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The Role of Tbx5 in Sinoatrial Node Differentiation in Mouse Embryonic Stem Cell Derived Cardiomyocytes Yunkai Dai Clemson University, [email protected]

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The Role of Tbx5 in Sinoatrial Node Differentiation in Mouse Embryonic Stem Cell Derived Cardiomyocytes Yunkai Dai Clemson University, Yunkaidai@Gmail.Com Clemson University TigerPrints All Dissertations Dissertations May 2019 The Role of Tbx5 in Sinoatrial Node Differentiation in Mouse Embryonic Stem Cell Derived Cardiomyocytes Yunkai Dai Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Recommended Citation Dai, Yunkai, "The Role of Tbx5 in Sinoatrial Node Differentiation in Mouse Embryonic Stem Cell Derived Cardiomyocytes" (2019). All Dissertations. 2377. https://tigerprints.clemson.edu/all_dissertations/2377 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. THE ROLE OF TBX5 IN SINOATRIAL NODE DIFFERENTIATION IN MOUSE EMBRYONIC STEM CELL DERIVED CARDIOMYOCYTES A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Bioengineering by Yunkai Dai May 2019 Accepted by: Dr Ann Foley, Committee Chair Dr Agneta Simionescu Dr Robin C. Muise-Helmericks Dr Ying Mei ABSTRACT The sinoatrial node of the mouse embryo arises from the wall of the right atrium near the border of the sinus venosus. Early in development this region expresses the transcription factor Tbx5. Because of this, Tbx5 is thought to sit at the apex of a transcriptional cascade leading to sinoatrial node (SAN) differentiation. To test this we produced a mouse embryonic stem cell line B1 (pTripZ-mTbx5; αMHC::GFP) that conditionally overexpresses Tbx5, to determine if this would lead to enhanced SAN differentiation. We found that ES cells overexpressing Tbx5 showed enhanced overall cardiac differentiation and that cardiac cells showed increased beat rates as compared control embryos. Despite this, key genes associated with SAN differentiation including HCN4 and Shox2 increased in cells overexpressing Tbx5, while the percent of HCN4 or Shox2 positive myocytes did not alter. Faster beating cells showed a decreased expression of the chamber specific marker Cx43 and increased expression of Tbx3 and Tbx18. These data suggest that Tbx5 overexpression is not sufficient for SAN differentiation, although it does activate part of the transcriptional cascade that directs early steps in the SAN. Together these data suggest that Tbx5 cannot activate SAN differentiation alone but instead must synergize with other factors. ii DEDICATION This work is dedicated to my parents, my father YUQING DAI, my mother WENYAN MAO. Without constant supports from you, I would never be eligible to fulfill my goal to pursue my PhD degree. In 2017, I had my low-back problem which needs immediate surgery, so my dad flied from Shanghai to Charleston over 10,000km to look after me before and after my surgery by Dr John Glaser. Their help brought me back to normal life. Thanks you so much. iii ACKNOWLEDGMENTS I would like to first thank my PI, Dr Ann.C.Foley, who has supported and guided me through the past six years. In 2014, when I was the first phD student in Foley lab, Dr Foley not only showed me the professional way to perform cellular and molecular experiments, but also drove me to supermarkets for grocery shopping over the weekends when I do not have a car at that time. Her famous saying is “Devil in the details”, each steps of protocols hides a devil inside when you do not take care of your experiments seriously. Working with my lab mate is so fun. Dr Andrew Hunter and Allison Reno built up MATLAB algorithm to calculate the beat rate automatically. Isabel Jia, she is a fast learner and a big help. Ray Deepe, he assisted me a lot for taking image on microscopy. I would also like to acknowledge other trainee of Foley lab and members of Mei lab and I really enjoyed discussing the projects with all these guys. I would like to thanks all of my thesis committee members, Dr Simionescu, Dr Muise-Helmericks and Dr Mei, who helped me and guided me through my phD study. Special thanks to Dr Robin Muise-Helmericks, she gave me a great overview of MAP3K7 signaling pathway. They gave me deep insight into my project. Finally, I would like to thanks my girlfriend, Bowen Sun, who is a beautiful girl from Harbin, China. We met together at Charleston, sounds like a miracle. She is a great cooker and always likes trying new recipe, so we both enjoyed the moment of tasting her delicious dinner. I hope this can stay forever. Love you so much, Bowen. iv TABLE OF CONTENTS Page TITLE PAGE .................................................................................................................... i ABSTRACT ..................................................................................................................... ii DEDICATION ................................................................................................................ iii ACKNOWLEDGMENTS .............................................................................................. iv LIST OF TABLES .......................................................................................................... ix LIST OF FIGURES ......................................................................................................... x CHAPTER I. LITERATURE REVIEW .............................................................................. 1 Abstract .................................................................................................... 1 Introduction .............................................................................................. 1 Characteristics of an ideal population for cardiac Graft ................................................................................................... 3 Studies elucidating the molecular mechanisms of cardiac differentiation .................................................................... 4 Cardiac Progenitors .................................................................................. 7 Progress and new challenges for cell-based therapies ........................................................................................... 10 Transcription Factor-Mediated Reprogramming ................................... 13 Engineering cardiovascular tissues ........................................................ 14 Heart Conduction System ...................................................................... 22 Developmental Origin of the Sinoatrial Node ....................................... 22 The transcriptional program that directs formation of the AVC and Purkinje system ..................................................... 26 Sinoatrial Node arrhythmias .................................................................. 27 Conclusions ............................................................................................ 27 II. SPECIFIC AIMS ......................................................................................... 28 Abstract .................................................................................................. 28 Specific aims .......................................................................................... 29 v Table of Contents (Continued) Page III. MATERIAL AND METHODS ................................................................... 33 Cell culture ............................................................................................. 33 Plasmid transfection and Lentivirus production .................................... 33 Lentivirus Transduction and establishment of clonal cell lines ................................................................................ 34 Embryoid Body (EB) Differentiation .................................................... 35 Construct of pTripZ Vector, doxycycline-inducible- Gene of Interest (GOI)-overexpression Vector ................................ 35 Construct of pTET-ON Vector, doxycycline-inducible- GOI-overexpression Vector Flow Cytometry .................................. 36 List of vectors and mESCs line made .................................................... 38 Real-time PCR ....................................................................................... 39 Flow Cytometry ..................................................................................... 42 Immunocytochemistry ........................................................................... 42 Beat Rate data ........................................................................................ 43 IV. TBX5 OVEREXPRESSION ENHANCES CARDIAC DIFFERENTIATION AND INCREASES BEAT RATE BUT DOES NOT INCREASE THE FORMATION OF FULLY DIFFERENTIATED SINOATRIAL NODE CELLS .............................................................. 44 Introduction ............................................................................................ 44 Four independent clonal lines that conditionally overexpress Tbx5 were established ................................................. 44 Addition of Doxycycline increased beat rate ......................................... 47 Relative Tbx5 RNA Expression during EB differentiation ................................................................................... 50 Tbx5 overexpression increases overall cardiac formation ..................... 55 Subtype of Differentiated Cardiomyocytes ........................................... 58 Representative Image of ICC staining ................................................... 62 Summary ...............................................................................................
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