VENTRICULAR TISSUE CULTURE MODEL FOR CARDIOVASCULAR RESEARCH KRISTY PURNAMAWATI (B. Eng., NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2015 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been previously submitted for any degree in any university. _________________________ Kristy Purnamawati 06 May 2015 ii ACKNOWLEDGEMENTS The completion of this thesis will not be possible without the kind and generous help from so many people both in Singapore and Germany. I wish to express my most sincere gratitude to my supervisors and mentors Dr. William Sun, Prof. E. Birgitte Lane, A/Prof. Cao Tong, Prof. Dr. Wolfram H. Zimmermann, Dr. Poh Loong Soong and Dr. Malte Tiburcy for their invaluable guidance, intellectual input and constant support throughout the course of my study. My special thanks go out to the Zimmermann lab in Göttingen for welcoming me to be part their lab for a good portion of the project. I would also like to thank A*STAR Graduate Academy (AGA), Deutsches Zentrum für Herz-Kreislauf-Forschung E.V. (DZHK) and Deutsche Forschungsgemeinschaft (DFG) for their financial support, as well as NGS, NUS for facilitating the multitudes of change that occurred throughout this PhD journey. This work was completed in 27 months, from conception to last day of data collection. I am very grateful to my family and friends for their love and encouragement. It kept me going. YHJ, this work is for you. RIP. iii TABLE OF CONTENT TITLE PAGE ................................................................................................................... i DECLARATION ............................................................................................................. ii ACKNOWLEDGEMENTS ............................................................................................... iii TABLE OF CONTENT .................................................................................................... iv SUMMARY ................................................................................................................. vi LIST OF FIGURES ......................................................................................................... ix LIST OF TABLES .......................................................................................................... xiii LIST OF APPENDICES ................................................................................................... xv LIST OF ABBREVIATIONS ............................................................................................ xvi PUBLICATIONS, PRESENTATIONS AND AWARDS .......................................................... xvii CHAPTER 1 ........................................................................................................................... 18 General Introduction 1.1. Introduction ........................................................................................................... 19 1.2. References ............................................................................................................. 23 CHAPTER 2 ...........................................................................................................................25 Differentiation of hPSCs to Cardiomyocytes and Generation of Engineered Heart Muscles (EHMs) 2.1. Abstract ................................................................................................................ 28 2.2. Introduction and Literature review ..................................................................... 29 2.3. Materials and Methods ........................................................................................ 43 2.4. Results .................................................................................................................. 54 2.5. Discussion ............................................................................................................. 67 2.6. References ........................................................................................................... 78 CHAPTER 3 ........................................................................................................................... 85 Engineering and Functional Validation of MYL2- Knock-In Reporter Line 3.1. Abstract .................................................................................................................. 88 3.2. Introduction and Literature review ....................................................................... 90 3.3. Materials and Methods .......................................................................................... 96 3.4. Results .................................................................................................................. 116 3.5. Discussion ............................................................................................................. 144 3.6. References ........................................................................................................... 150 iv CHAPTER 4 ......................................................................................................................... 153 In vitro Maturation of HESCs-derived Cardiomyocytes 4.1. Abstract ................................................................................................................ 156 4.2. Introduction and literature review ...................................................................... 158 4.3. Materials and Methods ........................................................................................ 172 4.4. Results .................................................................................................................. 175 4.5. Discussion ............................................................................................................. 219 4.6. References ........................................................................................................... 225 CHAPTER 5 ......................................................................................................................... 231 Utility of in vitro Ventricular Cardiomyocytes Model 5.1. Abstract ................................................................................................................ 233 5.2. Introduction and literature review ...................................................................... 235 5.3. Materials and Methods ........................................................................................ 239 5.4. Results .................................................................................................................. 241 5.5. Discussion ............................................................................................................. 269 5.6. References ........................................................................................................... 278 CHAPTER 6 ......................................................................................................................... 281 General Conclusion and Future Directions APPENDICES ............................................................................................................. 286 v SUMMARY The ventricles of the heart are the chambers responsible for pumping blood into the lungs as well as the entire body. In these chambers of the heart, the working myocytes are termed ventricular cardiomyocytes (vCMs). Diseases associated with the ventricular chambers of the heart such as ventricular fibrillation, myocardial infarction and cardiomyopathy would ultimately affect the properties and functions of these vCMs. Limited supply (Peeters et al., 1995) and the lack of regenerative capacity of adult human vCMs limit their utility for in vitro studies (Mummery, 2005). Therefore, a vCM-specific in vitro model would be a useful tool for the development of therapies for ventricular chambers-associated diseases. In addition, the vCM in vitro model can also be used in cardiotoxicity drug screening, to screen newly developed drugs for ventricular pro-arrhythmic liability. In this study, human embryonic stem cells (hESCs) are used as the source of cardiomyocytes (CMs). However, cardiac differentiation of hESCs gives rise to all three subtypes of CMs. In addition, these hESCs-derived CMs are phenotypically immature, closest to fetal or at best, neonatal CMs in characteristic. To facilitate the fractionation of vCMs from a pool of mixed hESCs-derived CMs, an MYL2 specific knock-in reporter line was developed. MYL2 has been reported to be specifically expressed in vCMs and not atrial cardiomyocytes (Bird, 2003; Zhang et al., 2011). The knock-in line was generated with homologous-recombination technique with the aid of a novel genome editing tool: transcription activator-like effector nuclease (TALEN). Two reporters (mCherry and Gaussia Luciferase) and one selectable marker (neomycin resistance) were knocked into the last exon of MYL2 gene, behind the coding sequence of the gene. PCR and Southern blotting confirmed that the targeting construct was inserted at the correct site. Immunohistochemistry and FACS analysis, together with karyotype data, also vi confirmed that the knock-in lines are pluripotent and do not show any gross chromosomal aberrations. Functional validation