Characterization of cardiac progenitor cell activity in engineered heart muscle Doctoral Thesis In partial fulfillment of the requirements for the degree “Doctor rerum naturalium (Dr. rer. nat.)” in the Molecular Medicine Study Program at the Georg-August University Göttingen Submitted by Elif Levent born in Karabük, Turkey Göttingen 2016 Members of the Thesis Committee: Prof. Dr. med. Wolfram-Hubertus Zimmermann (Supervisor) Email: [email protected] Phone: +49 (0) 551 39 5781 Postal Address: Institute of Pharmacology and Toxicology University Medical Center Göttingen Georg-August University Göttingen Robert-Koch-Str. 40 37075 Göttingen Germany Prof. Dr. Dörthe Katschinski (Second member of thesis committee) Email: [email protected] Phone: +49 (0) 551 39 5896 Postal Address: Institute of Cardiovascular Physiology Humboldtallee 23 37073 Göttingen Germany Prof. Dr. Viacheslav O. Nikolaev (Third member of thesis committee) Email: [email protected] Phone: +49 (0) 40 7410 51391 Postal Address: Institute of Experimental Cardiovascular Research University Medical Center Hamburg-Eppendorf (UKE) Martinistr. 52 20246 Hamburg Date of disputation: 13.06.16 AFFIDAVIT Here I declare that my doctoral thesis entitled: “Characterization of cardiac progenitor cell activity in engineered heart muscle” has been written independently with no other sources and aids than quoted. Elif Levent Göttingen, April 2016 List of poster presentations Elif Levent, Malte Tiburcy, Amke Hesse, Dörthe Katschinski, Mark Sussman and Wolfram-H Zimmermann (2013). Hypoxia/Reoxygenated engineered heart muscle as an injury model system to investigate cardiac progenitor cell function in heart repair in vitro. Cardiorepair European multidisciplinary initiative (CARE-MI) annual meeting Lisbon, Portugal. Elif Levent, Malte Tiburcy, Mark Sussman and Wolfram-H Zimmermann (2013). Engineered heart muscle as a model system to investigate cardiac progenitor cell function and heart regeneration in vitro. Deutsche Gesellschaft für Kardiologie (DGK) annual meeting, Mannheim, Germany. Elif Levent, Malte Tiburcy, Alberto Izarra, Antonio Bernad and Wolfram-H Zimmermann (2012). Engineered heart tissue to investigate cardiac progenitor cell function and heart regeneration in vitro. Cardiorepair European multidisciplinary initiative (CARE-MI) annual meeting, Leuven, Belgium. List of publications Vogler M, Zieseniss A, Hesse AR, Levent E, Tiburcy M, Heinze E, Burzlaff N, Schley G, Eckardt KU, Willam C, Katschinski DM. (2015). Pre- and post- conditional inhibition of prolyl-4-hydroxylase domain enzymes protects the heart from an ischemic insult. Pflugers Arch. 467:2141-9. Izarra A, Moscoso I, Levent E, Cañón S, Cerrada I, Díez-Juan A, Blanca V, Núñez-Gil IJ, Valiente I, Ruíz-Sauri A, Sepúlveda P, Tiburcy M, Zimmermann WH, Bernad A. (2014). mir-133a enhances the protective capacity of cardiac progenitors cells after myocardial infarction. Stem Cell Reports. 3:1029-42. Hesse AR*, Levent E*, Zieseniss A, Tiburcy M, Zimmermann WH, Katschinski DM. (2014). Lights on for HIF-1α: genetically enhanced mouse cardiomyocytes for heart tissue imaging. Cell Physiol Biochem. 34:455-62. (*equal contribution) Table of Contents Acknowledgements ................................................................................................... I Summary .................................................................................................................. III List of Figures .......................................................................................................... IV List of Tables .......................................................................................................... VII Abbreviations ........................................................................................................... IX 1 Introduction ........................................................................................................ 1 1.1 Evidence for cardiomyocyte renewal in the postnatal heart ........................... 1 1.2 Origin of new cardiomyocytes in the postnatal heart ..................................... 1 1.3 Stem/progenitor cell markers in the heart ...................................................... 4 1.4 Evidence for cardiac progenitors in the adult heart........................................ 6 1.5 Cardiogenic potential of endogenous CPCs .................................................. 8 1.5.1 c-Kit CPCs .............................................................................................. 8 1.5.2 Sca-1 CPCs .......................................................................................... 10 1.5.3 Vessel resident progenitor cells ............................................................ 11 1.6 Exogenous regeneration by CPCs .............................................................. 13 1.7 Cardiac stem cell based therapy ................................................................. 14 1.8 EHM as an in vitro cardiac muscle model .................................................... 18 1.9 Aims of the study ......................................................................................... 19 2 Materials and Methods .................................................................................... 22 2.1 Preparation of cardiomyocytes .................................................................... 22 2.1.1 Neonatal rat cardiomyocytes ................................................................. 22 2.1.2 Mouse embryonic stem cell-derived cardiomyocytes ............................ 22 2.1.3 Human embryonic stem cell-derived cardiomyocytes ........................... 24 2.2 Preparation of non-myocytes ....................................................................... 25 2.2.1 Mouse CPCs ......................................................................................... 25 2.2.2 Human CPCs ........................................................................................ 25 2.2.3 Mouse embryonic fibroblasts ................................................................ 25 2.2.4 Human foreskin fibroblasts.................................................................... 26 2.3 EHM ............................................................................................................ 26 2.3.1 Rat EHM ............................................................................................... 26 2.3.2 Mouse EHM .......................................................................................... 27 2.3.3 Human EHM ......................................................................................... 29 2.4 Isometric force measurement ...................................................................... 29 2.5 Immunostaining and microscopy ................................................................. 30 2.6 Flow cytometry ............................................................................................ 30 2.6.1 Dissociation of EHM .............................................................................. 30 2.6.2 Immunostaining for intracellular antigens .............................................. 31 2.6.3 Immunostaining for cell surface proteins ............................................... 31 2.6.4 Sorting of EHM-derived cells ................................................................. 31 2.7 Gene expression analysis ........................................................................... 32 2.7.1 RNA isolation ........................................................................................ 32 2.7.2 Reverse transcription ............................................................................ 33 2.7.3 Polymerase chain reaction .................................................................... 34 2.7.4 Agarose gel electrophoresis.................................................................. 36 2.7.5 Quantitative PCR .................................................................................. 37 2.7.6 RNA sequencing ................................................................................... 38 2.8 Cell based models of cardiomyocyte hypoxia .............................................. 38 2.8.1 Imaging hypoxia in mouse ODD-Luc cardiomyocytes ........................... 39 2.8.2 Imaging hypoxia in mouse ODD-Luc EHM ........................................... 39 2.8.3 Generation of ODD-Luc human embryonic stem cell line ..................... 40 2.8.4 Hypoxia imaging in ODD-Luc human cardiomyocytes .......................... 41 2.8.5 Hypoxia/Reoxygenation injury in human EHM ...................................... 41 2.8.6 Chronic hypoxia injury in human EHM .................................................. 42 2.9 Western blot analyses ................................................................................. 42 2.9.1 Protein isolation .................................................................................... 42 2.9.2 Bradford assay ...................................................................................... 42 2.9.3 SDS-gel electrophoresis ....................................................................... 43 2.9.4 Immunoblotting ..................................................................................... 43 2.10 Quantification of lactate release .................................................................. 44 2.11 Quantification of intracellular ATP ............................................................... 44 2.12 Statistics