Electropermeabilization of inner and outer cell membranes with microsecond pulsed electric fields : effective new tool to control mesenchymal stem cells spontaneous Ca2+ oscillations Hanna Hanna To cite this version: Hanna Hanna. Electropermeabilization of inner and outer cell membranes with microsecond pulsed electric fields : effective new tool to control mesenchymal stem cells spontaneous Ca2+ oscillations. Cellular Biology. Université Paris Saclay (COmUE), 2016. English. NNT : 2016SACLS493. tel- 01687535 HAL Id: tel-01687535 https://tel.archives-ouvertes.fr/tel-01687535 Submitted on 18 Jan 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. NNT : 2016SACLS493 THÈSE DE DOCTORAT DE L’UNIVERSITÉ PARIS-SACLAY PRÉPARÉE À L’UNIVERSITÉ PARIS-SUD 11 ÉCOLE DOCTORALE N˚ 569 Innovation Thérapeutique : du Fondamental à l’Appliqué Spécialité : Pharmacotechnie et Biopharmacie Par Mr. Hanna Hanna Electropermeabilization of inner and outer cell membranes with microsecond pulsed electric fields: effective new tool to control mesenchymal stem cells spontaneous calcium oscillations Thèse présentée et soutenue à Villejuif, le 13 Décembre 2016 Composition du Jury : M. Combettes Laurent Directeur de recherche, INSERM Président du jury M. O’Connor Rodney Professeur, Ecole des Mines de Saint Etienne Rapporteur M. Satkauskas Saulius Professeur, Vytautas Magnus University Rapporteur Mme Fromigué Olivia Chargé de recherche, INSERM Examinatrice M. Percherancier Yann Chargé de recherche, CNRS Examinateur M. Mir Lluis M. Directeur de recherche, CNRS Directeur de thèse M. André Franck M. Chargé de recherche, CNRS Co-encadrant de thèse Table of contents Abbreviations ____________________________________________________ 5 Preface _________________________________________________________ 9 I. State of the art _______________________________________________ 13 1 The Biomembranes _____________________________________________________ 15 1.1 The plasma membrane ____________________________________________________ 15 1.1.1 Historical view __________________________________________________________________ 15 1.1.1.1 The Fluid-Mosaic Model ______________________________________________________ 16 1.1.2 Plasma membrane composition ____________________________________________________ 17 1.1.2.1 Lipids _____________________________________________________________________ 17 1.1.2.1.1 Phospholipids ___________________________________________________________ 17 1.1.2.1.2 Glycolipids ______________________________________________________________ 19 1.1.2.1.3 Sterols _________________________________________________________________ 20 1.1.2.1.4 Lipid phases in plasma membrane ___________________________________________ 20 1.1.2.2 Proteins ___________________________________________________________________ 22 1.1.2.3 Carbohydrates _____________________________________________________________ 23 1.1.2.4 Lipid rafts _________________________________________________________________ 24 1.1.2.5 The asymmetry of the plasma membrane _______________________________________ 25 1.1.3 The dynamic nature of the plasma membrane ________________________________________ 26 1.1.4 The plasma membrane functions ___________________________________________________ 28 1.1.4.1 Lipids functions _____________________________________________________________ 28 1.1.4.1.1 Compartmentalization ____________________________________________________ 28 1.1.4.1.2 A selectively permeable barrier _____________________________________________ 28 1.1.4.1.3 Signal Transduction in response to external stimuli _____________________________ 29 1.1.4.2 Proteins functions __________________________________________________________ 29 1.1.4.2.1 Selective permeability: transport proteins _____________________________________ 29 1.1.4.2.2 Signal transduction: receptor proteins ________________________________________ 31 1.1.4.2.3 Other functions __________________________________________________________ 33 1.1.5 Resting membrane potential ______________________________________________________ 33 1.2 The organelles membranes _________________________________________________ 35 1.2.1 Eukaryotic cell compartmentalization _______________________________________________ 35 1.2.2 Organelles membranes composition ________________________________________________ 36 1.2.3 The endoplasmic reticulum ________________________________________________________ 38 1.2.3.1 The largest membranes network of the cell ______________________________________ 38 1.2.3.2 A highly permeable membrane? _______________________________________________ 39 1.2.3.3 The largest calcium store of the cell ____________________________________________ 40 1.2.3.4 The KDEL sequence _________________________________________________________ 41 1.3 The continuity of the cellular membranes _____________________________________ 42 1.3.1 Vesicular transport ______________________________________________________________ 42 2 Calcium regulation in the cell _____________________________________________ 45 2.1 The importance of the calcium in the organisms and the cell physiology _____________ 45 2.2 Calcium-signaling system __________________________________________________ 45 2.2.1 Principal components of the calcium increase step _____________________________________ 46 2.2.1.1 Voltage-operated calcium channels ____________________________________________ 47 1 2.2.1.2 Receptor-operated calcium channels ___________________________________________ 48 2.2.1.3 Inositol-1,4,5-triphosphate receptor ____________________________________________ 49 2.2.1.4 Ryanodine receptor _________________________________________________________ 50 2.2.1.5 Two-pore calcium channels ___________________________________________________ 50 2.2.1.6 Store-operated calcium channels ______________________________________________ 51 2.2.1.7 Calcium-induced calcium release (CICR) _________________________________________ 52 2.2.2 Principle components of the calcium decrease step ____________________________________ 52 2.2.2.1 Plasma membrane Ca2+-ATPase ________________________________________________ 53 2.2.2.2 Sarco/endoplasmic reticulum Ca2+-ATPase _______________________________________ 53 2.2.2.3 Plasma membrane NA+/Ca2+ exchanger _________________________________________ 54 2.2.2.4 Mitochondrial voltage-dependent anion channel _________________________________ 54 2.2.2.5 Mitochondrial calcium uniporter _______________________________________________ 55 2.2.3 Calcium buffers and effectors ______________________________________________________ 55 2.2.4 How the calcium spatial and temporal signaling is decoded by the cell? ____________________ 56 2.2.4.1 Calcium sparks, oscillations and waves __________________________________________ 56 2.2.4.2 Calcium-sensitive enzymes and transcription-factors ______________________________ 58 2.3 Different calcium-related mechanisms in the cell________________________________ 59 2.3.1 Mitochondria-associated membranes and apoptosis ___________________________________ 59 2.3.2 Other mechanisms and functions ___________________________________________________ 60 2.4 Studying calcium signaling: the calcium markers ________________________________ 61 2.4.1 Fluorescent Ca2+-sensitive dyes ____________________________________________________ 61 2.4.2 Genetically encoded Ca2+ indicators _________________________________________________ 63 3 Mesenchymal stem cells and differentiation _________________________________ 65 3.1 Mesenchymal stem cells ___________________________________________________ 65 3.1.1 Human adipose Mesenchymal stem cells ____________________________________________ 66 3.1.2 Calcium oscillations in MSCs _______________________________________________________ 68 3.1.3 Calcium oscillations between MSCs and differentiated cells _____________________________ 69 3.2 Mesenchymal stem cells differentiation _______________________________________ 70 3.2.1 Differentiation into osteoblasts ____________________________________________________ 71 3.2.1.1 Osteogenic-induction factors __________________________________________________ 72 3.2.1.2 The importance of the extracellular matrix in osteogenic differentiation _______________ 73 3.2.1.3 Osteodifferentiation markers _________________________________________________ 73 3.2.2 Differentiation into adipocytes _____________________________________________________ 74 3.2.2.1 Adipogenic-induction factors __________________________________________________ 74 3.2.2.2 Adipogenic markers _________________________________________________________ 75 3.2.3 Differentiation into neuronal cells __________________________________________________ 75 3.2.3.1 Neurogenic-induction factors _________________________________________________ 76 3.2.3.2 Neurogenic markers _________________________________________________________ 76 3.3 Effects of physical stimuli on the MSCs differentiation ___________________________ 77 3.3.1 Effect of mechanical stimulation ___________________________________________________ 77 3.3.2 Effect of