Using polyacrylamide hydrogels to explore differentiation and measure contractility of cardiomyocytes generated from human pluripotent stem cells

Human pluripotent stem cells (hPSCs) have the capacity to infinitely self-renew or differentiate into any somatic cell type. Since heart failure is the world’s leading cause of death, a great clinical need exists to differentiate hPSCs into cardiomyocytes, enabling exploration of new drugs or cell-based therapies. Various factors in the cellular microenvironment are known to influence self-renewal or differentiation trajectories of stem cells. In this work, we focused on substrate stiffness, as stiffening of the heart is a physiological phenomenon during embryonic development and progression of heart disease. To investigate effects of mechanics on differentiation to cardiomyocytes, we cultured hPSCs and their derivatives on polyacrylamide hydrogel substrates. In embryoid body and directed differentiation culture systems, hPSC differentiation to cardiomyocytes peaked on 49.4 kPa hydrogels. This was linked to an early peak in mesendoderm specification in hPSCs undergoing directed differentiation on hydrogels. Next we initiated differentiation on tissue culture polystyrene (TCPS) surfaces, split cells to hydrogels at the cardiac progenitor cell stage, ii and observed no ultimate difference in cardiomyocyte purity with stiffness. To manipulate cellular mechanics internally, we primed hPSCs on TCPS with Y27632, an inhibitor of cytoskeletal organization, and observed significant increases in cardiomyocyte purity. This work indicates that hPSCs are sensitive to mechanical modulation at early stages of differentiation, and proper mechanics can drastically alter their propensity to become cardiomyocytes. Although factors to obtain cardiomyocytes from hPSCs have been elucidated, the task remains to characterize the functionality of these beating cells. To this end, we developed an assay to quantify contraction stress of cardiomyocytes. We differentiated hPSCs to cardiomyocytes on TCPS and split them to polyacrylamide hydrogels embedded with fluorescent beads. Through traction force microscopy, we tracked bead displacements beneath contracting cardiomyocytes and converted them to contraction stresses. We found that, in hPSC-derived and neonatal rat cardiomyocytes, contraction stress increased with substrate stiffness. Next we demonstrated that hPSC-derived cardiomyocyte contractility responded appropriately to isoprenaline, a cardioactive drug, and remained stable in culture over a period of two months. This assay demonstrates appropriate functional responses of hPSC-derived cardiomyocytes and serves to motivate their use in drug studies and regenerative medicine. iii Acknowledgements There are many people who deserve recognition for their contributions to this work, or to my morale during the ups and downs of the past five years. First of all, thanks to my advisor, Sean Palecek, for giving me the opportunity to work in his lab and for his continuous support. Sean gave me the freedom to solve problems on my own but always had an open door for guidance, which maximized my growth as a researcher. I really appreciated his constructive criticism on every practice talk and draft, as well as his sense of humor and even temper that made him so approachable, and his grilling skills at our lab cookouts. Thanks to my inspirational friend and labmate Lance Lian for his willingness to share cells, protocols, and his encyclopedic knowledge of literature. Thanks to my considerate officemate Alicia Powers and all of the stem cell-culturing members of the Palecek lab (Samira Azarin, Elise Huang, Chet Hsiao, Josh Selekman, Amritava Das, Alex Laperle, Xiaoping Bao, and Matt Stebbins) for their constant help and friendship, and for taking good care of my cells anytime I was away. And absolutely, thanks to my three undergraduate research assistants (Mehmet Badur, Linc Han, and Stephanie Delgado) for their hard work and positive attitudes throughout good and bad phases of research. Thanks to Beth Pruitt and Chelsey Simmons for being terrific hosts during my visit to Stanford University and collaborators throughout the past three years. Thanks to Wendy Crone and Max Salick of the Engineering Physics department for performing mechanical testing, a crucial piece of this research. I am grateful to Ted Wakatsuki of InvivoSciences for providing iv cells and helpful discussions during the preparation of my first paper. In addition, thanks to Lance Rodenkirch of the W.M. Keck Laboratory for Biological Imaging for his microscopy expertise, and to WiCell Research Institute for providing cell culture space and core reagents, and for fostering a collaborative stem cell community on campus. Thanks to all of the friends that I made in Madison for some fantastic memories. My roommate Michelle Wilson is an extremely well-rounded person who introduced me to bike riding and cupcake baking, and I loved sharing my home with her. Elif Gurbuz and I had tons of carefree trips to the mall and movies as well as serious heart-to-heart talks, and she is a warm and thoughtful friend. Finally, thanks to my parents, sisters, brothers-in-law, nephews, and nieces for their support and unwavering confidence that I could succeed in this endeavor. They made each of my visits to Alabama very special and were understanding when my workload increased and my visits necessarily decreased, as long as I remembered to bring sufficient cheese and fairy food each time. It was comforting to have extended family in the Midwest, and I thank the Obukowiczes for relaxing weekends at their homes in Three Lakes and Minneapolis. To sum up my graduate school experience, the challenges and sacrifices but also the expansion of my horizons were greater than I could have anticipated. I am truly grateful to have been given this opportunity and will carry lessons and memories from Madison through all of my future endeavors. v Table of contents Abstract i Acknowledgements iii List of figures ix List of tables xi CHAPTER 1: INTRODUCTION 1 1.1 Origin of human pluripotent stem cells (hPSCs) 1 1.2 Maintenance and differentiation of human pluripotent stem cells 2 1.3 Motivation for deriving cardiomyocytes from hPSCs 3 1.4 Research objectives 4 1.4. 1 Investigate impact of substrate mechanics on differentiation of hPSCs to cardiomyocytes 5 1.4. 2 Develop an assay to quantify the functionality of hPSC-derived cardiomyocytes . 5 1.5 Outline of chapters 6 1.6 Figures and tables 7 1.7 References 8 CHAPTER 2: MANIPULATING THE CELLULAR MICROENVIRONMENT 11 2.