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Final Draft Masterthesis Gabbin MASTER THESIS Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Engineering at the University of Applied Sciences Technikum Wien Degree Program in Tissue Engineering and Regenerative Medicine Modeling of a Novel Filamin-C Mutation in Restrictive Cardiomyopathy using hiPSC- derived Cardiomyocytes By: Beatrice Gabbin, BSc Student Number: 1810692024 Supervisor 1: Dr. William Pu, MD Dr. Maksymilian Prondzynski, PhD Supervisor 2: Dorota Szwarc, MSc Boston, September 29, 2020 Declaration of Authenticity “As author and creator of this work to hand, I confirm with my signature knowledge of the relevant copyright regulations governed by higher education acts (see Urheberrechtsgesetz/ Austrian copyright law as amended as well as the Statute on Studies Act Provisions / Examination Regulations of the UAS Technikum Wien as amended). I hereby declare that I completed the present work independently and that any ideas, whether written by others or by myself, have been fully sourced and referenced. I am aware of any consequences I may face on the part of the degree program director if there should be evidence of missing autonomy and independence or evidence of any intent to fraudulently achieve a pass mark for this work (see Statute on Studies Act Provisions / Examination Regulations of the UAS Technikum Wien as amended). I further declare that up to this date I have not published the work to hand nor have I presented it to another examination board in the same or similar form. I affirm that the version submitted matches the version in the upload tool.” Boston, September 29, 2020 Place, Date Signature Abstract The purpose of this project was to establish an in vitro model for a novel FLNC missense mutation (c.7416_7418delGAA/p.Glu2472_Asn2473delAsp), which was identified at the Boston Children’s Hospital in a patient diagnosed with restrictive cardiomyopathy (RCM). The mutation was studied with the use of use of human induced pluripotent stem cell- (hiPSC) derived cardiomyocytes (hiPSC-CMs) by applying two- and three-dimensional (2D, 3D) models, with the ultimate goal of developing a new therapeutic approach for the affected patient as well as other RCM patients. The FLNC mutation was introduced by CRISPR/Cas9 genome editing into healthy control hiPSCs. CRISPR/Cas9-edited hiPSCs and their isogenic controls were differentiated into cardiomyocytes and assessed for differentiation efficiency and their genotype was determined. 2D and 3D disease modeling approaches recapitulated the restrictive phenotype caused by the novel FLNC mutation. Immunofluorescence assays showed and altered sarcomeric structure of mutant cardiomyocytes, whereby mRNA and protein expression analysis suggested that cells are undergoing extensive sarcomeric remodeling. 3D model of engineered heart tissues (EHTs) was able to give further insights on contraction parameters such as force, time and relaxation. Mutant EHTs showed lower contraction force and conduction deficiencies. These important parameters resembled an RCM phenotype in vitro that will lay the basis for further understanding of disease mechanisms that lead to RCM and the testing of novel therapeutic approaches. Acknowledgements In these past two years, science gave me the opportunity to get to know incredible people who accompanied me through my journey and enriched me day after day, both professionally and personally. I would like to start by thanking Dr. William Pu for having me as a student in his group at Boston Children’s Hospital. Coming to work for a prestigious Harvard Medical School’s associate laboratory was a privilege. Thank you to all the Pu lab team and staff who I worked with and always offered me a helping hand when needed. A special thank goes to Dr. Maksymilian Prondzynski. Dear Maks, you are one of those teachers who made an impact in my academic career. You were a great supervisor and an inspiring researcher. Your passion for science is tangible and your curiosity contagious. Working together was a pleasure and I had a lot to learn from you as a young scientist. I would like to thank the Austrian Marshall Plan Foundation for granting me the scholarship which allowed me to do research in the United States for my master thesis. Thank you to Dr. Andreas Teuschl, program director of the master program, for being supportive and involved with his students. Moreover, I would like to thank my supervisor Dorota Szwarc for her constructive feedback and her availability. Thank you to my friends: the old ones, the new ones and the ones who have been there for me even through hard times. I have always valued friendship, but never before this year I fully understood how important it is to have friends in your life. In particular, thank you to my friends from Italy for showing me love even across an ocean. Thank you to my amazing friends from Vienna and the memories we made together. Thank you to my friends from Boston and for making the lockdown caused by the global pandemic more tolerable. You were my family away from home. Lastly, my greatest thank goes to my family. Being away from you during such tough times was one of the hardest situations that life put me through. Nevertheless, you have always infused me with positivity and continued to be my biggest supporters. I would never be who I am today if it was not for you and what you taught me: the ones who never doubted either my stubborn ambitions or my abilities, even if they caused us to be far away from each other. Thank you for making me believe that anything is possible if you truly want it. Cara nonna, grazie per aver contato i giorni che ci separavano dal rivederci ogni volta che partivo per un viaggio. Grazie per avermi insegnato che è importante lavorare duro per ottenere ciò che si vuole nella vita. This year has been so far very challenging, but also full of experiences that led me to a huge self-improvement. Despite the difficulties, I consider myself proud of my achievements and grateful for being where I am now in life. Thanks to university years and internships, I opened my eyes to the beauty of science, and I could not be more excited to see what the future brings. Table of Contents 1 Introduction ....................................................................................................... 11 1.1 Restrictive cardiomyopathy ........................................................................................... 11 1.1.1 Diagnosis and treatment of RCM .............................................................................. 15 1.2 Filamin-C ......................................................................................................................... 17 1.3 Human induced pluripotent stem cells ........................................................................ 21 1.3.1 hiPSC-derived cardiomyocytes ................................................................................. 23 1.3.2 Disease modeling using hiPSCs-CMs ....................................................................... 24 1.4 Aim .................................................................................................................................... 24 1.5 Relevance ........................................................................................................................ 25 2 Methods ........................................................................................................... 26 2.1 Maintenance of WTC-Cas9 and FLNC clones cell lines ........................................... 26 2.2 Cardiomyocyte differentiation of hiPSCs .................................................................... 27 2.2.1 2D differentiation ....................................................................................................... 27 2.2.2 Lactate selection ........................................................................................................ 27 2.2.3 Enzymatic dissociation .............................................................................................. 28 2.3 Quality control ................................................................................................................. 28 2.3.1 Genotyping using polymerase chain reaction ........................................................... 28 2.3.2 Off-target analysis ..................................................................................................... 28 2.3.3 Amplicon sequencing ................................................................................................ 29 2.3.4 Gel electrophoresis .................................................................................................... 29 2.3.5 Karyotyping ................................................................................................................ 29 2.4 Fluorescence-activated cell sorting ............................................................................. 30 2.4.1 FACS analysis for pluripotency markers .................................................................... 30 2.4.2 FACS analysis for differentiated cardiomyocytes ...................................................... 30 2.5 Disease modeling with hiPSC-derived cardiomyocytes ............................................ 30 2.5.1 2D disease modeling ................................................................................................. 30 2.5.1.1 Fixation of 2D disease modeling plates ................................................................. 31 2.5.2 Generation of EHTs ..................................................................................................
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