Sweden)

    Mina Soufizomorrod

    2016-05-01

    Full Text Available Background: Hematopoietic stem cell transplantation (HSCT is a therapeutic approach for treatment of hematological malignancies and incompatibility of Bone marrow. Umbilical cord blood (UCB has known as an alternative for hematopoietic stem/progenitor cells (HPSC in allogeneic transplantation. The low volume of collected samples is the main hindrance in application of HPSC derived from umbilical cord blood. So, ex vivo expansion of HPSCs is the useful approach to overcome this restriction. The goal of using this system is to produce appropriate amount of hematopoietic stem cells, which have the ability of transplantation and long term haematopoiesis. Material & Methods: In current study CD133+ cells were isolated from cord blood (CB. Isolated cells were seeded on microwells. Then expanded cells proliferation rate and ability in colony formation were assessed and finally were compared with 2 Dimensional (2D culture systems. Results: Our findings demonstrated that CD133+ cells derived from UCB which were cultivated on microwells had significantly higher rate of proliferation in compared with routine cell culture systems. Conclusion: In Current study, it was shown that CD133+ cells’ proliferations which were seeded on PDMS microwells coated with collagen significantly increased. We hope that 3 dimensional (3D microenvironment which mimics the 3D structure of bone marrow can solve the problem of using UCB as an alternative source of bone marrow.

  8. Diminished Memory T-Cell Expansion Due to Delayed Kinetics of Antigen Expression by Lentivectors.

    Directory of Open Access Journals (Sweden)

    Karina Furmanov

    Full Text Available Memory CD8(+ T lymphocytes play a central role in protective immunity. In attempt to increase the frequencies of memory CD8(+ T cells, repeated immunizations with viral vectors are regularly explored. Lentivectors have emerged as a powerful vaccine modality with relatively low pre-existing and anti-vector immunity, thus, thought to be ideal for boosting memory T cells. Nevertheless, we found that lentivectors elicited diminished secondary T-cell responses that did not exceed those obtained by priming. This was not due to the presence of anti-vector immunity, as limited secondary responses were also observed following heterologous prime-boost immunizations. By dissecting the mechanisms involved in this process, we demonstrate that lentivectors trigger exceptionally slow kinetics of antigen expression, while optimal activation of lentivector-induced T cells relays on durable expression of the antigen. These qualities hamper secondary responses, since lentivector-encoded antigen is rapidly cleared by primary cytotoxic T cells that limit its presentation by dendritic cells. Indeed, blocking antigen clearance by cytotoxic T cells via FTY720 treatment, fully restored antigen presentation. Taken together, while low antigen expression is expected during secondary immunization with any vaccine vector, our results reveal that the intrinsic delayed expression kinetics of lentiviral-encoded antigen, further dampens secondary CD8(+ T-cell expansion.

  9. Atypical PKC-iota Controls Stem Cell Expansion via Regulation of the Notch Pathway

    Directory of Open Access Journals (Sweden)

    In Kyoung Mah

    2015-11-01

    Full Text Available The number of stem/progenitor cells available can profoundly impact tissue homeostasis and the response to injury or disease. Here, we propose that an atypical PKC, Prkci, is a key player in regulating the switch from an expansion to a differentiation/maintenance phase via regulation of Notch, thus linking the polarity pathway with the control of stem cell self-renewal. Prkci is known to influence symmetric cell division in invertebrates; however a definitive role in mammals has not yet emerged. Using a genetic approach, we find that loss of Prkci results in a marked increase in the number of various stem/progenitor cells. The mechanism used likely involves inactivation and symmetric localization of NUMB, leading to the activation of NOTCH1 and its downstream effectors. Inhibition of atypical PKCs may be useful for boosting the production of pluripotent stem cells, multipotent stem cells, or possibly even primordial germ cells by promoting the stem cell/progenitor fate.

  10. Telomere Attrition Occurs during Ex Vivo Expansion of Human Dental Pulp Stem Cells

    Directory of Open Access Journals (Sweden)

    Jaroslav Mokry

    2010-01-01

    Full Text Available We provide a detailed characteristic of stem cells isolated and expanded from the human dental pulp. Dental pulp stem cells express mesenchymal cell markers STRO-1, vimentin, CD29, CD44, CD73, CD90, CD166, and stem cell markers Sox2, nestin, and nucleostemin. They are multipotent as shown by their osteogenic and chondrogenic potential. We measured relative telomere length in 11 dental pulp stem cell lines at different passages by quantitative real-time PCR. Despite their large proliferative capacity, stable viability, phenotype, and genotype over prolonged cultivation, human dental pulp stem cells suffer from progressive telomere shortening over time they replicate in vitro. Relative telomere length (T/S was inversely correlated with cumulative doubling time. Our findings indicate that excessive ex vivo expansion of adult stem cells should be reduced at minimum to avoid detrimental effects on telomere maintenance and measurement of telomere length should become a standard when certificating the status and replicative age of stem cells prior therapeutic applications.

  11. Expansion in vitro and cytotoxicity of dendritic cells from patients with chronic myeloid leukemia.

    Science.gov (United States)

    Ji, Lei; Xing, Pei-Ni; Wei, Xu-Cang; Wang, Tong; Li, Mei-Sheng; Zhang, Wang-Gang

    2005-04-01

    The study was aimed to investigate the extensive amplification and the cytotoxicity of dendritic cells (DC) derived from chronic myeloid leukemia cells. DC were cultured in two steps: firstly, extensive amplification in primary culture of CD34(+) or mononuclear cells isolated from CML patients' bone marrow and peripheral blood with rhFlt3-L and rhTPO for 7 days; secondly, inducing culture of DC with rhGM-CSF, rhTNF and rhIL-4 for 14 days. A system inducing DC directly were established for comparison. DC were identified by immunophenotype with flow cytometry, chromosome analysis by displaying G banding and electric microscopy analysis. The function of stimulating T cells proliferation and cytotoxicity of CML cells were confirmed through MTT assay. The results showed that after first extensive amplification in primary culture with rhFlt3-L and rhTPO for 7 days, CD34(+) cells had a total cell number with (77 +/- 5) fold expansion, and DC were (39 +/- 8)% of total cell respectively after induction culture of DC with rhGM-CSF, rhTNF and rhIL-4 for 14 days. Both the amplification of cell number and yield of DC were higher than the system without extensively culture (P < 0.01). Such DC could stimulate T cells to proliferate and kill leukemia cells finally. In conclusion, two-step culture method can obviously improve the cell number of DC required, that is better than inducing them directly. DC derived from CML cells induce the generation of anti-leukemia immunization.

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

    Science.gov (United States)

    Butler, Colin R; Hynds, Robert E; 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; Janes, Sam M

    2016-07-15

    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. To define a scalable cell culture system to deliver airway epithelium to clinical grafts. 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. 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. Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.

  13. Characterization and ex vivo Expansion of Human Placenta-Derived Natural Killer Cells for Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Xiaokui eZhang

    2013-05-01

    Full Text Available Recent clinical studies suggest that adoptive transfer of donor-derived natural killer (NK cells may improve clinical outcome in hematological malignancies and some solid tumors by direct antitumor effects as well as by reduction of graft versus host disease (GVHD. NK cells have also been shown to enhance transplant engraftment during allogeneic hematopoietic stem cell transplantation (HSCT for hematological malignancies. The limited ex vivo expansion potential of NK cells from peripheral blood (PB or umbilical cord blood (UCB has however restricted their therapeutic potential. Here we define methods to efficiently generate NK cells from donor matched, full-term human placenta perfusate (termed Human Placenta-Derived Stem Cell, HPDSC and UCB. Following isolation from cryopreserved donor-matched HPDSC and UCB units, CD56+CD3- placenta-derived NK cells, termed pNK cells, were expanded in culture for up to 3 weeks to yield an average of 1.2 billion cells per donor that were >80% CD56+CD3-, comparable to doses previously utilized in clinical applications. Ex vivo-expanded pNK cells exhibited a marked increase in anti-tumor cytolytic activity coinciding with the significantly increased expression of NKG2D, NKp46 and NKp44 (p < 0.001, p < 0.001, and p < 0.05, respectively. Strong cytolytic activity was observed against a wide range of tumor cell lines in vitro. pNK cells display a distinct microRNA (miRNA expression profile, immunophenotype and greater antitumor capacity in vitro compared to PB NK cells used in recent clinical trials. With further development, pNK may represent a novel and effective cellular immunotherapy for patients with high clinical needs and few other therapeutic options.

  14. Fluorescence activated cell sorting of plant protoplasts.

    Science.gov (United States)

    Bargmann, Bastiaan O R; Birnbaum, Kenneth D

    2010-02-18

    High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce

  15. THE GENESIS OF PHOTOSYNTHESIS TYPES AS THE BASIS OF ECOLOGICAL EXPANSION OF HALOPHYTIC PLANTS

    Directory of Open Access Journals (Sweden)

    Pyurko O.Ye.

    2011-12-01

    Full Text Available The C3, C4, and CAM photosynthesis types are considerably differed by CO2 absorption intensity, its biochemistry, saturation level, water productivity, biological productivity, and other different features, which secure the plants survival at stress and extreme conditions. The aim of current research was to discover the photosynthesis peculiarities at halophytic plants species (Salicornia europaea L., Halimione pedunculata, Artemisia santonica L., Plantago lanceolata L. by salinity at model and natural conditions, and to generalize data in historical aspect. It was constituted that S. europaea L. was characterized by C3 photosynthesis passage which was switched on CAM CO2 fixation under soil salinity conditions till 4-4,5 %, but glycophyte A.santonica was immanent C4assimilation way of aspartate type.Analysis of literature data and own research allows to find out that in majority the C3photosynthesis dependence from environmental factors described by determinate curve with matched mathematical expression. It was suggested to generalize the data by Lagrange polynomial. The obtained results proved that the pattern of photosynthesis evolution is: C3 → C4 → CAM with commute possibilities: C3 → CAM; C4 → CAM.

  16. Gelatin–PMVE/MA composite scaffold promotes expansion of embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chhabra, Hemlata [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); Gupta, Priyanka [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); IITB-Monash Research Academy, Mumbai (India); Department of Chemical Engineering, Monash University, Melbourne (Australia); Verma, Paul J. [Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, South Australia (Australia); Jadhav, Sameer; Bellare, Jayesh R. [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India)

    2014-04-01

    We introduce a new composite scaffold of gelatin and polymethyl vinyl ether-alt-maleic anhydride (PMVE/MA) for expansion of embryonic stem cells (ESCs) in an in vitro environment. To optimize the scaffold, we prepared a gelatin scaffold (G) and three composite scaffolds namely GP-1, GP-2, and GP-3 with varying PMVE/MA concentrations (0.2–1%) and characterized them by scanning electron microscopy (SEM), swelling study, compression testing and FTIR. SEM micrographs revealed interconnected porous structure in all the scaffolds. The permissible hemolysis ratio and activation of platelets by scaffolds confirmed the hemocompatibility of scaffolds. Initial biocompatibility assessment of scaffolds was conducted using hepatocarcinoma (Hep G2) cells and adhesion, proliferation and infiltration of Hep G2 cells in depth of scaffolds were observed, proving the scaffold's biocompatibility. Further Oct4B2 mouse embryonic stem cells (mESCs), which harbor a green fluorescence protein transgene under regulatory control of the Oct4 promotor, were examined for expansion on scaffolds with MTT assay. The GP-2 scaffold demonstrated the best cell proliferation and was further explored for ESC adherence and infiltration in depth (SEM and confocal), and pluripotent state of mESCs was assessed with the expression of Oct4-GFP and stage-specific embryonic antigen-1 (SSEA-1). This study reports the first demonstration of biocompatibility of gelatin–PMVE/MA composite scaffold and presents this scaffold as a promising candidate for embryonic stem cell based tissue engineering. - Highlights: • Composite scaffolds of gelatin and PMVE/MA were prepared by freeze-drying method. • SEM micrographs showed porous structure in all scaffolds of varying pore dimension. • GP-2 composite exhibited better cellular response in comparison to other scaffolds. • mESCs proliferated and expressed Oct-4 and SSEA-1, when cultured on GP-2 scaffold.

  17. Histone deacetylase 11: A novel epigenetic regulator of myeloid derived suppressor cell expansion and function.

    Science.gov (United States)

    Sahakian, Eva; Powers, John J; Chen, Jie; Deng, Susan L; Cheng, Fengdong; Distler, Allison; Woods, David M; Rock-Klotz, Jennifer; Sodre, Andressa L; Youn, Je-In; Woan, Karrune V; Villagra, Alejandro; Gabrilovich, Dmitry; Sotomayor, Eduardo M; Pinilla-Ibarz, Javier

    2015-02-01

    Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of cells capable of suppressing anti-tumor T cell function in the tumor microenvironment, represent an imposing obstacle in the development of cancer immunotherapeutics. Thus, identifying elements essential to the development and perpetuation of these cells will undoubtedly improve our ability to circumvent their suppressive impact. HDAC11 has emerged as a key regulator of IL-10 gene expression in myeloid cells, suggesting that this may represent an important targetable axis through which to dampen MDSC formation. Using a murine transgenic reporter model system where eGFP expression is controlled by the HDAC11 promoter (Tg-HDAC11-eGFP), we provide evidence that HDAC11 appears to function as a negative regulator of MDSC expansion/function in vivo. MDSCs isolated from EL4 tumor-bearing Tg-HDAC11-eGFP display high expression of eGFP, indicative of HDAC11 transcriptional activation at steady state. In striking contrast, immature myeloid cells in tumor-bearing mice display a diminished eGFP expression, implying that the transition of IMC to MDSC's require a decrease in the expression of HDAC11, where we postulate that it acts as a gate-keeper of myeloid differentiation. Indeed, tumor-bearing HDAC11-knockout mice (HDAC11-KO) demonstrate a more suppressive MDSC population as compared to wild-type (WT) tumor-bearing control. Notably, the HDAC11-KO tumor-bearing mice exhibit enhanced tumor growth kinetics when compare to the WT control mice. Thus, through a better understanding of this previously unknown role of HDAC11 in MDSC expansion and function, rational development of targeted epigenetic modifiers may allow us to thwart a powerful barrier to efficacious immunotherapies.

  18. Ceratopteris richardii (C-fern: A model for investigating adaptive modification of vascular plant cell walls

    Directory of Open Access Journals (Sweden)

    Olivier eLeroux

    2013-09-01

    Full Text Available Plant cell walls are essential for most aspects of plant growth, development, and survival, including cell division, expansive cell growth, cell-cell communication, biomechanical properties, and stress responses. Therefore, characterising cell wall diversity contributes to our overall understanding of plant evolution and development. Recent biochemical analyses, concomitantly with whole genome sequencing of plants located at pivotal points in plant phylogeny, have helped distinguish between homologous characters and those which might be more derived. Most plant lineages now have at least one fully sequenced representative and although genome sequences for fern species are in progress they not yet available this group. Ferns offer key advantages for the study of developmental processes leading to vascularisation and complex organs as well as the specific differences between diploid sporophyte tissues and haploid gametophyte tissues and the interplay between them. Ceratopteris richardii has been well investigated building a body of knowledge which combined with the genomic and biochemical information available for other plants will progress our understanding of wall diversity and its impact on evolution and development.

  19. Microanalysis of Plant Cell Wall Polysaccharides

    Institute of Scientific and Technical Information of China (English)

    Nicolai Obel; Veronika Erben; Tatjana Schwarz; Stefan Kühne; Andrea Fodor; Markus Pauly

    2009-01-01

    Oligosaccharide Mass Profiling (OLIMP) allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry (MALDI-TOF MS). The short time required for sample preparation and analysis makes possible the study of a wide range of plant organs, revealing a high degree of heterogeneity in the substitution pattern of wall polymers such as the cross-linking glycan xyloglucan and the pectic polysaccharide homogalacturonan. The high sensitivity of MALDI-TOF allows the use of small amounts of samples, thus making it possible to investigate the wall structure of single cell types when material is collected by such methods as laser micro-dissection. As an example, the analysis of the xyloglucan structure in the leaf cell types outer epidermis layer, entire epidermis cell layer, palisade mesophyll cells, and vascular bundles were investigated. OLIMP is amenable to in situ wall analysis, where wall polymers are analyzed on unprepared plant tissue itself without first iso-lating cell walls. In addition, OLIMP enables analysis of wall polymers in Golgi-enriched fractions, the location of nascent matrix polysaccharide biosynthesis, enabling separation of the processes of wall biosynthesis versus post-deposition apo-plastic metabolism. These new tools will make possible a semi-quantitative analysis of the cell wall at an unprecedented level.

  20. Efficient Expansion of SALL4–Transduced Umbilical Cord Blood Derived CD133+Hematopoietic Stem Cells

    Directory of Open Access Journals (Sweden)

    Majid Mossahebi-Mohammadi

    2017-07-01

    Full Text Available Hematopoietic stem cells (HSCs were characterized by self-renewal and multilineage potential. Umbilical cord blood-derived (UCB as an alternative source of HSCs is widely used especially in children for stem cells transplant (SCT. The main limitation in using UCB for transplantation especially in adults is low cell dose. To overcome this limitation besides using double dose UCB, ex vivo expansion is the most important way to increase cell number for transplantation. HSCs are mainly isolated using CD133 or CD34. CD133, as the most primitive marker, shows important physiological role in maintenance and expansion of HSCs. SALL4 plays crucial role in the development and maintaining the pluripotency and self-renewal ability of embryonic stem cells (ESCs as well as HSCs. Moreover, SALL4 act as a regulator of HSCs expansion, normal hematopoiesis, and hematological malignancies. In the present study, CD133+ cells positively selected and ex vivo expanded in SALL-4 and GFP-transduced group. CD133 expression assessed using flow cytometry at day 0, 7 and 10. Moreover, multilineage differentiation and proliferation potential of expanded cells in both groups evaluated using colony forming unit (CFU assay, and cells count assay. Karyotyping analysis was performed to assess any chromosomal instability after 7 days of expansion. Obtained results demonstrated that SALL-4 transduced cells showed significant increase in cell number compared to control group. Moreover, immunophenotyping results showed higher expression level of CD133 at day 7 and 10 following expansion in SALL-4 transduced (62 % and 42% compared to control group (51% and 20.6%. Our results illustrated that SALL4 could act as a positive factor for the expansion of CD133+ derived UCB cells besides maintaining self-renewal and differentiation ability of expanded cell without any numerical and structural chromosomal aberrations .

  1. NFAT5 regulates T lymphocyte homeostasis and CD24-dependent T cell expansion under pathologic hypernatremia.

    Science.gov (United States)

    Berga-Bolaños, Rosa; Drews-Elger, Katherine; Aramburu, Jose; López-Rodríguez, Cristina

    2010-12-01

    Immune cells rely on the transcription factor NFAT5 to adapt to hypertonic stress. The hypertonicity-dependent role of NFAT5 in T cells in vivo remains unclear because mouse models of NFAT5 deficiency have produced substantially different T cell phenotypes. In this study, we analyzed the T cell compartment in NFAT5-null and T cell-specific NFAT5 knockout mice. We found that NFAT5-null mice had constitutive, pronounced hypernatremia and suffered a severe immunodeficiency, with T cell lymphopenia, altered CD8 naive/memory homeostasis, and inability to reject allogeneic tumors. By contrast, T cell-specific NFAT5 knockout mice had normal plasma tonicity, rejected allogeneic tumors, and exhibited only a mild, low-penetrance memory bias in CD8 cells. Notably, when T cells from these mice were cultured ex vivo in hypernatremic media, they exhibited features found in NFAT5-null mice, with pronounced naive/memory imbalance and impaired homeostatic survival in response to IL-7, as well as a severe inhibition of their mitogen-induced proliferation. By analyzing surface receptors whose expression might be affected in NFAT5-deficient cells, we identified CD24 as a novel NFAT5 target induced by hypertonicity both in vitro and in vivo, and required to sustain T cell expansion under osmostress. NFAT5 bound to the Cd24 promoter in response to hypertonicity facilitated the local derepression of chromatin and enhanced the expression of CD24 mRNA and protein. Altogether, our results indicate that the systemic hypernatremia of NFAT5-null mice is a major contributor to their immunodeficiency, and highlight the role of NFAT5 and CD24 in the homeostasis of T cells under osmostress in vivo.

  2. The Role of Amnion Membrane-Derived Mesenchymal Stem Cells on Differentiation and Expansion of Natural Killer Cell Progenitors Originated From Umbilical Cord Blood Mononuclear Cells

    Directory of Open Access Journals (Sweden)

    Ahmadi

    2015-11-01

    Full Text Available Background Natural killer (NK cells are members of the innate immune system. Their unique properties, including recognition of viral infected and tumor cells without major histocompatibility complex (MHC restriction or prior sensitization, make them a suitable choice for immunotherapy. Low numbers of NK cells in circulating blood is the most important obstacle for this goal. Objectives The aim of this study was to make an optimum in vitro condition to proliferate and differentiate cord blood (CB-NK cell progenitors to mature NK cells, which can be used for cell therapy. Materials and Methods In our study, CB-Mononuclear Cells’ (MNCs CD3+ lymphocytes were positive depleted using immunomagnetic microbeads. This CD3-depleted (CD3-dep CB - MNCs compartment was used for in vitro expansion with or without a layer of amnion membrane mesenchymal stem cells (MSCs in combination with cytokines that are essential for NK cells expansion (IL-2, IL-3, IL-15, and FLT3 ligand. The expansion period lasted for one week. On day seven, immunophenotype and fold expansion of differentiated cells were measured. Results Combination of cytokines and MSC layer yielded significant fold expansion in comparison with cytokines without feeder conditions (day 7: 5.2 ± 1.12 and 2 ± 0.78, respectively, P < 0.05. CD3-/CD56+ cells percentage increased during the culture period in MSCs/with cytokine and cytokine/without feeder, respectively (day 0: 4.4 ± 0.42% and day 7: 22.9 ± 3.6% and 13.9 ± 1.92 % for MSC/with cytokine and cytokine without feeder, respectively. Conclusions Our results suggested that CB-NK cells progenitors could proliferate and differentiate on feeder layer of amnion membrane MSCs in combination with specific cytokines to produce NK cells for immunotherapy.

  3. Ex vivo expansion and pluripotential differentiation of cryopreserved human bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    XIANG Ying; ZHENG Qiang; JIA Bing-bing; HUANG Guo-ping; Xu Yu-lin; WANG Jin-fu; PAN Zhi-jun

    2007-01-01

    This study is aimed at investigating the potentials of ex vivo expansion and pluri-differentiation of cryopreservation of adult human bone marrow mesenchymal stem cells (hMSCs) into chondrocytes, adipocytes and neurocytes. Cryopreserved hMSCs were resuscitated and cultured for 15 passages, and then induced into chondrocytes, adipocytes and neurocytes with corresponding induction medium. The induced cells were observed for morphological properties and detected for expressions of type II collagen, triglyceride or neuron-specific enolase and nestin. The result showed that the resuscitated cells could differentiate into chondrocytes after exposure to transforming growth factor β1 (TGF-β1), insulin-like growth factor I (IGF-I) and vitamin C (Vc), and uniformly changed morphologically from a spindle-like fibroblastic appearance to a polygonal shape in three weeks. The induced cells were heterochromatic to safranin O and expressed cartilage matrix-procollagenal (II) mRNA. The resuscitated cells cultured in induction medium consisting of dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin and IGF-I showed adipogenesis, and lipid vacuoles accumulation was detectable after 21 d. The resuscitated hMSCs were also induced into neurocytes and expressed nestin and neuron specific endolase (NSE) that were special surface markers associated with neural cells at different stage. This study suggested that the resuscitated hMSCs should be still a population of pluripotential cells and that it could be used for establishing an abundant hMSC reservoir for further experiment and treatment of various clinical diseases.

  4. Direct FuelCell/Turbine Power Plant

    Energy Technology Data Exchange (ETDEWEB)

    Hossein Ghezel-Ayagh

    2008-09-30

    This report summarizes the progress made in development of Direct FuelCell/Turbine (DFC/T{reg_sign}) power plants for generation of clean power at very high efficiencies. The DFC/T system employs an indirectly heated Turbine Generator to supplement fuel cell generated power. The concept extends the high efficiency of the fuel cell by utilizing the fuel cell's byproduct heat in a Brayton cycle. Features of the DFC/T system include: electrical efficiencies of up to 75% on natural gas, minimal emissions, reduced carbon dioxide release to the environment, simplicity in design, direct reforming internal to the fuel cell, and potential cost competitiveness with existing combined cycle power plants. Proof-of-concept tests using a sub-MW-class DFC/T power plant at FuelCell Energy's (FCE) Danbury facility were conducted to validate the feasibility of the concept and to measure its potential for electric power production. A 400 kW-class power plant test facility was designed and retrofitted to conduct the tests. The initial series of tests involved integration of a full-size (250 kW) Direct FuelCell stack with a 30 kW Capstone microturbine. The operational aspects of the hybrid system in relation to the integration of the microturbine with the fuel cell, process flow and thermal balances, and control strategies for power cycling of the system, were investigated. A subsequent series of tests included operation of the sub-MW Direct FuelCell/Turbine power plant with a Capstone C60 microturbine. The C60 microturbine extended the range of operation of the hybrid power plant to higher current densities (higher power) than achieved in initial tests using the 30kW microturbine. The proof-of-concept test results confirmed the stability and controllability of operating a fullsize (250 kW) fuel cell stack in combination with a microturbine. Thermal management of the system was confirmed and power plant operation, using the microturbine as the only source of fresh air supply

  5. GMP-Compliant Expansion of Clinical-Grade Human Mesenchymal Stromal/Stem Cells Using a Closed Hollow Fiber Bioreactor.

    Science.gov (United States)

    Barckhausen, Christina; Rice, Brent; Baila, Stefano; Sensebé, Luc; Schrezenmeier, Hubert; Nold, Philipp; Hackstein, Holger; Rojewski, Markus Thomas

    2016-01-01

    This chapter describes a method for GMP-compliant expansion of human mesenchymal stromal/stem cells (hMSC) from bone marrow aspirates, using the Quantum(®) Cell Expansion System from Terumo BCT. The Quantum system is a functionally closed, automated hollow fiber bioreactor system designed to reproducibly grow cells in either GMP or research laboratory environments. The chapter includes protocols for preparation of media, setup of the Quantum system, coating of the hollow fiber bioreactor, as well as loading, feeding, and harvesting of cells. We suggest a panel of quality controls for the starting material, the interim product, as well as the final product.

  6. Research Resource: A Dual Proteomic Approach Identifies Regulated Islet Proteins During β-Cell Mass Expansion In Vivo

    DEFF Research Database (Denmark)

    Horn, Signe; Kirkegaard, Jeannette S.; Hoelper, Soraya

    2016-01-01

    Diabetes is characterized by insulin insufficiency due to a relative paucity of functional β-cell mass. Thus, strategies for increasing β-cell mass in situ are sought-after for therapeutic purposes. Pregnancy is a physiological state capable of inducing robust β-cell mass expansion, however, the ...... as upstream regulators of the observed expressional changes. As the first characterization of the islet-proteome during pregnancy, this study provides novel insight into the mechanisms involved in promoting pregnancy-induced β-cell mass expansion and function.[on SciFinder (R)]...

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

    Energy Technology Data Exchange (ETDEWEB)

    Chatterjee, Somik [Center for Diabetes Research, Department of Medicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Yin, Hongshan [Center for Diabetes Research, Department of Medicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Department of Cardiovascular Medicine, Third Affiliated Hospital, Hebei Medical University, Shijiazhuang 050051, Hebei (China); Nam, Deokhwa [Center for Diabetes Research, Department of Medicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Li, Yong [Department of Pediatric Surgery, Center for Stem Cell Research and Regenerative Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030 (United States); Ma, Ke, E-mail: kma@houstonmethodist.org [Center for Diabetes Research, Department of Medicine, Houston Methodist Research Institute, Houston, TX 77030 (United States)

    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.

  8. Redox regulation in plant programmed cell death.

    Science.gov (United States)

    De Pinto, M C; Locato, V; De Gara, L

    2012-02-01

    Programmed cell death (PCD) is a genetically controlled process described both in eukaryotic and prokaryotic organisms. Even if it is clear that PCD occurs in plants, in response to various developmental and environmental stimuli, the signalling pathways involved in the triggering of this cell suicide remain to be characterized. In this review, the main similarities and differences in the players involved in plant and animal PCD are outlined. Particular attention is paid to the role of reactive oxygen species (ROS) as key inducers of PCD in plants. The involvement of different kinds of ROS, different sites of ROS production, as well as their interaction with other molecules, is crucial in activating PCD in response to specific stimuli. Moreover, the importance is stressed on the balance between ROS production and scavenging, in various cell compartments, for the activation of specific steps in the signalling pathways triggering this cell suicide process. The review focuses on the complexity of the interplay between ROS and antioxidant molecules and enzymes in determining the most suitable redox environment required for the occurrence of different forms of PCD. © 2011 Blackwell Publishing Ltd.

  9. Characterization of Plant Functions Using Cultured Plant Cells, and Biotechnological Applications

    National Research Council Canada - National Science Library

    SATO, Fumihiko

    2013-01-01

    .... On the other hand, the use of plant cell cultures for the more basic characterization of plant functions is rather limited due to the difficulties associated with functional differentiation in cell cultures...

  10. Plant thin cell layers: update and perspectives

    Directory of Open Access Journals (Sweden)

    Teixeira da Silva Jaime A.

    2015-12-01

    Full Text Available Thin cell layers (TCLs are small and versatile explants for the in vitro culture of plants. At face value, their morphogenic productivity may appear to be less than conventional explants, but once the plant growth correction factor and geometric factor have been applied, the true (potential productivity exceeds that of a conventional explant. It is for this reason that for almost 45 years, TCLs have been applied to the in vitro culture of almost 90 species or hybrids, mainly ornamentals and orchids, but also to field and vegetable crops and medicinal plants. Focusing on 12 new studies that have emerged in the recent past (2013-2015, this paper brings promise to other horticultural species that could benefit from the use of TCLs.

  11. Characterization of Cellulose Synthesis in Plant Cells

    Directory of Open Access Journals (Sweden)

    Samaneh Sadat Maleki

    2016-01-01

    Full Text Available Cellulose is the most significant structural component of plant cell wall. Cellulose, polysaccharide containing repeated unbranched β (1-4 D-glucose units, is synthesized at the plasma membrane by the cellulose synthase complex (CSC from bacteria to plants. The CSC is involved in biosynthesis of cellulose microfibrils containing 18 cellulose synthase (CesA proteins. Macrofibrils can be formed with side by side arrangement of microfibrils. In addition, beside CesA, various proteins like the KORRIGAN, sucrose synthase, cytoskeletal components, and COBRA-like proteins have been involved in cellulose biosynthesis. Understanding the mechanisms of cellulose biosynthesis is of great importance not only for improving wood production in economically important forest trees to mankind but also for plant development. This review article covers the current knowledge about the cellulose biosynthesis-related gene family.

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

    Science.gov (United States)

    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

  13. Methods of isolation, expansion, differentiating induction and preservation of human umbilical cord mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    LI Dong-rui; CAI Jian-hui

    2012-01-01

    Objective This literature review aims to summarize the methods of isolation,expansion,differentiation and preservation of human umbilical cord mesenchymal stem cells (hUCMSCs),for comprehensive understanding and practical use in preclinical research and clinical trials.Data sources All the literature reviewed was published over the last 10 years and is listed in PubMed and Chinese National Knowledge Infrastructure (CNKI).Studies were retrieved using the key word "human umbilical cord mesenchymal stem cells".Results Explants culture and enzymatic digestion are two methods to isolate hUCMSCs from WJ and there are modifications to improve these methods.Culture conditions may affect the expansion and differentiating orientations of hUCMSCs.In addition,hUCMSCs can maintain their multi-potential effects after being properly frozen and thawed.Conclusion Considering their multi-potential,convenient and non-invasive accessibility,low immunogenicity and the reported therapeutic effects in several different preclinical animal models,hUCMSCs have immense scope in regeneration medicine as a substitute for MSCs derived from bone marrow or umbilical cord blood.

  14. Controlling Expansion and Cardiomyogenic Differentiation of Human Pluripotent Stem Cells in Scalable Suspension Culture

    Directory of Open Access Journals (Sweden)

    Henning Kempf

    2014-12-01

    Full Text Available To harness the potential of human pluripotent stem cells (hPSCs, an abundant supply of their progenies is required. Here, hPSC expansion as matrix-independent aggregates in suspension culture was combined with cardiomyogenic differentiation using chemical Wnt pathway modulators. A multiwell screen was scaled up to stirred Erlenmeyer flasks and subsequently to tank bioreactors, applying controlled feeding strategies (batch and cyclic perfusion. Cardiomyogenesis was sensitive to the GSK3 inhibitor CHIR99021 concentration, whereas the aggregate size was no prevailing factor across culture platforms. However, in bioreactors, the pattern of aggregate formation in the expansion phase dominated subsequent differentiation. Global profiling revealed a culture-dependent expression of BMP agonists/antagonists, suggesting their decisive role in cell-fate determination. Furthermore, metallothionein was discovered as a potentially stress-related marker in hPSCs. In 100 ml bioreactors, the production of 40 million predominantly ventricular-like cardiomyocytes (up to 85% purity was enabled that were directly applicable to bioartificial cardiac tissue formation.

  15. Making the switch: alternatives to foetal bovine serum for adipose-derived stromal cell expansion

    Directory of Open Access Journals (Sweden)

    Carla Dessels

    2016-10-01

    Full Text Available Adipose-derived stromal cells (ASCs are being used extensively in clinical trials. These trials require that ASCs are prepared using good manufacturing procedures (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.

  16. Plant F-box protein evolution is determined by lineage-specific timing of major gene family expansion waves.

    Directory of Open Access Journals (Sweden)

    Aura Navarro-Quezada

    Full Text Available F-box proteins (FBPs represent one of the largest and fastest evolving gene/protein families in the plant kingdom. The FBP superfamily can be divided in several subfamilies characterized by different C-terminal protein-protein interaction domains that recruit targets for proteasomal degradation. Hence, a clear picture of their phylogeny and molecular evolution is of special interest for the general understanding of evolutionary histories of multi-domain and/or large protein families in plants. In an effort to further understand the molecular evolution of F-box family proteins, we asked whether the largest subfamily in Arabidopsis thaliana, which carries a C-terminal F-box associated domain (FBA proteins shares evolutionary patterns and signatures of selection with other FBPs. To address this question, we applied phylogenetic and molecular evolution analyses in combination with the evaluation of transcriptional profiles. Based on the 2219 FBA proteins we de novo identified in 34 completely sequenced plant genomes, we compared their evolutionary patterns to a previously analyzed large subfamily carrying C-terminal kelch repeats. We found that these two large FBP subfamilies generally tend to evolve by massive waves of duplication, followed by sequence conservation of the F-box domain and sequence diversification of the target recruiting domain. We conclude that the earlier in evolutionary time a major wave of expansion occurred, the more pronounced these selection signatures are. As a consequence, when performing cross species comparisons among FBP subfamilies, significant differences will be observed in the selective signatures of protein-protein interaction domains. Depending on the species, the investigated subfamilies comprise up to 45% of the complete superfamily, indicating that other subfamilies possibly follow similar modes of evolution.

  17. Expansion on stromal cells preserves the undifferentiated state of human hematopoietic stem cells despite compromised reconstitution ability.

    Directory of Open Access Journals (Sweden)

    Mattias Magnusson

    Full Text Available Lack of HLA-matched hematopoietic stem cells (HSC limits the number of patients with life-threatening blood disorders that can be treated by HSC transplantation. So far, insufficient understanding of the regulatory mechanisms governing human HSC has precluded the development of effective protocols for culturing HSC for therapeutic use and molecular studies. We defined a culture system using OP9M2 mesenchymal stem cell (MSC stroma that protects human hematopoietic stem/progenitor cells (HSPC from differentiation and apoptosis. In addition, it facilitates a dramatic expansion of multipotent progenitors that retain the immunophenotype (CD34+CD38-CD90+ characteristic of human HSPC and proliferative potential over several weeks in culture. In contrast, transplantable HSC could be maintained, but not significantly expanded, during 2-week culture. Temporal analysis of the transcriptome of the ex vivo expanded CD34+CD38-CD90+ cells documented remarkable stability of most transcriptional regulators known to govern the undifferentiated HSC state. Nevertheless, it revealed dynamic fluctuations in transcriptional programs that associate with HSC behavior and may compromise HSC function, such as dysregulation of PBX1 regulated genetic networks. This culture system serves now as a platform for modeling human multilineage hematopoietic stem/progenitor cell hierarchy and studying the complex regulation of HSC identity and function required for successful ex vivo expansion of transplantable HSC.

  18. CTLA-4 blockade during dendritic cell based booster vaccination influences dendritic cell survival and CTL expansion

    DEFF Research Database (Denmark)

    Pedersen, Anders E; Ronchese, Franca

    2007-01-01

    Dendritic cells (DCs) are potent antigen-presenting cells and critical for the priming of CD8+ T cells. Therefore the use of these cells as adjuvant cells has been tested in a large number of experimental and clinical vaccination studies, in particular cancer vaccine studies. A number of protocols...

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

    Science.gov (United States)

    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

  20. Expansion of Th17 Cells by Human Mast Cells Is Driven by Inflammasome-Independent IL-1β.

    Science.gov (United States)

    Suurmond, Jolien; Habets, Kim L L; Dorjée, Annemarie L; Huizinga, Tom W; Toes, René E M

    2016-12-01

    Mast cells (MC) are most well known for their role in innate immune responses. However, MC are increasingly recognized as important regulators of adaptive immune responses, especially in setting the outcome of T cell responses. In this study we determined the effect of MC on cytokine production by naive and memory human Th cells. CD4(+) T cells were cultured with MC supernatant or control medium, after which cytokine production by T cells was determined. Supernatant of activated MC specifically increased the number of IL-17-producing T cells. This enhancement of Th17 cell number was specifically observed for the memory CD4(+) T cell population and not for the naive CD4(+) T cell population. The effect of MC was inhibited for ∼80% by blocking Abs to IL-1β and the rIL-1R antagonist anakinra. Importantly, secretion of active IL-1β by MC was independent of caspase activity, indicating that Th17 cell expansion by MC occurred through inflammasome-independent IL-1β. Together, these studies reveal a role for human MC in setting the outcome of T cell responses through release of caspase-independent IL-1β, and provide evidence for a novel contribution of MC in boosting the Th17 axis in mucosal immune responses. Copyright © 2016 by The American Association of Immunologists, Inc.

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

    Science.gov (United States)

    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.

  2. Unique association of Waldenström macroglobulinemia with optic neuritis and monoclonal T cell expansion.

    Science.gov (United States)

    Morita, Ken; Yoshimi, Akihide; Masuda, Akiko; Ichikawa, Motoshi; Yatomi, Yutaka; Kurokawa, Mineo

    2013-08-01

    Waldenström macroglobulinemia is a lymphoplasmacytic lymphoma characterized by production of the immunoglobulin M (IgM) monoclonal protein. Commonly involved sites are the bone marrow, lymph nodes, and spleen. Lymphoplasmacytic infiltration of the central nervous system (CNS), in contrast, is referred to as Bing-Neel syndrome, and is an extremely rare phenomenon. Here, we present a unique case of Waldenström macroglobulinemia with optic neuritis accompanied by monoclonal expansion of T cells, which recovered after administration of CNS-targeting chemotherapy. Although the underlying causal relationships in this case remain obscure, aberrantly expanded T cells may have contributed to the development of optic neuritis, and we should be reminded that some types of cranial neuropathy in Waldenström macroglobulinemia may be reversible.

  3. Ex vivo expansion of haematopoietic cells in the treatment of accidental irradiation-induced aplasia. Feasibility Studies

    Energy Technology Data Exchange (ETDEWEB)

    Thierry, D.; Bertho, J.M.; Chapel, A.; Gourmelon, P. [Institut de Protection et de Surete Nucleaire, Fountenay-aux-Roses (France)

    2000-05-01

    The lessons learnt from the treatment of previous radiation accidents using either bone marrow transplantation or growth factor therapy suggest that it is of importance to investigate new therapeutic regiments. Ex vivo expansion of haematopoietic stem cells, precursors and differentiated cells is a new approach of growth factor therapy which may be of interest for the treatment of patients with irradiation-induced bone marrow aplasia. Ex vivo expanded maturing cells could be used to limit the early risks bound to aplasia (infections related to granulocytopaenia, bleedings associated with thrombocytopaenia), whereas expanded immature cells could hasten haematopoietic recovery. Indeed, it is possible to culture from the blood or bone marrow the cells able to proliferate and differentiate. A sufficient quantity of cells to cover the transfusion needs of a radiation victim through an aplasia episode can be produced, in presence of a specific growth factor combination. Qualitative studies shows that the expanded cells exhibit a close to normal functionality. Long-term culture techniques demonstrate the expansion of immature cells. We have set up a high dose total body irradiation non-human primate model in order to study the therapeutic potential of ex vivo expansion of autologous progenitors and differentiating cells. All the steps of the process (sampling, positive selection of the immature cells, ex vivo expansion, irradiation of the animals, reinjection of the cultured cells and study of the outcome) are established. In order to allow the long term follow up of the ex vivo expanded haematopoietic cells (homing to the bone marrow or localization to specific organs for example), a retroviral gene transfer technique for transduction of green fluorescence protein (GFP) gene toward the selected immature blood or bone marrow cells is under development in this model. Taken together these elements will allow establishing the feasibility of ex vivo expansion of

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

    Science.gov (United States)

    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

  5. Expansion of Umbilical Cord Blood Aldehyde Dehydrogenase Expressing Cells Generates Myeloid Progenitor Cells that Stimulate Limb Revascularization.

    Science.gov (United States)

    Putman, David M; Cooper, Tyler T; Sherman, Stephen E; Seneviratne, Ayesh K; Hewitt, Mark; Bell, Gillian I; Hess, David A

    2017-07-01

    Uncompromised by chronic disease-related comorbidities, human umbilical cord blood (UCB) progenitor cells with high aldehyde dehydrogenase activity (ALDH(hi) cells) stimulate blood vessel regeneration after intra-muscular transplantation. However, implementation of cellular therapies using UCB ALDH(hi) cells for critical limb ischemia, the most severe form of severe peripheral artery disease, is limited by the rarity (18-fold over 6-days under serum-free conditions. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, only 15.1% ± 1.3% of progeny maintained high ALDH-activity after culture. However, compared to fresh UCB cells, expansion increased the total number of ALDH(hi) cells (2.7-fold), CD34(+) /CD133(+) cells (2.8-fold), and hematopoietic colony forming cells (7.7-fold). Remarkably, injection of expanded progeny accelerated recovery of perfusion and improved limb usage in immunodeficient mice with femoral artery ligation-induced limb ischemia. At 7 or 28 days post-transplantation, mice transplanted with expanded ALDH(hi) cells showed augmented endothelial cell proliferation and increased capillary density compared to controls. Expanded cells maintained pro-angiogenic mRNA expression and secreted angiogenesis-associated growth factors, chemokines, and matrix modifying proteins. Coculture with expanded cells augmented human microvascular endothelial cell survival and tubule formation under serum-starved, growth factor-reduced conditions. Expanded UCB-derived ALDH(hi) cells represent an alternative to autologous bone marrow as an accessible source of pro-angiogenic hematopoietic progenitor cells for the refinement of vascular regeneration-inductive therapies. Stem Cells Translational Medicine 2017;6:1607-1619. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  6. Effects of nitrogen on development and growth of the leaves of vegetables. 1. Appearance, expansion growth and life span of leaves of Brussels sprouts plants.

    NARCIS (Netherlands)

    Biemond, H.; Vos, J.; Struik, P.C.

    1995-01-01

    In greenhouse pot experiments and field trials, leek cv. Albana plants were supplied with different amounts of N fertilizer at various growth stages. Leaf emergence, expansion, size and senescence were monitored. The rate of leaf appearance was not affected by N treatments and almost constant across

  7. Effects of nitrogen on development and growth of the leaves of vegetables. 2. Appearance, expansion growth and life span of leaves of leek plants.

    NARCIS (Netherlands)

    Biemond, H.

    1995-01-01

    In greenhouse pot experiments and field trials, leek cv. Albana plants were supplied with different amounts of N fertilizer at various growth stages. Leaf emergence, expansion, size and senescence were monitored. The rate of leaf appearance was not affected by N treatments and almost constant across

  8. Elevation-induced climate change as a dominant factor causing the late Miocene C(4) plant expansion in the Himalayan foreland.

    Science.gov (United States)

    Wu, Haibin; Guo, Zhengtang; Guiot, Joël; Hatté, Christine; Peng, Changhui; Yu, Yanyan; Ge, Junyi; Li, Qin; Sun, Aizhi; Zhao, Deai

    2014-05-01

    During the late Miocene, a dramatic global expansion of C4 plant distribution occurred with broad spatial and temporal variations. Although the event is well documented, whether subsequent expansions were caused by a decreased atmospheric CO2 concentration or climate change is a contentious issue. In this study, we used an improved inverse vegetation modeling approach that accounts for the physiological responses of C3 and C4 plants to quantitatively reconstruct the paleoclimate in the Siwalik of Nepal based on pollen and carbon isotope data. We also studied the sensitivity of the C3 and C4 plants to changes in the climate and the atmospheric CO2 concentration. We suggest that the expansion of the C4 plant distribution during the late Miocene may have been primarily triggered by regional aridification and temperature increases. The expansion was unlikely caused by reduced CO2 levels alone. Our findings suggest that this abrupt ecological shift mainly resulted from climate changes related to the decreased elevation of the Himalayan foreland.

  9. Isolation of plant cell wall proteins.

    Science.gov (United States)

    Jamet, Elisabeth; Boudart, Georges; Borderies, Giséle; Charmont, Stephane; Lafitte, Claude; Rossignol, Michel; Canut, Herve; Pont-Lezica, Rafael

    2008-01-01

    The quality of a proteomic analysis of a cell compartment strongly depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific drawbacks: (1) the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP) during the isolation procedure; (2) polysaccharide networks of cellulose, hemicelluloses, and pectins form potential traps for contaminants such as intracellular proteins; (3) the presence of proteins interacting in many different ways with the polysaccharide matrix require different procedures to elute them from the cell wall. Three categories of CWP are distinguished: labile proteins that have little or no interactions with cell wall components, weakly bound proteins extractable with salts, and strongly bound proteins. Two alternative protocols are decribed for cell wall proteomics: (1) nondestructive techniques allowing the extraction of labile or weakly bound CWP without damaging the plasma membrane; (2) destructive techniques to isolate cell walls from which weakly or strongly bound CWP can be extracted. These protocols give very low levels of contamination by intracellular proteins. Their application should lead to a realistic view of the cell wall proteome at least for labile and weakly bound CWP extractable by salts.

  10. Multivariate statistical data analysis as a tool to analyze ex vivo expansion dynamics of cytokine-induced killer cells.

    Science.gov (United States)

    Zanon, Cristina; Stocchero, Matteo; Albiero, Elena; Castegnaro, Silvia; Chieregato, Katia; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

    2014-07-01

    Cytokine-induced killer (CIK) cells, obtained after mononucleated cell stimulation with interferon-γ, interleukin-2, and anti-CD3 antibody, are constituted by CD3(+) CD56(+) (CIK) cells and a minority of natural killer (NK; CD3(-) CD56(+) ) cells and T-lymphocytes (CD3(+) CD56(-) ) with antitumor effect against hematological malignancies, thus representing a promising immunotherapy strategy. To ensure in vivo antitumor activity it is mandatory to maximize the percentage of CD3(+) 56(+) effector cells, which is highly variable depending on the starting sample and the harvesting day. Based on cytofluorimetric data, we have retrospectively applied multivariate statistical data analysis (MVDA) to 30 expansions building mathematical models able to predict the expansion fate and the optimal CIK harvesting day. Cell phenotype was monitored during culture; multivariate batch statistical process control was applied to monitor cell expansion and orthogonal projections to latent structures to predict CIK percentage. Ten expansions had CD3(+) CD56(+) cells ≥ 40% (good batches) and 20 had CD3(+) CD56(+) cells ≤ 40%. In 36.7%, CD3(+) CD56(+) cells reached the highest concentration at day 17 and the others at day 21. We built a highly predictive regression model for estimating CD3(+) CD56(+) cells during culture. Three variables resulted highly informative: NK % at day 0, cytotoxic T-lymphocytes % (CTLs, CD3(+) CD8(+) ) at day 4, and CIK % at day 7. "Good batches" are characterized by a high percentage of CTLs and CD3(+) CD56(+) cells at day 4 and day 7, respectively. By applying MVDA it is possible to optimize CIK expansion, deciding the optimal cell harvesting day. A predictive role for CTL and CIK was evidenced. © 2013 Clinical Cytometry Society.

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

    Science.gov (United States)

    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. Generation and In Vitro Expansion of Hepatic Progenitor Cells from Human iPS Cells.

    Science.gov (United States)

    Yanagida, Ayaka; Nakauchi, Hiromitsu; Kamiya, Akihide

    2016-01-01

    Stem cells have the unique properties of self-renewal and multipotency (producing progeny belonging to two or more lineages). Induced pluripotent stem (iPS) cells can be generated from somatic cells by simultaneous expression of pluripotent factors (Oct3/4, Klf4, Sox2, and c-Myc). They share the same properties as embryonic stem (ES) cells and can differentiate into several tissue cells, i.e., neurons, hematopoietic cells, and liver cells. Therefore, iPS cells are suitable candidate cells for regenerative medicine and analyses of disease mechanisms.The liver is the major organ that regulates a multitude of metabolic functions. Hepatocytes are the major cell type populating the liver parenchyma and express several metabolic enzymes that are necessary for liver functions. Although hepatocytes are essential for maintaining homeostasis, it is difficult to alter artificial and transplanted cells because of their multifunctionality, donor shortage, and immunorejection risk. During liver development, hepatic progenitor cells in the fetal liver differentiate into both mature hepatocytes and cholangiocytes. As hepatic progenitor cells have bipotency and high proliferation ability, they could present a potential source for generating transplantable cells or as a liver study model. Here we describe the induction and purification of hepatic progenitor cells derived from human iPS cells. These cells can proliferate for a long term under suitable culture conditions.

  13. Biomimetic postcapillary expansions for enhancing rare blood cell separation on a microfluidic chip.

    Science.gov (United States)

    Jain, Abhishek; Munn, Lance L

    2011-09-07

    Blood cells naturally auto-segregate in postcapillary venules, with the erythrocytes (red blood cells, RBCs) aggregating near the axis of flow and the nucleated cells (NCs)--which include leukocytes, progenitor cells and, in cancer patients, circulating tumor cells--marginating toward the vessel wall. We have used this principle to design a microfluidic device that extracts nucleated cells (NCs) from whole blood. Fabricated using polydimethylsiloxane (PDMS) soft lithography, the biomimetic cell extraction device consists of rectangular microchannels that are 20-400 μm wide, 11 μm deep and up to 2 cm long. The key design feature is the use of repeated expansions/contractions of triangular geometry mimicking postcapillary venules, which enhance margination and optimize the extraction. The device operates on unprocessed whole blood and is able to extract 94 ± 4.5% of NCs with 45.75 ± 2.5-fold enrichment in concentration at a rate of 5 nl s(-1). The device eliminates the need to preprocess blood via centrifugation or RBC lysis, and is ready to be implemented as the initial stage of lab-on-a-chip devices that require enriched nucleated cells. The potential downstream applications are numerous, encompassing all preclinical and clinical assays that operate on enriched NC populations and include on-chip flow cytometry (A. Y. Fu et al., Anal. Chem., 2002, 74, 2451-2457; A. Y. Fu et al., Nat. Biotechnol., 1999, 17, 1109-1111), genetic analyses (M. M. Wang et al., Nat. Biotechnol., 2005, 23, 83-87; L. C. Waters et al., Anal. Chem., 1998, 70, 5172-5176) and circulating tumor cell extraction (S. Nagrath et al., Nature, 2007, 450, 1235-1241; S. L. Stott et al., Proc. Natl. Acad. Sci. U. S. A., 2010, 18392-18397; H. K. Lin et al., Clin. Cancer Res., 2010, 16, 5011-5018).

  14. Whole transcriptome analysis for T cell receptor-affinity and IRF4-regulated clonal expansion of T cells

    Directory of Open Access Journals (Sweden)

    Wei Shi

    2014-12-01

    Full Text Available Clonal population expansion of T cells during an immune response is dependent on the affinity of the T cell receptor (TCR for its antigen [1]. However, there is little understanding of how this process is controlled transcriptionally. We found that the transcription factor IRF4 was induced in a manner dependent on TCR-affinity and was critical for the clonal expansion and maintenance of effector function of antigen-specific CD8+ T cells. We performed a genome-wide expression profiling experiment using RNA sequencing technology (RNA-seq to interrogate global expression changes when IRF4 was deleted in CD8+ T cells activated with either a low or high affinity peptide ligand. This allowed us not only to determine IRF4-dependent transcriptional changes but also to identify transcripts dependent on TCR-affinity [2]. Here we describe in detail the analyses of the RNA-seq data, including quality control, read mapping, quantification, normalization and assessment of differential gene expression. The RNA-seq data can be accessed from Gene Expression Omnibus database (accession number GSE49929.

  15. Todralazine protects zebrafish from lethal effects of ionizing radiation: role of hematopoietic cell expansion.

    Science.gov (United States)

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

    2015-02-01

    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 manifesting radioprotection.

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

    Science.gov (United States)

    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.

  17. The availability of filament ends modulates actin stochastic dynamics in live plant cells

    Science.gov (United States)

    Li, Jiejie; Staiger, Benjamin H.; Henty-Ridilla, Jessica L.; Abu-Abied, Mohamad; Sadot, Einat; Blanchoin, Laurent; Staiger, Christopher J.

    2014-01-01

    A network of individual filaments that undergoes incessant remodeling through a process known as stochastic dynamics comprises the cortical actin cytoskeleton in plant epidermal cells. From images at high spatial and temporal resolution, it has been inferred that the regulation of filament barbed ends plays a central role in choreographing actin organization and turnover. How this occurs at a molecular level, whether different populations of ends exist in the array, and how individual filament behavior correlates with the overall architecture of the array are unknown. Here we develop an experimental system to modulate the levels of heterodimeric capping protein (CP) and examine the consequences for actin dynamics, architecture, and cell expansion. Significantly, we find that all phenotypes are the opposite for CP-overexpression (OX) cells compared with a previously characterized cp-knockdown line. Specifically, CP OX lines have fewer filament–filament annealing events, as well as reduced filament lengths and lifetimes. Further, cp-knockdown and OX lines demonstrate the existence of a subpopulation of filament ends sensitive to CP concentration. Finally, CP levels correlate with the biological process of axial cell expansion; for example, epidermal cells from hypocotyls with reduced CP are longer than wild-type cells, whereas CP OX lines have shorter cells. On the basis of these and other genetic studies in this model system, we hypothesize that filament length and lifetime positively correlate with the extent of axial cell expansion in dark-grown hypocotyls. PMID:24523291

  18. Cell expansion not cell differentiation predominantly co-ordinates veins and stomata within and among herbs and woody angiosperms grown under sun and shade.

    Science.gov (United States)

    Carins Murphy, Madeline R; Jordan, Gregory J; Brodribb, Timothy J

    2016-11-01

    It has been proposed that modification of leaf size, driven by epidermal cell size, balances leaf water supply (determined by veins) with transpirational demand (generated by stomata) during acclimation to local irradiance. We aimed to determine whether this is a general pattern among plant species with contrasting growth habits. We compared observed relationships between leaf minor vein density, stomatal density, epidermal cell size and leaf size in four pairs of herbs and woody species from the same families grown under sun and shade conditions with modelled relationships assuming vein and stomatal densities respond passively to epidermal cell expansion. Leaf lignin content was also quantified to assess whether construction costs of herbaceous leaf veins differ from those of woody plants and the leaf mass fraction invested in veins. Modelled relationships accurately described observed relationships, indicating that in all species, co-ordinated changes to the density of minor veins and stomata were mediated by a common relationship between epidermal cell size, vein density and stomatal density, with little or no impact from stomatal index. This co-ordination was independent of changes in leaf size and is likely to be an adaptive process driven by the significant proportion of biomass invested in veins (13·1 % of sun leaf dry weight and 21·7 % of shade leaf dry weight). Relative costs of venation increased in the shade, intensifying selective pressure towards economizing investment in vein density. Modulation of epidermal cell size appears to be a general mechanism among our experimental species to maintain a constant ratio between leaf anatomical traits that control leaf water fluxes independently of habit. We propose that this process may co-ordinate plasticity in hydraulic supply and demand in the majority of eudicot angiosperms. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For

  19. [Effects of human mesenchymal stem cells and fibroblastoid cell line as feeder layers on expansion of umbilical cord blood CD34(+) cells in vitro].

    Science.gov (United States)

    Ma, Li-Jun; Gao, Lei; Zhou, Hong; Qiu, Hui-Ying; Hu, Xiao-Xia; Xie, Lin-Na; Wang, Jian-Min

    2006-10-01

    To investigate the effects of human mesenchymal stem cells (MSC) and human fibroblastoid cell line (HFCL) as feeder layer on expansion of umbilical cord blood CD34(+) cells in vitro, (60)Co gamma-ray irradiated MSC and HFCL were used as feeder layer to expand cord blood CD34(+) cells in culture. The efficiencies of MSC and HFCL on expansion of CD34(+) cells in culture with or without cytokines were compared. The results showed that no matter whether cytokines (rhFL, rhSCF, rhTPO) were added, the proliferation of nucleated cells after expansion for 12 days in HFCL group was statistically higher than that in MSC group, i.e. with cytokines (9797 +/- 361)% vs (7061 +/- 418)%; without cytokines (5305 +/- 354)% vs (1992 +/- 247)%, when the cell numbers at day 0 was accounted as 100%), P 0.05. However, in the presence of cytokines, the propagating rate of MSC group was lower than that of HFCL group (939 +/- 212)% vs (1617 +/- 222)%, P < 0.01. MSC was better than HFCL in maintaining the LTC-IC of UCB CD34(+) cells, i.e. the number of CFU-GM colonies in the fifth week was (129.95 +/- 8.73) /10(5) seeded cells vs (89.81 +/- 10.29) colonies/10(5) cells, P < 0.05; with addition of cytokines, the effect was more obvious, i.e. the number of CFU-GM colonies in the fifth week (192.93 +/- 4.95)/10(5) seeded cells vs (90.47 +/- 14.28) colonies/10(5) seeded cells, P < 0.01. MSC mixed with a certain proportion of HFCL facilitated maintaining the LTC-IC of UCB CD34(+) cells. When the proportion was 4:1, the number of CFU-GM colonies was the highest (186.89 +/- 11.11)/10(5) seeded cells, which was higher than that of both 3:2 group [(138.92 +/- 14.84) colonies/10(5) seeded cells] and MSC only group, i.e. (64.63 +/- 6.11) colonies/10(5) seeded cells, both P < 0.01. It is concluded that HFCL is better than MSC in maintaining the expansion of CD34(+) cells and cytokines can enhance this effect, while MSC are stronger than HFCL in maintaining the LTC-IC of UCB CD34(+) cells in vitro. MSC

  20. Plant Cell Adaptive Responses to Microgravity

    Science.gov (United States)

    Kordyum, Elizabeth; Kozeko, Liudmyla; Talalaev, Alexandr

    Microgravity is an abnormal environmental condition that plays no role in the functioning of biosphere. Nevertheless, the chronic effect of microgravity in space flight as an unfamiliar factor does not prevent the development of adaptive reactions at the cellular level. In real microgravity in space flight under the more or less optimal conditions for plant growing, namely temperature, humidity, CO2, light intensity and directivity in the hardware angiosperm plants perform an “reproductive imperative”, i.e. they flower, fruit and yield viable seeds. It is known that cells of a multicellular organism not only take part on reactions of the organism but also carry out processes that maintain their integrity. In light of these principles, the problem of the identification of biochemical, physiological and structural patterns that can have adaptive significance at the cellular and subcellular level in real and simulated microgravity is considered. Cytological studies of plants developing in real and simulated microgravity made it possible to establish that the processes of mitosis, cytokinesis, and tissue differentiation of vegetative and generative organs are largely normal. At the same time, under microgravity, essential reconstruction in the structural and functional organization of cell organelles and cytoskeleton, as well as changes in cell metabolism and homeostasis have been described. In addition, new interesting data concerning the influence of altered gravity on lipid peroxidation intensity, the level of reactive oxygen species, and antioxidant system activity, just like on the level of gene expression and synthesis of low-molecular and high-molecular heat shock proteins were recently obtained. So, altered gravity caused time-dependent increasing of the HSP70 and HSP90 levels in cells, that may indicate temporary strengthening of their functional loads that is necessary for re-establish a new cellular homeostasis. Relative qPCR results showed that

  1. Modification of gellan gum with nanocrystalline hydroxyapatite facilitates cell expansion and spontaneous osteogenesis.

    Science.gov (United States)

    Jamshidi, Parastoo; Chouhan, Gurpreet; Williams, Richard L; Cox, Sophie C; Grover, Liam M

    2016-07-01

    Nanocomposites composed of hydrogels and calcium phosphates are of great interest in the development of bone graft replacements since they may have a structural and compositional resemblance to bone. Culture beads formed from such materials could be used in stirred tank culture and thereby enable cell expansion in a sufficiently efficient manner to allow for the generation of enough large number of cells for large-scale bone reconstruction. Although combinations of materials such as alginate, collagens, and various calcium phosphates have been investigated as culture beads, these materials are unsuitable for application since they have been shown to rapidly degrade in physiological conditions and enable relatively little tailoring of mechanical properties. In this study, gellan gum-nano sized hydroxyapatite (nHA) composites, which have been shown to be resistant to degradation and easily modified with respect to modulus, were formulated and characterized as regards their ability to enable cell attachment and proliferation. It was shown that the addition of 5 wt% of nHA to the culture beads enabled cell attachment and that an increase in nHA concentration to up to 25 wt% enhanced the rate of cell proliferation. Most importantly, it was demonstrated that the addition of nHA to the cell culture beads enabled the formation of nodules in culture of MC3T3-E1 cells and strikingly stimulated the osteogenic differentiation of bone marrow stromal cells in the absence of osteogenic media when compared with tissue culture plastic (TCP) with the same condition. Biotechnol. Bioeng. 2016;113: 1568-1576. © 2016 Wiley Periodicals, Inc.

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

    Directory of Open Access Journals (Sweden)

    Michael J Osiecki

    Full Text Available 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.

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

    Science.gov (United States)

    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. PMID:26660475

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

    Science.gov (United States)

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

    Science.gov (United States)

    Rodrigo Albors, Aida; Tazaki, Akira; Rost, Fabian; Nowoshilow, Sergej; Chara, Osvaldo; Tanaka, Elly M

    2015-11-14

    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.

  6. Cutting Edge: Innate Lymphoid Cells Suppress Homeostatic T Cell Expansion in Neonatal Mice.

    Science.gov (United States)

    Bank, Ute; Deiser, Katrin; Finke, Daniela; Hämmerling, Günter J; Arnold, Bernd; Schüler, Thomas

    2016-05-01

    In adult mice, lymphopenia-induced proliferation (LIP) leads to T cell activation, memory differentiation, tissue destruction, and a loss of TCR diversity. Neonatal mice are lymphopenic within the first week of life. This enables some recent thymic emigrants to undergo LIP and convert into long-lived memory T cells. Surprisingly, however, most neonatal T cells do not undergo LIP. We therefore asked whether neonate-specific mechanisms prevent lymphopenia-driven T cell activation. In this study, we show that IL-7R-dependent innate lymphoid cells (ILCs) block LIP of CD8(+) T cells in neonatal but not adult mice. Importantly, CD8(+) T cell responses against a foreign Ag are not inhibited by neonatal ILCs. This ILC-based inhibition of LIP ensures the generation of a diverse naive T cell pool in lymphopenic neonates that is mandatory for the maintenance of T cell homeostasis and immunological self-tolerance later in life.

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

    Science.gov (United States)

    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.

  8. The potential of single-cell profiling in plants.

    Science.gov (United States)

    Efroni, Idan; Birnbaum, Kenneth D

    2016-04-05

    Single-cell transcriptomics has been employed in a growing number of animal studies, but the technique has yet to be widely used in plants. Nonetheless, early studies indicate that single-cell RNA-seq protocols developed for animal cells produce informative datasets in plants. We argue that single-cell transcriptomics has the potential to provide a new perspective on plant problems, such as the nature of the stem cells or initials, the plasticity of plant cells, and the extent of localized cellular responses to environmental inputs. Single-cell experimental outputs require different analytical approaches compared with pooled cell profiles and new tools tailored to single-cell assays are being developed. Here, we highlight promising new single-cell profiling approaches, their limitations as applied to plants, and their potential to address fundamental questions in plant biology.

  9. Replicate phylogenies and post-glacial range expansion of the pitcher-plant mosquito, Wyeomyia smithii, in North America.

    Directory of Open Access Journals (Sweden)

    Clayton Merz

    Full Text Available Herein we tested the repeatability of phylogenetic inference based on high throughput sequencing by increased taxon sampling using our previously published techniques in the pitcher-plant mosquito, Wyeomyia smithii in North America. We sampled 25 natural populations drawn from different localities nearby 21 previous collection localities and used these new data to construct a second, independent phylogeny, expressly to test the reproducibility of phylogenetic patterns. Comparison of trees between the two data sets based on both maximum parsimony and maximum likelihood with Bayesian posterior probabilities showed close correspondence in the grouping of the most southern populations into clear clades. However, discrepancies emerged, particularly in the middle of W. smithii's current range near the previous maximum extent of the Laurentide Ice Sheet, especially concerning the most recent common ancestor to mountain and northern populations. Combining all 46 populations from both studies into a single maximum parsimony tree and taking into account the post-glacial historical biogeography of associated flora provided an improved picture of W. smithii's range expansion in North America. In a more general sense, we propose that extensive taxon sampling, especially in areas of known geological disruption is key to a comprehensive approach to phylogenetics that leads to biologically meaningful phylogenetic inference.

  10. Haspin has Multiple Functions in the Plant Cell Division Regulatory Network.

    Science.gov (United States)

    Kozgunova, Elena; Suzuki, Takamasa; Ito, Masaki; Higashiyama, Tetsuya; Kurihara, Daisuke

    2016-04-01

    Progression of cell division is controlled by various mitotic kinases. In animal cells, phosphorylation of histone H3 at Thr3 by the kinase Haspin (haploid germ cell-specific nuclear protein kinase) promotes centromeric Aurora B localization to regulate chromosome segregation. However, less is known about the function of Haspin in regulatory networks in plant cells. Here, we show that inhibition of Haspin with 5-iodotubercidin (5-ITu) in Bright Yellow-2 (BY-2) cells delayed chromosome alignment. Haspin inhibition also prevented the centromeric localization of Aurora3 kinase (AUR3) and disrupted its function. This suggested that Haspin plays a role in the specific positioning of AUR3 on chromosomes in plant cells, a function conserved in animals. The results also indicated that Haspin and AUR3 are involved in the same pathway, which regulates chromosome alignment during prometaphase/metaphase. Remarkably, Haspin inhibition by 5-ITu also led to a severe cytokinesis defect, resulting in binuclear cells with a partially formed cell plate. The 5-ITu treatment did not affect microtubules, AUR1/2 or the NACK-PQR pathway; however, it did alter the distribution of actin filaments on the cell plate. Together, these results suggested that Haspin has several functions in regulating cell division in plant cells: in the localization of AUR3 on centromeres and in regulating late cell plate expansion during cytokinesis.

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

    Science.gov (United States)

    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.

  12. 三峡水电厂伸缩节导流板破坏机理%Damage Mechanism of Expansion Bend Guide Plate in Three Gorges Hydropower Plant

    Institute of Scientific and Technical Information of China (English)

    徐新田

    2011-01-01

    After the expansion bends of Three Gorges Hydropower Plant (TGHP) units are put into operation, incidents such as fracture of coupling bolts and splitting of expansion bend guide plate have have successively occurred. To ensure the long-term safe operation of the expansion bends, the damage mechanism of the expansion bend guide plate of TGHP was studied. Through field tests and theoretical calculations, the the forces and pulse pressures on the guide plate as well as the vibration characteristics of the expansion bends were highlighted. The research shows that the destruction of the expansion bend guide plate is mainly fatigued one.%三峡水电厂机组伸缩节投运后,先后出现部分伸缩节导流板连接螺栓断裂、导流板局部撕裂等现象.为确保伸缩节长期安全运行,三峡水电厂对伸缩节导流板破坏机理进行了研究.采用理论计算与现场试验相结合的方法,重点研究了机组伸缩节受力状况、脉动压力、导流板振动特性等.研究表明伸缩节导流板破坏主要是疲劳破坏.

  13. Ex Vivo Expansion or Manipulation of Stem Cells to Improve Outcome of Umbilical Cord Blood Transplantation.

    Science.gov (United States)

    Horwitz, Mitchell E

    2016-02-01

    The outcome of umbilical cord blood transplantation for adult patients with hematologic malignancies now rivals that of matched unrelated donor transplantation. However, delayed hematopoietic and immunologic recovery remains a source of significant morbidity and mortality. Multiple strategies are now being studied to overcome these limitations. One strategy involves ex vivo expansion of the umbilical cord blood unit prior to transplantation. A second strategy involves exposure of the umbilical cord blood graft to compounds aimed at improving homing and engraftment following transplantation. Such a strategy may also address the problem of slow hematopoietic recovery as well as the increased risk of graft failure. Many of these strategies are now being tested in late phase multi-center clinical trials. If proven cost-effective and efficacious, they may alter the landscape of donor options for allogeneic stem cell transplantation.

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

    Directory of Open Access Journals (Sweden)

    Xiaoqing Zhu

    2016-02-01

    Full Text Available 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.

  15. Low-density subculture: a technical note on the importance of avoiding cell-to-cell contact during mesenchymal stromal cell expansion.

    Science.gov (United States)

    Balint, Richard; Richardson, Stephen M; Cartmell, Sarah H

    2015-10-01

    Numerous scientific studies and clinical trials are carried out each year exploring the use of mesenchymal stromal cells in regenerative medicine and tissue engineering. However, the effective and reliable expansion of this very important cell type remains a challenge. In this study the importance of cell-to-cell contact during expansion has been explored on the proliferation and differentiation potential of the produced cells. Cells were cultured up to passage 5 under conditions where cell-to-cell contact was either probable (40-70% confluence; see supporting information, Protocol A) or where it was unlikely (10-50% confluence; see supporting information, Protocol B). The effect of the two different conditions on expansion efficiency; proliferation rate and tri-lineage differentiation potential was assessed. Differences in immunophenotype, cell size and senescence were also investigated. Protocol B cultures expanded twice as fast as those cultured with Protocol A. In passage 5 experiments low confluence expanded cells displayed a 10% higher overall proliferation rate, and produced 23% more cells in growth, 12% more in osteogenic, 77% more in adipogenic, but 27% less in chondrogenic medium. Differentiation potential wasn't decisively affected at the mRNA level. However, Protocol B favoured bone and cartilage differentiation at the secretional level. Protocol A populations showed reduced purity, expressing CD105 in only 76% compared to the 96.7% in Protocol B cultures. Protocol A populations also contained significantly more (+4.2%) senescent cells, however, no difference was found in cell size between the two protocols. The findings of this study suggest that cell-to-cell contact, and therefore high confluence levels, is detrimental to MSC quality.

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

    Science.gov (United States)

    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.

  17. Molecular regulation of plant cell wall extensibility

    Science.gov (United States)

    Cosgrove, D. J.

    1998-01-01

    Gravity responses in plants often involve spatial and temporal changes in cell growth, which is regulated primarily by controlling the ability of the cell wall to extend. The wall is thought to be a cellulose-hemicellulose network embedded in a hydrated matrix of complex polysaccharides and a small amount of structural protein. The wall extends by a form of polymer creep, which is mediated by expansins, a novel group of wall-loosening proteins. Expansins were discovered during a molecular dissection of the "acid growth" behavior of cell walls. Expansin alters the rheology of plant walls in profound ways, yet its molecular mechanism of action is still uncertain. It lacks detectable hydrolytic activity against the major components of the wall, but it is able to disrupt noncovalent adhesion between wall polysaccharides. The discovery of a second family of expansins (beta-expansins) sheds light on the biological role of a major group of pollen allergens and implies that expansins have evolved for diverse developmental functions. Finally, the contribution of other processes to wall extensibility is briefly summarized.

  18. Plant and animal stem cells: similar yet different

    NARCIS (Netherlands)

    Heidstra, R.; Sabatini, S.

    2014-01-01

    The astonishingly long lives of plants and their regeneration capacity depend on the activity of plant stem cells. As in animals, stem cells reside in stem cell niches, which produce signals that regulate the balance between self-renewal and the generation of daughter cells that differentiate into

  19. Plant and animal stem cells: similar yet different

    NARCIS (Netherlands)

    Heidstra, R.; Sabatini, S.

    2014-01-01

    The astonishingly long lives of plants and their regeneration capacity depend on the activity of plant stem cells. As in animals, stem cells reside in stem cell niches, which produce signals that regulate the balance between self-renewal and the generation of daughter cells that differentiate into n

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  2. Defect of CD8+ Memory T Cells Developed in Absence of IL-12 Priming for Secondary Expansion

    Institute of Scientific and Technical Information of China (English)

    Zhenmin Ye; Shulin Xu; Terence Moyana; Jicheng Yang; Jim Xiang

    2008-01-01

    IL-12 priming plays an important role in stimulation of CD8+ effector T cells and development of CD8+ memory T (Tm) cells. However, the functional alteration of CD8+ Tm cells developed in the absence of IL-12 priming is elusive.In this study, we investigated the capacity of secondary expansion of CD8+ Tm cells developed from transgenic OT I CD8+ T cells. The latter cells were in vitro and in vivo stimulated by ovalbumin (OVA)-puised dendritic cells [DCOVA and (IL-12-/-)DCOVA] derived from wild-type C57BL/6 and IL-12 gene knockout mice, respectively. We demonstrated that IL-12 priming is important not only in CD8+ T cell clonal expansion, but also in generation of CD8+ Tm cells with the capacity of secondary expansion upon antigen re-encounter. However, IL-12 signaling is not involved in CD8+ Tm cell survival and recall responses. Therefore, this study provides useful information for vaccine design and development.

  3. The potential of single-cell profiling in plants

    OpenAIRE

    Efroni, Idan; Birnbaum, Kenneth D

    2016-01-01

    Single-cell transcriptomics has been employed in a growing number of animal studies, but the technique has yet to be widely used in plants. Nonetheless, early studies indicate that single-cell RNA-seq protocols developed for animal cells produce informative datasets in plants. We argue that single-cell transcriptomics has the potential to provide a new perspective on plant problems, such as the nature of the stem cells or initials, the plasticity of plant cells, and the extent of localized ce...

  4. Optimising cell aggregate expansion in a perfused hollow fibre bioreactor via mathematical modelling.

    KAUST Repository

    Chapman, Lloyd A C

    2014-08-26

    The need for efficient and controlled expansion of cell populations is paramount in tissue engineering. Hollow fibre bioreactors (HFBs) have the potential to meet this need, but only with improved understanding of how operating conditions and cell seeding strategy affect cell proliferation in the bioreactor. This study is designed to assess the effects of two key operating parameters (the flow rate of culture medium into the fibre lumen and the fluid pressure imposed at the lumen outlet), together with the cell seeding distribution, on cell population growth in a single-fibre HFB. This is achieved using mathematical modelling and numerical methods to simulate the growth of cell aggregates along the outer surface of the fibre in response to the local oxygen concentration and fluid shear stress. The oxygen delivery to the cell aggregates and the fluid shear stress increase as the flow rate and pressure imposed at the lumen outlet are increased. Although the increased oxygen delivery promotes growth, the higher fluid shear stress can lead to cell death. For a given cell type and initial aggregate distribution, the operating parameters that give the most rapid overall growth can be identified from simulations. For example, when aggregates of rat cardiomyocytes that can tolerate shear stresses of up to 0:05 Pa are evenly distributed along the fibre, the inlet flow rate and outlet pressure that maximise the overall growth rate are predicted to be in the ranges 2.75 x 10(-5) m(2) s(-1) to 3 x 10(-5) m(2) s(-1) (equivalent to 2.07 ml min(-1) to 2.26 ml min(-1)) and 1.077 x 10(5) Pa to 1.083 x 10(5) Pa (or 15.6 psi to 15.7 psi) respectively. The combined effects of the seeding distribution and flow on the growth are also investigated and the optimal conditions for growth found to depend on the shear tolerance and oxygen demands of the cells.

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

    Science.gov (United States)

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

    Directory of Open Access Journals (Sweden)

    Robert C. Kauffman

    2016-12-01

    Full Text Available 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.

  7. Ex Vivo Expansion of Functional Human UCB-HSCs/HPCs by Coculture with AFT024-hkirre Cells

    Directory of Open Access Journals (Sweden)

    Muti ur Rehman Khan

    2014-01-01

    Full Text Available Kiaa1867 (human Kirre, hKirre has a critical role in brain development and/or maintenance of the glomerular slit diaphragm in kidneys. Murine homolog of this gene, mKirre expressed in OP9 and AFT024 cells could support hematopoietic stem cells/hematopoietic progenitor cells (HSC/HPC expansion in vitro. HKirre is also expressed in human FBMOB-hTERT cell line and fetal liver fibroblast-like cells but its function has remained unclear. In this paper, we cloned a hKirre gene from human fetal liver fibroblast-like cells and established a stably overexpressing hKirre-AFT024 cell line. Resultant cells could promote self-renewal and ex vivo expansion of HSCs/HPCs significantly higher than AFT024-control cells transformed with mock plasmid. The Expanded human umbilical cord blood (hUCB CD34+ cells retained the capacity of multipotent differentiation as long as 8 weeks and successfully repopulated the bone marrow of sublethally irradiated NOD/SCID mice, which demonstrated the expansion of long-term primitive transplantable HSCs/HPCs. Importantly, hkirre could upregulate the expressions of Wnt-5A, BMP4, and SDF-1 and downregulate TGF-β with other hematopoietic growth factors. By SDS-PAGE and Western Blot analysis, a ~89 kDa protein in total lysate of AFT024-hKirre was identified. Supernatants from AFT024-hkirre could also support CD34+CD38− cells expansion. These results demonstrated that the AFT024-hKirre cells have the ability to efficiently expand HSCs/HPCs.

  8. Bimodal ex vivo expansion of T cells from patients with head and neck squamous cell carcinoma: a prerequisite for adoptive cell transfer

    DEFF Research Database (Denmark)

    Junker, Niels; Wenandy, Lynn; Dombernowsky, Sarah Louise;

    2011-01-01

    Abstract Background aims. Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has proven effective in metastatic melanoma and should therefore be explored in other types of cancer. The aim of this study was to examine the feasibility of potentially expanding clinically relevant quantities...... tumors in high-dose interleukin (IL)-2. Secondly, selected bulk cultures were rapidly expanded using anti-CD3 antibody, feeder cells and high-dose IL-2. T-cell subsets were phenotypically characterized using flow cytometry. T-cell receptor (TCR) clonotype mapping was applied to examine clonotype dynamics....... Rapid expansions generated up to 3500-fold expansion of selected TIL cultures within 17 days. The cultures mainly consisted of T-effector memory cells, with varying distributions of CD8(+) and CD4(+) subtypes both among cultures and patients. TCR clonotype mapping demonstrated oligoclonal expanded...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  11. Leishmania infantum Exoproducts Inhibit Human Invariant NKT Cell Expansion and Activation

    Directory of Open Access Journals (Sweden)

    Renata Belo

    2017-06-01

    Full Text Available Leishmania infantum is one of the major parasite species associated with visceral leishmaniasis, a severe form of the disease that can become lethal if untreated. This obligate intracellular parasite has developed diverse strategies to escape the host immune response, such as exoproducts (Exo carrying a wide range of molecules, including parasite virulence factors, which are potentially implicated in early stages of infection. Herein, we report that L. infantum Exo and its two fractions composed of extracellular vesicles (EVs and vesicle-depleted-exoproducts (VDEs inhibit human peripheral blood invariant natural killer T (iNKT cell expansion in response to their specific ligand, the glycolipid α-GalactosylCeramide (α-GalCer, as well as their capacity to promptly produce IL-4 and IFNγ. Using plate-bound CD1d and α-GalCer, we found that Exo, EV, and VDE fractions reduced iNKT cell activation in a dose-dependent manner, suggesting that they prevented α-GalCer presentation by CD1d molecules. This direct effect on CD1d was confirmed by the observation that CD1d:α-GalCer complex formation was impaired in the presence of Exo, EV, and VDE fractions. Furthermore, lipid extracts from the three compounds mimicked the inhibition of iNKT cell activation. These lipid components of L. infantum exoproducts, including EV and VDE fractions, might compete for CD1-binding sites, thus blocking iNKT cell activation. Overall, our results provide evidence for a novel strategy through which L. infantum can evade immune responses of mammalian host cells by preventing iNKT lymphocytes from recognizing glycolipids in a TCR-dependent manner.

  12. Leishmania infantum Exoproducts Inhibit Human Invariant NKT Cell Expansion and Activation.

    Science.gov (United States)

    Belo, Renata; Santarém, Nuno; Pereira, Cátia; Pérez-Cabezas, Begoña; Macedo, Fátima; Leite-de-Moraes, Maria; Cordeiro-da-Silva, Anabela

    2017-01-01

    Leishmania infantum is one of the major parasite species associated with visceral leishmaniasis, a severe form of the disease that can become lethal if untreated. This obligate intracellular parasite has developed diverse strategies to escape the host immune response, such as exoproducts (Exo) carrying a wide range of molecules, including parasite virulence factors, which are potentially implicated in early stages of infection. Herein, we report that L. infantum Exo and its two fractions composed of extracellular vesicles (EVs) and vesicle-depleted-exoproducts (VDEs) inhibit human peripheral blood invariant natural killer T (iNKT) cell expansion in response to their specific ligand, the glycolipid α-GalactosylCeramide (α-GalCer), as well as their capacity to promptly produce IL-4 and IFNγ. Using plate-bound CD1d and α-GalCer, we found that Exo, EV, and VDE fractions reduced iNKT cell activation in a dose-dependent manner, suggesting that they prevented α-GalCer presentation by CD1d molecules. This direct effect on CD1d was confirmed by the observation that CD1d:α-GalCer complex formation was impaired in the presence of Exo, EV, and VDE fractions. Furthermore, lipid extracts from the three compounds mimicked the inhibition of iNKT cell activation. These lipid components of L. infantum exoproducts, including EV and VDE fractions, might compete for CD1-binding sites, thus blocking iNKT cell activation. Overall, our results provide evidence for a novel strategy through which L. infantum can evade immune responses of mammalian host cells by preventing iNKT lymphocytes from recognizing glycolipids in a TCR-dependent manner.

  13. Dynamic simulation of a direct carbonate fuel cell power plant

    Energy Technology Data Exchange (ETDEWEB)

    Ernest, J.B. [Fluor Daniel, Inc., Irvine, CA (United States); Ghezel-Ayagh, H.; Kush, A.K. [Fuel Cell Engineering, Danbury, CT (United States)

    1996-12-31

    Fuel Cell Engineering Corporation (FCE) is commercializing a 2.85 MW Direct carbonate Fuel Cell (DFC) power plant. The commercialization sequence has already progressed through construction and operation of the first commercial-scale DFC power plant on a U.S. electric utility, the 2 MW Santa Clara Demonstration Project (SCDP), and the completion of the early phases of a Commercial Plant design. A 400 kW fuel cell stack Test Facility is being built at Energy Research Corporation (ERC), FCE`s parent company, which will be capable of testing commercial-sized fuel cell stacks in an integrated plant configuration. Fluor Daniel, Inc. provided engineering, procurement, and construction services for SCDP and has jointly developed the Commercial Plant design with FCE, focusing on the balance-of-plant (BOP) equipment outside of the fuel cell modules. This paper provides a brief orientation to the dynamic simulation of a fuel cell power plant and the benefits offered.

  14. Adventage of mesenchymal stem cells (MSC expansion directly from purified bone marrow CD105+ and CD271+ cells.

    Directory of Open Access Journals (Sweden)

    Ewa Lukasiewicz

    2008-12-01

    Full Text Available Mesenchymal Stem Cells (MSC are employed in gene and cellular therapies. Routinely MSC are isolated from bone marrow mononuclear cells (MNC by plastic adherence. Here we compared new isolation strategies of bone marrow MSC including immunodepletion of hematopoietic cells and immunomagnetic isolation of CD105+ and CD271+ populations. Four fractions were obtained: MNC MSC, RosetteSep-isolated MSC, CD105+ and CD271+ sorted MSC. We evaluated i number of CFU-F colonies, ii cell phenotype, iii in vitro differentiation of expanded cells and iv expression of osteo/adipogenesis related genes. Results: Average number of day 9 CFU-F colonies was the highest for CD271 positive fraction. Real-Time PCR analysis revealed expression of RUNX2, PPARgamma and N-cadherin in isolated cells, particularly high in CD271+ cells. Expression of CD105, CD166, CD44, CD73 antigens was comparable for all expanded populations (over 90%. We observed various levels of hematopoietic contamination with the highest numbers of CD45+ cells in MNC-MSC fraction and the lowest in CD105+ and CD271+ fractions. Cells of all the fractions were CD34 antigen negative. Expanded CD105 and CD271 populations showed higher level of RUNX2, osteocalcin, PTHR, leptin, PPARgamma2 and aggrecan1 genes except for alpha1 collagen. After osteogenic differentiation CD105+ and CD271+ populations showed lower expression of RUNX, PPARgamma2 and also lower expression of osteocalcin and PTHR than MNC, with comparable alpha1-collagen expression. Chondrogenic and adipogenic gene expression was higher in MNC. More clonogenic CD105+ and particularly CD271+ cells, which seem to be the most homogenous fractions based on Real-Time PCR and immunostaining data, are better suited for MSC expansion.

  15. IL-21-dependent expansion of memory-like NK cells enhances protective immune responses against Mycobacterium tuberculosis.

    Science.gov (United States)

    Venkatasubramanian, S; Cheekatla, S; Paidipally, P; Tripathi, D; Welch, E; Tvinnereim, A R; Nurieva, R; Vankayalapati, R

    2017-07-01

    Natural killer (NK) cells are traditionally considered as innate cells, but recent studies suggest that NK cells can distinguish antigens, and that memory NK cells expand and protect against viral pathogens. Limited information is available about the mechanisms involved in memory-like NK cell expansion, and their role in bacterial infections and vaccine-induced protective immune responses. In the current study, using a mouse model of tuberculosis (TB) infection, we found that interferon-gamma producing CD3-NKp46+CD27+KLRG1+ memory-like NK cells develop during Bacille Calmette-Guérin vaccination, expand, and provide protection against challenge with Mycobacterium tuberculosis (M. tb). Using antibodies, short interfering RNA and gene-deleted mice, we found that expansion of memory-like NK cells depends on interleukin 21 (IL-21). NKp46+CD27+KLRG1+ NK cells expanded in healthy individuals with latent TB infection in an IL-21-dependent manner. Our study provides first evidence that memory-like NK cells survive long term, expansion depends on IL-21, and involved in vaccine-induced protective immunity against a bacterial pathogen.

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

    Science.gov (United States)

    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.

  17. Wnt1 Accelerates an Ex Vivo Expansion of Human Cord Blood CD34+CD38− Cells

    Directory of Open Access Journals (Sweden)

    Kamonnaree Chotinantakul

    2013-01-01

    Full Text Available Cord blood hematopoietic stem cells (CB-HSCs transplantation has been increasing gradually with facing the limitation of insufficient quantity of HSCs in each CB unit. Therefore, efficient expansion methods which can maintain stem cell characteristics are needed. In this study, umbilical CB-CD34+ cells were cultured in two different cytokine cocktails: 4 factors (4F = Flt3-L, SCF, IL-6, and TPO and 5 factors (5F = Wnt1 + 4F in both serum and serum-free media. The data revealed that the best condition to accelerate an expansion of CD34+CD38− cells was serum-free culture condition supplemented with 5F (5F KSR. This condition yielded 24.3 ± 2.1 folds increase of CD34+CD38− cells. The expanded cells exhibited CD34+ CD38− CD133+ CD71low CD33low CD3− CD19− markers, expressed nanog, oct3/4, c-myc, and sox2 genes, and maintained differentiation potential into lymphoid, erythroid and myeloid lineages. The achievement of CD34+CD38− cells expansion may overcome an insufficient quantity of the cells leading to the improvement of the stem cell transplantation. Altogether, our findings highlight the role of Wnt1 and the new culture condition in stimulating hematopoietic stem/progenitor cells expansion which may offer a new therapeutic avenue for cord blood transplantation, regenerative medicine, stem cell bank applications, and other clinical applications in the future.

  18. Overcoming the bottleneck of platelet lysate supply in large-scale clinical expansion of adipose-derived stem cells

    DEFF Research Database (Denmark)

    Glovinski, Peter V; Herly, Mikkel; Mathiasen, Anders B

    2017-01-01

    BACKGROUND: Platelet lysates (PL) represent a promising replacement for xenogenic growth supplement for adipose-derived stem cell (ASC) expansions. However, fresh platelets from human blood donors are not clinically feasible for large-scale cell expansion based on their limited supply. Therefore...... may be an efficient and reliable source of human growth supplement allowing for large-scale ASC expansion for clinical use....... stored in the platelet additive solution, InterSol. Three types of PLs were prepared from outdated PCs with platelets suspended in either (1) InterSol (not manipulated), (2) InterSol + supplemented with plasma or (3) plasma alone (InterSol removed). Using these PLs, we compared ASC population doubling...

  19. Brazilian Aluminium Company - new plant expansions will depend upon energy; Companhia Brasileira de Aluminio: novas expansoes vao depender de energia

    Energy Technology Data Exchange (ETDEWEB)

    Gorgueira, Cristina

    1996-08-01

    This document presents the perspectives for activities of the Brazilian Aluminium Company for the next future years, the investments to be applied and the expansion dependency on the increasing energy supplies. The expansion projects also take into account the environmental aspects.

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

    Science.gov (United States)

    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.

  1. Successful isolation, in vitro expansion and characterization of stem cells from Human Dental Pulp

    Directory of Open Access Journals (Sweden)

    Preethy SP

    2010-01-01

    acids (5 .Cell counting was done by Trypan Blue dye exclusion method and the cells were seeded in 6 well culture plates. The plates with cells were incubated at 37˚C with 5% CO2 for varying periods from 14 days-28 days. The cells were observed daily and media change was done every three days. RESULTS: Viable Dental Pulp tissue-cells were obtained after transportation of up to 48 hrs and the in vitro growth of cells was initially slow but colonies were identified from the 10th day onwards. The cells were harvested at different intervals of 14-28 days for each sample based on their growth and subjected to H & E staining .The H & E staining of the cultured cells of all the samples showed positive resultsCONCLUSION: We are able to transport extracted teeth and derive viable dental pulp tissue cells after enzymatic digestion and multiply them in culture after a maximum of 48 hrs after transportation. The cells could be grown in culture with a morphology resembling dental pulp stem cells while in culture expansion and in H&E studies. Further characterization of the cells is necessary to confirm their Stemness. References1.Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs in vitro and in vivo. Proc Natl Acad Sci U S A. 20002.Nosrat IV, Widenfalk J, Olson L, Nosrat CA. Dental pulp cells produce neurotrophic factors, interact with trigeminal neurons in vitro, and rescue motoneurons after spinal cord injury. Dev Biol. 2001 Oct 3.Iohara K, Zheng L, Ito M, Tomokiyo A, Matsushita K, Nakashima M. Side population cells isolated from porcine dental pulp tissue with self-renewal and multipotency for dentinogenesis, chondrogenesis, adipogenesis, and neurogenesis. Stem Cells. 2006 Nov4.Gandia C, Armiñan A, García-Verdugo JM, Lledó E, Ruiz A, Miñana MD, Sanchez-Torrijos J, Payá R, Mirabet V, Carbonell-Uberos F, Llop M, Montero JA, Sepúlveda P. Human dental pulp stem cells improve left ventricular function, induce angiogenesis, and reduce

  2. A nonlinear electromechanical coupling model for electropore expansion in cell electroporation

    Science.gov (United States)

    Deng, Peigang; Lee, Yi-Kuen; Zhang, Tong-Yi

    2014-11-01

    Under an electric field, the electric tractions acting on a cell membrane containing a pore-nucleus are investigated by using a nonlinear electromechanical coupling model, in which the cell membrane is treated as a hyperelastic material. Iterations between the electric field and the structure field are performed to reveal the electrical forces exerting on the pore region and the subsequent pore expansion process. An explicit exponential decay of the membrane’s edge energy as a function of pore radius is defined for a hydrophilic pore and the transition energy as a hydrophobic pore converts to a hydrophilic pore during the initial stage of pore formation is investigated. It is found that the edge energy for the creation of an electropore edge plays an important role at the atomistic scale and it determines the hydrophobic-hydrophilic transition energy barrier. Various free energy evolution paths are exhibited, depending on the applied electric field, which provides further insight towards the electroporation (EP) phenomenon. In comparison with previous EP models, the proposed model has the ability to predict the metastable point on the free energy curve that is relevant to the lipid ion channel. In addition, the proposed model can also predict the critical transmembrane potential for the activation of an effective electroporation that is in a good agreement with previously published experimental data.

  3. A nonlinear electromechanical coupling model for electropore expansion in cell electroporation

    KAUST Repository

    Deng, Peigang

    2014-10-15

    Under an electric field, the electric tractions acting on a cell membrane containing a pore-nucleus are investigated by using a nonlinear electromechanical coupling model, in which the cell membrane is treated as a hyperelastic material. Iterations between the electric field and the structure field are performed to reveal the electrical forces exerting on the pore region and the subsequent pore expansion process. An explicit exponential decay of the membrane\\'s edge energy as a function of pore radius is defined for a hydrophilic pore and the transition energy as a hydrophobic pore converts to a hydrophilic pore during the initial stage of pore formation is investigated. It is found that the edge energy for the creation of an electropore edge plays an important role at the atomistic scale and it determines the hydrophobic-hydrophilic transition energy barrier. Various free energy evolution paths are exhibited, depending on the applied electric field, which provides further insight towards the electroporation (EP) phenomenon. In comparison with previous EP models, the proposed model has the ability to predict the metastable point on the free energy curve that is relevant to the lipid ion channel. In addition, the proposed model can also predict the critical transmembrane potential for the activation of an effective electroporation that is in a good agreement with previously published experimental data.

  4. Regulatory activity of azabisphosphonate-capped dendrimers on human CD4+ T cell proliferation enhances ex-vivo expansion of NK cells from PBMCs for immunotherapy

    Directory of Open Access Journals (Sweden)

    Caminade Anne-Marie

    2009-09-01

    Full Text Available Abstract Background Adoptive cell therapy with allogenic NK cells constitutes a promising approach for the treatment of certain malignancies. Such strategies are currently limited by the requirement of an efficient protocol for NK cell expansion. We have developed a method using synthetic nanosized phosphonate-capped dendrimers allowing such expansion. We are showing here that this is due to a specific inhibitory activity towards CD4+ T cell which could lead to further medical applications of this dendrimer. Methods Mononuclear cells from human peripheral blood were used to investigate the immunomodulatory effects of nanosized phosphonate-capped dendrimers on interleukin-2 driven CD4+T cell expansion. Proliferation status was investigated using flow cytometry analysis of CFSE dilution and PI incorporation experiments. Magnetic bead cell sorting was used to address activity towards individual or mixed cell sub-populations. We performed equilibrium binding assay to assess the interaction of fluorescent dendrimers with pure CD4+ T cells. Results Phosphonate-capped dendrimers are inhibiting the activation, and therefore the proliferation; of CD4+ T cells in IL-2 stimulated PBMCs, without affecting their viability. This allows a rapid enrichment of NK cells and further expansion. We found that dendrimer acts directly on T cells, as their regulatory property is maintained when stimulating purified CD4+ T cells with anti-CD3/CD28 microbeads. Performing equilibrium binding assays using a fluorescent analogue, we show that the phosphonate capped-dendrimers are specifically interacting with purified CD4+ T cells. Ultimately, we found that our protocol prevents the IL-2 related expansion of regulatory T cells that would be deleterious for the activity of infused NK cells. Conclusion High yield expansion of NK cells from human PBMCs by phosphonate-capped dendrimers and IL-2 occurs through the specific inhibition of the CD4+ lymphocyte compartment. Given the

  5. BOLITA, an Arabidopsis AP2/ERF-like transcription factor that affects cell expansion and proliferation/differentiation pathways

    NARCIS (Netherlands)

    Marsch-Martinez, N.; Greco, R.; Becker, J.D.; Dixit, S.A.; Bergervoet, J.H.W.; Karaba, A.; Folter, de S.; Pereira, A.B.

    2006-01-01

    The BOLITA (BOL) gene, an AP2/ERF transcription factor, was characterized with the help of an activation tag mutant and overexpression lines in Arabidopsis and tobacco. The leaf size of plants overexpressing BOL was smaller than wild type plants due to a reduction in both cell size and cell number.

  6. Species-specific expansion and molecular evolution of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR gene family in plants.

    Directory of Open Access Journals (Sweden)

    Wei Li

    Full Text Available The terpene compounds represent the largest and most diverse class of plant secondary metabolites which are important in plant growth and development. The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR; EC 1.1.1.34 is one of the key enzymes contributed to terpene biosynthesis. To better understand the basic characteristics and evolutionary history of the HMGR gene family in plants, a genome-wide analysis of HMGR genes from 20 representative species was carried out. A total of 56 HMGR genes in the 14 land plant genomes were identified, but no genes were found in all 6 algal genomes. The gene structure and protein architecture of all plant HMGR genes were highly conserved. The phylogenetic analysis revealed that the plant HMGRs were derived from one ancestor gene and finally developed into four distinct groups, two in the monocot plants and two in dicot plants. Species-specific gene duplications, caused mainly by segmental duplication, led to the limited expansion of HMGR genes in Zea mays, Gossypium raimondii, Populus trichocarpa and Glycine max after the species diverged. The analysis of Ka/Ks ratios and expression profiles indicated that functional divergence after the gene duplications was restricted. The results suggested that the function and evolution of HMGR gene family were dramatically conserved throughout the plant kingdom.

  7. An Improved Harvest and in Vitro Expansion Protocol for Murine Bone Marrow-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Song Xu

    2010-01-01

    Full Text Available Compared to bone marrow (BM derived mesenchymal stem cells (MSCs from human origin or from other species, the in vitro expansion and purification of murine MSCs (mMSCs is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest, followed by an immunodepletion step using microbeads coated with CD11b, CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion, a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs are uniformly positive for stem cell antigen-1 (Sca-1, CD90, CD105 and CD73 cell surface markers, but negative for the hematopoietic surface markers CD14, CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic, osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time.

  8. Genome-wide Expansion and Expression Divergence of the Basic Leucine Zipper Transcription Factors in Higher Plants with an Emphasis on Sorghum

    Institute of Scientific and Technical Information of China (English)

    Jizhou Wang; Junxia Zhou; Baolan Zhang; Jeevanandam Vanitha; Srinivasan Ramachandran; Shu-Ye Jiang

    2011-01-01

    Plant bZIP transcription factors play crucial roles in multiple biological processes. However,little is known about the sorghum bZIP gene family although the sorghum genome has been completely sequenced. In this study,we have carried out a genome-wide identification and characterization of this gene family in sorghum.Our data show that the genome encodes at least 92 bZIP transcription factors. These bZIP genes have been expanded mainly by segmental duplication. Such an expansion mechanism has also been observed in rice,arabidopsis and many other plant organisms,suggesting a common expansion mode of this gene family in plants. Further investigation shows that most of the bZIP members have been present in the most recent common ancestor of sorghum and rice and the major expansion would occur before the sorghum-rice split era. Although these bZIP genes have been duplicated with a long history,they exhibited limited functional divergence as shown by nonsynonymous substitutions (Ka)/synonymous substitutions (Ks) analyses. Their retention was mainly due to the high percentages of expression divergence. Our data also showed that this gene family might play a role in multiple developmental stages and tissues and might be regarded as important regulators of various abiotic stresses and sugar signaling.

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

    Directory of Open Access Journals (Sweden)

    Vivas Yurena

    2011-12-01

    Full Text Available Abstract 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.

  10. Plant cortical microtubule dynamics and cell division plane orientation

    NARCIS (Netherlands)

    Chakrabortty, Bandan

    2017-01-01

    This thesis work aimed at a better understanding of the molecular basis of oriented cell division in plant cell. As, the efficiency of plant morphogenesis depends on oriented cell division, this work should contribute  towards a fundamental understanding of the  molecular basis of

  11. Pectin, a versatile polysaccharide present in plant cell walls

    NARCIS (Netherlands)

    Voragen, A.G.J.; Coenen, G.J.; Verhoef, R.P.; Schols, H.A.

    2009-01-01

    Pectin or pectic substances are collective names for a group of closely associated polysaccharides present in plant cell walls where they contribute to complex physiological processes like cell growth and cell differentiation and so determine the integrity and rigidity of plant tissue. They also pla

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

    Science.gov (United States)

    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.

  13. Teaching Practice and Expansion in Medicinal Plant Resources%药用植物资源的教学实践与拓展

    Institute of Scientific and Technical Information of China (English)

    王海英

    2012-01-01

    Teaching content arrangement, medicinal plant resources and natural products, medicinal plant resources teaching expansion, as well as creative thinking training were carried out teaching practice in medicinal plant resources of Plant Resource Science. Medicinal plant resources, such as Panax ginseng and Tarus brevifolia, were carried out teaching expansion practice, creative thinking training ways were explored in order to train student's creative ability.%从教学内容安排、药用植物资源与天然产物、药用植物资源教学拓展以及创新思维训练角度进行植物资源学课程中药用植物资源的教学实践,对药用资源植物如人参(Panax ginseng)和短叶红豆杉(Taxus brevifolia)进行教学拓展实践,探讨创新思维训练途径,培养大学生创新能力。

  14. Ultrastructure of autophagy in plant cells: a review.

    Science.gov (United States)

    van Doorn, Wouter G; Papini, Alessio

    2013-12-01

    Just as with yeasts and animal cells, plant cells show several types of autophagy. Microautophagy is the uptake of cellular constituents by the vacuolar membrane. Although microautophagy seems frequent in plants it is not yet fully proven to occur. Macroautophagy occurs farther away from the vacuole. In plants it is performed by autolysosomes, which are considerably different from the autophagosomes found in yeasts and animal cells, as in plants these organelles contain hydrolases from the onset of their formation. Another type of autophagy in plant cells (called mega-autophagy or mega-autolysis) is the massive degradation of the cell at the end of one type of programmed cell death (PCD). Furthermore, evidence has been found for autophagy during degradation of specific proteins, and during the internal degeneration of chloroplasts. This paper gives a brief overview of the present knowledge on the ultrastructure of autophagic processes in plants.

  15. Sulforaphane inhibits mitotic clonal expansion during adipogenesis through cell cycle arrest.

    Science.gov (United States)

    Choi, Kyeong-Mi; Lee, Youn-Sun; Sin, Dong-Mi; Lee, Seunghyun; Lee, Mi Kyeong; Lee, Yong-Moon; Hong, Jin-Tae; Yun, Yeo-Pyo; Yoo, Hwan-Soo

    2012-07-01

    Obesity is a risk factor for numerous metabolic disorders such as type 2 diabetes, hypertension, and coronary heart disease. Adipocyte differentiation is triggered by adipocyte hyperplasia, which leads to obesity. In this study, the inhibitory effect of sulforaphane, an isothiocyanate, on adipogenesis in 3T3-L1 cells was investigated. Sulforaphane decreased the accumulation of lipid droplets stained with Oil Red O and inhibited the elevation of triglycerides in the adipocytes (half-maximal inhibitory concentration = 7.3 µmol/l). The expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), major transcription factors for adipocyte differentiation, was significantly reduced by sulforaphane. The major effects of sulforaphane on the inhibition of adipocyte differentiation occurred during the early stage of adipogenesis. Thus, the expression of C/EBPβ, an early-stage biomarker of adipogenesis, decreased in a concentration-dependent manner when the adipocytes were exposed to sulforaphane (0, 5, 10, and 20 µmol/l). The proliferation of adipocytes treated with 20 µmol/l sulforaphane for 24 and 48 h was also suppressed. These results indicate that sulforaphane may specifically affect mitotic clonal expansion to inhibit adipocyte differentiation. Sulforaphane arrested the cell cycle at the G(0)/G(1) phase, increased p27 expression, and decreased retinoblastoma (Rb) phosphorylation. Additionally, sulforaphane modestly decreased the phosphorylation of ERK1/2 and Akt. Our results indicate that the inhibition of early-stage adipocyte differentiation by sulforaphane may be associated with cell cycle arrest at the G(0)/G(1) phase through upregulation of p27 expression.

  16. IGF1 stimulates crypt expansion via differential activation of 2 intestinal stem cell populations

    Science.gov (United States)

    Van Landeghem, Laurianne; Santoro, M. Agostina; Mah, Amanda T.; Krebs, Adrienne E.; Dehmer, Jeffrey J.; McNaughton, Kirk K.; Helmrath, Michael A.; Magness, Scott T.; Lund, P. Kay

    2015-01-01

    Insulin-like growth factor 1 (IGF1) has potent trophic effects on normal or injured intestinal epithelium, but specific effects on intestinal stem cells (ISCs) are undefined. We used Sox9-enhanced green fluorescent protein (EGFP) reporter mice that permit analyses of both actively cycling ISCs (Sox9-EGFPLow) and reserve/facultative ISCs (Sox9-EGFPHigh) to study IGF1 action on ISCs in normal intestine or during crypt regeneration after high-dose radiation-induced injury. We hypothesized that IGF1 differentially regulates proliferation and gene expression in actively cycling and reserve/facultative ISCs. IGF1 was delivered for 5 days using subcutaneously implanted mini-pumps in uninjured mice or after 14 Gy abdominal radiation. ISC numbers, proliferation, and transcriptome were assessed. IGF1 increased epithelial growth in nonirradiated mice and enhanced crypt regeneration after radiation. In uninjured and regenerating intestines, IGF1 increased total numbers of Sox9-EGFPLow ISCs and percentage of these cells in M-phase. IGF1 increased percentages of Sox9-EGFPHigh ISCs in S-phase but did not expand this population. Microarray revealed that IGF1 activated distinct gene expression signatures in the 2 Sox9-EGFP ISC populations. In vitro IGF1 enhanced enteroid formation by Sox9-EGFPHigh facultative ISCs but not Sox9-EGFPLow actively cycling ISCs. Our data provide new evidence that IGF1 activates 2 ISC populations via distinct regulatory pathways to promote growth of normal intestinal epithelium and crypt regeneration after irradiation.—Van Landeghem, L., Santoro, M. A., Mah, A. T., Krebs, A. E., Dehmer, J. J., McNaughton, K. K., Helmrath, M. A., Magness, S. T., Lund, P. K. IGF1 stimulates crypt expansion via differential activation of 2 intestinal stem cell populations. PMID:25837582

  17. End plate for e.g. solid oxide fuel cell stack, sets thermal expansion coefficient of material to predetermined value

    DEFF Research Database (Denmark)

    2011-01-01

    .05-0.3 mm. USE - End plate for solid oxide fuel cell stack (claimed). Can also be used in polymer electrolyte fuel cell stack and direct methanol fuel cell stack. ADVANTAGE - The robustness of the end plate is improved. The structure of the end plate is simplified. The risk of delamination of the stack......NOVELTY - The end plate is made of material whose thermal expansion coefficient is corresponding to that of material of a cell (103). The thermal expansion coefficient of material is 9asterisk10-6 K-1 to 14asterisk10-6 K11. The thickness of the end plate is within the range of 0.001-1 mm and 0...

  18. Expansion of myeloid-derived suppressor cells promotes differentiation of regulatory T cells in HIV-1+ individuals.

    Science.gov (United States)

    Wang, Ling; Zhao, Juan; Ren, Jun P; Wu, Xiao Y; Morrison, Zheng D; Elgazzar, Mohamed A; Ning, Shun B; Moorman, Jonathan P; Yao, Zhi Q

    2016-06-19

    Regulatory T cells (Tregs) contribute to HIV-1 disease progression by impairing antiviral immunity; however, the precise mechanisms responsible for the development of Tregs in the setting of HIV-1 infection are incompletely understood. In this study, we provide evidence that HIV-induced expansion of monocytic myeloid-derived suppressor cells (M-MDSCs) promote the differentiation of Foxp3 Tregs. We measured MDSC induction and cytokine expression by flow cytometry and analyzed their functions by coculturing experiments. We observed a dramatic increase in M-MDSC frequencies in the peripheral blood of HIV-1 seropositive (HIV-1) individuals, even in those on antiretroviral therapy with undetectable viremia, when compared with healthy participants. We also observed increases in M-MDSCs after incubating healthy peripheral mononuclear cells (PBMCs) with HIV-1 proteins (gp120 or Tat) or Toll-like receptor 4 ligand lipopolysaccharides in vitro, an effect that could be abrogated in the presence of the phosphorylated signal transducer and activator of transcription 3 inhibitor, STA-21. Functional analyses indicated that M-MDSCs from HIV-1 individuals express higher levels of IL-10, tumor growth factor-β, IL-4 receptor α, p47, programmed death-ligand 1, and phosphorylated signal transducer and activator of transcription 3 - all of which are known mediators of myelopoiesis and immunosuppression. Importantly, incubation of healthy CD4 T cells with MDSCs derived from HIV-1 individuals significantly increased differentiation of Foxp3 Tregs. In addition, depletion of MDSCs from PBMCs of HIV-1 individuals led to a significant reduction of Foxp3 Tregs and increase of IFNγ production by CD4 T effector cells. These results suggest that HIV-induced MDSCs promote Treg cell development and inhibit T cell function - a hallmark of many chronic infectious diseases.

  19. Auxin regulation of cell polarity in plants.

    Science.gov (United States)

    Pan, Xue; Chen, Jisheng; Yang, Zhenbiao

    2015-12-01

    Auxin is well known to control pattern formation and directional growth at the organ/tissue levels via the nuclear TIR1/AFB receptor-mediated transcriptional responses. Recent studies have expanded the arena of auxin actions as a trigger or key regulator of cell polarization and morphogenesis. These actions require non-transcriptional responses such as changes in the cytoskeleton and vesicular trafficking, which are commonly regulated by ROP/Rac GTPase-dependent pathways. These findings beg for the question about the nature of auxin receptors that regulate these responses and renew the interest in ABP1 as a cell surface auxin receptor, including the work showing auxin-binding protein 1 (ABP1) interacts with the extracellular domain of the transmembrane kinase (TMK) receptor-like kinases in an auxin-dependent manner, as well as the debate on this auxin binding protein discovered about 40 years ago. This review highlights recent work on the non-transcriptional auxin signaling mechanisms underscoring cell polarity and shape formation in plants.

  20. Tissue Source and Cell Expansion Condition Influence Phenotypic Changes of Adipose-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Lauren H. Mangum

    2017-01-01

    Full Text Available Stem cells derived from the subcutaneous adipose tissue of debrided burned skin represent an appealing source of adipose-derived stem cells (ASCs for regenerative medicine. Traditional tissue culture uses fetal bovine serum (FBS, which complicates utilization of ASCs in human medicine. Human platelet lysate (hPL is one potential xeno-free, alternative supplement for use in ASC culture. In this study, adipogenic and osteogenic differentiation in media supplemented with 10% FBS or 10% hPL was compared in human ASCs derived from abdominoplasty (HAP or from adipose associated with debrided burned skin (BH. Most (95–99% cells cultured in FBS were stained positive for CD73, CD90, CD105, and CD142. FBS supplementation was associated with increased triglyceride content and expression of adipogenic genes. Culture in hPL significantly decreased surface staining of CD105 by 31% and 48% and CD142 by 27% and 35% in HAP and BH, respectively (p<0.05. Culture of BH-ASCs in hPL also increased expression of markers of osteogenesis and increased ALP activity. These data indicate that application of ASCs for wound healing may be influenced by ASC source as well as culture conditions used to expand them. As such, these factors must be taken into consideration before ASCs are used for regenerative purposes.

  1. Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement.

    Science.gov (United States)

    Smith, Corey; Økern, Grethe; Rehan, Sweera; Beagley, Leone; Lee, Sau K; Aarvak, Tanja; Schjetne, Karoline W; Khanna, Rajiv

    2015-01-01

    The manufacture of clinical grade cellular products for adoptive immunotherapy requires ex vivo culture and expansion of human T cells. One of the key components in manufacturing of T cell therapies is human serum (HS) or fetal bovine serum (FBS), which can potentially expose immunotherapy recipient to adventitious infectious pathogens and are thus considered as non-cGMP compliant for adoptive therapy. Here we describe a novel xeno-free serum replacement (SR) with defined components that can be reproducibly used for the production of clinical grade T-cell therapies in combination with several different cell culture media. Dynabeads CD3/CD28 Cell Therapy System (CTS)-activated or antigen-specific T cells expanded using the xeno-free SR, CTS Immune Cell SR, showed comparable growth kinetics observed with cell culture media supplemented with HS or FBS. Importantly the xeno-free SR supplemented medium supported the optimal expansion of T cells specific for subdominant tumour-associated antigens and promoted expansion of T cells with central memory T-cell phenotype, which is favourable for in vivo survival and persistence following adoptive transfer. Furthermore, T cells expanded using xeno-free SR medium were highly amenable to lentivirus-mediated gene transduction for potential application for gene-modified T cells. Taken together, the CTS Immune Cell SR provides a novel platform strategy for the manufacture of clinical grade adoptive cellular therapies.

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

    Science.gov (United States)

    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.

  3. Plant and animal stem cells: similar yet different.

    Science.gov (United States)

    Heidstra, Renze; Sabatini, Sabrina

    2014-05-01

    The astonishingly long lives of plants and their regeneration capacity depend on the activity of plant stem cells. As in animals, stem cells reside in stem cell niches, which produce signals that regulate the balance between self-renewal and the generation of daughter cells that differentiate into new tissues. Plant stem cell niches are located within the meristems, which are organized structures that are responsible for most post-embryonic development. The continuous organ production that is characteristic of plant growth requires a robust regulatory network to keep the balance between pluripotent stem cells and differentiating progeny. Components of this network have now been elucidated and provide a unique opportunity for comparing strategies that were developed in the animal and plant kingdoms, which underlie the logic of stem cell behaviour.

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

    Science.gov (United States)

    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.

  5. Critical steps in the isolation and expansion of adipose-derived stem cells for translational therapy.

    Science.gov (United States)

    Riis, S; Zachar, V; Boucher, S; Vemuri, M C; Pennisi, C P; Fink, T

    2015-06-08

    Since the discovery of adipose-derived stem cells (ASCs), there have been high expectations of their putative clinical use. Recent advances support these expectations, and it is expected that the transition from pre-clinical and clinical studies to implementation as a standard treatment modality is imminent. However ASCs must be isolated and expanded according to good manufacturing practice guidelines and a basic assurance of quality, safety, and medical effectiveness is needed for authorisation by regulatory agencies, such as European Medicines Agency and US Food and Drug Administration. In this review, a collection of studies investigating the influence of different steps of the isolation and expansion protocol on the yield and functionality of ASCs has been presented in an attempt to come up with best recommendations that ensure potential beneficial clinical outcome of using ASCs in any therapeutic setting. If the findings confirm the initial observations of beneficial effects of ASCs, the path is paved for implementing these ASC-based therapies as standard treatment options.

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

    Science.gov (United States)

    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.

  7. Programmed cell death in the plant immune system.

    Science.gov (United States)

    Coll, N S; Epple, P; Dangl, J L

    2011-08-01

    Cell death has a central role in innate immune responses in both plants and animals. Besides sharing striking convergences and similarities in the overall evolutionary organization of their innate immune systems, both plants and animals can respond to infection and pathogen recognition with programmed cell death. The fact that plant and animal pathogens have evolved strategies to subvert specific cell death modalities emphasizes the essential role of cell death during immune responses. The hypersensitive response (HR) cell death in plants displays morphological features, molecular architectures and mechanisms reminiscent of different inflammatory cell death types in animals (pyroptosis and necroptosis). In this review, we describe the molecular pathways leading to cell death during innate immune responses. Additionally, we present recently discovered caspase and caspase-like networks regulating cell death that have revealed fascinating analogies between cell death control across both kingdoms.

  8. Generation, Expansion, and Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) Derived From the Umbilical Cords of Newborns.

    Science.gov (United States)

    Song, Richard S; Carroll, Jeanne M; Acevedo, Lisette; Wu, Dongmei; Liu, Yang; Snyder, Evan Y

    2014-05-16

    The umbilical cord is tissue that is normally discarded after the delivery of the infant, but it has been shown to be a rich source of stem cells from the cord blood, Wharton's jelly, and umbilical endothelial cells. Patient-specific human induced pluripotent stem cells (hiPSCs) reprogrammed from patient specific human umbilical vein endothelial cells in the neonatal intensive care unit (NICU) population (specifically, premature neonates) have not been shown in the literature. This unit describes a protocol for the generation and expansion of hiPSCs originating from umbilical cords collected from patients in the NICU.

  9. New plant-growth medium for increased power output of the Plant-Microbial Fuel Cell

    NARCIS (Netherlands)

    Helder, M.; Strik, D.P.B.T.B.; Hamelers, H.V.M.; Kuijken, R.C.P.; Buisman, C.J.N.

    2012-01-01

    In a Plant-Microbial Fuel Cell anode-conditions must be created that are favorable for plant growth and electricity production. One of the major aspects in this is the composition of the plant-growth medium. Hoagland medium has been used until now, with added phosphate buffer to reduce potential

  10. Umbilical Cord Blood Platelet Lysate as Serum Substitute in Expansion of Human Mesenchymal Stem Cells.

    Science.gov (United States)

    Shirzad, Negin; Bordbar, Sima; Goodarzi, Alireza; Mohammad, Monire; Khosravani, Pardis; Sayahpour, Froughazam; Baghaban Eslaminejad, Mohamadreza; Ebrahimi, Marzieh

    2017-10-01

    The diverse clinical applications for human mesenchymal stem cells (hMSCs) in cellular therapy and regenerative medicine warrant increased focus on developing adequate culture supplements devoid of animal-derived products. In the present study, we have investigated the feasibility of umbilical cord blood-platelet lysate (UCB-PL) as a standard substitute for fetal bovine serum (FBS) and human peripheral blood-PL (PB-PL). In this experimental study, platelet concentrates (PC) from UCB and human PB donors were frozen, melted, and sterilized to obtain PL. Quality control included platelet cell counts, sterility testing (viral and microbial), total protein concentrations, growth factor levels, and PL stability. The effects of UCB-PL and PB-PL on hMSCs proliferation and differentiation into osteocytes, chondrocytes, and adipocytes were studied and the results compared with FBS. UCB-PL contained high levels of protein content, platelet-derived growth factor- AB (PDGF-AB), and transforming growth factor (TGF) compared to PB-PL. All growth factors were stable for at least nine months post-storage at -70˚C. hMSCs proliferation enhanced following treatment with UCB-PL. With all three supplements, hMSCs could differentiate into all three lineages. PB-PL and UCB-PL both were potent in hMSCs proliferation. However, PB promoted osteoblastic differentiation and UCB-PL induced chondrogenic differentiation. Because of availability, ease of use and feasible standardization of UCB-PL, we have suggested that UCB-PL be used as an alternative to FBS and PB-PL for the cultivation and expansion of hMSCs in cellular therapy.

  11. Factors Promoting Tamoxifen Resistance in Breast Cancer via Stimulating Breast Cancer Stem Cell Expansion.

    Science.gov (United States)

    Ojo, Diane; Wei, Fengxiang; Liu, Yun; Wang, Enli; Zhang, Hongde; Lin, Xiaozeng; Wong, Nicholas; Bane, Anita; Tang, Damu

    2015-01-01

    Estrogen receptor-alpha positive (ER(+)) breast cancer constitutes 70-75% of the disease incidence. Tamoxifen has been the basis of endocrine therapy for patients with ER(+) breast cancer for more than three decades. The treatment reduces the annual mortality rate of breast cancer by 31%, and remains the most effective targeted cancer therapy. However, approximately one-third of patients treated with adjuvant tamoxifen suffer from aggressive recurrent disease. Resistance to tamoxifen, thus, remains a major challenge in providing effective treatments for these patients. In an effort to overcome the resistance, intensive research has been conducted to understand the underlying mechanisms; this has resulted in the identification of complex factors/pathways contributing to tamoxifen resistance, including modulations of the ERsignaling, upregulation of a set of growth factor receptor networks (HER2, EGFR, FGFR, and IGF1R), alterations of the PI3K-PTEN/AKT/mTOR pathway, and an elevation of the NF-κB signaling. Despite these advances, our understanding of the acquired resistance remains fragmented and there is a lack of a platform to integrate these diversified molecular factors/ pathways into a cohesive mechanistic model. Nonetheless, at the cellular level, it is becoming increasingly recongnized that cancer stem cells (CSCs) are key in driving cancer metastasis and therapy resistance. Likewise, evidence is emerging for the critical contributions of breast cancer stem cells (BCSCs) to tamoxifen resistance. In this review, we will discuss these recent developments of BCSC-mediated resistance to tamoxifen and the contributions of those demonstrated molecular factors/pathways to BCSC expansion during the emergency of tamoxifen resistance.

  12. Direct visualization of endogenous Salmonella-specific B cells reveals a marked delay in clonal expansion and germinal center development.

    Science.gov (United States)

    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.

  13. Experimental approaches to study plant cell walls during plant-microbe interactions.

    Science.gov (United States)

    Xia, Ye; Petti, Carloalberto; Williams, Mark A; DeBolt, Seth

    2014-01-01

    Plant cell walls provide physical strength, regulate the passage of bio-molecules, and act as the first barrier of defense against biotic and abiotic stress. In addition to providing structural integrity, plant cell walls serve an important function in connecting cells to their extracellular environment by sensing and transducing signals to activate cellular responses, such as those that occur during pathogen infection. This mini review will summarize current experimental approaches used to study cell wall functions during plant-pathogen interactions. Focus will be paid to cell imaging, spectroscopic analyses, and metabolic profiling techniques.

  14. Experimental approaches to study plant cell walls during plant-microbe interactions

    Directory of Open Access Journals (Sweden)

    Ye eXia

    2014-10-01

    Full Text Available Plant cell walls provide physical strength, regulate the passage of bio-molecules, and act as the first barrier of defense against biotic and abiotic stress. In addition to providing structural integrity, plant cell walls serve an important function in connecting cells to their extracellular environment by sensing and transducing signals to activate cellular responses, such as those that occur during pathogen infection. This mini review will summarize current experimental approaches used to study cell wall functions during plant-pathogen interactions. Focus will be paid to cell imaging, spectroscopic analyses, and metabolic profiling techniques

  15. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    Science.gov (United States)

    Bozzone, Donna M.

    1997-01-01

    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  16. Epigenetic memory and cell fate reprogramming in plants.

    Science.gov (United States)

    Birnbaum, Kenneth D; Roudier, François

    2017-02-01

    Plants have a high intrinsic capacity to regenerate from adult tissues, with the ability to reprogram adult cell fates. In contrast, epigenetic mechanisms have the potential to stabilize cell identity and maintain tissue organization. The question is whether epigenetic memory creates a barrier to reprogramming that needs to be erased or circumvented in plant regeneration. Early evidence suggests that, while chromatin dynamics impact gene expression in the meristem, a lasting constraint on cell fate is not established until late stages of plant cell differentiation. It is not yet clear whether the plasticity of plant cells arises from the ability of cells to erase identity memory or to deploy cells that may exhibit cellular specialization but still lack an epigenetic restriction on cell fate alteration.

  17. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    Science.gov (United States)

    Bozzone, Donna M.

    1997-01-01

    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  18. Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi.

    Science.gov (United States)

    Kubicek, Christian P; Starr, Trevor L; Glass, N Louise

    2014-01-01

    Approximately a tenth of all described fungal species can cause diseases in plants. A common feature of this process is the necessity to pass through the plant cell wall, an important barrier against pathogen attack. To this end, fungi possess a diverse array of secreted enzymes to depolymerize the main structural polysaccharide components of the plant cell wall, i.e., cellulose, hemicellulose, and pectin. Recent advances in genomic and systems-level studies have begun to unravel this diversity and have pinpointed cell wall-degrading enzyme (CWDE) families that are specifically present or enhanced in plant-pathogenic fungi. In this review, we discuss differences between the CWDE arsenal of plant-pathogenic and non-plant-pathogenic fungi, highlight the importance of individual enzyme families for pathogenesis, illustrate the secretory pathway that transports CWDEs out of the fungal cell, and report the transcriptional regulation of expression of CWDE genes in both saprophytic and phytopathogenic fungi.

  19. Formative cell divisions: principal determinants of plant morphogenesis.

    Science.gov (United States)

    Smolarkiewicz, Michalina; Dhonukshe, Pankaj

    2013-03-01

    Formative cell divisions utilizing precise rotations of cell division planes generate and spatially place asymmetric daughters to produce different cell layers. Therefore, by shaping tissues and organs, formative cell divisions dictate multicellular morphogenesis. In animal formative cell divisions, the orientation of the mitotic spindle and cell division planes relies on intrinsic and extrinsic cortical polarity cues. Plants lack known key players from animals, and cell division planes are determined prior to the mitotic spindle stage. Therefore, it appears that plants have evolved specialized mechanisms to execute formative cell divisions. Despite their profound influence on plant architecture, molecular players and cellular mechanisms regulating formative divisions in plants are not well understood. This is because formative cell divisions in plants have been difficult to track owing to their submerged positions and imprecise timings of occurrence. However, by identifying a spatiotemporally inducible cell division plane switch system applicable for advanced microscopy techniques, recent studies have begun to uncover molecular modules and mechanisms for formative cell divisions. The identified molecular modules comprise developmentally triggered transcriptional cascades feeding onto microtubule regulators that now allow dissection of the hierarchy of the events at better spatiotemporal resolutions. Here, we survey the current advances in understanding of formative cell divisions in plants in the context of embryogenesis, stem cell functionality and post-embryonic organ formation.

  20. Cell Fate Switch during In Vitro Plant Organogenesis

    Institute of Scientific and Technical Information of China (English)

    Xiang Yu Zhao; Ying Hua Su; Zhi Juan Cheng; Xian Sheng Zhang

    2008-01-01

    Plant mature cells have the capability to reverse their state of differenUation and produce new organs under cultured conditions. Two phases, dedifferentiation and redifferentiation, are commonly characterized during in vitro organogenesis.In these processes, cells undergo fate switch several times regulated by both extrinsic and intrinsic factors, which are associated with reentry to the cell cycle, the balance between euchromatin and heterochromatin, reprogramming of gene expression, and so forth. This short article reviews the advances in the mechanism of organ regeneration from plant somatic cells in molecular, genomic and epigenetic aspects, aiming to provide important information on the mechanism underlying cell fate switch during in vitro plant organogenesis.

  1. The First Observation on Plant Cell Fossils in China

    Institute of Scientific and Technical Information of China (English)

    WANG Xin; CUI Jinzhong

    2007-01-01

    For a long time, paleontologists have been focusing on hard parts of organisms during different geological periods while soft parts are rarely reported. Well-preserved plant cells, if found in fossils, are treated only as a rarity. Recent progress in research on fossil cytoplasm indicates that plant cytoplasm not only has excellent ultrastructures preserved but also may be a quite commonly seen fossil in strata. However, up to now there is no report of plant cell fossils in China yet. Here plant cell fossils are reported from Huolinhe Coal Mine (the early Cretaceous), Inner Mongolia, China. The presence of plant cytoplasm fossils in two cones on the same specimen not only provides further support for the recently proposed hypothesis on plant cytoplasm fossilization but also marks the first record of plant cytoplasm fossils in China, which suggests a great research potential in this new area.

  2. Progress and prospects for phosphoric acid fuel cell power plants

    Energy Technology Data Exchange (ETDEWEB)

    Bonville, L.J.; Scheffler, G.W.; Smith, M.J. [International Fuel Cells Corp., South Windsor, CT (United States)

    1996-12-31

    International Fuel Cells (IFC) has developed the fuel cell power plant as a new, on-site power generation source. IFC`s commercial fuel cell product is the 200-kW PC25{trademark} power plant. To date over 100 PC25 units have been manufactured. Fleet operating time is in excess of one million hours. Individual units of the initial power plant model, the PC25 A, have operated for more than 30,000 hours. The first model {open_quotes}C{close_quotes} power plant has over 10,000 hours of operation. The manufacturing, application and operation of this power plant fleet has established a firm base for design and technology development in terms of a clear understanding of the requirements for power plant reliability and durability. This fleet provides the benchmark against which power plant improvements must be measured.

  3. Involvement of IL-18 in the expansion of unique hepatic T cells with unconventional cytokine profiles during Schistosoma mansoni infection.

    Directory of Open Access Journals (Sweden)

    Keishi Adachi

    Full Text Available Infection with schistosomes invokes severe fibrotic granulomatous responses in the liver of the host. Schistosoma mansoni infection induces dramatic fluctuations in Th1 or Th2 cytokine responses systemically; Th1 reactions are provoked in the early phase, whilst Th2 responses become dominant after oviposition begins. In the liver, various unique immune cells distinct from those of conventional immune competent organs or tissues exist, resulting in a unique immunological environment. Recently, we demonstrated that S. mansoni infection induces unique CD4+ T cell populations exhibiting unconventional cytokine profiles in the liver of mice during the period between Th1- and Th2-phases, which we term the transition phase. They produce both IFN-γ and IL-4 or both IFN-γ and IL-13 simultaneously. Moreover, T cells secreting triple cytokines IFN-γ, IL-13 and IL-4 were also induced. We term these cells Multiple Cytokine Producing Hepatic T cells (MCPHT cells. During the transition phase, when MCPHT cells increase, IL-18 secretion was up-regulated in the liver and sera. In S. mansoni-infected IL-18-deficient mice, expansion of MCPHT cells was curtailed. Thus our data suggest that IL-18 produced during S. mansoni infection play a role in the expansion of MCPHT cells.

  4. Chloride regulates leaf cell size and water relations in tobacco plants.

    Science.gov (United States)

    Franco-Navarro, Juan D; Brumós, Javier; Rosales, Miguel A; Cubero-Font, Paloma; Talón, Manuel; Colmenero-Flores, José M

    2016-02-01

    Chloride (Cl(-)) is a micronutrient that accumulates to macronutrient levels since it is normally available in nature and actively taken up by higher plants. Besides a role as an unspecific cell osmoticum, no clear biological roles have been explicitly associated with Cl(-) when accumulated to macronutrient concentrations. To address this question, the glycophyte tobacco (Nicotiana tabacum L. var. Habana) has been treated with a basal nutrient solution supplemented with one of three salt combinations containing the same cationic balance: Cl(-)-based (CL), nitrate-based (N), and sulphate+phosphate-based (SP) treatments. Under non-saline conditions (up to 5 mM Cl(-)) and no water limitation, Cl(-) specifically stimulated higher leaf cell size and led to a moderate increase of plant fresh and dry biomass mainly due to higher shoot expansion. When applied in the 1-5 mM range, Cl(-) played specific roles in regulating leaf osmotic potential and turgor, allowing plants to improve leaf water balance parameters. In addition, Cl(-) also altered water relations at the whole-plant level through reduction of plant transpiration. This was a consequence of a lower stomatal conductance, which resulted in lower water loss and greater photosynthetic and integrated water-use efficiency. In contrast to Cl(-), these effects were not observed for essential anionic macronutrients such as nitrate, sulphate, and phosphate. We propose that the abundant uptake and accumulation of Cl(-) responds to adaptive functions improving water homeostasis in higher plants.

  5. Polarity in plant asymmetric cell division: Division orientation and cell fate differentiation

    OpenAIRE

    Shao, Wanchen; Dong, Juan

    2016-01-01

    Asymmetric cell division (ACD) is universally required for the development of multicellular organisms. Unlike animal cells, plant cells have a rigid cellulosic extracellular matrix, the cell wall, which provides physical support and forms communication routes. This fundamental difference leads to some unique mechanisms in plants for generating asymmetries during cell division. However, plants also utilize intrinsically polarized proteins to regulate asymmetric signaling and cell division, a s...

  6. Antigen-specific in vitro expansion of functional redirected NY-ESO-1-specific human CD8+ T-cells in a cell-free system.

    Science.gov (United States)

    Jakka, Gopinadh; Schuberth, Petra C; Thiel, Markus; Held, Gerhard; Stenner, Frank; Van Den Broek, Maries; Renner, Christoph; Mischo, Axel; Petrausch, Ulf

    2013-10-01

    Tumors can be targeted by the adoptive transfer of chimeric antigen receptor (CAR) redirected T-cells. Antigen-specific expansion protocols are needed to generate large quantities of redirected T-cells. We aimed to establish a protocol to expand functional active NY-ESO-1-specific redirected human CD8(+) T-cells. The anti-idiotypic Fab antibody A4 with specificity for HLA-A 0201/NY-ESO-1157-165 was tested by competition assays using a HLA-A 0201/NY-ESO-1157-165 tetramer. HLA-A 0201/NY-ESO-1157-165 redirected T-cells were generated, expanded and tested for CAR expression, cytokine release, in vitro cytolysis and protection against xenografted HLA-A 0201/NY-ESO-1157-165-positive multiple myeloma cells. A4 demonstrated antigen-specific binding to HLA-A 0201/NY-ESO-1157-165 redirected T-cells. Expansion with A4 resulted in 98% of HLA-A 0201/NY-ESO-1157-165 redirected T-cells. A4 induced strong proliferation, resulting in a 300-fold increase of redirected T-cells. After expansion protocols, redirected T-cells secreted Interleukin-2, (IL-2), interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) and lysed target cells in vitro and were protective in vivo. A4 expanded HLA-A 0201/NY-ESO-1157-165 redirected T-cells with preservation of antigen-specific function.

  7. Chemical- and pathogen-induced programmed cell death in plants

    NARCIS (Netherlands)

    Iakimova, E.T.; Atanassov, A.; Woltering, E.J.

    2005-01-01

    This review focuses on recent update in the understanding of programmed cell death regarding the differences and similarities between the diverse types of cell death in animal and plant systems and describes the morphological and some biochemical determinants. The role of PCD in plant development an

  8. Chemical- and pathogen-induced programmed cell death in plants

    NARCIS (Netherlands)

    Iakimova, E.T.; Atanassov, A.; Woltering, E.J.

    2005-01-01

    This review focuses on recent update in the understanding of programmed cell death regarding the differences and similarities between the diverse types of cell death in animal and plant systems and describes the morphological and some biochemical determinants. The role of PCD in plant development

  9. In vitro TNF blockade enhances ex vivo expansion of regulatory T cells in patients with immune thrombocytopenia

    Science.gov (United States)

    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

  10. Fibroblast Growth Factor-2 Enhances Expansion of Human Bone Marrow-Derived Mesenchymal Stromal Cells without Diminishing Their Immunosuppressive Potential

    Directory of Open Access Journals (Sweden)

    Jeffery J. Auletta

    2011-01-01

    Full Text Available Allogeneic hematopoietic stem cell transplantation is the main curative therapy for many hematologic malignancies. Its potential relies on graft-versus-tumor effects which associate with graft-versus-host disease. Mesenchymal stromal cells (MSCs possess immunomodulatory properties that make them attractive therapeutic alternatives. We evaluated the in vitro immunosuppressive activity of medium conditioned by human MSCs from 5 donors expanded 13 passages with or without FGF-2. FGF-2 supplementation increased expansion 3,500- and 240,000-fold by passages 7 and 13, respectively. There were no differences in immunosuppressive activity between media conditioned by passage-matched cells expanded under different conditions, but media conditioned by FGF-treated MSCs were superior to population doubling-matched controls. The immunosuppressive activity was maintained in three of the preparations but decreased with expansion in two. The proliferation induced by FGF-2 did not result in loss of immunosuppressive activity. However, because the immunosuppressive activity was not consistently preserved, caution must be exercised to ensure that the activity of the cells is sufficient after extensive expansion.

  11. Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves. II. Quantity and quality of light required

    Science.gov (United States)

    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.

  12. In vitro TNF blockade enhances ex vivo expansion of regulatory T cells in patients with immune thrombocytopenia.

    Science.gov (United States)

    Zhong, Hui; Bussel, James; Yazdanbakhsh, Karina

    2015-01-01

    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. © 2014 John Wiley & Sons Ltd.

  13. Meis1 Is Required for Adult Mouse Erythropoiesis, Megakaryopoiesis and Hematopoietic Stem Cell Expansion.

    Directory of Open Access Journals (Sweden)

    Michelle Erin Miller

    Full Text Available 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(ROSA26Sor(tm1(Cre/ERTNat/J or B6.Cg-Tg(Mx1-Cre1Cgn/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.

  14. The role of root border cells in plant defense.

    Science.gov (United States)

    Hawes, M C; Gunawardena, U; Miyasaka, S; Zhao, X

    2000-03-01

    The survival of a plant depends upon the capacity of root tips to sense and move towards water and other nutrients in the soil. Perhaps because of the root tip's vital role in plant health, it is ensheathed by large populations of detached somatic cells - root 'border' cells - which have the ability to engineer the chemical and physical properties of the external environment. Of particular significance, is the production by border cells of specific chemicals that can dramatically alter the behavior of populations of soilborne microflora. Molecular approaches are being used to identify and manipulate the expression of plant genes that control the production and the specialized properties of border cells in transgenic plants. Such plants can be used to test the hypothesis that these unusual cells act as a phalanx of biological 'goalies', which neutralize dangers to newly generated root tissue as the root tip makes its way through soil.

  15. Human placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34+ cells

    Institute of Scientific and Technical Information of China (English)

    YiZhanga; ChangdongLi; XiaoxiaJiang; ShuangxiZhang; YingWu; BingLiu; PeihsienTang; NingMao

    2005-01-01

    Objective. Allogeneic transplantation with umbilical cord blood (UCB) in adult recipients is limited mainly by a low CD34+ cell dose. To overcome this shortcoming, human placenta as a novel source of human mesenchymal progenitor cell (MPC) was incorporated in an attempt to expand CD34+ ceils from UCB in vitro.Materials and Methods. Human placenta MPC was isolated and characterized by morphologic,immunophenotypical, and functional analysis. UCB CD34+ cells were expanded by coculturewith placeutal MPC. Suitable aliquots of cells were used to monitor cell production, elonogenie activity, and tong-term culture-initiating culture (LTC-IC) output. Finally, the immunoregulatory effect of placental MPC was evaluated by T-cell proliferation assay.Results. In its undifferentiated state, placental MPC displayed fibroblastoid morphology; was CD73, CD105, CD29, CD44, HLA-ABC, and CD166 positive; produced fibronectin, laminin,and vimentin; but was negative for CD14, CD31, CD34, CD45, HLA-DR, and α-smooth muscle actin. Functionally, it could be induced into adipocytes, osteocytes, and chondrocytes.In vitro expansion of UCB hematopoietic cells, when cocultured with placental MPC in the presence of eytokines, was significantly enhanced: CD34+ cells by 14.89±2.32 fold; colonyforming cell (CFC) by 36.73±5.79 told; and LTC-IC by 7.43±2.66 fold. Moreover, placental MPC could suppress T-cell proliferation induced by cellular stimuli.Conclusion. These results strongly suggest that human placental MPC may be a suitable feeder layer for expansion of hematopoietic progenitors from UCB in vitro.

  16. Feasibility analysis in the expansion proposal of the nuclear power plant Laguna Verde: application of real options, binomial model; Analisis de viabilidad en la propuesta de expansion de la central nucleoelectrica Laguna Verde: aplicacion de opciones reales, modelo binomial

    Energy Technology Data Exchange (ETDEWEB)

    Hernandez I, S.; Ortiz C, E.; Chavez M, C., E-mail: lunitza@gmail.com [UNAM, Facultad de Ingenieria, Circuito Interior, Ciudad Universitaria, 04510 Mexico D. F. (Mexico)

    2011-11-15

    At the present time, is an unquestionable fact that the nuclear electrical energy is a topic of vital importance, no more because eliminates the dependence of the hydrocarbons and is friendly with the environment, but because is also a sure and reliable energy source, and represents a viable alternative before the claims in the growing demand of electricity in Mexico. Before this panorama, was intended several scenarios to elevate the capacity of electric generation of nuclear origin with a variable participation. One of the contemplated scenarios is represented by the expansion project of the nuclear power plant Laguna Verde through the addition of a third reactor that serves as detonator of an integral program that proposes the installation of more nuclear reactors in the country. Before this possible scenario, the Federal Commission of Electricity like responsible organism of supplying energy to the population should have tools that offer it the flexibility to be adapted to the possible changes that will be presented along the project and also gives a value to the risk to future. The methodology denominated Real Options, Binomial model was proposed as an evaluation tool that allows to quantify the value of the expansion proposal, demonstrating the feasibility of the project through a periodic visualization of their evolution, all with the objective of supplying a financial analysis that serves as base and justification before the evident apogee of the nuclear energy that will be presented in future years. (Author)

  17. THE EFFECT OF PHENYLACETATE ON THE EXPANSION AND CYTOTOXIC ACTIVITY OF ADHERENT LAK CELLS FROM PATIENTS WITH HEPATOCELLULAR CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    郑宁; 叶胜龙; 孙瑞霞; 赵燕; 汤钊猷

    2002-01-01

    Objective: To improve the preparation of adherent lymphokine-activated killer (A-LAK) cells and study the synergistic anti-tumor effect of phenylacetate (PA) and A-LAK cells. Methods:\tA-LAK cells were obtained from peripheral blood mononuclear cells (PBMC) of patients with hepatocellular carcinoma (HCC) by using L-phenylalanine methyl ester (PME) to deplete immunosuppressive monocytes. The proliferation of SMMC7721 cell line treated with PA was studied. A-LAK cells were treated with the supernatant of SMMC7721 cells which had been pretreated with PA and the changes of the proliferation and anti-tumor activity of A-LAK cells were investigated. Results: The expansion of A-LAK cells was significantly higher than that of non-adherent LAK (NA-LAK) cells as well as regular LAK cells. The growth of SMMC7721 cells was significantly suppressed by PA. The supernatant of cultured tumor cells intensively suppressed the proliferation and cytotoxicity of A-LAK cells, but the suppressive effect of supernatant treated with PA previously was decreased. Conclusion: A-LAK cells could be simply prepared by using PME, and showed a synergistic anti-tumor effect with the combination of PA.

  18. β-catenin is selectively required for the expansion and regeneration of mature pancreatic acinar cells in mice

    Directory of Open Access Journals (Sweden)

    Matthew D. Keefe

    2012-07-01

    The size of the pancreas is determined by intrinsic factors, such as the number of progenitor cells, and by extrinsic signals that control the fate and proliferation of those progenitors. Both the exocrine and endocrine compartments of the pancreas undergo dramatic expansion after birth and are capable of at least partial regeneration following injury. Whether the expansion of these lineages relies on similar mechanisms is unknown. Although we have shown that the Wnt signaling component β-catenin is selectively required in mouse embryos for the generation of exocrine acinar cells, this protein has been ascribed various functions in the postnatal pancreas, including proliferation and regeneration of islet as well as acinar cells. To address whether β-catenin remains important for the maintenance and expansion of mature acinar cells, we have established a system to follow the behavior and fate of β-catenin-deficient cells during postnatal growth and regeneration in mice. We find that β-catenin is continuously required for the establishment and maintenance of acinar cell mass, extending from embryonic specification through juvenile and adult self-renewal and regeneration. This requirement is not shared with islet cells, which proliferate and function normally in the absence of β-catenin. These results make distinct predictions for the relative role of Wnt–β-catenin signaling in the etiology of human endocrine and exocrine disease. We suggest that loss of Wnt–β-catenin activity is unlikely to drive islet dysfunction, as occurs in type 2 diabetes, but that β-catenin is likely to promote human acinar cell proliferation following injury, and might therefore contribute to the resolution of acute or chronic pancreatitis.

  19. Neonatal colonisation expands a specific intestinal antigen-presenting cell subset prior to CD4 T-cell expansion, without altering T-cell repertoire.

    Directory of Open Access Journals (Sweden)

    Charlotte F Inman

    Full Text Available Interactions between the early-life colonising intestinal microbiota and the developing immune system are critical in determining the nature of immune responses in later life. Studies in neonatal animals in which this interaction can be examined are central to understanding the mechanisms by which the microbiota impacts on immune development and to developing therapies based on manipulation of the microbiome. The inbred piglet model represents a system that is comparable to human neonates and allows for control of the impact of maternal factors. Here we show that colonisation with a defined microbiota produces expansion of mucosal plasma cells and of T-lymphocytes without altering the repertoire of alpha beta T-cells in the intestine. Importantly, this is preceded by microbially-induced expansion of a signal regulatory protein α-positive (SIRPα(+ antigen-presenting cell subset, whilst SIRPα(-CD11R1(+ antigen-presenting cells (APCs are unaffected by colonisation. The central role of intestinal APCs in the induction and maintenance of mucosal immunity implicates SIRPα(+ antigen-presenting cells as orchestrators of early-life mucosal immune development.

  20. Production of recombinant proteins in suspension-cultured plant cells.

    Science.gov (United States)

    Plasson, Carole; Michel, Rémy; Lienard, David; Saint-Jore-Dupas, Claude; Sourrouille, Christophe; de March, Ghislaine Grenier; Gomord, Véronique

    2009-01-01

    Plants have emerged in the past decade as a suitable alternative to the current production systems for recombinant pharmaceutical proteins and, today their potential for low-cost production of high quality, much safer and biologically active mammalian proteins is largely documented. Among various plant expression systems being explored, genetically modified suspension-cultured plant cells offer a promising system for production of biopharmaceuticals. Indeed, when compared to other plant-based production platforms that have been explored, suspension-cultured plant cells have the advantage of being totally devoid of problems associated with the vagaries of weather, pest, soil and gene flow in the environment. Because of short growth cycles, the timescale needed for the production of recombinant proteins in plant cell culture can be counted in days or weeks after transformation compared to months needed for the production in transgenic plants. Moreover, recovery and purification of recombinant proteins from plant biomass is an expensive and technically challenging business that may amount to 80-94% of the final product cost. One additional advantage of plant cell culture is that the recombinant protein fused with a signal sequence can be expressed and secreted into the culture medium, and therefore recovered and purified in the absence of large quantities of contaminating proteins. Consequently, the downstream processing of proteins extracted from plant cell culture medium is less expensive, which may/does balance the higher costs of fermentation. When needed for clinical use, recombinant proteins are easily produced in suspension-cultured plant cells under certified, controllable and sterile conditions that offer improved safety and provide advantages for good manufacturing practices and regulatory compliance. In this chapter, we present basic protocols for rapid generation of transgenic suspension-cultured cells of Nicotiana tabacum, Oriza sativa and Arabidopis

  1. Plant programmed cell death, ethylene and flower senescence

    NARCIS (Netherlands)

    Woltering, E.J.; Jong, de A.; Hoeberichts, F.A.; Iakimova, E.T.; Kapchina, V.

    2005-01-01

    Programmed cell death (PCD) applies to cell death that is part of the normal life of multicellular organisms. PCD is found throughout the animal and plant kingdoms; it is an active process in which a cell suicide pathway is activated resulting in controlled disassembly of the cell. Most cases of PCD

  2. Dynamics and Regulation of Actin Cytoskeleton in Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Ren Haiyun

    2007-01-01

    @@ The actin cytoskeleton constituted of globular actin (G-actin) is a ubiquitous component of eukaryotic cells and plays crucial roles in diverse physiological processes in plant cells, such as cytoplasmic streaming, organelle and nucleus positioning, cell morphogenesis, cell division, tip growth, etc.

  3. Plant programmed cell death, ethylene and flower senescence

    NARCIS (Netherlands)

    Woltering, E.J.; Jong, de A.; Hoeberichts, F.A.; Iakimova, E.T.; Kapchina, V.

    2005-01-01

    Programmed cell death (PCD) applies to cell death that is part of the normal life of multicellular organisms. PCD is found throughout the animal and plant kingdoms; it is an active process in which a cell suicide pathway is activated resulting in controlled disassembly of the cell. Most cases of PCD

  4. Plant cell wall proteomics: the leadership of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Cécile eALBENNE

    2013-05-01

    Full Text Available Plant cell wall proteins (CWPs progressively emerged as crucial components of cell walls although present in minor amounts. Cell wall polysaccharides such as pectins, hemicelluloses and cellulose represent more than 90% of primary cell wall mass, whereas hemicelluloses, cellulose and lignins are the main components of lignified secondary walls. All these polymers provide mechanical properties to cell walls, participate in cell shape and prevent water loss in aerial organs. However, cells walls need to be modified and customized during plant development and in response to environmental cues, thus contributing to plant adaptation. CWPs play essential roles in all these physiological processes and particularly in the dynamics of cell walls, which requires organization and rearrangements of polysaccharides as well as cell-to-cell communication. In the last ten years, plant cell wall proteomics has greatly contributed to a wider knowledge of CWPs. This update will deal with (i a survey of plant cell wall proteomics studies with a focus on Arabidopsis thaliana; (ii the main protein families identified and the still missing peptides; (iii the persistent issue of the non-canonical CWPs; (iv the present challenges to overcome technological bottlenecks; and (v the perspectives beyond cell wall proteomics to understand CWP functions.

  5. Plant cell wall proteomics: the leadership of Arabidopsis thaliana.

    Science.gov (United States)

    Albenne, Cécile; Canut, Hervé; Jamet, Elisabeth

    2013-01-01

    Plant cell wall proteins (CWPs) progressively emerged as crucial components of cell walls although present in minor amounts. Cell wall polysaccharides such as pectins, hemicelluloses, and cellulose represent more than 90% of primary cell wall mass, whereas hemicelluloses, cellulose, and lignins are the main components of lignified secondary walls. All these polymers provide mechanical properties to cell walls, participate in cell shape and prevent water loss in aerial organs. However, cell walls need to be modified and customized during plant development and in response to environmental cues, thus contributing to plant adaptation. CWPs play essential roles in all these physiological processes and particularly in the dynamics of cell walls, which requires organization and rearrangements of polysaccharides as well as cell-to-cell communication. In the last 10 years, plant cell wall proteomics has greatly contributed to a wider knowledge of CWPs. This update will deal with (i) a survey of plant cell wall proteomics studies with a focus on Arabidopsis thaliana; (ii) the main protein families identified and the still missing peptides; (iii) the persistent issue of the non-canonical CWPs; (iv) the present challenges to overcome technological bottlenecks; and (v) the perspectives beyond cell wall proteomics to understand CWP functions.

  6. Thermal Expansion of Three Closed Cell Polymeric Foams at Cryogenic Temperatures

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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; Wrammert, Jens; Harris, Jason B

    2016-12-20

    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. Cholera is a diarrheal disease that results in significant mortality. While oral cholera vaccines are beneficial, they do not achieve equivalent protection compared to infection with Vibrio cholerae Although antibodies likely mediate protection, the mechanisms of immunity following cholera are poorly understood, and a detailed understanding of antibody responses to cholera is of significance for human health. In this study, we characterized the human response to cholera at the single-plasmablast, monoclonal antibody level. Although this approach has not been widely applied to the study of human bacterial infection, we were

  8. Programmed cell death-1 deficiency exacerbates T cell activation and atherogenesis despite expansion of regulatory T cells in atherosclerosis-prone mice.

    Directory of Open Access Journals (Sweden)

    Clément Cochain

    Full Text Available T cell activation represents a double-edged sword in atherogenesis, as it promotes both pro-inflammatory T cell activation and atheroprotective Foxp3(+ regulatory T cell (Treg responses. Here, we investigated the role of the co-inhibitory receptor programmed cell death-1 (PD-1 in T cell activation and CD4(+ T cell polarization towards pro-atherogenic or atheroprotective responses in mice. Mice deficient for both low density lipoprotein receptor and PD-1 (Ldlr(-/-Pd1(-/- displayed striking increases in systemic CD4(+ and CD8(+ T cell activation after 9 weeks of high fat diet feeding, associated with an expansion of both pro-atherogenic IFNγ-secreting T helper 1 cells and atheroprotective Foxp3+ Tregs. Importantly, PD-1 deficiency did not affect Treg suppressive function in vitro. Notably, PD-1 deficiency exacerbated atherosclerotic lesion growth and entailed a massive infiltration of T cells in atherosclerotic lesions. In addition, aggravated hypercholesterolemia was observed in Ldlr(-/-Pd1(-/- mice. In conclusion, we here demonstrate that although disruption of PD-1 signaling enhances both pro- and anti-atherogenic T cell responses in Ldlr(-/- mice, pro-inflammatory T cell activation prevails and enhances dyslipidemia, vascular inflammation and atherosclerosis.

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

    Science.gov (United States)

    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.

  10. Optimization of culture conditions for the expansion of umbilical cord-derived mesenchymal stem or stromal cell-like cells using xeno-free culture conditions.

    Science.gov (United States)

    Hatlapatka, Tim; Moretti, Pierre; Lavrentieva, Antonina; Hass, Ralf; Marquardt, Nicole; Jacobs, Roland; Kasper, Cornelia

    2011-04-01

    First isolated from bone marrow, mesenchymal stem or stromal cells (MSC) were shown to be present in several postnatal and extraembryonic tissues as well as in a large variety of fetal tissues (e.g., fatty tissue, dental pulp, placenta, umbilical cord blood, and tissue). In this study, an optimized protocol for the expansion of MSC-like cells from whole umbilical cord tissue under xeno-free culture conditions is proposed. Different fetal calf sera and human serum (HS) were compared with regard to cell proliferation and MSC marker stability in long-term expansion experiments, and HS was shown to support optimal growth conditions. Additionally, the optimal concentration of HS during the cultivation was determined. With regard to cell proliferative potential, apoptosis, colony-forming unit fibroblast frequency, and cell senescence, our findings suggest that an efficient expansion of the cells is carried out best in media supplemented with 10% HS. Under our given xeno-free culture conditions, MSC-like cells were found to display in vitro immunoprivileged and immunomodulatory properties, which were assessed by co-culture and transwell culture experiments with carboxyfluorescein diacetate succinimidyl ester-labeled peripheral blood mononuclear cells. These findings may be of great value for the establishment of biotechnological protocols for the delivery of sufficient cell numbers of high quality for regenerative medicine purposes.

  11. Fluorescent Probes for Exploring Plant Cell Wall Deconstruction: A Review

    Directory of Open Access Journals (Sweden)

    Gabriel Paës

    2014-07-01

    Full Text Available Plant biomass is a potential resource of chemicals, new materials and biofuels that could reduce our dependency on fossil carbon, thus decreasing the greenhouse effect. However, due to its chemical and structural complexity, plant biomass is recalcitrant to green biological transformation by enzymes, preventing the establishment of integrated bio-refineries. In order to gain more knowledge in the architecture of plant cell wall to facilitate their deconstruction, many fluorescent probes bearing various fluorophores have been devised and used successfully to reveal the changes in structural motifs during plant biomass deconstruction, and the molecular interactions between enzymes and plant cell wall polymers. Fluorescent probes are thus relevant tools to explore plant cell wall deconstruction.

  12. Super-resolution Microscopy in Plant Cell Imaging.

    Science.gov (United States)

    Komis, George; Šamajová, Olga; Ovečka, Miroslav; Šamaj, Jozef

    2015-12-01

    Although the development of super-resolution microscopy methods dates back to 1994, relevant applications in plant cell imaging only started to emerge in 2010. Since then, the principal super-resolution methods, including structured-illumination microscopy (SIM), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion microscopy (STED), have been implemented in plant cell research. However, progress has been limited due to the challenging properties of plant material. Here we summarize the basic principles of existing super-resolution methods and provide examples of applications in plant science. The limitations imposed by the nature of plant material are reviewed and the potential for future applications in plant cell imaging is highlighted.

  13. The immune receptor Trem1 cooperates with diminished DNA damage response to induce preleukemic stem cell expansion.

    Science.gov (United States)

    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.

  14. Trichostatin A stabilizes the expression of pluripotent genes in human mesenchymal stem cells during ex vivo expansion.

    Directory of Open Access Journals (Sweden)

    Bing Han

    Full Text Available BACKGROUND: Mesenchymal stem cells (MSCs have been considered as ideal cells for the treatment of a variety of diseases. However, aging and spontaneous differentiation of MSCs during culture expansion dampen their effectiveness. Previous studies suggest that ex vivo aging of MSCs is largely caused by epigenetic changes particularly a decline of histone H3 acetylation levels in promoter regions of pluripotent genes due to inappropriate growth environment. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we examined whether histone deacetylase inhibitor trichostatin A (TSA could suppress the histone H3 deacetylation thus maintaining the primitive property of MSCs. We found that in regular adherent culture, human MSCs became flatter and larger upon successive passaging, while the expression of pluripotent genes such as Oct4, Sox2, Nanog, Rex-1, CD133 and TERT decreased markedly. Administration of low concentrations of TSA in culture significantly suppressed the morphological changes in MSCs otherwise occurred during culture expansion, increased their proliferation while retaining their cell contact growth inhibition property and multipotent differentiation ability. Moreover, TSA stabilized the expression of the above pluripotent genes and histone H3 acetylation levels in K9 and K14 in promoter regions of Oct4, Sox2 and TERT. CONCLUSIONS/SIGNIFICANCE: Our results suggest that TSA may serve as an effective culture additive to maintain the primitive feature of MSCs during culture expansion.

  15. Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

    Science.gov (United States)

    Chou, Ming-Li; Burnouf, Thierry; Wang, Tsung-Jen

    2014-01-01

    Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml) and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml) neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices and regulatory

  16. Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

    Directory of Open Access Journals (Sweden)

    Ming-Li Chou

    Full Text Available Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices

  17. Multidimensional solid-state NMR spectroscopy of plant cell walls.

    Science.gov (United States)

    Wang, Tuo; Phyo, Pyae; Hong, Mei

    2016-09-01

    Plant biomass has become an important source of bio-renewable energy in modern society. The molecular structure of plant cell walls is difficult to characterize by most atomic-resolution techniques due to the insoluble and disordered nature of the cell wall. Solid-state NMR (SSNMR) spectroscopy is uniquely suited for studying native hydrated plant cell walls at the molecular level with chemical resolution. Significant progress has been made in the last five years to elucidate the molecular structures and interactions of cellulose and matrix polysaccharides in plant cell walls. These studies have focused on primary cell walls of growing plants in both the dicotyledonous and grass families, as represented by the model plants Arabidopsis thaliana, Brachypodium distachyon, and Zea mays. To date, these SSNMR results have shown that 1) cellulose, hemicellulose, and pectins form a single network in the primary cell wall; 2) in dicot cell walls, the protein expansin targets the hemicellulose-enriched region of the cellulose microfibril for its wall-loosening function; and 3) primary wall cellulose has polymorphic structures that are distinct from the microbial cellulose structures. This article summarizes these key findings, and points out future directions of investigation to advance our fundamental understanding of plant cell wall structure and function.

  18. Characterization of a Novel Monoclonal Antibody to Human Stem Cell Factor and its Determination Effect on Ex Vivo Stem Cell Expansion

    Directory of Open Access Journals (Sweden)

    Fan Jie

    2013-06-01

    Full Text Available Background: Hematopoietic stem cell (HSC transplantation can be used to treat blood and immune system disorders. Fresh umbilical cord blood (UCB, a major source of HSC for potential clinical applications, contains a limited number of HSCs. Stem cell factor (SCF activates HSC self-renewal and is being used to stimulate ex vivo expansion of HSCs for treating various hematologic diseases in clinic. Yet, the mechanism by which SCF stimulates and supports HSCs expansion remains poorly understood. Thus, the purpose of the study is to obtain novel monoclonal antibodies for structural and functional SCF characterizations, as well as for the optimization of HSCs ex vivo expansion. Methods: Recombinant human stem cell factor (rhSCF was used for producing monoclonal antibody (mAb. High-titer mAb specific to rhSCF was selected by following immunochemical screening to various mAb cell lines. HSCs with CD34+ epitope were isolated from UCB using affinity chromatography. SCF activity was tested in an ex vivo HSC expansion assay, with use of flow cytometry for detection of CD34+ cell and total mononuclear cells. Part of rhSCF that contained the antibody-binding site was identified via immunoblot analysis of rhSCF tryptic peptides, rhSCF-specific mAb, and subsequent NH2-terminal amino acid sequence analysis of the detected peptides. Results: The mAb cell line 23C8 with a high titer was found to be specific for rhSCF. In ex vivo cord blood expansion assays, the ability of rhSCF to stimulate the expansion of CD34+ cells was significantly inhibited by 23C8 in a dose-dependet fashion(?. Through peptide mapping, the binding site of 23C8 on rhSCF was mapped to the first 104 amino acids.. Conclusion: The mAb cell line 23C8 produces specific and inhibitory anti-rhSCF mAb. The mAb appears to bind directly to a part of rhSCF that is critical for biological activity. This functionallyactive site of rhSCF is located in the first 104 amino acids from the NH2-terminus. The

  19. Characterization of a Novel Monoclonal Antibody to Human Stem Cell Factor and its Determination Effect on Ex Vivo Stem Cell Expansion

    Institute of Scientific and Technical Information of China (English)

    Jie Fan; Ding Xinxin; Jiang Yongping

    2013-01-01

    Background:Hematopoietic stem cell (HSC) transplantation can be used to treat blood and immune system disorders. Fresh umbilical cord blood (UCB), a major source of HSC for potential clinical applications, contains a limited number of HSCs. Stem cell factor (SCF) activates HSC self-renewal and is being used to stimulate ex vivo expansion of HSCs for treating various hematologic diseases in clinic. Yet, the mechanism by which SCF stimulates and supports HSCs expansion remains poorly understood. Thus, the purpose of the study is to obtain novel monoclonal antibodies for structural and functional SCF characterizations, as well as for the optimization of HSCs ex vivo expansion. Methods:Recombinant human stem cell factor (rhSCF) was used for producing monoclonal antibody (mAb). High-titer mAb speciifc to rhSCF was selected by following immunochemical screening to various mAb cell lines. HSCs with CD34+ epitope were isolated from UCB using affinity chromatography. SCF activity was tested in an ex vivo HSC expansion assay, with use of flow cytometry for detection of CD34+ cell and total mononuclear cells. Part of rhSCF that contained the antibody-binding site was identified via immunoblot analysis of rhSCF tryptic peptides, rhSCF-speciifc mAb, and subsequent NH2-terminal amino acid sequence analysis of the detected peptides. Results: The mAb cell line 23C8 with a high titer was found to be speciifc for rhSCF. In ex vivo cord blood expansion assays, the ability of rhSCF to stimulate the expansion of CD34+ cells was significantly inhibited by 23C8 in a dose-dependet fashion(?). Through peptide mapping, the binding site of 23C8 on rhSCF was mapped to the ifrst 104 amino acids.. Conclusion: The mAb cell line 23C8 produces speciifc and inhibitory anti-rhSCF mAb. The mAb appears to bind directly to a part of rhSCF that is critical for biological activity. This functionally active site of rhSCF is located in the ifrst 104 amino acids from the NH2-terminus. The novel anti

  20. Expansion of NK cells and reduction of NKG2D expression in chronic lymphocytic leukemia. Correlation with progressive disease.

    Directory of Open Access Journals (Sweden)

    Leticia Huergo-Zapico

    Full Text Available The immune system may mediate anti-tumor responses in chronic lymphocytic leukemia (CLL which may affect disease progression and survival. In this study, we analyzed the immune characteristics of 99 consecutive previously diagnosed CLL patients and 50 healthy controls. The distribution of lymphocyte subsets at diagnosis was retrospectively analyzed. Compared with controls, leukemia patients showed an expansion of NK and CD8 T cells at diagnosis. The relative number of CD8 T cells at diagnosis was associated with time to treatment, suggesting that CD8 T cells may modify disease progression. The distribution of lymphocyte subsets was analyzed again when patients were enrolled in this study. The median time since these patients were diagnosed was 277 weeks. Compared with diagnosis, the absolute number of CD8 T cells significantly decreased in these patients, reaching similar values to healthy controls; however NK cells kept significantly elevated overtime. Nevertheless, NK cells showed an impaired expression of NKG2D receptor and a defective cytotoxic activity. This down-regulation of NKG2D expression was further enhanced in patients with advanced and progressive disease. Additionally, membrane NKG2D levels significantly decreased on CD8 T cells, but a significant increase of NKG2D+CD4+ T cells was observed in CLL patients. The cytotoxic activity of NK cells was diminished in CLL patients; however the treatments with IL-2, IL-15, IL-21 and lenalidomide were able to restore their activity. The effect of IL-2 and IL-15 was associated with the increase of NKG2D expression on immune cells, but the effect of IL-21 and lenalidomide was not due to NKG2D up-regulation. The expansion of NK cells and the reversibility of NK cell defects provide new opportunities for the immunotherapeutic intervent