Characterization of Epithelial Progenitors in Normal Human Palatine Tonsils by Sung Yoon Catherine Kang M.Sc., Simon Fraser University, 2010 Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Department of Biomedical Physiology and Kinesiology Faculty of Science Sung Yoon Catherine Kang, 2016 SIMON FRASER UNIVERSITY Summer 2016 Approval Name: Sung Yoon Catherine Kang Degree: Doctor of Philosophy Title: Characterization of epithelial progenitors in normal human palatine tonsils Examining Committee: Chair: Will Cupples Professor Miriam P. Rosin Senior Supervisor Professor Lewei Zhang Supervisor Professor Department of Oral Biological and Medical Sciences University of British Columbia Connie J. Eaves Supervisor Professor Department of Medical Genetics University of British Columbia Sharon Gorski Internal Examiner Associate Professor Department of Molecular Biology and Biochemistry Rama Khokha External Examiner Professor Department of Medical Biophysics University of Toronto Date Defended/Approved: May 17, 2016 ii Ethics Statement iii Abstract The palatine tonsils are a collection of lymph nodes overlaid by stratified non-keratinized epithelium that invaginates deep into the tissue, forming tonsillar “crypts” where ingested and inhaled pathogens are collected and initiate an immune response followed by transepithelial lymphocyte infiltration. The dynamic nature of this site suggests the existence of primitive cells responsible for the constant tissue repair and regeneration; however, such cells in the tonsils have not been characterized. Human Papillomavirus (HPV)-associated cancer of oropharynx is a global health concern that is on the rise, with HPV16 oncoproteins E6/E7 frequently detected in the epithelium of tonsillar crypts. It is hypothesized that the long-term self-renewing progenitor cells are the target of HPV- induced malignancy, but the lack of a method to specifically study these critical cells has been a barrier to further investigation. In this study, I have developed and optimized the methodology to identify, isolate and quantitate epithelial progenitors from human palatine tonsil. I show that tonsillar progenitors that form colonies in vitro in 2D colony assays and differentiate into multilayered epithelial tissues in a 3D culture system are CD44+NGFR+ and present in both surface and crypt regions. Transcriptome analysis indicates a high similarity between CD44+NGFR+ cells in both regions, although those isolated from the crypt contained a higher proportion of the most primitive (holo)clonogenic cells. The method was then applied to study effects of HPV infection on purified CD44+NGFR+ cells from both regions. Lentiviral transduction of CD44+NGFR+ cells with HPV16 E6/E7 oncogenes prolonged their growth in 2D cultures and caused aberrant differentiation in 3D cultures. Interestingly, in the presence of the normal cells, the E6/E7-transduced cells proliferated more slowly in 2D cultures and formed uniquely heterogeneous epithelial structures displaying varying degrees of perturbation in 3D cultures suggesting possible inhibitory effects of the cocultured normal cells. The system developed and presented in this thesis allows for a targeted approach to study a specific subset of epithelial cells purified from the tonsillar crypts and their response to E6/E7 infection, setting the stage for addressing many unanswered questions pertaining to the early stages of tonsillar oncogenesis. Keywords: tonsil; epithelial progenitors; oropharyngeal cancer; HPV iv Dedication To my parents v Acknowledgements This thesis would not have come into existence without the help of a number of people I have been fortunate to work with. First, I would like to thank my supervisor, Dr. Miriam Rosin, for giving me the opportunity to take on this unique project and for her continuous support, thoughtfulness and encouragement throughout this journey. I was always inspired by her unceasing curiosity and her passion to make a difference. I also would like to thank my thesis committee members, Dr. Connie Eaves and Dr. Lewei Zhang, for their expertise and insightful comments that helped guide this research project, but also for the hard questions that taught me how to think and critique. I am truly indebted to my colleague, Dr. Nagarajan Kannan, for his resourcefulness, patience and support, scientifically and morally, from the very start of my doctoral training. I would not have made it this far if it had not been for all those hours of discussions and his timely, helpful replies to numerous emails. Thank you, Raj. I would like to acknowledge the excellent support of Ivan Sun with immunohistochemistry work and tonsil sample acquisition. I would like to thank the rest of the Rosin lab, Dr. Denise Laronde, Dr. Tarinee Lubpairee, Dr. Ajit Auluck, Leigha Rock, Jade Lavallee, Salima Walji, Marco Wu and anyone else that I may have missed, for their support, input and encouragement at every milestone. I thank Helen Chiu, Anita Fang, Huijun Jiang, Emily Morgan, Sarah Gustin, and Arla Yost for the administrative support. I would like to thank the surgeons, pathologists, and technologists for their assistance with obtaining tonsillectomy samples: Dr. Scott Durham, Dr. Ken Berean, Dr. Robert O’Connor, Dr. Matthew Dickson, Dr. Patricia Lee, Dr. Paul Mick, Dr. Donald Mintz, Shabniz Ramji and Margaret Luk. I thank Dr. Victor Martinez for his help with transcriptome analysis, and the staff of the FACS facility at the BC Cancer Research Centre, Gayle Thornbury, Wenbo Xu and David Ko, for their assistance with cell sorting and analysis. vi I also gratefully acknowledge the generous advice and assistance I received from the staff at the Eaves lab: Darcy Wilkinson for sharing her experience in tissue processing with me when I first started, Glenn Edin for preparing the lentivirus, and Margaret Hale for her help with some of the molecular biology experiments. I would also like to express my appreciation to the Mammary team in the Eaves lab, past and present, in particular, Dr. Maisam Makarem, Dr. Long Nguyen, Dr. Peter Eirew, Dr. Davide Pellacani, Dr. Sylvain Lefort and Sneha Balani, for the discussions, invaluable suggestions and input. Finally, my dearest gratitude goes to my parents, for all their sacrifice, support and prayers, especially during the last stage of my graduate studies when I had to face a major medical decision. I am deeply grateful for the health I have regained and for the successful completion of this dissertation in spite of the unexpected challenges, and I owe this to their love and support, physically and emotionally. Last but foremost, I thank God for giving me the strength to carry on even when it seemed impossible. vii Table of Contents Approval .......................................................................................................................... ii Ethics Statement ............................................................................................................ iii Abstract .......................................................................................................................... iv Dedication ....................................................................................................................... v Acknowledgements ........................................................................................................ vi Table of Contents .......................................................................................................... viii List of Tables .................................................................................................................. xi List of Figures................................................................................................................ xiii List of Acronyms ............................................................................................................ xv Chapter 1. Introduction ............................................................................................. 1 1.1. Palatine tonsils ....................................................................................................... 1 1.1.1. Anatomy and physiology of the palatine tonsils ......................................... 1 1.1.2. Development of the oral cavity ................................................................... 2 1.1.3. Development of the tonsils ........................................................................ 3 1.2. Oral and tonsillar epithelium ................................................................................... 3 1.2.1. Oral epithelium .......................................................................................... 3 1.2.2. Tonsillar epithelium .................................................................................... 4 1.3. Stem cells ............................................................................................................... 5 1.3.1. Adult stem cells ......................................................................................... 5 1.3.2. Stem cell niche .......................................................................................... 6 1.3.3. Stem cell hierarchy in other tissues ........................................................... 7 1.4. Detection, isolation and quantification of epithelial stem/progenitor cells ................ 8 1.4.1. Qualitative methods ................................................................................... 8 Label-retaining