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The Influence of Notch Over-Stimulation on Muscle Stem Cell Quiescence Versus Proliferation, and on Muscle Regeneration Can Ding

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The Influence of Notch Over-Stimulation on Muscle Stem Cell Quiescence Versus Proliferation, and on Muscle Regeneration Can Ding The influence of Notch over-stimulation on muscle stem cell quiescence versus proliferation, and on muscle regeneration Can Ding To cite this version: Can Ding. The influence of Notch over-stimulation on muscle stem cell quiescence versus proliferation, and on muscle regeneration. Cellular Biology. Université Pierre et Marie Curie - Paris VI; Freie Universität (Berlin). Fachbereich Biologie, 2015. English. NNT : 2015PA066399. tel-01316596 HAL Id: tel-01316596 https://tel.archives-ouvertes.fr/tel-01316596 Submitted on 6 Jul 2017 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. THESIS The influence of Notch over-stimulation on muscle stem cell quiescence versus proliferation, and on muscle regeneration Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) Submitted to The Department of Biology, Chemistry and Pharmacy Freie Universität, Berlin and Ecole doctorale ED515 «Complexité du vivant» Université Pierre et Marie Curie, Paris by Can Ding, M. Med. from the Peoples’ Republic of China 2015 The work was prepared between October 2011 and July 2015 under the supervision of Prof. Dr. med. Markus Schuelke in the Department of Neuropediatrics, Charité Univer- sitätsmedizin Berlin and Prof. Luis Garcia, PhD in INSERM U1179, Université de Ver- sailles St-Quentin. I conducted the main part of my thesis in the laboratory of Prof. Dr. med. Markus Schuelke at Charité in Berlin. During my PhD, I worked for 6 month in the laboratory of Prof. Luis Garcia, PhD in Paris, 3 months at Université Pierre et Marie Curie Paris in 2012 and 3 months at the Université de Versailles St-Quentin in 2014. Reviewers and Members of the Dissertation Committee PhD supervisor: Prof. Dr. med. Markus Schuelke Charité Universitätsmedizin Berlin PhD supervisor: Prof. Luis Garcia, PhD Université Pierre et Marie Curie Paris, and Université de Versailles St-Quentin 1st external scientific expert: PD Dr. rer. nat. Vera Kalscheuer Max-Planck-Institute for Molecular Genetics 2nd external scientific expert: Dr. med. Dipl. Chem. Sebahattin Cirak Universität zu Köln Date of thesis submission: 21.08.2015 Date of thesis defense: 06.11.2015 Affidavit I hereby declare that I wrote my doctoral thesis entitled "The influence of Notch over- stimulation on muscle stem cell quiescence versus proliferation, and on muscle regeneration” independently and with no other sources and aids than quoted. Berlin, August 2015 ____________________ (Signature) Table of Contents List of figures ................................................................................................................................... i List of tables ................................................................................................................................... ii List of abbreviations ...................................................................................................................... iii Summary ........................................................................................................................................ v Zusammenfassung ....................................................................................................................... vi Résumé ........................................................................................................................................ vii 1. General introduction .............................................................................................................. 1 1.1 Muscle tissue ........................................................................................................................ 1 1.1.1 Muscle development ................................................................................................. 2 1.2 Muscle stem cells (MuSCs) .................................................................................................. 7 1.2.1 Characterization of MuSCs ....................................................................................... 9 1.2.2 Human satellite cells ............................................................................................... 12 1.3 The stem cell niche ............................................................................................................. 13 1.3.1 Regulatory signaling pathways ............................................................................... 14 1.3.2 The satellite cell niche ............................................................................................. 17 1.4 Notch signaling ................................................................................................................... 18 1.4.1 Notch ligands and receptors ................................................................................... 18 1.4.2 Notch determines asymmetric division ................................................................... 20 1.4.3 Multiple roles of Notch in cells of the myogenic lineage ......................................... 21 1.4.4 Notch signaling in satellite cells .............................................................................. 22 1.5 Muscle stem cells for cell therapy ....................................................................................... 25 1.5.1 DMD model and treatment ...................................................................................... 25 1.5.2 Cell therapy ............................................................................................................. 25 1.5.3 Isolation of MuSCs .................................................................................................. 27 1.5.4 Adeno-associated virus (AAV) ................................................................................ 29 2. Aim of the Project ................................................................................................................ 32 3. Materials and methods ........................................................................................................ 33 3.1 Materials.............................................................................................................................. 33 3.1.1 Chemicals and consumables .................................................................................. 33 3.1.2 Plasmids and cell lines ............................................................................................ 33 3.1.3 Animals ................................................................................................................... 34 3.1.4 Antibodies ............................................................................................................... 35 3.1.5 Buffers, solutions and media ................................................................................... 36 3.1.6 Software .................................................................................................................. 37 3.2 Methods .............................................................................................................................. 38 3.2.1 Molecular biological methods.................................................................................. 38 3.2.2 Cell biology Methods ............................................................................................... 39 3.2.3 Biochemical methods .............................................................................................. 44 3.2.4 Histological methods ............................................................................................... 47 3.2.5 Transgenic animal experiments .............................................................................. 48 3.2.6 Statistical analysis ................................................................................................... 50 4. Results ................................................................................................................................ 51 4.1 Fiber-associated satellite cell culture .................................................................................. 51 4.2 Recombinant Dll1-Fc protein production ............................................................................ 54 4.3 The biological effect of Dll1-Fc on cultured myoblasts ....................................................... 55 4.4 The effect of Dll1-Fc on muscle progenitor cells (MPCs) ................................................... 59 4.5 The in vivo effect of Notch overstimulation ......................................................................... 64 4.5.1 The viral gene transfer system................................................................................ 64 4.5.2 The effect of Notch overstimulation on postnatal muscle growth ........................... 66 4.5.3 The effect of Dll1-overexpression during muscle regeneration .............................. 72 5. Discussion ........................................................................................................................... 77 5.1 Immobilization of Dll1-Fc and its effect in myoblasts.
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