MAKING MAMMALIAN STEM CELLS: IDENTIFYING AND OVERCOMING REPROGRAMMING BARRIERS By Anthony M. Parenti A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements for the degree of Cell and Molecular Biology - Doctor of Philosophy 2016 ABSTRACT MAKING MAMMALIAN STEM CELLS: IDENTIFYING AND OVERCOMING REPROGRAMMING BARRIERS By Anthony M. Parenti The field of stem cell biology had its first major boon when embryonic stem cells (ESCs) were derived from a mouse blastocyst in the 1980’s. ESCs have the potential to form any type of cell in the body, and thus represent a powerful new tool to study and treat a number of diseases that plague modern society. Despite the potential advantages ESCs offer, an embryo is destroyed in the derivation process, which leads to many ethical objections. Further, ESCs are not an exact genetic match to the patient they would be put into, which may lead to problems of graft rejection like we observe with organ transplantation. In 2006, a group of scientists made a revolutionary discovery when they expanded upon the trailblazing efforts of others, who employed somatic cell nuclear transfer and transcription factor based lineage conversion, to discover that a fully differentiated cell could be driven back to an embryonic state through forced expression of four transcription factors: Oct4, Sox2, Klf4, and cMyc (OSKM). These induced pluripotent stem cells (iPSCs) can also become any type of cell in the body and are identical to ESCs in many ways, but have the advantage of being derived from the patient they would be put back into, and do not require the destruction of an embryo. iPSCs offer the ideal tool to study and treat many different diseases including Alzheimer’s, Parkinson’s, diabetes, Huntington’s, and Huntington-Gilford Progeria Syndrome among many others. Despite the potential for iPSCs, they remain extremely hard to produce. Various reports have described “barriers to reprogramming” that inhibit the conversion of differentiated cells to iPSCs. In the chapters that follow, I present my work uncovering previously unknown barriers to iPSC reprogramming including the formation of a different stem cell type during OSKM mediated reprogramming. Further, I detail my findings that examine the impact of aging on iPSC reprogramming and my findings that cells derived from aged individuals are not rejuvenated during the iPSC reprogramming process as previously hypothesized, but instead maintain the functional defects of old cells. The work presented herein represents my efforts to uncover the mechanisms underlying OSKM reprogramming. Many previously-held conceptions about OSKM reprogramming are not supported by my findings and need to be reassessed. Further, my work should serve as the launching point for future studies aimed at improving iPSC reprogramming efficiency and quality. ACKNOWLEDGEMENTS Jennifer Parenti – You were by my side through the highs and lows and have a special way of helping me see the positive in life. Thank you for remaining remarkably calm when I came home one day to tell you that we would be moving our family to the Midwest and for being an amazing mother to our children throughout this endeavor. I know it has not been easy, but I could not have accomplished this without you and cannot imagine another person I would want to share this moment with. I love you more everyday and know that I am lucky to have found you. Mary and Lucy Parenti – You give me a reason to smile and something to look forward to each day. Watching you grow up has been the highlight of my life. I hope that you are happy and that I have set a good example for you to follow in whatever your futures hold. You will never know how much I love you. Paul and Kathy Parenti – You taught me the value of hard work and family, I strive everyday to live up to the example you set. The strength and grace you display while dealing with your personal struggle is inspiring and gives me a hope for the future. At various times on this graduate school journey, you put a roof over my head and took care of our children. I am forever grateful and will always love you. D.J. Parenti – You were my sounding board for various issues over the years and have been doing the duty of two sons in my absence. Thank you for being there for me, I love you. Jim and Karen Hall – At various times you offered Jen and I a place to live and cared for our children while we navigated this confusing and convoluted process and I am truly iv grateful. I don’t know what we would have done without your support. Please know that I will never forget the generosity and love you showed us. David Fernandes – Thank you for taking time to visit us throughout my time in graduate school, we enjoy every visit. You have a good natured attitude that brightens our moods. Your knowledge and skill were also invaluable throughout our home renovation process and we could not have completed our projects without your sweat and input. I admire your courage and work ethic and could not be happier to be you son in law. Dr. David Arnosti, Dr. Monique Floer, Dr. Jason Knott, and Dr. Keith Latham – Thank you for serving as members of my thesis committee. Each of you offered valuable insight, pushed me to progress as a scientist, and made suggestions that improved the quality of my work. Dr. Keith Latham and Dr. Kai Wang – You made valuable experimental and intellectual contributions to my thesis work and I do not know how I would have completed the in vivo developmental experiments without your help. Dr. Yi Zuo and Dr. Min Fu – Thank you for providing aged mice for my aging/reprogramming work. You saved us at least 2 years of time and allowed our research to progress. Dr. Stephanie Blij – Thank you for being my first true lab friend. I cherished our conversations and debates and hope we cross paths in the future. Dr. Tristan Frum – You served as my debate opponent, comedic foil, occasional golfing partner, and friend for the better part of 6 years. Thank you for helping to lighten my mood and remind me that science is fun. v Dr. Michael Halbisen – You were the first person in the lab to take me under your wing and show me how to be a scientist. I enjoyed working with you and arguing with you about topics that ranged from the origins of the U.S. Navy to “Which city sucks the most?”. I would also like to thank you for the opportunity to work on my physical fitness outside of the traditional gym environment when we moved trees, radiators, home contents, etc… Dr. Alyson Lokken – You are truly one of the nicest people I have ever met and it makes me feel like I am a better person because I can call you a friend. Thank you for being there to listen to my myriad complaints and always finding a positive way to spin the situation. I know your work ethic and intelligence will propel you to success in all your future endeavors. Dr. Amy Ralston – Working for you over the past 6 years has been an honor, thank you for taking a chance on me. You taught me how to think critically and helped to give me a sense of confidence I have often lacked. I do not know what compelled you to offer me a space in your lab, but I am glad to have played a part, however small, in your success. You are a brilliant scientist and I know you will find continued success in the future. vi TABLE OF CONTENTS LIST OF TABLES xi LIST OF FIGURES xii KEY TO ABBREVIATIONS xv Chapter 1 Mammalian Development, Stem Cells, Reprogramming, and Aging: A Historical Perspective 1 Abstract 2 Section 1. The Mammalian Blastocyst and Embryo Derived Stem Cells 4 The Mammalian Blastocyst 4 Embryo-Derived Stem Cells 5 Section 2. Somatic Cell Nuclear Transfer 7 Section 3. Cell Fate Conversion and the Discovery of Induced Pluripotency 9 Transcription Factor Based Cell Fate Conversion 9 The Discovery of Induced Pluripotency 10 Problems associated with iPSCs and Barriers to the Acquisition of Pluripotency 11 Section 4. The Role of OSKM in Development 13 Section 5. Aging and Questions of Stem Cell Quality 15 APPENDIX 18 REFERENCES 24 Chapter 2 OSKM induce extraembryonic endoderm stem (iXEN) cells in parallel to iPSCs 35 Abstract 36 Section 1. Introduction 37 Section 2. Materials and Methods 38 Mouse Strains 38 Fibroblast preparations 39 Reprogramming 39 XEN cell derivation and culture 40 Immunofluorescence and flow cytometry 40 RNA sequencing and qPCR 41 XEN/iXEN in vitro differentiation 43 iXEN/XEN/iPSC/ESC in vivo differentiation 43 Lineage Tracing 44 Viral Genotyping 44 shRNA Cloning and Testing 45 Proximity 45 vii Statistical Analyses 45 Section 3. Results 46 iXEN cells display XEN cell morphology and gene expression 46 MEF-derived XEN cells exhibit stem cell properties 48 iXEN cells are not derived from pre-existing iPSC colonies 49 All four reprogramming factors induce XEN cell fate 51 GATA6 and GATA4 facilitate iXEN, but not iPSC, cell formation 52 Section 4. Discussion 54 Comparison of Parenti et al. and Zhao et al. 55 What role does MEF heterogeneity play in the establishment of iXEN and iPSCs? 58 How do iXEN and cXEN compare to embryo-derived XEN? 59 Can iXEN contribute to Visceral Endoderm in vivo? 60 Can iXEN cells be derived from human cells and used as a model to study human extraembryonic development? 62 Acknowledgments 62 APPENDIX 63 REFERENCES 78 Chapter 3 iXEN and iPSC reprogramming is influenced by extrinsic and intrinsic factors 85 Abstract 86 Section 1.