Yennek S.,Institute Pasteur Paris |
Yennek S.,Paris-Sorbonne University |
Burute M.,French National Institute for Agricultural Research |
Burute M.,CYTOO SA |
And 4 more authors.
Cell Reports | Year: 2014
Cells of several metazoan species have been shown to non-randomly segregate their DNA such that older template DNA strands segregate to one daughter cell. The mechanisms that regulate this asymmetry remain undefined. Determinants of cell fate are polarized during mitosis and partitioned asymmetrically as the spindle pole orients during cell division. Chromatids align along the pole axis; therefore, it is unclear whether extrinsic cues that determine spindle pole position also promote non-random DNA segregation. To mimic the asymmetric divisions seen in the mouse skeletal stem cell niche, we used micropatterns coated with extracellular matrix in asymmetric and symmetric motifs. We show that the frequency of non-random DNA segregation and transcription factor asymmetry correlates with the shape of the motif and that these events can be uncoupled. Furthermore, regulation of DNA segregation by cell adhesion occurs within a defined time interval. Thus, cell adhesion cues have a major impact on determining both DNA segregation patterns and cell fates. © 2014 The Authors.
Liu J.,CEA Grenoble |
Liu J.,CYTOO SA |
Bombera R.,CEA Grenoble |
Leroy L.,CEA Grenoble |
And 8 more authors.
PLoS ONE | Year: 2013
Background: Solid-state micropores have been widely employed for 6 decades to recognize and size flowing unlabeled cells. However, the resistive-pulse technique presents limitations when the cells to be differentiated have overlapping dimension ranges such as B and T lymphocytes. An alternative approach would be to specifically capture cells by solid-state micropores. Here, the inner wall of 15-μm pores made in 10 μm-thick silicon membranes was covered with antibodies specific to cell surface proteins of B or T lymphocytes. The selective trapping of individual unlabeled cells in a bio-functionalized micropore makes them recognizable just using optical microscopy. Methodology/Principal Findings: We locally deposited oligodeoxynucleotide (ODN) and ODN-conjugated antibody probes on the inner wall of the micropores by forming thin films of polypyrrole-ODN copolymers using contactless electro-functionalization. The trapping capabilities of the bio-functionalized micropores were validated using optical microscopy and the resistive-pulse technique by selectively capturing polystyrene microbeads coated with complementary ODN. B or T lymphocytes from a mouse splenocyte suspension were specifically immobilized on micropore walls functionalized with complementary ODN-conjugated antibodies targeting cell surface proteins. Conclusions/Significance: The results showed that locally bio-functionalized micropores can isolate target cells from a suspension during their translocation throughout the pore, including among cells of similar dimensions in complex mixtures. © 2013 Liu et al.
PubMed | French Institute of Health and Medical Research, Institut Universitaire de France, French National Institute for Agricultural Research, University Paris Diderot and CYTOO SA
Type: | Journal: Developmental cell | Year: 2017
During epithelial-to-mesenchymal transition (EMT), cells lining the tissue periphery break up their cohesion to migrate within the tissue. This dramatic reorganization involves a poorly characterized reorientation of the apicobasal polarity of static epithelial cells into the front-rear polarity of migrating mesenchymal cells. To investigate the spatial coordination of intracellular reorganization with morphological changes, we monitored centrosome positioning during EMT invivo, in developing mouse embryos and mammary gland, and invitro, in cultured 3D cell aggregates and micropatterned cell doublets. In all conditions, centrosomes moved from their off-centered position next to intercellular junctions toward extracellular matrix adhesions on the opposite side of the nucleus, resulting in an effective internal polarity reversal. This move appeared to be supported by controlled microtubule network disassembly. Sequential release of cell confinement using dynamic micropatterns, and modulation of microtubule dynamics, confirmed that centrosome repositioning was responsible for further cell disengagement and scattering.
Rodriguez-Fraticelli A.E.,Autonomous University of Madrid |
Auzan M.,CYTOO SA |
Alonso M.A.,Autonomous University of Madrid |
Bornens M.,University Pierre and Marie Curie |
Martin-Belmonte F.,Autonomous University of Madrid
Journal of Cell Biology | Year: 2012
Epithelial organ morphogenesis involves sequential acquisition of apicobasal polarity by epithelial cells and development of a functional lumen. In vivo, cells perceive signals from components of the extracellular matrix (ECM), such as laminin and collagens, as well as sense physical conditions, such as matrix stiffness and cell confinement. Alteration of the mechanical properties of the ECM has been shown to promote cell migration and invasion in cancer cells, but the effects on epithelial morphogenesis have not been characterized. We analyzed the effects of cell confinement on lumen morphogenesis using a novel, micropatterned, three-dimensional (3D) Madin-Darby canine kidney cell culture method. We show that cell confinement, by controlling cell spreading, limits peripheral actin contractility and promotes centrosome positioning and lumen initiation after the first cell division. In addition, peripheral actin contractility is mediated by master kinase Par-4/LKB1 via the RhoA-Rho kinase-myosin II pathway, and inhibition of this pathway restores lumen initiation in minimally confined cells. We conclude that cell confinement controls nuclear-centrosomal orientation and lumen initiation during 3D epithelial morphogenesis. © 2012 Rodríguez-Fraticelli et al.
Agency: European Commission | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2011.1.1-1 | Award Amount: 5.90M | Year: 2011
The MEHTRICS proposal aims to challenge the current limits of HT cell-based screening by combining HT-RNAi with an emerging new technology for normalizing cultured cell behaviors, namely the growth of cells on adhesive micropatterns. Initial applications of this technology have already demonstrated its potential for enhancing the quality of existing high content analyses by radically reducing the cell populations phenotypic variability, resulting in much lower cell sampling requirements. This approach also promises to open up major new assay development space by broadening the range of analysis strategies that drive the novel cell-based assay designs, whose evolution has otherwise stagnated in recent years. The consortium will carry out several parallel and complementary lines of development to diversify the applicability of the MEHTRICS platform for both academic and industrial uses, ultimately validating the new capabilities through proof of principle screens. Our key objectives are 1) to optimize micropattern geometries and compositions to accommodate extended timelines typical of siRNA assays, 2) to integrate the promising Transfected Cell Array (TCA) technique to decrease cost and increase throughput, 3) to develop novel cell models of key diseases based on micropatterned adult stem cells and polarized epithelia/endothelial architectures and 4) to validate each of these implementations in key industry-relevant siRNA screening applications. Since experimental designs required to run RNAi screens are among the most demanding of all HT/HC studies and encompass virtually all technical challenges also encountered in compound screens, we expect the proposed scope of activities to deliver the maximal potential for impactful innovation, widespread adoption and clear relevance for all major applications of HT/HC cell screening. The resulting new tools and methodologies will be incorporated into the commercial offerings of the consortiums two key SMEs, CYTOO and CENIX.
Czondor K.,Institut Universitaire de France |
Czondor K.,French National Center for Scientific Research |
Garcia M.,Institut Universitaire de France |
Garcia M.,French National Center for Scientific Research |
And 10 more authors.
Nature Communications | Year: 2013
Studying the roles of different proteins and the mechanisms involved in synaptogenesis is hindered by the complexity and heterogeneity of synapse types, and by the spatial and temporal unpredictability of spontaneous synapse formation. Here we demonstrate a robust and high-content method to induce selectively presynaptic or postsynaptic structures at controlled locations. Neurons are cultured on micropatterned substrates comprising arrays of micron-scale dots coated with various synaptogenic adhesion molecules. When plated on neurexin-1β-coated micropatterns, neurons expressing neuroligin-1 exhibit specific dendritic organization and selective recruitment of the postsynaptic scaffolding molecule PSD-95. Furthermore, functional AMPA receptors are trapped at neurexin-1β dots, as revealed by live-imaging experiments. In contrast, neurons plated on SynCAM1-coated substrates exhibit strongly patterned axons and selectively assemble functional presynapses. N-cadherin coating, however, is not able to elicit synapses, indicating the specificity of our system. This method opens the way to both fundamental and therapeutic studies of various synaptic systems. © 2013 Macmillan Publishers Limited. All rights reserved.
Monjaret F.,CYTOO SA |
Fernandes M.,CYTOO SA |
Duchemin-Pelletier E.,CYTOO SA |
Argento A.,CYTOO SA |
And 2 more authors.
Journal of Laboratory Automation | Year: 2016
Adoption of spheroids within high-content screening (HCS) has lagged behind high-throughput screening (HTS) due to issues with running complex assays on large three-dimensional (3D) structures. To enable multiplexed imaging and analysis of spheroids, different cancer cell lines were grown in 3D on micropatterned 96-well plates with automated production of nine uniform spheroids per well. Spheroids achieve diameters of up to 600 µm, and reproducibility was experimentally validated (interwell and interplate CVdiameter <5%). Biphoton imaging confirmed that micropatterned spheroids exhibit characteristic cell heterogeneity with distinct microregions. Furthermore, central necrosis appears at a consistent spheroid size, suggesting standardized growth. Using three reference compounds (fluorouracil, irinotecan, and staurosporine), we validated HT-29 micropatterned spheroids on an HCS platform, benchmarking against hanging-drop spheroids. Spheroid formation and imaging in a single plate accelerate assay workflow, and fixed positioning prevents structures from overlapping or sticking to the well wall, augmenting image processing reliability. Furthermore, multiple spheroids per well increase the statistical confidence sufficiently to discriminate compound mechanisms of action and generate EC50 values for endpoints of cell death, architectural change, and size within a single-pass read. Higher quality data and a more efficient HCS work chain should encourage integration of micropatterned spheroid models within fundamental research and drug discovery applications. © 2015, 2015 Society for Laboratory Automation and Screening.
Agency: European Commission | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2013.1.4-1 | Award Amount: 7.86M | Year: 2014
Despite progress in producing beta cells from human pluripotent stem cells (hPSCs) in recent years, full differentiation cannot be obtained in vitro. The HumEn project hypothesises that a fundamental understanding of the coupling between endodermal progenitor expansion and differentiation is relevant for elucidating how to a) generate glucose-responsive beta cells from hPSCs in vitro, and b) generate sufficient number of beta cells to meet future clinical needs in cell therapy in diabetes. Thus, the overall aim of HumEn is to identify, understand, and expand human endodermal progenitors as a consistent and renewable source of cells for pancreatic beta cells differentiation. We will focus on precursors from two stages of pancreatic differentiation; anterior definitive endoderm (ADE) and pancreatic endoderm (PE) progenitors, providing mechanistic insight into the signalling pathways and downstream targets that control their expansion and functional maturation into human beta cells. Rigorous in vitro (regulated insulin-release) and in vivo (protection against experimentally induced diabetes in mice) testing of insulin-producing cells will ensure a functional end product. The consortium proposes to address these problems by a unique combination of models and experimental approaches, including genetic, surface/biomaterial screens (3D), and cell surface antibody screens as well as cell signalling-to-transcription factor/chromatin effectors. In the end, HumEn aims to deliver a reliable and scalable protocol for directed differentiation of hPSCs into bona fide beta cells. The results of the project will not only provide answers to fundamental questions, but also deliver new concepts and knowledge of general importance for coordination of cell cycle progression and regulation of cell fate specification in stem cells/progenitors. HumEn is highly innovative and carries excellent potential for translational output.
PubMed | CYTOO SA
Type: Journal Article | Journal: Journal of laboratory automation | Year: 2016
Adoption of spheroids within high-content screening (HCS) has lagged behind high-throughput screening (HTS) due to issues with running complex assays on large three-dimensional (3D) structures.To enable multiplexed imaging and analysis of spheroids, different cancer cell lines were grown in 3D on micropatterned 96-well plates with automated production of nine uniform spheroids per well. Spheroids achieve diameters of up to 600 m, and reproducibility was experimentally validated (interwell and interplate CV(diameter) <5%). Biphoton imaging confirmed that micropatterned spheroids exhibit characteristic cell heterogeneity with distinct microregions. Furthermore, central necrosis appears at a consistent spheroid size, suggesting standardized growth.Using three reference compounds (fluorouracil, irinotecan, and staurosporine), we validated HT-29 micropatterned spheroids on an HCS platform, benchmarking against hanging-drop spheroids. Spheroid formation and imaging in a single plate accelerate assay workflow, and fixed positioning prevents structures from overlapping or sticking to the well wall, augmenting image processing reliability. Furthermore, multiple spheroids per well increase the statistical confidence sufficiently to discriminate compound mechanisms of action and generate EC50 values for endpoints of cell death, architectural change, and size within a single-pass read. Higher quality data and a more efficient HCS work chain should encourage integration of micropatterned spheroid models within fundamental research and drug discovery applications.
PubMed | The Interdisciplinary Center, Institute Pasteur Paris and CYTOO SA
Type: Journal Article | Journal: Proceedings of the National Academy of Sciences of the United States of America | Year: 2015
Neuronal growth cones move forward by dynamically connecting actin-based motility to substrate adhesion, but the mechanisms at the individual molecular level remain unclear. We cultured primary neurons on N-cadherin-coated micropatterned substrates, and imaged adhesion and cytoskeletal proteins at the ventral surface of growth cones using single particle tracking combined to photoactivated localization microscopy (sptPALM). We demonstrate transient interactions in the second time scale between flowing actin filaments and immobilized N-cadherin/catenin complexes, translating into a local reduction of the actin retrograde flow. Normal actin flow on micropatterns was rescued by expression of a dominant negative N-cadherin construct competing for the coupling between actin and endogenous N-cadherin. Fluorescence recovery after photobleaching (FRAP) experiments confirmed the differential kinetics of actin and N-cadherin, and further revealed a 20% actin population confined at N-cadherin micropatterns, contributing to local actin accumulation. Computer simulations with relevant kinetic parameters modeled N-cadherin and actin turnover well, validating this mechanism. Such a combination of short- and long-lived interactions between the motile actin network and spatially restricted adhesive complexes represents a two-tiered clutch mechanism likely to sustain dynamic environment sensing and provide the force necessary for growth cone migration.