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Delineating the Cardio-Myogenic Hierarchy During Mouse Embryonic Development

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Delineating the Cardio-Myogenic Hierarchy during Mouse Embryonic Development by Charles Yoon A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Biomaterials and Biomedical Engineering University of Toronto © Copyright by Charles Yoon 2018 Delineating the Cardio-Myogenic Hierarchy during Mouse Embryonic Development Charles Yoon Doctor of Philosophy Institute of Biomaterials and Biomedical Engineering University of Toronto 2018 Abstract The identification of cell surface proteins on stem cells or stem cell derivatives is a key strategy for the functional characterization, isolation, and understanding of stem cell population dynamics. We have turned to cell surface mass spectrometry to increase the candidate pool of membrane proteins on cardiac progenitor cells (CPCs). Specifically, we examined the expression of surface markers on CPCs using an integrated mass spectrometry and microarray-based approach. We analyzed the genome and surface proteome of cardiac progenitors generated from the stage- specific differentiation of mouse (m) and human (h) pluripotent stem cells (PSCs). We have identified and characterized Frizzled 4 (FZD4) as a new marker for lateral plate mesoderm (LPM). We also utilized FZD4 as a marker, in conjunction with fetal liver kinase 1 (FLK1) and platelet derived growth factor alpha (PDGFRA) and demonstrated an increase in CPC purity and a subsequent increase in cardiomyocyte (CM) enrichment. Additionally, we have found FZD4 is also expressed in the hPSC system and results in a similar enrichment in CM. Furthermore, we showed that NORRIN can be presented to the FZD4 receptor to induce Wingless-related integration site (WNT) signaling-mediated proliferation, resulting in an increase in CM output ii from CPCs. This demonstrates the value in knowing the set of surface markers present on a cell at a specific stage of development and the potential to leverage that knowledge into more efficient cell differentiation protocols. Further validation of the function of FZD4 is also being established in a preliminary in vivo study. The identified surface markers also have the potential to isolate cell types not only within the CPC stage, but within the PSC, epiblast, and primitive streak stages as well. These markers have been compiled into a preliminary cell-cell communication network model overlaid with an initial alternative slicing analysis to determine potential mechanisms that impact cell signaling during early CPC development. In summary, the application of a systems biology approach as demonstrated in this thesis, greatly expanded the number of surface markers available and upon further characterization and validation, can improve our understanding of cardiac biology. iii Acknowledgments I would like to thank all those who have made this long journey a memorable one. This dissertation would not have been possible without all your support, kindness, and goodwill. First, I would to thank my supervisor, Peter Zandstra, for providing me with the opportunity to pursue a prestigious degree in a leading world-class academic environment. I also would like to thank him for his support and mentorship, and especially his infinite patience, for he has given me countless chances to learn from my mistakes and to improve upon myself. Being in this supportive environment gave me room to grow, not just academically, but personally as well, and I will keep these lessons with me always. Second, to my committee members, Gordon Keller, Andrew Emili, and Anthony Gramolini, as well as my collaborator, Bernd Wollscheid, for giving me your time and valuable feedback in guiding me through my degree as you helped me develop my critical thinking skills. Also, the support from your respective labs were instrumental in the development of my project and I would also like to thank your lab members, especially Damaris Bausch-Fluck, Andreas Frei, Steve Kattman, Nicole Dubois, Alec Witty, Johannes Hewel, and Hongbo Guo, for their time and assistance. Third, to my friends and lab mates, there isn’t much to say other than it has been an absolute blast. Special thanks to Emanuel Nazareth, Joel Ostblom, Manu Tewary, Shreya Shukla, Nimalan Thavandiran, Nika Shakiba, Dave Fluri, Liz Csaszar, Jen Ma, and everyone else for making the lab environment a fun and happy place to spend countless hours fussing over experiments and poring over data. A special thank you to Ting Yin for being our lab mom and making sure we were all well fed and clean up after ourselves. The lab wouldn’t have been able to run without you. Finally, to my parents John and Agnes Yoon, thank you for always being there through thick and thin throughout my personal journey and always giving me support when I need. Your tutelage has made me to be what I am today and your insistence on higher levels of education have provided me the opportunity to pursue my passions. iv Table of Contents Acknowledgments.......................................................................................................................... iv Table of Contents .............................................................................................................................v List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix List of Appendices ......................................................................................................................... xi List of Abbreviations .................................................................................................................... xii INTRODUCTION ......................................................................................................................1 1.1 Motivation ............................................................................................................................2 1.2 Mouse Cardiac Development ...............................................................................................3 1.2.1 Cardiac Morphogenesis in vivo ................................................................................3 1.2.2 Signaling during Heart Development ......................................................................5 1.2.3 Transcriptional Regulation of Cardiac Cells..........................................................11 1.2.4 Identification of Cardiac Progenitors and CPC Markers in vitro ..........................12 1.3 Bioreactor Technology and Stem Cell Production ............................................................14 1.4 Mass Spectrometry-Based Surface Profiling of Pluripotent Stem Cells and Derivatives ............................................................................................................................................17 1.4.1 Liquid Chromatography - Tandem Mass Spectrometry ........................................17 1.4.2 Methods to Isolate Cell Surface Proteins ...............................................................19 1.4.3 Cell Surface Proteomics in Stem Cells ..................................................................21 1.5 Transcriptomic Analysis of Pluripotent Stem Cells and their Derivatives ........................24 1.5.1 Microarray Analysis and the Advent of Deep Sequencing ....................................24 1.5.2 Expression Profiling in Cardiac Development ......................................................26 1.6 Thesis Goals and Approach ...............................................................................................27 v FZD4 marks lateral plate mesoderm and signals with NORRIN to increase cardiomyocyte induction from pluripotent stem cell-derived cardiac progenitors ............................................28 2.1 Abstract ..............................................................................................................................29 2.2 Introduction ........................................................................................................................29 2.3 Results ................................................................................................................................31 2.3.1 Integrated Mass Spectrometry and Microarray Analysis Identifies Early Mesoderm Surface Markers ...................................................................................31 2.3.2 FZD4 Identified as a Potential Marker of a Sub-Population Enriched for Cardiac Progenitors .............................................................................................................35 2.3.3 Gene Expression Analysis on Sorted CPC Sub-Populations Indicates that FZD4 Marks Pre-Cardiac Mesoderm in CPCs .................................................................38 2.3.4 FZD4-Expressing CPCs Yield Higher CM Outputs ..............................................40 2.3.5 Greater CM Output from FZD4+ CPCs is Also Observed during hPSC Differentiation ........................................................................................................43 2.3.6 Canonical FZD4-NORRIN Signaling Enhances CM Output ................................45 2.4 Discussion ..........................................................................................................................47 2.5 Conclusion .........................................................................................................................50
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