THE ROLE OF OSTEOCYTES IN DISUSE AND MICROGRAVITY-INDUCED BONE LOSS byMASCUETINTTT AFTCHNOG Jordan Matthew Spatz B.S., M.S. University of Colorado at Boulder SEP 2 4 2015 Submitted to the LIBRARIES Harvard-MIT Program in Health Sciences and Technology in Partial Fulfillment of the Requirements for the Degree of I DOCTOR OF PHILOSOPHY IN HEALTH SCIENCES AND TECHNOLOGY at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY September 2015 2015 Massachusetts Institute of Technology. All rights reserved. Signature of Author: Signature redacted Harvard-MIT Program in Health Sciences and Technology September 1st, 2015 Certified by: Signature redacted Mary L. Bouxsein, Ph.D. Associate Professor of Orthopedic Surgery, Harvard Medical School Thesis Supervisor A A Certified by: Signature redacted ___ i aola Divieti Pajevic, MD, Ph.D. Ass ciate Professor of Iolecular and Cell, Boston University Thesiy Supervisor Signature redacted Accepted byr. Emery N. Brown, MD, Ph.D. Director, Harv d-MIT Program in Health Sciences and Technology Professor of Computational Neuroscience & Health Sciences and Technology 2 The Role of Osteocytes in Disuse and Microgravity-Induced Bone Loss by Jordan Matthew Spatz B.S., M.S. University of Colorado at Boulder Submitted to the Harvard-MIT Health Sciences and Technology September 2015, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Health Sciences and Technology Abstract A human mission to Mars will be physically demanding and presents a variety of medical risks to crewmembers. It has been recognized for over a century that loading is fundamental for bone health, and that reduced loading, as in prolonged bed rest or space flight, leads to bone loss. Osteocytes, the most abundant bone cell type, are thought to be key mechanical sensors in bone, yet the molecular mechanism of this action remains poorly understood. Improved understanding of how osteocytes regulate skeletal responses to mechanical loading and unloading could have significant implications for treatment of bone disorders related to disuse or immobilization. Thus, we conducted in vitro and in vivo studies on osteocytes exposed to unloading to investigate their role in disuse and microgravity-induced bone loss. Specifically, we generated and characterized a novel osteocytic cell line that recapitulates the response to hormonal and mechanical stimuli of osteocytes in vivo. This novel cell line provided the first evidence of a cell-autonomous increase in sclerostin, a potent inhibitor of Wnt- signaling, following exposure to simulated microgravity. These cells were also used for a spaceflight mission after demonstrating their ability to maintain an osteocytic phenotype when cultured in a fully automated flight-certified system. Finally, we utilized murine models of unloading to show that pharmacologic inhibition of sclerostin induces bone formation and prevents disuse-induced bone loss. Thesis Supervisor: Mary L. Bouxsein, Ph.D. Associate Professor of Orthopedic Surgery, Harvard Medical School Thesis Supervisor: Paola Divieti Pajevic, MD, Ph.D. Associate Professor of Molecular and Cell Biology, Boston University 3 Thesis Committee Laurence R. Young, Sc.D (Chair) Apollo Program Professor of Astronautics and Professor of Health Sciences and Technology, Director of HST PhD Program in Bioastronautics Mary L. Bouxsein, Ph.D. (Thesis Supervisor) Associate Professor of Orthopedic Surgery, Harvard Medical School Associate Biologist, Endocrine Division, Massachusetts General Hospital Adjunct Assistant Professor, Department of Mechanical Engineering, Boston University Faculty Member, Bioastronautics Program, Harvard-MIT Division of Health Sciences and Technology Paola Divieti Pajevic, MD, Ph.D. (Thesis Supervisor) Associate Professor of Molecular and Cell Biology, Goldman School of Dental Medicine, Boston University Associate Biologist, Endocrine Unit, Massachusetts General Hospital Jeffrey M Karp, Ph.D. (Thesis Reader) Associate Professor, Harvard Medical School Co-Director, Center for Regenerative Therapeutics, Brigham and Women's Hospital 4 Acknowledgments In an effort that spans so many years and milestones there are many to be thanked. My graduate school years would be nothing were it not for those who encouraged me to follow my dreams, supported my development academically, personally, and as a scientist throughout this process. To my advisors: for always having an open-door, putting my career at the top of your list, and answering my endless questions no matter your other priorities. Mary Bouxsein: I will always remember our afternoon advisor conversations and hope to live up to your passion for science to do my part to make the world a better place. Your dedication to always have my back, my career development as a person, and a scientist has been something I will carry forward with me throughout my life and career. Paola Divieti Pajevic: I could not have known from coming to your lab as engineer for help to learn to grow osteocytes how much I would learn from you. I cannot thank you enough for passionately teaching me the amazing science of biology that I gained from working side-by-side with you at the bench and how to always do the best science. Thank you for the courage to allow me to try crazy ideas and for sticking with me throughout this learning journey. Jeff Karp: Thank you for imparting to me your forward thinking and progressive perspective on advancing science throughout my M.I.T. graduate studies. I will carry with, throughout my career, your advice to change the calculus when presented with an otherwise intractable problem and remember your advice on how to think outside the box. 5 Larry Young: My passion for bioastronautics would not have led to me to M.I.T. without your foresight to believe in me. Thank you for enriching and enhancing my passion for aerospace medicine and the human exploration of the universe. To my funding sources: Northrop Grumman Aerospace Systems Ph.D. Training Fellowship, M.I.T. Hugh Hampton Young Fellowship, the National Space Biomedical Research Institute through NASA NCC 9-58, Beth Israel Deaconess Medical Center Translational Research in Aging Training Program, and the U.S. Army Institute for Environmental Medicine Oak Ridge Science Institute for Science and Education fellowship program. To my NASA mentors: Jean Sibonga, Scott Smith, Zara Smith, and Honglu Wu thank you for mentorship and hosting my research at the NASA Johnson Spaceflight Center. To my U.S. Army Institute of Environmental Medicine mentors: Julie Hughes and Wayne Matheny thank you for the mentorship, friendship, and support. To my colleagues: that helped make this thesis a reality by conducting experiments with me throughout my graduate work, but especially Rachel Ellman, Keertik Fulzele, Yili Qu, Shawn Liu, Chris Dedic, Forest Lai, Jonathan Gooi, Alison Cloutier, Leeann Louis, Miranda Van Vliet, Daniel Brooks, and Jenna Garr thank you from the bottom of my heart for the long hours and tireless dedication. To Hank Kronenberg: thank you for always spending time to provide deep insight and teachings, shadowing in clinic, and for advice all things endocrine over the many years at M.G.H. To Marc Wein: your starting to work with the osteocyte cell line during the mid- stretch of my thesis studies was like having an all-star baseball reliever in 6 baseball coming out of the bullpen with the bases loaded and nobody out. Thank you for all the career mentorship, opportunity to work with you both at the bench and the bedside, and always spending time to help make me a better scientist. To Chris Adamson, Lowell Misener, Margaret Eberle, and the rest of the Calm Technologies team: thank for just being awesome for many years of dedication and answering of my endless questions on the Osteo-4 project. To my fellow bioastronautics and M.I.T. manned vehicle colleagues: Dava Newman, Alan Natapoff, Leia Sterling, Alexander Bruno, Dan Buckland, Katelyn Burkhart, Dustin Kendrick, Conor Culliane, Nikhil Vadhavkar, Justin Kaderka, Aaron Johnson, Allie Anderson, Torin Clark, and Erika Wagner thank you for your friendship and long conversations about human space exploration and aerospace medicine. To my M.G.H. endocrine unit colleagues: Lynn Moulton, Julia Maclaughlin, Latanya Turner, Leslie Johnson, Marie Demay, Tatsuya Kobayashi, John Potts, Henry Keutmann, Tom Gardella, Harald Jueppner, Kelly Lauter, Melissa Putnam, Paula Cohen at Beth Israel Deaconess Medical Center, and Elizabeth Zotos at M.I.T. this thesis would not have occurred without your tireless efforts of support over the many years of my graduate studies. To my Northrop Grumman colleagues: thank you for the continued dedicated support throughout my graduate school years at M.I.T. To the National Space Biomedical Research Institute (NSBRI): thanks to educational support and encouragement from Dr. Jeff Sutton, Dr. John Clark, and Amanda Hackler. And last but first in my heart, to my friends and family: for all your love and support! 7 8 Biographical Note Jordan Matthew Spatz was born in Fontana, California and raised in Los Angeles. From 2001 to 2006, he attended the University of Colorado at Boulder, earning Masters and Bachelors degrees in Aerospace Engineering Sciences. In 2006, Jordan joined Northrop Grumman Corporation as an aerospace systems engineer for two years prior to starting his doctoral studies at the Massachusetts Institute of Technology. During his tenure at M.I.T. Jordan also lead an Engineers Without Borders project to build a preschool in the Palaung hill tribe village, Ban Nor Lae, in the province of Chiang Mai, Thailand. He held fellowships from Northrop Grumman Corporation, M.I.T. Hugh Hampton Young, the National Space Biomedical Research Institute, Beth Israel Deaconess Medical Center Translational Research in Aging Training Program, and the U.S. Army Institute for Environmental Medicine Oak Ridge Science Institute for Science and Education. 9 "We found ourselves bidding goodbye to the old learn-by-heart method, and beginning the study of observing the facts and laws of nature. We learned from experiment and experience what might be expected to happen if a given set of forces started to act.