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Engineering of Tools for De Novo Assembly of Human Cells by Chung-Yun (George) Chao B.S

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Engineering of Tools for De Novo Assembly of Human Cells by Chung-Yun (George) Chao B.S Engineering of Tools for De Novo Assembly of Human Cells By Chung-Yun (George) Chao B.S. Genetics, Cell Biology, and Development, University of Minnesota – Twin Cities, 2013 B.S. Computer Science, University of Minnesota – Twin Cities, 2013 SUBMITTED TO THE HARVARD-MIT PROGRAM IN HEALTH SCIENCES AND TECHNOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN MEDICAL ENGINEERING AND MEDICAL PHYSICS AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY SEPTEMBER 2020 ©2020 George Chao. All rights reserved. The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now known or hereafter created. Signature of Author: ____________________________________________________________ Harvard-MIT Program in Health Sciences and Technology September 10, 2020 Certified by: ___________________________________________________________________ George M. Church, Ph.D. Professor of Genetics, Harvard Medical School Thesis Supervisor Accepted by: __________________________________________________________________ Emery N. Brown, M.D., Ph.D. Director, Harvard-MIT Program in Health Sciences and Technology Professor of Computational Neuroscience and Health Sciences and Technology, MIT Page Intentionally Left Black for Double-Sided Printing 2 Engineering of Tools for De Novo Assembly of Human Cells By Chung-Yun (George) Chao Submitted to the Department of Health Sciences and Technology on September 10, 2020 in Partial Fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Engineering and Medical Physics ABSTRACT Organs for transplantation has continuously been in short supply and, given COVID-19’s propensity to adversely impact solid organs, the shortage will likely become exacerbated. For decades, the field of tissue engineering has developed innovative methods to generate model tissues de novo. Top-down approaches, such as microfluidics and 3D bioprinting, provide spatial control by patterning cell types with high resolution, but face challenges in reproducing physiologically accurate cell types and interactions. Bottom-up methods, such as organoids, induce pluripotent cells to differentiate into aggregates that resemble their in vivo counterparts, yet the size and complexity of these structures are limited by nutrient diffusion and the morphology cannot be controlled. An ideal system would allow for high spatial control while retaining native cell-cell interactions formed through developmental progression. To approach this capability, we aimed to create a sequential gene expression system that programmatically aggregate and differentiate cells, merging both top-down and bottom-up characteristics. First, we curated and characterized 28 recombinases to determine efficiency and pairwise compatibility for use in mammalian recombinase genetic circuits (RGC). From this set, we designed an RGC capable of expressing 12 genes in sequence, providing a framework for simulating the gene expression cascades of development. To elucidate the temporal dynamics of recombinase action in mammalian cells, we formulated a mathematical model for recombinase expression and catalysis and validated it with experimental data. We found that recombinases have variable expression levels, catalytic rates, and binding affinities, which should be accounted for when designing RGCs. Separately, we designed a platform for engineering novel membrane proteins for inducing specific cell-cell interactions using coiled-coils, called helixCAM. We demonstrated that helixCAMs are capable of inducing patterned cell binding in E. coli, yeast, and human cells, and further utilized a library-on-library approach to engineer new helixCAM-optimized coiled- coils. Taken together, the genetic tools described in this thesis establish groundwork towards hybrid tissue engineering strategies capable of high-resolution patterning while enabling endogenous cell differentiation and cell-cell interactions to form, ultimately serving as a template for engineering large-scale tissue and organs de novo. Thesis Supervisor: Dr. George M. Church Title: Professor of Genetics, Harvard Medical School 3 Page Intentionally Left Black for Double-Sided Printing 4 This thesis is dedicated to the people fighting to maintain truth in a dark era. It is on their shoulders that science can exist. 5 Page Intentionally Left Black for Double-Sided Printing 6 Acknowledgements I still remember my goals from when I first joined the HST Program in 2013: to develop a novel, groundbreaking technology on my own, and to obtain my PhD in 4 years. Here I am, 7 years later, having achieved neither, but certainly better for it. Like it or not, completing a PhD in health sciences is a marathon, a team sport, and a lifestyle. The path to building a body of work is long and cannot be sprinted. Contemporary science is best done collaboratively, sharing expertise and passion. Finally, multiple years of solely performing research is no way to live life, and finding interests outside of the lab, such as traveling and social dancing for me, paradoxically improves productivity within it. Of course, you cannot start a fire without a spark, and, likewise my passion for science can be directly attributed to a nurturing environment from my parents, teachers, and professors along the way who went above and beyond to encourage my curiosity and interests. As such, I would like to thank my teachers and professors who believed in me and nudged me in the right path during my academic journey. Thank you to Mrs. Keshvala, one of my first teachers when we immigrated to the U.S. and perhaps the first teacher I enjoyed learning from. She emphasized the importance of having sound mathematic and scientific foundations, and the impact she had on me by screening Gattaca and October’s Sky in class cannot be overstated. Thank you to Mrs. Roberts, who directed my love for math and engineering towards biological applications and opened my eyes to collaborating with diverse teams. Taking a step back, I would like to thank the all of the WFBHS and NCHS teachers I had the pleasure of learning from, who saw something in me and never provided me with anything less than their full support in my academic pursuits. My confidence in my academic pursuits are to your credit. Throughout my college career at the University of Minnesota – Twin Cities, I had the great fortune to be mentored by incredibly intelligent and caring faculty. The biggest call-out has to be to Dr. Thomas Neufeld, who opened his lab to a freshman even before he took his first class. To this day, I am not sure that was a strategically sound decision, but from that kindness, I was able to learn about the lab environment and become knowledgeable in Drosophila genetics. Through this experience, I was also able to meet Dr. Nakato and through that, have the opportunity to be a visiting researcher at the lab of Dr. Takashi Adachi-Yamada at Gakushuin University in Japan. I also have to thank Dr. Jeffery Gralnick, who is the epitome of the “chill professor.” The research directions that I developed during his class, Engineering Living Systems, have had an outsized impact in defining my future in research (as reflected in this thesis work). He also advised my 2012 iGEM team, hosting us in his lab, helping us fundraise, and bringing us to the iGEM Jamboree East in Pittsburgh. I am proud to have Dr. G. as a colleague and a friend to this day. I would be remiss not to thank Dr. David Matthes for his enthusiasm for science and support for my more ambitious ideas. I am not sure if he remembers this, but his short advice on the value of creating platform technologies when I ran into him outside of Moos Tower, about to bike home, remains with me to this day. I would like to thank Dr. Chad Myers for opening my eyes to how computer science can be applied to intimately understand biology – I still use the concepts and algorithms from his Functional Genomics and Bioinformatics course to this day. Thank you to Dr. Daniel Keefe, who gave me a look into a what “pure” computer 7 science research is like. I would like to thank some of my wonderful administrators from the U. Dean Robert Elde, who made time to meet directly with students, had multiple coffees with me, and even wrote me a letter of recommendation. Sarah Corrigan, my academic advisor, always allowed me to take the courses I wanted and found a way to make it all work. Thank you to Meaghan Stein and Nikki Shultz for the Dean’s Scholars leadership training curriculum. To be honest, I was not expecting the experience to be used so soon, but time and again I find myself breaking out the DiSC assessment, icebreakers, and introspective leadership tools as I work with teams from small to large. Last but not least, an undying gratitude to the Barry Goldwater Scholarship and Excellence in Education Foundation for opening countless doors in my life. I would also like to thank my amazing host family in Japan. Yoshihiro, Etsuko, Yusuke, Sousuke, and Kaoru Fujita. Thank you to all of you opening your door to me and taking much better care of me than I do myself. I will always consider you my family. Also, a big thanks to my CIEE friends, Jaren Shigeta, Korin Redig, and my Aikido friends Kyohei Tada and Chiemi Enomoto. I would like to thank the two professors that opened their door for me to rotate in at the beginning of my PhD journey: Dr. Tim Lu and Dr. Tina Stankovic. From both labs, I took away invaluable knowledge and experience. I would also like to thank Dr. Rick Mitchell and Dr. Bobby Padera for their infectious enthusiasm in teaching the Human Pathology course, which is truly a life changing experience for HST M.D. and PhD students alike. Also, a big thank-you to Dr. Pete Szolovitz for his wonderful course, Medical Informatics, and to Dr. Randy Gollub and Dr.
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