Induced pluripotency and reprogramming by defined factors

The process by which the totipotent zygote undergoes development into an adult organism using a single genome is the foundation for epigenetics and cellular differentiation. Somatic cell nuclear transfer experiments (SCNT) provided unequivocal proof of nuclear equivalency between adult somatic cells. However the molecular mechanisms of somatic cell reprogramming have remained largely a mystery. Recent advancements in epigenetic reprogramming by defined factors provide new opportunities to explore factors that regulate induction of pluripotency in somatic cells. Nuclear reprogramming by SCNT occurs in an 'indirect' manner by unidentified components within oocyte cytoplasm and requires the destruction of embryos. The introduction of induced pluripotent stem cells (iPS cells) and 'direct' reprogramming methods created a tractable system to both study of the process in vitro and potentially derive personalized pluripotent stem cells free of the practical and ethical concerns surrounding embryonic stem (ES) cells and SCNT. Herein we study mouse somatic cell reprogramming by defined factors and develop novel tools to compare the induced pluripotent state to the gold standard of pluripotency, ES cells. First, we designed reprogramming vectors that minimize the number of viruses required to generate iPS cells, yielding pluripotent cells with minimal genomic alterations from reprogramming factors. This allowed the creation of transgenic "reprogrammable mouse" strains after gene targeting in ES cells, providing a renewable source of somatic cells that can be induced to pluripotency by addition of a drug. In addition we can easily introduce or mate these strains to study unique genetic variants during reprogramming. Third, we study factors that influence the induced pluripotent state, specifically how to generate pluripotent cells with all properties of embryonic stem cells including derivation of "all iPS cell mice" by tetraploid complementation assays. In contrast to previous reports, we find the majority (80%) of iPS cell lines derived from adult somatic cells of varying organs contain the developmental potential of ES cells. This outcome correlated with high expression of Oct4 and Klf4 and low expression of Sox2 and c-Myc during reprogramming. In addition we report that adult mice derived from iPS cells are healthy and do not develop tumors. Together these results suggest in vitro reprogramming to pluripotency by defined factors holds great promise for regenerative medicine. Thesis supervisor: Dr. Rudolf Jaenisch Title: Professor of Biology Dedication: I would like to dedicate this to my father James R. Carey for his endless encouragement and support of my pursuits towards a life conducting scientific research. He endures as a model for me both personally and academically as someone who lives with great integrity, absolute honesty and strength of character. Acknowledgements: I would like to first thank Rudolf for taking me into his lab. It has been a remarkable experience I will never forget. He and all lab members were extremely welcoming upon arrival and provided me with a fantastic, highly supportive environment to conduct my thesis research. Although conducted in a super charged, internationally competitive time surrounding the discovery of induced pluripotent stem cells, Rudolf always maintained his patience, support, and encouragement no matter what was going on around us. His steadfast leadership and long-term commitment ensured the success of all of my projects. I also have had the privilege of working alongside a good friend and colleague Jacob Hanna, who I would like to thank. His keen insights and vision on many projects kept me from veering off course numerous times. His focus and determination to do the right experiment were an inspiration throughout my time in the Jaenisch lab. These experiments would also not have been possible without several other close colleagues who have also become good friends. I would not have got off the ground without the help of Marius Wernig. His efforts to establish reprogramming technologies in the Jaenisch lab propelled many careers forward including my own, teaching me my first reprogramming experiment and helping me design my first reprogramming vectors. I would like to thank Stella Markoulaki who I worked with on every project to generate mice. She remains a strong supporter and someone who I can always rely on. Caroline Beard provided invaluable help in the beginning stages of my projects; her keen advice and unparalled knowledge of molecular biology will always be remembered as an invaluable asset to the Jaenisch lab. Kibibi Gantz is also someone who helped me in the mouse-generating department. I appreciate her dedication and feel she is a great new addition to the Jaenisch Lab. I would also like to thank Maya Mitalipova who introduced me to human stem cell work. She provided crucial support in the early years of my project. In addition I held close collaborations with Menno Creyghton and Albert Cheng who taught me genome-wide mapping technologies, always showing patience and dedication to our questions. Along the way I was supported experimentally or stimulated intellectually from many post-docs and grad students such as Frank Soldner, Chris Lengner, Kris Saha, JP Kim, Mike Lodato, Leah Medieros, John Cassady, Dina Faddah, Grant Welstead and always received support in some form or another by anyone who I asked for help. This is something that is often taken for granted in the Jaenisch lab! Most importantly I want to thank the person who kept me sane throughout this process, my fianc Leah Cohen. She has been my most steadfast supporter at all times and continually helped me stay balanced outside of lab. I feel so very lucky to have her in my life. Finally I want to thank my thesis committee members Tyler Jacks, Peter Reddien and Laurie Boyer. They always encouraged me to keep at it and to be patient so I can do the right experiment. Their skepticism also helped me stay on course at critical junctures. In addition I want to thank Alex Meissner for returning to the Whitehead to generously serve as my outside committee member.