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A Multi-Angled Approach to Discover and Improve Skeletal Muscle Stem Cell Therapies

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A Multi-Angled Approach to Discover and Improve Skeletal Muscle Stem Cell Therapies A Multi-Angled Approach to Discover and Improve Skeletal Muscle Stem Cell Therapies by Sadegh Davoudi 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 Sadegh Davoudi 2019 A Multi-Angled Approach to Discover and Improve Skeletal Muscle Stem Cell Therapies Sadegh Davoudi Doctor of Philosophy Institute of Biomaterials and Biomedical Engineering University of Toronto 2019 Abstract Skeletal muscle plays an essential role in locomotion, metabolism, and thermoregulation. An intriguing characteristic of skeletal muscle is its remarkable regenerative capacity: a highly orchestrated cellular process, in which the resident muscle stem cells (MuSCs) play a central role. Upon tissue injury, quiescent MuSCs are activated and give rise to a population of primary myoblasts (pMBs). pMBs undergo several rounds of division and ultimately fuse with one another to form multi-nucleated myofibers and repair the muscle. As observed in a wide range of conditions such as muscular dystrophies and aging, disruptions in the repair process can lend to impaired regeneration and progressive muscle wasting. Strategies to restore strength and function to pathological muscle include cell-based therapies to replace defective myogenic cells, and treatments to restore the endogenous repair process. Despite substantial advances, these treatment options are still in the early stages of clinical translation. The specific goal of this thesis is to improve upon currently available strategies as well as to lay the groundwork for the emergence of new therapeutic entry-points for skeletal muscle treatments. In the area of cell transplantation therapy, challenges remain in producing clinically-relevant numbers of cells that, as a population, possess high regenerative potency to produce skeletal muscle and repopulate the stem cell niche. We demonstrate that by using a bioactive hydrogel ii (HAMC) as the cell delivery vehicle, it is possible to improve MuSC transplantation outcomes, and thereby reduce the overall number of required MuSCs. Next, we address the issue of producing sufficient numbers of highly regenerative myogenic cells. Using a high-throughput drug screen and in-vivo intramuscular transplantation assay validation, we identify epidermal growth factor receptor (Egfr) and vascular endothelial growth factor receptor 2 (Vegfr2, Kdr) as new druggable targets, that upon inhibition, produce a population of cultured MuSCs with greater regenerative potency than control treated. Finally, using single-cell RNA sequencing, we shed light on the diversity and intercommunication of cells present in skeletal muscle. This dataset serves as a valuable resource through which new regulators of MuSCs and other cells in skeletal muscle can be evaluated with an eye towards skeletal muscle regenerative medicine applications. iii Acknowledgments First and foremost, I would like to thank my supervisor, Dr. Penney Gilbert, for her continuous support, advice, and encouragement throughout the past few years. Penney’s enthusiasm for research and her thoughtful feedback and guidance inspired me to be a better scholar and allowed me to find my place in the field at my own pace while ensuring I develop my capabilities as a researcher. Thank you, Penney, for taking a chance on me, continuing to challenge me, and for being a shining example of a great scientist. I am also indebted to my supervisory committee members: Drs. Craig Simmons and Edmond Young. Your time and constructive scientific critique provided me with valuable feedback on my work and helped sharpen my research and analysis. Throughout my time in the Gilbert lab, I have watched the lab grow from a handful of enthusiastic students to a full-fledged powerhouse producing state-of-the-art research. I want to convey my gratitude to my lab members, current and past, without whom I could not have completed this journey. Mohsen, we began and completed this journey together; thank you for being there during my highs and my lows, and always willing to lend a helping hand. Gini, thanks being an amazing undergraduate student! Gini, Bella, Mohammad, Majid, Aliyah, Richard, Ben, Min, Olivia, Louise, and all others, thank you for bringing your amazing positive energy to the lab and making it such a collaborative and welcoming environment. To my parents, Hadi and Monir, thank you for sparking my interest in research and your continuous support throughout both my personal and academic life. I will be forever grateful for all the sacrifices you’ve made for me. And to my siblings, Mohammad and Mahtab, thank you for being the wonderful people you are and the memorable memories we’ve built together. Without all of you, I would not be where I am today. And lastly, I want to acknowledge the contributions of my wife, Fatemeh. We started our journey together at the same time I began my PhD and throughout this time, you have motivated me, challenged me, made me smile, and lead by example how to live a good life. Your love, support, and encouragement have made this dissertation a reality. Thank you! iv Table of Contents Acknowledgments.......................................................................................................................... iv Table of Contents .............................................................................................................................v List of Abbreviations .......................................................................................................................x List of Tables ............................................................................................................................... xiv List of Figures ................................................................................................................................xv Chapter 1 Introduction .....................................................................................................................1 Introduction .................................................................................................................................1 1.1 Skeletal muscle ....................................................................................................................1 1.2 Skeletal muscle homeostasis ................................................................................................3 1.2.1 Muscle stem cells in quiescent muscle ....................................................................4 1.2.2 Vasculature and endothelial cells .............................................................................7 1.2.3 Interstitial cells .........................................................................................................7 1.2.4 Motor neurons ..........................................................................................................8 1.3 Skeletal muscle regeneration ...............................................................................................9 1.3.1 Muscle stem cells in skeletal muscle regeneration ................................................11 1.3.2 Immune response ...................................................................................................17 1.3.3 Fibro-adipogenic progenitors .................................................................................18 1.3.4 Endothelial cells .....................................................................................................19 1.4 Skeletal muscle pathology and degeneration .....................................................................20 1.4.1 Aging......................................................................................................................20 1.4.2 Muscular dystrophies .............................................................................................22 1.5 Skeletal muscle treatments .................................................................................................25 1.5.1 Cell based therapies ...............................................................................................25 1.5.2 Enabling endogenous repair ...................................................................................28 1.6 Thesis Motivation, aims, and approach .............................................................................30 v 1.6.1 Thesis motivation and aims ...................................................................................30 1.6.2 Thesis Overview ....................................................................................................32 Chapter 2 Muscle stem cell intramuscular delivery within hyaluronan methylcellulose improves engraftment efficiency and dispersion ......................................................................34 Muscle stem cell intramuscular delivery within hyaluronan methylcellulose improves engraftment efficiency and dispersion ......................................................................................35 2.1 Abstract ..............................................................................................................................35 2.2 Introduction ........................................................................................................................36 2.3 Results ................................................................................................................................39 2.3.1 Muscle stem cell delivery within HAMC improves
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