WorldWideScience

Sample records for mechanotransduction cells durotaxis

  1. Probing the mystery of Chinese medicine meridian channels with special emphasis on the connective tissue interstitial fluid system, mechanotransduction, cells durotaxis and mast cell degranulation

    Directory of Open Access Journals (Sweden)

    Fung Peter

    2009-05-01

    Full Text Available Abstract This article hypothesizes that the Chinese medicine meridian system is a special channel network comprising of skin with abundant nerves and nociceptive receptors of various types, and deeper connective tissues inside the body with the flowing interstitial fluid system. These meridian channels provide efficient migratory tracks mainly due to durotaxis (also including chemotaxis for mast cells, fibroblasts and other cells to migrate and carry out a number of physiological functions. Acupuncture acting on meridian channel causes cytoskeletal remodeling through mechanotransduction, leading to regulation of gene expression and the subsequent production of related proteins. Also, stimulation on cell surface can trigger Ca2+ activities, resulting in a cascade of intra- and inter-cellular signaling. Moreover, nerve endings in the meridian channels interact with mast cells and induce the degranulation of these cells, leading to the release of many specific biomolecules needed for homeostasis, immune surveillance, wound healing and tissue repair. Acupoint along a meridian channel is a functional site to trigger the above functions with specificity and high efficiency.

  2. Mechanotransduction in Endothelial Cells Studied with Fluorescence Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Chien Shu [Departments of Bioengineering and Medicine and Institute of Engineering in Medicine, University of California, San Diego, La Jolla, California 92093-0427 (United States)

    2011-01-01

    Mechanotransduction involves the conversion of mechanical stimuli to intracellular signaling to modulate gene and protein expressions and hence cellular functions in endothelial cells, thus playing importance roles in the regulation of homeostasis in health and disease. The aim of this paper is to investigate the dynamics of mechanotransduction in endothelial cells by the use of fluorescent resonance energy transfer (FRET) to study the temporal and spatial activation of Src kinase and focal adhesion kinase, both of which play critical roles in many cellular processes. The results have contributed to the elucidation of the roles of these two important signaling molecules and their interactions in mediating mechanotransduction.

  3. Signal transduction pathways involved in mechanotransduction in bone cells

    International Nuclear Information System (INIS)

    Liedert, Astrid; Kaspar, Daniela; Blakytny, Robert; Claes, Lutz; Ignatius, Anita

    2006-01-01

    Several in vivo and in vitro studies with different loading regimens showed that mechanical stimuli have an influence on proliferation and differentiation of bone cells. Prerequisite for this influence is the transduction of mechanical signals into the cell, a phenomenon that is termed mechanotransduction, which is essential for the maintenance of skeletal homeostasis in adults. Mechanoreceptors, such as the integrins, cadherins, and stretch-activated Ca 2+ channels, together with various signal transduction pathways, are involved in the mechanotransduction process that ultimately regulates gene expression in the nucleus. Mechanotransduction itself is considered to be regulated by hormones, the extracellular matrix of the osteoblastic cells and the mode of the mechanical stimulus

  4. Roles of the cytoskeleton, cell adhesion and rho signalling in mechanosensing and mechanotransduction.

    Science.gov (United States)

    Ohashi, Kazumasa; Fujiwara, Sachiko; Mizuno, Kensaku

    2017-03-01

    All cells sense and respond to various mechanical forces in and mechanical properties of their environment. To respond appropriately, cells must be able to sense the location, direction, strength and duration of these forces. Recent progress in mechanobiology has provided a better understanding of the mechanisms of mechanoresponses underlying many cellular and developmental processes. Various roles of mechanoresponses in development and tissue homeostasis have been elucidated, and many molecules involved in mechanotransduction have been identified. However, the whole picture of the functions and molecular mechanisms of mechanotransduction remains to be understood. Recently, novel mechanisms for sensing and transducing mechanical stresses via the cytoskeleton, cell-substrate and cell-cell adhesions and related proteins have been identified. In this review, we outline the roles of the cytoskeleton, cell-substrate and cell-cell adhesions, and related proteins in mechanosensing and mechanotransduction. We also describe the roles and regulation of Rho-family GTPases in mechanoresponses. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  5. Biomaterials and bone mechanotransduction

    Science.gov (United States)

    Sikavitsas, V. I.; Temenoff, J. S.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Bone is an extremely complex tissue that provides many essential functions in the body. Bone tissue engineering holds great promise in providing strategies that will result in complete regeneration of bone and restoration of its function. Currently, such strategies include the transplantation of highly porous scaffolds seeded with cells. Prior to transplantation the seeded cells are cultured in vitro in order for the cells to proliferate, differentiate and generate extracellular matrix. Factors that can affect cellular function include the cell-biomaterial interaction, as well as the biochemical and the mechanical environment. To optimize culture conditions, good understanding of these parameters is necessary. The new developments in bone biology, bone cell mechanotransduction, and cell-surface interactions are reviewed here to demonstrate that bone mechanotransduction is strongly influenced by the biomaterial properties.

  6. The role of apical cell-cell junctions and associated cytoskeleton in mechanotransduction.

    Science.gov (United States)

    Sluysmans, Sophie; Vasileva, Ekaterina; Spadaro, Domenica; Shah, Jimit; Rouaud, Florian; Citi, Sandra

    2017-04-01

    Tissues of multicellular organisms are characterised by several types of specialised cell-cell junctions. In vertebrate epithelia and endothelia, tight and adherens junctions (AJ) play critical roles in barrier and adhesion functions, and are connected to the actin and microtubule cytoskeletons. The interaction between junctions and the cytoskeleton is crucial for tissue development and physiology, and is involved in the molecular mechanisms governing cell shape, motility, growth and signalling. The machineries which functionally connect tight and AJ to the cytoskeleton comprise proteins which either bind directly to cytoskeletal filaments, or function as adaptors for regulators of the assembly and function of the cytoskeleton. In the last two decades, specific cytoskeleton-associated junctional molecules have been implicated in mechanotransduction, revealing the existence of multimolecular complexes that can sense mechanical cues and translate them into adaptation to tensile forces and biochemical signals. Here, we summarise the current knowledge about the machineries that link tight and AJ to actin filaments and microtubules, and the molecular basis for mechanotransduction at epithelial and endothelial AJ. © 2017 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

  7. Heparan sulfate proteoglycans mediate interstitial flow mechanotransduction regulating MMP-13 expression and cell motility via FAK-ERK in 3D collagen.

    Directory of Open Access Journals (Sweden)

    Zhong-Dong Shi

    2011-01-01

    Full Text Available Interstitial flow directly affects cells that reside in tissues and regulates tissue physiology and pathology by modulating important cellular processes including proliferation, differentiation, and migration. However, the structures that cells utilize to sense interstitial flow in a 3-dimensional (3D environment have not yet been elucidated. Previously, we have shown that interstitial flow upregulates matrix metalloproteinase (MMP expression in rat vascular smooth muscle cells (SMCs and fibroblasts/myofibroblasts via activation of an ERK1/2-c-Jun pathway, which in turn promotes cell migration in collagen. Herein, we focused on uncovering the flow-induced mechanotransduction mechanism in 3D.Cleavage of rat vascular SMC surface glycocalyx heparan sulfate (HS chains from proteoglycan (PG core proteins by heparinase or disruption of HS biosynthesis by silencing N-deacetylase/N-sulfotransferase 1 (NDST1 suppressed interstitial flow-induced ERK1/2 activation, interstitial collagenase (MMP-13 expression, and SMC motility in 3D collagen. Inhibition or knockdown of focal adhesion kinase (FAK also attenuated or blocked flow-induced ERK1/2 activation, MMP-13 expression, and cell motility. Interstitial flow induced FAK phosphorylation at Tyr925, and this activation was blocked when heparan sulfate proteoglycans (HSPGs were disrupted. These data suggest that HSPGs mediate interstitial flow-induced mechanotransduction through FAK-ERK. In addition, we show that integrins are crucial for mechanotransduction through HSPGs as they mediate cell spreading and maintain cytoskeletal rigidity.We propose a conceptual mechanotransduction model wherein cell surface glycocalyx HSPGs, in the presence of integrin-mediated cell-matrix adhesions and cytoskeleton organization, sense interstitial flow and activate the FAK-ERK signaling axis, leading to upregulation of MMP expression and cell motility in 3D. This is the first study to describe a flow-induced mechanotransduction

  8. The cellular mastermind(?) – Mechanotransduction and the nucleus

    Science.gov (United States)

    Kaminski, Ashley; Fedorchak, Gregory R.; Lammerding, Jan

    2015-01-01

    Cells respond to mechanical stimulation by activation of specific signaling pathways and genes that allow the cell to adapt to its dynamic physical environment. How cells sense the various mechanical inputs and translate them into biochemical signals remains an area of active investigation. Recent reports suggest that the cell nucleus may be directly implicated in this cellular mechanotransduction process. In this chapter, we discuss how forces applied to the cell surface and cytoplasm induce changes in nuclear structure and organization, which could directly affect gene expression, while also highlighting the complex interplay between nuclear structural proteins and transcriptional regulators that may further modulate mechanotransduction signaling. Taken together, these findings paint a picture of the nucleus as a central hub in cellular mechanotransduction—both structurally and biochemically—with important implications in physiology and disease. PMID:25081618

  9. Proteomic Dissection of Nanotopography-Sensitive Mechanotransductive Signaling Hubs that Foster Neuronal Differentiation in PC12 Cells

    Directory of Open Access Journals (Sweden)

    Elisa Maffioli

    2018-01-01

    Full Text Available Neuronal cells are competent in precisely sensing nanotopographical features of their microenvironment. The perceived microenvironmental information will be “interpreted” by mechanotransductive processes and impacts on neuronal functioning and differentiation. Attempts to influence neuronal differentiation by engineering substrates that mimic appropriate extracellular matrix (ECM topographies are hampered by the fact that profound details of mechanosensing/-transduction complexity remain elusive. Introducing omics methods into these biomaterial approaches has the potential to provide a deeper insight into the molecular processes and signaling cascades underlying mechanosensing/-transduction but their exigence in cellular material is often opposed by technical limitations of major substrate top-down fabrication methods. Supersonic cluster beam deposition (SCBD allows instead the bottom-up fabrication of nanostructured substrates over large areas characterized by a quantitatively controllable ECM-like nanoroughness that has been recently shown to foster neuron differentiation and maturation. Exploiting this capacity of SCBD, we challenged mechanosensing/-transduction and differentiative behavior of neuron-like PC12 cells with diverse nanotopographies and/or changes of their biomechanical status, and analyzed their phosphoproteomic profiles in these settings. Versatile proteins that can be associated to significant processes along the mechanotransductive signal sequence, i.e., cell/cell interaction, glycocalyx and ECM, membrane/f-actin linkage and integrin activation, cell/substrate interaction, integrin adhesion complex, actomyosin organization/cellular mechanics, nuclear organization, and transcriptional regulation, were affected. The phosphoproteomic data suggested furthermore an involvement of ILK, mTOR, Wnt, and calcium signaling in these nanotopography- and/or cell mechanics-related processes. Altogether, potential nanotopography

  10. Mechanotransduction across the cell surface and through the cytoskeleton

    Science.gov (United States)

    Wang, N.; Butler, J. P.; Ingber, D. E.

    1993-01-01

    Mechanical stresses were applied directly to cell surface receptors with a magnetic twisting device. The extracellular matrix receptor, integrin beta 1, induced focal adhesion formation and supported a force-dependent stiffening response, whereas nonadhesion receptors did not. The cytoskeletal stiffness (ratio of stress to strain) increased in direct proportion to the applied stress and required intact microtubules and intermediate filaments as well as microfilaments. Tensegrity models that incorporate mechanically interdependent struts and strings that reorient globally in response to a localized stress mimicked this response. These results suggest that integrins act as mechanoreceptors and transmit mechanical signals to the cytoskeleton. Mechanotransduction, in turn, may be mediated simultaneously at multiple locations inside the cell through force-induced rearrangements within a tensionally integrated cytoskeleton.

  11. HCN channels are not required for mechanotransduction in sensory hair cells of the mouse inner ear.

    Directory of Open Access Journals (Sweden)

    Geoffrey C Horwitz

    Full Text Available The molecular composition of the hair cell transduction channel has not been identified. Here we explore the novel hypothesis that hair cell transduction channels include HCN subunits. The HCN family of ion channels includes four members, HCN1-4. They were originally identified as the molecular correlates of the hyperpolarization-activated, cyclic nucleotide gated ion channels that carry currents known as If, IQ or Ih. However, based on recent evidence it has been suggested that HCN subunits may also be components of the elusive hair cell transduction channel. To investigate this hypothesis we examined expression of mRNA that encodes HCN1-4 in sensory epithelia of the mouse inner ear, immunolocalization of HCN subunits 1, 2 and 4, uptake of the transduction channel permeable dye, FM1-43 and electrophysiological measurement of mechanotransduction current. Dye uptake and transduction current were assayed in cochlear and vestibular hair cells of wildtype mice exposed to HCN channel blockers or a dominant-negative form of HCN2 that contained a pore mutation and in mutant mice that lacked HCN1, HCN2 or both. We found robust expression of HCNs 1, 2 and 4 but little evidence that localized HCN subunits in hair bundles, the site of mechanotransduction. Although high concentrations of the HCN antagonist, ZD7288, blocked 50-70% of the transduction current, we found no reduction of transduction current in either cochlear or vestibular hair cells of HCN1- or HCN2- deficient mice relative to wild-type mice. Furthermore, mice that lacked both HCN1 and HCN2 also had normal transduction currents. Lastly, we found that mice exposed to the dominant-negative mutant form of HCN2 had normal transduction currents as well. Taken together, the evidence suggests that HCN subunits are not required for mechanotransduction in hair cells of the mouse inner ear.

  12. Mechanotransduction mechanisms in growing spherically structured tissues

    Science.gov (United States)

    Littlejohns, Euan; Dunlop, Carina M.

    2018-04-01

    There is increasing experimental interest in mechanotransduction in multi-cellular tissues as opposed to single cells. This is driven by a growing awareness of the importance of physiologically relevant three-dimensional culture and of cell–cell and cell–gel interactions in directing growth and development. The paradigm biophysical technique for investigating tissue level mechanobiology in this context is to grow model tissues in artificial gels with well-defined mechanical properties. These studies often indicate that the stiffness of the encapsulating gel can significantly alter cellular behaviours. We demonstrate here potential mechanisms linking tissue growth with stiffness-mediated mechanotransduction. We show how tissue growth in gel systems generates points at which there is a significant qualitative change in the cellular stress and strain experienced. We show analytically how these potential switching points depend on the mechanical properties of the constraining gel and predict when they will occur. Significantly, we identify distinct mechanisms that act separately in each of the stress and strain fields at different times. These observations suggest growth as a potential physical mechanism coupling gel stiffness with cellular mechanotransduction in three-dimensional tissues. We additionally show that non-proliferating areas, in the case that the constraining gel is soft compared with the tissue, will expand and contract passively as a result of growth. Central compartment size is thus seen to not be a reliable indicator on its own for growth initiation or active behaviour.

  13. A chemo-mechanical free-energy-based approach to model durotaxis and extracellular stiffness-dependent contraction and polarization of cells.

    Science.gov (United States)

    Shenoy, Vivek B; Wang, Hailong; Wang, Xiao

    2016-02-06

    We propose a chemo-mechanical model based on stress-dependent recruitment of myosin motors to describe how the contractility, polarization and strain in cells vary with the stiffness of their surroundings and their shape. A contractility tensor, which depends on the distribution of myosin motors, is introduced to describe the chemical free energy of the cell due to myosin recruitment. We explicitly include the contributions to the free energy that arise from mechanosensitive signalling pathways (such as the SFX, Rho-Rock and MLCK pathways) through chemo-mechanical coupling parameters. Taking the variations of the total free energy, which consists of the chemical and mechanical components, in accordance with the second law of thermodynamics provides equations for the temporal evolution of the active stress and the contractility tensor. Following this approach, we are able to recover the well-known Hill relation for active stresses, based on the fundamental principles of irreversible thermodynamics rather than phenomenology. We have numerically implemented our free energy-based approach to model spatial distribution of strain and contractility in (i) cells supported by flexible microposts, (ii) cells on two-dimensional substrates, and (iii) cells in three-dimensional matrices. We demonstrate how the polarization of the cells and the orientation of stress fibres can be deduced from the eigenvalues and eigenvectors of the contractility tensor. Our calculations suggest that the chemical free energy of the cell decreases with the stiffness of the extracellular environment as the cytoskeleton polarizes in response to stress-dependent recruitment of molecular motors. The mechanical energy, which includes the strain energy and motor potential energy, however, increases with stiffness, but the overall energy is lower for cells in stiffer environments. This provides a thermodynamic basis for durotaxis, whereby cells preferentially migrate towards stiffer regions of the

  14. Lung heparan sulfates modulate Kfc during increased vascular pressure: evidence for glycocalyx-mediated mechanotransduction

    Science.gov (United States)

    Cluff, Mark; Kingston, Joseph; Hill, Denzil; Chen, Haiyan; Hoehne, Soeren; Malleske, Daniel T.; Kaur, Rajwinederjit

    2012-01-01

    Lung endothelial cells respond to changes in vascular pressure through mechanotransduction pathways that alter barrier function via non-Starling mechanism(s). Components of the endothelial glycocalyx have been shown to participate in mechanotransduction in vitro and in systemic vessels, but the glycocalyx's role in mechanosensing and pulmonary barrier function has not been characterized. Mechanotransduction pathways may represent novel targets for therapeutic intervention during states of elevated pulmonary pressure such as acute heart failure, fluid overload, and mechanical ventilation. Our objective was to assess the effects of increasing vascular pressure on whole lung filtration coefficient (Kfc) and characterize the role of endothelial heparan sulfates in mediating mechanotransduction and associated increases in Kfc. Isolated perfused rat lung preparation was used to measure Kfc in response to changes in vascular pressure in combination with superimposed changes in airway pressure. The roles of heparan sulfates, nitric oxide, and reactive oxygen species were investigated. Increases in capillary pressure altered Kfc in a nonlinear relationship, suggesting non-Starling mechanism(s). nitro-l-arginine methyl ester and heparanase III attenuated the effects of increased capillary pressure on Kfc, demonstrating active mechanotransduction leading to barrier dysfunction. The nitric oxide (NO) donor S-nitrosoglutathione exacerbated pressure-mediated increase in Kfc. Ventilation strategies altered lung NO concentration and the Kfc response to increases in vascular pressure. This is the first study to demonstrate a role for the glycocalyx in whole lung mechanotransduction and has important implications in understanding the regulation of vascular permeability in the context of vascular pressure, fluid status, and ventilation strategies. PMID:22160307

  15. Piezo1 regulates mechanotransductive release of ATP from human RBCs.

    Science.gov (United States)

    Cinar, Eyup; Zhou, Sitong; DeCourcey, James; Wang, Yixuan; Waugh, Richard E; Wan, Jiandi

    2015-09-22

    Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca(2+)) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca(2+) influx decrease significantly. Remarkably, a critical extracellular Ca(2+) concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease.

  16. Mechanotransduction through Cytoskeleton

    Science.gov (United States)

    Ingber, Donald

    2002-01-01

    The goal of this project was to characterize the molecular mechanism by which cells recognize and respond to physical forces in their local environment. The project was based on the working hypothesis that cells sense mechanical stresses, such as those due to gravity, through their cell surface adhesion receptors (e.g., integrins) and that they respond as a result of structural arrangements with their internal cytoskeleton (CSK) which are orchestrated through use of tensegrity architecture. In this project, we carried out studies to define the architectural and molecular basis of cellular mechanotransduction. Our major goal was to define the molecular pathway that mediates mechanical force transfer between integrins and the CSK and to determine how mechanical deformation of integrin-CSK linkages is transduced into a biochemical response. Elucidation of the mechanism by which cells sense mechanical stresses through integrins and translate them into a biochemical response should help us to understand the molecular basis of the cellular response to gravity as well as many other forms of mechanosensation and tissue regulation. The specific aims of this proposal were: 1. To define the molecular basis of mechanical coupling between integrins, vinculin, and the actin CSK; 2. To develop a computer simulation of how mechanical stresses alter CSK structure and test this model in living cells; 3. To determine how mechanical deformation of integrin-CSK linkages is transduced into a biochemical response.

  17. Mechanotransduction and Growth Factor Signalling to Engineer Cellular Microenvironments.

    Science.gov (United States)

    Cipitria, Amaia; Salmeron-Sanchez, Manuel

    2017-08-01

    Engineering cellular microenvironments involves biochemical factors, the extracellular matrix (ECM) and the interaction with neighbouring cells. This progress report provides a critical overview of key studies that incorporate growth factor (GF) signalling and mechanotransduction into the design of advanced microenvironments. Materials systems have been developed for surface-bound presentation of GFs, either covalently tethered or sequestered through physico-chemical affinity to the matrix, as an alternative to soluble GFs. Furthermore, some materials contain both GF and integrin binding regions and thereby enable synergistic signalling between the two. Mechanotransduction refers to the ability of the cells to sense physical properties of the ECM and to transduce them into biochemical signals. Various aspects of the physics of the ECM, i.e. stiffness, geometry and ligand spacing, as well as time-dependent properties, such as matrix stiffening, degradability, viscoelasticity, surface mobility as well as spatial patterns and gradients of physical cues are discussed. To conclude, various examples illustrate the potential for cooperative signalling of growth factors and the physical properties of the microenvironment for potential applications in regenerative medicine, cancer research and drug testing. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Ion Permeation and Mechanotransduction Mechanisms of Mechanosensitive Piezo Channels.

    Science.gov (United States)

    Zhao, Qiancheng; Wu, Kun; Geng, Jie; Chi, Shaopeng; Wang, Yanfeng; Zhi, Peng; Zhang, Mingmin; Xiao, Bailong

    2016-03-16

    Piezo proteins have been proposed as the long-sought-after mechanosensitive cation channels in mammals that play critical roles in various mechanotransduction processes. However, the molecular bases that underlie their ion permeation and mechanotransduction have remained functionally undefined. Here we report our finding of the miniature pore-forming module of Piezo1 that resembles the pore architecture of other trimeric channels and encodes the essential pore properties. We further identified specific residues within the pore module that determine unitary conductance, pore blockage and ion selectivity for divalent and monovalent cations and anions. The non-pore-containing region of Piezo1 confers mechanosensitivity to mechano-insensitive trimeric acid-sensing ion channels, demonstrating that Piezo1 channels possess intrinsic mechanotransduction modules separate from their pore modules. In conclusion, this is the first report on the bona fide pore module and mechanotransduction components of Piezo channels, which define their ion-conducting properties and gating by mechanical stimuli, respectively. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. IGF-1 signaling mediated cell-specific skeletal mechano-transduction.

    Science.gov (United States)

    Tian, Faming; Wang, Yongmei; Bikle, Daniel D

    2018-02-01

    Mechanical loading preserves bone mass and stimulates bone formation, whereas skeletal unloading leads to bone loss. In addition to osteocytes, which are considered the primary sensor of mechanical load, osteoblasts, and bone specific mesenchymal stem cells also are involved. The skeletal response to mechanical signals is a complex process regulated by multiple signaling pathways including that of insulin-like growth factor-1 (IGF-1). Conditional osteocyte deletion of IGF-1 ablates the osteogenic response to mechanical loading. Similarly, osteocyte IGF-1 receptor (IGF-1R) expression is necessary for reloading-induced periosteal bone formation. Transgenic overexpression of IGF-1 in osteoblasts results in enhanced responsiveness to in vivo mechanical loading in mice, a response which is eliminated by osteoblastic conditional disruption of IGF-1 in vivo. Bone marrow derived stem cells (BMSC) from unloaded bone fail to respond to IGF-1 in vitro. IGF-1R is required for the transduction of a mechanical stimulus to downstream effectors, transduction which is lost when the IGF-1R is deleted. Although the molecular mechanisms are not yet fully elucidated, the IGF signaling pathway and its interactions with potentially interlinked signaling cascades involving integrins, the estrogen receptor, and wnt/β-catenin play an important role in regulating adaptive response of cancer bone cells to mechanical stimuli. In this review, we discuss recent advances investigating how IGF-1 and other interlinked molecules and signaling pathways regulate skeletal mechano-transduction involving different bone cells, providing an overview of the IGF-1 signaling mediated cell-specific response to mechanical stimuli. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:576-583, 2018. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  20. Adding dimension to cellular mechanotransduction: Advances in biomedical engineering of multiaxial cell-stretch systems and their application to cardiovascular biomechanics and mechano-signaling.

    Science.gov (United States)

    Friedrich, O; Schneidereit, D; Nikolaev, Y A; Nikolova-Krstevski, V; Schürmann, S; Wirth-Hücking, A; Merten, A L; Fatkin, D; Martinac, B

    2017-11-01

    Hollow organs (e.g. heart) experience pressure-induced mechanical wall stress sensed by molecular mechano-biosensors, including mechanosensitive ion channels, to translate into intracellular signaling. For direct mechanistic studies, stretch devices to apply defined extensions to cells adhered to elastomeric membranes have stimulated mechanotransduction research. However, most engineered systems only exploit unilateral cellular stretch. In addition, it is often taken for granted that stretch applied by hardware translates 1:1 to the cell membrane. However, the latter crucially depends on the tightness of the cell-substrate junction by focal adhesion complexes and is often not calibrated for. In the heart, (increased) hemodynamic volume/pressure load is associated with (increased) multiaxial wall tension, stretching individual cardiomyocytes in multiple directions. To adequately study cellular models of chronic organ distension on a cellular level, biomedical engineering faces challenges to implement multiaxial cell stretch systems that allow observing cell reactions to stretch during live-cell imaging, and to calibrate for hardware-to-cell membrane stretch translation. Here, we review mechanotransduction, cell stretch technologies from uni-to multiaxial designs in cardio-vascular research, and the importance of the stretch substrate-cell membrane junction. We also present new results using our IsoStretcher to demonstrate mechanosensitivity of Piezo1 in HEK293 cells and stretch-induced Ca 2+ entry in 3D-hydrogel-embedded cardiomyocytes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Zyxin Is Involved In Regulation Of Mechanotransduction In Arteriole Smooth Muscle Cells

    Directory of Open Access Journals (Sweden)

    Zhe eSun

    2012-12-01

    Full Text Available Zyxin is a focal adhesion protein that has been implicated in the modulation of cell adhesion and motility, and is hypothesized to be a mechano-sensor in integrin-mediated responses to mechanical force. To test the functional role of zyxin in the mechanotransduction of microvascular smooth muscle cells (VSMC, we utilized atomic force microscopy (AFM to apply localized pulling forces to VSMC through a fibronectin (FN focal adhesion induced by a FN-coated bead on cell surface. Application of force with the AFM induced an increase of zyxin accumulation at the site of the FN-bead focal adhesion that accompanied the VSMC contractile response. Whereas, reduction of zyxin expression by using a zyxin-shRNA construct abolished the VSMC contractile response to AFM pulling forces, even though the zyxin-silenced VSMCs displayed increased adhesion to FN in both AFM adhesion assays and cell adhesion assays. The reduced zyxin expression significantly impaired cell spreading and reorganization of the actin cytoskeleton that could indicate a possible underlying reason for the loss of a contractile response to mechanical force. Consistent with these observations, zyxin silencing also resulted in reduced expression of Rac1, which plays an important role in the actin reorganization in VSMC, but increased TRIP6 and FAK expression, the latter being a major protein that promote cell adhesion. In conclusion, these data support an important enabling role for zyxin in VSMCs ability to mechanically respond to applied force.

  2. Functional Assay of Cancer Cell Invasion Potential Based on Mechanotransduction of Focused Ultrasound

    Directory of Open Access Journals (Sweden)

    Andrew C. Weitz

    2017-08-01

    Full Text Available Cancer cells undergo a number of biophysical changes as they transform from an indolent to an aggressive state. These changes, which include altered mechanical and electrical properties, can reveal important diagnostic information about disease status. Here, we introduce a high-throughput, functional technique for assessing cancer cell invasion potential, which works by probing for the mechanically excitable phenotype exhibited by invasive cancer cells. Cells are labeled with fluorescent calcium dye and imaged during stimulation with low-intensity focused ultrasound, a non-contact mechanical stimulus. We show that cells located at the focus of the stimulus exhibit calcium elevation for invasive prostate (PC-3 and DU-145 and bladder (T24/83 cancer cell lines, but not for non-invasive cell lines (BPH-1, PNT1A, and RT112/84. In invasive cells, ultrasound stimulation initiates a calcium wave that propagates from the cells at the transducer focus to other cells, over distances greater than 1 mm. We demonstrate that this wave is mediated by extracellular signaling molecules and can be abolished through inhibition of transient receptor potential channels and inositol trisphosphate receptors, implicating these proteins in the mechanotransduction process. If validated clinically, our technology could provide a means to assess tumor invasion potential in cytology specimens, which is not currently possible. It may therefore have applications in diseases such as bladder cancer, where cytologic diagnosis of tumor invasion could improve clinical decision-making.

  3. Endothelial Mechanotransduction, Redox Signaling and the Regulation of Vascular Inflammatory Pathways

    Directory of Open Access Journals (Sweden)

    Shampa Chatterjee

    2018-06-01

    Full Text Available The endothelium that lines the interior of blood vessels is directly exposed to blood flow. The shear stress arising from blood flow is “sensed” by the endothelium and is “transduced” into biochemical signals that eventually control vascular tone and homeostasis. Sensing and transduction of physical forces occur via signaling processes whereby the forces associated with blood flow are “sensed” by a mechanotransduction machinery comprising of several endothelial cell elements. Endothelial “sensing” involves converting the physical cues into cellular signaling events such as altered membrane potential and activation of kinases, which are “transmission” signals that cause oxidant production. Oxidants produced are the “transducers” of the mechanical signals? What is the function of these oxidants/redox signals? Extensive data from various studies indicate that redox signals initiate inflammation signaling pathways which in turn can compromise vascular health. Thus, inflammation, a major response to infection or endotoxins, can also be initiated by the endothelium in response to various flow patterns ranging from aberrant flow to alteration of flow such as cessation or sudden increase in blood flow. Indeed, our work has shown that endothelial mechanotransduction signaling pathways participate in generation of redox signals that affect the oxidant and inflammation status of cells. Our goal in this review article is to summarize the endothelial mechanotransduction pathways that are activated with stop of blood flow and with aberrant flow patterns; in doing so we focus on the complex link between mechanical forces and inflammation on the endothelium. Since this “inflammation susceptible” phenotype is emerging as a trigger for pathologies ranging from atherosclerosis to rejection post-organ transplant, an understanding of the endothelial machinery that triggers these processes is very crucial and timely.

  4. Integrins and extracellular matrix in mechanotransduction

    Directory of Open Access Journals (Sweden)

    Ramage L

    2011-12-01

    Full Text Available Lindsay RamageQueen’s Medical Research Institute, University of Edinburgh, Edinburgh, UKAbstract: Integrins are a family of cell surface receptors which mediate cell–matrix and cell–cell adhesions. Among other functions they provide an important mechanical link between the cells external and intracellular environments while the adhesions that they form also have critical roles in cellular signal-transduction. Cell–matrix contacts occur at zones in the cell surface where adhesion receptors cluster and when activated the receptors bind to ligands in the extracellular matrix. The extracellular matrix surrounds the cells of tissues and forms the structural support of tissue which is particularly important in connective tissues. Cells attach to the extracellular matrix through specific cell-surface receptors and molecules including integrins and transmembrane proteoglycans. Integrins work alongside other proteins such as cadherins, immunoglobulin superfamily cell adhesion molecules, selectins, and syndecans to mediate cell–cell and cell–matrix interactions and communication. Activation of adhesion receptors triggers the formation of matrix contacts in which bound matrix components, adhesion receptors, and associated intracellular cytoskeletal and signaling molecules form large functional, localized multiprotein complexes. Cell–matrix contacts are important in a variety of different cell and tissue properties including embryonic development, inflammatory responses, wound healing, and adult tissue homeostasis. This review summarizes the roles and functions of integrins and extracellular matrix proteins in mechanotransduction.Keywords: ligand binding, α subunit, ß subunit, focal adhesion, cell differentiation, mechanical loading, cell–matrix interaction

  5. In Touch With the Mechanosensitive Piezo Channels: Structure, Ion Permeation, and Mechanotransduction.

    Science.gov (United States)

    Geng, J; Zhao, Q; Zhang, T; Xiao, B

    2017-01-01

    Mechanotransduction, the conversion of mechanical forces into biological signals, plays critical roles in various physiological and pathophysiological processes in mammals, such as conscious sensing of touch, pain, and sound, as well as unconscious sensing of blood flow-associated shear stress, urine flow, and bladder distention. Among the various molecules involved in mechanotransduction, mechanosensitive (MS) cation channels have long been postulated to represent one critical class of mechanotransducers that directly and rapidly converts mechanical force into electrochemical signals. Despite the awareness of their functional significance, the molecular identities of MS cation channels in mammals had remained elusive for decades till the groundbreaking finding that the Piezo family of genes, including Piezo1 and Piezo2, constitutes their essential components. Since their identification about 6years ago, tremendous progress has been made in understanding their physiological and pathophysiological importance in mechanotransduction and their structure-function relationships of being the prototypic class of mammalian MS cation channels. On the one hand, Piezo proteins have been demonstrated to serve as physiologically and pathophysiologically important mechanotransducers for most, if not all, mechanotransduction processes. On the other hand, they have been shown to form a remarkable three-bladed, propeller-shaped homotrimeric channel complex comprising a separable ion-conducting pore module and mechanotransduction modules. In this chapter, we review the major advancements, with a particular focus on the structural and biophysical features that enable Piezo proteins to serve as sophisticated MS cation channels for force sensing, transduction, and ion conduction. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Multi-scale and multi-physics model of the uterine smooth muscle with mechanotransduction.

    Science.gov (United States)

    Yochum, Maxime; Laforêt, Jérémy; Marque, Catherine

    2018-02-01

    Preterm labor is an important public health problem. However, the efficiency of the uterine muscle during labor is complex and still poorly understood. This work is a first step towards a model of the uterine muscle, including its electrical and mechanical components, to reach a better understanding of the uterus synchronization. This model is proposed to investigate, by simulation, the possible role of mechanotransduction for the global synchronization of the uterus. The electrical diffusion indeed explains the local propagation of contractile activity, while the tissue stretching may play a role in the synchronization of distant parts of the uterine muscle. This work proposes a multi-physics (electrical, mechanical) and multi-scales (cell, tissue, whole uterus) model, which is applied to a realistic uterus 3D mesh. This model includes electrical components at different scales: generation of action potentials at the cell level, electrical diffusion at the tissue level. It then links these electrical events to the mechanical behavior, at the cellular level (via the intracellular calcium concentration), by simulating the force generated by each active cell. It thus computes an estimation of the intra uterine pressure (IUP) by integrating the forces generated by each active cell at the whole uterine level, as well as the stretching of the tissue (by using a viscoelastic law for the behavior of the tissue). It finally includes at the cellular level stretch activated channels (SACs) that permit to create a loop between the mechanical and the electrical behavior (mechanotransduction). The simulation of different activated regions of the uterus, which in this first "proof of concept" case are electrically isolated, permits the activation of inactive regions through the stretching (induced by the electrically active regions) computed at the whole organ scale. This permits us to evidence the role of the mechanotransduction in the global synchronization of the uterus. The

  7. Endothelial mechanotransduction proteins and vascular function are altered by dietary sucrose supplementation in healthy young male subjects.

    Science.gov (United States)

    Gliemann, Lasse; Rytter, Nicolai; Lindskrog, Mads; Slingsby, Martina H Lundberg; Åkerström, Thorbjörn; Sylow, Lykke; Richter, Erik A; Hellsten, Ylva

    2017-08-15

    Mechanotransduction in endothelial cells is a central mechanism in the regulation of vascular tone and vascular remodelling Mechanotransduction and vascular function may be affected by high sugar levels in plasma because of a resulting increase in oxidative stress and increased levels of advanced glycation end-products (AGE). In healthy young subjects, 2 weeks of daily supplementation with 3 × 75 g of sucrose was found to reduce blood flow in response to passive lower leg movement and in response to 12 W of knee extensor exercise. This vascular impairment was paralleled by up-regulation of platelet endothelial cell adhesion molecule (PECAM)-1, endothelial nitric oxide synthase, NADPH oxidase and Rho family GTPase Rac1 protein expression, an increased basal phosphorylation status of vascular endothelial growth factor receptor 2 and a reduced phosphorylation status of PECAM-1. There were no measurable changes in AGE levels. The findings of the present study demonstrate that daily high sucrose intake markedly affects mechanotransduction proteins and has a detrimental effect on vascular function. Endothelial mechanotransduction is important for vascular function but alterations and activation of vascular mechanosensory proteins have not been investigated in humans. In endothelial cell culture, simple sugars effectively impair mechanosensor proteins. To study mechanosensor- and vascular function in humans, 12 young healthy male subjects supplemented their diet with 3 × 75 g sucrose day -1 for 14 days in a randomized cross-over design. Before and after the intervention period, the hyperaemic response to passive lower leg movement and active knee extensor exercise was determined by ultrasound doppler. A muscle biopsy was obtained from the thigh muscle before and after acute passive leg movement to allow assessment of protein amounts and the phosphorylation status of mechanosensory proteins and NADPH oxidase. The sucrose intervention led to a reduced flow

  8. Fluctuation-driven mechanotransduction regulates mitochondrial-network structure and function

    Science.gov (United States)

    Bartolák-Suki, Erzsébet; Imsirovic, Jasmin; Parameswaran, Harikrishnan; Wellman, Tyler J.; Martinez, Nuria; Allen, Philip G.; Frey, Urs; Suki, Béla

    2015-10-01

    Cells can be exposed to irregular mechanical fluctuations, such as those arising from changes in blood pressure. Here, we report that ATP production, assessed through changes in mitochondrial membrane potential, is downregulated in vascular smooth muscle cells in culture exposed to monotonous stretch cycles when compared with cells exposed to a variable cyclic stretch that incorporates physiological levels of cycle-by-cycle variability in stretch amplitude. Variable stretch enhances ATP production by increasing the expression of ATP synthase’s catalytic domain, cytochrome c oxidase and its tyrosine phosphorylation, mitofusins and PGC-1α. Such a fluctuation-driven mechanotransduction mechanism is mediated by motor proteins and by the enhancement of microtubule-, actin- and mitochondrial-network complexity. We also show that, in aorta rings isolated from rats, monotonous stretch downregulates--whereas variable stretch maintains--physiological vessel-wall contractility through mitochondrial ATP production. Our results have implications for ATP-dependent and mechanosensitive intracellular processes.

  9. The imperative for controlled mechanical stresses in unraveling cellular mechanisms of mechanotransduction

    Directory of Open Access Journals (Sweden)

    Sorkin Adam M

    2006-05-01

    Full Text Available Abstract Background In vitro mechanotransduction studies are designed to elucidate cell behavior in response to a well-defined mechanical signal that is imparted to cultured cells, e.g. through fluid flow. Typically, flow rates are calculated based on a parallel plate flow assumption, to achieve a targeted cellular shear stress. This study evaluates the performance of specific flow/perfusion chambers in imparting the targeted stress at the cellular level. Methods To evaluate how well actual flow chambers meet their target stresses (set for 1 and 10 dyn/cm2 for this study at a cellular level, computational models were developed to calculate flow velocity components and imparted shear stresses for a given pressure gradient. Computational predictions were validated with micro-particle image velocimetry (μPIV experiments. Results Based on these computational and experimental studies, as few as 66% of cells seeded along the midplane of commonly implemented flow/perfusion chambers are subjected to stresses within ±10% of the target stress. In addition, flow velocities and shear stresses imparted through fluid drag vary as a function of location within each chamber. Hence, not only a limited number of cells are exposed to target stress levels within each chamber, but also neighboring cells may experience different flow regimes. Finally, flow regimes are highly dependent on flow chamber geometry, resulting in significant variation in magnitudes and spatial distributions of stress between chambers. Conclusion The results of this study challenge the basic premise of in vitro mechanotransduction studies, i.e. that a controlled flow regime is applied to impart a defined mechanical stimulus to cells. These results also underscore the fact that data from studies in which different chambers are utilized can not be compared, even if the target stress regimes are comparable.

  10. Substrate and mechanotransduction influence SERCA2a localization in human pluripotent stem cell-derived cardiomyocytes affecting functional performance

    Directory of Open Access Journals (Sweden)

    Sebastian Martewicz

    2017-12-01

    In this work, we show involvement of the mechanotransduction pathway RhoA/ROCK in the structural reorganization of hPSC-derived cardiomyocytes after adhesion plating. These structural changes have a major impact on the intracellular localization of SERCA2 pumps and concurrent improvement in calcium cycling. The process is triggered by cell interaction with the culture substrate, which mechanical cues drive sarcomeric alignment and SERCA2a spreading and relocalization from a perinuclear to a whole-cell distribution. This structural reorganization is mediated by the mechanical properties of the substrate, as shown by the process failure in hPSC-CMs cultured on soft 4 kPa hydrogels as opposed to physiologically stiff 16 kPa hydrogels and glass. Finally, pharmacological inhibition of Rho-associated protein kinase (ROCK by different compounds identifies this specific signaling pathway as a major player in SERCA2 localization and the associated improvement in hPSC-CMs calcium handling ability in vitro.

  11. Rheological Influence Upon the Glomerular Podocyte and Resultant Mechanotransduction

    Directory of Open Access Journals (Sweden)

    Simona Pichler Sekulic

    2015-03-01

    Full Text Available The glomerular podocyte is exposed to numerous mechanical forces as a constituent of the glomerular filtration apparatus. This includes fluid shear stress (FSS displaced upon the podocytic foot process's apical, lateral, and basal surfaces. Even in the face of continuous flow the podocyte is capable of contributing to physiologic filtration, however with pathologic levels of hyperfiltration there is increased FSS placed upon the cell. The mechanisms by which the podocyte detects and responds to FSS are topics of recent investigations, with the aim to clarify the way these cells are injured and/or adapt in times of hyperfiltration and disease states. As the pathogenesis of numerous glomerulopathies is contingent on the status of the podocyte, understanding the manner that these cells can be modified by FSS is essential. Likewise, determination of the effect of such mechanical forces upon other resident cells of the renal corpuscle would reveal the contribution of FSS in the progression of glomerular diseases. The biochemical manner in which podocytes sense and respond to FSS, that is mechanotransduction, will be discussed.

  12. The roles of P2Y2 purinergic receptors in osteoblasts and mechanotransduction.

    Directory of Open Access Journals (Sweden)

    Yanghui Xing

    Full Text Available We previously demonstrated, using osteoblastic MC3T3-E1 cells, that P2Y2 purinergic receptors are involved in osteoblast mechanotransduction. In this study, our objective was to further investigate, using a knockout mouse model, the roles of P2Y2 receptors in bone mechanobiology. We first examined bone structure with micro-CT and measured bone mechanical properties with three point bending experiments in both wild type mice and P2Y2 knockout mice. We found that bones from P2Y2 knockout mice have significantly decreased bone volume, bone thickness, bone stiffness and bone ultimate breaking force at 17 week old age. In order to elucidate the mechanisms by which P2Y2 receptors contribute to bone biology, we examined differentiation and mineralization of bone marrow cells from wild type and P2Y2 knockout mice. We found that P2Y2 receptor deficiency reduces the differentiation and mineralization of bone marrow cells. Next, we compared the response of primary osteoblasts, from both wild type and P2Y2 knockout mice, to ATP and mechanical stimulation (oscillatory fluid flow, and found that osteoblasts from wild type mice have a stronger response, in terms of ERK1/2 phosphorylation, to both ATP and fluid flow, relative to P2Y2 knockout mice. However, we did not detect any difference in ATP release in response to fluid flow between wild type and P2Y2 knock out osteoblasts. Our findings suggest that P2Y2 receptors play important roles in bone marrow cell differentiation and mineralization as well as in bone cell mechanotransduction, leading to an osteopenic phenotype in P2Y2 knockout mice.

  13. Evidence for the involvement of ASIC3 in sensory mechanotransduction in proprioceptors

    Science.gov (United States)

    Lin, Shing-Hong; Cheng, Yuan-Ren; Banks, Robert W.; Min, Ming-Yuan; Bewick, Guy S.; Chen, Chih-Cheng

    2016-01-01

    Acid-sensing ion channel 3 (ASIC3) is involved in acid nociception, but its possible role in neurosensory mechanotransduction is disputed. We report here the generation of Asic3-knockout/eGFPf-knockin mice and subsequent characterization of heterogeneous expression of ASIC3 in the dorsal root ganglion (DRG). ASIC3 is expressed in parvalbumin (Pv+) proprioceptor axons innervating muscle spindles. We further generate a floxed allele of Asic3 (Asic3f/f) and probe the role of ASIC3 in mechanotransduction in neurite-bearing Pv+ DRG neurons through localized elastic matrix movements and electrophysiology. Targeted knockout of Asic3 disrupts spindle afferent sensitivity to dynamic stimuli and impairs mechanotransduction in Pv+ DRG neurons because of substrate deformation-induced neurite stretching, but not to direct neurite indentation. In behavioural tasks, global knockout (Asic3−/−) and Pv-Cre::Asic3f/f mice produce similar deficits in grid and balance beam walking tasks. We conclude that, at least in mouse, ASIC3 is a molecular determinant contributing to dynamic mechanosensitivity in proprioceptors. PMID:27161260

  14. Intermediate Filaments at the Junction of Mechanotransduction, Migration, and Development

    Directory of Open Access Journals (Sweden)

    Rucha Sanghvi-Shah

    2017-09-01

    Full Text Available Mechanically induced signal transduction has an essential role in development. Cells actively transduce and respond to mechanical signals and their internal architecture must manage the associated forces while also being dynamically responsive. With unique assembly-disassembly dynamics and physical properties, cytoplasmic intermediate filaments play an important role in regulating cell shape and mechanical integrity. While this function has been recognized and appreciated for more than 30 years, continually emerging data also demonstrate important roles of intermediate filaments in cell signal transduction. In this review, with a particular focus on keratins and vimentin, the relationship between the physical state of intermediate filaments and their role in mechanotransduction signaling is illustrated through a survey of current literature. Association with adhesion receptors such as cadherins and integrins provides a critical interface through which intermediate filaments are exposed to forces from a cell's environment. As a consequence, these cytoskeletal networks are posttranslationally modified, remodeled and reorganized with direct impacts on local signal transduction events and cell migratory behaviors important to development. We propose that intermediate filaments provide an opportune platform for cells to both cope with mechanical forces and modulate signal transduction.

  15. Design of a nanocomposite substrate inducing adult stem cell assembly and progression toward an Epiblast-like or Primitive Endoderm-like phenotype via mechanotransduction.

    Science.gov (United States)

    Morena, Francesco; Armentano, Ilaria; Montanucci, Pia; Argentati, Chiara; Fortunati, Elena; Montesano, Simona; Bicchi, Ilaria; Pescara, Teresa; Pennoni, Ilaria; Mattioli, Samantha; Torre, Luigi; Latterini, Loredana; Emiliani, Carla; Basta, Giuseppe; Calafiore, Riccardo; Kenny, Josè Maria; Martino, Sabata

    2017-11-01

    This work shows that the active interaction between human umbilical cord matrix stem cells and Poly (l-lactide)acid (PLLA) and PLLA/Multi Walled Carbon Nanotubes (MWCNTs) nanocomposite films results in the stem cell assembly as a spheroid conformation and affects the stem cell fate transition. We demonstrated that spheroids directly respond to a tunable surface and the bulk properties (electric, dielectric and thermal) of plain and nanocomposite PLLA films by triggering a mechanotransduction axis. This stepwise process starts from tethering of the cells' focal adhesion proteins to the surface, together with the adherens junctions between cells. Both complexes transmit traction forces to F-Actin stress fibres that link Filamin-A and Myosin-IIA proteins, generating a biological scaffold, with increased stiffening conformation from PLLA to PLLA/MWCNTs, and enable the nucleoskeleton proteins to boost chromatin reprogramming processes. Herein, the opposite expression of NANOG and GATA6 transcription factors, together with other lineage specification related proteins, steer spheroids toward an Epiblast-like or Primitive Endoderm-like lineage commitment, depending on the absence or presence of 1 wt% MWCNTs, respectively. This work represents a pioneering effort to create a stem cell/material interface that can model the stem cell fate transition under growth culture conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Mechanics rules cell biology

    Directory of Open Access Journals (Sweden)

    Wang James HC

    2010-07-01

    Full Text Available Abstract Cells in the musculoskeletal system are subjected to various mechanical forces in vivo. Years of research have shown that these mechanical forces, including tension and compression, greatly influence various cellular functions such as gene expression, cell proliferation and differentiation, and secretion of matrix proteins. Cells also use mechanotransduction mechanisms to convert mechanical signals into a cascade of cellular and molecular events. This mini-review provides an overview of cell mechanobiology to highlight the notion that mechanics, mainly in the form of mechanical forces, dictates cell behaviors in terms of both cellular mechanobiological responses and mechanotransduction.

  17. Mechanotransduction-modulated fibrotic microniches reveal the contribution of angiogenesis in liver fibrosis

    Science.gov (United States)

    Liu, Longwei; You, Zhifeng; Yu, Hongsheng; Zhou, Lyu; Zhao, Hui; Yan, Xiaojun; Li, Dulei; Wang, Bingjie; Zhu, Lu; Xu, Yuzhou; Xia, Tie; Shi, Yan; Huang, Chenyu; Hou, Wei; Du, Yanan

    2017-12-01

    The role of pathological angiogenesis on liver fibrogenesis is still unknown. Here, we developed fibrotic microniches (FμNs) that recapitulate the interaction of liver sinusoid endothelial cells (LSECs) and hepatic stellate cells (HSCs). We investigated how the mechanical properties of their substrates affect the formation of capillary-like structures and how they relate to the progression of angiogenesis during liver fibrosis. Differences in cell response in the FμNs were synonymous of the early and late stages of liver fibrosis. The stiffness of the early-stage FμNs was significantly elevated due to condensation of collagen fibrils induced by angiogenesis, and led to activation of HSCs by LSECs. We utilized these FμNs to understand the response to anti-angiogenic drugs, and it was evident that these drugs were effective only for early-stage liver fibrosis in vitro and in an in vivo mouse model of liver fibrosis. Late-stage liver fibrosis was not reversed following treatment with anti-angiogenic drugs but rather with inhibitors of collagen condensation. Our work reveals stage-specific angiogenesis-induced liver fibrogenesis via a previously unrevealed mechanotransduction mechanism which may offer precise intervention strategies targeting stage-specific disease progression.

  18. Endothelial mechanotransduction proteins and vascular function are altered by dietary sucrose supplementation in healthy young male subjects

    DEFF Research Database (Denmark)

    Gliemann, Lasse; Rytter, Nicolai; Lindskrog, Mads

    2017-01-01

    Endothelial mechanotransduction is important for vascular function but alterations and activation of vascular mechanosensory proteins have not been investigated in humans. In endothelial cell culture, simple sugars effectively impair mechanosensor proteins. To study mechanosensor- and vascular...... by ultrasound doppler. A muscle biopsy was obtained from the thigh muscle before and after acute passive leg movement, to asses the protein amount and phosphorylation status of mechanosensory proteins and NADPH oxidase. The sucrose intervention led to a reduced flow response to passive movement (by 17 ± 2...... %) and to 12 watts of active exercise (by 9 ± 1 %), indicating impaired vascular function. Reduced flow response to passive and active exercise was paralleled by a significant upregulation of Platelet endothelial cell adhesion molecule (PECAM-1), endothelial nitric oxide synthase, NADPH oxidase and the Rho...

  19. Control of cell behaviour through nanovibrational stimulation: nanokicking

    Science.gov (United States)

    Robertson, Shaun N.; Campsie, Paul; Childs, Peter G.; Madsen, Fiona; Donnelly, Hannah; Henriquez, Fiona L.; Mackay, William G.; Salmerón-Sánchez, Manuel; Tsimbouri, Monica P.; Williams, Craig; Dalby, Matthew J.; Reid, Stuart

    2018-05-01

    Mechanical signals are ubiquitous in our everyday life and the process of converting these mechanical signals into a biological signalling response is known as mechanotransduction. Our understanding of mechanotransduction, and its contribution to vital cellular responses, is a rapidly expanding field of research involving complex processes that are still not clearly understood. The use of mechanical vibration as a stimulus of mechanotransduction, including variation of frequency and amplitude, allows an alternative method to control specific cell behaviour without chemical stimulation (e.g. growth factors). Chemical-independent control of cell behaviour could be highly advantageous for fields including drug discovery and clinical tissue engineering. In this review, a novel technique is described based on nanoscale sinusoidal vibration. Using finite-element analysis in conjunction with laser interferometry, techniques that are used within the field of gravitational wave detection, optimization of apparatus design and calibration of vibration application have been performed. We further discuss the application of nanovibrational stimulation, or `nanokicking', to eukaryotic and prokaryotic cells including the differentiation of mesenchymal stem cells towards an osteoblast cell lineage. Mechanotransductive mechanisms are discussed including mediation through the Rho-A kinase signalling pathway. Optimization of this technique was first performed in two-dimensional culture using a simple vibration platform with an optimal frequency and amplitude of 1 kHz and 22 nm. A novel bioreactor was developed to scale up cell production, with recent research demonstrating that mesenchymal stem cell differentiation can be efficiently triggered in soft gel constructs. This important step provides first evidence that clinically relevant (three-dimensional) volumes of osteoblasts can be produced for the purpose of bone grafting, without complex scaffolds and/or chemical induction

  20. Pannexin 1 channels play essential roles in urothelial mechanotransduction and intercellular signaling.

    Directory of Open Access Journals (Sweden)

    Hiromitsu Negoro

    Full Text Available Urothelial cells respond to bladder distension with ATP release, and ATP signaling within the bladder and from the bladder to the CNS is essential for proper bladder function. In other cell types, pannexin 1 (Panx1 channels provide a pathway for mechanically-induced ATP efflux and for ATP-induced ATP release through interaction with P2X7 receptors (P2X7Rs. We report that Panx1 and P2X7R are functionally expressed in the bladder mucosa and in immortalized human urothelial cells (TRT-HU1, and participate in urothelial ATP release and signaling. ATP release from isolated rat bladders induced by distention was reduced by the Panx1 channel blocker mefloquine (MFQ and was blunted in mice lacking Panx1 or P2X7R expression. Hypoosmotic shock induced YoPro dye uptake was inhibited by MFQ and the P2X7R blocker A438079 in TRT-HU1 cells, and was also blunted in primary urothelial cells derived from mice lacking Panx1 or P2X7R expression. Rinsing-induced mechanical stimulation of TRT-HU1 cells triggered ATP release, which was reduced by MFQ and potentiated in low divalent cation solution (LDPBS, a condition known to enhance P2X7R activation. ATP signaling evaluated as intercellular Ca2+ wave radius was significantly larger in LDPBS, reduced by MFQ and by apyrase (ATP scavenger. These findings indicate that Panx1 participates in urothelial mechanotransduction and signaling by providing a direct pathway for mechanically-induced ATP release and by functionally interacting with P2X7Rs.

  1. Mechano-adaptation of the stem cell nucleus.

    Science.gov (United States)

    Heo, Su-Jin; Cosgrove, Brian D; Dai, Eric N; Mauck, Robert L

    2018-01-01

    Exogenous mechanical forces are transmitted through the cell and to the nucleus, initiating mechanotransductive signaling cascades with profound effects on cellular function and stem cell fate. A growing body of evidence has shown that the force sensing and force-responsive elements of the nucleus adapt to these mechanotransductive events, tuning their response to future mechanical input. The mechanisms underlying this "mechano-adaptation" are only just beginning to be elucidated, and it remains poorly understood how these components act and adapt in tandem to drive stem cell differentiation. Here, we review the evidence on how the stem cell nucleus responds and adapts to physical forces, and provide a perspective on how this mechano-adaptation may function to drive and enforce stem cell differentiation.

  2. Mechanotransduction in cortical bone and the role of piezoelectricity: a numerical approach.

    Science.gov (United States)

    Stroe, M C; Crolet, J M; Racila, M

    2013-01-01

    This paper is a contribution to a plausible explanation of the mechanotransduction phenomenon in cortical bone during its remodelling. Our contribution deals only with the mechanical processes and the biological aspects have not been taken into account. It is well known that osteoblasts are able to generate bone in a suitable bony substitute only under fluid action. But the bone created in this manner is not organised to resist specific mechanical stress. Our aim was to suggest the nature of the physical information that can be transmitted - directly or via a biological or biochemical process - to the cell to initiate a cellular activity inducing the reconstruction of the osteon that is best adapted to local mechanical stresses. For this, the cell must have, from our point of view, a good knowledge of its structural environment. But this knowledge exists at the cellular scale while the bone is loaded at the macroscopic scale. This study is based on the SiNuPrOs model that allows exchange of information between the different structural scales of cortical bone. It shows that more than the fluid, the collagen - via its piezoelectric properties - plays an essential role in the transmission of information between the macroscopic and nanoscopic scales. Moreover, this process allows us to explain various dysfunctions and even some diseases.

  3. Using Magnets and Magnetic Beads to Dissect Signaling Pathways Activated by Mechanical Tension Applied to Cells

    Science.gov (United States)

    Marjoram, R.J.; Guilluy, C; Burridge, K.

    2015-01-01

    Cellular tension has implications in normal biology and pathology. Membrane adhesion receptors serve as conduits for mechanotransduction that lead to cellular responses. Ligand-conjugated magnetic beads are a useful tool in the study of how cells sense and respond to tension. Here we detail methods for their use in applying tension to cells and strategies for analyzing the results. We demonstrate the methods by analyzing mechanotransduction through VE-cadherin on endothelial cells using both permanent magnets and magnetic tweezers. PMID:26427549

  4. Varicose Veins: Role of Mechanotransduction of Venous Hypertension

    Science.gov (United States)

    Atta, Hussein M.

    2012-01-01

    Varicose veins affect approximately one-third of the adult population and result in significant psychological, physical, and financial burden. Nevertheless, the molecular pathogenesis of varicose vein formation remains unidentified. Venous hypertension exerted on veins of the lower extremity is considered the principal factor in varicose vein formation. The role of mechanotransduction of the high venous pressure in the pathogenesis of varicose vein formation has not been adequately investigated despite a good progress in understanding the mechanomolecular mechanisms involved in transduction of high blood pressure in the arterial wall. Understanding the nature of the mechanical forces, the mechanosensors and mechanotransducers in the vein wall, and the downstream signaling pathways will provide new molecular targets for the prevention and treatment of varicose veins. This paper summarized the current understanding of mechano-molecular pathways involved in transduction of hemodynamic forces induced by blood pressure and tries to relate this information to setting of venous hypertension in varicose veins. PMID:22489273

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

    Directory of Open Access Journals (Sweden)

    David W. Holdsworth

    2012-03-01

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

  6. Red blood cell dynamics: from cell deformation to ATP release.

    Science.gov (United States)

    Wan, Jiandi; Forsyth, Alison M; Stone, Howard A

    2011-10-01

    The mechanisms of red blood cell (RBC) deformation under both static and dynamic, i.e., flow, conditions have been studied extensively since the mid 1960s. Deformation-induced biochemical reactions and possible signaling in RBCs, however, were proposed only fifteen years ago. Therefore, the fundamental relationship between RBC deformation and cellular signaling dynamics i.e., mechanotransduction, remains incompletely understood. Quantitative understanding of the mechanotransductive pathways in RBCs requires integrative studies of physical models of RBC deformation and cellular biochemical reactions. In this article we review the physical models of RBC deformation, spanning from continuum membrane mechanics to cellular skeleton dynamics under both static and flow conditions, and elaborate the mechanistic links involved in deformation-induced ATP release. This journal is © The Royal Society of Chemistry 2011

  7. Biomechanics of Meniscus Cells: Regional Variation and Comparison to Articular Chondrocytes and Ligament Cells

    OpenAIRE

    Sanchez-Adams, Johannah; Athanasiou, Kyriacos A.

    2012-01-01

    Central to understanding mechanotransduction in the knee meniscus is the characterization of meniscus cell mechanics. In addition to biochemical and geometric differences, the inner and outer regions of the meniscus contain cells that are distinct in morphology and phenotype. This study investigated the regional variation in meniscus cell mechanics in comparison to articular chondrocytes and ligament cells. It was found that the meniscus contains two biomechanically distinct cell populations,...

  8. Rat Merkel cells are mechanoreceptors and osmoreceptors.

    Directory of Open Access Journals (Sweden)

    Nicholas Boulais

    Full Text Available Merkel cells (MCs associated with nerve terminals constitute MC-neurite complexes, which are involved in slowly-adapting type I mechanoreception. Although MCs are known to express voltage-gated Ca2+ channels and hypotonic-induced membrane deformation is known to lead to Ca2+ transients, whether MCs initiate mechanotransduction is currently unknown. To answer to this question, rat MCs were transfected with a reporter vector, which enabled their identification.Their properties were investigated through electrophysiological studies. Voltage-gated K+, Ca2+ and Ca2+-activated K+ (KCachannels were identified, as previously described. Here, we also report the activation of Ca2+ channels by histamine and their inhibition by acetylcholine. As a major finding, we demonstrated that direct mechanical stimulations induced strong inward Ca2+ currents in MCs. Depolarizations were dependent on the strength and the length of the stimulation. Moreover, touch-evoked currents were inhibited by the stretch channel antagonist gadolinium. These data confirm the mechanotransduction capabilities of MCs. Furthermore, we found that activation of the osmoreceptor TRPV4 in FM1-43-labeled MCs provoked neurosecretory granule exocytosis. Since FM1-43 blocks mechanosensory channels, this suggests that hypo-osmolarity activates MCs in the absence of mechanotransduction. Thus, mechanotransduction and osmoreception are likely distinct pathways.

  9. Piezo Is Essential for Amiloride-Sensitive Stretch-Activated Mechanotransduction in Larval Drosophila Dorsal Bipolar Dendritic Sensory Neurons.

    Science.gov (United States)

    Suslak, Thomas J; Watson, Sonia; Thompson, Karen J; Shenton, Fiona C; Bewick, Guy S; Armstrong, J Douglas; Jarman, Andrew P

    2015-01-01

    Stretch-activated afferent neurons, such as those of mammalian muscle spindles, are essential for proprioception and motor co-ordination, but the underlying mechanisms of mechanotransduction are poorly understood. The dorsal bipolar dendritic (dbd) sensory neurons are putative stretch receptors in the Drosophila larval body wall. We have developed an in vivo protocol to obtain receptor potential recordings from intact dbd neurons in response to stretch. Receptor potential changes in dbd neurons in response to stretch showed a complex, dynamic profile with similar characteristics to those previously observed for mammalian muscle spindles. These profiles were reproduced by a general in silico model of stretch-activated neurons. This in silico model predicts an essential role for a mechanosensory cation channel (MSC) in all aspects of receptor potential generation. Using pharmacological and genetic techniques, we identified the mechanosensory channel, DmPiezo, in this functional role in dbd neurons, with TRPA1 playing a subsidiary role. We also show that rat muscle spindles exhibit a ruthenium red-sensitive current, but found no expression evidence to suggest that this corresponds to Piezo activity. In summary, we show that the dbd neuron is a stretch receptor and demonstrate that this neuron is a tractable model for investigating mechanisms of mechanotransduction.

  10. Magnetically actuated mechanical stimuli on Fe3O4/mineralized collagen coatings to enhance osteogenic differentiation of the MC3T3-E1 cells.

    Science.gov (United States)

    Zhuang, Junjun; Lin, Suya; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

    2018-04-15

    Mechanical stimuli at the bone-implant interface are considered to activate the mechanotransduction pathway of the cell to improve the initial osseointegration establishment and to guarantee clinical success of the implant. However, control of the mechanical stimuli at the bone-implant interface still remains a challenge. In this study, we have designed a strategy of a magnetically responsive coating on which the mechanical stimuli is controlled because of coating deformation under static magnetic field (SMF). The iron oxide nanoparticle/mineralized collagen (IOP-MC) coatings were electrochemically codeposited on titanium substrates in different quantities of IOPs and distributions; the resulting coatings were verified to possess swelling behavior with flexibility same as that of hydrogel. The relative quantity of IOP to collagen and the IOP distribution in the coatings were demonstrated to play a critical role in mediating cell behavior. The cells present on the outer layer of the distributed IOP-MC (O-IOP-MC) coating with a mass ratio of 0.67 revealed the most distinct osteogenic differentiation activity being promoted, which could be attributed to the maximized mechanical stimuli with exposure to SMF. Furthermore, the enhanced osteogenic differentiation of the stimulated MC3T3-E1 cells originated from magnetically actuated mechanotransduction signaling pathway, embodying the upregulated expression of osteogenic-related and mechanotransduction-related genes. This work therefore provides a promising strategy for implementing mechanical stimuli to activate mechanotransduction on the bone-implant interface and thus to promote osseointegration. The magnetically actuated coating is designed to produce mechanical stimuli to cells for promoting osteogenic differentiation based on the coating deformation. Iron oxide nanoparticles (IOPs) were incorporated into the mineralized collagen coatings (MC) forming the composite coatings (IOP-MC) with spatially distributed IOPs

  11. Fetal jaw movement affects Ihh signaling in mandibular condylar cartilage development: the possible role of Ihh as mechanotransduction mediator.

    Science.gov (United States)

    Jahan, Esrat; Matsumoto, Akihiro; Rafiq, Ashiq Mahmood; Hashimoto, Ryuju; Inoue, Takayuki; Udagawa, Jun; Sekine, Joji; Otani, Hiroki

    2014-10-01

    Jaw movement is an important mechanical factor for prenatal development of the condylar cartilage of mandible. Fetal jaw movement restriction has been shown to cause deformity of the mandibular condyle. We hypothesized that this treatment affects the expression of mechanosensitive molecules, namely Indian hedgehog (Ihh) and Parathyroid hormone related protein (PTHrP) in the condyle. We restrained jaw movement by suturing the jaw of E15.5 mouse embryos and allowed them to develop until E18.5 using exo utero system, and analyzed them by immunohistochemistry and in situ hybridization methods. Morphological, histomorphometric and immunohistochemical study showed that the mandibular condylar cartilage was reduced and deformed, the volume and total cell numbers in the condylar cartilage were also reduced, and number and/or distribution of 5-bromo-2'-deoxyuridine-positive cells, Ihh-positive cells in the mesenchymal and pre-hypertrophic zones were significantly and correspondingly decreased in the sutured group. Using in situ hybridization, reduced expression of Ihh, PTHrP and their related receptors were observed in condylar cartilage of the sutured embryos. Our results revealed that the altered mechanical stress induced by prenatal jaw movement restriction decreased proliferating cells, the amount of cartilage, and altered expression of the Ihh and PTHrP, suggesting that Ihh act as mechanotransduction mediators in the development of mandibular condylar cartilage. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Documentation and localization of force-mediated filamin A domain perturbations in moving cells

    Science.gov (United States)

    Nakamura, Fumihiko; Song, Mia; Hartwig, John H.; Stossel, Thomas P.

    2014-08-01

    Endogenously and externally generated mechanical forces influence diverse cellular activities, a phenomenon defined as mechanotransduction. Deformation of protein domains by application of stress, previously documented to alter macromolecular interactions in vitro, could mediate these effects. We engineered a photon-emitting system responsive to unfolding of two repeat domains of the actin filament (F-actin) crosslinker protein filamin A (FLNA) that binds multiple partners involved in cell signalling reactions and validated the system using F-actin networks subjected to myosin-based contraction. Expressed in cultured cells, the sensor-containing FLNA construct reproducibly reported FLNA domain unfolding strikingly localized to dynamic, actively protruding, leading cell edges. The unfolding signal depends upon coherence of F-actin-FLNA networks and is enhanced by stimulating cell contractility. The results establish protein domain distortion as a bona fide mechanism for mechanotransduction in vivo.

  13. Collective epithelial cell sheet adhesion and migration on polyelectrolyte multilayers with uniform and gradients of compliance

    Energy Technology Data Exchange (ETDEWEB)

    Martinez, Jessica S. [Department of Biological Science, Florida State University, Tallahassee, FL 32306 (United States); Schlenoff, Joseph B. [Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306 (United States); Keller, Thomas C.S., E-mail: tkeller@bio.fsu.edu [Department of Biological Science, Florida State University, Tallahassee, FL 32306 (United States)

    2016-08-01

    surfaces. Interestingly, cells throughout the interior region of the sheets on uncrosslinked PEMUs retained their actin and vinculin organization at adherens junctions after treatment with Blebbistatin. Like Blebbistatin, a Rho-kinase (ROCK) inhibitor, Y27632, promoted loss of cell-cell connections between edge cells, whereas a Rac1 inhibitor, NSC23766, primarily altered the lamellipodial protrusion in edge cells. Compliance gradient PAH-PEMUs promoted durotaxis of the cell sheets but not of individual keratocytes, demonstrating durotaxis, like plithotaxis, is an emergent property of cell sheet organization. - Highlights: • Fish scale cell sheets migrate on PAH-PAABp polyelectrolyte multilayers. • Sheets migrating on softer PEMUs periodically retract. • Sheets durotax on modulus gradients. • Myosin II inhibitors inhibit sheet integrity and migration.

  14. Collective epithelial cell sheet adhesion and migration on polyelectrolyte multilayers with uniform and gradients of compliance

    International Nuclear Information System (INIS)

    Martinez, Jessica S.; Schlenoff, Joseph B.; Keller, Thomas C.S.

    2016-01-01

    surfaces. Interestingly, cells throughout the interior region of the sheets on uncrosslinked PEMUs retained their actin and vinculin organization at adherens junctions after treatment with Blebbistatin. Like Blebbistatin, a Rho-kinase (ROCK) inhibitor, Y27632, promoted loss of cell-cell connections between edge cells, whereas a Rac1 inhibitor, NSC23766, primarily altered the lamellipodial protrusion in edge cells. Compliance gradient PAH-PEMUs promoted durotaxis of the cell sheets but not of individual keratocytes, demonstrating durotaxis, like plithotaxis, is an emergent property of cell sheet organization. - Highlights: • Fish scale cell sheets migrate on PAH-PAABp polyelectrolyte multilayers. • Sheets migrating on softer PEMUs periodically retract. • Sheets durotax on modulus gradients. • Myosin II inhibitors inhibit sheet integrity and migration.

  15. Role of TRP Channels in Dinoflagellate Mechanotransduction.

    Science.gov (United States)

    Lindström, J B; Pierce, N T; Latz, M I

    2017-10-01

    Transient receptor potential (TRP) ion channels are common components of mechanosensing pathways, mainly described in mammals and other multicellular organisms. To gain insight into the evolutionary origins of eukaryotic mechanosensory proteins, we investigated the involvement of TRP channels in mechanosensing in a unicellular eukaryotic protist, the dinoflagellate Lingulodinium polyedra. BLASTP analysis of the protein sequences predicted from the L. polyedra transcriptome revealed six sequences with high similarity to human TRPM2, TRPM8, TRPML2, TRPP1, and TRPP2; and characteristic TRP domains were identified in all sequences. In a phylogenetic tree including all mammalian TRP subfamilies and TRP channel sequences from unicellular and multicellular organisms, the L. polyedra sequences grouped with the TRPM, TPPML, and TRPP clades. In pharmacological experiments, we used the intrinsic bioluminescence of L. polyedra as a reporter of mechanoresponsivity. Capsaicin and RN1734, agonists of mammalian TRPV, and arachidonic acid, an agonist of mammalian TRPV, TRPA, TRPM, and Drosophila TRP, all stimulated bioluminescence in L. polyedra. Mechanical stimulation of bioluminescence, but not capsaicin-stimulated bioluminescence, was inhibited by gadolinium (Gd 3+ ), a general inhibitor of mechanosensitive ion channels, and the phospholipase C (PLC) inhibitor U73122. These pharmacological results are consistent with the involvement of TRP-like channels in mechanosensing by L. polyedra. The TRP channels do not appear to be mechanoreceptors but rather are components of the mechanotransduction signaling pathway and may be activated via a PLC-dependent mechanism. The presence and function of TRP channels in a dinoflagellate emphasize the evolutionary conservation of both the channel structures and their functions.

  16. Changes in muscle cell metabolism and mechanotransduction are associated with myopathic phenotype in a mouse model of collagen VI deficiency.

    Directory of Open Access Journals (Sweden)

    Sara De Palma

    Full Text Available This study identifies metabolic and protein phenotypic alterations in gastrocnemius, tibialis anterior and diaphragm muscles of Col6a1(-/- mice, a model of human collagen VI myopathies. All three muscles of Col6a1(-/- mice show some common changes in proteins involved in metabolism, resulting in decreased glycolysis and in changes of the TCA cycle fluxes. These changes lead to a different fate of α-ketoglutarate, with production of anabolic substrates in gastrocnemius and tibialis anterior, and with lipotoxicity in diaphragm. The metabolic changes are associated with changes of proteins involved in mechanotransduction at the myotendineous junction/costameric/sarcomeric level (TN-C, FAK, ROCK1, troponin I fast and in energy metabolism (aldolase, enolase 3, triose phosphate isomerase, creatine kinase, adenylate kinase 1, parvalbumin, IDH1 and FASN. Together, these change may explain Ca(2+ deregulation, impaired force development, increased muscle-relaxation-time and fiber damage found in the mouse model as well as in patients. The severity of these changes differs in the three muscles (gastrocnemius

  17. Role for Mechanotransduction in Macrophage and Dendritic Cell Immunobiology

    NARCIS (Netherlands)

    Mennens, S.F.B.J.; Dries, K. van den; Cambi, A.

    2017-01-01

    Tissue homeostasis is not only controlled by biochemical signals but also through mechanical forces that act on cells. Yet, while it has long been known that biochemical signals have profound effects on cell biology, the importance of mechanical forces has only been recognized much more recently.

  18. A device for real-time live-cell microscopy during dynamic dual-modal mechanostimulation

    Science.gov (United States)

    Lorusso, D.; Nikolov, H. N.; Chmiel, T.; Beach, R. J.; Sims, S. M.; Dixon, S. J.; Holdsworth, D. W.

    2017-03-01

    Mechanotransduction - the process by which cells sense and respond to mechanical stimuli - is essential for several physiological processes including skeletal homeostasis. Mammalian cells are thought to be sensitive to different modes of mechanical stimuli, including vibration and fluid shear. To better understand the mechanisms underlying the early stages of mechanotransduction, we describe the development of devices for mechanostimulation (by vibration and fluid shear) of live cells that can be integrated with real-time optical microscopy. The integrated system can deliver up to 3 Pa of fluid shear simultaneous with high-frequency sinusoidal vibrations up to 1 g. Stimuli can be applied simultaneously or independently to cells during real-time microscopic imaging. A custom microfluidic chamber was prepared from polydimethylsiloxane on a glass-bottom cell culture dish. Fluid flow was applied with a syringe pump to induce shear stress. This device is compatible with a custom-designed motion control vibration system. A voice coil actuates the system that is suspended on linear air bushings. Accelerations produced by the system were monitored with an on-board accelerometer. Displacement was validated optically using particle tracking digital high-speed imaging (1200 frames per second). During operation at nominally 45 Hz and 0.3 g, displacements were observed to be within 3.56% of the expected value. MC3T3-E1 osteoblast like cells were seeded into the microfluidic device and loaded with the calcium sensitive fluorescent probe fura-2, then mounted onto the dual-modal mechanostimulation platform. Cells were then imaged and monitored for fluorescence emission. In summary, we have developed a system to deliver physiologically relevant vibrations and fluid shear to live cells during real-time imaging and photometry. Monitoring the behavior of live cells loaded with appropriate fluorescent probes will enable characterization of the signals activated during the initial

  19. Cell Adhesions: Actin-Based Modules that Mediate Cell-Extracellular Matrix and Cell-Cell Interactions

    Science.gov (United States)

    Bachir, Alexia; Horwitz, Alan Rick; Nelson, W. James; Bianchini, Julie M.

    2018-01-01

    Cell adhesions link cells to the extracellular matrix (ECM) and to each other, and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping functional modules. These modules establish physical association with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as sense and translate the mechanical properties of the cellular environment to changes in cell organization and behavior. In this chapter we discuss the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions, and how adhesion molecules mediate crosstalk between cell-ECM and cell-cell adhesion sites. PMID:28679638

  20. A small scale cell culture system to analyze mechanobiology using reporter gene constructs and polyurethane dishes

    DEFF Research Database (Denmark)

    Seefried, Lothar; Mueller-Deubert, Sigrid; Wentzer, Thomas Schwarz

    2010-01-01

    of mechanotransduction and its crosstalk with biochemically induced signal transduction, AP1 and SP1 luciferase reporter gene constructs were cloned and transfected into various cell lines and primary cells. A newly developed bioreactor and small-scale 24-well polyurethane dishes were used to apply cyclic stretching...

  1. Mechanotransductive Regulation of Gap-Junction Activity Between MLO-Y4 Osteocyte-Like and MC3T3-E1 Osteoblast-Like Cells in Three-Dimensional Co-Culture.

    Science.gov (United States)

    Juran, C. M.; Blaber, E. A.; Almeida, E. A. C.

    2016-01-01

    immunohistological observation. Increased CX43 expression is also observed with the three-dimensional co-cultures with MC3T3-E1 MCS stimulation but the increased gap-junction protein presence was limited to the osteoblast-osteocyte interface region. Previously reported PCR evaluation of osteogenic markers further corroborate that the co-cultured populations communicative networks play a role in translating mechanical signals to molecular messaging. These findings suggests an osteocyte-osteoblast gap-junction signaling feedback mechanism may regulate mechanotransduction of apoptosis initiation and transcription of cytokine signaling proteins responsible for stem cell niche recruitment much more directly than previously believed.

  2. Persistence-Driven Durotaxis: Generic, Directed Motility in Rigidity Gradients

    Science.gov (United States)

    Novikova, Elizaveta A.; Raab, Matthew; Discher, Dennis E.; Storm, Cornelis

    2017-02-01

    Cells move differently on substrates with different rigidities: the persistence time of their motion is higher on stiffer substrates. We show that this behavior—in and of itself—results in a net flux of cells directed up a soft-to-stiff gradient. Using simple random walk models with varying persistence and stochastic simulations, we characterize the propensity to move in terms of the durotactic index also measured in experiments. A one-dimensional model captures the essential features and highlights the competition between diffusive spreading and linear, wavelike propagation. Persistence-driven durokinesis is generic and may be of use in the design of instructive environments for cells and other motile, mechanosensitive objects.

  3. Cell-Type-Specific Splicing of Piezo2 Regulates Mechanotransduction

    Directory of Open Access Journals (Sweden)

    Marcin Szczot

    2017-12-01

    Full Text Available Summary: Piezo2 is a mechanically activated ion channel required for touch discrimination, vibration detection, and proprioception. Here, we discovered that Piezo2 is extensively spliced, producing different Piezo2 isoforms with distinct properties. Sensory neurons from both mice and humans express a large repertoire of Piezo2 variants, whereas non-neuronal tissues express predominantly a single isoform. Notably, even within sensory ganglia, we demonstrate the splicing of Piezo2 to be cell type specific. Biophysical characterization revealed substantial differences in ion permeability, sensitivity to calcium modulation, and inactivation kinetics among Piezo2 splice variants. Together, our results describe, at the molecular level, a potential mechanism by which transduction is tuned, permitting the detection of a variety of mechanosensory stimuli. : Szczot et al. find that the mechanoreceptor Piezo2 is extensively alternatively spliced, generating multiple distinct isoforms. Their findings indicate that these splice products have specific tissue and cell type expression patterns and exhibit differences in receptor properties. Keywords: Piezo, touch, sensation, ion-channel, splicing

  4. Innovative approaches to cell biomechanics from cell migration to on-chip manipulation

    CERN Document Server

    Okeyo, Kennedy Omondi; Adachi, Taiji

    2015-01-01

    This book covers topics on mechanosensing, mechanotransduction, and actin cytoskeletal dynamics in cell motility. It will contribute to a better understanding of how cells functionally adapt to their mechanical environment as well as highlighting fundamental concepts for designing material niches for cell manipulation. With topics from multidisciplinary fields of the life sciences, medicine, and engineering, the book is the first of its kind, providing comprehensive, integrated coverage of innovative approaches to cell biomechanics. It provides a valuable resource for seniors and graduate students studying cell biomechanics, and is also suitable for researchers interested in the application of methods and strategies in connection with the innovative approaches discussed. Each section of the book has been supplemented with concrete examples and illustrations to facilitate understanding even for readers unfamiliar with cell biomechanics.

  5. Usher protein functions in hair cells and photoreceptors.

    Science.gov (United States)

    Cosgrove, Dominic; Zallocchi, Marisa

    2014-01-01

    The 10 different genes associated with the deaf/blind disorder, Usher syndrome, encode a number of structurally and functionally distinct proteins, most expressed as multiple isoforms/protein variants. Functional characterization of these proteins suggests a role in stereocilia development in cochlear hair cells, likely owing to adhesive interactions in hair bundles. In mature hair cells, homodimers of the Usher cadherins, cadherin 23 and protocadherin 15, interact to form a structural fiber, the tip link, and the linkages that anchor the taller stereocilia's actin cytoskeleton core to the shorter adjacent stereocilia and the elusive mechanotransduction channels, explaining the deafness phenotype when these molecular interactions are perturbed. The conundrum is that photoreceptors lack a synonymous mechanotransduction apparatus, and so a common theory for Usher protein function in the two neurosensory cell types affected in Usher syndrome is lacking. Recent evidence linking photoreceptor cell dysfunction in the shaker 1 mouse model for Usher syndrome to light-induced protein translocation defects, combined with localization of an Usher protein interactome at the periciliary region of the photoreceptors suggests Usher proteins might regulate protein trafficking between the inner and outer segments of photoreceptors. A distinct Usher protein complex is trafficked to the ribbon synapses of hair cells, and synaptic defects have been reported in Usher mutants in both hair cells and photoreceptors. This review aims to clarify what is known about Usher protein function at the synaptic and apical poles of hair cells and photoreceptors and the prospects for identifying a unifying pathobiological mechanism to explain deaf/blindness in Usher syndrome. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Merkel cells and neurons keep in touch

    Science.gov (United States)

    Woo, Seung-Hyun; Lumpkin, Ellen A.; Patapoutian, Ardem

    2014-01-01

    The Merkel cell-neurite complex is a unique vertebrate touch receptor comprising two distinct cell types in the skin. Its presence in touch-sensitive skin areas was recognized more than a century ago, but the functions of each cell type in sensory transduction have been unclear. Three recent studies demonstrate that Merkel cells are mechanosensitive cells that function in touch transduction via Piezo2. One study concludes that Merkel cells rather than sensory neurons are principal sites of mechanotransduction, whereas the other two studies report that both Merkel cells and neurons encode mechanical inputs. Together, these studies settle a longstanding debate on whether Merkel cells are mechanosensory cells, and enable future investigations of how these skin cells communicate with neurons. PMID:25480024

  7. 17β estradiol regulation of connexin 43-based gap junction and mechanosensitivity through classical estrogen receptor pathway in osteocyte-like MLO-Y4 cells.

    KAUST Repository

    Ren, Jian; Wang, Xuhui; Wang, Guangchao; Wu, Junhua

    2013-01-01

    Connexin 43 (Cx43) plays an essential role in osteocyte mechanotransduction. Although estrogen involves in the adaptive responses of bone cells to mechanical loadings, its effects on osteocytic Cx43-based gap junction intercellular communication

  8. Mechanosensing of matrix by stem cells: From matrix heterogeneity, contractility, and the nucleus in pore-migration to cardiogenesis and muscle stem cells in vivo.

    Science.gov (United States)

    Smith, Lucas; Cho, Sangkyun; Discher, Dennis E

    2017-11-01

    Stem cells are particularly 'plastic' cell types that are induced by various cues to become specialized, tissue-functional lineages by switching on the expression of specific gene programs. Matrix stiffness is among the cues that multiple stem cell types can sense and respond to. This seminar-style review focuses on mechanosensing of matrix elasticity in the differentiation or early maturation of a few illustrative stem cell types, with an intended audience of biologists and physical scientists. Contractile forces applied by a cell's acto-myosin cytoskeleton are often resisted by the extracellular matrix and transduced through adhesions and the cytoskeleton ultimately into the nucleus to modulate gene expression. Complexity is added by matrix heterogeneity, and careful scrutiny of the evident stiffness heterogeneity in some model systems resolves some controversies concerning matrix mechanosensing. Importantly, local stiffness tends to dominate, and 'durotaxis' of stem cells toward stiff matrix reveals a dependence of persistent migration on myosin-II force generation and also rigid microtubules that confer directionality. Stem and progenitor cell migration in 3D can be further affected by matrix porosity as well as stiffness, with nuclear size and rigidity influencing niche retention and fate choices. Cell squeezing through rigid pores can even cause DNA damage and genomic changes that contribute to de-differentiation toward stem cell-like states. Contraction of acto-myosin is the essential function of striated muscle, which also exhibit mechanosensitive differentiation and maturation as illustrated in vivo by beating heart cells and by the regenerative mobilization of skeletal muscle stem cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Role of YAP/TAZ in cell-matrix adhesion-mediated signalling and mechanotransduction

    International Nuclear Information System (INIS)

    Dupont, Sirio

    2016-01-01

    Signalling from the extracellular matrix (ECM) is a fundamental cellular input that sustains proliferation, opposes cell death and regulates differentiation. Through integrins, cells perceive both the chemical composition and physical properties of the ECM. In particular, cell behaviour is profoundly influenced by the mechanical elasticity or stiffness of the ECM, which regulates the ability of cells to develop forces through their contractile actomyosin cytoskeleton and to mature focal adhesions. This mechanosensing ability affects fundamental cellular functions, such that alterations of ECM stiffness is nowadays considered not a simple consequence of pathology, but a causative input driving aberrant cell behaviours. We here discuss recent advances on how mechanical signals intersect nuclear transcription and in particular the activity of YAP/TAZ transcriptional coactivators, known downstream transducers of the Hippo pathway and important effectors of ECM mechanical cues.

  10. Role of YAP/TAZ in cell-matrix adhesion-mediated signalling and mechanotransduction

    Energy Technology Data Exchange (ETDEWEB)

    Dupont, Sirio, E-mail: sirio.dupont@unipd.it

    2016-04-10

    Signalling from the extracellular matrix (ECM) is a fundamental cellular input that sustains proliferation, opposes cell death and regulates differentiation. Through integrins, cells perceive both the chemical composition and physical properties of the ECM. In particular, cell behaviour is profoundly influenced by the mechanical elasticity or stiffness of the ECM, which regulates the ability of cells to develop forces through their contractile actomyosin cytoskeleton and to mature focal adhesions. This mechanosensing ability affects fundamental cellular functions, such that alterations of ECM stiffness is nowadays considered not a simple consequence of pathology, but a causative input driving aberrant cell behaviours. We here discuss recent advances on how mechanical signals intersect nuclear transcription and in particular the activity of YAP/TAZ transcriptional coactivators, known downstream transducers of the Hippo pathway and important effectors of ECM mechanical cues.

  11. Mechanotransduction by bone cells in vitro: mechanobiology of bone tissue

    NARCIS (Netherlands)

    Mullender, M.; El Haj, A.J.; Yang, Y.; van Duin, M.A.; Burger, E.H.; Klein-Nulend, J.

    2004-01-01

    Mechanical force plays an important role in the regulation of bone remodelling in intact bone and bone repair. In vitro, bone cells demonstrate a high responsiveness to mechanical stimuli. Much debate exists regarding the critical components in the load profile and whether different components, such

  12. Biochemical analysis of force-sensitive responses using a large-scale cell stretch device.

    Science.gov (United States)

    Renner, Derrick J; Ewald, Makena L; Kim, Timothy; Yamada, Soichiro

    2017-09-03

    Physical force has emerged as a key regulator of tissue homeostasis, and plays an important role in embryogenesis, tissue regeneration, and disease progression. Currently, the details of protein interactions under elevated physical stress are largely missing, therefore, preventing the fundamental, molecular understanding of mechano-transduction. This is in part due to the difficulty isolating large quantities of cell lysates exposed to force-bearing conditions for biochemical analysis. We designed a simple, easy-to-fabricate, large-scale cell stretch device for the analysis of force-sensitive cell responses. Using proximal biotinylation (BioID) analysis or phospho-specific antibodies, we detected force-sensitive biochemical changes in cells exposed to prolonged cyclic substrate stretch. For example, using promiscuous biotin ligase BirA* tagged α-catenin, the biotinylation of myosin IIA increased with stretch, suggesting the close proximity of myosin IIA to α-catenin under a force bearing condition. Furthermore, using phospho-specific antibodies, Akt phosphorylation was reduced upon stretch while Src phosphorylation was unchanged. Interestingly, phosphorylation of GSK3β, a downstream effector of Akt pathway, was also reduced with stretch, while the phosphorylation of other Akt effectors was unchanged. These data suggest that the Akt-GSK3β pathway is force-sensitive. This simple cell stretch device enables biochemical analysis of force-sensitive responses and has potential to uncover molecules underlying mechano-transduction.

  13. Cell and Tissue Engineering

    CERN Document Server

    2012-01-01

    Cell and Tissue Engineering” introduces the principles and new approaches in cell and tissue engineering. It includes both the fundamentals and the current trends in cell and tissue engineering, in a way useful both to a novice and an expert in the field. The book is composed of 13 chapters all of which are written by the leading experts. It is organized to gradually assemble an insight in cell and tissue function starting form a molecular nano-level, extending to a cellular micro-level and finishing at the tissue macro-level. In specific, biological, physiological, biophysical, biochemical, medical, and engineering aspects are covered from the standpoint of the development of functional substitutes of biological tissues for potential clinical use. Topics in the area of cell engineering include cell membrane biophysics, structure and function of the cytoskeleton, cell-extracellular matrix interactions, and mechanotransduction. In the area of tissue engineering the focus is on the in vitro cultivation of ...

  14. Materials as stem cell regulators

    Science.gov (United States)

    Murphy, William L.; McDevitt, Todd C.; Engler, Adam J.

    2014-01-01

    The stem cell/material interface is a complex, dynamic microenvironment in which the cell and the material cooperatively dictate one another's fate: the cell by remodelling its surroundings, and the material through its inherent properties (such as adhesivity, stiffness, nanostructure or degradability). Stem cells in contact with materials are able to sense their properties, integrate cues via signal propagation and ultimately translate parallel signalling information into cell fate decisions. However, discovering the mechanisms by which stem cells respond to inherent material characteristics is challenging because of the highly complex, multicomponent signalling milieu present in the stem cell environment. In this Review, we discuss recent evidence that shows that inherent material properties may be engineered to dictate stem cell fate decisions, and overview a subset of the operative signal transduction mechanisms that have begun to emerge. Further developments in stem cell engineering and mechanotransduction are poised to have substantial implications for stem cell biology and regenerative medicine. PMID:24845994

  15. Regulation of fibrochondrogenesis of mesenchymal stem cells in an integrated microfluidic platform embedded with biomimetic nanofibrous scaffolds.

    Directory of Open Access Journals (Sweden)

    Weiliang Zhong

    Full Text Available In native fibrocartilage, mechanotransduction allows the cells to perceive the physical microenvironment not only through topographical cues from the extracellular matrix, but also through mechanical cues, such as interstitial flow. To create a microenvironment that simultaneously integrates nanotopography and flow stimulus, we developed a biomimetic microfluidic device embedded with aligned nanofibers to contain microchambers of different angles, which enabled the flow direction to form different angles with the fibers. Using this device, we investigated the effects of microfluidic and nanotopographical environment on the morphology and fibrochondrogenesis of mesenchymal stem cells (MSCs and the involvement of RhoA/ROCK pathway and Yes-associated protein (YAP/transcriptional co-activator with PDZ-binding motif (TAZ. The results showed that the flow direction perpendicular to aligned nanofibers was conducive to fibrochondrogenesis of MSCs. In addition, ROCK inhibitor and knockdown of YAP/TAZ disrupted fibrochondrogenic differentiation of MSCs. In conclusion, our data suggest the crucial role of mechanotransduction in regulating fibrochondrogenic differentiation of MSCs, which may be mediated by RhoA/ROCK pathway and YAP/TAZ.

  16. Nuclear Mechanics and Stem Cell Differentiation.

    Science.gov (United States)

    Mao, Xinjian; Gavara, Nuria; Song, Guanbin

    2015-12-01

    Stem cells are characterized by their self-renewal and multi-lineage differentiation potential. Stem cell differentiation is a prerequisite for the application of stem cells in regenerative medicine and clinical therapy. In addition to chemical stimulation, mechanical cues play a significant role in regulating stem cell differentiation. The integrity of mechanical sensors is necessary for the ability of cells to respond to mechanical signals. The nucleus, the largest and stiffest cellular organelle, interacts with the cytoskeleton as a key mediator of cell mechanics. Nuclear mechanics are involved in the complicated interactions of lamins, chromatin and nucleoskeleton-related proteins. Thus, stem cell differentiation is intimately associated with nuclear mechanics due to its indispensable role in mechanotransduction and mechanical response. This paper reviews several main contributions of nuclear mechanics, highlights the hallmarks of the nuclear mechanics of stem cells, and provides insight into the relationship between nuclear mechanics and stem cell differentiation, which may guide clinical applications in the future.

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

    Science.gov (United States)

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

    2016-02-08

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

  18. A protein interaction mechanism for suppressing the mechanosensitive Piezo channels

    OpenAIRE

    Zhang, Tingxin; Chi, Shaopeng; Jiang, Fan; Zhao, Qiancheng; Xiao, Bailong

    2017-01-01

    Piezo proteins are bona fide mammalian mechanotransduction channels for various cell types including endothelial cells. The mouse Piezo1 of 2547 residues forms a three-bladed, propeller-like homo-trimer comprising a central pore-module and three propeller-structures that might serve as mechanotransduction-modules. However, the mechanogating and regulation of Piezo channels remain unclear. Here we identify the sarcoplasmic /endoplasmic-reticulum Ca2+ ATPase (SERCA), including the widely expres...

  19. Non-lethal heat treatment of cells results in reduction of tumor initiation and metastatic potential

    International Nuclear Information System (INIS)

    Kim, Yoo-Shin; Lee, Tae Hoon; O'Neill, Brian E.

    2015-01-01

    Non-lethal hyperthermia is used clinically as adjuvant treatment to radiation, with mixed results. Denaturation of protein during hyperthermia treatment is expected to synergize with radiation damage to cause cell cycle arrest and apoptosis. Alternatively, hyperthermia is known to cause tissue level changes in blood flow, increasing the oxygenation and radiosensitivity of often hypoxic tumors. In this study, we elucidate a third possibility, that hyperthermia alters cellular adhesion and mechanotransduction, with particular impact on the cancer stem cell population. We demonstrate that cell heating results in a robust but temporary loss of cancer cell aggressiveness and metastatic potential in mouse models. In vitro, this heating results in a temporary loss in cell mobility, adhesion, and proliferation. Our hypothesis is that the loss of cellular adhesion results in suppression of cancer stem cells and loss of tumor virulence and metastatic potential. Our study suggests that the metastatic potential of cancer is particularly reduced by the effects of heat on cellular adhesion and mechanotransduction. If true, this could help explain both the successes and failures of clinical hyperthermia, and suggest ways to target treatments to those who would most benefit. - Highlights: • Non-lethal hyperthermia treatment of cancer cells is shown to cause a reduction in rates of tumor initiation and metastasis. • Dynamic imaging of cells during heat treatment shows temporary changes in cell shape, cell migration, and cell proliferation. • Loss of adhesion may lead to the observed effect, which may disproportionately impact the tumor initiating cell fraction. • Loss or suppression of the tumor initiating cell fraction results in the observed loss of metastatic potential in vivo. • This result may lead to new approaches to synergizing hyperthermia with surgery, radiation, and chemotherapy

  20. Non-lethal heat treatment of cells results in reduction of tumor initiation and metastatic potential

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yoo-Shin; Lee, Tae Hoon; O' Neill, Brian E., E-mail: BEOneill@houstonmethodist.org

    2015-08-14

    Non-lethal hyperthermia is used clinically as adjuvant treatment to radiation, with mixed results. Denaturation of protein during hyperthermia treatment is expected to synergize with radiation damage to cause cell cycle arrest and apoptosis. Alternatively, hyperthermia is known to cause tissue level changes in blood flow, increasing the oxygenation and radiosensitivity of often hypoxic tumors. In this study, we elucidate a third possibility, that hyperthermia alters cellular adhesion and mechanotransduction, with particular impact on the cancer stem cell population. We demonstrate that cell heating results in a robust but temporary loss of cancer cell aggressiveness and metastatic potential in mouse models. In vitro, this heating results in a temporary loss in cell mobility, adhesion, and proliferation. Our hypothesis is that the loss of cellular adhesion results in suppression of cancer stem cells and loss of tumor virulence and metastatic potential. Our study suggests that the metastatic potential of cancer is particularly reduced by the effects of heat on cellular adhesion and mechanotransduction. If true, this could help explain both the successes and failures of clinical hyperthermia, and suggest ways to target treatments to those who would most benefit. - Highlights: • Non-lethal hyperthermia treatment of cancer cells is shown to cause a reduction in rates of tumor initiation and metastasis. • Dynamic imaging of cells during heat treatment shows temporary changes in cell shape, cell migration, and cell proliferation. • Loss of adhesion may lead to the observed effect, which may disproportionately impact the tumor initiating cell fraction. • Loss or suppression of the tumor initiating cell fraction results in the observed loss of metastatic potential in vivo. • This result may lead to new approaches to synergizing hyperthermia with surgery, radiation, and chemotherapy.

  1. Impact of mechanical stretch on the cell behaviors of bone and surrounding tissues

    Directory of Open Access Journals (Sweden)

    Hye-Sun Yu

    2016-02-01

    Full Text Available Mechanical loading is recognized to play an important role in regulating the behaviors of cells in bone and surrounding tissues in vivo. Many in vitro studies have been conducted to determine the effects of mechanical loading on individual cell types of the tissues. In this review, we focus specifically on the use of the Flexercell system as a tool for studying cellular responses to mechanical stretch. We assess the literature describing the impact of mechanical stretch on different cell types from bone, muscle, tendon, ligament, and cartilage, describing individual cell phenotype responses. In addition, we review evidence regarding the mechanotransduction pathways that are activated to potentiate these phenotype responses in different cell populations.

  2. Impact of mechanical stretch on the cell behaviors of bone and surrounding tissues

    Science.gov (United States)

    Yu, Hye-Sun; Kim, Jung-Ju; Kim, Hae-Won; Lewis, Mark P; Wall, Ivan

    2016-01-01

    Mechanical loading is recognized to play an important role in regulating the behaviors of cells in bone and surrounding tissues in vivo. Many in vitro studies have been conducted to determine the effects of mechanical loading on individual cell types of the tissues. In this review, we focus specifically on the use of the Flexercell system as a tool for studying cellular responses to mechanical stretch. We assess the literature describing the impact of mechanical stretch on different cell types from bone, muscle, tendon, ligament, and cartilage, describing individual cell phenotype responses. In addition, we review evidence regarding the mechanotransduction pathways that are activated to potentiate these phenotype responses in different cell populations. PMID:26977284

  3. Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development.

    Science.gov (United States)

    Guan, Yingjie; Yang, Xu; Yang, Wentian; Charbonneau, Cherie; Chen, Qian

    2014-10-01

    Mechanical stress regulates development by modulating cell signaling and gene expression. However, the cytoplasmic components mediating mechanotransduction remain unclear. In this study, elimination of muscle contraction during chicken embryonic development resulted in a reduction in the activity of mammalian target of rapamycin (mTOR) in the cartilaginous growth plate. Inhibition of mTOR activity led to significant inhibition of chondrocyte proliferation, cartilage tissue growth, and expression of chondrogenic genes, including Indian hedgehog (Ihh), a critical mediator of mechanotransduction. Conversely, cyclic loading (1 Hz, 5% matrix deformation) of embryonic chicken growth plate chondrocytes in 3-dimensional (3D) collagen scaffolding induced sustained activation of mTOR. Mechanical activation of mTOR occurred in serum-free medium, indicating that it is independent of growth factor or nutrients. Treatment of chondrocytes with Rapa abolished mechanical activation of cell proliferation and Ihh gene expression. Cyclic loading of chondroprogenitor cells deficient in SH2-containing protein tyrosine phosphatase 2 (Shp2) further enhanced mechanical activation of mTOR, cell proliferation, and chondrogenic gene expression. This result suggests that Shp2 is an antagonist of mechanotransduction through inhibition of mTOR activity. Our data demonstrate that mechanical activation of mTOR is necessary for cell proliferation, chondrogenesis, and cartilage growth during bone development, and that mTOR is an essential mechanotransduction component modulated by Shp2 in the cytoplasm. © FASEB.

  4. Mutations in ap1b1 cause mistargeting of the Na(+/K(+-ATPase pump in sensory hair cells.

    Directory of Open Access Journals (Sweden)

    Rachel Clemens Grisham

    Full Text Available The hair cells of the inner ear are polarized epithelial cells with a specialized structure at the apical surface, the mechanosensitive hair bundle. Mechanotransduction occurs within the hair bundle, whereas synaptic transmission takes place at the basolateral membrane. The molecular basis of the development and maintenance of the apical and basal compartments in sensory hair cells is poorly understood. Here we describe auditory/vestibular mutants isolated from forward genetic screens in zebrafish with lesions in the adaptor protein 1 beta subunit 1 (ap1b1 gene. Ap1b1 is a subunit of the adaptor complex AP-1, which has been implicated in the targeting of basolateral membrane proteins. In ap1b1 mutants we observed that although the overall development of the inner ear and lateral-line organ appeared normal, the sensory epithelium showed progressive signs of degeneration. Mechanically-evoked calcium transients were reduced in mutant hair cells, indicating that mechanotransduction was also compromised. To gain insight into the cellular and molecular defects in ap1b1 mutants, we examined the localization of basolateral membrane proteins in hair cells. We observed that the Na(+/K(+-ATPase pump (NKA was less abundant in the basolateral membrane and was mislocalized to apical bundles in ap1b1 mutant hair cells. Accordingly, intracellular Na(+ levels were increased in ap1b1 mutant hair cells. Our results suggest that Ap1b1 is essential for maintaining integrity and ion homeostasis in hair cells.

  5. Nanotopographical Control of Stem Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Laura E. McNamara

    2010-01-01

    Full Text Available Stem cells have the capacity to differentiate into various lineages, and the ability to reliably direct stem cell fate determination would have tremendous potential for basic research and clinical therapy. Nanotopography provides a useful tool for guiding differentiation, as the features are more durable than surface chemistry and can be modified in size and shape to suit the desired application. In this paper, nanotopography is examined as a means to guide differentiation, and its application is described in the context of different subsets of stem cells, with a particular focus on skeletal (mesenchymal stem cells. To address the mechanistic basis underlying the topographical effects on stem cells, the likely contributions of indirect (biochemical signal-mediated and direct (force-mediated mechanotransduction are discussed. Data from proteomic research is also outlined in relation to topography-mediated fate determination, as this approach provides insight into the global molecular changes at the level of the functional effectors.

  6. Low-Intensity Pulsed Ultrasound Enhances Nerve Growth Factor-Induced Neurite Outgrowth through Mechanotransduction-Mediated ERK1/2-CREB-Trx-1 Signaling.

    Science.gov (United States)

    Zhao, Lu; Feng, Yi; Hu, Hong; Shi, Aiwei; Zhang, Lei; Wan, Mingxi

    2016-12-01

    Enhancing the action of nerve growth factor (NGF) is a potential therapeutic approach to neural regeneration. To facilitate neural regeneration, we investigated whether combining low-intensity pulsed ultrasound (LIPUS) and NGF could promote neurite outgrowth, an essential process in neural regeneration. In the present study, PC12 cells were subjected to a combination of LIPUS (1 MHz, 30 or 50 mW/cm 2 , 20% duty cycle and 100-Hz pulse repetition frequency, 10 min every other day) and NGF (50 ng/mL) treatment, and then neurite outgrowth was compared. Our findings indicated that the combined treatment with LIPUS (50 mW/cm 2 ) and NGF (50 ng/mL) promotes neurite outgrowth that is comparable to that achieved by NGF (100 ng/mL) treatment alone. LIPUS significantly increased NGF-induced neurite length, but not neurite branching. These effects were attributed to the enhancing effects of LIPUS on NGF-induced phosphorylation of ERK1/2 and CREB and the expression of thioredoxin (Trx-1). Furthermore, blockage of stretch-activated ion channels with Gd 3+ suppressed the stimulating effects of LIPUS on NGF-induced neurite outgrowth and the downstream signaling activation. Taken together, our findings suggest that LIPUS enhances NGF-induced neurite outgrowth through mechanotransduction-mediated signaling of the ERK1/2-CREB-Trx-1 pathway. The combination of LIPUS and NGF could potentially be used for the treatment of nerve injury and neurodegenerative diseases. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  7. Computational simulation of static/cyclic cell stimulations to investigate mechanical modulation of an individual mesenchymal stem cell using confocal microscopy

    International Nuclear Information System (INIS)

    Alihemmati, Zakieh; Vahidi, Bahman; Haghighipour, Nooshin; Salehi, Mohammad

    2017-01-01

    It has been found that cells react to mechanical stimuli, while the type and magnitude of these cells are different in various physiological and pathological conditions. These stimuli may affect cell behaviors via mechanotransduction mechanisms. The aim of this study is to evaluate mechanical responses of a mesenchymal stem cell (MSC) to a pressure loading using finite elements method (FEM) to clarify procedures of MSC mechanotransduction. The model is constructed based on an experimental set up in which statics and cyclic compressive loads are implemented on a model constructed from a confocal microscopy 3D image of a stem cell. Both of the applied compressive loads are considered in the physiological loading regimes. Moreover, a viscohyperelastic material model was assumed for the cell through which the finite elements simulation anticipates cell behavior based on strain and stress distributions in its components. As a result, high strain and stress values were captured from the viscohyperelastic model because of fluidic behavior of cytosol when compared with the obtained results through the hyperelastic models. It can be concluded that the generated strain produced by cyclic pressure is almost 8% higher than that caused by the static load and the von Mises stress distribution is significantly increased to about 150 kPa through the cyclic loading. In total, the results does not only trace the efficacy of an individual 3D model of MSC using biomechanical experiments of cell modulation, but these results provide knowledge in interpretations from cell geometry. The current study was performed to determine a realistic aspect of cell behavior. - Graphical abstract: Based on confocal microscopy images and through finite elements analysis, we simulate mechanical behavior of the stem cell components (the cell membrane, cytoplasm and nucleus) under a compressive load. A major novelty of this investigation is the usage of viscohyperelastic behavior for the realistic stem

  8. Cell and Tissue Organization in the Circulatory and Ventilatory Systems Volume 1 Signaling in Cell Organization, Fate, and Activity, Part A Cell Structure and Environment

    CERN Document Server

    Thiriet, Marc

    2011-01-01

    The volumes in this authoritative series present a multidisciplinary approach to modeling and simulation of flows in the cardiovascular and ventilatory systems, especially multiscale modeling and coupled simulations. The cardiovascular and respiratory systems are tightly coupled, as their primary function is to supply oxygen to and remove carbon dioxide from the body's cells. Because physiological conduits have deformable and reactive walls, macroscopic flow behavior and prediction must be coupled to nano- and microscopic events in a corrector scheme of regulated mechanisms. Therefore, investigation of flows of blood and air in physiological conduits requires an understanding of the biology, chemistry, and physics of these systems together with the mathematical tools to describe their functioning.  The present volume is devoted to cellular events that allow adaptation to environmental conditions, particularly mechanotransduction. It begins with cell organization and a survey of cell types in the vasculatur...

  9. Micromechanical and surface adhesive properties of single saccharomyces cerevisiae cells

    Science.gov (United States)

    Farzi, Bahman; Cetinkaya, Cetin

    2017-09-01

    The adhesion and mechanical properties of a biological cell (e.g. cell membrane elasticity and adhesiveness) are often strong indicators for the state of its health. Many existing techniques for determining mechanical properties of cells require direct physical contact with a single cell or a group of cells. Physical contact with the cell can trigger complex mechanotransduction mechanisms, leading to cellular responses, and consequently interfering with measurement accuracy. In the current work, based on ultrasonic excitation and interferometric (optical) motion detection, a non-contact method for characterizing the adhesion and mechanical properties of single cells is presented. It is experimentally demonstrated that the rocking (rigid body) motion and internal vibrational resonance frequencies of a single saccharomyces cerevisiae (SC) (baker’s yeast) cell can be acquired with the current approach, and the Young’s modulus and surface tension of the cell membrane as well as surface adhesion energy can be extracted from the values of these acquired resonance frequencies. The detected resonance frequency ranges for single SC cells include a rocking (rigid body) frequency of 330  ±  70 kHz and two breathing resonance frequencies of 1.53  ±  0.12 and 2.02  ±  0.31 MHz. Based on these values, the average work-of-adhesion of SC cells on a silicon substrate in aqueous medium is extracted, for the first time, as WASC-Si=16.2+/- 3.8 mJ {{m}-2} . Similarly, the surface tension and the Young’s modulus of the SC cell wall are predicted as {{σ }SC}=0.16+/- 0.02 N {{m}-1} and {{E}SC}= 9.20  ±  2.80 MPa, respectively. These results are compared to those reported in the literature by utilizing various methods, and good agreements are found. The current approach eliminates the measurement inaccuracies associated with the physical contact. Exciting and detecting cell dynamics at micro-second time-scales is significantly faster than the

  10. Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Kim, Jong Hyun; Kim, Hyung Woo; Cha, Kyoung Je; Han, Jiyou; Jang, Yu Jin; Kim, Dong Sung; Kim, Jong-Hoon

    2016-03-22

    Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells.

  11. [CHARACTERISTICS OF OSTEOCYTE CELL LINES FROM BONES FORMED AS A RESULT OF MEMBRANOUS (SKULL BONES) AND CHONDRAL (LONG BONES) OSSIFICATION].

    Science.gov (United States)

    Avrunin, A S; Doktorov, A A

    2016-01-01

    The aim of this work was to analyze the literature data and the results of authors' own research, to answer the question--if the osteocytes of bone tissues resulting from membranous and chondral ossification, belong to one or to different cell lines. The differences between the cells of osteocyte lines derived from bones resulting from membranous and chondral ossification were established in: 1) the magnitude of the mechanical signal, initiating the development of the process of mechanotransduction; 2) the nature of the relationship between the magnitude of the mechanical signal that initiates the reorganization of the architecture of bone structures and the resource of their strength; in membranous bones significantly lower mechanical signal caused a substantially greater increment of bone strength resource; 3) the biological activity of bone structures, bone fragments formed from membranous tissue were more optimal for transplantation; 4) the characteristics of expression of functional markers of bone cells at different stages of their differentiation; 5) the nature of the reaction of bone cells to mechanical stress; 6) the sensitivity of bone cells to one of the factors controlling the process of mechanotransduction (PGI2); 7) the functioning of osteocytes during lactation. These differences reflect the functional requirements to the bones of the skeleton--the supporting function in the bones of the limbs and the shaping and protection in the bones of the cranial vault. These data suggest that the results of research conducted on the bones of the skull, should not be transferred to the entire skeleton as a whole.

  12. Stimulation of hair cells with ultraviolet light

    Science.gov (United States)

    Azimzadeh, Julien B.; Fabella, Brian A.; Hudspeth, A. J.

    2018-05-01

    Hair bundles are specialized organelles that transduce mechanical inputs into electrical outputs. To activate hair cells, physiologists have resorted to mechanical methods of hair-bundle stimulation. Here we describe a new method of hair-bundle stimulation, irradiation with ultraviolet light. A hair bundle illuminated by ultraviolet light rapidly moves towards its tall edge, a motion typically associated with excitatory stimulation. The motion disappears upon tip-link rupture and is associated with the opening of mechanotransduction channels. Hair bundles can be induced to move sinusoidally with oscillatory modulation of the stimulation power. We discuss the implications of ultraviolet stimulation as a novel hair-bundle stimulus.

  13. A protein interaction mechanism for suppressing the mechanosensitive Piezo channels.

    Science.gov (United States)

    Zhang, Tingxin; Chi, Shaopeng; Jiang, Fan; Zhao, Qiancheng; Xiao, Bailong

    2017-11-27

    Piezo proteins are bona fide mammalian mechanotransduction channels for various cell types including endothelial cells. The mouse Piezo1 of 2547 residues forms a three-bladed, propeller-like homo-trimer comprising a central pore-module and three propeller-structures that might serve as mechanotransduction-modules. However, the mechanogating and regulation of Piezo channels remain unclear. Here we identify the sarcoplasmic /endoplasmic-reticulum Ca 2+ ATPase (SERCA), including the widely expressed SERCA2, as Piezo interacting proteins. SERCA2 strategically suppresses Piezo1 via acting on a 14-residue-constituted intracellular linker connecting the pore-module and mechanotransduction-module. Mutating the linker impairs mechanogating and SERCA2-mediated modulation of Piezo1. Furthermore, the synthetic linker-peptide disrupts the modulatory effects of SERCA2, demonstrating the key role of the linker in mechanogating and regulation. Importantly, the SERCA2-mediated regulation affects Piezo1-dependent migration of endothelial cells. Collectively, we identify SERCA-mediated regulation of Piezos and the functional significance of the linker, providing important insights into the mechanogating and regulation mechanisms of Piezo channels.

  14. Actin retrograde flow controls natural killer cell response by regulating the conformation state of SHP-1.

    Science.gov (United States)

    Matalon, Omri; Ben-Shmuel, Aviad; Kivelevitz, Jessica; Sabag, Batel; Fried, Sophia; Joseph, Noah; Noy, Elad; Biber, Guy; Barda-Saad, Mira

    2018-03-01

    Natural killer (NK) cells are a powerful weapon against viral infections and tumor growth. Although the actin-myosin (actomyosin) cytoskeleton is crucial for a variety of cellular processes, the role of mechanotransduction, the conversion of actomyosin mechanical forces into signaling cascades, was never explored in NK cells. Here, we demonstrate that actomyosin retrograde flow (ARF) controls the immune response of primary human NK cells through a novel interaction between β-actin and the SH2-domain-containing protein tyrosine phosphatase-1 (SHP-1), converting its conformation state, and thereby regulating NK cell cytotoxicity. Our results identify ARF as a master regulator of the NK cell immune response. Since actin dynamics occur in multiple cellular processes, this mechanism might also regulate the activity of SHP-1 in additional cellular systems. © 2018 The Authors.

  15. Mechanically induced intracellular calcium waves in osteoblasts demonstrate calcium fingerprints in bone cell mechanotransduction.

    Science.gov (United States)

    Godin, Lindsay M; Suzuki, Sakiko; Jacobs, Christopher R; Donahue, Henry J; Donahue, Seth W

    2007-11-01

    An early response to mechanical stimulation of bone cells in vitro is an increase in intracellular calcium concentration ([Ca (2+)](i)). This study analyzed the [Ca (2+)](i) wave area, magnitude, duration, rise time, fall time, and time to onset in individual osteoblasts for two identical bouts of mechanical stimulation separated by a 30-min rest period. The area under the [Ca (2+)](i) wave increased in the second loading bout compared to the first. This suggests that rest periods may potentiate mechanically induced intracellular calcium signals. Furthermore, many of the [Ca (2+)](i) wave parameters were strongly, positively correlated between the two bouts of mechanical stimulation. For example, in individual primary osteoblasts, if a cell had a large [Ca (2+)](i) wave area in the first bout it was likely to have a large [Ca (2+)](i) wave area in the second bout (r (2) = 0.933). These findings support the idea that individual bone cells have "calcium fingerprints" (i.e., a unique [Ca (2+)](i) wave profile that is reproducible for repeated exposure to a given stimulus).

  16. Cellular Mechanosensing: Getting to the nucleus of it all

    Science.gov (United States)

    Fedorchak, Gregory R.; Kaminski, Ashley; Lammerding, Jan

    2014-01-01

    Cells respond to mechanical forces by activating specific genes and signaling pathways that allow the cells to adapt to their physical environment. Examples include muscle growth in response to exercise, bone remodeling based on their mechanical load, or endothelial cells aligning under fluid shear stress. While the involved downstream signaling pathways and mechanoresponsive genes are generally well characterized, many of the molecular mechanisms of the initiating ‘mechanosensing’ remain still elusive. In this review, we discuss recent findings and accumulating evidence suggesting that the cell nucleus plays a crucial role in cellular mechanotransduction, including processing incoming mechanoresponsive signals and even directly responding to mechanical forces. Consequently, mutations in the involved proteins or changes in nuclear envelope composition can directly impact mechanotransduction signaling and contribute to the development and progression of a variety of human diseases, including muscular dystrophy, cancer, and the focus of this review, dilated cardiomyopathy. Improved insights into the molecular mechanisms underlying nuclear mechanotransduction, brought in part by the emergence of new technologies to study intracellular mechanics at high spatial and temporal resolution, will not only result in a better understanding of cellular mechanosensing in normal cells but may also lead to the development of novel therapies in the many diseases linked to defects in nuclear envelope proteins. PMID:25008017

  17. Mechanotransduction and the Cytoskeleton

    Science.gov (United States)

    Pickard, Barbara G.

    1996-01-01

    Two intellectual developments in the lab suggested a more powerful approach to the problem defined in the original statement. These were discussed with the "Program Chief', who agreed that an alteration of approach was desirable. Second expansion of our work was on the computational optical sectioning microscope (COSM). Additionally, we hoped to visualize the channels with respect to cytoskeletal entities. We have identified major here-to-fore unknown cytoskeletal structure in our representative experimental system, the onion epidermal cell, and have named this structure the endomembrane grant.

  18. A supramolecular look at microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny

    Directory of Open Access Journals (Sweden)

    Marcela Aldrovani

    Full Text Available ABSTRACT Various approaches have been taken to improve our knowledge of the microenvironmental regulation of limbal epithelial stem cells. Researchers have extensively investigated the roles of growth factors, survival factors, cytokines, enzymes, and permeable molecules secreted by the limbal cells. However, recent evidence suggests that stem cell fate (i.e., self-renewal or differentiation can also be influenced by biophysical and mechanical cues related to the supramolecular organization and the liquid crystalline (mesophase nature of the stromal extracellular matrix. These cues can be sensed by stem cells and transduced into intracellular biochemical and functional responses, a process known as mechanotransduction. The objective of this review is to offer perspectives on the supramolecular microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny.

  19. Classification of Hydrogels Based on Their Source: A Review and Application in Stem Cell Regulation

    Science.gov (United States)

    Khansari, Maziyar M.; Sorokina, Lioudmila V.; Mukherjee, Prithviraj; Mukhtar, Farrukh; Shirdar, Mostafa Rezazadeh; Shahidi, Mahnaz; Shokuhfar, Tolou

    2017-08-01

    Stem cells are recognized by their self-renewal ability and can give rise to specialized progeny. Hydrogels are an established class of biomaterials with the ability to control stem cell fate via mechanotransduction. They can mimic various physiological conditions to influence the fate of stem cells and are an ideal platform to support stem cell regulation. This review article provides a summary of recent advances in the application of different classes of hydrogels based on their source (e.g., natural, synthetic, or hybrid). This classification is important because the chemistry of substrate affects stem cell differentiation and proliferation. Natural and synthetic hydrogels have been widely used in stem cell regulation. Nevertheless, they have limitations that necessitate a new class of material. Hybrid hydrogels obtained by manipulation of the natural and synthetic ones can potentially overcome these limitations and shape the future of research in application of hydrogels in stem cell regulation.

  20. SUN1 splice variants, SUN1_888, SUN1_785, and predominant SUN1_916, variably function in directional cell migration

    OpenAIRE

    Nishioka, Yu; Imaizumi, Hiromasa; Imada, Junko; Katahira, Jun; Matsuura, Nariaki; Hieda, Miki

    2016-01-01

    The LINC complex is a multifunctional protein complex that is involved in various processes at the nuclear envelope, such as nuclear migration, mechanotransduction and chromatin tethering in the meiotic phase. However, it remains unknown how these functions are regulated in different cell contexts. An inner nuclear membrane component of the LINC complex, SUN1, is ubiquitously expressed. The human SUN1 gene produces over 10 variants by alternative splicing. Although functions of SUN1 are relat...

  1. Extracellular matrix elasticity and topography: material-based cues that affect cell function via conserved mechanisms

    Science.gov (United States)

    Janson, Isaac A.; Putnam, Andrew J.

    2014-01-01

    Chemical, mechanical, and topographic extracellular matrix (ECM) cues have been extensively studied for their influence on cell behavior. These ECM cues alter cell adhesion, cell shape, and cell migration, and activate signal transduction pathways to influence gene expression, proliferation, and differentiation. ECM elasticity and topography, in particular, have emerged as material properties of intense focus based on strong evidence these physical cue can partially dictate stem cell differentiation. Cells generate forces to pull on their adhesive contacts, and these tractional forces appear to be a common element of cells’ responses to both elasticity and topography. This review focuses on recently published work that links ECM topography and mechanics and their influence on differentiation and other cell behaviors, We also highlight signaling pathways typically implicated in mechanotransduction that are (or may be) shared by cells subjected to topographic cues. Finally, we conclude with a brief discussion of the potential implications of these commonalities for cell based therapies and biomaterial design. PMID:24910444

  2. The modulation of stem cell behaviors by functionalized nanoceramic coatings on Ti-based implants

    Directory of Open Access Journals (Sweden)

    Xiangmei Liu

    2016-09-01

    Full Text Available Nanoceramic coating on the surface of Ti-based metallic implants is a clinical potential option in orthopedic surgery. Stem cells have been found to have osteogenic capabilities. It is necessary to study the influences of functionalized nanoceramic coatings on the differentiation and proliferation of stem cells in vitro or in vivo. In this paper, we summarized the recent advance on the modulation of stem cells behaviors through controlling the properties of nanoceramic coatings, including surface chemistry, surface roughness and microporosity. In addition, mechanotransduction pathways have also been discussed to reveal the interaction mechanisms between the stem cells and ceramic coatings on Ti-based metals. In the final part, the osteoinduction and osteoconduction of ceramic coating have been also presented when it was used as carrier of BMPs in new bone formation.

  3. Individually programmable cell stretching microwell arrays actuated by a Braille display.

    Science.gov (United States)

    Kamotani, Yoko; Bersano-Begey, Tommaso; Kato, Nobuhiro; Tung, Yi-Chung; Huh, Dongeun; Song, Jonathan W; Takayama, Shuichi

    2008-06-01

    Cell culture systems are often static and are therefore nonphysiological. In vivo, many cells are exposed to dynamic surroundings that stimulate cellular responses in a process known as mechanotransduction. To recreate this environment, stretchable cell culture substrate systems have been developed, however, these systems are limited by being macroscopic and low throughput. We have developed a device consisting of 24 miniature cell stretching chambers with flexible bottom membranes that are deformed using the computer-controlled, piezoelectrically actuated pins of a Braille display. We have also developed efficient image capture and analysis protocols to quantify morphological responses of the cells to applied strain. Human dermal microvascular endothelial cells (HDMECs) were found to show increasing degrees of alignment and elongation perpendicular to the radial strain in response to cyclic stretch at increasing frequencies of 0.2, 1, and 5 Hz, after 2, 4, and 12h. Mouse myogenic C2C12 cells were also found to align in response to the stretch, while A549 human lung adenocarcinoma epithelial cells did not respond to stretch.

  4. Arctigenin protects against neuronal hearing loss by promoting neural stem cell survival and differentiation.

    Science.gov (United States)

    Huang, Xinghua; Chen, Mo; Ding, Yan; Wang, Qin

    2017-03-01

    Neuronal hearing loss has become a prevalent health problem. This study focused on the function of arctigenin (ARC) in promoting survival and neuronal differentiation of mouse cochlear neural stem cells (NSCs), and its protection against gentamicin (GMC) induced neuronal hearing loss. Mouse cochlea was used to isolate NSCs, which were subsequently cultured in vitro. The effects of ARC on NSC survival, neurosphere formation, differentiation of NSCs, neurite outgrowth, and neural excitability in neuronal network in vitro were examined. Mechanotransduction ability demonstrated by intact cochlea, auditory brainstem response (ABR), and distortion product optoacoustic emissions (DPOAE) amplitude in mice were measured to evaluate effects of ARC on GMC-induced neuronal hearing loss. ARC increased survival, neurosphere formation, neuron differentiation of NSCs in mouse cochlear in vitro. ARC also promoted the outgrowth of neurites, as well as neural excitability of the NSC-differentiated neuron culture. Additionally, ARC rescued mechanotransduction capacity, restored the threshold shifts of ABR and DPOAE in our GMC ototoxicity murine model. This study supports the potential therapeutic role of ARC in promoting both NSCs proliferation and differentiation in vitro to functional neurons, thus supporting its protective function in the therapeutic treatment of neuropathic hearing loss in vivo. © 2017 Wiley Periodicals, Inc.

  5. Supplementation of exogenous adenosine 5'-triphosphate enhances mechanical properties of 3D cell-agarose constructs for cartilage tissue engineering.

    Science.gov (United States)

    Gadjanski, Ivana; Yodmuang, Supansa; Spiller, Kara; Bhumiratana, Sarindr; Vunjak-Novakovic, Gordana

    2013-10-01

    Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

  6. The structure of cell-matrix adhesions: the new frontier.

    Science.gov (United States)

    Hanein, Dorit; Horwitz, Alan Rick

    2012-02-01

    Adhesions between the cell and the extracellular matrix (ECM) are mechanosensitive multi-protein assemblies that transmit force across the cell membrane and regulate biochemical signals in response to the chemical and mechanical environment. These combined functions in force transduction, signaling and mechanosensing contribute to cellular phenotypes that span development, homeostasis and disease. These adhesions form, mature and disassemble in response to actin organization and physical forces that originate from endogenous myosin activity or external forces by the extracellular matrix. Despite advances in our understanding of the protein composition, interactions and regulation, our understanding of matrix adhesion structure and organization, how forces affect this organization, and how these changes dictate specific signaling events is limited. Insights across multiple structural levels are acutely needed to elucidate adhesion structure and ultimately the molecular basis of signaling and mechanotransduction. Here we describe the challenges and recent advances and prospects for unraveling the structure of cell-matrix adhesions and their response to force. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. The wall traction induced by flowing red blood cells in model microvessels and its potential mechanotransduction

    Science.gov (United States)

    Freund, Jonathan; Vermot, Julien

    2013-11-01

    There is evidence in early embryonic development, even well before advective oxygen transport is important, that the presence of red bloods cells per se trigger essential steps of normal vascular development. For example, showed that sequestration of blood cells early in the development of a mouse, such that the hematocrit is reduced, suppresses normal vascular network development. Vascular development also provides a model for remodeling and angiogenesis. We consider the transient stresses associated with blood cells flowing in model microvessels of comparable diameter to those at early stages of development (6 μm to 12 μm). A detailed simulation tool is used to show that passing blood cells present a significant fluctuating traction signature on the vessel wall, well above the mean stresses. This is particularly pronounced for slow flows (layer is also considered. NSF supported.

  8. Coupling primary and stem cell–derived cardiomyocytes in an in vitro model of cardiac cell therapy

    Science.gov (United States)

    Aratyn-Schaus, Yvonne; Pasqualini, Francesco S.; Yuan, Hongyan; McCain, Megan L.; Ye, George J.C.; Sheehy, Sean P.; Campbell, Patrick H.

    2016-01-01

    The efficacy of cardiac cell therapy depends on the integration of existing and newly formed cardiomyocytes. Here, we developed a minimal in vitro model of this interface by engineering two cell microtissues (μtissues) containing mouse cardiomyocytes, representing spared myocardium after injury, and cardiomyocytes generated from embryonic and induced pluripotent stem cells, to model newly formed cells. We demonstrated that weaker stem cell–derived myocytes coupled with stronger myocytes to support synchronous contraction, but this arrangement required focal adhesion-like structures near the cell–cell junction that degrade force transmission between cells. Moreover, we developed a computational model of μtissue mechanics to demonstrate that a reduction in isometric tension is sufficient to impair force transmission across the cell–cell boundary. Together, our in vitro and in silico results suggest that mechanotransductive mechanisms may contribute to the modest functional benefits observed in cell-therapy studies by regulating the amount of contractile force effectively transmitted at the junction between newly formed and spared myocytes. PMID:26858266

  9. Cadherin-23 may be dynamic in hair bundles of the model sea anemone Nematostella vectensis.

    Directory of Open Access Journals (Sweden)

    Pei-Ciao Tang

    Full Text Available Cadherin 23 (CDH23, a component of tip links in hair cells of vertebrate animals, is essential to mechanotransduction by hair cells in the inner ear. A homolog of CDH23 occurs in hair bundles of sea anemones. Anemone hair bundles are located on the tentacles where they detect the swimming movements of nearby prey. The anemone CDH23 is predicted to be a large polypeptide featuring a short exoplasmic C-terminal domain that is unique to sea anemones. Experimentally masking this domain with antibodies or mimicking this domain with free peptide rapidly disrupts mechanotransduction and morphology of anemone hair bundles. The loss of normal morphology is accompanied, or followed by a decrease in F-actin in stereocilia of the hair bundles. These effects were observed at very low concentrations of the reagents, 0.1-10 nM, and within minutes of exposure. The results presented herein suggest that: (1 the interaction between CDH23 and molecular partners on stereocilia of hair bundles is dynamic and; (2 the interaction is crucial for normal mechanotransduction and morphology of hair bundles.

  10. Sensory hair cell death and regeneration in fishes

    Directory of Open Access Journals (Sweden)

    Jerry D. Monroe

    2015-04-01

    Full Text Available Sensory hair cells are specialized mechanotransductive receptors required for hearing and vestibular function. Loss of hair cells in humans and other mammals is permanent and causes reduced hearing and balance. In the early 1980’s, it was shown that hair cells continue to be added to the inner ear sensory epithelia in cartilaginous and bony fishes. Soon thereafter, hair cell regeneration was documented in the chick cochlea following acoustic trauma. Since then, research using chick and other avian models has led to great insights into hair cell death and regeneration. However, with the rise of the zebrafish as a model organism for studying disease and developmental processes, there has been an increased interest in studying sensory hair cell death and regeneration in its lateral line and inner ears. Advances derived from studies in zebrafish and other fish species include understanding the effect of ototoxins on hair cells and finding otoprotectants to mitigate ototoxin damage, the role of cellular proliferation versus direct transdifferentiation during hair cell regeneration, and elucidating cellular pathways involved in the regeneration process. This review will summarize research on hair cell death and regeneration using fish models, indicate the potential strengths and weaknesses of these models, and discuss several emerging areas of future studies.

  11. XIRP2, an Actin-Binding Protein Essential for Inner Ear Hair-Cell Stereocilia

    Directory of Open Access Journals (Sweden)

    Déborah I. Scheffer

    2015-03-01

    Full Text Available Hair cells of the inner ear are mechanoreceptors for hearing and balance, and proteins highly enriched in hair cells may have specific roles in the development and maintenance of the mechanotransduction apparatus. We identified XIRP2/mXinβ as an enriched protein likely to be essential for hair cells. We found that different isoforms of this protein are expressed and differentially located: short splice forms (also called XEPLIN are targeted more to stereocilia, whereas two long isoforms containing a XIN-repeat domain are in both stereocilia and cuticular plates. Mice lacking the Xirp2 gene developed normal stereocilia bundles, but these degenerated with time: stereocilia were lost and long membranous protrusions emanated from the nearby apical surfaces. At an ultrastructural level, the paracrystalline actin filaments became disorganized. XIRP2 is apparently involved in the maintenance of actin structures in stereocilia and cuticular plates of hair cells, and perhaps in other organs where it is expressed.

  12. Mechanosensitive promoter region in the human HB-GAM gene

    DEFF Research Database (Denmark)

    Liedert, Astrid; Kassem, Moustapha; Claes, Lutz

    2009-01-01

    Mechanical loading is essential for maintaining bone mass in the adult skeleton. However, the underlying process of the transfer of the physical stimulus into a biochemical response, which is termed mechanotransduction is poorly understood. Mechanotransduction results in the modulation of gene...... cells. Analysis of the human HB-GAM gene upstream regulatory region with luciferase reporter gene assays revealed that the upregulation of HB-GAM expression occurred at the transcriptional level and was mainly dependent on the HB-GAM promoter region most upstream containing three potential AP-1 binding...

  13. Causes and Consequences of Sensory Hair Cell Damage and Recovery in Fishes.

    Science.gov (United States)

    Smith, Michael E; Monroe, J David

    2016-01-01

    Sensory hair cells are the mechanotransductive receptors that detect gravity, sound, and vibration in all vertebrates. Damage to these sensitive receptors often results in deficits in vestibular function and hearing. There are currently two main reasons for studying the process of hair cell loss in fishes. First, fishes, like other non-mammalian vertebrates, have the ability to regenerate hair cells that have been damaged or lost via exposure to ototoxic chemicals or acoustic overstimulation. Thus, they are used as a biomedical model to understand the process of hair cell death and regeneration and find therapeutics that treat or prevent human hearing loss. Secondly, scientists and governmental natural resource managers are concerned about the potential effects of intense anthropogenic sounds on aquatic organisms, including fishes. Dr. Arthur N. Popper and his students, postdocs and research associates have performed pioneering experiments in both of these lines of fish hearing research. This review will discuss the current knowledge regarding the causes and consequences of both lateral line and inner ear hair cell damage in teleost fishes.

  14. PIEZO channel protein naturally expressed in human breast cancer cell MDA-MB-231 as probed by atomic force microscopy

    Science.gov (United States)

    Weng, Yuanqi; Yan, Fei; Chen, Runkang; Qian, Ming; Ou, Yun; Xie, Shuhong; Zheng, Hairong; Li, Jiangyu

    2018-05-01

    Mechanical stimuli drives many physiological processes through mechanically activated channels, and the recent discovery of PIEZO channel has generated great interests in its mechanotransduction. Many previous researches investigated PIEZO proteins by transcribing them in cells that originally have no response to mechanical stimulation, or by forming PIEZO-combined complexes in vitro, and few studied PIEZO protein's natural characteristics in cells. In this study we show that MDA-MB-231, a malignant cell in human breast cancer cell line, expresses the mechanosensitive behavior of PIEZO in nature without extra treatment, and we report its characteristics in response to localized mechanical stimulation under an atomic force microscope, wherein a correlation between the force magnitude applied and the channel opening probability is observed. The results on PIEZO of MDA-MB-231 can help establish a basis of preventing and controlling of human breast cancer cell via mechanical forces.

  15. A Biophysical-Computational Perspective of Breast Cancer Pathogenesis and Treatment Response

    Science.gov (United States)

    2009-03-01

    oriented silk biopolymers with "tuned" diameters, orientations and stiffness. A. Gradient mechanically compliant substrata to investigate durotaxis...the most exciting discovery/application we have made this past year is adoptation of biopolymer silk . The biopolymer silk is an attractive...this unique durotactic behavior. B. Silk microfibers with tunable diameter and mechanical properties: 1. Background and Rationale: Probably however

  16. The apelin receptor influences biomechanical and morphological properties of endothelial cells.

    Science.gov (United States)

    Strohbach, Anne; Pennewitz, Malte; Glaubitz, Michael; Palankar, Raghavendra; Groß, Stefan; Lorenz, Florian; Materzok, Ilka; Rong, Alena; Busch, Mathias C; Felix, Stephan B; Delcea, Mihaela; Busch, Raila

    2018-08-01

    The adaption of endothelial cells to local flow conditions is a multifunctional process which leads to distinct alterations in cell shape, the subcellular distribution of structural proteins, and cellular function. G-protein-coupled receptors (GPCRs) have been identified to be fundamentally involved in such processes. Recently, we and others have shown that the expression of the endothelial GPCR apelin receptor (APJ) is regulated by fluid flow and that activation of APJ participates in signaling pathways which are related to processes of mechanotransduction. The present study aims to illuminate these findings by further visualization of APJ function. We show that APJ is located to the cellular junctions and might thus be associated with platelet endothelial cell adhesion molecule-1 (PECAM-1) in human umbilical vein endothelial cells (HUVEC). Furthermore, siRNA-mediated silencing of APJ expression influences the shear-induced adaption of HUVEC in terms of cytoskeletal remodeling, cellular elasticity, cellular motility, attachment, and distribution of adhesion complexes. Taken together, our results demonstrate that APJ is crucial for complemented endothelial adaption to local flow conditions. © 2018 Wiley Periodicals, Inc.

  17. Monitoring intracellular calcium ion dynamics in hair cell populations with Fluo-4 AM.

    Directory of Open Access Journals (Sweden)

    Kateri J Spinelli

    Full Text Available We optimized Fluo-4 AM loading of chicken cochlea to report hair-bundle Ca(2+ signals in populations of hair cells. The bundle Ca(2+ signal reported the physiological state of the bundle and cell; extruding cells had very high bundle Fluo-4 fluorescence, cells with intact bundles and tip links had intermediate fluorescence, and damaged cells with broken tip links had low fluorescence. Moreover, Fluo-4 fluorescence in the bundle correlated with Ca(2+ entry through transduction channels; mechanically activating transduction channels increased the Fluo-4 signal, while breaking tip links with Ca(2+ chelators or blocking Ca(2+ entry through transduction channels each caused bundle and cell-body Fluo-4 fluorescence to decrease. These results show that when tip links break, bundle and soma Ca(2+ decrease, which could serve to stimulate the hair cell's tip-link regeneration process. Measurement of bundle Ca(2+ with Fluo-4 AM is therefore a simple method for assessing mechanotransduction in hair cells and permits an increased understanding of the interplay of tip links, transduction channels, and Ca(2+ signaling in the hair cell.

  18. Matrix mechanics and fluid shear stress control stem cells fate in three dimensional microenvironment.

    Science.gov (United States)

    Chen, Guobao; Lv, Yonggang; Guo, Pan; Lin, Chongwen; Zhang, Xiaomei; Yang, Li; Xu, Zhiling

    2013-07-01

    Stem cells have the ability to self-renew and to differentiate into multiple mature cell types during early life and growth. Stem cells adhesion, proliferation, migration and differentiation are affected by biochemical, mechanical and physical surface properties of the surrounding matrix in which stem cells reside and stem cells can sensitively feel and respond to the microenvironment of this matrix. More and more researches have proven that three dimensional (3D) culture can reduce the gap between cell culture and physiological environment where cells always live in vivo. This review summarized recent findings on the studies of matrix mechanics that control stem cells (primarily mesenchymal stem cells (MSCs)) fate in 3D environment, including matrix stiffness and extracellular matrix (ECM) stiffness. Considering the exchange of oxygen and nutrients in 3D culture, the effect of fluid shear stress (FSS) on fate decision of stem cells was also discussed in detail. Further, the difference of MSCs response to matrix stiffness between two dimensional (2D) and 3D conditions was compared. Finally, the mechanism of mechanotransduction of stem cells activated by matrix mechanics and FSS in 3D culture was briefly pointed out.

  19. Release of tensile strain on engineered human tendon tissue disturbs cell adhesions, changes matrix architecture, and induces an inflammatory phenotype

    DEFF Research Database (Denmark)

    Bayer, Monika L; Schjerling, Peter; Herchenhan, Andreas

    2014-01-01

    Mechanical loading of tendon cells results in an upregulation of mechanotransduction signaling pathways, cell-matrix adhesion and collagen synthesis, but whether unloading removes these responses is unclear. We investigated the response to tension release, with regard to matrix proteins, pro......-inflammatory mediators and tendon phenotypic specific molecules, in an in vitro model where tendon-like tissue was engineered from human tendon cells. Tissue sampling was performed 1, 2, 4 and 6 days after surgical de-tensioning of the tendon construct. When tensile stimulus was removed, integrin type collagen receptors...... were upregulated. Stimulation with the cytokine TGF-β1 had distinct effects on some tendon-related genes in both tensioned and de-tensioned tissue. These findings indicate an important role of mechanical loading for cellular and matrix responses in tendon, including that loss of tension leads...

  20. Expression data from SPHINX (SPaceflight of Huvec: an INtegrated eXperiment)

    Data.gov (United States)

    National Aeronautics and Space Administration — Changes in the physical environment modulate cell responses and may lead to the impairment or even failure of tissue function as a result of mechanotransduction...

  1. Role of substrate biomechanics in controlling (stem) cell fate: Implications in regenerative medicine.

    Science.gov (United States)

    Macri-Pellizzeri, Laura; De-Juan-Pardo, Elena M; Prosper, Felipe; Pelacho, Beatriz

    2018-04-01

    Tissue-specific stem cells reside in a specialized environment known as niche. The niche plays a central role in the regulation of cell behaviour and, through the concerted action of soluble molecules, supportive somatic cells, and extracellular matrix components, directs stem cells to proliferate, differentiate, or remain quiescent. Great efforts have been done to decompose and separately analyse the contribution of these cues in the in vivo environment. Specifically, the mechanical properties of the extracellular matrix influence many aspects of cell behaviour, including self-renewal and differentiation. Deciphering the role of biomechanics could thereby provide important insights to control the stem cells responses in a more effective way with the aim to promote their therapeutic potential. In this review, we provide a wide overview of the effect that the microenvironment stiffness exerts on the control of cell behaviour with a particular focus on the induction of stem cells differentiation. We also describe the process of mechanotransduction and the molecular effectors involved. Finally, we critically discuss the potential involvement of tissue biomechanics in the design of novel tissue engineering strategies. Copyright © 2017 John Wiley & Sons, Ltd.

  2. Zebrafish hair cell mechanics and physiology through the lens of noise-induced hair cell death

    Science.gov (United States)

    Coffin, Allison B.; Xu, Jie; Uribe, Phillip M.

    2018-05-01

    Hair cells are exquisitely sensitive to auditory stimuli, but also to damage from a variety of sources including noise trauma and ototoxic drugs. Mammals cannot regenerate cochlear hair cells, while non-mammalian vertebrates exhibit robust regenerative capacity. Our research group uses the lateral line system of larval zebrafish to explore the mechanisms underlying hair cell damage, identify protective therapies, and determine molecular drivers of innate regeneration. The lateral line system contains externally located sensory organs called neuromasts, each composed of ˜8-20 hair cells. Lateral line hair cells are homologous to vertebrate inner ear hair cells and share similar susceptibility to ototoxic damage. In the last decade, the lateral line has emerged as a powerful model system for understanding hair cell death mechanisms and for identifying novel protective compounds. Here we demonstrate that the lateral line is a tractable model for noise-induced hair cell death. We have developed a novel noise damage system capable of inducing over 50% loss of lateral line hair cells, with hair cell death occurring in a dose- and time-dependent manner. Cell death is greatest 72 hours post-exposure. However, early signs of hair cell damage, including changes in membrane integrity and reduced mechanotransduction, are apparent within hours of noise exposure. These features, early signs of damage followed by delayed hair cell death, are consistent with mammalian data, suggesting that noise acts similarly on zebrafish and mammalian hair cells. In our future work we will use our new model system to investigate noise damage events in real time, and to develop protective therapies for future translational research.

  3. Cell type-specific suppression of mechanosensitive genes by audible sound stimulation.

    Science.gov (United States)

    Kumeta, Masahiro; Takahashi, Daiji; Takeyasu, Kunio; Yoshimura, Shige H

    2018-01-01

    Audible sound is a ubiquitous environmental factor in nature that transmits oscillatory compressional pressure through the substances. To investigate the property of the sound as a mechanical stimulus for cells, an experimental system was set up using 94.0 dB sound which transmits approximately 10 mPa pressure to the cultured cells. Based on research on mechanotransduction and ultrasound effects on cells, gene responses to the audible sound stimulation were analyzed by varying several sound parameters: frequency, wave form, composition, and exposure time. Real-time quantitative PCR analyses revealed a distinct suppressive effect for several mechanosensitive and ultrasound-sensitive genes that were triggered by sounds. The effect was clearly observed in a wave form- and pressure level-specific manner, rather than the frequency, and persisted for several hours. At least two mechanisms are likely to be involved in this sound response: transcriptional control and RNA degradation. ST2 stromal cells and C2C12 myoblasts exhibited a robust response, whereas NIH3T3 cells were partially and NB2a neuroblastoma cells were completely insensitive, suggesting a cell type-specific response to sound. These findings reveal a cell-level systematic response to audible sound and uncover novel relationships between life and sound.

  4. Pulmonary Arterial Stiffness: Toward a New Paradigm in Pulmonary Arterial Hypertension Pathophysiology and Assessment.

    Science.gov (United States)

    Schäfer, Michal; Myers, Cynthia; Brown, R Dale; Frid, Maria G; Tan, Wei; Hunter, Kendall; Stenmark, Kurt R

    2016-01-01

    Stiffening of the pulmonary arterial bed with the subsequent increased load on the right ventricle is a paramount feature of pulmonary hypertension (PH). The pathophysiology of vascular stiffening is a complex and self-reinforcing function of extracellular matrix remodeling, driven by recruitment of circulating inflammatory cells and their interactions with resident vascular cells, and mechanotransduction of altered hemodynamic forces throughout the ventricular-vascular axis. New approaches to understanding the cell and molecular determinants of the pathophysiology combine novel biopolymer substrates, controlled flow conditions, and defined cell types to recapitulate the biomechanical environment in vitro. Simultaneously, advances are occurring to assess novel parameters of stiffness in vivo. In this comprehensive state-of-art review, we describe clinical hemodynamic markers, together with the newest translational echocardiographic and cardiac magnetic resonance imaging methods, to assess vascular stiffness and ventricular-vascular coupling. Finally, fluid-tissue interactions appear to offer a novel route of investigating the mechanotransduction processes and disease progression.

  5. Time dependency of morphological remodeling of endothelial cells in response to substrate stiffness

    Science.gov (United States)

    Goli-Malekabadi, Zahra; Tafazzoli-shadpour, Mohammad; Tamayol, Ali; Seyedjafari, Ehsan

    2017-01-01

    Introduction: Substrate stiffness regulates cellular behavior as cells experience different stiffness values of tissues in the body. For example, endothelial cells (ECs) covering the inner layer of blood vessels are exposed to different stiffness values due to various pathologic and physiologic conditions. Despite numerous studies, cells by time span sense mechanical properties of the substrate, but the response is not well understood. We hypothesized that time is a major determinant influencing the behavior of cells seeded on substrates of varying stiffness. Methods: We monitored cell spreading, internal structure, 3D topography, and the viability of ECs over 24 hours of culture on polydimethylsiloxane (PDMS) substrates with two different degrees of elastic modulus. Results: Despite significant differences in cell spreading after cell seeding, cells showed a similar shape and internal structure after 24 hours of culture on both soft and stiff substrates. However, 3D topographical images confirmed existence of rich lamellipodia and filopodia around the cells cultured on stiffer PDMS substrates. Conclusion: It was concluded that the response of ECs to the substrate stiffness was time dependent with initial enhanced cellular spreading and viability on stiffer substrates. Results can provide a better comprehension of cell mechanotransduction for tissue engineering applications. PMID:28546952

  6. Vascular mechanotransduction data in a rodent model of diabetes: Pressure-induced regulation of SHP2 and associated signaling in the rat inferior vena cava

    Directory of Open Access Journals (Sweden)

    Kevin M. Rice

    2017-12-01

    Full Text Available The effect of diabetes on vascular mechano-transductive response is of great concern. Given the higher rate of vein graft failures associated with diabetes, understanding the multiple cellular and molecular events associated with vascular remodeling is of vital importance. This article represents data related to a study published in Cardiovascular Diabetology [1] (Rice et al., 2006 and Open Journal of Endocrine and Metabolic Diseases [2] (Rice et al., 2015 evaluating the effect of pressurization on rat inferior venae cavae (IVC. Provided within this articles is information related to the method and processing of raw data related to our prior publish work and Data in Brief articles [3,4] (Rice et al., 2017, as well as the evaluation of alternation in SHP-2 signaling and associated proteins in response to mechanical force. IVC from lean and obese animals were exposed to a 30 min perfusion of 120 mm Hg pressure and evaluated for changes in expression of SHP2, BCL-3, BCL-XL, HSP 27, HSP 70, and PI3K p85, along with the phosphorylation of SHP-2 (Tyr 542.

  7. A mathematical model of breast cancer cell motion through a microfluidic device

    Science.gov (United States)

    Barber, Jared

    2017-11-01

    Deaths due to breast cancer are usually caused by metastases at other locations (e.g. bone), not by the primary tumor. Much research has targeted understanding how to lower the metastatic potential of individual breast cancer cells with the end goal being the mitigation of the effects of breast cancer on the 3.5 million people in the US affected by the disease. Experiments show that metastatic potential correlates well with the physical properties of a cell and its surrounding environment. Biology also suggests that mechanotransduction of cellular pathways (e.g. apoptosis, division) can affect metastatic potential. Because of these insights, we are developing a mechanical model of breast cancer cell translocation in microvessels. Our first model is a two-dimensional model with interconnected viscoelastic elements submersed in a surrounding Stokes flow. This model has been used to consider breast cancer cell translocation through a microfluidic device that was designed as a diagnostic tool for assessing the metastatic potential of breast cells. We will present this current model and share results. We believe that further development of this model will allow consideration of metastatic potential in both in vitro and in vivo settings.

  8. Membrane tension controls adhesion positioning at the leading edge of cells.

    Science.gov (United States)

    Pontes, Bruno; Monzo, Pascale; Gole, Laurent; Le Roux, Anabel-Lise; Kosmalska, Anita Joanna; Tam, Zhi Yang; Luo, Weiwei; Kan, Sophie; Viasnoff, Virgile; Roca-Cusachs, Pere; Tucker-Kellogg, Lisa; Gauthier, Nils C

    2017-09-04

    Cell migration is dependent on adhesion dynamics and actin cytoskeleton remodeling at the leading edge. These events may be physically constrained by the plasma membrane. Here, we show that the mechanical signal produced by an increase in plasma membrane tension triggers the positioning of new rows of adhesions at the leading edge. During protrusion, as membrane tension increases, velocity slows, and the lamellipodium buckles upward in a myosin II-independent manner. The buckling occurs between the front of the lamellipodium, where nascent adhesions are positioned in rows, and the base of the lamellipodium, where a vinculin-dependent clutch couples actin to previously positioned adhesions. As membrane tension decreases, protrusion resumes and buckling disappears, until the next cycle. We propose that the mechanical signal of membrane tension exerts upstream control in mechanotransduction by periodically compressing and relaxing the lamellipodium, leading to the positioning of adhesions at the leading edge of cells. © 2017 Pontes et al.

  9. Modeling and predictions of biphasic mechanosensitive cell migration altered by cell-intrinsic properties and matrix confinement.

    Science.gov (United States)

    Pathak, Amit

    2018-04-12

    Motile cells sense the stiffness of their extracellular matrix (ECM) through adhesions and respond by modulating the generated forces, which in turn lead to varying mechanosensitive migration phenotypes. Through modeling and experiments, cell migration speed is known to vary with matrix stiffness in a biphasic manner, with optimal motility at an intermediate stiffness. Here, we present a two-dimensional cell model defined by nodes and elements, integrated with subcellular modeling components corresponding to mechanotransductive adhesion formation, force generation, protrusions and node displacement. On 2D matrices, our calculations reproduce the classic biphasic dependence of migration speed on matrix stiffness and predict that cell types with higher force-generating ability do not slow down on very stiff matrices, thus disabling the biphasic response. We also predict that cell types defined by lower number of total receptors require stiffer matrices for optimal motility, which also limits the biphasic response. For a cell type with robust biphasic migration on 2D surface, simulations in channel-like confined environments of varying width and height predict faster migration in more confined matrices. Simulations performed in shallower channels predict that the biphasic mechanosensitive cell migration response is more robust on 2D micro-patterns as compared to the channel-like 3D confinement. Thus, variations in the dimensionality of matrix confinement alters the way migratory cells sense and respond to the matrix stiffness. Our calculations reveal new phenotypes of stiffness- and topography-sensitive cell migration that critically depend on both cell-intrinsic and matrix properties. These predictions may inform our understanding of various mechanosensitive modes of cell motility that could enable tumor invasion through topographically heterogeneous microenvironments. © 2018 IOP Publishing Ltd.

  10. Citoesqueleto e mecanotransdução na fisiopatologia da lesão pulmonar induzida por ventilador Cytoskeleton and mechanotransduction in the pathophysiology of ventilator-induced lung injury

    Directory of Open Access Journals (Sweden)

    Leandro Utino Taniguchi

    2010-06-01

    Full Text Available A ventilação mecânica é uma terapia importante, mas pode resultar em complicações. Uma das mais relevantes é a lesão pulmonar induzida por ventilador. Devido à hiperdistensão alveolar, o pulmão inicia um processo inflamatório, com infiltrado neutrofílico, formação de membrana hialina, fibrogênese e prejuízo de troca gasosa. Nesse processo, a mecanotransdução da hiperdistensão celular é mediada através do citoesqueleto da célula e de suas interações com a matriz extracelular e com as células vizinhas, de modo que o estímulo mecânico da ventilação se traduz em sinalização bioquímica intracelular, desencadeando ativação endotelial, permeabilidade vascular pulmonar, quimiotaxia leucocitária, produção de citocinas e, possivelmente, lesão de órgãos à distância. Estudos clínicos demonstram essa relação entre distensão pulmonar e mortalidade em pacientes com lesão pulmonar induzida por ventilador. Entretanto, apesar de o citoesqueleto ter um papel fundamental na patogênese da lesão pulmonar induzida por ventilador, a literatura carece de estudos utilizando modelos in vivo sobre as alterações do citoesqueleto e de suas proteínas associadas durante esse processo patológico.Although mechanical ventilation is an important therapy, it can result in complications. One major complication is ventilator-induced lung injury, which is caused by alveolar hyperdistension, leading to an inflammatory process, with neutrophilic infiltration, hyaline membrane formation, fibrogenesis and impaired gas exchange. In this process, cellular mechanotransduction of the overstretching stimulus is mediated by means of the cytoskeleton and its cell-cell and cell-extracellular matrix interactions, in such a way that the mechanical stimulus of ventilation is translated into an intracellular biochemical signal, inducing endothelial activation, pulmonary vascular permeability, leukocyte chemotaxis, cytokine production and, possibly

  11. Mechanically Gated Ion Channels in Mammalian Hair Cells

    Directory of Open Access Journals (Sweden)

    Xufeng Qiu

    2018-04-01

    Full Text Available Hair cells in the inner ear convert mechanical stimuli provided by sound waves and head movements into electrical signal. Several mechanically evoked ionic currents with different properties have been recorded in hair cells. The search for the proteins that form the underlying ion channels is still in progress. The mechanoelectrical transduction (MET channel near the tips of stereociliary in hair cells, which is responsible for sensory transduction, has been studied most extensively. Several components of the sensory mechanotransduction machinery in stereocilia have been identified, including the multi-transmembrane proteins tetraspan membrane protein in hair cell stereocilia (TMHS/LHFPL5, transmembrane inner ear (TMIE and transmembrane channel-like proteins 1 and 2 (TMC1/2. However, there remains considerable uncertainty regarding the molecules that form the channel pore. In addition to the sensory MET channel, hair cells express the mechanically gated ion channel PIEZO2, which is localized near the base of stereocilia and not essential for sensory transduction. The function of PIEZO2 in hair cells is not entirely clear but it might have a role in damage sensing and repair processes. Additional stretch-activated channels of unknown molecular identity and function have been found to localize at the basolateral membrane of hair cells. Here, we review current knowledge regarding the different mechanically gated ion channels in hair cells and discuss open questions concerning their molecular composition and function.

  12. 3,4-Dihydroxy-L-Phenylalanine as a Novel Covalent Linker of Extracellular Matrix Proteins to Polyacrylamide Hydrogels with a Tunable Stiffness

    NARCIS (Netherlands)

    Wouters, Olaf Y.; Ploeger, Diana T. A.; van Putten, Sander M.; Bank, Ruud A.

    Cells acquire mechanical information from their surrounding and convert this into biochemical activity. The concept and mechanism behind this cellular mechanosensing and mechanotransduction are often studied by means of two-dimensional hydrogels. Polyacrylamide hydrogels (PAAMs) offer chemical,

  13. Laminar shear stress modulates endothelial luminal surface stiffness in a tissue-specific manner.

    Science.gov (United States)

    Merna, Nick; Wong, Andrew K; Barahona, Victor; Llanos, Pierre; Kunar, Balvir; Palikuqi, Brisa; Ginsberg, Michael; Rafii, Shahin; Rabbany, Sina Y

    2018-04-17

    Endothelial cells form vascular beds in all organs and are exposed to a range of mechanical forces that regulate cellular phenotype. We sought to determine the role of endothelial luminal surface stiffness in tissue-specific mechanotransduction of laminar shear stress in microvascular mouse cells and the role of arachidonic acid in mediating this response. Microvascular mouse endothelial cells were subjected to laminar shear stress at 4 dynes/cm 2 for 12 hours in parallel plate flow chambers that enabled real-time optical microscopy and atomic force microscopy measurements of cell stiffness. Lung endothelial cells aligned parallel to flow, while cardiac endothelial cells did not. This rapid alignment was accompanied by increased cell stiffness. The addition of arachidonic acid to cardiac endothelial cells increased alignment and stiffness in response to shear stress. Inhibition of arachidonic acid in lung endothelial cells and embryonic stem cell-derived endothelial cells prevented cellular alignment and decreased cell stiffness. Our findings suggest that increased endothelial luminal surface stiffness in microvascular cells may facilitate mechanotransduction and alignment in response to laminar shear stress. Furthermore, the arachidonic acid pathway may mediate this tissue-specific process. An improved understanding of this response will aid in the treatment of organ-specific vascular disease. © 2018 John Wiley & Sons Ltd.

  14. Phase imaging of mechanical properties of live cells (Conference Presentation)

    Science.gov (United States)

    Wax, Adam

    2017-02-01

    The mechanisms by which cells respond to mechanical stimuli are essential for cell function yet not well understood. Many rheological tools have been developed to characterize cellular viscoelastic properties but these typically require direct mechanical contact, limiting their throughput. We have developed a new approach for characterizing the organization of subcellular structures using a label free, noncontact, single-shot phase imaging method that correlates to measured cellular mechanical stiffness. The new analysis approach measures refractive index variance and relates it to disorder strength. These measurements are compared to cellular stiffness, measured using the same imaging tool to visualize nanoscale responses to flow shear stimulus. The utility of the technique is shown by comparing shear stiffness and phase disorder strength across five cellular populations with varying mechanical properties. An inverse relationship between disorder strength and shear stiffness is shown, suggesting that cell mechanical properties can be assessed in a format amenable to high throughput studies using this novel, non-contact technique. Further studies will be presented which include examination of mechanical stiffness in early carcinogenic events and investigation of the role of specific cellular structural proteins in mechanotransduction.

  15. Effects of Inflammation on Multiscale Biomechanical Properties of Cartilaginous Cells and Tissues.

    Science.gov (United States)

    Nguyen, Q T; Jacobsen, T D; Chahine, N O

    2017-11-13

    Cells within cartilaginous tissues are mechanosensitive and thus require mechanical loading for regulation of tissue homeostasis and metabolism. Mechanical loading plays critical roles in cell differentiation, proliferation, biosynthesis, and homeostasis. Inflammation is an important event occurring during multiple processes, such as aging, injury, and disease. Inflammation has significant effects on biological processes as well as mechanical function of cells and tissues. These effects are highly dependent on cell/tissue type, timing, and magnitude. In this review, we summarize key findings pertaining to effects of inflammation on multiscale mechanical properties at subcellular, cellular, and tissue level in cartilaginous tissues, including alterations in mechanotransduction and mechanosensitivity. The emphasis is on articular cartilage and the intervertebral disc, which are impacted by inflammatory insults during degenerative conditions such as osteoarthritis, joint pain, and back pain. To recapitulate the pro-inflammatory cascades that occur in vivo, different inflammatory stimuli have been used for in vitro and in situ studies, including tumor necrosis factor (TNF), various interleukins (IL), and lipopolysaccharide (LPS). Therefore, this review will focus on the effects of these stimuli because they are the best studied pro-inflammatory cytokines in cartilaginous tissues. Understanding the current state of the field of inflammation and cell/tissue biomechanics may potentially identify future directions for novel and translational therapeutics with multiscale biomechanical considerations.

  16. Neurite extension and neuronal differentiation of human induced pluripotent stem cell derived neural stem cells on polyethylene glycol hydrogels containing a continuous Young's Modulus gradient.

    Science.gov (United States)

    Mosley, Matthew C; Lim, Hyun Ju; Chen, Jing; Yang, Yueh-Hsun; Li, Shenglan; Liu, Ying; Smith Callahan, Laura A

    2017-03-01

    Mechanotransduction in neural cells involves multiple signaling pathways that are not fully understood. Differences in lineage and maturation state are suggested causes for conflicting reports on neural cell mechanosensitivity. To optimize matrices for use in stem cell therapy treatments transplanting human induced pluripotent stem cell derived neural stem cells (hNSC) into lesions after spinal cord injury, the effects of Young's Modulus changes on hNSC behavior must be understood. The present study utilizes polyethylene glycol hydrogels containing a continuous gradient in Young's modulus to examine changes in the Young's Modulus of the culture substrate on hNSC neurite extension and neural differentiation. Changes in the Young's Modulus of the polyethylene glycol hydrogels was found to affect neurite extension and cellular organization on the matrices. hNSC cultured on 907 Pa hydrogels were found to extend longer neurites than hNSC cultured on other tested Young's Moduli hydrogels. The gene expression of β tubulin III and microtubule-associated protein 2 in hNSC was affected by changes in the Young's Modulus of the hydrogel. The combinatory method approach used in the present study demonstrates that hNSC are mechanosensitive and the matrix Young's Modulus should be a design consideration for hNSC transplant applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 824-833, 2017. © 2016 Wiley Periodicals, Inc.

  17. Visualizing the interior architecture of focal adhesions with high-resolution traction maps.

    Science.gov (United States)

    Morimatsu, Masatoshi; Mekhdjian, Armen H; Chang, Alice C; Tan, Steven J; Dunn, Alexander R

    2015-04-08

    Focal adhesions (FAs) are micron-sized protein assemblies that coordinate cell adhesion, migration, and mechanotransduction. How the many proteins within FAs are organized into force sensing and transmitting structures is poorly understood. We combined fluorescent molecular tension sensors with super-resolution light microscopy to visualize traction forces within FAs with <100 nm spatial resolution. We find that αvβ3 integrin selectively localizes to high force regions. Paxillin, which is not generally considered to play a direct role in force transmission, shows a higher degree of spatial correlation with force than vinculin, talin, or α-actinin, proteins with hypothesized roles as force transducers. These observations suggest that αvβ3 integrin and paxillin may play important roles in mechanotransduction.

  18. Touch, Tension, and Transduction – the Function and Regulation of Piezo Ion Channels

    Science.gov (United States)

    Wu, Jason; Lewis, Amanda; Grandl, Jörg

    2016-01-01

    In 2010, two proteins, Piezo1 and Piezo2, were identified as the long-sought molecular carriers of an excitatory mechanically activated current found in many cells. This discovery has opened the floodgates for studying a vast number of mechanotransduction processes. Over the past six years, groundbreaking research has identified Piezos as ion channels that sense light touch, proprioception, and vascular blood flow, ruled out roles for Piezos in several other mechanotransduction processes, and revealed the basic structural and functional properties of the channel. Here, we review these findings and discuss the many aspects of Piezo function that remain mysterious, including how Piezos convert a variety of mechanical stimuli into channel activation and subsequent inactivation, and what molecules and mechanisms modulate Piezo function. PMID:27743844

  19. Role of Piezo Channels in Joint Health and Injury.

    Science.gov (United States)

    Lee, W; Guilak, F; Liedtke, W

    2017-01-01

    Cartilage is an intrinsically mechanically sensitive tissue composed of chondrocytes as the only cell type. Chondrocyte mechanotransduction is not well understood, but recently we identified critical components of the mechanotransduction machinery demonstrating how mechanical stimulation of these cells can be converted into cellular calcium signals. Physiologic mechanical cues induce anabolic responses of (post-mitotic) chondrocytes via transient receptor potential vanilloid 4 ion channels, whereas injurious mechanical stress is transduced by Piezo1 jointly with Piezo2 ion channels. This chapter sheds light on the latter discovery and provides a rationale for follow-up questions, such as the nature of interaction between Piezo1 and Piezo2, and their tethering to the cytoskeleton. These recent insights can be leveraged toward translational medical progress to benefit diagnosis and treatment of osteoarthritis, representing a large and growing unmet medical need in the United States and large parts of the world. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Piezo proteins are pore-forming subunits of mechanically activated channels.

    Science.gov (United States)

    Coste, Bertrand; Xiao, Bailong; Santos, Jose S; Syeda, Ruhma; Grandl, Jörg; Spencer, Kathryn S; Kim, Sung Eun; Schmidt, Manuela; Mathur, Jayanti; Dubin, Adrienne E; Montal, Mauricio; Patapoutian, Ardem

    2012-02-19

    Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezo1 (MmPiezo1) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. Here we show that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezo1 assembles as a ∼1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction.

  1. Characterisation of stromal-cellular mechanotransduction through syndecan-4

    DEFF Research Database (Denmark)

    Hollosi, Peter; Grossi, Alberto; Couchman, John Robert

    phenotype and rate of cell migration. We map the binding site for syndecan-4 cytoplasmic domain in a-actinin to spectrin repeat4, utilising solid phase binding assays and recombinant peptides. Moreover, phosphorylation of syndecan-4 on its sole serine residue, known to influence cytoplasmic domain...

  2. Defective fluid shear stress mechanotransduction mediates hereditary hemorrhagic telangiectasia

    Science.gov (United States)

    Baeyens, Nicolas; Larrivée, Bruno; Ola, Roxana; Hayward-Piatkowskyi, Brielle; Dubrac, Alexandre; Huang, Billy; Ross, Tyler D.; Coon, Brian G.; Min, Elizabeth; Tsarfati, Maya; Tong, Haibin; Eichmann, Anne

    2016-01-01

    Morphogenesis of the vascular system is strongly modulated by mechanical forces from blood flow. Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal-dominant disease in which arteriovenous malformations and telangiectasias accumulate with age. Most cases are linked to heterozygous mutations in Alk1 or Endoglin, receptors for bone morphogenetic proteins (BMPs) 9 and 10. Evidence suggests that a second hit results in clonal expansion of endothelial cells to form lesions with poor mural cell coverage that spontaneously rupture and bleed. We now report that fluid shear stress potentiates BMPs to activate Alk1 signaling, which correlates with enhanced association of Alk1 and endoglin. Alk1 is required for BMP9 and flow responses, whereas endoglin is only required for enhancement by flow. This pathway mediates both inhibition of endothelial proliferation and recruitment of mural cells; thus, its loss blocks flow-induced vascular stabilization. Identification of Alk1 signaling as a convergence point for flow and soluble ligands provides a molecular mechanism for development of HHT lesions. PMID:27646277

  3. The genetics of hair-cell function in zebrafish.

    Science.gov (United States)

    Nicolson, Teresa

    2017-09-01

    Our ears are remarkable sensory organs, providing the important senses of balance and hearing. The complex structure of the inner ear, or 'labyrinth', along with the assorted neuroepithelia, have evolved to detect head movements and sounds with impressive sensitivity. The rub is that the inner ear is highly vulnerable to genetic lesions and environmental insults. According to National Institute of Health estimates, hearing loss is one of the most commonly inherited or acquired sensorineural diseases. To understand the causes of deafness and balance disorders, it is imperative to understand the underlying biology of the inner ear, especially the inner workings of the sensory receptors. These receptors, which are termed hair cells, are particularly susceptible to genetic mutations - more than two dozen genes are associated with defects in this cell type in humans. Over the past decade, a substantial amount of progress has been made in working out the molecular basis of hair-cell function using vertebrate animal models. Given the transparency of the inner ear and the genetic tools that are available, zebrafish have become an increasingly popular animal model for the study of deafness and vestibular dysfunction. Mutagenesis screens for larval defects in hearing and balance have been fruitful in finding key components, many of which have been implicated in human deafness. This review will focus on the genes that are required for hair-cell function in zebrafish, with a particular emphasis on mechanotransduction. In addition, the generation of new tools available for the characterization of zebrafish hair-cell mutants will be discussed.

  4. Touch, Tension, and Transduction - The Function and Regulation of Piezo Ion Channels.

    Science.gov (United States)

    Wu, Jason; Lewis, Amanda H; Grandl, Jörg

    2017-01-01

    In 2010, two proteins, Piezo1 and Piezo2, were identified as the long-sought molecular carriers of an excitatory mechanically activated current found in many cells. This discovery has opened the floodgates for studying a vast number of mechanotransduction processes. Over the past 6 years, groundbreaking research has identified Piezos as ion channels that sense light touch, proprioception, and vascular blood flow, ruled out roles for Piezos in several other mechanotransduction processes, and revealed the basic structural and functional properties of the channel. Here, we review these findings and discuss the many aspects of Piezo function that remain mysterious, including how Piezos convert a variety of mechanical stimuli into channel activation and subsequent inactivation, and what molecules and mechanisms modulate Piezo function. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Remodelling of Membrane Rafts Expression in Lung Cells as an Early Sign of Mechanotransduction-Signalling in Pulmonary Edema

    Directory of Open Access Journals (Sweden)

    Paola Palestini

    2011-01-01

    Full Text Available Membrane rafts (MRs are clusters of lipids, organized in a “quasicrystalline” liquid-order phase, organized on the cell surface and whose pattern of molecules and physicochemical properties are distinct from those of the surrounding plasma membrane. MRs may be considered an efficient and fairly rapid cell-activated mechanism to express or mask surface receptors aimed at triggering specific response pathways. This paper reports observations concerning the role of MRs in the control of lung extravascular water that ought to be kept at minimum to assure gas diffusion, supporting the hypothesis that MRs expression is a potential mechanism of sensing minor changes in the volume of extravascular water. We present the evidence that MRs expression specifically relates to signal-transduction processes evoked by mechanical stimuli arising in the interstitial lung compartment when a small increase in extravascular volume occurs. We further hypothesize that a differential expression of MRs might also reflect the damage to precise components of the extracellular matrix caused by the perturbation in water balance and thus can trigger a molecule-oriented specific matrix remodelling.

  6. Glycocalyx Degradation Induces a Proinflammatory Phenotype and Increased Leukocyte Adhesion in Cultured Endothelial Cells under Flow.

    Directory of Open Access Journals (Sweden)

    Karli K McDonald

    Full Text Available Leukocyte adhesion to the endothelium is an early step in the pathogenesis of atherosclerosis. Effective adhesion requires the binding of leukocytes to their cognate receptors on the surface of endothelial cells. The glycocalyx covers the surface of endothelial cells and is important in the mechanotransduction of shear stress. This study aimed to identify the molecular mechanisms underlying the role of the glycocalyx in leukocyte adhesion under flow. We performed experiments using 3-D cell culture models, exposing human abdominal aortic endothelial cells to steady laminar shear stress (10 dynes/cm2 for 24 hours. We found that with the enzymatic degradation of the glycocalyx, endothelial cells developed a proinflammatory phenotype when exposed to uniform steady shear stress leading to an increase in leukocyte adhesion. Our results show an up-regulation of ICAM-1 with degradation compared to non-degraded controls (3-fold increase, p<0.05 and we attribute this effect to a de-regulation in NF-κB activity in response to flow. These results suggest that the glycocalyx is not solely a physical barrier to adhesion but rather plays an important role in governing the phenotype of endothelial cells, a key determinant in leukocyte adhesion. We provide evidence for how the destabilization of this structure may be an early and defining feature in the initiation of atherosclerosis.

  7. Toward compression of small cell population: harnessing stress in passive regions of dielectric elastomer actuators

    Science.gov (United States)

    Poulin, Alexandre; Rosset, Samuel; Shea, Herbert

    2014-03-01

    We present a dielectric elastomer actuator (DEA) for in vitro analysis of mm2 biological samples under periodic compressive stress. Understanding how mechanical stimuli affect cell functions could lead to significant advances in diseases diagnosis and drugs development. We previously reported an array of 72 micro-DEAs on a chip to apply a periodic stretch to cells. To diversify our cell mechanotransduction toolkit we have developed an actuator for periodic compression of small cell populations. The device is based on a novel design which exploits the effects of non-equibiaxial pre-stretch and takes advantage of the stress induced in passive regions of DEAs. The device consists of two active regions separated by a 2mm x 2mm passive area. When connected to an AC high-voltage source, the two active regions periodically compress the passive region. Due to the non-equibiaxial pre-stretch it induces uniaxial compressive strain greater than 10%. Cells adsorbed on top of this passive gap would experience the same uniaxial compressive stain. The electrodes configuration confines the electric field and prevents it from reaching the biological sample. A thin layer of silicone is casted on top of the device to ensure a biocompatible environment. This design provides several advantages over alternative technologies such as high optical transparency of the area of interest (passive region under compression) and its potential for miniaturization and parallelization.

  8. X-ray-enhanced cancer cell migration requires the linker of nucleoskeleton and cytoskeleton complex.

    Science.gov (United States)

    Imaizumi, Hiromasa; Sato, Katsutoshi; Nishihara, Asuka; Minami, Kazumasa; Koizumi, Masahiko; Matsuura, Nariaki; Hieda, Miki

    2018-04-01

    The linker of nucleoskeleton and cytoskeleton (LINC) complex is a multifunctional protein complex that is involved in various processes at the nuclear envelope, including nuclear migration, mechanotransduction, chromatin tethering and DNA damage response. We recently showed that a nuclear envelope protein, Sad1 and UNC84 domain protein 1 (SUN1), a component of the LINC complex, has a critical function in cell migration. Although ionizing radiation activates cell migration and invasion in vivo and in vitro, the underlying molecular mechanism remains unknown. Here, we examined the involvement of the LINC complex in radiation-enhanced cell migration and invasion. A sublethal dose of X-ray radiation promoted human breast cancer MDA-MB-231 cell migration and invasion, whereas carbon ion beam radiation suppressed these processes in a dose-dependent manner. Depletion of SUN1 and SUN2 significantly suppressed X-ray-enhanced cell migration and invasion. Moreover, depletion or overexpression of each SUN1 splicing variant revealed that SUN1_888 containing 888 amino acids of SUN1 but not SUN1_916 was required for X-ray-enhanced migration and invasion. In addition, the results suggested that X-ray irradiation affected the expression level of SUN1 splicing variants and a SUN protein binding partner, nesprins. Taken together, our findings supported that the LINC complex contributed to photon-enhanced cell migration and invasion. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  9. Single-cell mechanics--An experimental-computational method for quantifying the membrane-cytoskeleton elasticity of cells.

    Science.gov (United States)

    Tartibi, M; Liu, Y X; Liu, G-Y; Komvopoulos, K

    2015-11-01

    novelty of this study is the development of new technology for quantifying the elastic stiffness of the membrane-cytoskeleton system of cells. This capability could have immense implications in cell biology, particularly in establishing correlations between various cell diseases, mortality, and differentiation with membrane-cytoskeleton elasticity, examining through-tissue cell migration, and understanding cell infiltration in porous scaffolds. The present method can be further extended to analyze membrane-cytoskeleton viscous behavior, identify the contribution of other subcellular components (e.g., nucleus envelope) to load sharing, and elucidate mechanotransduction effects due to repetitive compressive loading and unloading on cell differentiation and motility. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  10. AGE-modified basement membrane cooperates with Endo180 to promote epithelial cell invasiveness and decrease prostate cancer survival

    DEFF Research Database (Denmark)

    Rodriguez-Teja, Mercedes; Gronau, Julian H; Breit, Claudia

    2015-01-01

    Biomechanical strain imposed by age-related thickening of the basal lamina and augmented tissue stiffness in the prostate gland coincides with increased cancer risk. Here we hypothesized that the structural alterations in the basal lamina associated with age can induce mechanotransduction pathways...... in prostate epithelial cells (PECs) to promote invasiveness and cancer progression. To demonstrate this, we developed a 3D model of PEC acini in which thickening and stiffening of basal lamina matrix was induced by advanced glycation end-product (AGE)-dependent non-enzymatic crosslinking of its major......(Δ) (Ex2-6/) (Δ) (Ex2-6) mice, with constitutively exposed CTLD2 and decreased survival of men with early (non-invasive) prostate cancer with high epithelial Endo180 expression and levels of AGE. These findings indicate that AGE-dependent modification of the basal lamina induces invasive behaviour...

  11. Synchronizing atomic force microscopy force mode and fluorescence microscopy in real time for immune cell stimulation and activation studies

    Energy Technology Data Exchange (ETDEWEB)

    Cazaux, Séverine; Sadoun, Anaïs; Biarnes-Pelicot, Martine; Martinez, Manuel; Obeid, Sameh [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France); Bongrand, Pierre [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France); APHM, Hôpital de la Conception, Laboratoire d’Immunologie, Marseille F-13385 (France); Limozin, Laurent [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France); Puech, Pierre-Henri, E-mail: pierre-henri.puech@inserm.fr [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France)

    2016-01-15

    A method is presented for combining atomic force microscopy (AFM) force mode and fluorescence microscopy in order to (a) mechanically stimulate immune cells while recording the subsequent activation under the form of calcium pulses, and (b) observe the mechanical response of a cell upon photoactivation of a small G protein, namely Rac. Using commercial set-ups and a robust signal coupling the fluorescence excitation light and the cantilever bending, the applied force and activation signals were very easily synchronized. This approach allows to control the entire mechanical history of a single cell up to its activation and response down to a few hundreds of milliseconds, and can be extended with very minimal adaptations to other cellular systems where mechanotransduction is studied, using either purely mechanical stimuli or via a surface bound specific ligand. - Highlights: • A signal coupling AFM and fluorescence microscopy was characterized for soft cantilevers. • It can be used as an intrinsic timer to synchronize images and forces. • Mechanical stimulation of single immune cells while recording calcium fluxes was detailed. • Light-induced mechanical modifications of lymphocytes using a PA-Rac protein were demonstrated. • The precautions and limitations of use of this effect were presented.

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

    Science.gov (United States)

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

    2018-01-23

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

  13. Stress relaxing hyaluronic acid-collagen hydrogels promote cell spreading, fiber remodeling, and focal adhesion formation in 3D cell culture.

    Science.gov (United States)

    Lou, Junzhe; Stowers, Ryan; Nam, Sungmin; Xia, Yan; Chaudhuri, Ovijit

    2018-02-01

    The physical and architectural cues of the extracellular matrix (ECM) play a critical role in regulating important cellular functions such as spreading, migration, proliferation, and differentiation. Natural ECM is a complex viscoelastic scaffold composed of various distinct components that are often organized into a fibrillar microstructure. Hydrogels are frequently used as synthetic ECMs for 3D cell culture, but are typically elastic, due to covalent crosslinking, and non-fibrillar. Recent work has revealed the importance of stress relaxation in viscoelastic hydrogels in regulating biological processes such as spreading and differentiation, but these studies all utilize synthetic ECM hydrogels that are non-fibrillar. Key mechanotransduction events, such as focal adhesion formation, have only been observed in fibrillar networks in 3D culture to date. Here we present an interpenetrating network (IPN) hydrogel system based on HA crosslinked with dynamic covalent bonds and collagen I that captures the viscoelasticity and fibrillarity of ECM in tissues. The IPN hydrogels exhibit two distinct processes in stress relaxation, one from collagen and the other from HA crosslinking dynamics. Stress relaxation in the IPN hydrogels can be tuned by modulating HA crosslinker affinity, molecular weight of the HA, or HA concentration. Faster relaxation in the IPN hydrogels promotes cell spreading, fiber remodeling, and focal adhesion (FA) formation - behaviors often inhibited in other hydrogel-based materials in 3D culture. This study presents a new, broadly adaptable materials platform for mimicking key ECM features of viscoelasticity and fibrillarity in hydrogels for 3D cell culture and sheds light on how these mechanical and structural cues regulate cell behavior. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Merkel cells and Meissner's corpuscles in human digital skin display Piezo2 immunoreactivity.

    Science.gov (United States)

    García-Mesa, Y; García-Piqueras, J; García, B; Feito, J; Cabo, R; Cobo, J; Vega, J A; García-Suárez, O

    2017-12-01

    The transformation of mechanical energy into electrical signals is the first step in mechanotransduction in the peripheral sensory nervous system and relies on the presence of mechanically gated ion channels within specialized sensory organs called mechanoreceptors. Piezo2 is a vertebrate stretch-gated ion channel necessary for mechanosensitive channels in mammalian cells. Functionally, it is related to light touch, which has been detected in murine cutaneous Merkel cell-neurite complexes, Meissner-like corpuscles and lanceolate nerve endings. To the best of our knowledge, the occurrence of Piezo2 in human cutaneous mechanoreceptors has never been investigated. Here, we used simple and double immunohistochemistry to investigate the occurrence of Piezo2 in human digital glabrous skin. Piezo2 immunoreactivity was detected in approximately 80% of morphologically and immunohistochemically characterized (cytokeratin 20 + , chromogranin A + and synaptophisin + ) Merkel cells. Most of them were in close contact with Piezo2 - nerve fibre profiles. Moreover, the axon, but not the lamellar cells, of Meissner's corpuscles was also Piezo2 + , but other mechanoreceptors, i.e. Pacinian or Ruffini's corpuscles, were devoid of immunoreactivity. Piezo2 was also observed in non-nervous tissue, especially the basal keratinocytes, endothelial cells and sweat glands. The present results demonstrate the occurrence of Piezo2 in cutaneous sensory nerve formations that functionally work as slowly adapting (Merkel cells) and rapidly adapting (Meissner's corpuscles) low-threshold mechanoreceptors and are related to fine and discriminative touch but not to vibration or hard touch. These data offer additional insight into the molecular basis of mechanosensing in humans. © 2017 Anatomical Society.

  15. Physically based principles of cell adhesion mechanosensitivity in tissues

    International Nuclear Information System (INIS)

    Ladoux, Benoit; Nicolas, Alice

    2012-01-01

    The minimal structural unit that defines living organisms is a single cell. By proliferating and mechanically interacting with each other, cells can build complex organization such as tissues that ultimately organize into even more complex multicellular living organisms, such as mammals, composed of billions of single cells interacting with each other. As opposed to passive materials, living cells actively respond to the mechanical perturbations occurring in their environment. Tissue cell adhesion to its surrounding extracellular matrix or to neighbors is an example of a biological process that adapts to physical cues. The adhesion of tissue cells to their surrounding medium induces the generation of intracellular contraction forces whose amplitude adapts to the mechanical properties of the environment. In turn, solicitation of adhering cells with physical forces, such as blood flow shearing the layer of endothelial cells in the lumen of arteries, reinforces cell adhesion and impacts cell contractility. In biological terms, the sensing of physical signals is transduced into biochemical signaling events that guide cellular responses such as cell differentiation, cell growth and cell death. Regarding the biological and developmental consequences of cell adaptation to mechanical perturbations, understanding mechanotransduction in tissue cell adhesion appears as an important step in numerous fields of biology, such as cancer, regenerative medicine or tissue bioengineering for instance. Physicists were first tempted to view cell adhesion as the wetting transition of a soft bag having a complex, adhesive interaction with the surface. But surprising responses of tissue cell adhesion to mechanical cues challenged this view. This, however, did not exclude that cell adhesion could be understood in physical terms. It meant that new models and descriptions had to be created specifically for these biological issues, and could not straightforwardly be adapted from dead matter

  16. Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development

    OpenAIRE

    Guan, Yingjie; Yang, Xu; Yang, Wentian; Charbonneau, Cherie; Chen, Qian

    2014-01-01

    Mechanical stress regulates development by modulating cell signaling and gene expression. However, the cytoplasmic components mediating mechanotransduction remain unclear. In this study, elimination of muscle contraction during chicken embryonic development resulted in a reduction in the activity of mammalian target of rapamycin (mTOR) in the cartilaginous growth plate. Inhibition of mTOR activity led to significant inhibition of chondrocyte proliferation, cartilage tissue growth, and express...

  17. The role of Drosophila Piezo in mechanical nociception.

    Science.gov (United States)

    Kim, Sung Eun; Coste, Bertrand; Chadha, Abhishek; Cook, Boaz; Patapoutian, Ardem

    2012-02-19

    Transduction of mechanical stimuli by receptor cells is essential for senses such as hearing, touch and pain. Ion channels have a role in neuronal mechanotransduction in invertebrates; however, functional conservation of these ion channels in mammalian mechanotransduction is not observed. For example, no mechanoreceptor potential C (NOMPC), a member of transient receptor potential (TRP) ion channel family, acts as a mechanotransducer in Drosophila melanogaster and Caenorhabditis elegans; however, it has no orthologues in mammals. Degenerin/epithelial sodium channel (DEG/ENaC) family members are mechanotransducers in C. elegans and potentially in D. melanogaster; however, a direct role of its mammalian homologues in sensing mechanical force has not been shown. Recently, Piezo1 (also known as Fam38a) and Piezo2 (also known as Fam38b) were identified as components of mechanically activated channels in mammals. The Piezo family are evolutionarily conserved transmembrane proteins. It is unknown whether they function in mechanical sensing in vivo and, if they do, which mechanosensory modalities they mediate. Here we study the physiological role of the single Piezo member in D. melanogaster (Dmpiezo; also known as CG8486). Dmpiezo expression in human cells induces mechanically activated currents, similar to its mammalian counterparts. Behavioural responses to noxious mechanical stimuli were severely reduced in Dmpiezo knockout larvae, whereas responses to another noxious stimulus or touch were not affected. Knocking down Dmpiezo in sensory neurons that mediate nociception and express the DEG/ENaC ion channel pickpocket (ppk) was sufficient to impair responses to noxious mechanical stimuli. Furthermore, expression of Dmpiezo in these same neurons rescued the phenotype of the constitutive Dmpiezo knockout larvae. Accordingly, electrophysiological recordings from ppk-positive neurons revealed a Dmpiezo-dependent, mechanically activated current. Finally, we found that Dmpiezo

  18. 17β estradiol regulation of connexin 43-based gap junction and mechanosensitivity through classical estrogen receptor pathway in osteocyte-like MLO-Y4 cells.

    KAUST Repository

    Ren, Jian

    2013-04-01

    Connexin 43 (Cx43) plays an essential role in osteocyte mechanotransduction. Although estrogen involves in the adaptive responses of bone cells to mechanical loadings, its effects on osteocytic Cx43-based gap junction intercellular communication (GJIC) remain obscure. We found that 17β estradiol (E2) up-regulated Cx43, and enhanced GJIC in osteocyte-like MLO-Y4 cells in fluorescence recovery after photobleaching (FRAP) assay. Combination of E2 pre-treatment and oscillating fluid flow (OFF) further enhanced Cx43 expression and mitogen-activated protein kinase (MAPK) phosphorylation, comparing to E2 or OFF treatment alone. Both blocking of classical estrogen receptors (ERα/β) by fulvestrant and ERα knockdown by small interfering RNA inhibited E2-mediated Cx43 increase, while a GPR30-specific agonist G-1 failed to promote Cx43 expression. Our results suggest that the presence of E2 enhanced Cx43-based GJIC mainly via ERα/β pathway, and sensitized osteocytes to mechanical loading. © 2012 Elsevier Inc. All rights reserved.

  19. Mammalian Auditory Hair Cell Bundle Stiffness Affects Frequency Tuning by Increasing Coupling along the Length of the Cochlea.

    Science.gov (United States)

    Dewey, James B; Xia, Anping; Müller, Ulrich; Belyantseva, Inna A; Applegate, Brian E; Oghalai, John S

    2018-06-05

    The stereociliary bundles of cochlear hair cells convert mechanical vibrations into the electrical signals required for auditory sensation. While the stiffness of the bundles strongly influences mechanotransduction, its influence on the vibratory response of the cochlear partition is unclear. To assess this, we measured cochlear vibrations in mutant mice with reduced bundle stiffness or with a tectorial membrane (TM) that is detached from the sensory epithelium. We found that reducing bundle stiffness decreased the high-frequency extent and sharpened the tuning of vibratory responses obtained postmortem. Detaching the TM further reduced the high-frequency extent of the vibrations but also lowered the partition's resonant frequency. Together, these results demonstrate that the bundle's stiffness and attachment to the TM contribute to passive longitudinal coupling in the cochlea. We conclude that the stereociliary bundles and TM interact to facilitate passive-wave propagation to more apical locations, possibly enhancing active-wave amplification in vivo. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. Mechanical stimulation of cyclic tensile strain induces reduction of pluripotent related gene expressions via activation of Rho/ROCK and subsequent decreasing of AKT phosphorylation in human induced pluripotent stem cells

    International Nuclear Information System (INIS)

    Teramura, Takeshi; Takehara, Toshiyuki; Onodera, Yuta; Nakagawa, Koichi; Hamanishi, Chiaki; Fukuda, Kanji

    2012-01-01

    Highlights: ► Mechanical stimulation is an important factor for regulation of stem cell fate. ► Cyclic stretch to human induced pluripotent stem cells activated small GTPase Rho. ► Rho-kinase activation attenuated pluripotency via inhibition of AKT activation. ► This reaction could be reproduced only by transfection of dominant active Rho. ► Rho/ROCK are important molecules in mechanotransduction and control of stemness. -- Abstract: Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the stemness or related molecular mechanisms have not been well determined. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, AKT phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the AKT phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator.

  1. Involvement of YAP, TAZ and HSP90 in contact guidance and intercellular junction formation in corneal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Vijay Krishna Raghunathan

    Full Text Available The extracellular environment possesses a rich milieu of biophysical and biochemical signaling cues that are simultaneously integrated by cells and influence cellular phenotype. Yes-associated protein (YAP and transcriptional co-activator with PDZ-binding motif (WWTR1; TAZ, two important signaling molecules of the Hippo pathway, have been recently implicated as nuclear relays of cytoskeletal changes mediated by substratum rigidity and topography. These proteins intersect with other important intracellular signaling pathways (e.g. Wnt and TGFβ. In the cornea, epithelial cells adhere to the stroma through a 3-dimensional topography-rich basement membrane, with features in the nano-submicron size-scale that are capable of profoundly modulating a wide range of fundamental cell behaviors. The influences of substratum-topography, YAP/TAZ knockdown, and HSP90 inhibition on cell morphology, YAP/TAZ localization, and the expression of TGFβ2 and CTGF, were investigated. The results demonstrate (a that knockdown of TAZ enhances contact guidance in a YAP dependent manner, (b that CTGF is predominantly regulated by YAP and not TAZ, and (c that TGFβ2 is regulated by both YAP and TAZ in these cells. Additionally, inhibition of HSP90 resulted in nuclear localization and subsequent transcriptional-activation of YAP, formation of cell-cell junctions and co-localization of E-cadherin and β-catenin at adherens junctions. Results presented in this study reflect the complexities underlying the molecular relationships between the cytoskeleton, growth factors, heat shock proteins, and co-activators of transcription that impact mechanotransduction. The data reveal the importance of YAP/TAZ on the cell behaviors, and gene and protein expression.

  2. Rapid and Localized Mechanical Stimulation and Adhesion Assay: TRPM7 Involvement in Calcium Signaling and Cell Adhesion.

    Directory of Open Access Journals (Sweden)

    Wagner Shin Nishitani

    Full Text Available A cell mechanical stimulation equipment, based on cell substrate deformation, and a more sensitive method for measuring adhesion of cells were developed. A probe, precisely positioned close to the cell, was capable of a vertical localized mechanical stimulation with a temporal frequency of 207 Hz, and strain magnitude of 50%. This setup was characterized and used to probe the response of Human Umbilical Endothelial Vein Cells (HUVECs in terms of calcium signaling. The intracellular calcium ion concentration was measured by the genetically encoded Cameleon biosensor, with the Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7 expression inhibited. As TRPM7 expression also regulates adhesion, a relatively simple method for measuring adhesion of cells was also developed, tested and used to study the effect of adhesion alone. Three adhesion conditions of HUVECs on polyacrylamide gel dishes were compared. In the first condition, the substrate is fully treated with Sulfo-SANPAH crosslinking and fibronectin. The other two conditions had increasingly reduced adhesion: partially treated (only coated with fibronectin, with no use of Sulfo-SANPAH, at 5% of the normal amount and non-treated polyacrylamide gels. The cells showed adhesion and calcium response to the mechanical stimulation correlated to the degree of gel treatment: highest for fully treated gels and lowest for non-treated ones. TRPM7 inhibition by siRNA on HUVECs caused an increase in adhesion relative to control (no siRNA treatment and non-targeting siRNA, but a decrease to 80% of calcium response relative to non-targeting siRNA which confirms the important role of TRPM7 in mechanotransduction despite the increase in adhesion.

  3. Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Horiuchi, Rie; Akimoto, Takayuki; Hong, Zhang; Ushida, Takashi

    2012-01-01

    Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17 Hz. The expression of Nanog was reduced during ES cell differentiation in response to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling. -- Highlights: ► The expression of Nanog, which is an essential regulator of “stemness” was reduced during embryonic stem (ES) cell differentiation. ► Cyclic mechanical strain attenuated the reduction of Nanog expression. ► Cyclic mechanical strain promoted PI3K-Akt signaling and mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor and an inhibitor of actin polymerization.

  4. Structure of a force-conveying cadherin bond essential for inner-ear mechanotransduction.

    Science.gov (United States)

    Sotomayor, Marcos; Weihofen, Wilhelm A; Gaudet, Rachelle; Corey, David P

    2012-12-06

    Hearing and balance use hair cells in the inner ear to transform mechanical stimuli into electrical signals. Mechanical force from sound waves or head movements is conveyed to hair-cell transduction channels by tip links, fine filaments formed by two atypical cadherins known as protocadherin 15 and cadherin 23 (refs 4, 5). These two proteins are involved in inherited deafness and feature long extracellular domains that interact tip-to-tip in a Ca(2+)-dependent manner. However, the molecular architecture of this complex is unknown. Here we combine crystallography, molecular dynamics simulations and binding experiments to characterize the protocadherin 15-cadherin 23 bond. We find a unique cadherin interaction mechanism, in which the two most amino-terminal cadherin repeats (extracellular cadherin repeats 1 and 2) of each protein interact to form an overlapped, antiparallel heterodimer. Simulations predict that this tip-link bond is mechanically strong enough to resist forces in hair cells. In addition, the complex is shown to become unstable in response to Ca(2+) removal owing to increased flexure of Ca(2+)-free cadherin repeats. Finally, we use structures and biochemical measurements to study the molecular mechanisms by which deafness mutations disrupt tip-link function. Overall, our results shed light on the molecular mechanics of hair-cell sensory transduction and on new interaction mechanisms for cadherins, a large protein family implicated in tissue and organ morphogenesis, neural connectivity and cancer.

  5. Mechanical analysis of single myocyte contraction in a 3-D elastic matrix.

    Directory of Open Access Journals (Sweden)

    John Shaw

    Full Text Available Cardiac myocytes experience mechanical stress during each heartbeat. Excessive mechanical stresses under pathological conditions cause functional and structural remodeling that lead to heart diseases, yet the precise mechanisms are still incompletely understood. To study the cellular and molecular level mechanotransduction mechanisms, we developed a new 'cell-in-gel' experimental system to exert multiaxial (3-D stresses on a single myocyte during active contraction.Isolated myocytes are embedded in an elastic hydrogel to simulate the mechanical environment in myocardium (afterload. When electrically stimulated, the in-gel myocyte contracts while the matrix resists shortening and broadening of the cell, exerting normal and shear stresses on the cell. Here we provide a mechanical analysis, based on the Eshelby inclusion problem, of the 3-D strain and stress inside and outside the single myocyte during contraction in an elastic matrix.(1 The fractional shortening of the myocyte depends on the cell's geometric dimensions and the relative stiffness of the cell to the gel. A slender or softer cell has less fractional shortening. A myocyte of typical dimensions embedded in a gel of similar elastic stiffness can contract only 20% of its load-free value. (2 The longitudinal stress inside the cell is about 15 times the transverse stress level. (3 The traction on the cell surface is highly non-uniform, with a maximum near its ends, showing 'hot spots' at the location of intercalated disks. (4 The mechanical energy expenditure of the myocyte increases with the matrix stiffness in a monotonic and nonlinear manner.Our mechanical analyses provide analytic solutions that readily lend themselves to parametric studies. The resulting 3-D mapping of the strain and stress states serve to analyze and interpret ongoing cell-in-gel experiments, and the mathematical model provides an essential tool to decipher and quantify mechanotransduction mechanisms in cardiac

  6. The Distinct Effects of Estrogen and Hydrostatic Pressure on Mesenchymal Stem Cells Differentiation: Involvement of Estrogen Receptor Signaling.

    Science.gov (United States)

    Zhao, Ying; Yi, Fei-Zhou; Zhao, Yin-Hua; Chen, Yong-Jin; Ma, Heng; Zhang, Min

    2016-10-01

    This study aimed to investigate the differential and synergistic effects of mechanical stimulation and estrogen on the proliferation and osteogenic or chondrogenic differentiation potential of bone marrow mesenchymal stem cells (BMSCs) and the roles of estrogen receptor (ER) in them. BMSCs were isolated and cultured using the whole bone marrow adherence method, and flow cytometry was used to identify the surface marker molecules of BMSCs. Cells were pre-treated with 1 nM 17β-estradiol or 1 nM of the estrogen receptor antagonist tamoxifen. Then, the cells were stimulated with hydrostatic pressure. Assessment included flow cytometry analysis of the cell cycle; immunofluorescent staining for F-actin; protein quantification for MAPK protein; and mRNA analysis for Col I, OCN, OPN and BSP after osteogenic induction and Sox-9, Aggrecan and Col-II after chondrogenic induction. Hydrostatic pressure (90 kPa/1 h) and 1 nM 17β-estradiol enhanced the cellular proliferation ability and the cytoskeleton activity but without synergistic biological effects. Estrogen activated ERKs and JNKs simultaneously and promoted the osteogenic differentiation, whereas the pressure just caused JNK-1/2 activation and promoted the chondrogenic differentiation of BMSCs. Estrogen had antagonism effect on chondrogenic promotion of hydrostatic pressure. Mechanobiological effects of hydrostatic pressure are closely associated with ERα activity. MAPK molecules and F-actin were likely to be important mediator molecules in the ER-mediated mechanotransduction of BMSCs.

  7. The Extracellular Environment's Effect on Cellular Processes: An In Vitro Study of Mechanical and Chemical Cues on Human Mesenchymal Stem Cells and C17.2 Neural Stem Cells

    Science.gov (United States)

    Casey, Meghan E.

    Stem cells are widely used in the area of tissue engineering. The ability of cells to interact with materials on the nano- and micro- level is important in the success of the biomaterial. It is well-known that cells respond to their micro- and nano-environments through a process termed chemo-mechanotransduction. It is important to establish standard protocols for cellular experiments, as chemical modifications to maintenance environments can alter long-term research results. In this work, the effects of different media compositions on human mesenchymal stem cells (hMSCs) throughout normal in vitro maintenance are investigated. Changes in RNA regulation, protein expression and proliferation are studied via quantitative polymerase chain reaction (qPCR), immunocytochemistry (ICC) and cell counts, respectively. Morphological differences are also observed throughout the experiment. Results of this study illustrate the dynamic response of hMSC maintenance to differences in growth medium and passage number. These experiments highlight the effect growth medium has on in vitro experiments and the need of consistent protocols in hMSC research. A substantial opportunity exists in neuronal research to develop a material platform that allows for both the proliferation and differentiation of stem cells into neurons and the ability to quantify the secretome of neuronal cells. Anodic aluminum oxide (AAO) membranes are fabricated in a two-step anodization procedure where voltage is varied to control the pore size and morphology of the membranes. C17.2 neural stem cells are differentiated on the membranes via serum-withdrawal. Cellular growth is characterized by scanning electron microscopy (SEM), ICC and qPCR. ImageJ software is used to obtain phenotypic cell counts and neurite outgrowth lengths. Results indicate a highly tunable correlation between AAO nanopore sizes and differentiated cell populations. By selecting AAO membranes with specific pore size ranges, control of neuronal

  8. Effects of Neuropeptides and Mechanical Loading on Bone Cell Resorption in Vitro

    Directory of Open Access Journals (Sweden)

    Yeong-Min Yoo

    2014-04-01

    Full Text Available Neuropeptides such as vasoactive intestinal peptide (VIP and calcitonin gene-related peptide (CGRP are present in nerve fibers of bone tissues and have been suggested to potentially regulate bone remodeling. Oscillatory fluid flow (OFF-induced shear stress is a potent signal in mechanotransduction that is capable of regulating both anabolic and catabolic bone remodeling. However, the interaction between neuropeptides and mechanical induction in bone remodeling is poorly understood. In this study, we attempted to quantify the effects of combined neuropeptides and mechanical stimuli on mRNA and protein expression related to bone resorption. Neuropeptides (VIP or CGRP and/or OFF-induced shear stress were applied to MC3T3-E1 pre-osteoblastic cells and changes in receptor activator of nuclear factor kappa B (NF-κB ligand (RANKL and osteoprotegerin (OPG mRNA and protein levels were quantified. Neuropeptides and OFF-induced shear stress similarly decreased RANKL and increased OPG levels compared to control. Changes were not further enhanced with combined neuropeptides and OFF-induced shear stress. These results suggest that neuropeptides CGRP and VIP have an important role in suppressing bone resorptive activities through RANKL/OPG pathway, similar to mechanical loading.

  9. Tuning Piezo ion channels to detect molecular-scale movements relevant for fine touch

    Science.gov (United States)

    Poole, Kate; Herget, Regina; Lapatsina, Liudmila; Ngo, Ha-Duong; Lewin, Gary R.

    2014-01-01

    In sensory neurons, mechanotransduction is sensitive, fast and requires mechanosensitive ion channels. Here we develop a new method to directly monitor mechanotransduction at defined regions of the cell-substrate interface. We show that molecular-scale (~13 nm) displacements are sufficient to gate mechanosensitive currents in mouse touch receptors. Using neurons from knockout mice, we show that displacement thresholds increase by one order of magnitude in the absence of stomatin-like protein 3 (STOML3). Piezo1 is the founding member of a class of mammalian stretch-activated ion channels, and we show that STOML3, but not other stomatin-domain proteins, brings the activation threshold for Piezo1 and Piezo2 currents down to ~10 nm. Structure–function experiments localize the Piezo modulatory activity of STOML3 to the stomatin domain, and higher-order scaffolds are a prerequisite for function. STOML3 is the first potent modulator of Piezo channels that tunes the sensitivity of mechanically gated channels to detect molecular-scale stimuli relevant for fine touch. PMID:24662763

  10. Synergy between Piezo1 and Piezo2 channels confers high-strain mechanosensitivity to articular cartilage

    Science.gov (United States)

    Lee, Whasil; Leddy, Holly A.; Chen, Yong; Lee, Suk Hee; Zelenski, Nicole A.; McNulty, Amy L.; Wu, Jason; Beicker, Kellie N.; Coles, Jeffrey; Zauscher, Stefan; Grandl, Jörg; Sachs, Frederick; Liedtke, Wolfgang B.

    2014-01-01

    Diarthrodial joints are essential for load bearing and locomotion. Physiologically, articular cartilage sustains millions of cycles of mechanical loading. Chondrocytes, the cells in cartilage, regulate their metabolic activities in response to mechanical loading. Pathological mechanical stress can lead to maladaptive cellular responses and subsequent cartilage degeneration. We sought to deconstruct chondrocyte mechanotransduction by identifying mechanosensitive ion channels functioning at injurious levels of strain. We detected robust expression of the recently identified mechanosensitive channels, PIEZO1 and PIEZO2. Combined directed expression of Piezo1 and -2 sustained potentiated mechanically induced Ca2+ signals and electrical currents compared with single-Piezo expression. In primary articular chondrocytes, mechanically evoked Ca2+ transients produced by atomic force microscopy were inhibited by GsMTx4, a PIEZO-blocking peptide, and by Piezo1- or Piezo2-specific siRNA. We complemented the cellular approach with an explant-cartilage injury model. GsMTx4 reduced chondrocyte death after mechanical injury, suggesting a possible therapy for reducing cartilage injury and posttraumatic osteoarthritis by attenuating Piezo-mediated cartilage mechanotransduction of injurious strains. PMID:25385580

  11. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures

    Science.gov (United States)

    Juhász, Tamás; Szentléleky, Eszter; Szűcs Somogyi, Csilla; Takács, Roland; Dobrosi, Nóra; Engler, Máté; Tamás, Andrea; Reglődi, Dóra; Zákány, Róza

    2015-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load. PMID:26230691

  12. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures

    Directory of Open Access Journals (Sweden)

    Tamás Juhász

    2015-07-01

    Full Text Available Pituitary adenylate cyclase activating polypeptide (PACAP is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load.

  13. A novel Atoh1 "self-terminating" mouse model reveals the necessity of proper Atoh1 level and duration for hair cell differentiation and viability.

    Directory of Open Access Journals (Sweden)

    Ning Pan

    Full Text Available Atonal homolog1 (Atoh1 is a bHLH transcription factor essential for inner ear hair cell differentiation. Targeted expression of Atoh1 at various stages in development can result in hair cell differentiation in the ear. However, the level and duration of Atoh1 expression required for proper hair cell differentiation and maintenance remain unknown. We generated an Atoh1 conditional knockout (CKO mouse line using Tg(Atoh1-cre, in which the cre expression is driven by an Atoh1 enhancer element that is regulated by Atoh1 protein to "self-terminate" its expression. The mutant mice show transient, limited expression of Atoh1 in all hair cells in the ear. In the organ of Corti, reduction and delayed deletion of Atoh1 result in progressive loss of almost all the inner hair cells and the majority of the outer hair cells within three weeks after birth. The remaining cells express hair cell marker Myo7a and attract nerve fibers, but do not differentiate normal stereocilia bundles. Some Myo7a-positive cells persist in the cochlea into adult stages in the position of outer hair cells, flanked by a single row of pillar cells and two to three rows of disorganized Deiters cells. Gene expression analyses of Atoh1, Barhl1 and Pou4f3, genes required for survival and maturation of hair cells, reveal earlier and higher expression levels in the inner compared to the outer hair cells. Our data show that Atoh1 is crucial for hair cell mechanotransduction development, viability, and maintenance and also suggest that Atoh1 expression level and duration may play a role in inner vs. outer hair cell development. These genetically engineered Atoh1 CKO mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with Atoh1 therapy.

  14. Mechanical stimulation of cyclic tensile strain induces reduction of pluripotent related gene expressions via activation of Rho/ROCK and subsequent decreasing of AKT phosphorylation in human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Teramura, Takeshi, E-mail: teramura@med.kindai.ac.jp [Institute of Advanced Clinical Medicine, Kinki University, Faculty of Medicine, Osaka (Japan); Takehara, Toshiyuki; Onodera, Yuta [Institute of Advanced Clinical Medicine, Kinki University, Faculty of Medicine, Osaka (Japan); Nakagawa, Koichi; Hamanishi, Chiaki [Department of Orthopaedic Surgery, Kinki University, Faculty of Medicine, Osaka (Japan); Fukuda, Kanji [Institute of Advanced Clinical Medicine, Kinki University, Faculty of Medicine, Osaka (Japan); Department of Orthopaedic Surgery, Kinki University, Faculty of Medicine, Osaka (Japan)

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Mechanical stimulation is an important factor for regulation of stem cell fate. Black-Right-Pointing-Pointer Cyclic stretch to human induced pluripotent stem cells activated small GTPase Rho. Black-Right-Pointing-Pointer Rho-kinase activation attenuated pluripotency via inhibition of AKT activation. Black-Right-Pointing-Pointer This reaction could be reproduced only by transfection of dominant active Rho. Black-Right-Pointing-Pointer Rho/ROCK are important molecules in mechanotransduction and control of stemness. -- Abstract: Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the stemness or related molecular mechanisms have not been well determined. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, AKT phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the AKT phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator.

  15. Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Horiuchi, Rie [Division of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan); Akimoto, Takayuki, E-mail: akimoto@m.u-tokyo.ac.jp [Division of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan); Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Waseda-tsurumaki, Shinjuku, Tokyo 162-0041 (Japan); Hong, Zhang [Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Waseda-tsurumaki, Shinjuku, Tokyo 162-0041 (Japan); Ushida, Takashi [Division of Regenerative Medical Engineering, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan)

    2012-08-15

    Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17 Hz. The expression of Nanog was reduced during ES cell differentiation in response to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling. -- Highlights: Black-Right-Pointing-Pointer The expression of Nanog, which is an essential regulator of 'stemness' was reduced during embryonic stem (ES) cell differentiation. Black-Right-Pointing-Pointer Cyclic mechanical strain attenuated the reduction of Nanog expression. Black-Right-Pointing-Pointer Cyclic mechanical strain promoted PI3K-Akt signaling and mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor and an inhibitor of actin polymerization.

  16. The effects of osmotic stress on the structure and function of the cell nucleus.

    Science.gov (United States)

    Finan, John D; Guilak, Farshid

    2010-02-15

    Osmotic stress is a potent regulator of the normal function of cells that are exposed to osmotically active environments under physiologic or pathologic conditions. The ability of cells to alter gene expression and metabolic activity in response to changes in the osmotic environment provides an additional regulatory mechanism for a diverse array of tissues and organs in the human body. In addition to the activation of various osmotically- or volume-activated ion channels, osmotic stress may also act on the genome via a direct biophysical pathway. Changes in extracellular osmolality alter cell volume, and therefore, the concentration of intracellular macromolecules. In turn, intracellular macromolecule concentration is a key physical parameter affecting the spatial organization and pressurization of the nucleus. Hyper-osmotic stress shrinks the nucleus and causes it to assume a convoluted shape, whereas hypo-osmotic stress swells the nucleus to a size that is limited by stretch of the nuclear lamina and induces a smooth, round shape of the nucleus. These behaviors are consistent with a model of the nucleus as a charged core/shell structure pressurized by uneven partition of macromolecules between the nucleoplasm and the cytoplasm. These osmotically-induced alterations in the internal structure and arrangement of chromatin, as well as potential changes in the nuclear membrane and pores are hypothesized to influence gene transcription and/or nucleocytoplasmic transport. A further understanding of the biophysical and biochemical mechanisms involved in these processes would have important ramifications for a range of fields including differentiation, migration, mechanotransduction, DNA repair, and tumorigenesis. (c) 2009 Wiley-Liss, Inc.

  17. Biomechanics and mechanical signaling in the ovary: a systematic review.

    Science.gov (United States)

    Shah, Jaimin S; Sabouni, Reem; Cayton Vaught, Kamaria C; Owen, Carter M; Albertini, David F; Segars, James H

    2018-04-24

    Mammalian oogenesis and folliculogenesis share a dynamic connection that is critical for gamete development. For maintenance of quiescence or follicular activation, follicles must respond to soluble signals (growth factors and hormones) and physical stresses, including mechanical forces and osmotic shifts. Likewise, mechanical processes are involved in cortical tension and cell polarity in oocytes. Our objective was to examine the contribution and influence of biomechanical signaling in female mammalian gametogenesis. We performed a systematic review to assess and summarize the effects of mechanical signaling and mechanotransduction in oocyte maturation and folliculogenesis and to explore possible clinical applications. The review identified 2568 publications of which 122 met the inclusion criteria. The integration of mechanical and cell signaling pathways in gametogenesis is complex. Follicular activation or quiescence are influenced by mechanical signaling through the Hippo and Akt pathways involving the yes-associated protein (YAP), transcriptional coactivator with PDZ-binding motif (TAZ), phosphatase and tensin homolog deleted from chromosome 10 (PTEN) gene, the mammalian target of rapamycin (mTOR), and forkhead box O3 (FOXO3) gene. There is overwhelming evidence that mechanical signaling plays a crucial role in development of the ovary, follicle, and oocyte throughout gametogenesis. Emerging data suggest the complexities of mechanotransduction and the biomechanics of oocytes and follicles are integral to understanding of primary ovarian insufficiency, ovarian aging, polycystic ovary syndrome, and applications of fertility preservation.

  18. Micro and nanotechnology for biological and biomedical applications.

    Science.gov (United States)

    Lim, Chwee Teck; Han, Jongyoon; Guck, Jochen; Espinosa, Horacio

    2010-10-01

    This special issue contains some of the current state-of-the-art development and use of micro and nanotechnological tools, devices and techniques for both biological and biomedical research and applications. These include nanoparticles for bioimaging and biosensing, optical and biophotonic techniques for probing diseases at the nanoscale, micro and nano-fabricated tools for elucidating molecular mechanisms of mechanotransduction in cell and molecular biology and cell separation microdevices and techniques for isolating and enriching targeted cells for disease detection and diagnosis. Although some of these works are still at the research stage, there is no doubt that some of the important outcomes will eventually see actual biomedical applications in the not too distant future.

  19. Loss of Slc4a1b chloride/bicarbonate exchanger function protects mechanosensory hair cells from aminoglycoside damage in the zebrafish mutant persephone.

    Directory of Open Access Journals (Sweden)

    Dale W Hailey

    Full Text Available Mechanosensory hair cell death is a leading cause of hearing and balance disorders in the human population. Hair cells are remarkably sensitive to environmental insults such as excessive noise and exposure to some otherwise therapeutic drugs. However, individual responses to damaging agents can vary, in part due to genetic differences. We previously carried out a forward genetic screen using the zebrafish lateral line system to identify mutations that alter the response of larval hair cells to the antibiotic neomycin, one of a class of aminoglycoside compounds that cause hair cell death in humans. The persephone mutation confers resistance to aminoglycosides. 5 dpf homozygous persephone mutants are indistinguishable from wild-type siblings, but differ in their retention of lateral line hair cells upon exposure to neomycin. The mutation in persephone maps to the chloride/bicarbonate exchanger slc4a1b and introduces a single Ser-to-Phe substitution in zSlc4a1b. This mutation prevents delivery of the exchanger to the cell surface and abolishes the ability of the protein to import chloride across the plasma membrane. Loss of function of zSlc4a1b reduces hair cell death caused by exposure to the aminoglycosides neomycin, kanamycin, and gentamicin, and the chemotherapeutic drug cisplatin. Pharmacological block of anion transport with the disulfonic stilbene derivatives DIDS and SITS, or exposure to exogenous bicarbonate, also protects hair cells against damage. Both persephone mutant and DIDS-treated wild-type larvae show reduced uptake of labeled aminoglycosides. persephone mutants also show reduced FM1-43 uptake, indicating a potential impact on mechanotransduction-coupled activity in the mutant. We propose that tight regulation of the ionic environment of sensory hair cells, mediated by zSlc4a1b activity, is critical for their sensitivity to aminoglycoside antibiotics.

  20. Integrins and small GTPases as modulators of phagocytosis.

    Science.gov (United States)

    Sayedyahossein, Samar; Dagnino, Lina

    2013-01-01

    Phagocytosis is the mechanism whereby cells engulf large particles. This process has long been recognized as a critical component of the innate immune response, which constitutes the organism's defense against microorganisms. In addition, phagocytic internalization of apoptotic cells or cell fragments plays important roles in tissue homeostasis and remodeling. Phagocytosis requires target interactions with receptors on the plasma membrane of the phagocytic cell. Integrins have been identified as important mediators of particle clearance, in addition to their well-established roles in cell adhesion, migration and mechanotransduction. Indeed, these ubiquitously expressed proteins impart phagocytic capacity to epithelial, endothelial and mesenchymal cell types. The importance of integrins in particle internalization is emphasized by the ability of microbial and viral pathogens to exploit their signaling pathways to invade host cells, and by the wide variety of disorders that arise from abnormalities in integrin-dependent phagocytic uptake. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Can resistive breathing injure the lung? Implications for COPD exacerbations

    Directory of Open Access Journals (Sweden)

    Vassilakopoulos T

    2016-09-01

    Full Text Available Theodoros Vassilakopoulos, Dimitrios Toumpanakis Pulmonary and Critical Care Medicine, Medical School, National and Kapodistrian University of Athens, Greece Abstract: In obstructive lung diseases, airway inflammation leads to bronchospasm and thus resistive breathing, especially during exacerbations. This commentary discusses experimental evidence that resistive breathing per se (the mechanical stimulus in the absence of underlying airway inflammation leads to lung injury and inflammation (mechanotransduction. The potential implications of resistive breathing-induced mechanotrasduction in COPD exacerbations are presented along with the available clinical evidence. Keywords: resistive breathing, COPD, mechanotransduction, bronchoconstriction, inflammation

  2. The Emergence of Physiology and Form: Natural Selection Revisited

    Science.gov (United States)

    Torday, John S.

    2016-01-01

    Natural Selection describes how species have evolved differentially, but it is descriptive, non-mechanistic. What mechanisms does Nature use to accomplish this feat? One known way in which ancient natural forces affect development, phylogeny and physiology is through gravitational effects that have evolved as mechanotransduction, seen in the lung, kidney and bone, linking as molecular homologies to skin and brain. Tracing the ontogenetic and phylogenetic changes that have facilitated mechanotransduction identifies specific homologous cell-types and functional molecular markers for lung homeostasis that reveal how and why complex physiologic traits have evolved from the unicellular to the multicellular state. Such data are reinforced by their reverse-evolutionary patterns in chronic degenerative diseases. The physiologic responses of model organisms like Dictyostelium and yeast to gravity provide deep comparative molecular phenotypic homologies, revealing mammalian Target of Rapamycin (mTOR) as the final common pathway for vertical integration of vertebrate physiologic evolution; mTOR integrates calcium/lipid epistatic balance as both the proximate and ultimate positive selection pressure for vertebrate physiologic evolution. The commonality of all vertebrate structure-function relationships can be reduced to calcium/lipid homeostatic regulation as the fractal unit of vertebrate physiology, demonstrating the primacy of the unicellular state as the fundament of physiologic evolution. PMID:27534726

  3. The hemodynamically-regulated vascular microenvironment promotes migration of the steroidogenic tissue during its interaction with chromaffin cells in the zebrafish embryo.

    Directory of Open Access Journals (Sweden)

    Chih-Wei Chou

    Full Text Available BACKGROUND: While the endothelium-organ interaction is critical for regulating cellular behaviors during development and disease, the role of blood flow in these processes is only partially understood. The dorsal aorta performs paracrine functions for the timely migration and differentiation of the sympatho-adrenal system. However, it is unclear how the adrenal cortex and medulla achieve and maintain specific integration and whether hemodynamic forces play a role. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, the possible modulation of steroidogenic and chromaffin cell integration by blood flow was investigated in the teleostean counterpart of the adrenal gland, the interrenal gland, in the zebrafish (Danio rerio. Steroidogenic tissue migration and angiogenesis were suppressed by genetic or pharmacologic inhibition of blood flow, and enhanced by acceleration of blood flow upon norepinephrine treatment. Repressed steroidogenic tissue migration and angiogenesis due to flow deficiency were recoverable following restoration of flow. The regulation of interrenal morphogenesis by blood flow was found to be mediated through the vascular microenvironment and the Fibronectin-phosphorylated Focal Adhesion Kinase (Fn-pFak signaling. Moreover, the knockdown of krüppel-like factor 2a (klf2a or matrix metalloproteinase 2 (mmp2, two genes regulated by the hemodynamic force, phenocopied the defects in migration, angiogenesis, the vascular microenvironment, and pFak signaling of the steroidogenic tissue observed in flow-deficient embryos, indicating a direct requirement of mechanotransduction in these processes. Interestingly, epithelial-type steroidogenic cells assumed a mesenchymal-like character and downregulated β-Catenin at cell-cell junctions during interaction with chromaffin cells, which was reversed by inhibiting blood flow or Fn-pFak signaling. Blood flow obstruction also affected the migration of chromaffin cells, but not through

  4. Voluntary wheel running increases satellite cell abundance and improves recovery from disuse in gastrocnemius muscles from mice.

    Science.gov (United States)

    Brooks, Matthew J; Hajira, Ameena; Mohamed, Junaith S; Alway, Stephen E

    2018-02-22

    Reloading of atrophied muscles after hindlimb suspension unloading (HSU) can induce injury and prolong recovery. Low-impact exercise, such as voluntary wheel running, has been identified as a non-damaging rehabilitation therapy in rodents, but its effects on muscle function, morphology, and satellite cell activity after HSU are unclear. This study tested the hypothesis that low impact wheel running would increase satellite cell proliferation and improve recovery of muscle structure and function after HSU in mice. Young adult male and female C57BL/6 mice (n=6/group) were randomly placed into 5 groups. These included HSU without recovery (HSU), normal ambulatory recovery for 14 days after HSU (HSU+NoWR), and voluntary wheel running recovery for 14 days after HSU (HSU+WR). Two control groups were used: non-suspended mice-cage controls (Control) and voluntary wheel running controls (ControlWR). Satellite cell activation, was evaluated by providing mice 5-bromo-2'-deoxyuridine (BrdU) in their drinking water. As expected, HSU significantly reduced in vivo maximal force and decreased the in vivo fatigability and decreased type I and IIa myosin heavy chain (MHC) abundance in plantarflexor muscles. HSU+WR mice significantly improved plantarflexor fatigue resistance, increased type type I and IIa MHC abundance, increased fiber cross sectional area (CSA), and an increased the percentage of type I and IIA muscle fibers in the gastrocnemius muscle. HSU+WR mice also had a significantly greater percentage of BrdU-positive and Pax 7 positive nuclei inside muscle fibers and a greater MyoD to Pax 7 protein ratio when compared to HSU+NoWR mice. The mechanotransduction protein Yes-associated protein (YAP) was elevated with reloading after HSU, but HSU+WR had lower levels of the inactive phosphorylated YAP serine127 which may have contributed to increased satellite cell activation creased with reloading after HSU. These results indicate that voluntary wheel running increased YAP

  5. Mechanical confinement regulates cartilage matrix formation by chondrocytes

    Science.gov (United States)

    Lee, Hong-Pyo; Gu, Luo; Mooney, David J.; Levenston, Marc E.; Chaudhuri, Ovijit

    2017-12-01

    Cartilage tissue equivalents formed from hydrogels containing chondrocytes could provide a solution for replacing damaged cartilage. Previous approaches have often utilized elastic hydrogels. However, elastic stresses may restrict cartilage matrix formation and alter the chondrocyte phenotype. Here we investigated the use of viscoelastic hydrogels, in which stresses are relaxed over time and which exhibit creep, for three-dimensional (3D) culture of chondrocytes. We found that faster relaxation promoted a striking increase in the volume of interconnected cartilage matrix formed by chondrocytes. In slower relaxing gels, restriction of cell volume expansion by elastic stresses led to increased secretion of IL-1β, which in turn drove strong up-regulation of genes associated with cartilage degradation and cell death. As no cell-adhesion ligands are presented by the hydrogels, these results reveal cell sensing of cell volume confinement as an adhesion-independent mechanism of mechanotransduction in 3D culture, and highlight stress relaxation as a key design parameter for cartilage tissue engineering.

  6. Biomechanics trends in modeling and simulation

    CERN Document Server

    Ogden, Ray

    2017-01-01

    The book presents a state-of-the-art overview of biomechanical and mechanobiological modeling and simulation of soft biological tissues. Seven well-known scientists working in that particular field discuss topics such as biomolecules, networks and cells as well as failure, multi-scale, agent-based, bio-chemo-mechanical and finite element models appropriate for computational analysis. Applications include arteries, the heart, vascular stents and valve implants as well as adipose, brain, collagenous and engineered tissues. The mechanics of the whole cell and sub-cellular components as well as the extracellular matrix structure and mechanotransduction are described. In particular, the formation and remodeling of stress fibers, cytoskeletal contractility, cell adhesion and the mechanical regulation of fibroblast migration in healing myocardial infarcts are discussed. The essential ingredients of continuum mechanics are provided. Constitutive models of fiber-reinforced materials with an emphasis on arterial walls ...

  7. Microengineered in vitro model of cardiac fibrosis through modulating myofibroblast mechanotransduction

    International Nuclear Information System (INIS)

    Zhao, Hui; Li, Xiaokang; Zhao, Shan; Zeng, Yang; Ding, Haiyan; Du, Yanan; Zhao, Long; Sun, Wei

    2014-01-01

    Cardiac fibrosis greatly impairs normal heart function post infarction and there is no effective anti-fibrotic drug developed at present. The current therapies for cardiac infarction mainly take effect by eliminating occlusion in coronary artery by thrombolysis drugs, vascular stent grafting or heart bypass operation, which are capable to provide sufficient blood flow for intact myocardium yet showed subtle efficacy in ameliorating fibrosis condition. The advances of in vitro cell/tissue models open new avenues for drug assessment due to the low cost, good controllability and availability as well as the convenience for operation as compared to the animal models. To our knowledge, no proper biomimetic in vitro cardiac fibrosis model has been reported yet. Here we engineered an in vitro cardiac fibrosis model using heart-derived fibroblasts, and the fibrogenesis was recapitulated by patterning the substrate rigidity which mimicked the mechanical heterogeneity of myocardium post-infarction. Various biomarkers for cardiac fibrosis were assayed to validate the biomimicry of the engineered platform. Subsequent addition of Rho-associated protein kinase (ROCK) pathway inhibitor reduced the ratio of myofibroblasts, indicating the feasibility of applying this platform in screening anti-fibrosis drugs. (paper)

  8. Chemo-mechanical control of neural stem cell differentiation

    Science.gov (United States)

    Geishecker, Emily R.

    Cellular processes such as adhesion, proliferation, and differentiation are controlled in part by cell interactions with the microenvironment. Cells can sense and respond to a variety of stimuli, including soluble and insoluble factors (such as proteins and small molecules) and externally applied mechanical stresses. Mechanical properties of the environment, such as substrate stiffness, have also been suggested to play an important role in cell processes. The roles of both biochemical and mechanical signaling in fate modification of stem cells have been explored independently. However, very few studies have been performed to study well-controlled chemo-mechanotransduction. The objective of this work is to design, synthesize, and characterize a chemo-mechanical substrate to encourage neuronal differentiation of C17.2 neural stem cells. In Chapter 2, Polyacrylamide (PA) gels of varying stiffnesses are functionalized with differing amounts of whole collagen to investigate the role of protein concentration in combination with substrate stiffness. As expected, neurons on the softest substrate were more in number and neuronal morphology than those on stiffer substrates. Neurons appeared locally aligned with an expansive network of neurites. Additional experiments would allow for statistical analysis to determine if and how collagen density impacts C17.2 differentiation in combination with substrate stiffness. Due to difficulties associated with whole protein approaches, a similar platform was developed using mixed adhesive peptides, derived from fibronectin and laminin, and is presented in Chapter 3. The matrix elasticity and peptide concentration can be individually modulated to systematically probe the effects of chemo-mechanical signaling on differentiation of C17.2 cells. Polyacrylamide gel stiffness was confirmed using rheological techniques and found to support values published by Yeung et al. [1]. Cellular growth and differentiation were assessed by cell counts

  9. Force control for mechanoinduction of impedance variation in cellular organisms

    International Nuclear Information System (INIS)

    Nam, Joo Hoo; Chen, Peter C Y; Lu, Zhe; Luo, Hong; Lin, Wei; Ge, Ruowen

    2010-01-01

    Constantly exposed to various forms of mechanical forces inherent in their physical environment (such as gravity, stress induced by fluid flow or cell–cell interactions, etc), cellular organisms sense such forces and convert them into biochemical signals through the processes of mechanosensing and mechanotransduction that eventually lead to biological changes. The effect of external forces on the internal structures and activities in a cellular organism may manifest in changes its physical properties, such as impedance. Studying variation in the impedance of a cellular organism induced by the application of an external mechanical force represents a meaningful endeavor (from a biosystems perspective) in exploring the complex mechanosensing and mechanotransduction mechanisms that govern the behavior of a cellular organism under the influence of external mechanical stimuli. In this paper we describe the development of an explicit force-feedback control system for exerting an indentation force on a cellular organism while simultaneously measuring its impedance. To demonstrate the effectiveness of this force-control system, we have conducted experiments using zebrafish embryos as a test model of a cellular organism. We report experimental results demonstrating that the application of a properly controlled external force leads to a significant change in the impedance of a zebrafish embryo. These results offer support for a plausible explanation that activities of pore canals in the chorion are responsible for the observed change in impedance.

  10. The force-sensing device region of α-catenin is an intrinsically disordered segment in the absence of intramolecular stabilization of the autoinhibitory form.

    Science.gov (United States)

    Hirano, Yoshinori; Amano, Yu; Yonemura, Shigenobu; Hakoshima, Toshio

    2018-05-01

    Mechanotransduction by α-catenin facilitates the force-dependent development of adherens junctions (AJs) by recruiting vinculin to reinforce actin anchoring of AJs. The α-catenin mechanotransducing action is facilitated by its force-sensing device region that autoinhibits the vinculin-binding site 1 (VBS1). Here, we report the high-resolution structure of the force-sensing device region of α-catenin, which shows the autoinhibited form comprised of helix bundles E, F and G. The cryptic VBS1 is embedded into helix bundle E stabilized by direct interactions with the autoinhibitory region forming helix bundles F and G. Our molecular dissection study showed that helix bundles F and G are stable in solution in each isolated form, whereas helix bundle E that contains VBS1 is unstable and intrinsically disordered in solution in the isolated form. We successfully identified key residues mediating the autoinhibition and produced mutated α-catenins that display variable force sensitivity and autoinhibition. Using these mutants, we demonstrate both in vitro and in vivo that, in the absence of this stabilization, the helix bundle containing VBS1 would adopt an unfolded form, thus exposing VBS for vinculin binding. We provide evidence for importance of mechanotransduction with the intrinsic force sensitivity for vinculin recruitment to adherens junctions of epithelial cell sheets with mutated α-catenins. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  11. Effects of mechanical loading on human mesenchymal stem cells for cartilage tissue engineering.

    Science.gov (United States)

    Choi, Jane Ru; Yong, Kar Wey; Choi, Jean Yu

    2018-03-01

    Today, articular cartilage damage is a major health problem, affecting people of all ages. The existing conventional articular cartilage repair techniques, such as autologous chondrocyte implantation (ACI), microfracture, and mosaicplasty, have many shortcomings which negatively affect their clinical outcomes. Therefore, it is essential to develop an alternative and efficient articular repair technique that can address those shortcomings. Cartilage tissue engineering, which aims to create a tissue-engineered cartilage derived from human mesenchymal stem cells (MSCs), shows great promise for improving articular cartilage defect therapy. However, the use of tissue-engineered cartilage for the clinical therapy of articular cartilage defect still remains challenging. Despite the importance of mechanical loading to create a functional cartilage has been well demonstrated, the specific type of mechanical loading and its optimal loading regime is still under investigation. This review summarizes the most recent advances in the effects of mechanical loading on human MSCs. First, the existing conventional articular repair techniques and their shortcomings are highlighted. The important parameters for the evaluation of the tissue-engineered cartilage, including chondrogenic and hypertrophic differentiation of human MSCs are briefly discussed. The influence of mechanical loading on human MSCs is subsequently reviewed and the possible mechanotransduction signaling is highlighted. The development of non-hypertrophic chondrogenesis in response to the changing mechanical microenvironment will aid in the establishment of a tissue-engineered cartilage for efficient articular cartilage repair. © 2017 Wiley Periodicals, Inc.

  12. Integrated strain array for cellular mechanobiology studies

    International Nuclear Information System (INIS)

    Simmons, C S; Sim, J Y; Baechtold, P; Chung, C; Borghi, N; Pruitt, B L; Gonzalez, A

    2011-01-01

    We have developed an integrated strain array for cell culture enabling high-throughput mechano-transduction studies. Biocompatible cell culture chambers were integrated with an acrylic pneumatic compartment and microprocessor-based control system. Each element of the array consists of a deformable membrane supported by a cylindrical pillar within a well. For user-prescribed waveforms, the annular region of the deformable membrane is pulled into the well around the pillar under vacuum, causing the pillar-supported region with cultured cells to be stretched biaxially. The optically clear device and pillar-based mechanism of operation enables imaging on standard laboratory microscopes. Straightforward fabrication utilizes off-the-shelf components, soft lithography techniques in polydimethylsiloxane and laser ablation of acrylic sheets. Proof of compatibility with basic biological assays and standard imaging equipment were accomplished by straining C2C12 skeletal myoblasts on the device for 6 h. At higher strains, cells and actin stress fibers realign with a circumferential preference

  13. Tissue Functioning and Remodeling in the Circulatory and Ventilatory Systems

    CERN Document Server

    Thiriet, Marc

    2013-01-01

    The volumes in this authoritative series present a multidisciplinary approach to modeling and simulation of flows in the cardiovascular and ventilatory systems, especially multiscale modeling and coupled simulations. Volume 5 is devoted to cells, tissues, and organs of the cardiovascular and ventilatory systems with an emphasis on mechanotransduction-based regulation of flow. The blood vessel wall is a living tissue that quickly reacts to loads applied on it by the flowing blood. In any segment of a blood vessel, the endothelial and smooth muscle cells can sense unusual time variations in small-magnitude wall shear stress and large-amplitude wall stretch generated by abnormal hemodynamic stresses. These cells respond with a short-time scale (from seconds to hours) to adapt the vessel caliber. Since such adaptive cell activities can be described using mathematical models, a key objective of this volume is to identify the mesoscopic agents and nanoscopic mediators required to derive adequate mathematical models...

  14. Mechanotransduction and Metabolism in Cardiomyocyte Microdomains.

    Science.gov (United States)

    Pasqualini, Francesco S; Nesmith, Alexander P; Horton, Renita E; Sheehy, Sean P; Parker, Kevin Kit

    2016-01-01

    Efficient contractions of the left ventricle are ensured by the continuous transfer of adenosine triphosphate (ATP) from energy production sites, the mitochondria, to energy utilization sites, such as ionic pumps and the force-generating sarcomeres. To minimize the impact of intracellular ATP trafficking, sarcomeres and mitochondria are closely packed together and in proximity with other ultrastructures involved in excitation-contraction coupling, such as t-tubules and sarcoplasmic reticulum junctions. This complex microdomain has been referred to as the intracellular energetic unit. Here, we review the literature in support of the notion that cardiac homeostasis and disease are emergent properties of the hierarchical organization of these units. Specifically, we will focus on pathological alterations of this microdomain that result in cardiac diseases through energy imbalance and posttranslational modifications of the cytoskeletal proteins involved in mechanosensing and transduction.

  15. Quantitative characterization of 3D deformations of cell interactions with soft biomaterials

    Science.gov (United States)

    Franck, Christian

    -induced or externally applied deformations. This method is validated by comparing experimentally measured non-uniform deformation fields near hard and soft spherical inclusions under uniaxial compression with the corresponding analytical solution. Utilization of a newly developed computationally efficient stretch-correlation and deconvolution algorithm is shown to improve the overall measurement accuracy, in particular under large deformations. Using this technique, the full three-dimensional substrate displacement fields are experimentally determined during the migration of individual fibroblast cells on polyacrylamide gels. This is the first study to show the highly three-dimensional structure of cell-induced displacement and traction fields. These new findings suggest a three-dimensional push-pull cell motility, which differs from the traditional theories based on two-dimensional data. These results provide new insight into the dynamic cell-matrix force exchange or mechanotransduction of migrating cells, and will aid in the development of new three-dimensional cell motility and adhesion models. As this study reveals, the mechanical interactions of cells and their extracellular matrix appear to be highly three-dimensional. It also shows that the LSCM-DVC technique is well suited for investigating the mechanics of cell-matrix interactions while providing a platform to access detailed information of the intricate biomechanical coupling for many cellular responses. Thus, this method has the capability to provide direct quantitative experimental data showing how cells interact with their surroundings in three dimensions and might stimulate new avenues of scientific thought in understanding the fundamental role physical forces play in regulating cell behavior.

  16. Three-dimensional micro-scale strain mapping in living biological soft tissues.

    Science.gov (United States)

    Moo, Eng Kuan; Sibole, Scott C; Han, Sang Kuy; Herzog, Walter

    2018-04-01

    Non-invasive characterization of the mechanical micro-environment surrounding cells in biological tissues at multiple length scales is important for the understanding of the role of mechanics in regulating the biosynthesis and phenotype of cells. However, there is a lack of imaging methods that allow for characterization of the cell micro-environment in three-dimensional (3D) space. The aims of this study were (i) to develop a multi-photon laser microscopy protocol capable of imprinting 3D grid lines onto living tissue at a high spatial resolution, and (ii) to develop image processing software capable of analyzing the resulting microscopic images and performing high resolution 3D strain analyses. Using articular cartilage as the biological tissue of interest, we present a novel two-photon excitation imaging technique for measuring the internal 3D kinematics in intact cartilage at sub-micrometer resolution, spanning length scales from the tissue to the cell level. Using custom image processing software, we provide accurate and robust 3D micro-strain analysis that allows for detailed qualitative and quantitative assessment of the 3D tissue kinematics. This novel technique preserves tissue structural integrity post-scanning, therefore allowing for multiple strain measurements at different time points in the same specimen. The proposed technique is versatile and opens doors for experimental and theoretical investigations on the relationship between tissue deformation and cell biosynthesis. Studies of this nature may enhance our understanding of the mechanisms underlying cell mechano-transduction, and thus, adaptation and degeneration of soft connective tissues. We presented a novel two-photon excitation imaging technique for measuring the internal 3D kinematics in intact cartilage at sub-micrometer resolution, spanning from tissue length scale to cellular length scale. Using a custom image processing software (lsmgridtrack), we provide accurate and robust micro

  17. Deletion of PDZD7 disrupts the Usher syndrome type 2 protein complex in cochlear hair cells and causes hearing loss in mice.

    Science.gov (United States)

    Zou, Junhuang; Zheng, Tihua; Ren, Chongyu; Askew, Charles; Liu, Xiao-Ping; Pan, Bifeng; Holt, Jeffrey R; Wang, Yong; Yang, Jun

    2014-05-01

    Usher syndrome type 2 (USH2) is the predominant form of USH, a leading genetic cause of combined deafness and blindness. PDZD7, a paralog of two USH causative genes, USH1C and USH2D (WHRN), was recently reported to be implicated in USH2 and non-syndromic deafness. It encodes a protein with multiple PDZ domains. To understand the biological function of PDZD7 and the pathogenic mechanism caused by PDZD7 mutations, we generated and thoroughly characterized a Pdzd7 knockout mouse model. The Pdzd7 knockout mice exhibit congenital profound deafness, as assessed by auditory brainstem response, distortion product otoacoustic emission and cochlear microphonics tests, and normal vestibular function, as assessed by their behaviors. Lack of PDZD7 leads to the disorganization of stereocilia bundles and a reduction in mechanotransduction currents and sensitivity in cochlear outer hair cells. At the molecular level, PDZD7 determines the localization of the USH2 protein complex, composed of USH2A, GPR98 and WHRN, to ankle links in developing cochlear hair cells, likely through its direct interactions with these three proteins. The localization of PDZD7 to the ankle links of cochlear hair bundles also relies on USH2 proteins. In photoreceptors of Pdzd7 knockout mice, the three USH2 proteins largely remain unchanged at the periciliary membrane complex. The electroretinogram responses of both rod and cone photoreceptors are normal in knockout mice at 1 month of age. Therefore, although the organization of the USH2 complex appears different in photoreceptors, it is clear that PDZD7 plays an essential role in organizing the USH2 complex at ankle links in developing cochlear hair cells. GenBank accession numbers: KF041446, KF041447, KF041448, KF041449, KF041450, KF041451.

  18. The purinergic P2X7 ion channel receptor — a ‘repair’ receptor in bone

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye

    2018-01-01

    A strong skeleton relies on adaptation to varying physical demands and on maintenance of the bone tissue in order to avoid accumulation of micro-damage. In bone, the purinergic P2X7 ion channel receptor is expressed on both cells of the stromal lineage such as the bone forming osteoblasts...... and the mechano-sensing osteocytes and on cells belonging to the immune-related monocyte–macrophage lineage, the bone resorbing osteoclasts. Recent studies have demonstrated that the receptor plays important roles in the anabolic responses to mechanical loading on bone and, together with the pannexin1 hemi......-channel, in the process of initiating bone remodeling in response to micro-damage. Thus, the receptor is crucial in skeletal mechano-transduction and in the continuous repair process. However, under pathophysiological conditions such as diabetes with high glucose concentrations or glucocorticoid-treatment the receptor...

  19. Mathematical Modeling of Ischemia-Reperfusion Injury and Postconditioning Therapy.

    Science.gov (United States)

    Fong, D; Cummings, L J

    2017-11-01

    Reperfusion (restoration of blood flow) after a period of ischemia (interruption of blood flow) can paradoxically place tissues at risk of further injury: so-called ischemia-reperfusion injury or IR injury. Recent studies have shown that postconditioning (intermittent periods of further ischemia applied during reperfusion) can reduce IR injury. We develop a mathematical model to describe the reperfusion and postconditioning process following an ischemic insult, treating the blood vessel as a two-dimensional channel, lined with a monolayer of endothelial cells that interact (respiration and mechanotransduction) with the blood flow. We investigate how postconditioning affects the total cell density within the endothelial layer, by varying the frequency of the pulsatile flow and the oxygen concentration at the inflow boundary. We find that, in the scenarios we consider, the pulsatile flow should be of high frequency to minimize cellular damage, while oxygen concentration at the inflow boundary should be held constant, or subject to only low-frequency variations, to maximize cell proliferation.

  20. The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking

    International Nuclear Information System (INIS)

    Rossier, Olivier; Giannone, Grégory

    2016-01-01

    Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.

  1. The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking.

    Science.gov (United States)

    Rossier, Olivier; Giannone, Grégory

    2016-04-10

    Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells. Copyright © 2015. Published by Elsevier Inc.

  2. The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking

    Energy Technology Data Exchange (ETDEWEB)

    Rossier, Olivier; Giannone, Grégory [Univ. Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux (France); CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux (France)

    2016-04-10

    Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.

  3. Mechanical stretching for tissue engineering: two-dimensional and three-dimensional constructs.

    Science.gov (United States)

    Riehl, Brandon D; Park, Jae-Hong; Kwon, Il Keun; Lim, Jung Yul

    2012-08-01

    Mechanical cell stretching may be an attractive strategy for the tissue engineering of mechanically functional tissues. It has been demonstrated that cell growth and differentiation can be guided by cell stretch with minimal help from soluble factors and engineered tissues that are mechanically stretched in bioreactors may have superior organization, functionality, and strength compared with unstretched counterparts. This review explores recent studies on cell stretching in both two-dimensional (2D) and three-dimensional (3D) setups focusing on the applications of stretch stimulation as a tool for controlling cell orientation, growth, gene expression, lineage commitment, and differentiation and for achieving successful tissue engineering of mechanically functional tissues, including cardiac, muscle, vasculature, ligament, tendon, bone, and so on. Custom stretching devices and lab-specific mechanical bioreactors are described with a discussion on capabilities and limitations. While stretch mechanotransduction pathways have been examined using 2D stretch, studying such pathways in physiologically relevant 3D environments may be required to understand how cells direct tissue development under stretch. Cell stretch study using 3D milieus may also help to develop tissue-specific stretch regimens optimized with biochemical feedback, which once developed will provide optimal tissue engineering protocols.

  4. GPR68 Senses Flow and Is Essential for Vascular Physiology.

    Science.gov (United States)

    Xu, Jie; Mathur, Jayanti; Vessières, Emilie; Hammack, Scott; Nonomura, Keiko; Favre, Julie; Grimaud, Linda; Petrus, Matt; Francisco, Allain; Li, Jingyuan; Lee, Van; Xiang, Fu-Li; Mainquist, James K; Cahalan, Stuart M; Orth, Anthony P; Walker, John R; Ma, Shang; Lukacs, Viktor; Bordone, Laura; Bandell, Michael; Laffitte, Bryan; Xu, Yan; Chien, Shu; Henrion, Daniel; Patapoutian, Ardem

    2018-04-19

    Mechanotransduction plays a crucial role in vascular biology. One example of this is the local regulation of vascular resistance via flow-mediated dilation (FMD). Impairment of this process is a hallmark of endothelial dysfunction and a precursor to a wide array of vascular diseases, such as hypertension and atherosclerosis. Yet the molecules responsible for sensing flow (shear stress) within endothelial cells remain largely unknown. We designed a 384-well screening system that applies shear stress on cultured cells. We identified a mechanosensitive cell line that exhibits shear stress-activated calcium transients, screened a focused RNAi library, and identified GPR68 as necessary and sufficient for shear stress responses. GPR68 is expressed in endothelial cells of small-diameter (resistance) arteries. Importantly, Gpr68-deficient mice display markedly impaired acute FMD and chronic flow-mediated outward remodeling in mesenteric arterioles. Therefore, GPR68 is an essential flow sensor in arteriolar endothelium and is a critical signaling component in cardiovascular pathophysiology. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Mitochondria-targeting nanomedicine: An effective and potent strategy against aminoglycosides-induced ototoxicity.

    Science.gov (United States)

    Zhou, Shuang; Sun, Yanhui; Kuang, Xiao; Hou, Shanshan; Yang, YinXian; Wang, Zhenjie; Liu, Hongzhuo

    2018-04-21

    We report a proof-of-concept for the development of mitochondria-targeting nanoparticles (NPs) loaded with geranylgeranylacetone (GGA) to protect against a wide range of gentamicin-induced ototoxicity symptoms in a zebrafish model. The polymeric NPs were functionalized with a mitochondrial-homing peptide (d‑Arg‑Dmt‑Orn‑Phe‑NH 2 ) and exhibited greater mitochondrial uptake and lower gentamicin uptake in hair cells via mechanotransduction (MET) channels and tuned machinery in the hair bundle than the ordinary NPs did. Blockade of MET channels rapidly reversed this effect, indicating the reversible responses of hair cells to the targeting NPs were mediated by MET channels. Pretreatment of hair cells with mitochondria-targeting GGA-loaded NPs exhibited a superior acute or chronic protective efficacy against subsequent exposure to gentamicin compared with unmodified formulations. Mitochondrial delivery regulating the death pathway of hair cells appeared to cause the therapeutic failure of untargeted NPs. Thus, peptide-directed mitochondria-targeting NPs may represent a novel therapeutic strategy for mitochondrial dysfunction-linked diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Characterisation of cellular adhesion reinforcement by multiple bond force spectroscopy in alveolar epithelial cells.

    Science.gov (United States)

    Nguyen, Ngoc-Minh; Angely, Christelle; Andre Dias, Sofia; Planus, Emmanuelle; Filoche, Marcel; Pelle, Gabriel; Louis, Bruno; Isabey, Daniel

    2017-07-01

    Integrin-mediated adhesion is a key process by which cells physically connect with their environment, and express sensitivity and adaptation through mechanotransduction. A critical step of cell adhesion is the formation of the first bonds which individually generate weak contacts (∼tens pN) but can sustain thousand times higher forces (∼tens nN) when associated. We propose an experimental validation by multiple bond force spectroscopy (MFS) of a stochastic model predicting adhesion reinforcement permitted by non-cooperative, multiple bonds on which force is homogeneously distributed (called parallel bond configuration). To do so, spherical probes (diameter: 6.6 μm), specifically coated by RGD-peptide to bind integrins, are used to statically indent and homogenously stretch the multiple bonds created for short contact times (2 s) between the bead and the surface of epithelial cells (A549). Using different separation speeds (v = 2, 5, 10 μm/s) and measuring cellular Young's modulus as well as the local stiffness preceding local rupture events, we obtain cell-by-cell the effective loading rates both at the global cell level and at the local level of individual constitutive bonds. Local rupture forces are in the range: f*=60-115 pN , whereas global rupture (detachment) forces reach F*=0.8-1.7 nN . Global and local rupture forces both exhibit linear dependencies with the effective loading rate, the slopes of these two linear relationships providing an estimate of the number of independent integrin bonds constituting the tested multiple bond structure (∼12). The MFS method enables to validate the reinforcement of integrin-mediated adhesion induced by the multiple bond configuration in which force is homogeneously distributed amongst parallel bonds. Local rupture events observed in the course of a spectroscopy manoeuver (MFS) lead to rupture force values considered in the literature as single-integrin bonds. Adhesion reinforcement permitted by the parallel

  7. Textural versus electrostatic exclusion-enrichment effects in the effective chemical transport within the cortical bone: a numerical investigation.

    Science.gov (United States)

    Lemaire, T; Kaiser, J; Naili, S; Sansalone, V

    2013-11-01

    Interstitial fluid within bone tissue is known to govern the remodelling signals' expression. Bone fluid flow is generated by skeleton deformation during the daily activities. Due to the presence of charged surfaces in the bone porous matrix, the electrochemical phenomena occurring in the vicinity of mechanosensitive bone cells, the osteocytes, are key elements in the cellular communication. In this study, a multiscale model of interstitial fluid transport within bone tissues is proposed. Based on an asymptotic homogenization method, our modelling takes into account the physicochemical properties of bone tissue. Thanks to this multiphysical approach, the transport of nutrients and waste between the blood vessels and the bone cells can be quantified to better understand the mechanotransduction of bone remodelling. In particular, it is shown that the electrochemical tortuosity may have stronger implications in the mass transport within the bone than the purely morphological one. Copyright © 2013 John Wiley & Sons, Ltd.

  8. Molecular mechanisms of the angiogenic effects of low-energy shock wave therapy: roles of mechanotransduction.

    Science.gov (United States)

    Hatanaka, Kazuaki; Ito, Kenta; Shindo, Tomohiko; Kagaya, Yuta; Ogata, Tsuyoshi; Eguchi, Kumiko; Kurosawa, Ryo; Shimokawa, Hiroaki

    2016-09-01

    We have previously demonstrated that low-energy extracorporeal cardiac shock wave (SW) therapy improves myocardial ischemia through enhanced myocardial angiogenesis in a porcine model of chronic myocardial ischemia and in patients with refractory angina pectoris. However, the detailed molecular mechanisms for the SW-induced angiogenesis remain unclear. In this study, we thus examined the effects of SW irradiation on intracellular signaling pathways in vitro. Cultured human umbilical vein endothelial cells (HUVECs) were treated with 800 shots of low-energy SW (1 Hz at an energy level of 0.03 mJ/mm(2)). The SW therapy significantly upregulated mRNA expression and protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS). The SW therapy also enhanced phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) and Akt. Furthermore, the SW therapy enhanced phosphorylation of caveolin-1 and the expression of HUTS-4 that represents β1-integrin activity. These results suggest that caveolin-1 and β1-integrin are involved in the SW-induced activation of angiogenic signaling pathways. To further examine the signaling pathways involved in the SW-induced angiogenesis, HUVECs were transfected with siRNA of either β1-integrin or caveolin-1. Knockdown of either caveolin-1 or β1-integrin suppressed the SW-induced phosphorylation of Erk1/2 and Akt and upregulation of VEGF and eNOS. Knockdown of either caveolin-1 or β1-integrin also suppressed SW-induced enhancement of HUVEC migration in scratch assay. These results suggest that activation of mechanosensors on cell membranes, such as caveolin-1 and β1-integrin, and subsequent phosphorylation of Erk and Akt may play pivotal roles in the SW-induced angiogenesis. Copyright © 2016 the American Physiological Society.

  9. The 133-kDa N-terminal domain enables myosin 15 to maintain mechanotransducing stereocilia and is essential for hearing

    Science.gov (United States)

    Fang, Qing; Indzhykulian, Artur A; Mustapha, Mirna; Riordan, Gavin P; Dolan, David F; Friedman, Thomas B; Belyantseva, Inna A; Frolenkov, Gregory I; Camper, Sally A; Bird, Jonathan E

    2015-01-01

    The precise assembly of inner ear hair cell stereocilia into rows of increasing height is critical for mechanotransduction and the sense of hearing. Yet, how the lengths of actin-based stereocilia are regulated remains poorly understood. Mutations of the molecular motor myosin 15 stunt stereocilia growth and cause deafness. We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different stereocilia rows. Using an isoform-specific knockout mouse, we show that hair cells expressing only the small isoform remarkably develop normal stereocilia bundles. However, a critical subset of stereocilia with active mechanotransducer channels subsequently retracts. The larger isoform with the 133-kDa N-terminal domain traffics to these specialized stereocilia and prevents disassembly of their actin core. Our results show that myosin 15 isoforms can navigate between functionally distinct classes of stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture. DOI: http://dx.doi.org/10.7554/eLife.08627.001 PMID:26302205

  10. Viscoelastic response of a model endothelial glycocalyx

    International Nuclear Information System (INIS)

    Nijenhuis, Nadja; Spaan, Jos A E; Mizuno, Daisuke; Schmidt, Christoph F

    2009-01-01

    Many cells cover themselves with a multifunctional polymer coat, the pericellular matrix (PCM), to mediate mechanical interactions with the environment. A particular PCM, the endothelial glycocalyx (EG), is formed by vascular endothelial cells at their luminal side, forming a mechanical interface between the flowing blood and the endothelial cell layer. The glycosaminoglycan (GAG) hyaluronan (HA) is involved in the main functions of the EG, mechanotransduction of fluid shear stress and molecular sieving. HA, due to its length, is the only GAG in the EG or any other PCM able to form an entangled network. The mechanical functions of the EG are, however, impaired when any one of its components is removed. We here used microrheology to measure the effect of the EG constituents heparan sulfate, chondroitin sulfate, whole blood plasma and albumin on the high-bandwidth mechanical properties of a HA solution. Furthermore, we probed the effect of the hyaldherin aggrecan, a constituent of the PCM of chondrocytes, and very similar to versican (present in the PCM of various cells, and possibly in the EG). We show that components directly interacting with HA (chondroitin sulfate and aggrecan) can increase the viscoelastic shear modulus of the polymer composite

  11. Cyclic tensile strain enhances human mesenchymal stem cell Smad 2/3 activation and tenogenic differentiation in anisotropic collagen-glycosaminoglycan scaffolds

    Directory of Open Access Journals (Sweden)

    WK Grier

    2017-03-01

    Full Text Available Orthopaedic injuries, particularly those involving ligaments and tendons, are some of the most commonly treated ailments in the United States and are associated with both high costs and poor outcomes. Regenerative medicine strategies for tendon injuries could be enhanced by three-dimensional biomaterials that can promote cell alignment and pro-tenogenic differentiation of patient-derived MSCs. We have previously described a collagen-glycosaminoglycan (CG scaffold possessing aligned structural features able to promote bone marrow MSC differentiation towards a tenogenic lineage, in the absence of growth factor supplementation. We aimed to employ a bioreactor to enhance MSC tenogenic differentiation within the aligned CG scaffold via cyclic tensile strain (CTS, and further to evaluate the relative effects of strain cycle duration and extended application of repeated cycles of CTS on MSC response. Human MSCs were cultured in CG scaffolds for up to 6 d under static (unloaded or cyclic tensile strain (1 Hz for 10 min every 6 h. Time-dependent activation of ERK 1/2 and p38 mechanotransduction pathways was observed within each 6 h strain cycle. MSCs remained viable throughout the experiment and application of CTS robustly upregulated the expression of tendon-specific extracellular matrix proteins and phenotypic markers. Simultaneously, CTS promoted increased phosphorylation of Smad 2/3, suggesting a link between tensile stimulation and TGF-β family growth factor production. Together, we demonstrated the design, fabrication and validation of a high-throughput tensile stimulation bioreactor to increase MSC tenogenic differentiation in porous CG scaffolds.

  12. Structural, biochemical, cellular, and functional changes in skeletal muscle extracellular matrix with aging

    DEFF Research Database (Denmark)

    Kragstrup, Tue Wenzel; Kjaer, M; Mackey, A L

    2011-01-01

    The extracellular matrix (ECM) of skeletal muscle is critical for force transmission and for the passive elastic response of skeletal muscle. Structural, biochemical, cellular, and functional changes in skeletal muscle ECM contribute to the deterioration in muscle mechanical properties with aging......-links and a buildup of advanced glycation end-product cross-links. Altered mechanotransduction, poorer activation of satellite cells, poorer chemotactic and delayed inflammatory responses, and a change in modulators of the ECM are important cellular changes. It is possible that the structural and biochemical changes...... in skeletal muscle ECM contribute to the increased stiffness and impairment in force generated by the contracting muscle fibers seen with aging. The cellular interactions provide and potentially coordinate an adaptation to mechanical loading and ensure successful regeneration after muscle injury. Some...

  13. Deficient Mechanical Activation of Anabolic Transcripts and Post-Traumatic Cartilage Degeneration in Matrilin-1 Knockout Mice.

    Directory of Open Access Journals (Sweden)

    Yupeng Chen

    Full Text Available Matrilin-1 (Matn1, a cartilage-specific peri-cellular and extracellular matrix (ECM protein, has been hypothesized to regulate ECM interactions and transmit mechanical signals in cartilage. Since Matn1 knock-out (Matn1-/- mice exhibit a normal skeleton, its function in vivo is unclear. In this study, we found that the anabolic Acan and Col2a transcript levels were significantly higher in wildtype (Matn1+/+ mouse cartilage than that of MATN1-/- mice in vivo. However, such difference was not observed between Matn1+/+ and MATN1-/- chondrocytes cultured under stationary conditions in vitro. Cyclic loading significantly stimulated Acan and Col2a transcript levels in Matn1+/+ but not in MATN1-/- chondrocytes. This suggests that, while Matn1+/+ chondrocytes increase their anabolic gene expression in response to mechanical loading, the MATN1-/- chondrocytes fail to do so because of the deficiency in mechanotransduction. We also found that altered elastic modulus of cartilage matrix in Matn1-/- mice, suggesting the mechanotransduction has changed due to the deficiency of Matn1. To understand the impact of such deficiency on joint disease, mechanical loading was altered in vivo by destabilization of medial meniscus. While Matn1+/+ mice exhibited superficial fissures and clefts consistent with mechanical damage to the articular joint, Matn1-/- mice presented more severe cartilage lesions characterized by proteoglycan loss and disorganization of cells and ECM. This suggests that Matn1 deficiency affects pathogenesis of post-traumatic osteoarthritis by failing to up-regulate anabolic gene expression. This is the first demonstration of Matn1 function in vivo, which suggests its protective role in cartilage degeneration under altered mechanical environment.

  14. High resolution, large deformation 3D traction force microscopy.

    Directory of Open Access Journals (Sweden)

    Jennet Toyjanova

    Full Text Available Traction Force Microscopy (TFM is a powerful approach for quantifying cell-material interactions that over the last two decades has contributed significantly to our understanding of cellular mechanosensing and mechanotransduction. In addition, recent advances in three-dimensional (3D imaging and traction force analysis (3D TFM have highlighted the significance of the third dimension in influencing various cellular processes. Yet irrespective of dimensionality, almost all TFM approaches have relied on a linear elastic theory framework to calculate cell surface tractions. Here we present a new high resolution 3D TFM algorithm which utilizes a large deformation formulation to quantify cellular displacement fields with unprecedented resolution. The results feature some of the first experimental evidence that cells are indeed capable of exerting large material deformations, which require the formulation of a new theoretical TFM framework to accurately calculate the traction forces. Based on our previous 3D TFM technique, we reformulate our approach to accurately account for large material deformation and quantitatively contrast and compare both linear and large deformation frameworks as a function of the applied cell deformation. Particular attention is paid in estimating the accuracy penalty associated with utilizing a traditional linear elastic approach in the presence of large deformation gradients.

  15. A New Approach to Heart Valve Tissue Engineering Based on Modifying Autologous Human Pericardium by 3D Cellular Mechanotransduction

    Czech Academy of Sciences Publication Activity Database

    Straka, František; Schorník, David; Mašín, J.; Filová, Elena; Miřejovský, T.; Burdíková, Z.; Švindrych, Z.; Chlup, H.; Horný, L.; Veselý, J.; Pirk, J.; Bačáková, Lucie

    2017-01-01

    Roč. 7, č. 7 (2017), s. 527-543 ISSN 2157-9083 R&D Projects: GA MZd(CZ) NV15-29153A; GA MZd(CZ) NT11270 Institutional support: RVO:67985823 Keywords : autologous human pericardium * pericardial interstitial cells * heart valve * 3D mechanotranduction * bioreactor Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery OBOR OECD: Cardiac and Cardiovascular systems Impact factor: 1.383, year: 2016

  16. Identification of mechanosensitive genes during skeletal development: alteration of genes associated with cytoskeletal rearrangement and cell signalling pathways.

    Science.gov (United States)

    Rolfe, Rebecca A; Nowlan, Niamh C; Kenny, Elaine M; Cormican, Paul; Morris, Derek W; Prendergast, Patrick J; Kelly, Daniel; Murphy, Paula

    2014-01-20

    Mechanical stimulation is necessary for regulating correct formation of the skeleton. Here we test the hypothesis that mechanical stimulation of the embryonic skeletal system impacts expression levels of genes implicated in developmentally important signalling pathways in a genome wide approach. We use a mutant mouse model with altered mechanical stimulation due to the absence of limb skeletal muscle (Splotch-delayed) where muscle-less embryos show specific defects in skeletal elements including delayed ossification, changes in the size and shape of cartilage rudiments and joint fusion. We used Microarray and RNA sequencing analysis tools to identify differentially expressed genes between muscle-less and control embryonic (TS23) humerus tissue. We found that 680 independent genes were down-regulated and 452 genes up-regulated in humeri from muscle-less Spd embryos compared to littermate controls (at least 2-fold; corrected p-value ≤0.05). We analysed the resulting differentially expressed gene sets using Gene Ontology annotations to identify significant enrichment of genes associated with particular biological processes, showing that removal of mechanical stimuli from muscle contractions affected genes associated with development and differentiation, cytoskeletal architecture and cell signalling. Among cell signalling pathways, the most strongly disturbed was Wnt signalling, with 34 genes including 19 pathway target genes affected. Spatial gene expression analysis showed that both a Wnt ligand encoding gene (Wnt4) and a pathway antagonist (Sfrp2) are up-regulated specifically in the developing joint line, while the expression of a Wnt target gene, Cd44, is no longer detectable in muscle-less embryos. The identification of 84 genes associated with the cytoskeleton that are down-regulated in the absence of muscle indicates a number of candidate genes that are both mechanoresponsive and potentially involved in mechanotransduction, converting a mechanical stimulus

  17. Microstructural heterogeneity directs micromechanics and mechanobiology in native and engineered fibrocartilage

    Science.gov (United States)

    Han, Woojin M.; Heo, Su-Jin; Driscoll, Tristan P.; Delucca, John F.; McLeod, Claire M.; Smith, Lachlan J.; Duncan, Randall L.; Mauck, Robert L.; Elliott, Dawn M.

    2016-04-01

    Treatment strategies to address pathologies of fibrocartilaginous tissue are in part limited by an incomplete understanding of structure-function relationships in these load-bearing tissues. There is therefore a pressing need to develop micro-engineered tissue platforms that can recreate the highly inhomogeneous tissue microstructures that are known to influence mechanotransductive processes in normal and diseased tissue. Here, we report the quantification of proteoglycan-rich microdomains in developing, ageing and diseased fibrocartilaginous tissues, and the impact of these microdomains on endogenous cell responses to physiologic deformation within a native-tissue context. We also developed a method to generate heterogeneous tissue-engineered constructs (hetTECs) with non-fibrous proteoglycan-rich microdomains engineered into the fibrous structure, and show that these hetTECs match the microstructural, micromechanical and mechanobiological benchmarks of native tissue. Our tissue-engineered platform should facilitate the study of the mechanobiology of developing, homeostatic, degenerating and regenerating fibrous tissues.

  18. An operating principle of the turtle utricle to detect wide dynamic range.

    Science.gov (United States)

    Nam, Jong-Hoon

    2018-03-01

    The utricle encodes both static information such as head orientation, and dynamic information such as vibrations. It is not well understood how the utricle can encode both static and dynamic information for a wide dynamic range (from 2 times the gravitational acceleration; from DC to > 1000 Hz vibrations). Using computational models of the hair cells in the turtle utricle, this study presents an explanation on how the turtle utricle encodes stimulations over such a wide dynamic range. Two hair bundles were modeled using the finite element method-one representing the striolar hair cell (Cell S), and the other representing the medial extrastriolar hair cell (Cell E). A mechano-transduction (MET) channel model was incorporated to compute MET current (i MET ) due to hair bundle deflection. A macro-mechanical model of the utricle was used to compute otoconial motions from head accelerations (a Head ). According to known anatomical data, Cell E has a long kinocilium that is embedded into the stiff otoconial layer. Unlike Cell E, the hair bundle of Cell S falls short of the otoconial layer. Considering such difference in the mechanical connectivity between the hair cell bundle and the otoconial layer, three cases were simulated: Cell E displacement-clamped, Cell S viscously-coupled, and Cell S displacement-clamped. Head accelerations at different amplitude levels and different frequencies were simulated for the three cases. When a realistic head motion was simulated, Cell E was responsive to head orientation, while the viscously-coupled Cell S was responsive to fast head motion imitating the feeding strike of a turtle. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Upregulation of Ih expressed in IB4-negative Aδ nociceptive DRG neurons contributes to mechanical hypersensitivity associated with cervical radiculopathic pain

    OpenAIRE

    Da-Lu Liu; Na Lu; Wen-Juan Han; Rong-Gui Chen; Rui Cong; Rou-Gang Xie; Yu-Fei Zhang; Wei-Wei Kong; San-Jue Hu; Ceng Luo

    2015-01-01

    Cervical radiculopathy represents aberrant mechanical hypersensitivity. Primary sensory neuron?s ability to sense mechanical force forms mechanotransduction. However, whether this property undergoes activity-dependent plastic changes and underlies mechanical hypersensitivity associated with cervical radiculopathic pain (CRP) is not clear. Here we show a new CRP model producing stable mechanical compression of dorsal root ganglion (DRG), which induces dramatic behavioral mechanical hypersensit...

  20. Blood and interstitial flow in the hierarchical pore space architecture of bone tissue.

    Science.gov (United States)

    Cowin, Stephen C; Cardoso, Luis

    2015-03-18

    There are two main types of fluid in bone tissue, blood and interstitial fluid. The chemical composition of these fluids varies with time and location in bone. Blood arrives through the arterial system containing oxygen and other nutrients and the blood components depart via the venous system containing less oxygen and reduced nutrition. Within the bone, as within other tissues, substances pass from the blood through the arterial walls into the interstitial fluid. The movement of the interstitial fluid carries these substances to the cells within the bone and, at the same time, carries off the waste materials from the cells. Bone tissue would not live without these fluid movements. The development of a model for poroelastic materials with hierarchical pore space architecture for the description of blood flow and interstitial fluid flow in living bone tissue is reviewed. The model is applied to the problem of determining the exchange of pore fluid between the vascular porosity and the lacunar-canalicular porosity in bone tissue due to cyclic mechanical loading and blood pressure. These results are basic to the understanding of interstitial flow in bone tissue that, in turn, is basic to understanding of nutrient transport from the vasculature to the bone cells buried in the bone tissue and to the process of mechanotransduction by these cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. TRPV1 and Endocannabinoids: Emerging Molecular Signals that Modulate Mammalian Vision

    Directory of Open Access Journals (Sweden)

    Daniel A. Ryskamp

    2014-09-01

    Full Text Available Transient Receptor Potential Vanilloid 1 (TRPV1 subunits form a polymodal cation channel responsive to capsaicin, heat, acidity and endogenous metabolites of polyunsaturated fatty acids. While originally reported to serve as a pain and heat detector in the peripheral nervous system, TRPV1 has been implicated in the modulation of blood flow and osmoregulation but also neurotransmission, postsynaptic neuronal excitability and synaptic plasticity within the central nervous system. In addition to its central role in nociception, evidence is accumulating that TRPV1 contributes to stimulus transduction and/or processing in other sensory modalities, including thermosensation, mechanotransduction and vision. For example, TRPV1, in conjunction with intrinsic cannabinoid signaling, might contribute to retinal ganglion cell (RGC axonal transport and excitability, cytokine release from microglial cells and regulation of retinal vasculature. While excessive TRPV1 activity was proposed to induce RGC excitotoxicity, physiological TRPV1 activity might serve a neuroprotective function within the complex context of retinal endocannabinoid signaling. In this review we evaluate the current evidence for localization and function of TRPV1 channels within the mammalian retina and explore the potential interaction of this intriguing nociceptor with endogenous agonists and modulators.

  2. Modulation of Huh7.5 spheroid formation and functionality using modified PEG-based hydrogels of different stiffness.

    Directory of Open Access Journals (Sweden)

    Bae Hoon Lee

    Full Text Available Physical cues, such as cell microenvironment stiffness, are known to be important factors in modulating cellular behaviors such as differentiation, viability, and proliferation. Apart from being able to trigger these effects, mechanical stiffness tuning is a very convenient approach that could be implemented readily into smart scaffold designs. In this study, fibrinogen-modified poly(ethylene glycol-diacrylate (PEG-DA based hydrogels with tunable mechanical properties were synthesized and applied to control the spheroid formation and liver-like function of encapsulated Huh7.5 cells in an engineered, three-dimensional liver tissue model. By controlling hydrogel stiffness (0.1-6 kPa as a cue for mechanotransduction representing different stiffness of a normal liver and a diseased cirrhotic liver, spheroids ranging from 50 to 200 μm were formed over a three week time-span. Hydrogels with better compliance (i.e. lower stiffness promoted formation of larger spheroids. The highest rates of cell proliferation, albumin secretion, and CYP450 expression were all observed for spheroids in less stiff hydrogels like a normal liver in a healthy state. We also identified that the hydrogel modification by incorporation of PEGylated-fibrinogen within the hydrogel matrix enhanced cell survival and functionality possibly owing to more binding of autocrine fibronectin. Taken together, our findings establish guidelines to control the formation of Huh7.5 cell spheroids in modified PEGDA based hydrogels. These spheroids may serve as models for applications such as screening of pharmacological drug candidates.

  3. A Tour de Force: The Discovery, Properties, and Function of Piezo Channels.

    Science.gov (United States)

    Gottlieb, P A

    2017-01-01

    Mechanical transducers appear throughout cell biology and are used to convert mechanical stress into chemical or electrical signals that allow the cell to respond to environmental changes. In the past six years, a eukaryotic mechanical channel family with two members, Piezo1 and Piezo2, has been identified. Piezo1 was shown to be a cation-selective channel that does not require ancillary proteins for activity. Mouse Piezo1 is large, with over 2500 amino acids, and is not homologous to other ion channels. Both piezo channels have rapid voltage-dependent inactivation with a reversal potential near 0mV. The CryoEm structure of Piezo1 at 4.8Å shows trimer stoichiometry. Since the discovery of the piezo channels, their roles in the physiological response of cells have started to emerge. Significant progress has been made in understanding the intrinsic properties of the channels and how these properties are modulated by cytoskeletal elements. Specific diseases, such as hereditary xerocytosis affecting red blood cells, have mutations in Piezo1 that alter the cell's response to force, typically slowing inactivation and introducing a latency for activation. A number of physiological functions for piezo channels have been identified. These range from sensing the stiffness of surrounding substrate, to the response to light touch, to serotonin release from the gut. This review provides a general overview of the properties and roles of Piezo1 and Piezo2 in eukaryotic mechanotransduction. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Segmentation of nanotomographic cortical bone images for quantitative characterization of the osteoctyte lacuno-canalicular network

    Energy Technology Data Exchange (ETDEWEB)

    Ciani, A.; Kewish, C. M. [Synchrotron Soleil, L’Orme des Merisiers, 91192 Saint-Aubin (France); Guizar-Sicairos, M.; Diaz, A.; Holler, M. [Paul Scherrer Institut, 5232 Villigen PSI (Switzerland); Pallu, S.; Achiou, Z.; Jennane, R.; Toumi, H.; Lespessailles, E. [Univ Orléans, I3MTO, Ea 4708, 45000 Orléans (France)

    2016-01-28

    A newly developed data processing method able to characterize the osteocytes lacuno-canalicular network (LCN) is presented. Osteocytes are the most abundant cells in the bone, living in spaces called lacunae embedded inside the bone matrix and connected to each other with an extensive network of canals that allows for the exchange of nutrients and for mechanotransduction functions. The geometrical three-dimensional (3D) architecture is increasingly thought to be related to the macroscopic strength or failure of the bone and it is becoming the focus for investigating widely spread diseases such as osteoporosis. To obtain 3D LCN images non-destructively has been out of reach until recently, since tens-of-nanometers scale resolution is required. Ptychographic tomography was validated for bone imaging in [1], showing clearly the LCN. The method presented here was applied to 3D ptychographic tomographic images in order to extract morphological and geometrical parameters of the lacuno-canalicular structures.

  5. Computational exploration of single-protein mechanics by steered molecular dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Sotomayor, Marcos [Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio (United States)

    2015-12-31

    Hair cell mechanotransduction happens in tens of microseconds, involves forces of a few picoNewtons, and is mediated by nanometer-scale molecular conformational changes. As proteins involved in this process become identified and their high resolution structures become available, multiple tools are being used to explore their “single-molecule responses” to force. Optical tweezers and atomic force microscopy offer exquisite force and extension resolution, but cannot reach the high loading rates expected for high frequency auditory stimuli. Molecular dynamics (MD) simulations can reach these fast time scales, and also provide a unique view of the molecular events underlying protein mechanics, but its predictions must be experimentally verified. Thus a combination of simulations and experiments might be appropriate to study the molecular mechanics of hearing. Here I review the basics of MD simulations and the different methods used to apply force and study protein mechanics in silico. Simulations of tip link proteins are used to illustrate the advantages and limitations of this method.

  6. Endothelium derived nitric oxide synthase negatively regulates the PDGF-survivin pathway during flow-dependent vascular remodeling.

    Directory of Open Access Journals (Sweden)

    Jun Yu

    Full Text Available Chronic alterations in blood flow initiate structural changes in vessel lumen caliber to normalize shear stress. The loss of endothelial derived nitric oxide synthase (eNOS in mice promotes abnormal flow dependent vascular remodeling, thus uncoupling mechanotransduction from adaptive vascular remodeling. However, the mechanisms of how the loss of eNOS promotes abnormal remodeling are not known. Here we show that abnormal flow-dependent remodeling in eNOS knockout mice (eNOS (-/- is associated with activation of the platelet derived growth factor (PDGF signaling pathway leading to the induction of the inhibitor of apoptosis, survivin. Interfering with PDGF signaling or survivin function corrects the abnormal remodeling seen in eNOS (-/- mice. Moreover, nitric oxide (NO negatively regulates PDGF driven survivin expression and cellular proliferation in cultured vascular smooth muscle cells. Collectively, our data suggests that eNOS negatively regulates the PDGF-survivin axis to maintain proportional flow-dependent luminal remodeling and vascular quiescence.

  7. Microstructural Heterogeneity in Native and Engineered Fibrocartilage Directs Micromechanics and Mechanobiology

    Science.gov (United States)

    Han, Woojin M; Heo, Su-Jin; Driscoll, Tristan P; Delucca, John F; McLeod, Claire M; Smith, Lachlan J; Duncan, Randall L; Mauck, Robert L; Elliott, Dawn M

    2015-01-01

    Treatment strategies to address pathologies of fibrocartilaginous tissue are in part limited by an incomplete understanding of structure-function relationships in these load-bearing tissues. There is therefore a pressing need to develop microengineered tissue platforms that can recreate the highly inhomogeneous tissue microstructures that are known to influence mechanotransductive processes in normal and diseased tissue. Here, we report the quantification of proteoglycan-rich microdomains in developing, aging, and diseased fibrocartilaginous tissues, and the impact of these microdomains on endogenous cell responses to physiologic deformation within a native-tissue context. We also developed a method to generate heterogeneous tissue engineered constructs (hetTECs) with microscale non-fibrous proteoglycan-rich microdomains engineered into the fibrous structure, and show that these hetTECs match the microstructural, micromechanical, and mechanobiological benchmarks of native tissue. Our tissue engineered platform should facilitate the study of the mechanobiology of developing, homeostatic, degenerating, and regenerating fibrous tissues. PMID:26726994

  8. The Role of PPK26 in Drosophila Larval Mechanical Nociception

    Directory of Open Access Journals (Sweden)

    Yanmeng Guo

    2014-11-01

    Full Text Available In Drosophila larvae, the class IV dendritic arborization (da neurons are polymodal nociceptors. Here, we show that ppk26 (CG8546 plays an important role in mechanical nociception in class IV da neurons. Our immunohistochemical and functional results demonstrate that ppk26 is specifically expressed in class IV da neurons. Larvae with mutant ppk26 showed severe behavioral defects in a mechanical nociception behavioral test but responded to noxious heat stimuli comparably to wild-type larvae. In addition, functional studies suggest that ppk26 and ppk (also called ppk1 function in the same pathway, whereas piezo functions in a parallel pathway. Consistent with these functional results, we found that PPK and PPK26 are interdependent on each other for their cell surface localization. Our work indicates that PPK26 and PPK might form heteromeric DEG/ENaC channels that are essential for mechanotransduction in class IV da neurons.

  9. Role of BAG3 in cancer progression: A therapeutic opportunity.

    Science.gov (United States)

    De Marco, Margot; Basile, Anna; Iorio, Vittoria; Festa, Michelina; Falco, Antonia; Ranieri, Bianca; Pascale, Maria; Sala, Gianluca; Remondelli, Paolo; Capunzo, Mario; Firpo, Matthew A; Pezzilli, Raffaele; Marzullo, Liberato; Cavallo, Pierpaolo; De Laurenzi, Vincenzo; Turco, Maria Caterina; Rosati, Alessandra

    2018-06-01

    BAG3 is a multifunctional protein that can bind to heat shock proteins (Hsp) 70 through its BAG domain and to other partners through its WW domain, proline-rich (PXXP) repeat and IPV (Ile-Pro-Val) motifs. Its intracellular expression can be induced by stressful stimuli, while is constitutive in skeletal muscle, cardiac myocytes and several tumour types. BAG3 can modulate the levels, localisation or activity of its partner proteins, thereby regulating major cell pathways and functions, including apoptosis, autophagy, mechanotransduction, cytoskeleton organisation, motility. A secreted form of BAG3 has been identified in studies on pancreatic ductal adenocarcinoma (PDAC). Secreted BAG3 can bind to a specific receptor, IFITM2, expressed on macrophages, and induce the release of factors that sustain tumour growth and the metastatic process. BAG3 neutralisation therefore appears to constitute a novel potential strategy in the therapy of PDAC and, possibly, other tumours. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Oscillatory fluid flow influences primary cilia and microtubule mechanics.

    Science.gov (United States)

    Espinha, Lina C; Hoey, David A; Fernandes, Paulo R; Rodrigues, Hélder C; Jacobs, Christopher R

    2014-07-01

    Many tissues are sensitive to mechanical stimuli; however, the mechanotransduction mechanism used by cells remains unknown in many cases. The primary cilium is a solitary, immotile microtubule-based extension present on nearly every mammalian cell which extends from the basal body. The cilium is a mechanosensitive organelle and has been shown to transduce fluid flow-induced shear stress in tissues, such as the kidney and bone. The majority of microtubules assemble from the mother centriole (basal body), contributing significantly to the anchoring of the primary cilium. Several studies have attempted to quantify the number of microtubules emanating from the basal body and the results vary depending on the cell type. It has also been shown that cellular response to shear stress depends on microtubular integrity. This study hypothesizes that changing the microtubule attachment of primary cilia in response to a mechanical stimulus could change primary cilia mechanics and, possibly, mechanosensitivity. Oscillatory fluid flow was applied to two different cell types and the microtubule attachment to the ciliary base was quantified. For the first time, an increase in microtubules around primary cilia both with time and shear rate in response to oscillatory fluid flow stimulation was demonstrated. Moreover, it is presented that the primary cilium is required for this loading-induced cellular response. This study has demonstrated a new role for the cilium in regulating alterations in the cytoplasmic microtubule network in response to mechanical stimulation, and therefore provides a new insight into how cilia may regulate its mechanics and thus the cells mechanosensitivity. Copyright © 2014 Wiley Periodicals, Inc.

  11. Mechanobiology of the Meniscus

    Science.gov (United States)

    McNulty, Amy L.; Guilak, Farshid

    2015-01-01

    The meniscus plays a critical biomechanical role in the knee, providing load support, joint stability, and congruity. Importantly, growing evidence indicates that the mechanobiologic response of meniscal cells plays a critical role in the physiologic, pathologic, and repair responses of the meniscus. Here we review experimental and theoretical studies that have begun to directly measure the biomechanical effects of joint loading on the meniscus under physiologic and pathologic conditions, showing that the menisci are exposed to high contact stresses, resulting in a complex and nonuniform stress-strain environment within the tissue. By combining microscale measurements of the mechanical properties of meniscal cells and their pericellular and extracellular matrix regions, theoretical and experimental models indicate that the cells in the meniscus are exposed to a complex and inhomogeneous environment of stress, strain, fluid pressure, fluid flow, and a variety of physicochemical factors. Studies across a range of culture systems from isolated cells to tissues have revealed that the biological response of meniscal cells is directly influenced by physical factors, such as tension, compression, and hydrostatic pressure. In addition, these studies have provided new insights into the mechanotransduction mechanisms by which physical signals are converted into metabolic or pro/anti-inflammatory responses. Taken together, these in vivo and in vitro studies show that mechanical factors play an important role in the health, degeneration, and regeneration of the meniscus. A more thorough understanding of the mechanobiologic responses of the meniscus will hopefully lead to therapeutic approaches to prevent degeneration and enhance repair of the meniscus. PMID:25731738

  12. UTP-induced ATP release is a fine-tuned signalling pathway in osteocytes

    DEFF Research Database (Denmark)

    Kringelbach, Tina M.; Aslan, Derya; Novak, Ivana

    2014-01-01

    Osteocytes reside as a cellular network throughout the mineralised matrix of bone and are considered the primary mechanosensors of this tissue. They sense mechanical stimulation such as fluid flow and are able to regulate osteoblast and osteoclast functions on the bone surface. Previously, we fou...... signals may be propagated by P2 receptor activation and further ATP release in the osteocyte network and implicate purinergic signalling as a central signalling pathway in osteocyte mechanotransduction....

  13. The interplay between tissue growth and scaffold degradation in engineered tissue constructs

    KAUST Repository

    O’Dea, R. D.

    2012-09-18

    In vitro tissue engineering is emerging as a potential tool to meet the high demand for replacement tissue, caused by the increased incidence of tissue degeneration and damage. A key challenge in this field is ensuring that the mechanical properties of the engineered tissue are appropriate for the in vivo environment. Achieving this goal will require detailed understanding of the interplay between cell proliferation, extracellular matrix (ECM) deposition and scaffold degradation. In this paper, we use a mathematical model (based upon a multiphase continuum framework) to investigate the interplay between tissue growth and scaffold degradation during tissue construct evolution in vitro. Our model accommodates a cell population and culture medium, modelled as viscous fluids, together with a porous scaffold and ECM deposited by the cells, represented as rigid porous materials. We focus on tissue growth within a perfusion bioreactor system, and investigate how the predicted tissue composition is altered under the influence of (1) differential interactions between cells and the supporting scaffold and their associated ECM, (2) scaffold degradation, and (3) mechanotransduction-regulated cell proliferation and ECM deposition. Numerical simulation of the model equations reveals that scaffold heterogeneity typical of that obtained from μCT scans of tissue engineering scaffolds can lead to significant variation in the flow-induced mechanical stimuli experienced by cells seeded in the scaffold. This leads to strong heterogeneity in the deposition of ECM. Furthermore, preferential adherence of cells to the ECM in favour of the artificial scaffold appears to have no significant influence on the eventual construct composition; adherence of cells to these supporting structures does, however, lead to cell and ECM distributions which mimic and exaggerate the heterogeneity of the underlying scaffold. Such phenomena have important ramifications for the mechanical integrity of

  14. Osteogenic Transcription Regulated by Exaggerated Stretch Loading via Convergent Wnt Signaling

    Science.gov (United States)

    Juran, Cassandra M.; Blaber, Elizabeth A.; Almeida, Eduardo A. C.

    2017-01-01

    Cell and animal studies conducted onboard the International Space Station and formerly the Shuttle flights have provided data illuminating the deleterious biological response of bone to mechanical unloading. Down regulation of proliferative mechanisms within stem cell populations of the osteogenic niche is a suggested mechanism for loss of bone mass. However the intercellular communicative cues from osteoblasts and osteocytes in managing stem cell proliferation and osteogenic differentiation are largely unknown. In this investigation, MLO-Y4 osteocyte-like and MC3T3-E1 osteoblast-like cells, are co-culture under dynamic tensile conditions and evaluated for phenotypic expression of biochemical signaling proteins influential in driving stem cell differentiation. MLO-Y4 and MC3T3-E1 were co-cultured on polyethersulfone membrane with a 0.45m porosity to permit soluble factor transfer and direct cell-cell gap junction signaling. Cyclic tensile stimulation was applied for 48 h at a frequency of 0.1Hz and strain of 0.1. Total Live cell counts indicate mechanical activation of MC3T3-E1s inhibits proliferation while MLO-Y4s increase in number. However, the percent of live MLO-Y4s within the population is low (46.3 total count, *p0.05, n4) suggesting a potential apoptotic signaling cascade. Immunofluorescence demonstrated that stimulation of co-cultures elicits increased gap junction communication. Previously reported PCR evaluation of osteogenic markers further corroborate that the co-cultured populations communicative networks play a role in translating mechanical signals to molecular messaging. These findings suggest that an osteocyte-osteoblast signaling feedback mechanism may regulate mechanotransduction of an apoptotic cascade within osteocytes and transcription of cytokine signaling proteins responsible for stem cell niche recruitment much more directly than previously believed.

  15. Role of Bioreactor Technology in Tissue Engineering for Clinical Use and Therapeutic Target Design

    Directory of Open Access Journals (Sweden)

    Clare Selden

    2018-04-01

    Full Text Available Micro and small bioreactors are well described for use in bioprocess development in pre-production manufacture, using ultra-scale down and microfluidic methodology. However, the use of bioreactors to understand normal and pathophysiology by definition must be very different, and the constraints of the physiological environment influence such bioreactor design. This review considers the key elements necessary to enable bioreactors to address three main areas associated with biological systems. All entail recreation of the in vivo cell niche as faithfully as possible, so that they may be used to study molecular and cellular changes in normal physiology, with a view to creating tissue-engineered grafts for clinical use; understanding the pathophysiology of disease at the molecular level; defining possible therapeutic targets; and enabling appropriate pharmaceutical testing on a truly representative organoid, thus enabling better drug design, and simultaneously creating the potential to reduce the numbers of animals in research. The premise explored is that not only cellular signalling cues, but also mechano-transduction from mechanical cues, play an important role.

  16. The osteogenic capacity of biomimetic hierarchical micropore/nanorod-patterned Sr-HA coatings with different interrod spacings.

    Science.gov (United States)

    Zhou, Jianhong; Li, Bo; Han, Yong; Zhao, Lingzhou

    2016-07-01

    Advanced titanium based bone implant with fast established, rigid and stable osseointegration is stringently needed in clinic. Here the hierarchical micropore/nanorod-patterned strontium doped hydroxyapatite (Ca9Sr1(PO4)6(OH)2, Sr1-HA) coatings (MNRs) with different interrod spacings varying from about 300 to 33nm were developed. MNRs showed dramatically differential biological performance closely related to the interrod spacing. Compared to micropore/nanogranule-patterned Sr1-HA coating (MNG), MNRs with an interrod spacing of larger than 137nm resulted in inhibited in vitro mesenchymal stem cell functions and in vivo osseointegration, while those of smaller than 96nm gave rise to dramatically enhanced the biological effect, especially those of mean 67nm displayed the best effect. The differential biological effect of MNRs was related to their modulation on the focal adhesion mediated mechanotransduction. These results suggest that MNRs with a mean interrod spacing of 67nm may give rise to an advanced implant of improved clinical performance. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Correlation between vortices and wall shear stress in a curved artery model under pulsatile flow conditions

    Science.gov (United States)

    Cox, Christopher; Plesniak, Michael W.

    2017-11-01

    One of the most physiologically relevant factors within the cardiovascular system is the wall shear stress. The wall shear stress affects endothelial cells via mechanotransduction and atherosclerotic regions are strongly correlated with curvature and branching in the human vasculature, where the shear stress is both oscillatory and multidirectional. Also, the combined effect of curvature and pulsatility in cardiovascular flows produces unsteady vortices. In this work, our goal is to assess the correlation between multiple vortex pairs and wall shear stress. To accomplish this, we use an in-house high-order flux reconstruction Navier-Stokes solver to simulate pulsatile flow of a Newtonian blood-analog fluid through a rigid 180° curved artery model. We use a physiologically relevant flow rate and generate results using both fully developed and uniform entrance conditions, the latter motivated by the fact that flow upstream to a curved artery may not be fully developed. Under these two inflow conditions, we characterize the evolution of various vortex pairs and their subsequent effect on several wall shear stress metrics. Supported by GW Center for Biomimetics and Bioinspired Engineering.

  18. Construction and characterization of a novel vocal fold bioreactor.

    Science.gov (United States)

    Zerdoum, Aidan B; Tong, Zhixiang; Bachman, Brendan; Jia, Xinqiao

    2014-08-01

    In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 µm. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

  19. Mechanosensory and ATP Release Deficits following Keratin14-Cre-Mediated TRPA1 Deletion Despite Absence of TRPA1 in Murine Keratinocytes.

    Directory of Open Access Journals (Sweden)

    Katherine J Zappia

    Full Text Available Keratinocytes are the first cells that come into direct contact with external tactile stimuli; however, their role in touch transduction in vivo is not clear. The ion channel Transient Receptor Potential Ankyrin 1 (TRPA1 is essential for some mechanically-gated currents in sensory neurons, amplifies mechanical responses after inflammation, and has been reported to be expressed in human and mouse skin. Other reports have not detected Trpa1 mRNA transcripts in human or mouse epidermis. Therefore, we set out to determine whether selective deletion of Trpa1 from keratinocytes would impact mechanosensation. We generated K14Cre-Trpa1fl/fl mice lacking TRPA1 in K14-expressing cells, including keratinocytes. Surprisingly, Trpa1 transcripts were very poorly detected in epidermis of these mice or in controls, and detection was minimal enough to preclude observation of Trpa1 mRNA knockdown in the K14Cre-Trpa1fl/fl mice. Unexpectedly, these K14Cre-Trpa1fl/fl mice nonetheless exhibited a pronounced deficit in mechanosensitivity at the behavioral and primary afferent levels, and decreased mechanically-evoked ATP release from skin. Overall, while these data suggest that the intended targeted deletion of Trpa1 from keratin 14-expressing cells of the epidermis induces functional deficits in mechanotransduction and ATP release, these deficits are in fact likely due to factors other than reduction of Trpa1 expression in adult mouse keratinocytes because they express very little, if any, Trpa1.

  20. Progressive hearing loss and gradual deterioration of sensory hair bundles in the ears of mice lacking the actin-binding protein Eps8L2.

    Science.gov (United States)

    Furness, David N; Johnson, Stuart L; Manor, Uri; Rüttiger, Lukas; Tocchetti, Arianna; Offenhauser, Nina; Olt, Jennifer; Goodyear, Richard J; Vijayakumar, Sarath; Dai, Yuhai; Hackney, Carole M; Franz, Christoph; Di Fiore, Pier Paolo; Masetto, Sergio; Jones, Sherri M; Knipper, Marlies; Holley, Matthew C; Richardson, Guy P; Kachar, Bechara; Marcotti, Walter

    2013-08-20

    Mechanotransduction in the mammalian auditory system depends on mechanosensitive channels in the hair bundles that project from the apical surface of the sensory hair cells. Individual stereocilia within each bundle contain a core of tightly packed actin filaments, whose length is dynamically regulated during development and in the adult. We show that the actin-binding protein epidermal growth factor receptor pathway substrate 8 (Eps8)L2, a member of the Eps8-like protein family, is a newly identified hair bundle protein that is localized at the tips of stereocilia of both cochlear and vestibular hair cells. It has a spatiotemporal expression pattern that complements that of Eps8. In the cochlea, whereas Eps8 is essential for the initial elongation of stereocilia, Eps8L2 is required for their maintenance in adult hair cells. In the absence of both proteins, the ordered staircase structure of the hair bundle in the cochlea decays. In contrast to the early profound hearing loss associated with an absence of Eps8, Eps8L2 null-mutant mice exhibit a late-onset, progressive hearing loss that is directly linked to a gradual deterioration in hair bundle morphology. We conclude that Eps8L2 is required for the long-term maintenance of the staircase structure and mechanosensory function of auditory hair bundles. It complements the developmental role of Eps8 and is a candidate gene for progressive age-related hearing loss.

  1. pix-1 controls early elongation in parallel with mel-11 and let-502 in Caenorhabditis elegans.

    Science.gov (United States)

    Martin, Emmanuel; Harel, Sharon; Nkengfac, Bernard; Hamiche, Karim; Neault, Mathieu; Jenna, Sarah

    2014-01-01

    Cell shape changes are crucial for metazoan development. During Caenorhabditis elegans embryogenesis, epidermal cell shape changes transform ovoid embryos into vermiform larvae. This process is divided into two phases: early and late elongation. Early elongation involves the contraction of filamentous actin bundles by phosphorylated non-muscle myosin in a subset of epidermal (hypodermal) cells. The genes controlling early elongation are associated with two parallel pathways. The first one involves the rho-1/RHOA-specific effector let-502/Rho-kinase and mel-11/myosin phosphatase regulatory subunit. The second pathway involves the CDC42/RAC-specific effector pak-1. Late elongation is driven by mechanotransduction in ventral and dorsal hypodermal cells in response to body-wall muscle contractions, and involves the CDC42/RAC-specific Guanine-nucleotide Exchange Factor (GEF) pix-1, the GTPase ced-10/RAC and pak-1. In this study, pix-1 is shown to control early elongation in parallel with let-502/mel-11, as previously shown for pak-1. We show that pix-1, pak-1 and let-502 control the rate of elongation, and the antero-posterior morphology of the embryos. In particular, pix-1 and pak-1 are shown to control head, but not tail width, while let-502 controls both head and tail width. This suggests that let-502 function is required throughout the antero-posterior axis of the embryo during early elongation, while pix-1/pak-1 function may be mostly required in the anterior part of the embryo. Supporting this hypothesis we show that low pix-1 expression level in the dorsal-posterior hypodermal cells is required to ensure high elongation rate during early elongation.

  2. A mechanical design principle for tissue structure and function in the airway tree.

    Science.gov (United States)

    LaPrad, Adam S; Lutchen, Kenneth R; Suki, Béla

    2013-01-01

    With every breath, the dynamically changing mechanical pressures must work in unison with the cells and soft tissue structures of the lung to permit air to efficiently traverse the airway tree and undergo gas exchange in the alveoli. The influence of mechanics on cell and tissue function is becoming apparent, raising the question: how does the airway tree co-exist within its mechanical environment to maintain normal cell function throughout its branching structure of diminishing dimensions? We introduce a new mechanical design principle for the conducting airway tree in which mechanotransduction at the level of cells is driven to orchestrate airway wall structural changes that can best maintain a preferred mechanical microenvironment. To support this principle, we report in vitro radius-transmural pressure relations for a range of airway radii obtained from healthy bovine lungs and model the data using a strain energy function together with a thick-walled cylinder description. From this framework, we estimate circumferential stresses and incremental Young's moduli throughout the airway tree. Our results indicate that the conducting airways consistently operate within a preferred mechanical homeostatic state, termed mechanical homeostasis, that is characterized by a narrow range of circumferential stresses and Young's moduli. This mechanical homeostatic state is maintained for all airways throughout the tree via airway wall dimensional and mechanical relationships. As a consequence, cells within the airway walls throughout the airway tree experience similar oscillatory strains during breathing that are much smaller than previously thought. Finally, we discuss the potential implications of how the maintenance of mechanical homeostasis, while facilitating healthy tissue-level alterations necessary for maturation, may lead to airway wall structural changes capable of chronic asthma.

  3. A mechanical design principle for tissue structure and function in the airway tree.

    Directory of Open Access Journals (Sweden)

    Adam S LaPrad

    Full Text Available With every breath, the dynamically changing mechanical pressures must work in unison with the cells and soft tissue structures of the lung to permit air to efficiently traverse the airway tree and undergo gas exchange in the alveoli. The influence of mechanics on cell and tissue function is becoming apparent, raising the question: how does the airway tree co-exist within its mechanical environment to maintain normal cell function throughout its branching structure of diminishing dimensions? We introduce a new mechanical design principle for the conducting airway tree in which mechanotransduction at the level of cells is driven to orchestrate airway wall structural changes that can best maintain a preferred mechanical microenvironment. To support this principle, we report in vitro radius-transmural pressure relations for a range of airway radii obtained from healthy bovine lungs and model the data using a strain energy function together with a thick-walled cylinder description. From this framework, we estimate circumferential stresses and incremental Young's moduli throughout the airway tree. Our results indicate that the conducting airways consistently operate within a preferred mechanical homeostatic state, termed mechanical homeostasis, that is characterized by a narrow range of circumferential stresses and Young's moduli. This mechanical homeostatic state is maintained for all airways throughout the tree via airway wall dimensional and mechanical relationships. As a consequence, cells within the airway walls throughout the airway tree experience similar oscillatory strains during breathing that are much smaller than previously thought. Finally, we discuss the potential implications of how the maintenance of mechanical homeostasis, while facilitating healthy tissue-level alterations necessary for maturation, may lead to airway wall structural changes capable of chronic asthma.

  4. Like a slippery fish, a little slime is a good thing: the glycocalyx revealed.

    Science.gov (United States)

    Biddle, Chuck

    2013-12-01

    The glycocalyx is a dynamic network of multiple membrane-bound complexes lining the vascular endothelium. Its role in maintaining vascular homeostasis includes regulating vascular permeability as well as a range of vital functions, such as mechanotransduction, hemostasis, modulation of inflammatory processes, and serving as an antiatherogenic. Revisionist thinking about the Starling principle is discussed in terms of the major influence of the glycocalyx on capillary and tissue fluid homeostasis. The clinical and pathophysiologic threats to the glycocalyx are reviewed as well as strategies to maintain its integrity.

  5. Do metabolites that are produced during resistance exercise enhance muscle hypertrophy?

    Science.gov (United States)

    Dankel, Scott J; Mattocks, Kevin T; Jessee, Matthew B; Buckner, Samuel L; Mouser, J Grant; Loenneke, Jeremy P

    2017-11-01

    Many reviews conclude that metabolites play an important role with respect to muscle hypertrophy during resistance exercise, but their actual physiologic contribution remains unknown. Some have suggested that metabolites may work independently of muscle contraction, while others have suggested that metabolites may play a secondary role in their ability to augment muscle activation via inducing fatigue. Interestingly, the studies used as support for an anabolic role of metabolites use protocols that are not actually designed to test the importance of metabolites independent of muscle contraction. While there is some evidence in vitro that metabolites may induce muscle hypertrophy, the only study attempting to answer this question in humans found no added benefit of pooling metabolites within the muscle post-exercise. As load-induced muscle hypertrophy is thought to work via mechanotransduction (as opposed to being metabolically driven), it seems likely that metabolites simply augment muscle activation and cause the mechanotransduction cascade in a larger proportion of muscle fibers, thereby producing greater muscle growth. A sufficient time under tension also appears necessary, as measurable muscle growth is not observed after repeated maximal testing. Based on current evidence, it is our opinion that metabolites produced during resistance exercise do not have anabolic properties per se, but may be anabolic in their ability to augment muscle activation. Future studies are needed to compare protocols which produce similar levels of muscle activation, but differ in the magnitude of metabolites produced, or duration in which the exercised muscles are exposed to metabolites.

  6. Function and morphology correlates of rectal nerve mechanoreceptors innervating the guinea pig internal anal sphincter.

    Science.gov (United States)

    Lynn, P A; Brookes, S J H

    2011-01-01

    Mechanoreceptors to the internal anal sphincter (IAS) contribute to continence and normal defecation, yet relatively little is known about their function or morphology. We investigated the function and structure of mechanoreceptors to the guinea pig IAS. Extracellular recordings from rectal nerve branches to the IAS in vitro, combined with anterograde labeling of recorded nerve trunks, were used to characterize extrinsic afferent nerve endings activated by circumferential distension. Slowly adapting, stretch-sensitive afferents were recorded in rectal nerves to the IAS. Ten of 11 were silent under basal conditions and responded to circumferential stretch in a saturating linear manner. Rectal nerve afferents responded to compression with von Frey hairs with low thresholds (0.3-0.5 mN) and 3.4 ± 0.5 discrete, elongated mechanosensitive fields of innervation aligned parallel to circular muscle bundles (length = 62 ± 16 mm, n = 10). Anterogradely labeled rectal nerve axons typically passed through sparse irregular myenteric ganglia adjacent to the IAS, before ending in extensive varicose arrays within the circular muscle and, to a lesser extent, the longitudinal muscle overlying the IAS. Few (8%) IAS myenteric ganglia contained intraganglionic laminar endings. In eight preparations, mechanotransduction sites were mapped in combination with successful anterograde fills. Mechanotransduction sites were strongly associated with extensive fine varicose arrays within the circular muscle (P IAS are likely to correspond to extensive fine varicose arrays within the circular muscle. © 2010 Blackwell Publishing Ltd.

  7. The Influence of Bioreactor Geometry and the Mechanical Environment on Engineered Tissues

    KAUST Repository

    Osborne, J. M.; O’ Dea, R. D.; Whiteley, J. P.; Byrne, H. M.; Waters, S. L.

    2010-01-01

    A three phase model for the growth of a tissue construct within a perfusion bioreactor is examined. The cell population (and attendant extracellular matrix), culture medium, and porous scaffold are treated as distinct phases. The bioreactor system is represented by a two-dimensional channel containing a cell-seeded rigid porous scaffold (tissue construct), which is perfused with a culture medium. Through the prescription of appropriate functional forms for cell proliferation and extracellular matrix deposition rates, the model is used to compare the influence of cell density-, pressure-, and culture medium shear stress-regulated growth on the composition of the engineered tissue. The governing equations are derived in O'Dea et al. "A Three Phase Model for Tissue Construct Growth in a Perfusion Bioreactor," Math. Med. Biol., in which the long-wavelength limit was exploited to aid analysis; here, finite element methods are used to construct two-dimensional solutions to the governing equations and to investigate thoroughly their behavior. Comparison of the total tissue yield and averaged pressures, velocities, and shear stress demonstrates that quantitative agreement between the two-dimensional and long-wavelength approximation solutions is obtained for channel aspect ratios of order 10 -2 and that much of the qualitative behavior of the model is captured in the long-wavelength limit, even for relatively large channel aspect ratios. However, we demonstrate that in order to capture accurately the effect of mechanotransduction mechanisms on tissue construct growth, spatial effects in at least two dimensions must be included due to the inherent spatial variation of mechanical stimuli relevant to perfusion bioreactors, most notably, fluid shear stress, a feature not captured in the long-wavelength limit. Copyright © 2010 by ASME.

  8. The Influence of Bioreactor Geometry and the Mechanical Environment on Engineered Tissues

    KAUST Repository

    Osborne, J. M.

    2010-01-01

    A three phase model for the growth of a tissue construct within a perfusion bioreactor is examined. The cell population (and attendant extracellular matrix), culture medium, and porous scaffold are treated as distinct phases. The bioreactor system is represented by a two-dimensional channel containing a cell-seeded rigid porous scaffold (tissue construct), which is perfused with a culture medium. Through the prescription of appropriate functional forms for cell proliferation and extracellular matrix deposition rates, the model is used to compare the influence of cell density-, pressure-, and culture medium shear stress-regulated growth on the composition of the engineered tissue. The governing equations are derived in O\\'Dea et al. "A Three Phase Model for Tissue Construct Growth in a Perfusion Bioreactor," Math. Med. Biol., in which the long-wavelength limit was exploited to aid analysis; here, finite element methods are used to construct two-dimensional solutions to the governing equations and to investigate thoroughly their behavior. Comparison of the total tissue yield and averaged pressures, velocities, and shear stress demonstrates that quantitative agreement between the two-dimensional and long-wavelength approximation solutions is obtained for channel aspect ratios of order 10 -2 and that much of the qualitative behavior of the model is captured in the long-wavelength limit, even for relatively large channel aspect ratios. However, we demonstrate that in order to capture accurately the effect of mechanotransduction mechanisms on tissue construct growth, spatial effects in at least two dimensions must be included due to the inherent spatial variation of mechanical stimuli relevant to perfusion bioreactors, most notably, fluid shear stress, a feature not captured in the long-wavelength limit. Copyright © 2010 by ASME.

  9. Caveolins and caveolae in ocular physiology and pathophysiology.

    Science.gov (United States)

    Gu, Xiaowu; Reagan, Alaina M; McClellan, Mark E; Elliott, Michael H

    2017-01-01

    Caveolae are specialized, invaginated plasma membrane domains that are defined morphologically and by the expression of signature proteins called, caveolins. Caveolae and caveolins are abundant in a variety of cell types including vascular endothelium, glia, and fibroblasts where they play critical roles in transcellular transport, endocytosis, mechanotransduction, cell proliferation, membrane lipid homeostasis, and signal transduction. Given these critical cellular functions, it is surprising that ablation of the caveolae organelle does not result in lethality suggesting instead that caveolae and caveolins play modulatory roles in cellular homeostasis. Caveolar components are also expressed in ocular cell types including retinal vascular cells, Müller glia, retinal pigment epithelium (RPE), conventional aqueous humor outflow cells, the corneal epithelium and endothelium, and the lens epithelium. In the eye, studies of caveolae and other membrane microdomains (i.e., "lipid rafts") have lagged behind what is a substantial body of literature outside vision science. However, interest in caveolae and their molecular components has increased with accumulating evidence of important roles in vision-related functions such as blood-retinal barrier homeostasis, ocular inflammatory signaling, pathogen entry at the ocular surface, and aqueous humor drainage. The recent association of CAV1/2 gene loci with primary open angle glaucoma and intraocular pressure has further enhanced the need to better understand caveolar functions in the context of ocular physiology and disease. Herein, we provide the first comprehensive review of literature on caveolae, caveolins, and other membrane domains in the context of visual system function. This review highlights the importance of caveolae domains and their components in ocular physiology and pathophysiology and emphasizes the need to better understand these important modulators of cellular function. Copyright © 2016 Elsevier Ltd. All

  10. Nucleoskeleton dynamics and functions in health and disease

    Directory of Open Access Journals (Sweden)

    Meinke P

    2015-01-01

    Full Text Available Peter Meinke, Alexandr A Makarov, Phú Lê Thành, Daina Sadurska, Eric C SchirmerWellcome Trust Centre for Cell Biology and Institute of Cell Biology, University of Edinburgh, Edinburgh, UKAbstract: It is a common misconception to view the "cyto"-skeleton as just the filament systems in the "cyto"-plasm. In fact, the cytoskeleton extends into the nucleus where the complex network connects to chromatin, and it also connects through the plasma membrane to the cytoskeleton of adjacent cells and to the "exo"-skeleton of the extracellular matrix. This review will focus principally on the subcomplex of the cytoskeleton associated with the nucleus, often referred to as the nucleoskeleton, but in the context of its extensive interconnectivity with the rest of the nucleus and with cytoplasmic filament systems all the way to the exoskeleton. The nucleoskeleton, made principally of type-V intermediate filament lamins, connects across the double membrane system of the nuclear envelope to likely all three primary cytoplasmic filament systems. It provides structural stability to the nucleus, and also incredible flexibility. In both its core structural aspect and through specificity gained by tissue-specific partner proteins, it contributes to genome organization and regulation as well as to signal transduction, both through chemical signaling cascades and likely through mechanotransduction. Defects in the nucleoskeleton have far-ranging effects due to its interactions with cytoplasmic filament systems, from mispositioning of nuclei to disruption of cell polarity and both decreased and increased cell migration depending on the defect. Accordingly, it is not surprising that many nucleoskeletal components are linked to a wide range of human diseases from specific types of cancer to muscular dystrophies, neuropathies, dermopathies, and premature aging syndromes.Keywords: nuclear envelope, lamin, LINC complex, cytoskeleton, nuclear envelopathies

  11. Investigation of the Effects of Extracellular Osmotic Pressure on Morphology and Mechanical Properties of Individual Chondrocyte.

    Science.gov (United States)

    Nguyen, Trung Dung; Oloyede, Adekunle; Singh, Sanjleena; Gu, YuanTong

    2016-06-01

    It has been demonstrated that most cells of the body respond to osmotic pressure in a systematic manner. The disruption of the collagen network in the early stages of osteoarthritis causes an increase in water content of cartilage which leads to a reduction of pericellular osmolality in chondrocytes distributed within the extracellular environment. It is therefore arguable that an insight into the mechanical properties of chondrocytes under varying osmotic pressure would provide a better understanding of chondrocyte mechanotransduction and potentially contribute to knowledge on cartilage degeneration. In this present study, the chondrocyte cells were exposed to solutions with different osmolality. Changes in their dimensions and mechanical properties were measured over time. Atomic force microscopy (AFM) was used to apply load at various strain-rates and the force-time curves were logged. The thin-layer elastic model was used to extract the elastic stiffness of chondrocytes at different strain-rates and at different solution osmolality. In addition, the porohyperelastic (PHE) model was used to investigate the strain-rate-dependent responses under the loading and osmotic pressure conditions. The results revealed that the hypo-osmotic external environment increased chondrocyte dimensions and reduced Young's modulus of the cells at all strain-rates tested. In contrast, the hyper-osmotic external environment reduced dimensions and increased Young's modulus. Moreover, using the PHE model coupled with inverse FEA simulation, we established that the hydraulic permeability of chondrocytes increased with decreasing extracellular osmolality which is consistent with previous work in the literature. This could be due to a higher intracellular fluid volume fraction with lower osmolality.

  12. Osteoarthritis Year in Review 2015: Mechanics

    Science.gov (United States)

    Varady, Nathan H.; Grodzinsky, Alan J.

    2015-01-01

    Motivated by the conceptual framework of multi-scale biomechanics, this narrative review highlights recent major advances with a focus on gait and joint kinematics, then tissue-level mechanics, cell mechanics and mechanotransduction, matrix mechanics, and finally the nanoscale mechanics of matrix macromolecules. A literature review was conducted from January 2014 to April 2015 using PubMed to identify major developments in mechanics related to osteoarthritis (OA). Studies of knee adduction, flexion, rotation, and contact mechanics have extended our understanding of medial compartment loading. In turn, advances in measurement methodologies have shown how injuries to both the meniscus and ligaments, together, can alter joint kinematics. At the tissue scale, novel findings have emerged regarding the mechanics of the meniscus as well as cartilage superficial zone. Moving to the cell level, poroelastic and poroviscoelastic mechanisms underlying chondrocyte deformation have been reported, along with the response to osmotic stress. Further developments have emerged on the role of calcium signaling in chondrocyte mechanobiology, including exciting findings on the function of mechanically activated cation channels newly found to be expressed in chondrocytes. Finally, AFM-based nano-rheology systems have enabled studies of thin murine tissues and brush layers of matrix molecules over a wide range of loading rates including high rates corresponding to impact injury. With OA acknowledged to be a disease of the joint as an organ, understanding mechanical behavior at each length scale helps to elucidate the connections between cell biology, matrix biochemistry and tissue structure/function that may play a role in the pathomechanics of OA. PMID:26707990

  13. The very large G-protein-coupled receptor VLGR1: a component of the ankle link complex required for the normal development of auditory hair bundles.

    Science.gov (United States)

    McGee, Joann; Goodyear, Richard J; McMillan, D Randy; Stauffer, Eric A; Holt, Jeffrey R; Locke, Kirsten G; Birch, David G; Legan, P Kevin; White, Perrin C; Walsh, Edward J; Richardson, Guy P

    2006-06-14

    Sensory hair bundles in the inner ear are composed of stereocilia that can be interconnected by a variety of different link types, including tip links, horizontal top connectors, shaft connectors, and ankle links. The ankle link antigen is an epitope specifically associated with ankle links and the calycal processes of photoreceptors in chicks. Mass spectrometry and immunoblotting were used to identify this antigen as the avian ortholog of the very large G-protein-coupled receptor VLGR1, the product of the Usher syndrome USH2C (Mass1) locus. Like ankle links, Vlgr1 is expressed transiently around the base of developing hair bundles in mice. Ankle links fail to form in the cochleae of mice carrying a targeted mutation in Vlgr1 (Vlgr1/del7TM), and the bundles become disorganized just after birth. FM1-43 [N-(3-triethylammonium)propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] dye loading and whole-cell recordings indicate mechanotransduction is impaired in cochlear, but not vestibular, hair cells of early postnatal Vlgr1/del7TM mutant mice. Auditory brainstem recordings and distortion product measurements indicate that these mice are severely deaf by the third week of life. Hair cells from the basal half of the cochlea are lost in 2-month-old Vlgr1/del7TM mice, and retinal function is mildly abnormal in aged mutants. Our results indicate that Vlgr1 is required for formation of the ankle link complex and the normal development of cochlear hair bundles.

  14. Polycystins, calcium signaling, and human diseases

    International Nuclear Information System (INIS)

    Delmas, Patrick; Padilla, Francoise; Osorio, Nancy; Coste, Bertrand; Raoux, Matthieu; Crest, Marcel

    2004-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is a major, inherited nephropathy affecting over 1:1000 of the worldwide population. It is a systemic condition with frequent hepatic and cardiovascular manifestations in addition to the progressive development of fluid-filled cysts from the tubules and collecting ducts of affected kidneys. The pathogenesis of cyst formation is currently thought to involve increased proliferation of epithelial cells, mild dedifferentiation, and fluid accumulation. In the past decade, study of ADPKD led to the discovery of a unique family of highly complex proteins, the polycystins. Loss-of-function mutations in either of two polycystin proteins, polycystin-1 or polycystin-2, give rise to ADPKD. These proteins are thought to function together as part of a multiprotein complex that may initiate Ca 2+ signals, directing attention to the regulation of intracellular Ca 2+ as a possible misstep that participates in cyst formation. Here we review what is known about the Ca 2+ signaling functions of polycystin proteins and focus on findings that have significantly advanced our physiological insight. Special attention is paid to the recently discovered role of these proteins in the mechanotransduction of the renal primary cilium and the model it suggests

  15. Human Digital Meissner Corpuscles Display Immunoreactivity for the Multifunctional Ion Channels Trpc6 and Trpv4.

    Science.gov (United States)

    Alonso-González, Paula; Cabo, Roberto; San José, Isabel; Gago, Angel; Suazo, Iván C; García-Suárez, Olivia; Cobo, Juan; Vega, José A

    2017-06-01

    Ion channels are at the basis of the sensory processes including mechanosensing. Some members of the transient receptor potential (TRP) ion channel superfamily have been proposed as mechanosensors, but their putative role in mechanotransduction is controversial. Among them there are TRP canonical 6 (TRPC6) and TRP vanilloid 4 (TRPV4) ion channels, which are known to cooperate in mechanical hyperalgesia. Here, we investigated the occurrence, distribution, and possible colocalization of TRPC6 and TRPV4 in human digital Meissner sensory corpuscles using immunohistochemistry and double immunofluorescence (associate with markers for specific corpuscular constituents). TRPC6 immunoreactivity was restricted to the axon of Meissner corpuscles, whereas TRPV4 was detected in the axon but also in the lamellar cells. Moreover, axonal colocalization of TRPV4 and TRPC6 was found in the digital Meissner corpuscles. Present results demonstrate for the first time the occurrence and colocalization of two ion channels candidates to mechanosensors in human cutaneous mechanoreceptors. The functional significance of these ion channels in that place remains to be clarified, but should be related to different properties of mechanosensitivity. Anat Rec, 300:1022-1031, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  16. Integrin-linked kinase is required for TGF-β1 induction of dermal myofibroblast differentiation.

    Science.gov (United States)

    Vi, Linda; de Lasa, Cristina; DiGuglielmo, Gianni M; Dagnino, Lina

    2011-03-01

    Cutaneous repair after injury requires activation of resident dermal fibroblasts and their transition to myofibroblasts. The key stimuli for myofibroblast formation are activation of transforming growth factor-β (TGF-β) receptors and mechanotransduction mediated by integrins and associated proteins. We investigated the role of integrin-linked kinase (ILK) in TGF-β1 induction of dermal fibroblast transition to myofibroblasts. ILK-deficient fibroblasts treated with TGF-β1 exhibited attenuation of Smad 2 and 3 phosphorylation, accompanied by impaired transcriptional activation of Smad targets, such as α-smooth muscle actin. These alterations were not limited to Smad-associated TGF-β1 responses, as stimulation of noncanonical mitogen-activated protein kinase pathways by this growth factor was also diminished in the absence of ILK. ILK-deficient fibroblasts exhibited abnormalities in the actin cytoskeleton, and did not form supermature focal adhesions or contractile F-actin stress fibers, indicating a severe impairment in their capacity to differentiate into myofibroblasts. These defects extended to the inability of cells to contract extracellular matrices when embedded in collagen lattices. We conclude that ILK is necessary to transduce signals implicated in the transition of dermal fibroblasts to myofibroblasts originating from matrix substrates and TGF-β1.

  17. Mechano- and Chemo-Sensory Polycystins

    Science.gov (United States)

    Patel, Amanda; Delmas, Patrick; Honoré, Eric

    Polycystins belong to the superfamily of transient receptor potential (TRP) channels and comprise five PKD1-like and three PKD2-like (TRPP) subunits. In this chapter, we review the general properties of polycystins and discuss their specific role in both mechanotransduction and chemoreception. The heteromer PKD1/PKD2 expressed at the membrane of the primary cilium of kidney epithelial cells is proposed to form a mechano-sensitive calcium channel that is opened by physiological fluid flow. Dysfunction or loss of PKD1 or PKD2 polycystin genes may be responsible for the inability of epithelial cells to sense mechanical cues, thus provoking autosomal dominant polycystic kidney disease (ADPKD), one of the most prevalent genetic kidney disorders. pkd1 and pkd2 knock-out mice recapitulate the human disease. Similarly, PKD2 may function as a mechanosensory calcium channel in the immotile monocilia of the developing node transducing leftward flow into an increase in calcium and specifying the left-right axis. pkd2, unlike pkd1 knock-out embryos are characterized by right lung isomerism (situs inversus). Mechanical stimuli also induce cleavage and nuclear translocation of the PKD1 C-terminal tail, which enters the nucleus and initiates signaling processes involving the AP-1, STAT6 and P100 pathways. This intraproteolytic mechanism is implicated in the transduction of a change in renal fluid flow to a transcriptional long-term response. The heteromer PKD1L3/PKD2L1 is the basis for acid sensing in specialised sensory cells including the taste bud cells responsible for sour taste. Moreover, PKD1L3/PKD2L1 may be implicated in the chemosensitivity of neurons surrounding the spinal cord canal, sensing protons in the cerebrospinal fluid. These recent results demonstrate that polycystins fulfill a major sensory role in a variety of cells including kidney epithelial cells, taste buds cells and spinal cord neurons. Such mechanisms are involved in short- and long-term physiological

  18. Regulation of cell cycle progression by cell-cell and cell-matrix forces

    NARCIS (Netherlands)

    Uroz, Marina; Wistorf, Sabrina; Serra-Picamal, Xavier; Conte, Vito; Sales-Pardo, Marta; Roca-Cusachs, Pere; Guimerà, Roger; Trepat, Xavier

    2018-01-01

    It has long been proposed that the cell cycle is regulated by physical forces at the cell-cell and cell-extracellular matrix (ECM) interfaces 1-12 . However, the evolution of these forces during the cycle has never been measured in a tissue, and whether this evolution affects cell cycle progression

  19. Well-Controlled Cell-Trapping Systems for Investigating Heterogeneous Cell-Cell Interactions.

    Science.gov (United States)

    Kamiya, Koki; Abe, Yuta; Inoue, Kosuke; Osaki, Toshihisa; Kawano, Ryuji; Miki, Norihisa; Takeuchi, Shoji

    2018-03-01

    Microfluidic systems have been developed for patterning single cells to study cell-cell interactions. However, patterning multiple types of cells to understand heterogeneous cell-cell interactions remains difficult. Here, it is aimed to develop a cell-trapping device to assemble multiple types of cells in the well-controlled order and morphology. This device mainly comprises a parylene sheet for assembling cells and a microcomb for controlling the cell-trapping area. The cell-trapping area is controlled by moving the parylene sheet on an SU-8 microcomb using tweezers. Gentle downward flow is used as a driving force for the cell-trapping. The assembly of cells on a parylene sheet with round and line-shaped apertures is demonstrated. The cell-cell contacts of the trapped cells are then investigated by direct cell-cell transfer of calcein via connexin nanopores. Finally, using the device with a system for controlling the cell-trapping area, three different types of cells in the well-controlled order are assembled. The correct cell order rate obtained using the device is 27.9%, which is higher than that obtained without the sliding parylene system (0.74%). Furthermore, the occurrence of cell-cell contact between the three cell types assembled is verified. This cell-patterning device will be a useful tool for investigating heterogeneous cell-cell interactions. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Relationship between micro-porosity, water permeability and mechanical behavior in scaffolds for cartilage engineering.

    Science.gov (United States)

    Vikingsson, L; Claessens, B; Gómez-Tejedor, J A; Gallego Ferrer, G; Gómez Ribelles, J L

    2015-08-01

    In tissue engineering the design and optimization of biodegradable polymeric scaffolds with a 3D-structure is an important field. The porous scaffold provide the cells with an adequate biomechanical environment that allows mechanotransduction signals for cell differentiation and the scaffolds also protect the cells from initial compressive loading. The scaffold have interconnected macro-pores that host the cells and newly formed tissue, while the pore walls should be micro-porous to transport nutrients and waste products. Polycaprolactone (PCL) scaffolds with a double micro- and macro-pore architecture have been proposed for cartilage regeneration. This work explores the influence of the micro-porosity of the pore walls on water permeability and scaffold compliance. A Poly(Vinyl Alcohol) with tailored mechanical properties has been used to simulate the growing cartilage tissue inside the scaffold pores. Unconfined and confined compression tests were performed to characterize both the water permeability and the mechanical response of scaffolds with varying size of micro-porosity while volume fraction of the macro-pores remains constant. The stress relaxation tests show that the stress response of the scaffold/hydrogel construct is a synergic effect determined by the performance of the both components. This is interesting since it suggests that the in vivo outcome of the scaffold is not only dependent upon the material architecture but also the growing tissue inside the scaffold׳s pores. On the other hand, confined compression results show that compliance of the scaffold is mainly controlled by the micro-porosity of the scaffold and less by hydrogel density in the scaffold pores. These conclusions bring together valuable information for customizing the optimal scaffold and to predict the in vivo mechanical behavior. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Ultra-fast optical manipulation of single proteins binding to the actin cytoskeleton

    Science.gov (United States)

    Capitanio, Marco; Gardini, Lucia; Pavone, Francesco Saverio

    2014-02-01

    In the last decade, forces and mechanical stresses acting on biological systems are emerging as regulatory factors essential for cell life. Emerging evidences indicate that factors such as applied forces or the rigidity of the extracellular matrix (ECM) determine the shape and function of cells and organisms1. Classically, the regulation of biological systems is described through a series of biochemical signals and enzymatic reactions, which direct the processes and cell fate. However, mechanotransduction, i.e. the conversion of mechanical forces into biochemical and biomolecular signals, is at the basis of many biological processes fundamental for the development and differentiation of cells, for their correct function and for the development of pathologies. We recently developed an in vitro system that allows the investigation of force-dependence of the interaction of proteins binding the actin cytoskeleton, at the single molecule level. Our system displays a delay of only ~10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. Our assay allows direct measurements of load-dependence of lifetimes of single molecular bonds and conformational changes of single proteins and molecular motors. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.

  2. Meningeal mast cell-T cell crosstalk regulates T cell encephalitogenicity.

    Science.gov (United States)

    Russi, Abigail E; Walker-Caulfield, Margaret E; Guo, Yong; Lucchinetti, Claudia F; Brown, Melissa A

    2016-09-01

    GM-CSF is a cytokine produced by T helper (Th) cells that plays an essential role in orchestrating neuroinflammation in experimental autoimmune encephalomyelitis, a rodent model of multiple sclerosis. Yet where and how Th cells acquire GM-CSF expression is unknown. In this study we identify mast cells in the meninges, tripartite tissues surrounding the brain and spinal cord, as important contributors to antigen-specific Th cell accumulation and GM-CSF expression. In the absence of mast cells, Th cells do not accumulate in the meninges nor produce GM-CSF. Mast cell-T cell co-culture experiments and selective mast cell reconstitution of the meninges of mast cell-deficient mice reveal that resident meningeal mast cells are an early source of caspase-1-dependent IL-1β that licenses Th cells to produce GM-CSF and become encephalitogenic. We also provide evidence of mast cell-T cell co-localization in the meninges and CNS of recently diagnosed acute MS patients indicating similar interactions may occur in human demyelinating disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2005-05-01

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

  4. Osteoarthritis year in review 2015: mechanics.

    Science.gov (United States)

    Varady, N H; Grodzinsky, A J

    2016-01-01

    Motivated by the conceptual framework of multi-scale biomechanics, this narrative review highlights recent major advances with a focus on gait and joint kinematics, then tissue-level mechanics, cell mechanics and mechanotransduction, matrix mechanics, and finally the nanoscale mechanics of matrix macromolecules. A literature review was conducted from January 2014 to April 2015 using PubMed to identify major developments in mechanics related to osteoarthritis (OA). Studies of knee adduction, flexion, rotation, and contact mechanics have extended our understanding of medial compartment loading. In turn, advances in measurement methodologies have shown how injuries to both the meniscus and ligaments, together, can alter joint kinematics. At the tissue scale, novel findings have emerged regarding the mechanics of the meniscus as well as cartilage superficial zone. Moving to the cell level, poroelastic and poro-viscoelastic mechanisms underlying chondrocyte deformation have been reported, along with the response to osmotic stress. Further developments have emerged on the role of calcium signaling in chondrocyte mechanobiology, including exciting findings on the function of mechanically activated cation channels newly found to be expressed in chondrocytes. Finally, AFM-based nano-rheology systems have enabled studies of thin murine tissues and brush layers of matrix molecules over a wide range of loading rates including high rates corresponding to impact injury. With OA acknowledged to be a disease of the joint as an organ, understanding mechanical behavior at each length scale helps to elucidate the connections between cell biology, matrix biochemistry and tissue structure/function that may play a role in the pathomechanics of OA. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  5. Induction of Functional Hair-Cell-Like Cells from Mouse Cochlear Multipotent Cells

    Directory of Open Access Journals (Sweden)

    Quanwen Liu

    2016-01-01

    Full Text Available In this paper, we developed a two-step-induction method of generating functional hair cells from inner ear multipotent cells. Multipotent cells from the inner ear were established and induced initially into progenitor cells committed to the inner ear cell lineage on the poly-L-lysine substratum. Subsequently, the committed progenitor cells were cultured on the mitotically inactivated chicken utricle stromal cells and induced into hair-cell-like cells containing characteristic stereocilia bundles. The hair-cell-like cells exhibited rapid permeation of FM1-43FX. The whole-cell patch-clamp technique was used to measure the membrane currents of cells differentiated for 7 days on chicken utricle stromal cells and analyze the biophysical properties of the hair-cell-like cells by recording membrane properties of cells. The results suggested that the hair-cell-like cells derived from inner ear multipotent cells were functional following differentiation in an enabling environment.

  6. Cell Cycle Related Differentiation of Bone Marrow Cells into Lung Cells

    Energy Technology Data Exchange (ETDEWEB)

    Dooner, Mark; Aliotta, Jason M.; Pimental, Jeffrey; Dooner, Gerri J.; Abedi, Mehrdad; Colvin, Gerald; Liu, Qin; Weier, Heinz-Ulli; Dooner, Mark S.; Quesenberry, Peter J.

    2007-12-31

    Green-fluorescent protein (GFP) labeled marrow cells transplanted into lethally irradiated mice can be detected in the lungs of transplanted mice and have been shown to express lung specific proteins while lacking the expression of hematopoietic markers. We have studied marrow cells induced to transit cell cycle by exposure to IL-3, IL-6, IL-11 and steel factor at different times of culture corresponding to different phases of cell cycle. We have found that marrow cells at the G1/S interface have a 3-fold increase in cells which assume a lung phenotype and that this increase is no longer seen in late S/G2. These cells have been characterized as GFP{sup +} CD45{sup -} and GFP{sup +} cytokeratin{sup +}. Thus marrow cells with the capacity to convert into cells with a lung phenotype after transplantation show a reversible increase with cytokine induced cell cycle transit. Previous studies have shown the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse cell cycle, leading to a continuum model of stem cell regulation. The present studies indicate that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.

  7. Deletion of Notch1 converts pro-T cells to dendritic cells and promotes thymic B cells by cell-extrinsic and cell-intrinsic mechanisms.

    Science.gov (United States)

    Feyerabend, Thorsten B; Terszowski, Grzegorz; Tietz, Annette; Blum, Carmen; Luche, Hervé; Gossler, Achim; Gale, Nicholas W; Radtke, Freddy; Fehling, Hans Jörg; Rodewald, Hans-Reimer

    2009-01-16

    Notch1 signaling is required for T cell development and has been implicated in fate decisions in the thymus. We showed that Notch1 deletion in progenitor T cells (pro-T cells) revealed their latent developmental potential toward becoming conventional and plasmacytoid dendritic cells. In addition, Notch1 deletion in pro-T cells resulted in large numbers of thymic B cells, previously explained by T-to-B cell fate conversion. Single-cell genotyping showed, however, that the majority of these thymic B cells arose from Notch1-sufficient cells by a cell-extrinsic pathway. Fate switching nevertheless exists for a subset of thymic B cells originating from Notch1-deleted pro-T cells. Chimeric mice lacking the Notch ligand delta-like 4 (Dll4) in thymus epithelium revealed an essential role for Dll4 in T cell development. Thus, Notch1-Dll4 signaling fortifies T cell commitment by suppressing non-T cell lineage potential in pro-T cells, and normal Notch1-driven T cell development repels excessive B cells in the thymus.

  8. Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

    International Nuclear Information System (INIS)

    Varga, Nóra; Veréb, Zoltán; Rajnavölgyi, Éva; Német, Katalin; Uher, Ferenc; Sarkadi, Balázs; Apáti, Ágota

    2011-01-01

    Highlights: ► MSC like cells were derived from hESC by a simple and reproducible method. ► Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. ► MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  9. Involvement of plant stem cells or stem cell-like cells in dedifferentiation

    Directory of Open Access Journals (Sweden)

    Fangwei eJiang

    2015-11-01

    Full Text Available Dedifferentiation is the transformation of cells from a given differentiated state to a less differentiated or stem cell-like state. Stem cell-related genes play important roles in dedifferentiation, which exhibits similar histone modification and DNA methylation features to stem cell maintenance. Hence, stem cell-related factors possibly synergistically function to provide a specific niche beneficial to dedifferentiation. During callus formation in Arabidopsis petioles, cells adjacent to procambium cells (stem cell-like cells are dedifferentiated and survive more easily than other cell types. This finding indicates that stem cells or stem cell-like cells may influence the dedifferentiating niche. In this paper, we provide a brief overview of stem cell maintenance and dedifferentiation regulation. We also summarize current knowledge of genetic and epigenetic mechanisms underlying the balance between differentiation and dedifferentiation. Furthermore, we discuss the correlation of stem cells or stem cell-like cells with dedifferentiation.

  10. Cell-cell interactions mediate cytoskeleton organization and collective endothelial cell chemotaxis.

    Science.gov (United States)

    Shamloo, Amir

    2014-09-01

    This study investigates the role of cell-cell and cell-ligand interactions in cytoskeleton organization of endothelial cells (ECs) and their directional migration within a microfluidic device. The migration of ECs in response to a biochemical factor was studied. Mathematical analysis of the cell migration pathways and cellular cytoskeleton revealed that directional migration, migration persistence length, migration speed, and cytoskeletal stress fiber alignment can be mediated by the level of cell contacts as well as the presence or absence of a biochemical polarizing factor. It was shown that in the presence of a biochemical polarizing factor, higher cell density and more frequent cell contacts has a reinforcing effect on collective cell chemotaxis. In contrast, in the absence of a polarizing factor, high cell density can decrease or suppress the ability of the cells to migrate. Also, the correlation of actin stress fiber organization and alignment with directional migration of ECs was investigated. It was shown that in the presence of a biochemical polarizing factor, stress fibers within the cytoskeleton of ECs can be significantly aligned parallel to the gradient direction when the cells have higher level of contacts. The results also show that the organization and alignment of actin stress fibers is mediated by cell adhesion junctions during collective cell migration and introduce cell-cell interactions as a key factor during collective cell chemotaxis. © 2014 Wiley Periodicals, Inc.

  11. Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Varga, Nora [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Vereb, Zoltan; Rajnavoelgyi, Eva [Department of Immunology, Medical and Health Science Centre, University of Debrecen, Debrecen (Hungary); Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Apati, Agota, E-mail: apati@kkk.org.hu [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  12. Computational cell model based on autonomous cell movement regulated by cell-cell signalling successfully recapitulates the "inside and outside" pattern of cell sorting

    Directory of Open Access Journals (Sweden)

    Ajioka Itsuki

    2007-09-01

    Full Text Available Abstract Background Development of multicellular organisms proceeds from a single fertilized egg as the combined effect of countless numbers of cellular interactions among highly dynamic cells. Since at least a reminiscent pattern of morphogenesis can be recapitulated in a reproducible manner in reaggregation cultures of dissociated embryonic cells, which is known as cell sorting, the cells themselves must possess some autonomous cell behaviors that assure specific and reproducible self-organization. Understanding of this self-organized dynamics of heterogeneous cell population seems to require some novel approaches so that the approaches bridge a gap between molecular events and morphogenesis in developmental and cell biology. A conceptual cell model in a computer may answer that purpose. We constructed a dynamical cell model based on autonomous cell behaviors, including cell shape, growth, division, adhesion, transformation, and motility as well as cell-cell signaling. The model gives some insights about what cellular behaviors make an appropriate global pattern of the cell population. Results We applied the model to "inside and outside" pattern of cell-sorting, in which two different embryonic cell types within a randomly mixed aggregate are sorted so that one cell type tends to gather in the central region of the aggregate and the other cell type surrounds the first cell type. Our model can modify the above cell behaviors by varying parameters related to them. We explored various parameter sets with which the "inside and outside" pattern could be achieved. The simulation results suggested that direction of cell movement responding to its neighborhood and the cell's mobility are important for this specific rearrangement. Conclusion We constructed an in silico cell model that mimics autonomous cell behaviors and applied it to cell sorting, which is a simple and appropriate phenomenon exhibiting self-organization of cell population. The model

  13. Real-time TIRF observation of vinculin recruitment to stretched α-catenin by AFM.

    Science.gov (United States)

    Maki, Koichiro; Han, Sung-Woong; Hirano, Yoshinori; Yonemura, Shigenobu; Hakoshima, Toshio; Adachi, Taiji

    2018-01-25

    Adherens junctions (AJs) adaptively change their intensities in response to intercellular tension; therefore, they integrate tension generated by individual cells to drive multicellular dynamics, such as morphogenetic change in embryos. Under intercellular tension, α-catenin, which is a component protein of AJs, acts as a mechano-chemical transducer to recruit vinculin to promote actin remodeling. Although in vivo and in vitro studies have suggested that α-catenin-mediated mechanotransduction is a dynamic molecular process, which involves a conformational change of α-catenin under tension to expose a cryptic vinculin binding site, there are no suitable experimental methods to directly explore the process. Therefore, in this study, we developed a novel system by combining atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF). In this system, α-catenin molecules (residues 276-634; the mechano-sensitive M 1 -M 3 domain), modified on coverslips, were stretched by AFM and their recruitment of Alexa-labeled full-length vinculin molecules, dissolved in solution, were observed simultaneously, in real time, using TIRF. We applied a physiologically possible range of tensions and extensions to α-catenin and directly observed its vinculin recruitment. Our new system could be used in the fields of mechanobiology and biophysics to explore functions of proteins under tension by coupling biomechanical and biochemical information.

  14. Histopathologic study of human vocal fold mucosa unphonated over a decade.

    Science.gov (United States)

    Sato, Kiminori; Umeno, Hirohito; Ono, Takeharu; Nakashima, Tadashi

    2011-12-01

    Mechanotransduction caused by vocal fold vibration could possibly be an important factor in the maintenance of extracellular matrices and layered structure of the human adult vocal fold mucosa as a vibrating tissue after the layered structure has been completed. Vocal fold stellate cells (VFSCs) in the human maculae flavae of the vocal fold mucosa are inferred to be involved in the metabolism of extracellular matrices of the vocal fold mucosa. Maculae flavae are also considered to be an important structure in the growth and development of the human vocal fold mucosa. Tension caused by phonation (vocal fold vibration) is hypothesized to stimulate the VFSCs to accelerate production of extracellular matrices. A human adult vocal fold mucosa unphonated over a decade was investigated histopathologically. Vocal fold mucosa unphonated for 11 years and 2 months of a 64-year-old male with cerebral hemorrhage was investigated by light and electron microscopy. The vocal fold mucosae (including maculae flavae) were atrophic. The vocal fold mucosa did not have a vocal ligament, Reinke's space or a layered structure. The lamina propria appeared as a uniform structure. Morphologically, the VFSCs synthesized fewer extracellular matrices, such as fibrous protein and glycosaminoglycan. Consequently, VFSCs appeared to decrease their level of activity.

  15. Endothelial cell subpopulations in vitro: cell volume, cell cycle, and radiosensitivity

    International Nuclear Information System (INIS)

    Rubin, D.B.; Drab, E.A.; Bauer, K.D.

    1989-01-01

    Vascular endothelial cells (EC) are important clinical targets of radiation and other forms of free radical/oxidant stresses. In this study, we found that the extent of endothelial damage may be determined by the different cytotoxic responses of EC subpopulations. The following characteristics of EC subpopulations were examined: (1) cell volume; (2) cell cycle position; and (3) cytotoxic indexes for both acute cell survival and proliferative capacity after irradiation (137Cs, gamma, 0-10 Gy). EC cultured from bovine aortas were separated by centrifugal elutriation into subpopulations of different cell volumes. Through flow cytometry, we found that cell volume was related to the cell cycle phase distribution. The smallest EC were distributed in G1 phase and the larger cells were distributed in either early S, middle S, or late S + G2M phases. Cell cycle phase at the time of irradiation was not associated with acute cell loss. However, distribution in the cell cycle did relate to cell survival based on proliferative capacity (P less than 0.01). The order of increasing radioresistance was cells in G1 (D0 = 110 cGy), early S (135 cGy), middle S (145 cGy), and late S + G2M phases (180 cGy). These findings (1) suggest an age-related response to radiation in a nonmalignant differentiated cell type and (2) demonstrate EC subpopulations in culture

  16. Quantitative imaging of epithelial cell scattering identifies specific inhibitors of cell motility and cell-cell dissociation

    NARCIS (Netherlands)

    Loerke, D.; le Duc, Q.; Blonk, I.; Kerstens, A.; Spanjaard, E.; Machacek, M.; Danuser, G.; de Rooij, J.

    2012-01-01

    The scattering of cultured epithelial cells in response to hepatocyte growth factor (HGF) is a model system that recapitulates key features of metastatic cell behavior in vitro, including disruption of cell-cell adhesions and induction of cell migration. We have developed image analysis tools that

  17. Excitation-contraction coupling and mechano-sensitivity in denervated skeletal muscles

    Directory of Open Access Journals (Sweden)

    Fabio Francini

    2010-09-01

    Full Text Available Skeletal muscle atrophy can be defined as a wasting or decrease in muscle mass and muscle force generation owing lack of use, ageing, injury or disease. Thus, the etiology of atrophy can be different. Atrophy in denervated muscle is a consequence of two factors: 1 the complete lack of motoneuron activity inducing the deficiency of neurotransmitter release and 2 the muscles disuse. The balance of the muscular functions depends on extra- and intra-muscular signals. In the balance are involved the excitation-contraction coupling (ECC, local growth factors, Ca2+-dependent and independent intracellular signals, mechano-sensitivity and mechano-transduction that activate Ca2+-dependent signaling proteins and cytoskeleton- nucleus pathways to the nucleus, that regulate the gene expression. Moreover, retrograde signal from intracellular compartments and cytoskeleton to the sarcolemma are additional factors that regulate the muscle function. Proteolytic systems that operate in atrophic muscles progressively reduce the muscle protein content and so the sarcolemma, ECC and the force generation. In this review we will focus on the more relevant changes of the sarcolemma, excitation-contraction coupling, ECC and mechano-transduction evaluated by electrophysiological methods and observed from early- to long-term denervated skeletal muscles. This review put in particular evidence that long-term denervated muscle maintain a sub-population of fibers with ECC and contractile machinery able to be activated, albeit in lesser amounts, by electrical and mechanical stimulation. Accordingly, this provides a potential molecular explanation of the muscle recovery that occurs in response to rehabilitation strategy as transcutaneous electrical stimulation and passive stretching of denervated muscles, which wre developed as a result of empirical clinical observations.

  18. The cell cycle as a brake for β-cell regeneration from embryonic stem cells.

    Science.gov (United States)

    El-Badawy, Ahmed; El-Badri, Nagwa

    2016-01-13

    The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.

  19. Pancreatic stellate cells enhance stem cell-like phenotypes in pancreatic cancer cells

    International Nuclear Information System (INIS)

    Hamada, Shin; Masamune, Atsushi; Takikawa, Tetsuya; Suzuki, Noriaki; Kikuta, Kazuhiro; Hirota, Morihisa; Hamada, Hirofumi; Kobune, Masayoshi; Satoh, Kennichi; Shimosegawa, Tooru

    2012-01-01

    Highlights: ► Pancreatic stellate cells (PSCs) promote the progression of pancreatic cancer. ► Pancreatic cancer cells co-cultured with PSCs showed enhanced spheroid formation. ► Expression of stem cell-related genes ABCG2, Nestin and LIN28 was increased. ► Co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells in vivo. ► This study suggested a novel role of PSCs as a part of the cancer stem cell niche. -- Abstract: The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Recent studies have identified that a portion of cancer cells, called “cancer stem cells”, within the entire cancer tissue harbor highly tumorigenic and chemo-resistant phenotypes, which lead to the recurrence after surgery or re-growth of the tumor. The mechanisms that maintain the “stemness” of these cells remain largely unknown. We hypothesized that PSCs might enhance the cancer stem cell-like phenotypes in pancreatic cancer cells. Indirect co-culture of pancreatic cancer cells with PSCs enhanced the spheroid-forming ability of cancer cells and induced the expression of cancer stem cell-related genes ABCG2, Nestin and LIN28. In addition, co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells in vivo. These results suggested a novel role of PSCs as a part of the cancer stem cell niche.

  20. In silico characterization of cell-cell interactions using a cellular automata model of cell culture.

    Science.gov (United States)

    Kihara, Takanori; Kashitani, Kosuke; Miyake, Jun

    2017-07-14

    Cell proliferation is a key characteristic of eukaryotic cells. During cell proliferation, cells interact with each other. In this study, we developed a cellular automata model to estimate cell-cell interactions using experimentally obtained images of cultured cells. We used four types of cells; HeLa cells, human osteosarcoma (HOS) cells, rat mesenchymal stem cells (MSCs), and rat smooth muscle A7r5 cells. These cells were cultured and stained daily. The obtained cell images were binarized and clipped into squares containing about 10 4 cells. These cells showed characteristic cell proliferation patterns. The growth curves of these cells were generated from the cell proliferation images and we determined the doubling time of these cells from the growth curves. We developed a simple cellular automata system with an easily accessible graphical user interface. This system has five variable parameters, namely, initial cell number, doubling time, motility, cell-cell adhesion, and cell-cell contact inhibition (of proliferation). Within these parameters, we obtained initial cell numbers and doubling times experimentally. We set the motility at a constant value because the effect of the parameter for our simulation was restricted. Therefore, we simulated cell proliferation behavior with cell-cell adhesion and cell-cell contact inhibition as variables. By comparing growth curves and proliferation cell images, we succeeded in determining the cell-cell interaction properties of each cell. Simulated HeLa and HOS cells exhibited low cell-cell adhesion and weak cell-cell contact inhibition. Simulated MSCs exhibited high cell-cell adhesion and positive cell-cell contact inhibition. Simulated A7r5 cells exhibited low cell-cell adhesion and strong cell-cell contact inhibition. These simulated results correlated with the experimental growth curves and proliferation images. Our simulation approach is an easy method for evaluating the cell-cell interaction properties of cells.

  1. Host cell reactivation in mammalian cells

    International Nuclear Information System (INIS)

    Lytle, C.D.; Benane, S.G.; Stafford, J.E.

    1976-01-01

    The survival of UV-irradiated herpes simplex virus was determined in cultured Potoroo (a marsupial) and human cells under lighting conditions which promoted photereactivation. Photoreactivation was readily demonstrated for herpes virus in two lines of Potoroo cells with dose reduction factors of 0.7 to 0.8 for ovary cells and 0.5 to 0.7 for kidney cells. Light from Blacklite (near UV) lamps was more effective than from Daylight (mostly visible) lamps, suggesting that near UV radiation was more effecient for photoreactivation in Potoroo cells. The quantitative and qualitative aspects of this photoreactivation were similar to those reported for a similar virus infecting chick embryo cells. UV-survival curves of herpes virus in Potoroo cells indicated a high level of 'dark' host cell reactivation. No photoreactivation was found for UV-irradiated vaccinia virus in Potoroo cells. A similar photoreactivation study was done using special control lighting (lambda>600 nm) and human cells with normal repair and with cells deficient in excision repair (XP). No photoreactivation was found for UV-irradiated herpes virus in either human cell with either Blacklite or Daylight lamps as the sources of photoreactivating light. This result contrasts with a report of photoreactivation for a herpes virus in the same XP cells using incandescent lamps. (author)

  2. Local cell metrics: a novel method for analysis of cell-cell interactions

    Directory of Open Access Journals (Sweden)

    Chen Chien-Chiang

    2009-10-01

    Full Text Available Abstract Background The regulation of many cell functions is inherently linked to cell-cell contact interactions. However, effects of contact interactions among adherent cells can be difficult to detect with global summary statistics due to the localized nature and noise inherent to cell-cell interactions. The lack of informatics approaches specific for detecting cell-cell interactions is a limitation in the analysis of large sets of cell image data, including traditional and combinatorial or high-throughput studies. Here we introduce a novel histogram-based data analysis strategy, termed local cell metrics (LCMs, which addresses this shortcoming. Results The new LCM method is demonstrated via a study of contact inhibition of proliferation of MC3T3-E1 osteoblasts. We describe how LCMs can be used to quantify the local environment of cells and how LCMs are decomposed mathematically into metrics specific to each cell type in a culture, e.g., differently-labelled cells in fluorescence imaging. Using this approach, a quantitative, probabilistic description of the contact inhibition effects in MC3T3-E1 cultures has been achieved. We also show how LCMs are related to the naïve Bayes model. Namely, LCMs are Bayes class-conditional probability functions, suggesting their use for data mining and classification. Conclusion LCMs are successful in robust detection of cell contact inhibition in situations where conventional global statistics fail to do so. The noise due to the random features of cell behavior was suppressed significantly as a result of the focus on local distances, providing sensitive detection of cell-cell contact effects. The methodology can be extended to any quantifiable feature that can be obtained from imaging of cell cultures or tissue samples, including optical, fluorescent, and confocal microscopy. This approach may prove useful in interpreting culture and histological data in fields where cell-cell interactions play a critical

  3. Local cell metrics: a novel method for analysis of cell-cell interactions.

    Science.gov (United States)

    Su, Jing; Zapata, Pedro J; Chen, Chien-Chiang; Meredith, J Carson

    2009-10-23

    The regulation of many cell functions is inherently linked to cell-cell contact interactions. However, effects of contact interactions among adherent cells can be difficult to detect with global summary statistics due to the localized nature and noise inherent to cell-cell interactions. The lack of informatics approaches specific for detecting cell-cell interactions is a limitation in the analysis of large sets of cell image data, including traditional and combinatorial or high-throughput studies. Here we introduce a novel histogram-based data analysis strategy, termed local cell metrics (LCMs), which addresses this shortcoming. The new LCM method is demonstrated via a study of contact inhibition of proliferation of MC3T3-E1 osteoblasts. We describe how LCMs can be used to quantify the local environment of cells and how LCMs are decomposed mathematically into metrics specific to each cell type in a culture, e.g., differently-labelled cells in fluorescence imaging. Using this approach, a quantitative, probabilistic description of the contact inhibition effects in MC3T3-E1 cultures has been achieved. We also show how LCMs are related to the naïve Bayes model. Namely, LCMs are Bayes class-conditional probability functions, suggesting their use for data mining and classification. LCMs are successful in robust detection of cell contact inhibition in situations where conventional global statistics fail to do so. The noise due to the random features of cell behavior was suppressed significantly as a result of the focus on local distances, providing sensitive detection of cell-cell contact effects. The methodology can be extended to any quantifiable feature that can be obtained from imaging of cell cultures or tissue samples, including optical, fluorescent, and confocal microscopy. This approach may prove useful in interpreting culture and histological data in fields where cell-cell interactions play a critical role in determining cell fate, e.g., cancer, developmental

  4. Single-cell sequencing in stem cell biology.

    Science.gov (United States)

    Wen, Lu; Tang, Fuchou

    2016-04-15

    Cell-to-cell variation and heterogeneity are fundamental and intrinsic characteristics of stem cell populations, but these differences are masked when bulk cells are used for omic analysis. Single-cell sequencing technologies serve as powerful tools to dissect cellular heterogeneity comprehensively and to identify distinct phenotypic cell types, even within a 'homogeneous' stem cell population. These technologies, including single-cell genome, epigenome, and transcriptome sequencing technologies, have been developing rapidly in recent years. The application of these methods to different types of stem cells, including pluripotent stem cells and tissue-specific stem cells, has led to exciting new findings in the stem cell field. In this review, we discuss the recent progress as well as future perspectives in the methodologies and applications of single-cell omic sequencing technologies.

  5. NKT Cell Responses to B Cell Lymphoma.

    Science.gov (United States)

    Li, Junxin; Sun, Wenji; Subrahmanyam, Priyanka B; Page, Carly; Younger, Kenisha M; Tiper, Irina V; Frieman, Matthew; Kimball, Amy S; Webb, Tonya J

    2014-06-01

    Natural killer T (NKT) cells are a unique subset of CD1d-restricted T lymphocytes that express characteristics of both T cells and natural killer cells. NKT cells mediate tumor immune-surveillance; however, NKT cells are numerically reduced and functionally impaired in lymphoma patients. Many hematologic malignancies express CD1d molecules and co-stimulatory proteins needed to induce anti-tumor immunity by NKT cells, yet most tumors are poorly immunogenic. In this study, we sought to investigate NKT cell responses to B cell lymphoma. In the presence of exogenous antigen, both mouse and human NKT cell lines produce cytokines following stimulation by B cell lymphoma lines. NKT cell populations were examined ex vivo in mouse models of spontaneous B cell lymphoma, and it was found that during early stages, NKT cell responses were enhanced in lymphoma-bearing animals compared to disease-free animals. In contrast, in lymphoma-bearing animals with splenomegaly and lymphadenopathy, NKT cells were functionally impaired. In a mouse model of blastoid variant mantle cell lymphoma, treatment of tumor-bearing mice with a potent NKT cell agonist, α-galactosylceramide (α-GalCer), resulted in a significant decrease in disease pathology. Ex vivo studies demonstrated that NKT cells from α-GalCer treated mice produced IFN-γ following α-GalCer restimulation, unlike NKT cells from vehicle-control treated mice. These data demonstrate an important role for NKT cells in the immune response to an aggressive hematologic malignancy like mantle cell lymphoma.

  6. Endermologie New Aproach in the Medicine Treatment

    Science.gov (United States)

    Mezencevová, Viktória; Torok, Jozef; Czánová, Tatiana; Zajac, Ján

    2017-10-01

    Using the effect of mechanical forces affecting cellular response in the treatment of post-traumatic, postoperative, post-imlantation conditions through the application of Endermologie®- mechanotransduction represents a revolutionary solution in tissue-rehabilitation and positive target tissue influencing, with faster regeneration (1). Endermologie® is a noninvasive, painless, natural method of treatments of all connective tissue transformations, muscle and circulation pathologies. The aim of our study is investigation and explanation the mechanism of action by observing the physiological effects of Endermologie® based on human studies. The paper is focused on monitoring of possitive effect tissue regeneration using endermologie as a tools mechanostimulation improvements of systems integridy and health improvement.

  7. Syndecan-4 shedding impairs macrovascular angiogenesis in diabetes mellitus

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ran; Xie, Jun; Wu, Han; Li, Guannan; Chen, Jianzhou; Chen, Qinhua; Wang, Lian; Xu, Biao, E-mail: xubiao@medmail.com.cn

    2016-05-20

    Purpose: Syndecan-4 (synd4) is a ubiquitous heparan sulfate proteoglycan cell surface receptor that modulates cell proliferation, migration, mechanotransduction, and endocytosis. The extracellular domain of synd4 sheds heavily in acute inflammation, but the shedding of synd4 in chronic inflammation, such as diabetes mellitus (DM), is still undefined. We investigated the alterations of synd4 endothelial expression in DM and the influence of impaired synd4 signaling on angiogenesis in human umbilical vein endothelial cells (HUVECs), diabetic rats, synd4 null mice, and db/db mice. Material and methods: HUVECs were incubated with advanced glycation end products (AGEs). Western blot analysis was used to determine synd4 protein expression and ELISA was used to detect soluble synd4 fragments. The concentration of synd4 in the aortic endothelia of diabetic rats was detected by immunohistochemical staining. Aortic ring assays were performed to study the process of angiogenesis in the diabetic rats and in synd4 null and db/db mice. Recombinant adenoviruses containing the synd4 gene or null were constructed to enhance synd4 aortic expression in db/db mice. Results: Western blot analysis showed decreased expression of the synd4 extracellular domain in HUVECs, and ELISA detected increased soluble fragments of synd4 in the media. Synd4 endothelial expression in the aortas of diabetic rats was decreased. Aortic ring assay indicated impaired angiogenesis in synd4 null and db/db mice, which was partially reversed by synd4 overexpression in db/db mice. Conclusion: Synd4 shedding from vascular endothelial cells played an important role in the diabetes-related impairment of angiogenesis. -- Highlights: •Synd4 shedding from endothelial cells is accelerated under the stimulation of AGEs. •Extracellular domain of synd4 is diminished in the endothelium of DM rats. •Aortic rings of synd4 null mice showed impaired angiogenesis. •Overexpression of synd4 partly rescues macrovascular

  8. Automated Cell-Cutting for Cell Cloning

    Science.gov (United States)

    Ichikawa, Akihiko; Tanikawa, Tamio; Matsukawa, Kazutsugu; Takahashi, Seiya; Ohba, Kohtaro

    We develop an automated cell-cutting technique for cell cloning. Animal cells softened by the cytochalasin treatment are injected into a microfluidic chip. The microfluidic chip contains two orthogonal channels: one microchannel is wide, used to transport cells, and generates the cutting flow; the other is thin and used for aspiration, fixing, and stretching of the cell. The injected cell is aspirated and stretched in the thin microchannel. Simultaneously, the volumes of the cell before and after aspiration are calculated; the volumes are used to calculate the fluid flow required to aspirate half the volume of the cell into the thin microchannel. Finally, we apply a high-speed flow in the orthogonal microchannel to bisect the cell. This paper reports the cutting process, the cutting system, and the results of the experiment.

  9. Different cell fates from cell-cell interactions: core architectures of two-cell bistable networks.

    Science.gov (United States)

    Rouault, Hervé; Hakim, Vincent

    2012-02-08

    The acquisition of different fates by cells that are initially in the same state is central to development. Here, we investigate the possible structures of bistable genetic networks that can allow two identical cells to acquire different fates through cell-cell interactions. Cell-autonomous bistable networks have been previously sampled using an evolutionary algorithm. We extend this evolutionary procedure to take into account interactions between cells. We obtain a variety of simple bistable networks that we classify into major subtypes. Some have long been proposed in the context of lateral inhibition through the Notch-Delta pathway, some have been more recently considered and others appear to be new and based on mechanisms not previously considered. The results highlight the role of posttranscriptional interactions and particularly of protein complexation and sequestration, which can replace cooperativity in transcriptional interactions. Some bistable networks are entirely based on posttranscriptional interactions and the simplest of these is found to lead, upon a single parameter change, to oscillations in the two cells with opposite phases. We provide qualitative explanations as well as mathematical analyses of the dynamical behaviors of various created networks. The results should help to identify and understand genetic structures implicated in cell-cell interactions and differentiation. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Solo and keratin filaments regulate epithelial tubule morphology.

    Science.gov (United States)

    Nishimura, Ryosuke; Kato, Kagayaki; Fujiwara, Sachiko; Ohashi, Kazumasa; Mizuno, Kensaku

    2018-04-28

    Epithelial tubules, consisting of the epithelial cell sheet with a central lumen, are the basic structure of many organs. Mechanical forces play an important role in epithelial tubulogenesis; however, little is known about the mechanisms controlling the mechanical forces during epithelial tubule morphogenesis. Solo (also known as ARHGEF40) is a RhoA-targeting guanine-nucleotide exchange factor that is involved in mechanical force-induced RhoA activation and stress fiber formation. Solo binds to keratin-8/keratin-18 (K8/K18) filaments, and this interaction plays a crucial role in mechanotransduction. In this study, we examined the roles of Solo and K8/K18 filaments in epithelial tubulogenesis using MDCK cells cultured in 3D collagen gels. Knockdown of either Solo or K18 resulted in rounder tubules with increased lumen size, indicating that Solo and K8/K18 filaments play critical roles in forming the elongated morphology of epithelial tubules. Moreover, knockdown of Solo or K18 decreased the level of diphosphorylated myosin light chain (a marker of contractile force) at the luminal and outer surfaces of tubules, suggesting that Solo and K8/K18 filaments are involved in the generation of the myosin II-mediated contractile force during epithelial tubule morphogenesis. In addition, K18 filaments were normally oriented along the long axis of the tubule, but knockdown of Solo perturbed their orientation. These results suggest that Solo plays crucial roles in forming the elongated morphology of epithelial tubules and in regulating myosin II activity and K18 filament organization during epithelial tubule formation.

  11. Combined Effects of Ventilation Mode and Positive End-Expiratory Pressure on Mechanics, Gas Exchange and the Epithelium in Mice with Acute Lung Injury

    Science.gov (United States)

    Thammanomai, Apiradee; Hamakawa, Hiroshi; Bartolák-Suki, Erzsébet; Suki, Béla

    2013-01-01

    The accepted protocol to ventilate patients with acute lung injury is to use low tidal volume (VT) in combination with recruitment maneuvers or positive end-expiratory pressure (PEEP). However, an important aspect of mechanical ventilation has not been considered: the combined effects of PEEP and ventilation modes on the integrity of the epithelium. Additionally, it is implicitly assumed that the best PEEP-VT combination also protects the epithelium. We aimed to investigate the effects of ventilation mode and PEEP on respiratory mechanics, peak airway pressures and gas exchange as well as on lung surfactant and epithelial cell integrity in mice with acute lung injury. HCl-injured mice were ventilated at PEEPs of 3 and 6 cmH2O with conventional ventilation (CV), CV with intermittent large breaths (CVLB) to promote recruitment, and a new mode, variable ventilation, optimized for mice (VVN). Mechanics and gas exchange were measured during ventilation and surfactant protein (SP)-B, proSP-B and E-cadherin levels were determined from lavage and lung homogenate. PEEP had a significant effect on mechanics, gas exchange and the epithelium. The higher PEEP reduced lung collapse and improved mechanics and gas exchange but it also down regulated surfactant release and production and increased epithelial cell injury. While CVLB was better than CV, VVN outperformed CVLB in recruitment, reduced epithelial injury and, via a dynamic mechanotransduction, it also triggered increased release and production of surfactant. For long-term outcome, selection of optimal PEEP and ventilation mode may be based on balancing lung physiology with epithelial injury. PMID:23326543

  12. Modeling the tumor extracellular matrix: Tissue engineering tools repurposed towards new frontiers in cancer biology.

    Science.gov (United States)

    Gill, Bartley J; West, Jennifer L

    2014-06-27

    Cancer progression is mediated by complex epigenetic, protein and structural influences. Critical among them are the biochemical, mechanical and architectural properties of the extracellular matrix (ECM). In recognition of the ECM's important role, cancer biologists have repurposed matrix mimetic culture systems first widely used by tissue engineers as new tools for in vitro study of tumor models. In this review we discuss the pathological changes in tumor ECM, the limitations of 2D culture on both traditional and polyacrylamide hydrogel surfaces in modeling these characteristics and advances in both naturally derived and synthetic scaffolds to facilitate more complex and controllable 3D cancer cell culture. Studies using naturally derived matrix materials like Matrigel and collagen have produced significant findings related to tumor morphogenesis and matrix invasion in a 3D environment and the mechanotransductive signaling that mediates key tumor-matrix interaction. However, lack of precise experimental control over important matrix factors in these matrices have increasingly led investigators to synthetic and semi-synthetic scaffolds that offer the engineering of specific ECM cues and the potential for more advanced experimental manipulations. Synthetic scaffolds composed of poly(ethylene glycol) (PEG), for example, facilitate highly biocompatible 3D culture, modular bioactive features like cell-mediated matrix degradation and complete independent control over matrix bioactivity and mechanics. Future work in PEG or similar reductionist synthetic matrix systems should enable the study of increasingly complex and dynamic tumor-ECM relationships in the hopes that accurate modeling of these relationships may reveal new cancer therapeutics targeting tumor progression and metastasis. © 2013 Published by Elsevier Ltd.

  13. Identification of a conserved set of upregulated genes in mouse skeletal muscle hypertrophy and regrowth.

    Science.gov (United States)

    Chaillou, Thomas; Jackson, Janna R; England, Jonathan H; Kirby, Tyler J; Richards-White, Jena; Esser, Karyn A; Dupont-Versteegden, Esther E; McCarthy, John J

    2015-01-01

    The purpose of this study was to compare the gene expression profile of mouse skeletal muscle undergoing two forms of growth (hypertrophy and regrowth) with the goal of identifying a conserved set of differentially expressed genes. Expression profiling by microarray was performed on the plantaris muscle subjected to 1, 3, 5, 7, 10, and 14 days of hypertrophy or regrowth following 2 wk of hind-limb suspension. We identified 97 differentially expressed genes (≥2-fold increase or ≥50% decrease compared with control muscle) that were conserved during the two forms of muscle growth. The vast majority (∼90%) of the differentially expressed genes was upregulated and occurred at a single time point (64 out of 86 genes), which most often was on the first day of the time course. Microarray analysis from the conserved upregulated genes showed a set of genes related to contractile apparatus and stress response at day 1, including three genes involved in mechanotransduction and four genes encoding heat shock proteins. Our analysis further identified three cell cycle-related genes at day and several genes associated with extracellular matrix (ECM) at both days 3 and 10. In conclusion, we have identified a core set of genes commonly upregulated in two forms of muscle growth that could play a role in the maintenance of sarcomere stability, ECM remodeling, cell proliferation, fast-to-slow fiber type transition, and the regulation of skeletal muscle growth. These findings suggest conserved regulatory mechanisms involved in the adaptation of skeletal muscle to increased mechanical loading. Copyright © 2015 the American Physiological Society.

  14. Rescuing loading induced bone formation at senescence.

    Directory of Open Access Journals (Sweden)

    Sundar Srinivasan

    2010-09-01

    Full Text Available The increasing incidence of osteoporosis worldwide requires anabolic treatments that are safe, effective, and, critically, inexpensive given the prevailing overburdened health care systems. While vigorous skeletal loading is anabolic and holds promise, deficits in mechanotransduction accrued with age markedly diminish the efficacy of readily complied, exercise-based strategies to combat osteoporosis in the elderly. Our approach to explore and counteract these age-related deficits was guided by cellular signaling patterns across hierarchical scales and by the insight that cell responses initiated during transient, rare events hold potential to exert high-fidelity control over temporally and spatially distant tissue adaptation. Here, we present an agent-based model of real-time Ca(2+/NFAT signaling amongst bone cells that fully described periosteal bone formation induced by a wide variety of loading stimuli in young and aged animals. The model predicted age-related pathway alterations underlying the diminished bone formation at senescence, and hence identified critical deficits that were promising targets for therapy. Based upon model predictions, we implemented an in vivo intervention and show for the first time that supplementing mechanical stimuli with low-dose Cyclosporin A can completely rescue loading induced bone formation in the senescent skeleton. These pre-clinical data provide the rationale to consider this approved pharmaceutical alongside mild physical exercise as an inexpensive, yet potent therapy to augment bone mass in the elderly. Our analyses suggested that real-time cellular signaling strongly influences downstream bone adaptation to mechanical stimuli, and quantification of these otherwise inaccessible, transient events in silico yielded a novel intervention with clinical potential.

  15. Low Doses of Curcuma longa Modulates Cell Migration and Cell-Cell Adhesion.

    Science.gov (United States)

    de Campos, Paloma Santos; Matte, Bibiana Franzen; Diel, Leonardo Francisco; Jesus, Luciano Henrique; Bernardi, Lisiane; Alves, Alessandro Menna; Rados, Pantelis Varvaki; Lamers, Marcelo Lazzaron

    2017-09-01

    Cell invasion and metastasis are involved in clinical failures in cancer treatment, and both events require the acquisition of a migratory behavior by tumor cells. Curcumin is a promising natural product with anti-proliferative activity, but its effects on cell migration are still unclear. We evaluated the effects of curcumin on the proliferation, apoptosis, migration, and cell-cell adhesion of keratinocyte, oral squamous cell carcinoma (OSCC), and fibroblast cell lines, as well as in a xenograft model of OSCC. Curcumin (2 μM) decreased cell proliferation in cell lines with mesenchymal characteristics, while cell death was detected only at 50 μM. We observed that highly migratory cells showed a decrease on migration speed and directionality when treated with 2 or 5 μM of curcumin (50% and 40%, respectively, p curcumin dose dependently decreased cell-cell adhesion, especially on tumor-derived spheroids. Also, in a xenograft model with patient-derived OSCC cells, the administration of curcumin decreased tumor growth and aggressiveness when compared with untreated tumors, indicating the potential antitumor effect in oral cancer. These results suggest that lower doses of curcumin can influence several steps involved in tumorigenesis, including migration properties, suggesting a possible use in cancer therapy. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  16. Nonimmune cells equipped with T-cell-receptor-like signaling for cancer cell ablation.

    Science.gov (United States)

    Kojima, Ryosuke; Scheller, Leo; Fussenegger, Martin

    2018-01-01

    The ability to engineer custom cell-contact-sensing output devices into human nonimmune cells would be useful for extending the applicability of cell-based cancer therapies and for avoiding risks associated with engineered immune cells. Here we have developed a new class of synthetic T-cell receptor-like signal-transduction device that functions efficiently in human nonimmune cells and triggers release of output molecules specifically upon sensing contact with a target cell. This device employs an interleukin signaling cascade, whose OFF/ON switching is controlled by biophysical segregation of a transmembrane signal-inhibitory protein from the sensor cell-target cell interface. We further show that designer nonimmune cells equipped with this device driving expression of a membrane-penetrator/prodrug-activating enzyme construct could specifically kill target cells in the presence of the prodrug, indicating its potential usefulness for target-cell-specific, cell-based enzyme-prodrug cancer therapy. Our study also contributes to the advancement of synthetic biology by extending available design principles to transmit extracellular information to cells.

  17. Proliferating cells in psoriatic dermis are comprised primarily of T cells, endothelial cells, and factor XIIIa+ perivascular dendritic cells

    International Nuclear Information System (INIS)

    Morganroth, G.S.; Chan, L.S.; Weinstein, G.D.; Voorhees, J.J.; Cooper, K.D.

    1991-01-01

    Determination of the cell types proliferating in the dermis of patients with psoriasis should identify those cells experiencing activation or responding to growth factors in the psoriatic dermal milieu. Toward that end, sections of formalin-fixed biopsies obtained from 3H-deoxyuridine (3H-dU)-injected skin of eight psoriatic patients were immunostained, followed by autoradiography. Proliferating dermal cells exhibit silver grains from tritium emissions. The identity of the proliferating cells could then be determined by simultaneous visualization with antibodies specific for various cell types. UCHL1+ (CD45RO+) T cells (recall antigen-reactive helper T-cell subset) constituted 36.6 +/- 3.1% (mean +/- SEM, n = 6) of the proliferating dermal cells in involved skin, whereas Leu 18+ (CD45RA+) T cells (recall antigen naive T-cell subsets) comprised only 8.7 +/- 1.5% (n = 6). The Factor XIIIa+ dermal perivascular dendritic cell subset (24.9 +/- 1.5% of proliferating dermal cells, n = 6) and Factor VIII+ endothelial cells represented the two other major proliferating populations in lesional psoriatic dermis. Differentiated tissue macrophages, identified by phase microscopy as melanophages or by immunostaining with antibodies to Leu M1 (CD15) or myeloid histiocyte antigen, comprised less than 5% of the proliferating population in either skin type. In addition to calculating the relative proportions of these cells to each other as percent, we also determined the density of cells, in cells/mm2 of tissue. The density of proliferating cells within these populations was increased in involved versus uninvolved skin: UCHL1+, 9.0 +/- 1.7 cells/mm2 versus 1.8 +/- 0.6 cells/mm2, p less than 0.01; Factor XIIIa+, 6.0 +/- 0.7 cells/mm2 versus 1.5 +/- 0.5 cells/mm2, p less than 0.01; Factor VIII+, 5.5 +/- 1.4 cells/mm2 versus 0.0 cells/mm2, p less than 0.05

  18. Pathological significance and prognostic roles of densities of CD57+ cells, CD68+ cells, and mast cells, and their ratios in clear cell renal cell carcinoma.

    Science.gov (United States)

    Nakanishi, Hiromi; Miyata, Yasuyoshi; Mochizuki, Yasushi; Yasuda, Takuji; Nakamura, Yuichiro; Araki, Kyohei; Sagara, Yuji; Matsuo, Tomohiro; Ohba, Kojiro; Sakai, Hideki

    2018-05-19

    The immune system is closely associated with malignant behavior in renal cell carcinoma (RCC). Therefore, understanding the pathological roles of immune cells in tumor stroma is essential to discuss the pathological characteristics of RCC. In this study, the clinical significance of densities of CD57+ cells, CD68+ cells, and mast cells, and their ratios were investigated in patients with clear cell RCC. The densities of CD57+, CD68+, and mast cells were evaluated by immunohistochemical techniques in 179 patients. Proliferation index (PI), apoptotic index (AI), and microvessel density (MVD) were evaluated by using anti-Ki-67, anti-cleaved caspase-3, and anti-CD31 antibodies, respectively. The density of CD57+ cell was negatively correlated with grade, pT stage, and metastasis, although densities of CD68+ cell and mast cell were positively correlated. Ratios of CD68+ cell/CD57+ cell and mast cell/CD57+ cell were significantly correlated with grade, pT stage, and metastasis. Survival analyses showed that the CD68+ cell/CD57+ cell ratio was a significant predictor for cause-specific survival by multi-variate analyses (hazard ratio=1.41, 95% confidential interval=1.03-1.93, P=.031), and was significantly correlated with PI, AI, and MVD (r=.47; P <. 001, r=-.31, P<.001, and r=.40, P<.001, respectively). In conclusion, CD57+ cell, CD68+ cell, and mast cell played important roles in malignancy in clear cell RCC. The CD68+ cell/CD57+ cell ratio was strongly correlated with pathological features and prognosis in these patients because this ratio reflected the status of cancer cell proliferation, apoptosis, and angiogenesis. Copyright © 2018. Published by Elsevier Inc.

  19. Cell-extracellular matrix and cell-cell adhesion are linked by syndecan-4

    DEFF Research Database (Denmark)

    Pakideeri Karat, Sandeep Gopal; Multhaupt, Hinke A B; Pocock, Roger

    2017-01-01

    Cell-extracellular matrix (ECM) and cell-cell junctions that employ microfilaments are sites of tension. They are important for tissue repair, morphogenetic movements and can be emblematic of matrix contraction in fibrotic disease and the stroma of solid tumors. One cell surface receptor, syndecan...... calcium. While it is known that cell-ECM and cell-cell junctions may be linked, possible roles for syndecans in this process are not understood. Here we show that wild type primary fibroblasts and those lacking syndecan-4 utilize different cadherins in their adherens junctions and that tension is a major...... factor in this differential response. This corresponds to the reduced ability of fibroblasts lacking syndecan-4 to exert tension on the ECM and we now show that this may extend to reduced tension in cell-cell adhesion....

  20. MARCKS-related protein regulates cytoskeletal organization at cell-cell and cell-substrate contacts in epithelial cells.

    Science.gov (United States)

    Van Itallie, Christina M; Tietgens, Amber Jean; Aponte, Angel; Gucek, Marjan; Cartagena-Rivera, Alexander X; Chadwick, Richard S; Anderson, James M

    2018-02-02

    Treatment of epithelial cells with interferon-γ and TNF-α (IFN/TNF) results in increased paracellular permeability. To identify relevant proteins mediating barrier disruption, we performed proximity-dependent biotinylation (BioID) of occludin and found that tagging of MARCKS-related protein (MRP; also known as MARCKSL1) increased ∼20-fold following IFN/TNF administration. GFP-MRP was focused at the lateral cell membrane and its overexpression potentiated the physiological response of the tight junction barrier to cytokines. However, deletion of MRP did not abrogate the cytokine responses, suggesting that MRP is not required in the occludin-dependent IFN/TNF response. Instead, our results reveal a key role for MRP in epithelial cells in control of multiple actin-based structures, likely by regulation of integrin signaling. Changes in focal adhesion organization and basal actin stress fibers in MRP-knockout (KO) cells were reminiscent of those seen in FAK-KO cells. In addition, we found alterations in cell-cell interactions in MRP-KO cells associated with increased junctional tension, suggesting that MRP may play a role in focal adhesion-adherens junction cross talk. Together, our results are consistent with a key role for MRP in cytoskeletal organization of cell contacts in epithelial cells. © 2018. Published by The Company of Biologists Ltd.

  1. Stem cells engineering for cell-based therapy.

    Science.gov (United States)

    Taupin, Philippe

    2007-09-01

    Stem cells carry the promise to cure a broad range of diseases and injuries, from diabetes, heart and muscular diseases, to neurological diseases, disorders and injuries. Significant progresses have been made in stem cell research over the past decade; the derivation of embryonic stem cells (ESCs) from human tissues, the development of cloning technology by somatic cell nuclear transfer (SCNT) and the confirmation that neurogenesis occurs in the adult mammalian brain and that neural stem cells (NSCs) reside in the adult central nervous system (CNS), including that of humans. Despite these advances, there may be decades before stem cell research will translate into therapy. Stem cell research is also subject to ethical and political debates, controversies and legislation, which slow its progress. Cell engineering has proven successful in bringing genetic research to therapy. In this review, I will review, in two examples, how investigators are applying cell engineering to stem cell biology to circumvent stem cells' ethical and political constraints and bolster stem cell research and therapy.

  2. Dissecting engineered cell types and enhancing cell fate conversion via CellNet

    Science.gov (United States)

    Morris, Samantha A.; Cahan, Patrick; Li, Hu; Zhao, Anna M.; San Roman, Adrianna K.; Shivdasani, Ramesh A.; Collins, James J.; Daley, George Q.

    2014-01-01

    SUMMARY Engineering clinically relevant cells in vitro holds promise for regenerative medicine, but most protocols fail to faithfully recapitulate target cell properties. To address this, we developed CellNet, a network biology platform that determines whether engineered cells are equivalent to their target tissues, diagnoses aberrant gene regulatory networks, and prioritizes candidate transcriptional regulators to enhance engineered conversions. Using CellNet, we improved B cell to macrophage conversion, transcriptionally and functionally, by knocking down predicted B cell regulators. Analyzing conversion of fibroblasts to induced hepatocytes (iHeps), CellNet revealed an unexpected intestinal program regulated by the master regulator Cdx2. We observed long-term functional engraftment of mouse colon by iHeps, thereby establishing their broader potential as endoderm progenitors and demonstrating direct conversion of fibroblasts into intestinal epithelium. Our studies illustrate how CellNet can be employed to improve direct conversion and to uncover unappreciated properties of engineered cells. PMID:25126792

  3. cgCorrect: a method to correct for confounding cell-cell variation due to cell growth in single-cell transcriptomics

    Science.gov (United States)

    Blasi, Thomas; Buettner, Florian; Strasser, Michael K.; Marr, Carsten; Theis, Fabian J.

    2017-06-01

    Accessing gene expression at a single-cell level has unraveled often large heterogeneity among seemingly homogeneous cells, which remains obscured when using traditional population-based approaches. The computational analysis of single-cell transcriptomics data, however, still imposes unresolved challenges with respect to normalization, visualization and modeling the data. One such issue is differences in cell size, which introduce additional variability into the data and for which appropriate normalization techniques are needed. Otherwise, these differences in cell size may obscure genuine heterogeneities among cell populations and lead to overdispersed steady-state distributions of mRNA transcript numbers. We present cgCorrect, a statistical framework to correct for differences in cell size that are due to cell growth in single-cell transcriptomics data. We derive the probability for the cell-growth-corrected mRNA transcript number given the measured, cell size-dependent mRNA transcript number, based on the assumption that the average number of transcripts in a cell increases proportionally to the cell’s volume during the cell cycle. cgCorrect can be used for both data normalization and to analyze the steady-state distributions used to infer the gene expression mechanism. We demonstrate its applicability on both simulated data and single-cell quantitative real-time polymerase chain reaction (PCR) data from mouse blood stem and progenitor cells (and to quantitative single-cell RNA-sequencing data obtained from mouse embryonic stem cells). We show that correcting for differences in cell size affects the interpretation of the data obtained by typically performed computational analysis.

  4. Single cell time-lapse analysis reveals that podoplanin enhances cell survival and colony formation capacity of squamous cell carcinoma cells.

    Science.gov (United States)

    Miyashita, Tomoyuki; Higuchi, Youichi; Kojima, Motohiro; Ochiai, Atsushi; Ishii, Genichiro

    2017-01-06

    Tumor initiating cells (TICs) are characterized by high clonal expansion capacity. We previously reported that podoplanin is a TIC-specific marker for the human squamous cell carcinoma cell line A431. The aim of this study is to explore the molecular mechanism underlying the high clonal expansion potential of podoplanin-positive A431cells using Fucci imaging. Single podoplanin-positive cells created large colonies at a significantly higher frequency than single podoplanin-negative cells, whereas no difference was observed between the two types of cells with respect to cell cycle status. Conversely, the cell death ratio of progenies derived from podoplanin-positive single cell was significantly lower than that of cells derived from podoplanin-negative cells. Single A431 cells, whose podoplanin expression was suppressed by RNA interference, exhibited increased cell death ratios and decreased frequency of large colony forming. Moreover, the frequency of large colony forming decreased significantly when podoplanin-positive single cells was treated with a ROCK (Rho-associated coiled-coil kinase) inhibitor, whereas no difference was observed in single podoplanin-negative cells. Our current study cleared that high clonal expansion capacity of podoplanin-positive TICs populations was the result of reduced cell death by podoplanin-mediated signaling. Therefore, podoplanin activity may be a therapeutic target in the treatment of squamous cell carcinomas.

  5. Modeling cell-in-cell structure into its biological significance

    OpenAIRE

    He, M-f; Wang, S; Wang, Y; Wang, X-n

    2013-01-01

    Although cell-in-cell structure was noted 100 years ago, the molecular mechanisms of ?entering' and the destination of cell-in-cell remain largely unclear. It takes place among the same type of cells (homotypic cell-in-cell) or different types of cells (heterotypic cell-in-cell). Cell-in-cell formation affects both effector cells and their host cells in multiple aspects, while cell-in-cell death is under more intensive investigation. Given that cell-in-cell has an important role in maintainin...

  6. Cell volume change through water efflux impacts cell stiffness and stem cell fate

    NARCIS (Netherlands)

    Guo, Ming; Pegoraro, Adrian F.; Mao, Angelo; Zhou, Enhua H.; Arany, Praveen R.; Han, Yulong; Burnette, Dylan T.; Jensen, Mikkel H.; Kasza, Karen E.; Moore, Jeffrey R.; Mackintosh, Frederick C.; Fredberg, Jeffrey J.; Mooney, David J.; Lippincott-Schwartz, Jennifer; Weitz, David A.

    2017-01-01

    Cells alter their mechanical properties in response to their local microenvironment; this plays a role in determining cell function and can even influence stem cell fate. Here, we identify a robust and unified relationship between cell stiffness and cell volume. As a cell spreads on a substrate, its

  7. Retinal stem cells and potential cell transplantation treatments

    Directory of Open Access Journals (Sweden)

    Tai-Chi Lin

    2014-11-01

    Full Text Available The retina, histologically composed of ten delicate layers, is responsible for light perception and relaying electrochemical signals to the secondary neurons and visual cortex. Retinal disease is one of the leading clinical causes of severe vision loss, including age-related macular degeneration, Stargardt's disease, and retinitis pigmentosa. As a result of the discovery of various somatic stem cells, advances in exploring the identities of embryonic stem cells, and the development of induced pluripotent stem cells, cell transplantation treatment for retinal diseases is currently attracting much attention. The sources of stem cells for retinal regeneration include endogenous retinal stem cells (e.g., neuronal stem cells, Müller cells, and retinal stem cells from the ciliary marginal zone and exogenous stem cells (e.g., bone mesenchymal stem cells, adipose-derived stem cells, embryonic stem cells, and induced pluripotent stem cells. The success of cell transplantation treatment depends mainly on the cell source, the timing of cell harvesting, the protocol of cell induction/transplantation, and the microenvironment of the recipient's retina. This review summarizes the different sources of stem cells for regeneration treatment in retinal diseases and surveys the more recent achievements in animal studies and clinical trials. Future directions and challenges in stem cell transplantation are also discussed.

  8. Hybrid cell adhesive material for instant dielectrophoretic cell trapping and long-term cell function assessment.

    Science.gov (United States)

    Reyes, Darwin R; Hong, Jennifer S; Elliott, John T; Gaitan, Michael

    2011-08-16

    Dielectrophoresis (DEP) for cell manipulation has focused, for the most part, on approaches for separation/enrichment of cells of interest. Advancements in cell positioning and immobilization onto substrates for cell culture, either as single cells or as cell aggregates, has benefited from the intensified research efforts in DEP (electrokinetic) manipulation. However, there has yet to be a DEP approach that provides the conditions for cell manipulation while promoting cell function processes such as cell differentiation. Here we present the first demonstration of a system that combines DEP with a hybrid cell adhesive material (hCAM) to allow for cell entrapment and cell function, as demonstrated by cell differentiation into neuronlike cells (NLCs). The hCAM, comprised of polyelectrolytes and fibronectin, was engineered to function as an instantaneous cell adhesive surface after DEP manipulation and to support long-term cell function (cell proliferation, induction, and differentiation). Pluripotent P19 mouse embryonal carcinoma cells flowing within a microchannel were attracted to the DEP electrode surface and remained adhered onto the hCAM coating under a fluid flow field after the DEP forces were removed. Cells remained viable after DEP manipulation for up to 8 d, during which time the P19 cells were induced to differentiate into NLCs. This approach could have further applications in areas such as cell-cell communication, three-dimensional cell aggregates to create cell microenvironments, and cell cocultures.

  9. Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

    Science.gov (United States)

    Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

    2013-03-01

    Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

  10. Cell patterning without chemical surface modification: Cell cell interactions between printed bovine aortic endothelial cells (BAEC) on a homogeneous cell-adherent hydrogel

    Science.gov (United States)

    Chen, C. Y.; Barron, J. A.; Ringeisen, B. R.

    2006-10-01

    Cell printing offers the unique ability to directly deposit one or multiple cell types directly onto a surface without the need to chemically pre-treat the surface with lithographic methods. We utilize biological laser printing (BioLP ™) to form patterns of bovine aortic endothelial cells (BAECs) onto a homogeneous cell adherent hydrogel surface. These normal cells are shown to retain near-100% viability post-printing. In order to determine whether BAECs encountered shear and/or heat stress during printing, immunocytochemical staining experiments were performed to detect potential expression of heat shock proteins (HSP) by the deposited cells. Printed BAECs expressed HSP at levels similar to negative control cells, indicating that the BioLP process does not expose cells to damaging levels of stress. However, HSP expression was slightly higher at the highest laser energy studied, suggesting more stress was present under these extreme conditions. Printed BAECs also showed preferential asymmetric growth and migration towards each other and away from the originally printed pattern, demonstrating a retained ability for the cells to communicate post-printing.

  11. Llgl1 Connects Cell Polarity with Cell-Cell Adhesion in Embryonic Neural Stem Cells.

    Science.gov (United States)

    Jossin, Yves; Lee, Minhui; Klezovitch, Olga; Kon, Elif; Cossard, Alexia; Lien, Wen-Hui; Fernandez, Tania E; Cooper, Jonathan A; Vasioukhin, Valera

    2017-06-05

    Malformations of the cerebral cortex (MCCs) are devastating developmental disorders. We report here that mice with embryonic neural stem-cell-specific deletion of Llgl1 (Nestin-Cre/Llgl1 fl/fl ), a mammalian ortholog of the Drosophila cell polarity gene lgl, exhibit MCCs resembling severe periventricular heterotopia (PH). Immunohistochemical analyses and live cortical imaging of PH formation revealed that disruption of apical junctional complexes (AJCs) was responsible for PH in Nestin-Cre/Llgl1 fl/fl brains. While it is well known that cell polarity proteins govern the formation of AJCs, the exact mechanisms remain unclear. We show that LLGL1 directly binds to and promotes internalization of N-cadherin, and N-cadherin/LLGL1 interaction is inhibited by atypical protein kinase C-mediated phosphorylation of LLGL1, restricting the accumulation of AJCs to the basolateral-apical boundary. Disruption of the N-cadherin-LLGL1 interaction during cortical development in vivo is sufficient for PH. These findings reveal a mechanism responsible for the physical and functional connection between cell polarity and cell-cell adhesion machineries in mammalian cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Snail modulates cell metabolism in MDCK cells

    Energy Technology Data Exchange (ETDEWEB)

    Haraguchi, Misako, E-mail: haraguci@m3.kufm.kagoshima-u.ac.jp [Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Indo, Hiroko P. [Department of Maxillofacial Radiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Iwasaki, Yasumasa [Health Care Center, Kochi University, Kochi 780-8520 (Japan); Iwashita, Yoichiro [Department of Maxillofacial Radiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Fukushige, Tomoko [Department of Dermatology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Majima, Hideyuki J. [Department of Maxillofacial Radiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Izumo, Kimiko; Horiuchi, Masahisa [Department of Environmental Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Kanekura, Takuro [Department of Dermatology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Furukawa, Tatsuhiko [Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Ozawa, Masayuki [Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan)

    2013-03-22

    Highlights: ► MDCK/snail cells were more sensitive to glucose deprivation than MDCK/neo cells. ► MDCK/snail cells had decreased oxidative phosphorylation, O{sub 2} consumption and ATP content. ► TCA cycle enzyme activity, but not expression, was lower in MDCK/snail cells. ► MDCK/snail cells showed reduced PDH activity and increased PDK1 expression. ► MDCK/snail cells showed reduced expression of GLS2 and ACLY. -- Abstract: Snail, a repressor of E-cadherin gene transcription, induces epithelial-to-mesenchymal transition and is involved in tumor progression. Snail also mediates resistance to cell death induced by serum depletion. By contrast, we observed that snail-expressing MDCK (MDCK/snail) cells undergo cell death at a higher rate than control (MDCK/neo) cells in low-glucose medium. Therefore, we investigated whether snail expression influences cell metabolism in MDCK cells. Although gylcolysis was not affected in MDCK/snail cells, they did exhibit reduced pyruvate dehydrogenase (PDH) activity, which controls pyruvate entry into the tricarboxylic acid (TCA) cycle. Indeed, the activity of multiple enzymes involved in the TCA cycle was decreased in MDCK/snail cells, including that of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDH2), succinate dehydrogenase (SDH), and electron transport Complex II and Complex IV. Consequently, lower ATP content, lower oxygen consumption and increased survival under hypoxic conditions was also observed in MDCK/snail cells compared to MDCK/neo cells. In addition, the expression and promoter activity of pyruvate dehydrogenase kinase 1 (PDK1), which phosphorylates and inhibits the activity of PDH, was increased in MDCK/snail cells, while expression levels of glutaminase 2 (GLS2) and ATP-citrate lyase (ACLY), which are involved in glutaminolysis and fatty acid synthesis, were decreased in MDCK/snail cells. These results suggest that snail modulates cell metabolism by altering the expression and activity of

  13. nduced pluripotent stem cells and cell therapy

    Directory of Open Access Journals (Sweden)

    Banu İskender

    2013-12-01

    Full Text Available Human embryonic stem cells are derived from the inner cell mass of a blastocyst-stage embryo. They hold a huge promise for cell therapy with their self-renewing ability and pluripotency, which is known as the potential to differentiate into all cell types originating from three embryonic germ layers. However, their unique pluripotent feature could not be utilised for therapeutic purposes due to the ethical and legal problems during derivation. Recently, it was shown that the cells from adult tissues could be reverted into embryonic state, thereby restoring their pluripotent feature. This has strenghtened the possiblity of directed differentition of the reprogrammed somatic cells into the desired cell types in vitro and their use in regenerative medicine. Although these cells were termed as induced pluripotent cells, the mechanism of pluripotency has yet to be understood. Still, induced pluripotent stem cell technology is considered to be significant by proposing novel approaches in disease modelling, drug screening and cell therapy. Besides their self-renewing ability and their potential to differentiate into all cell types in a human body, they arouse a great interest in scientific world by being far from the ethical concerns regarding their embryonic counterparts and their unique feature of being patient-specific in prospective cell therapies. In this review, induced pluripotent stem cell technology and its role in cell-based therapies from past to present will be discussed. J Clin Exp Invest 2013; 4 (4: 550-561

  14. Cell-mediated mutagenesis and cell transformation of mammalian cells by chemical carcinogens

    International Nuclear Information System (INIS)

    Huberman, E.; Langenbach, R.

    1977-01-01

    We have developed a cell-mediated mutagenesis assay in which cells with the appropriate markers for mutagenesis are co-cultivated with either lethally irradiated rodent embryonic cells that can metabolize carcinogenic hydrocarbons or with primary rat liver cells that can metabolize chemicals carcinogenic to the liver. During co-cultivation, the reactive metabolites of the procarcinogen appear to be transmitted to the mutable cells and induce mutations in them. Assays of this type make it possible to demonstrate a relationship between carcinogenic potency of the chemicals and their ability to induce mutations in mammalian cells. In addition, by simultaneously comparing the frequencies of transformation and mutation induced in normal diploid hamster cells by benzo(a)pyrene (BP) and one of its metabolites, it is possible to estimate the genetic target size for cell transformation in vitro

  15. Gastric stem cells and gastric cancer stem cells

    OpenAIRE

    Han, Myoung-Eun; Oh, Sae-Ock

    2013-01-01

    The gastric epithelium is continuously regenerated by gastric stem cells, which give rise to various kinds of daughter cells, including parietal cells, chief cells, surface mucous cells, mucous neck cells, and enteroendocrine cells. The self-renewal and differentiation of gastric stem cells need delicate regulation to maintain the normal physiology of the stomach. Recently, it was hypothesized that cancer stem cells drive the cancer growth and metastasis. In contrast to conventional clonal ev...

  16. Hybrid clone cells derived from human breast epithelial cells and human breast cancer cells exhibit properties of cancer stem/initiating cells.

    Science.gov (United States)

    Gauck, Daria; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S; Dittmar, Thomas

    2017-08-02

    The biological phenomenon of cell fusion has been associated with cancer progression since it was determined that normal cell × tumor cell fusion-derived hybrid cells could exhibit novel properties, such as enhanced metastatogenic capacity or increased drug resistance, and even as a mechanism that could give rise to cancer stem/initiating cells (CS/ICs). CS/ICs have been proposed as cancer cells that exhibit stem cell properties, including the ability to (re)initiate tumor growth. Five M13HS hybrid clone cells, which originated from spontaneous cell fusion events between M13SV1-EGFP-Neo human breast epithelial cells and HS578T-Hyg human breast cancer cells, and their parental cells were analyzed for expression of stemness and EMT-related marker proteins by Western blot analysis and confocal laser scanning microscopy. The frequency of ALDH1-positive cells was determined by flow cytometry using AldeRed fluorescent dye. Concurrently, the cells' colony forming capabilities as well as the cells' abilities to form mammospheres were investigated. The migratory activity of the cells was analyzed using a 3D collagen matrix migration assay. M13HS hybrid clone cells co-expressed SOX9, SLUG, CK8 and CK14, which were differently expressed in parental cells. A variation in the ALDH1-positive putative stem cell population was observed among the five hybrids ranging from 1.44% (M13HS-7) to 13.68% (M13HS-2). In comparison to the parental cells, all five hybrid clone cells possessed increased but also unique colony formation and mammosphere formation capabilities. M13HS-4 hybrid clone cells exhibited the highest colony formation capacity and second highest mammosphere formation capacity of all hybrids, whereby the mean diameter of the mammospheres was comparable to the parental cells. In contrast, the largest mammospheres originated from the M13HS-2 hybrid clone cells, whereas these cells' mammosphere formation capacity was comparable to the parental breast cancer cells. All M13HS

  17. PINCH1 regulates cell-matrix and cell-cell adhesions, cell polarity and cell survival during the peri-implantation stage

    DEFF Research Database (Denmark)

    Li, Shaohua; Bordoy, Randi; Stanchi, Fabio

    2005-01-01

    PINCH1 is composed of 5 LIM domains, binds integrin-linked kinase (ILK) and locates to integrin-mediated adhesion sites. In order to investigate PINCH1 function we generated mice and embryonic stem (ES) cell-derived embryoid bodies (EBs) lacking the PINCH1 gene. Similar to mice lacking beta1...... integrin or Ilk, loss of PINCH1 arrested development at the peri-implantation stage. In contrast to beta1 integrin or Ilk mutants, however, disruption of the PINCH1 gene produced implantation chambers with visible cell clumps even at embryonic day 9.5. In order to define the phenotype leading to the peri...... not observed in beta1 integrin- or ILK-deficient mice or EBs, included abnormal cell-cell adhesion of endoderm and epiblast as well as the presence of apoptotic cells in the endodermal cell layer. Although ILK and PINCH1 were shown to be involved in the phosphorylation of serine-473 of PKB/Akt, immunostaining...

  18. In vitro differentiation of primordial germ cells and oocyte-like cells from stem cells.

    Science.gov (United States)

    Costa, José J N; Souza, Glaucinete B; Soares, Maria A A; Ribeiro, Regislane P; van den Hurk, Robert; Silva, José R V

    2018-02-01

    Infertility is the result of failure due to an organic disorder of the reproductive organs, especially their gametes. Recently, much progress has been made on generating germ cells, including oocytes, from various types of stem cells. This review focuses on advances in female germ cell differentiation from different kinds of stem cells, with emphasis on embryonic stem cells, adult stem cells, and induced pluripotent stem cells. The advantages and disadvantages of the derivation of female germ cells from several types of stem cells are also highlighted, as well as the ability of stem cells to generate mature and functional female gametes. This review shows that stem cell therapies have opened new frontiers in medicine, especially in the reproductive area, with the possibility of regenerating fertility.

  19. Cell cycle control by components of cell anchorage

    OpenAIRE

    Gad, Annica

    2005-01-01

    Extracellular factors, such as growth factors and cell anchorage to the extracellular matrix, control when and where cells may proliferate. This control is abolished when a normal cell transforms into a tumour cell. The control of cell proliferation by cell anchorage was elusive and less well studied than the control by growth factors. Therefore, we aimed to clarify at what points in the cell cycle and through which molecular mechanisms cell anchorage controls cell cycle pro...

  20. Casticin impairs cell growth and induces cell apoptosis via cell cycle arrest in human oral cancer SCC-4 cells.

    Science.gov (United States)

    Chou, Guan-Ling; Peng, Shu-Fen; Liao, Ching-Lung; Ho, Heng-Chien; Lu, Kung-Wen; Lien, Jin-Cherng; Fan, Ming-Jen; La, Kuang-Chi; Chung, Jing-Gung

    2018-02-01

    Casticin, a polymethoxyflavone, present in natural plants, has been shown to have biological activities including anti-cancer activities. Herein, we investigated the anti-oral cancer activity of casticin on SCC-4 cells in vitro. Viable cells, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS) production, and Ca 2+ production, levels of ΔΨ m and caspase activity were measured by flow cytometric assay. Cell apoptosis associated protein expressions were examined by Western blotting and confocal laser microscopy. Results indicated that casticin induced cell morphological changes, DNA condensation and damage, decreased the total viable cells, induced G 2 /M phase arrest in SCC-4 cells. Casticin promoted ROS and Ca 2+ productions, decreases the levels of ΔΨ m , promoted caspase-3, -8, and -9 activities in SCC-4 cells. Western blotting assay demonstrated that casticin affect protein level associated with G2/M phase arrest and apoptosis. Confocal laser microscopy also confirmed that casticin increased the translocation of AIF and cytochrome c in SCC-4 cells. In conclusion, casticin decreased cell number through G 2 /M phase arrest and the induction of cell apoptosis through caspase- and mitochondria-dependent pathways in SCC-4 cells. © 2017 Wiley Periodicals, Inc.

  1. Skin Stem Cells in Skin Cell Therapy

    Directory of Open Access Journals (Sweden)

    Mollapour Sisakht

    2015-12-01

    Full Text Available Context Preclinical and clinical research has shown that stem cell therapy is a promising therapeutic option for many diseases. This article describes skin stem cells sources and their therapeutic applications. Evidence Acquisition Compared with conventional methods, cell therapy reduces the surgical burden for patients because it is simple and less time-consuming. Skin cell therapy has been developed for variety of diseases. By isolation of the skin stem cell from the niche, in vitro expansion and transplantation of cells offers a surprising healing capacity profile. Results Stem cells located in skin cells have shown interesting properties such as plasticity, transdifferentiation, and specificity. Mesenchymal cells of the dermis, hypodermis, and other sources are currently being investigated to promote regeneration. Conclusions Because skin stem cells are highly accessible from autologous sources and their immunological profile is unique, they are ideal for therapeutic approaches. Optimization of administrative routes requires more investigation own to the lack of a standard protocol.

  2. Cell biology of mesangial cells: the third cell that maintains the glomerular capillary.

    Science.gov (United States)

    Kurihara, Hidetake; Sakai, Tatsuo

    2017-03-01

    The renal glomerulus consists of glomerular endothelial cells, podocytes, and mesangial cells, which cooperate with each other for glomerular filtration. We have produced monoclonal antibodies against glomerular cells in order to identify different types of glomerular cells. Among these antibodies, the E30 clone specifically recognizes the Thy1.1 molecule expressed on mesangial cells. An injection of this antibody into rats resulted in mesangial cell-specific injury within 15 min, and induced mesangial proliferative glomerulonephritis in a reproducible manner. We examined the role of mesangial cells in glomerular function using several experimental tools, including an E30-induced nephritis model, mesangial cell culture, and the deletion of specific genes. Herein, we describe the characterization of E30-induced nephritis, formation of the glomerular capillary network, mesangial matrix turnover, and intercellular signaling between glomerular cells. New molecules that are involved in a wide variety of mesangial cell functions are also introduced.

  3. Natural killer cells for immunotherapy – Advantages of cell lines over blood NK cells

    Directory of Open Access Journals (Sweden)

    Hans eKlingemann

    2016-03-01

    Full Text Available Natural killer cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells form a patient’s blood since they represent only 10% of the lymphocytes. Especially, cancer patients are known to have dysfunctional NK cells. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T-cells. Establishing cell lines from donor blood NK cells have not been successful, in contrast to blood NK cells obtained from patients with a clonal NK cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. However, except for the NK-92 cell line none of the other six known cell lines has consistent and reproducibly high anti-tumor cytotoxicity, nor can they be easily genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through ADCC. NK-92 is also the only cell line product that has been widely given to patients with advanced cancer with demonstrated efficiency and minimal side effects.

  4. Cell sheet technology and cell patterning for biofabrication

    Energy Technology Data Exchange (ETDEWEB)

    Hannachi, Imen Elloumi; Yamato, Masayuki; Okano, Teruo [Institute of Advanced Biomedical Engineering and Science, Tokyo Women' s Medical University, 8-1 Kawada-cho, Shinjuku, Tokyo (Japan)

    2009-06-01

    We have developed cell sheet technology as a modern method for the fabrication of functional tissue-like and organ-like structures. This technology allows for a sheet of interconnected cells and cells in full contact with their natural extracellular environment to be obtained. A cell sheet can be patterned and composed according to more than one cell type. The key technology of cell sheet engineering is that a fabricated cell sheet can be harvested and transplanted utilizing temperature-responsive surfaces. In this review, we summarize different aspects of cell sheet engineering and provide a survey of the application of cell sheets as a suitable material for biofabrication and clinics. Moreover, since cell micropatterning is a key tool for cell sheet engineering, in this review we focus on the introduction of our approaches to cell micropatterning and cell co-culture to the principles of automation and how they can be subjected to easy robotics programming. Finally, efforts towards making cell sheet technology suitable for biofabrication and robotic biofabrication are also summarized. (topical review)

  5. Free-zone electrophoresis of animal cells. 1: Experiments on cell-cell interactions

    Science.gov (United States)

    Todd, P. W.; Hjerten, S.

    1985-01-01

    The electrophoretically migrating zones wasa monitored. The absence of fluid flows in the direction of migration permits direct measurement of electrophoretic velocities of any material. Sedimentation is orthogonal to electrokinetic motion and the effects of particle-particle interaction on electrophoretic mobility is studied by free zone electrophoresis. Fixed erythrocytes at high concentrations, mixtures of fixed erythrocytes from different animal species, and mixtures of cultured human cells were studied in low ionic strength buffers. The electrophoretic velocity of fixed erythrocytes was not altered by increasing cell concentration or by the mixing of erythrocytes from different species. When zones containing cultured human glial cells and neuroblastoma cells are permitted to interact during electrophoresis, altered migration patterns occur. It is found that cell-cell interactions depends upon cell type.

  6. Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

    Science.gov (United States)

    Velásquez, Celestino; Amako, Yutaka; Harold, Alexis; Toptan, Tuna; Chang, Yuan; Shuda, Masahiro

    2018-01-01

    Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

  7. c-Myc-Dependent Cell Competition in Human Cancer Cells.

    Science.gov (United States)

    Patel, Manish S; Shah, Heta S; Shrivastava, Neeta

    2017-07-01

    Cell Competition is an interaction between cells for existence in heterogeneous cell populations of multicellular organisms. This phenomenon is involved in initiation and progression of cancer where heterogeneous cell populations compete directly or indirectly for the survival of the fittest based on differential gene expression. In Drosophila, cells having lower dMyc expression are eliminated by cell competition through apoptosis when present in the milieu of cells having higher dMyc expression. Thus, we designed a study to develop c-Myc (human homolog) dependent in vitro cell competition model of human cancer cells. Cells with higher c-Myc were transfected with c-myc shRNA to prepare cells with lower c-Myc and then co-cultured with the same type of cells having a higher c-Myc in equal ratio. Cells with lower c-Myc showed a significant decrease in numbers when compared with higher c-Myc cells, suggesting "loser" and "winner" status of cells, respectively. During microscopy, engulfment of loser cells by winner cells was observed with higher expression of JNK in loser cells. Furthermore, elimination of loser cells was prevented significantly, when co-cultured cells were treated with the JNK (apoptosis) inhibitor. Above results indicate elimination of loser cells in the presence of winner cells by c-Myc-dependent mechanisms of cell competition in human cancer cells. This could be an important mechanism in human tumors where normal cells are eliminated by c-Myc-overexpressed tumor cells. J. Cell. Biochem. 118: 1782-1791, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. β-Cell regeneration through the transdifferentiation of pancreatic cells: Pancreatic progenitor cells in the pancreas.

    Science.gov (United States)

    Kim, Hyo-Sup; Lee, Moon-Kyu

    2016-05-01

    Pancreatic progenitor cell research has been in the spotlight, as these cells have the potential to replace pancreatic β-cells for the treatment of type 1 and 2 diabetic patients with the absence or reduction of pancreatic β-cells. During the past few decades, the successful treatment of diabetes through transplantation of the whole pancreas or isolated islets has nearly been achieved. However, novel sources of pancreatic islets or insulin-producing cells are required to provide sufficient amounts of donor tissues. To overcome this limitation, the use of pancreatic progenitor cells is gaining more attention. In particular, pancreatic exocrine cells, such as duct epithelial cells and acinar cells, are attractive candidates for β-cell regeneration because of their differentiation potential and pancreatic lineage characteristics. It has been assumed that β-cell neogenesis from pancreatic progenitor cells could occur in pancreatic ducts in the postnatal stage. Several studies have shown that insulin-producing cells can arise in the duct tissue of the adult pancreas. Acinar cells also might have the potential to differentiate into insulin-producing cells. The present review summarizes recent progress in research on the transdifferentiation of pancreatic exocrine cells into insulin-producing cells, especially duct and acinar cells.

  9. 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.

  10. Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells.

    Science.gov (United States)

    Sánchez-Martínez, Diego; Lanuza, Pilar M; Gómez, Natalia; Muntasell, Aura; Cisneros, Elisa; Moraru, Manuela; Azaceta, Gemma; Anel, Alberto; Martínez-Lostao, Luis; Villalba, Martin; Palomera, Luis; Vilches, Carlos; García Marco, José A; Pardo, Julián

    2016-01-01

    Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV ) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.

  11. Lung cells support osteosarcoma cell migration and survival.

    Science.gov (United States)

    Yu, Shibing; Fourman, Mitchell Stephen; Mahjoub, Adel; Mandell, Jonathan Brendan; Crasto, Jared Anthony; Greco, Nicholas Giuseppe; Weiss, Kurt Richard

    2017-01-25

    Osteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined. Three human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment. Lung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased alkaline

  12. Human regulatory B cells control the TFH cell response.

    Science.gov (United States)

    Achour, Achouak; Simon, Quentin; Mohr, Audrey; Séité, Jean-François; Youinou, Pierre; Bendaoud, Boutahar; Ghedira, Ibtissem; Pers, Jacques-Olivier; Jamin, Christophe

    2017-07-01

    Follicular helper T (T FH ) cells support terminal B-cell differentiation. Human regulatory B (Breg) cells modulate cellular responses, but their control of T FH cell-dependent humoral immune responses is unknown. We sought to assess the role of Breg cells on T FH cell development and function. Human T cells were polyclonally stimulated in the presence of IL-12 and IL-21 to generate T FH cells. They were cocultured with B cells to induce their terminal differentiation. Breg cells were included in these cultures, and their effects were evaluated by using flow cytometry and ELISA. B-cell lymphoma 6, IL-21, inducible costimulator, CXCR5, and programmed cell death protein 1 (PD-1) expressions increased on stimulated human T cells, characterizing T FH cell maturation. In cocultures they differentiated B cells into CD138 + plasma and IgD - CD27 + memory cells and triggered immunoglobulin secretions. Breg cells obtained by Toll-like receptor 9 and CD40 activation of B cells prevented T FH cell development. Added to T FH cell and B-cell cocultures, they inhibited B-cell differentiation, impeded immunoglobulin secretions, and expanded Foxp3 + CXCR5 + PD-1 + follicular regulatory T cells. Breg cells modulated IL-21 receptor expressions on T FH cells and B cells, and their suppressive activities involved CD40, CD80, CD86, and intercellular adhesion molecule interactions and required production of IL-10 and TGF-β. Human Breg cells control T FH cell maturation, expand follicular regulatory T cells, and inhibit the T FH cell-mediated antibody secretion. These novel observations demonstrate a role for the Breg cell in germinal center reactions and suggest that deficient activities might impair the T FH cell-dependent control of humoral immunity and might lead to the development of aberrant autoimmune responses. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  13. A method to quantify mechanobiologic forces during zebrafish cardiac development using 4-D light sheet imaging and computational modeling.

    Directory of Open Access Journals (Sweden)

    Vijay Vedula

    2017-10-01

    Full Text Available Blood flow and mechanical forces in the ventricle are implicated in cardiac development and trabeculation. However, the mechanisms of mechanotransduction remain elusive. This is due in part to the challenges associated with accurately quantifying mechanical forces in the developing heart. We present a novel computational framework to simulate cardiac hemodynamics in developing zebrafish embryos by coupling 4-D light sheet imaging with a stabilized finite element flow solver, and extract time-dependent mechanical stimuli data. We employ deformable image registration methods to segment the motion of the ventricle from high resolution 4-D light sheet image data. This results in a robust and efficient workflow, as segmentation need only be performed at one cardiac phase, while wall position in the other cardiac phases is found by image registration. Ventricular hemodynamics are then quantified by numerically solving the Navier-Stokes equations in the moving wall domain with our validated flow solver. We demonstrate the applicability of the workflow in wild type zebrafish and three treated fish types that disrupt trabeculation: (a chemical treatment using AG1478, an ErbB2 signaling inhibitor that inhibits proliferation and differentiation of cardiac trabeculation; (b injection of gata1a morpholino oligomer (gata1aMO suppressing hematopoiesis and resulting in attenuated trabeculation; (c weak-atriumm58 mutant (wea with inhibited atrial contraction leading to a highly undeveloped ventricle and poor cardiac function. Our simulations reveal elevated wall shear stress (WSS in wild type and AG1478 compared to gata1aMO and wea. High oscillatory shear index (OSI in the grooves between trabeculae, compared to lower values on the ridges, in the wild type suggest oscillatory forces as a possible regulatory mechanism of cardiac trabeculation development. The framework has broad applicability for future cardiac developmental studies focused on quantitatively

  14. Single-cell protein secretomic signatures as potential correlates to tumor cell lineage evolution and cell-cell interaction

    Directory of Open Access Journals (Sweden)

    Minsuk eKwak

    2013-02-01

    Full Text Available Secreted proteins including cytokines, chemokines and growth factors represent important functional regulators mediating a range of cellular behavior and cell-cell paracrine/autocrine signaling, e.g. in the immunological system, tumor microenvironment or stem cell niche. Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically-identical cell population can give rise to diverse phenotypic differences. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this Perspective Article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer.

  15. Mesenchymal Stem Cells Induce Epithelial to Mesenchymal Transition in Colon Cancer Cells through Direct Cell-to-Cell Contact

    Directory of Open Access Journals (Sweden)

    Hidehiko Takigawa

    2017-05-01

    Full Text Available We previously reported that in an orthotopic nude mouse model of human colon cancer, bone marrow–derived mesenchymal stem cells (MSCs migrated to the tumor stroma and promoted tumor growth and metastasis. Here, we evaluated the proliferation and migration ability of cancer cells cocultured with MSCs to elucidate the mechanism of interaction between cancer cells and MSCs. Proliferation and migration of cancer cells increased following direct coculture with MSCs but not following indirect coculture. Thus, we hypothesized that direct contact between cancer cells and MSCs was important. We performed a microarray analysis of gene expression in KM12SM colon cancer cells directly cocultured with MSCs. Expression of epithelial-mesenchymal transition (EMT–related genes such as fibronectin (FN, SPARC, and galectin 1 was increased by direct coculture with MSCs. We also confirmed the upregulation of these genes with real-time polymerase chain reaction. Gene expression was not elevated in cancer cells indirectly cocultured with MSCs. Among the EMT-related genes upregulated by direct coculture with MSCs, we examined the immune localization of FN, a well-known EMT marker. In coculture assay in chamber slides, expression of FN was seen only at the edges of cancer clusters where cancer cells directly contacted MSCs. FN expression in cancer cells increased at the tumor periphery and invasive edge in orthotopic nude mouse tumors and human colon cancer tissues. These results suggest that MSCs induce EMT in colon cancer cells via direct cell-to-cell contact and may play an important role in colon cancer metastasis.

  16. Activated allogeneic NK cells preferentially kill poor prognosis B-cell chronic lymphocytic leukemia cells

    Directory of Open Access Journals (Sweden)

    Diego Sanchez-Martinez

    2016-10-01

    Full Text Available Mutational status of TP53 together with expression of wild type (wt IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL patients. Adoptive cell therapy using allogeneic HLA mismatched Natural Killer (NK cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs the most effective stimulus to activate NK cells. Here we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell activating receptors (NKG2D and NCRs and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.□

  17. Mast cells dysregulate apoptotic and cell cycle genes in mucosal squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Davis Paul

    2006-12-01

    Full Text Available Abstract Background Mucosal squamous cell carcinoma of the head and neck is a disease of high mortality and morbidity. Interactions between the squamous cell carcinoma and the host's local immunity, and how the latter contributes to the biological behavior of the tumor are unclear. In vivo studies have demonstrated sequential mast cell infiltration and degranulation during squamous cell carcinogenesis. The degree of mast cell activation correlates closely with distinct phases of hyperkeratosis, dysplasia, carcinoma in-situ and invasive carcinoma. However, the role of mast cells in carcinogenesis is unclear. Aim This study explores the effects of mast cells on the proliferation and gene expression profile of mucosal squamous cell carcinoma using human mast cell line (HMC-1 and human glossal squamous cell carcinoma cell line (SCC25. Methods HMC-1 and SCC25 were co-cultured in a two-compartment chamber, separated by a polycarbonate membrane. HMC-1 was stimulated to degranulate with calcium ionophore A23187. The experiments were done in quadruplicate. Negative controls were established where SCC25 were cultured alone without HMC-1. At 12, 24, 48 and 72 hours, proliferation and viability of SCC25 were assessed with MTT colorimetric assay. cDNA microarray was employed to study differential gene expression between co-cultured and control SCC25. Results HMC-1/SCC25 co-culture resulted in suppression of growth rate for SCC-25 (34% compared with 110% for the control by 72 hours, p Conclusion We show that mast cells have a direct inhibitory effect on the proliferation of mucosal squamous cell carcinoma in vitro by dysregulating key genes in apoptosis and cell cycle control.

  18. Plant cell wall polysaccharide analysis during cell elongation

    DEFF Research Database (Denmark)

    Guo, Xiaoyuan

    Plant cell walls are complex structures whose composition and architecture are important to various cellular activities. Plant cell elongation requires a high level of rearrangement of the cell wall polymers to enable cell expansion. However, the cell wall polysaccharides dynamics during plant cell...... elongation is poorly understood. This PhD project aims to elucidate the cell wall compositional and structural change during cell elongation by using Comprehensive Microarray Polymer Profiling (CoMPP), microscopic techniques and molecular modifications of cell wall polysaccharide. Developing cotton fibre......, pea and Arabidopsis thaliana were selected as research models to investigate different types of cell elongation, developmental elongation and tropism elongation. A set of comprehensive analysis covering 4 cotton species and 11 time points suggests that non-cellulosic polysaccharides contribute...

  19. Estrogen enhanced cell-cell signalling in breast cancer cells exposed to targeted irradiation

    International Nuclear Information System (INIS)

    Shao, Chunlin; Folkard, Melvyn; Held, Kathryn D; Prise, Kevin M

    2008-01-01

    Radiation-induced bystander responses, where cells respond to their neighbours being irradiated are being extensively studied. Although evidence shows that bystander responses can be induced in many types of cells, it is not known whether there is a radiation-induced bystander effect in breast cancer cells, where the radiosensitivity may be dependent on the role of the cellular estrogen receptor (ER). This study investigated radiation-induced bystander responses in estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231 breast cancer cells. The influence of estrogen and anti-estrogen treatments on the bystander response was determined by individually irradiating a fraction of cells within the population with a precise number of helium-3 using a charged particle microbeam. Damage was scored as chromosomal damage measured as micronucleus formation. A bystander response measured as increased yield of micronucleated cells was triggered in both MCF-7 and MDA-MB-231 cells. The contribution of the bystander response to total cell damage in MCF-7 cells was higher than that in MDA-MB-231 cells although the radiosensitivity of MDA-MB-231 was higher than MCF-7. Treatment of cells with 17β-estradiol (E2) increased the radiosensitivity and the bystander response in MCF-7 cells, and the effect was diminished by anti-estrogen tamoxifen (TAM). E2 also increased the level of intracellular reactive oxygen species (ROS) in MCF-7 cells in the absence of radiation. In contrast, E2 and TAM had no influence on the bystander response and ROS levels in MDA-MB-231 cells. Moreover, the treatment of MCF-7 cells with antioxidants eliminated both the E2-induced ROS increase and E2-enhanced bystander response triggered by the microbeam irradiation, which indicates that ROS are involved in the E2-enhanced bystander micronuclei formation after microbeam irradiation. The observation of bystander responses in breast tumour cells may offer new potential targets for radiation

  20. Follow-the-leader cell migration requires biased cell-cell contact and local microenvironmental signals

    Science.gov (United States)

    Wynn, Michelle L.; Rupp, Paul; Trainor, Paul A.; Schnell, Santiago; Kulesa, Paul M.

    2013-06-01

    Directed cell migration often involves at least two types of cell motility that include multicellular streaming and chain migration. However, what is unclear is how cell contact dynamics and the distinct microenvironments through which cells travel influence the selection of one migratory mode or the other. The embryonic and highly invasive neural crest (NC) are an excellent model system to study this question since NC cells have been observed in vivo to display both of these types of cell motility. Here, we present data from tissue transplantation experiments in chick and in silico modeling that test our hypothesis that cell contact dynamics with each other and the microenvironment promote and sustain either multicellular stream or chain migration. We show that when premigratory cranial NC cells (at the pre-otic level) are transplanted into a more caudal region in the head (at the post-otic level), cells alter their characteristic stream behavior and migrate in chains. Similarly, post-otic NC cells migrate in streams after transplantation into the pre-otic hindbrain, suggesting that local microenvironmental signals dictate the mode of NC cell migration. Simulations of an agent-based model (ABM) that integrates the NC cell behavioral data predict that chain migration critically depends on the interplay of biased cell-cell contact and local microenvironment signals. Together, this integrated modeling and experimental approach suggests new experiments and offers a powerful tool to examine mechanisms that underlie complex cell migration patterns.

  1. Hilar mossy cell circuitry controlling dentate granule cell excitability

    Directory of Open Access Journals (Sweden)

    Seiichiro eJinde

    2013-02-01

    Full Text Available Glutamatergic hilar mossy cells of the dentate gyrus can either excite or inhibit distant granule cells, depending on whether their direct excitatory projections to granule cells or their projections to local inhibitory interneurons dominate. However, it remains controversial whether the net effect of mossy cell loss is granule cell excitation or inhibition. Clarifying this controversy has particular relevance to temporal lobe epilepsy, which is marked by dentate granule cell hyperexcitability and extensive loss of dentate hilar mossy cells. Two diametrically opposed hypotheses have been advanced to explain this granule cell hyperexcitability – the dormant basket cell and the irritable mossy cell hypotheses. The dormant basket cell hypothesis proposes that mossy cells normally exert a net inhibitory effect on granule cells and therefore their loss causes dentate granule cell hyperexcitability. The irritable mossy cell hypothesis takes the opposite view that mossy cells normally excite granule cells and that the surviving mossy cells in epilepsy increase their activity, causing granule cell excitation. The inability to eliminate mossy cells selectively has made it difficult to test these two opposing hypotheses. To this end, we developed a transgenic toxin-mediated, mossy cell-ablation mouse line. Using these mutants, we demonstrated that the extensive elimination of hilar mossy cells causes granule cell hyperexcitability, although the mossy cell loss observed appeared insufficient to cause clinical epilepsy. In this review, we focus on this topic and also suggest that different interneuron populations may mediate mossy cell-induced translamellar lateral inhibition and intralamellar recurrent inhibition. These unique local circuits in the dentate hilar region may be centrally involved in the functional organization of the dentate gyrus.

  2. Cell Biochips

    Science.gov (United States)

    Pioufle, B. Le; Picollet-D'Hahan, N.

    A cell biochip is a microsystem, equipped with electronic and microfluidic functions, designed to manipulate or analyse living cells. The first publications in this emerging area of research appeared toward the end of the 1980s. In 1989 Washizu described a biochip designed to fuse two cells by electropermeabilisation of the cytoplasmic membrane [1]. Research centers have devised a whole range of cell chip structures, for simultaneous or sequential analysis of single cells, cell groups, or cell tissues reconstituted on the chip. The cells are arranged in a square array on a parallel cell chip for parallel analysis, while they are examined and processed one by one in a microchannel in the case of a series cell chip. In contrast to these biochips for high-throughput analysis of a large number of cells, single-cell chips focus on the analysis of a single isolated cell. As in DNA microarrays, where a large number of oligonucleotides are ordered in a matrix array, parallel cell chips order living cells in a similar way. At each point of the array, the cells can be isolated, provided that the cell type allows this, e.g., blood cells, or cultivated in groups (most adhesion cells can only survive in groups). The aim is to allow massively parallel analysis or processing. Le Pioufle et al. describe a microdevice for the culture of single cells or small groups of cells in a micropit array [2]. Each pit is equipped to stimulate the cell or group of cells either electrically or fluidically. Among the applications envisaged are gene transfer, cell sorting, and screening in pharmacology. A complementary approach, combining the DNA microarray and cell biochip ideas, has been put forward by Bailey et al. [3]. Genes previously arrayed on the chip transfect the cultured cells on the substrate depending on their position in the array (see Fig. 19.1). This way of achieving differential lipofection on a chip was then taken up again by Yoshikawa et al. [4] with primary cells, more

  3. Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

    Science.gov (United States)

    Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

    2000-04-01

    Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

  4. The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines

    International Nuclear Information System (INIS)

    Fujiwara, Daisuke; Kato, Kazunori; Nohara, Shigeo; Iwanuma, Yoshimi; Kajiyama, Yoshiaki

    2013-01-01

    Highlights: •Spheroids were created from esophageal carcinoma cells using NanoCulture® Plates. •The proportion of strongly ALDH-positive cells increased in 3-D culture. •Expression of cancer stem cell-related genes was enhanced in 3-D culture. •CA-9 expression was enhanced, suggesting hypoxia had been induced in 3-D culture. •Drug resistance was increased. 3-D culture is useful for inducing cancer stem cells. -- Abstract: In recent years, research on resistance to chemotherapy and radiotherapy in cancer treatment has come under the spotlight, and researchers have also begun investigating the relationship between resistance and cancer stem cells. Cancer stem cells are assumed to be present in esophageal cancer, but experimental methods for identification and culture of these cells have not yet been established. To solve this problem, we created spheroids using a NanoCulture® Plate (NCP) for 3-dimensional (3-D) cell culture, which was designed as a means for experimentally reproducing the 3-D structures found in the body. We investigated the potential for induction of cancer stem cells from esophageal cancer cells. Using flow cytometry we analyzed the expression of surface antigen markers CD44, CD133, CD338 (ABCG2), CD318 (CDCP1), and CD326 (EpCAM), which are known cancer stem cell markers. None of these surface antigen markers showed enhanced expression in 3-D cultured cells. We then analyzed aldehyde dehydrogenase (ALDH) enzymatic activity using the ALDEFLUOR reagent, which can identify immature cells such as stem cells and precursor cells. 3-D-cultured cells were strongly positive for ALDH enzyme activity. We also analyzed the expression of the stem cell-related genes Sox-2, Nanog, Oct3/4, and Lin28 using RT-PCR. Expression of Sox-2, Nanog, and Lin28 was enhanced. Analysis of expression of the hypoxic surface antigen marker carbonic anhydrase-9 (CA-9), which is an indicator of cancer stem cell induction and maintenance, revealed that CA-9 expression

  5. A novel cell division factor from tobacco 2B-13 cells that induced cell division in auxin-starved tobacco BY-2 cells

    Science.gov (United States)

    Shimizu, Takashi; Eguchi, Kentaro; Nishida, Ikuo; Laukens, Kris; Witters, Erwin; van Onckelen, Harry; Nagata, Toshiyuki

    2006-06-01

    Effects of auxin as plant hormones are widespread; in fact in almost all aspects of plant growth and development auxin plays a pivotal role. Although auxin is required for propagating cell division in plant cells, its effect upon cell division is least understood. If auxin is depleted from the culture medium, cultured cells cease to divide. It has been demonstrated in this context that the addition of auxin to auxin-starved nondividing tobacco BY-2 cells induced semisynchronous cell division. On the other hand, there are some cell lines, named habituated cells, that can grow without auxin. The cause and reason for the habituated cells have not been clarified. A habituated cell line named 2B-13 is derived from the tobacco BY-2 cell line, which has been most intensively studied among plant cell lines. When we tried to find the difference between two cell lines of BY-2 and 2B-13 cells, we found that the addition of culture filtrated from the auxin-habituated 2B-13 cells induced semisynchronous cell division in auxin-starved BY-2 cells. The cell division factor (CDF) that is responsible for inducing cell division in auxin-starved BY-2 cells was purified to near-homogeneity by sequential passage through a hydroxyapatite column, a ConA Sepharose column and a Sephadex gel filtration column. The resulting purified fraction appeared as a single band of high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by silver staining and was able to induce cell division in auxin-starved BY-2 cells. Identification of the protein by MALD-TOF-MS/MS revealed that it is structurally related to P-glycoprotein from Gossypioides kirkii, which belongs to ATP-binding cassette (ABC)-transporters. The significance of CDF as a possible ABC-transporter is discussed in relationship to auxin-autotrophic growth and auxin-signaling pathway.

  6. Cell damage evaluation of mammalian cells in cell manipulation by amplified femtosecond ytterbium laser

    Science.gov (United States)

    Hong, Z.-Y.; Iino, T.; Hagihara, H.; Maeno, T.; Okano, K.; Yasukuni, R.; Hosokawa, Y.

    2018-03-01

    A micrometer-scale explosion with cavitation bubble generation is induced by focusing a femtosecond laser in an aqueous solution. We have proposed to apply the explosion as an impulsive force to manipulate mammalian cells especially in microfluidic chip. Herein, we employed an amplified femtosecond ytterbium laser as an excitation source for the explosion and evaluated cell damage in the manipulation process to clarify the application potential. The damage of C2C12 myoblast cell prepared as a representative mammalian cell was investigated as a function of distance between cell and laser focal point. Although the cell received strong damage on the direct laser irradiation condition, the damage sharply decreased with increasing distance. Since the threshold distance, above which the cell had no damage, was consistent with radius of the cavitation bubble, impact of the cavitation bubble would be a critical factor for the cell damage. The damage had strong nonlinearity in the pulse energy dependence. On the other hand, cell position shift by the impact of the cavitation bubble was almost proportional to the pulse energy. In balance between the cell viability and the cell position shift, we elucidated controllability of the cell manipulation in microfluidic chip.

  7. Force-induced bone growth and adaptation: A system theoretical approach to understanding bone mechanotransduction

    International Nuclear Information System (INIS)

    Maldonado, Solvey; Findeisen, Rolf

    2010-01-01

    The modeling, analysis, and design of treatment therapies for bone disorders based on the paradigm of force-induced bone growth and adaptation is a challenging task. Mathematical models provide, in comparison to clinical, medical and biological approaches an structured alternative framework to understand the concurrent effects of the multiple factors involved in bone remodeling. By now, there are few mathematical models describing the appearing complex interactions. However, the resulting models are complex and difficult to analyze, due to the strong nonlinearities appearing in the equations, the wide range of variability of the states, and the uncertainties in parameters. In this work, we focus on analyzing the effects of changes in model structure and parameters/inputs variations on the overall steady state behavior using systems theoretical methods. Based on an briefly reviewed existing model that describes force-induced bone adaptation, the main objective of this work is to analyze the stationary behavior and to identify plausible treatment targets for remodeling related bone disorders. Identifying plausible targets can help in the development of optimal treatments combining both physical activity and drug-medication. Such treatments help to improve/maintain/restore bone strength, which deteriorates under bone disorder conditions, such as estrogen deficiency.

  8. Can resting B cells present antigen to T cells

    International Nuclear Information System (INIS)

    Ashwell, J.D.; DeFranco, A.L.; Paul, W.E.; Schwartz, R.H.

    1985-01-01

    Antigen stimulation of T lymphocytes can occur only in the presence of an antigen-presenting cell (APC). An ever-increasing number of cell types have been found to act as APCs; these include macrophages, splenic and lymph node dendritic cells, and Langerhans cells of the skin. Although activated B lymphocytes and B cell lymphomas are known to serve as APCs, it has been generally believed that resting B cells cannot perform this function. However, in recent studies the authors have found that resting B cells can indeed present soluble antigen to T cell clones as well as to antigen-primed T cells. The previous difficulty in demonstrating this activity can be explained by the finding that, in contrast to macrophages and dendritic cells, the antigen-presenting ability of resting B cells is very radiosensitive. Macrophages are usually irradiated with 2000-3300 rads to prevent them from incorporating [ 3 H]thymidine in the T cell proliferation assay. Resting B cells, however, begin to lose presenting function at 1500 rads and have completely lost this activity at 3300 rads. It was also possible to distinguish two distinct T cell clonal phenotypes when resting B cells were used as APCs on the basis of two different assays (T cell proliferation, and B cell proliferation resulting from T cell activation). The majority of T cell clones tested were capable of both proliferating themselves and inducing the proliferation of B cells. Some T cells clones, however, could not proliferate in the presence of antigen and B cell APCs, although they were very good at inducing the proliferation of B cells

  9. Analysis of individual cells identifies cell-to-cell variability following induction of cellular senescence.

    Science.gov (United States)

    Wiley, Christopher D; Flynn, James M; Morrissey, Christapher; Lebofsky, Ronald; Shuga, Joe; Dong, Xiao; Unger, Marc A; Vijg, Jan; Melov, Simon; Campisi, Judith

    2017-10-01

    Senescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single-cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell-to-cell variability resulted in a loss of correlation among the expression of several senescence-associated genes. Many genes encoding senescence-associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  10. Dynamic imaging of cell-free and cell-associated viral capture in mature dendritic cells.

    Science.gov (United States)

    Izquierdo-Useros, Nuria; Esteban, Olga; Rodriguez-Plata, Maria T; Erkizia, Itziar; Prado, Julia G; Blanco, Julià; García-Parajo, Maria F; Martinez-Picado, Javier

    2011-12-01

    Dendritic cells (DCs) capture human immunodeficiency virus (HIV) through a non-fusogenic mechanism that enables viral transmission to CD4(+) T cells, contributing to in vivo viral dissemination. Although previous studies have provided important clues to cell-free viral capture by mature DCs (mDCs), dynamic and kinetic insight on this process is still missing. Here, we used three-dimensional video microscopy and single-particle tracking approaches to dynamically dissect both cell-free and cell-associated viral capture by living mDCs. We show that cell-free virus capture by mDCs operates through three sequential phases: virus binding through specific determinants expressed in the viral particle, polarized or directional movements toward concrete regions of the cell membrane and virus accumulation in a sac-like structure where trapped viral particles display a hindered diffusive behavior. Moreover, real-time imaging of cell-associated viral transfer to mDCs showed a similar dynamics to that exhibited by cell-free virus endocytosis leading to viral accumulation in compartments. However, cell-associated HIV type 1 transfer to mDCs was the most effective pathway, boosted throughout enhanced cellular contacts with infected CD4(+) T cells. Our results suggest that in lymphoid tissues, mDC viral uptake could occur either by encountering cell-free or cell-associated virus produced by infected cells generating the perfect scenario to promote HIV pathogenesis and impact disease progression. © 2011 John Wiley & Sons A/S.

  11. Role of Cell-Cell bond for the viability and the function of vascular smooth muscle cells

    Directory of Open Access Journals (Sweden)

    M. Mura

    2010-01-01

    Full Text Available Vascular smooth muscle cell (VSMC viability and homeostasis is regulated by cell-matrix and cell-cell contact: disruption of these interactions are responsible of a switch from a mature to a high proliferative phenotype. VSMCs migration, rate of growth and apoptosis, and the extent of their extracellular matrix (ECM deposition can be also modulated by proatherogenic peptides. Among them, ATII induces the transactivation of IGF I R, which, together with the binding protein IGFBP3, represents a determinant of cell survival, growth and proliferation. Aim of our in vitro study was to verify the role of elective cell-cell bond in moulating the response to ATII. Thus, we evaluated viability, proliferation, IGFIR, IGFBP3 expression and the long term survival and production of ECM in a provisional tissue. A7r5 cell-line was used in adherent cultures or incubated in agarose-coated culture plates to inhibit cell-matrix interactions. Cells, treated or not with ATII 100 nM, were evaluated for apoptosis rate, cell cycle, IGFIR and IGFBP3 protei expression. Fibrin provisional tissue was developed polymerizing a fibrin solution. cantaining A7r5 cells with thrombin. Histological stainings for ECM components were performed on sections of prvisional tissue. An exclusive cell-cell contact resulted to monolayer cell cultures. ATII did not affect the cell survival in both culture conditions, but promoted a 10% decrease in "S" phase and an increases IGFIR expression only in adherent cells. while suspended cell aggregates were resistant to ATII administration; IGFBP3 was reduced both in ATII treated adherent cells and in floating clustered cells, irrespective of the treatmentn. VSMC conditioning in agarose-coated plates before seeding in fibrin provisional matrix reduced, but not abolished, the cell ability to colonize the clot and to produce ECM. This study demonstrates that the elective cell-cell contact induces a quiescent status in cells lacking of cell

  12. Semiallogenic fusions of MSI+ tumor cells and activated B cells induce MSI-specific T cell responses

    International Nuclear Information System (INIS)

    Garbe, Yvette; Klier, Ulrike; Linnebacher, Michael

    2011-01-01

    Various strategies have been developed to transfer tumor-specific antigens into antigen presenting cells in order to induce cytotoxic T cell responses against tumor cells. One approach uses cellular vaccines based on fusions of autologous antigen presenting cells and allogeneic tumor cells. The fusion cells combine antigenicity of the tumor cell with optimal immunostimulatory capacity of the antigen presenting cells. Microsatellite instability caused by mutational inactivation of DNA mismatch repair genes results in translational frameshifts when affecting coding regions. It has been shown by us and others that these mutant proteins lead to the presentation of immunogenic frameshift peptides that are - in principle - recognized by a multiplicity of effector T cells. We chose microsatellite instability-induced frameshift antigens as ideal to test for induction of tumor specific T cell responses by semiallogenic fusions of microsatellite instable carcinoma cells with CD40-activated B cells. Two fusion clones of HCT116 with activated B cells were selected for stimulation of T cells autologous to the B cell fusion partner. Outgrowing T cells were phenotyped and tested in functional assays. The fusion clones expressed frameshift antigens as well as high amounts of MHC and costimulatory molecules. Autologous T cells stimulated with these fusions were predominantly CD4 + , activated, and reacted specifically against the fusion clones and also against the tumor cell fusion partner. Interestingly, a response toward 6 frameshift-derived peptides (of 14 tested) could be observed. Cellular fusions of MSI + carcinoma cells and activated B cells combine the antigen-presenting capacity of the B cell with the antigenic repertoire of the carcinoma cell. They present frameshift-derived peptides and can induce specific and fully functional T cells recognizing not only fusion cells but also the carcinoma cells. These hybrid cells may have great potential for cellular immunotherapy and

  13. NK Cells and Other Innate Lymphoid Cells in Hematopoietic Stem Cell Transplantation.

    Science.gov (United States)

    Vacca, Paola; Montaldo, Elisa; Croxatto, Daniele; Moretta, Francesca; Bertaina, Alice; Vitale, Chiara; Locatelli, Franco; Mingari, Maria Cristina; Moretta, Lorenzo

    2016-01-01

    Natural killer (NK) cells play a major role in the T-cell depleted haploidentical hematopoietic stem cell transplantation (haplo-HSCT) to cure high-risk leukemias. NK cells belong to the expanding family of innate lymphoid cells (ILCs). At variance with NK cells, the other ILC populations (ILC1/2/3) are non-cytolytic, while they secrete different patterns of cytokines. ILCs provide host defenses against viruses, bacteria, and parasites, drive lymphoid organogenesis, and contribute to tissue remodeling. In haplo-HSCT patients, the extensive T-cell depletion is required to prevent graft-versus-host disease (GvHD) but increases risks of developing a wide range of life-threatening infections. However, these patients may rely on innate defenses that are reconstituted more rapidly than the adaptive ones. In this context, ILCs may represent important players in the early phases following transplantation. They may contribute to tissue homeostasis/remodeling and lymphoid tissue reconstitution. While the reconstitution of NK cell repertoire and its role in haplo-HSCT have been largely investigated, little information is available on ILCs. Of note, CD34(+) cells isolated from different sources of HSC may differentiate in vitro toward various ILC subsets. Moreover, cytokines released from leukemia blasts (e.g., IL-1β) may alter the proportions of NK cells and ILC3, suggesting the possibility that leukemia may skew the ILC repertoire. Further studies are required to define the timing of ILC development and their potential protective role after HSCT.

  14. NK cells and other innate lymphoid cells in haematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Paola eVacca

    2016-05-01

    Full Text Available Natural Killer (NK cells play a major role in the T-cell depleted haploidentical haematopoietic stem cell transplantation (haplo-HSCT to cure high-risk leukemias. NK cells belong to the expanding family of innate lymphoid cells (ILC. At variance with NK cells, the other ILC populations (ILC1/2/3 are non-cytolytic, while they secrete different patterns of cytokines. ILC provide host defences against viruses, bacteria and parasites, drive lymphoid organogenesis, and contribute to tissue remodelling. In haplo-HSCT patients, the extensive T-cell depletion is required to prevent graft-versus-host disease (GvHD but increases risks of developing a wide range of life-threatening infections. However, these patients may rely on innate defences that are reconstituted more rapidly than the adaptive ones. In this context, ILC may represent important players in the early phases following transplantation. They may contribute to tissue homeostasis/remodelling and lymphoid tissue reconstitution. While the reconstitution of NK cell repertoire and its role in haplo-HSCT have been largely investigated, little information is available on ILC. Of note, CD34+ cells isolated from different sources of HSC, may differentiate in vitro towards various ILC subsets. Moreover, cytokines released from leukemia blasts (e.g. IL-1β may alter the proportions of NK cells and ILC3, suggesting the possibility that leukemia may skew the ILC repertoire. Further studies are required to define the timing of ILC development and their potential protective role after HSCT.

  15. Cell-substrate impedance fluctuations of single amoeboid cells encode cell-shape and adhesion dynamics.

    Science.gov (United States)

    Leonhardt, Helmar; Gerhardt, Matthias; Höppner, Nadine; Krüger, Kirsten; Tarantola, Marco; Beta, Carsten

    2016-01-01

    We show systematic electrical impedance measurements of single motile cells on microelectrodes. Wild-type cells and mutant strains were studied that differ in their cell-substrate adhesion strength. We recorded the projected cell area by time-lapse microscopy and observed irregular oscillations of the cell shape. These oscillations were correlated with long-term variations in the impedance signal. Superposed to these long-term trends, we observed fluctuations in the impedance signal. Their magnitude clearly correlated with the adhesion strength, suggesting that strongly adherent cells display more dynamic cell-substrate interactions.

  16. Cell-substrate impedance fluctuations of single amoeboid cells encode cell-shape and adhesion dynamics

    Science.gov (United States)

    Leonhardt, Helmar; Gerhardt, Matthias; Höppner, Nadine; Krüger, Kirsten; Tarantola, Marco; Beta, Carsten

    2016-01-01

    We show systematic electrical impedance measurements of single motile cells on microelectrodes. Wild-type cells and mutant strains were studied that differ in their cell-substrate adhesion strength. We recorded the projected cell area by time-lapse microscopy and observed irregular oscillations of the cell shape. These oscillations were correlated with long-term variations in the impedance signal. Superposed to these long-term trends, we observed fluctuations in the impedance signal. Their magnitude clearly correlated with the adhesion strength, suggesting that strongly adherent cells display more dynamic cell-substrate interactions.

  17. Cell division orientation is coupled to cell-cell adhesion by the E-cadherin/LGN complex

    NARCIS (Netherlands)

    Gloerich, Martijn; Bianchini, Julie M.; Siemers, Kathleen A.; Cohen, Daniel J.; Nelson, W. James

    2017-01-01

    Both cell-cell adhesion and oriented cell division play prominent roles in establishing tissue architecture, but it is unclear how they might be coordinated. Here, we demonstrate that the cell-cell adhesion protein E-cadherin functions as an instructive cue for cell division orientation. This is

  18. Stem Cells

    Science.gov (United States)

    Stem cells are cells with the potential to develop into many different types of cells in the body. ... the body. There are two main types of stem cells: embryonic stem cells and adult stem cells. Stem ...

  19. Inflammatory Cell Distribution in Primary Merkel Cell Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Wheat, Rachel [School of Cancer Sciences and CR UK Centre for Cancer Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT (United Kingdom); Roberts, Claudia [School of Cancer Sciences and CR UK Centre for Cancer Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT (United Kingdom); University Hospitals Birmingham NHS Foundation Trust, New Queen Elizabeth Hospital Birmingham, Mindelsohn Way, Edgbaston, Birmingham, B15 2WB (United Kingdom); Waterboer, Tim [Infection and Cancer Program, DKFZ (German Cancer Research Centre), 69120 Heidelberg (Germany); Steele, Jane [Human Biomaterials Resource Centre, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT (United Kingdom); Marsden, Jerry [University Hospitals Birmingham NHS Foundation Trust, New Queen Elizabeth Hospital Birmingham, Mindelsohn Way, Edgbaston, Birmingham, B15 2WB (United Kingdom); Steven, Neil M., E-mail: n.m.steven@bham.ac.uk [School of Cancer Sciences and CR UK Centre for Cancer Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT (United Kingdom); University Hospitals Birmingham NHS Foundation Trust, New Queen Elizabeth Hospital Birmingham, Mindelsohn Way, Edgbaston, Birmingham, B15 2WB (United Kingdom); Blackbourn, David J., E-mail: n.m.steven@bham.ac.uk [Department of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH (United Kingdom)

    2014-05-06

    Merkel cell carcinoma (MCC) is an aggressive poorly differentiated neuroendocrine cutaneous carcinoma associated with older age, immunodeficiency and Merkel cell polyomavirus (MCPyV) integrated within malignant cells. The presence of intra-tumoural CD8+ lymphocytes reportedly predicts better MCC-specific survival. In this study, the distribution of inflammatory cells and properties of CD8+ T lymphocytes within 20 primary MCC specimens were characterised using immunohistochemistry and multicolour immunofluorescent staining coupled to confocal microscopy. CD8+ cells and CD68+ macrophages were identified in 19/20 primary MCC. CD20+ B cells were present in 5/10, CD4+ cells in 10/10 and FoxP3+ cells in 7/10 specimens. Only two specimens had almost no inflammatory cells. Within specimens, inflammatory cells followed the same patchy distribution, focused at the edge of sheets and nodules and, in some cases, more intense in trabecular areas. CD8+ cells were outside vessels on the edge of tumour. Those few within malignant sheets typically lined up in fine septa not contacting MCC cells expressing MCPyV large T antigen. The homeostatic chemokine CXCL12 was expressed outside malignant nodules whereas its receptor CXCR4 was identified within tumour but not on CD8+ cells. CD8+ cells lacked CXCR3 and granzyme B expression irrespective of location within stroma versus malignant nodules or of the intensity of the intra-tumoural infiltrate. In summary, diverse inflammatory cells were organised around the margin of malignant deposits suggesting response to aberrant signaling, but were unable to penetrate the tumour microenvironment itself to enable an immune response against malignant cells or their polyomavirus.

  20. Inflammatory Cell Distribution in Primary Merkel Cell Carcinoma

    International Nuclear Information System (INIS)

    Wheat, Rachel; Roberts, Claudia; Waterboer, Tim; Steele, Jane; Marsden, Jerry; Steven, Neil M.; Blackbourn, David J.

    2014-01-01

    Merkel cell carcinoma (MCC) is an aggressive poorly differentiated neuroendocrine cutaneous carcinoma associated with older age, immunodeficiency and Merkel cell polyomavirus (MCPyV) integrated within malignant cells. The presence of intra-tumoural CD8+ lymphocytes reportedly predicts better MCC-specific survival. In this study, the distribution of inflammatory cells and properties of CD8+ T lymphocytes within 20 primary MCC specimens were characterised using immunohistochemistry and multicolour immunofluorescent staining coupled to confocal microscopy. CD8+ cells and CD68+ macrophages were identified in 19/20 primary MCC. CD20+ B cells were present in 5/10, CD4+ cells in 10/10 and FoxP3+ cells in 7/10 specimens. Only two specimens had almost no inflammatory cells. Within specimens, inflammatory cells followed the same patchy distribution, focused at the edge of sheets and nodules and, in some cases, more intense in trabecular areas. CD8+ cells were outside vessels on the edge of tumour. Those few within malignant sheets typically lined up in fine septa not contacting MCC cells expressing MCPyV large T antigen. The homeostatic chemokine CXCL12 was expressed outside malignant nodules whereas its receptor CXCR4 was identified within tumour but not on CD8+ cells. CD8+ cells lacked CXCR3 and granzyme B expression irrespective of location within stroma versus malignant nodules or of the intensity of the intra-tumoural infiltrate. In summary, diverse inflammatory cells were organised around the margin of malignant deposits suggesting response to aberrant signaling, but were unable to penetrate the tumour microenvironment itself to enable an immune response against malignant cells or their polyomavirus

  1. Skeletal Muscle Cell Induction from Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Yusaku Kodaka

    2017-01-01

    Full Text Available Embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs have the potential to differentiate into various types of cells including skeletal muscle cells. The approach of converting ESCs/iPSCs into skeletal muscle cells offers hope for patients afflicted with the skeletal muscle diseases such as the Duchenne muscular dystrophy (DMD. Patient-derived iPSCs are an especially ideal cell source to obtain an unlimited number of myogenic cells that escape immune rejection after engraftment. Currently, there are several approaches to induce differentiation of ESCs and iPSCs to skeletal muscle. A key to the generation of skeletal muscle cells from ESCs/iPSCs is the mimicking of embryonic mesodermal induction followed by myogenic induction. Thus, current approaches of skeletal muscle cell induction of ESCs/iPSCs utilize techniques including overexpression of myogenic transcription factors such as MyoD or Pax3, using small molecules to induce mesodermal cells followed by myogenic progenitor cells, and utilizing epigenetic myogenic memory existing in muscle cell-derived iPSCs. This review summarizes the current methods used in myogenic differentiation and highlights areas of recent improvement.

  2. Cell-cell transmission of VSV-G pseudotyped lentivector particles.

    Directory of Open Access Journals (Sweden)

    Amy M Skinner

    Full Text Available Many replicating viruses, including HIV-1 and HTLV-1, are efficiently transmitted from the cell surface of actively infected cells upon contact with bystander cells. In a previous study, we reported the prolonged cell surface retention of VSV-G replication-deficient pseudotyped lentivector prior to endocytic entry. However, the competing kinetics of cell surface versus dissociation, neutralization or direct transfer to other cells have received comparatively little attention. Here we demonstrate that the relative efficiency of cell-cell surface transmission can outpace "cell-free" transduction at limiting vector input. This coincides with the prolonged half-life of cell bound vector but occurs, unlike HTLV-1, without evidence for particle aggregation. These studies suggest that cell-surface attachment stabilizes particles and alters neutralization kinetics. Our experiments provide novel insight into the underexplored cell-cell transmission of pseudotyped particles.

  3. Chromosome aberrations and cell survival in irradiated mammalian cells

    International Nuclear Information System (INIS)

    Tremp, J.

    1981-01-01

    A possible correlation between chromosome aberrations and reduced proliferation capacity or cell death was investigated. Synchronized Chinese hamster fibroblast cells were irradiated with 300 rad of x rays in early G 1 . Despite synchronization the cells reached the subsequent mitosis at different times. The frequency of chromosome aberrations was determined in the postirradiation division at 2-h intervals. The highest frequency occurred in cells with a first cell cycle of medium length. The colony-forming ability of mitotic cells was measured in parallel samples by following the progress of individual mitoses. The proportion of cells forming macrocolonies decreased with increasing cell cycle length, and the number of non-colony-forming cells increased. Irrespective of various first cell cycle lengths and different frequencies of chromosome aberrations, the number of cells forming microcolonies remained constant. A correlation was found between the absence of chromosome aberrations and the ability of cells to form macrocolonies. However, cells with a long first cell cycle formed fewer macrocolonies than expected

  4. The role of Rap1 in cell-cell junction formation

    NARCIS (Netherlands)

    Kooistra, M.R.H.

    2008-01-01

    Both epithelial and endothelial cells form cell-cell junctions at the cell-cell contacts to maintain tissue integrity. Proper regulation of cell-cell junctions is required for the organisation of the tissue and to prevent leakage of blood vessels. In endothelial cells, the cell-cell junctions are

  5. Recipient dendritic cells, but not B cells, are required antigen-presenting cells for peripheral alloreactive CD8+ T-cell tolerance.

    Science.gov (United States)

    Mollov, J L; Lucas, C L; Haspot, F; Gaspar, J Kurtz C; Guzman, A; Sykes, M

    2010-03-01

    Induction of mixed allogeneic chimerism is a promising approach for achieving donor-specific tolerance, thereby obviating the need for life-long immunosuppression for solid organ allograft acceptance. In mice receiving a low dose (3Gy) of total body irradiation, allogeneic bone marrow transplantation combined with anti-CD154 tolerizes peripheral CD4 and CD8 T cells, allowing achievement of mixed chimerism with specific tolerance to donor. With this approach, peripheral CD8 T-cell tolerance requires recipient MHC class II, CD4 T cells, B cells and DCs. Recipient-type B cells from chimeras that were tolerant to donor still promoted CD8 T-cell tolerance, but their role could not be replaced by donor-type B cells. Using recipients whose B cells or DCs specifically lack MHC class I and/or class II or lack CD80 and CD86, we demonstrate that dendritic cells (DCs) must express CD80/86 and either MHC class I or class II to promote CD8 tolerance. In contrast, B cells, though required, did not need to express MHC class I or class II or CD80/86 to promote CD8 tolerance. Moreover, recipient IDO and IL-10 were not required. Thus, antigen presentation by recipient DCs and not by B cells is critical for peripheral alloreactive CD8 T cell tolerance.

  6. Pseudomonas aeruginosa Transmigrates at Epithelial Cell-Cell Junctions, Exploiting Sites of Cell Division and Senescent Cell Extrusion.

    Directory of Open Access Journals (Sweden)

    Guillaume Golovkine

    2016-01-01

    Full Text Available To achieve systemic infection, bacterial pathogens must overcome the critical and challenging step of transmigration across epithelial barriers. This is particularly true for opportunistic pathogens such as Pseudomonas aeruginosa, an agent which causes nosocomial infections. Despite extensive study, details on the mechanisms used by this bacterium to transmigrate across epithelial tissues, as well as the entry sites it uses, remain speculative. Here, using real-time microscopy and a model epithelial barrier, we show that P. aeruginosa employs a paracellular transmigration route, taking advantage of altered cell-cell junctions at sites of cell division or when senescent cells are expelled from the cell layer. Once a bacterium transmigrates, it is followed by a cohort of bacteria using the same entry point. The basal compartment is then invaded radially from the initial penetration site. Effective transmigration and propagation require type 4 pili, the type 3 secretion system (T3SS and a flagellum, although flagellum-deficient bacteria can occasionally invade the basal compartment from wounded areas. In the basal compartment, the bacteria inject the T3SS toxins into host cells, disrupting the cytoskeleton and focal contacts to allow their progression under the cells. Thus, P. aeruginosa exploits intrinsic host cell processes to breach the epithelium and invade the subcellular compartment.

  7. Stem Cell Therapy: Repurposing Cell-Based Regenerative Medicine Beyond Cell Replacement.

    Science.gov (United States)

    Napoli, Eleonora; Lippert, Trenton; Borlongan, Cesar V

    2018-02-27

    Stem cells exhibit simple and naive cellular features, yet their exact purpose for regenerative medicine continues to elude even the most elegantly designed research paradigms from developmental biology to clinical therapeutics. Based on their capacity to divide indefinitely and their dynamic differentiation into any type of tissue, the advent of transplantable stem cells has offered a potential treatment for aging-related and injury-mediated diseases. Recent laboratory evidence has demonstrated that transplanted human neural stem cells facilitate endogenous reparative mechanisms by initiating multiple regenerative processes in the brain neurogenic areas. Within these highly proliferative niches reside a myriad of potent regenerative molecules, including anti-inflammatory cytokines, proteomes, and neurotrophic factors, altogether representing a biochemical cocktail vital for restoring brain function in the aging and diseased brain. Here, we advance the concept of therapeutically repurposing stem cells not towards cell replacement per se, but rather exploiting the cells' intrinsic properties to serve as the host brain regenerative catalysts.

  8. Parallel and convergent processing in grid cell, head-direction cell, boundary cell, and place cell networks.

    Science.gov (United States)

    Brandon, Mark P; Koenig, Julie; Leutgeb, Stefan

    2014-03-01

    The brain is able to construct internal representations that correspond to external spatial coordinates. Such brain maps of the external spatial topography may support a number of cognitive functions, including navigation and memory. The neuronal building block of brain maps are place cells, which are found throughout the hippocampus of rodents and, in a lower proportion, primates. Place cells typically fire in one or few restricted areas of space, and each area where a cell fires can range, along the dorsoventral axis of the hippocampus, from 30 cm to at least several meters. The sensory processing streams that give rise to hippocampal place cells are not fully understood, but substantial progress has been made in characterizing the entorhinal cortex, which is the gateway between neocortical areas and the hippocampus. Entorhinal neurons have diverse spatial firing characteristics, and the different entorhinal cell types converge in the hippocampus to give rise to a single, spatially modulated cell type-the place cell. We therefore suggest that parallel information processing in different classes of cells-as is typically observed at lower levels of sensory processing-continues up into higher level association cortices, including those that provide the inputs to hippocampus. WIREs Cogn Sci 2014, 5:207-219. doi: 10.1002/wcs.1272 Conflict of interest: The authors have declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website. © 2013 John Wiley & Sons, Ltd.

  9. α-Mangostin Induces Apoptosis and Cell Cycle Arrest in Oral Squamous Cell Carcinoma Cell

    Directory of Open Access Journals (Sweden)

    Hyun-Ho Kwak

    2016-01-01

    Full Text Available Mangosteen has long been used as a traditional medicine and is known to have antibacterial, antioxidant, and anticancer effects. Although the effects of α-mangostin, a natural compound extracted from the pericarp of mangosteen, have been investigated in many studies, there is limited data on the effects of the compound in human oral squamous cell carcinoma (OSCC. In this study, α-mangostin was assessed as a potential anticancer agent against human OSCC cells. α-Mangostin inhibited cell proliferation and induced cell death in OSCC cells in a dose- and time-dependent manner with little to no effect on normal human PDLF cells. α-Mangostin treatment clearly showed apoptotic evidences such as nuclear fragmentation and accumulation of annexin V and PI-positive cells on OSCC cells. α-Mangostin treatment also caused the collapse of mitochondrial membrane potential and the translocation of cytochrome c from the mitochondria into the cytosol. The expressions of the mitochondria-related proteins were activated by α-mangostin. Treatment with α-mangostin also induced G1 phase arrest and downregulated cell cycle-related proteins (CDK/cyclin. Hence, α-mangostin specifically induces cell death and inhibits proliferation in OSCC cells via the intrinsic apoptosis pathway and cell cycle arrest at the G1 phase, suggesting that α-mangostin may be an effective agent for the treatment of OSCC.

  10. Two subpopulations of stem cells for T cell lineage

    International Nuclear Information System (INIS)

    Katsura, Y.; Amagai, T.; Kina, T.; Sado, T.; Nishikawa, S.

    1985-01-01

    An assay system for the stem cell that colonizes the thymus and differentiates into T cells was developed, and by using this assay system the existence of two subpopulations of stem cells for T cell lineage was clarified. Part-body-shielded and 900-R-irradiated C57BL/6 (H-2b, Thy-1.2) recipient mice, which do not require the transfer of pluripotent stem cells for their survival, were transferred with cells from B10 X Thy-1.1 (H-2b, Thy-1.1) donor mice. The reconstitution of the recipient's thymus lymphocytes was accomplished by stem cells in the donor cells and those spared in the shielded portion of the recipient that competitively colonize the thymus. Thus, the stem cell activity of donor cells can be evaluated by determining the proportion of donor-type (Thy-1.1+) cells in the recipient's thymus. Bone marrow cells were the most potent source of stem cells. By contrast, when the stem cell activity was compared between spleen and bone marrow cells of whole-body-irradiated (800 R) C57BL/6 mice reconstituted with B10 X Thy-1.1 bone marrow cells by assaying in part-body-shielded and irradiated C57BL/6 mice, the activity of these two organs showed quite a different time course of development. The results strongly suggest that the stem cells for T cell lineage in the bone marrow comprise at least two subpopulations, spleen-seeking and bone marrow-seeking cells

  11. Nanodiamond internalization in cells and the cell uptake mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Perevedentseva, E. [National Dong Hwa University, Department of Physics (China); Hong, S.-F.; Huang, K.-J. [National Dong Hwa University, Department of Life Sciences (China); Chiang, I.-T.; Lee, C.-Y. [National Dong Hwa University, Department of Physics (China); Tseng, Y.-T. [National Dong Hwa University, Department of Life Sciences (China); Cheng, C.-L., E-mail: clcheng@mail.ndhu.edu.tw [National Dong Hwa University, Department of Physics (China)

    2013-08-15

    Cell type-dependent penetration of nanodiamond in living cells is one of the important factors for using nanodiamond as cellular markers/labels, for drug delivery as well as for other biomedical applications. In this work, internalization of 100 nm nanodiamonds by A549 lung human adenocarcinoma cell, Beas-2b non-tumorigenic human bronchial epithelial cell, and HFL-1 fibroblast-like human fetal lung cell is studied and compared. The penetration of nanodiamond into the cells was observed using confocal fluorescence imaging and Raman imaging methods. Visualization of the nanodiamond in cells allows comparison of the internalization for diamond nanoparticles in cancer A549 cell, non-cancer HFL-1, and Beas-2b cells. The dose-dependent and time-dependent behavior of nanodiamond uptake is observed in both cancer as well as non-cancer cells. The mechanism of nanodiamond uptake by cancer and non-cancer cells is analyzed by blocking different pathways. The uptake of nanodiamond in both cancer and non-cancer cells was found predominantly via clathrin-dependent endocytosis. In spite of observed similarity in the uptake mechanism for cancer and non-cancer cells, the nanodiamond uptake for cancer cell quantitatively exceeds the uptake for non-cancer cells, for the studied cell lines. The observed difference in internalization of nanodiamond by cancer and non-cancer cells is discussed.

  12. Nanodiamond internalization in cells and the cell uptake mechanism

    International Nuclear Information System (INIS)

    Perevedentseva, E.; Hong, S.-F.; Huang, K.-J.; Chiang, I.-T.; Lee, C.-Y.; Tseng, Y.-T.; Cheng, C.-L.

    2013-01-01

    Cell type-dependent penetration of nanodiamond in living cells is one of the important factors for using nanodiamond as cellular markers/labels, for drug delivery as well as for other biomedical applications. In this work, internalization of 100 nm nanodiamonds by A549 lung human adenocarcinoma cell, Beas-2b non-tumorigenic human bronchial epithelial cell, and HFL-1 fibroblast-like human fetal lung cell is studied and compared. The penetration of nanodiamond into the cells was observed using confocal fluorescence imaging and Raman imaging methods. Visualization of the nanodiamond in cells allows comparison of the internalization for diamond nanoparticles in cancer A549 cell, non-cancer HFL-1, and Beas-2b cells. The dose-dependent and time-dependent behavior of nanodiamond uptake is observed in both cancer as well as non-cancer cells. The mechanism of nanodiamond uptake by cancer and non-cancer cells is analyzed by blocking different pathways. The uptake of nanodiamond in both cancer and non-cancer cells was found predominantly via clathrin-dependent endocytosis. In spite of observed similarity in the uptake mechanism for cancer and non-cancer cells, the nanodiamond uptake for cancer cell quantitatively exceeds the uptake for non-cancer cells, for the studied cell lines. The observed difference in internalization of nanodiamond by cancer and non-cancer cells is discussed

  13. CD34+ cells cultured in stem cell factor and interleukin-2 generate CD56+ cells with antiproliferative effects on tumor cell lines

    Directory of Open Access Journals (Sweden)

    Hensel Nancy

    2005-04-01

    Full Text Available Abstract In vitro stimulation of CD34+ cells with IL-2 induces NK cell differentiation. In order to define the stages of NK cell development, which influence their generation from CD34 cells, we cultured G-CSF mobilized peripheral blood CD34+ cells in the presence of stem cell factor and IL-2. After three weeks culture we found a diversity of CD56+ subsets which possessed granzyme A, but lacked the cytotoxic apparatus required for classical NK-like cytotoxicity. However, these CD56+ cells had the unusual property of inhibiting proliferation of K562 and P815 cell lines in a cell-contact dependent fashion.

  14. Pluripotent stem cells and reprogrammed cells in farm animals.

    Science.gov (United States)

    Nowak-Imialek, Monika; Kues, Wilfried; Carnwath, Joseph W; Niemann, Heiner

    2011-08-01

    Pluripotent cells are unique because of their ability to differentiate into the cell lineages forming the entire organism. True pluripotent stem cells with germ line contribution have been reported for mice and rats. Human pluripotent cells share numerous features of pluripotentiality, but confirmation of their in vivo capacity for germ line contribution is impossible due to ethical and legal restrictions. Progress toward derivation of embryonic stem cells from domestic species has been made, but the derived cells were not able to produce germ line chimeras and thus are termed embryonic stem-like cells. However, domestic animals, in particular the domestic pig (Sus scrofa), are excellent large animals models, in which the clinical potential of stem cell therapies can be studied. Reprogramming technologies for somatic cells, including somatic cell nuclear transfer, cell fusion, in vitro culture in the presence of cell extracts, in vitro conversion of adult unipotent spermatogonial stem cells into germ line derived pluripotent stem cells, and transduction with reprogramming factors have been developed with the goal of obtaining pluripotent, germ line competent stem cells from domestic animals. This review summarizes the present state of the art in the derivation and maintenance of pluripotent stem cells in domestic animals.

  15. Animal Models of Congenital Cardiomyopathies Associated With Mutations in Z-Line Proteins.

    Science.gov (United States)

    Bang, Marie-Louise

    2017-01-01

    The cardiac Z-line at the boundary between sarcomeres is a multiprotein complex connecting the contractile apparatus with the cytoskeleton and the extracellular matrix. The Z-line is important for efficient force generation and transmission as well as the maintenance of structural stability and integrity. Furthermore, it is a nodal point for intracellular signaling, in particular mechanosensing and mechanotransduction. Mutations in various genes encoding Z-line proteins have been associated with different cardiomyopathies, including dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, restrictive cardiomyopathy, and left ventricular noncompaction, and mutations even within the same gene can cause widely different pathologies. Animal models have contributed to a great advancement in the understanding of the physiological function of Z-line proteins and the pathways leading from mutations in Z-line proteins to cardiomyopathy, although genotype-phenotype prediction remains a great challenge. This review presents an overview of the currently available animal models for Z-line and Z-line associated proteins involved in human cardiomyopathies with special emphasis on knock-in and transgenic mouse models recapitulating the clinical phenotypes of human cardiomyopathy patients carrying mutations in Z-line proteins. Pros and cons of mouse models will be discussed and a future outlook will be given. J. Cell. Physiol. 232: 38-52, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  16. Extracorporeal shockwaves as regenerative therapy in orthopedic traumatology: a narrative review from basic research to clinical practice.

    Science.gov (United States)

    D Agostino, M C; Frairia, R; Romeo, P; Amelio, E; Berta, L; Bosco, V; Gigliotti, S; Guerra, C; Messina, S; Messuri, L; Moretti, B; Notarnicola, A; Maccagnano, G; Russo, S; Saggini, R; Vulpiani, M C; Buselli, P

    2016-01-01

    Extracorporeal Shock Wave Therapy (ESWT), after its first medical application in the urological field for lithotripsy, nowadays represents a valid therapeutical tool also for many musculoskeletal diseases, as well as for regenerative medicine applications. This is possible thanks to its mechanisms of action, which in the non-urological field are not related to mechanical disruption (as for renal stones), but rather to the capacity, by mechanotransduction, to induce neoangiogenesis, osteogenesis and to improve local tissue trophism, regeneration and remodeling, through stem cell stimulation. On the basis of these biological assumptions, it becomes clear that ESWT can represent a valid therapeutic tool also for all those pathological conditions that derive from musculoskeletal trauma, and are characterized by tissue loss and/or delayed healing and regeneration (mainly bone and skin, but not only). As a safe, repeatable and non–invasive therapy, in many cases it can represent a first–line therapeutic option, as an alternative to surgery (for example, in bone and skin healing disorders), or in combination with some other treatment options. It is hoped that with its use in daily practice also the muscle–skeletal field will grow, not only for standard indications, but also in post–traumatic sequelae, in order to improve recovery and shorten healing time, with undoubted advantages for the patients and lower health service expenses.

  17. Possible role of mechanical force in regulating regeneration of the vascularized fat flap inside a tissue engineering chamber.

    Science.gov (United States)

    Ye, Yuan; Yuan, Yi; Lu, Feng; Gao, Jianhua

    2015-12-01

    In plastic and reconstructive surgery, adipose tissue is widely used as effective filler for tissue defects. Strategies for treating soft tissue deficiency, which include free adipose tissue grafts, use of hyaluronic acid, collagen injections, and implantation of synthetic materials, have several clinical limitations. With the aim of overcoming these limitations, researchers have recently utilized tissue engineering chambers to produce large volumes of engineered vascularized fat tissue. However, the process of growing fat tissue in a chamber is still relatively limited, and can result in unpredictable or dissatisfactory final tissue volumes. Therefore, detailed understanding of the process is both necessary and urgent. Many studies have shown that mechanical force can change the function of cells via mechanotransduction. Here, we hypothesized that, besides the inflammatory response, one of the key factors to control the regeneration of vascularized fat flap inside a tissue engineering chamber might be the balance of mechanical forces. To test our hypothesis, we intend to change the balance of forces by means of measures in order to make the equilibrium point in favor of the direction of regeneration. If those measures proved to be feasible, they could be applied in clinical practice to engineer vascularized adipose tissue of predictable size and shape, which would in turn help in the advancement of tissue engineering. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Mechanosensitive channels: feeling tension in a world under pressure

    Directory of Open Access Journals (Sweden)

    Peyronnet eRemi

    2014-10-01

    Full Text Available Plants, like other organisms, are facing multiple mechanical constraints generated both in their tissues and by the surrounding environment. They need to sense and adapt to these forces throughout their lifetime. To do so, different mechanisms devoted to force transduction have emerged. Here we focus on fascinating proteins: the mechanosenstive (MS channels. Mechanosensing in plants has been described for centuries but the molecular identification of MS channels occurred only recently. This review is aimed at plant biologists and plant biomechanists who want to be introduced to MS channel identity, how do they work and what they might do in planta? In this review, electrophysiological properties, regulations and functions of well characterized MS channels belonging to bacteria and animals are compared with those of plant . Common and specific properties are discussed. We deduce which tools and concepts from animal and bacterial fields could be helpful for improving our understanding of plant mechanotransduction. MS channel embedded in its plasma membrane is sandwiched between the cell wall and the cytoskeleton. The consequences of this peculiar situation are analyzed and discussed. We also stress how important it is to probe mechanical forces at cellular and subcellular levels in planta in order to reveal the intimate relationship linking the membrane with MS channel activity. Finally we will propose new tracks to help to reveal their physiological functions at tissue and plant levels.

  19. Epithelial cell polarity, stem cells and cancer

    DEFF Research Database (Denmark)

    Martin-Belmonte, Fernando; Perez-Moreno, Mirna

    2011-01-01

    , deregulation of adhesion and polarity proteins can cause misoriented cell divisions and increased self-renewal of adult epithelial stem cells. In this Review, we highlight some advances in the understanding of how loss of epithelial cell polarity contributes to tumorigenesis.......After years of extensive scientific discovery much has been learned about the networks that regulate epithelial homeostasis. Loss of expression or functional activity of cell adhesion and cell polarity proteins (including the PAR, crumbs (CRB) and scribble (SCRIB) complexes) is intricately related...

  20. Regulatory T cells and B cells: implication on autoimmune diseases

    OpenAIRE

    Wang, Ping; Zheng, Song Guo

    2013-01-01

    The regulatory T (Treg) cells play an important role in the maintenance of homeostasis and the prevention of autoimmune diseases. Although most studies are focusing on the role of Treg cells in T cells and T cells-mediated diseases, these cells also directly affect B cells and other non-T cells. This manuscript updates the role of Treg cells on the B cells and B cell-mediated diseases. In addition, the mechanisms whereby Treg cells suppress B cell responses have been discussed.

  1. Sertoli cells maintain Leydig cell number and peritubular myoid cell activity in the adult mouse testis.

    Directory of Open Access Journals (Sweden)

    Diane Rebourcet

    Full Text Available The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.

  2. Selection of radioresistant cells by vitamin A deficiency in a small cell lung cancer cell line

    International Nuclear Information System (INIS)

    Terasaki, Takeo; Shimosato, Yukio; Wada, Makio; Yokota, Jun; Terada, Masaaki

    1990-01-01

    Radiation sensitivity of a human small cell lung cancer cell line, Lu-134-B cells, cultured in serum-supplemented medium and of cells transferred to and cultured in delipidized serum-supplemented (vitamin A-deficient) medium was studied. The cells cultured in serum-supplemented medium showed the phenotype of classic small cell lung cancer sensitive to radiation, while cells transferred to delipidized serum-supplemented medium showed partial squamous cell differentiation and became resistant to radiation. These results suggest that some small cell lung cancer cells in vitro change their morphology and radiosensitivity depending on the culture conditions. The change in radiosensitivity was reproducible, and was not reversible by culture of the radioresistant cells in delipidized serum-supplemented medium with addition of retinoic acid (vitamin A-sufficient medium) for two months, although squamous cells disappeared. Acquisition of radioresistancy was considered to occur as the result of clonal selective growth in delipidized medium of a minor cell population in the original cell culture, based on a study of chromosome number. It was also found that there was no association of myc-family oncogenes with the changes of radiosensitivity in this cell line. (author)

  3. Mast-Cell-Derived TNF Amplifies CD8+ Dendritic Cell Functionality and CD8+ T Cell Priming

    Directory of Open Access Journals (Sweden)

    Jan Dudeck

    2015-10-01

    Full Text Available Mast cells are critical promoters of adaptive immunity in the contact hypersensitivity model, but the mechanism of allergen sensitization is poorly understood. Using Mcpt5-CreTNFFL/FL mice, we show here that the absence of TNF exclusively in mast cells impaired the expansion of CD8+ T cells upon sensitization and the T-cell-driven adaptive immune response to elicitation. T cells primed in the absence of mast cell TNF exhibited a diminished efficiency to transfer sensitization to naive recipients. Specifically, mast cell TNF promotes CD8+ dendritic cell (DC maturation and migration to draining lymph nodes. The peripherally released mast cell TNF further critically boosts the CD8+ T-cell-priming efficiency of CD8+ DCs, thereby linking mast cell effects on T cells to DC modulation. Collectively, our findings identify the distinct potential of mast cell TNF to amplify CD8+ DC functionality and CD8+ T-cell-dominated adaptive immunity, which may be of great importance for immunotherapy and vaccination approaches.

  4. The Emerging Cell Biology of Thyroid Stem Cells

    Science.gov (United States)

    Latif, Rauf; Minsky, Noga C.; Ma, Risheng

    2011-01-01

    Context: Stem cells are undifferentiated cells with the property of self-renewal and give rise to highly specialized cells under appropriate local conditions. The use of stem cells in regenerative medicine holds great promise for the treatment of many diseases, including those of the thyroid gland. Evidence Acquisition: This review focuses on the progress that has been made in thyroid stem cell research including an overview of cellular and molecular events (most of which were drawn from the period 1990–2011) and discusses the remaining problems encountered in their differentiation. Evidence Synthesis: Protocols for the in vitro differentiation of embryonic stem cells, based on normal developmental processes, have generated thyroid-like cells but without full thyrocyte function. However, agents have been identified, including activin A, insulin, and IGF-I, which are able to stimulate the generation of thyroid-like cells in vitro. In addition, thyroid stem/progenitor cells have been identified within the normal thyroid gland and within thyroid cancers. Conclusions: Advances in thyroid stem cell biology are providing not only insight into thyroid development but may offer therapeutic potential in thyroid cancer and future thyroid cell replacement therapy. PMID:21778219

  5. Oral epithelial cells are susceptible to cell-free and cell-associated HIV-1 infection in vitro

    International Nuclear Information System (INIS)

    Moore, Jennifer S.; Rahemtulla, Firoz; Kent, Leigh W.; Hall, Stacy D.; Ikizler, Mine R.; Wright, Peter F.; Nguyen, Huan H.; Jackson, Susan

    2003-01-01

    Epithelial cells lining the oral cavity are exposed to HIV-1 through breast-feeding and oral-genital contact. Genital secretions and breast milk of HIV-1-infected subjects contain both cell-free and cell-associated virus. To determine if oral epithelial cells can be infected with HIV-1 we exposed gingival keratinocytes and adenoid epithelial cells to cell-free virus and HIV-1-infected peripheral blood mononuclear cells and monocytes. Using primary isolates we determined that gingival keratinocytes are susceptible to HIV-1 infection via cell-free CD4-independent infection only. R5 but not X4 viral strains were capable of infecting the keratinocytes. Further, infected cells were able to release infectious virus. In addition, primary epithelial cells isolated from adenoids were also susceptible to infection; both cell-free and cell-associated virus infected these cells. These data have potential implications in the transmission of HIV-1 in the oral cavity

  6. Nonmalignant T cells stimulate growth of T-cell lymphoma cells in the presence of bacterial toxins

    DEFF Research Database (Denmark)

    Woetmann, Anders; Lovato, Paola; Eriksen, Karsten W

    2007-01-01

    Bacterial toxins including staphylococcal enterotoxins (SEs) have been implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCLs). Here, we investigate SE-mediated interactions between nonmalignant T cells and malignant T-cell lines established from skin and blood of CTCL patients....... The malignant CTCL cells express MHC class II molecules that are high-affinity receptors for SE. Although treatment with SE has no direct effect on the growth of the malignant CTCL cells, the SE-treated CTCL cells induce vigorous proliferation of the SE-responsive nonmalignant T cells. In turn, the nonmalignant...... T cells enhance proliferation of the malignant cells in an SE- and MHC class II-dependent manner. Furthermore, SE and, in addition, alloantigen presentation by malignant CTCL cells to irradiated nonmalignant CD4(+) T-cell lines also enhance proliferation of the malignant cells. The growth...

  7. The CEA−/lo colorectal cancer cell population harbors cancer stem cells and metastatic cells

    Science.gov (United States)

    Zhang, Bo; Mu, Lei; Huang, Kaiyu; Zhao, Hui; Ma, Chensen; Li, Xiaolan; Tao, Deding; Gong, Jianping; Qin, Jichao

    2016-01-01

    Serum carcinoembryonic antigen (CEA) is the most commonly used tumor marker in a variety of cancers including colorectal cancer (CRC) for tumor diagnosis and monitoring. Recent studies have shown that colonic crypt cells expressing little or no CEA may enrich for stem cells. Numerous studies have clearly shown that there exist CRC patients with normal serum CEA levels during tumor progression or even tumor relapse, although CEA itself is considered to promote metastasis and block cell differentiation. These seemingly contradictory observations prompted us to investigate, herein, the biological properties as well as tumorigenic and metastatic capacity of CRC cells that express high (CEA+) versus low CEA (CEA−/lo) levels of CEA. Our findings show that the abundance of CEA−/lo cells correlate with poor differentiation and poor prognosis, and moreover, CEA−/lo cells form more spheres in vitro, generate more tumors and exhibit a higher potential in developing liver and lung metastases than corresponding CEA+ cells. Applying RNAi-mediated approach, we found that IGF1R mediated tumorigenic and capacity of CEA−/lo cells but did not mediate those of CEA+ cells. Notably, our data demonstrated that CEA molecule was capable of protecting CEA−/lo cells from anoikis, implying that CEA+ cells, although themselves possessing less tumorigenic and metastatic capacity, may promote metastasis of CEA−/lo cells via secreting CEA molecule. Our observations suggest that, besides targeting CEA molecule, CEA−/lo cells may represent a critical source of tumor progression and metastasis, and should therefore be the target of future therapies. PMID:27813496

  8. Basal cell carcinoma of the skin with areas of squamous cell carcinoma: a basosquamous cell carcinoma?

    OpenAIRE

    de Faria, J

    1985-01-01

    The diagnosis of basosquamous cell carcinoma is controversial. A review of cases of basal cell carcinoma showed 23 cases that had conspicuous areas of squamous cell carcinoma. This was distinguished from squamous differentiation and keratotic basal cell carcinoma by a comparative study of 40 cases of compact lobular and 40 cases of keratotic basal cell carcinoma. Areas of intermediate tumour differentiation between basal cell and squamous cell carcinoma were found. Basal cell carcinomas with ...

  9. The cell biology of T-dependent B cell activation

    DEFF Research Database (Denmark)

    Owens, T; Zeine, R

    1989-01-01

    The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...... includes multipoint intermolecular interactions that probably involve aggregation of both polymorphic and monomorphic T cell surface molecules. Such aggregations have been shown in vitro to markedly enhance and, in some cases, induce T cell activation. The production of T-derived lymphokines that have been...... implicated in B cell activation is dependent on the T cell receptor for antigen and its associated CD3 signalling complex. T-dependent help for B cell activation is therefore similarly MHC-restricted and involves T-B intercellular interaction. Recent reports that describe antigen-independent B cell...

  10. Activation of glioma cells generates immune tolerant NKT cells.

    Science.gov (United States)

    Tang, Bo; Wu, Wei; Wei, Xiaowei; Li, Yang; Ren, Gang; Fan, Wenhai

    2014-12-12

    Therapeutic outcomes of glioma are currently not encouraging. Tumor tolerance plays an important role in the pathogenesis of glioma. It is reported that micro RNAs (miR) are associated with tumor development. This study aims to investigate the role of miR-92a in the development of tolerant natural killer T (NKT) cells. In this study, U87 cells (a human glioma cell line) and primary glioma cells were prepared. The assessment of miR-92a was performed by real time RT-PCR. The expression of interleukin (IL)-10 and IL-6 in NKT cells was evaluated by flow cytometry. Results showed that abundant IL-6(+) IL-10(+) NKT cells were detected in glioma tissue. Cultures of glioma cells and NKT cells induced the expression of IL-6 and IL-10 in NKT cells. Glioma cells expressed miR-92a; the latter played a critical role in the induction of IL-6 and IL-10 expression in NKT cells. The expression of the antitumor molecules, including perforin, Fas ligand, and interferon-γ, was significantly attenuated compared with control NKT cells. The IL-6(+) IL-10(+) NKT cells showed less capability in the induction of apoptosis in glioma cells, but showed the immune suppressor functions on CD8(+) T cell activities. We conclude that glioma-derived miR-92a induces IL-6(+) IL-10(+) NKT cells; this fraction of NKT cells can suppress cytotoxic CD8(+) T cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Metformin inhibits cell cycle progression of B-cell chronic lymphocytic leukemia cells.

    Science.gov (United States)

    Bruno, Silvia; Ledda, Bernardetta; Tenca, Claudya; Ravera, Silvia; Orengo, Anna Maria; Mazzarello, Andrea Nicola; Pesenti, Elisa; Casciaro, Salvatore; Racchi, Omar; Ghiotto, Fabio; Marini, Cecilia; Sambuceti, Gianmario; DeCensi, Andrea; Fais, Franco

    2015-09-08

    B-cell chronic lymphocytic leukemia (CLL) was believed to result from clonal accumulation of resting apoptosis-resistant malignant B lymphocytes. However, it became increasingly clear that CLL cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. Drugs interfering with CLL cell cycle entry would be greatly beneficial in the treatment of this disease. 1, 1-Dimethylbiguanide hydrochloride (metformin), the most widely prescribed oral hypoglycemic agent, inexpensive and well tolerated, has recently received increased attention for its potential antitumor activity. We wondered whether metformin has apoptotic and anti-proliferative activity on leukemic cells derived from CLL patients. Metformin was administered in vitro either to quiescent cells or during CLL cell activation stimuli, provided by classical co-culturing with CD40L-expressing fibroblasts. At doses that were totally ineffective on normal lymphocytes, metformin induced apoptosis of quiescent CLL cells and inhibition of cell cycle entry when CLL were stimulated by CD40-CD40L ligation. This cytostatic effect was accompanied by decreased expression of survival- and proliferation-associated proteins, inhibition of signaling pathways involved in CLL disease progression and decreased intracellular glucose available for glycolysis. In drug combination experiments, metformin lowered the apoptotic threshold and potentiated the cytotoxic effects of classical and novel antitumor molecules. Our results indicate that, while CLL cells after stimulation are in the process of building their full survival and cycling armamentarium, the presence of metformin affects this process.

  12. Non-Small Cell Lung Cancer Cells Expressing CD44 Are Enriched for Stem Cell-Like Properties

    Science.gov (United States)

    Leung, Elaine Lai-Han; Fiscus, Ronald R.; Tung, James W.; Tin, Vicky Pui-Chi; Cheng, Lik Cheung; Sihoe, Alan Dart-Loon; Fink, Louis M.; Ma, Yupo; Wong, Maria Pik

    2010-01-01

    Background The cancer stem cell theory hypothesizes that cancers are perpetuated by cancer stem cells (CSC) or tumor initiating cells (TIC) possessing self-renewal and other stem cell-like properties while differentiated non-stem/initiating cells have a finite life span. To investigate whether the hypothesis is applicable to lung cancer, identification of lung CSC and demonstration of these capacities is essential. Methodology/Principal Finding The expression profiles of five stem cell markers (CD34, CD44, CD133, BMI1 and OCT4) were screened by flow cytometry in 10 lung cancer cell lines. CD44 was further investigated by testing for in vitro and in vivo tumorigenecity. Formation of spheroid bodies and in vivo tumor initiation ability were demonstrated in CD44+ cells of 4 cell lines. Serial in vivo tumor transplantability in nude mice was demonstrated using H1299 cell line. The primary xenografts initiated from CD44+ cells consisted of mixed CD44+ and CD44− cells in similar ratio as the parental H1299 cell line, supporting in vivo differentiation. Semi-quantitative Real-Time PCR (RT-PCR) showed that both freshly sorted CD44+ and CD44+ cells derived from CD44+-initiated tumors expressed the pluripotency genes OCT4/POU5F1, NANOG, SOX2. These stemness markers were not expressed by CD44− cells. Furthermore, freshly sorted CD44+ cells were more resistant to cisplatin treatment with lower apoptosis levels than CD44− cells. Immunohistochemical analysis of 141 resected non-small cell lung cancers showed tumor cell expression of CD44 in 50.4% of tumors while no CD34, and CD133 expression was observed in tumor cells. CD44 expression was associated with squamous cell carcinoma but unexpectedly, a longer survival was observed in CD44-expressing adenocarcinomas. Conclusion/Significance Overall, our results demonstrated that stem cell-like properties are enriched in CD44-expressing subpopulations of some lung cancer cell lines. Further investigation is required to clarify

  13. Satellite Cells and the Muscle Stem Cell Niche

    Science.gov (United States)

    Yin, Hang; Price, Feodor

    2013-01-01

    Adult skeletal muscle in mammals is a stable tissue under normal circumstances but has remarkable ability to repair after injury. Skeletal muscle regeneration is a highly orchestrated process involving the activation of various cellular and molecular responses. As skeletal muscle stem cells, satellite cells play an indispensible role in this process. The self-renewing proliferation of satellite cells not only maintains the stem cell population but also provides numerous myogenic cells, which proliferate, differentiate, fuse, and lead to new myofiber formation and reconstitution of a functional contractile apparatus. The complex behavior of satellite cells during skeletal muscle regeneration is tightly regulated through the dynamic interplay between intrinsic factors within satellite cells and extrinsic factors constituting the muscle stem cell niche/microenvironment. For the last half century, the advance of molecular biology, cell biology, and genetics has greatly improved our understanding of skeletal muscle biology. Here, we review some recent advances, with focuses on functions of satellite cells and their niche during the process of skeletal muscle regeneration. PMID:23303905

  14. TOPICAL REVIEW: Stem cells engineering for cell-based therapy

    Science.gov (United States)

    Taupin, Philippe

    2007-09-01

    Stem cells carry the promise to cure a broad range of diseases and injuries, from diabetes, heart and muscular diseases, to neurological diseases, disorders and injuries. Significant progresses have been made in stem cell research over the past decade; the derivation of embryonic stem cells (ESCs) from human tissues, the development of cloning technology by somatic cell nuclear transfer (SCNT) and the confirmation that neurogenesis occurs in the adult mammalian brain and that neural stem cells (NSCs) reside in the adult central nervous system (CNS), including that of humans. Despite these advances, there may be decades before stem cell research will translate into therapy. Stem cell research is also subject to ethical and political debates, controversies and legislation, which slow its progress. Cell engineering has proven successful in bringing genetic research to therapy. In this review, I will review, in two examples, how investigators are applying cell engineering to stem cell biology to circumvent stem cells' ethical and political constraints and bolster stem cell research and therapy.

  15. CellNet: Network Biology Applied to Stem Cell Engineering

    Science.gov (United States)

    Cahan, Patrick; Li, Hu; Morris, Samantha A.; da Rocha, Edroaldo Lummertz; Daley, George Q.; Collins, James J.

    2014-01-01

    SUMMARY Somatic cell reprogramming, directed differentiation of pluripotent stem cells, and direct conversions between differentiated cell lineages represent powerful approaches to engineer cells for research and regenerative medicine. We have developed CellNet, a network biology platform that more accurately assesses the fidelity of cellular engineering than existing methodologies and generates hypotheses for improving cell derivations. Analyzing expression data from 56 published reports, we found that cells derived via directed differentiation more closely resemble their in vivo counterparts than products of direct conversion, as reflected by the establishment of target cell-type gene regulatory networks (GRNs). Furthermore, we discovered that directly converted cells fail to adequately silence expression programs of the starting population, and that the establishment of unintended GRNs is common to virtually every cellular engineering paradigm. CellNet provides a platform for quantifying how closely engineered cell populations resemble their target cell type and a rational strategy to guide enhanced cellular engineering. PMID:25126793

  16. CellNet: network biology applied to stem cell engineering.

    Science.gov (United States)

    Cahan, Patrick; Li, Hu; Morris, Samantha A; Lummertz da Rocha, Edroaldo; Daley, George Q; Collins, James J

    2014-08-14

    Somatic cell reprogramming, directed differentiation of pluripotent stem cells, and direct conversions between differentiated cell lineages represent powerful approaches to engineer cells for research and regenerative medicine. We have developed CellNet, a network biology platform that more accurately assesses the fidelity of cellular engineering than existing methodologies and generates hypotheses for improving cell derivations. Analyzing expression data from 56 published reports, we found that cells derived via directed differentiation more closely resemble their in vivo counterparts than products of direct conversion, as reflected by the establishment of target cell-type gene regulatory networks (GRNs). Furthermore, we discovered that directly converted cells fail to adequately silence expression programs of the starting population and that the establishment of unintended GRNs is common to virtually every cellular engineering paradigm. CellNet provides a platform for quantifying how closely engineered cell populations resemble their target cell type and a rational strategy to guide enhanced cellular engineering. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

    Directory of Open Access Journals (Sweden)

    Tongfang Tang

    Full Text Available BACKGROUND: Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD through alternation of liver innate immune response. AIMS: The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. METHODS: Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. RESULTS: High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4 expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. CONCLUSION: High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

  18. Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

    Science.gov (United States)

    Tang, Tongfang; Sui, Yongheng; Lian, Min; Li, Zhiping; Hua, Jing

    2013-01-01

    Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD) through alternation of liver innate immune response. The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4) expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

  19. T cell-B cell interactions in primary immunodeficiencies.

    Science.gov (United States)

    Tangye, Stuart G; Deenick, Elissa K; Palendira, Umaimainthan; Ma, Cindy S

    2012-02-01

    Regulated interactions between cells of the immune system facilitate the generation of successful immune responses, thereby enabling efficient neutralization and clearance of pathogens and the establishment of both cell- and humoral-mediated immunological memory. The corollary of this is that impediments to efficient cell-cell interactions, normally necessary for differentiation and effector functions of immune cells, underly the clinical features and disease pathogenesis of primary immunodeficiencies. In affected individuals, these defects manifest as impaired long-term humoral immunity and susceptibility to infection by specific pathogens. In this review, we discuss the importance of, and requirements for, effective interactions between B cells and T cells during the formation of CD4(+) T follicular helper cells and the elicitation of cytotoxic function of virus-specific CD8(+) T cells, as well as how these processes are abrogated in primary immunodeficiencies due to loss-of-function mutations in defined genes. © 2012 New York Academy of Sciences.

  20. Are cancer cells really softer than normal cells?

    Science.gov (United States)

    Alibert, Charlotte; Goud, Bruno; Manneville, Jean-Baptiste

    2017-05-01

    Solid tumours are often first diagnosed by palpation, suggesting that the tumour is more rigid than its surrounding environment. Paradoxically, individual cancer cells appear to be softer than their healthy counterparts. In this review, we first list the physiological reasons indicating that cancer cells may be more deformable than normal cells. Next, we describe the biophysical tools that have been developed in recent years to characterise and model cancer cell mechanics. By reviewing the experimental studies that compared the mechanics of individual normal and cancer cells, we argue that cancer cells can indeed be considered as softer than normal cells. We then focus on the intracellular elements that could be responsible for the softening of cancer cells. Finally, we ask whether the mechanical differences between normal and cancer cells can be used as diagnostic or prognostic markers of cancer progression. © 2017 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

  1. Exosome-Based Cell-Cell Communication in the Tumor Microenvironment

    Directory of Open Access Journals (Sweden)

    Joana Maia

    2018-02-01

    Full Text Available Tumors are not isolated entities, but complex systemic networks involving cell-cell communication between transformed and non-transformed cells. The milieu created by tumor-associated cells may either support or halt tumor progression. In addition to cell-cell contact, cells communicate through secreted factors via a highly complex system involving characteristics such as ligand concentration, receptor expression and integration of diverse signaling pathways. Of these, extracellular vesicles, such as exosomes, are emerging as novel cell-cell communication mediators in physiological and pathological scenarios. Exosomes, membrane vesicles of endocytic origin released by all cells (both healthy and diseased, ranging in size from 30 to 150 nm, transport all the main biomolecules, including lipids, proteins, DNAs, messenger RNAs and microRNA, and perform intercellular transfer of components, locally and systemically. By acting not only in tumor cells, but also in tumor-associated cells such as fibroblasts, endothelium, leukocytes and progenitor cells, tumor- and non-tumor cells-derived exosomes have emerged as new players in tumor growth and invasion, tumor-associated angiogenesis, tissue inflammation and immunologic remodeling. In addition, due to their property of carrying molecules from their cell of origin to the peripheral circulation, exosomes have been increasingly studied as sources of tumor biomarkers in liquid biopsies. Here we review the current literature on the participation of exosomes in the communication between tumor and tumor-associated cells, highlighting the role of this process in the setup of tumor microenvironments that modulate tumor initiation and metastasis.

  2. Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

    Directory of Open Access Journals (Sweden)

    José R Sotelo

    Full Text Available To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells at the site of injury to promote regeneration.

  3. Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

    Science.gov (United States)

    Sotelo, José R; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A

    2013-01-01

    To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

  4. Cell fusion induced by ionizing radiation in various cell lines

    International Nuclear Information System (INIS)

    Khair, M.B.

    1994-07-01

    Cell fusion induced by ionizing radiation has been studied in rat's hepatocytes in vivo and in different cell lines in vitro. These cell lines were: Hela cells, V-79 fibroblasts, human and rat lymphocytes. For irradiation, 0.85 MeV fission neutrons and 14 MeV fast neutrons were used. Cell analyses were performed by fluorescent dyes using immunofluorescent microscope and flow cytometre. Our results in vivo showed that, regardless the dose-rate, a dose of 1 Gy approximately was enough to induce a significant level of cell fusion depending on neutron energy and the age of rats. The level of cell fusion was also significant in Hela cells at a dose of 0.5 Gy. Similar effect, but to a lesser extent, was observed in V-79 cells. Whereas, in lymphocytes insignificant cell fusion was noticed. The varying levels of cell-fusion in different cell lines could be attributed to the type of cells and mutual contact between cells. Furthermore irradiation did not show any influence on cell division ability in both hepatocytes and Hela cells and that fused cells were also able to divide forming a new generation of cells. (author). 36 refs., 8 figs., 10 tabs

  5. Cell proliferation alterations in Chlorella cells under stress conditions

    International Nuclear Information System (INIS)

    Rioboo, Carmen; O'Connor, Jose Enrique; Prado, Raquel; Herrero, Concepcion; Cid, Angeles

    2009-01-01

    Very little is known about growth and proliferation in relation to the cell cycle regulation of algae. The lack of knowledge is even greater when referring to the potential toxic effects of pollutants on microalgal cell division. To assess the effect of terbutryn, a triazine herbicide, on the proliferation of the freshwater microalga Chlorella vulgaris three flow cytometric approaches were used: (1) in vivo cell division using 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining was measured, (2) the growth kinetics were determined by cytometric cell counting and (3) cell viability was evaluated with the membrane-impermeable double-stranded nucleic acid stain propidium iodide (PI). The results obtained in the growth kinetics study using CFSE to identify the microalgal cell progeny were consistent with those determined by cytometric cell counting. In all C. vulgaris cultures, each mother cell had undergone only one round of division through the 96 h of assay and the cell division occurred during the dark period. Cell division of the cultures exposed to the herbicide was asynchronous. Terbutryn altered the normal number of daughter cells (4 autospores) obtained from each mother cell. The number was only two in the cultures treated with 250 nM. The duration of the lag phase after the exposure to terbutryn could be dependent on the existence of a critical cell size to activate cytoplasmic division. Cell size, complexity and fluorescence of chlorophyll a of the microalgal cells presented a marked light/dark (day/night) cycle, except in the non-dividing 500 nM cultures, where terbutryn arrested cell division at the beginning of the cycle. Viability results showed that terbutryn has an algastatic effect in C. vulgaris cells at this concentration. The rapid and precise determination of cell proliferation by CFSE staining has allowed us to develop a model for assessing both the cell cycle of C. vulgaris and the in vivo effects of pollutants on growth and

  6. Cell proliferation alterations in Chlorella cells under stress conditions

    Energy Technology Data Exchange (ETDEWEB)

    Rioboo, Carmen [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain); O' Connor, Jose Enrique [Laboratorio de Citomica, Unidad Mixta de Investigacion CIPF-UVEG, Centro de Investigacion Principe Felipe, Avda. Autopista del Saler, 16, 46013 Valencia (Spain); Prado, Raquel; Herrero, Concepcion [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain); Cid, Angeles, E-mail: cid@udc.es [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain)

    2009-09-14

    Very little is known about growth and proliferation in relation to the cell cycle regulation of algae. The lack of knowledge is even greater when referring to the potential toxic effects of pollutants on microalgal cell division. To assess the effect of terbutryn, a triazine herbicide, on the proliferation of the freshwater microalga Chlorella vulgaris three flow cytometric approaches were used: (1) in vivo cell division using 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining was measured, (2) the growth kinetics were determined by cytometric cell counting and (3) cell viability was evaluated with the membrane-impermeable double-stranded nucleic acid stain propidium iodide (PI). The results obtained in the growth kinetics study using CFSE to identify the microalgal cell progeny were consistent with those determined by cytometric cell counting. In all C. vulgaris cultures, each mother cell had undergone only one round of division through the 96 h of assay and the cell division occurred during the dark period. Cell division of the cultures exposed to the herbicide was asynchronous. Terbutryn altered the normal number of daughter cells (4 autospores) obtained from each mother cell. The number was only two in the cultures treated with 250 nM. The duration of the lag phase after the exposure to terbutryn could be dependent on the existence of a critical cell size to activate cytoplasmic division. Cell size, complexity and fluorescence of chlorophyll a of the microalgal cells presented a marked light/dark (day/night) cycle, except in the non-dividing 500 nM cultures, where terbutryn arrested cell division at the beginning of the cycle. Viability results showed that terbutryn has an algastatic effect in C. vulgaris cells at this concentration. The rapid and precise determination of cell proliferation by CFSE staining has allowed us to develop a model for assessing both the cell cycle of C. vulgaris and the in vivo effects of pollutants on growth and

  7. Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization.

    Science.gov (United States)

    Hazim, Roni A; Karumbayaram, Saravanan; Jiang, Mei; Dimashkie, Anupama; Lopes, Vanda S; Li, Douran; Burgess, Barry L; Vijayaraj, Preethi; Alva-Ornelas, Jackelyn A; Zack, Jerome A; Kohn, Donald B; Gomperts, Brigitte N; Pyle, April D; Lowry, William E; Williams, David S

    2017-10-02

    Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation. We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions. Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function. This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by

  8. Cell Cycle Regulation of Stem Cells by MicroRNAs.

    Science.gov (United States)

    Mens, Michelle M J; Ghanbari, Mohsen

    2018-06-01

    MicroRNAs (miRNAs) are a class of small non-coding RNA molecules involved in the regulation of gene expression. They are involved in the fine-tuning of fundamental biological processes such as proliferation, differentiation, survival and apoptosis in many cell types. Emerging evidence suggests that miRNAs regulate critical pathways involved in stem cell function. Several miRNAs have been suggested to target transcripts that directly or indirectly coordinate the cell cycle progression of stem cells. Moreover, previous studies have shown that altered expression levels of miRNAs can contribute to pathological conditions, such as cancer, due to the loss of cell cycle regulation. However, the precise mechanism underlying miRNA-mediated regulation of cell cycle in stem cells is still incompletely understood. In this review, we discuss current knowledge of miRNAs regulatory role in cell cycle progression of stem cells. We describe how specific miRNAs may control cell cycle associated molecules and checkpoints in embryonic, somatic and cancer stem cells. We further outline how these miRNAs could be regulated to influence cell cycle progression in stem cells as a potential clinical application.

  9. Tetherin restricts productive HIV-1 cell-to-cell transmission.

    Directory of Open Access Journals (Sweden)

    Nicoletta Casartelli

    2010-06-01

    Full Text Available The IFN-inducible antiviral protein tetherin (or BST-2/CD317/HM1.24 impairs release of mature HIV-1 particles from infected cells. HIV-1 Vpu antagonizes the effect of tetherin. The fate of virions trapped at the cell surface remains poorly understood. Here, we asked whether tetherin impairs HIV cell-to-cell transmission, a major means of viral spread. Tetherin-positive or -negative cells, infected with wild-type or DeltaVpu HIV, were used as donor cells and cocultivated with target lymphocytes. We show that tetherin inhibits productive cell-to-cell transmission of DeltaVpu to targets and impairs that of WT HIV. Tetherin accumulates with Gag at the contact zone between infected and target cells, but does not prevent the formation of virological synapses. In the presence of tetherin, viruses are then mostly transferred to targets as abnormally large patches. These viral aggregates do not efficiently promote infection after transfer, because they accumulate at the surface of target cells and are impaired in their fusion capacities. Tetherin, by imprinting virions in donor cells, is the first example of a surface restriction factor limiting viral cell-to-cell spread.

  10. Ion channels involved in cell volume regulation: effects on migration, proliferation, and programmed cell death in non adherent EAT cells and adherent ELA cells.

    Science.gov (United States)

    Hoffmann, Else Kay

    2011-01-01

    This mini review outlines studies of cell volume regulation in two closely related mammalian cell lines: nonadherent Ehrlich ascites tumour cells (EATC) and adherent Ehrlich Lettre ascites (ELA) cells. Focus is on the regulatory volume decrease (RVD) that occurs after cell swelling, the volume regulatory ion channels involved, and the mechanisms (cellular signalling pathways) that regulate these channels. Finally, I shall also briefly review current investigations in these two cell lines that focuses on how changes in cell volume can regulate cell functions such as cell migration, proliferation, and programmed cell death. Copyright © 2011 S. Karger AG, Basel.

  11. Cholesterol inhibits entotic cell-in-cell formation and actomyosin contraction.

    Science.gov (United States)

    Ruan, Banzhan; Zhang, Bo; Chen, Ang; Yuan, Long; Liang, Jianqing; Wang, Manna; Zhang, Zhengrong; Fan, Jie; Yu, Xiaochen; Zhang, Xin; Niu, Zubiao; Zheng, You; Gu, Songzhi; Liu, Xiaoqing; Du, Hongli; Wang, Jufang; Hu, Xianwen; Gao, Lihua; Chen, Zhaolie; Huang, Hongyan; Wang, Xiaoning; Sun, Qiang

    2018-01-01

    Cell-in-cell structure is prevalent in human cancer, and associated with several specific pathophysiological phenomena. Although cell membrane adhesion molecules were found critical for cell-in-cell formation, the roles of other membrane components, such as lipids, remain to be explored. In this study, we attempted to investigate the effects of cholesterol and phospholipids on the formation of cell-in-cell structures by utilizing liposome as a vector. We found that Lipofectamine-2000, the reagent commonly used for routine transfection, could significantly reduce entotic cell-in-cell formation in a cell-specific manner, which is correlated with suppressed actomyosin contraction as indicated by reduced β-actin expression and myosin light chain phosphorylation. The influence on cell-in-cell formation was likely dictated by specific liposome components as some liposomes affected cell-in-cell formation while some others didn't. Screening on a limited number of lipids, the major components of liposome, identified phosphatidylethanolamine (PE), stearamide (SA), lysophosphatidic acid (LPA) and cholesterol (CHOL) as the inhibitors of cell-in-cell formation. Importantly, cholesterol treatment significantly inhibited myosin light chain phosphorylation, which resembles the effect of Lipofectamine-2000, suggesting cholesterol might be partially responsible for liposomes' effects on cell-in-cell formation. Together, our findings supporting a role of membrane lipids and cholesterol in cell-in-cell formation probably via regulating actomyosin contraction. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Selfish cells in altruistic cell society - a theoretical oncology.

    Science.gov (United States)

    Chigira, M

    1993-09-01

    In multicellular organisms, internal evolution of individual cells is strictly forbidden and 'evolutional' DNA replication should be performed only by the sexual reproduction system. Wholistic negative control system called 'homeostasis' serves all service to germ line cells. All somatic cells are altruistic to the germ line cells. However, in malignant tumors, it seems that individual cells replicate and behave 'selfishly' and evolve against the internal microenvironment. Tumor cells only express the occult selfishness which is programmed in normal cells a priori. This phenomenon is based on the failure of identical DNA replication, and results in 'autonomy' and 'anomie' of cellular society as shown in tumor cells. Genetic programs of normal cells connote this cellular autonomy and anomie introduced by the deletion of regulators on structure genes. It is rather paradoxical that the somatic cells get their freedom from wholistic negative regulation programmed internally. However, this is not a true paradox, since multicellular organisms have clearly been evolved from 'monads' in which cells proliferate without wholistic regulation. Somatic cells revolt against germ cell DNA, called 'selfish replicator' by Dawkins. It is an inevitable destiny that the 'selfishness' coded in genome should be revenged by itself. Selfish replicator in germ cell line should be revolted by its selfishness in the expansion of somatic cells, since they have an orthogenesis to get more selfishness in order to increase their genome. Tumor heterogeneity and progression can be fully explained by this self-contradictory process which produces heterogeneous gene copies different from the original clone in the tumor, although 'selfish' gene replication is the final target of being. Furthermore, we have to discard the concept of clonality of tumor cells since genetic instability is a fundamental feature of tumors. Finally, tumor cells and proto-oncogenes can be considered as the ultimate parasite

  13. The Antigen Presenting Cells Instruct Plasma Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Wei eXu

    2014-01-01

    Full Text Available The professional antigen presenting cells (APCs, including many subsets of dendritic cells and macrophages, not only mediate prompt but nonspecific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells, which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only signal 1 (the antigen, but also signal 2 to directly instruct the differentiation process of plasma cells in a T cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

  14. Cell cycle variation in x-ray survival for cells from spheroids measured by volume cell sorting

    International Nuclear Information System (INIS)

    Freyer, J.P.; Wilder, M.E.; Raju, M.R.

    1984-01-01

    Considerable work has been done studying the variation in cell survival as a function of cell cycle position for monolayers or single cells exposed to radiation. Little is known about the effects of multicellular growth on the relative radiation sensitivity of cells in different cell cycle stages. The authors have developed a new technique for measuring the response of cells, using volume cell sorting, which is rapid, non-toxic, and does not require cell synchronization. By combining this technique with selective spheroid dissociation,they have measured the age response of cells located at various depths in EMT6 and Colon 26 spheroids. Although cells in the inner region had mostly G1-phase DNA contents, 15-20% had S- and G2-phase DNA contents. Analysis of these cells using BrdU labeling and flow cytometric analysis with a monoclonal antibody to BrdU indicated that the inner region cells were not synthesizing DNA. Thus, the authors were able to measure the radiation response of cells arrested in G1, S and G2 cell cycle phases. Comparison of inner and outer spheroid regions, and monolayer cultures, indicates that it is improper to extrapolate age response data in standard culture conditions to the situation in spheroids

  15. Combination cell therapy with mesenchymal stem cells and neural stem cells for brain stroke in rats.

    Science.gov (United States)

    Hosseini, Seyed Mojtaba; Farahmandnia, Mohammad; Razi, Zahra; Delavari, Somayeh; Shakibajahromi, Benafsheh; Sarvestani, Fatemeh Sabet; Kazemi, Sepehr; Semsar, Maryam

    2015-05-01

    Brain stroke is the second most important events that lead to disability and morbidity these days. Although, stroke is important, there is no treatment for curing this problem. Nowadays, cell therapy has opened a new window for treating central nervous system disease. In some previous studies the Mesenchymal stem cells and neural stem cells. In this study, we have designed an experiment to assess the combination cell therapy (Mesenchymal and Neural stem cells) effects on brain stroke. The Mesenchymal stem cells were isolated from adult rat bone marrow and the neural stem cells were isolated from ganglion eminence of rat embryo 14 days. The Mesenchymal stem cells were injected 1 day after middle cerebral artery occlusion (MCAO) and the neural stem cells transplanted 7 day after MCAO. After 28 days, the neurological outcomes and brain lesion volumes were evaluated. Also, the activity of Caspase 3 was assessed in different groups. The group which received combination cell therapy had better neurological examination and less brain lesion. Also the combination cell therapy group had the least Caspase 3 activity among the groups. The combination cell therapy is more effective than Mesenchymal stem cell therapy and neural stem cell therapy separately in treating the brain stroke in rats.

  16. Tissue-resident natural killer (NK) cells are cell lineages distinct from thymic and conventional splenic NK cells

    Science.gov (United States)

    Sojka, Dorothy K; Plougastel-Douglas, Beatrice; Yang, Liping; Pak-Wittel, Melissa A; Artyomov, Maxim N; Ivanova, Yulia; Zhong, Chao; Chase, Julie M; Rothman, Paul B; Yu, Jenny; Riley, Joan K; Zhu, Jinfang; Tian, Zhigang; Yokoyama, Wayne M

    2014-01-01

    Natural killer (NK) cells belong to the innate immune system; they can control virus infections and developing tumors by cytotoxicity and producing inflammatory cytokines. Most studies of mouse NK cells, however, have focused on conventional NK (cNK) cells in the spleen. Recently, we described two populations of liver NK cells, tissue-resident NK (trNK) cells and those resembling splenic cNK cells. However, their lineage relationship was unclear; trNK cells could be developing cNK cells, related to thymic NK cells, or a lineage distinct from both cNK and thymic NK cells. Herein we used detailed transcriptomic, flow cytometric, and functional analysis and transcription factor-deficient mice to determine that liver trNK cells form a distinct lineage from cNK and thymic NK cells. Taken together with analysis of trNK cells in other tissues, there are at least four distinct lineages of NK cells: cNK, thymic, liver (and skin) trNK, and uterine trNK cells. DOI: http://dx.doi.org/10.7554/eLife.01659.001 PMID:24714492

  17. The recruitability and cell-cycle state of intestinal stem cells

    International Nuclear Information System (INIS)

    Potten, C.S.; Chadwick, C.; Ijiri, K.; Tsubouchi, S.; Hanson, W.R.

    1984-01-01

    Evidence is presented which suggests that the crypts of the small intestine contain at least two discrete but interdependent classes of stem cells, some with discrete cell kinetic properties and some with discrete radiation responses or radiosensitivities. Very low doses of X rays or gamma rays, or neutrons, kill a few cells in the stem cell regions of the crypt in a sensitive dose-dependent manner. Similar doses generate several different cell kinetic responses within either the clonogenic fraction or the cells at the stem cell position within the crypt. The cell kinetic responses range from apparent recruitment of G0 clonogenic cells into cycle, to a marked shortening of the average cell cycle of the cells at the stem cell position. It is suggested that the cell kinetic changes may be the consequence of the cell destruction

  18. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

    Directory of Open Access Journals (Sweden)

    Chen Lei

    2011-06-01

    Full Text Available Abstract Background Cancer stem cells (CSCs are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44. Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

  19. Th17 cell-mediated immune responses promote mast cell proliferation by triggering stem cell factor in keratinocytes

    International Nuclear Information System (INIS)

    Cho, Kyung-Ah; Park, Minhwa; Kim, Yu-Hee; Woo, So-Youn

    2017-01-01

    Although mast cells are traditionally thought to function as effector cells in allergic responses, they have increasingly been recognized as important regulators of various immune responses. Mast cells mature locally; thus, tissue-specific influences are important for promoting mast cell accumulation and survival in the skin and the gastrointestinal tract. In this study, we determined the effects of keratinocytes on mast cell accumulation during Th17-mediated skin inflammation. We observed increases in dermal mast cells in imiquimod-induced psoriatic dermatitis in mice accompanied by the expression of epidermal stem cell factor (SCF), a critical mast cell growth factor. Similar to mouse epidermal keratinocytes, SCF was highly expressed in the human HaCaT keratinocyte cell line following stimulation with IL−17. Further, keratinocytes promoted mast cell proliferation following stimulation with IL−17 in vitro. However, the effects of keratinocytes on mast cells were significantly diminished in the presence of anti−CD117 (stem cell factor receptor) blocking antibodies. Taken together, our results revealed that the Th17-mediated inflammatory environment promotes mast cell accumulation through keratinocyte-derived SCF. - Highlights: • Psoriasis-like skin inflammation increase dermal mast cells. • Keratinocyte produce stem cell factor in psoriasis-like skin inflammation. • Keratinocyte promote mast cell proliferation by stem cell factor dependent manner

  20. NKp46+CD3+ cells - a novel non-conventional T-cell subset in cattle exhibiting both NK cell and T-cell features

    Science.gov (United States)

    Connelley, Timothy K.; Longhi, Cassandra; Burrells, Alison; Degnan, Kathryn; Hope, Jayne; Allan, Alasdair; Hammond, John A.; Storset, Anne K.; Morrison, W. Ivan

    2014-01-01

    The NKp46 receptor demonstrates a high degree of lineage-specificity, being expressed almost exclusively in natural killer cells. Previous studies have demonstrated NKp46 expression by T-cells, but NKp46+CD3+ cells are rare and almost universally associated with NKp46 acquisition by T-cells following stimulation. In this study we demonstrate the existence of a population of NKp46+CD3+ cells resident in normal bovine PBMC which include cells of both the αβ TCR+ and γδ TCR+ lineages and is present at a frequency of 0.1-1.7%. NKp46+CD3+ cells express transcripts for a broad repertoire of both natural killer (NKR) and T-cell receptors (TCR) and also the CD3ζ, DAP10 and FcεR1γ but not DAP12 adaptor proteins. In vitro functional analysis of NKp46+CD3+ cells confirm that NKp46, CD16 and CD3 signalling pathways are all functionally competent and capable of mediating-re-direct cytolysis. However, only CD3 cross-ligation elicits IFN-γ release. NKp46+CD3+ cells exhibit cytotoxic activity against autologous Theileria parva infected cells in vitro and during in vivo challenge with this parasite an expansion of NKp46+CD3+ cells was observed in some animals, indicating the cells have the potential to act as an anti-pathogen effector population. The results presented herein identifies and describes a novel non-conventional NKp46+CD3+ T-cell subset that is phenotypically and functionally distinct from conventional NK and T-cells. The ability to exploit both NKR and TCR suggests these cells may fill a functional niche at the interface of innate and adaptive immune responses. PMID:24639352

  1. Optimizing cell viability in droplet-based cell deposition

    NARCIS (Netherlands)

    Hendriks, Jan; Visser, C.W.; Henke, S.J.; Leijten, Jeroen Christianus Hermanus; Saris, Daniël B.F.; Sun, Chao; Lohse, Detlef; Karperien, Hermanus Bernardus Johannes

    2015-01-01

    Biofabrication commonly involves the use of liquid droplets to transport cells to the printed structure. However, the viability of the cells after impact is poorly controlled and understood, hampering applications including cell spraying, inkjet bioprinting, and laser-assisted cell transfer. Here,

  2. Pituitary cell differentiation from stem cells and other cells: toward restorative therapy for hypopituitarism?

    Science.gov (United States)

    Willems, Christophe; Vankelecom, Hugo

    2014-01-01

    The pituitary gland, key regulator of our endocrine system, produces multiple hormones that steer essential physiological processes. Hence, deficient pituitary function (hypopituitarism) leads to severe disorders. Hypopituitarism can be caused by defective embryonic development, or by damage through tumor growth/resection and traumatic brain injury. Lifelong hormone replacement is needed but associated with significant side effects. It would be more desirable to restore pituitary tissue and function. Recently, we showed that the adult (mouse) pituitary holds regenerative capacity in which local stem cells are involved. Repair of deficient pituitary may therefore be achieved by activating these resident stem cells. Alternatively, pituitary dysfunction may be mended by cell (replacement) therapy. The hormonal cells to be transplanted could be obtained by (trans-)differentiating various kinds of stem cells or other cells. Here, we summarize the studies on pituitary cell regeneration and on (trans-)differentiation toward hormonal cells, and speculate on restorative therapies for pituitary deficiency.

  3. Single-Cell RNA Sequencing of Glioblastoma Cells.

    Science.gov (United States)

    Sen, Rajeev; Dolgalev, Igor; Bayin, N Sumru; Heguy, Adriana; Tsirigos, Aris; Placantonakis, Dimitris G

    2018-01-01

    Single-cell RNA sequencing (sc-RNASeq) is a recently developed technique used to evaluate the transcriptome of individual cells. As opposed to conventional RNASeq in which entire populations are sequenced in bulk, sc-RNASeq can be beneficial when trying to better understand gene expression patterns in markedly heterogeneous populations of cells or when trying to identify transcriptional signatures of rare cells that may be underrepresented when using conventional bulk RNASeq. In this method, we describe the generation and analysis of cDNA libraries from single patient-derived glioblastoma cells using the C1 Fluidigm system. The protocol details the use of the C1 integrated fluidics circuit (IFC) for capturing, imaging and lysing cells; performing reverse transcription; and generating cDNA libraries that are ready for sequencing and analysis.

  4. Como a célula óssea reconhece o estímulo mecânico? How do bone cells sense mechanical loading?

    Directory of Open Access Journals (Sweden)

    Carlos Vinícius Buarque de Gusmão

    2009-01-01

    . Activities resulting in impact are particularly known to stimulate osteogenesis, thus reducing bone mass loss. Knowing how bone cells recognize the mechanical deformation imposed to the bone and trigger a series of biochemical chain reactions is of crucial importance for the development of therapeutic and preventive practices in orthopaedic activity. There is still a long way to run until we can understand the whole process, but current knowledge has shown a strong progression, with researches being conducted focused on therapies. For a mechanical sign to be transformed into a biological one (mechanotransduction, it must be amplified at cell level by the histological structure of bone tissue, producing tensions in cell membrane proteins (integrins and changing their spatial structure. Such change activates bindings between these and the cytoskeleton, producing focal adhesions, where cytoplasmatic proteins are recruited to enable easier biochemical reactions. Focal adhesion kinase (FAK is the most important one being self-activated when its structure is changed by integrins. Activated FAK triggers a cascade of reactions, resulting in the activation of ERK-1/2 and Akt, which are proteins that, together with FAK, regulate the production of bone mass. Osteocytes are believed to be the mechanosensor cells of the bone and to transmit the mechanical deformation to osteoblasts and osteoclasts. Ionic channels and gap junctions are considered as intercellular communication means for biochemical transmission of a mechanical stimulus. These events occur continuously on bone tissue and regulate bone remodeling.

  5. Relation Between the Cell Volume and the Cell Cycle Dynamics in Mammalian cell

    International Nuclear Information System (INIS)

    Magno, A.C.G.; Oliveira, I.L.; Hauck, J.V.S.

    2016-01-01

    The main goal of this work is to add and analyze an equation that represents the volume in a dynamical model of the mammalian cell cycle proposed by Gérard and Goldbeter (2011) [1]. The cell division occurs when the cyclinB/Cdkl complex is totally degraded (Tyson and Novak, 2011)[2] and it reaches a minimum value. At this point, the cell is divided into two newborn daughter cells and each one will contain the half of the cytoplasmic content of the mother cell. The equations of our base model are only valid if the cell volume, where the reactions occur, is constant. Whether the cell volume is not constant, that is, the rate of change of its volume with respect to time is explicitly taken into account in the mathematical model, then the equations of the original model are no longer valid. Therefore, every equations were modified from the mass conservation principle for considering a volume that changes with time. Through this approach, the cell volume affects all model variables. Two different dynamic simulation methods were accomplished: deterministic and stochastic. In the stochastic simulation, the volume affects every model's parameters which have molar unit, whereas in the deterministic one, it is incorporated into the differential equations. In deterministic simulation, the biochemical species may be in concentration units, while in stochastic simulation such species must be converted to number of molecules which are directly proportional to the cell volume. In an effort to understand the influence of the new equation a stability analysis was performed. This elucidates how the growth factor impacts the stability of the model's limit cycles. In conclusion, a more precise model, in comparison to the base model, was created for the cell cycle as it now takes into consideration the cell volume variation (paper)

  6. Cell-substrate interaction with cell-membrane-stress dependent adhesion.

    Science.gov (United States)

    Jiang, H; Yang, B

    2012-01-10

    Cell-substrate interaction is examined in a two-dimensional mechanics model. The cell and substrate are treated as a shell and an elastic solid, respectively. Their interaction through adhesion is treated using nonlinear springs. Compared to previous cell mechanics models, the present model introduces a cohesive force law that is dependent not only on cell-substrate distance but also on internal cell-membrane stress. It is postulated that a living cell would establish focal adhesion sites with density dependent on the cell-membrane stress. The formulated mechanics problem is numerically solved using coupled finite elements and boundary elements for the cell and the substrate, respectively. The nodes in the adhesion zone from either side are linked by the cohesive springs. The specific cases of a cell adhering to a homogeneous substrate and a heterogeneous bimaterial substrate are examined. The analyses show that the substrate stiffness affects the adhesion behavior significantly and regulates the direction of cell adhesion, in good agreement with the experimental results in the literature. By introducing a reactive parameter (i.e., cell-membrane stress) linking biological responses of a living cell to a mechanical environment, the present model offers a unified mechanistic vehicle for characterization and prediction of living cell responses to various kinds of mechanical stimuli including local extracellular matrix and neighboring cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Dendritic cells fused with different pancreatic carcinoma cells induce different T-cell responses

    Directory of Open Access Journals (Sweden)

    Andoh Y

    2013-01-01

    Full Text Available Yoshiaki Andoh,1,2 Naohiko Makino,2 Mitsunori Yamakawa11Department of Pathological Diagnostics, 2Department of Gastroenterology, Yamagata University School of Medicine, Yamagata, JapanBackground: It is unclear whether there are any differences in the induction of cytotoxic T lymphocytes (CTL and CD4+CD25high regulatory T-cells (Tregs among dendritic cells (DCs fused with different pancreatic carcinomas. The aim of this study was to compare the ability to induce cytotoxicity by human DCs fused with different human pancreatic carcinoma cell lines and to elucidate the causes of variable cytotoxicity among cell lines.Methods: Monocyte-derived DCs, which were generated from peripheral blood mononuclear cells (PBMCs, were fused with carcinoma cells such as Panc-1, KP-1NL, QGP-1, and KP-3L. The induction of CTL and Tregs, and cytokine profile of PBMCs stimulated by fused DCs were evaluated.Results: The cytotoxicity against tumor targets induced by PBMCs cocultured with DCs fused with QGP-1 (DC/QGP-1 was very low, even though PBMCs cocultured with DCs fused with other cell lines induced significant cytotoxicity against the respective tumor target. The factors causing this low cytotoxicity were subsequently investigated. DC/QGP-1 induced a significant expansion of Tregs in cocultured PBMCs compared with DC/KP-3L. The level of interleukin-10 secreted in the supernatants of PBMCs cocultured with DC/QGP-1 was increased significantly compared with that in DC/KP-3L. Downregulation of major histocompatibility complex class I expression and increased secretion of vascular endothelial growth factor were observed with QGP-1, as well as in the other cell lines.Conclusion: The present study demonstrated that the cytotoxicity induced by DCs fused with pancreatic cancer cell lines was different between each cell line, and that the reduced cytotoxicity of DC/QGP-1 might be related to the increased secretion of interleukin-10 and the extensive induction of Tregs

  8. Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

    Energy Technology Data Exchange (ETDEWEB)

    Kelly, Catriona, E-mail: catriona.kelly@qub.ac.uk [SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine (United Kingdom); Flatt, Peter R.; McClenaghan, Neville H. [SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine (United Kingdom)

    2010-08-20

    Research highlights: {yields} TGP52 cells display enhanced functionality in pseudoislet form. {yields} Somatostatin content was reduced, but secretion increased in high glucose conditions. {yields} Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mM glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.

  9. Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

    International Nuclear Information System (INIS)

    Kelly, Catriona; Flatt, Peter R.; McClenaghan, Neville H.

    2010-01-01

    Research highlights: → TGP52 cells display enhanced functionality in pseudoislet form. → Somatostatin content was reduced, but secretion increased in high glucose conditions. → Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mM glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.

  10. Microencapsulating and Banking Living Cells for Cell-Based Medicine

    Directory of Open Access Journals (Sweden)

    Wujie Zhang

    2011-01-01

    Full Text Available A major challenge to the eventual success of the emerging cell-based medicine such as tissue engineering, regenerative medicine, and cell transplantation is the limited availability of the desired cell sources. This challenge can be addressed by cell microencapsulation to overcome the undesired immune response (i.e., to achieve immunoisolation so that non-autologous cells can be used to treat human diseases, and by cell/tissue preservation to bank living cells for wide distribution to end users so that they are readily available when needed in the future. This review summarizes the status quo of research in both cell microencapsulation and banking the microencapsulated cells. It is concluded with a brief outlook of future research directions in this important field.

  11. Effect of cell-phone radiofrequency on angiogenesis and cell invasion in human head and neck cancer cells.

    Science.gov (United States)

    Alahmad, Yaman M; Aljaber, Mohammed; Saleh, Alaaeldin I; Yalcin, Huseyin C; Aboulkassim, Tahar; Yasmeen, Amber; Batist, Gerald; Moustafa, Ala-Eddin Al

    2018-05-13

    Today, the cell phone is the most widespread technology globally. However, the outcome of cell-phone radiofrequency on head and neck cancer progression has not yet been explored. The chorioallantoic membrane (CAM) and human head and neck cancer cell lines, FaDu and SCC25, were used to explore the outcome of cell-phone radiofrequency on angiogenesis, cell invasion, and colony formation of head and neck cancer cells, respectively. Western blot analysis was used to investigate the impact of the cell phone on the regulation of E-cadherin and Erk1/Erk2 genes. Our data revealed that cell-phone radiofrequency promotes angiogenesis of the CAM. In addition, the cell phone enhances cell invasion and colony formation of human head and neck cancer cells; this is accompanied by a downregulation of E-cadherin expression. More significantly, we found that the cell phone can activate Erk1/Erk2 in our experimental models. Our investigation reveals that cell-phone radiofrequency could enhance head and neck cancer by stimulating angiogenesis and cell invasion via Erk1/Erk2 activation. © 2018 Wiley Periodicals, Inc.

  12. Stabilization Of Apoptotic Cells: Generation Of Zombie Cells

    Directory of Open Access Journals (Sweden)

    José A. Sánchez Alcázar

    2015-08-01

    Stabilization of apoptotic cells can be used for reliable detection and quantification of apoptosis in cultured cells and may allow a safer administration of apoptotic cells in clinical applications. Furthermore, it opens new avenues in the functional reconstruction of apoptotic cells for longer preservation.

  13. Single cell transcriptome profiling of developing chick retinal cells.

    Science.gov (United States)

    Laboissonniere, Lauren A; Martin, Gregory M; Goetz, Jillian J; Bi, Ran; Pope, Brock; Weinand, Kallie; Ellson, Laura; Fru, Diane; Lee, Miranda; Wester, Andrea K; Liu, Peng; Trimarchi, Jeffrey M

    2017-08-15

    The vertebrate retina is a specialized photosensitive tissue comprised of six neuronal and one glial cell types, each of which develops in prescribed proportions at overlapping timepoints from a common progenitor pool. While each of these cells has a specific function contributing to proper vision in the mature animal, their differential representation in the retina as well as the presence of distinctive cellular subtypes makes identifying the transcriptomic signatures that lead to each retinal cell's fate determination and development challenging. We have analyzed transcriptomes from individual cells isolated from the chick retina throughout retinogenesis. While we focused our efforts on the retinal ganglion cells, our transcriptomes of developing chick cells also contained representation from multiple retinal cell types, including photoreceptors and interneurons at different stages of development. Most interesting was the identification of transcriptomes from individual mixed lineage progenitor cells in the chick as these cells offer a window into the cell fate decision-making process. Taken together, these data sets will enable us to uncover the most critical genes acting in the steps of cell fate determination and early differentiation of various retinal cell types. © 2017 Wiley Periodicals, Inc.

  14. Mechanisms for Cell-to-Cell Transmission of HIV-1

    Science.gov (United States)

    Bracq, Lucie; Xie, Maorong; Benichou, Serge; Bouchet, Jérôme

    2018-01-01

    While HIV-1 infection of target cells with cell-free viral particles has been largely documented, intercellular transmission through direct cell-to-cell contact may be a predominant mode of propagation in host. To spread, HIV-1 infects cells of the immune system and takes advantage of their specific particularities and functions. Subversion of intercellular communication allows to improve HIV-1 replication through a multiplicity of intercellular structures and membrane protrusions, like tunneling nanotubes, filopodia, or lamellipodia-like structures involved in the formation of the virological synapse. Other features of immune cells, like the immunological synapse or the phagocytosis of infected cells are hijacked by HIV-1 and used as gateways to infect target cells. Finally, HIV-1 reuses its fusogenic capacity to provoke fusion between infected donor cells and target cells, and to form infected syncytia with high capacity of viral production and improved capacities of motility or survival. All these modes of cell-to-cell transfer are now considered as viral mechanisms to escape immune system and antiretroviral therapies, and could be involved in the establishment of persistent virus reservoirs in different host tissues. PMID:29515578

  15. Cell origami: self-folding of three-dimensional cell-laden microstructures driven by cell traction force.

    Directory of Open Access Journals (Sweden)

    Kaori Kuribayashi-Shigetomi

    Full Text Available This paper describes a method of generating three-dimensional (3D cell-laden microstructures by applying the principle of origami folding technique and cell traction force (CTF. We harness the CTF as a biological driving force to fold the microstructures. Cells stretch and adhere across multiple microplates. Upon detaching the microplates from a substrate, CTF causes the plates to lift and fold according to a prescribed pattern. This self-folding technique using cells is highly biocompatible and does not involve special material requirements for the microplates and hinges to induce folding. We successfully produced various 3D cell-laden microstructures by just changing the geometry of the patterned 2D plates. We also achieved mass-production of the 3D cell-laden microstructures without causing damage to the cells. We believe that our methods will be useful for biotechnology applications that require analysis of cells in 3D configurations and for self-assembly of cell-based micro-medical devices.

  16. Cell origami: self-folding of three-dimensional cell-laden microstructures driven by cell traction force.

    Science.gov (United States)

    Kuribayashi-Shigetomi, Kaori; Onoe, Hiroaki; Takeuchi, Shoji

    2012-01-01

    This paper describes a method of generating three-dimensional (3D) cell-laden microstructures by applying the principle of origami folding technique and cell traction force (CTF). We harness the CTF as a biological driving force to fold the microstructures. Cells stretch and adhere across multiple microplates. Upon detaching the microplates from a substrate, CTF causes the plates to lift and fold according to a prescribed pattern. This self-folding technique using cells is highly biocompatible and does not involve special material requirements for the microplates and hinges to induce folding. We successfully produced various 3D cell-laden microstructures by just changing the geometry of the patterned 2D plates. We also achieved mass-production of the 3D cell-laden microstructures without causing damage to the cells. We believe that our methods will be useful for biotechnology applications that require analysis of cells in 3D configurations and for self-assembly of cell-based micro-medical devices.

  17. Short-lived, transitory cell-cell interactions foster migration-dependent aggregation.

    Directory of Open Access Journals (Sweden)

    Melissa D Pope

    Full Text Available During embryonic development, motile cells aggregate into cohesive groups, which give rise to tissues and organs. The role of cell migration in regulating aggregation is unclear. The current paradigm for aggregation is based on an equilibrium model of differential cell adhesivity to neighboring cells versus the underlying substratum. In many biological contexts, however, dynamics is critical. Here, we provide evidence that multicellular aggregation dynamics involves both local adhesive interactions and transport by cell migration. Using time-lapse video microscopy, we quantified the duration of cell-cell contacts among migrating cells that collided and adhered to another cell. This lifetime of cell-cell interactions exhibited a monotonic decreasing dependence on substratum adhesivity. Parallel quantitative measurements of cell migration speed revealed that across the tested range of adhesive substrata, the mean time needed for cells to migrate and encounter another cell was greater than the mean adhesion lifetime, suggesting that aggregation dynamics may depend on cell motility instead of the local differential adhesivity of cells. Consistent with this hypothesis, aggregate size exhibited a biphasic dependence on substratum adhesivity, matching the trend we observed for cell migration speed. Our findings suggest a new role for cell motility, alongside differential adhesion, in regulating developmental aggregation events and motivate new design principles for tuning aggregation dynamics in tissue engineering applications.

  18. Radiation-induced cell death in embryogenic cells of coniferous plants

    International Nuclear Information System (INIS)

    Watanabe, Yoshito; Homma-Takeda, Shino; Yukawa, Masae; Nishimura, Yoshikazu; Sasamoto, Hamako; Takahagi, Masahiko

    2004-01-01

    Reproductive processes are particularly radiosensitive in plant development, which was clearly illustrated in reduction of seed formation in native coniferous plants around Chernobyl after the nuclear accident. For the purpose to investigate the effects of ionizing radiation on embryonic formation in coniferous plants, we used an embryo-derived embryogenic cell culture of a Japanese native coniferous plant, Japanese cedar (Cryplomeria japonica). The embryogenic cells were so radiosensitive that most of the cells died by X-ray irradiation of 5 Gy. This indicated that the embryogenic cells are as radiosensitive as some mammalian cells including lymphocytes. We considered that this type of radiosensitive cell death in the embryogenic cells should be responsible for reproductive damages of coniferous plants by low dose of ionizing radiation. The cell death of the embryogenic cells was characteristic of nuclear DNA fragmentation, which is typically observed in radiation-induced programmed cell death, i.e. apoptosis, in mammalian cells. On the other hand, cell death with nuclear DNA fragmentation did not develop by X-ray irradiation in vegetative cells including meristematic cells of Japanese cedar. This suggests that an apoptosis-like programmed cell death should develop cell-specifically in embryogenic cells by ionizing radiation. The abortion of embryogenic cells may work to prevent transmission of radiation-induced genetic damages to the descendants. (author)

  19. The Measles Virus Receptor SLAMF1 Can Mediate Particle Endocytosis.

    Science.gov (United States)

    Gonçalves-Carneiro, Daniel; McKeating, Jane A; Bailey, Dalan

    2017-04-01

    The signaling lymphocyte activation molecule F1 (SLAMF1) is both a microbial sensor and entry receptor for measles virus (MeV). Herein, we describe a new role for SLAMF1 to mediate MeV endocytosis that is in contrast with the alternative, and generally accepted, model that MeV genome enters cells only after fusion at the cell surface. We demonstrated that MeV engagement of SLAMF1 induces dramatic but transient morphological changes, most prominently in the formation of membrane blebs, which were shown to colocalize with incoming viral particles, and rearrangement of the actin cytoskeleton in infected cells. MeV infection was dependent on these dynamic cytoskeletal changes as well as fluid uptake through a macropinocytosis-like pathway as chemical inhibition of these processes inhibited entry. Moreover, we identified a role for the RhoA-ROCK-myosin II signaling axis in this MeV internalization process, highlighting a novel role for this recently characterized pathway in virus entry. Our study shows that MeV can hijack a microbial sensor normally involved in bacterial phagocytosis to drive endocytosis using a complex pathway that shares features with canonical viral macropinocytosis, phagocytosis, and mechanotransduction. This uptake pathway is specific to SLAMF1-positive cells and occurs within 60 min of viral attachment. Measles virus remains a significant cause of mortality in human populations, and this research sheds new light on the very first steps of infection of this important pathogen. IMPORTANCE Measles is a significant disease in humans and is estimated to have killed over 200 million people since records began. According to current World Health Organization statistics, it still kills over 100,000 people a year, mostly children in the developing world. The causative agent, measles virus, is a small enveloped RNA virus that infects a broad range of cells during infection. In particular, immune cells are infected via interactions between glycoproteins found

  20. Stem Cell Lineages: Between Cell and Organism

    Directory of Open Access Journals (Sweden)

    Melinda Bonnie Fagan

    2017-01-01

    Full Text Available Ontologies of living things are increasingly grounded on the concepts and practices of current life science. Biological development is a process, undergone by living things, which begins with a single cell and (in an important class of cases ends with formation of a multicellular organism. The process of development is thus prima facie central for ideas about biological individuality and organismality. However, recent accounts of these concepts do not engage developmental biology. This paper aims to fill the gap, proposing the lineage view of stem cells as an ontological framework for conceptualizing organismal development. This account is grounded on experimental practices of stem cell research, with emphasis on new techniques for generating biological organization in vitro. On the lineage view, a stem cell is the starting point of a cell lineage with a specific organismal source, time-interval of existence, and ‘tree topology’ of branch-points linking the stem to developmental termini. The concept of ‘enkapsis’ accommodates the cell-organism relation within the lineage view; this hierarchical notion is further explicated by considering the methods and results of stem cell experiments. Results of this examination include a (partial characterization of stem cells’ developmental versatility, and the context-dependence of developmental processes involving stem cells.

  1. A millifluidic study of cell-to-cell heterogeneity in growth-rate and cell-division capability in populations of isogenic cells of Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Shima P Damodaran

    Full Text Available To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell populations of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers and a significant subpopulation of slowly dividing cells (slow-growers. These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, populations dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.

  2. Stem cells in dentistry--part I: stem cell sources.

    Science.gov (United States)

    Egusa, Hiroshi; Sonoyama, Wataru; Nishimura, Masahiro; Atsuta, Ikiru; Akiyama, Kentaro

    2012-07-01

    Stem cells can self-renew and produce different cell types, thus providing new strategies to regenerate missing tissues and treat diseases. In the field of dentistry, adult mesenchymal stem/stromal cells (MSCs) have been identified in several oral and maxillofacial tissues, which suggests that the oral tissues are a rich source of stem cells, and oral stem and mucosal cells are expected to provide an ideal source for genetically reprogrammed cells such as induced pluripotent stem (iPS) cells. Furthermore, oral tissues are expected to be not only a source but also a therapeutic target for stem cells, as stem cell and tissue engineering therapies in dentistry continue to attract increasing clinical interest. Part I of this review outlines various types of intra- and extra-oral tissue-derived stem cells with regard to clinical availability and applications in dentistry. Additionally, appropriate sources of stem cells for regenerative dentistry are discussed with regard to differentiation capacity, accessibility and possible immunomodulatory properties. Copyright © 2012 Japan Prosthodontic Society. Published by Elsevier Ltd. All rights reserved.

  3. T cell receptor-engineered T cells to treat solid tumors: T cell processing toward optimal T cell fitness

    NARCIS (Netherlands)

    C.H.J. Lamers (Cor); S. van Steenbergen-Langeveld (Sabine); M. van Brakel (Mandy); C.M. Groot-van Ruijven (Corrien); P.M.M.L. van Elzakker (Pascal); B.A. van Krimpen (Brigitte); S. Sleijfer (Stefan); J.E.M.A. Debets (Reno)

    2014-01-01

    textabstractTherapy with autologous T cells that have been gene-engineered to express chimeric antigen receptors (CAR) or T cell receptors (TCR) provides a feasible and broadly applicable treatment for cancer patients. In a clinical study in advanced renal cell carcinoma (RCC) patients with CAR T

  4. Interaction of Stellate Cells with Pancreatic Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Marco Siech

    2010-09-01

    Full Text Available Pancreatic cancer is characterized by its late detection, aggressive growth, intense infiltration into adjacent tissue, early metastasis, resistance to chemo- and radiotherapy and a strong “desmoplastic reaction”. The dense stroma surrounding carcinoma cells is composed of fibroblasts, activated stellate cells (myofibroblast-like cells, various inflammatory cells, proliferating vascular structures, collagens and fibronectin. In particular the cellular components of the stroma produce the tumor microenvironment, which plays a critical role in tumor growth, invasion, spreading, metastasis, angiogenesis, inhibition of anoikis, and chemoresistance. Fibroblasts, myofibroblasts and activated stellate cells produce the extracellular matrix components and are thought to interact actively with tumor cells, thereby promoting cancer progression. In this review, we discuss our current understanding of the role of pancreatic stellate cells (PSC in the desmoplastic response of pancreas cancer and the effects of PSC on tumor progression, metastasis and drug resistance. Finally we present some novel ideas for tumor therapy by interfering with the cancer cell-host interaction.

  5. Bio optofluidics cell sorter: cell-BOCS concept and applications

    Science.gov (United States)

    Roth, Tue; Glückstad, Jesper

    2012-03-01

    The cell-BOCS is a novel microfluidics based cell-sorting instrument utilizing next generation optical trapping technology developed at the Technical University of Denmark. It is targeted emerging bio-medical research and diagnostics markets where it for certain applications offers a number of advantages over conventional fluorescence activated cell-sorting (FACSTM) technology. Advantages include gentle handling of cells, sterile sorting, easy operation, small footprint and lower cost allowing out-of-core-facility use. Application examples are found within sorting of fragile transfected cells, high value samples and primary cell lines, where traditional FACS technology has limited application due to it's droplet-based approach to cell-sorting. In the diagnostics field, in particular applying the cell-BOCS for isolating pure populations of circulating tumor cells is an area that has generated a lot of interest.

  6. HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell.

    Directory of Open Access Journals (Sweden)

    Mikaël Boullé

    2016-11-01

    Full Text Available Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses.

  7. Primitive human hematopoietic cells give rise to differentially specified daughter cells upon their initial cell division.

    NARCIS (Netherlands)

    Giebel, B.; Zhang, T.; Beckmann, J.; Spanholtz, J.; Wernet, P.; Ho, A.; Punzel, M.

    2006-01-01

    It is often predicted that stem cells divide asymmetrically, creating a daughter cell that maintains the stem-cell capacity, and 1 daughter cell committed to differentiation. While asymmetric stem-cell divisions have been proven to occur in model organisms (eg, in Drosophila), it remains illusive

  8. A Dominant-Negative PPARγ Mutant Promotes Cell Cycle Progression and Cell Growth in Vascular Smooth Muscle Cells

    Directory of Open Access Journals (Sweden)

    Joey Z. Liu

    2009-01-01

    Full Text Available PPARγ ligands have been shown to have antiproliferative effects on many cell types. We herein report that a synthetic dominant-negative (DN PPARγ mutant functions like a growth factor to promote cell cycle progression and cell proliferation in human coronary artery smooth muscle cells (CASMCs. In quiescent CASMCs, adenovirus-expressed DN-PPARγ promoted G1→S cell cycle progression, enhanced BrdU incorporation, and increased cell proliferation. DN-PPARγ expression also markedly enhanced positive regulators of the cell cycle, increasing Rb and CDC2 phosphorylation and the expression of cyclin A, B1, D1, and MCM7. Conversely, overexpression of wild-type (WT or constitutively-active (CA PPARγ inhibited cell cycle progression and the activity and expression of positive regulators of the cell cycle. DN-PPARγ expression, however, did not up-regulate positive cell cycle regulators in PPARγ-deficient cells, strongly suggesting that DN-PPARγ effects on cell cycle result from blocking the function of endogenous wild-type PPARγ. DN-PPARγ expression enhanced phosphorylation of ERK MAPKs. Furthermore, the ERK specific-inhibitor PD98059 blocked DN-PPARγ-induced phosphorylation of Rb and expression of cyclin A and MCM7. Our data thus suggest that DN-PPARγ promotes cell cycle progression and cell growth in CASMCs by modulating fundamental cell cycle regulatory proteins and MAPK mitogenic signaling pathways in vascular smooth muscle cells (VSMCs.

  9. MCF-10A-NeoST: A New Cell System for Studying Cell-ECM and Cell-Cell Interactions in Breast Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Zantek, Nicole Dodge; Walker-Daniels, Jennifer; Stewart, Jane; Hansen, Rhonda K.; Robinson, Daniel; Miao, Hui; Wang, Bingcheng; Kung, Hsing-Jien; Bissell, Mina J.; Kinch, Michael S.

    2001-08-22

    There is a continuing need for genetically matched cell systems to model cellular behaviors that are frequently observed in aggressive breast cancers. We report here the isolation and initial characterization of a spontaneously arising variant of MCF-10A cells, NeoST, which provides a new model to study cell adhesion and signal transduction in breast cancer. NeoST cells recapitulate important biological and biochemical features of metastatic breast cancer, including anchorage-independent growth, invasiveness in threedimensional reconstituted membranes, loss of E-cadherin expression, and increased tyrosine kinase activity. A comprehensive analysis of tyrosine kinase expression revealed overexpression or functional activation of the Axl, FAK, and EphA2 tyrosine kinases in transformed MCF-10A cells. MCF-10A and these new derivatives provide a genetically matched model to study defects in cell adhesion and signaling that are relevant to cellular behaviors that often typify aggressive breast cancer cells.

  10. Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method.

    Science.gov (United States)

    Nakajima, Ryota; Takeda, Shizu

    2014-01-01

    The use of murine 3T3 feeder cells needs to be avoided when fabricating corneal epithelial cell sheets for use in treating ocular surface diseases. However, the expression level of the epithelial stem/progenitor cell marker, p63, is down-regulated in feeder-free culture systems. In this study, in order to fabricate corneal epithelial cell sheets that maintain colony-forming cells without using any feeder cells, we investigated the use of an oxygen-controlled method that was developed previously to fabricate cell sheets efficiently. Rabbit limbal epithelial cells were cultured under hypoxia (1-10% O2) and under normoxia during stratification after reaching confluence. Multilayered corneal epithelial cell sheets were fabricated using an oxygen-controlled method, and immunofluorescence analysis showed that cytokeratin 3 and p63 was expressed in appropriate localization in the cell sheets. The colony-forming efficiency of the cell sheets fabricated by the oxygen-controlled method without feeder cells was significantly higher than that of cell sheets fabricated under 20% O2 without feeder cells. These results indicate that the oxygen-controlled method has the potential to achieve a feeder-free culture system for fabricating corneal epithelial cell sheets for corneal regeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. iNKT cells suppress the CD8+ T cell response to a murine Burkitt's-like B cell lymphoma.

    Directory of Open Access Journals (Sweden)

    Ryan L Bjordahl

    Full Text Available The T cell response to B cell lymphomas differs from the majority of solid tumors in that the malignant cells themselves are derived from B lymphocytes, key players in immune response. B cell lymphomas are therefore well situated to manipulate their surrounding microenvironment to enhance tumor growth and minimize anti-tumor T cell responses. We analyzed the effect of T cells on the growth of a transplantable B cell lymphoma and found that iNKT cells suppressed the anti-tumor CD8(+ T cell response. Lymphoma cells transplanted into syngeneic wild type (WT mice or Jalpha18(-/- mice that specifically lack iNKT cells grew initially at the same rate, but only the mice lacking iNKT cells were able to reject the lymphoma. This effect was due to the enhanced activity of tumor-specific CD8(+ T cells in the absence of iNKT cells, and could be partially reversed by reconstitution of iNKT cells in Jalpha 18(-/- mice. Treatment of tumor-bearing WT mice with alpha -galactosyl ceramide, an activating ligand for iNKT cells, reduced the number of tumor-specific CD8(+ T cells. In contrast, lymphoma growth in CD1d1(-/- mice that lack both iNKT and type II NKT cells was similar to that in WT mice, suggesting that type II NKT cells are required for full activation of the anti-tumor immune response. This study reveals a tumor-promoting role for iNKT cells and suggests their capacity to inhibit the CD8(+ T cell response to B cell lymphoma by opposing the effects of type II NKT cells.

  12. Advances in reprogramming somatic cells to induced pluripotent stem cells.

    Science.gov (United States)

    Patel, Minal; Yang, Shuying

    2010-09-01

    Traditionally, nuclear reprogramming of cells has been performed by transferring somatic cell nuclei into oocytes, by combining somatic and pluripotent cells together through cell fusion and through genetic integration of factors through somatic cell chromatin. All of these techniques changes gene expression which further leads to a change in cell fate. Here we discuss recent advances in generating induced pluripotent stem cells, different reprogramming methods and clinical applications of iPS cells. Viral vectors have been used to transfer transcription factors (Oct4, Sox2, c-myc, Klf4, and nanog) to induce reprogramming of mouse fibroblasts, neural stem cells, neural progenitor cells, keratinocytes, B lymphocytes and meningeal membrane cells towards pluripotency. Human fibroblasts, neural cells, blood and keratinocytes have also been reprogrammed towards pluripotency. In this review we have discussed the use of viral vectors for reprogramming both animal and human stem cells. Currently, many studies are also involved in finding alternatives to using viral vectors carrying transcription factors for reprogramming cells. These include using plasmid transfection, piggyback transposon system and piggyback transposon system combined with a non viral vector system. Applications of these techniques have been discussed in detail including its advantages and disadvantages. Finally, current clinical applications of induced pluripotent stem cells and its limitations have also been reviewed. Thus, this review is a summary of current research advances in reprogramming cells into induced pluripotent stem cells.

  13. Tuft (caveolated) cells in two human colon carcinoma cell lines.

    Science.gov (United States)

    Barkla, D H; Whitehead, R H; Foster, H; Tutton, P J

    1988-09-01

    The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting from the apical surface. The microvilli are attached by a core of long microfilaments passing deep into the apical cytoplasm. Between the microvilli are parallel arrays of vesicles (caveoli) containing flocculent material. Two different but not mutually exclusive explanations for the presence of tuft cells are proposed. The first explanation is that tuft cells came from the resected tumour and have survived by mitotic division during subsequent passages. The second explanation suggests that tuft cells are the progeny of undifferentiated tumour cells. Descriptions of tuft cells in colon carcinomas are uncommon and possible reasons for this are presented. The morphology of tuft cells is consistent with that of a highly differentiated cell specialised for absorption, and these new models provide an opportunity to further investigate the structure and function of tuft cells.

  14. Promotion of initiated cells by radiation-induced cell inactivation.

    Science.gov (United States)

    Heidenreich, W F; Paretzke, H G

    2008-11-01

    Cells on the way to carcinogenesis can have a growth advantage relative to normal cells. It has been hypothesized that a radiation-induced growth advantage of these initiated cells might be induced by an increased cell replacement probability of initiated cells after inactivation of neighboring cells by radiation. Here Monte Carlo simulations extend this hypothesis for larger clones: The effective clonal expansion rate decreases with clone size. This effect is stronger for the two-dimensional than for the three-dimensional situation. The clones are irregular, far from a circular shape. An exposure-rate dependence of the effective clonal expansion rate could come in part from a minimal recovery time of the initiated cells for symmetric cell division.

  15. Cre-mediated cell ablation contests mast cell contribution in models of antibody- and T cell-mediated autoimmunity.

    Science.gov (United States)

    Feyerabend, Thorsten B; Weiser, Anne; Tietz, Annette; Stassen, Michael; Harris, Nicola; Kopf, Manfred; Radermacher, Peter; Möller, Peter; Benoist, Christophe; Mathis, Diane; Fehling, Hans Jörg; Rodewald, Hans-Reimer

    2011-11-23

    Immunological functions of mast cells remain poorly understood. Studies in Kit mutant mice suggest key roles for mast cells in certain antibody- and T cell-mediated autoimmune diseases. However, Kit mutations affect multiple cell types of both immune and nonimmune origin. Here, we show that targeted insertion of Cre-recombinase into the mast cell carboxypeptidase A3 locus deleted mast cells in connective and mucosal tissues by a genotoxic Trp53-dependent mechanism. Cre-mediated mast cell eradication (Cre-Master) mice had, with the exception of a lack of mast cells and reduced basophils, a normal immune system. Cre-Master mice were refractory to IgE-mediated anaphylaxis, and this defect was rescued by mast cell reconstitution. This mast cell-deficient strain was fully susceptible to antibody-induced autoimmune arthritis and to experimental autoimmune encephalomyelitis. Differences comparing Kit mutant mast cell deficiency models to selectively mast cell-deficient mice call for a systematic re-evaluation of immunological functions of mast cells beyond allergy. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. In vitro germ cell differentiation from cynomolgus monkey embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Kaori Yamauchi

    Full Text Available BACKGROUND: Mouse embryonic stem (ES cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. METHODS AND FINDINGS: To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis. VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. CONCLUSION: VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the

  17. Mesenchymal stem cells: cell biology and potential use in therapy

    DEFF Research Database (Denmark)

    Kassem, Moustapha; Kristiansen, Malthe; Abdallah, Basem M

    2004-01-01

    Mesenchymal stem cells are clonogenic, non-haematopoietic stem cells present in the bone marrow and are able to differentiate into multiple mesoderm-type cell lineages e.g. osteoblasts, chondrocytes, endothelial-cells and also non-mesoderm-type lineages e.g. neuronal-like cells. Several methods...... are currently available for isolation of the mesenchymal stem cells based on their physical and immunological characteristics. Because of the ease of their isolation and their extensive differentiation potential, mesenchymal stem cells are among the first stem cell types to be introduced in the clinic. Recent...... studies have demonstrated that the life span of mesenchymal stem cells in vitro can be extended by increasing the levels of telomerase expression in the cells and thus allowing culture of large number of cells needed for therapy. In addition, it has been shown that it is possible to culture the cells...

  18. Altered development of NKT cells, γδ T cells, CD8 T cells and NK cells in a PLZF deficient patient.

    Directory of Open Access Journals (Sweden)

    Maggie Eidson

    Full Text Available In mice, the transcription factor, PLZF, controls the development of effector functions in invariant NKT cells and a subset of NKT cell-like, γδ T cells. Here, we show that in human lymphocytes, in addition to invariant NKT cells, PLZF was also expressed in a large percentage of CD8+ and CD4+ T cells. Furthermore, PLZF was also found to be expressed in all γδ T cells and in all NK cells. Importantly, we show that in a donor lacking functional PLZF, all of these various lymphocyte populations were altered. Therefore, in contrast to mice, PLZF appears to control the development and/or function of a wide variety of human lymphocytes that represent more than 10% of the total PBMCs. Interestingly, the PLZF-expressing CD8+ T cell population was found to be expanded in the peripheral blood of patients with metastatic melanoma but was greatly diminished in patients with autoimmune disease.

  19. Fuel Cell/Electrochemical Cell Voltage Monitor

    Science.gov (United States)

    Vasquez, Arturo

    2012-01-01

    A concept has been developed for a new fuel cell individual-cell-voltage monitor that can be directly connected to a multi-cell fuel cell stack for direct substack power provisioning. It can also provide voltage isolation for applications in high-voltage fuel cell stacks. The technology consists of basic modules, each with an 8- to 16-cell input electrical measurement connection port. For each basic module, a power input connection would be provided for direct connection to a sub-stack of fuel cells in series within the larger stack. This power connection would allow for module power to be available in the range of 9-15 volts DC. The relatively low voltage differences that the module would encounter from the input electrical measurement connection port, coupled with the fact that the module's operating power is supplied by the same substack voltage input (and so will be at similar voltage), provides for elimination of high-commonmode voltage issues within each module. Within each module, there would be options for analog-to-digital conversion and data transfer schemes. Each module would also include a data-output/communication port. Each of these ports would be required to be either non-electrical (e.g., optically isolated) or electrically isolated. This is necessary to account for the fact that the plurality of modules attached to the stack will normally be at a range of voltages approaching the full range of the fuel cell stack operating voltages. A communications/ data bus could interface with the several basic modules. Options have been identified for command inputs from the spacecraft vehicle controller, and for output-status/data feeds to the vehicle.

  20. Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer.

    Science.gov (United States)

    Sung, Li-Ying; Gao, Shaorong; Shen, Hongmei; Yu, Hui; Song, Yifang; Smith, Sadie L; Chang, Ching-Chien; Inoue, Kimiko; Kuo, Lynn; Lian, Jin; Li, Ao; Tian, X Cindy; Tuck, David P; Weissman, Sherman M; Yang, Xiangzhong; Cheng, Tao

    2006-11-01

    Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

  1. Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

    Science.gov (United States)

    Wang, Zhongde

    2011-01-01

    Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

  2. Airway Basal Cell Heterogeneity and Lung Squamous Cell Carcinoma.

    Science.gov (United States)

    Hynds, Robert E; Janes, Sam M

    2017-09-01

    Basal cells are stem/progenitor cells that maintain airway homeostasis, enact repair following epithelial injury, and are a candidate cell-of-origin for lung squamous cell carcinoma. Heterogeneity of basal cells is recognized in terms of gene expression and differentiation capacity. In this Issue, Pagano and colleagues isolate a subset of immortalized basal cells that are characterized by high motility, suggesting that they might also be heterogeneous in their biophysical properties. Motility-selected cells displayed an increased ability to colonize the lung in vivo The possible implications of these findings are discussed in terms of basal cell heterogeneity, epithelial cell migration, and modeling of metastasis that occurs early in cancer evolution. Cancer Prev Res; 10(9); 491-3. ©2017 AACR See related article by Pagano et al., p. 514 . ©2017 American Association for Cancer Research.

  3. Studies on ADCC (antibody-dependent cell-mediated cytotoxicity) using sheep red blood cells as target cells, 2

    International Nuclear Information System (INIS)

    Ichikawa, Yukinobu; Takaya, Masatoshi; Arimori, Shigeru

    1979-01-01

    A non-specific cytotoxic mediator from effector cells (human peripheral blood leukocytes) was investigated in the ADCC (antibody-dependent cell-mediated cytotoxicity) system using antibody-coated sheep red blood cells (SRBC) as target cells. 51 Cr-labelled homologous (sheep) or heterologous (human) red blood cells were used as adjacent cells. Either crude lymphocyte fraction, phagocyte depleted fraction or granulocyte rich fraction separated from human peripheral leukocytes showed moderate cytotoxic effect on homologous adjacent cells, however no cytotoxic activity on heterologous adjacent cells was demonstrated in any leukocyte fraction. This suggests that the cytotoxic effects on homologous adjacent cells were resulted from the translocation of antibody molecules to adjacent cells from antibody-coated target cells. We concluded that the cytotoxic mechanism in this ADCC system was not mediated by non-specific soluble factors released from either human peripheral lymphocytes, monocytes or granulocytes. (author)

  4. Galvanic cells: setting up the Daniell cell.

    OpenAIRE

    Milla González, Miguel

    2008-01-01

    With the reagents (0.05M copper nitrate solution, 0.05M zinc nitrate solution) and material (glassware, potentiometer, electric wire) availabe in the laboratory, the user must set up the Daniell cell. Different configurations can be possible if the half cells are filled with either electrolyte solution. The cell connections to the measuring device can also be changed. In all instances, an explanation of the set up cell is obtained as well as of the measured potential difference.

  5. Relative Contributions of B Cells and Dendritic Cells from Lupus-Prone Mice to CD4+ T Cell Polarization.

    Science.gov (United States)

    Choi, Seung-Chul; Xu, Zhiwei; Li, Wei; Yang, Hong; Roopenian, Derry C; Morse, Herbert C; Morel, Laurence

    2018-05-01

    Mouse models of lupus have shown that multiple immune cell types contribute to autoimmune disease. This study sought to investigate the involvement of B cells and dendritic cells in supporting the expansion of inflammatory and regulatory CD4 + T cells that are critical for lupus pathogenesis. We used lupus-prone B6.NZM2410.Sle1.Sle2.Sle3 (TC) and congenic C57BL/6J (B6) control mice to investigate how the genetic predisposition of these two cell types controls the activity of normal B6 T cells. Using an allogeneic in vitro assay, we showed that TC B1-a and conventional B cells expanded Th17 cells significantly more than their B6 counterparts. This expansion was dependent on CD86 and IL-6 expression and mapped to the Sle1 lupus-susceptibility locus. In vivo, TC B cells promoted greater differentiation of CD4 + T cells into Th1 and follicular helper T cells than did B6 B cells, but they limited the expansion of Foxp3 regulatory CD4 + T cells to a greater extent than did B6 B cells. Finally, when normal B6 CD4 + T cells were introduced into Rag1 -/- mice, TC myeloid/stromal cells caused their heightened activation, decreased Foxp3 regulatory CD4 + T cell differentiation, and increased renal infiltration of Th1 and Th17 cells in comparison with B6 myeloid/stromal cells. The results show that B cells from lupus mice amplify inflammatory CD4 + T cells in a nonredundant manner with myeloid/stromal cells. Copyright © 2018 by The American Association of Immunologists, Inc.

  6. Electrorefining cell evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Bronson, M.C.; Thomas, R.L. (ed.)

    1989-04-14

    Operational characteristics of the LANL electrorefining cell, a modified LANL electrorefining cell, and an advanced electrorefining cell (known as the CRAC cell) were determined. Average process yields achieved were: 75% for the LANL cell, 82% for the modified LANL cell, and 86% for the CRAC cell. All product metal from the LANL and modified LANL cells was within foundry specifications. Metal from one run in the CRAC cell exceeded foundry specifications for tantalum. The LANL and modified LANL cells were simple in design and operation, but product separation was more labor intensive than with the CRAC cell. The CRAC cell was more complicated in design but remained relatively simple in operation. A decision analysis concluded that the modified LANL cell was the preferred cell. It was recommended that the modified LANL cell be implemented by the Plutonium Recovery Project at Rocky Flats and that development of the CRAC cell continue. 8 refs., 22 figs., 12 tabs.

  7. Canthin-6-one induces cell death, cell cycle arrest and differentiation in human myeloid leukemia cells.

    Science.gov (United States)

    Vieira Torquato, Heron F; Ribeiro-Filho, Antonio C; Buri, Marcus V; Araújo Júnior, Roberto T; Pimenta, Renata; de Oliveira, José Salvador R; Filho, Valdir C; Macho, Antonio; Paredes-Gamero, Edgar J; de Oliveira Martins, Domingos T

    2017-04-01

    Canthin-6-one is a natural product isolated from various plant genera and from fungi with potential antitumor activity. In the present study, we evaluate the antitumor effects of canthin-6-one in human myeloid leukemia lineages. Kasumi-1 lineage was used as a model for acute myeloid leukemia. Cells were treated with canthin-6-one and cell death, cell cycle and differentiation were evaluated in both total cells (Lin + ) and leukemia stem cell population (CD34 + CD38 - Lin -/low ). Among the human lineages tested, Kasumi-1 was the most sensitive to canthin-6-one. Canthin-6-one induced cell death with apoptotic (caspase activation, decrease of mitochondrial potential) and necrotic (lysosomal permeabilization, double labeling of annexin V/propidium iodide) characteristics. Moreover, canthin-6-one induced cell cycle arrest at G 0 /G 1 (7μM) and G 2 (45μM) evidenced by DNA content, BrdU incorporation and cyclin B1/histone 3 quantification. Canthin-6-one also promoted differentiation of Kasumi-1, evidenced by an increase in the expression of myeloid markers (CD11b and CD15) and the transcription factor PU.1. Furthermore, a reduction of the leukemic stem cell population and clonogenic capability of stem cells were observed. These results show that canthin-6-one can affect Kasumi-1 cells by promoting cell death, cell cycle arrest and cell differentiation depending on concentration used. Canthin-6-one presents an interesting cytotoxic activity against leukemic cells and represents a promising scaffold for the development of molecules for anti-leukemic applications, especially by its anti-leukemic stem cell activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. The antigen presenting cells instruct plasma cell differentiation.

    Science.gov (United States)

    Xu, Wei; Banchereau, Jacques

    2014-01-06

    The professional antigen presenting cells (APCs), including many subsets of dendritic cells and macrophages, not only mediate prompt but non-specific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells (PCs), which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only "signal 1" (the antigen), but also "signal 2" to directly instruct the differentiation process of PCs in a T-cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching, and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

  9. Stem cell biology and cell transplantation therapy in the retina.

    Science.gov (United States)

    Osakada, Fumitaka; Hirami, Yasuhiko; Takahashi, Masayo

    2010-01-01

    Embryonic stem (ES) cells, which are derived from the inner cell mass of mammalian blastocyst stage embryos, have the ability to differentiate into any cell type in the body and to grow indefinitely while maintaining pluripotency. During development, cells undergo progressive and irreversible differentiation into specialized adult cell types. Remarkably, in spite of this restriction in potential, adult somatic cells can be reprogrammed and returned to the naive state of pluripotency found in the early embryo simply by forcing expression of a defined set of transcription factors. These induced pluripotent stem (iPS) cells are molecularly and functionally equivalent to ES cells and provide powerful in vitro models for development, disease, and drug screening, as well as material for cell replacement therapy. Since functional impairment results from cell loss in most central nervous system (CNS) diseases, recovery of lost cells is an important treatment strategy. Although adult neurogenesis occurs in restricted regions, the CNS has poor potential for regeneration to compensate for cell loss. Thus, cell transplantation into damaged or diseased CNS tissues is a promising approach to treating various neurodegenerative disorders. Transplantation of photoreceptors or retinal pigment epithelium cells derived from human ES cells can restore some visual function. Patient-specific iPS cells may lead to customized cell therapy. However, regeneration of retinal function will require a detailed understanding of eye development, visual system circuitry, and retinal degeneration pathology. Here, we review the current progress in retinal regeneration, focusing on the therapeutic potential of pluripotent stem cells.

  10. RNAi screen reveals host cell kinases specifically involved in Listeria monocytogenes spread from cell to cell.

    Directory of Open Access Journals (Sweden)

    Ryan Chong

    Full Text Available Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin

  11. β-Cell dedifferentiation, reduced duct cell plasticity, and impaired β-cell mass regeneration in middle-aged rats.

    Science.gov (United States)

    Téllez, Noèlia; Vilaseca, Marina; Martí, Yasmina; Pla, Arturo; Montanya, Eduard

    2016-09-01

    Limitations in β-cell regeneration potential in middle-aged animals could contribute to the increased risk to develop diabetes associated with aging. We investigated β-cell regeneration of middle-aged Wistar rats in response to two different regenerative stimuli: partial pancreatectomy (Px + V) and gastrin administration (Px + G). Pancreatic remnants were analyzed 3 and 14 days after surgery. β-Cell mass increased in young animals after Px and was further increased after gastrin treatment. In contrast, β-cell mass did not change after Px or after gastrin treatment in middle-aged rats. β-Cell replication and individual β-cell size were similarly increased after Px in young and middle-aged animals, and β-cell apoptosis was not modified. Nuclear immunolocalization of neurog3 or nkx6.1 in regenerative duct cells, markers of duct cell plasticity, was increased in young but not in middle-aged Px rats. The pancreatic progenitor-associated transcription factors neurog3 and sox9 were upregulated in islet β-cells of middle-aged rats and further increased after Px. The percentage of chromogranin A+/hormone islet cells was significantly increased in the pancreases of middle-aged Px rats. In summary, the potential for compensatory β-cell hyperplasia and hypertrophy was retained in middle-aged rats, but β-cell dedifferentiation and impaired duct cell plasticity limited β-cell regeneration. Copyright © 2016 the American Physiological Society.

  12. Estimating cell capacity for multi-cell electrical energy system

    Science.gov (United States)

    Hashemi, Iman Ahari

    A Multi-Cell Electrical Energy System is a set of batteries that are connected in series. The series batteries provide the required voltage necessary for the contraption. After using the energy that is provided by the batteries, some cells within the system tend to have a lower voltage than the other cells. Also, other factors, such as the number of times a battery has been charged or discharged, how long it has been within the system and many other factors, result in some cells having a lesser capacity compared to the other cells within the system. The outcome is that it lowers the required capacity that the electrical energy system is required to provide. By having an unknown cell capacity within the system, it is unknown how much of a charge can be provided to the system so that the cells are not overcharged or undercharged. Therefore, it is necessary to know the cells capacity within the system. Hence, if we were dealing with a single cell, the capacity could be obtained by a full charge and discharge of the cell. In a series system that contains multiple cells a full charging or discharging cannot happen as it might result in deteriorating the structure of some cells within the system. Hence, to find the capacity of a single cell within an electrical energy system it is required to obtain a method that can estimate the value of each cell within the electrical energy system. To approach this method an electrical energy system is required. The electrical energy system consists of rechargeable non-equal capacity batteries to provide the required energy to the system, a battery management system (BMS) board to monitor the cells voltages, an Arduino board that provides the required communication to BMS board, and the PC, and a software that is able to deliver the required data obtained from the Arduino board to the PC. The outcome, estimating the capacity of a cell within a multi-cell system, can be used in many battery related technologies to obtain unknown

  13. Cross-talk between cd1d-restricted nkt cells and γδ cells in t regulatory cell response

    Directory of Open Access Journals (Sweden)

    Huber Sally A

    2011-01-01

    Full Text Available Abstract CD1d is a non-classical major histocompatibility class 1-like molecule which primarily presents either microbial or endogenous glycolipid antigens to T cells involved in innate immunity. Natural killer T (NKT cells and a subpopulation of γδ T cells expressing the Vγ4 T cell receptor (TCR recognize CD1d. NKT and Vγ4 T cells function in the innate immune response via rapid activation subsequent to infection and secrete large quantities of cytokines that both help control infection and modulate the developing adaptive immune response. T regulatory cells represent one cell population impacted by both NKT and Vγ4 T cells. This review discusses the evidence that NKT cells promote T regulatory cell activation both through direct interaction of NKT cell and dendritic cells and through NKT cell secretion of large amounts of TGFβ, IL-10 and IL-2. Recent studies have shown that CD1d-restricted Vγ4 T cells, in contrast to NKT cells, selectively kill T regulatory cells through a caspase-dependent mechanism. Vγ4 T cell elimination of the T regulatory cell population allows activation of autoimmune CD8+ effector cells leading to severe cardiac injury in a coxsackievirus B3 (CVB3 myocarditis model in mice. CD1d-restricted immunity can therefore lead to either immunosuppression or autoimmunity depending upon the type of innate effector dominating during the infection.

  14. [gammadelta T cells stimulated by zoledronate kill osteosarcoma cells].

    Science.gov (United States)

    Jiang, Hui; Xu, Qiang; Yang, Chao; Cao, Zhen-Guo; Li, Zhao-Xu; Ye, Zhao-Ming

    2010-12-01

    To investigate the cytotoxicity of human γδT cells from PBMCs stimulated by zoledronate against osteosarcoma cell line HOS in vitro and in vivo and evaluate the relavent pathways. The peripheral blood mononuclear cells (PBMCs)of healthy donors were stimulated by single dose zoledronate and cultured in the present of IL-2 for two weeks, analysising the percentage of γδT cells on a FACSCalibur cytometer.Study the cytotoxicity of γδT cells against the osteosarcoma line HOS using LDH release assay kit. Pre-treatment of γδT cells with anti-human γδTCR antibody, anti-human NKG2D antibody and concanamycin A to bolck the relavent pathways for evaluating the mechenisms of its cytotoxicity. In vivo, BALB/c mice were inoculated subcutaneously osteosarcoma cell HOS for developing hypodermal tumors. And they were randomized into two groups: unteated group, γδT cell therapy group. Tumor volume and weight of the two groups were compared. After two weeks of culture, γδT cells from zoledronate-stimulated PBMCs could reach (95±3)%. When the E:T as 6:1, 12:1, 25:1, 50:1, the percentage of osteosarcoma cell HOS killed by γδT cells was 26.8%, 31.5%, 37.8%, 40.9%, respectively.When anti-huma γδTCR antibody, anti-human NKG2D antibody and concanamycin A blocked the relavent pathways, the percentage was 32.3%, 4.7%, 16.7% ( E:T as 25:1), respectively. In vivo, the tumor inhibition rate of the group of γδT cell therapy was 42.78%. γδT cells derived from PBMCs stimulated by zoledronate can acquired pure γδT cells. And they show strong cytoxicity against osteosarcoma cell line HOS in vitro and in vivo.

  15. Tuft (caveolated) cells in two human colon carcinoma cell lines.

    OpenAIRE

    Barkla, D. H.; Whitehead, R. H.; Foster, H.; Tutton, P. J.

    1988-01-01

    The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting f...

  16. Identification of Human Cutaneous Basal Cell Carcinoma Cancer Stem Cells.

    Science.gov (United States)

    Morgan, Huw; Olivero, Carlotta; Patel, Girish K

    2018-04-20

    The cancer stem cell model states that a subset of tumor cells, called "cancer stem cells," can initiate and propagate tumor growth through self-renewal, high proliferative capacity, and their ability to recreate tumor heterogeneity. In basal cell carcinoma (BCC), we have shown that tumor cells that express the cell surface protein CD200 fulfill the cancer stem cell hypothesis. CD200+ CD45- BCC cells represent 0.05-3.96% of all BCC cells and reside in small clusters at the tumor periphery. Using a novel, reproducible in vivo xenograft growth assay, we determined that tumor-initiating cell (TIC) frequencies are approximately 1 per 1.5 million unsorted BCC cells. The CD200+ CD45- BCC subpopulation recreated BCC tumor growth in vivo with typical histological architecture and expression of sonic hedgehog-regulated genes. Reproducible in vivo BCC growth was achieved with as few as 10,000 CD200+ CD45- cells, representing ~1500-fold enrichment. The methods used to identify and purify CD200+ CD45- BCC cells, as well as characterize gene expression, are described herein.

  17. Radiation sensitivity of Merkel cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

    1995-07-30

    Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

  18. Radiation sensitivity of Merkell cell carcinoma cell lines

    International Nuclear Information System (INIS)

    Leonard, J. Helen; Ramsay, Jonathan R.; Kearsley, John H.; Birrell, Geoff W.

    1995-01-01

    Purpose: Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Methods and Materials: Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after γ irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. Results: We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to γ irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Conclusion: Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution

  19. Hakai reduces cell-substratum adhesion and increases epithelial cell invasion

    International Nuclear Information System (INIS)

    Rodríguez-Rigueiro, Teresa; Valladares-Ayerbes, Manuel; Haz-Conde, Mar; Aparicio, Luis A; Figueroa, Angélica

    2011-01-01

    The dynamic regulation of cell-cell adhesions is crucial for developmental processes, including tissue formation, differentiation and motility. Adherens junctions are important components of the junctional complex between cells and are necessary for maintaining cell homeostasis and normal tissue architecture. E-cadherin is the prototype and best-characterized protein member of adherens junctions in mammalian epithelial cells. Regarded as a tumour suppressor, E-cadherin loss is associated with poor prognosis in carcinoma. The E3 ubiquitin-ligase Hakai was the first reported posttranslational regulator of the E-cadherin complex. Hakai specifically targetted E-cadherin for internalization and degradation and thereby lowered epithelial cell-cell contact. Hakai was also implicated in controlling proliferation, and promoted cancer-related gene expression by increasing the binding of RNA-binding protein PSF to RNAs encoding oncogenic proteins. We sought to investigate the possible implication of Hakai in cell-substratum adhesions and invasion in epithelial cells. Parental MDCK cells and MDCK cells stably overexpressing Hakai were used to analyse cell-substratum adhesion and invasion capabilities. Western blot and immunofluoresecence analyses were performed to assess the roles of Paxillin, FAK and Vinculin in cell-substratum adhesion. The role of the proteasome in controlling cell-substratum adhesion was studied using two proteasome inhibitors, lactacystin and MG132. To study the molecular mechanisms controlling Paxillin expression, MDCK cells expressing E-cadherin shRNA in a tetracycline-inducible manner was employed. Here, we present evidence that implicate Hakai in reducing cell-substratum adhesion and increasing epithelial cell invasion, two hallmark features of cancer progression and metastasis. Paxillin, an important protein component of the cell-matrix adhesion, was completely absent from focal adhesions and focal contacts in Hakai-overexpressing MDCK cells. The

  20. Single-Cell Transcriptomics and Fate Mapping of Ependymal Cells Reveals an Absence of Neural Stem Cell Function.

    Science.gov (United States)

    Shah, Prajay T; Stratton, Jo A; Stykel, Morgan Gail; Abbasi, Sepideh; Sharma, Sandeep; Mayr, Kyle A; Koblinger, Kathrin; Whelan, Patrick J; Biernaskie, Jeff

    2018-05-03

    Ependymal cells are multi-ciliated cells that form the brain's ventricular epithelium and a niche for neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ). In addition, ependymal cells are suggested to be latent NSCs with a capacity to acquire neurogenic function. This remains highly controversial due to a lack of prospective in vivo labeling techniques that can effectively distinguish ependymal cells from neighboring V-SVZ NSCs. We describe a transgenic system that allows for targeted labeling of ependymal cells within the V-SVZ. Single-cell RNA-seq revealed that ependymal cells are enriched for cilia-related genes and share several stem-cell-associated genes with neural stem or progenitors. Under in vivo and in vitro neural-stem- or progenitor-stimulating environments, ependymal cells failed to demonstrate any suggestion of latent neural-stem-cell function. These findings suggest remarkable stability of ependymal cell function and provide fundamental insights into the molecular signature of the V-SVZ niche. Copyright © 2018 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-07-12

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

  3. Establishment of cell lines from adult T-cell leukemia cells dependent on negatively charged polymers.

    Science.gov (United States)

    Kagami, Yoshitoyo; Uchiyama, Susumu; Kato, Harumi; Okada, Yasutaka; Seto, Masao; Kinoshita, Tomohiro

    2017-07-05

    Growing adult T-cell leukemia/lymphoma (ATLL) cells in vitro is difficult. Here, we examined the effects of static electricity in the culture medium on the proliferation of ATLL cells. Six out of 10 ATLL cells did not proliferate in vitro and thus had to be cultured in a medium containing negatively charged polymers. In the presence of poly-γ-glutamic acid (PGA) or chondroitin sulfate (CDR), cell lines (HKOX3-PGA, HKOX3-CDR) were established from the same single ATLL case using interleukin (IL)-2, IL-4, and feeder cells expressing OX40L (OX40L + HK). Dextran sulfate inhibited growth in both HKOX3 cell lines. Both PGA and OX40L + HK were indispensable for HKOX3-PGA growth, but HKOX3-CDR could proliferate in the presence of CDR or OX40L + HK alone. Thus, the specific action of each negatively charged polymer promoted the growth of specific ATLL cells in vitro.

  4. Direct Cell-Cell Contact between Mesenchymal Stem Cells and Endothelial Progenitor Cells Induces a Pericyte-Like Phenotype In Vitro

    Directory of Open Access Journals (Sweden)

    Markus Loibl

    2014-01-01

    Full Text Available Tissue engineering techniques for the regeneration of large bone defects require sufficient vascularisation of the applied constructs to ensure a sufficient supply of oxygen and nutrients. In our previous work, prevascularised 3D scaffolds have been successfully established by coculture of bone marrow derived stem cells (MSCs and endothelial progenitor cells (EPCs. We identified stabilising pericytes (PCs as part of newly formed capillary-like structures. In the present study, we report preliminary data on the interactions between MSCs and EPCs, leading to the differentiation of pericyte-like cells. MSCs and EPCs were seeded in transwell cultures, direct cocultures, and single cultures. Cells were cultured for 10 days in IMDM 10% FCS or IMDM 5% FCS 5% platelet lysate medium. Gene expression of PC markers, CD146, NG2, αSMA, and PDGFR-β, was analysed using RT-PCR at days 0, 3, 7, and 10. The upregulation of CD146, NG2, and αSMA in MSCs in direct coculture with EPCs advocates the MSCs’ differentiation towards a pericyte-like phenotype in vitro. These results suggest that pericyte-like cells derive from MSCs and that cell-cell contact with EPCs is an important factor for this differentiation process. These findings emphasise the concept of coculture strategies to promote angiogenesis for cell-based tissue engineered bone grafts.

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

    Science.gov (United States)

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

    2017-11-15

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

  6. Industrial n-type solar cells with >20% cell efficiency

    Energy Technology Data Exchange (ETDEWEB)

    Romijn, I.G.; Anker, J.; Burgers, A.R.; Gutjahr, A.; Koppes, M.; Kossen, E.J.; Lamers, M.W.P.E.; Heurtault, Benoit; Saynova-Oosterling, D.S.; Tool, C.J.J. [ECN Solar Energy, Petten (Netherlands)

    2013-03-15

    To realize high efficiencies at low costs, ECN has developed the n-Pasha solar cell concept. The n-Pasha cell concept is a bifacial solar cell concept on n-Cz base material, with which average efficiencies of above 20% have been demonstrated. In this paper recent developments at ECN to improve the cost of ownership (lower Euro/Wp) of the n-Pasha cell concept are discussed. Two main drivers for the manufacturing costs of n-type solar cells are addressed: the n-type Cz silicon material and the silver consumption. We show that a large resistivity range between 2 and 8 cm can be tolerated for high cell efficiency, and that the costs due to the silver metallization can be significantly reduced while increasing the solar cell efficiency. Combining the improved efficiency and cost reduction makes the n-Pasha cell concept a very cost effective solution to manufacture high efficient solar cells and modules.

  7. Demonstration of NK cell-mediated lysis of varicella-zoster virus (VZV)-infected cells: characterization of the effector cells

    Energy Technology Data Exchange (ETDEWEB)

    Tilden, A.B.; Cauda, R.; Grossi, C.E.; Balch, C.M.; Lakeman, A.D.; Whitley, R.J.

    1986-06-01

    Infection with varicella-zoster virus (VZV) rendered RAJI cells more susceptible to lysis by non-adherent blood lymphocytes. At an effector to target ratio of 80:1 the mean percentage of /sup 51/Cr release of VZV-infected RAJI cells was 41 +/- 12%, whereas that of uninfected RAJI cells was 15 +/- 6%. The increased susceptibility to lysis was associated with increased effector to target conjugate formation in immunofluorescence binding assays. The effector cells cytotoxic for VZV-infected RAJI cells were predominantly Leu-11a/sup +/ Leu-4/sup -/ granular lymphocytes as demonstrated by fluorescence-activated cell sorting. The effector cell active against VZV-infected RAJI cells appeared similar to those active against herpes simplex virus (HSV)-infected cells, because in cold target competition experiments the lysis of /sup 51/Cr-labeled VZV-infected RAJI cells was efficiently inhibited by either unlabeled VZV-infected RAJI cells (mean 71% inhibition, 2:1 ratio unlabeled to labeled target) or HSV-infected RAJI cells (mean 69% inhibition) but not by uninfected RAJI cells (mean 10% inhibition). In contrast, competition experiments revealed donor heterogeneity in the overlap between effector cells for VZV- or HSV-infected RAJI vs K-562 cells.

  8. Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research.

    Science.gov (United States)

    Kniss, Douglas A; Summerfield, Taryn L

    2014-08-01

    Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research. © The Author(s) 2014.

  9. Chemically Induced Reprogramming of Somatic Cells to Pluripotent Stem Cells and Neural Cells.

    Science.gov (United States)

    Biswas, Dhruba; Jiang, Peng

    2016-02-06

    The ability to generate transplantable neural cells in a large quantity in the laboratory is a critical step in the field of developing stem cell regenerative medicine for neural repair. During the last few years, groundbreaking studies have shown that cell fate of adult somatic cells can be reprogrammed through lineage specific expression of transcription factors (TFs)-and defined culture conditions. This key concept has been used to identify a number of potent small molecules that could enhance the efficiency of reprogramming with TFs. Recently, a growing number of studies have shown that small molecules targeting specific epigenetic and signaling pathways can replace all of the reprogramming TFs. Here, we provide a detailed review of the studies reporting the generation of chemically induced pluripotent stem cells (ciPSCs), neural stem cells (ciNSCs), and neurons (ciN). We also discuss the main mechanisms of actions and the pathways that the small molecules regulate during chemical reprogramming.

  10. Cell-in-Shell Hybrids: Chemical Nanoencapsulation of Individual Cells.

    Science.gov (United States)

    Park, Ji Hun; Hong, Daewha; Lee, Juno; Choi, Insung S

    2016-05-17

    Nature has developed a fascinating strategy of cryptobiosis ("secret life") for counteracting the stressful, and often lethal, environmental conditions that fluctuate sporadically over time. For example, certain bacteria sporulate to transform from a metabolically active, vegetative state to an ametabolic endospore state. The bacterial endospores, encased within tough biomolecular shells, withstand the extremes of harmful stressors, such as radiation, desiccation, and malnutrition, for extended periods of time and return to a vegetative state by breaking their protective shells apart when their environment becomes hospitable for living. Certain ciliates and even higher organisms, for example, tardigrades, and others are also found to adopt a cryptobiotic strategy for survival. A common feature of cryptobiosis is the structural presence of tough sheaths on cellular structures. However, most cells and cellular assemblies are not "spore-forming" and are vulnerable to the outside threats. In particular, mammalian cells, enclosed with labile lipid bilayers, are highly susceptible to in vitro conditions in the laboratory and daily life settings, making manipulation and preservation difficult outside of specialized conditions. The instability of living cells has been a main bottleneck to the advanced development of cell-based applications, such as cell therapy and cell-based sensors. A judicious question arises: can cellular tolerance against harmful stresses be enhanced by simply forming cell-in-shell hybrid structures? Experimental results suggest that the answer is yes. A micrometer-sized "Iron Man" can be generated by chemically forming an ultrathin (cell. Since the report on silica nanoencapsulation of yeast cells, in which cytoprotective yeast-in-silica hybrids were formed, several synthetic strategies have been developed to encapsulate individual cells in a cytocompatible fashion, mimicking the cryptobiotic cell-in-shell structures found in nature, for example

  11. Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures.

    Science.gov (United States)

    Juhász, Tamás; Matta, Csaba; Somogyi, Csilla; Katona, Éva; Takács, Roland; Soha, Rudolf Ferenc; Szabó, István A; Cserháti, Csaba; Sződy, Róbert; Karácsonyi, Zoltán; Bakó, Eva; Gergely, Pál; Zákány, Róza

    2014-03-01

    Biomechanical stimuli play important roles in the formation of articular cartilage during early foetal life, and optimal mechanical load is a crucial regulatory factor of adult chondrocyte metabolism and function. In this study, we undertook to analyse mechanotransduction pathways during in vitro chondrogenesis. Chondroprogenitor cells isolated from limb buds of 4-day-old chicken embryos were cultivated as high density cell cultures for 6 days. Mechanical stimulation was carried out by a self-designed bioreactor that exerted uniaxial intermittent cyclic load transmitted by the culture medium as hydrostatic pressure and fluid shear to differentiating cells. The loading scheme (0.05 Hz, 600 Pa; for 30 min) was applied on culturing days 2 and 3, when final commitment and differentiation of chondroprogenitor cells occurred in this model. The applied mechanical load significantly augmented cartilage matrix production and elevated mRNA expression of several cartilage matrix constituents, including collagen type II and aggrecan core protein, as well as matrix-producing hyaluronan synthases through enhanced expression, phosphorylation and nuclear signals of the main chondrogenic transcription factor Sox9. Along with increased cAMP levels, a significantly enhanced protein kinase A (PKA) activity was also detected and CREB, the archetypal downstream transcription factor of PKA signalling, exhibited elevated phosphorylation levels and stronger nuclear signals in response to mechanical stimuli. All the above effects were diminished by the PKA-inhibitor H89. Inhibition of the PKA-independent cAMP-mediators Epac1 and Epac2 with HJC0197 resulted in enhanced cartilage formation, which was additive to that of the mechanical stimulation, implying that the chondrogenesis-promoting effect of mechanical load was independent of Epac. At the same time, PP2A activity was reduced following mechanical load and treatments with the PP2A-inhibitor okadaic acid were able to mimic the effects of

  12. Biomek Cell Workstation: A Variable System for Automated Cell Cultivation.

    Science.gov (United States)

    Lehmann, R; Severitt, J C; Roddelkopf, T; Junginger, S; Thurow, K

    2016-06-01

    Automated cell cultivation is an important tool for simplifying routine laboratory work. Automated methods are independent of skill levels and daily constitution of laboratory staff in combination with a constant quality and performance of the methods. The Biomek Cell Workstation was configured as a flexible and compatible system. The modified Biomek Cell Workstation enables the cultivation of adherent and suspension cells. Until now, no commercially available systems enabled the automated handling of both types of cells in one system. In particular, the automated cultivation of suspension cells in this form has not been published. The cell counts and viabilities were nonsignificantly decreased for cells cultivated in AutoFlasks in automated handling. The proliferation of manual and automated bioscreening by the WST-1 assay showed a nonsignificant lower proliferation of automatically disseminated cells associated with a mostly lower standard error. The disseminated suspension cell lines showed different pronounced proliferations in descending order, starting with Jurkat cells followed by SEM, Molt4, and RS4 cells having the lowest proliferation. In this respect, we successfully disseminated and screened suspension cells in an automated way. The automated cultivation and dissemination of a variety of suspension cells can replace the manual method. © 2015 Society for Laboratory Automation and Screening.

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

    Directory of Open Access Journals (Sweden)

    Daniel Joyce

    2018-05-01

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

  14. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  15. NK cells and T cells: mirror images?

    NARCIS (Netherlands)

    Versteeg, R.

    1992-01-01

    The expression of MHC class I molecules protects cells against lysis by natural killer (NK) cells. It is possible that NK cells are 'educated' to recognize self MHC class I molecules and that the combination of self peptide and MHC class I molecule blocks NK-mediated lysis. Here, Rogier Versteeg

  16. Inhibition of PTP1B disrupts cell-cell adhesion and induces anoikis in breast epithelial cells.

    Science.gov (United States)

    Hilmarsdottir, Bylgja; Briem, Eirikur; Halldorsson, Skarphedinn; Kricker, Jennifer; Ingthorsson, Sævar; Gustafsdottir, Sigrun; Mælandsmo, Gunhild M; Magnusson, Magnus K; Gudjonsson, Thorarinn

    2017-05-11

    Protein tyrosine phosphatase 1B (PTP1B) is a well-known inhibitor of insulin signaling pathways and inhibitors against PTP1B are being developed as promising drug candidates for treatment of obesity. PTP1B has also been linked to breast cancer both as a tumor suppressor and as an oncogene. Furthermore, PTP1B has been shown to be a regulator of cell adhesion and migration in normal and cancer cells. In this study, we analyzed the PTP1B expression in normal breast tissue, primary breast cells and the breast epithelial cell line D492. In normal breast tissue and primary breast cells, PTP1B is widely expressed in both epithelial and stromal cells, with highest expression in myoepithelial cells and fibroblasts. PTP1B is widely expressed in branching structures generated by D492 when cultured in 3D reconstituted basement membrane (3D rBM). Inhibition of PTP1B in D492 and another mammary epithelial cell line HMLE resulted in reduced cell proliferation and induction of anoikis. These changes were seen when cells were cultured both in monolayer and in 3D rBM. PTP1B inhibition affected cell attachment, expression of cell adhesion proteins and actin polymerization. Moreover, epithelial to mesenchymal transition (EMT) sensitized cells to PTP1B inhibition. A mesenchymal sublines of D492 and HMLE (D492M and HMLEmes) were more sensitive to PTP1B inhibition than D492 and HMLE. Reversion of D492M to an epithelial state using miR-200c-141 restored resistance to detachment induced by PTP1B inhibition. In conclusion, we have shown that PTP1B is widely expressed in the human breast gland with highest expression in myoepithelial cells and fibroblasts. Inhibition of PTP1B in D492 and HMLE affects cell-cell adhesion and induces anoikis-like effects. Finally, cells with an EMT phenotype are more sensitive to PTP1B inhibitors making PTP1B a potential candidate for further studies as a target for drug development in cancer involving the EMT phenotype.

  17. High throughput single-cell and multiple-cell micro-encapsulation.

    Science.gov (United States)

    Lagus, Todd P; Edd, Jon F

    2012-06-15

    Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 μm polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency P(k=1) of 83.7% and a double-particle encapsulation efficiency P(k=2) of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify

  18. Investigation of the response of low-dose irradiated cells. Pt. 2. Radio-adaptive response of human embryonic cells is related to cell-to-cell communication

    International Nuclear Information System (INIS)

    Ishii, Keiichiro; Watanabe, Masami.

    1994-01-01

    To clarify the radio-adaptive response of normal cells to low-dose radiation, we irradiated human embryonic cells and HeLa cells with low-dose X-ray and examined the changes in sensitivity to subsequent high-dose X-irradiation. The results obtained were as follows; (1) When HE cells were irradiated by a high-dose of 200 cGy, the growth ratio of the living cells five days after the irradiation decreased to 37% of that of the cells which received no X-irradiation. When the cells received a preliminary irradiation of 10 to 20 cGy four hours before the irradiation of 200 cGy, the relative growth ratios increased significantly to 45-53%. (2) This preliminary irradiation effect was not observed in HeLa cells, being cancer cells. (3) When the HE cells suspended in a Ca 2+ iron-free medium or TPA added medium while receiving the preliminary irradiation of 13 cGy, the effect of the preliminary irradiation in increasing the relative growth ratio of living cells was not observed. (4) This indicates that normal cells shows an adaptive response to low-dose radiation and become more radioresistant. This phenomenon is considered to involve cell-to-cell communication maintained in normal cells and intracellular signal transduction in which Ca 2+ ion plays a role. (author)

  19. Autologous blood cell therapies from pluripotent stem cells

    Science.gov (United States)

    Lengerke, Claudia; Daley, George Q.

    2010-01-01

    Summary The discovery of human embryonic stem cells (hESCs) raised promises for a universal resource for cell based therapies in regenerative medicine. Recently, fast-paced progress has been made towards the generation of pluripotent stem cells (PSCs) amenable for clinical applications, culminating in reprogramming of adult somatic cells to autologous PSCs that can be indefinitely expanded in vitro. However, besides the efficient generation of bona fide, clinically safe PSCs (e.g. without the use of oncoproteins and gene transfer based on viruses inserting randomly into the genome), a major challenge in the field remains how to efficiently differentiate PSCs to specific lineages and how to select for cells that will function normally upon transplantation in adults. In this review, we analyse the in vitro differentiation potential of PSCs to the hematopoietic lineage discussing blood cell types that can be currently obtained, limitations in derivation of adult-type HSCs and prospects for clinical application of PSCs-derived blood cells. PMID:19910091

  20. CD8+ T cells induce thyroid epithelial cell hyperplasia and fibrosis.

    Science.gov (United States)

    Yu, Shiguang; Fang, Yujiang; Sharav, Tumenjargal; Sharp, Gordon C; Braley-Mullen, Helen

    2011-02-15

    CD8(+) T cells can be important effector cells in autoimmune inflammation, generally because they can damage target cells by cytotoxicity. This study shows that activated CD8(+) T cells induce thyroid epithelial cell hyperplasia and proliferation and fibrosis in IFN-γ(-/-) NOD.H-2h4 SCID mice in the absence of CD4(+) T cells. Because CD8(+) T cells induce proliferation rather than cytotoxicity of target cells, these results describe a novel function for CD8(+) T cells in autoimmune disease. In contrast to the ability of purified CD8(+) T cells to induce thyrocyte proliferation, CD4(+) T cells or CD8 T cell-depleted splenocytes induced only mild thyroid lesions in SCID recipients. T cells in both spleens and thyroids highly produce TNF-α. TNF-α promotes proliferation of thyrocytes in vitro, and anti-TNF-α inhibits development of thyroid epithelial cell hyperplasia and proliferation in SCID recipients of IFN-γ(-/-) splenocytes. This suggests that targeting CD8(+) T cells and/or TNF-α may be effective for treating epithelial cell hyperplasia and fibrosis.